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Epithelial cell integrin phenotype in drug-induced gingival overgrowth tissue Walsh, Priscilla M. 2000

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EPITHELIAL CELL INTEGRIN PHENOTYPE IN DRUG-INDUCED GINGIVAL OVERGROWTH TISSUE by PRISCILLA M. WALSH BSc, University of Alberta, 1994 DDS., University of Alberta, 1996 A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Oral Biological and Medical Sciences) We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA June 2000 © Priscilla M. Walsh, 2000 In presenting this thesis in partial fulfilment 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. Department of fWjL k'iCLr&\C0&+ MjfjjJi&Q ScKQy\0)£> The University of British Columbia Vancouver, Canada Date r V u 28 /gJOOk , DE-6 (2/88) ABSTRACT Gingival overgrowth can be an unpleasant side effect of the commonly prescribed medications nifedipine and cyclosporin. Integrins are transmembrane glycoproteins that mediate cell-to-cell and cell-to-extracellular matrix cell adhesion events. The aim of this study was to characterize epithelial cell integrin phenotype in drug-induced gingival overgrowth tissue. Human gingival biopsies of patients taking nifedipine, cyclosporin, or a combination of both medications were used. Integrins and their ligands were localized in frozen sections using immunohistochemistry. Comparisons between integrin expression in normal and the drug-induced gingival tissue were made. The drug-induced gingival overgrowth tissue exhibited an increase in suprabasal expression of integrins ocvpi, a5pi, and avp6 which is similar to the integrin expression during wound healing. The integrin avp6 was expressed, however, with similar frequency in both the control and the drug-induced gingival overgrowth groups. Fibronectin, a possible ligand for the cc5pi and avp6 integrins, was not only expressed within the connective tissue of all groups, but was also expressed around some of the basal keratinocytes of the control, nifedipine-, and cyclosporin-induced gingival overgrowth groups. No relationship between inflammation and integrin expression could be identified. The results suggest that the epithelium of drug-induced gingival overgrowth tissue exhibit certain changes in its integrin repetoire. This upregulation of certain integrins, which is consistent to one seen during wound healing, could result in controlling the formation of the connective tissue. T A B L E OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vii ACKNOWLEDGEMENTS x CHAPTER ONE - REVIEW OF THE LITERATURE 1 1.1 Clinical presentation and prevalence of nifedipine and cyclosporin -induced gingival enlargement 1 1.2 Histological features of nifedipine- and cyclosporin-induced gingival overgrowth compared to normal gingiva 5 1.3 Possible pathogenesis of nifedipine- and cyclosporin-induced gingival overgrowth 10 1.4 Treatment of nifedipine-and cyclosporin-induced gingival overgrowth 21 1.5 Integrins 23 1.6 Ligands associated with keratinocyte integrins 25 1.7 Integrin expression during wound healing, chronic inflammation, and in oral leukoplakia 28 1.8 Transforming growth factor-beta (TGF-P) and integrin expression 33 CHAPTER TWO - AIM OF THE STUDY 35 CHAPTER THREE - MATERIALS AND METHODS 36 3.1 Gingival samples 36 3.2 Immunofluorescence stainings 39 3.3 Morphological analysis 40 iii CHAPTER FOUR - RESULTS 44 4.1 Analysis of patient information 44 4.2 Histological analysis of the gingival samples 44 4.3 Correlation of inflammation adjacent to the oral and sulcular epithelium in the different tissue types 46 4.4 Frequency and expression of the integrins in each sample 47 4.5 Expression of the fibronectin ligand 47 4.6 Expression of the integrins ct5pi, avp6, avpi and fibronectin 47 4.7 Identification of the components of the basement membrane: laminin-5 and type IV collagen, and the integrin a6p4 50 CHAPTER FIVE - DISCUSSION 72 5.1 Comparison of integrin localization in drug-induced gingival overgrowth tissue to those previously reported in the literature 73 5.2 Integrin expression and inflammation 77 5.3 Integrin expression and fibronectin 77 5.4 Integrin expression and transforming growth factor-p 78 5.5 Location of the tissue biopsies and integrin expression 80 5.6 Basement membrane components 80 5.7 Effect of the medications on the epithelium 80 5.8 Limitations of the study 82 CHAPTER SIX - CONCLUSIONS 84 REFERENCES 86 LIST OF TABLES Table 1 - Patient characteristics 38 Table 2 - Antibodies used against the integrin subunits and their ligands 40 Table 3 - Degree of inflammation 41 Table 4 - Degree of acanthosis 41 Table 5 - Rete ridge length, epithelial thickness and acanthosis of the samples 45 Table 6 - Frequency of expression of a5 integrin 51 Table 7 - Frequency of expression of av integrin 51 Table 8 - Frequency of expression of P1 integrin 51 Table 9 - Frequency of expression of pi active integrin 51 Table 10- Frequency of expression of P4 integrin 52 Table 11- Frequency of expression of P6 integrin 52 Table 12- Frequency of expression of EDA-FN.... 52 Table 13- Frequency of expression of laminin-5 ligand 53 Table 14-Frequency of expression of type IV collagen ligand 53 Table 15- Integrin expression in the control samples -sulcular epithelium (SE) 54 Table 16- Integrin expression in the control samples -oral epithelium (OE) 54 Table 17- Integrin expression in the nifedipine-induced overgrowth samples (SE) 55 Table 18- Integrin expression in the nifedipine-induced overgrowth samples (OE) 55 Table 19- Integrin expression in the cyclosporin-induced overgrowth samples (SE) 56 Table 20- Integrin expression in the cyclosporin-induced overgrowth samples (OE) 56 Table 21- Integrin expression in the combined nifedipine and cyclosporin-induced overgrowth samples (SE) 57 Table 22- Integrin expression in the combined nifedipine and cyclosporin-induced overgrowth samples (OE) 57 Table 23- Comparison of frequency of integrin expression in different tissue types 58 Table 24 -Frequency of av(31 and fibronectin (EDA-FN) in each tissue group 58 Table 25- Frequency of a5pi and fibronectin (EDA-FN) in the tissue_samples 59 Table 26- Frequency of ocvp6 and fibronectin (EDA-FN) in the tissue samples 59 LIST OF FIGURES Figure 1 - Hematoxylin and eosin stainings of tissue samples demonstrating the degree of inflammatory infiltrate. Panel A-minimal infiltration of inflammatory cells, panel B-mild infiltration of inflammatory cells, panel C-moderate infiltration of inflammatory cells, panel D-severe infiltration of inflammatory cells. Bar = 1 mm. E = epithelium, CT = connective tissue 42 Figure 2 - Hematoxylin and eosin stainings of tissue samples demonstrating the degree of acanthosis. Panel A-mild acanthosis, panel B-moderate acanthosis, panel C-severe acanthosis. Bar = 1 mm. E = epithelium CT = connective tissue 43 Figure 3 - Inflammation adjacent to oral epithelium in different tissue types 46 Figure 4 - Inflammation adjacent to sulcular epithelium in different tissue types 46 Figure 5 - Immunolocalization of a5 integrin around the basal keratinocytes of the oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 urn. Arrowhead shows a.5 integrin around the basal keratinocytes. Arrow with tail indicates suprabasal expression of oc5 integrin. E = epithelium, CT = connective tissue 60 Figure 6 - Immunolocalization of av integrin around the basal keratinocytes of the oral epithelium. Panel A-control tissue, panel B- nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 um. Arrow with tail indicates suprabasal expression of av integrin. E = epithelium, CT = connective tissue 61 Figure 7 - Immunolocalization of P1 integrin around the basal keratinocytes of the oral Epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 um. E = epithelium, CT = connective tissue 62 Figure 8 -Immunolocalization of p4 integrin, oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 urn. Arrow with tail indicates suprabasal expression of p4 integrin. E = epithelium, CT = connective tissue 63 V l l Figure 9 - Immune-localization of P6 integrin. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth group. Arrowheads show 06 integrin around the basal keratinocytes of the oral epithelium. Bar = 50 pm. E = epithelium, CT = connective tissue 64 Figure 10-Immunolocalization of laminin-5 in the oral epithelium of the different groups. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced tissue. Bar = 200 pm. E = epithelium, CT = connective tissue 65 Figure 11-Immunolocalizationof EDA-FN. Expression of EDA-FN was demonstrated around the basal keratinocytes of some samples, and only within the connective tissue of other samples. Panels A and B-control tissue, panels C and D-nifedipine-induced overgrowth tissue, panels E and F-cyclosporin-induced overgrowth tissue, panel G-combined drug-induced overgrowth group. Bar = 200 pm. Arrow with tail indicates suprabasal expression of EDA-FN. E = epithelium, CT= connective tissue 66 Figure 12-Immunolocalization of av, pi active, P6 integrin, and EDA-FN within the oral epithelium of the control tissue. Panel A-av integrin, panel B-pi-active integrin, panel. C-EDA-FN, panel D-negative stain for P6 integrin. Panels A, B and C demonstrate colocalization of the integrin avpi with EDA-FN within parallel sections of the same specimen. Panels E (a5 integrin), F (P1 integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pi with EDA-FN within parallel sections of the same specimen. Bar = 200 pm. E = epithelium, CT = connective tissue 67 Figure 13-Immunolocalization of av, pi-active, p6 integrin, and EDA-FN within the oral epithelium of nifedipine-induced overgrowth tissue. Panel A-av integrin, panel B-pi-active integrin, panel. C-EDA-FN, panel D-P6 integrin. Panels A, B, C and D demonstrate colocalization of the integrins avpi and avp6 with EDA-FN in parallel sections of the same specimen (see arrowheads). Panels E (a5 integrin), F (pi integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pi with EDA-FN in parallel sections of the same specimen (see arrowheads). Bar = 100 pm. E = epithelium, CT = connective tissue 68 Figure 14-Immunolocalization of av, pi, P6 integrin, and EDA-FN around the basal keratinocytes of the oral epithelium of the cyclosporin-induced overgrowth tissue. Panel A-av integrin, panel B-pi-active integrin, panel. C-viii EDA-FN, panel D-P6 integrin. Panels A, B, C and D demonstrate colocalization of the integrins av(31 and avp6 with EDA-FN. Panels E (oc5 integrin), F (pi integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pi with EDA-FN (see arrowheads). All panels are from parallel sections of the same specimen. Bar =100 pm. E = epithelium, CT = connective tissue 69 Figure 15- Immunolocalization of av, P6 integrin, and EDA-FN around the basal keratinocytes of the oral epithelium of the combined drug-induced overgrowth tissue. Panel A-av integrin, panel B-P6 integrin, panel C-EDA-FN (Bar = 200 pm). Panel D-av integrin, panel E-P6 integrin, panel F-EDA-FN (Bar = 50 pm). Panels A, B, C, D, E, F demonstrate colocalization of the integrin avp6 with EDA-FN (arrows with tail show expression of the integrin and/or EDA-FN within parallel sections of the same specimen). E = epithelium, CT = connective tissue 70 Figure 16-Immunolocalization of a5 and pi-active integrin around the basal keratinocytes of the oral epithelium of the combined drug-induced overgrowth tissue. Panel A (a5 integrin), panel B (pi active integrin) and panel C (EDA-FN) demonstrate colocalization of the integrin a5pi with EDA-FN (arrows with tail indicates the parallel sections within the same specimen). Bar = 100 pm. E = epithelium, CT = connective tissue 71 ix ACKNOWLEDGEMENTS I would like to express my gratitude and thanks to Dr. Hannu Larjava, my thesis supervisor, for his guidance and support during the course of this project. Thank you to Dr. H. Pernu and the University of Oulu, as this project would have not been possible if it were not for the donation of slides from the University of Oulu, Finland. Thank you to the members of my committee, Dr. Edward Putnins, Dr. Doug Waterfield and Dr. Lewei Zhang. Very special thanks to Dr. Lari Hakkinen for all his time and assistance with this project, and to Mr. Christian Sperantia for all his technical support. Thank you to Dr. Reza Ghaeli for his help with the statistical analysis. To my classmates, Dr. Nazanin Narani and Dr. Chris Hildebrand, thank you for all your help over the past three years both with this project, and with the program. Finally, to Dr. Gerry Pochynok, thank you for all your support and help over the past years, I am eternally grateful. Chapter One -Review of the literature 1.1 Clinical presentation and prevalence of nifedipine and cyclosporin-induced gingival enlargement. Nifedipine Nifedipine is a calcium channel blocker that is used in the treatment of angina and hypertension. Its function is to inhibit calcium ion entry into cells. The reduction of available calcium reduces myocardial cell contractility and oxygen consumption. This results in the relaxation of coronary vascular smooth muscle, dilation of coronary and peripheral arteries, and an increase in myocardial oxygen delivery in angina patients (Gage and Pickett, 1997; Seymour and Heasman, 1988). At a cellular level, nifedipine blocks the breakdown of ATP by calcium-dependent ATPase, which results in a decrease of high energy phosphate consumption, mechanical tension, and oxygen requirements of the myocardium (Hillis, 1980). The systemic side effects of nifedipine include dizziness, flushing, headaches, weakness, nausea, muscle cramps, tremor, peripheral edema, joint stiffness, dermatitis, pruritus, and urticaria (Lederman et al., 1984). In addition, gingival enlargement has been documented as an oral side effect caused by the administration of nifedipine (Lederman et al., 1984; Ramon et al., 1984). The incidence of nifedipine-induced gingival enlargement has been reported as 6.4% (Ellis et al., 1999), 15% (Barak et al., 1987), 20% (Barclay et al., 1992), 29% (Tavassoli et al., 1998), and 44% (Nery et al., 1995). 