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The role of αvβ6 integrin in epidermal wound healing Al-Dahlawi, Salwa 2004

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THE R O L E OF avf6 INTEGRIN IN EPIDERMAL WOUND HEALING  by SALWA A L DAHLAWI  BDS., King Abdulaziz University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biological and Medical Sciences) We accept this thosi$ as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA June 2004 © Salwa Al Dahlawi, 2004  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Name of Author (please print)  Title of Thesis:  Degree:  Date (dd/mm/yyyy)  of  ^ ve< oE QS  «.Mp>,C  vAecyf;^  S^c^.  Department of o/qi h i c ^ o / y c ^ The University of British Columbia Vancouver, BC Canada  in  Y  o.™A w ^ ; ^  Ae<^a'  e  a  Sc^ceA).  r  :  HecLv^  ?- ^ eo  ABSTRACT The avp6 integrin is an exclusively epithelial integrin that is highly expressed during fetal development. In adult tissue av(36 integrin is re-expressed during inflammation, carcinogenesis and in wound healing. T h e objective o f this study was to investigate whether compromised wound healing by hydrocortisone treatment is altered in mice deficient or overexpressing the p6 subunit in comparison to wild type littermates. Three groups o f age and sex matched mice were used; wild type ( W T ) , p6 integrin deficient (p6~ ), and human 06 integrin overexpressing transgenic mice (h|36Fl). E a c h A  group was  subdivided into untreated (control) and treatment  groups. Treatment  groups received a daily intraperitoneal injection o f 1 m g o f hydrocortisone for 13 days. Excisional 4 m m wounds were made on the dorsal surface o f the mice and wound healing was evaluated daily. Comparisons between different groups were made from the histological analysis o f three and ten days wound healing. A g e d (22month-old) 06 integrin deficient (P6" ) animals showed a significant delay in wound /_  healing when compared to their aged matched wild type animals. The most significant delay was observed at the stages where significant granulation tissue formation is occurring (four to seven days post wounding). Hydrocortisone treatment significantly delayed wound healing in w i l d type and P6 integrin deficient mice in comparison to the untreated controls. However, hydrocortisone treatment in human P6 integrin overexpressing ( h p 6 F l ) animals did not cause a significant delay in wound healing. The results o f this study indicated that the presence  of  avP6  integrin plays an  important role in wound healing in animals compromised by either age or stress.  ii  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF FIGURES  .  .  ACKNOWLEDGMENTS  vii xii  CHAPTER 1-REVIEW OF THE LITERATURE  1  1.1 Epithelial integrins  1  1.2 Epithelial integrins and inflammation  7  1.3 Growth factors and wound healing  10  1.4 Introduction to wound healing  28  1.5 Re-epithelialization  29  1.6 Fibroplasia  32  1.7 Psychological stress and the immune system  36  1.8 Stress and wound healing  42  CHAPTER 2-ATM OF T H E STUDY  48  CHAPTER 3-MATERIAL AND METHODS  49  3.1 Excisional wound healing  49  3.2 Clinical wound healing  50  iii  3.3 Histology and immunohistochemical staining  50  CHAPTER 4-RESULTS  53  4.1 Wound healing in adult wild type animals (6-month-old)  53  4.2 Wound healing in aged wild type animals (22-month-old)  56  4.3 Comparison of wound healing in wild type animals in relation to age. ..58 4.4 Wound healing in adult |36 integrin deficient (p6 ~) animals (6-month_/  old)  61  4.5 Wound healing in aged p6 integrin deficient (P6 ~) animals (22-monthV  old)  66  4.6 Comparison of wound healing in p6 integrin deficient (P6" ) animals in A  relation to age  71  4.7 Wound healing in human P6 integrin overexpressing (hp6Fl) animals (22-month-old)  74  4.8 Histology of 3-day-old wound healing  79  4.9 Histology of 10-day-old wound healing  88  4.10 Expression of type IV collagen  93  4.11 Expression of P6 integrin subunit  93  4.12 Immunolocalization of TGFP isoforms  94  CHAPTER 5-DISCUSSION  101  5.1 Wound healing and glucocorticoid treatment  101  iv  5.2 Wound healing in 0 6 deficient animals  104  5.3 Wound healing in 0 6 integrin overexpressing animals  109  5.4 Role of mononuclear cells in wound healing  113  5.5 Limitations of the study  116  CHAPTER 6-CONCLUSIONS  117  APPENDIX A  119  A - l Animals information, the control group (6-month-old)  119  A-2 Animals information, the experimental group (6-month-old)  120  A-3 Animals information, the control group (22-month-old)  121  A-4 Animals information, the experimental group (22-month-old)  122  APPENDIX B  123  B - l .Relative wound area in untreated adult wild type animals  123  B-2 Relative wound area in treated adult wild type animals  124  B-3 Relative wound area in untreated aged wild type animals  125  B-4 Relative wound area in treated aged wild type animals  126  B-5 Relative wound area in untreated adult 0 6 integrin deficient animals  (06 ) 7  127  B-6 Relative wound area in treated adult 06 integrin deficient (06" ) /_  animals  128  v  B-7 Relative wound area in untreated aged |36 integrin deficient (p6" ) /_  animals  129  B-8 Relative wound area in treated aged p6 integrin deficient (fJ6" ) A  animals  130  B-9 Relative wound area in untreated human p6 integrin overexpressing (hp6Fl) animals  131  B - l Relative wound area in treated human P6 integrin overexpressing (hp6Fl) animals  132  REFERENCES  ..133  vi  LIST OF FIGURES Figure 1. Clinical photographs showing wound healing over time in adult (6month-old) wild type (WT) animals in control and treatment groups 55 Figure 2. Clinical photographs showing wound healing over time in aged (22-month-old) wild type (WT) animals in control and treatment groups 57 Figure 3. Mean and standard error of wound size changes over time in wild type animals. Comparison between the adult (6-month-old) and the aged (22-month-old) mice in control and treatment groups 59 Figure 4. H 2 O 2 test in wild type animals comparing different age groups. Percentage of wounds reacting positively to the test over time 60 Figure 5. Clinical photographs showing wound healing over time in adult (6month-old) 06 integrin deficient (p6 ~) animals in control and treatment groups 63 _/  Figure 6. Mean and standard error of wound size changes over time in adult (6-month-old) wild type and p6 integrin deficient (p6~ ) animals. Comparison between control and treatment groups 64 /_  Figure 7. H 0 test in adult (6-month-old) f>6 integrin deficient (p6~ ) animals in comparison to age matched wild type animals 65 A  2  2  Figure 8. Clinical photographs showing wound healing over time in aged /_ (22-month-old) p6 integrin deficient (P6" ) animals in control and treatment groups 68 Figure 9. Mean and standard error of wound size changes over time in aged (22-month-old) wild type and P6 integrin deficient (P6") animals. Comparison between control and treatment groups .69 /_  Figure 10. H 0 test in aged (22-month-old) p6 integrin deficient (p6 ~) animals in comparison to age matched wild type animals 70 _/  2  2  Vll  Figure 11. Mean and standard error of wound size changes over time in (36 integrin deficient (P6" ) animals. Comparison between the adult (6-monthold) and the aged (22-month-old) mice in control and treatment groups 72 7  Figure 12. H 2 O 2 test in (36 integrin deficient (p6" ) animals comparing different age groups. Percentage of wounds reacting positively to the test overtime 73 A  Figure 13. Clinical photographs showing wound healing over time in human (36 integrin overexpressing (h(36Fl) animals in control and treatment groups 76 Figure 14. Mean and standard error of wound size changes over time in human P6 integrin overexpressing (hp6Fl) and wild type mice. Comparison between the control and treatment groups 77 Figure 15. Percentage of wounds positive to H 2 O 2 over time. Comparison between aged wild type and human P6 integrin overexpressing (hp6Fl) mice 78 Figure 16. Hematoxylin stained sections of 3-day-old wounds in untreated wild type mice (WTcon). An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, CT: Connective tissue, BC: Blood clot, F: Fat cells, arrows point to the tip of migrating epithelium 81 Figure 17. Hematoxylin stained sections of 3-day-old wounds in untreated P6 integrin deficient (P6" con) mice. An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, HF: Hair follicle, arrows point to the tip of migrating epithelium 82 A  Figure 18. Hematoxylin stained sections of 3-day-old wounds in untreated human P6 integrin transgenic (hp6Flcon) mice. An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, arrows point to the tip of migrating epithelium 83  viii  Figure 19. Hematoxylin stained sections of 3-day-old wounds in wild type mice treated by hydrocortisone (WTTx). An overview of the wound (A) showing the minimum migration of the epithelium at the wound edges; magnified sections showing the tip of migrating epithelium (B and C). E : Epithelium, CT: Connective tissue, B C : Blood clot, F: Fat cells, arrows point to the tip of migrating epithelium 84 Figure 20. Hematoxylin stained sections of 3-day-old wounds in 0 6 integrin deficient mice treated by hydrocortisone (06 ~Tx). An overview of the wound (A) showing the minimum migration of the epithelium at the wound edges; magnified sections showing the tip of migrating epithelium (B and C). E : Epithelium, GT: Granulation tissue, CT: Connective tissue, B C : Blood clot, arrows point to the tip of migrating epithelium 85 _/  Figure 21. Hematoxylin stained sections of 3-day-old in (36 integrin overexpressing mice treated by hydrocortisone (06FlTx). An overview of the wound (A) showing advanced epithelial migration as compared to the other treatment groups. Magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, arrows point to the tip of migrating epithelium 86 Figure 22. Mean and standard error of epithelial cell migration in 3-day-old wounds 87 Figure 23. Histological sections of 10-day-old wounds in wild type (WT) mice. Untreated wounds (A and B) and treated wounds (C and D ) . Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D ) . Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer, F: Fat cells 90 Figure 24. Histological section in 10-day-old wounds in 06 integrin deficient ( 0 6 " ) mice. Untreated wounds (A and B) and treated wounds (C and D ) . Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D ) . Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer, F: Fat cells 91 A  IX  Figure 25. Histological sections of 10-day-old wounds in 06 integrin overexpressing (h06Fl) mice. Untreated wounds (A and B) and treated wounds (C and D). Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D). Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer 92 Figure 26. hnmunohistochemical localization of type IV collagen in 3-dayold wounds in treated (A, C, E) and untreated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h|36Fl) mice (C and D), and 06 integrin deficient (06~ ) mice (E and F). E: Epithelium, CT: Connective tissue. Arrows point to the tip of migrating epithelium, arrow heads point to the end of positive staining for type IV collagen 96 A  Figure 27. hnmunohistochemical localization of type IV collagen in 10-dayold wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D), and 06 integrin deficient (06") mice (E and F). Results show the complete restoration of type IV collagen staining at the basement membrane zone. E: Epithelium, CT: Connective tissue 97 Figure 28. hnmunohistochemical localization of 06 integrin in 3-day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D), and 06 integrin deficient (06"~) mice (E and F). E : Epithelium, CT: Connective tissue, HF: Hair follicle, arrows point to the tip of migrating epithelium, arrow heads point to cells with positive staining 98 /  Figure 29. hnmunohistochemical localization of 06 integrin in 10-day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D), and 06 integrin deficient (06" ) mice (E and F). E: Epithelium, CT: Connective tissue 99 /_  Figure 30. hnmunohistochemical localization of TGF0 in 10-day-old wounds. Untreated (A, C, E) and treated (B, D , F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D),  and 06 integrin deficient (p6 ) mice (E and F). E: Epithelium, CT Connective tissue, WCT: Wound connective tissue 100  XI  ACKNOWLEDGEMENTS  Dedicated to my father whose love and nurturing made this all possible And to Hani for all his support and love  Chapter 1-Review of the literature 1.1 Epithelial integrins Integrins are a large family o f heterodimeric transmembrane glycoproteins that were initially defined as receptors for the extracellular matrix ( E C M ) . Integrins are now known to function as signalling receptors, participating in many cellular activities  including spreading,  migration, proliferation and  differentiation.  Furthermore, integrin-ligand binding effects the expression o f a number of other genes including cytokines and metalloproteinase (Watt, 2002). Members o f the integrins family are expressed in every cell except red blood cells. It is likely that different integrins can direct different cellular responses to a single ligand or that the same integrin can perform different functions when expressed on different cells.  Epithelial  interactions  with  cells perform a number of functions the  extracellular  matrix  including  that involve wound  unique  healing  and  modulation o f local inflammation. A t least eight different integrins are expressed by  keratinocytes during their life span (Watt, 2002). In normal undamaged  epidermis, integrin expression is confined to the basal layer and outer root sheath of hair follicles, with the exception o f ctvp8 which is absent from the basal layer of adult epidermis and found exclusively in the supra basal layer (Stepp, 1999; Cambier et al., 2000; Watt, 2002). a2f31, a301 and a6J34 are three integrins that are known to bind ligand found in the basement membrane i.e. collagen type I V and  laminin-5 (Hertle et a l , 1992; Watt, 2002). The a6p4 integrin is a major  component o f the hemidesmosomes, providing stability by binding to anchoring filaments made o f laminin-5 (Stepp et al., 1990; Watt, 2002). Animals deficient  1  in a.6 integrin die during the neonatal period due to massive epidermolysis bullosa which is characterized by blister formation due to separation o f the basal epithelial cells from a normal formed basement membrane (Georges-Labouesse et al., 1996). A n identical phenotype is seen in the null mutation o f the 04 integrin subunit (Sheppard, 2000). The altered expression o f ct604 integrin was observed in human and mouse squamous cell carcinoma and correlates with tumour invasion and poor prognosis (Witkowski et al., 2000). The integrin a301 is concentrated on the basal surface o f basal keratinocytes. It also expressed at lower levels around the lateral and apical surfaces o f cells through the epithelium (Sheppard, 1996). The ct3 integrin knockout mice develop blistering o f the skin as a result o f severe disorganization o f the basement membrane which may suggest that  a301  collaborates  with  cc6p4  in binding to  laminin-5  and  maintaining the integrity of the basement membrane (DiPersio et al., 1997). Keratinocytes cultured from these mice demonstrate increased cell motility on fibronectin and type I V collagen which suggests .that the expression o f a301 integrin in keratinocytes might inhibit cell migration (Hodivala-Dilke et al., 1998). In contrast to a301, a2(31 integrin is expressed predominantly at local adhesion plaques and serves as a collagen receptor which binds to collagen types I, III and I V (Sheppard,  1996). During wound healing,  a201  integrin is  concentrated at the forward tip o f migrating keratinocytes (Hakkinen et al., 2000b). Binding o f ct2pl to type I collagen at the migrating edge has the ability to activate matrix metalloproteinase-1  ( M M P - 1 ) (Pilcher et al., 1997) which is  believed to be the mechanism by which keratinocytes determine and maintain  2  their direction during re-epithelialization (Parks, 1999). M i c e deficient in the p i integrin subunit die early during embryogenesis. However, mice where the p i subunit is under the control o f a promoter (keratin 5 or 14) that is active in the basal cell layer o f the epidermis have severe hair loss and lack hair follicles and sebaceous glands.  These mice  also display epidermal and dermal blisters,  disruption o f the basement membrane with reduced expression of a6p4, dermal fibrosis and inflammation and abnormal wound healing, suggesting the p i subunit has an important role in normal epidermal proliferation (Brakebusch et al., 2000; Watt, 2002). The finding that stem keratinocytes, which have a continuous proliferation ability show a higher expression o f the p i integrin subunit than other keratinocytes subunit  in  experiments,  regulating  cell  further supports the importance o f the p i  proliferation  (Watt,  2002).  In  wound  healing  p i null keratinocytes show impaired migration and were more  densely packed in the hyperproliferative epithelium than control cells (Grose et al., 2002a). Ultimately, p i deficient epidermis did not cover the wound bed and epithelial architecture was abnormal. In addition to constitutively expressed integrins described earlier, keratinocytes can be induced to express other integrins by simply placing keratinocytes in culture or wounding (Breuss et al., 1995). ct5pi integrin is one o f the principle fibronectin receptors that appears on the surface o f epithelial cells as early as 24 hours after wounding. Its expression continues up to 14 days after wounding although it become limited to basal cells (Clark, 1990). The primary function o f a5pi  integrin is to enhance cell mobility on  fibronectin  and facilitate  cell  3  migration during re-epithelialization of cutaneous wounds. Secondarily, a5|31 integrin may serve as a signal for the termination of keratinocytes differentiation (Watt, 2002). The other integrin which is believed to be a characteristic of a migratory phenotype is ocv05. The ctv05 integrin is expressed by wound keratinocytes as early as 3 days after wounding. By day seven, ctv05 is found around the perimeter of the basal cells of the migrating epidermis, but by day 14, keratinocytes covering the wound area express other integrins but not ccv05 (Larjava et al., 1993; Clark et al., 1996a). The av05 integrin may play a role in controlling cell mobility since the retrieval of av05 integrin in malignant keratinocyte cell lines was associated with decreased cell mobility and invasion capacity (Thomas et al., 2001). However, re-epithelialization of dermal wounds is associated with a switch from av05 integrin to av06 integrin (Clark et al., 1996a).  The ccv06 integrin is expressed exclusively by epithelial cells in  developing fetal tissue but is downregulated in differentiated adult epithelia. It is re-expressed in injured and inflamed tissue and during carcinogenesis (Breuss et al., 1995). The av06 integrin is considered a receptor of two extracellular matrix proteins, fibronectin and tenascin; both are a component of the provisional matrix laid by wounded keratinocytes and thought to influence keratinocytes migration (Hakkinen et al., 2000a). Cell lines derived from 06 integrin subunit deficient mice showed a decreased migration ability on fibronectin (and to some extent vitronectin) as compared to wild type cell lines, and the addition of ocv06 monoclonal antibodies to wild type cell culture reduced their migration on fibronectin to a rate comparable to that of cells derived from 06"'" cell line  4  (Huang et al., 1998a). During wound healing,  av06  integrin is expressed by  migrating keratinocytes as early as two days after wounding. However, it reaches its  maximum  expression  when  epithelial  edges are united  and basement  membrane organization is started (Haapasalmi et al., 1996). Interestingly, 05, and the  05-06 integrin  06  double knockout mice developed normally and show no  significant difference in wound closure rates when compared with wild type littermates (Huang et al., 2000). However, 06 integrin knockout mice manifest inflammatory baldness and lung inflammation, which seems to be dependant on environmental insults (Huang et al., 1996). This suggests that ocv06 integrin normally  contributes  in  the  local  regulation  of  inflammation.  However,  transgenic mice that constitutively expressed human 06 (h06) integrin in the epithelial tissues tended to develop  spontaneous chronic wounds that were  surrounded by areas o f progressive fibrosis and ulceration (Hakkinen et al., 2004). Histologically, these lesions are characterized by numerous  activated  fibroblasts and macrophages. The epithelium at the edges o f the ulcers expressed  av06  integrin in the basal and suprabasal layer. Myofibroblasts in the collagen  rich matrix were found in the granulation tissue o f these wounds. Chronic lesions also contained significantly higher levels of transforming growth factor  01  ( T G F 0 1 ) in comparison to healthy skin from the transgenic mice and the wild type animals (Hakkinen et al., 2004). Interestingly, the  av06 integrin  to be induced in human chronic nonhealing wounds  e.g.  was found  infected  wounds,  diabetic ulcers, and venous leg ulcers (Hakkinen et al., 2004). Whether the overexpression  o f a v 0 6 integrin is actually a factor in the development  of  5'  chronic wounds or induced by the chronic status is still unclear, but the ability of  ctv06 to  activate TGF01 (Munger et al., 1999) may provide a mechanism by  which excessive matrix formation and constant activation of inflammatory cells results in a nonhealing status (Pierce, 2001). De novo expression of 06 integrin was associated with increased expression of M M P - 9 in normal oral keratinocytes. M M P - 9 degrades type IV collagen and is considered an important step in migration of keratinocytes as it facilitate the breakdown of the basement membrane (Thomas et al., 2001). The switch from ctv05 integrin into av06 integrin and the overexpression of MMP-9 was also demonstrated in epithelial tumours e.g.  squamous cell carcinoma, colon  carcinoma and ovarian carcinoma (Thomas and Speight, 2001). In oral squamous cell carcinoma, the overexpressed ctv06 integrin was found to be at its highest level at the invading edge of the tumour cell island (Jones et al., 1997). In colon cancer cells, the expression of av06 integrin induces gelatinase B (MMP-9) secretion through the C-terminal cytoplasmic extension unique to the 06 subunit, which has a major implication in tumour growth (Agrez et al., 1999). The cytoplasmic terminal also plays an important role in the regulation of other integrin expression in colon cancer cells (Niu et al., 2002). One explanation for the role  ctv06 plays in epithelial  tumour growth is its ability to  bind and activate TGF01 (Munger et al., 1999), which is a multifunctional cytokine regulating connective tissue formation, inflammation, and has been involved in epithelial tumour progression (Thomas et al., 2002). In fact, expressing  oral  leukoplakia  lesions  had  a  higher  rate  of  av06  malignant  6  transformation over a one to four year period when compared to a v p 6 negative lesions (Hamidi et al., 2000). It seems that a v 0 6 has multiple roles in malignant transformation through its  effect  on cell migration, spreading,  inflammation and matrix degradation.  1.2 Epithelial integrins and inflammation  Given  the  role  of  integrins  in  regulating  keratinocyte  adhesion  and  differentiation, it is not surprising that integrin expression is altered in both benign and neoplastic keratinocyte disorders. Although integrin expression is normally confined to the basal layer o f the epidermis, suprabasal integrin expression is a feature o f hyperproliferative epidermis, as found, for example, after wound closure or in lesions o f benign human skin disorders e.g. psoriasis (Hertle et al., 1992; Romero et al., 1999). Suprabasal integrin expression can play a causal role in the onset o f psoriasis and has been demonstrated by creating transgenic mice in which various integrin subunits are expressed under the control o f the involucrin promoter (Carroll et al., 1995). In these mice, overexpression o f the 01 subunit suprabasally resulted in sporadic epidermal hyperproliferation with  accompanying  histological  features  of  psoriasis,  including a lymphocytic infiltrate (Carroll et al., 1995).  The role o f mitogen activated protein kinase ( M A P K ) in the pathogenesis o f psoriasis has been well investigated. Activation o f M A P K in cultured human keratinocytes  results in increased  cell proliferation and delayed  onset o f  7  terminal differentiation which are features o f psoriasis. One way o f activating M A P K is through the binding o f 01 integrins subunit to their ligand (Haase et al., 2001). The second mechanism by which suprabasal integrin can activate M A P K is through stimulating keratinocytes to release I L - l a (Haase et al., 2001). I L - l a  production by keratinocytes  induces  a dermal mononuclear  infiltrate leading to further cytokine and growth factor release, which could account for the inflammation seen in transgenic mice expressing suprabasal integrins (Haase et al., 2001). M A P K is not the only pathway that is altered in psoriasis; IL-1 is known to activate other cellular signalling pathways including the nuclear factor K B ( N F - K B ) pathway. A m o n g genes that are regulated by N F - K B in the skin are those central to the initiation o f inflammation e.g. genes that stimulate the secretion o f a wide variety o f chemokines and cytokines from keratinocytes and fibroblasts. N F - K B also activates E-Selectin, I C A M - 1 and VCAM-1  on the surface o f endothelial cells, which are needed for lymphocyte  homing to the skin. Once activated, cutaneous lymphocyte antigen-T cells ( C L A - T ) produce a T h l response and stimulate further recruitment o f C L A - T cells to the area (Robert and Kupper, 1999). I L - l a is certainly not the only cytokine that might be involved in the pathogenesis o f psoriasis; IL-12 is one of  the  candidate  cytokines  and its role in stimulating inflammatory cell  infiltrate is currently under investigation (Robert and Kupper, 1999).  In vitro 01 integrin null keratinocytes shows impaired migration and failure in re-epithelialization associated with prolonged inflammatory response in an incisional wound healing model (Grose et al., 2002a). Other integrin that is  8  dramatically induced in response to injury and inflammation is expressing  the  null  mutation  of  ctv06  integrin  develop  av06.  Mice  exaggerated  inflammation o f the skin and lungs. Inflammation o f the skin is localized to areas o f minor trauma and characterized histologically by large numbers o f macrophages infiltrating the dermis, destroyed hair follicles and baldness. L u n g inflammation is characterized by a large number o f infiltrating macrophages, lymphocytes,  neutrophils and eosinophils,  and it seems to be related to  environmental insult since it is more prominent in animals kept in unventilated cages than those kept in well-ventilated ones (Sheppard, 2000). Evidence that inactivation o f the 06 subunit is indeed responsible for the exaggerated airway inflammation seen in these mice was demonstrated by Huang and co-workers, (1998) and suggests that limited expression o f  av06 on alveolar type  II cells or  bronchiolar cells is sufficient to reverse most o f the inflammatory infiltrate (Huang et al., 1998b). Persistent lung inflammation was thought to be the leading cause for progressive pulmonary fibrosis. M u c h o f the information regarding the development o f pulmonary fibrosis was derived from animal models where fibrosis is induced by a single intra tracheal administration o f the cytotoxic drug bleomycin. Bleomycin induces lung inflammation followed by a progressive destruction o f normal lung architecture. A s expected, 06 integrin knockout mice developed a greater degree o f lung inflammation compared to wild type mice at every time point examined. However, knockout mice did not develop the exaggerated fibrosis that the wild type mice did (Sheppard, 2001).  9  This lack o f response to the drug can be explained by the fact that av06 integrin has the ability to bind and activate latent TGF0 (Munger et al., 1999).  