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Oxidized low density lipoprotein mediated macrophage survival : an essential role for PI3K/PKB pathway Hundal, Rajinder Singh 2001

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OXIDIZED L O W DENSITY LIPOPROTEIN M E D I A T E D M A C R O P H A G E SURVIVAL: A N ESSENTIAL R O L E FOR PI3K/PKB P A T H W A Y  by RAJINDER SINGH H U N D A L B.Sc, University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL F U L F U L L M E N T OF T H E REQUIREMENT FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Experimental Medicine Program :  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A July 2001 © Rajinder Singh Hundal, 2001  Wednesday, August 22,  2001  UBC Special Collections - Thesis Authorisation Form  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . It i s understood that c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department of  7\ArA .Vi'n  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  g Columbia  http://www.library.ubc.ca/spcoll/thesauth.html  ABSTRACT Oxidized L D L (ox-LDL) has previously been shown to induce endothelial adhesion molecule, MCP-1 and M - C S F expression thereby leading to monocyte recruitment, adherence and macrophage differentiation and has recently been shown to induce macrophage proliferation.  The purpose of this study was to examine an additional  mechanism by which ox-LDL might increase macrophage populations - enhanced macrophage survival. I found that ox-LDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages following M - C S F withdrawal. Incubation of macrophages with either native L D L or acetylated L D L had no effect on survival, thereby suggesting an oxidation specific event. The pro-survival effect of ox-LDL was not inhibited by neutralizing antibodies to G M - C S F , was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. This suggested that ox-LDL activated cytokine-independent intracellular survival signaling pathways. Ox-LDL caused activation of the M A P kinases ERK1/2 as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI3K). Furthermore, there was phosphorylation of two important PKB downstream targets lKB-a-Ser-32 and BadSer-136, both of which act at the level of the pro-survival Bcl-2 protein, Bcl-X to inhibit L  apoptosis. The M E K inhibitors PD 98059 and U0126 blocked ERK1/2 activation, but did not diminish survival. Conversely, the PI3K inhibitors L Y 294002 and wortmannin blocked PKB activation, and the ability of ox-LDL to promote macrophage survival.  ii  I also demonstrated that ox-LDL prevented the elaboration of ceramide following the withdrawal of M - C S F , and pre-treatment with soluble ceramides attenuated ox-LDL mediated macrophage survival and PKB activation. The source of ceramide elaboration was through the metabolism of sphingomyelin to ceramide rather than de novo synthesis as determined by inhibitor studies with fumonisin BI and desipramine; as well as by determination of levels of sphingomyelin. Ox-LDL was also able to prevent the decline in the levels of Bcl-X that occur following M - C S F withdrawal. Taken together, these results L  indicate that ox-LDL prevents ceramide elaboration, activates a PI3K/PKB pathway that reduces cytosolic sequestration of N F - K B , as well as phosphorylates Bad and thereby promotes macrophage survival in the absence of growth factors at the level of B c l - X and L  caspase 3 activation.  iii  TABLE OF  CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  x  ACKNOWLEDGEMENTS  xii  1.  INTRODUCTION  1  1.1  ATHEROSCLEROSIS  1  1.1.1  The Pathogenesis of Atherosclerosis  1  1.1.2  Mechanisms of Oxidation and Structural Changes Associated with L D L  8  1.1.3  The Biological Effects of Oxidized L D L  1.2  M A C R O P H A G E SURVIVAL 1.2.1  1.3  14  M-CSF Signaling  14  PI3K/PKB SIGNALING C A S C A D E  17  1.3.1  PI3K  17  1.3.2  PKB  20  1.4  APOPTOSIS  24  1.4.1  Caspases  24  1.4.2  Bcl-2 Family Members  28  C E R A M I D E A N D APOPTOSIS  32  1.5 1.5.1  Ceramide Generation and Metabolism  32  1.5.2  Ceramide Induced Apoptosis  35  1.6  O X - L D L M E D I A T E D M A C R O P H A G E D E A T H , PROLIFERATION A N D S U R V I V A L SIGNALING  1.7  11  37  1.6.1  Ox-LDL and Death Signaling  39  1.6.2  Ox-LDL and Proliferation Signaling  43  1.6.3  Ox-LDL and Survival Signaling  47  OBJECTIVES A N D HYPOTHESES  50  iv  2.  MATERIALS AND METHODS  52  2.1  Materials  52  2.2  Lipoprotein Isolation and Oxidation  53  2.3  Characterization of L D L  54  2.4  Cell Culture  54  2.5  M T S Cell Viability Assay  55  2.6  Cell Counts  56  2.7  D N A Fragmentation and Cell Cycle Analysis  56  2.8  Western Blotting  56  2.9  Morphological Observations  57  2.10  In-Vitro PKB Kinase Assay  58  2.11  Ceramide and Sphingomyelin Determination  58  2.12  Lipid/Protein Seperation of Ox-LDL  59  2.13  Retroviral Infection of B M D M  60  2.14  Statistical Analysis  61  3.  O X - L D L PROMOTES MACROPHAGE SURVIVAL BY INHIBITING  :  APOPTOSIS  62  3.1  R A T I O N A L E A N D HYPOTHESIS  62  3.2  RESULTS A N D DISCUSSION  63  4.  OX-LDL PROMOTES CYTOKINE INDEPENDENT SURVIVAL THROUGH ACTIVATION OF T H E PI3K/PKB PATHWAY  79  4.1  R A T I O N A L E A N D HYPOTHESIS  79  4.2  RESULTS A N D DISCUSSION  80  5.  MECHANISMS OF OX-LDL MEDIATED M A C R O P H A G E SURVIVAL: AN IMPORTANT R O L E FOR B C L - X AND CERAMIDE  98  5.1  R A T I O N A L E A N D HYPOTHESIS  98  5.2  RESULTS A N D DISCUSSION  99  6.  SUMMARY  115  7.  BIBLIOGRAPHY  118  L  v  LIST O F T A B L E S  C H A P T E R 1. INTRODUCTION Table 1. Biological properties of ox-LDL on monocyte/macrophagi  vi  LIST OF FIGURES C H A P T E R 1. INTRODUCTION Figure 1.1. Ox-LDL and early atherogenesis  3  Figure 1.2. Phosphatidylinositol structure and the point of action of PI3K  19  Figure 1.3. Ceramide generation and metabolism  34  C H A P T E R 3.  O X - L D L PROMOTES M A C R O P H A G E S U R V I V A L B Y INHIBITING  APOPTOSIS Figure 3.1. Time dependent inhibition of cell death in B M D M by ox-LDL  69  Figure 3.2. Dose dependent inhibition of cell death in B M D M by ox-LDL  70  Figure 3.3. Electrophoretic mobilities of L D L with varying degrees of oxidation  71  Figure 3.4. Oxidation dependent inhibition of cell death in B M D M by ox-LDL  72  Figure 3.5. O x - L D L does not lead to an increase in cyclin D l  73  Figure 3.6. Ox-LDL does not increase S phase of the D N A cycle  74  Figure 3.7. Ox-LDL does not increase macrophage cell numbers  75  Figure 3.8. O x - L D L inhibits D N A fragmentation in B M D M  76  Figure 3.9. Ox-LDL inhibits caspase 3 activation in B M D M  77  Figure 3.10. Ox-LDL maintains phosphatidylserine asymmetry  78  C H A P T E R 4. O X - L D L P R O M O T E S C Y T O K I N E I N D E P E N D E N T T H R O U G H A C T I V A T I O N OF THE PI3K/PKB P A T H W A Y  SURVIVAL  Figure 4.1. Ox-LDL promotes cytokine independent survival  87  Figure 4.2. Ox-LDL promotes GM-CSF independent survival  88  vii  Figure 4.3. Ox-LDL promotes M-CSF independent survival  89  Figure 4.4. Ox-LDL activates ERK1/2 and PKB  90  Figure 4.5. PI3K inhibitors block PKB activation and M E K inhibitors block ERK1/2 activation by ox-LDL  91  Figure 4.6. PI3K inhibitors block ox-LDL mediated macrophage survival  92  Figure 4.7. Kinetics of PKB activation by ox-LDL  93  Figure 4.8. Ox-LDL phosphorylates the PKB targets, IKB and Bad  94  Figure 4.9. Total lipid component of ox-LDL prevents macrophage apoptosis  95  Figure 4.10. Total lipid component of ox-LDL activates PKB  96  Figure 4.11. Retroviral infection of B M D M  97  C H A P T E R 5. MECHANISMS OF O X - L D L M E D I A T E D M A C R O P H A G E S U R V I V A L : A N IMPORTANT R O L E FOR B C L - X A N D C E R A M I D E L  Figure 5.1. B M D M apoptosis is associated with ceramide generation  104  Figure 5.2. Ceramide pre-treatment blocks ox-LDL mediated macrophage survival  105  Figure 5.3. Desipramine blocks sphingomyelin hydrolysis to ceramide  106  Figure 5.4. Desipramine prevents macrophage apoptosis  107  Figure 5.5. Ceramide blocks ox-LDL mediated PKB activation  108  Figure 5.6. Ox-LDL maintains Bcl-X levels  109  L  Figure 5.7. PI3K inhibitors block ox-LDL mediated phosphorylation of IKB and Bcl-X maintenance  L  110  viii  Figure 5.8. N F - K B inhibitors prevent ox-LDL maintenance of Bcl-X levels  Ill  Figure 5.9. N F - K B inhibitors prevent ox-LDL mediated macrophage survival  112  Figure 5.10. Ox-LDL prevents the activation of caspase 9-caspase 3 cascade  113  Figure 5.11. Caspase 9 and caspase 3 inhibitors block survival  114  L  C H A P T E R 6. S U M M A R Y Figure 6.1. Ox-LDL induced macrophage survival: a working model  ix  117  LIST O F ABBREVIATIONS  4-HT  4-hydroxytamoxifen  ac-LDL  Acetyl L D L  apoB  Apolipoprotein B -100  AIF  Apoptosis inducing factor  Apaf  Apoptotic protease activating factor  BH  Bcl-2 homology  BMDM  Bone marrow derived macrophages  CAPE  Caffeic acid phenylethyl ester  EDTA  Ethylenediaminetetraacetic acid  ERK  Extracellular regulated kinase  FBS  Fetal bovine serum  GSK-3P  Glycogen synthase kinase P  GM-CSF  Granulocyte macrophage colony stimulating factor  GFP  Green fluorescent protein  HDL  High density lipoprotein  h  Hours  IKK  I K B kinase  JNK  c-Jun NH2-terminal protein kinase  LDL  Low density lipoprotein  LysoPC  Lysophosphatidylcholine  M-CSF  Macrophage colony stimulating factor  MARCO  Macrophage receptor with collagenous structure  MMP  Matrix metalloprotease  mm-LDL  Minimally modified L D L  min  Minutes  MAP  Mitogen activated protein  MCP-1  Monocyte chemotactic protein-1  MTS  [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4sulphophenyl)-2H-tetrazolium, inner salt]  n-LDL  Native L D L  NF-KB  Nuclear factor-K B  ox-LDL  oxidized L D L  PT  Permeability transition  PTEN  Phosphatase with homology to tensin  PBS  Phosphate buffered saline  PC  Phosphatidylcholine  x  PI3K  Phosphatidylinositol 3-kinase  PDGF  Platlet derived growth factor  PH  Pleckstrin homology  PARP  Poly (ADP-ribose) polymerase  PKB  Protein kinase B  PKC  Protein kinase C  Ser  Serine  SR-AI/II  Scavenger receptor class A type I/II  SHIP  SH2 domain containing inositol phosphatase  SH2  src homology 2  SCF  Stem cell factor  SAPK  Stress activated protein kinase  Thr  Threonine  TLC  Thin layer chromatography  TBS  Tris buffered saline  TNF-a  Tumor necrosis factor-a  xi  ACKNOWLEDGEMENTS The perseverance of a Ph.D. program is not merely an accomplishment of the individual, but also a reflection of the supportive environment required for successful completion.  I extend my sincere gratitude to the many people that have supported my  research endeavors and whom I hope to maintain as friends and colleagues in the years to come. In particular, I would like to thank Urs Steinbrecher for providing me the opportunity to develop both scientifically and socially in his laboratory, as well as providing an important balance between academic guidance and individuality. I would also like to thank my immediate family, Baljinder Dhaliwal, Shannon Dhaliwal, Kuljit Parhar, Maryam Moussavi and Myriam Farah for teaching me the true meaning of friendship ... of which the learning process never ceases to continue ...  xii  1.  INTRODUCTION  1.1  ATHEROSCLEROSIS  1.1.1  T h e Pathogenesis of Atherosclerosis  Atherosclerosis is a chronic progressive disease that affects large and medium-sized elastic and muscular arteries.  Although a multifactorial disease process, the lesions of  atherosclerosis represent the common endpoint of repeated cycles of injury, inflammation, and repair (1-4). The name is derived from the Greek roots athero meaning paste and sclerosis meaning hardening. During the early stages of the disease or atherogenesis proper, lipid (principally cholesterol) is deposited in the arterial intima subendothelial space. Lipoprotein transport across the endothelial barrier occurs in a concentration-dependent process and does not require receptor-mediated endocytosis (5). Once in the subendothelial space, low density lipoprotein (LDL) particles become associated with proteoglycans within the intima (6, 7). In the sequestered "microenvironment" of the subendothelium, L D L can undergo progressive oxidation since the intima lacks many of the antioxidant defense mechanisms that are present in the bloodstream (8).  Initially, macrophages may play a  protective role by removing the modified L D L molecules through scavenger receptormediated endocytosis (9-11). However, continued uptake of modified L D L results in the histologic hallmark of the early atherosclerotic lesion, the macrophage derived foam cell (1214). These cells play an important role in the development and progression of atherosclerosis through the release of potent cytokines/growth factors and oxidizing agents which can lead to  1  monocyte recruitment, smooth muscle cell migration from the media, smooth muscle cell proliferation, continued free radical mediated oxidation of incoming L D L and retention of monocyte derived macrophages into the expanding intimal space (1, 6, 7, 15). Some of these important pro-atherogenic changes are summarized in Figure 1.1. According to this model, these and other inflammatory events culminate in exacerbated lesion progression, resulting ultimately in the formation of atheromatous plaques or atheromas. In addition to their role in plaque formation, recent studies have shown that macrophage-derived foam cells destabilize advanced atherosclerotic lesions and favor plaque disruption, dissection, or ulceration (16-18). Foam cells and extracellular lipid deposits have negligible tensile strength and their presence in a lesion creates a potential fracture plane. In addition, macrophages secrete cytokines such as interferon y that inhibit collagen synthesis by smooth muscle cells, and create an environment favoring L D L oxidation which in turn can induce smooth muscle cell apoptosis (19, 20).  Macrophages also play a key role in  matrix degradation through the action of matrix-degrading enzymes known as matrix metalloproteases (MMP). Unstable atherosclerotic lesions frequently exhibit increased levels of matrix-degrading enzymes (21-23). Furthermore, macrophages isolated from atheromas constitutively secrete the metalloproteases, MMP-1 and MMP-3 (22, 24) and macrophages are responsible for the increased levels of MMP-1 and MMP-3 in unstable plaques (25). Macrophage density is inversely related to plaque stability (26) and macrophage infiltration of the fibrous cap is a ubiquitous finding in unstable lesions (17, 27, 28). Therefore, these  2  1.1. O x - L D L  Once in the intimal space, LDL can become trapped by proteoglycans and subsequently oxidized by a free radical mediated reaction that converts unsaturated fatty acids to reactive lipid hydroperoxides and aldehydes. These reactive lipid substances then further accelerate oxidation of the L D L particle, leading to the depletion of polyunsaturated fatty acids and antioxidants, and the fragmentation of apolipoprotein B. Ox-LDL stimulates the expression of cell adhesion molecules (VCAM, ICAM-1, and GRO) and the secretion of MCP-1 and M-CSF from endothelial cells, causing monocytes to enter the arterial intima and differentiate into macrophages. Ox-LDL also becomes endocytosed via scavenger receptors on the surface of macrophages, resulting in lipid accumulation and ultimately foam cell formation. Ox-LDL has a number of proatherogenic and pro-atherosclerotic properties on monocyte/macrophages, some of which are summarized in Table 1. Figure  and early atherogenesis.  3  studies provide a strong link between macrophage infiltration and fibrous cap weakening. There is also compelling evidence that cholesterol-lowering therapy leads to a reduction in coronary events not because of a decrease in lesion size, but rather at the level of increased plaque stability consequent to a reduction in the number of macrophage derived foam cells in superficial regions of plaques (3, 4, 16, 29-31). As such, understanding factors that regulate the formation of the macrophage derived foam cell, as well as the survival, proliferation and death of these cells is of obvious importance in understanding the etiology of atherosclerosis. Atherosclerosis remains the principal cause of death in North America, Europe, and much of Asia (32, 33). Numerous risk factors for atherosclerosis have been identified. For example, four non-modifiable risk factors are age, gender, family history of vascular disease, and history of symptomatic vascular disease. Modifiable risk factors that are also causally related to atherosclerosis and its clinical sequelae include smoking, hypertension, diabetes mellitus, obesity, sedentary lifestyle and psychosocial stress (34).  However, perhaps the  most important and best studied of the major risk factors for atherosclerosis  is  hypercholesterolemia. In humans, L D L is the major transporter of cholesterol in blood, and it is the source of most of the cholesterol found in atherosclerotic lesions (1, 35-38). In contrast, high density lipoprotein (HDL) acts as an extracellular cholesterol acceptor, thereby allowing extrahepatic cells to remove excess cholesterol through reverse cholesterol transport, and this is consistent with the known protective effect of H D L cholesterol (39, 40). Another putative mechanism by which H D L may be protective is through the inhibition of  4  L D L oxidation. For example, paraoxonase, present on HDL, has been shown to inhibit L D L oxidation and thereby protect against atherosclerosis (41). In early lesions, much of the cholesterol is found as cytoplasmic lipid droplets within monocyte derived macrophages, and it is believed that cholesterol accumulation within these cells plays an important role in the development of more advanced lesions (42-46). The mechanism by which macrophages in the arterial intima accumulate massive amounts of cholesterol in patients with hypercholesterolemia is not readily apparent, however, as the activity of the L D L receptor is regulated by intracellular cholesterol content, and incubation of cultured macrophages with high concentrations of native L D L does not result in gross lipid accumulation (47, 48). Subsequent studies showed that certain chemical modifications of L D L lysine residues, such as acetylation, converted it to a form that was recognized by a specific receptor on macrophages termed the acetyl L D L receptor or scavenger receptor class A type I/II (SR-AI/II) (48, 49). This scavenger receptor, unlike the native L D L receptor, is not down-regulated when the cholesterol content of the cell increases and thereby allows for cholesterol engorgement in macrophages giving rise to macrophage derived foam cells (48, 49). In principle, acetylation of L D L could account for foam cell formation, however, there is no evidence that such a modification occurs in vivo, therefore, the search began for a biologically plausible modification of L D L that could account for foam cell formation. Another modified form of L D L emerged as a suitable candidate when it was shown that  5  simply incubating L D L overnight with a monolayer of endothelial cells converted it to a form that was taken up more readily by macrophages and capable of increasing cholesterol content (50-52).  