1 The clinical presentation of nifedipine-induced enlargement closely resembles the presentation of dilantin-induced gingival enlargement (Seymour and Heasman, 1988). The overgrowth usually occurs within one to two months after the initiation of the medication (Seymour, 1991). The site of enlargement is typically the labial gingiva of the upper and lower teeth (Seymour and Heasman, 1988). Although many authors have reported no evidence of enlargement in edentulous areas (Barclay et al., 1992; Ramon et al., 1984; Seymour and Heasman 1988), a recent study reported one case of nifedipine-induced gingival overgrowth in an edentulous area (Tavassoli et al., 1998). The pattern of overgrowth, which is limited to the attached gingival tissue, begins as a swelling of the interdental papillae. As the enlargement progresses, the papillae may then coalesce and cover the surface of the tooth. The enlarged tissues, which are separated from the gingival margin by a linear groove, appear edematous and hyperemic. In severe cases, the tissue can extend over the crowns of the teeth and consequently interfere with eating (Seymour, 1991). It has been reported that a higher incidence of enlargement is evident in patients in their fourth decade compared to patients in their fifth and sixth decade (Nery et al., 1995; Tavassoli et al., 1998). Males appear to have a three times greater chance of developing clinically significant nifedipine-induced gingival overgrowth compared to females (Ellis et al., 1999). Cyclosporin Cyclosporin, discovered by Borel in 1972, is a fungal metabolite produced by Trichoderma polysporum Rifai and Cyclindrocarpon lucidum (Borel et al., 1976). The pharmaceutical company's initial goal was to create a potent antimicrobial compound, but studies later 2 revealed that cyclosporin had poor antimicrobial activity. It was serendipitously discovered that cyclosporin had an inhibitory effect on the subpopulation of T-lymphocytes, no effect on the humoral response, and low myelotoxicity (Borel et al., 1976; Britton and Palacios, 1982). The pathway through which T-lymphocyte function is impaired is via specific inhibition of the synthesis of interleukin-2. Other growth factors, which include interleukin-l(IL-l), lymphokine-3(IL-3), migration inhibitory factor(MIF), gamma interferon (IFN-y), lymphocyte directed chemotactic factor (LDCF), and macrophage activation factor (MAF), have also been shown to be inhibited by cyclosporin (CPS, 1998). The principle use of cyclosporin is the prevention of graft rejection in organ transplantation, but it is also being used in other diseases which may have an altered cell-mediated immunological mechanism. Such diseases include: type I diabetes mellitus (Stiller et al., 1983), primary biliary cirrhosis (Routhier et al., 1980), psoriasis (Mueller and Herrmann, 1979), rheumatoid arthritis, erosive lichen planus, ulcerative colitis, Crohn's disease (Daley and Wysocki, 1984), and Behcets disease (French-Constant et al., 1983). Gingival overgrowth was first recognized in association with cyclosporin use while the medication was being evaluated in patients that had received organ transplants (Calne et al., 1981; Starzl et al., 1980). It was observed that the pattern of overgrowth was identical to that of phenytoin-induced overgrowth (Wysocki et al., 1983). The enlargement, which typically occurs within three months of taking cyclosporin (Seymour et al., 1987), begins in the papillae area, and like phenytoin-induced overgrowth, the papillae appear to coalesce. The overgrowth is limited to the attached gingiva, and in severe cases, can extend coronally and interfere with speech, mastication, and occlusion (Tyldesley and Rotter, 1984). It appears that the overgrowth is more common on the labial surfaces of the teeth, and in the anterior regions 3 of the mouth (Daley and Wysocki, 1984). Specifically, the overgrowth is more common on the labial aspect of the canine teeth compared to the overgrowth around the central incisors (Thomason et al., 1996a). The tissues are different from phenytoin-induced overgrowth in that the cyclosporin-induced overgrowth appears more hyperemic (Seymour and Jacobs, 1992). Cyclosporin-induced gingival overgrowth has not been shown to occur in edentulous spaces (Somacarrera et al., 1994), nor does it occur in edentulous patients (Friskopp and Klintman, 1986). The incidence of cyclosporin-induced overgrowth has been reported as high as 70% (Daley et al., 1986), though most authors concur that the incidence is approximately 30% (Seymour et al., 1987; Wysocki et al., 1983; McGaw et al., 1987). There is a lower incidence of gingival overgrowth in bone marrow transplant patients treated with cyclosporin than in renal transplant patients (Beveridge, 1983). Controversy exists regarding a higher incidence of cyclosporin-induced overgrowth in children and adolescent patients. Some authors believe that there is a higher incidence of overgrowth in this subset of patients (Daley and Wysocki, 1984; Karpinia et al., 1996), whereas other authors disagree (Seymour and Smith, 1991). Cyclosporin-induced gingival overgrowth does not appear to have any gender predilection (McGaw et al., 1987). Nifedipine and cyclosporin-induced gingival overgrowth Nifedipine and cyclosporin are frequently prescribed together for use by renal and cardiac transplant patients (Thomason et al., 1993). In these cases, nifedipine is used to treat either primary or secondary hypertension. The incidence of nifedipine and cyclosporin-induced overgrowth is greater than the incidence of overgrowth in patients taking only one of the 4 medications. Reports on incidence range from 48% to 60% (King et al., 1993; Margiotta et al., 1996; Thomason et al., 1993; Slavin and Taylor, 1987). The prevalence of the overgrowth appears to be similar when comparing gingival overgrowth in patients taking cyclosporin alone to patients taking both cyclosporin and nifedipine, but the severity of the overgrowth in the combination patients is increased (Thomason et al., 1993; Thomason et al., 1995). A study which reported on cardiac transplant patients taking both cyclosporin and nifedipine found that males were more likely than females to have clinically significant overgrowth. It was also shown that there was a significantly greater need (25-fold) to perform gingival surgery on patients taking both medications, compared to patients only taking one medication (Thomason et al., 1995). The increased risk for progression and recurrence of gingival overgrowth due to the administration of both cyclosporin and nifedipine has been confirmed by other studies (Bokenkamp et al., 1994; O'Valle et al., 1995). 1.2 Histological features of nifedipine and cyclosporin-induced gingival overgrowth compared to normal gingiva. Normal gingiva The histological appearance of normal gingiva includes the stratified squamous epithelium and the underlying gingival connective tissue. The epithelium is comprised of four strata: basale, spinosum, granulosum, and corneum. The stratum basale is comprised of mostly keratinocytes, which are attached laterally by gap junctions and hemidesmosomes, and to the underlying basal lamina via hemidesmosomes. The keratinocytes undergo cell division in the stratum basale and then migrates across the epithelium to reach the stratum corneum. As the 5 keratinocyte crosses the epithelium, various changes occur within the cell. These changes include: loss of ability to undergo mitosis, elevated production of proteins and accumulation of keratohyalin granules, loss of organelles which are responsible for protein and energy production, subsequent degeneration and intracellular keratinization without loss of the cell to cell attachments, and finally, disintegration of the cell to cell attachments and loss of the cell into the oral cavity (Schroeder and Page, 1990). Other cells including Langerhans cells, Merkel cells, and melanocytes are evident within the epithelium. Langerhans cells are derived from bone marrow progenitor cells. It has been shown that they possess surface antigens that are similar to that found on lymphocytes and macrophages, as well as receptors for complement and immunoglobulin. It has therefore been proposed that the function of Langerhans cells is primarily defensive (Newcomb et al., 1982). Merkel cells possess nerve endings, and are attached to adjacent cells via desmosomes. These cells are considered tactile perceptors (Ness et al., 1987). Another cell found in the gingival epithelium is the melanocyte. This stellate cell possesses dendritic processes, and it is not attached to adjacent cells. The primary function of melanocytes is the production of melanin, which is transferred to keratinocytes by phagocytosis (Schroeder, 1969). The epithelial extracellular matrix does not contain fibrous protein components, but the nonfibrous epithelial components include: water, glycoproteins, proteoglycans, and lipids (Bartold, 1987). The connective tissue compartment is comprised of a cellular and non-cellular component. Cells comprise approximately 8% of the volume of the connective tissue compartment (Schroeder et al., 1973). Fibroblasts, polymorphonuclear leukocytes, plasma cells, mast cells, endothelial cells and macrophages are all found in healthy gingival tissue (Hassell, 1993). The fibroblast, which is the most common cell in the connective tissue, is responsible for the 6 production of collagen, elastin, glycoproteins, glycosaminoglycans, as well as matrix metalloproteinases (gelatinases, collagenases, stromelysins, matrilysin) (Birkedal-Hansen, 1988). There is evidence to support the concept that phenotypically distinct and functionally different subpopulations of fibroblasts exist within one individual (Hakkinen and Larjava, 1992; Hassell and Stanek, 1983). Clinical normalcy may be the result of a mixture of cell subpopulations within the healthy tissue. Insults to the tissue may result in a shift in the composition of the mixture, therefore resulting in connective tissue fibrosis or connective tissue loss (Hassell, 1993). The non-cellular component of the connective tissue compartment consists of collagen, elastin, fibronectin, laminin, tenascin, osteonectin, glycosaminoglycans, and proteoglycans (Mariotti, 1993). In healthy gingiva, type I collagen is the most common collagen (Narayanan et al., 1985). Other collagens found in the gingival connective tissue include types III, IV, V, VI (Becker et al., 1986; Chavrier et al., 1984). Together, collagens account for approximately 60% of the total protein (Page, 1972). The elastic fiber system, which accounts for 6% of the total gingival protein (Chavrier, 1990), is partially responsible for the elastic properties of the gingiva. Other noncollagenous proteins include fibronectin, which is distributed with collagen and is found throughout the connective tissue (Narayanan et al., 1985), laminin, and tenascin, which is found in and near the subepithelial basement membrane in the connective tissue (Steffensen et al., 1992). Osteonectin has been identified in the gingival connective tissue, but its role has not been elucidated (Salonen et al., 1990). Various glycosaminoglycans and proteoglycans in the gingival connective tissue have been described. Dermatan sulfate is the most common glycosaminoglycan, and it accounts for 60% of the total gingival glycosaminoglycans (Bartold et al., 1981). Chondroitan-4-sulfate accounts for 28%) of total glycosaminoglycans, whereas heparan sulfate (the most common 7 gingival epithelial glycosaminoglycan) accounts for 5%. Hyaluronic acid is also found both the gingival connective tissue and the epithelium (Bartold et al., 1981; Tammi et al., 1988). The proteoglycans that have been discovered in the gingiva include decorin, biglycan, versican, syndecan, and CD44 (Hakkinen et al.,1993; Larjava et al., 1992a). The gingival epithelium is separated from the underlying connective tissue by the basement membrane. The basement membrane is also responsible for separating the connective tissue from nerves and from endothelial cells of the blood vessels (Bartold and Narayanan, 1998). Type IV collagen and laminin are major components of the basement membrane (Chavrier et al., 1984; Narayanan et al., 1985). The basement membrane consists of two layers: the lamina densa and lamina lucida. The lamina lucida is attached to the basal keratinocytes via hemidesmosomes which contain type XVII collagen (also known as bullous pemphigoid antigen-2, BAPG-2), BAPG-1, and the integrin cc6p4 (Jones et al., 1991; Uitto and Christiano, 1992). The lamina densa is attached to the connective tissue via anchoring fibrils, which contain type VII collagen (Keene et al., 1987; Ooya and Tooya, 1981). Nifedipine-induced gingival overgrowth tissue. The histological appearance of nifedipine-induced gingival overgrowth tissue is similar to that seen in phenytoin overgrowth (Marshall and Bartold, 1998). The features commonly seen in phenytoin overgrowth include: acanthosis, evident as elongated rete ridges, hyperkeratosis and parakeratosis, and an increase in frequency of epithelial islands in the connective tissue due to sectioning of the tissue. A ten-fold increase in the epithelial width (normal width ranges between 0.3 to 0.5mm) has been reported (Van der Wall et al., 1985). The appearance of the connective tissue varies according to the degree of inflammation and 8 the stage of development of the overgrowth. There appears to be an increase in the number of fibroblasts as well as an increase in the formation of collagen fibers. Chronic inflammatory cells, specifically lymphocytes and plasma cells, are visible in most phenytoin cases (Angelopoulos, 1975). Nifedipine-induced overgrowth shares the following features with phenytoin-induced gingival overgrowth: slight to moderate hyperkeratosis, thickened spinous layer of the epithelium, elongation of the rete ridges, fibrotic connective tissue with fibroblast proliferation, increased numbers of capillaries with chronic perivascular inflammation, and abundant plasma cell, mast cell and lymphocyte infiltration (Barak et al., 1987; Lederman et al., 1984; Lucas et al., 1985; Ramon et al., 1984). The number of CD la-labelled Langerhans cells has been shown to be significantly lower in the sulcular epithelium of nifedipine-induced gingival overgrowth samples compared to normal and immunosuppressive medication induced gingival overgrowth samples (Nurmenniemi et al., 1999). The fibroblasts in the connective tissue are considered "active" due to the presence of prominent rough endoplasmic reticulum and large cytoplasmic secretory granules (Lucas et al., 1985). Cyclosporin-induced gingival overgrowth tissue Histologically, cyclosporin-induced gingival overgrowth tissue is very similar to phenytoin and nifedipine-induced overgrowth tissue (Wysocki et al., 1983). The epithelium demonstrates variable thickness, areas of parakeratosis, and elongated rete ridges (Deliliers et al., 1986; Wysocki et al., 1983). Needle-like crystallite structures, presumed to represent accumulated cyclosporin, as well as intact and degranulated mast cells, have been identified within the gingival epithelium of patients exhibiting cyclosporin-induced gingival overgrowth (Pisanty et al., 1990). The basal lamina is interrupted in areas where the epithelium has 9 invaded the underlying connective tissue, and it is in direct contact with a layer of collagen fibers (Mariani et al., 1996). The connective tissue demonstrates increased numbers of fibroblasts, mast cells, plasma cells, as well as an increase in the degree of vascularization (Deliliers et al., 1986; Mariani et al., 1996; Rateitschak-Pliiss et al., 1983). The helper/inducer T lymphocyte (CD4) and the cytotoxic/suppressor T lymphocyte (CD8) ratio has been shown to be higher in patients with cyclosporin-induced gingival overgrowth (O'Valle et al., 1994). In addition, the number of epithelial cells with intraepithelial deposits of cyclosporin were found to be positively correlated with the degree of inflammatory infiltrate of CD4+, CD8+ and CD68+ cells (O'Valle et al., 1994). The majority of mononuclear cells found in this form of overgrowth tissue included monocytes and T-lymphocytes; virtually no B-lymphocytes were seen (Friskopp et al., 1986). The fibroblasts, which have been described as resembling myofibroblasts (Yamasaki et al., 1987), contained dilated ergastoplasmatic cisternae, which is characteristic of productive behavior (Mariani et al., 1993). The amount of amorphous ground substance produced by the fibroblasts in cyclosporin-induced overgrowth tissue was higher than that found in normal gingival samples (Mariani etal., 1996). 