TGF0 as explained earlier is a potent stimulator o f extracellular matrix formation. TGF|3 itself has the ability to regulate the expression o f ccv06 integrin (Koivisto et al., 1999) Further support for this hypothesis can be found in the work o f Kaminski and co-workers,(2000)  who were able to identify a  cluster o f 66 genes that appeared to be involved in bleomycin induced fibrosis. These genes were expressed at similar levels at baseline in wild type and 06 integrin subunit knockout animals, but were induced to a higher level in wild type after bleomycin injection. Interestingly, most o f these genes are known to be induced by TGF0 and their expression was found to be higher in bleomycin treated wild type mice compared to 06 knockout animals (Kaminski et al., 2000). It is believed that integrin-mediated activation o f TGF0 is a process by which the cells offer tight spatial regulation and by which activity o f this potent profibrotic cytokine is restricted to sites were it is most needed. Thus av06 or any other cellular regulator o f this process can be attractive new targets for the treatment o f lung fibrosis (Kaminski et al., 2000).  1.3 Growth factors and wound healing  Growth factors are proteins that serve as signalling agents for the cells. They function as part o f the cellular communication network which critical  functions  such  as  cell  division,  matrix  production,  influences and  tissue  10  differentiation. Growth factors may have effects on multiple cell types and may induce different spectrums o f cellular function. Once a growth factor binds to a target cell  receptor, it induces  an intracellular transduction system  that  ultimately reaches the nucleus and induces the expression o f a new gene or a set o f genes which ultimately change the characteristic o f that cell.  Transforming Growth Factor-P: Transforming growth factor-P refers to a highly homologous family o f peptides that are differentially expressed and exert their multifunctional effect on a wide range o f cells.  Three different mammalian isoforms o f T G F p  has  been  identified and named as p i , P2 and P3. They seem to show sequence similarity between 70-80% and they display similar functions (Roberts and Sporn, 1993). A l l three isoforms act on two different transmembrane serine-threonine kinase protein receptors named, type I (RI) and type II (RII) (Takehara, 2000). T o activate the signalling cascade, RII associates with betaglycan bound to RI, forming a heteromeric complex with T G F p . RII is unable to signal without RI, and R I itself is unable to bind T G F p in the absence o f RII (Shukla et al., 1999). A l l T G F p isoforms are formed from a precursor protein that results in the formation o f a latent associated peptide ( L A P ) - T G F p  complex and can be  secreted by the cell. Before or after its secretion, T G F p has the ability to associate with different proteins to form a high molecular weight complex. The large latent complex consists o f the small latent complex ( T G F P  and its  propeptide) and a high molecular weight protease resistant binding protein,  11  latent  TGF0 binding protein  and secretion o f  TGF0  ( L T B P ) . L T B P is required for the proper folding  and deposition to the extracellular matrix (Koli et al.,  2001).Therefore, the normal function o f  TGF0  is controlled by its activation  from the latent state. Recent evidence suggests that cell surface molecule or secreted extracellular molecules can activate T G F p \ The activation o f  TGF0  involves the disruption o f the non-covalent interaction between L A P and TGF0 enabling  TGF0 to  association to  bind to its receptor. L A P has to be either released from its  TGF0 or undergo  a conformational change such that L A P is not  released but TGF|3 receptor site is exposed. In vitro activation o f  TGF0 can  be  achieved by proteolysis or enzymatic deglycosylation and acid treatment (Koli et al., 2001). Plasmin mediated proteolysis appears to be a major mechanism by which  TGF0 is  activated in vivo (Huber et al.,  is neutralized by a feedback inhibition since  1991; Godar  TGF0  plasminogen activator inhibitor-1 (Lyons et al.,  TGF0  activates  et al.,  1999)  and it  induces the production o f  1990). Thrombospondin  also  via a mechanism that does not involve the cell surface or  proteases but rather it involves a conformational change in L A P , thus releasing the active  TGF0. Thrombospondin  then reacts with the L A P to form an active  complex and prevent the reformation o f the LAP-TGF0 complex (MurphyUllrich  and Poczatek, 2000). Thrombospondin is induced during wound  healing and this may provide a mechanism by which local controlled (Murphy-Ullrich and Poczatek,  2000).  TGF0  level is  Thrombospondin deficient  mice display many phenotypical alterations similar to those seen in  TGF01  12  deficient mice (Crawford et al., 1998). The a v integrin subunit recognizes the R G D motif on  LAP-TGF0 and  mediates integrin-LAP binding, but so far only  a v 0 6 integrin can mediate the activation o f T G F p through 06  cytoplasmic  domain dependant conformational changes (Munger et al., 1999). Recently M u and co workers (2002) provided evidence that a v 0 8 has the ability to bind and activate  TGFP  via  an  M M P dependant  mechanism  which,  provides  a  mechanism by which a v p 8 mediates epithelial homeostasis ( M u et al., 2002). During wound healing T G F p has the ability to upregulate the expression o f several epithelial integrins that are important in wound healing, including a5pl,  av05  and a 2 p l  (Zambruno et al., 1995). In addition, T G F p l  is  considered a ligand for the a v p 6 integrin and has the ability to induce its de novo expression during wound healing, as HaCat keratinocytes exposed to  TGF01  have demonstrated a three-fold increase in the expression of a v P 6  integrin (Koivisto et al., 1999),which explains the profound effect o f T G F p on epithelial cell migration. The signal pathways activated by T G F p are transduced by a series o f S M A D proteins (Massague, 1998). It is S M A D 3 and S M A D 4 which translocate the signal to the nucleus and activate target gene expression by directly binding to specific D N A sites or indirectly interacting with other transcription factors. The type o f the cell, the degree o f the differentiation o f a particular cell, and the different molecules that are associated with the T G F p complex are responsible  for the wide variety o f effect that  TGFp  contradictory (Ling and Robinson,  TGFp  exerts on cells which sometime seems  2002).  plays an important part in regulating the immune response. It is released  from degranulated platelets at the early phase o f inflammation, and mediates leukocyte recruitment and activation, and stimulates the production o f proinflammatory cytokines Simultaneously,  including  TGFp  itself (Ling and Robinson,  it stimulates cell apoptosis and activates macrophages  downregulate the inflammatory response (Ling and Robinson, deficient  in  2002).  TGF01  to  2002). Animals  are b o m normally but develop massive inflammatory  lesions in multiple organs, which is thought to be due to the lack o f the regulatory effect that  TGFp  TGFP3  and  knockout mice die rapidly postnatally. In the case o f  TGFP3,  animals die due to cleft palate and delayed pulmonary development  TGF(32  (Proetzel et al.,  TGFP  exerts on immune response (Shull et al.,  1995; Sanford et  1992).  al., 1997).  plays an important role in wound healing partially due to its immune-  stimulating effects including its ability to chemoattract  a wide variety o f  immune cells such as monocytes, leukocytes and mast cells, and increasing cell apoptosis and cleaning by macrophages.  It also induces angiogenesis and  controls the structure and production o f extracellular matrix  components  including collagen, fibronectin, vitronectin, tenascin and proteoglycans (Koli et al.,  2001). TGFp  also suppresses matrix degradation  by  decreasing  synthesis o f proteases such as plasminogen activator (Laiho et al., 1986)  the and  increasing the synthesis o f protease inhibitors such as tissue inhibitors o f  14  metalloproteinase-1  (Edwards  et  al.,  1987;  McKaig  et  al.,  2003)  and  plasminogen activator inhibitors-1 (PAI-1) (Laiho et al., 1986). Exogenous application o f T G F p shows a potent stimulatory effect on granulation tissue formation (Roberts and Sporn, 1993). Transient upregulation o f T G F p  is  needed for tissue repair as dysregulation/sustained overproduction results in tissue  fibrosis.  TGFp  over-expression  has  been  linked to  scleroderma,  hypertrophic scars and keloid (Schmid et al., 1998). Tissue scarring may be related to overproduction o f T G F p 1 whereas T G F 0 2 and T G F p 3 may inhibit this effect (Shah et al., 1995). However, a careful balance between all three isoforms is needed for optimal healing. In  an excisional  wound healing model, wounded tissue shows a strong  expression o f T G F p i  m R N A within 24 hours o f injury. Whereas T G F 0 3  m R N A expression was low in the first three days, it increases up to twelve-fold to reach its maximum level by day seven (Frank et al., 1996). The levels o f the three T G F p isoforms decline by day 13 but do not reach baseline level. M o s t o f the expression was at the edges of the wound and T G F p 2 was abundant below the hyperprolifrative epithelium (Frank et al., 1996). In a rat wound healing model, almost 38% o f latent T G F p was activated within the first hour after wounding; another peak o f T G F p activation (about 17% of the total T G F P ) was noticed at five days (Yang et al., 1999). The predominant forms were T G F p i and T G F p 2 with only a minor amount o f T G F P 3 present (Yang et al., 1999). In normal human skin, the expression o f R I and RII receptors is visible in the epidermis, epidermal appendages and vascular cells. Only a small number o f  15  dermal fibroblasts showed T G F p receptor expression (Schmid et al., 1998). In contrast, granulation tissue fibroblasts demonstrate a strong expression o f RI and RII and that expression markedly decreased as the repair process advances (later than three weeks) (Schmid et al., 1998). O n the other hand, fibroblasts within the nodular structure o f collagen fibres in  hyperkeloid scars revealed  abundant immune staining o f RI and RII (Schmid et al., 1998), which may mean that failure to remove T G F p i receptor overexpressing fibroblasts during tissue remodelling results in overproduction o f the extracellular matrix and fibrosis. Interestingly, nonscarring fetal skin is relatively T G F p i deficient since neither T G F p i m R N A nor. TGF01 protein was detected in wounded fetal skin (Lin  and Adzick, 1996). However, when exogenous T G F p i  wounded human fetal skin, induction o f T G F p i  was added to  m R N A expression in fetal  fibroblasts occurred and an adult like inflammatory response was detected (Lin and Adzick, 1996; Cowin et al., 2001). Studies  on  knockout  animals  revealed  interesting  results  regarding  the  functions o f T G F p . M i c e null for S M A D 2 die early in embryogenesis whereas S M A D 3 null mice survive to adulthood but die at six months o f age due to the development o f multiple abscesses and impaired neutrophil chemotaxis, which reveals a compromised mucosal immunity (Datto et al., 1999). Interestingly, S M A D 3 knockout animals seem to be protected from radiation induced fibrosis with less influx o f inflammatory cells to the skin o f irradiated areas (Roberts et al., 2003). A l s o the wounds o f irradiated skin reepithelialized faster with less myofibroblast and eventually less fibrosis in S M A D 3 knockout compared to  16  normal mice (Roberts et al., 2003). Full thickness wounds in the back o f S M A D 3 knockout mice completely reepithelialized in two days as compared to five days in healthy controls (Ashcroft et al., 1999b). The wound surface area was  smaller with less infiltrating inflammatory cells and fibroblasts and  characterized by the absence  o f P M N s and monocytes  in knockout mice  (Ashcroft et al., 1999b). The addition o f exogenous T G F p i prior to wounding results in increased numbers o f monocytes in the wound area, but only in the heterozygous mice, not in the null ones. However, the null mice showed an increase in granulation tissue formation without increasing the number o f fibroblasts in the wound bed (Ashcroft et al., 1999b). It was concluded from this work that inhibition o f re-epithelialization is S M A D 3 dependent and the enhanced re-epithelialization seen in those animals may be due to increases proliferation or enhanced migration due to the effects o f other growth factors in a SMAD3  independent way. Since the reduced granulation tissue can be  reversed by the addition o f exogenous T G F p i , it may mean that fibroblast matrix formation can be S M A D 3 independent. In agreement with these results are the findings that laser burn wounds in mice overexpressing T G F p 1 locally in the basal keratinocytes showed inhibition o f wound re-epithelialization up to twelve days after wounding and that wounded tissue in  TGF01 transgenic  mice  showed increased expression o f type I procollagen as compared to the control mice (Yang e t a l , 2001)  T G F p and glucocorticoids interaction  17  TGFp  is a multi-functional cytokine which is considered a key regulatory  molecule  in the  control o f the  activity o f fibroblasts  and regulates  the  production o f multiple molecules of the extracellular matrix including collagen, fibronectin and tenascin. The fibrinogenic potential o f T G F p makes it the prime candidate as a mediator in tissue fibrosis and a target for drug therapy in the treatment o f such a condition (Shukla et al., 1999). Glucocorticoids, on the other hand, control collagen synthesis in the opposite direction to T G F p . They decrease  collagen  synthesis  and  selectively  decrease  the  synthesis  of  hydroxylated collagen peptides (Shukla et al., 1999). The observed decline in total  procollagen  synthesis  in cells by dexamethasone  treatment  reflection o f the decrease in total cellular content o f procollagen  was  a  specific  m R N A and was not due to decreased functional activity o f those m R N A s (Cockayne et al., 1986). It is believed that glucocorticoid mediated regulation of the type 1 procollagen gene is directed by D N A binding proteins acting through T G F p i regulatory element (Meisler et al., 1995). Dexamethasone has been shown to be capable o f blocking the fibrotic effects o f T G F p i both by reversing the T G F p i - m e d i a t e d increase in collagen production in fibroblast cell  cultures  Furthermore,  and by reducing scarring in vivo dexamethasone  treated  (Meisler et  rat lung fibroblasts  al.,  showed  1997). a dose  dependant decrease in T G F p i m R N A and T G F p i activity within two hours o f exposure and was sustained for 24 hours (Shull et al., 1995). The treatment o f human fetal lung fibroblasts with glucocorticoids did not only significantly inhibit T G F p i  and T G F 0 2 production but also reduced the upregulation o f  18  TGFpi  and T G F 0 2 m R N A induced by exogenous application o f  TGFpi,  T G F p 2 and T G F P 3 (Wen et al., 2002, 2003). There are several mechanisms by which glucocorticoids can modulate the effect o f T G F p production; the first one is through a T G F P independent mechanism. Consistent with that is the ability o f glucocorticoids to inhibit the basal production o f T G F p i  in a  concentration and time dependent manner without affecting T G F p 2 . T h e fact that a glucocorticoid response element is found on the promotor region o f human  TGFpi  supports  such  a  mechanism  (Parrelli  et  al,  1998).  Glucocorticoids can modulate T G F p induced production by either altering receptor expression or altering signal transduction. It has been documented that glucocorticoids target transcriptional activation o f S M A D 3 (Song et a l , 1999). Based upon these results, it is proposed that less T G F P is released by the cells under glucocorticoid treatment to react with cell membrane receptors, resulting in decreased signal transduction and protein binding to react with the T G F p element in procollagen genes.  Platelet-derived growth factor Platelet derived growth factor ( P D G F ) is one o f the principle mitogens and a potent  activator for mesenchymal  cells (Heldin and Westermark, 1999).  Originally purified from platelets, P D G F is believed to be secreted by many cells including platelets, vascular smooth muscle cells and activated monocytes and macrophages (Ross et a l , 1990). P D G F is stored in a granules in blood platelets and it consists o f two polypeptide chains linked together by disulfide  19  bonds. Three different isoforms o f P D G F were identified: A A , A B , and B B . The A B heterodimer is the most abundant species that can be isolated from blood platelets. T w o different tyrosine kinase receptor binding P D G F were identified which are expressed on overlapping but distinct cell types. The a receptor binds both a and P chains o f P D G F with high affinity, whereas the (3 receptor binds only p chain with high affinity (Meyer-Ingold and Eichner, 1995). This is believed to be responsible for the wide spectrum o f physiological activity o f P D G F  which includes  its effect on cell growth,  chemotactic  stimulus, actin fiber organization and prevention o f apoptosis. In the wound environment, P D G F is released by platelets, activated macrophages (Heldin and Westermark, 1999) and from the smooth muscles o f the damaged arterioles as well as epidermal cells (Ansel et al., 1993). It has proven to be mitogenic and  chemotactic to fibroblasts and smooth muscle cells. In addition to its  chemotactic  effects  on  macrophages  and  neutrophils,  it  stimulates  the  production o f other growth factors by macrophages (Heldin and Westermark, 1999), and stimulates the production o f extracellular matrix components by fibroblasts  including  fibronectin,  collagen  (Blatti  et  al.,  1988)  and  proteoglycans, and hyaluronic acid (Heldin et al., 1989). A t later stages o f wound healing, P D G F may play a role in wound contraction since it is able to induce contraction o f collagen gel populated by fibroblasts (Kuwahara et al., 2000; Han, 2002).  Incubation o f dermal fibroblasts with P D G F - A B resulted in a 2.5 fold increase in cell migration on type I collage and fibronectin matrix. Cells  showed  20  enhanced expression o f cc2, cc3 and a 5  as well as 01  integrin subunits  (Kirchberg et al., 1995). T h e P D G F enhanced migration on type I collagen was blocked by adding monoclonal antibodies against 01 or a 2 subunits to the cell culture. A s well as ct5 and 01 antibodies blocked P D G F enhanced migration on fibronectin (Kirchberg et al., 1995). Wound healing studies showed that the application o f r h P D G F - B B increases healing rate o f incisional wounds in rat skin (Pierce et al., 1989b), excisional ischemic wounds in rabbits ears (Tyrone et al., 2000) and partial and full thickness burns in pigs (Danilenko et al., 1995). A single application to wounds resulted in a significant increase in wound breaking strength up to 150% compared to control wounds (Pierce et al.,  1989b) and increased granulation tissue formation (which was rich in  fibronectin and glycosaminoglycan). It also increased rate o f epithelialization, and neovascularization (Tyrone et al., 2000). It seems that P D G F does not alter the normal sequence o f healing but rather increases its rate. Recently P D G F treatment was approved in the U . S . by F D A as a treatment for diabetic ulcers (Takehara, 2000).The effect o f P D G F on bone and fracture healing is still not completely understood. Although P D G F is a stimulant o f osteoblasts, P D G F seems to inhibit osteogenin induced bone regeneration by 17-53% (Marden et al., 1993). Healing o f unilateral tibial osteotomies treated with 80 ug P D G F did not show a difference in three point bending test at two and four weeks (Lieberman et al., 2002).It appears from these studies that P D G F promotes soft tissue healing rather than bone regeneration.  21  Fibroblast growth factors Fibroblast growth factors are a family o f at least twenty-three structurally similar polypeptides. The first one to be identified was acidic F G F (a-FGF) in 1974 (Gospodarowicz et al., 1974), which was found in high levels in neural tissue. The family also includes basic F G F and F G F - 7 . Acidic F G F (a-FGF) is now known as F G F - 1 and is an anionic mitogen o f molecular weight o f 15,00017,000 K D . It is not as widely distributed as basic F G F ( b - F G F ) (known as F G F - 2 ) . F G F - 2 is found in many tissues including the brain, kidney, adrenal gland, and activated macrophages. F G F - 2 is a cationic mitogen at physiological p H with a molecular weight o f 18,000 K D . F G F - 1 and F G F - 2 are single chain polypeptides which share 55% sequence homology (Bohlen et al., 1985). Both factors are heparin binding peptides, which protects them from degradation and damage, potentiates and control their biological activity, and helps to create a local reservoir (Heldin and Westermark, 1999). F G F interacts with specific cell surface  receptors  known F G F R 1 - 4  (Heldin and Westermark, 1999). F G F  family members are released from fibroblasts and endothelial cells (Werner et al., 1991) and exert their effects on a wide variety o f cells and are involved in multiple physiological and pathological processes including limb development, angiogenesis, wound healing, and tumour growth (Heldin and Westermark, 1999). Their most profound effect was through their ability to  stimulate  angiogenesis and neovascularization (Slavin, 1995). F G F - 1 and F G F - 2 are chemotactic to endothelial cells and stimulate their migration (Besser et al., 1995). F G F s interact with  av03  integrin (Sepp et al., 1994) which acts as a  22  vitronectin-fibrinogen receptor and has the ability to localized M M P s activity on the surface o f endothelial cells (Brooks et al., 1996). In addition to their action on endothelial cells, F G F - 1 and F G F - 2 are chemotactic for fibroblasts, and reduce collagen production by dermal fibroblasts. Thus F G F - 2 is a potent negative regulator o f collagen metabolism (Slavin et al., 1992). Histologically, F G F - 2 treated wounds angiogenesis,  show a greater amount o f granulation tissue and  more glycosaminoglycan and fibronectin content, and greater  resistance to disturbances (Ono, 2002). A s well, the application o f r h F G F - 2 to full thickness wounds was able to partially reverse healing defect in diabetic mice  (Tsuboi and Rifkin,  1990).  The application o f r b - F G F had  90%  effectiveness in improving the healing o f chronic wounds in 28 patients who did not respond to regular care (Fu et al., 2002). Clinical healing o f bums, donor sites and chronic dermal ulcers in 1,024  patents was  significantly  accelerated by topical application o f r b - F G F compared to placebo group treatment methods (Fu et al., 2000). Fracture healing in response to a single dose o f rhFGF-2 injected locally at segmental tibial defects in rabbits showed a dose dependent effect on healing. Bone volume and mineral content increased by 95% and 36% respectively (Lieberman et al., 2002). Histologically, at two weeks treated wounds showed a higher number o f periosteal mesenchymal cells and a higher number o f differentiated osteocytes and condrocytes when compared to control fractures. B y eight weeks, fracture strength recovered in r h F G F - 2 treated fractures compared to very low fracture strength in controls (Lieberman et al., 2002). F G F - 2 knockout mice develop normally, fertile and  23  phenotypically indistinguishable from F G F - 2  + / +  mice. However, histological  and immunohistochemical investigation showed neural cell defects and delayed healing o f excisional wounds. Only 10% o f wounds in the knockout mice healed by day 14 compared to a 100% healing rate in the control group (Ortega et al., 1998). Histologically, there was three days delay in healing in the F G F - 2 knockout  mice,  with  larger surface  area and  less  epithelialization  and  granulation tissue formation in wound area (Ortega et al., 1998).  Keratinocvte growth factor Keratinocyte growth factor ( K G F ) is a member o f heparin-binding fibroblast growth  factor  specificity.  family  (FGF-7)  with  a distinctive  pattern  o f target  K G F is solely produced by cells o f mesenchymal  cell  origin e.g.  fibroblasts, and it acts only on epithelial cells, leading to the hypothesis that it functions as a paracrine mediator o f mesenchymal-epithelial  communication  (Rubin et al., 1995). K G F was purified from fibroblast culture fluid as a monomeric polypeptide with a molecular weight o f 26-28 kDa. Its main function is to stimulate epithelial cell proliferation and terminal differentiation. It also stimulates epithelial cell migration on collagen and fibronectin (Putnins et al., 1999). Although epithelial cells themselves are unable to secrete K G F , they can control its expression through the release of DL-1 which is a potent inducer o f K G F (Brauchle et a l , 1994; Sanale et a l , 2002). In 1996, F G F - 1 0 was discovered (Emoto et a l , 1997). Initial studies showed that it has a significant homology to K G F , so it was named K G F - 2 . L i k e K G F , F G F - 1 0 is  24  mitogenic to keratinocytes but not to fibroblasts and is highly induced in the skin after wounding (Tagashira et al., 1997). K G F receptor is a tyrosine kinase isoform that binds F G F - 1 and F G F - 2 in addition to K G F . K G F knockout mice develop normally with no abnormality in wound healing capacity (Guo et al., 1996). However, truncated K G F receptor transgenic mice demonstrated severe epidermal  atrophy, reduced  basal  cell  proliferation and cornification  of  suprabasal keratinocytes, and abnormal hair structure and growth. They also show impaired wound healing (Werner et al., 1994; Werner and M u n z , 1998). Wound thickness increase  healing  studies reported that treatment  and ischemic in  the  wound models  re-epithelialization  o f full  thickness,  with K G F resulted in  rate,  and  the  new  partial  significant  epithelium  was  significantly thicker with deep rete ridge patterns (Staiano-Coico et al., 1993; Pierce et al., 1994; X i a et al., 1999). Those studies also reported that the effect o f K G F may indirectly extend beyond the epithelium to affect granulation tissue formation as acute wounds in rats receiving K G F c D N A showed not only was  improved  construct  epidermal regeneration but dermal regeneration  improved by increasing collagen  deposition  and evidence  o f more  neovascularization (Jeschke et al., 2002). The use o f K G F - 2 (FGF-10) in ischemic ear model in rabbits significantly increased epithelialization and enhanced granulation tissue without scar formation ( X i a et al., 1999).  Epidermal growth factors and transforming growth factor a  25  The  epidermal growth  factors  ( E G F ) family  is  compromised  of  several  different members o f growth factors including E G F , T G F a , heparin binding growth factor, epiregulin, betacelluin and others (Werner and Grose, 2003). A l l members  share  arrangement  the  o f six  so-called  E G F domain  cysteine residues.  which  A l l the  characterized  residue  participate  by  an  in the  formation o f intramolecular disulfide bond which seems to be important for receptor binding (Werner and Grose, 2003). The membrane-anchored precursor of the E G F family molecules is enzymatically processed externally to release the mature soluble form which acts as an autocrine growth factor. A l l o f the different  E G F family members  act in cross induction pathways  i.e.  the  activation o f one family member has the ability to induce the activation o f the others. E G F family members bind to one of four tyrosine kinase receptors found in human keratinocytes known as E G F R / E r b l - 4 . The overexpression o f those receptors is often  found in human cancer and may play a role in  resistance to treatment (Bujia et al., 1996; Mariotti et al., 2001; Yano et al., 2003). In addition, in vivo and in vitro studies confirmed the role that E G F receptors play in wound healing and maintenance o f epidermal integrity. In early stages o f healing o f partial and full thickness burns, E G F R was found in undifferentiated marginal keratinocytes and hyperproliferative epithelium and hair follicles.  