Interestingly, this cell mediated modification turned out to be oxidative  modification (53). Other modifications of L D L that have been reported to facilitate foam cell formation include glycation, aggregation, association with proteoglycans, and incorporation into immune complexes (54-57). While there is evidence that all of these modifications may occur in vivo, oxidative modification has been the most extensively studied and there are several lines of evidence indicating that it plays a significant role in the pathogenesis of atherosclerosis (15, 35, 58, 59). The first line of evidence is that arterial endothelial cells, smooth muscle cells and macrophages are capable of inducing oxidative modification of L D L , and L D L modified in this fashion has been shown to bind with high affinity to scavenger receptors on macrophages (50-53, 60-63). A complete discussion of other putative scavenger receptors, including SRAI/II, macrophage receptor with collagenous structure (MARCO), CD36, macrosialin/CD68 and LOX-1 is beyond the scope of this introduction [see (64) for review]. However, it has been shown that about one-third of ox-LDL uptake by macrophages is mediated by SR-AI/II (65), and CD36 may contribute a similar amount (66-69).  A second line of evidence  implicating o x - L D L in atherosclerosis is that immunohistochemical analyses  of  atherosclerotic lesions demonstrated the presence of ox-LDL using antibodies directed against ox-LDL (70-72). Moreover, L D L extracted from atherosclerotic lesions has been  6  shown to have alterations consistent with oxidative modification (73, 74). A third line of evidence is that oxidized L D L has been shown to have numerous pro-atherogenic biological actions in vitro and in vivo, as discussed in section 1.1.3. Finally, a critical line of evidence that indicates a causal role for ox-LDL in atherogenesis is that antioxidants can inhibit progression of atherosclerosis in some animal models (75).  7  1.1.2  Mechanisms of Oxidation and Structural Changes Associated with LDL The physical and chemical changes that occur in the L D L particle that impart  recognition by one or more scavenger receptors for oxidized L D L are very complex and incompletely understood [reviewed in (76)]. Oxidation of L D L by cells involves a transition metal-catalyzed,  free radical-mediated lipid peroxidation process in which  the  polyunsaturated fatty acyl residues in the L D L lipids are degraded to a variety of lipid peroxidation products. These products include hydroperoxy and hydroxy fatty acids (77-79), as well as aldehydes and hydroxyaldehydes (80, 81).  The aidehydie lipid peroxidation  products that are formed are highly reactive and can derivatize the free amino groups of apolipoprotein B (apoB)(10). Derivatization of the lysine e-amino groups on apoB results in an increased negative charge on the L D L particle and prevents interaction with the native L D L receptor, but it permits binding to and unregulated uptake by scavenger receptors on phagocytic cells (9, 10, 53, 81, 82). Although the altered protein moiety had been identified as an important component in scavenger receptor recognition (81, 82), recent evidence has demonstrated that the lipid fraction of ox-LDL can also compete for the uptake and degradation of ox-LDL by macrophages (83, 84).  Interestingly, it has been shown that  oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for receptor recognition (85, 86). Furthermore, during metal-catalyzed oxidation, apoB is also modified through radical-mediated scission of peptide bonds, and probably also by direct reaction with fatty acyl hydroperoxides (87).  8  Oxidation of L D L by metal ions is also  accompanied by loss or destruction of endogenous antioxidants (ubiquinone, vitamin E , carotenoids), oxidation of cholesterol esters and free cholesterol, and by hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (lysoPC) through the action of a lipoprotein-associated phospholipase A acetylhydrolase (88).  2  known as platelet activating factor (PAF)  Therefore, both the lipid and protein moieties can be oxidatively  modified, and in fact, most of the components, major and minor, including antioxidants are modified yielding a radically different physical and biochemical lipoprotein from native L D L . In addition to depletion of antioxidants and loss of polyunsaturated fatty acids, other changes may include aggregation (89-91) increased density and altered particle diameter (9, 92). Interestingly, most of the atherogenic effects of ox-LDL (discussed in section 1.1.3) have been attributed to the oxidized lipid components. These "active" lipid components may include esterified and unesterified peroxidized lipids, lysoPC, cholesterol oxidation products, aldehydes derived from the breakdown of oxidized fatty acids and peroxidized lipids that may associate with apolipoprotein B-100. For example, lysoPC has been shown to be a direct chemotactic factor for monocytes and T-lymphocytes (93, 94) and the importance of oxysterol derivatives in apoptosis is well established (95-99). Arterial cells (endothelial, smooth muscle cells, monocytes and macrophages) have all been shown to be capable of enhancing the rate of oxidation of L D L , in vitro (53). The role of cells in the oxidative modification of L D L appears to be simply to accelerate the rate of peroxidation at low (nanomolar) free metal ion concentrations, either by providing thiols  9  which can reduce transition metal ions to a catalyticaly active form (60, 100) or by "seeding" L D L in the medium with lipid hydroperoxides to initiate a peroxidation reaction (101). In fact, identical changes occur when L D L is oxidized in the absence of cells by incubation with micromolar concentrations of transition metal ions such as copper (79).  Typically, both  cellular oxidation of L D L and transition metal ion oxidation of L D L result in depletion of more than 70% of the L D L linoleic acid and all of the arachidonic acid content, complete loss of endogenous antioxidants, hydrolysis of about half of L D L PC to lysoPC, and more than 30% of apoB lysine residues being derivatized by lipid peroxide decomposition products, resulting in an electrophoretic mobility greater than 2.5 times that of native L D L (10).  Lipoprotein oxidation by cells or transition metal in vitro is blocked by small  concentrations of plasma or plasma proteins (102), such as albumin (103, 104).  As  mentioned previously, oxidation of L D L is more likely to occur within the sequestered "microenvironment" of the subendothelium, where increased levels of copper and iron ions are generally found and antioxidant levels are low (105). Although there is debate on the actual in vivo oxidation event that mediates oxidative modification of L D L , there is evidence implicating myeloperoxidase (106) and/or lipoxygenase pathways (107, 108) as contributing to oxidative stress in the intima. Interestingly, disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis  in apolipoprotein E-deficient mice (109).  Similarly,  immunoreactive myeloperoxidase and HOCl-modified epitopes on L D L have been demonstrated in human atherosclerotic lesions (110-112).  10  1.1.3  The Biological Effects of Oxidized L D L Oxidized L D L has been found to exhibit a number of interesting pro-atherogenic  properties that are absent in native L D L as shown in Figure 1.1.  However, it should be  emphasized that a given biologic action can vary not only in magnitude but even in direction depending on the nature and extent of oxidative modification of L D L as well as the cell type. In some studies, the extent of L D L oxidation was not controlled and/or inadequately characterized. For example, mildly oxidized or "minimally modified" L D L (mm-LDL) has an increased hydroperoxide content compared to native L D L , and unlike extensively oxidized L D L it retains at least some of its endogenous antioxidants. Furthermore, less than 5% of the unsaturated fatty acids are consumed in mm-LDL, and it exhibits minimal derivatization of free amino groups on its apoB component. As a result, the electrophoretic mobility of mm-LDL is similar to native L D L . Biologic effects seen with mildly oxidized L D L include induction of monocyte chemotactic protein-1 (MCP-1) expression by endothelial cells and smooth muscle cells (113), enhanced adhesiveness of cultured endothelial cells for monocytes (114), and expression of the inflammatory cytokines G M CSF, M - C S F (115), and the chemokine G R O (116).  Conversely, extensive L D L  modification is necessary to induce macrophage proliferation (117). Therefore, it becomes apparent that there is a great deal of molecular heterogeneity in what is broadly termed "oxidized L D L " , with a continuous spectrum of oxidative modification from minimal to maximal modification as well as different biological properties thereof. The remainder of  11  this thesis will focus on the atherogenic properties of extensively or maximally oxidized L D L ; referred to as oxidized L D L from this point forward.  The chemical/physical  characteristics and properties of oxidized L D L were discussed in section 1.1.2.  As  mentioned previously, one of the most important properties of oxidized L D L that makes it pro-atherogenic is that it is recognized by scavenger receptors on the surface of macrophages and can potentially give rise to foam cells, which are one of the earliest hallmarks of atherosclerosis (9, 12, 14, 24, 42, 44-46, 118).  A growing list of well over 30 biological  actions have been attributed to oxidized L D L on smooth muscle cells, macrophages and endothelial cells (35).  Table 1 provides a non-exhaustive list of some of the biological  actions of oxidized L D L on monocyte/macrophages that account for foam cell formation as well as some of the pathological complications of atherosclerosis mentioned above.  12  Table 1. Biological properties of ox-LDL on monocyte/macrophages  Biologic Action  References  Atherogenesis Foam cell formation  (12, 14,119-126)  Scavenger receptor expression  (127-131)  Monocyte/macrophage retention  (132-134)  Monocyte adhesion  (135, 136)  Monocyte differentiation  (131, 137)  L D L oxidation  (138-141)  Monocyte chemotaxis  (134, 142)  MCP-1 expression  (113, 143, 144)  Lesion Progression Macrophage survival  (145)  Macrophage proliferation  (117, 146-157)  Plaque Destabilization and Pro-Thrombotic Macrophage death  (95, 158-167)  Metalloprotease expression  (168)  Tissue factor expression  (169, 170)  13  1.2  M A C R O P H A G E SURVIVAL  1.2.1  M - C S F Signaling Macrophage colony stimulating factor (M-CSF) controls the survival, proliferation  and differentiation of cells belonging to the monocyte-macrophage lineage both in vivo and in vitro. For example, M-CSF enables the proliferation and differentiation of committed bone marrow progenitors into macroscopic colonies of macrophages. M-CSF also stimulates the survival of monocytes and macrophages (171, 172). The key role of M - C S F in monocyte/macrophage development has been demonstrated in the osteopetrotic mutant mice which lack functional M-CSF and are deficient in osteoclasts and macrophages, however, these populations can be restored upon exogenous M - C S F injections (173). M - C S F is a growth factor that has its biological activity as a disulfide-linked dimer (174, 175). A l l of the biological effects are mediated by a unique receptor (M-CSFR), encoded by the protooncogene c-ftns. M-CSF dimers cross-link two tyrosine kinase M-CSF receptors, resulting in auto and transphosphorylation of tyrosine residues on the cytoplasmic domains of the M-CSF receptor. The receptor for M-CSF is homologous to receptors for platelet derived growth factor (PDGF) and stem cell factor (SCF) which possess intrinsic tyrosine kinase activity (176). Following ligand interaction, phosphotyrosine residues in the cytoplasmic domain of the receptor induce the translocation of intracellular signaling molecules through SH2 domains (src homology 2) and activate two key intracellular pathways, the PI3K-dependent and Ras/Raf/MEK/ERK M A P kinase-dependent pathway (174, 175, 177).  14  Although  advances have been made in describing the signal transduction molecules activated in response to M-CSF, the details of the pathways leading to their activation are still relatively unknown. The role of M-CSF in controlling G, progression has been shown and continued stimulation by M-CSF is required for the synthesis of D-type cyclins as is the regulation of cmyc (178-180). Conversely, relatively little is known about the signal transduction pathways activated by M-CSF involved in maintaining macrophage survival. Numerous mammalian M A P kinase members have been discovered including p42/p44 M A P kinase or extracellular regulated kinases 1/2 (ERK 1/2), c-Jun NH(2)-terminal protein kinase (JNK) or stress activated protein kinases (SAPK) and p38 kinases [see (181, 182) for review]. In a prototypical M A P kinase-signaling module, the kinase-core consists of a minimum of three kinases, an upstream serine/threonine kinase, a middle dual specificity kinase and a downstream serine/threonine kinase. For example, one linear phosphorylation signaling cascade is the Raf->MEKl/2->ERKl/2 kinase module. A number of recent studies have suggested roles for different members of the M A P kinase family in promoting either cell survival and/or proliferation in response to growth factor stimulation by E R K 1/2, or apoptosis, in response to various stress stimuli through activation of JNK/SAPK or p38 (183185).  Of particular relevance, Jaworowski and colleagues found that M - C S F weakly  activated p38 M A P K , and did not activate JNK in bone marrow derived macrophages. Moreover, it was found that there was no role for either p38 or JNK in macrophage apoptosis induced following M-CSF withdrawal (186). Although M-CSF activated E R K activity, the  15  upstream dual specificity M E K inhibitor PD 98059 did not promote apoptosis, thereby excluding the M E K l / 2 - > E R K l / 2 pathway as essential for the M - C S F survival signal. In contrast, the importance of the PI3K/PKB cascade in macrophage survival has been identified recently.  Mice deficient in either SHIP (SH2 domain containing inositol  phosphatase) or P T E N (phosphatase with homology to tensin) have expansion of tissue macrophage populations (187).  More importantly, Kelley and colleagues recently  demonstrated in human monocytes and in NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors, that M-CSF promoted survival in a PI3K/PKB-dependent manner (188). However, the downstream targets of PKB that mediate survival by M - C S F remain to be identified.  16  1.3  PI3K/PKB SIGNALING CASCADE  1.3.1  PI3K Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric lipid kinase consisting of a  p85 regulatory subunit and a p i 10 catalytic subunit and is capable of triggering a plethora of biological responses, including cell survival, cell proliferation/mitogenesis, cell cycle regulation, membrane trafficking, glucose transport, cell metabolism,  cytoskeletal  rearrangement, and membrane ruffling. PI3K can be divided into 3 categories (Class I and A  Class I , Class II and Class III) based on sequence homologies, lipid substrate specificity and B  structure [see (189) for review]. For example, Class I PI3Ks use phosphatidylinositol (4,5)P  2  as their preferred substrate whereas Class III only use phosphatidylinositol as a substrate. PI3K catalyzes the transfer of the y-phosphate from ATP to the D-3 position of the inositol ring of phosphatidylinositol to yield increases in phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P ) and phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P ) as shown in Figure 1.2. 3  2  These phospholipid intermediates may act as second messengers and/or regulators of proteinprotein and protein-lipid interactions as will be discussed for P K B through binding to pleckstrin homology (PH) domains. The levels of PI(3,4,5)P are negatively regulated in 3  cells by the lipid phosphatases, the inositol 5-phosphatase SHIP yielding PI(3,4,)P (187) and 2  the inositol 3-phosphatase PTEN yielding PI(4,5)P (190). Through the dephosphorylation of 2  PI(3,4,5)P , PTEN and SHIP constitute an important class of regulatory enzymes. Although, 3  PI3K has many pleiotropic properties, it was first identified as an important regulator of  17  ...  growth-factor survival in PC 12 cells, Rat-1 and REF52 cells that underwent apoptosis following either serum-deprivation or treatment with the PI3K inhibitors wortmannin and LY294002 (191). Further studies revealed that PI3K is required for promoting cell survival in a variety of cell types including fibroblasts (192) hematopoietic cells (193-195), neurons (196, 197) and macrophages (198) as well as a host of others. Furthermore, the ability of constitutively active PI3K to attenuate cytokine withdrawal induced apoptosis further supports a role of PI3K in survival (199). Although many PI3K downstream targets may be involved mediating survival responses (200), including protein kinase C (PKC) (201-207), recent attention has focussed on protein kinase B (PKB).  18  ADP  phosphatidyl-inositol * Ptdlns  Figure  1.2.  Phosphatidylinositol structure and the point of action of PI3K.  Vanhaesebroeck B., and Alessi D.R. (2000). Biochem. J., 346, 561-576.  