Joseph L. Witztum - US grants

In humans, apolipoprotein E (apoE) deficiency causes hyperlipidemia and vascular disease. We have recently determined that harbor seals and sea lions do not have detectable levels of plasma apoE, yet they do not develop hyperlipidemia or remnant lipoproteins typically seen in apoE deficient humans. The diet of seals and sea lions is rich in omega-3 fatty acids and may suppress VLDL synthesis, thus "masking" the impact of the apoE deficiency. The objectives of this study are 1) to determine the basis for the apoE deficiency in harbor seals and sea lions, 2) to determine the mechanism by which seals efficiently metabolize triglyceride-rich lipoproteins, and 3) to determine the role of dietary omega-3 fatty in maintaining normolipemia. First, we will determine which apoprotein(s) are responsible for receptor-mediated uptake of seal lipoproteins using both cell culture techniques and in vivo turnover studies. Second, experiments will be conducted to determine whether harbor seals and sea lions have the apoE gene and apoE mRNA using Southern and Northern blotting techniques. respectively. Third, we will assess the role of the fish-diet by feeding seals an alternative isocaloric diet high in cholesterol and saturated fat. These studies of lipoprotein metabolism are unique in that harbor seals and sea lions are animal models in which the full impact of apoE deficiency can be studied. Because their diets are composed entirely of fish, they offer a unique opportunity to determine the maximal effect of such a diet, especially in the face of apoE deficiency. Thus these studies are highly relevant to the goal of understanding the relationship of diet and lipoprotein metabolism to the causation of atherosclerosis in man.

There is now considerable evidence, both in animals and in humans, that oxidative modification of LDL is ongoing in vivo and that it plays an important role in the atherogenic process. Ultimately, the crucial test of this hypothesis in man will come from controlled clinical studies in which therapies known to protect LDL from oxidation are tested as to their efficacy in inhibiting the atherogenic process and its clinical manifestations. The primary goal of this unit is to develop and evaluate effective and practical therapeutic modalities that will protect LDL from oxidation so that a clinical trial can be rationally designed to test the role of oxidation of LDL in the atherogenic process. In addition we will test the hypothesis that in patients whose LDL are more susceptible to oxidation and/or in whom monocytes have an increased potential to oxidize LDL may be at high risk for atherosclerosis and its complications. We will also test the hypothesis that markers for the presence of early changes indicative of LDL oxidation can be detected in the plasma of such high risk subjects and that the presence of autoantibodies to epitopes of oxidized LDL are one such marker system. Specifically, we will test whether dietary supplementation with one or more natural antioxidants, such as beta-carotene, vitamin E, or vitamin C, given alone or in combination, will lead to increased protection of LDL against oxidative modification. Because unsaturated fatty acids in the LDL are the initial substrate for oxidative attack, we will test the hypothesis that replacement of unsaturated fatty acids with monounsaturated, or selected saturated fatty acids, will be another strategy that will effectively inhibit LDL susceptibility to modification. We will also continue our studies with the potent lipophilic antioxidant drug, probucol, to determine its optimal use with respect to protection of LDL, as well as to test the possibility that enrichment of the cellular content with probucol may inhibit the ability of cells to modify LDL. In addition, we will compare the susceptibility to oxidation of LDL isolated from patients with known CAD, or at increased risk for CAD, and determine if there are differences from a normal population. We will also study selected kindreds with increased risk for CAD to determine if steps in the oxidative modification pathway, might be uniquely enhanced.

antigens; immunologic assay /test; immunization; biomedical facility; low density lipoprotein; guinea pigs;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This protocol allows for systematic evaluation of patients with hyperlipoproteinemia and unusual forms of lipid transport abnormalities, including provisions for family screening.

DESCRIPTION (provided by the applicant): Despite recent advances in lifestyles and hypolipidemic therapy, atherosclerosis remains the major cause of morbidity and mortality. Among the many factors that go "beyond cholesterol," much evidence supports the oxidation of LDL (OxLDL) as playing a key role in the atherogenic process. While the original interest in OxLDL occurred because it led to rapid macrophage uptake and foam-cell formation, almost 20 years later it is now clear that OxLDL and its many oxidatively modified products contribute to atherogenesis by its proinflammatory, immunogenic and cytotoxic properties, consistent with the concept that atherosclerosis is a chronic inflammatory process that involves complex interactions between the endogenous cells of the artery and cells recruited from the blood, chiefly monocytes and T cells. The goal of the La Jolla SCOR is to investigate mechanisms by which oxidation influences these processes and specifically focuses on the role of monocyte/macrophages and the role of T cells and immune responses. Because the recruitment of monocytes into the artery is rate limiting, we will study the extracellular and intracellular mechanisms for the regulation of chemokine receptors and adhesion molecules which determine rates of recruitment and retention. We will utilize new methods to quantify the rates of recruitment of monocytes into lesions and the impact of various interventions, such as such as deletion of CCR2, or MCP-1, or the use of vitamin E or agonists for PPARgamma. We will study the structure and function of receptors involved in the binding and internalization of OxLDL, including CD36, and CD68, and define ligands on OxLDL and apoptotic cells for these receptors. Nuclear receptors such as PPARs, LXRs and possibly ESRs appear to play pivotal roles in expression of genes known to affect atherogenesis, such as scavenger receptors, cytokines, and cholesterol homeostasis. We will determine the molecular mechanisms by which activation of these receptors modulate macrophage gene expression, and determine the role of each receptor in vivo in inflammation and atherosclerosis models through the use of novel gene targeted murine models. Oxidation of LDL renders it immunogenic and we will determine the consequences to atherogenesis of the immune response. In particular, we will determine the mechanisms by which immunization of mice with OxLDL ameliorates atherosclerosis and specifically determine the role of T cells in this process. We will utilize immunological techniques to measure the relative rates of oxidation of LDL in vivo in animals and determine the impact of various interventions, such as vitamin E. Finally, we will determine the epidemiologic relationship of various plasma markers of OxLDL, including a measure of OxLDL (mmLDL) itself, with respect to clinical and morphological measures of atherosclerosis and the ability of these measures to predict disease. In summary, the La Jolla SCOR proposes a multi-disciplinary approach that focus on the consequences to atherogenesis of the immune response to OxLDL and on the effects of OxLDL on monocyte/macrophage biology. These insights may lead to novel approaches to the treatment and prevention of atherosclerosis.