A t later stages, the receptor was found in hyperprolifrative  epithelium and in all skin appendages (Wenczak et al., 1992). The addition o f E G F R inhibitor OS48-1 to full thickness wounds resulted in strong retardation of re-epithelialization(Tokumaru et al., 2000). E G F supplemented cell cultures  26  showed enhanced migration and proliferation and epidermal growth (Gibbs et al., 2000). Animal models showed significant acceleration o f wound healing o f partial and full thickness wounds in pigs (Breuing et al., 1997) and increased wound tensile strength in rat skin (Brown et al., 1988; Celebi et al., 1994) and in  rabbit cornea (Beaubien et al., 1994;  Szaflik et al., 1999). A n E G F  supplement in the drinking water helped to accelerate the healing rate of tongue punch wounds in diabetic mice to a rate comparable to that o f normal mice (Nagy et al., 2001). A s well, the addition of E G F to rabbit gastric epithelium in culture helped to reverse the aspirin induced inhibition o f cell proliferation and accelerated  wound  repair (Yoshizawa  et  al., 2000).  Interestingly, E G F  knockout mice have no phenotypical abnormalities and were found to heal normally (Luetteke et al., 1999), in contrast to E G F R knockout mice  which  showed striking abnormalities that proved to be lethal (Sibilia et al., 2003). Transforming  growth  factor a  (TGFa),  is the  first polypeptide  mitogen  discovered in 1962, is widely expressed in developing embryo and adult tissue (Rappolee et al., 1988a). It is one o f the main regulators o f normal growth and development. It has also been implicated in wound healing and homeostasis. T G F a is produced by platelets, salivary and lacrimal glands in addition to activated macrophages and eosinophils, suggesting an immunological function (Rappolee et al., 1988b; Wong, 1993). Keratinocytes respond to T G F a by increasing the proliferation and migration rate. It is also a potent promoter o f angiogenesis through its ability to induce the expression o f vascular endothelial growth factor ( V E G F ) (Gibbs et al., 2000). Transgenic mice lacking the T G F a  27  gene develop normally, and although wavy hair and whiskers have been reported, their wound healing capacity is normal (Luetteke et al., 1993). O n the other hand, newborn mice overexpressing T G F a in the basal cells have flaky skin and a thickened epidermis due to basal and spinal cell hypertrophy. However, these abnormality resolve at five weeks o f age (Vassar and Fuchs, 1991).  1.4 Introduction to wound healing Wound healing whether initiated by trauma, microbes or foreign material, proceeds  via  an  overlapping  pattern  of  events  including  coagulation,  inflammation, epithelialization, formation o f granulation tissue, and matrix and tissue remodelling. The process o f repair is largely mediated by interacting molecules signals,  primarily cytokines  that motivate  and control cellular  activities (Clark, 2003). The initial injury triggers coagulation and an acute inflammatory reaction followed by mesenchymal cell recruitment, proliferation and matrix formation. Failure to resolve inflammation can lead to chronic nonhealing wounds (Pierce, 2001) while uncontrolled matrix formation leads to excessive scarring and fibrosis (Mutsaers et a l , 1997). Damage to blood vessels will stimulate the coagulation cascade to initiate homeostasis. The adhesion, aggregation and degranulation o f blood platelets release a plethora o f mediators that includes T G F P , P D G F , and V E G F which influences tissue edema and inflammation (Martin, 1997). Active T G F p elicits a  rapid  chemotactic  response by  neutrophils  and monocytes.  Autocrine  28  induction o f T G F p by leukocytes and fibroblasts in turn induces those cells to generate additional cytokines  e.g. T N F a , T L - i p and P D G F , which further  augment the immune response (Martin, 1997). A m o n g the first cells to respond are endothelial cells in which adhesion molecules are expressed to allow the transmigration o f leukocytes to the site of tissue injury. Migrating through the matrix, leukocytes release proteases and engage in essential functions phagocytosis  of  cellular  and  tissue  debris  (Martin,  1997).  of  Neutrophil  recruitment typically peaks around 24-48 hours after wounding followed by increased  representation  o f monocytes/macrophages.  activated, further cytokine secretion,  Once those cells are  recruitment and reinforcement o f the  immune response is induced (Hubner et al., 1996).  1.5 Re-epithelialization Re-epithelialization is a term used to describe the resurfacing o f the skin with new  epithelium  after wounding.  It involves  multiple steps including the  formation o f a new provisional matrix, migration o f epidermal keratinocytes from the edges o f the wound, proliferation of keratinocytes  to feed  the  migrating epithelial tongue, maturation o f the new epithelium, and formation o f the new basement membrane. After wounding, keratinocytes at the wound edges start to migrate within 24 hours (Clark, 1996), and epidermal appendages throughout the wound bed contribute to the re-epithelialization process (Clark, 1996). A s cells migrate, they change their morphology and become flat and elongated, hemidesmosomal attachments are lost and desmosomes and the gap  29  junctions become more prominent (Oldand and Ross, 1968). Epithelial cells develop cytoplasmic extensions named lamellipodia and redistribute the actin cytoskeleton into them ( O T o o l e , 2001). Keratinocytes are highly phagocytic (Woodley, 1996) and, as they migrate, a pathway through tissue debris and blood is created. Cell migration is controlled by the ability o f keratinocytes to focalize proteolytic activity at the lamellipodia tips ( O T o o l e , 2001). In order to do so, keratinocytes  express many proteolytic enzymes (Steffensen et al.,  2001). In fact, the expression o f urokinase plasminogen  activator and its  receptors is upregulated during wound healing ( O T o o l e , 2001) and wounds on knockout mice lacking plasminogen activator fail to re-epithelialize (Romer et al., 1996). Histologically, fibrinogen rich deposits were found in front o f the migrating keratinocytes  which, theoretically  limit their ability to  migrate  (Romer et al., 1996). The M M P family o f enzymes are also overexpressed in basal keratinocytes at the migration front (Parks, 1999; Steffensen et al., 2001). M M P - 9 (gelatinase B ) is needed for the degradation o f type I V and VII collagen  found  in the basal  lamina. M M P - 1 (interstitial  collagenase)  is  upregulated in basal keratinocytes beyond the free edge o f the basal lamina and is needed to degrade the type I collagen found in the dermal matrix (Pilcher et al., 1997). It thought that the interaction o f cc2pi with collagen at the exposed dermal edge is responsible for the activation o f M M P - 1 which helps to free keratinocytes from the attachment to collagen and allows them to migrate. Through this process, cells can maintain their direction as they move on the exposed dermal collagen (Pilcher et al., 1997; Parks, 1999; Larjava et al.,  30  2002).  Other M M P s  that  are  upregulated  during  wound  healing  are  stromelysin-1 and-2 (Pilcher et al., 1999). Stromelysin-1 and-2 are also found in the epidermis o f normal acute wounds. Stromelysin-2 co-localizes  with  collagenase-1 and may facilitate cell migration over non-collagenous matrices o f the dermis (Pilcher et al., 1999). In contrast, stromelysin-1 is expressed by keratinocytes behind the migrating front which remain on basal lamina (Pilcher et al., 1999). Studies with stromelysin-1 deficient mice suggest that this M M P plays  a role  in keratinocyte  detachment  from the  underlying  basement  membrane to initiate cell migration (Bullard et al., 1999). Since most chronic non-healing wounds show a higher concentration o f M M P s in the wound fluids, a balanced control and tight regulation o f M M P s is needed during the healing process(Steffensen et al., 2001). T w o models o f epithelial cell migration have been proposed (Stenn and Malhotra, 1992). In the sliding model, cells at the leading edge actively pull the other cells behind them. In the leap frog model, as cells contact the provisional matrix, they become nonmotile and adhere to the matrix. Cells behind them crawl over the newly attached ones until they contact the provisional matrix (Laplante  et  al., 2001).  As  cells  migrate,  they  interact  with  different  extracellular matrix molecules. Some are found in the blood clot but most are secreted  by the migrating keratinocytes  to support their own  movement  (Hakkinen et al., 2000b). These molecule includes fibrin, fibronectin, tenascinC , vitronectin and laminin-5 (Gailit and Clark, 1994; Larjava et al., 2002). Cells not only secret E C M components but also express different integrins  31  during different phases o f healing that allow them to interact with the E C M and affect not only the  function o f keratinocytes  but also granulation tissue  formation (Clark, 2003). In early phases after wounding, the cells use a 5 p i and a2|31  integrins to attach and migrate on E D A fibronectin and laminin-5  respectively (Larjava et al., 2002). a v 0 6 integrin is another fibronectin receptor that is expressed at a later stage when epithelial edges meet (Haapasalmi et al., 1996) and has the ability to bind and activate T G F p and stimulate granulation tissue formation (Munger et al., 1999). cc2pi integrin has high affinity to the unprocessed  form o f laminin-5, which seems to stimulate  cell migration  (Nguyen et al., 2000). Laminin-5 is transformed into the processed form by removal o f the a3chain by plasminogen activator (Nguyen et al., 2000). The processed  form supports  a6p4  mediated  hemidesmosome  formation  and  retards migration as the basement membrane is being reformed ( O T o o l e et al., 1997) . The switch between fibronectin receptors a 5 p i and  avP6 at the later  stage in wound healing (Clark et al., 1996a) may be a signal for epithelial stratification and differentiation as a 5 p i  integrin is known to inhibit cell  differentiation (Watt, 2002).  1.6 Fibroplasia When the injury to the tissue is extended beyond the epidermis to reach the dermis, a reddish, granular material forms and is referred to as granulation tissue. Granulation tissue consists o f a dense population o f macrophages,  32  fibroblasts  and  neovascularization  embedded  within  a  loose  matrix  of  fibronectin, collagen and hyaluronic acid (Mutsaers et al., 1997). Granulation tissue formation involves  multiple steps beginning with  the  transformation o f fibroblasts from quiescent to proliferating and subsequently migratory, and the development o f a matrix producing and contractile cells. In the first two days after injury, the change in the E C M and growth factors released by platelets e.g. P D G F , connective tissue growth factor ( C T G F ) and T G F p , all help to activate residual fibroblasts in areas surrounding the wound (Takehara,  2000).  Local  fibroblasts  fibroblasts,  as some evidence  are not the  only source  suggests that pericytes  of  wound  in the surrounding  vascular endothelium are also induced to proliferate and migrate to the wound area. Bone marrow stem cells can serve as progenitor for various mesenchymal tissue (Bucala et al., 1994). After a lag period o f two to four days, fibroblasts start to invade the wound area and completely fill the defect by day seven (McClain et al., 1996). At this stage, fibroblasts adjust their integrin profile to allow for the migration and matrix production phase. Human dermal fibroblasts use  avP3 and avP5 integrins  for attachment to vitronectin and ctvpi and a v p 3  integrins for attachment to fibronectin (Clark et al., 1996b). Interestingly, the classical fibronectin receptor  a5pi  is not expressed until the late phases o f  healing. Its expression is thought to be a rate limiting step in the formation o f granulation ( M c C l a i n et al., 1996). This supports in vitro studies showing that the overexpression o f  a5pi  integrin in fibroblast cells retards cell movement  over fibronectin (Chan et al., 1992). Human dermal fibroblasts in surgical  33  wound healing with primary intention show upregulated expression o f a 2 , a v and p i subunits as early as six days after wounding The expression reaches its peak at nineteen days but decreases around 27 days after injury (Noszczyk et al., 2002). The upregulation o f the a v integrin subunit was found to be in coincidence  with  the  upregulation  o f vitronectin,  which  is  again  most  pronounced at day 19 (Noszczyk et al., 2002). The other conclusion that can be drawn from the study is that human dermal fibroblasts use a 2 p i integrin as their primary collagen receptor. The finding that a 2 p i integrin is needed for the contraction o f a three-dimensional  collagen  lattice supports this view  (Langholz et al., 1995). Moreover, the increased expression o f a 2 p i integrin could also contribute to the collagen matrix organization as it was shown that this integrin mediates the induction o f M M P - 1 (Langholz et al., 1995). O f importance is the interaction between the E C M and the growth factors in controlling and modifying the expression o f integrins on fibroblast surfaces. In vitro studies found that a3 and a 5 integrin subunits are upregulated when P D G F was added to fibroblast cell lines in fibronectin matrix, whereas the a 2 integrin subunit was overexpressed when cells were kept in collagen gel ( X u and Clark, 1996). Once fibroblasts establish residence in the wound area, they begin to produce E C M ; first cellular fibronectin and then type I collagen is laid down (Gabbiani, 2003). Type I collagen  interacts with a 2 p i  integrin to  promote cell proliferation, but as collagen fibers mature and fibrillar collagen forms, its ability to interact with a 2 p i integrin decreases and cellular activity is downregulated. The peak matrix production occurs around day seven, which  34  coincides with the peak activation o f T G F p and the induction o f a v 0 6 integrin (Hakkinen et al., 2000b). T G F p does not only controls and induces type I collagen production by fibroblasts but also induces the synthesis o f a smooth muscle  ( a - S M ) actin filaments  in fibroblastic cells (Desmouliere,  1995;  Gabbiani, 2003). The appearance o f a - S M filaments results in the formation o f a specified  type  o f fibroblasts  called myofibroblasts.  Myofibroblasts are  characterized by loosely organized bundles in the periphery o f the cells and gap/adherence junction representing the cellular connection. (Badid et al., 2000). a - S M is considered the first marker to identify myofibroblasts and is related to their contractile phenotype as in vivo and in vitro studies show a direct  correlation  between  the  expression  of  a-SM  expression  and  myofibroblast contraction (Hinz et al., 2001). W o u n d contraction results as myofibroblasts in the central granulation tissue pull the new matrix together; further contraction o f the wound edges occur as fibroblasts organize the E C M that occurs between days seven and fourteen (Hinz et al., 2001). In addition to T G F p i , the differentiation o f myofibroblasts is controlled by a 2 p l integrin, the composition o f the extracellular matrix e.g. E D A fibronectin (Serini et al., 1998; Gabbiani, 2003), and mechanical stress (Hinz et al., 2001; Lorena et al., 2002; Tomasek et al., 2002).  A final phase o f healing occurs when the wound is completely closed, as many fibroblast develops pyknotic nuclei, which are markers for cellular apoptosis (Desmouliere,  1995). Apoptosis mark the transition from a cellular rich  granulation tissue into an acellular scar. Factors that control cellular apoptosis  35  are not clear, but b - F G F , T N F a and IFNy may play an important roles as they are found to decrease the expression o f a - S M actin m R N A and protein in cultured fibroblasts (Lorena et al., 2002). In addition, hypertrophic scars treated with y U N showed a decrease in size and severity (Pittet et al., 1994). Mechanical stress is another factor in controlling apoptosis as pressure therapy was found to partially restore E C M organization and induce the disappearance o f myofibroblasts in second and third degrees burn (Costa et al., 1999). The role o f integrins in the initiation and control o f cellular apoptosis  is not  completely understood but in vivo studies showed that fibroblasts undergo apoptosis when kept in contractile collagen gel but not when kept in fibrin gel (Fluck  et  al.,  1998).  When granulation tissue cells are not  eliminated,  myofibroblasts will continue to deposit E C M , leading to the development o f hypertrophic scar or keloid (Lorena et al., 2002).  1.7 Psychological stress and the immune system T h e central nervous system ( C N S ) and the immune system communicate v i a multiple neuroanatomical and hormonal routes and molecular pathways. The interaction between the two systems provides a regulatory pathway required for health; disturbance at any level might lead to changes in susceptibility to autoimmune or inflammatory disease (Eskandari and Sternberg, 2002). Several brain centers are involved in immune regulation, but the two major pathways involved are the hormonal stress response  through the activation o f the  hypothalamus-pituitary-adrenal ( H P A ) pathway and the autonomic nervous  36  system through the release o f epinephrine and norepinephrine. H P A stimulation results in an overall increase in glucocorticoid ( G C ) production, which in turns regulates a wide variety o f immune cell functions e.g. modulates the expression of  cytokines  and adhesion  molecule,  regulates  immune  cell  trafficking,  maturation and differentiation, and regulates the expression o f chemoattractants and cell migration and production o f inflammatory mediators  and other  molecules (Barnes, 1998; Adcock, 2000). Initially, H P A mediated stress was believed to be immunosuppressive, but accumulating evidence suggests that long term glucocorticoid production causes a shift in immune response from a pro-inflammatory cytokine pattern to anti-inflammatory cytokines (Glaser et al., 2001; Elenkov and Chrousos, 2002). The molecular pathway for this shift is complex but one o f the possible mechanisms is through the inhibition o f IL-12 secretion from antigen presenting cells ( A P C ) (Trinchieri, 1995). IL-12 is a potent inducer o f IFNy production and a potent inhibitor o f IL-4 synthesis (Trinchieri,  1995).  Thus  Glucocorticoid  treated  monocytes/macrophages  produce significantly less IL-12 (Blotta et al., 1997), leading to decreased INFy and an increase in the production o f IL-4 by T cells, probably due to blocking the suppressive effect o f IL-12 (DeKruyff et al., 1998). Glucocorticoid also downregulates the expression o f IL-12 receptors on T and natural killer ( N K ) cells and has the ability to upregulates the production o f IL-10 by lymphocytes, all o f which favour Th2 response (Ramierz et al., 1996). Immune cells also carry neurotransmitter receptors e.g. adrenergic receptors on lymphocytes, which allow them to respond to neurotransmittors released by the  37  sympathetic  nerve  ending.  The  systemic  release  of  epinephrine  and  norepinephrine results in overall inhibition o f pro-inflammatory cytokines and stimulation o f anti-inflammatory cytokines  release (Elenkov et al., 2000).  Through this mechanism,  system may selectively inhibit  the sympathetic  cellular immunity and enhance the humoral immunity in the form o f increased Th2 response. Experiments with 02 adrenergic receptor agonists showed that norepinephrine has the ability to downregulate the production o f IL-12 and enhance the production o f IL-10, which promotes T h 2 cell (Panina-Bordignon et al., 1997). Stress hormones  differentiation  inhibit the function  of  cellular immunity through the inhibition o f the activity o f natural killer cells, which seems to be most sensitive cell to the effect o f stress (Elenkov and Chrousos, 1999). Another possible mechanism explaining the immune modulating effect o f stress is through the ability o f peripherally secreted corticotropin releasing hormone ( C R H ) to stimulate mast cell degranulation (Webster et al., 1998). The release o f histamine stimulates H 2 receptors on peripheral monocytes and inhibit the secretion o f IL-12 while stimulating the release o f IL-10 (Elenkov and Chrousos, 1999). H 2 receptor stimulation results in inhibition o f IFNy production but has no effect on IL-4 production, which may appear to drive a Th2 shift (LeResche and Dworkin, 2002). Further support to this hypothesis comes from studies looking at the effect o f academic stress on immune system function which found that even a short term stressor (e.g. examination stress) was associated with a shift in the T h l / T h 2 balance towards Th2 (Marucha et  38  al., 1998; Y a n g and Glaser, 2002). Peripheral blood leukocytes from medical students during examinations period showed decreased synthesis o f INFy and increased production o f IL-10 compared to pre-exams periods (Marucha et al., 1998). Examination stress also resulted in decreased natural killer ( N K ) cell activity; decreased response o f peripheral blood leukocytes (PBLs) to mitogen, and decreased C o n - A stimulated production o f IFN-y by P B L s (Yang and Glaser, 2002). This was confirmed in animal models where restraint stress resulted in a significant decrease in N K cell activity, T h l cytokine production, production o f TNFct, and decreased leukocyte migration (Iwakabe et al., 1998). Chronic  stress associated  with  caring for a person with dementia  was  associated with a higher percentage o f IL-10 positive cells in lymphocytes obtained from caregivers when compared to controls (Glaser et al., 2001). N K cell response to rhEL-2 and I N F a was decreased in caregivers and the incidence o f upper respiratory tract ( U R T ) infection was increased in comparison to controls (Kiecolt-Glaser et al., 2002a). Decreased cell mediated immunity has been associated with increased susceptibility to viral, fungal and mycobactrial infection  (Elenkov  et  al.,  2000).  Recently,  an  association  between  psychological stress and susceptibility to the common cold among healthy volunteers intentionally inoculated with five different strains o f live respiratory viruses was shown as the high stress group had an increased risk o f developing acute U R T infection and greater expression o f illness judged by the severity o f symptoms (Cohen et al., 1999). B l o o d analysis showed higher production o f IL-6 by P B L s (Cohen et al., 1999). Interestingly, elevated levels o f IL-6 were  39  associated with stress and depression in humans and animals exposed to varies kind o f stressors. Plasma levels o f IL-6 paralleled the increase in the production o f corticosteroids which implicate a feedback controlling mechanism (Maes et al., 1998). Accelerated progression o f H I V infection was related to stress and decreased social support in a group o f 96 H I V seropositive asymptomatic adults followed over a period o f 9 years (Leserman et al., 2002). Progression o f H I V has been characterized by increased  Cortisol secretion on early and late stages o f the  disease and in that previous study, serum  Cortisol levels predicted disease  progression (Leserman et al., 2002). H I V infected men who completed a 10 week stress management intervention had higher C D 4 counts at follow up than did control subjects, and this difference was independent o f initial number o f naive T cells and H I V viral load (Antoni et al., 2002). The competence  o f the cellular immune response  is a critical factor in  controlling and maintaining virus latency. Stress results in less control over the expression o f the latent virus in vitro and in vivo (Glaser et al., 1991; Cacioppo et al., 2002; Yang and Glaser, 2002). In animal models, social disturbance results in activation o f latent herpes simplex virus type-1 ( H S V - 1 ) in more than 40 % o f latency infected mice (Padgett et al., 1998b). Data from series o f studies evaluated the response o f stressed individuals to different forms o f vaccines e.g. pneumonia (Glaser et al., 2000), influenza (Kiecolt-Glaser et al., 1996; Vedhara et al., 1999) and hepatitis B vaccine (Glaser et al., 1992) help to confirm the apparent shift in the T h l / T h 2 response. These individuals tend to  40  have a decreased antibody titer with late seroconversion and decreased virus specific T cell response (Cohen et al., 2001). These studies suggest that psychological stress can modulate immune responses to increase the risk for the development and severity o f infectious disease. Moreover, psychological stress can be involved in the development o f autoimmune disease and in tumour progression (Elenkov and Chrousos, 1999). Immune modulation and disturbance in the T h l / T h 2 balance is being related to development and or severity o f autoimmune diseases e.g. arthritis, type I diabetes, multiple sclerosis and S L E (Elenkov and Chrousos, 2002). The development o f animal strains with hyper-reactive H P A (e.g. Fischer rats) or hypo-reactive H P A (e.g. Lewis rats) and the finding that those animals can be resistant or susceptible to the development o f experimentally induced disease based on their reactivity level further supports the role o f stress in modulating autoimmune diseases (Webster et al., 2002; Wilder, 2002). In addition, the remission o f T h l type mediated autoimmune diseases e.g. arthritis in pregnant women during the third trimester (when plasma  Cortisol levels are increased) can be explained by the cortisol-  induced shift towards a Th2 response and potentiation o f anti-inflammatory cytokines (Elenkov et al., 2001; Munoz-Valle et al., 2003). Through the same mechanism, a Th2 mediated disease e.g. S L E can flareup during periods o f high  Cortisol production e.g. stress (Pawlak et al., 2003) or pregnancy (Elenkov  etal., 2001; Meyer, 2003). The amount o f IL-12 available at tumour sites appears to be important for the tumour progression and metastases as lower levels o f IL-12 were related to  41  tumour growth (Kobayashi et al., 2002; Shibata et al., 2002). The inhibitory effect o f stress on the secretion o f IL-12 explains the relationship between psychological stress and incidence, progression and metastases o f certain types o f tumours (Kiecolt-Glaser et al., 2002b).  1.8 Stress and wound healing The inflammatory phase is important in order to protect the site of injury from infection and to prepare it for subsequent repair. Cytokines (e.g. IL-1, EL-8 and T N F a ) play an important part of the inflammatory phase and any disruption to this role  results  in  impairment to  wound  repair (Martin,  1997).  