19  1.3.2  P K B  PKB (c-Akt) is a 57 kDa protein serine/threonine kinase that is the cellular homologue of the transforming oncogene of the AKT8 retrovirus (208, 209). There are three known mammalian isoforms of PKB: PKBoc, PKB|3 and PKBy. These three isoforms are encoded by three independently regulated but closely related genes that have a greater than 85% sequence identity and the same structural organization of the protein products (210212). The A G C family proteins contain a central kinase domain with specificity for serine or threonine residues on substrate proteins. The amino terminus of PKB includes a PH domain (213-215), which mediates lipid-protein and/or protein-protein interactions, and the carboxyl terminus has hydrophobic and proline-rich domains (209, 216). PI3K-generated  phospholipids,  phosphatidylinositol  3,4-bisphosphate  and  phosphatidylinositol 3,4,5-trisphosphate act by multiple mechanisms to regulate P K B activity. One mechanism is through the direct binding of phosphoinositides with the PKB PH domain (217-219).  This interaction results in the translocation of PKB from the  cytoplasm to the inner surface of the plasma membrane, where the products of PI3Kgenerated 3'-phosphorylated phospholipids reside (189). Translocation of PKB to the plasma membrane brings PKB in close proximity to regulatory kinases that phosphorylate and activate PKB. Alessi and colleagues identified four sites (Ser-124, Thr-308, Thr-450 and Ser-473) on PKB that are phosphorylated in vivo. Whereas the Thr-308 and Ser-473 are inducibly phosphorylated following stimulation, Ser-124 and Thr-450 are constitutively  20  phosphorylated. Mutational analysis revealed that phosphorylation of Ser-473 and Thr-308 are both necessary for full enzymatic activity (220), and therefore phospho-immunoblotting for Ser-473 or Thr-308 serves as an excellent marker for PKB activation. Thr-308 is located in the activation loop of the central kinase domain of PKB and is phosphorylated by phosphoinositide dependent kinase 1 (PDK1) (221, 222). PDK1 is a 63 kDa serine/threonine kinase that upon activation, binds phosphoinositides to its PH domain which then anchor it to the plasma membrane where it then phosphorylates members of the A G C subfamily of kinases, such as PKB that are also recruited to the plasma membrane by phosphoinositides (221-225).  Because the in vivo phosphorylation of Thr-308 by PDK1 is abolished by  mutations to the PH domain of PKB, it has been suggested that binding of phospholipids to the PH domain of PKB may induce a conformational change that permits phosphorylation of Thr-308 by PDK1 (223, 226).  The identity of the kinase that phosphorylates Ser-473,  operationally termed PDK2, remains unknown. A number of putative PDK2 molecules have been suggested, including M A P K A P kinase-2 (227), integrin linked kinase (ILK) (228), P D K - l / P R K - 2 complex (229) or autophosphorylation following Thr-308 phosphorylation (230). As discussed above, the inositol 5-phosphatase SHIP and the inositol 3-phosphatase PTEN act to down regulate PKB activity by down regulating PI3K activity, and therefore constitute an important class of negative regulatory enzymes.  Given the importance of PI3K in mediating survival, there has been significant interest in identifying the molecular targets of PI3K that block apoptosis.  21  As mentioned  above, a number of these PI3K downstream targets have been implicated in the suppression of apoptosis, including P K C (201-207). Recent data, however, has suggested that P K B activity is sufficient to block apoptosis induced by a number of death stimuli, including cytokine-withdrawal (193, 231-234), T N F - a (235), CD95-and Fas/FasL (236).  As well,  PKB is reported to mediate survival induced by a plethora of survival/growth factors (237243).  Of particular interest, it has been shown that the pro-survival effect of M - C S F is  mediated through activation of the PI3K/PKB signaling cascade (188), thereby implicating an important role of PKB in macrophage survival. One mechanism by which PKB may mediate cell survival is by inactivating members of the intrinsic cell death machinery. Several components  of the apoptotic machinery containing the P K B consensus  phosphorylation sequence, R X R X X S / T have been demonstrated to be PKB downstream targets (224, 226).  These may include an inhibitory phosphorylation of the pro-apoptotic  Bcl-2 protein Bad (233, 244-246), caspase 9 (247, 248), transcription factors of the Forkhead family (249-258), and two kinases, glycogen synthase kinase (GSK-3p) (228, 259-264) and I K B kinase (IKK), which regulates N F - K B transcription factor through its cytoplasmic inhibitor I K B (265-269). PKB has also been shown to inhibit the release of cytochrome c from the mitochondria (270). PKB may act through serine/threonine phosphorylation of various substrates to prevent apoptosis (274). One phosphorylation-dependent mechanism involves 14-3-3 proteins, which associate with phosphorylated proteins and prevents their  22  interaction with targets.  For example, consequent to phosphorylation the Forkhead  transcription factors are sequestered in the cytosol through interaction with 14-3-3 proteins, away from their nuclear targets, such as the FasL gene, which in turn can lead to apoptotic signaling through the activation of caspase 8 and effector caspases as discussed in section 1.4 (250, 275).  Work by others has suggested the existence of PKB-independent pathways  leading to Bad phosphorylation (271-273), thereby suggesting both P K B dependent and independent pathways mediating survival at the level of Bad.  23  1.4  APOPTOSIS  1.4.1  Caspases Apoptosis, or programmed cell death is essential to normal development and tissue  homeostasis, and alterations in apoptosis has been implicated in many disease processes including cancer and atherosclerosis (4, 276). The apoptotic phenotype is characterized by a number of distinct features including ruffling or blebbing of the cell membrane, loss of phospholipid asymmetry of phosphatidylserine, cytoplasmic shrinkage, chromatin condensation and D N A fragmentation.  These hallmark features generally distinguish  apoptosis from other forms of cell death such as necrosis (277). Of particular importance in apoptosis, is the activation of a group of aspartate-specific cysteine proteases known as caspases. In 1993, researchers discovered that the Caenorhabditis  elegans cell death gene,  ced-3 had remarkable sequence similarity to interleukin- l(3-converting enzyme (ICE or caspase 1), a mammalian protease responsible for proteolytic maturation of pre-interleukin1(3 (278, 279). This seminal finding identified the first two members of the caspase family and suggested that these proteases might function during apoptosis.  Many subsequent  studies have identified over a dozen caspase family members important in apoptosis and/or inflammation [reviewed in (280, 281)].  Sequence analysis and x-ray crystallography data suggest that all caspases share a common structure and are expressed as pro-enzymes or zymogens that contain an N-terminal  24  prodomain, a large (-20 kDa) subunit containing the active site cysteine within a conserved Q A C X G motif, and a small (-10 kDa) subunit (282). An aspartate cleavage site separates the prodomain from the large subunit, and an interdomain linker containing one or two aspartate cleavage sites separates the large and small subunits. Caspase activation follows proteolysis of the interdomain linker allowing the large and small subunit to associate to form a heterodimeric enzyme, consisting of two large/small subunit heterodimers. The individual caspases have two major structural differences: substrate preference sequences and length of caspase prodomain. Furthermore, caspases can be further divided into two functional groups: the initiators, such as caspase 8 and caspase 9, which in turn lead to the activation of the effectors, such as caspase 3, caspase 6 and caspase 7 (281, 282). Caspase 3 is regarded as one of the central executioners in the apoptotic cascade and is activated in many cell types after exposure to apoptotic stimuli and is responsible for cleaving a variety of different proteins, including nuclear proteins, such as poly (ADP-ribose) polymerase (PARP), proteins involved in signal transduction, such as M E K K 1 and PKB, and cytoskeletal targets (283-286). This proteolysis of proteins essential for structural, metabolic and repair processes commits the cell to die. Apoptosis differs from other forms of cell death such as necrosis in that it is a coordinated energy dependent process, and ends with the clearance of apoptotic cells and apoptotic bodies by phagocytic cells, such as macrophages rather than mere disintegration and leakage of cytoplasmic contents into the interstitium (287-290). Caspase activation has also been shown to lead to the loss of phosphatidylserine  25  asymmetry in the plasma membrane, resulting in the exposure of phosphatidylserine to the outer leaflet of the plasma membrane. In most cells, an asymmetry of phospholipids is maintained with phosphatidylserine and phosphatidylethanolamine being confined to the inner leaflet of the cell membrane (291). This loss of asymmetry may be critical in the recognition and clearance of apoptotic bodies by macrophages (292-298). Interaction of phosphatidylserine on the cell surface of the apoptotic cell with phosphatidylserine receptors on the phagocytosing cell constitutes one important mechanism of removal (294, 299, 300). Caspase 3 activation may take place either within death receptor complexes of the cytoplasmic membrane in a caspase 8 dependent manner or by a mitochondria-dependent mechanism within the cytosol through caspase 9 activation (282). The initiation of caspase 3 activation by death receptors involves the binding of extracellular death signal proteins, such as T N F - a and FasL to their cognate cell surface receptors. The death receptors contain a distinct cytoplasmic domain comprising about 80 amino acids known as the "death domain" that is critical for their apoptotic function by recruiting intracellular adapter proteins to the cell membrane and subsequently interacting with and leading to the activation of caspase 8, which in turn activates caspase 3 through cleavage (301). The mitochondrial death signal involves the release of three apoptotic protease-activating factors (Apafs) from the mitochondria that lead to activation of caspase 3. Apaf-1, the mammalian homologue of Ced-4 (302), acts downstream of Bcl-2 anti-apoptotic proteins but upstream of caspase 3. Apaf-1 contains a conserved amino acid sequence in its prodomain, known as the caspase  26  recruitment domain (CARD), which interacts with the C A R D domain of caspase 9 (SOSSOS). In the presence of cytochrome c (Apaf-2) and deoxyadenosine triphosphate (dATP), Apaf-1 binds to and activates caspase 9, which subsequently cleaves and activates caspase 3 (306). Together, Apaf-1, cytochrome c, dATP and caspase 9 form a functional complex known as the apoptosome that leads to the activation of caspase 3.  27  1.4.2  Bcl-2 Family Members Genetic analysis of C. elegans Ced-9 gene revealed that it is homologous to the  mammalian protein Bcl-2. Bcl-2 was initially discovered as an over-expressed protein in human B-cell lymphomas by virtue of its reciprocal translocation (tl4:18), where the gene fell under the control of the immunoglobulin heavy chain intron enhancer (307, 308), resulting in a transcriptional upregulation. Bcl-2 was initially found to inhibit cell death induced by IL-3 deprivation in a lymphoid and a myeloid cell line (309). Subsequently, it was shown that over-expression of Bcl-2 protects many cell types against apoptosis in response to a diverse array of apoptotic stimuli, including oxysterol induced apoptosis of macrophages (95). A large number of Bcl-2 related proteins have been isolated; these can be classified into three categories: 1. Anti-apoptotic members, such as Bcl-2, Bcl-X , Bcl-w, Mcl-1, A l (Bfl-1) and Boo, all of L  which exert anti-death activity and share sequence homology, particularly within four regions, Bcl-2 homology (BH) 1 through BH4. These domains have been shown to mediate protein interactions. For example, BH1 and BH2 domain are required for Bcl-2 and Bcl-X to suppress apoptosis (310). L  2. Pro-apoptotic members such as Bax, Bak, Bad, Mtd/Bok, Diva, which share sequence homology in BH1, BH2 and BH3, but not in BH4, although significant homology at BH4 has been seen in some members.  28  3. The "BH3-only" members, are pro-apoptotic proteins which include Bik, Bid, Bim, Hrk/DP5, Blk and Bnip3 and share sequence homology only in BH3. The Bcl-2 family members have been shown to be membrane associated and cytosolic (311). One of the unique features of Bcl-2 family proteins is heterodimerization between anti-apoptotic and pro-apoptotic proteins, which is considered to inhibit the biological activity of their partners (312, 313). This dimerization is mediated by the insertion of a BH3 region of a pro-apoptotic protein into a hydrophobic cleft composed of BH1, BH2 and BH3 from an anti-apoptotic protein (314). A possible function of the Bcl-2 family members came from the three-dimensional structure of B c l - X (315). The protein showed a structural homology to the pore-forming L  domains of certain bacterial toxins, such as diphtheria toxin and the colicins A and E l . These bacterial toxins are pore forming proteins which function as pore forming proteins/channels for ions or small proteins.  The structure of B c l - X , lacking the L  hydrophobic C-terminal domain, consists of two central hydrophobic helices (a5 and a 6) surrounded by five amphipathic helices, as well as a 60-residue flexible loop. The BH1, BH2 and BH3 domains are all located in close proximity on the surface of the protein and form an elongated hydrophobic cleft which has been shown to represent the binding site for the BH3 domain of Bak. Although this notion as channel forming proteins is consistent with the finding that some Bcl-2 members such as Bcl-2, Bcl-X and Bax, can form ion channels in L  synthetic lipid membranes (316-320), it still remains to be determined whether Bcl-2 family  29  members actually form ion channels in vivo and whether these proteins regulate apoptosis through the creation of such channels. As discussed above, mitochondria play an important role in many forms of apoptosis by releasing apoptogenic factors such as cytochrome c and Apafs (321-323) and apoptosisinducing factor (AIF) (324) from the intermembranous space of the mitochondria. Three models have been proposed for the release of cytochrome c from the mitochondria during apoptosis: (A) swelling and physical rupture of the outer mitochondrial membrane, (B) a channel formed by pro-apoptotic Bcl-2 family members, such as Bax, and (C) cytochrome c release regulated by the membrane permeability transition (PT) pore (311).  The  mitochondrial PT is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes. The PT pore participates in the regulation of matrix C a , voltage-, transmembrane potential (At)/) and volume. 2+  Opening of the  mitochondrial PT pore leads to the uncoupling of the respiratory chain, cessation of A T P synthesis and production of free radicals (superoxide anions). Moreover, opening of the PT pore causes an increase in mitochondrial matrix volume, resulting in local mechanical disruption of the outer mitochondrial membrane with consequent release of soluble intermembranous proteins, such as cytochrome c and AIF, resulting in the activation of caspases (325).  The pro-survival Bcl-2 family proteins may function by suppressing  caspase-dependent apoptosis by inhibiting cytochrome c release (326, 327). In addition, BclX b u t probably not Bcl-2 has an additional ability to prevent caspase activation by L  30  sequestering Apaf-1 (328, 329).  Therefore, both anti-apoptotic and pro-apoptotic Bcl-2  members have been shown to play the role of mitochondrial "gatekeepers" in regulating mitochondrial release of cytochrome c and activation of the downstream execution phase of apoptosis (311).  31  1.5 C E R A M I D E A N D APOPTOSIS 1.5.1 Ceramide Generation and Metabolism The membrane sphingolipid, ceramide serves as a second messenger for cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis. The effect of ceramide generation in a given setting depends on the rate and magnitude of ceramide formation, coupling to downstream effectors, and the activity of enzymes that convert ceramide into other metabolites, some of which have opposing actions to ceramide [see (330) for review]. Evidence of a supportive role for ceramide in apoptosis includes the ability of cell-permeable ceramide analogs to induce apoptosis (331-335), as well as the strong correlation between production of ceramide and subsequent cell death (336). The generation of ceramide may constitute an important stress-induced apoptotic signaling event. Examples of cellular stresses that have been linked to ceramide production are T N F - a , (337341), ionizing radiation (342, 343), chemotherapeutic drugs (336, 344), as well as oxidized L D L (96, 161). There are two potential sources of ceramide: sphingomyelin hydrolysis by sphingomyelinases or de novo synthesis via ceramide synthase.  Sphingomyelinases are  sphingomyelin specific forms of phospholipase C, which hydrolyze the phosphodiester bond of sphingomyelin to produce ceramide and choline phosphate (345-347). There are five different forms of sphingomyelinases, distinguished by different pH optima and referred to as acid, neutral and alkaline (330). De novo synthesis of ceramide is through the enzymatic  32  condensation of serine and palmitoyl-CoA to from 3-ketosphinganine. 3-ketosphinganine is reduced to dihydrosphingosine by an NADPH-dependent reductase and then acylated by ceramide synthase to yield dihydroceramide. Introduction of a trans-4,5, double bond generates ceramide (330).  Once generated, ceramide may be converted to various  metabolites. Phosphorylation by ceramide kinase (348-350) generates ceramide 1-phosphate, while deacylation by various ceramidases yield sphingosine, which may then be phosphorylated to sphingosine 1-phosphate (345). Ceramide may also be converted back into sphingomyelin by the transfer of phosphocholine from phosphatidylcholine to ceramide by sphingomyelin synthase (346). Furthermore, ceramide can be glycosylated by various enzymes in the Golgi apparatus to form complex glycosphingolipids. Figure 1.3 summarizes the generation and metabolism of ceramide.  