Patients with the antiphospholipid antibody syndrome (APS) have autoantibodies to certain phospholipids (aPL) such as cardiolipin and/or the lupus anticoagulant and clinically experience recurrent venous or arterial thrombosis, history of fetal death and autoimmune thrombocytopenia. Increased aPL also appear to predict increased risk of stroke and myocardial infarction in otherwise healthy men as well. However, controversy exists about the target antigens of aPL, and even university laboratories cannot agree who has elevated aPL titers. In turn, clinical management is hampered by lack of an underlying hypothesis to explain why antibodies should form to such ubiquitous compounds as PL. We have developed the novel hypothesis that many aPL are directed against epitopes of oxidized PL (OxPL) and/or against covalent adducts of OxPL and associated PL binding proteins, such as beta2GPI. Our hypothesis suggests that states of enhanced lipid peroxidation, as occurs in inflammation or atherosclerosis, leads to oxidation of PL (such as in LDL or in membranes of apoptotic or dying cells) which creates neo self-determinants and immunogenic epitopes. The resultant autoantibodies can then target such neoepitopes in many tissues, and may have a variety of biological consequences. Cardiolipin (CL) is the most common PL used to test for aPL. We have shown that APS plasma bind exclusively to OxCL, or to OxCL adducts with beta2GPI, and not to native CL. We propose to further test our hypothesis by determining if antibodies to other OxPL are also present in sera from patients and mice with lupus- like syndromes. We will generate a panel of such aOxPL murine monoclonals from (NZWxBXSB) F1 males. Similar Fab and scFv antibodies will be generated from a human phage-display library. We will determine the epitopes to which they bind and their impact on in vitro and in vivo coagulation, with an emphasis on the Protein C pathway. We will treat lupus-prone mice with potent antioxidants to see if changes in aPL titers and/or other clinical parameters occur. Understanding the etiology of even some of the aPL should lead not only to development of more standardized assays, which should improve our ability to detect high risk individuals, but also to consideration of new therapeutic modalities for patients with aPL and APS (e.g. aggressive anti-inflammatory and/or antioxidant interventions).

Oxidation of LDL (OxLDL) renders it immunogenic and both a humoral and cell-mediated response occurs. The overall goal of this unit is to explore the role of the immune response to OxLDL, with emphasis on the humoral immunity that occurs in human populations. We will measure the autoantibody titers to epitopes of oxidized LDL in plasma of human subjects already enrolled in a variety of well established epidemiologic studies containing both men and women of various ethnic backgrounds. We will determine the relationship of the titers to clinical and anatomical measures of atherosclerosis and to a variety of risk factors such as age, smoking hyperlipidemia, gender, diabetes and/or ethnic background. In particular, we will determine if the titer of these antibodies is predictive of future clinical events and whether interventions, such as hypolipidemic or antioxidant therapy in humans and animal models will alter autoantibody titers. We will determine if immune complexes with LDL are found in plasma in animals and humans, and if there relate to the extent of atherosclerosis. We will utilize sensitive immunological techniques to determine if epitopes of OxLDL found in atherosclerotic lesions are also present on circulating LDL and if the levels of these differ in various patient populations. We will clone and characterize naturally occurring monoclonal antibodies to epitopes of OxLDL derived from apo E- derived from apo E-deficient with extensive atherosclerosis. Because these mice have not been immunized exogenously, the monoclonal antibodies to OxLDL presumably define epitopes actually formed in vivo. These antibodies will be used to characterize in detail a spectrum of epitopes present in OxLDL and, in turn, these defined antigens will be used as immunogens in Unit 3 to determine if immunization of mice with these epitopes will augment the humoral immune response and ameliorate atherosclerosis, as was previously observed in WHHL rabbits immunized with autologous malondialdehyde-modified LDL. Finally, together with Unit 2, we will also utilize these antibodies to define the epitopes on OxLDL that bind to the various OxLDL receptors present on macrophages. Thus, these studies should provide both clinical and basic information that will help to better understand the role of the immune response to oxidation of LDL, and may provide useful diagnostic tools and outline new therapeutic options.