1.3 Possible pathogenesis of nifedipine and cyclosporin-induced gingival overgrowth. The pathogenesis of nifedipine and cyclosporin-induced gingival overgrowth is not fully known or understood (Marshall and Bartold, 1998; Seymour et al., 1996). The multifactorial nature of this process can be related to certain factors: genetic predisposition, 10 pharmacokinetic variables, alterations in the gingival connective tissue homeostasis, including the non-collagenous matrix and the connective tissue metabolism, the indirect effect of nifedipine, and the presence of dental plaque, inflammation and gingival bleeding (Pernu et al., 1992; Seymour et al., 1996). Nifedipine-induced gingival overgrowth Genetic predisposition may play a role in the occurrence of nifedipine-induced gingival overgrowth because it has been shown that not all patients taking nifedipine will show signs of gingival overgrowth. Patients are referred to as "responders" if they demonstrate medication-induced gingival changes (Seymour et al., 1996). Responders may show differences in fibroblast function, drug receptor binding and drug metabolism. Fibroblasts within one individual may show phenotypically distinct and functionally different subpopulations (Hakkinen and Larjava, 1992; Hassell and Stanek, 1983). In vitro studies utilizing fibroblasts from nifedipine-induced gingival overgrowth samples showed increased cell proliferation when fibroblasts were exposed to 1 ug/ml nifedipine for short (72 hours) and long (six weeks) term experiments. The increase in cell proliferation at six weeks was concomitant with a decrease in proteoglycan synthesis (Willershausen-Zonnchen et al., 1994). The results of this study support the findings of other in vitro experiments (Fujii et al., 1994; McKevitt and Irwin, 1995). Conversely, other in vitro experiments have shown that fibroblasts from nifedipine-induced gingival overgrowth samples exposed to nifedipine did not proliferate at a rate any different from normal fibroblasts (Nishikawa et al., 1991; Tipton et al., 1994). The production of glycosaminoglycans and fibronectin was not different from that produced by normal fibroblasts, and the production of collagenase was decreased (Tipton 11 et al., 1994). Collagen production by fibroblasts derived from nifedipine-induced gingival overgrowth has been shown to increase (Fujii et al., 1994; McKevitt and Irwin, 1995; Tipton et al., 1994), remain unchanged (Nishikawa et al., 1991), and decrease (Salo et al., 1990; Zebrowski et al., 1986). The high variability of the results of these in vitro studies may be due to the genetic heterogeneity of the fibroblasts that can exist within one patient. This has been confirmed by one study that demonstrated that individual fibroblast cell lines possess many differing responses to external stimuli (Larjava and Uitto, 1987). The shortcoming of many of the in vitro studies is that very few fibroblast cell lines were utilized, making the interpretation of the results difficult. The determination of human lymphocyte antigen (HLA) expression has been examined in patients who exhibit nifedipine-induced gingival overgrowth. These studies showed opposing results. One study did not find a correlation between HLA expression and overgrowth (Margiotta et al., 1996), whereas the other found a trend for the HLA-DR2+ patients to have more severe gingival overgrowth (Pernu et al., 1994). More studies are required to definitively link HLA expression and fibroblast phenotype (Seymour et al., 1996). Other genetic variations may include the function of the cytochrome P450 group (Seymour et al., 1996). It has been determined that the cytochrome P450 genes display genetic polymorphism, which can result in interindividual variation in drug metabolism (Gonzales, 1992). Such variation may have some influence on the development of gingival overgrowth in certain individuals (Seymour et al., 1996). Pharmacokinetic variables may play a role in the development of nifedipine-induced gingival overgrowth. The primary action of nifedipine is the inhibition of calcium ion influx. It may be through this direct action on the gingival cells that gingival overgrowth may result (Marshall and Bartold, 1998; Seymour et al., 1988). It has been postulated that the physico-12 chemical profile of nifedipine (highly lipophilic) enhances its ability to interact with the cells within the gingival tissue (Ellis et al., 1999). This hypothesis requires further studies to determine its validity. Nifedipine is metabolized into two pharmacologically inactive metabolites. The primary metabolite (95%) is dehydronifedipine, the secondary metabolite (5%>) is the lactone derivative of nifedipine (CPS, 1999). An examination of the effect of nifedipine and its metabolites revealed that the metabolites were not a risk factor for the severity of gingival overgrowth in patients taking both cyclosporin and nifedipine (Thomason et al., 1997). The dose and duration of nifedipine and its relationship to the prevalence of overgrowth has not been fully determined. It has been reported that the dose and duration of nifedipine is both related (Tavassoli et al, 1998), and not related (Barclay et al., 1992; Nery et al., 1995) to the presence of overgrowth. Most authors agree that individual sensitivity in metabolizing nifedipine is likely the main reason for the presence of overgrowth (King et al., 1993; Tavassoli et al., 1998). It was proposed that the sequestration of nifedipine in the gingival tissues may predispose patients to developing gingival overgrowth. This was supported by a study which demonstrated nifedipine at higher concentrations in the gingival crevicular fluid (15-316 times) compared to that found in the plasma in patients demonstrating nifedipine-induced gingival overgrowth (Ellis et al., 1992). A subsequent study revealed that there was no difference between responders and non-responders with respect to the detection and level of nifedipine concentration in the gingival crevicular fluid (Ellis et al., 1993). It was later determined that the presence of inflammation and plaque were significantly related to the sequestration of nifedipine in the gingival crevicular fluid of patients with gingival overgrowth (Ellis et al., 1995). In view of the current findings, the 13 concentration of nifedipine in the gingival tissues may provide some information regarding the pathogenesis and manifestation of gingival overgrowth (Seymour et al., 1996). The alterations in the gingival connective tissue homeostasis may include the effect of nifedipine on fibroblasts, as previously discussed. Other effects may include the effect of the medication on gene expression. The expression of type VI collagen genes and the deposition of type VI collagen was found to be increased in patients exhibiting nifedipine-induced gingival overgrowth (Shikata et al., 1993). The effect of nifedipine on gene expression may play an important role in the pathogenesis of the overgrowth, but further studies are required in this area (Seymour et al., 1996). In vitro experiments analyzing the effect of nifedipine on proteoglycan synthesis have reported both increased synthesis (Saito et al., 1996), and decreased deposition of proteoglycans by fibroblasts (Tipton et al., 1994; Willershausen-Zonnchen et al., 1994). Normal gingival tissues exhibit a level of homeostasis in which collagen synthesis and degradation are balanced. In medication-induced gingival overgrowth the normal metabolism of the connective tissue can be altered in many different ways (Barclay et al., 1992). The collagenolytic signal from inflammatory cells to the fibroblasts and the synthesis of metalloproteinases are calcium dependent. It has been hypothesized that the reduction in free calcium results in impaired collagen resorption (Barclay et al., 1992). A different mechanism may include the production of collagenase. This is supported by a study that demonstrated increased collagenase production by normal gingival fibroblasts when exposed to nifedipine (Zebrowski et al., 1988). Nifedipine has been shown to have indirect effects on components of the gingival tissues. The reduction of free calcium by nifedipine can result in the prevention of phytohemagglutinin (PHA)-induced lymphocyte proliferation, and the reduction of both 14 lectin-induced increase of free cytosolic calcium and interleukin-2 secretion (Gelfand et al., 1986). Another indirect effect of nifedipine includes the production of higher levels of active androgen metabolites by fibroblasts from patients with nifedipine-induced gingival overgrowth (Sooriyamoorthy et al., 1990). Nifedipine at a physiological level may stimulate the production of interleukin-2 by T-lymphocytes, or the production of active androgen metabolites, and this could then result in the proliferation and subsequent increase in collagen synthesis by fibroblasts (Nishikawa et al., 1991). Other indirect effects of nifedipine include the increased synthesis of growth factors such as transforming growth factor-p and basic fibroblast growth factor. Both growth factors, their receptors, and heparan sulphate glycosaminoglycan were increased in nifedipine-induced overgrowth tissue (Saito et al., 1996). The in vivo levels of interleukin-ip have been shown to be inversely proportional to the in vitro responsiveness of the fibroblasts from nifedipine-induced gingival overgrowth, which helps support the concept that nifedipine affects the metabolism of fibroblasts (Henderson et al., 1997). Medullasin, a neutrophil elastase-like proteinase, was increased in nifedipine-induced gingival overgrowth tissue. This enzyme may participate in the pathogenesis of the overgrowth, possibly through host defence and immunoregulation, but the mechanism through which it acts is not fully known (Kunimatsu et al., 1996). Together, it is evident that nifedipine may act indirectly to contribute to the overgrowth tissue. The presence of dental plaque and inflammation and its role in the pathogenesis of nifedipine-induced gingival overgrowth is unclear (Marshall and Bartold, 1998). One study has reported that plaque is not related to the presence of gingival overgrowth (Barclay et al., 1992). This is contrary to the findings of other studies and case reports which demonstrate that plaque is a cofactor necessary for nifedipine-induced gingival overgrowth (Ellis et al., 15 1999; Hancock and Swan 1992; Nery et al., 1995; Nishikawa et al., 1991). A study performed on pathogen-free rats showed that nifedipine induced gingival overgrowth occurred in the presence or absence of inflammation and/or dental plaque, and that both factors could augment the effect of nifedipine on the gingiva (Morisaki et al., 1993). One study has examined the microflora associated with nifedipine-induced gingival overgrowth. It was determined that the percent and frequency of certain gram-positive and gram-negative rods including Eubacterium alactolyticum, Capnocytophaga gingivalis, Capnocytophaga ochracea, Capnocytophaga sputigena, and Fusobacterium nucleatum, were increased. It was further postulated that the combination of impaired host response due to the medication, and the presence of enlarged gingiva, allowed for the establishment of certain gram-negative anaerobic species (Nakou et al., 1998). Cyclosporin-induced gingival overgrowth Fibroblast heterogeneity and the direct and indirect effect of cyclosporin and its metabolites on the fibroblasts are likely the main reasons for cyclosporin-induced gingival overgrowth (Marshall and Bartold, 1998). Cyclosporin undergoes extensive hepatic metabolism, but it is not metabolized in one single pathway. The major metabolic pathways include hydroxylation and N-demethylation of the amino-acids of the parent compound. Of the 20 metabolites, 14 have been identified. The major metabolites, which can be determined by high-pressure liquid chromatography (HPLC) include M17, M l , M18, and M21 (CPS, 1998; Garraffo, 1992; Khoschsorur et al., 1998). There appears to be high individual variations with respect to resorption and metabolization rates, but the recommended range of blood and plasma/serum levels of 16 cyclosporin is between 100-400 ng/ml and 50-200 ng/ml, respectively (CPS, 1998; Seymour and Jacobs, 1992). The relationship between plasma and/or salivary concentrations of cyclosporin has been reported to be a factor that is both positively related (McGaw et al., 1987; Rostock et al., 1986; Seymour et al., 1987; Somacarrera et al, 1994), and not related (Daley et al., 1986; Ross et al., 1989; Seymour and Smith, 1991) in the pathogenesis of cyclosporin-induced gingival overgrowth. A study utilizing multiple sclerosis patients receiving cyclosporin demonstrated that the odds ratio was 17.3 for having a cyclosporin blood level of >400 ng/ml and the incidence of overgrowth (Hefti et al., 1994). The relationship between plasma/blood levels and overgrowth is unclear, and unfortunately, most of the studies did not employ the use of high-pressure liquid chromatography to determine the plasma levels (Marshall and Bartold., 1998). In addition to the dose of cyclosporin possibly having an influence on the incidence and severity of the overgrowth, the duration of cyclosporin has been implicated as a factor that can increase the chance of gingival overgrowth (Thomason et al., 1995). Multiple in vitro studies have examined the effect of cyclosporin on fibroblast growth, as well as collagen and proteoglycan synthesis. Both increased DNA synthesis and proliferation of fibroblasts (Bartold, 1989; Mariotti et al., 1998; Willershausen-Zonnchen et al., 1992) and decreased DNA and reduction of total protein production by fibroblasts (Barber et al., 1992) have been observed when exposed to cyclosporin. Collagen synthesis has been reported to either decrease (Mariotti et al., 1998) or increase (Willershausen-Zonnchen et al., 1992). Proteoglycan synthesis has been observed to either decrease (Barber et al., 1992; Willershausen-Zonnchen et al., 1992), remain unchanged (Bartold, 1989), or increase (Newell and Irwin, 1997). One observation that appears to be consistent is the ability of cyclosporin to 17 retain its ability to induce proliferative activity despite the presence of lipopolysaccharide (Barber et al., 1992; Bartold, 1989). The disparity between the in vitro studies may be due to fibroblast heterogeneity, and the use of only a limited number of fibroblast cell lines. One in vitro study has demonstrated that fibroblasts derived from healthy gingiva showed heterogeneity in basal levels of collagenase activity. When the fibroblasts were exposed to cyclosporin, the production of collagenase was decreased in some strains, and was unchanged in other strains. This variation in collagenase production was coincident with unchanged or increased production of tissue inhibitor of metalloproteinase (TIMP). This study concluded that the heterogeneity of the collagenolytic response of the fibroblast strains may explain why only some individuals exhibit cyclosporin-induced gingival overgrowth (Tipton et al., 1991). The determination of human leukocyte antigen (HLA) phenotype as a risk factor for developing cyclosporin-induced gingival overgrowth has been examined. It was determined that the HLA-B37 allele was statistically significant as a risk factor for developing drug-induced overgrowth (Thomason et al., 1996b), and that the allele HLA-DR1 was significantly higher in patients who did not develop drug-induced overgrowth (Cebeci et al., 1996). These results add further support to the concept of fibroblast heterogeneity. The role of plaque and inflammation in the pathogenesis of cyclosporin-induced gingival overgrowth is unclear (Marshall and Bartold, 1998). Various studies have reported that plaque and inflammation are factors influencing the incidence and severity of the overgrowth (Daley et al., 1986; McGaw et al., 1987; Pernu et al., 1992; Somacarrera et al., 1994; Wysocki et al., 1983). This has been supported by a study which demonstrated that cyclosporin can concentrate locally in plaque (Niimi et al., 1990). Conversely, other studies have reported that plaque and inflammation were not important determinants for gingival overgrowth 18 (Seymour et al., 1987; Seymour and Smith, 1991). Another factor that may play a role in the pathogenesis of cyclosporin-induced gingival overgrowth is the effect of cytokine expression. It has been shown that medication-induced overgrowth tissue contains significantly higher levels of interleukin-6 (IL-6) mRNA when compared to control tissues (Williamson et al., 1994). These findings were supported by the results of another study that showed that fibroblasts from cyclosporin-induced gingival overgrowth tissue produced higher levels of IL-6 compared to controls when cultured with cyclosporin in vitro (Myrillas et al., 1999). It was also demonstrated in the same study that interleukin-lp (IL-ip) was not significantly greater in the overgrown gingiva compared to the normal gingiva. Since IL-ip induces connective tissue degradation by increasing the expression of collagenase and decreasing levels of TIMP (Birkedal-Hansen, 1993), and IL-6 enhances connective tissue accumulation by increasing levels of TIMP (Sato et al., 1990), it may be concluded that the altered expression of cytokines could play a critical role in the pathogenesis of cyclosporin-induced gingival