33  Ceramide synthase  Sphinganine  Dihydroceramide desaturase  ^* Dihydroceramide  3-Oxosphinganine reductase  ^ Ceramide ^  Ceramide synthase  3 -Oxosphinganine A  SMase  tl  ^ Sphingomyelin  SM synthase  | | Ceramidase  Sphingosine Serine palmitoyltransferase  Serine + palmitoyl-CoA  SIP phosphatase  Sphingosine kinase  Sphingosine-1 -Phosphate ^  SIP lyase  Phosphoethanolamine + hexadecanol  Figure 1.3. Ceramide generation and metabolism. Adopted from Mathias, S., Pena, L . A . , and Kolesnick, R.N. (1998). Biochem. J., 335, 465-480.  34  1.5.2  Ceramide Induced Apoptosis The mechanisms by which ceramide promotes apoptosis remain unresolved.  However, a number of mechanisms have been postulated including activation of SAPK/JNK and inhibition of survival pathways such as PKB. The JNK pathway is activated in response to various stress stimuli and leads to the trans-activation of various transcription factors, such as c-jun and jun-D (351) and the subsequent transcription of death promoting genes, such as FasL and T N F - a (275, 350). Treatment of cells with cell-permeable ceramides activates the JNK pathway, and disruption of JNK signaling by over-expression of dominant-negative cjun attenuates ceramide-induced apoptosis (352). Ceramide may activate the JNK signaling pathway either via transforming-growth-factor(3-activated kinase (TAK1) (353) or via the small G-protein Rac-1 (354).  Furthermore, it has recently been shown that J N K can  translocate to the mitochondria and phosphorylate Bcl-2 and B c l - X at an inhibitory site, L  thereby preventing their anti-apoptotic functions (355). These and other studies suggest an important role for the JNK signaling pathway in ceramide-mediated apoptosis (341, 356359).  Ceramide has also been shown to inhibit the pro-survival PKB pathway (360-363).  Therefore, it becomes apparent that ceramide may induce apoptosis by increasing proapoptotic signaling and/or by decreasing anti-apoptotic signaling in a given cell system in response to a given stimulus. Other mechanisms by which ceramide may induce apoptosis include ceramide induced mitochondrial permeability and the release of cytochrome c or AIF (364-369), the generation of reactive oxygen species (ROS) (369-373) and ceramide induced 35  Bcl-2 dephosphorylation (374). For example, ceramide has been shown to disrupt the mitochondrial membrane potential (Av|/) (375-378). A y disruption seems to be an obligatory step during early apoptosis and is stabilized by Bcl-2 and Bcl-X (379). Furthermore, Bcl-2 L  or B c l - X prevent ceramide formation by repressing neutral sphingomyelinase as well as L  ceramide-induced cytochrome c release (380).  Therefore, ceramide may lead to the  activation of caspases through the release of cytochrome c and AIF (324). Indeed, a number of studies have implicated that ceramide acts proximal to the activation caspase 3 (381-385). The biological actions mediated by ceramide are believed to act via the activation of three putative targets, ceramide activated protein phosphatases (CAPP), ceramide activated protein kinase (CAPK) and protein kinase Ct, (330). For example, both the ceramide mediated dephosphorylation of Bcl-2 (374) as well as inhibition of P K B (360, 361) have been attributed to the action of ceramide activated protein phosphatases.  36  1.6  OX-LDL MEDIATED MACROPHAGE DEATH, PROLIFERATION AND SURVIVAL SIGNALING The discussion in section 1.1.1, highlighted the role of macrophages in the  inflammatory arteriopathy known as atherosclerosis through their effects on vessel diameter, endothelial function, smooth muscle migration, and destabilization through metalloproteasemediated matrix dissolution (15, 34).  Therefore, a comprehensive understanding of the  factors that regulate macrophage numbers and/or function in atherosclerotic lesions is of obvious importance and could lead to new targets for therapeutic intervention.  The  importance of oxidized L D L on regulating macrophage density has centered principally on two cellular processes: proliferation and survival at low concentrations and cytotoxicity at higher concentrations. This bipartite effect of ox-LDL is compatible with the notion that oxL D L plays a role in atherogenesis by leading to the formation and survival of the histologic hallmark of atherosclerotic lesions, the macrophage derived foam cell, but also more extensively in the development of the structure typical of the atherosclerotic lesion, with focal excessive proliferation alternating with cell death. Whereas ox-LDL induces a wide range of cellular responses in macrophages including death, proliferation and survival, the factors determining these responses are not clearly understood. However, it has been shown that minimally oxidized L D L induced proliferation and macrophage activation whereas extensive modification induced cell death at the same concentration (386). Moreover, oxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending  37  on the strength and exposure time (386). Therefore, one important variable in determining the biological action of ox-LDL, may relate to the physical/chemical nature of what is broadly termed "ox-LDL" as well as its concentration.  For example, the oxysterol  component of ox-LDL has been shown to be cytotoxic (95, 98, 99, 387), whereas the lysoPC component has been implicated in mediating macrophage growth in some studies (151, 153, 155, 388). Therefore, the importance of the observed biological response may depend on the activation of death, proliferation and/or survival intracellular signaling events in response to these factors.  38  1.6.1  Ox-LDL and Death Signaling The existence of ox-LDL as a cytotoxic factor for macrophages has been established  since 1993 (166, 167) and an abundance of literature has examined the role of ox-LDL in promoting macrophage apoptosis (20, 95, 160, 161, 163, 164, 389-391) as well as necrosis (386). Furthermore, the oxysterols, 7(3-hydroxycholesterol and 7-ketocholesterol have been shown to have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts (97). Although the existence of macrophage apoptosis in atherosclerotic plaques is well established (4, 16), to date there is a paucity of information pertaining to the role of intracellular signaling cascades translating these events. O x - L D L has been shown to induce apoptosis in the monocyte cell line THP-1 (158) in a caspase dependent manner, whereas after differentiation to macrophage-like phenotype induced by P M A , THP-1 cells are resistant. One of the important changes associated with the differentiation of monocytes to macrophages is an increase in the expression of the scavenger receptors, such as SR-AI/II (130, 392, 393).  Recent studies have shown that  expression of these receptors confers resistance of macrophages to the apoptotic stimulation by ox-LDL and well as its cytotoxic lipid component, 7-ketocholesterol.  These results  suggest that by preventing apoptosis, SR-AI/II may contribute to the long-term survival of macrophages and macrophage-derived lipid-laden foam cells in atherosclerotic lesions (98). This effect, however, may be cell type specific as scavenger-receptor gene transfer to rabbit  39  aortic smooth muscle cells leads to functional endocytotic receptor expression, foam cell formation, and increased susceptibility to apoptosis (394). To date, there is no published information on apoptotic signaling cascades in macrophage cell death. With this in mind, however, JNK are thought to be crucial in transmitting transmembrane signals required for cell apoptosis in vitro in a number of cell systems (341). Recently, Metzler and colleagues identified the localization and activity of SAPK/JNK to both smooth muscle cells and macrophages in atherosclerotic lesions (395). It has also been shown that Chinese hamster ovary (CHO)-Kl cells expressing human CD36 rendered these cells susceptible to killing by ox-LDL (99). CD36 may also be involved in the mediation of intracellular signaling through the activation of N F - K B (396). Interestingly, the uptake of ox-LDL has been reported to be diminished in macrophages from CD36-deficient patients; macrophages may also have an impaired response of ox-LDL induced N F - K B activation and subsequent expression of cytokines, such as T N F - a and IL-lp that may mediate apoptosis (397). The role of ox-LDL in activating N F - K B in macrophages is controversial with certain groups reporting activation. For example, short-term incubation of THP-1 monocytes with ox-LDL activated N F - K B and induced the expression of the target gene IL-8. This activation of N F - K B , however, was inhibited by long-term treatment with ox-LDL (398).  O x - L D L has also been reported to  inhibit lipopolysaccharide-induced binding of N F - K B to D N A and the subsequent expression of T N F - a and I L - l p in macrophages (399-401).  40  Oxidized L D L induced apoptosis in  endothelial cells following hypoxia-reoxygenation involves the activation of P K C and protein tyrosine kinases (PTK) (402). The activation of both PKC (149, 403-405) and PTK (117) by ox-LDL is well established in macrophages. Furthermore, activation of p38 M A P kinase by oxidized L D L in vascular smooth muscle cells is associated with oxidized L D L induced cytotoxicity (406). It has also been reported that ox-LDL leads to the activation of p38 M A P K in THP-1 monocyte cell line (142). The role of these and other intracellular signaling pathways involved in translating a death signal in macrophages remains to be elucidated. As discussed above, ceramide is an important mediator of the apoptotic response in many cells types.  It has also been reported that neutral lipids in ox-LDL, but neither  phospholipid nor lysoPC, induced endothelial apoptosis by activating membrane sphingomyelinase and causing ceramide generation in a superoxide-dependent manner (96), thereby implicating an important role of the lipid second messenger in this process. With respect to macrophages, Kinscherf and colleagues showed that ox-LDL-induced apoptosis in human macrophages is associated with a concomitant induction of p53 and manganese superoxide dismutase, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway (161). Conversely, work in our laboratory has suggested that ox-LDL mediated macrophage survival is associated with a reduction in the generation of ceramide and that ceramide pre-treatment markedly diminishes survival, thereby  41  suggesting an important role of ceramide regulation in the apoptotic response in macrophagi (manuscript in preparation).  42  1.6.2  Ox-LDL and Proliferation Signaling The mechanism that has by far received the most attention with respect to  contributing to macrophage density is macrophage proliferation. Oxidized L D L has been shown to induce the proliferation of both mouse peritoneal macrophages (117, 146-152, 155157, 403) and human monocyte-derived macrophages (153). Until recently, it was thought that smooth muscle cells are the major proliferating cell type found in atherosclerotic lesions. However, immunocytochemical studies of human atherosclerotic lesions have shown that macrophages are the predominant cell type expressing proliferating cell nuclear antigen, even in lesions containing cells derived mainly from smooth muscle cells (407, 408). Therefore, it is reasonable to expect that oxidized L D L induced macrophage foam cell proliferation may contribute to lesion formation and progression in vivo. The first work on ox-LDL mediated macrophage proliferation, was attributed to scavenger receptor mediated internalization of lysoPC, a phospholipid which is generated during oxidation through the enzymatic conversion of phosphatidylcholine (155). It was also shown that oxidized L D L induced cell growth was effectively inhibited by anti-GM-CSF antibodies,  suggesting that oxidized L D L promoted growth indirectly through  paracrine/autocrine mechanisms (155). Biwa and colleagues demonstrated that effective endocytosis of lysoPC of ox-LDL by macrophages through SR-AI/II and subsequent P K C activation lead to G M - C S F release into the medium which played a "first-step" or priming role in conjunction with other cytokines in promoting macrophage growth (148).  43  Furthermore, an important role for the PI3K pathway was identified as part of the mitogenic effect accounting for 40-50% of macrophage proliferation induced by oxidized L D L in murine peritoneal macrophages (117). Interestingly, in contrast to previous reports, it was shown that oxidatively modified apoB and not lysoPC is the main growth-stimulating component of oxidized L D L , but that oxidized phospholipids may play a secondary role (157). Thereafter, Biwa and colleagues subsequently showed that PKC plays a role upstream in the signaling pathway to G M - C S F induction, whereas PI3K is involved, at least in part, downstream in the signaling pathway after GM-CSF induction (403). Others have suggested the presence of two intracellular signaling pathways for activation of P K C and ox-LDL mediated macrophage growth, a rise in calcium that was mediated by pertussis toxinsensitive G protein and the internalization of lysoPC through the scavenger receptors (149). Conversely, Hamilton and colleagues reported that neither G M - C S F nor M - C S F play an important role in macrophage survival induced by ox-LDL in macrophage survival (145), but rather that ox-LDL "primes" macrophage proliferation in response to other factors, such as M - C S F or G M - C S F .  As the expression of G M - C S F and M - C S F has been shown in  atherosclerotic lesions, this may also suggest an important "priming" role for oxidized L D L (115, 409-411). M A P kinases represent important intracellular signaling pathways that have been shown to induce proliferation in a number of cell systems (181, 182).  Oxidized L D L  mediated activation of the p42/p44 M A P kinases (ERK 1/2) has been shown in macrophages  44  (142, 412-414); although inhibitory studies have not demonstrated a functional role for activation of these kinases in macrophage proliferation. However, the M E K inhibitor PD 98059 was recently shown to block SMC proliferation (415). In addition, it has been shown that ox-LDL leads to the activation of other mitogenic signaling pathways, such as phospholipase D in macrophages (416). Phospholipase D activation leads to generation of important second messengers such as phosphatidate, lysophosphatidate, and diacylglycerol, which are known to regulate many cellular functions including ox-LDL induced mitogenesis of vascular smooth muscle cells (417). Although, the lipid second messenger ceramide has been reported to induce apoptosis in a number of cell types, Auge and colleagues have recently identified an important role for the lipid second messenger ceramide in ox-LDL mediated smooth muscle proliferation (418, 419).  Furthermore, work on smooth muscle  cells has shown the importance of ceramide turnover to a number of sphingolipid metabolites, such as sphingosine 1-phosphate in the mitogenic effect of ox-LDL (420). A similar mechanism for ceramide or ceramide metabolites may also exist in macrophages as exposure of human monocyte derived macrophages to both oxidized and acetylated L D L induced an approximately 40% elevation of the endogenous level of ceramide (421). Although in this thesis I present data that suggests that ox-LDL blocks the generation of ceramide involved in macrophage apoptosis following M - C S F withdrawal, under the appropriate conditions, ceramide may act to promote survival or induce proliferation either directly or through the conversion to ceramide metabolites, such as ceramide 1-phosphate  45  (422, 423) or sphingosine 1-phosphate (420, 424-427). currently being investigated in our laboratory.  46  This interesting possibility  1.6.3  Ox-LDL  and Survival Signaling  Hamilton and colleagues were the first to demonstrate a pro-survival action for oxLDL on bone marrow derived macrophages (BMDM) (145). The effect of ox-LDL occurred even in the absence of endogenous M-CSF or GM-CSF, thereby suggesting a direct action of ox-LDL rather than an indirect process mediated through cytokine elaboration. To date, this is the only work examining a role of oxidized LDL in mediated macrophage survival. By promoting the survival of local macrophage populations, ox-LDL may also "prime" them so that they may proliferate in response to mitogenic factors such as M-CSF Or GM-CSF that have been shown to be present in atheromas (115, 409-411). Ox-LDL has been reported to cause the release of GM-CSF from macrophages (148, 152, 156, 428-430) as well as from endothelial cells (115). Monocyte proliferation induced by modified serum has also been associated with endogenous M-CSF production (431).  Furthermore, the developing  atherosclerotic lesion has also been shown to be associated with the expression of GM-CSF and M-CSF (410, 432). Interestingly, Shi and colleagues recently demonstrated that the expression of M-CSF from the endothelium by minimally modified L D L varied from susceptible and resistant strains of mice, thereby providing a molecular explanation on the genetic variability to atherosclerosis (428). Furthermore, the advent of genetic mice has also elucidated the importance of M-CSF and macrophages in the atherosclerotic process. For example, it was shown that heterozygous osteopetrotic mutation (op/+) in LDL receptor deficient mice exhibited a 2-fold reduction in M-CSF expression, but had reduced lesion size 47  approximately 100-fold relative to control mice (+/+) (433). As the number of circulating monocytes was approximately the same in both groups, these studies suggest that M - C S F plays an important role in fatty streak formation and progression to more complex lesions. Therefore, by promoting macrophage survival, ox-LDL may create the appropriate "environment" leading to macrophage proliferation, through G M - C S F and/or M-CSF. From a teleological perspective, the ability of ox-LDL to promote macrophage survival, may be related to the role of macrophages as "housekeepers" involved in the removal of apoptotic cells (292-298). Interestingly, ox-LDL but not acetyl L D L inhibited the binding and phagocytosis of non-opsonized, oxidatively damaged red blood cells by mouse peritoneal macrophages (434).  