Test whether sustained release form of simvastatin administered at a dose of 160mg/day for 24 weeks will be more effective in lowering plasma LDL cholesterol levels than the currently available conventional simvastatin administered at 80 mg/day. Test whether sustained release drug will reduce LDL cholesterol to a greater extent than regular drug without significant side effects.

We have developed monoclonal antibodies, both induced and autoantibodies, that bind to oxidized LDL and specifically to oxidized phospholipids. These epitopes are present on OxLDL in both the lipid and protein phase (the latter as conjugates of oxidized phospholipids to protein.) By far analysis we have shown these bind not only to OxLDL but also to apoptotic cells (which undergo enhanced oxidative stress.) We are using confocal microscopy to help document the location and cellular binding of these monoclonal antibodies. Thus far, we have determined that these epitopes are present on the cell membrane of apoptotic cells.

There is now much evidence to implicate the immune system in atherogenesis and the purpose of this grant is to explore the role of B-lymphocyte associated immune mechanisms. We have previously demonstrated that oxidation of LDL (OxLDL) renders it immunogenic and cloned spontaneously arising IgM autoantibodies (Abs) from the spleens of apoE-/- mice (EO Abs) that were selected for binding OxLDL. All were subsequently shown to bind oxidized phospholipids present as free lipids or as adducts with proteins. We demonstrated that the EO Abs (such as EO6) bound to apoptotic cells, which are known to be under oxidative stress. EO6 block macrophage uptake of OxLDL, as well as phagocytosis of apoptotic cells. These data indicate that oxidized phospholipids serve as ligands that mediate macrophage recognition of oxidatively modified cells and lipoproteins. To gain insight into the structure of these Abs, we cloned and sequenced their variable region genes and discovered a 100 percent homology of both VH/VL gene usage with T15 anti-phosphorylcholine (PC) Abs that have been extensively studied for over 30 years. T15 Abs bind to S. pneumonia, which contains PC on its cell wall polysaccharide, and confers "innate" protection against this pathogen. However, the T15 clone expands even in germ-free mice suggesting that it is selected based on an innate "housekeeping role," although the neo-antigens to which it binds have not been defined, prior to our studies. The fact that T15 B-cells are greatly expanded in the apoE-/- mice suggests a specific immune response to oxidation-specific epitopes generated by their atherosclerotic burden. In this proposal, we will define the natural history of T15 Ig and T15 B-cells in plasma, lesions and B-cell compartments during the course of development of atherogenesis in murine models. We will determine their impact on atherogenesis through passive transfer experiments in mice, with T15/EO6 Abs themselves or B-1 cells. In addition, we will determine if humans develop anti OxLDL Abs following pneumococcal infection, or after pneumococcal vaccination, and determine experimentally in mice whether pneumococcal exposure or immunization impacts the atherogenic process. Finally, we will attempt to clone and characterize similar autoAbs in man. These data should provide novel information on T15 B-cell mediated mechanisms in atherogenesis and for the first time characterize "neo-self" ligands to which anti-OxLDL/T15 B cells respond.

Atherosclerosis is a chronic inflammatory disease of the macrovasculature, leading to myocardial infraction as well as heart failure (HF). Oxidation of LDL (OxLDL) leads to its unregulated uptake by macrophages, causing foam cells, the initial lesion of atherosclerosis. OxLDL is also immunogenic, leading to the generation of autoantibodies to epitopes of OxLDL. We have cloned both murine and human monoclonal autoantibodies to OxLDL and shown that when they are radiolabelled and injected intravenously, they target atherosclerotic lesions and that some block the binding and uptake of OxLDL by macrophages. We propose to develop gene transfer techniques that would allow for the systematic expression from ectopic organs, such as liver of muscle, of such recombinant single chain antibodies (scFv) to OxLDL to affect the atherogenic process, for example by blocking OxLDL uptake by macrophages or by delivery to the lesion (which is rich in OxLDL) of important therapeutic molecules. The successful development of these techniques might provide novel therapeutic modalities to retard atherogenesis and improve contractility of the failing heart. There are increasing examples of beneficial effects in humans of the infection of monoclonal antibodies in a variety of different disease states. Therefore, these techniques may be of general utility and could be models by which constant and sufficient delivery of a recombinant antibody could be delivered extracellularly or intracellularly for a wide variety of therapeutic purposes.