overgrowth (Myrillas et al., 1999). An in vivo study, which determined the levels of IL-ip, TNF-a, and IL-6 in gingival crevicular fluid in patients with cyclosporin-induced gingival overgrowth, reported that the levels of IL-ip were elevated, and the levels of IL-6 and TNF-a were unchanged. It was concluded that the cyclosporin therapy did not increase the levels of IL-1P and IL-6, rather, the presence of gingival inflammation may be responsible for these elevated cytokine levels (Atilla and Ktitukculer, 1998). Prostaglandin I2, which exerts an antiproliferative effect via cAMP-elevation, has been found to be decreased in gingival overgrowth tissue (Nell et al., 1996). In addition, immunolocalization studies have demonstrated increased transforming growth factor-P (TGF-p) in the connective tissue papillae of cyclosporin-induced gingival 19 overgrowth samples (James et al., 1998). The authors isolated the fibroblasts from this tissue and determined that increased matrix production by the fibroblasts occurred in response to TGF-P (James et al, 1998). The expression of both platelet-derived growth factor-P (PDGF-P) and its gene have been found to be increased in cyclosporin-induced gingival overgrowth tissue (Iacopino et al., 1997; Nares et al., 1996; Plemons et al., 1996). An in vivo study determined that the production of PDGF-P was primarily by the macrophages, which followed a non-uniform distribution throughout the gingiva (Nares et al., 1996). The production of cytokine interleukin-1 by fibroblasts did not seem to increase when cultured with cyclosporin (Iacopino et al., 1997). Cyclosporin appears to potentiate tumor necrosis factor-a (TNF-a) induced prostaglandin E2 (PGE2) formation by gingival fibroblasts in a concentration dependent manner, which may play a role in the pathogenesis of gingival overgrowth (Wondimu and Modeer, 1997). It is evident that cytokines may play a critical role in the pathogenesis of cyclosporin-induced gingival overgrowth, but further studies may be required in order to form definitive conclusions. Effect of nifedipine and cyclosporin on the epithelium Very few studies have examined the effect of both nifedipine and cyclosporin on the epithelium. The basal keratinocytes of cyclosporin and phenytoin-induced gingival overgrowth samples exhibited less DNA polymerase a compared to normal samples. The authors felt that the acanthosis could be attributed to augmented keratinocyte life span, rather than increased keratinocyte proliferation (Niimi et al., 1990). Needle-like crystallites (presumed to be cyclosporin) embedded at the base of the acanthotic projections of epithelium in patients taking cyclosporin for up to 20 months, have been described (Pisanty et al., 1990). 20 The authors concluded that cyclosporin affects the gingival epithelium and that the observed overgrowth was due to the cyclosporin-epithelial interaction (Pisanty et al., 1990). The expression of p53 protein and Ki-67 antigen in nifedipine-induced gingival overgrowth samples has been reported. Fifty percent of the nifedipine-induced gingival overgrowth samples expressed p53 protein in the nuclei of the epithelial cells, and the expression of Ki-67 antigen was more than 10% higher than the controls. The expression of Ki-67 was suppressed in the elongated rete pegs, but was upregulated in the fibroblasts of the connective tissue. The authors concluded that the pathogenesis of the overgrowth was partially related to impaired DNA, and the arrested growth in the epithelium may be the result of the Ki-67 antigen, which may prevent the invasive expansion of epithelial cells that are undergoing DNA damage (Saito et al., 1999). The immunolocalization of c-Myc proto-oncogene product, which augments cellular proliferation of growing tissue, and the bcl-2 proto-oncogene product, which inhibits apoptopic responses in various cell types, have been recently described in the literature (Saito et al., 2000). The authors demonstrated that, when compared to the control specimens, the nifedipine-induced gingival overgrowth specimens overexpressed both the c-Myc and the bcl-2 oncoprotein within the epithelium. The authors felt that the expression of both c-Myc and bcl-2 oncoprotein may play a role in the pathogenesis of drug-induced gingival overgrowth (Saito et al., 2000). 1.4 Treatment of nifedipine and cyclosporin-induced gingival overgrowth. The treatment of nifedipine and cyclosporin-induced gingival overgrowth has been described as a combination of good oral hygiene, initial debridement followed by frequent 21 prophylaxis visits, and a gingivectomy procedure (Daley and Wysocki, 1984; Nishikawa et al., 1991; Rateitschak-Pluss et al., 1983; Tyldesley and Rotter, 1984). Ideally, a preventive program should be instituted prior to or at the onset of drug administration (Modeer and Dahlldf, 1987). A gingivectomy procedure may be necessary for patients taking cyclosporin as good oral hygiene alone does not prevent the occurrence of overgrowth (Seymour and Smith, 1991). A recent study, however, demonstrated that a combination of supra- and subgingival scaling and good oral hygiene reduced the number of patients with clinically significant (>30% of the tooth surface) cyclosporin-induced overgrowth by 47% (Kantarci et al., 1999). Patients taking nifedipine may not require surgery if the patient follows a program which includes an extensive oral hygiene regimen, thorough scaling and root planing, and chlorhexidine gluconate 0.12% rinses for one month (Hancock and Swan, 1992). Gingivectomy procedures may be successfully performed using a C O 2 laser, which can reduce bleeding and post-operative pain and discomfort (Barak and Kaplan, 1988). A one-year study which compared a periodontal flap technique to a gingivectomy procedure for the treatment of gingival overgrowth showed that the periodontal flap technique resulted in more shallow probing depths after one year (Pilloni et al., 1998). In all cases, recurrence of some degree of gingival enlargement was evident at one year, which is contrary to other reports (Bennett and Christian, 1985; Lederman et al., 1984). An important factor in preventing recurrence of nifedipine and/or cyclosporine-induced gingival overgrowth after surgery is to ensure that the patient follows a regular maintenance schedule for plaque control. This has been supported by studies which have shown that overgrowth tends to recur after surgery if a regular recall schedule (every three months) has not been followed (Ilgenli et al., 1999; Pernu et al., 1993). 22 Spontaneous regression of nifedipine-induced gingival overgrowth has been reported to occur after the replacement of nifedipine with thiazide (Nishikawa et al., 1991). Replacing cyclosporin with a new immunosuppressive, tacrolimus, has been shown to reduce the amount of overgrowth by approximately 50% (Bader et al., 1998). Tacrolimus is currently prescribed as a drug of second choice, so it is recommended that replacement of cyclosporin with tacrolimus only occur if ordered by the patients' physician (Platz et al., 1995). 1.5 Integrins Integrins are cell surface glycoproteins that mediate cell-to-extracellular matrix and cell-to-cell adhesion events (Hynes, 1987; Hynes 1992; Miyamoto et al, 1998). Integrins are composed of an a and a p subunit which are non-covalently linked together (Watt and Hertle, 1994). Currently, 18 a and 8 P subunits have been identified. These subunits can combine to form 24 different cell surface receptors that possess ligand-binding specificities. Each subunit is composed of three parts: a large extracellular domain, a hydrophobic transmembrane component, and a short cytoplasmic domain (Bartold and Narayanan, 1998; Hynes, 1992). The extracellular domains of integrins appear as a globular head consisting of an a and P subunit (Carrell et al., 1985). The a subunits share between 20% and 70% homology with each other. At the extracellular amino terminus of the a subunit, there are seven repeating homologous domains, and it is the last three or four sequences which contribute to the divalent cation binding property of the subunit. A very short conserved region is present on the a chain. Certain chains may undergo a post-translational proteolytic cleavage at the conserved region, but a disulfide bond maintains segments if cleavage occurs 23 (Hynes, 1992). The extracellular amino terminus of the p subunits are relatively well-conserved, and the overall homology of the different P subunits approaches 40% (Bartold and Narayanan, 1998; Tozer et al., 1996). The cysteine rich area of the p subunit closer to the cell membrane is comprised of four EGF-like domains. The cytoplasmic domain is short (<50 amino acids) in most integrins except P4 ; which possesses a cytoplasmic domain which is over 1000 amino acids (Giancotti et al., 1992; Hynes, 1992). The cytoplasmic domain interacts with cytoskeletal proteins including talin and a-actinin (Horwitz et al., 1986; Otey and Burridge, 1990). The a and P subunits associate through noncovalent interactions which are dependent upon the binding of the extracellular domains and divalent cations. It is the nature of the cations (calcium, magnesium) that can affect the specificity and affinity for the ligands that are required for the ap subunit association (Gailit and Ruoslahti, 1988). Besides the specific cation, other specific areas of the integrin are required for integrin-ligand binding. The highly conserved regions on the extracellular portion of the a and P chain may be involved in the binding process (Tuckwell and Humphries, 1993). A specific sequence of amino acids has been identified in the ligands that bind with specific integrins. This sequence contains a tripeptide sequence of Arg-Gly-Asp (RGD), which is recognized by many integrins (Hynes, 1992). The specificity and affinity of an integrin receptor is not always stable. Integrin up- and down-regulation as well as activation and deactivation has been observed (Hynes, 1992). Signaling through integrin receptors can occur in two different ways. The first, described as inside-out signaling, involves the change of the receptor from the inside of the cell. The second, described as outside-in signaling, involves the activation of certain intracellular events which include tyrosine phosphorylation, cytoplasmic alkalinization through binding of 24 a ligand with the receptor (for review see Yamada, 1997). Integrins can exist in three states of activation: active, partially active, and inactive. The inside-out or outside-in signaling determines the state in which the integrin will function. If an integrin is "active" then it can mediate adhesion more effectively (Yamada et al., 1996). The function of integrins includes an important role in matrix deposition and maturation, as well as a communication link between the extracellular matrix and the cytoskeleton (Schwartz, 1992). Integrin upregulation and downregulation has been demonstrated in wound healing, tumorigenesis, and chronic inflammation of the human periodontium, respectively (Albelda 1993; Haapasalmi et al., 1996). 1.6 Ligands associated with keratinocyte integrins. Integrins facilitate the attachment of keratinocytes to the basement membrane, which delineates the epidermis from the dermis. The integrins and their ligands that have been identified in keratinocytes include: oc2pi (collagens, tenascin), a3pi(laminin-5), a5pl (fibronectin), a9pi (tenascin), ct6p4(laminin-5, collagen XVII), avP5 (vitronectin), avp6 (fibronectin), avpi (fibronectin) (Adams and Watt 1990; Adams and Watt 1991; Carter et al., 1990; Hertle et al., 1991; Koivisto et al., 1999; Larjava et al, 1993; Marchisio et al., 1991; Steppetal., 1990). Fibronectin Fibronectin is a multifunctional extracellular matrix glycoprotein that possesses many binding sites for cellular receptors and extracellular molecules. Fibronectin has been shown 25 to play a role in cell adhesion, cell spreading and migration, and is critical for growth and development, wound repair, and oncogenic transformation (Yamada and Clark, 1996 for review). Fibronectin has been identified in two forms. Plasma fibronectin (pFn) which is synthesized by hepatocytes, exists as a soluble dimeric form in the plasma. Cellular fibronectin (cFn) is produced by many cells including epithelial cells and fibroblasts, and it exists as a dimeric or multimeric form deposited as fibrils in the extracellular matrix of most tissues (Bartold and Narayanan, 1998). Cellular fibronectins, which are found in increased numbers in healing wounds, contain higher proportions of alternately spliced sequences (ED-A, ED-B) compared to plasma fibronectin (Brown et al., 1993a; ffrench-Constant et al, 1989). The fibronectin gene is highly conserved, but as a result of mRNA splicing, many different forms of fibronectin have been identified (Kornblihtt et al., 1996). Fibronectin is comprised of three types of structural modules identified as type I, II, and III. Alternative splicing of mRNA results in the inclusion or deletion of type III molecules identified as ED-A (EIIIA), ED-B (EIIIB) and IIICS. Cell binding to fibronectin may occur at the heparin II region, the alternatively spliced IIICS region, or at the central cell-binding region (Piershbacher and Ruoslahti, 1984; Yamada and Clark, 1996). The centrally located cell-binding region consists of an RGD (Arg-Gly-Asp) site, and a synergy region PHSRN (Pro-His-Ser-Arg-Asn) which provides the RGD sequence with high affinity and specificity for the a5pl integrin (Adams and Watt 1990; Kim et al., 1992; Miyamoto et al., 1998; Singer et al., 1988). The alternatively spliced IIICS region preferentially binds cells including activated lymphocytes and cells which are of embryonic neural crest origin; fibroblasts cannot adhere to this region. Cell adhesion in this area, as well as in the heparin-binding sites, is regulated by the integrin oc4pi (McCarthy et al., 1988, 1990; Yamada and Clark, 1996). 26 Other fibronectin-binding integrins which are expressed by human keratinocytes include av(36 (Adams and Watt 1991; Busk et a l , 1992; Larjava et a l , 1996). The integrin av(36, which is not present in normal epidermis but is upregulated during inflammation, injury or neoplasms, binds fibronectin at the RGD site (Breuss et al., 1995; Busk et al., 1992). Collagens Collagens are a group of proteins that play a crucial role in the structural component of tissues as well as cellular activities, shape and differentiation. Certain features of collagen include the presence of an a chain, within which the repetitive amino acid sequence Gly-X-Y is found. The presence of G ly -X-Y allows for collagen triple-helical formation. The 19 different collagen types can be categorized into three groups according to their ability to form fibrils (for review see Bartold and Narayanan, 1998; Eckes et al., 1996). The first group, also known as the fibril forming collagens, consists of type I, II, III, V , and XI collagens. The second group, which contains collagens type IX, XII, X IV, and XVI , are considered the fibril-associated collagens with interrupted triple helices (FACIT). These collagens are interrupted by noncollagenous sequences, such as glycosaminoglycans, which are covalently linked to the protein. The last group is considered the nonfibrillar group within which are the network forming collagens (type IV, VII, and X), the beaded collagens (type VI), and the anchoring fibrils (type VII). The integrin a2p i is the primary receptor for collagen (Carter et al., 1990a). Other receptors include the integrins ccipi and a3pi (Hynes, 1992). Type IV collagen is the most abundant collagen of the dermoepidermal junction (Eckes et al., 1996). The formation of type IV collagen starts at the C-termini, which then progresses to 27 tetramer formation, and finally results in a mesh-like network of fibrils. This network is a major component of the basement membrane, and it acts as an anchor for other cells and other components of the basement membrane. Laminin-5 Laminin-5 is a member of the laminin family, which is the major noncollagenous protein component of the basement membrane (Beck et al., 1990). Laminin-5, previously known as nicein, kalinin, and epiligrin, is distributed in the anchoring filaments which traverses the basement membrane (Carter et al., 1991; Rousselle et al., 1991; Verrando et al., 1987). The composition of laminin-5 includes three subunits (a3, p3, y2) which form a cross shaped structure. Laminin-5 is synthesized by basal keratinocytes, and it facilitates keratinocyte attachment to the basement membrane. Certain regions of the laminin-5 molecule interact with the lamina densa, whereas other sites interact with the keratinocyte surface (Uitto et al., 1996). The integrins ct3pi and a6p4 (hemidesmosome protein) have been shown to interact with laminin-5 (Carter et al., 1990; Niessen et al., 1994; Peltonen et al.,T989). 