Studies  in  psychosomatic medicine have shown that greater stress before surgical procedures is associated with poorer outcome, more complications and longer hospitalization (Kiecolt-Glaser et al., 1998; Rojas et al., 2002). Stress induced delayed wound healing is well documented in both animal models and in human, even a transient, predictable and benign stress such as examination stress results in a delayed wound healing by 40% in dental student (Marucha et al., 1998). Chronic stress associated with caring for a spouse with Alzheimer's disease resulted in a 24% (three days) delay in the healing of a 3.5 mm punch wound (Kiecolt-Glaser et al., 1995). In both studies, peripheral blood leukocytes (PBL) produced less DL-ip in response to L P S stimulation (Kiecolt-Glaser and Glaser, 2001). The healing of naturally occurring leg ulcers evaluated in 53 patients treated for persistent leg ulcer over 3 month showed that delayed wound healing was associated with higher hospital anxiety and depression ( H A D ) scale and that patients scoring in the top 50% of total H A D scores were four times more likely to have delayed wound  42  healing than those scoring in the bottom 50% (Cole-King and Harding, 2001). The biological mechanism of delayed wound healing has been studied in humans and animal since early the 1960s (Sandberg, 1964; Ehrlich and Hunt, 1968). Animal studies showed that pharmacological doses of steroids inhibit neutrophil and monocyte recruitment, macrophage phagocytosis (Leibovich and Ross, 1975) and bacterial killing (Ehrlich and Hunt, 1968). Steroids also delay epithelialization (Lenco et al., 1975), fibroblast proliferation, neuvascularization, and synthesis of glycosaminoglycan and collagen (Salmela, 1981; Oishi et al., 2002). Because wound tensile strength is dependent on wound collagen content, G C treatment impairs wound tensile strength (Ehrlich and Hunt, 1969; Gupta et al., 1999). G C affects other extracellular matrix molecule as the production of fibronectin is increased by dermal fibroblasts (Babu et al., 1989). On the other hand, tenascin-c and hyalurnic acid production is decreased by wound fibroblasts (Sarnstrand et al., 1982; Ekblom et al., 1993; Fassler et al., 1996). The most common explanation of the inhibitory effect of G C on healing is that they prevent the early inflammation phase. Administration of steroids before wounding causes inhibition of wound repair where as administration of steroids  three days after wounding  was  ineffective (Sandberg, 1964). In addition to reducing inflammation, G C reduces vascular permeability, which in turn reduces the availability of chemoattractants and inflammatory cell recruitment to the injury site (Wahl and Wahl, 1985). In vivo administration of higher doses of hydrocortisone results in inhibition of angiogenesis and fibroplasia due to decreased chemoattractant and growth factors and as a direct inhibitory effect on fibroblast proliferation and collagen synthesis (Wahl  and Wahl,  1985).  G C treatment  drastically reduces  the  level  of  43  troprocollagen HI and I, with tropocollagen UI being most severally  affected  (Oishi et al., 2002). The local wound environment was evaluated by the creation of suction wounds in the forearms of 36 women. Women with higher perceived stress had significantly lower levels of IL-loc and IL-8 (Glaser et al., 1999). Pro-inflammatory cytokines  IL-la, IL-1J3 and T N F a m R N A were significantly reduced in three-day-old wounds in mice treated with G C in comparison to normal mice. Levels of IL-{3 were almost 30% less in G C treated mice, whereas I L l - a and T N F a were reduced by 50 % and 70 % respectively (Hubner et al., 1996). Restraint stress significantly downregulated the expression of IL-1 p m R N A in one-day-old wounds (Mercado et al., 2002b).That suppression was completely restored to a level comparable to control animals by treating the animals with RU486, which is a G C receptor antagonist (Mercado et al., 2002b). Reduction in the level of IL-1 can be explained by G C mediated reduction in the number of inflammatory cells reaching the site of injury. At five days, wounds from restraint animals had a significantly higher level of m R N A of both I L - l a and IL-1 p in comparison to control animals. This shift in the kinetic pro-inflammatory cytokine expression can be a major contributory factor in delayed wound healing (Mercado et al., 2002b). Delayed inflammatory response may alter the transition to the proliferative phase where fibroblast proliferation, collagen synthesis and neovascularization are needed. Reduced levels of pro-inflammatory cytokines in early wound healing may affect the subsequent steps by altering the level of expression of other growth factors, for example,  P D G F - A and P D G F - B  mRNA  and P D G F R - B were significantly reduced not only in early phases of wound  44  healing but also in the nonwounded skin of dexamethasone treated mice as well as in cultured fibroblasts, demonstrating that G C treatment affects P D G F production at the gene level (Beer et al., 1997). K G F m R N A expression in G C treated animals was increased by only 10-13 fold in one-day-old wounds whereas in normal animals, wounded skin show a 65-fold increase (Brauchle et al., 1995). This inhibition could be the results of a direct inhibition of the K G F gene by G C or indirectly through the inhibition of other cytokine production. However, treatment with RU486 did not restore K G F m R N A expression even when I L l - p response was recovered (Mercado et al., 2002a). Among the other growth factors that are downregulated by G C treatment are T G F P isoforms, where p i and P2 isoforms were reduced by 65% in three day and five day wounds, and the P3 isoform was highly expressed in the early phase of wound repair (Frank et al., 1996). T G F p receptors were regulated by G C treatment in a complex manner, RI expression was slightly induced and RII was slightly reduced. These data suggests that correct regulation of T G F P and T G F p receptor expression is important for normal wound healing, and may help to explain the beneficial effect of exogenous T G F p application on healing in G C treated animals (Frank et al., 1996). Recently Ishimoto and Ishibashi (2002) documented that the expression of K G F , T G F a and b F G F m R N A during healing of tympanic membrane perforation in G C treated rats is reduced and concluded that the exogenous application of K G F only improves epithelial migration in the tympanic membrane (Ishimoto and Ishibashi, 2002). Grose  and  co-workers  (2002)  recently  studied  the  effect  of  endogenous  glucocorticoid on wound healing using a mice model with defective D N A binding  45  G C receptor ( G R  dim)  . In G R  d i m  mice, large wound granulation tissue was observed  in the early phases of healing. Tissues had higher density of cellular infdtrate, but no visible effect on re-epithelialization at any time point. Expression of IL-1 p m R N A was enhanced in one-day-old wounds (Grose et al., 2002b). In addition, TGFJ32 m R N A increased 1.5-3 fold and T G F p 3 1.2-2 fold. B y day 13, T G F p 2 levels was still elevated but T G F p 3 levels were comparable to those of control mice. Fibronectin expression increased by 1.5 times in fibroblast derived from GR  d u n  m i c e as compared to control cells (Grose et al., 2002b). In vivo, the rate of  collagen gel contraction by wound fibroblasts was enhanced, and eventually wounds healed normally. From this model it can be concluded that endogenous G C exerts a supportive effect in early wound healing and can play a useful regulatory role in circumstances where the inflammatory response is excessive, for example in infected wounds. In addition, part of the effect that G C have on wound environment is the selective modulation of M M P action. In a full thickness wound healing model, systemic treatment of mice with dexamethasone resulted in almost complete downregulation of macrophage specific M M P expression, most likely due to reduced infiltration and activation of monocytes (Madlener et al., 1998). However, the expression levels of collagenase, gelatinase and stromelysin-1 were weakly regulated by G C treatment (Madlener et a l , 1998). However, other studies showed that these enzymes are affected by G C treatment e.g. administration of steroids decreased gelatinase in a suction blister model in human skin (Oikarinen et al., 1993) and in cultured fibroblasts (Salo and Oikarinen, 1985). Collagenase production was reduced by dexamethasone  treatment in human skin explants  (Koob et al., 1980) and in cultured skin fibroblasts (Oikarinen et al., 1987).  46  Interestingly stromelysin-2  was upregulated in unwounded skin and in full  thickness wounds of mice treated with dexamethasone compared to untreated controls (Madlener et al., 1996), which indicates that G C treatment may effect wound healing by affecting the balance of M M P in the wound environment.  47,  Chapter 2-Aim of the Study T o investigate whether wound healing under stress (induced by hydrocortisone) is altered in transgenic mice lacking or overexpressing the 06 integrin subunit in comparison to wild type mice.  Hypothesis:  The 06 integrin subunit protects and positively improves the re-epithlialization o f epidermal wounds in wound healing model compromised by hydrocortisone treatment.  48  Chapter 3-M ate rial and Methods This study consisted of two parts that followed the same protocol. For the first part, two groups of mice were used, wild type (WT) and 06 integrin deficient (06" ) (Huang et al., 1996). Each group consisted of 10 age (6-months-old) and /_  sex matched animals (Appendix A) and each group was subdivided into treatment (Tx) and untreated control (con) groups with n=5 mice.  In the second part, three groups of mice were used, wild type mice (WT),,06 integrin deficient (P6" ), and human 06 integrin overexpressing transgenic mice A  (h06Fl) (Hakkinen et al., 2004). Each group consisted of 10 age (22 months old) and sex matched mice (Appendix A) and each group was subdivided into treatment (Tx) and untreated control (con) groups. The experiment protocol was approved by Animal Care Committee at the University of British Columbia.  3.1 Excisional wound healing The experimental group received intraperitoneal injections of 1 mg/0.2 ml of hydrocortisone 21-Hemisuccinate (Sigma AlDrich Co; St Louis, USA) (Dostal and Gamelli, 1990) daily at 9 a.m. until the end of the experiment. The control group received intraperitoneal injections of 0.2 ml of phosphate buffered saline (PBS) daily. On the third day, mice were anaesthetized with inhalation of halothane (MTC Pharmaceuticals, Cambridge, ON, Canada) and the dorsal skin was shaved and depilated (Veet™). Four full thickness, 4 mm punch biopsy  49  wounds were created on the dorsal surface of the mice. Wounds were left to heal for three and ten days and, during that period; mice were caged separately and had free access to water and food.  3.2 Clinical wound healing  A t the time o f wounding and at every day post-wounding, standardized images o f the wound were recorded using a digital camera to analyse the wound surface area. The surface area was quantified using an N T H image program, and the wound size at each time point was expressed relative to the original size at day zero.  Evaluation o f wound closure was done daily using 3% hydrogen peroxide ( H 0 ) (Fisher Scientific, N e w jersey, U S A ) . Each wound received 40u.l o f 2  H 0 2  2  2  that was allowed to react for one minute. Bubbles forming in the center o f  the wound were recorded as a positive test indicating incomplete  wound  closure. This test is based on the fact that the underlying connective tissue contains  the enzyme  catalase.  Catalase liberates  oxygen  and water from  hydrogen peroxide. If the epithelial barrier is intact, then hydrogen peroxide does not diffuse into the underlying connective tissue and oxygen is not liberated (Marucha et al., 1998; Padgett et al., 1998a).  3.3 Histology and immunohistochemistry  M i c e were euthanized via C 0 inhalation. Wounds were collected with 3 m m 2  of healthy skin margin. Ear samples were collected for verification o f the  50 r  transgene expression in control and human f36 integrin overexpressing animals. Immediately after collection, each wound was divided into two halves. H a l f o f the wound was fixed using 10% formalin for 24 hours, processed for paraffin embedding and stored at 4 ° C . The other half o f the wound was mounted in T i s s u e - T E K (Sakura inetechnical, U S A ) and snap frozen in liquid nitrogen. Tissue blocks were stored at - 8 0 ° C .  Frozen sections (10 urn) were cut and fixed with acetone for five minutes and stored at - 8 0 ° C until used for staining. Each fifth section was stained with Harris's hematoxylin and mounted using Entellan. Three days wound sections were photographed with standard techniques using a digital camera. For each wound, the distance from the wound edge to the tip o f epithelial tongue was measured using the N I H program. A n average epithelial migration distance was recorded for each wound.  In  a set o f frozen sections, the 06  integrin subunit was  monoclonal rabbit antibody ( B l ) against human  localized using  ccv06 integrin  (Huang et al.,  1998b). In addition, type I V collagen was also localized using rabbit antihuman monoclonal antibodies (Monosan, Cedarlane L a b , Hornby, O N , Canada) (Sakai et al., 1982).  TGF0-l,-2 and-3 were localized with a monoclonal antibody  recognizing all three isoforms ( R & D System, Inc., Minneapolis, M N , U S A ) ( D a s c h e t a l . , 1989).  A l l frozen sections were rinsed and incubated with normal blocking serum (Vectastain, Vector Laboratories, Burlinghame, C A , U S A ) for 20 minutes at room temperature and then incubated with the primary antibody in P B S / B S A in a humid chamber at 4 ° C overnight. After washing with P B S / P S A , sections were incubated with the appropriate biotinylated secondary antibody for 60 minutes and then reacted with A B C Avidin/peroxidase agent (Vectastain Elite Kit, Vector Laboratories Inc). Chromogen was developed using V I P (Vector Laboratories Inc) as chromogenic substrate. The reaction was monitored until significant  colour over the background developed  and then washed  with  ••distilled water to stop the reaction. Sections were mounted using Vecta mount (Vector L a b , C A , U S A ) .  A set o f paraffin blocks were cut (10 um). Each fifth slide was stained with Hematoxylin and Eosin and each sixth slide was  stained with Masson's  Trichrome for evaluation o f tissue healing.  Statistical analysis  The wound size o f different groups at each day o f healing was compared using Student's t-test. The same test was used to compare the rate o f epithelial migration in the histological sections o f three-day-old wounds. The response to H 0 2  2  test was evaluated using Chi-square test. Analysis was  done using  Microsoft excel program and a p value < 0.05 was considered statistically significant.  52  Chapter 4-Results r  All animals except three survived the experiment; one animal in the untreated human 06 integrin overexpressing (h06Fl) group, one in the treated human 06 integrin overexpressing (h06FlTx) group, and one in the treated adult wild type (WTTx)  group. A total o f five wounds were excluded from the analysis, one  wound in each of the following groups: untreated human 06 integrin overexpressing  (h06Flcon),  treated human 06  integrin overexpressing  (h06FlTx), untreated 06 integrin deficient (06~ con), treated 06 integrin /_  deficient (06" Tx), and treated wild type ( W T T x ) . The reason for exclusion of /_  these wounds was constant biting of the wound. The average weight loss at the end of the experiment was 3 g in both control and treatment groups.  4.1 Wound healing in adult wild type animals (6-month-old) All wounds in the wild type control group (WTcon) followed the expected rate of healing. Wound size was considered to be 100% at day zero; after three days, the average wound size was 48% of the original wound. Wound size continued to decrease in the following days. At day seven the average wound size was 9% with most wounds showing clinical healing. At day ten, all wounds were completely closed. The wound area resembled normal unwounded skin and the wound margin was difficult to identify from adjacent tissue (Figure 1). Wild type group treated with hydrocortisone (WTTx) showed the same pattern of healing but at a slower rate. As the wound size continued to  53  decrease over time, wounds in this group presented with a larger size at all time points. This size difference was statistically significant at all time points. A t day three, wound size was 63.6% o f the original wound compared to 48% in the control group (p=0.0008). A t day seven, some wounds started to show clinical healing but the majority were still unhealed. The average wound size at seven days was 20% which was significantly larger than that o f the control (p=0.02). A t day ten, wound margins were still clearly identifiable from normal skin (Figure 1). The average wound size was reduced to 2% o f the original. In spite o f the fact that most wounds were closed at this time point, the difference in wound size between the control and the experimental group was still significant (p= 0.03) (Figure 3). A peroxide test (absences o f bubbling in one minute in response to  H 0 2  2  application) was used as a biochemical indicator of wound closure. A l l control wounds were positive for the first four days o f healing. B y day seven, 80% o f the control wounds and 37% o f the treated wounds gave negative results to the test i.e. wounds were closed. A t day ten, 20% o f the treated wounds were still positive (open wounds) while all control wounds were negative by day nine (Figure 4).  54  Day 0 Control animals  Day 1  Day 2  4£  %  Day 3  Day 4  h  Hydrocortisone-treated animals  Day 5  Day 6  Day 7  Day 8  Day 9  Day 10  Control animals  Hydrocortisone-treated animals  Figure 1. Clinical photographs showing wound healing over time in adult (6month-old) wild type (WT) animals in control and hydrocortisone-treated groups.  55  4.2 Wound healing in aged wild type animals (22-month-old) A l l wounds in untreated wild type (WTcon) animals healed normally. The average wound size at day three was 50% o f the original wound size at day zero (Figure 3). B y day seven, wound size was 6% o f the original, and by day ten, wounds were hardly identifiable clinically and the wound area looked like normal unwounded skin to the naked eye (Figure 2). B y that time point, wounds were 100% closed. In the treated group, hydrocortisone treatment significantly delayed healing and the average wound size was larger when compared to the control wounds at all time points. The most significant delay was observed in the early phases o f healing where wounds in the treated wild type ( W T T x ) group showed only a 25% reduction in wound size by the third day ( p O . O O l ) (Figure 3). A t seven days post wounding, wound size was 23% (p=0.03). B y ten days most wounds showed 95% reduction in their size as compared to the original wound area. However, most wounds were clinically identifiable with distinct margins (Figure 2) and the difference in wound size as a  results  of  hydrocortisone  treatment  was  still  statistically  significant  (pO.OOl). A l l control wounds were positive for the peroxide test during the first four days o f healing. B y day seven, 80% o f the control wounds and 50% o f the treated wounds gave negative results to the test i.e. wounds were closed. A t day ten, 40% o f the treated wounds were still positive (open wounds) while all control wounds were negative by day nine (p<0.05) (Figure 4).  56  Day 0  Day 1  ^M^^MHjl  ^ ^ -—r m  m  Day 2  Day 3  Day 4  ^^^^^^M  ^^^^HIP  ^HI^^HI  Day 8  Day 9  Control animals  Hydrocortisone-treated animals  1  Day 5  Day 6  Day 7  Day 10  Control animals  Hydrocortisone-treated animals  Figure 2. Clinical photographs showing wound healing over time in aged (22-month-old) wild type (WT) animals in control and Hydrocortisonetreated groups.  57  4.3 Comparison of wound healing in wild type animals in relation to age Wounds in both control groups followed the same pattern o f healing. Most o f the reduction in the wound size was achieved by day three. Average wound size was 52% in adult and 50% in the aged group. Wound healing continued at the same rate over time and was 100 % closed by day nine. Analyzing the wound size changes in both groups failed to show any statistically significant difference at any time point. In the treatment groups, older animals presented with a larger wound size at all time points. However, there was no statistically significant difference between the two groups (Figure 3). A l l wounds were positive to the peroxide test in the first four days. B y day seven, 20% o f wounds in both adult and aged animals were positive to the test. A l l wounds were completely  closed by day nine in both groups. In the  hydrocortisone treated groups, 43% and 50% o f wounds were positive at day seven in adult and aged animals respectively. B y day ten 40% o f aged and 20% of adult animals were still positive to the test (Figure 4). The results above indicate that the wound healing rate is not significantly affected by age. However, in wound healing compromised by stress, there was a tendency for the older animals to have larger wounds, although the difference did not reach statistically significant level.  58  Relative wound size in wild type animals 120  -i  ,oo J  r  1  2  3  4  5  6  7  8  9  10  Day  Figure 3. Mean and standard error of wound size changes over time in wild type animals. Comparison between the adult (6-month-old) and the aged (22-month-old) mice in control and treatment group. * WTcon (6-month-old)/WTTx (6-month-old): p=0.0002,0.009,0.0008,0.05,0.02, 0.01,0.005,0.03 for day 1,2,3, 5,7, 8, 9, and 10. t WTcon (22-month-old)/WTTx (22-month-old): p=001,0.002,0.001,0.02,0.01,0.03,0.01,0.0001 for day 1,2, 3,4,5,7,8, and 10.  59  H2Q2 test in wild type animals 120  Figure 4. H 0 test in wild type animals comparing different age groups. Percentage of 2  2  wounds reacting positively to the test over time. * WTcon(6-month-old)/WTcon(22-month-old): pO05.  60  4.4 W o u n d healing in adult 66 integrin deficient mice (6-month-old) The wound healing rate in the adult 06 integrin deficient control mice (06"~ 7  con) was comparable to that o f adult wild type control (WTcon) animals (Figure 5). A t day three, the average wound size was 54% and 48% in 06 integrin deficient and wild type animals, respectively. Over the time, the same trend o f comparable healing rates and wound size changes was  observed  between the two groups and complete healing was reached by day nine (Figure 6). Hydrocortisone treatment slowed the healing rate in both experimental groups (adult wild type and 06 integrin deficient).  The degree o f delayed  healing was not different when the two groups were compared clinically and statistically (Figure 6). However, wounds in the 06 integrin deficient animals treated with hydrocortisone presented with a larger wound size at all time points when compared to wounds in the untreated 06 integrin deficient animals (Figure 5). For example, at day three the average wound size was 65% which was significantly larger than the wound size in the untreated 06  integrin  deficient mice (p=0.01). The largest difference in wound size occurred at day seven as wound size in the control group was 5% and 18% in the treated group (p=0.0001). A t day ten wound size was only 2% o f the original but when compared to the wound size in the control animals, the difference  was  statistically significant (p= 0.02) (Figure 6). Wound size in treated 06 integrin deficient animals (06" Tx) was significantly /_  larger at all time points when compared to the wound size o f the age matched untreated wild type animals (WTcon). F o r example, at day three wound size in  61  the untreated wild type group (WTcon) was reduced by 62% whereas wound size in the  06 integrin  deficient wounds  (06 Tx) had only a 35% reduction _/  (p=0.0003). B y day eight, wounds in the untreated wild type animals (WTcon) had a reduction o f  96%  compared to  89%  in the  06  integrin deficient  (06"Tx) /  wounds (p=0.01) (Figure 6). The peroxide test was positive for all the wounds in all 06 integrin deficient animals for the first four days. B y day seven, all control wounds were negative to the test while 30% o f the hydrocortisone treated wounds were still positive. E v e n after ten days o f healing, 20% o f the treated wounds still tested positive to the peroxides test (Figure 7). In summary, the results demonstrated that adult 06 integrin deficient animals healed favourably and at a comparable rate to that o f age matched wild type animals. Animals stressed by hydrocortisone treatment responded with the same delayed healing regardless o f their 06 integrin status.  62  DayO  Dayl  Day 2  Day 3  Day 4  Day 5  Day 6  Day 7  Day 8  Day 9  Control animals  Hydrocortisone-treated animals  Control animals  Day 10  ||  Hydrocortisone-treated animals  Figure 5. Clinical photographs showing wound healing over time in adult (6month-old) p6 integrin deficient (p6 ) animals in control and hydrocortisone-treated groups. _yL  63  Relative wound size in adult wild type and beta 6 integrin deficient mice 100  1  2  3  4  5  6  7  8  9  10  Day  Figure 6. Mean and standard error of wound size changes over time in adult (6-month-old) wild type and P6 integrin deficient animals. Comparison between control and treatment groups. * WTcon (6-monfo-old)/pV"con (6-month-old): p= 0.02 for day 1. t p^con (6-month-old)/pVTx (6-month-old): p= 0.01,0.03,0.03,0.0001,0.0001,0.007,0.02 for day 3,4, 5, 7, 8, 9, and 10. § WTcon (6-month-old)/p6'Tx (6-month-old): p= 0.01,0.0,0.0003,0.02, 0.01, 0.02,0.02 for day 1, 2,3, 7, 8,9, and 10.  64  H202 test in adult mice 120  T—  Day  Figure 7. H 0 test in adult (6-month-old) P6 integrin deficient animals in comparison to 2  2  age matched wild type animals. * WTcon (6-month-old) / p6 " con (6-month-old): p<0.025. v  § WTcon (6-month-old) / p6 Tx (6-month-old): p<0.05. v  65  4.5 Wound healing in aged B6 integrin deficient animals (22-month-old) Wounds in aged untreated 06 integrin deficient mice (06 con) (Figure 8) /_  demonstrated a significantly slower healing rate compared to age matched untreated wild type animals. A t day three, the average wound size in 06 integrin deficient animals was 65% o f the original size, which was statistically larger than the wound size in the wild type group (p=0.0008) (Figure 9). Healing continued at a slower rate in the 06 integrin deficient animals and the most significant difference in wound size occurred between day three and day seven, which coincides with the time where maximum granulation tissue formation is expected (Figure 9). A t later stages (days eight and on) wound size became  comparable to that o f the wild type group. B y the end of the  experiment (day 10) wound size was not statistically different between the two control groups (Figure 9). In the aged 06  integrin deficient (06" ) group /_  receiving hydrocortisone, six to ten-day-old wounds  showed  significantly  reduced healing compared to untreated controls (Figure 9). In fact, the slowest healing rate o f all animals was found in this group. For example, 3-day-old wounds in 22-month-old untreated wild type animals showed a 50% reduction in  wound  size  while  22-month-old  06  integrin  deficient  mice  in  the  hydrocortisone group demonstrated only a 30% reduction in wound size (p=0.0005). At day eight, wound size in the aged untreated wild type animals was reduced by 97% compared to only a 77% reduction in the 06 integrin deficient animals (p=0.001) (Figure 9).  66  \  The peroxide test was positive for all the wounds in the aged 06 integrin deficient animals for the first four days. B y day seven, 33% o f control wounds in the aged 06 integrin deficient animals were positive to the test (compared to 20% in aged wild type animals) and by day 10 only 11% were still positive (Figure 10). Interestingly, 06 integrin deficient mice in the hydrocortisone group showed positive results until day six which was the most response  among all animals groups and was  statistically  delayed  different  when  compared to wild type control animals (p<0.025). B y day 10 only 22% o f the wounds were still positive for this test (Figure 10) In summary, wound healing in older 06 integrin deficient animals showed a significant delay when compared to the age matched wild type animals. Wounds in the 06 integrin deficient experimental group reacted similarly to the wild type experimental group in terms o f healing rate.  67  DayO  Dayl  Day 2  Day 3  Day 4  Day 6  Day 7  Day 8  Day 9  Control animals  Hydrocortisone-treated animals  WM  Day 5 Control animals  Day 10  mm mm mm mm mm  Hydrocortisone-treated animals  Figure 8. Clinical photographs showing wound healing over time in aged (22month-old) p6 integrin deficient (p6" ) animals in control and hydrocortisonetreated groups. /_  68  Relative wound size in aged mice 100  1  2  3  4  5  6  7  8  9  10  Figure 9. Mean and standard error of wound size changes over time in aged (22-month-old) wild type and p6 integrin deficient animals. Comparison between control and treatment groups. * WTcon (22-month-old)/p6" "con (22-month-old): p=0.002, 0.004, 0.0008, 0.007,0.002, 0.004, 0.003 for day /  1,2,3,4,5, 6, and 7. § WTcon (22-month-old)/P6"TX (22-month-old): p=0.002, 0.0006, 0.0005, 0.01, 0.0001, 0.001, 0.0007, 0.001 for day 1,2, 3,4, 5, 8,9, and 10. t p6'"con (22-month-old)/p6^Tx(22-month-old): p=0.006,0.05,0.01,0.05,0.003 for day 6,7, 8,9, and 10.  