DESCRIPTION (provided by applicant): There is now much evidence to implicate the immune system in atherogenesis and the purpose of this grant is to explore the role of B-lymphocyte associated immune mechanisms. We have shown that oxidation of LDL (OxLDL) renders it immunogenic and cloned spontaneously arising IgM autoantibodies (Abs) from spleens of apo +mice (EO Abs) that were selected for binding to OxLDL. All were subsequently shown to bind oxidized phospholipids (OxPL) present as free lipids or as adducts with proteins. We demonstrated that the EO Abs (such as EO6) bound to apoptotic cells, which are known to be under oxidative stress. EO6 blocked macrophage uptake of OxLDL, as well as phagocytosis of apoptotic cells. These data indicate that OxPL serve as ligands that mediate macrophage recognition of oxidatively modified cells and lipoproteins. To gain insight into the structure of these Abs, we cloned and sequenced their variable region genes and discovered a 100% homology of both VH/VL gene usage with T15 anti-phosphorylcholine (PC) Abs that have been extensively studied for over 30 years. T15 Abs bind to S. pneumonia, which contains PC on its cell wall polysaccharide, and confers "innate" protection against this pathogen. However, the T15 clone expands even in germ-free mice suggesting that it is selected based on an innate "housekeeping role," although the neo-antigens to which it binds had not been defined, prior to our studies. The fact that T15 B-cells are greatly expanded in the apoE-/-mice suggests a specific immune response to oxidation-specific epitopes generated by their atherosclerotic burden. In this proposal, we will define the natural history of T15 Abs and T15 B-cells in plasma, lesions and B-cell compartments during the course of development of atherogenesis in murine models. We will determine their impact on atherogenesis through passive transfer experiments in mice, with T15/EO6 Abs themselves or B-1 cells. We will determine the impact of similar experiments in mice genetically unable to make the T15 clone and in mice that overexpress B-1 cell Abs. In addition, we will determine if humans develop anti OxLDL Abs following pneumococcal infection, or after pneumococcal vaccination, and determine experimentally in mice whether pneumococcal exposure or immunization impacts the atherogenic process. Finally, we will attempt to clone and characterize similar autoAbs in man. These data should provide novel information on T15 B-cell mediated mechanisms in atherogenesis and for the first time characterize oxidation-specific "neo-self" ligands to which anti-OxLDL/T15 B cells respond.

DESCRIPTION (provided by applicant): There is now much evidence to implicate the immune system in atherogenesis and the purpose of this grant is to explore the role of B-lymphocyte associated immune mechanisms. We have shown that oxidation of LDL (OxLDL) renders it immunogenic and cloned spontaneously arising IgM autoantibodies (Abs) from spleens of apo +mice (EO Abs) that were selected for binding to OxLDL. All were subsequently shown to bind oxidized phospholipids (OxPL) present as free lipids or as adducts with proteins. We demonstrated that the EO Abs (such as EO6) bound to apoptotic cells, which are known to be under oxidative stress. EO6 blocked macrophage uptake of OxLDL, as well as phagocytosis of apoptotic cells. These data indicate that OxPL serve as ligands that mediate macrophage recognition of oxidatively modified cells and lipoproteins. To gain insight into the structure of these Abs, we cloned and sequenced their variable region genes and discovered a 100% homology of both VH/VL gene usage with T15 anti-phosphorylcholine (PC) Abs that have been extensively studied for over 30 years. T15 Abs bind to S. pneumonia, which contains PC on its cell wall polysaccharide, and confers "innate" protection against this pathogen. However, the T15 clone expands even in germ-free mice suggesting that it is selected based on an innate "housekeeping role," although the neo-antigens to which it binds had not been defined, prior to our studies. The fact that T15 B-cells are greatly expanded in the apoE-/-mice suggests a specific immune response to oxidation-specific epitopes generated by their atherosclerotic burden. In this proposal, we will define the natural history of T15 Abs and T15 B-cells in plasma, lesions and B-cell compartments during the course of development of atherogenesis in murine models. We will determine their impact on atherogenesis through passive transfer experiments in mice, with T15/EO6 Abs themselves or B-1 cells. We will determine the impact of similar experiments in mice genetically unable to make the T15 clone and in mice that overexpress B-1 cell Abs. In addition, we will determine if humans develop anti OxLDL Abs following pneumococcal infection, or after pneumococcal vaccination, and determine experimentally in mice whether pneumococcal exposure or immunization impacts the atherogenic process. Finally, we will attempt to clone and characterize similar autoAbs in man. These data should provide novel information on T15 B-cell mediated mechanisms in atherogenesis and for the first time characterize oxidation-specific "neo-self" ligands to which anti-OxLDL/T15 B cells respond.