1.7 Integrin expression during wound healing, chronic inflammation, and in oral leukoplakia. Integrin expression during wound healing During wound healing, keratinocytes detach from the basement membrane, migrate through the blood clot, over a provisional matrix into the wound bed, and finally regenerate the basement membrane. The provisional matrix consists of fibronectin, tenascin, vitronectin 28 and different collagen types (Gailit and Clark, 1993). The expression of keratinocyte integrins tends to change during wound healing. The expression of the integrin p 1 has been shown to be strongly upregulated during wound healing (Cavani et al., 1993; Gailit et al., 1994; Hertle et al., 1992; Juhasz et al., 1993; Larjava et al., 1993). Experiments utilizing a human mucosal incisional wound model demonstrated that the integrin P1 was present at the wound margin in one-day old wounds. At three days post- wounding, a 1.5 fold increase in the relative amount of the integrin pi was shown at the leading front of the keratinocytes. The distribution of the integrin pi changed with wound healing. The non-wounded areas expressed the p 1 integrin in the intercellular areas of the basal keratinocytes, whereas the keratinocytes in the wounded areas demonstrated strong positive pi-integrin filopodia-like extensions into the wound bed (Larjava et al., 1993). At seven days post-wounding, the distribution of the p 1 integrin started to return to normal, yet the granulation tissue showed continued strong expression of this integrin. The ct-subunits which were associated with the P1 integrins during wound healing included those present in the epithelium of normal mucosa (ct2 and a3), and also the integrin oc5. The integrin a5pi (fibronectin receptor) was only expressed in the keratinocytes of the migrating epithelial sheet and was not seen in the non-wounded area (Larjava et al., 1993). The integrin subunits a6 and p4 were present around the basal surfaces of the keratinocytes in the non-wounded areas. One-day old wounds showed both integrin subunits around the keratinocytes at the leading edge of the wound margin. Three-day old wounds showed the integrin subunit a6 in several cell layers and in the filopodia of the basal keratinocytes of the migrating epithelial sheet. At seven days, the distribution of the integrin cc6p4 was like that of non-wounded mucosa (Larjava et al., 1993). 29 The distribution of the a604 integrin changes from a hemidesmosomal expression, to a more diffuse expression during cell migration (Kurpakus et al., 1991). The expression of the integrin subunit av changes during wound healing. The subunit av is not expressed in non-wounded normal human mucosa, however, the basal aspect of the keratinocytes in the migrating epithelial sheet in the wounded samples expressed the av integrin subunit. The expression of the av subunit was in the same location as the a5pi fibronectin receptor, but it was found not to be colocalized with the P3 integrin subunit. The seven-day wounds also demonstrated the av integrin around the basal keratinocytes that were not contacting the newly regenerated basement membrane (Larjava et al., 1993). The expression of the integrin avP6 integrin has been shown in three - and seven-day old human mucosal wounds. The expression of avp6 was present in the basal cell layer of the wound area, but it was not evident in the non-wounded area of the tissue (Haapasalmi et al., 1996). Conversely, the expression of avp5 integrin was not shown in any of the wounded samples. The expression of the integrin avp6 was maximal in the seven-day old wound, and this was in conjunction with maximal expression of the possible ligand, tenascin, within the granulation tissue (Haapasalmi et al., 1996; Prieto et al., 1993). The other possible avp6 ligand, fibronectin, was also found throughout the normal connective tissue and in the wound granulation tissue matrix (Haapasalmi et al., 1996). The early stages of wound healing seem to coincide with the expression of the a5pl fibronectin receptor, which may mediate binding of the keratinocytes to serum fibronectin. During the later stages, however, the integrin avp6 may serve as the fibronectin, and/or tenascin receptor. The expression of the integrin a6p4 as well as a3pi during wound healing 30 may mediate the interaction between keratinocytes and laminin-5. Finally, the integrin a2p i , also present during wound healing, may mediate keratinocyte adhesion to collagen and tenascin (Larjava et al., 1996). Integrin expression during chronic inflammation of the human periodontium Chronically inflamed human periodontal tissue is remarkable for the apical extension of the junctional epithelium. Epithelial cells are exposed to new matrix components of this chronically inflamed tissue. As a consequence, this type of tissue demonstrates some changes in integrin expression (Haapasalmi et al., 1995). The integrin p i demonstrated both focal losses and strong expression throughout the thickness of the epithelium in chronically inflamed tissue. The integrins a2pi and a3p l demonstrated variable expression including focal losses and areas of upregulation throughout the samples of chronically inflamed tissue. The pattern of distribution of these integrins changed from localization around the basal cells to a circumscribed pattern around the cells in all cell layers in areas of inflammation. The integrin a6p4 showed areas of focal losses in inflamed areas, whereas the non-inflamed areas had a continuous pattern of ot6p4 around the basal keratinocytes. The strong expression of the integrin a6p4 and p i was found in conjunction with streak-like patterns of type IV and VII collagen and laminin-5. The av integrin family (avp5 and avP6) was not seen in the healthy or inflamed epithelium samples (Haapasalmi et al., 1995). Integrin expression in oral leukoplakia, lichen planus, and inflammatory hyperplasia. The integrin expression in oral leukoplakia, lichen planus and inflammatory hyperplasia has been investigated and characterized (Hamidi et al., 2000). The integrins p i and P4 31 showed similar expression in the samples of leukoplakia as that found in normal mucosa. The integrin ocvp6 was expressed in 41% of leukoplakia samples, 27% of dysplasia samples, and 86%o of hyperkeratotic, hyperplastic, and atypic tissue. Expression of av|36 was limited to the basal keratinocytes at the tips of the rete ridges. The integrin subunits (33 and P 5 were not seen in the leukoplakia samples. The distribution of fibronectin and tenascin (ocvp6 ligands) was similar in the leukoplakia samples as that found in normal mucosa (Hamidi et al., 2000). The samples of lichen planus demonstrated focal losses of the integrins pi and P4, however, when these integrins were present, they were localized around the basal keratinocytes (Haapalainen et al., 1995; Hamidi et al., 2000). The integrin avp6 was found in both the basal and suprabasal layers in 85%) of the lichen planus samples. Similar to the oral leukoplakia samples, the lichen planus samples did not express the integrins P3 and P5 (Hamidi et al., 2000). The squamous cell carcinoma samples were remarkable for suprabasal expression of the integrins pi and p4. The integrin avp6 was present in 80%) of the samples (Hamidi et al.,2000). It was postulated that the absence of the integrin avp6 could be a marker for nonprogressive leukoplakia lesions (ie. those without malignant change). However, due to the small sample size used in the study, this is not a definitive test for a nonprogressive lesion. The high rate of expression of the integrin avp6 may be associated with a combination of factors including the selection of a certain phenotype of lymphocytes, the presence of inflammation, and the potential for malignant transformation (Hamidi et al., 2000). 32 1.8 Transforming growth factor-beta (TGF-B) and integrin expression. TGF-P is a homodimeric peptide which is produced by cell types including activated macrophages, fibroblasts, lymphocytes, epithelial and endothelial cells, and platelets (for review see Roberts and Sporn, 1996; Slavin, 1996). The predominant form of this growth factor is TGF-p 1. The primary effect of TGF-p is in the reparation of injured tissue (wound healing). Specific effects exerted upon one cell type depends upon the specific cell type beinj examined. For example, TGF-P stimulates fibroblast division at low concentrations, but at high concentrations it results in the differentiation of a fibroblast phenotype associated with increased collagen and matrix production (Ignotz and Massague, 1986). TGF-P is also involved with chemotaxis, proliferation, angiogenesis, matrix synthesis, and decreased synthesis of collagenase and increased production of the tissue inhibitor of metalloproteinases-1 (Bartold and Narayanan, 1998; Edwards et al., 1987). Conversely, an in vitro study utilizing cultured human keratinocytes demonstrated that TGF-p increased the expression of type IV collagenase and mRNA levels of type IV collagenase (Salo et al., 1991) TGF-p has been shown, in vitro, to upregulate the expression of the fibronectin receptor integrin a5pi, the vitronectin receptor ctvp5, and the collagen receptor a2pl (Zambruno et al., 1995). In addition, the downregulation of ct3pi and the de novo expression of ccvp6 was seen. The results of this study concluded that TGF-p reproduced the integrin expression pattern seen on migrating keratinocytes during wound healing (Zambruno et al., 1995). TGF-P has also been shown to stimulate fibronectin synthesis in keratinocytes, and regulate the different forms of fibronectin which include fibronectin ED-A (Nickoloff et al., 1988; Balza 33 et al., 1988). It has also been demonstrated that TGF-pl is a ligand for the integrin av|36. This integrin can bind TGF-P through its latency activated peptide that contains an Arg-Gly-Asp (RGD) peptide. Activation of this growth factor by the integrin avP6 produced lung and skin inflammation and pulmonary fibrosis in mice (Munger et al., 1998; Munger et al., 1999). A three-fold increase in the expression of the integrin avp6 has been shown in HaCaT keratinocytes exposed to TGF-pi (Koivisto et al., 1999). In similar conditions, there was up regulation of a2pi, cc3pi, cc5pi, and avp5 integrins. It was concluded that for HaCaT keratinocytes, avP6 was the major fibronectin-binding integrin which was upregulated by TGF-P 1, and that a v p i and oc5pi collaborated in cell adhesion and migration on fibronectin (Koivisto et al., 1999). In the same study, it was shown that pretreatment with TGF-P 1 accelerated HaCaT cell spreading and haptotaxis on a fibronectin-coated substratum. Antibodies against oc5pl integrins did not have any effect on the maximal spreading of the cells on fibronectin, and the increased expression of avp6 by itself was sufficient to maintain maximum spreading (Koivisto et al., 1999). 34 Chapter Two - Aim of the study. Integrins function in regulating cell growth, differentiation, migration, and gene expression. Epithelial integrin avB6 may be involved in controlling extracellular matrix deposition through its capacity to activate TGF-P. Drug-induced gingival overgrowth demonstrates alterations in the epithelium (acanthosis, long rete ridges) as well as in the accumulation of extracellular matrix. The aim of the study is to characterize epithelial cell integrin phenotype in drug-induced gingival overgrowth and compare it to that expressed by the normal gingival tissue. The association between number of inflammatory cells and integrin expression was also examined. We hypothesize: -Epithelial integrin phenotype is altered in drug-induced gingival overgrowth to one consistent with wound healing and therefore could participate in regulating the formation of the connective tissue matrix. 35 Chapter Three - Materials and Methods 3.1 Gingival samples Human gingival tissue biopsies were collected from 19 control patients (no medication), 12 nifedipine-treated cardiac outpatients, 22 immunosuppression-treated organ transplant patients (11 patients taking cyclosporin and 11 patients taking both cyclosporin and nifedipine). The cardiac outpatients were referred by their physician or dentist for periodontal surgery due to nifedipine-induced gingival overgrowth. The immunosuppression-treated organ transplant patients were treated as per the treatment protocol followed by organ transplant patients in the Oulu University Hospital, Finland. The immunosuppressive treatment of the transplant patients consisted of triple medication including cyclosporin, methylprednisolone and azathioprine in 14 patient, and double medication (cyclosporin and methylprednisolone or cyclosporin and azathioprine) in five cases. Al l samples represented moderate to severe gingival overgrowth which extended between the middle 1/3 and coronal 2/3 of the clinical crown (Pernu et al., 1992). The mean whole blood cyclosporin concentration was determined according to values measured during the six months before gingival biopsy. The blood tests were standardized as morning values prior to CsA medication. Patients gave their informed consent to participation in this investigation. Patients were placed into four groups according to medication: 1) Immunosuppression, 2) Immunosuppression and Nifedipine, 3) Nifedipine and 4) Control groups. Patient characteristics are illustrated in Table 1. Gingival biopsies were taken during the required gingivectomy procedures. The biopsies 36 were rinsed in physiological saline, embedded in Tissuetek (Miles Inc., Elkhart, IN), and immediately frozen in liquid nitrogen. The tissue samples were stored at -70°C until the frozen sections were prepared. Serial cryostat sections (5um) were prepared at -25°C from each specimen in order to obtain cross sections of the oral and sulcular epithelium and connective tissue. The sections were collected on 3-aminopropyltriethoxysilane (Fluka Chemie, Buchs, Switzerland) -coated glass slides and stored at -70°C until they were used. One section of each specimen was stained with hematoxylin and eosin to allow for morphological analysis of the tissue. The hematoxylin and eosin stained sections were evaluated using a Zeiss Axiolab E microscope. The frozen sections were used for immunolocalization of integrins and their putative ligands. 37 Table 1. Patient characteristics. Values are means ± s.e.mean, except in relation to gender. Immunosuppression Immunosuppression Nifedipine and Nifedipine Control N= 11 N= 11 N= 12 N= 19 Gender (men/women) 7/4 9/2 8/4 9/10 Age (years) 44 ± 2 36 ± 3 42 ± 4 47 + 2 Duration of medication (months) - nifedipine - immunosuppression 29.2 ± 7.5 29.8 ± 4.9a 29.2 ± 4.5 18.4 ±3.7 -Daily oral dose of medication (mg) - nifedipine - cyclosporin-A - methylprednisolone - azathioprine 158 ± 2 4 4.7 ± 1.4 42.1 ± 10.1 40.0 ±5.2 2 2 5 ± 1 8 b 5.0 ± 1.3 52.3 ±8.6 54.2 ±4.3 -Mean whole blood concentration of cyclosporine-A (Hg/ml) 132 ± 12 127 ± 16 - -Mann-Whitney U-test: a p = 0.079 b p = 0.012 Statistical analysis Before statistical analysis, normalities of the data distributions were tested with the Shapiro -Wilks procedure. Significances of differences between the groups were determined with the Kruskall-Wallis test, and significances of differences between pairs of results with the Mann-Whitney U-test. The calculations were made using the SPSS® for Windows statistical package. 