69  H202 test in aged mice  Figure 10. H 0 test in aged (22-month-old) P6 integrin deficient animals in comparison to 2  2  age matched wild type animals. * WTTx (22-month-old) / p ^ ' T x (22-month-old): P=0.025. § WTcon (22-month-old) / p V T x (22-month-old): p=0.025.  70  4.6 C o m p a r i s o n of w o u n d healing in B6 deficient animals in relation to age Control wounds in older animals presented with a larger wound size when compared to younger animals in all time points. The delay in healing was statistically significant (Figure 11). For example, at days four and seven, wound size was 39% and 5% respectively in the adult animals, whereas the wound size in the aged animals at the same time points was 62% and 21% respectively (p=0.002). This difference in wound size continued to be obvious clinically and statistically until day ten (Figure 11). The same trend was observed in the experimental group in which older animals had larger wound sizes and a significant delay in healing when compared to the younger animals at all time points (Figure 11). The peroxide test was positive for all the wounds in the control groups for the first four days. B y day seven, all wounds in the adult animals were negative to the test while 33% of the wounds in the aged animals were still positive to the test (p<0.01). B y day ten only 11% o f wounds in that group were still positive to the test ( p O . O O l ) . In the treated groups, all wounds in the aged animals were still positive to the test by day six, while 66% o f wounds in the mature animals were positive at that time point (p<0.05). B y day ten, 20% o f wounds in both treatment groups were still positive to the test (Figure 12). The results showed that older animals deficient in the p6 integrin subunit had delayed healing in both normal and compromised wound healing models when compared to the younger age group.  Relative wound size in beta 6 integrin deficient mice  1  2  3  4  5  6  7  8  9  10  Day  Figure 11. Mean and standard error of wound size changes over time in f36 integrin deficient animals. Comparison between adult (6-month-old) and aged (22-month-old) mice in control and treatment groups. * Po^cont (6-month-old)/p6"'" con (22-month-old): p=0.02,0.002,0.01,0.009, 0.002,0.02 for day 3,4, 5, 6,7, and 9. § P 6 ' T X (6-month-old)/p6" Tx (22-month-old): p=0.01, 0.05,0.01, 0.001, 0.006, 0.03, 0.03 for day 2,4, 5, 6, /  7, 8, and 10.  72  H202  Figure 12.  H 0 2  2  test in beta 6 Integrin deficient mice  test in P6 integrin deficient animals comparing different age groups.  Percentage of wounds reacting positively to the test over time. § p V T x (6-month-old) / p V T x (22-month-old): pO.05. *P6"'"con (6-month-old) /pVcon(22-month-old): pO.OOl.  73  4.7 W o u n d healing in aged B6 integrin overexpressing animals (hB6Fl)  Because the absence of the 06 integrin subunit appeared to be associated with slower wound healing rates, especially in aged animals, we investigated whether overexpression of this integrin will positively affect the healing rate. Wounds in untreated 06 integrin overexpressing animals (h06Fl) healed with same rate as the untreated wild type animals. At day three, wound size was reduced by 51%, and complete wound closure was achieved by day nine in this group (Figure 13 and 14). As expected, wounds in the  06  integrin overexpressing animal  (h06FlTx)  group receiving hydrocortisone showed a slower healing rate when compared to wounds in untreated 06 integrin overexpressing animals (h06Flcon) and the most significant delay occurred by days nine and ten (p=0.005, 0.05) (Figure 14). Interestingly, when comparing treated 06 integrin overexpressing animals (h06FlTx) to treated wild type (WTTx) animals, the treated 06 integrin overexpressing animals  (h06Fl) healed  faster at almost all time points. The  most significant difference occurred during the early healing phases with the average wound size being 60% in the treated 06 integrin overexpressing animal (h06FlTx) group and 75% in the wild type (WTTx) group (p=0.05) at day three. By day ten, wound sizes in treated 06 integrin overexpressing animals  (h06FlTx)  and wild type ( W T T x ) mice were 0% and 5% respectively  (p=0.003) (Figure 14).  The same tendency towards improved wound closure rate in the treated 06 integrin overexpressing (h06FlTx) animals compared to the treated wild type 74  ones was observed in the peroxide test. B y days nine and ten, 40% o f wounds in the treated wild type group were positive as compared to only 12% in h p 6 F l animals (Figure 15). In  summary, mice  overexpressing  human  06  integrin were  moderately  protected from the delayed wound closure caused by hydrocortisone treatment.  75  DayO  Dayl  Day 2  Day 3  Day 4  Control animals  # •  Hydrocortisone-treated animlas  Day 5  Day 6  Day 7  Day 8  Day 9  Day 10  Control animals  Hydrocortisone-treated animals  Figure 13. Clinical photographs showing wound healing over time in human (36 integrin overexpressing (hp6Fl) animals in control and hydrocortisonetreated groups.  76  Relative wound size in wild type and human beta 6 integrin overexpressing mice  Figure 14. Mean and standard error of wound size changes over time in human 06 integrin overexpressing (hp6Fl) and wild type (WT) mice. Comparison between the control and treatment group. * WTTx (22-month-old)/hp6FlTx (22-month-old): p=0.008,0.009,0.05, 0.003 for day 1,2,3 and 10. § hp6FlTx(22-month-old)/hp6Flcon (22-month-old): p=0.005,0.01 for day 9 and 10.  77  H202 test in wild type and human beta 6 integrin overexpressing mice 120  2  3  4  5  6  7  8  9  Day  Figure 15. Percentage of wounds positive to  H2O2 over  time. Comparison between aged  wild type and human p"6 integrin overexpressing mice. § WTcon (22-month-old) / WTTx (22-month-old): p=005. * hp6Flcon (22-month-old) / hpoTTTx (22-month-old): p=0.05. t WTcon (22-month-old) / hp6Flcon (22-month-old): p=O.05.  78  4.8 Histology of 3-day-old wounds Histological sections from the center o f 3-day-old wounds o f the untreated animals showed the epithelial tongue migrating to a variable distance (Figures 16, 17, and 18). Analysis o f the average distance migrated by the epithelial cells in the three untreated animal groups failed to show a statistically significant difference (Figure 22). However, when the distance migrated by epithelial cells in wounds o f the animals receiving hydrocortisone treatment was analysed, a clear inhibition o f cell migration was observed. In the treated wild type ( W T T x ) group, keratinocytes at the wound edges were proliferative but minimal migration was detected (Figure 19). The same observation was seen in the wounds from treated 06 integrin deficient (P6 Tx) animals (Figure _/  20). The difference in the average distance migrated by wound keratinocytes in control and experimental groups was significant (p =0.003, p=0.0002 for W T and P6 " animals, respectively), while the distance migrated by keratinocytes in V  both  treatment  groups  was  not  statistically  significant.  Analyzing  the  histological section from the hydrocortisone-treated P6 integrin overexpressing animals ( h p 6 F l T x ) , it was observed that epithelial cells migrated to a distance comparable to that o f the untreated animals and the difference in migration between this group and the hydrocortisone-treated wild type ( W T T x ) group was statistically significant (p=0.01) (Figures 21 and 22). This supports the clinical observation that wounds in the treated p6 integrin overexpressing ( h p 6 F l T x ) group healed at a faster rate than corresponding wounds in the treated wild type ( W T T x ) group.  79  Wounds from the control animals were still covered by blood clots. In addition to the epithelial tongue migrating to variable distances into the wound area, a significant amount o f granulation tissue had formed. The wound area showed a heavy inflammatory infiltrate consisting mostly o f mononuclear cells. In the experimental group (with the exception o f the h p 6 F l group), a significant delay in keratinocyte migration as explained above was seen and granulation tissue was hardly detectable. Degree o f inflammation varied, although the general impression was that wounds belonging to the experimental groups showed less inflammation. In the treated p6 integrin overexpressing ( h p 6 F l T x ) group, the histological picture was similar to that of the control group rather than the experimental group. Advanced epithelial migration with larger amounts o f granulation tissue formation were seen, however less inflammatory reaction was seen in the wound area.  80  Figure 16. Hematoxylin stained sections of 3-day-old wounds in untreated wild type mice (WTcon). An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, CT: Connective tissue, BC: Blood clot, F: Fat cells, arrows point to the tip of migrating epithelium.  Figure 17. Hematoxylin stained sections of 3-day-old wounds in untreated p6 integrin deficient (P6"^"con) mice. An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, HF: Hair follicle, arrows point to the tip of migrating epithelium.  82  Figure 18. Hematoxylin stained sections of 3-day-old wounds in untreated human p6 integrin transgenic (hp6Flcon) mice. An overview of the wound (A); magnified sections showing the tip of migrating epithelium (B and C). E : Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, arrows point to the tip of migrating epithelium.  83  Figure 19. Hematoxylin stained sections of 3-day-old wounds in wild type mice treated by hydrocortisone (WTTx). An overview of the wound (A) showing the minimum migration of the epithelium at the wound edges; magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, CT: Connective tissue, B C : Blood clot, F: Fat cells, arrows point to the tip of migrating epithelium.  84  Figure 20. Hematoxylin stained sections of 3-day-old wounds in p6 integrin deficient mice treated by hydrocortisone (p6'^"Tx). An overview of the wound (A) showing the minimum migration of the epithelium at the wound edges; magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, arrows point to the tip of migrating epithelium.  85  Figure 21. Hematoxylin stained sections of 3-day-old in 06 integrin overexpressing mice treated by hydrocortisone (P6FlTx). An overview of the wound (A) showing advanced epithelial migration as compared to the other treatment groups. Magnified sections showing the tip of migrating epithelium (B and C). E: Epithelium, GT: Granulation tissue, CT: Connective tissue, BC: Blood clot, arrows point to the tip of migrating epithelium.  86  Epithelial cell migration in 3-day-old wounds  *T  T  »T  —  T  T  , WTcon  p6-/-con  1  hB6F1con  WTTx  ,  1  BfrJ-Tx  hp6F1Tx  Animal group  Figure 22. Mean and standard error of epithelial cell migration in 3-day-old wounds. * WTcon/WTTx: p=0.003. § p6 con/p6-'Tx: p=0.0002. v  t hp6FlTx/WTTx: p=0.01.  87  4.9 Histology of 10-day-old wounds All  10-day-old wounds  /  in the untreated wild type (WTcon) group were  completely re-epithelialized. However, wound epithelium was still thicker than the surrounding tissue (Figure 23). Wound granulation tissue was rich with spindle shaped cells resembling fibroblasts embedded in a lightly stained fibrillar matrix similar to collagen fibers. Fibers were less organized and more wavy in comparison to the darkly stained collagen fibers in the connective tissue  of  adjacent,  unwounded  skin.  Inflammatory  cell  infiltrate  was  significantly decreased when compared to that seen in the 3-day-old wounds. Multiple blood vessels were present in the granulation tissue, and fat and muscle layers were not completely restored in the wound area. In the treated wild type ( W T T x ) group, all wounds were also completely reepithelialized after ten days. However, the epithelium in the center o f the wound was still hyperproliferative with prominent rete ridges extending deep into granulation tissue. Cell rich granulation tissue was seen with numerous blood vessels surrounded by very lightly stained fibers (Figure 23). In  untreated  {36  epithelialization  integrin rate,  deficient  inflammatory  (P6" ) group, no A  reaction  or  differences  organization  of  in  re-  wound  granulation tissue were detected (Figure 24). Similarly, 10-day-old wounds in the treated p6 integrin deficient (P6 "Tx) group were all re-epithelialized and v  the granulation tissue formation resembled that seen in the treated wild type animals (Figure 24).  88  10-day-old wounds in the untreated 06 integrin overexpressing  (h06Flcon)  group were completely re-epithelialized and resembled those seen in the wild type mice (Figure 25). In the treated 06 integrin overexpressing ( h 0 6 F l T x ) group,  10-day-wounds  demonstrated higher organisation in the wound epithelium compared to those in the untreated wild type animals. Minimal rete ridge formation was present, the  epithelium  thickness  resembled  that o f untreated  controls,  and  the  granulation tissue appeared to have more organized collagen fibers (Figure 25) In summary, complete  re-epithelialization o f all 10-day-old wounds  observed,  with  combined  significant  granulation  tissue  was  formation.  Hydrocortisone appeared to delay the organization o f wound epithelium as long extensions (rete ridges) were visible in all treated animals except in the 06 integrin overexpressing mice. The histological delay is therefore in agreement with the clinical observations and the histology o f the 3-day-old wounds. 06 integrin overexpression may in fact protect from delayed the wound healing caused by hydrocortisone treatment.  89  Figure 23. Histological sections of 10-day-old wounds in wild type (WT) mice. Untreated wounds (A and B) and treated wounds (C and D). Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D). Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer, F: Fat cells.  90  Figure 24. Histological section in 10-day-old wounds in 06 integrin deficient (P6"/") mice. Untreated wounds (A and B) and treated wounds (C and D). Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D). Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer, F: Fat cells.  91  Figure 25. Histological sections of 10-day-old wounds in 06 integrin overexpressing (h06Fl) mice. Untreated wounds (A and B) and treated wounds (C and D). Hematoxylin and eosin stained sections (A and C), Masson's trichrome stained sections (B and D). Complete re-epithelialization of the wound and newly formed collagen fibers and blood vessels can be seen in the wound area. E: Epithelium, GT: Granulation tissue, CT: Connective tissue, M : Muscle layer.  92  4.10 Expression of type IV collagen In 3-day-old wounds, strong staining for type I V collagen was seen at the basement membrane zone in nonwounded skin in all different groups. The staining was absent at the migrating epithelial tip, indicating the lack o f a normal basement membrane under migrating cells (Figure 26). In 10-day-old wounds, type I V collagen staining was observed at the basement membrane zone in all samples (Figure 27).  4.11 Expression of B6 integrin 06 integrin was immunolocalized in wounds and animal tissue with an antibody reacting with both endogenous mouse and human (transgene) 06 integrin. Overexpression o f human  06  integrin in  h06Fl  animals was confirmed by  strong staining in the basal keratinocytes o f the ear that typically express higher levels o f the  transgene (Hakkinen et al., 2004).  In wild type  animals,  keratinocytes in intact epidermis o f nonwounded tissue did not express 06 integrin except occasionally around the hair follicles. In contrast, the migrating epithelial tongue showed relatively strong staining but it was confined to the basal cell layer (Figure 28 A ) . Epidermis in wild type animals treated with hydrocortisone ( W T T x ) showed only faint staining at the edges o f the wound which demonstrated minimal epidermal migration (Figure 28 B ) . Animals deficient in  06  integrin  (06" ) A  showed complete lack of staining in  unwounded and wounded skin regardless of the amount of epithelial migration and regardless of the treatment (Figure 28 E and F).  93  Animals overexpressing the human 06 integrin in addition to their own (h06Fl) showed strong staining for 06 integrin even in nonwounded skin and around the hair follicles. In wounded skin, darkly stained cells were seen in basal and suprabasal layers along the wound edges. The pattern of expression was similar in the treated group (h06F!Tx), suggesting that hydrocortisone did not directly interfere with 06 integrin expression in this group (Figure 28 C and D). In 10-day-old wounds in both wild type untreated and treated animals, 06 integrin was still expressed in wound epithelium. The expression was confined to the center of the wounds with no staining at the periphery (Figure 29 A and B). Tips of the rete ridges of the wounds in the treated animals still show strong 06 integrin expression remaining (Figure 29 B). Animals overexpressing the human 06 integrin (h06Fl) continued to show high 06 integrin expression in basal and suprabasal cell layers through out the wounded skin (Figure 29 C and D), and no staining was seen in the wounds of animals deficient in 06 integrin (p6" ) (Figure A  29 E and F).  4.12 Immunolocalization of TGFp* isoforms We used a monoclonal antibody that recognizes all three isoforms of TGF-01,02, and -03. Strong staining of basal and suprabasal keratinocytes was seen in all 10-day-old untreated wounds. However, dermal tissue showed minimal staining in the deeper tissue. Wounds in the treated wild type and 06 deficient animals (WTTx and 06"Tx) showed variable intensity of staining in the dermal /  tissue with minimal staining under the epithelium. Wounds in the treated  94  human p6 integrin overexpression group ( h p 6 F l T x ) showed strongly stained cells in the wound connective tissue (Figure 30).  95  Figure 26. Immunohistochemical localization of type IV collagen in 3-dayold wounds in treated (A, C, E) and untreated (B, D, F) animals. Wild type (WT) mice (A and B), human (36 integrin overexpressing (hp6Fl) mice (C and D), and p6 integrin deficient (P6''~) mice (E and F). E: Epithelium, CT: Connective tissue. Arrows point to the tip of migrating epithelium, arrow heads point to the end of positive staining for type IV collagen.  96  Figure 27. Immunohistochemical localization of type IV collagen in 10day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human p6 integrin overexpressing (hp6Fl) mice (C and D), and p6 integrin deficient (p6" ) mice (E and F). Results show the complete restoration of type IV collagen staining at the basement membrane zone. E: Epithelium, CT: Connective tissue. /_  97  Figure 28. Immunohistochemical localization of 06 integrin in 3-day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D), and 06 integrin deficient (06~/~) mice (E and F). E: Epithelium, CT: Connective tissue, HF: Hair follicle, arrows point to the tip of migrating epithelium, arrow heads point to cells with positive staining.  98  Figure 29. Immunohistochemical localization of 06 integrin in 10-day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human 06 integrin overexpressing (h06Fl) mice (C and D), and 06 integrin deficient (06"^") mice (E and F). E : Epithelium, CT: Connective tissue.  99  E  Figure 30. Immunohistochemical localization of TGFp in 10-day-old wounds. Untreated (A, C, E) and treated (B, D, F) animals. Wild type (WT) mice (A and B), human p6 integrin overexpressing (hp6Fl) mice (C and D), and p6 integrin deficient (p6 ) mice (E and F). E: Epithelium, CT: Connective tissue, WCT: Wound connective tissue. _/_  100  Chapter 5 - Discussion 5.1 Wound healing and glucocorticoid treatment Our results showed that there is a significant delay in wound healing as a result o f hydrocortisone treatment. This delay was present in both adult and aged mice of all genetic backgrounds used. The most significant delay in our study was seen in the three day old wounds. A t this time, wounds exhibited larger size clinically while histologically, hydrocortisone treatment slowed epithelial cell migration and decreased the amount o f early granulation tissue formation. This result is in agreement with previously published results (Lenco et al., 1975; Salmela, 1981; Brauchle et al., 1995). A t ten days, all wounds were completely re-epithelialized. However, wounds in the treated groups showed more distinct wound margins clinically. Histologically, epithelial morphology was less organized with abundant inflammatory reaction and angiogenesis in wound  granulation  tissue.  This  indicates  delayed  inflammatory  and  proliferative phases in the treated wounds as compared to wounds in the untreated groups. Similar findings were reported in mice after a single injection o f dexamethasone (Durmus et al., 2003). Previous data indicated that wounds in  animals  treated  epithelialization,  with  collagen  hydrocortisone formation,  show  wound  significant  contraction  delayed  and  re-  decreased  fibroblast numbers and hydroxyproline content. This ultimately results in a significant decrease in wound strength at all time point (Sandberg,  1964;  Ehrlich and Hunt, 1968; Lenco et al., 1975; Salmela, 1981; Gupta et al., 1999). Initially,  delayed  wound  healing  was  thought  to  be  due  to  the  anti-  101  inflammatory action of hydrocortisone because delayed hydrocortisone administration beyond the initial inflammatory phase (first three days after wounding) did not result in a significant difference in healing (Sandberg, 1964; Stephens et al., 1971; Salmela et al., 1980). When different glucocorticoids with equivalent doses of anti-inflammatory action were compared, the most significant delay in healing was seen in the hydrocortisone group (Lenco et al., 1975; Dostal and Gamelli, 1990), suggesting that the cellular effects of glucocorticoids on wound healing extend beyond their anti-inflammatory action. Over the years, mechanisms for delayed wound healing caused by GC have been explored. Systemic hydrocortisone injection in guinea pigs induced a prolonged monocytopenia and reduced the macrophage levels to one-third that of controls. This resulted in partial inhibition of wound debridement leading to delayed healing; however, these wounds eventually healed (Leibovich and Ross, 1975). Wounds in prednisolone treated mice showed total absence of neutrophils and macrophages (Salmela et al., 1980; Pierce et al., 1989a). Dexamethasone treatment to dermal fibroblasts resulted in a dose dependant downregulation of monocyte chemoattractant protein (MCP-1) mRNA, which is responsible for the recruitment and activation of monocytes to the wound area (Slavin et al., 1995; Dipietro et al., 2001). Interestingly, delayed reepithelialization in wounds from MCP-1 deficient (MCP-1 ") mice was 7  reported (Low et al., 2001) and the greatest delay was in the first three days after wounding. In addition, collagen synthesis and hydroxyproline content of the wound were reduced. However, by day ten, the difference in wound healing  102  between MCP-1"" mice and wild type littermates no longer persisted (Low et al., 2001). In addition to a decreased number of macrophages in glucocorticoid treated wounds, the functional activity of those macrophages is reduced e.g. GC treatment downregulates the production of inflammatory cytokine such as TNFa, IL-1 (3 and IL-12frommacrophages (Bendrups et al., 1993; Blotta et al., 1997). Cytokines and growth factors production is reduced in response to glucocorticoid treatment as an indirect effect of decreased inflammatory cell recruitment to the site of injury (Beer et al., 2000) and decreased angiogenesis (Folkman, 1983). Direct inhibitory effects on the cells can not, however, be excluded. The ability of GC to bind and inhibit N F - K B , which is a regulatory factor for many cytokine and cell adhesion molecules, could explain the profound effect that GC have on almost all cytokines (Auphan et al., 1995). For example, dexamethasone treatment reduced the normal induction of IL-10, ILla, and TNFa mRNA in three day wounds by 50-70% (Hubner et al., 1996). Moreover, growth factors like KGF and PDGF A, B mRNA and their receptors were found to be reduced significantly in three day old wounds of dexamethasone treated animals (Brauchle et al., 1995; Beer et al., 1997). Other growth factors that have been reported to be negatively affected by dexamethasone treatment were TGFpM and -2 and TGFJ3 type II receptor whereas, TGFp-3 show a mild increase in the early phases of healing in wounds derived from glucocorticoid animals (Frank et al., 1996). Prednisone treatment significantly reduced TGFp levels in wound fluid and resulted in a 1  decreased hydroxyproline content (Wicke et al., 2000). This may give a  103  molecular explanation o f the beneficial effect o f exogenous TGFJ31 application to the wounds in glucocorticoid treated animals (Pierce et al., 1989a; Beck et al.,  1991; Beck et al., 1993). Systemic T G F p i  application resulted in an  increased number o f wound fibroblasts and increased procollagen production, which ultimately increased wound strength to a level comparable to that o f untreated animals (Beck et al., 1993). Topical T G F p i application also resulted in increased matrix formation and re-epithelialization o f ischemic open wounds in G C treated rabbits (Beck et al., 1991). In addition, T G F P treatment o f dermal fibroblasts upregulated M C P - 1 m R N A levels and reversed the inhibitory effect of dexamethasone on dermal fibroblasts (Slavin et al., 1995).  5.2 Wound healing in B6 deficient animals Our results showed that P6 deficiency did not impair wound healing in young animals, which was in agreement with published results previously (Huang et al.,  1996). These findings suggest that the role o f avp6 integrin in normal  wound healing in young animals is limited and its function can be compensated by other receptors. This observation is not surprising given the fact that av06 integrin is expressed relatively late in wound healing when the epithelial edges have already joined (Haapasalmi et al., 1996). In addition T G F p activation may need to be spatially and temporally well controlled as constitive overexpression of  epithelial T G F P  seems to delay epithelialization (Yang et al., 2001).  Knockout animals to many receptors and cytokines important in wound healing have mild or no wound healing defects (Scheid et al., 2000). F o r example,  104  young  TGF01  animals (less than 30 days old) have a healing  capacity  comparable to their wild type littermates. However, studies on T G F p i " Shid " 7  7  animals which lack T G F p i and T and B lymphocytes, show a major delay (up to one week) o f all phases o f healing compared to Shid " mice with normal 7  T G F p i (Crowe et al., 2000). Interestingly, aged untreated P6 deficient animals show a significant delay in wound healing and a larger wound size when compared to younger P6 integrin deficient and to age matched wild type mice. M a n y studies show the effects o f age on wound healing. Older age  was  associated with significantly delayed re-epithelialization (Van de Kerkhof et al., 1994; Ballas and Davidson, 2001; Gosain and DiPietro, 2004; Mogford et al., 2004), decreased extracellular matrix deposition and reduced angiogenesis (Ashcroft et al., 1997c; Swift et al., 1999). In vivo studies showed that collagen and  fibronectin protein levels in normal skin, acute wounds and chronic  wounds are reduced in the elderly (Ashcroft et al., 1997c; Herrick et al., 1997). Decreased levels o f T I M P - 1 and -2 in normal skin and in response to acute injury associated with upregulation o f M M P - 9 and M M P - 2 may explain the tendency o f the elderly to develop chronic wounds (Ashcroft et al., 1997b). Age related changes in the morphology o f the skin might affect the course o f healing.  