laboratory mouse; low density lipoprotein; tissue /cell culture

DESCRIPTION (provided by applicant): Evidence continues to accumulate that immunological mechanisms are of central importance in atherogenesis, consistent with our current understanding of atherosclerosis as a chronic inflammatory disease. Our lab first demonstrated that epitopes of oxidized LDL (OxLDL) were immunodominant within the atherosclerotic plaque and that immunization of animals with a model of OxLDL, malondialdehyde modified LDL (MDA-LDL), was atheroprotective. We also demonstrated that immunization with a model oxidized phospholipid analogue found in OxLDL also provided atheroprotection. This suggests that an appropriate immunization strategy could be developed to inhibit atherogenesis. However, immunization with modified autologous LDL would not be practical for large populations. To develop a generalized vaccine requires the identification of the oxidation-specific epitope(s) that provides the atheroprotective immunity, but the actual chemical moieties generated by MDA modification of LDL are complex. The overall goal of these studies is to improve our understanding of immune responses to OxLDL and to initiate the development of a vaccine approach for the amelioration of atherosclerosis. To accomplish this we will identify specific immunogenic oxidation-specific epitope(s) that provide atheroprotective immunity, which could then be formulated into one or more vaccine approaches. We will synthesize a panel of MDA-lysine derived adducts, and a panel of oxidized phospholipid derived adducts, and use these to screen murine and human sera to determine the immunodominant chemical moieties. We will determine which of these candidate epitopes occur in vivo in atherosclerotic lesions and select candidates to test for their ability to be atheroprotective in cholesterol-fed LDL receptor deficient mice. We will develop a synthetic single epitope immunization strategy to test the immunogenicity and atheroprotective properties of selected epitopes in mice and we will determine mechanisms by which successful immunotherapy occurs. This information will improve our understanding of immune responses to OxLDL and could lead to the development of an immunization strategy that could be applied widely to ameliorate the progression of atherosclerosis. Lay Abstract: The immune system plays an important role in the development of atherosclerosis. We are trying to develop a vaccine that could be used to help prevent the progression of atherosclerosis

The Administrative Core will perform the operations necessary for the smooth and efficient operation of the PPG program. This core will be responsible for the personnel, administration of each ofthe projects and cores, will order joint supplies, equipment and services necessary to further the scientific goals ofthe various scientific projects. The Administrative Core will also be responsible for recording expenses associated with these functions, and will perform bookkeeping tasks necessary to reconcile the Program Project Accounts with the University ledgers. The Core will be responsible for the organization and execution of the annual retreat that will include an External Advisory committee, and will plan for and facilitate the more frequent quarterly meetings of Project Investigators to ensure maximal scientific collaborations.. The Administrative Core will also serve to coordinate and prepare the Annual Reports for the Program Office. Finally it will serve to foster communication between the Project scientists and staff and assist them in any way possible to foster the productive scientific environment.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We propose to submit a new PPG application to facilitate study of the role of the innate immune system in atherogenesis. Once initiated, atherosclerosis has all the characteristics of a chronic inflammatory disease and each Project Leader of the proposed PPG has contributed importantly to the recognition that immunological mechanisms play a central role in modulating disease progression. In vivo studies and our own data suggest that TLRs, which play critical roles in pathogen recognition, also modify atherosclerosis by mediating inflammatory responses to modified lipoproteins and proatherogenic ligands. We propose to use a combination of in vitro and in vivo approaches to understand the regulation of innate immune responses to relevant "pathogens", and their impact on inflammation and atherosclerosis. [unreadable] [unreadable] There is extensive evidence that PPARg and PPARd ligands inhibit inflammatory processes, including TLR-dependent mechanisms, and we will use a combination of molecular, cellular and genomics approaches to understand how they control programs of inflammatory gene expression in macrophages and other cells in the artery. Specifically, we will test the hypothesis that NCoR/SMRT/SUMOylation-dependent pathway plays an important role in vitro and in vivo in mediating the anti-inflammatory and anti-atherogenic effects of PPARg and that PPARd regulates the inflammatory state by control of the concentrations of free and nuclear receptor-bound fractions of the co-repressors BCL-6 and SMRT. The relevance of these observations for atherogenesis will be tested using a variety of unique gene targeted murine models. TLRs of innate immunity sense pathogens, both exogenous and endogenous and induce proinflammatory, proatherogenic responses in macrophages and other cells. Using a variety of unique genetic models, we will determine the coreceptors that pair with TR2 to promote atherosclerosis, the ligands with which they interact, and the molecular and cellular mechanisms responsible. A third focus on innate immunity will be on innate B-1 cells and the IgM natural antibodies (NAbs) they secrete, which appear to target oxidation-specific epitopes as found on OxLDL and apoptotic cells. Using reconstituted mice in which all plasma IgM are NAbs, we will explore their role in atherosclerosis and homeostasis. We will explore the regulation of B-1 cells by TLRs and by nuclear receptors and determine the molecular pathways by which this occurs. In summary, our studies will lead to an increased understanding of the innate network of immune regulation, which could lead to novel therapeutic options to control inflammation and atherosclerosis. [unreadable]