Furthermore, phosphatidylserine liposome binding to  macrophages is also inhibited by ox-LDL but not acetyl L D L (435).  As mentioned  previously, phosphatidylserine expression on the outer leaflet of the plasma membrane, may play a key role in macrophage recognition of oxidatively damaged and apoptotic cells. Although it is still not clear which receptors mediate phosphatidylserine recognition on apoptotic cells, several interesting candidates have been proposed, including two scavenger receptors, CD36 and CD68 (294). Mouse peritoneal macrophages lacking SR-AI/II bound oxidatively damaged red blood cells just as effectively as wild-type macrophages, whereas their binding and uptake of acetyl L D L was reduced by more than 80% suggesting a role other than SR-AI/II in this process (436). Similarly, ox-LDL is also rapidly cleared by the liver in mice with disruption of SR-AI/II gene via Kupffer cells, suggesting a role other than  48  SR-AI/II in this process (437). Furthermore, recent studies implicate the importance for the lipid moiety of ox-LDL in the competitive binding to resident mouse peritoneal macrophages as microemulsions prepared from the lipids extracted from ox-LDL are very effective in inhibiting the binding of oxidatively damaged red blood cells and also, to a lesser extent, apoptotic thymocytes to macrophages (84). The observations that apoptotic cells are under increased oxidative stress with associated oxidative changes to the plasma membrane, such as exposure of phosphatidylserine, and that ox-LDL competes with apoptotic cells for macrophage binding, suggests that both ox-LDL and apoptotic cells may share oxidatively modified moieties, such as oxidized or anionic phospholipids on their surfaces that serve as ligands for macrophage recognition (438). These fascinating series of observations, formed the theoretical framework for this thesis in that ox-LDL may promote the survival of macrophages which are responsible for the clearance of cells undergoing apoptosis or cells in an apoptotic/highly oxidative environment through the activation of intracellular survival signaling pathways, such as M A P kinases and PI3K/PKB.  49  1.7  OBJECTIVES AND HYPOTHESES The role of ox-LDL as a macrophage growth factor is well established and the  signaling events involved are rapidly being elucidated. The preliminary study by Hamilton and colleagues demonstrated a novel mechanism that might account for the persistence of macrophage derived foam cells in the atherosclerotic lesion, namely enhanced macrophage survival (145). The mechanism(s) by which ox-LDL promotes survival, however, remains to be defined. The main objective of this study was to further elucidate the role of oxidized L D L in promoting macrophage survival in B M D M . Bone marrow derived macrophages, represent a homogenous cell population that have an absolute requirement for the macrophage survival cytokine, M-CSF (171, 172), and therefore can be used as a powerful experimental tool to examine macrophage survival in the absence of a proliferation background. Namely, mouse peritoneal macrophages (which our laboratory previously used to demonstrate growth induction by oxidized L D L over a 7-day timecourse) are not optimal for studying effects of oxidized L D L on apoptosis because they die slowly in the absence of growth factors and to a significant extent by non-apoptotic mechanisms. In contrast, B M D M reproducibly undergo apoptosis within 48 hr of cytokine withdrawal, and so effects on apoptosis can readily be studied at an incubation time where significant cell proliferation does not occur. To address this specific objective, the following hypotheses were examined:  50  1. Ox-LDL promotes macrophage survival and does not induce proliferation by specifically preventing apoptotic cell death induced in B M D M following the withdrawal of the macrophage survival cytokine, M-CSF. 2. O x - L D L promotes survival in a cytokine independent manner by directly activating similar intracellular signaling pathways as those activated by M-CSF, namely ERK1/2 andPBK/PKB. 3. Ox-LDL promotes survival by inhibiting the generation of ceramide and the subsequent activation of the caspase 3 apoptotic cascade, through the upregulation or maintenance of the pro-survival member of the Bcl-2 family of proteins, Bcl-X in a PI3K/PKB/NF-KB L  dependent manner.  51  2.  MATERIALS AND METHODS  2.1  Materials D M E M and RPMI 1640 medium were purchased from Canadian Life Technologies.  Hyclone defined fetal bovine serum (FBS) was purchased from Hyclone. M T S [3-(4,5dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium,  inner  salt] was purchased from Promega. Propidium iodide, RNAse and phenazine methosulfate (PMS), fumonisin B I , desipramine, caffeic acid phenylethyl ester (CAPE) and polybrene (hexadimethrine bromide) were purchased from Sigma. C -ceramide, C -ceramide and C 2  ceramide were purchased from Avanti Polar Lipids.  6  8  [ H]palmitate was purchased from 3  Mandel Scientific. Cyclin DI, caspase 3, caspase 8, caspase 9, Bcl-2, Bax, Bad, PKB and ERK1 antibodies were purchased from Stressgen.  G A P D H antibody was purchased from  Advanced Immunochemical. Annexin-V-FITC, Poly (ADP-ribose) polymerase (PARP) and Bad were purchased from B D Pharmingen/Transduction. Centricon microconcentrators were purchased from Amicon.  Phospho-PKB-Ser-473, phospho-ERKl/2-Thr-202/Tyr-204,  phospho-lKB-a-Ser-32 and phospho-Bad-Ser-136 were purchased from New England Biolabs Inc.  Goat anti-rabbit IgG and goat anti-mouse IgG, horseradish peroxidase  secondary antibodies, M E K inhibitors (PD 98059 and U0126), PI3K inhibitors ( L Y 294002 and wortmannin), DEVD-FMK,  NF-KB  inhibitory peptide SN50, and caspase inhibitors ( Z - V A D - F M K , Z -  Z-IETD-FMK,  Z-LEHD-FMK)  52  were purchased from Calbiochem.  Nitrocellulose membrane and low molecular weight protein standards for immunoblotting were purchased from Bio-Rad Laboratories. Reagents for enhanced chemiluminescence were purchased from Amersham International. Molecular Biochemicals.  G M - C S F was purchased from Roche  Anti-GM-CSF antibodies were provided by Dr. John Schrader  (Biomedical Research Centre, Vancouver). Osteopetrotic mice homozygous for a mutation in M-CSF (B6C3Fe-a/a-Csfl ) (op/op) were purchased from the Jackson Laboratory. Bosc op  23 retrovirus packing cell line was kindly provided by Dr. Vince Duronio (Jack Bell Research Centre, Vancouver). R A W 264.7 cells were purchased from American Type Culture Collection. Effectene transfection reagent and Qiagen mini-plasmid purification kits were purchased from Qiagen.  2.2  Lipoprotein Isolation and Oxidation L D L (d= 1.019-1.063) was isolated by sequential ultracentrifugation of EDTA-anti-  coagulated fasting plasma obtained from healthy normolipidemic volunteers (439). The concentrations of E D T A in L D L preparations were reduced prior to oxidation by dialysis against Dulbecco's phosphate-buffered saline (PBS) containing 10 | i M E D T A . Oxidation was performed using 200 |ig/ml L D L in Dulbecco's PBS containing 5 pJVI C u S 0 incubated 4  at 37°C for 24 h and the reaction stopped with 200 u M E D T A and 50 u M B H T (10, 53). Acetylation of L D L was performed by the sequential addition of acetic anhydride (48).  53  2.3  Characterization of LDL Lipoprotein electrophoresis was performed using a Ciba-Corning apparatus and  T I T A N G E L lipoprotein agarose gel in 50 m M barbital buffer (pH 8.6) according to the manufacturer's instructions. Bovine serum albumin (BSA) was added to lipoprotein samples to ensure reproducible migration distances. Lipoprotein bands were visualized by staining with Fat Red 7B. The relative electrophoretic mobility of modified L D L was calculated by dividing the distance traveled during electrophoresis by the distance traveled by native L D L , and satisfactory acetylation or oxidation was confirmed if the relative electrophoretic mobility (R ) was greater than or equal to 3 (extensively modified LDL). O x - L D L protein f  was assayed by the method of Lowry (440) in presence of 0.05% sodium deoxycholate to minimize turbidity using B S A as a standard.  2.4  Cell Culture Bone marrow cells were isolated from the femurs of female C D - I mice or female  op/op mice (B6C3Fe-a/a-Csfl ) mice as described (145). Cells were plated for 24 h in op  RPMI 1640 containing 10% FBS and 10% L-cell conditioned medium (LCM); a crude source of M - C S F , kindly provided by Dr. John Schrader (Biomedical Research Centre, Vancouver) corresponding to approximately 10 000 U/ml of M-CSF. The non-adherent cells were removed and cultured in the above medium until confluence was reached (5-7 days). Thereafter, the cells were harvested using a teflon cell lifter and seeded at 10xl0 cells/well 3  in either 96 well plates, lxlO cells/well in 6 well plate dishes, or 5xl0 cells/100 mm dishes 6  6  54  in RPMI 1640 with 10% FBS, but without M-CSF for 24 h prior to use to render the cells quiescent.  Bone marrow-derived macrophages are a relatively pure and homogenous  population with more than 95% of the adherent cells binding M - C S F (171, 172). R A W 264.7 were cultured in D M E M containing 10% FBS.  2.5  MTS Cell Viability Assay Macrophage survival was determined by the MTS formazan method. This assay is  based on the cellular bioreduction of MTS by mitochondrial dehydrogenase enzymes in metabolically active cells. The quantity of formazan product formed was measured by the amount of 490 nm absorbance and is directly proportional to the number of viable cells in culture. 20 ulAvell of MTS/PMS solution was added to wells containing 100 pi of culture medium in 96 well plates 3 h prior to terminating the experiment.  This resulted in final  concentrations of 333 [ig/ml MTS and 25 u M PMS. After 3 h at 37°C in a humidified 5% C 0 atmosphere, the absorbance at 490 nm was recorded using an ELISA plate reader. For 2  conditioned medium studies, control or ox-LDL treated medium was removed and filtered with 100 kDa microconcentrators prior to reapplication. For inhibitor studies, the cells were pre-incubated with the indicated concentrations of either the M E K , PI3K, or soluble ceramides for 1 h prior to treatment with ox-LDL.  55  NF-KB  inhibitors  2.6  Cell Counts Macrophage cell numbers were determined by culturing B M D M at 0.5xl0 cells/well 6  in 12 well plates as described above. Following treatment, the culture medium was aspirated, cells were washed twice with in 4 ° C PBS, and the adherent cells scraped into a known volume of PBS and counted using a hemocytometer in at least 4 random fields of view. Total adherent cells were counted.  2.7  DNA Fragmentation and Cell Cycle Analysis Macrophages were harvested by scraping and then washed twice in 4°C PBS. Cells  were fixed in ice-cold 70% ethanol for 1 h at - 2 0 ° C , washed three times with 4°C PBS and resuspended in hypotonic fluorochrome buffer consisting of 0.1% Triton X-100, 0.1% sodium citrate, 25 |ig/ml RNAse and 50 |ig/ml propidium iodide. After 24 h incubation at 4°C, fluorescence was measured using a fluorescence-activated cell sorter (Beckman Coulter Epics X L - M C L ) .  Subdiploid D N A and cell cycle analysis was performed on singlet  populations using Expo X L software or WinMDI 3.8 (Joseph Trotter). At least 10xl0  3  cellular events were counted.  2.8  Western Blotting Macrophages were harvested as above and lysed in ice cold homogenization buffer  (20 m M MOPS, pH 7.2, 1% Triton X-100, 50 m M (3-glycerol phosphate, 5 m M E G T A , 2 m M E D T A , 1 m M sodium vanadate, 25 |J,M (3-methyl aspartic acid, 1 m M D T T , 1 m M  56  phenylmethylsulfonyl fluoride, 10 )iig/ml aprotinin and 10 u.g/ml leupeptin). Lysates were centrifuged at 14 000 rpm for 10 min and the protein content of supernatants was quantified with the Bradford protein assay (Bio-Rad). 50 u.g of protein from each sample was loaded and separated by SDS-PAGE using a 10% separating gel.  Gels were calibrated using  prestained SDS-PAGE low molecular weight standards (Bio-Rad).  Proteins were then  transferred to nitrocellulose paper and blocked for 1 h with 4% skim milk, 0.01% NaN in 3  TBS/0.1% Tween 20 followed by incubation with the primary antibody overnight in TBS/0.1% Tween 20 at room temperature.  After three 10 min washes with TBS/0.1%  Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at a 1:5000 dilution for 1 h. Thereafter, the proteins were visualized by using enhanced chemiluminescence.  For inhibitor studies, the cells were pre-incubated with the  indicated concentrations of M E K , PI3K,  NF-KB  inhibitors, or soluble ceramides for 1 h prior  to treatment with ox-LDL.  2.9  Morphological Observations Morphological observations of cultured B M D M and Bosc 23 cells was performed  using a fluorescent and phase microscopy (Nikon ECLIPSE TE300 Inverted Microscope). Immunocytochemistry for Annexin-V was performed according to the supplier's information, by incubating the B M D M for 15 min at room temperature with Annexin-V-conjugated FITC,  57  followed by two washes with PBS and examination by FITC fluorescence in 3 random 200 X fields of view.  2.10  In-Vitro PKB Kinase Assay PKB was immunoprecipitated from 500 fig of protein lysate as determined by  Bradford protein assay and 2 |ig of anti-PKB antibody for 18 h at 4 ° C followed by 30 [ll of protein A Sepharose for 1 h at 4°C. Beads with immunoprecipitated kinases were washed three times with homogenization buffer and once with kinase wash buffer (100 m M Hepes pH 7.0, 2 m M M g C l , 2 m M M n C l , and 2 mM sodium ortho vanadate). 2  2  The beads were  pelleted and the reaction was started by the addition of 25 |il of the kinase reaction buffer (50 mM Hepes pH 7.0, 1 m M MgCl , 1 m M MnCl , and 1 m M sodium orthovanadate, 2 m M 2  2  NaF, 5 tig of H2B and 0.5 |ig of A T P (250 ^tM ATP, 1 p,Ci [y- P] ATP) at 30°C for 30 min. 32  The reaction was terminated with the addition of 10 (ll of 4X samples buffer and resolved on SDS-PAGE using a 15% separating gel, followed by autoradiography for [y- P] labelled 32  histone H2B..  2.11  Ceramide and Sphingomyelin Determination [ H]ceramides were determined following labeling of B M D M with 5 |iCi/ml of 3  [ H]palmitate for 24 h in RPMI 1640 with 10% FBS and 5% L C M . The radioactive medium 3  was aspirated and the cells washed twice with PBS. The macrophages were then incubated for 24 h without M - C S F , 25 |ig/ml ox-LDL, 50 n M fumonisin BI or 10 \xM desipramine.  58  Following termination of the experiments, lipids were extracted by the method of Bligh and Dyer (441). Briefly, cells were scraped into 1 ml methanol, mixed with 1 ml chloroform and 0.9 ml of 2 M KC1/0.2 M H P 0 and the lower chloroform phase was dried under nitrogen 3  4  and ceramides separated by thin layer chromatography (TLC) using Silica Gel 60 coated glass plates. T L C plates were developed with chloroform/methanol/acetic acid (9:1:1 v/v/v) for half their length and then dried. ether/diethylether/acetic  The plates were further separated with petroleum  acid (60:40:1 v/v/v).  Ceramides were identified by co-  chromatography with ceramide standards followed by iodine staining.  The levels of  [ H] sphingomyelin were determined as described above followed by T L C in the following 3  separation solvent: chloroform/methanol/acetic v/v/v/v/v)  with sphingomyelin standards.  acid/formic acid/water (35:15:6:2:1  Levels of radiolabeled ceramide and  sphingomyelin were determined following scraping from T L C plates and liquid scintillation counting.  2.72  LipiaVProtein Separation of Ox-LDL To 0.5 ml of an aqueous solution containing 200 (Xg of ox-LDL in PBS, 1.88 ml of  chloroform:methanol (1:2) was added and vortexed for 5 min at room temperature. 1.25 ml of chloroform followed by 1.25 ml of 2 M KC1/0.2 M H P0 was added. The lower lipid 3  4  containing chloroform layer was dried under nitrogen and then resuspended in 5 pi D M S O and then RPMI 1640/10 % FBS.  The protein component of ox-LDL was extracted from  59  1.8 ml containing 200 |ig of ox-LDL by adding 2 ml of ice-cold methanol followed by 2 ml of chloroform. The mixture was centrifuged at 1800 x g for 10 min to separate the phases and to allow the protein to form a dense interphase. The two phases were removed, leaving the protein band adhering to the wall of the glass tube. The protein was washed twice with 2 ml of ice-cold water and once with acetone. After a final wash with 2 ml of water, 1 ml of aqueous solution of octyl glucoside (6.0 mg/ml) was added to the protein and the mixture vortexed for 5 min at room temperature. The detergent was removed by dialysis against PBS, and the protein concentration determined by the method of Lowry (440).  2.13  Retroviral Infection of BMDM Ecotropic virus packaging cell line BOSC 23 (442), kindly provided by Dr. Vince  Duronio (Jack Bell Research Centre, Vancouver) was grown in 6 well plate dishes in D M E M supplemented with 10 % FBS. These cells were transfected with an active version of PKB that was constructed by fusing the PKB containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor (ER) that selectively binds 4hydroxytamoxifen (4-HT) and tagged with green fluorescent protein (GFP), kindly provided by Dr. Richard Roth (Department of Pharmacology, Stanford) using the Effectene transfection reagent and plasmid D N A purified by Q I A G E N plasmid mini-kit at 50 % confluence (443). The medium was then replaced with RPMI 1640 containing 10% FBS for 24 h after transfection. After an additional 48 h, the retrovirus containing supernatant was sterile filtered and used to infect B M D M cultured in 6 well plate dishes in RPMI 1640  60  containing 10% FBS and 10% L C M and 4 (ig/ml polybrene following centrifugation at 1800 x g for 30 min at room temperature.  The transfection efficiency of Bosc 23 cells and  infection of B M D M with 4-HT/PKB-ER construct was confirmed fluorescent microscopy for GFP as described above.  2.14  Statistical Analysis Results were expressed as mean ± SEM. Statistical analysis was done with A N O V A  or Student's t-test as appropriate. A p value of less than 0.05 was taken as significant.  