However,  these  changes  likely  represent  a  response  to  an  environmental insult rather than a direct effect o f age (Ashcroft et al., 2002). Altered inflammatory responses in older animals have been reported (Ashcroft et al., 1998), as older mice showed a 40% decrease in the biological functions of macrophages in terms o f phagocytic capacity and cytokine production (Swift  105  et al., 1999; Swift et al., 2001). Young (6-month-old) mice injected with rabbit anti-mouse macrophages serum for four days after wounding had a significant delay in wound closure rates similar to that seen in older animals (26-monthold) (Cohen et al., 1987). Cutaneous wounds in older animals injected with peritoneal macrophages derived from younger ones showed wound healing accelerated to a rate similar to that of younger animals (Danon et al., 1989). These data suggest that reduced macrophage function may play a role in reduced healing at older age. In humans and in animal models, an early influx of PMNs in epidermal wounds in older individuals has been reported (Ashcroft et al., 1998). This was associated with increased PMN-derived elastase and increased fibronectin breakdown (Herrick et al., 1997). Recently, delayed T cell appearance in the wounds of aged mice was reported (Swift et al., 2001). The reversal of age induced delayed wound healing was possible by systemic and topical application of estrogen downregulates  (Ashcroft et al., 1997a). Estrogen  neutrophil function through decreasing the expression of  adhesion molecules, inhibition of chemotaxsis and dampening the immune response in general (Ashcroft et al., 2003). In vivo studies showed age decreases cellular activity as human dermal fibroblast migration, integrin function and cytoskeletal organization was decreased in cell lines derived from 50% of the volunteers over the age of 80 years (Reed et al., 2001). Slower rate of epithelial migration and increased keratinocyte response to the negative regulator of migration such as INFy was reported in both in vivo and in vitro studies (Swift et al., 1999; Yao et al., 2001). The molecular mechanisms of  106  decreased cell function are complex but TGFJ3 may play an important role as dermal fibroblasts from older volunteers showed a decreased expression o f T G F P receptors in response to hypoxia and an overall decreased response to T G F p stimulation as measured by activation o f the p42>p44 M A P K pathway (Mogford et al., 2002). When cell migration was evaluated as an outcome o f TGFpi  stimulation, dermal fibroblasts from older individuals had a lower  response (i.e. reduced migration capacity) in comparison to fibroblasts from younger  volunteers  (Mogford  et  al.,  2002).  Keratinocytes  from  older  individuals fail to upregulate T G F p receptor expression in response to hypoxia as the cells derived from younger volunteers do ( X i a et al., 2001; Y a o et al., 2001). Aged rabbits had a major decline in T G F p i m R N A levels in wound area under both ischemic and non-ischemic conditions (Wu et al., 1999) and the number o f positive cells and expression levels o f T G F p i in the dermis o f old mice 24 hours after thermal injury was reduced when compared to young mice (Schmid et al., 1993). In humans, delayed wound healing was associated with decreased levels o f T G F P - 1 and-2 in the wound fluid o f older individuals when compared to younger subjects. Decreased T G F p i staining in the wounds o f older females was reported when compared to age matched controls (Ashcroft et al., 1997a). Interestingly, T G F p i administration systemically reversed age induced defects in healing in old mice and topical estrogen application on cutaneous wounds in elderly males and females was associated with decreased wound size, faster epithelialization and increased collagen matrix deposition, and  increased T G F p  expression in wound areas (Ashcroft et al., 1999a).  107  Estrogen increased the secretion of T G F P 1 by dermal fibroblasts regardless of donor age (Ashcroft et al., 1997a). Alteration in other cytokines can not be ignored as delayed and decreased secretion of P D G F - A and-B isoforms and their receptors expression during wound healing was reported (Ashcroft et al., 1997d; Yao et al., 2001), as well as alteration in E G F levels and the expression of its receptor on aged murine keratinocytes (Ashcroft et al., 1997d; Yao et al., 2001). Other growth factors being affected by age were V E G F and b-FGF which resulted in delayed angiogenesis (Swift et al., 1999; Swift et al., 2001). Age  related increase in collagen gel remodelling and contraction, with  increased expression of MMP-2 by skin fibroblasts derived from aged mice, was reported and may provide the basis for the alteration in the tissue remodelling phase. However, the number of apoptotic cells and myofibroblasts were not different between young and older mice (Ballas and Davidson, 2001). Interestingly, wounds in older humans and animals were reported to heal with a better quality of scarring compared to their younger counterpart. Dermal organization was reported to closely resemble that of normal skin (Ashcroft et al., 1997c). Reduced local TGF|3 levels may be responsible for the decreased scarring seen in the elderly (Ashcroft et al., 1997d). Estrogen administration reversed age induced defect in healing in both humans and animals but reduced scar quality (Ashcroft et al., 1997a). Decreased levels of TGF(3 in the wound area as a direct effect of age on macrophages and dermal fibroblasts, in addition to decreased local activation of  TGF0 due to the  deletion of the p6 integrin subunit (Sheppard, 2001), might  108  be responsible for the delayed wound healing in aged p6 integrin deficient mice. However, all wounds in our study closed by day ten regardless o f their treatment status. Wound closure is achieved by the migration o f activated keratinocytes  from the edges o f the wound coupled by enhanced mitotic  activity behind the migration front (Clark, 1996). Wound contraction reduces the size o f the defect by pulling the wound edges together, decreasing the distance the cells has to migrate in order to re-establish epidermal integrity (Martin, 1997). In rodent (mice in particular) wound contraction plays a significant role in healing. Almost 70% of wound closure in mice is due to contraction, compared to only 20% in human skin (Snowden, 1984; Olsen et al., 1995). Because of that, an adult mouse has the ability to close a full thickness, 1.5 cm, circular wound in a period o f 10 days (Mellin et al., 1995). M i c e and rabbits show an early closure phase while this phase was lacking in animals with fixed skin like pigs (Kennedy and Cliff,  1979). In fact the  removal o f panniculus carnosus muscle prior to wounding significantly delayed healing in rabbits by three days (Snowden et al., 1982). In our study, inspite o f the initial contraction phase, wounds in aged animals had a larger size when compared to control wounds, which suggests that the defect in healing might be even bigger in mammals with fixed skin like humans.  5.3 Wound healing in 66 integrin overexpressing animals Wounds  in animals overexpressing  the  p6 subunit show a healing  rate  comparable to that o f age matched wild type animals. Clinically, a 50 %  109  reduction in wound size was achieved in the first three days. Histologically, the re-epithelialization rate and granulation tissue formation phase was similar to untreated wild type ( W T con) mice. This is in agreement with previously published  data  (Hakkinen  et  al.,  2004).  However,  in  wound  healing  compromised by hydrocortisone treatment, overexpression o f the 06 subunit seems to play a protective role. The finding that the treatment group showed an epithelial migration comparable to that o f the control animals at three days post wounding and that the wound size was reduced at a much faster rate when compared to the healing rate o f the wounds in the experimental wild type was interesting. While  av06 integrin expression was downregulated by day ten in  wild type animals in a previous study (Hakkinen et al., 2004) as well as this study,  <xv06 was highly expressed in the wound epithelium o f transgenic  animals during the later stages o f wound healing (Hakkinen et al., 2004), which may provide an explanation for its protective role in the compromised wound healing model. One possible explanation is via the ability o f activate  av06 to bind and  TGF01. Newly released TGFP peptide is rendered inactive due to its  association with glycosylated latency associated peptide ( L A P ) and the latent  TGFp binding protein ( L T B P ) (Koli et al., 2001). L T B P function as an anchoring protein that localize presenting  TGFp to matrix and play an important role in  TGFP to activating proteins such as plasmin and thrombospondin  (Murphy-Ullrich  and Poczatek,  2000).  avP6 integrin activates TGFp by  inducing conformational changes leading to the exposure o f receptor binding site on  TGFP molecules (Munger et al., 1999). Recently M u and co-workers  110  (2002) presented evidence that avf38 integrin was able to bind and activate T G F P through an M M P dependant mechanism. However, the cell surface appears to be required for the interaction between <xvP8-TGFp complex and MMP  since the secreted  forms o f avp8 and M T 1 - M M P  do not  mediate  activation o f T G F P ( M u et al., 2002). Once activated T G F P plays an important role as a chemoattractant and activating factor for monocytes/macrophages, resulting  in the  secretion  o f inflammatory cytokines  and growth  factors  (McCartney - Francis et al., 1990). Monocytes/macrophages themselves are able to produce T G F p  and control its activation (Assoian et al., 1987).  Subsequently, active T G F P acts on wide variety o f cells including autocrine effects on inflammatory cells and paracrine effects on mesenchymal cells. In addition, T G F p i can stimulate epithelial cell function directly and indirectly. It is known to induce epidermal expression of several integrins that are critical for wound epithelialization (Gailit et al., 1994; Zambruno et al., 1995; Koivisto et al., 1999). Indirectly, T G F p i stimulates the secretion o f other cytokines by a wide variety o f cells in the wound area; for example, it stimulates the secretion of V E G F (Berse et a l , 1999). V E G F , in addition to its effect on angiogenesis, stimulates  endothelial cells to secrete P D G F ,  activation o f fibroblasts.  which is important for the  Activated fibroblasts secrete growth factors that  stimulate epidermal proliferation e.g. IGF-1 (Hodak et a l , 1996) and K G F - 1 and -2 (Werner, 1998). K G F - 1 had the ability to induce plasmonigen activating factor, which serves as a mechanism to activate T G F P and thus amplify the proliferation signal (Tsuboi et a l , 1993). In addition to the ability o f av06  111  integrin to activate T G F p , <xvP6 integrin is important in stimulating epithelial cell adhesion and migration on fibronectin and tenascin .(Busk et al., 1992; Hakkinen et al., 2000a), both o f which are extracellular matrix proteins that are expressed during wound healing. Cells derived from p 6 " A mice show a marked decrease in migration on fibronectin and to some extent vitronectin (Huang et al.,  1998a). In addition, antibodies  against the P6 subunit decreased  the  migration ability o f wild type cells to a degree similar to that o f P6 " cells _/  (Huang et al., 1998a). The fact that  avp6  integrin is expressed at the leading  edge o f migrating epithelium and between tumour cells and adjacent stroma supports avP6 being important in cell migration (Haapasalmi et al., 1996; Jones et al., 1997). Its ability to stimulate the production o f M M P - 9 in human oral keratinocytes and in colon cancer cell lines gives a molecular mechanism by which  avp6  integrin is important in cell migration (Thomas et al., 2001). In  addition, cells expressing  av06  integrin show enhanced proliferation in three  dimensional collagen matrixes and in nude mice (Agrez et al., 1994). Those functions may provide an explanation of the added advantage o f P6 integrin expression  in the compromised wound model. In addition to its role in  promoting cell adhesion and migration, to inflammation (Breuss  ocvP6 integrin  acts as a local modulator  et al., 1995). It is re-expressed  in response to  inflammation, for example in human chronic wounds (Hakkinen et al., 2004), and in the lungs o f Rhesus monkeys exposed to ozone pollution (Miller et al., 2001). The fact that P6""mice show lymphatic and macrophage infiltrate to the /  skin and lungs suggest that the  avp6  receptor may play a role in down  112  regulating inflammation (Huang et al., 1996). Its ability to bind and activate T G F P which was able to block the production o f pro-inflammatory cytokine (EL-ip, T N F a ,  IL-8) from alveolar macrophages in vivo provides further  support to its regulating effects (Bogdan et al., 1992). p6" mice were protected A  from bleomycin induced lung fibrosis and yet lymphocytic infiltration to the lung in response to bleomycin was not inhibited (Sheppard, 2001). The ability o f limited expression o f this integrin to reverse lung inflammation further supports its effect in regulating the inflammatory response (Huang et al., 1998b). Recently, mice homogenous to the null mutation in P6 subunit showed marked  induction  of  macrophage  MMP-12  secretion,  resulting  in  the  development o f age dependant lung emphysema (Morris et al., 2003). This effect can be abrogated either by transgenic expression o f the version o f P6 subunit that support T G F P activation or by loss o f macrophage M M P - 1 2 (Morris et al., 2003).  5.4 Role o f mononuclear cells in w o u n d healing Experiments conducted by Leibovitch and Ross phagocytes  depleted  guinea  pigs  provide the  (1975) in mononuclear  evidence  of  the  role  of  macrophages in tissue repair. Monocyte depletion by hydrocortisone treatment leads  to  a  defective  wound  debridement  and  delayed  healing  process  (Leibovich and Ross, 1975). In addition to their role in wound debridement, macrophages are a vital source o f cytokines and growth factors, all o f which are important in the repair process (McCartney - Francis et al., 1990). A t later  113  stages o f healing, macrophages limit tissue injury by their ability to recognize and clear apoptotic neutrophils (Newman et al., 1982). This step is important in the resolution of inflammation and return o f tissue to its normal state. T G F p interaction with monocyte/macrophage Initially T G F p  acts as  in wound healing is bidirectional.  a pro-inflammatory mediator that results  in  the  stimulation o f monocyte-derived cytokines (McCartney - Francis et al., 1990). At  later stages o f healing, T G F P  response  (Ling  and  Robinson,  acts to downregulate the inflammatory 2002).  The  effect  of  TGFp  on  monocytes/macrophages is dependant on the concentration gradient and on the stage o f differentiation o f the cells (Ling and Robinson, 2002). Resting monocytes  responded  to  TGFp  activation  transcription and activation, a response  by  upregulation  of  TGFp  that is reduced in activated cells  (Brandes et al., 1991). In addition to its chemoattractant action on monocytes, T G F p enhances the expression o f surface integrins (e.g. a 3 p l and a 5 p i ) that are needed for the attachment to the provisional matrix laid down during wound healing (Wahl et al., 1993). T G F p also stimulates the secretion of M M P - 2 and M M P - 9 needed for the migration o f monocytes through the matrix (Wahl et al., 1993). A t wound site, T G F P stimulates the secretion o f various cytokines and growth factors (e.g. IL-1, IL-6, P D G F and b - F G F ) all o f which are important in the repair process (McCartney - Francis et al., 1990). It also enhances phagocytosis  by increasing the expression o f C D 16 receptors on  monocyte cell surfaces, which allow the cells to recognize bound I g G (Welch et a l , 1990). Interestingly, fetal wound healing is characterized by a low level  114  of T G F p and the absence o f monocytes ( L i n and Adzick, 1996; C o w i n et al., 2001). Neutralizing the effect o f T G F P by topical antibodies resulted in scar free healing associated with a reduced number o f monocytes, all o f which supports the pro-inflammatory effects o f T G F p (Shah et al., 1992, However,  transgenic  animals  lacking T G F p i  suffer  from  1995).  a multiorgan  inflammatory infiltrate resulting in death early in life (Shull et al., 1992). This highlights the role o f T G F p on downregulating the inflammatory response. Activated macrophages downregulate the expression o f T G F p receptors on their surface and decrease their sensitivity to T G F P stimulation (Ashcroft, 1999). Removal o f apoptotic neutrophils by macrophages not only protects the tissue from potentially toxic intracellular content, but also produces antiinflammatory phenotypes  as  it increases  the production o f T G F p  which  subsequently suppresses the production o f pro-inflammatory cytokines (Fadok et al., 1998). Macrophage deactivation is further achieved by the effect o f T G F P on reactive oxygen and nitrogen intermediates (Warwick-Davies et al., 1995) . In addition, T G F p i knockout animals show increase in the production of nitrous oxide and enhanced expression on NO2 synthas (Vodovotz et al., 1996) .  5.5 Limitations o f the study This experimental study has several limitations. The primary investigator was not blinded to the different groups o f animals during the analysis. D u e to the nature o f the study, some parts o f the experiment were done at different time  115  points, which might affect the interpretation o f the results. However, to reduce the bias, additional experienced examiners ( H L & L H ) were utilized to confirm the findings. T o avoid magnification error during clinical wound assessment, camera settings and position were recorded and kept constant during all phases o f the experiments.  A measurement ruler was placed beside the wounds to  ensure all photographs were taken at the same magnification. Analysis was done by a computer program and images were cropped and sharpened for best visualization o f wound margin. If the wound margin was not identified, wounds were considered be closed. For the analysis o f keratinocyte migration in threeday-old wounds, histological sections o f the wound center were photographed. Wound margins and the tip o f the migrating keratinocytes were identified by the primary investigator and confirmed by L H and the distance was measured by the N T H program.  116  Chapter 6-Conclusions The role of av06 integrin during compromised wound healing was investigated in this study. Comparisons were made between wound healing in different animal groups in relation to their genetic background, age, and stress level. The following observations were made: -  Stress as induced by hydrocortisone injection delays wound healing significantly. In addition to all of the previously reported effects of glucocorticoid treatment on granulation tissue formation, the most significant delay observed in our study was in the early phases of healing. At three days post wounding, keratinocytes at the wound edges showed significant delay in migration resulting in larger open wound size and an overall delay in the healing process.  -  Although 06 integrin deficiency did not affect the wound healing capacity in younger animals, older animals showed a larger wound size and delayed healing, particularly in the stage where granulation tissue formation was expected. However, eventually all wounds healed, indicating that 06 deficiency did not result in a permanent damage to the healing process.  -  06 integrin overexpression by keratinocytes did not alter the normal wound healing rate or capacity. However, in wounds compromised by stress, 06 integrin overexpressing animals showed only a modest delay in the healing rate. Keratinocyte migration after three days of healing was comparable to that of controls. This indicated that 06 integrin  117  overexpression protected and partially recovered the delaying effects o f stress on wound healing. A possible explanation o f the differences in wound healing between the wild type and the transgenic mice may relate to the ability o f  avP6 integrin to bind  and activate T G F p . This may serve as a mechanism by which compromised wound healing is improved in animals overexpressing the P6 integrin subunit. It also explains the delayed wound healing seen in older p6 integrin deficient mice. However, since wound healing is a complex process, other mechanisms may also be involved. W e propose that  avP6 integrin serves as a rescue receptor when  wound healing is compromised.  118  Appendix A  A . 1-Animal information, the untreated group (6-month-old). Date of birth  Sex  Weight start  at of  the  Weight  the  end  of  the the  experiment (g)  experiment (g)  WT  July 11/03  Male  34.20  34.00  WT  July 4/03  Female  32.30  36.75  WT  July 4/03  Female  41.90  38.78  WT  July 4/03  Female  39.00  38.70  WT  July 4/03  Female  32..05  30.60  6-  A p r i l 20/03  Female  30.44  29.44  P6'  A p r i l 20/03  Female  26.47  30.04  6-  A p r i l 20/03  Female  30.00  29.42  P  6-  A p r i l 20/03  Female  30.60  29.57  P  P6^  A p r i l 20/03  Male  34.60  33.36  P  at  119  A.2-Animal information, the treatment group (6-month-old). Date of birth  Sex  Weight start  at the of  the  Weight end  at the of  the  experiment (g)  experiment (g)  WT  July 4/03  Male  39.20  30.70  WT  July 4/03  Female  37.80  Died at day 0  WT  July 11/03  Female  31.70  34.09  WT  July 4/03  Female  37.90  32.23  WT  July 4/03  Female  25.20  25.00  p6^  April 20/03  Female  27.10  25.24  P6"  April 20/03  Female  28.80  27.78  P6'  April 20/03  Female  27.00  25.07  P6'  April 20/03  Male  34.00  31.25  P6-  April 20/03  Male  32.30  31.95  120  A.3-Animal information, the untreated group (22-month-old). Date of birth  Sex  Weight start  at the of  the  Weight end  at the of  experiment (g).  experiment (g).  WT  November 13/00  Female  31.50  26.13  WT  November 13/00  Female  34.20  30.45  WT  November 21/00  Male  39.30  37.49  WT  November 13/00  Male  34.10  31.05  WT  July 12/00  Female  32.19  27.00  P  6-'-  October 25/01  Female  36.34  34.08  P  6-'-  July 27/01  Male  27.63  24.32  P6-'"  November 27/01  Female  35.11  35.00  6-'-  October 25/01  Female  29.58  28.50  P6-'"  October 25/01  Female  36.28  33.96  P6F1  October 29/00  Female  32.65  29.58  P6F1  October 29/00  Female  26.74  27.58  P6F1  June 25/00  Female  34.20  28.93  P6F1  June 21/00  Male  27.52  Died day 6  P6F1  November 21/00  Female  33.59  30.60  P  the  121  A.4-Animal information, the experimental group (22-month-old). Date of birth  Sex  Weight start  at the of  the  Weight end  at the of  the  experiment (g).  experiment (g)  WT  July 12/00  Female  29.66  26.23  WT  June 21/00  Female  27.72  26.93  WT  July 12/00  Female  30.88  26.93  WT  October 25/00  Female  31.57  28.64  WT  June 21/00  Male  30.68  27.15  is"  October 25/01  Female  27.13  25.05  July 22/01  Female  36.55  32.71  pV-  October 25/01  Female  30.06  30.06  P6'-  July 27/01  Male  28.21  27.63  P  6-'-  July 27/01  Male  38.16  32.25  P6F1  June 21/00  Male  31.10  Died day 9  P6F1  June 21/00  Male  34.29  33.00  P6F1  October 29/00  Female  27.53  26.58  P6F1  October 29/00  Female  25.05  23.27  P6F1  June 25/00  Female  35.45  30.35  122  2  00  co  oo 00  JO cp_ m  to <  CD  o c 3 Q.  co  cn N'  CD  oo  c  3 — 1 CD to CD CL  to  <n  cn  i  3  o 3 i—+  3" i  o  oo  Q.  oo  olo  123  71 r 71  -  T  1—  f-  *  1 Animal -Dav 1  MEAN SD  IsE  71 r 71 k 71  30 r- 71  r-  I 53.5 87.5 I 74.64286 I 65.64286 I 3.937004 1 13.2061 I 13.3797 I 16.74216I 6.86 I I 3.57 I 1.05 OJ bi  I 28.16667 I 20.33333 13.66667 7.333333 2.333333 16.7332 I 13.16688 I 8.164966 7.659417 5.428321 3.386247 I 1.381 6.86 to  cn  CO CO CO - J CO ot> CD CO CO CO CD - J CD CD OJ CD CO to o o - J CO o o CO o CD  t  o  < 1  Ol CD Ol  1 i  Ol Ol Ol Ol OJ  OD CO ~ J CD 4k Ol --4 OJ OJ k -J o  o cn  OJ  -J  •fe. 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CD  To  CD  ca  a.  CD CL  rco  t  C D CO  C DCO  CD  CO  CD  130  73  30  NJ  co  C•D  co  7J  co  CD  N>  to <• CD  i  Ol  c  CO  CO  3  Q .  S£ N' CD  c  co  to  3  CO  OJ » — * CD  CL  3" C  co co  co  3 to  NJ  Ol 3  cr CD CD  oo  co  co  o <  CD  3  NJ  CO  CD  NJ  oo  NJ NJ  O CD NJ NJ i  3  CO  o CL  131  03  cn  o>  co  CD  cn  o>  00  cn  co  oo  ro CD  CD  CD  oo  cn  a.  co  co  cr CD  D3  CD  CO  co  oo  cn  cn  to  cn 3  ro  co  to  CD CO  cn  cn  co  cn  co  co  CD  to to 3 o 3  cn CD  cn  co  to o>  o Q.  cn  o co  132  References Adcock I. M. (2000). "Molecular mechanisms of glucocorticosteroid actions." Pulm Pharmacol Ther 13(3): 115-26. Agrez M., Chen A., Cone R. I., Pytela R. and Sheppard D. (1994). "The alpha v beta 6 integrin promotes proliferation of colon carcinoma cells through a unique region of the beta 6 cytoplasmic domain." J Cell Biol 127(2): 547-56. Agrez M., Gu X., Turton J., Meldrum C , Niu J., Antalis T. and Howard E. W. (1999). "The alpha v beta 6 mtegrin induces gelatinase B secretion in colon cancer cells." Int J Cancer 81(1): 90-7. Ansel J. C , Tiesman J. P., Olerud J. E., Krueger J. G., Krane J. F., Tara D. C , Shipley G. D., Gilbertson D., Usui M. L. and Hart C. E. (1993). "Human keratinocytes are a major source of cutaneous platelet-derived growth factor." J Clin Invest 92(2): 671-8. Antoni M. H., Cruess D. G., Klimas N., Maher K., Cruess S., Kumar M., Lutgendorf S., Ironson G., Schneiderman N. and Fletcher M. A. (2002). "Stress management and immune system reconstitution in symptomatic HIV-infected gay men over time: effects on transitional naive T cells (CD4(+)CD45RA(+)CD29(+))." Am J Psychiatry 159(1): 143-5. Ashcroft G. S. (1999). "Bidirectional regulation of macrophage function by TGF-beta." Microbes Infect 1(15): 1275-82. Ashcroft G. S., Dodsworth J., van Boxtel E., Tarnuzzer R. W., Horan M. A., Schultz G. S. and Ferguson M. W. (1997a). "Estrogen accelerates cutaneous wound healing associated with an increase in TGF-betal levels." Nat Med 3(11): 1209-15. Ashcroft G. S., Greenwell-Wild T., Horan M. A., Wahl S. M. and Ferguson M. W. (1999a). "Topical estrogen accelerates cutaneous  133  wound healing in aged humans associated with an altered inflammatory response." Am J Pathol 155(4): 1137-46. Ashcroft G. S., Herrick S. E., Tarnuzzer R. W., Horan M. A., Schultz G. S. and Ferguson M. W. (1997b). "Human ageing impairs injuryinduced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-l and -2 proteins and mRNA." J Pathol 183(2): 169-76. Ashcroft G. S., Horan M. A. and Ferguson M. W. (1997c). "Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model." J Invest Dermatol 108(4): 430-7. Ashcroft G. S., Horan M. A. and Ferguson M. W. (1997d). "The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model." J An at 190 ( Pt 3): 351-65. Ashcroft G. S., Horan M. A. and Ferguson M. W. (1998). "Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing." Lab Invest 78(1): 47-58. Ashcroft G. S., Mills S. J. and Ashworth J. J. (2002). "Ageing and wound healing." Biogerontologv 3(6): 337-45. Ashcroft G. S., Mills S. J., Lei K., Gibbons L., Jeong M. J., Taniguchi M., Burow M., Horan M. A., Wahl S. M. and Nakayama T. (2003). "Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor." J Clin Invest 111(9). 1309-18. Ashcroft G. S., Yang X., Glick A. B., Weinstein M., Letterio J. L., Mizel D. E., Anzano M., Greenwell-Wild T., Wahl S. M., Deng C. and Roberts A. B. (1999b). "Mice lacking Smad3 show accelerated 134  wound healing and an impaired local inflammatory response." Nat Cell Biol 1(5): 260-6. Assoian R. K., Fleurdelys B. E., Stevenson H. C , Miller P. J., Madtes D. K., Raines E. W., Ross R. and Sporn M. B. (1987). "Expression and secretion of type beta transforming growth factor by activated human macrophages." Proc Natl Acad Sci U S A 84(17): 6020-4. Auphan N., DiDonato J. A., Rosette C , Helmberg A. and Karin M. (1995). "Immunosuppression by glucocorticoids: inhibition ofNFkappa B activity through induction of I kappa B synthesis." Science 270(5234): 286-90. Babu M., Diegelmann R. and Oliver N. (1989). "Fibronectin is overproduced by keloid fibroblasts during abnormal wound healing." Mol Cell Biol 9(4): 1642-50. Badid C , Mounier N., Costa A. M. and Desmouliere A. (2000). "Role of myofibroblasts during normal tissue repair and excessive scarring: interest of their assessment in nephropathies." Histol Histopathol 15(1): 269-80. Ballas C. B. and Davidson J. M. (2001). "Delayed wound healing in aged rats is associated with increased collagen gel remodeling and contraction by skin fibroblasts, not with differences in apoptotic or myofibroblast cell populations." Wound Repair Regen 9(3): 22337. Barnes P. J. (1998). "Anti-inflammatory actions of glucocorticoids: molecular mechanisms." Clin Sci (Lond) 94(6): 557-72. Beaubien J., Boisjoly H. M., Gagnon P. and Guidoin R. (1994). "Mechanical properties of the rabbit cornea during wound healing after treatment with epidermal growth factor." Can J Ophthalmol 2 9 ( 2 ) : 61-5.  