Evidence continues to accumulate that immunological mechanisms are of central importance in atherogenesis. Our lab first demonstrated that oxidation-specific epitopes of oxidized LDL (OxLDL) are immunogenic and that adaptive and innate immune responses to these neoepitopes lead not only to proatherogenic but atheroprotective responses as well. Innate immune responses provide the first line of defense against pathogens, and of necessity its receptors are preformed and selected by conservation. Our recent work supports the hypothesis that innate immune responses to neoepitopes of OxLDL have been conserved because common oxidation-specific epitopes of OxLDL are also present on apoptotic cells and display molecular mimicry with epitopes on common pathogens. Thus, these innate responses were conserved to play important roles in homeostasis in general. The overall aim of this Project is to explore the consequences of innate immunological responses to oxidation-specific epitopes of OxLDL. We will specifically test the hypothesis that oxidation-specific epitopes are a major target of innate immunity in general, and of natural antibodies (NAbs) in particular, and that these NAbs play important roles in health and disease. Our Specific Aims are to test the following hypotheses: 1) That in mice, oxidation-specific epitopes are a major target of B-1 cell derived IgM NAbs, which play important roles in health (to clear apoptotic cells/apoptotic bodies and/or neutralize proinflammatory effects) and disease, (e.g. to block uptake of OxLDL and prevent atherogenesis). 2) That vital innate immune functions of B-1 cells, such as secretion of NAbs and cytokines, are regulated by ligation of pattern recognition receptors, (such as TLRs and CD36) as well as by nuclear receptors expressed in these cells (PPARg and delta), reflecting part of a primary integrated response of the innate immune network to inflammation and infection. 3) That oxidationspecific NAbs are also prevalent in humans and play similar important roles. In summary, in general these studies should provide an improved understanding of the role of innate immunity in atherogenesis and specifically define the role of oxidation-specific NAbs and the B-1 cells that secrete them in health and disease. Insights from these studies could lead to novel diagnostic and therapeutic options for patients with CVD and other inflammatory conditions.

DESCRIPTION (provided by applicant): We propose to submit a new PPG application to facilitate study of the role of the innate immune system in atherogenesis. Once initiated, atherosclerosis has all the characteristics of a chronic inflammatory disease and each Project Leader of the proposed PPG has contributed importantly to the recognition that immunological mechanisms play a central role in modulating disease progression. In vivo studies and our own data suggest that TLRs, which play critical roles in pathogen recognition, also modify atherosclerosis by mediating inflammatory responses to modified lipoproteins and proatherogenic ligands. We propose to use a combination of in vitro and in vivo approaches to understand the regulation of innate immune responses to relevant "pathogens", and their impact on inflammation and atherosclerosis. There is extensive evidence that PPARg and PPARd ligands inhibit inflammatory processes, including TLR-dependent mechanisms, and we will use a combination of molecular, cellular and genomics approaches to understand how they control programs of inflammatory gene expression in macrophages and other cells in the artery. Specifically, we will test the hypothesis that NCoR/SMRT/SUMOylation-dependent pathway plays an important role in vitro and in vivo in mediating the anti-inflammatory and anti-atherogenic effects of PPARg and that PPARd regulates the inflammatory state by control of the concentrations of free and nuclear receptor-bound fractions of the co-repressors BCL-6 and SMRT. The relevance of these observations for atherogenesis will be tested using a variety of unique gene targeted murine models. TLRs of innate immunity sense pathogens, both exogenous and endogenous and induce proinflammatory, proatherogenic responses in macrophages and other cells. Using a variety of unique genetic models, we will determine the coreceptors that pair with TR2 to promote atherosclerosis, the ligands with which they interact, and the molecular and cellular mechanisms responsible. A third focus on innate immunity will be on innate B-1 cells and the IgM natural antibodies (NAbs) they secrete, which appear to target oxidation-specific epitopes as found on OxLDL and apoptotic cells. Using reconstituted mice in which all plasma IgM are NAbs, we will explore their role in atherosclerosis and homeostasis. We will explore the regulation of B-1 cells by TLRs and by nuclear receptors and determine the molecular pathways by which this occurs. In summary, our studies will lead to an increased understanding of the innate network of immune regulation, which could lead to novel therapeutic options to control inflammation and atherosclerosis.

The Analytical Core will provide a variety of specialized analytical services for the investigators of the Program Project to assist in the conduct ofthe molecular, cellular and in vivo studies. The Analytical Core will be a collaborative effort between units at the University of California, San Diego under Dr. Joseph Witztum and one at the Salk Institute under the direction of Dr. Ronald Evans. The overall goal of the Analytical Core is to take advantage of specialized resources and expertise to provide investigators with selected core services that will assist in the research mission of each Project. The Aims of this Core are: Specific Aim 1: To provide high-throughput nano-scale quantitative PCR analysis (Nano-QPCR) profiling of gene signatures and pathways. Here we describe the equipment and operation procedures for high-throughput nano-scale quantitative, real-time, reverse-transcription PCR (Nano-QPCR) for surveying SNP genotyping and profiling the expression of particular transcripts in samples provided by PPG investigators. Specific Aim 2: To provide high throughput, high sensitivity analysis of cytokines and chemokines with the use of Luminex Bio-Plex technology. The Bio-Plex suspension array component ofthe core has been in operation in the Evans laboratory for over 10 years and consists of a Luminex Bio-Plex workstation that will facilitate proteomic profiling of cytokines and chemokines in sufficiently small sample volumes compatible with plasma or culture fluids in a high throughput format. Specific Aim 3: To provide immunological support for investigators of the PPG. The Immunology Core will generate antisera against desired antigens and prepare a wide variety of immunological reagents to be used by the other Projects as requested, including primary or secondary antibodies to be used in immunoassays or Western blots or FACS analysis. Specific Aim 4: To provide quantitative and qualitative analysis of lipid and lipoprotein levels and provision of lipoprotein fractions. The core will provide measures of total cholesterol and triglycerides and will analyze FPLC profiles of murine plasma for these analytes and provide human LDL and/or other lipoproteins as needed