61  3.  O X - L D L P R O M O T E S M A C R O P H A G E SURVIVAL B Y INHIBITING APOPTOSIS  3.1.  RATIONALE A N D HYPOTHESIS  Previous results from two independent laboratories have shown that oxidized L D L can induce growth in murine peritoneal macrophages. Sakai and colleagues were the first to describe the stimulation of macrophage growth by oxidized L D L . This group concluded that the effect of oxidized L D L required internalization through scavenger receptor class A type I/II, and was due to release of G M - C S F from macrophages by lysoPC in ox-LDL (148, 151, 155). Conversely, Martens and colleagues found that the stimulation of macrophage growth by oxidized L D L depends on structural changes in apoB associated with very extensive oxidation, and that activation of PI3K was required for this effect (117, 157).  Recently,  Hamilton and colleagues demonstrated that ox-LDL promotes the survival of bone marrow derived macrophages.  The mechanism of this effect was not defined, but appears to be  independent of G M - C S F or M-CSF (145). The overall goal of this study was to define the mechanism for the pro-survival role of ox-LDL in macrophages, and to ascertain if this mechanism was the same as that responsible for the growth-inducing effect. To begin, it was important to establish whether the effect of ox-LDL involved inhibition of apoptosis or of other types of cell death, such as necrosis.  Accordingly, initial experiments were done to test the hypothesis that ox-LDL  specifically prevents apoptotic cell death induced by the withdrawal of M-CSF from B M D M .  62  3.2  RESULTS AND DISCUSSION My initial experiments were done to establish optimal conditions for studying the  effect of o x - L D L on the survival of B M D M .  Survival was monitored using the  mitochondrial reduction of a soluble tetrazolium salt (MTS) to a colored formazan product, which has previously been validated as a marker for cell viability (155). Incubation of macrophages in the absence of M-CSF caused a progressive decline in cell viability induced over a 96 h time course as illustrated in Figure 3.1. This decline was completely inhibited by 25 |ig/ml of ox-LDL, whereas native L D L (n-LDL) and acetylated L D L (ac-LDL) were without effect.  This latter control indicated that the pro-survival effect is an oxidation-  specific event and is not related simply to the delivery of cholesterol or phospholipids to macrophages.  The concentration response curve for o x - L D L shown in Figure 3.2  demonstrated a biphasic effect on viability, in that the pro-survival effect was lost at concentrations above 100 |ig/ml. Other investigators have reported that ox-LDL (typically at concentrations >100 |0g/ml) was cytotoxic and caused apoptotic cell death in macrophages (20, 95, 160, 161, 163, 164, 166, 167, 386, 390), vascular smooth muscle cells (19, 20, 394, 406, 444-448) and endothelial cells (96, 402, 449-455). My results are consistent with these reports. At these concentrations, ox-LDL was cytotoxic even in the presence of M - C S F (data not shown), and so it is likely that the cytotoxic effect of ox-LDL is mediated by different components and/or different intracellular signaling pathways than the pro-survival effect  63  (456). Much of the cytotoxicity of ox-LDL can be accounted for by oxysterols (95, 97, 99, 446, 448, 449). Although lysoPC has been shown to promote macrophage proliferation by some investigators (148, 149, 153, 155), at high concentrations it can be toxic by increasing permeability of the cell membrane. Hence, the overall effect of ox-LDL is likely to depend on the nature and extent of L D L oxidation, as well as the type of cell and conditions of isolation and culture. Incubation in the absence of fetal bovine serum (FBS) increased the apparent susceptibility of the cells to cytotoxic effects of ox-LDL, while lowering the threshold concentration for maximal viability. There are several potential explanations for this effect of serum, but the simplest may be that albumin binds to potentially toxic polar lipids in ox-LDL and reduces their rate of transfer to the cell membrane. Alternatively, FBS components such as growth factors like insulin-like growth factor-1 (IGF-1) may also offer some buffering capacity to the cytotoxic effects of ox-LDL. To define the extent of modification necessary for ox-LDL mediated macrophage survival, ox-LDL was oxidized to varying degrees by exposure to 5 pJVI C u  2 +  for 2-24 h  (Figure 3.3) and each preparation of ox-LDL was tested for ability to promote macrophage survival. Results in Figure 3.4 show that oxidized L D L promoted macrophage survival with only minimal degrees of modification as reflected in less than 1.5-fold increase in electrophoretic mobility (Figure 3.3). However, more extensively modified L D L was more potent in promoting survival (Figure 3.4). Mildly oxidized L D L is not modified enough to interact with SR-AI/II. The observation that mildly oxidized L D L significantly improved  64  macrophage survival together with the finding that the prototypic SR-AI/II ligand acetyl L D L did not suggested that this receptor was not required for survival signaling by oxidize L D L . This was confirmed by the demonstration that there was no significant difference in the prosurvival effects of oxidized L D L in SR-AI/II -/- macrophages compared to wild-type macrophages (data not shown).  It remains possible that ox-LDL-mediated macrophage  survival involves other scavenger receptors such as CD36, which can bind to mildly as well as extensively oxidized L D L (9). Interestingly, CD36 is linked to the activation of  NF-KB  (396), and this was shown to be important in ox-LDL mediated macrophage survival (see Chapter 4). Oxidized L D L has been shown to be mitogenic towards several types of cells, including murine peritoneal macrophages, human monocyte-derived macrophages and smooth muscle cells (117, 152-154, 417). To determine if the preservation of cell viability by ox-LDL as assessed with the MTS assay was due only to an effect on cell survival or whether there was also proliferation of macrophages under these conditions, three indices of proliferation were examined. First, immunoblotting for cyclin D l was performed on four treatment conditions. D-type cyclins are considered to be growth factor sensors and are rate limiting and essential for G l progression (457-459). Moreover, D-type cyclins are essential cofactors for the cyclin-dependent kinases (cdk), cdk4 and cdk6, and a key substrate for the cyclin D/cdk holoenzyme is the retinoblastoma protein (pRB).  Phosphorylation of pRB  releases the transcriptional repression of a variety of genes required for proliferation (460).  65  As shown in Figure 3.5. cells starved of M - C S F overnight prior to treatment with ox-LDL maintained levels of cyclin DI, whereas cells starved for an additional 24 h or cultured with 25 |i.g/ml of ox-LDL for 24 h had reduced levels of cyclin DI. The addition of 5 000 U/ml of M - C S F served as a positive control for macrophage proliferation and caused an increase of cyclin DI levels as expected. Second, as shown in Figure 3.6 cell cycle analysis of ox-LDL treated cells revealed that these cells did not have an increase in the S-phase (2.34 ± 0.07 % of total D N A content) of the cell cycle compared to quiescent cells starved overnight of M CSF (2.54 ± 0.08 % of total D N A content), whereas cells treated with 5 000 U/ml of M - C S F had an increase in S-phase (15.61 ± 0.09 % of D N A content). Lastly, there was no change in the total number of adherent cells following ox-LDL treatment for 24 h, whereas 5 000 U/ml of M - C S F for 24 h produced an increase in cell number (Figure 3.71 Taken together, these results supply compelling evidence that ox-LDL promotes the survival of B M D M without inducing proliferation. These results, however, do not exclude the possibility that ox-LDL might induce cell proliferation under other conditions or at later time points. For example, treatment of macrophages with 25 u,g/ml ox-LDL without first inducing quiescence by overnight M - C S F starvation induces macrophage proliferation (data not shown). Therefore, enhanced macrophage survival may act as a "first-step" priming macrophages for proliferation as well as lengthening the tenure of the macrophage derived foam cell in the plaque.  66  Cell death can occur by necrosis (oncosis) or by apoptosis.  Apoptotic cell death  involves a number of characteristic biochemical and morphologic features that distinguish it from necrosis. These include loss of plasma membrane asymmetry of phosphatidylserine, reduced mitochondrial metabolism, activation of caspase 3, and D N A fragmentation (276, 277, 456, 461). To determine if the pro-survival effect of ox-LDL was due specifically to the inhibition of apoptosis, B M D M were assayed for three classical markers of apoptotic cell death: D N A fragmentation, caspase 3 activation and loss of plasma membrane phospholipid asymmetry of phosphatidylserine. Figure 3.8 shows that ox-LDL attenuated the increase in subdiploid (sub-Gl) D N A content, a marker of D N A fragmentation, induced by the removal of M - C S F , whereas n-LDL and ac-LDL were ineffective.  5000 U / m l of M - C S F also  prevented D N A fragmentation. Figure 3.9 depicts results of immunoblotting for pro-caspase 3 and poly (ADP-ribose) polymerase (PARP), a substrate of active caspase 3. O x - L D L prevented both the activation of caspase 3 as shown by the more intense pro-enzyme form of caspase 3 and prevented the caspase 3 mediated degradation of the 116 kDa PARP substrate to its 85 kDa cleaved fragment. Phase-contrast micrographs shown in Figure 3.10 A and B demonstrate that cells treated without ox-LDL were small and rounded, characteristic of apoptotic cells, whereas ox-LDL treated cells retain a normal adherent morphology. Panels B and D are fluorescence micrographs after staining macrophages with Annexin V-FITC. Annexin V- is a Ca -dependent phospholipid-binding protein that has a high affinity for 2+  phosphatidylserine, and is therefore a useful tool for visualizing apoptotic cells as these lose  67  the ability to maintain phospholipid asymmetry across the plasma membrane. The results indicate that control cells have higher levels of phosphatidylserine expression on the outer leaflet of the plasma membrane compared to ox-LDL treated cells. Taken together, these results supply compelling evidence that ox-LDL promotes the survival of B M D M by attenuating the apoptosis induced by the removal of M-CSF.  Importantly, neither native  L D L nor acetylated L D L had an effect on these markers of apoptosis, indicating that the enhanced survival was not due simply to delivery of cholesterol or phospholipids to cells, but rather to oxidative modification.  68  Figure 3.1. Time dependent inhibition of cell death in B M D M by ox-LDL. B M D M were seeded at 10x103 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 0-96 h with this medium alone (open squares), or with addition of 25 u,g/ml ox-LDL (closed squares), nL D L (open circles) or ac-LDL (closed circles). Macrophage viability was measured by the bioreduction of the soluble tetrazolium salt, MTS as described in the Materials and Methods. Results are expressed relative to control cells treated without ox-LDL at 0 h. Data are representative of mean ± S E M of quadruplicate samples of 3 separate experiments (* P<0.05 vs. 0 h). 69  120n  Figure 3.2. Dose dependent inhibition of cell death in B M D M by o x - L D L . B M D M were seeded at lOxlO cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. B M D M were treated for 24 h with increasing concentrations of ox-LDL (1.56-200 p.g/ml) in RPMI 1640 containing 10% FBS (open squares) or no FBS (closed squares). Macrophage viability was measured by the bioreduction of the soluble tetrazolium salt, MTS as described in the Materials and Methods. Results are expressed relative to control cells treated without ox-LDL at 0 h. Data are representative of mean ± S E M of quadruplicate samples of 3 separate experiments. 3  70  R : 1.00 f  1.04  1.21  2.56  2.96  4.08  4.40  + 0  0  o  1  2  4  8  16  24 h oxidation —  Figure 3.3. Electrophoretic mobilities of L D L with varying degrees of oxidation. Native L D L , isolated from human plasma as described in the Materials and Methods, was incubated at 200 | i g / m l with 5 | i M C u in sterile P B S containing 10 m M C a for the indicated periods of time at 37°C. Aliquots of each sample were run on agarose gel electrophoresis (1-2 p,g/lane) and stained with Fat Red. The electrophoretic mobility (R ) of o x - L D L is expressed relative to the migration of native L D L (0 h oxidation). Data are representative of 2 separate experiments. 2 +  2 +  f  71  120  20 H  4  8  1 6  24  Oxidation (Hours)  Figure 3.4.  Oxidation dependent inhibition of cell death in B M D M by ox-LDL.  B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone or with the addition of 25 p:g/ml ox-LDL oxidized for 2, 4, 8, 16 or 24 h. Macrophage viability was measured by the bioreduction of the soluble tetrazolium salt, MTS as described in the Materials and Methods. Results are expressed relative to cells treated without ox-LDL at 0 h. Data are representative of mean ± SEM of quadruplicate samples of 3 separate experiments (* P<0.05 vs. 0 h). 3  72  COh  C24h  Ox24h M-CSF 24 h  Figure 3.5. O x - L D L does not lead to an increase in cyclin D I . B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h (C 0 h). Macrophages were then incubated for an additional 24 h with this medium alone (C 24 h), with the addition of 25 u.g/ml ox-LDL (Ox 24 h) or 5 000 U/ml of M-CSF (M-CSF 24 h). Immunoblotting for cyclin DI was performed as described in the Materials and Methods. GAPDH was used as a control to monitor protein loading. Data are representative of 2 separate experiments. 6  73  Ox-LDL 24 h  Control 0 h  G1  G2/M  s  "i  i  r DNA Content i  i  r  i  1  1023  0  I  I  I  I  I  A I  I  I  I  1  0  2  3  DNA Content  M-CSF 24 h G1  G2/M S "1  I  I  I  I  I  I  I  1023  DNA Content  3.6. O x - L D L does not increase S phase of the D N A cycle. B M D M were seeded at lxlO cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h (Control 0 h). Macrophages were then incubated for 24 h with 25 Ug/ml ox-LDL (Ox-LDL 24 h) or 5 000 U/ml of M-CSF (M-CSF 24 h) and cell cycle analysis was performed as described in the Materials and Methods. Data are representative of 2 separate experiments done in triplicate. 74 Figure 6  80  -i  * I 60 H  o X  o  40 H  "53  O  20 H  B M D M were seeded at 0.5xl0 cells/well in 12 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h (Control 0 h). Macrophages were then incubated for 24 h with 25 Ug/ml ox-LDL (Ox-LDL 24 h) or 5 000 U/ml of M-CSF (M-CSF 24 h). Total adherent cells were counted following two washes with PBS and scraped in a known volume of PBS as described in the Materials and Methods. Data are representative of mean ± SEM of quadruplicate samples of 2 separate experiments (* P<0.05 vs Control 0 h). Figure 3.7.  O x - L D L does not increase macrophage cell numbers.  6  75  cs C  3  rH . >/">  D  oi  G1  G1  *  i  4.3%  51.8% Sub-G1  Sub-G1  G2/M  S  G2/M  i 10  1  10 10 FL3 L O G 2  3  10  10  4  1  10 FL3 L O G 2  10  3  10  4  E rH  G1  9.8% Sub-G1 10"  10  1  10 FL3 L O G 2  G2/M 10  3  ~lo  4  Figure 3.8. Ox-LDL inhibits DNA fragmentation in BMDM. B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M - C S F for 24 h followed by 24 h in RPMI 1640/10% FBS alone (A), or with 25 |ig/ml n-LDL (B), ac-LDL (C), ox-LDL (D) or 5000 U/ml M-CSF (E). D N A fragmentation was analyzed by flow cytometry using propidium iodide staining cells as described in the Materials and Methods. Data are representative of 3 separate experiments (* P<0.05 vs. A ) . 6  76  Figure 3.9. O x - L D L inhibits caspase 3 activation in B M D M . B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in R P M I 1640 with 10% F B S , but without M - C S F for 24 h followed by 24 h in R P M I 1640/10% F B S alone, or with 25 |ig/ml n - L D L , a c - L D L or o x - L D L . Immunoblotting for pro-caspase 3 and poly (ADP-ribose) polymerase ( P A R P ) was done as described in the Materials and Methods. G A P D H was used as a control to monitor protein loading. Data are representative of 3 separate experiments. 6  77  B  A  Figure 3.10. O x - L D L maintains phosphatidylserine asymmetry. B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h followed by 24 h in RPMI 1640/10% FBS alone (A and C), or with 25 Lig/ml ox-LDL (B and D). Phase (A and B) and fluorescent microscopy with FITClabeled Annexin V (C and D) were done as described in the Materials and Methods at 200 X magnification. Data are representative of 3 separate 200 X fields of magnification from 2 separate experiments. 6  78  4.  OX-LDL PROMOTES CYTOKINE INDEPENDENT SURVIVAL THROUGH ACTIVATION OF THE PI3K/PKB PATHWAY  4.1  RATIONALE AND HYPOTHESIS B M D M are dependent on the cytokines GM-CSF or M-CSF for survival (171, 172).  The receptor for G M - C S F belongs to the type I, cytokine receptor family that includes receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15 and G M - C S F . A l l of these have a conserved extracellular motif and lack an intracellular receptor tyrosine kinase domain (462). In contrast, the receptor for M-CSF is homologous to receptors for platelet-derived growth factor and stem cell factor which possess intrinsic tyrosine kinase activity (175).  Despite these differences, both G M - C S F and M - C S F activate similar  intracellular survival pathways, i.e. the ras-raf-ERKl/2, M A P kinase pathway and the PI3K pathway.  The M A P kinase pathway has been shown to be involved in growth factor-  independent survival in B M D M (463), as well as transforming growth factor (3-mediated rescue of serum deprivation-induced apoptosis in macrophages (464).  Similarly, the  PI3K/PKB pathway has been recently shown to mediate macrophage survival by M - C S F (188).  P K B might promote survival at a post-transcriptional level by phosphorylating  components of the apoptotic machinery, such as forkhead transcription factors or Bad, or indirectly by changing the level of expression of the genes that encode components of cell death machinery, such as the anti-apoptotic members of the Bcl-2 family members through I K B phosphorylation and subsequent release of N F - K B (274).  