38 3.2 Immunofluorescence stainings Immunofluorescence stainings were performed as previously described (Larjava et al., 1993). Sections were incubated with phosphate buffered saline (PBS) containing lmg/ml bovine serum albumin (BSA) for 60 minutes at room temperature. Primary antibodies (see table 2) diluted in the PBS/BSA solution was added to the sections and incubated at 4°C for a minimum of 12 hours. The sections were then washed twice for five minutes with PBS/BSA, and then incubated with affinity-purified rhodamine-conjugated secondary antibodies (dilution 1:50, anti-mouse and anti-rabbit, both supplied from Boehringer-Mannheim Biochemicals, Indianapolis, IN., Volsen, 1984; or Alexa 546 goat anti-rabbit, Yeh et al., 1987) for 60 minutes at room temperature. Sections were then washed twice for five minutes with PBS/BSA, rinsed with distilled water, air dried, and mounted using cyanoacrylate adhesive (Borden Co., Willowdale, Ontario, Canada). The sections were then examined using a Zeiss Axioskop 20 fluorescence microscope, and photographed using an MC 80 Zeiss microscope camera. Control stainings for nonspecific binding were performed with the use of nonimmune rabbit serum (Chemicon) and without the use of primary antibodies. All control specimens stained were negative. 39 Table 2. Antibodies used against the integrin subunits and their ligands. Integrin/ligand Antibody Reference cc5 mAb 1986 te Veldeetal., 1988 av mAb L230 Houghton etal., 1982 (31 mAb 3847 Roberts et al., 1988 (31 HUTS-4 Luqueetal., 1996 P4 mAb AA3 Tamura et al., 1990 P6 mAb E7P6 Weinacker et al., 1994 Fibronectin EDA Chen etal., 1985. Type IV collagen mAb PS057 Visser etal., 1986 Laminin-5 mAb GB3 Verrando et al., 1987 3.3 Morphological analysis The hematoxylin and eosin sections were evaluated using a Zeiss Axiolab E microscope to determine the degree of acanthosis, thickness of the epithelium, and inflammation. The grid used was 100=lmm (10pm), and the magnification was 10 X /0.25 with a 10 X lens piece. The criterion for inflammation was based upon the degree of inflammatory infiltrate (see below, table 3, figure 1). The criterion for acanthosis was based upon the definition that acanthosis is the abnormal thickening of the spinous layer for a particular location (see below, table 4, figure 2). Severe acanthosis was defined as elongation, thickened, blunted, and confluence of the rete pegs (Shafer et al., 1983). Table 3. Degree of inflammation Score Degree of inflammatory infiltrate within the connective tissue 0 Minimal infiltration (Figure 1, panel A) 1 Mi ld infiltration (Figure 1, panel B) 2 Moderate infiltration (Figure 1, panel C) 3 Severe infiltration (Figure 1, panel D) Table 4. Degree of acanthosis Score Degree of acanthosis Mi ld mild elongation of the rete pegs (Figure 2, panel A) Moderate moderate elongation of the rete pegs (Figure 2, panel B) Severe severe elongation and thickening of the rete pegs (Figure 2, panel C) 41 Figure 1. Hematoxylin and eosin staining of tissue samples demonstrating degree of inflammatory infiltrate. Panel A-minimal infiltration of inflammatory cells, panel B-mild infiltration of inflammatory cells, panel C-moderate infiltration of inflammatory cells, panel D-severe infiltration of inflammatory cells. Bar = 1 mm. E = epithelium, C T = connective tissue 42 Figure 2. Hematoxylin and eosin stainings of tissue samples demonstrating the degree of acanthosis. Panel A - m i l d acanthosis, panel B-moderate acanthosis, panel C-severe acanthosis. Bar = 1mm. E = epithelium, CT = connective tissue. 43 Chapter Four - Results 4.1 Analysis of patient information. The four patient groups were similar with respect to age; the three medication groups were similar with respect to gender distribution. Eleven of the transplant recipients were taking nifedipine. The duration that nifedipine medication was taken was longer in the immunosuppression and nifedipine group than in nifedipine group (p=0.079), but the mean oral dose of nifedipine was comparable in both groups. Among transplant recipients no intergroup differences were found with respect to the duration of CsA medication, the mean whole blood CsA-concentration, the mean oral doses of methylprednisolone and azathioprine medication. The mean oral dose of CsA was significantly higher in immunosuppression and nifedipine group as compared with immunosuppression group (p=0.012) (Table 1). 4.2 Histological analysis of the gingival samples. Rete ridge length and epithelial thickness on average was shorter in the control specimens than the drug-induced specimens. The degree of acanthosis ranged from mild-moderate for the control specimens, whereas the drug-induced specimens had a degree of acanthosis ranging from moderate to severe (table 5). Table 5. Rete ridge length, epithelial thickness and acanthosis of the samples. Type of tissue Average length of rete ridge um Average epithelial thickness um Degree of acanthosis Control 26 ±11.53 21 ± 14.65 Mild-moderate Cyclosporin 49 ±20.71* 29 ± 12.21 Moderate -severe Nifedipine 40 ± 10.22* 32 ± 18.64* Moderate-severe Cyclosporin-Nifedipine combination 40.5 ±14.1* 28 ± 14.21 Moderate-severe *p<0.05, significant at a level of 95% confidence interval. 45 4.3 Correlation of inflammation adjacent to the oral and sulcular epithelium in the different tissue types. There is no correlation between the inflammation adjacent to the oral and sulcular epithelium and the different tissue types. (Figures 3 and 4) Figure 3 c M - O O '43 (0 D) E 0) re Inflammation adjacent to oral epithelium in different tissue types 3 2.5 2 1.5 1 0.5 0 CsA Nif Combined Control Tissue types Figure 4 c <+- o o *= o S £ | 0) re Inflammation adjacent to sulcular epithelium in different tissue types 3.5 3 2.5 2 1.5 1 0.5 0 CsA Nif Combined Control Tissue types 46 4.4 Frequency and expression of the integrins in each sample. The integrins were documented as being positive or negative in specific areas of the tissue. This included a localized or generalized distribution in the basal keratinocytes of the sulcular or oral epithelium (see tables 6-11). 4.5 Expression of EDA fibronectin (EDA-FN). EDA fibronectin was expressed, with a low frequency (17-36%), around the basal keratinocytes of the oral and sulcular epithelium in all the groups except the combined drug group (table 12). Two specimens had high suprabasal expression of EDA-FN around the suprabasal keratinocytes of the oral epithelium in the CsA group. The connective tissue distribution of EDA-FN was similar in all groups (>73%). Figure 11 illustrates the expression of EDA-FN. 4.6 Expression of the integrins a531, ctv36, ccvBl and fibronectin. a5(31 and fibronectin. The a5(31 integrin was expressed with low frequency (9-15%) in the control tissue and those obtained from patients with single drug regimens. Expression was around the basal keratinocytes in localized areas of the oral epithelium of all groups (see figures 5, 12, 13, 14, and 16). The combined drug group, however, displayed a higher frequency of expression of this integrin in the sulcular and oral epithelium (see tables 6, 8, 9 and 25). The sulcular epithelium of two control samples (15%) expressed the a5(31 integrin. Of these two samples, only one expressed EDA-FN around the basal keratinocytes of the sulcular epithelium. The oral epithelium of the control samples expressed the integrin 47 a5pi in four samples (two samples with generalized expression and two samples with localized expression in the oral epithelium). Of these samples, only one expressed EDA-FN in the same area. The remaining samples did not express EDA-FN around the basal keratinocytes of the oral epithelium, rather, EDA-FN was within the connective tissue (see tables 15, 16, 25, and figure 12). The sulcular epithelium of one of the nifedipine samples demonstrated the presence of the oc5pi integrin with no colocalization with EDA-FN. In the oral epithelium, two samples showed the ct5pi integrin. Of these two samples, only one showed colocalization with EDA-FN around the basal keratinocytes (see tables 17, 18, 25, and figure 13). The cyclosporin samples showed the a5pi integrin around the basal keratinocytes of the sulcular epithelium in only one sample, with colocalization with EDA-FN around the basal keratinocytes. In the oral epithelium, two samples expressed both the oc5pi integrin and EDA-FN around the basal keratinocytes of the oral epithelium (see tables 19, 20, 25, and figure 14). Interestingly, all the combined drug-induced samples, except one sample, demonstrated the ct5pi integrin around the keratinocytes of the sulcular and the oral epithelium. The expression of EDA-FN in this group was seen exclusively in the connective tissue (see tables 21, 22,25, and figure 16). avP6 and fibronectin The ocvp6 integrin was expressed with high frequency in all specimens either locally or generally (table 11, table 26, figure 9). The combined drug-induced group 48 demonstrated higher frequency of the generalized type of expression. In addition, the avP6 integrin was frequently present in the suprabasal layers of the epithelium of this group (table 11). EDA-FN colocalized with the avP6 integrin around the basal cells of either the sulcular or oral epithelium in 23-45% of the cases (figures 12,13, 14). Interestingly, EDA-FN was never present in the combined drug-induced group and therefore no colocalization of these two molecules was seen (table 26, figure 15). In these specimens, EDA-FN was present in the connective tissue immediately adjacent to the epithelium (figure 15). avp 1 and fibronectin. The av integrin can combine with the either the P6, P5 or pi subunit in keratinocytes (Hakkinen et al., 2000). No clear staining was obtained in any of the samples using an antibody against P5 integrin subunit (not shown). Distribution of the av integrin was often much more extensive than that of the avP6 integrin, suggesting that the av integrin could pair with another p-subunit. The only possible pairing P-subunit which was localized to the same area was the pi-subunit. Generalized expression of the av-integrin was seen in most samples. There was no difference in the frequency of expression of the av-integrin between the groups. However, av-integrin was more frequently present in the suprabasal layers of the epithelium of the combined drug-induced group (table 7, figure 6). EDA-FN colocalized with av-integrin in 25-36%) of the specimens (table 24). 49 4.7 Identification of the components of the basement membrane: laminin-5 and type IV collagen, and the integrin ct6fi4. The basement membrane was identified using antibodies recognizing laminin-5 and type IV collagen (tables 13 and 14, figure 10). Type IV collagen localized as a continuous line under the epithelium in all specimens studied (table 14). Laminin-5, however, showed discontinuations in a majority of specimens representing the sulcular epithelium. Localized breaks of laminin-5 were also present in the basement membrane zone of the oral epithelium (table 13). The main receptor for laminin-5 is the integrin a6p4 which was shown to be expressed in the basal portion of the basal keratinocytes of the oral and sulcular epithelium in all tissue samples. The combined-drug induced tissue samples showed more frequent suprabasal staining of the oc6p4 integrin of the oral epithelial keratinocytes (see figure 8 and table 10). 5 0 Table 6. Frequency of expression of oc5 integrin Tissue Loc. SE Loc. OE Gen. Supra- Supra- Gen. SE+OE basal SE basal OE Supra-basal Control 0/13 2/13 2/13 0 0 0 Nif 0/10 1/10 1/10 0 0 0 CsA 0/11 1/11 1/11 0 0 0 Nif+CsA 5/11* 5/11 5/11 1/11 1/11 3/11* *p<0.05, significant at a level of 95% confidence interval. Table 7. Frequency of expression of av integrin. Tissue Loc. SE Loc. OE Gen. Suprabasal Suprabasal Gen. SE + OE SE OE Suprabasal Control 2/19 1/19 14/19 0 1/19 1/19 Nif 1/12 1/12 10/12 1/12 1/12 1/12 CsA 1/11 1/11 9/11 0 4/11* 0 Nif+CsA 3/11 2/11 8/11 7/11* 3/11* 2/11 *p<0.05, significant at a level of 95% confidence interval. Table 8. Frequency of expression of pi integrin Tissue Loc. SE Loc. OE Gen. Supra- Supra- Gen. SE+OE basal SE basal OE Supra-basal Control 1/19 3/19 16/19 0 0 0 Nif 0 1/12 10/12 0 2/12* 0 CsA 0 1/11 10/11 0 1/11 0 Nif+CsA 0 0 11/11 0 6/11* 3/11* *p<0.05, significant at a level of 95% confidence interval. Table 9. Frequency of expression of pi active integrin Tissue Loc. SE Loc. OE Gen. Supra- Supra- Gen. SE+OE basal SE basal OE Supra-basal Control 3/14 6/14 8/14 0 2/14 1/14 Nif 1/12 4/12 5/12 0 1/12 0 CsA 2/10 7/10 2/10 0 0 0 Nif 0 0 11/11* 0 6/11* 0 +CsA *p<0.05, significant at a level of 95% confidence interval. 51 Table 10. Frequency of expression of P4 integrin Tissue Loc. SE Loc. OE Gen. Supra- Supra- Gen. SE+OE basal SE basal OE Supra-basal Control 0 0 19/19* 2/19 2/19 1/19 Nif 1/12 3/12* 8/12 2/12 3/12 2/12 CsAHO 0 3/10* 7/10 0 3/10 1/10 stained Nif+CsA 0 0 11/11 2/11 6/11* 1/11 *p<0.05, significant at a level of 95% confidence interval. Table 11. Frequency of expression of avp6 integrin Tissue Loc. SE Loc. OE Gen. SE+OE Supra-basal Supra-basal Gen. Supra-SE OE basal Control *14 11/14 8/14 3/14 0 0 1/14 slides stained Nif 3/12 6/12 6/12 0 1/12 0 CsA 3/10 5/10 4/10 0 0 0 Nif+CsA 4/11 2/11 • 7/11* 9/11* 4/11* 0 *p<0.05, significant at a level of 95% confidence interval. • p<0.05, significant at a level of 95% confidence interval. Table 12. Frequency of expression of EDA-FN Tissue Loc. SE Loc. OE Gen. Supra- Supra- Gen. C. T. SE+OE basal SE basal OE Supra-basal Control 0 2/15 4/15 0 0 1/15 13/15 Nif 2/12 3/12 2/12 0 0 0 10/12 CsA 0 1/11 4/11 0 2/11* 0 8/11 Nif+CsA 0 0 0/11* 0 0 0 11/11 *p<0.05, significant at a level of 95% confidence interval. 