Atherosclerosis is now recognized as a chronic inflammatory disease, and Project Leaders of our PPG have contributed seminal information to the widespread recognition that immune mechanisms play a central role in modulating atherogenesis. Leveraging information gained in the first cycle, we propose studies of the roles of macrophages, T cells, B cells, Natural antibodies (NAbs) and innate TLRs on inflammation and atherogenesis. Project 1 will pursue their seminal observations that foam cell formation in the peritoneum of cholesterol-fed mice exhibited an unexpected suppressed inflammatory gene phenotype, which was due to accumulation of desmosterol, a potent LXR ligand, leading to inhibition of inflammatory gene expression. They will study the transcriptional mechanisms by which this occurs, and determine if novel therapeutic approaches can be exploited based on use of desmosterol-like agents that inhibit inflammatory activity. Project 2 will pursue their findings that PPARy is expressed in Treg cells in peri-aortic adipose tissue, which surrounds the aorta at key anatomical sites where atherogenesis is enhanced. They will explore the hypothesis that this is mediated by a proinflammatory gene network that can be modulated at the transcriptional level by PPARy and REVERBa/p, regulating vital functions of Treg and Th17 cells. Project 3 will pursue their recent identification that oxidation specific epitopes (OSE), as occur on OxLDL or apoptotic cells, are major targets of innate NAbs. They will focus on defining the prevalence of OSE-NAbs in humans and mice, the mechanisms by which they are atheroprotective, and define transcriptional mechanisms by which GR and LXR regulate B-1 cells, which generate NAbs. Overall, these studies should provide vital insights into novel and as yet unexplored mechanisms by which adaptive and innate immunity regulates inflammation and atherosclerosis, and may lead to novel diagnostic and therapeutic approaches for cardiovascular disease.

Innate Immunity plays a fundamental role in atherogenesis and our work has provided an improved understanding of this by demonstrating that oxidation-specific epitopes (OSE), which are generated on oxidatively damaged molecular complexes, such as OxLDL and apoptotic cells, are major targets of innate pattern recognition receptors, such as lgM Natural Antibodies (NAbs). During the first cycle, we demonstrated that 20-30% of all lgM NAbs bind to OSE in both mice and humans, and we proposed that lgM OSE-NAbs have been conserved to provide homeostasis to the many OSE generated in both health and disease. Considerable data support an atheroprotective role for lgM in murine models and lgM titers in humans are inversely related to cardiovascular disease (CVD). In the renewal, we will focus on understanding the role of NAbs in mice and humans, and define the mechanisms that regulate 8-1 cells that generate NAbs. Specific Aim 1 will define the repertoire and prevalence of OSE NAbs in mice and humans. We will generate a B-1 cell derived database of lgM NAb heavy chain variable (IGHV) CDR3 sequences and their relative expression in both humans and mice. We will then sort OSE-B-1 cells to annotate OSE NAbs, and will examine their relative expression under experimental models of inflammation and atherosclerosis in mice, and in epidemiological studies in humans. Specific Aim 2 will define the roles of OSE NAbs and B-1 cells in inflammation and atherogenesis. Using transgenic mice expressing OSE antibodies, we will seek to define the mechanisms by which OSE-Abs influence inflammation and atherosclerosis. Because these NAbs target prevalent oxidized lipids in atherosclerotic lesions, these studies should define the importance of these oxidized moieties in mediating inflammation and atherogenesis. Specific aim 3 will test the hypothesis that vital functions of B-1 cells, such as proliferation and secretion of NAbs are positively regulated by TLRs, while the nuclear receptors GRand LXR negatively regulate B-1 cells. We will determine the impact of these immune modulators on transcriptional regulation of B-1 cells in comparison to B-2 cells and macrophages, to provide an improved understanding of the integrated responses to these immune cell regulators. These studies should yield new insights into the important role that innate immunity plays in inflammation and atherosclerosis, and may lead to novel diagnostic and therapeutic approaches for CVD.