135  Beck L. S., Deguzman L., Lee W. P., Xu Y., McFatridge L. A. and Amento E. P. (1991). "TGF-beta 1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits." Growth Factors 5(4): 295-304. Beck L. S., DeGuzman L., Lee W. P., Xu Y., Siegel M. W. and Amento E. P. (1993). "One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing." J Clin Invest 92(6): 2841-9. Beer H. D., Fassler R. and Werner S. (2000). "Glucocorticoid regulated gene expresion during cautaneous wound repair." Vitm Horm 59: 217-39. Beer H. D., Longaker M. T. and Werner S. (1997). "Reduced expression of PDGF and PDGF receptors during impaired wound healing." J Invest Dermatol 109(2): 132-8. Bendrups A., Hilton A., Meager A. and Hamilton J. A. (1993). "Reduction of tumor necrosis factor alpha and interleukin-1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid." Rheumatol Int 12(6): 217-20. Berse B., Hunt J. A., Diegel R. J., Morganelli P., Yeo K., Brown F. and Fava R. A. (1999). "Hypoxia augments cytokine (transforming growth factor-beta (TGF-beta) and IL-l)-induced vascular endothelial growth factor secretion by human synovial fibroblasts." Clin Exp Immunol 115(1): 176-82. Besser D., Presta M. and Nagamine Y. (1995). "Elucidation of a signaling pathway induced by FGF-2 leading to uPA gene expression in NIH 3T3 fibroblasts." Cell Growth Differ 6(8): 1009-17. Blatti S. P., Foster D. N., Ranganathan G., Moses H. L. and Getz M. J. (1988). "Induction of fibronectin gene transcription and mRNA is a  136  primary response to growth-factor stimulation of AKR-2B cells." Proc Natl Acad Sci U S A 85(4): 1119-23. Blotta M. H., DeKruyff R. H. and Umetsu D. T. (1997). "Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4+ lymphocytes." J Immunol 158(12): 5589-95. Bogdan C , Paik J., Vodovotz Y. and Nathan C. (1992). "Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-beta and interleukin-10." J Biol Chem 267(32): 23301-8. Bohlen P., Esch F., Baird A. and Gospodarowicz D. (1985). "Acidic fibroblast growth factor (FGF)frombovine brain: ammo-terminal sequence and comparison with basic FGF." Embo J 4(8): 1951-6. Brakebusch C , Grose R., Quondamatteo F., Ramirez A., Jorcano J. L., Pirro A., Svensson M., Herken R., Sasaki T., Timpl R., Werner S. and Fassler R. (2000). "Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes." Embo J 19(15): 3990-4003. Brandes M. E., Wakefield L. M. and Wahl S. M. (1991). "Modulation of monocyte type Ifransforminggrowth factor-beta receptors by inflammatory stimuli." J Biol Chem 266(29): 19697-703. Brauchle M., Angermeyer K., Hubner G. and Werner S. (1994). "Large induction of keratinocyte growth factor expression by serum growth factors and pro-inflammatory cytokines in cultured fibroblasts." Oncogene 9(11): 3199-204. Brauchle M., Fassler R. and Werner S. (1995). "Suppression of keratinocyte growth factor expression by glucocorticoids in vitro and during wound healing." J Invest Dermatol 105(4): 579-84.  137  Breuing K., Andree C , Helo G., Slama J., Liu P. Y. and Eriksson E. (1997).  "Growth factors in the repair of partial thickness porcine  skin wounds." Plast Reconstr Surg 100(3):  657r64.  Breuss J. M , Gallo J., DeLisser H. M., Klimanskaya I. V., Folkesson H. G., Pittet J. F., NisMmura S. L., Aldape K., Landers D. V., Carpenter W. and et al.  (1995).  "Expression of the beta 6 integrin  subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling." J Cell Sci 108  ( Pt  6):  2241-51.  Brooks P. C , Stromblad S., Sanders L. C , von Schalscha T. L., Aimes R. T., Stetler-Stevenson W. G., Quigley J. P. and Cheresh D. A. (1996).  "Localization of matrix metalloproteinase M M P - 2 to the  surface of invasive cells by interaction with integrin alpha v beta 3."  Cell 85(5):  683-93.  Brown G. L., Curtsinger L. J., White M., Mitchell R. O., Pietsch J., Nordquist R., von Fraunhofer A. and Schultz G. S.  (1988).  "Acceleration of tensile strength of incisions treated with EGF and TGF-beta." Ann Surg 208(6):  788-94.  Bucala R., Spiegel L. A., Chesney J., Hogan M. and Cerami A.  (1994).  "Ckculating fibrocytes define a new leukocyte subpopulation that mediates tissue repair." Mol Med  1(1):  71-81.  Bujia J., Kim C , Holly A., Sudhoff H., Ostos P. and Kastenbauer E. (1996).  "Epidermal growth factor receptor (EGF-R) in human  middle ear cholesteatoma: an analysis of protein production and gene expression." Am J Qtol 17(2):  203-6.  Bullard K. M., Lund L., Mudgett J. S., Mellin T. N., Hunt T. K., Murphy B., Ronan J., Werb Z. and Banda M. J.  (1999).  "Impaired wound  contraction in stromelysin-1-deficient mice." Ann Surg 230(2): 260-5.  138  Busk M., Pytela R. and Sheppard D. (1992). "Characterization of the integrin alpha v beta 6 as afibronectm-bindingprotein." J Biol Chem 267(9): 5790-6. Cacioppo J. T., Kiecolt-Glaser J. K., Malarkey W. B., Laskowski B. F., Rozlog L. A., poehlmann K. M., Burleson M. H. and Glaser R. (2002). "Autonomic and glucocorticoid association with study state espression of latent Epstein-Barr virus." Horm behav 42(1): 32-41. Cambier S., Mu D. Z., O'Connell D., Boylen K., Travis W., Liu W. H., Broaddus V. C. andNishimura S. L. (2000). "A role for the integrin alphavbeta8 in the negative regulation of epithelial cell growth." Cancer Res 60(24): 7084-93. Carroll J. M., Romero M. R. and Watt F. M. (1995). "Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and aphenotype resembling psoriasis." Cell 83(6): 957-68. Celebi N., Erden N., Gonul B. and Koz M. (1994). "Effects of epidermal growth factor dosage forms on dermal wound strength in mice." J Pharm Pharmacol 46(5): 386-7. Chan B. M., Kassner P. D., Schiro J. A., Byers R., Kupper T. S. and Hemler M. E. (1992). "Distinct cellular function mediated by different V L A integrin alpha subuint cytoplasmic domains." Cell 68: 1051-1060. Clark R. A. (1990). "Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin." J Invest Dermatol 94(6 Suppl): 128S-134S. Clark R. A. (1996). Wound repair: Overview and general considerations. The molecular and cellular biology of wound repair. R. A. Clark. New York and London, Plenum: 3-35.  139  Clark R. A. (2003). "Epithelial-mesenchymal networks in wounds: a hierarchical view." J Invest Dermatol 120(6): ix-xi. Clark R. A., Ashcroft G. S., Spencer M. j . , Larjava H. and Ferguson M. W. (1996a). "Re-epithelialization of normal human excisional wounds is associated with a switch from alpha v beta 5 to alpha v beta 6 integrins." Br J Dermatol 135(1): 46-51. Clark R. A., Tonnesen M. G., Gailit J. and Cheresh D. A. (1996b). "Transient functional expression of alphaVbeta 3 on vascular cells during wound repair." Am J Pathol 148(5): 1407-21. Cockayne D., Sterling K. M., Jr., Shull S., Mintz K. P., Illeyne S. and Cutroneo K. R. (1986). "Glucocorticoids decrease the synthesis of type I procollagen mRNAs." Biochemistry 25(11): 3202-9. Cohen B. J., Danon D. and Roth G. S. (1987). "Wound repair in mice as influenced by age and antimacrophage serum." J Gerontol 42(3): 295-301. Cohen S., Doyle W. J. and Skoner D. P. (1999). "Psychological stress, cytokine production, and severity of upper respiratory illness." Psychosom Med 61(2): 175-80. Cohen S., Miller G. E. and Rabin B. S. (2001). "Psychological stress and antibody response to immunization: a critical review of the human literature." Psychosom Med 63(1): 7-18. Cole-King A. and Harding K. G. (2001). "Psychological factors and delayed healing in chronic wounds." Psychosom Med 63(2): 21620. Costa A. M., Peyrol S., Porto L. C , Comparin J. P., Foyatier J. L. and Desmouliere A. (1999). "Mechanical forces induce scar remodeling. Study in non-pressure-treated versus pressure-treated hypertrophic scars." Am J Pathol 155(5): 1671-9.  140  Cowin A. J., Holmes T. M., Brosnan P. and Ferguson M. W. (2001). "Expression of TGF-beta and its receptors in murine fetal and adult dermal wounds." Eur J Dermatol 11(5): 424-31. Crawford S. E., Stellmach V., Murphy-Ullrich J. E., Ribeiro S. M., Lawler J., Hynes R. O., Boivin G. P. and Bouck N. (1998). "Thrombospondin-1 is a major activator of TGF-betal in vivo." Cell 93(7): 1159-70. Crowe M. J., Doetschman T. and Greenhalgh D. G. (2000). "Delayed wound healing in immunodeficient TGF-beta 1 knockout mice." J Invest Dermatol 115(1): 3-11. Danilenko D. M., Ring B. D., Tarpley J. E., Morris B., Van G. Y., Morawiecki A., Callahan W., Goldenberg M., Hershenson S. and Pierce G. F. (1995). "Growth factors in porcine full and partial thickness bum repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor." Am J Pathol Nov;147( (5):): 1261-77. Danon D., Kowatch M. A. and Roth G. S. (1989). "Promotion of wound repair in old mice by local injection of macrophages." Proc Natl Acad Sci U S A 86(6): 2018-20. Dasch J. R., Pace D. R., Waegell W., Inenaga D. and Ellingsworth L. (1989). "Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transfonning growth factor beta 2 affinity purification." J Immunol 142(5): 1536-41. Datto M. B., Frederick J. P., Pan L., Borton A. J., Zhuang Y. and Wang X. F. (1999). "Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction." Mol Cell Biol 19(4): 2495-504.  141  DeKruyff R. H., Fang Y. and Umetsu D. T. (1998). "Corticosteroids enhance the capacity of macrophages to induce Th2 cytokine synthesis in CD4+ lymphocytes by inhibiting IL-12 production." J Immunol 160(5): 2231-7. Desmouliere A. (1995). "Factors influencing myofibroblast differentiation during wound healing and fibrosis." Cell Biol Int 19(5): 471-6. DiPersio C. M., Hodivala-Dilke K. M., Jaenisch R., Kreidberg J. A. and Hynes R. O. (1997). "alpha3betal Integrin is required for normal development of the epidermal basement membrane." J Cell Biol 137(3): 729-42. Dipietro L. A., Reintjes M. G., Low Q. E., Levi B. and Gamelli R. L. (2001). "Modulation of macrophage recruitment into wounds by monocyte chemoattractant protein-1." Wound Repair Regen 9( 1) : 28-33. Dostal G. H. and Gamelli R. L. (1990). "The differential effect of corticosteroids on wound disruption strength in mice." Arch Surg 125(5): 636-40. Durmus M., Karaaslan E., Ozturk E., Gulec M., Iraz M., Edali N. and Ersoy M. O. (2003). "The effects of single-dose dexamethasone on wound healing in rats." Anesth Analg 97(5): 1377-80. Edwards D. R., Murphy G., Reynolds J. J., Whitham S. E., Docherty A. J., Angel P. and Heath J. K. (1987). "Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor." Embo J 6(7): 1899-904. Ehrlich H. P. and Hunt T. K. (1968). "Effects of cortisone and vitamin A on wound healing." Ann Surg 167(3): 324-8.  142  Ehrlich H. P. and Hunt T. K. (1969). "The effects of cortisone and anabolic steroids on the tensile strength of healing wounds." Ann Surg 170(2): 203-6. Ekblom M , Fassler R., Tomasini-Johansson B., Nilsson K. and Ekblom P. (1993). "Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts." J Cell Biol 123(4): 1037-45. Elenkov I. J. and Chrousos G. P. (1999). "Stress Hormones, Thl/Th2 patterns, Pro/Anti-inflammatory Cytokines and Susceptibility to Disease." Trends Endocrinol Metab 10(9): 359-368. Elenkov I. J. and Chrousos G. P. (2002). "Stress hormones, proinflammatory and antimflammatory cytokines, and autoimmunity." Ann N Y Acad Sci 966: 290-303. Elenkov I. J., Chrousos G. P. and Wilder R. L. (2000). "Neuroendocrine regulation of IL-12 and TNF-alpha/IL-10 balance. Clinical implications." Ann N Y Acad Sci 917: 94-105. Elenkov I. J., Wilder R. L., Bakalov V. K., Link A. A., Dimitrov M. A., Fisher S., Crane M., Kanik K. S. and Chrousos G. P. (2001). "IL12, TNF-alpha, and hormonal changes during late pregnancy and early postpartum: implications for autoimmune disease activity during these times." J Clin Endocrinol Metab 86(10): 4933-8. Emoto H., Tagashira S., Mattei M. G , Yamasaki M., Hashimoto G., Katsumata T., Negoro T., Nakatsuka M., Birnbaum D., Coulier F. and Itoh N. (1997). "Structure and expression of human fibroblast growth factor-10." J Biol Chem 272(37): 23191-4. Eskandari F. and Sternberg E. M. (2002). "Neural-immune interactions in health and disease." Ann N Y Acad Sci 966: 20-7. Fadok V. A., Bratton D. L., Konowal A., Freed P. W., Westcott J. Y. and Henson P. M. (1998). "Macrophages that have ingested apoptotic 143  cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF." J Clin Invest 101(4): 890-8. Fassler R., Sasaki T., Timpl R., Chu M. L. and Werner S. (1996). "Differential regulation offibulin,tenascin-C, and nidogen expression during wound healing of normal and glucocorticoidtreated mice." Exp Cell Res 222(1): 111-6. Fluck J., Querfeld C , Cremer A., Niland S., Krieg T. and Sollberg S. (1998). "Normal human primaryfibroblastsundergo apoptosis in three-dimensional contractile collagen gels." J Invest Dermatol 110(2): 153-7. Folkman J. (1983). "Angiogenesis: initiation and modulation." Symp Fundam Cancer Res 36: 201-8. Frank S., Madlener M. and Werner S. (1996). "Transforming growth factors betal, beta2, and beta3 and their receptors are differentially regulated during normal and impaired wound healing." J Biol Chem 271(17): 10188-93. Fu X., Shen Z., Chen Y., Xie J., Guo Z., Zhang M. and Sheng Z. (2000). "Recombinant bovine basicfibroblastgrowth factor accelerates wound healing in patients with bums, donor sites and chronic dermal ulcers." Chin Med J (Engl) 113(4): 367-71. Fu X., Shen Z., Guo Z , Zhang M. and Sheng Z. (2002). "Healing of chronic cutaneous wounds by topical treatment with basic fibroblast growth factor." Chin Med J (Engl) 115(3): 331-5. Gabbiani G. (2003). "The myofibroblast in wound healing and fibrocontractive diseases." J Pathol 200(4): 500-3. Gailit J. and Clark R. A. (1994). "Wound repair in the context of extracellular matrix." Curr Opin Cell Biol 6(5): 717-25.  144  Gailit J., Welch M. P. and Clark R. A. (1994). "TGF-beta 1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds." J Invest Dermatol 103(2): 221-7. Georges-Labouesse E., Messaddeq N., Yehia G., Cadalbert L., Dierich A. and Le Meur M. (1996). "Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice." Nat Genet 13(3): 370-3. Gibbs S., Silva Pinto A. N., Murli S., Huber M., Hohl D. and Ponec M. (2000). "Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation." Wound Repair Regen 8(3): 192-203. Glaser R., Kiecolt-Glaser J. K., Bonneau R. H., Malarkey W., Kennedy S. and Hughes J. (1992). "Stress-induced modulation of the immune response to recombinant hepatitis B vaccine." Psychosom Med 54(1): 22-9. Glaser R., Kiecolt-Glaser J. K., Marucha P. T., MacCallum R. C , Laskowski B. F. and Malarkey W. B. (1999). "Stress-related changes in proinflammatory cytokine production in wounds." Arch Gen Psychiatry 56(5): 450-6. Glaser R., MacCallum R. C , Laskowski B. F., Malarkey W. B., Sheridan J. F. and Kiecolt-Glaser J. K. (2001). "Evidence for a shift in the Th-1 to Th-2 cytokine response associated with chronic stress and aging." J Gerontol A Biol Sci Med Sci 56(8): M477-82. Glaser R., Pearson G. R., Jones J. F., Hillhouse J., Kennedy S., Mao H. Y. and Kiecolt-Glaser J. K. (1991). "Stress-related activation of Epstein-Barr virus." Brain Behav Immun 5(2): 219-32. Glaser R., Sheridan J., Malarkey W. B., MacCallum R. C. and KiecoltGlaser J. K. (2000). "Chronic stress modulates the immune  145  response to a pneumococcal pneumonia vaccine." Psychosom Med 62(6): 804-7. Godar S., Horejsi V., Weidle U. H., Binder B. R., Hansmann C. and Stockinger H. (1999). "M6P/IGFII-receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor-betal." Eur J Immunol 29(3): 1004-13. Gosain A. and DiPietro L. A. (2004). "Aging and wound healing." World J Surg 28(3): 321-6. Gospodarowicz D., Jones K. L. and Sato G. (1974). "Purification of a growth factor for ovarian cellsfrombovine pituitary glands." Proc Natl Acad Sci U S A 71(6): 2295-9. Grose R , Hutter C , Bloch W., Thorey I., Watt F. M., Fassler R., Brakebusch C. and Werner S. (2002a). "A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair." Development 129(9): 2303-15. Grose R., Werner S., Kessler D., Tuckermann J., Huggel K., Durka S., Reichardt H. M. and Werner S. (2002b). "A role for endogenous glucocorticoids in wound repair." EMBO Reports 3(6): 575-82. Guo L., Degenstein L. and Fuchs E. (1996). "Keratinocyte growth factor is required for hair development but not for wound healing." Genes Devl0(2): 165-75. Gupta A., Jain G. K. and Raghubir R. (1999). "A time course study for the development of an immunocompromised wound model, using hydrocortisone." J Pharmacol Toxicol Methods 41(4): 183-7. Haapasalmi K., Zhang K., Tonnesen M., Olerud J., Sheppard D., Salo T., Kramer R., Clark R. A., Uitto V. J. and Larjava H. (1996). "Keratinocytes in human wounds express alpha v beta 6 integrin." J Invest Dermatol 106(1): 42-8.  146  Haase I., Hobbs R. M., Romero M. R., Broad S. and Watt F. M. (2001). "A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis." J Clin Invest 108(4): 527-36. Hakkinen L., Hildebrand H. C , Berndt A., Kosmehl H. and Larjava H. (2000a). "hnmunolocalization of tenascin-C, alpha integrin 0  subunit, and alphavbeta6 integrin during wound healing in human oral mucosa." J Histochem Cvtochem 48(7): 985-98. Hakkinen L., Koivisto L., Gardner H., Saarialho-Kere U., Carroll J. M., Lakso M., Rauvala H., Laato M., Heino J. and Larjava H. (2004). "Increased expression of beta6-mtegrin in skin leads to spontaneous development of chronic wounds." Am J Pathol 164(1): 229-42. Hakkinen L., Uitto V. J. and Larjava H. (2000b). "Cell biology of gingival wound healing." Periodontol 2000 24: 127-52. Hamidi S., Salo T., Kainulainen T., Epstein J., Lerner K. and Larjava H. (2000). "Expression of alpha(v)beta6 integrin in oral leukoplakia." Br J Cancer 82(8): 1433-40. Han K. S. (2002). "The effect of an integrated stress management program on the psychologic and physiologic stress reactions of peptic ulcer in Korea." Int J Nurs Stud 39(5): 539-48. Heldin C. H. and Westermark B. (1999). "Mechanism of action and in vivo role of platelet-derived growth factor." Physiol Rev 79(4): 1283-316. Heldin P., Laurent T. C. and Heldin C. H. (1989). "Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts." BiochemJ 258(3): 919-22. Herrick S., Ashcroft G., Ireland G., Horan M., McCollum C. and Ferguson M. (1997). "Up-regulation of elastase in acute wounds of  147  healthy aged humans and chronic venous leg ulcers are associated with matrix degradation." Lab Invest 77(3): 281-8. Hertle M. D., Kubler M. D., Leigh I. M. and Watt F. M. (1992). "Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis." J Clin Invest 89(6): 1892-901. Hinz B., Mastrangelo D., Iselin C. E., Chaponnier C. and Gabbiani G. (2001). "Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation." Am J Pathol 159(3): 1009-20. Hodak E., Gottlieb A. B., Anzilotti M. and Krueger J. G. (1996). "The insulin-like growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: correlation of increased expression with epidermal hyperplasia." J Invest Dermatol 106(3): 564-70. Hodivala-Dilke K. M., DiPersio C. M., Kreidberg J. A. and Hynes R. O. (1998). "Novel roles for alpha3betal integrin as a regulator of cytoskeletal assembly and as a trans-dorninant inhibitor of integrin receptor function in mouse keratinocytes." J Cell Biol 142(5): 1357-69. Huang X., Griffiths M , Wu J., Farese R. V., Jr. and Sheppard D. (2000). "Normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice." Mol Cell Biol 20(3): 755-9. Huang X , Wu J., Spong S. and Sheppard D. (1998a). "The integrin alphavbeta6 is critical for keratinocyte migration on both its known ligand,fibronectin,and on vitronectin." J Cell Sci 111 ( Pt 15): 2189-95. Huang X., Wu J., Zhu W., Pytela R. and Sheppard D. (1998b). "Expression of the human integrin beta6 subunit in alveolar type II  148  cells and bronchiolar epithelial cells reverses lung mflammation in beta6 knockout mice." Am J Respir Cell Mol Biol 19(4): 636-42. Huang X. Z., Wu J. F., Cass D., Erie D. J., Cony D., Young S. G., Farese R. V., Jr. and Sheppard D. (1996). "Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial mtegrins in regulating inflammation in the lung and skin." J Cell Biol 133(4): 921-8. Huber D., Fontana A. and Bodmer S. (1991). "Activation of human platelet-derived latent transfoiming growth factor-beta 1 by human glioblastoma cells. Comparison with proteolytic and glycosidic enzymes." Biochem J 277 ( Pt 1): 165-73. Hubner G., Brauchle M., SmolaH., Madlener M., Fassler R. and Werner S. (1996). "Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice." Cytokine 8(7): 548-56. Ishimoto S. and Ishibashi T. (2002). "Induction of growth factor expression is reduced during healing of tympanic membrane perforations in glucocorticoid-treated rats." Ann Qtol Rhinol Laryngoi 111(10): 947-53. Iwakabe K., Shimada M., Ohta A., Yahata T., Ohmi Y., Habu S. and Nishimura T. (1998). "The restraint stress drives a shift in Thl/Th2 balance toward Th2-dominant immunity in mice." Immunol Lett 62(1): 39-43. Jeschke M. G., Richter G., Hofstadter F., Hemdon D. N., Perez-Polo J. R. and Jauch K. W. (2002). "Non-viral liposomal keratinocyte growth factor (KGF) cDNA gene transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors." Gene Ther 9(16): 1065-74.  149  Jones J., Watt F. M. and Speight P. M. (1997). "Changes in the expression of alpha v integrins in oral squamous cell carcinomas." J Oral Pathol Med 26(2): 63-8. Kaminski N., Allard J. D., Pittet J. F., Zuo F., Griffiths M. J., Morris D., Huang X., Sheppard D. and Heller R. A. (2000). "Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis." Proc Natl Acad Sci U S A 97(4): 1778-83. Kennedy D. F. and Cliff W. J. (1979). "A systematic study of wound contraction in mammalian skin." Pathology 11(2): 207-22. Kiecolt-Glaser J. K. and Glaser R. (2001). "Psychological stress and wound healing: Kiecolt-Glaser et al. (1995)." Adv Mind Body Med 17(1): 15-6. Kiecolt-Glaser J. K., Glaser R., Gravenstein S., Malarkey W. B. and Sheridan J. (1996). "Chronic stress alters the immune response to influenza virus vaccine in older adults." Proc Natl Acad Sci U S A 93(7): 3043-7. Kiecolt-Glaser J. K., Marucha P. T., Malarkey W. B., Mercado A. M. and Glaser R. (1995). "Slowing of wound healing by psychological stress." Lancet 346(8984): 1194-6. Kiecolt-Glaser J. K., McGuire L., Robles T. F. and Glaser R. (2002a). "Psychoneuroimmunology and psychosomatic medicine: back to the future." Psvchosom Med 64(1): 15-28. Kiecolt-Glaser J. K., Page G. G., Marucha P. T., MacCallum R. C. and Glaser R. (1998). "Psychological influences on surgical recovery. Perspectives from psychoneuroimmunology." Am Psychol 53(11): 1209-18. Kiecolt-Glaser J. K., Robles T. F., Heffher K. L., Loving T. J. and Glaser R. (2002b). "Psycho-oncology and cancer: 150  psychoneuroimmunology and cancer." Ann Oncol 13 Suppl 4: 165-9. Kirchberg K., Lange T. S., Klein E. C , Jungtaubl H., Heinen G., MeyerIngold W. and Scharffetter-Kochanek K. (1995). "Induction of beta 1 integrin synthesis by recombinant platelet-derived growth factor (PDGF-AB) correlates with an enhanced migratory response of human dermal fibroblasts to various extracellular matrix proteins." Exp Cell Res 220(1): 29-35. Kobayashi T., Shiiba K., Satoh M., Hashimoto W., Mizoi T., Matsuno S. and Takeda K. (2002). "Interleukin-12 administration is more effective for preventing metastasis than for inhibiting primary established tumors in a murine model of spontaneous hepatic metastasis." Surg Today 32(3): 236-42. Koivisto L., Larjava H., Hakkinen L., Uitto V. J., Heino J. and Larjava H. (1999). "Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes onfibronectin."Cell Adhes Commun 7(3): 245-57. Koli K., Saharinen J., Hyytiainen M., Penttinen C. and Keski-Oja J. (2001). "Latency, activation, and binding proteins of TGF-beta." Microsc Res Tech 52(4): 354-62. Koob T. J., Jeffrey J. J., Eisen A. Z. and Bauer E. A. (1980). "Hormonal interactions in mammalian collagenase regulation. Comparative studies in human skin and rat uterus." Biochim Biophys Acta 629(1): 13-23. Kuwahara H., Mitchell A. T., Macklin M. D., Zhao J., Listengarten D. and Phillips L. G. (2000). "Transfer of platelet-derived growth factor-BB gene by gene gun increases contraction of collagen lattice by fibroblasts in diabetic and non-diabetic human skin." Scand J Plast Reconstr Surg Hand Surg 34(4): 301-7. 151  Laiho M., Saksela O. and Keski-Oja J. (1986). "Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts." Exp Cell Res 164(2): 399-407. Langholz O., Rockel D., Mauch C , Kozlowska E., Bank I., Krieg T. and Eckes B. (1995). "Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 mtegrins." J Cell Biol 131(6 Pt 2): 1903-15. Laplante A. F., Germain L., Auger F. A. and Moulin V. (2001). "Mechanisms of wound reepithelialization: hintsfroma tissueengineered reconstructed skin to long-standing questions." Faseb J 15(13): 2377-89. Larjava H., Koivisto L. and Halddnen L. (2002). Keratinocyte interactions withfibronectinduring wound healing. Cell Invasion. J. Heino and V.-M. Kahari. Landes Biosciences, Eurecah. com Georgetown: 42-64. Larjava H., Salo T., Haapasalmi K., Kramer R. H. and Heino J. (1993). "Expression of integrins and basement membrane components by wound keratinocytes." J Clin Invest 92(3): 1425-35. Leibovich S. J. and Ross R. (1975). "The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum." Am J Pathol 78(1): 71-100. Lenco W., McKnight M. and Macdonald A. S. (1975). "Effects of cortisone acetate, methylprednisolone and medroxyprogesterone on wound contracture and epithelization in rabbits." Ann Surg 181(1): 67-73. LeResche L. and Dworkin S. F. (2002). "The role of stress in inflammatory disease, including periodontal disease: review of concepts and currentfindings."Periodontol 2000 30: 91-103. 152  Leserman J., Petitto J. M., Gu H., Gaynes B. N., Barroso J., Golden R. N., Perkins D. O., Folds J. D. and Evans D. L. (2002). "Progression to AIDS, a clinical AIDS condition and mortality: psychosocial and physiological predictors." Psychol Med 32(6): 1059-73. Lieberman J. R., Daluiski A. and Einhorn T. A. (2002). "The role of growth factors in the repair of bone. Biology and clinical applications." J Bone Joint Surg Am 84-A(6): 1032-44. Lin R. Y. and Adzick N. S. (1996). "The role of the fetal fibroblast and transforniing growth factor-beta in a model of human fetal wound repair." Semin Pediatr Surg 5(3): 165-74. Ling E. and Robinson D. S. (2002). "Transforming growth factor-betal: its anti-inflammatory and pro-fibrotic effects." Clin Exp Allergy 32(2): 175-8. Lorena D., Uchio K., Costa A. M. and Desmouliere A. (2002). "Normal scarring: importance of myofibroblasts." Wound Repair Regen 10(2): 86-92. Low Q. E., Drugea I. A., Duffher L. A., Quinn D. G., Cook D. N., Rollins B. J., Kovacs E. J. and DiPietro L. A. (2001). "Wound healing in MIP-lalpha(-/-) and MCP-1 (-/-) mice." Am J Pathol 159(2): 45763. Luetteke N. C , Qiu T. H., Fenton S. E., Troyer K. L., Riedel R. F., Chang A. and Lee D. C. (1999). "Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development." Development 126(12): 2739-50. Luetteke N. C , Qiu T. Ff., Peiffer R. L., Oliver P., Smithies O. and Lee D. C. (1993). "TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice." Cell 73(2): 263-78.  153  Lyons R. M., Gentry L. E. and Moses H. L. (1990). "Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin." J cell Biol 110: 1361-7. Madlener M., Mauch C , Conca W., Brauchle M., Parks W. C. and Werner S. (1996). "Regulation of the expression of stromelysin-2 by growth factors in keratinocytes: implications for normal and impaired wound healing." Biochem J 320 ( Pt 2): 659-64. Madlener M., Parks W. C. and Werner S. (1998). "Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair." Exp Cell Res 242(1): 201-10. Maes M., Song C , Lin A., De Jongh R., Van Gastel A., Kenis G., Bosnians E., De Meester I., Benoy I., Neels H., Demedts P., Janca A., Scharpe S. and Smith R. S. (1998). "The effects of psychological stress on humans: increased production of proinflammatory cytokines and a Thl-like response in stress-induced anxiety." Cytokine 10(4): 313-8. Marden L. J., Fan R. S., Pierce G. F., Reddi A. H. and Hollinger J. O. (1993). "Platelet-derived growth factor inhibits bone regeneration induced by osteogenin, a bone morphogenetic protein, in rat craniotomy defects." J Clin Invest 92(6): 2897-905. Mariotti A., Kedeshian P. A., Dans M., Curatola A. M., GagnouxPalacios L. and Giancotti F. G. (2001). "EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion." J Cell Biol 155(3): 447-58. Martin P. (1997). "Wound healmg--auning for perfect skin regeneration." Science 276(5309): 75-81.  