52 Table 13. Frequency of expression of Laminin-5 ligand Tissue Loc. Breaks in SE Loc. Breaks in OE Control 7/11 2/11 Nif 7/8 3/8 CsA 4/9 4/9 Nif 6/11 3/11 +CsA *p<0.05, significant at a level of 95% confidence interval. Table 14. Frequency of expression of Type IV Collagen ligand Tissue Loc. Breaks in Loc. Breaks in OE SE Control 0/2 0/2 Nif 0/0 0/0 CsA 0/0 0/0 Nif 0/11 0/11 +CsA *p<0.05, significant at a level of 95% confidence interval. Localized SE-localized expression of integrin around basal keratinocytes of the sulcular epithelium. Localized OE-localized expression of integrin around basal keratinocytes of the oral epithelium. Generalized SE + OE -generalized expression of integrin around basal keratinocytes of the oral and sulcular epithelium Suprabasal SE-expression of integrin around the suprabasal keratinocytes of the sulcular epithelium. Suprabasal OE-expression of integrin around the suprabasal keratinocytes of the oral epithelium. Generalized suprabasal -expression of integrin around the suprabasal keratinocytes of the oral and sulcular epithelium 53 Table 15- Integrin expression in the control samples -sulcular epithelium (SE) Slide oc5 av PI PI active P4 P6 Fn 82 - + + + + + -103 + + + + + + + 97 - + + + + + -64 - + + - + + -161 Ns + + Ns + Ns + 170 Ns + + + + + -163 Ns Ns + Ns + Ns Ns 164 - + + + + + + 216 Ns + + Ns + Ns Ns 61 Ns Ns + Ns + Ns Ns 54 - + - - + + -59 + + + + + + -105 - + + - + + -217 - + + + + - -218 - + + + + + + 162 - + + + + + -99 - + + + + + -55 Ns - - Ns + Ns Ns 220 - + + + + + -Table 16 - integrin expression in the control samples- oral epithelium (OE) Slide a5 av PI PI active P4 P6 Fn 82 - + + + + + + 103 + + + + + + + 97 - + + + + + -64 - + + + + + -161 Ns - + Ns + Ns + 170 Ns + + + + + -163 Ns Ns + Ns Ns Ns Ns 164 - + + + + + + 216 Ns + + Ns + Ns Ns 61 Ns Ns + Ns + Ns Ns 54 + + + + + - -59 + + + + + -105 - + + + + + + 217 - + + + + - -218 - + + + + + + 162 + + + + + - -99 - + + + + + -55 Ns - + Ns + Ns Ns 220 - + + + + + -+ = positive stain, - = negative stain, Ns=not stained Table 17 - integrin expression in the nifedipine-overgrowth samples - sulcular epithelium (SE) Slide oc5 av PI PI active P4 P6 Fn 84 - + + - + + + 140 - + + + + + -197 Ns - + + + + + 138 Ns + - - - + -171 - + + + + + + 176 + + + + + + -150 - + + + + - -137 - + + + + + -44 - + + + + + -36 - + + Ns + - + 74 - + + - + + -215 - + + + + - -Table 18 - integrin expression in the nifedipine-overgrowth samples - oral epithelium (OE) Slide a5 av PI PI active P4 P6 Fn 84 - + + + + + + 140 - - + + + + -197 Ns + + + + + + 138 Ns + + + + + + 171 - + + + + + -176 + + + + + + -150 - + • + + + + -137 - + + + + + -44 - + + - + + -36 + + Ns + - + 74 - + + - - + -215 - + + + + + -+ = positive stain, - = negative stain, Ns=not stained 55 Table 19- integrin expression in the cyclosporin-overgrowth samples - sulcular epithelium (SE) Slide oc5 av PI PI active P4 P6 Fn 68 + + + + + - + 90 + - - + + + 141 + + - - + -145 + + Ns + -187 + + Ns" - Ns -167 + + - + + + 179 + + - + + -73 + - + + - -78 + + + + + 113 - + + - + -116 + + - + - -Table 20 - integrin expression in the cyclosporin-overgrowth samples - oral epithelium (OE) Slide a5 av PI PI active P4 P6 Fn 68 + + + + + + + 90 - + + + + + + 141 + + + - + + + 145 - + + + Ns + -187 - + + Ns + Ns -167 - + + + + + + 179 - + + + + + -73 - + + + + - -78 - + + + + + -113 - + + + + + -116 - + + + + + -+ = positive stain, - = negative stain, Ns=not stained 56 Table 21 - integrin expression in the combined nifedipine and cyclosporin-overgrowth samples- sulcular epithelium (SE) Slide ct5 av P I P I active P4 P6 Fn 147 - + + + + + -190 + + + + + + -192 + + + + + + -191 + + + + + + -199 + + + + + + -158 + + + + + + -157 + + + + + + -146 + + + + + + -32 + + + + + -110 + + + + + + -160 + + + + + + -Table 22 - integrin expression in the combined nifedipine and cyclosporin-overgrowth samples - oral epithelium (OE) Slide a5 av P I P I active P4 P6 Fn 147 - - + + + -190 + + + + + -192 + + + + + + 191 + + + + + + 199 + + + + + + 158 + + + + + + 157 + + + + + + 146 + + + + + + 32 + + + + + + 110 + + + + + + 160 + + + + + + + = positive stain, - = negative stain, Ns=not stained Table 23. Comparison of frequency of integrin expression in different tissue types. Integrin Control Nifedipine Cyclosporin CsA + Nif ct5 = = = +SE(sb) +OE (sb) av =SE +OE (sb) +SE(sb) +OE(sb) PI = =SE +OE (sb) +SE(sb) +OE(sb) pi active = = +SE(b) +OE(b/sb) P4 +SE(b) =SE =SE +OE (sb) +OE(b) +OE (b) +OE(b/sb) P6 = = +SE(sb) +OE (b/sb) = similar frequency compared to other groups b - basal keratinocytes - reduced frequency compared to other groups sb - suprabasal keratinocytes + increased frequency compared to other groups Table 24. Frequency of putative avpi integrin and fibronectin (EDA-FN) in each tissue group. The frequency of EDA-FN is from the specimens positive for the putative av(31 integrin. Tissue Samples avpi Fibronectin SE OE SE OE CT E CT E Control 15/17 15/17 10/15 4/15 9/14 5/14 88% 88% 67% 27% 64% 36% Nifedipine 10/12 11/12 7/10 3/10 7/11 4/11 83% 92% 70% 30% 64% 36% Cyclosporin 8/11 11/11 6/8 2/8 7/11 4/11 73% 100% 75% 25% 64% 36% Nif+CsA 11/11 10/11 11/11 0/11 10/10 0/10 100% 91% 100% * • 100% * • SE-sulcular epithelium, OE-oral epithelium, CT-connective tissue, E-epithelium *p<0.05, significant at a level of 95% confidence interval. •p<0.05, significant at a level of 95% confidence interval. 58 Table 25. Frequency of cc5pi integrin and fibronectin (EDA-FN) in the tissue samples. The frequency of EDA-FN is from the specimens which are positive for the cdpl integrin. Tissue Samples ct5pl Fibronectin SE O E SE O E C T E C T E Control 2/13 4/13 1 12 1 12 3/4 1/4 15% 31% 50% 50% 75% 25% Nifedipine 1/10 2/10 1/1 0/1 1/2 1/2 10% 20% 100% 0% 50% 50% Cyclosporin 1/11 2/11 0 1/1 0 2/2 9% 18% 0% 100 0% 100 % %* N i f + C s A 10/11 10/11 10/10 0 10/10 0 91% * 91%* 100% 100% SE-sulcular epithelium, OE-oral epithelium, CT-connective tissue, E-epithelium *p<0.05, significant at a level of 95% confidence interval. •p<0.05, significant at a level of 95% confidence interval. Table 26. Frequency of avP6 integrin and fibronectin (EDA-FN) in the tissue samples. The frequency of EDA-FN is from the specimens positive for the avP6 integrin. Tissue samples rxv 36 Fibronectin SE O E SE O E C T E C T E Control 13/14, 93% 11/14, 79% 10/13 3/13 6/11 5/11 77% 23% 55% 45% Nifedipine 8/12, 58% • 10/12, 83% 5/7 2/7 7/10 3/10 71% 29% 70% 30% Cyclosporin 6/10, 60% • 9/10, 90% 4/6 2/6, 5/9 4/9 67% 33% 56% 44% N i f + C s A 11/11, 100% 9/11,82% 11/11 0 • 11/11 0 • 100%* 100% * SE-sulcular epithelium, OE-oral epithelium, CT-connective tissue, E-epithelium *p<0.05, significant at a level of 95% confidence interval. • p<0.05, significant at a level of 95% confidence interval. 59 Figure 5. Immunolocalization of a5 integrin around the basal keratinocytes of the oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 urn. Arrowhead shows a5 integrin around the basal keratinocytes. Arrow with tail indicates suprabasal expression of a5 integrin. E = epithelium, CT = connective tissue. 60 Figure 6. Immune-localization of av integrin around the basal keratinocytes of the oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar - 200 pm. Arrow with tail indicates suprabasal expression of av integrin. E = epithelium, CT = connective tissue. 61 Figure 7. Immunolocalization of P1 integrin around the basal keratinocytes of the oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 um. E = epithelium, CT = connective tissue. 62 Figure 8. Immunolocalization of p4 integrin, oral epithelium. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 jam. Arrow with tail indicates suprabasal expression of p4 integrin. E = epithelium, CT = connective tissue. 63 Figure 9. Immune-localization of p6 integrin. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 50 pm. Arrowheads show P6 integrin around the basal keratinocytes of the oral epithelium. E = epithelium, CT = connective tissue. 64 mi mm M 1 Figure 10. Immunolocalization of laminin-5 in the oral epithelium of the different tissue groups. Panel A-control tissue, panel B-nifedipine-induced overgrowth tissue, panel C-cyclosporin-induced overgrowth tissue, panel D-combined drug-induced overgrowth tissue. Bar = 200 urn. E = epithelium, CT = connective tissue. 65 Figure 11. Immune-localization of EDA-FN. Expression of EDA-FN was demonstrated around the basal keratinocytes of the oral epithelium of some samples, and only within the connective tissue of other samples. Panel A and B-control tissue, panel C and D-nifedipine-induced overgrowth tissue, panel E and F-cyclosporin-induced overgrowth tissue, panel G-combined drug-induced overgrowth tissue. Bar = 200 pm. Arrow with tail indicates suprabasal expression of EDA-FN. E = epithelium, CT = connective tissue. 66 %0> Figure 12. Immunolocalization of av, BI-active, B6 integrin, and EDA-FN within the oral epithelium of the control tissue. Panel A-av integrin, panel B-pi-active integrin, panel C-EDA-FN, panel D-negative stain for 06 integrin. Panels A, B and C demonstrate colocalization of the integrin avp 1 with EDA-FN within parallel sections of the same specimen. Panels E (a5 integrin), F (p 1 integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pl with EDA-FN within parallel sections of the same specimen. Bar = 200 pm. E = epithelium, CT = connective tissue. 67 Figure 13. Immunolocalization of av, (31-active, p6 integrin, and EDA-FN within the oral epithelium of nifedipine-induced overgrowth tissue. Panel A-av integrin, panel B-pi-active integrin, panel C-EDA-FN, panel D-P6 integrin. Panels A, B, C and D demonstrate colocalization of the integrins avp 1 and avP6 with EDA-FN in parallel sections of the same specimen (see arrowheads). Panels E (a5 integrin), F (pi integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pi with EDA-FN in parallel sections of the same specimen (see arrowheads). Bar = 100 um. E = epithelium, CT = connective tissue. 68 Figure 14. Immune-localization of av, BI, 06 integrin, and EDA-FN around the basal keratinocytes of the oral epithelium of the cyclosporin-induced overgrowth tissue. Panel A-av integrin, panel B-pl- integrin, panel C-EDA-FN, panel D-06 integrin. Panels A, B, C and D demonstrate colocalization of putative integrins avpi and avP6 with EDA-FN. Panels E (a5 integrin), F (P1 integrin) and G (EDA-FN) demonstrate colocalization of the integrin a5pl with the EDA-FN (see arrowheads). All panels are from parallel sections of the same specimen. Bar = 100 um for panels A, B, C, D, and 50 pm for panels E, F, and G. E = epithelium, CT = connective tissue. 69 Figure 15. Immunolocalization of av, (36 integrin, and EDA-FN around the basal keratinocytes of the oral epithelium of the combined drug-induced overgrowth tissue. Panel A-av integrin, panel B-P6 integrin, panel C-EDA-FN (Bar = 200 urn). Panel D-av integrin, panel E-P6 integrin, panel F-EDA-FN (Bar = 50 um). Panels A, B, C, D, E, F demonstrate colocalization of the integrins avp 1 and avP6 with EDA-FN (arrows with tail show expression of the integrin and/or EDA-FN within parallel sections of the same specimen). E = epithelium, CT = connective tissue. 70 Figure 16. Immunolocalization of cc5 and pi-active integrin around the basal keratinocytes of the oral epithelium of the combined drug-induced overgrowth tissue. Panel A (a5 integrin), B (pi-active integrin) and panel C (EDA-FN within the connective tissue) demonstrate colocalization of the integrin cc5pl with EDA-FN (arrows with tail indicates the parallel sections within the same specimen). Bar = 100 urn. E = epithelium, CT = connective tissue. 71 Chapter 5 - Discussion Gingival overgrowth has been documented as one of the oral side effects of medications which include phenytoin, nifedipine, cyclosporin, or a combination of both (Lederman et al., 1984; Slavin and Taylor 1987; Starzl et al., 1980). The pathogenesis of drug-induced gingival overgrowth is multifactorial. Genetic predisposition, pharmacokinetic variables, alterations in gingival connective tissue homeostasis, and the presence of dental plaque may all contribute to the changes seen in the gingival tissue. Histological changes in drug-induced gingival overgrowth tissue include acanthosis evident as elongated rete ridges, hyperkeratosis, and an increase in epithelial width (Marshall and Bartold, 1998; Van der Wall et al., 1985). Integrins are cell surface glycoproteins that mediate cell-cell and cell-extracellular matrix adhesion events (Hynes 1992; Miyamoto et al., 1998). Integrins mediate the attachment of keratinocytes to the basement membrane. Fibronectin has been shown to bind to integrins a5pi, avpi and avp6 (Koivisto et al., 1999; Larjava et al., 1996; Singer et al., 1988). Alterations in integrin expression during wound healing, chronically inflamed periodontal tissue, and in oral leukoplakia and normal oral epithelium has been described (Haapasalmi et al., 1995; Hamidi et al., 2000; Larjava et al., 1993; Thorup et al., 1997). This study demonstrated the changes that occur in integrin expression in drug-induced gingival overgrowth tissue may resemble that of the integrin expression found during wound healing. 5.1 Comparison of integrin localization in drug-induced gingival overgrowth tissue to those previously reported in the literature. Our results demonstrate that, in general, the integrins ct5, av, pi, and P6 were expressed with greater frequency or altered distribution (suprabasal expression) in the drug-induced gingival overgrowth tissues than the control tissues. One possible explanation for the upregulation of certain integrins may be related to differing expression of cytokines as a result of the influence of the medication, or due to the direct influence of the medications on the epithelium. The overall trend reflects increased integrin expression in the combined drug-induced gingival overgrowth group, which may be a result of the additive effect of the medications. Compared to previous literature, the upregulation of the pi integrin occurs during wound healing (Cavani et al., 1993; Gailit et al., 1994). Specifically, the upregulation of the a5pl integrin by basal keratinocytes during wound healing is apparent (Larjava et al., 1993). In the present study increased expression of the a5pi integrin in the drug-induced gingival overgrowth samples was found. Colocalization of the integrins av and p 1 was noted in all groups, which suggests that they may combine to form another fibronectin binding integrin in keratinocytes. This was because no other putative P-subunits were available for av (areas where avp6 was absent but av was present) due to lack of expression of either P5 and P3 subunits in the gingival epithelia. The avpl integrin has not been previously shown to be present in the gingiva, however, it has been shown to be present in cultured keratinocytes (Koivisto et al., 1999) and possibly gingival wounds 73 (Hakkinen, 2000, unpublished data). These findings taken together may support the hypothesis that the drug-induced gingival overgrowth tissue integrin expression resembles that of wound healing. The ct5pi integrin exhibited greater expression in the combined drug-induced overgrowth samples compared to the other drug-induced overgrowth groups and the control group. This integrin has been shown to be expressed by wound keratinocytes, and not expressed by normal resting keratinocytes (Larjava et al., 1993). The expression of the ct5pi integrin by the control group is a new finding and deserves further discussion. The a5pi integrin has been previously reported as being absent from normal gingiva (Larjava et al., 1993) as well as from gingival tissue containing oral epithelium from periodontitis patients (Haapasalmi et al., 1995). In these studies, the tissue was obtained mainly from palatal gingiva or non-papillary gingiva, respectively. There are no studies available which address the expression of the ct5pi integrin in papillary gingiva or sulcular/junctional epithelium. Therefore, the presence of the a5pi integrin may be a reflection of the unique phenotype of the papillary gingival epithelium. The increased expression of the a5pi integrin in the combined drug-induced overgrowth samples may resemble that seen in wound healing. Generalized suprabasal expression of the integrin a5pi was also seen more frequently in the combined drug-induced overgrowth group. In general, suprabasal expression of integrins has been shown to occur due to keratinocyte hyperproliferation or abnormal terminal differentiation, but not as a result of inflammation (Hertle et al., 1995). The suprabasal expression of cc5pi integrin in the combined drug-induced overgrowth group may be a function of perturbed keratinocyte differentiation or hyperproliferation. Conversely, one study demonstrated that the 74 suprabasal expression of integrins in transgenic mice had no direct effect on the proliferation of cells in the underlying basal layer (Romero et al., 1999). Further studies are required to clarify the role of a5(31 integrin expression in overgrowth tissue. One study has reported evidence that may support the theory that psoriasis may be a result of altered keratinocyte integrin expression rather than an immunological disease (Carroll et al., 1995). Transgenic mice that expressed suprabasal ct5pi integrin exhibited features consistent with psoriasis which included epidermal hyperproliferation, perturbed keratinocyte differentiation, and skin inflammation. The mechanism that explains why suprabasal integrin expression triggers hyperproliferation is not known, but it was speculated that the functional status (active vs. inactive) of the receptors may be involved (Carroll et al., 1995). In relation to drug-induced overgrowth, it may be possible that the keratinocytes play a role in the clinical appearance of gingival overgrowth as evident in the suprabasal expression of the a501 integrin. The underlying mechanism to explain why the keratinocytes express this integrin in unknown, but it may be related to the additive effect of the medications and/or the epithelial cell phenotype. The av06 integrin is strongly expressed by wound keratinocytes, but it is not expressed by non-wounded tissue nor is it expressed by healthy or inflamed human periodontal tissue samples (Haapasalmi et al., 1995; Haapasalmi et al., 1996). This integrin is, however, expressed by epithelial cells in oral leukoplakia, dysplasia, and by hyperkeratotic, hyperplastic, and atypic tissue (Hamidi et al., 2000). In this study, the av(36 integrin was seen with similar frequency in all groups, including the control group. This finding contradicts those of previous findings in that the control group displayed an usually high frequency of expression of the av06 integrin, and this is not seen in normal 75 oral mucosa (Haapasalmi et al., 1995; Haapasalmi et al., 1996). Explanations for this finding may include a difference in tissue location as discussed above. The combined drug-induced group demonstrated higher frequency of generalized and suprabasal expression of avp6 integrin. The integrin p4 showed the greatest frequency of generalized expression in both the oral and sulcular epithelium of the control group when compared to the drug-induced groups. Previous literature has shown that the a6p4 integrin demonstrates focal losses in inflamed areas and a continuous pattern in non-inflamed areas (Haapasalmi et al., 1995). The a6p4 integrin is likely responsible in both the establishment of adhesion complexes during wound healing and the maintenance of adhesion complex integrity in non-wounded areas of epithelial-connective tissue interaction (Kurpakus et al., 1991). Wound studies indicate that the a6p4 integrin is expressed by the keratinocytes around the leading edge of the wound margin, and in seven-day old wounds, the distribution resembles that of normal mucosa (Larjava et al, 1993). The finding that the control group demonstrates normal distribution of a6p4 integrin supports that of previous studies (Thorup et al., 1997). The drug-induced groups also exhibited generalized distribution of a6p4 integrin in the oral and sulcular epithelium. An additional finding in our study was that the combined drug-induced gingival overgrowth group demonstrated greater suprabasal expression of a6p4 integrin by the keratinocytes of the oral epithelium compared to the other groups. Other gingival tissue that also exhibits suprabasal expression of the P4 integrin includes squamous cell carcinoma samples (Hamidi et al., 2000). 