79  It was recently reported that o x - L D L promotes the proliferation of peritoneal macrophages by elaborating the release of G M - C S F (148).  Conversely, Hamilton and  colleagues found that o x - L D L promoted macrophage survival independent of G M - C S F or M - C S F (145). T o address this issue, I formulated the working hypothesis that o x - L D L promotes the survival of B M D M by directly activating the p42/44 M A P kinase ( E R K 1 / 2 ) and/or the P I 3 K / P K B signaling cascades, and thereby subverting the need for cytokines to activate these pathways and maintain cell viability. 4.2  R E S U L T S A N D DISCUSSION To clarify whether or not the pro-survival effect of o x - L D L required the secretion of  soluble mediators or cytokines other than G M - C S F or M - C S F , medium from cells treated with o x - L D L for various time intervals was removed, ultrafiltered through a 100,000 M . W . cutoff membrane to remove any remaining o x - L D L , and tested for its effect on macrophage viability.  A s shown i n Figure 4.1. conditioned medium from o x - L D L treated cells was  unable to prevent cell death at any time point, suggesting that stable soluble mediators were not sufficient for the pro-survival effect. A positive control using 5000 U / m l of M - C S F was able to promote macrophage proliferation, therefore, ruling out an effect of cytokine absorption on the membrane during filtration (data not shown). Interestingly, simply treating B M D M with o x - L D L for 2 h or more was able to induce a statistically significant increase in viability. T o determine i f soluble factors were necessary for this effect, I added neutralizing antibodies against G M - C S F and found that 1:20 dilution of a n t i - G M - C S F failed to block the  80  anti-apoptotic effect of ox-LDL, but completely blocked the effect of G M - C S F on macrophage survival (Figure 4.2). In addition, as shown in Figure 4.3. ox-LDL was effective at promoting survival of B M D M from osteopetrotic B6C3Fe-a/a-Csfl mice, which carry a op  homozygous null mutation in the M-CSF gene. Similar results were reported previously by Hamilton and colleagues (145). These experiments are strong evidence that neither G M CSF, M - C S F nor other soluble mediators are responsible for mediating B M D M survival in response to ox-LDL. Moreover, these results are consistent with a direct action of ox-LDL on macrophages rather than an indirect effect mediated by cytokine release as responsible for promoting B M D M survival. Immunoblotting using antibodies that only detect the phosphorylated (active) form of these kinases indicated that ox-LDL activated both ERK1/2 and PKB whereas native L D L or acetyl L D L had no effect (Figure 4.4 A). The activation of PKB by ox-LDL was confirmed by an in-vitro kinase assay (Figure 4.4 B). As expected, the M E K inhibitors, PD 98059 and U0126, inhibited ERK1/2 activation and the PI3K inhibitors, L Y 294002 and wortmannin, blocked PKB activation by ox-LDL (Figure 4.5). Wortmannin is a fungal metabolite that is a potent and irreversible inhibitor of mammalian class I PI3K (465). Wortmannin forms A  covalent bonds with Lys-802 in the ATP-binding site of PI3K, alkylating the nucleophilic residue and inhibiting PI3K activity. Conversely, L Y 294002 functions as a competitive inhibitor for the ATP-binding site of PI3K (466).  81  To determine if activation of either of these pathways was essential for the inhibition of apoptotic cell death, I tested the effect of these inhibitors on cell survival in the presence of ox-LDL. As shown in Figure 4.6. pre-treatment with L Y 294002 and wortmannin blocked the effect of ox-LDL on macrophage survival, whereas PD 98059 and U0126 were without effect. The activation of ERK1/2 by ox-LDL has been previously described in macrophages and smooth muscle cells (412-415, 419, 467-469), but in this study it was shown that these kinases were not essential for the anti-apoptotic effect of ox-LDL. These results provide the first evidence for the activation of PKB by ox-LDL, and demonstrate a direct role for the activation of the PI3K/PKB pathway in the enhancement of macrophage survival. Moreover, these findings are consistent with previous findings supporting a role of PI3K/PKB in macrophage survival, including survival mediated by M - C S F (188) as opposed to M A P kinase activation (186). Interestingly, the kinetics of ox-LDL mediated P K B activation differed in the Raw 264.7 murine macrophage cell line compared to primary B M D M .  As  shown in Figure 4.7A. PKB activation in Raw 264.7 cells was transient with a maximum at 5 min and returning to baseline levels by 15 min. Conversely, PKB activation in B M D M was sustained for up to 12 h of exposure (Figure 4.7B). These results highlight that there may be important differences in agonist-induced activation of signal transduction pathways in primary cell cultures compared to immortalized cell lines, where proliferation and survival pathways are intrinsically activated.  82  Several targets of the PI3K/PKB signaling cascade have been identified that might underlie the ability of this pathway to promote survival (274). These include components of the apoptotic machinery, including Bad (the pro-apoptotic member of the Bcl-2 family of proteins), transcription factors of the forkhead family; and two kinases, glycogen synthase kinase-3(3 (GSK-3J3) and I K B kinase (IKK). cleavage by a specific protease, and freeing  I K K phosphorylates I K B - a , triggering its  NF-KB  to translocate to the nucleus and activate  target survival genes. Oxidized L D L has been previously shown to promote the activation of NF-KB  in some (397, 398, 470-472), but not all (399, 473) reports. Active N F - K B has been  shown to be present in atherosclerotic lesions, suggesting that  NF-KB  could play a role in  atherosclerosis (474-477). In the present study, I demonstrate that ox-LDL leads to  IKB-CC  phosphorylation (Figure 4.8). The phosphorylation of lKB-oc-Ser-32 targets it for proteosome mediated degradation, and therefore is an excellent marker of  NF-KB  activation (478-480).  As shown in Figure 4.8. following phosphorylation and subsequent proteosome mediated degradation the levels of I K B - a decline in ox-LDL treated cells. The mechanism by which NF-KB  promotes cell survival may involve the upregulation of anti-apoptotic genes (478,  479). In particular, the Bcl-2 family of proteins includes some of the most important cellular regulators of apoptosis (310, 311, 481). Although bcl-2 itself does not appear to be a target for N F - K B , the anti-apoptotic Bcl-2 like protein A l / B f l l (482, 483) and Bcl-X (484-487) L  contain functional upstream  NF-KB  binding sites within their promoters. Moreover,  83  activation of the PI3K/PKB pathway has been shown to lead to the upregulation of Bcl-X by L  activation of  NF-KB  (488, 489). Similarly, PKB mediated phosphorylation of Bad on Ser-  136, sequesters it in the cytoplasm with 14-3-3 protein and prevents its inhibition of the antiapoptotic Bcl-X through heterodimerization (490, 491). Therefore, in the present study oxL  L D L may act through at least two PKB downstream targets at the level of Bcl-X to promote L  survival (Chapter 5). O x - L D L also caused a detectable increase in the phosphorylation of the forkhead transcription factor, F K H R and  GSK-3P (data not shown). GSK-3(3 phosphorylation has  been shown to inhibit apoptosis (260, 492) although the mechanism is not well defined. Phosphorylation of Forkhead members by PKB stimulates their nuclear export, thereby preventing the transcription of pro-apoptotic proteins, such as the FasL (249, 250, 253, 255257). The importance of phosphorylation of these factors remains to be established in oxL D L mediated macrophage survival, and warrants further investigation. Because the role of GSK-3p\ F K H R or the phosphorylation of other factors in the inhibition of apoptosis are unclear,  I  focussed only on  IKB-OC  and Bad phosphorylation. lKB-a-Ser-32 and Bad-Ser-136  phosphorylation are of obvious interest because of their role in preventing the release of cytochrome c from the mitochondria and subsequent activation of caspase 3 at the level of the anti-apoptotic member of the Bcl-2 family, Bcl-X . As such, the novel findings that oxL  L D L leads to the activation of PKB and phosphorylation of these two downstream targets  84  formed the basis for the hypothesis that ox-LDL may lead to the upregulation of Bcl-X and L  prevention of caspase 3 activation examined in Chapter 5. Previous reports have identified lysophosphatidylcholine as playing an important role in ox-LDL mediated macrophage growth (149, 151, 155).  Conversely, Martens and  coworkers showed that protein modification plays a critical role in ox-LDL mediated macrophage growth (157).  To test whether the lipid or protein component of ox-LDL  mediated macrophage survival, I separated the lipid and protein component of L D L using the Bligh and Dyer method and treated cells with either the total lipid or with the octylglucosideresolubilized protein. Interestingly, the total lipid extract from 25 |ig/ml of ox-LDL was equally effective in promoting macrophage survival as 25 (ig/ml of ox-LDL (Figure 4.9). Conversely, the reconstituted protein extract from 25 u,g/ml of ox-LDL was unable to promote macrophage survival. Neither component from 25 u.g/ml of ac-LDL had an effect. Moreover, as shown in Figure 4.10. the total lipid extract was as potent as intact ox-LDL in activating PKB, whereas the reconstituted protein component was ineffective. These results suggest the lipid component of ox-LDL plays an important role in mediating macrophage survival. These findings, however, do not exclude a role for the protein component, as the delipidation/reconstitution procedure undoubtedly results in significant conformational changes and possibly loss of some components that could adversely affect its biological activity.  85  To confirm the importance of PKB in ox-LDL mediated B M D M survival, retroviral infection was attempted using the Bosc 23 retrovirus packaging cell line and a 4-HT activated PKB-ER construct tagged with a green fluorescent protein (GFP) (443). As shown in Figure 4.1 IB. transfection of Bosc cells with the GFP-tagged 4-HT/PKB-ER construct was successful.  However, the infection of primary B M D M with the retrovirus containing  supernatant from Bosc cells yielded very few positively infected cells (Figure 4.1 ID) and so the result is inconclusive.  The difficulty of introducing foreign D N A into primary  macrophages is well established (493, 494). Work is currently being undertaken to enhance the retroviral infection rate of B M D M .  86  120 n  20 H 0  1  1  4  1  1  8  '  1  12  i  1  16  1  1  20  1  1  24  Time (Hours)  Figure 4.1.  O x - L D L promotes cytokine independent survival. B M D M were seeded at  10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% F B S , but without M - C S F for 24 h. Macrophages were then incubated with this medium alone or with the addition of 25 |ig/ml ox-LDL for the indicated periods of time, the medium was removed, filtered with a 100 kDa microconcentrator to remove non-internalized ox-LDL and then the conditioned medium applied to control cells (closed squares) and ox-LDL treated cells were replaced with RPMI 1640/10% FBS alone (open squares). Macrophage viability was measured after 24 h by the bioreduction of the soluble tetrazolium salt, M T S as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL at 0 h. Data are representative of mean ± S E M of quadruplicate samples of 3 separate experiments (* P<0.05 vs 0 h). 3  87  120 -|  Figure 4.2. O x - L D L promotes G M - C S F independent survival. B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were incubated with control medium, 25 p:g/ml oxL D L or 5 units of GM-CSF in the presence or absence of 1:20 anti-GM-CSF neutralization antibody (AB). Macrophage viability was measured after 24 h by the bioreduction of the soluble tetrazolium salt, MTS as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL at 0 h. Data are representative of mean ± S E M of quadruplicate samples of 2 separate experiments (* P<0.05 vs. A B treatment). 3  88  100 -i  o-i  «  1  0  50  '  1  '  100  ox-LDL |Ig/ml  1  150  '  1  200  Figure 4.3. Ox-LDL promotes M - C S F independent survival. B M D M from op/op mice were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone or with increasing concentrations of ox-LDL (1.56-200 (ig/ml). Macrophage viability was measured after 24 h by the bioreduction of the soluble tetrazolium salt, M T S as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL at 0 h. Data are mean ± S E M of quadruplicate samples of 2 separate experiments. 3  89  A  B  Histone 2B Control  n-LDL  ac-LDL  ox-LDL  Figure  4 . 4 . O x - L D L activates E R K 1 / 2 and P K B . (A) Raw 264.7 macrophages were seeded at l x l O cells in 6 well plate dishes in D M E M with 10% F B S , starved of FBS overnight and then incubated with D M E M alone, 25 ^tg/ml n-LDL, ac-LDL or ox-LDL for 10 min and the activation of the intracellular survival signaling pathways, ERK1/2 and P K B examined by phospho-specific immunoblotting as described in the Materials and Methods. ERK1 and P K B served as controls to monitor protein loading. Data are representative of 3 separate experiments. (B) In-order to confirm the activation of P K B , Raw 264.7 macrophages were seeded at 5 x l 0 cells/100 mm dishes in D M E M with 10% FBS, starved of FBS overnight and then incubated with D M E M alone, 25 p.g/ml n-LDL, ac-LDL or oxL D L for 10 min followed by in vitro kinase assay as described in the Materials and Methods. Data are representative of 2 separate experiments. 6  6  90  Phospho-PKB-Ser-473  PKB  -~~-jjr\ m  Phospho-ERKl Phospho-ERK2  ERK 1  + +  +  25 ng/ml ox-LDL 10 u M PD 98059 2 u M U0126 5 \iM L Y 294002 100 nM wortmannin  Figure 4.5. PI3K inhibitors block P K B activation and M E K inhibitors block ERK1/2 activation by o x - L D L . Raw 264.7 macrophages were seeded at 1x10 cells in 6 well plate dishes in D M E M with 10% FBS, starved of FBS overnight and then macrophages were preincubated with the M E K inhibitors (PD 98059 or U0126) and the PI3K inhibitors (LY 294002 or wortmannin) for 1 h prior to treatment with 25 u\g/ml of ox-LDL for 10 min. E R K 1/2 and PKB activation were examined by phospho-specific immunoblotting as described in the Materials and Methods. E R K 1 and PKB served as controls to monitor protein loading. Data are representative of 3 separate experiments. s  91  120'  100 H  80 H  J3 >  601  40 H  20 H  +  + +  +  +  +  + +  +  25 U-g/ml ox-LDL 10 u M PD 98059 2 u M U0126 5 (iM L Y 294002 100 nM wortmannin  Figure 4.6. PI3K inhibitors block ox-LDL mediated macrophage survival. B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone or with the addition of 25 u,g/ml ox-LDL following pre-incubation with the M E K inhibitors (PD 98059 or U0126) and the PI3K inhibitors (LY 294002 or wortmannin) for 1 h prior to treatment with ox-LDL. Macrophage viability was measured after 24 h by the bioreduction of the soluble tetrazolium salt, MTS as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL for 0 h. Data are representative of mean ± S E M of quadruplicate samples of 3 separate experiments. 3  92  A  Figure 4.7. Kinetics of PKB activation by ox-LDL.  A) Raw 264.7 macrophages were seeded at l x l O cells in 6 well plate dishes in D M E M with 10% FBS, starved of FBS overnight and then treated with 25 ug/ml ox-LDL for the time intervals indicated. B) B M D M were seeded at 5xl0 cells/100 mm dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h and were incubated with 25 ug/ml ox-LDL for the time intervals indicated. PKB activation was examined by phospho-specific immunoblotting as described in the Materials and Methods. P K B served as a control for protein loading. Data are representative of 2 separate experiments. 6  6  93  Phospho-PKB-Ser-473  PKB  Phospho-lKB-a-Ser-32  IKB  Phospho-Bad-Ser-136  Bad  Control  ox-LDL  Figure 4.8. O x - L D L phosphorylates the P K B targets, I K B and Bad. B M D M were seeded at 5xl0 cells/100 mm dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M - C S F for 24 h and were incubated with 25 Ug/ml ox-LDL for 1 h and the phosphorylation of two putative PKB downstream targets, I K B - O C and Bad examined by phospho-specific immunoblotting as described in the Materials and Methods. PKB and Bad served as controls to monitor protein loading. Data are representative of 2 separate experiments. 6  94  120  80 H  20  i  o O  x o  X  o  •g 'a. co o  o  X  CO  o c 'a) o Q.  o  CO  •g _co o  o h-  o  CO  c 'a) o CL  aj o  Figure 4.9. Total lipid component of ox-LDL prevents macrophage apoptosis. B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone, 25 |ig/ml ox-LDL, total lipid from 25 Ug/ml ox-LDL and total reconstituted protein from 25 |lg/ml ox-LDL or ac-LDL controls. Macrophage viability was measured after 24 h by the bioreduction of the soluble tetrazolium salt, MTS as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL for 0 h. Data are representative of mean ± S E M of quadruplicate samples of 2 separate experiments. 3  95  o-PKB-Ser-473  Control  ox-LDL  ox-Lipid  ox-Protein  Figure 4.10. Total lipid component of ox-LDL activates P K B . B M D M were seeded at 5xl0 cells/100 mm dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M CSF for 24 h and were incubated with control medium, 25 Ug/ml ox-LDL, total lipid from 25 Ug/ml ox-LDL and total reconstituted protein from 25 Ug/ml ox-LDL for 1 h. PKB activation was examined by phospho-specific immunoblotting as described in the Materials and Methods. PKB served as a control to monitor protein loading. Results are representative of 3 separate experiments. 6  96  Figure 4.11. Retroviral infection of B M D M . Bosc 23 retroviral packaging cells (A) were transfected with an 4-HT activated PKB-ER construct tagged with GFP (B) as described in the Materials and Methods. Next, B M D M (C) were infected with the retroviral containing supernatant from Bosc cells (D). Phase contrast (A and C) and fluorescent microscopy (B and D) were performed at 200 X as described in the Materials and Methods. Data are representative of 3 separate 200 X fields of magnification from 2 separate experiments. 