154  Marucha P. T., Kiecolt-Glaser J. K. and Favagehi M. (1998). "Mucosal wound healing is impaired by examination stress." Psychosom Med 60(3): 362-5. Massague J. (1998). "TGF-beta signal transduction." Annu Rev Biochem 67: 753-91. McCartney - Francis N., Mizel D., Wong H. and Wahl L. (1990). "TGf beta regulates production of growth factors and TGf beta by human peripheral blood monocytes." Growth factors 4: 27-35. McClain S. A., Simon M., Jones E., Nandi A., Gailit J. O., Tonnesen M. G., Newman D. and Clark R. A. (1996). "Mesenchymal cell activation is the rate-limiting step of granulation tissue induction." Am J Pathol 149(4): 1257-70. McKaig B. C , McWilliams D., Watson S. A. and Mahida Y. R. (2003). "Expression and regulation of tissue inhibitor of metalloproteinase1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease." Am J Pathol 162(4): 1355-60. Meisler N., Keefer K. A., Ehrlich H. P., Yager D. R., Myers-Parrelli J. and Cutroneo K. R. (1997). "Dexamethasone abrogates the fibrogenic effect of transforming growth factor-beta in rat granuloma and granulation tissuefibroblasts."J Invest Dermatol 108(3): 285-9. Meisler N., Shull S., Xie R., Long G. L., Absher M., Connolly J. P. and Cutroneo K. R. (1995). "Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid and transforming growth factor beta response elements: a novel mechanism of glucocorticoid regulation of eukaryotic genes." J Cell Biochem 59(3): 376-88. Mellin T. N., Cashen D. E., Ronan J. J., Murphy B. S., DiSalvo J. and Thomas K. A. (1995). "Acidic fibroblast growth factor accelerates 155  dermal wound healing in diabetic mice." J Invest Dermatol 104(5): 850-5. Mercado A. M., Padgett D. A., Sheridan J. F. and Marucha P. T. (2002a). "Altered kinetics of IL-1 alpha, IL-1 beta, and KGF-1 gene expression in early wounds of restrained mice." Brain Behav Immun 16(2): 150-62. Mercado A. M., Quan N., Padgett D. A., Sheridan J. F. and Marucha P. T. (2002b). "Restraint stress alters the expression of interleukin-1 and keratinocyte growth factor at the wound site: an in situ hybridization study." J Neuroimmunol 129(1-2): 74-83. Meyer O. (2003). "[Hormonal life in systemic lupus and other connective tissue diseases]." Gynecol Obstet Fertil 31(9): 746-56. Meyer-Ingold W. and Eichner W. (1995). "Platelet-derived growth factor." Cell Biol Int 19(5): 389-98. Miller L. A , Barnett N. L., Sheppard D. and Hyde D. M. (2001). "Expression of the beta6 mtegrin subunit is associated with sites of neutrophil influx in lung epithelium." J Histochem Cytochem 49(1): 41-8. Mogford J. E„ Sisco M., Bonomo S. R., Robinson A. M. and Mustoe T. A. (2004). "Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing." J Surg Res 118(2): 190-6. Mogford J. E., Tawil N., Chen A., Gies D., Xia Y. and Mustoe T. A. (2002). "Effect of age and hypoxia on TGFbetal receptor expression and signal transduction in human dermal fibroblasts: impact on cell migration." J Cell Physiol 190(2): 259-65. Morris D. G., Huang X., Kaminski N., Wang Y., Shapiro S. D , Dolganov G., Glick A. and Sheppard D. (2003). "Loss of integrin alpha(v)beta6-mediated TGF-beta activation causes Mmpl2dependent emphysema." Nature 422(6928): 169-73. 156  Mu D., Cambier S., Fjellbirkeland L., Baron J. L., Munger J. S., Kawakatsu H., Sheppard D., Broaddus V. C. and Nishimura S. L. (2002) . "The integrin alpha(v)beta8 mediates epithelial homeostasis through MTl-MMP-dependent activation of TGFbetal." J Cell Biol 157(3): 493-507. Munger s., Huang X. and Kawakatsu H. (1999). "The integrin alphavbeta6 binds and activaites latent TGFbetal :a mechanism for regulating pulmonary inflammation and fibrosis." Cell 96: 319328. Munoz-Valle J. F., Vazquez-Del Mercado M., Garcia-Iglesias T., Orozco-Barocio G., Bernard-Medina G., Martinez-Bonilla G., Bastidas-Ramirez B. E., Navarro A. D., Bueno M., Martinez-Lopez E., Best-Aguilera C. R., Kamachi M. and Armendariz-Borunda J. (2003) . "T(H)1/T(H)2 cytokine profile, metalloprotease-9 activity and hormonal status in pregnant rheumatoid arthritis and systemic lupus erythematosus patients." Clin Exp Immunol 131(2): 377-84. Murphy-Ullrich J. E. and Poczatek M. (2000). "Activation of latent TGFbetaby thrombospondin-1: mechanisms and physiology." Cytokine Growth Factor Rev 11(1-2): 59-69. Mutsaers S. E., Bishop J. E., McGrouther G. and Laurent G. J. (1997). "Mechanisms of tissue repair:fromwound healing tofibrosis."Int J Biochem Cell Biol 29(1): 5-17. Nagy A., Nagasliima H., Cha S., Oxford G. E., Zelles T., Peck A. B. and Humphreys-Beher M. G. (2001). "Reduced oral wound healing in the NOD mouse model for type 1 autoimmune diabetes and its reversal by epidermal growth factor supplementation." Diabetes 50(9): 2100-4.  157  Newman S. L., Henson J. E. and Henson P. M. (1982). "Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages." J Exp Med 156(2): 430-42. Nguyen B. P., Gil S. G. and Carter W. G. (2000). "Deposition of laminin 5 by keratinocytes regulates integrin adhesion and signaling." J Biol Chem 275(41): 31896-907. Niu J., Dorahy D. J., Gu X., Scott R. J., Draganic B., Ahmed N. and Agrez M. V. (2002). "Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the beta6 integrin subunit." Int J Cancer 99(4): 529-37. Noszczyk B. H., Klein E., Holtkoetter O., Krieg T. and Majewski S. (2002). "Integrin expression in the dermis during scar formation in humans." Exp Dermatol 11(4): 311-8. Oikarinen A., Kylmaniemi M., Autio-Harmainen H., Autio P. and Salo T. (1993). "Demonstration of 72-kDa and 92-kDa forms of type IV collagenase in human skin: variable expression in various blistering diseases, induction during re-epithelialization, and decrease by topical glucocorticoids." J Invest Dermatol 101(2): 205-10. Oikarinen A., Oikarinen H., Tan E. M. and Uitto J. (1987). "Modulation of collagen metabolism in cultured human skin fibroblasts by dexamethasone: correlation with glucocorticoid receptor density." Acta Derm Venereol 67(2): 106-15. Oishi Y., Fu Z. W., Ohnuki Y., Kato H. and Noguchi T. (2002). "Molecular basis of the alteration in skin collagen metabolism in response to in vivo dexamethasone treatment: effects on the synthesis of collagen type I and III, collagenase, and tissue inhibitors of metalloproteinases." Br J Dermatol 147(5): 859-68.  158  Oldand G. and Ross R. (1968). "Human wound repair, epidermal regeneration." J cell Biol 39: 135-51. Olsen L., Sherratt J. A. and Maini P. K. (1995). "A mechanochemical model for adult dermal wound contraction and the permanence of the contracted tissue displacement profile." J Theor Biol 177(2): 113-28. Ono I. (2002). "The effects of basic fibroblast growth factor (bFGF) on the breaking strength of acute incisional wounds." J Dermatol Sci 29(2): 104-13. Ortega S., Ittmann M., Tsang S. H., Ehrlich M. and Basilico C. (1998). "Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2." Proc Natl Acad Sci U S A 95(10): 5672-7. OTooleE. A. (2001). "Extracellular matrix and keratinocyte migration." Clin Exp Dermatol 26(6): 525-30. O'Toole E. A., Marinkovich M. P., Hoeffler W. K., Furthmayr H. and WoodleyD. T. (1997). "Laminin-5 inhibits human keratinocyte migration." Exp Cell Res 233(2): 330-9. Padgett D. A., Marucha P. T. and Sheridan J. F. (1998a). "Restraint stress slows cutaneous wound healing in mice." Brain Behav Immun 12(1): 64-73. Padgett D. A., Sheridan J. F., Dome J., Berntson G. G., Candelora J. and Glaser R. (1998b). "Social stress and the reactivation of latent herpes simplex virus type 1." Proc Natl Acad Sci U S A 95(12): 7231-5. Panina-Bordignon P., Mazzeo D., Lucia P. D., D'Ambrosio D., Lang R., Fabbri L., Self C. and Sinigaglia F. (1997). "Beta2-agonists prevent Thl development by selective inhibition of interleukin 12." J Clin Invest 100(6): 1513-9. 159  Parks W. C. (1999). "matrix metalloprotinase in repair." Wound Repair Regen 7: 423-32. Parrelli J. M., Meisler N. and Cutroneo K. R. (1998). "Identification of a glucocorticoid response element in the human transforming growth factor beta 1 gene promoter." Int J Biochem Cell Biol 30(5): 623-7. Pawlak C. R., Witte T., Heiken H., Hundt M., Schubert J., Wiese B., Bischoff-Renken A., Gerber K., Licht B., Goebel M. U . , Heijnen C. J., Schmidt R. E. and Schedlowski M. (2003). "Flares in patients with systemic lupus erythematosus are associated with daily psychological stress." Psychother Psychosom 72(3): 159-65. Pierce G. F. (2001). "Inflammation in nonhealing diabetic wounds: the space-time continuum does matter." Am J Pathol 159(2): 399-403. Pierce G. F., Mustoe T. A., Lingelbach J., Masakowski V. R., Gramates P. and Deuel T. F. (1989a). "Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats: possible regulation in macrophages by platelet-derived growth factor." Proc Natl Acad Sci U S A 86(7): 2229-33. Pierce G. F., Mustoe T. A., Lingelbach J., Masakowski V. R, Griffin G. L., Senior R. M. and Deuel T. F. (1989b). "Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms." J Cell Biol 109(1): 429-40. Pierce G. F., Yanagihara D., Klopchin K., Danilenko D. M., Hsu E., Kenney W. C. and Morris C. F. (1994). "Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor." J Exp Med 179(3): 831-40. Pilcher B. K., Dumin J. A., Sudbeck B. D., Krane S. M., Welgus H. G. and Parks W. C. (1997). "The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix." J Cell Biol 137(6): 1445-57.  160  Pilcher B. K., Wang M., Qin X. J , Parks W. C , Senior R. M. and Welgus H. G. (1999). "Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity." Ann N Y Acad Sci 878: 12-24. Pittet B., Rubbia-Brandt L., Desmouliere A., Sappino A. P., Roggero P., Guerret S., Grimaud J. A., Lacher R., Montandon D. and Gabbiani G. (1994). "Effect of gamma-interferon on the clinical and biologic evolution of hypertrophic scars and Dupuytren's disease: an open pilot study." Plast Reconstr Surg 93(6): 1224-35. Proetzel G., Pawlowski S. A., Wiles M. V., Yin M., Boivin G. P., Howies P. N., Ding J., Ferguson M. W. and Doetschman T. (1995). "Transforming growth factor-beta 3 is required for secondary palate fusion." Nat Genet 11(4): 409-14. Putnins E. E., Firth J. D., Lohachitranont A., Uitto V. J. and Larjava H. (1999). "Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen andfibronectin."Cell Adhes Commun 7(3): 211-21. Ramierz F., Fowell D. J., Puklavec M , Simmonds S. and Mason D. (1996). "Glucocorticoids promote a TH2 cytokine response by CD4+ T cells in vitro." J Immunol 156(7): 2406-12. Rappolee D. A., Brenner C. A., Schultz R., Mark D. and Werb Z. (1988a). "Developmental expression of PDGF, TGF-alpha, and TGF-beta genes in preimplantation mouse embryos." Science 241(4874): 1823-5. Rappolee D. A., Mark D., Banda M. J. and Werb Z. (1988b). "Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping." Science 241(4866): 708-12. Reed M. J., Ferara N. S. and Vernon R. B. (2001). "Impaired migration, integrin function, and actin cytoskeletal organization in dermal 161  fibroblastsfroma subset of aged human donors." Mech Ageing Dev 122(11): 1203-20. Robert C. and Kupper T. S. (1999). "Inflammatory skin diseases, T cells, and immune surveillance." N Engl J Med 341(24): 1817-28. Roberts A. B., Russo A., Felici A. and Flanders K. C. (2003). "Smad3: a key player in pathogenetic mechanisms dependent on TGF-beta." Ann N Y Acad Sci 995: 1-10. Roberts A. B. and Sporn M. B. (1993). "Physiological actions and clinical applications offransforminggrowth factor-beta (TGF-beta)." Growth Factors 8(1): 1-9. Rojas I. G., Padgett D. A., Sheridan J. F. and Marucha P. T. (2002). "Stress-induced susceptibility to bacterial infection during cutaneous wound healing." Brain Behav Immun 16(1): 74-84. Romer J., Bugge T. H., Pyke C , Lund L. R., Flick M. J., Degen J. L. and Dano K. (1996). "Impaired wound healing in mice with a disrupted plasminogen gene." Nat Med 2(3): 287-92. Romero M. R., Carroll J. M. and Watt F. M. (1999). "Analysis of cultured keratinocytesfroma transgenic mouse model of psoriasis: effects of suprabasal integrin expression on keratinocyte adhesion, proliferation and terminal differentiation." Exp Dermatol 8(1): 5367. Ross R., Masuda J., Raines E. W., Gown A. M., Katsuda S., Sasahara M., Maiden L. T., Masuko H. and Sato H. (1990). "Localization of PDGF-B protein in macrophages in all phases of atherogenesis." Science 248(4958): 1009-12. Rubin J. S., Bottaro D. P., Chedid M., Miki T., Ron D., Cunha G. R. and Finch P. W. (1995). "Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction." Exs 74: 191-214.  162  Sakai L. Y., Engvall E., Hollister D. W. and Burgeson R. E. (1982). "Production and characterization of a monoclonal antibody to human Type IV collagen." Am J Pathol 108(3): 310-8. Salmela K. (1981). "Comparison of the effects of methylprednisolone and hydrocortisone on granulation tissue development. An experimental study in rat." Scand J Plast Reconstr Surg 15(2): 8791. Salmela K., Roberts P. J., Lautenschlager I. and Ahonen J. (1980). "The effect of local methylprednisolone on granulation tissue formation. II. Mechanisms of action." Acta Chir Scand 146(8): 541-4. Salo T. and Oikarinen J. (1985). "Regulation of type IV collagen degrading enzyme by Cortisol during human skin fibroblast growth." Biochem Biophvs Res Commun 130(2): 588-95. Sanale A. R., Firth J. D., Uitto V. J. and Putnins E. E. (2002). "Keratinocyte growth factor (KGF)-l and -2 protein and gene expression in human gingival fibroblasts." J Periodontal Res 37(1): 66-74. Sandberg N. (1964). "Time Relationship between Administration of Cortisone and Wound Healing in Rats." Acta Chir Scand 127: 44655. Sanford L. P., Ormsby I., Gittenberger-de Groot A. C , Sariola H., Friedman R., Boivin G. P., Cardell E. L. and Doetschman T. (1997). "TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes." Development 124(13): 2659-70. Sarnstrand B., Brattsand R. and Malmstrom A. (1982). "Effect of glucocorticoids on glycosaminoglycan metabolism in cultured human skin fibroblasts." J Invest Dermatol 79(6): 412-7.  163  Scheid A., Meuli M , Gassmann M. and Wenger R. H. (2000). "Genetically modified mouse models in studies on cutaneous wound healing." E X P Phvsiol 85(6): 687-704. Schmid P., Itin p., Cherry G., Bi C. and Cox D. (1998). "Enhanced expression of transforming growth factor beta type I and type II receptors in wound granulation tissue and hypertrophic scar." Am J Pathol 152(2): 485-93. Schmid P., Kunz S., Cerletti N., McMaster G. and Cox D. (1993). "Injury induced expression of TGF-beta 1 mRNA is enhanced by exogenously applied TGF-beta S." Biochem Biophys Res Commun 194(1): 399-406. Sepp N. T., Li L. J., Lee K. H., Brown E. J., Caughman S. W., Lawley T. J. and Swerlick R. A. (1994). "Basic fibroblast growth factor increases expression of the alpha v beta 3 mtegrin complex on human microvascular endothelial cells." J Invest Dermatol 103(3): 295-9. Serini G., Bochaton-Piallat M. L., Ropraz P., Geinoz A., Borsi L., Zardi L. and Gabbiani G. (1998). "The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by fransforming growth factor-betal." J Cell Biol 142(3): 873-81. Shah M., Foreman D. M. and Ferguson M. W. (1992). "Control of scarring in adult wounds by neutralising antibody to transforming growth factor beta." Lancet 339(8787): 213-4. Shah M., Foreman D. M. and Ferguson M. W. (1995). "Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring." J Cell Sci 108 ( Pt 3): 985-1002. Sheppard D. (1996). "Epithelial integrins." Bioessavs 18(8): 655-60.  164  Sheppard D. (2000). "In vivo functions of integrins: lessonsfromnull mutations in mice." Matrix Biol 19(3): 203-9. Sheppard D. (2001). "Integrin-mediated activation of fransforming growth factor-beta(l) in pulmonary fibrosis." Chest 120(1 Suppl): 49S-53S. Shibata M., Nezu T., Kanou H., Abe H., Takekawa M. and Fukuzawa M. (2002). "Decreased production of interleukin-12 and type 2 immune responses are marked in cachectic patients with colorectal and gastric cancer." J Clin Gastroenterol 34(4): 416-20. Shukla A., Meisler N. and Cutroneo K. R. (1999). "Perspective article: fransforming growth factor-beta: crossroad of glucocorticoid and bleomycin regulation of collagen synthesis in lung fibroblasts." Wound Repair Regen 7(3): 133-40. Shull M. M., Ormsby I., Kier A. B., Pawlowski S., Diebold R. J., Yin M., Allen R., Sidman C , Proetzel G., Calvin D. and et al. (1992). "Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease." Nature 359(6397): 693-9. Shull S., Meisler N., Absher M., Phan S. and Cutroneo K. (1995). "Glucocorticoid-induced down regulation offransforminggrowth factor-beta 1 in adult rat lung fibroblasts." Lung 173(2): 71-8. Sibilia M., Wagner B., Hoebertz A , Elliott C , Marino S., Jochum W. and Wagner E. F. (2003). "Mice humanised for the EGF receptor display hypomorphic phenotypes in skin, bone and heart." Development 130(19): 4515-25. Slavin J. (1995). "Fibroblast growth factors: at the heart of angiogenesis." Cell Biol Int 19(5): 431-44. Slavin J., Hunt J. A., Nash J. R., Williams D. F. and Kingsnorth A. N. (1992). "Recombinant basic fibroblast growth factor in red blood 165  cell ghosts accelerates incisional wound healing." Br J Surg 79(9). 918-21. Slavin J., Unemori E., Hunt T. K. and Amento E. (1995). "Monocyte chemotactic protein-1 (MCP-1) mRNA is down-regulated in human dermal fibroblasts by dexamethasone: differential regulation by TGF-beta." Growth Factors 12(2): 151-7. Snowden J. M. (1984). "Wound closure: an analysis of the relative contributions of contraction and epithelialization." J Surg Res 37(6): 453-63. Snowden J. M., Kennedy D. F. and Cliff W. J. (1982). "Wound contraction. The effects of scab formation and the nature of the wound bed." Aust J Exp Biol Med Sci 60(Pt 1): 73-82. Song C. Z., Tian X. and Gelehrter T. D. (1999). "Glucocorticoid receptor inhibits transforming growth factor-beta signaling by directly targeting the transcriptional activation function of Smad3." Proc Natl Acad Sci U S A 96(21): 11776-81. Staiano-Coico L., Krueger J. G., Rubin J. S., D'Limi S., Vallat V. P., Valentino L., Fahey T., 3rd, Hawes A., Kingston G., Madden M. R. and et al. (1993). "Human keratinocyte growth factor effects in a porcine model of epidermal wound healing." J Exp Med 178(3): 865-78. Steffensen B., Hakkinen L. and Larjava H. (2001). "Proteolytic events of wound-healing—coordinated interactions among matrix metalloproteinases (MMPs), integrins, and extracellular matrix molecules." P i t Rev Oral Biol Med 12(5): 373-98. Stenn K. and Malhotra R. (1992). "Chapter 1:epithelialization in wound healing . biochemical and clinical aspects." INB Saunders Co: 115127.  166  Stephens F. O., Dunphy J. E. and Hunt T. K. (1971). "Effect of delayed ao!rninistration of corticosteroids on wound contraction." Ann Surg 173(2): 214-8. Stepp M., Spurr-Michaud S. and Tisdale A. (1990). "alpha6beta4 integrin heterodimer is a component of hemidesmosomes." proc natl acad sci usa 87: 8970-8974. Stepp M. A. (1999). "Alpha9 andbeta8 integrin expression correlates with the merger of the developing mouse eyelids." Dev Dyn 214(3): 216-28. Swift M. E., Burns A. L., Gray K. L. and DiPietro L. A. (2001). "Agerelated alterations in the inflammatory response to dermal injury." J Invest Dermatol 117(5): 1027-35. Swift M. E., Kleinman H. K. and DiPietro L. A. (1999). "Impaired wound repair and delayed angiogenesis in aged mice." Lab Invest 79(12): 1479-87. Szaflik J., Fryczkowski A. W., Liberek I., Czubak M., Brix M., Broniek G. and Fryczkowski P. (1999). "[Corneal wound healing after penetrating keratoplasty with EGF application. Experimental studies]." Klin Oczna 101(6): 409-16. Tagashira S., Harada H., Katsumata T., Itoh N. and Nakatsuka M. (1997). "Cloning of mouse FGF 10 and up-regulation of its gene expression during wound healing." Gene 197(1-2): 399-404. Takehara K. (2000). "Growth regulation of skin fibroblasts." J Dermatol Sd Dec 24(s 1): S70-7. Thomas G. J., Hart I. R., Speight P. M. and Marshall J. F. (2002). "Binding of TGF-betal latency-associated peptide (LAP) to alpha(v)beta6 integrin modulates behaviour of squamous carcinoma cells." Br J Cancer 87(8): 859-67.  167  Thomas G. J., Poomsawat S., Lewis M. P., Hart I. R., Speight P. M. and Marshall J. F. (2001). "alpha v beta 6 Integrin upregulates matrix metalloproteinase 9 and promotes migration of normal oral keratinocytes." J Invest Dermatol 116(6): 898-904. Thomas G. J. and Speight P. M. (2001). "Cell adhesion molecules and oral cancer." Crit Rev Oral Biol Med 12(6): 479-98. Tokumaru S., Higashiyama S., Endo T., Nakagawa T., Miyagawa J. I., Yamamori K., Hanakawa Y., Ohmoto H., Yoshino K., Shirakata Y., Matsuzawa Y., Hashimoto K. and Taniguchi N. (2000). "Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing." J Cell Biol 151(2): 209-20. Tomasek J. J., Gabbiani G., Hinz B., Chaponnier C. and Brown R. A. (2002). "Myofibroblasts and mechano-regulation of connective tissue remodelling." Nat Rev Mol Cell Biol 3(5): 349-63. Trinchieri G. (1995). "Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity." Annu Rev Immunol 13: 25176. Tsuboi R. and Rifkin D. B. (1990). "Recombinant basic fibroblast growth factor stimulates wound healing in healmg-impaired db/db mice." J Exp Med 172(1): 245-51.  Tsuboi R., Sato C , Kurita Y., Ron D., Rubin J. S. and Ogawa H. (1993). "Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes." J Invest Dermatol 101(1): 49-53. Tyrone J. W., Mogford J. E., Chandler L. A., Ma C , Xia Y., Pierce G. F. and Mustoe T. A. (2000). "Collagen-embedded platelet-derived  168  growth factor DNA plasmid promotes wound healing in a dermal ulcer model." J Surg Res Gct;93((2)): 230-6. Van de Kerkhof P. C , Van Bergen B., Spruijt K. and Kuiper J. P. (1994). "Age-related changes in wound healing." Clin Exp Dermatol 19(5): 369-74. Vassar R. and Fuchs E. (1991). "Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation." Genes Dev 5(5): 714-27. Vedhara K., Cox N. K., Wilcock G. K., Perks P., Hunt M , Anderson S., Lightman S. L. and Shanks N. M. (1999). "Chronic stress in elderly carers of dementia patients and antibody response to influenza vaccination." Lancet 353(9153): 627-31. Vodovotz Y., Geiser A. G., Chesler L., Letterio J. J., Campbell A., Lucia M. S., Sporn M. B. and Roberts A. B. (1996). "Spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse." J Exp Med 183(5): 2337-42. Wahl S. M., Allen J. B , Weeks B. S., Wong H. L. and Klotman P. E. (1993). "Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes." Proc Natl Acad Sci U S A 90(10): 4577-81. Wahl S. M. and Wahl L. M. (1985). "Regulation of macrophage collagenase, prostaglandin, and fibroblast-activating-factor production by anti-inflammatory agents: different regulatory mechanisms for tissue injury and repair." Cell Immunol 92(2): 30212. Warwick-Davies J., Lowrie D. B. and Cole P. J. (1995). "Selective deactivation of human rnpnocyte functions by TGF-beta." J Immunol 155(6): 3186-93. 169  Watt F. M. (2002). "Role of mtegrins in regulating epidermal adhesion, growth and differentiation." Embo J 21(15): 3919-26. Webster E. L., Barrientos R. M., Contoreggi C , Isaac M. G., Ligier S., Gabry K. E., Chrousos G. P., McCarthy E. F., Rice K. C , Gold P. W. and Sternberg E. M. (2002). "Corticotropin releasing hormone (CRH) antagonist attenuates adjuvant induced arthritis: role of CRH in peripheral inflammation." J Rheumatol 29(6): 1252-61. Webster E. L., Torpy D. J., Elenkov I. J. and Chrousos G. P. (1998). "Corticotropin-releasing hormone and inflammation." Ann N Y Acad Sci 840: 21-32. Welch G. R., Wong H. L. and Wahl S. M. (1990). "Selective induction of Fc gamma RIII on human monocytes by transforming growth factor-beta." J Immunol 144(9): 3444-8. Wen F. Q., Kohyama T., Skold C. M., Zhu Y. K., Liu X., Romberger D. J., Stoner J. and Rennard S. I. (2002). "Glucocorticoids modulate TGF-beta production." Inflammation 26(6): 279-90. Wen F. Q., Kohyama T., Skold C. M., Zhu Y. K., Liu X., Romberger D. J., Stoner J. and Rennard S. I. (2003). "Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts." Inflammation 27(1): 9-19. Wenczak B. A., Lynch J. B. and Nanney L. B. (1992). "Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair." J Clin Invest 90(6): 2392-401. Werner S. (1998). "Keratinocyte growth factor: a unique player in epithelial repair processes." Cytokine Growth Factor Rev 9(2): 153-65. Werner S. and Grose R. (2003). "Regulation of wound healing by growth factors and cytokines." Physiol Rev 83(3): 835-70.  170  Werner S. and Munz B. (1998). "[Molecular biology contributions to wound healing and practical applications]." Langenbecks Arch Chir Suppl Kongressbd 115: 678-82. Werner S., Roth W. K., Bates B., Goldfarb M. and Hofschneider P. H. (1991). "Fibroblast growth factor 5 proto-oncogene is expressed in normal humanfibroblastsand induced by serum growth factors." Oncogene 6(11): 2137-44. Werner S., SmolaH., Liao X., Longaker M. T., Krieg T., Hofschneider P. H. and Williams L. T. (1994). "The function of KGF in morphogenesis of epithelium and reepithelialization of wounds." Science 266(5186): 819-22. Wicke C , Halliday B., Allen D., Roche N. S., Scheuenstuhl H., Spencer M. M., Roberts A. B. and Hunt T. K. (2000). "Effects of steroids and retinoids on wound healing." Arch Surg 135(11): 1265-70. Wilder R. L. (2002). "Neuroinrniunoendocrinology of the rheumatic diseases: past, present, and future." Ann N Y Acad Sci 966: 13-9. Witkowski C. M., Bowden G. T., Nagle R. B. and Cress A. E. (2000). "Altered surface expression and increased turnover of the alpha6beta4 integrin in an undifferentiated carcinoma." Carcinogenesis 21(2): 325-30. Wong D. T. (1993). "TGF-alpha and oral carcinogenesis." Eur J Cancer B Oral Oncol 29B(1): 3-7. Woodley D. T. (1996). Reepithelialization. The molecular and celluar biology of wound repair. R. A. Clark. New york and London, Plenum: 339-50. Wu L., Xia Y. P., Roth S. I., Gruskin E. and Mustoe T. A. (1999). "Transforming growth factor-beta 1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic  171  ulcer model: importance of oxygen and age." Am J Pathol 154(1): 301-9. Xia Y. P., Zhao Y., Marcus J., Jimenez P. A , Ruben S. M., Moore P. A., Khan F. and Mustoe T. A. (1999). "Effects of keratinocyte growth factor-2 (KGF-2) on wound healing in an ischaemia-impaired rabbit ear model and on scar formation." J Pathol 188(4): 431-8. Xia Y. P., Zhao Y., Tyrone J. W., Chen A. and Mustoe T. A. (2001). "Differential activation of migration by hypoxia in keratinocytes isolatedfromdonors of increasing age: implication for chronic wounds in the elderly." J Invest Dermatol 116(1): 50-6. Xu J. and Clark R. A. (1996). "Extracellular matrix alters PDGF regulation offibroblastintegrins." J Cell Biol 132(1-2): 239-49. Yang E. V. and Glaser R. (2002). "Stress-induced immunomodulation and the implications for health." Int Immunopharmacol 2(2-3): 315-24. Yang L., Chan T., Demare J., Iwashina T., Ghahary A., Scott P. G. and Tredget E. E. (2001). "Healing of bum wounds in transgenic mice overexpressingfransforminggrowth factor-beta 1 in the epidermis." Am J Pathol 159(6): 2147-57. Yang L., Qiu C. X., Ludlow A., Ferguson M. W. and Brunner G. (1999). "Activefransforminggrowth factor-beta in wound repair: determination using a new assay." Am J Pathol 154(1): 105-11. Yano S., Kondo K., Yamaguchi M., Richmond G., Hutchison M., Wakeling A., Averbuch S. and Wadsworth P. (2003). "Distribution and function of EGFR in human tissue and the effect of EGFR tyrosine kinase inhibition." Anticancer Res 23(5A): 3639-50. Yao F., Visovatti S., Johnson C. S., Chen M., Slama J., Wenger A. and Eriksson E. (2001). "Age and growth factors in porcine fullthickness wound healing." Wound Repair Regen 9(5): 371-7. 172  Yoshizawa T., Watanabe S., Hirose M , Yamamoto J., Osada T., Sato K., Oide H., Kitamura T., Takei Y., Ogihara T., Miwa H., Miyazaki A. and Sato N. (2000). "Effects of growth factors on aspirin-induced inhibition of wound repair in a rabbit gastric epithelial cell model." Aliment Pharmacol Ther 14 Suppl 1: 176-82. Zambruno G., Marchisio P. C , Marconi A., Vaschieri C , Melchiori A., Giannetti A. and De Luca M. (1995). "Transforming growth factor beta 1 modulates betal and beta 5 integrin receptors and induces the de novo expression of the alphav beta6 heterodimer in normal human keratinocytes: implication for wound healing." J Cell Biol 129(3): 853-65.  173  

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