76 5.2 Integrin expression and inflammation Previous studies have shown that focal losses of integrins which include a2(31, a3Bl, and a6p4 occur in inflamed areas of human periodontal tissue (Haapasalmi et al., 1995). Our study showed that the degree of inflammation was similar in all tissue types (see figures 3 and 4), yet the distribution of integrins differed between the groups. No specific pattern of losses of integrins was evident within the samples. It may be concluded that inflammation, in the case of drug-induced gingival overgrowth tissue, may not play a major role in the regulation of expression of various integrins in this type of tissue. 5.3 Integrin expression and fibronectin Integrins that are known to bind fibronectin include avp6, avpi and ct5pi (Koivisto et al., 1999; Larjava et al., 1996; Singer et al., 1988). Results from our study demonstrated that the combined drug-induced overgrowth group exhibited strong expression of cellular fibronectin (EDA) within the connective tissue, but lacked expression around the basal keratinocytes. Distribution of cellular fibronectin (EDA) in the connective tissue as well as around the basal keratinocytes of the control, nifedipine-and cyclosporin-induced overgrowth groups were also observed. These findings were confirmed by numerous repeated stainings. Additional studies have identified plasma fibronectin around the basal cells of the epidermis from perilesional uninvolved psoriasis skin sections (Bata-Csorgo et al., 1998; Pelligrini et al., 1992). This pattern of staining was also found around the keratinocytes from intralesional areas of psoriasis. One study demonstrated higher expression of the a5pi integrin in the non-lesional psoriatic epidermis compared to normal keratinocytes (Bata-Csorgo et al., 1998). The 77 upregulation of the a5(31 integrin was thought to occur as a result of genetic susceptibility or due to altered regulation of the in vivo environment. The authors hypothesized that the basement membrane fenestrations in psoriatic lesions may have allowed for fibronectin to penetrate into the epidermis, resulting in an upregulation of a5(31 integrin by the keratinocytes, which then allows the keratinocytes to become hyperproliferative if exposed to the appropriate cytokines (Bata-Csorgo et al., 1998). In a more recent study, cellular fibronectin (EDA) was shown to exist at the dermal-epidermal junction in uninvolved psoriasis specimens, but was not present in the control samples. This study showed that the fibronectin type present in the keratinocyte cell layer did not leak from the plasma, but rather, was synthesized in situ (Ting et al., 2000). In the case of the control, nifedipine-and cyclosporin-drug induced overgrowth groups, the oc5pl integrin was not upregulated, yet fibronectin was evident around the basal keratinocytes of some of the samples. Conversely, the combined drug-induced overgrowth groups showed upregulation of the a5pi integrin, with no evidence of fibronectin around the basal keratinocytes. An explanation as to why this pattern of expression of fibronectin occurred in those specific groups is unknown. Possibly keratinocyte phenotype, location of the tissue (papillary or marginal), and influence of the medications may play a role. Additional studies with larger sample sizes are required to confirm these findings. 5.4 Integrin expression and transforming growth factor-|3 Latent transforming growth factor-P has been shown to bind to the ccvp6 integrin (Munger et al., 1999). Transforming growth factor-P has also been shown to upregulate 78 the expression of the a5pi integrin as well as stimulate fibronectin synthesis by keratinocytes (Hashiro et al, 1991; Nickoloff et al., 1988; Zambruno et al., 1995). It has been observed that the avp6 integrin can activate endogenous latent TGF-P, and that this mechanism may regulate pulmonary inflammation and fibrosis in mice (Munger et al., 1999). Mice that did not express the avp6 integrin developed inflammation and were protected from fibrosis, whereas those mice that expressed the avp6 integrin by the airway epithelial cells developed lung fibrosis. Experiments using both keratinocytes and human bronchial epithelial cells were also shown to activate latent TGF-p (Munger et al., 1999). One of the hypotheses of the study was that avp6 would be present in the overgrowth tissue providing a putative mechanism for TGF-P activation. TGF-P has been shown to be present in overgrowth tissue (Saito et al., 1996). The expression of the ccvp6 integrin by the control group was surprising. More suprabasal expression of the ocvp6 integrin was evident in combined overgrowth tissue, but no conclusions can be drawn about the putative role of avp6 integrin expression and the promotion of connective tissue formation in gingival overgrowth tissue. Another consideration is that the avpi integrin can activate TGF-P (Munger et al., 1998), and more suprabasal expression of av integrin was also seen in the combined drug-induced group. Clearly, descriptional studies like this must be supported with functional studies before any conclusions in this regard can be drawn. ; 79 5.5 Location of the tissue biopsies and integrin expression The biopsies received included 33 from the interdental papilla area and 19 from the marginal gingiva area. Previous wound healing studies (Larjava et al., 1993) have examined tissue from palatal keratinized gingiva, therefore direct comparison of results may not be applicable. The different cytokeratin patterns and resultant phenotypic variations that exist between different tissue types may explain the differences in integrin expression. As mentioned previously, the transition between junctional, sulcular and oral epithelium may affect the interpretation of integrin expression. It may be possible that the location of the gingiva, and the difference in phenotype may influence the expression of the integrins, but further studies are required to confirm this. 5.6 Basement membrane components The expression of both laminin-5 and type IV collagen was similar in all groups. Laminin-5 demonstrated localized breaks in the continuity in all the groups with the same frequency. This is similar to the finding in diseased human periodontal tissue (Haapasalmi et al., 1995). It may be possible that inflammation, rather than the effect of nifedipine and/or cyclosporin, is responsible for this pattern of staining of Laminin-5. The expression of type IV collagen was similar to that seen in normal mucosa (Larjava et al., 1993). 5.7 Effect of the medications on the epithelium A review of the literature revealed one study that identified the effects of cyclosporin on human keratinocytes in culture (Prignano et al., 1996). This study showed that 80 cyclosporin inhibited the growth of keratinocytes, and an organized network of tonofibrils was not evident in this group compared to the normal keratinocytes. The authors proposed that the cytoskeleton of the keratinocytes could be a target of cyclosporin, and that it may effect the growth of keratinocytes (Prignano et al., 1996). A complex relationship exists between growth factors, integrin expression, the extracellular matrix and keratinocytes. This study did not address these factors so the results must be interpreted with caution. No studies were identified which examined the direct effect of nifedipine and a combination of nifedipine and cyclosporin on human keratinocytes. It is possible that these medications may have a direct effect upon keratinocytes, that effect is unknown, but it may contribute to the appearance of gingival overgrowth in susceptible individuals, and/or a change in integrin expression. Previous studies have shown that the c-Myc and bcl-2 proto-oncogene products were over-expressed within the epithelium of nifedipine-induced gingival overgrowth specimens (Saito et al., 2000). The trend shown in this study was that suprabasal expression of integrins was seen in the drug-induced specimens. A relationship between the suprabasal expression of integrins and the over-expression of the c-Myc and bcl-2 proto-oncogene products within the epithelium of drug-induced gingival overgrowth tissue may exist, but further studies are required to confirm this hypothesis. The cyclosporin and the combined drug-induced overgrowth groups from our study also received methylprednisone and azathioprine as part of their treatment regimen (see table 1). It is possible that these additional medications may have had an effect on integrin expression. Based upon the overall trend of integrin expression in our study, one would expect to have seen similarities between the cyclosporin and the combined drug-81 induced overgrowth group if the additional medications exerted any influence. This was not evident, as the major trend was that the combined drug induced overgrowth group exhibited more frequent expression of certain integrins. Additional studies identifying the effects of nifedipine, cyclosporin, methylprednisone, and azathioprine on human keratinocytes would be of use. A clinical study has compared the appearance of drug-induced gingival overgrowth and the effect of prednisolone and azathioprine in conjunction with cyclosporin and nifedipine treatment in both juvenile and adult renal transplant patients. Their findings identified an inverse relationship between prednisolone and azathioprine and the degree of drug-induced gingival overgrowth (Wilson et al., 1998). Although these findings cannot be directly related to our study, it may provide some evidence that prednisolone and azathioprine do not have an effect on drug-induced gingival overgrowth. 5.8 Limitations of this study This study was of an observational nature, and there are inherent problems associated with observational studies. Our small sample size is likely not representative of all drug-induced gingival overgrowth tissues, therefore, results must be interpreted with caution. The tissue processing and sectioning may have changed the profile of integrin expression. In areas with very localized expression of one particular integrin, the matching subunit may have not been identified due to the sectioning or due to the limited number of sections from that particular patient. The limited numbers of sections precluded us from repeating all the stainings, however, those that were performed did confirm the existing findings. 82 Certain factors were controlled for in order to reduce the degree of systematic error. This included the use of monoclonal antibodies, which provided greater specificity compared to a polyclonal antibody as it reduces the likelihood of binding to multiple isoforms of the target (Mighell, 1998). In addition, a routine staining method was used (Larjava et al., 1993), which has been successfully used with the same antibodies and verified to be accurate by other authors (Haapasalmi et al., 1995; Haapasalmi et al., 1996; Hamidi et al., 2000). Determinate error was reduced utilizing additional examiners. The sections were first analyzed for presence or absence of integrins by one examiner (PW). In order to reduce the examiner bias, the examiner was calibrated, and a criterion for positive stain was established. The calibration consisted of visual examination of the slides stained with different antibodies, and having the results verified by two experienced examiners (LH and HL). Positive criteria were developed based upon the negative control slides, and those positive slides used during the calibration trial. Positive criteria included intense fluorescence located in the area of the cell membrane, visible at a minimum of 20X magnification, and adjacent to a dark, non-fluorescent area. Negative criteria included no fluorescent stain located in the area of the cell membrane. The calibration trial presumably ensured that the repeated analysis of the sections confirmed the identical location of the integrin in each sample, thereby ensuring precision. In this study, the use of specific monoclonal antibodies and the use of calibrated examiners negated the need to quantify the immunofluorescence of the antibodies. 83 Chapter 6 - Conclusions The expression of keratinocyte integrins in drug-induced gingival overgrowth tissue was characterized in this study. Comparisons were made between integrin expression in both normal tissue and wound healing. The following trends were noted: -the integrin cc5pi was found to be upregulated in the combined-drug induced gingival overgrowth samples. This pattern of expression has been shown to exist in both wound healing and psoriasis. -the expression of the integrin av06 was evident, with similar frequency, in all groups. The expression of this integrin in the drug-induced groups is similar to that found in wound healing, however, increased suprabasal expression was noted within the drug-induced specimens. The expression of av06 by the keratinocytes in the control group is a novel finding, which may be partially explained by difference in epithelial phenotype. -the expression of the integrin a6pM was similar to normal mucosa, however the suprabasal expression of this integrin in the oral epithelium of the combined-drug induced overgrowth group is like that seen in squamous cell carcinoma. -cellular fibronectin (EDA-FN) was identified around the basal keratinocytes of some of the specimens in the control, nifedipine-, and cyclosporin-induced gingival overgrowth groups. This has been shown to occur in psoriatic lesions. -basement membrane components (laminin-5 and type IV collagen) were expressed in a pattern similar to that found in human periodontal tissue and normal mucosa. -no relationship could be identified between inflammation and integrin expression in this study. 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