97  5.  MECHANISMS OF OX-LDL MEDIATED MACROPHAGE SURVIVAL: AN IMPORTANT ROLE FOR BCL-X AND CERAMIDE L  5.1  RATIONALE AND HYPOTHESIS This section deals with studies to define upstream and downstream mechanisms by  which ox-LDL might block apoptosis.  The lipid second messenger ceramide has been  implicated in a number of cellular processes, including growth arrest and apoptosis. Apoptotic signaling mediated by ceramide can occur by several mechanisms. For example, ceramide may induce apoptosis by activating pro-death pathways, such as the JNK/SAPK pathway (341, 356-359), by promoting dephosphorylation of the pro-apoptotic protein Bad which would then by free to act to inhibit Bcl-X (495) or by the inactivation of pro-survival L  pathways, such as PKB (360-363). Given the importance of the PI3K/PKB signaling cascade in ox-LDL mediated macrophage survival, the importance of the latter was of particular interest. Zundel and colleagues recently demonstrated the down-regulation of PI3K activity by ceramide results in inhibition of PKB and decreased phosphorylation of Bad (363). More recently, however, other reports have demonstrated that ceramide-mediated PKB inhibition occurs in a PI3K-independent manner (360-362). For example, Schubert and colleagues demonstrated that ceramide decreases PKB activity, not through PI3K modulation, but rather by selectively accelerating the dephosphorylation of Ser-473, a residue necessary for full PKB enzymatic activity (360).  98  Given the importance of the PI3K/PKB signaling cascade in ox-LDL mediated macrophage survival from Chapter 4, I hypothesized that ox-LDL leads to sustained PKB activation by blocking ceramide generation and by therefore removing the inhibitory effects of ceramide on PKB. With regard to downstream mechanisms, I hypothesized that ox-LDL prevents apoptosis by blocking the activation of caspase 9-caspase 3 cascade at the level of the pro-survival member of the Bcl-2 family of proteins, Bcl-X . This latter hypothesis was L  based on the findings from Chapter 4 that ox-LDL leads to the activation of N F - K B through IicB-a-Ser-32 phosphorylation. As the Bcl-X promoter contains N F - K B binding sites, it L  seemed reasonable to propose that the activation of N F - K B by ox-LDL would maintain or upregulate levels of Bcl-X relative to viable cells. L  5.2.  RESULTS AND DISCUSSION An  important mediator in the induction of apoptosis in response to a number of  apoptotic stimuli involves the elaboration of the lipid second messenger, ceramide. To examine the effects of ceramide on B M D M apoptosis, I measured ceramide levels by radiolabeling cells with [ H]-palmitate and isolating ceramide by thin layer chromatography. 3  As shown in Figure 5.1. the withdrawal of M - C S F from B M D M was associated with an increase in ceramide levels whereas the treatment of B M D M with 25 |ig/ml of ox-LDL attenuated the increase in ceramide levels. As well, 1 h pre-treatment of B M D M with the soluble ceramides, C -ceramide, C -ceramide and C -ceramide abrogated the pro-survival 2  6  8  99  effect of ox-LDL (Figure 5.2). Interestingly, it has been reported that overexpression of PKB prevents the formation of ceramide (496). Therefore, the prolonged activation of PKB in B M D M observed with ox-LDL may have directly inhibited ceramide generation. An important aspect in the identification of the ceramide pathway in apoptosis centers on the enzymes involved in ceramide generation. For example, T N F - a and the FasL activate acid sphingomyelinase in many cell types (337, 338), whereas ionizing radiation induces apoptosis in endothelial cells by activating neutral sphingomyelinase (343, 352). De novo ceramide generation in daunorubicin-induced apoptosis occurs via ceramide synthase (336). To define the metabolic origin of ceramide in growth factor-deprived B M D M , selective inhibitors of enzymes involved in sphingolipid metabolism were used. As shown in Figure 5.3. the sphingomyelinase inhibitor desipramine (497) but not the fungal metabolite fumonisin BI (FBI), a specific inhibitor of ceramide synthase (498, 499), prevented the decrease in sphingomyelin and the generation of ceramide following the withdrawal of M CSF. The effect of desipramine was similar to that of ox-LDL. These results suggest the source of ceramide is through the metabolism of sphingomyelin rather than de novo synthesis.  On a functional level, desipramine was equivalent to ox-LDL in preventing the  decline in cell viability induced by the removal of M-CSF, whereas FB1 was without effect (Figure 5.4).  Lastly, in this study I demonstrate that ceramide pre-treatment prevents the  activation of PKB as examined by PKB-Ser-473 phosphorylation (Figure 5.5). These results suggest that the anti-apoptotic effect of ox-LDL could be fully explained by the inhibition of  100  ceramide generation, and makes the prevention of sphingomyelin hydrolysis an attractive candidate as the primary target of ox-LDL formation. The finding that ox-LDL results in the phosphorylation of I K B - a and therefore the subsequent release and activation of N F - K B , suggested that ox-LDL may lead to the upregulation of the pro-survival member Bcl-X . L  Bcl-X is a anti-apoptotic member of the L  Bcl-2 family of proteins, located at the outer mitochondrial membrane and is thought to inhibit mitochondrial membrane permeability transition and the subsequent release of proapoptotic factors, such as cytochrome c and apoptosis inducing factor (316, 327-329, 379). As outlined previously, release of these factors would lead to the activation of effector proteases involved in apoptosis, such as caspase 3 through the activation of caspase 9caspase 3 cascade. To investigate this hypothesis, immunoblotting was performed for two pro-survival members of Bcl-2 family of proteins, Bcl-2 and Bcl-X and two pro-apoptotic L  members of the Bcl-2 family of proteins, Bad and Bax. Figure 5.6. illustrates that ox-LDL prevents the decline in levels of Bcl-X following the withdrawal of M-CSF, whereas the L  other members of the Bcl-2 family examined remain unchanged. Control B M D M at 0 h represent quiescent but viable cells starved overnight of M-CSF.  Pre-treatment with the  PI3K inhibitors (LY 294002 and wortmannin) prevented PKB activation, phosphorylation of I K B and the maintenance of Bcl-X (Figure 5.7). whereas the M A P kinase inhibitors (PD L  98059 and U0126) were without effect. These results emphasize the important connection  101  between the PI3K/PKB pathway, nuclear events involving B c l - X i n ox-LDL treated cells. L  Two  NF-KB  NF-KB  and the maintenance of  inhibitors caffeic acid phenylethyl ester  (CAPE) (500) and a peptide inhibitor (SN50) blocked the ability of ox-LDL to maintain BclX  L  levels, (Figure 5.8). as well as its effect on macrophage survival (Figure 5.9). Therefore,  these results suggest an important role of B c l - X  L  in mediating macrophage survival in  response to ox-LDL. As both the inhibitory phosphorylation of Bad and upregulation of Bcl-X have been L  shown to act at the level of mitochondria by preventing the release of cytochrome c and therefore the activation of caspase 9 and formation of the functional apoptosome (328, 329). I tested the hypothesis that ox-LDL prevents the activation of caspase 9, which in turn would lead to caspase 3 activation. This was tested in B M D M by examining the level of expression of the pro-enzyme forms of caspases 3, 8 and 9 by immunoblotting. As shown in Figure 5.10. ox-LDL prevents the activation of both caspase 9 and caspase 3 and cleavage of PARP, as shown by higher levels of the pro-enzyme; levels of caspase 8 remain unchanged. This indicates that the caspase 9 (mitochondrial pathway) and not the caspase 8 (death receptor pathway) is involved in apoptosis in these experiments. These studies were complemented with caspase inhibitor studies on B M D M viability. Figure 5.11 shows that treating B M D M with broad base caspase inhibitor (Z-VAD-FMK), caspase 3 inhibitor ( Z - D E V D - F M K ) and caspase 9 inhibitor ( Z - L E H D - F M K ) prevented the decline in viability following the withdrawal of M-CSF, whereas the caspase 8 inhibitor (Z-IETD-FMK) was without effect. 102  These results supply compelling evidence that ox-LDL promotes macrophage survival by inhibiting the activation of caspase 3; a key executioner in the apoptotic cascade and that Bcl-X may play an important role in ox-LDL mediated macrophage survival by preventing L  the activation of caspase 9-caspase 3 apoptotic cascade. These results are in agreement with previous results showing an important role of caspase 9 in apoptosis of human monocytes through the PI3K/PKB pathway (188).  103  *  3n  > 0  2H  CD  •g "E CO  1_  CD  o  CD > CD  rr  o o -•—• c o O  CvJ  c o O  CO  X  o  Figure 5.1. BMDM apoptosis is associated with ceramide generation. B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M - C S F for 24 h (Control 0 h). Macrophages were then incubated for 24 h with this medium alone (Control 24 h) or 25 jig/ml of ox-LDL (ox-LDL 24 h), and the levels of ceramide determined as described in the Materials and Methods. Data are representative of mean ± S E M of triplicate samples of 2 separate experiments (* P<0.05 vs. 0 h). 6  104  120  0)  a>  E  E  2 a5 o  cu "a E  CO  a3  Q3  o  o  co  cb  O  O  O  +  +  +  _l Q  _i D _i  _j Q _i  X  X  o  c o O  CO  C\J  _J  o  X  o  o  Figure 5.2. Ceramide pre-treatment blocks ox-LDL mediated macrophage survival. B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone or with the addition of 25 |ig/ml ox-LDL following 1 h pretreatment with 20 u M of the soluble ceramides (C , C and C -ceramide). Macrophage viability was measured after 24 h by the bioreduction of MTS as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL for 0 h. Data are representative of mean ± S E M of quadruplicate samples of 2 separate experiments (* P<0.05 vs. Control). 3  2  105  6  8  Figure 5.3. Desipramine blocks sphingomyelin hydrolysis to ceramide. B M D M were seeded at l x l O cells/well in 6 well plate dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M - C S F for 24 h. Macrophages were then incubated for 24 h with this medium alone or with the addition of 50 nM fumonosin B I , 10 pJVI desipramine or 25 |0-g/ml ox-LDL and the generation of ceramide (open bars) and sphingomyelin (closed bars) levels determined as described in the Materials and Methods. Data are representative of mean ± S E M of triplicate samples of 2 separate experiments. 6  106  120  -i  100 H  80 H  CO  60 H  > 40 H  20 H  CO  CD C  LL  "E  X  o  CO  i  c o O  Q_ CO CD  Q  Figure 5.4. Desipramine prevents macrophage apoptosis. B M D M were seeded at lOxlO cells/well in 96 well plates and pre-incubated in R P M I 1640 with 10% FBS, but without M - C S F for 24 h. Macrophages were then incubated for 24 h with this medium alone, 50 n M fumonosin B I , 10 uM desipramine or 25 Ug/ml ox-LDL. Macrophage viability was measured after 24 h by the bioreduction of MTS as described in Materials and Methods. Results are expressed relative to control cells treated without ox-LDL for 0 h. Data are representative of mean ± S E M of quadruplicate samples of 2 separate experiments. 3  107  Phospho-PKB-Ser-473  "  PKB  +  +  25 |ig/ml ox-LDL  +  20 | i M C -ceramide 2  Figure 5.5. Ceramide blocks o x - L D L mediated P K B activation. Raw 264.7 macrophages were seeded at l x l O cells in 6 well plate dishes in D M E M with 10% FBS, starved of FBS overnight and then treated with 25 )ig/ml ox-LDL for 10 min following 1 h pre-treatment with 20 p M C -ceramide. PKB activation was examined by phospho-specific immunoblotting as described in the Materials and Methods. PKB served as a control to monitor protein loading. Data are representative of 3 separate experiments. 6  2  108  Figure 5.6. Ox-LDL maintains Bcl-X levels. B M D M were seeded at 5 x l 0 cells/100 m m dishes and pre-incubated in R P M I 1640 with 10% F B S , but without M - C S F for 24 h (C 0 h). Macrophages were then incubated for an additional 24 h with this medium alone (C 24 h) or with the addition of 25 ug/ml o x - L D L for 24 h (Ox 24 h) and the levels of the Bcl-2 members: B c l - 2 , B c l - X , B a d and Bax determined by immunoblotting as described in the Materials and Methods. G A P D H served as a control to monitor protein loading. Data are representative of 2 separate experiments. 6  L  L  109  + +  + + -  + -  + -  + -  +  25 |lg/ml ox-LDL 10 | i M P D 98059 2^MU0126 5 U M L Y 294002 100 nM wortmannin  Figure 5.7. PI3K inhibitors block ox-LDL mediated phosphorylation of I K B and BclX maintenance. B M D M were seeded at 5xl0 cells/100 mm dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h and were incubated with the M E K inhibitors (PD 98059 or U0126) or the PI3K inhibitors (LY 294002 or wortmannin) for 1 h prior to treatment with 25 p,g/ml ox-LDL for 1 h and the phosphorylation of PKB and I K B examined by phospho-specific immunoblotting as described in the Materials and Methods. PKB and G A P D H served as controls to monitor protein loading. B c l - X levels were determined following 24 h. Data are representative of 2 separate experiments. 6  L  L  110  + -  + + -  +  25 Ug/ml o x - L D L 12.5 ug/ml C A P E 20UMSN50  +  Figure 5.8. N F - K B inhibitors prevent ox-LDL maintenance of B c l - X levels. B M D M were seeded at 5 x l 0 cells/100 m m dishes and pre-incubated in R P M I 1640 with 10% F B S , but without M - C S F for 24 h. Macrophages were then pre-incubated with 12.5 Ug/ml C A P E or 20 uM of the N F - K B inhibitor peptide S N 50 for 1 h and then treated with 25 Ug/ml oxL D L for 24 h and the levels of B c l - X determined by immunoblotting as described in the Materials and Methods. G A P D H served as a control to monitor protein loading. Data are representative of 2 separate experiments. L  6  L  Ill  120  -i  100  H  80  H  60  H  40  H  20  H  .Q  CO  >  +  + +  + +  5.9.  25 ug/ml ox-LDL 12.5 ug/ml CAPE 20uMSN50  BMDM were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF. Macrophages were then pre-incubated with 10 uM CAPE or 20 uM of the inhibitor peptide, SN 50 for 1 h and then treated with 25 Ug/ml ox-LDL for 24 h Macrophage viability was measured after 24 h by the bioreduction of MTS as described in Materials and Methods. Results are expressed relative to control cells treated without oxLDL for 0 h. Data are representative of mean ± SEM of quadruplicate samples of 2 separate experiments. Figure  N F - K B inhibitors prevent o x - L D L mediated macrophage survival. 3  112  Caspase 8 (55/57 kDa)  Caspase 9 (49 kDa)  Caspase 3 (32 kDa) PARP(116kDa) Cleaved PARP (85 kDa)  GAPDH . ....  COh  C 24 h  Ox 24 h  F i g u r e 5.10. O x - L D L prevents the activation of caspase 9-caspase 3 cascade. BMDM  were seeded at 5xl0 cells/100 mm dishes and pre-incubated in RPMI 1640 with 10% FBS, but without M - C S F for 24 h (C 0 h). Macrophages were then pre-incubated with (Ox 24 h) or without (C 24 h) 25 Ug/ml ox-LDL for 24 h and the levels of the pro-enzyme forms of caspase 8, caspase 9, caspase 3, and the caspase 3 substrate, PARP determined by immunoblotting as described in the Materials and Methods. GAPDH served as a control to monitor protein loading. Data are representative of 2 separate experiments. 6  113  12CH  20H  + + + +  100 uM Z-VAD-FMK 100 uM Z-DEVD-FMK 100 uM Z-IETD-FMK 100 uM Z-LEHD-FMK 25 ng/ml ox-LDL  B M D M were seeded at 10xl0 cells/well in 96 well plates and pre-incubated in RPMI 1640 with 10% FBS, but without M-CSF for 24 h. Macrophages were then incubated for 24 h with this medium alone, 100 uM of the broad base caspase inhibitor (Z-VAD-FMK), 100 uM of the caspase 3 inhibitor (Z-DEVD-FMK), 100 fiM of the caspase 8 inhibitor (Z-IETD-FMK), 100 uM of the caspase 9 inhibitor (Z-LEHD-FMK), or 25 Ug/ml ox-LDL. Macrophage viability was measured after 24 h by the bioreduction of MTS as described in the Materials and Methods. Results are expressed relative to cells treated without ox-LDL for 0 h. Data are representative of mean ± SEM of quadruplicate samples of 2 separate experiments (* P<0.05 vs. no treatment). Figure  5.11.  Caspase  9  and caspase  3 inhibitors block  3  114  survival.  6.  SUMMARY In summary, ox-LDL promotes the survival of cultured bone marrow derived  macrophages by specifically blocking apoptotic cell death induced by the withdrawal of M CSF. Oxidized LDL was shown to activate both ERK1/2 MAP kinases and PKB, but only the activation of the PI3K/PKB pathway was essential for the pro-survival effect. Moreover, ox-LDL promotes the phosphorylation of two important PKB downstream targets:  IKB-OC-  Ser-32 and Bad-Ser-136. Both of these targets act to prevent apoptosis at the level of Bcl-X , L  and by preventing the activation of the key effector protease, caspase 3 through the caspase 9-caspase 3 cascade. Finally, these studies reveal that ox-LDL prevents the elaboration of apoptotic lipid second messenger, ceramide, which may act to inhibit cell survival through an inhibition of PKB activity. These studies provide interesting insights into the proatherogenic properties of ox-LDL with respect to macrophage survival. Like many scientific endeavors, the work in this doctoral thesis represents ... "an end to a beginning" and provides the necessary framework to address the following experimental questions: 1. Which receptor(s) are involved in ox-LDL mediated macrophage survival?' 2. Which pathway(s) regulate the upstream activation of PKB by ox-LDL? 3. Which lipid component(s) of ox-LDL promote survival? 4. What is the importance of other PKB downstream targets, such as GSK-3J3 and FKHR in ox-LDL mediated macrophage survival? 5. How does ceramide prevent ox-LDL mediated PKB activation? 115  6. Does ox-LDL regulate the activity of sphingomyelinases? 7. Which signaling pathways determine the final biological outcome between survival, proliferation and death at various concentrations of ox-LDL? 8. Which component(s) of ox-LDL regulate those pathways?  116  Figure 6.1. O x - L D L induced macrophage survival: a working model. 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