DESCRIPTION (provided by applicant): The overall purpose of this program is the training of new scientists capable of performing high quality biomedical research in Endocrinology and Metabolism emphasizing techniques of molecular, cellular and physical biology to solve fundamental problems in regulatory biology. A group of 17 dedicated faculty provide an integrated interdisciplinary program for training M.D.'s and Ph.D.'s in modern research relevant to hormonal signaling and clinical problems related to thyroid disease, atherosclerosis, cancer, developmental defects, diabetes and reproduction. Support is requested for 3 postdoctoral positions per year that will be filled by selected M.D. and Ph. D. candidates. Trainees will be selected from a pool of applicants who apply for training in the Division of Endocrinology and Metabolism and who apply to faculty directly. An average training period of 2-3 years will focus on research training with a faculty preceptor. The program will also include: (1) intensive laboratory and/or clinical research training, (2) seminars and other conferences, and (3) formal course instruction. The primary focus of the training program is the research undertaken by each trainee in association with a member of the training grant faculty. Under close supervision by the faculty member, the trainee will be encouraged, and expected, to assume an increasingly independent scientific role in all aspects of the research. In addition, the training program will foster and encourage a scholarly exchange of ideas and intellectual cross fertilization. M.D. trainees will receive appropriate clinical training through the Division of Endocrinology and Metabolism training program and Ph.D. trainees will be exposed to relevant clinical problems through divisional conferences and seminars. The training grant faculty will tailor the educational program to the needs of each trainee and provide assessment of progress and guidance in career development. All trainees are required to take 4 quarters of formal course work including a required course entitled Biomedical Research Ethics offered through the School of Medicine. Special efforts will continue to recruit individuals from underrepresented racial/ethnic groups and to increase diversity. This training program will bring together a diverse group of trainees and faculty members at UCSD emphasizing the essential role of hormonal control mechanisms. The trainees will be well prepared for careers as faculty in schools of medicine, in basic science departments, and in the biotechnology industry.

Summary Lipid peroxidation, a central event in atherogenesis and NASH, results in formation of oxidation-specific epitopes (OSE) such as oxidized phospholipids (OxPL), malondialdehyde (MDA) and complex MDA adducts termed MAA, which we termed ?oxidation-specific-epitopes? (OSE). They are proinflammatory and promote chronic inflammation. Because OSE are mostly products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them were unavailable until now and their actual roles in vivo in disease states such as atherosclerosis and NASH are unknown. Project 3 is focused on understanding the role of OSE in both atherosclerosis and NASH and the common (or unique) mechanisms by which they contribute to these diseases. This is now feasible based on our recent development of transgenic mice that constitutively express single chain antibodies that target OxPL--the E06-scFv mice?or target MDA/MAA?the IK17-scFv mice. In recently published and new preliminary data we demonstrate that targeting OxPL in mice consuming NASH producing diets decreases both atherosclerosis and NASH. Preliminary studies demonstrate that targeting of MDA/MAA can also reduce the progression of atherosclerosis and hepatic inflammation in a NASH model. Patients with NASH are at increased risk for CVD, and share common risk factors. However, NASH confers additional risk for CVD above that due to known shared risk factors. This Application will test the hypothesis that OSE are a previously unrecognized common risk factor. Uniquely, this project will simultaneously focus on the role of OSE in the pathogenesis of atherosclerosis and NASH and define common (or distinct) mechanisms by which OSE promote these diseases. Specific Aim 1 will use the E06-scFv transgenic mice to study the Role of OxPL in Atherosclerosis and Hepatic Steatosis/NASH/Fibrosis in mouse models and determine if targeting OxPL can simultaneously reduce disease burden in both the artery and liver. We will use the E06-scFv mice to investigate the specific mechanisms by which neutralization of OxPL impacts atherogenesis and liver disease. Specific Aim 2 will study the Role of MDA/MAA in Atherosclerosis and Hepatic Steatosis/NASH/ Fibrosis in mouse models in parallel studies to Aim 1. Specific Aim 3 are Translational Studies of the Role of OSE in CVD and NAFLD/NASH and will seek to determine if targeting OxPL and/or MDA/MAA can not only prevent progression of disease but cause regression of existing disease. This will be accomplished by generating transgenic mice that conditionally express the antibodies targeting OSE so that enhanced titers can be achieved after disease is established. This will allow studies to determine if targeting OSE can be therapeutic and reduce disease burden. These studies should provide an understanding of novel common risk factors connecting NASH and atherogenesis. Because an antibody-mediated approach to neutralize OSE could target both NASH and atherogenesis simultaneously, these studies may lead to innovative translational applications.

PROJECT SUMMARY Core A. Phenotyping The mission of the Phenotyping Core A is to provide standardized analytical methodologies and services to assist the Project Investigators in efficiently achieving the Scientific Aims of their respective projects. The Core will provide targeted phenotyping of both mouse models and human subjects, as well as providing selected reagents used by the Projects. The Core is organized into 5 Sub-Cores, which will provide the following services: 1) An Atherosclerosis Morphology Sub-Core to provide qualitative and quantitative analyses of mouse atherosclerosis. 2) Liver Morphology Sub-Core to provide qualitative and quantitative analysis of mouse liver histology in the context of NASH and fibrosis. 3) Targeted Lipidomic Analysis Sub-Core to provide analyses of plasma and tissues in the UCSD LIPID MAPS Lipidomic Core. 4) Targeted Proteomic Sub-Core to provide high- throughput, high- sensitivity analysis of cytokines, chemokines and other analytes with the use of Luminex Bio- Plex technology and 5). Lipoprotein Sub-Core, which will measure lipid and lipoprotein levels in mouse plasma and also provide standardized preparations of LDL and modified LDLs (and other needed lipoproteins) for investigators? use. The Core will be centered and organized in Dr. Witztum's laboratory at UCSD, who will be the overall director and responsible to see that all of the Sub-Cores fulfill their missions. The Atherosclerosis Morphology and Liver Morphology Sub-Cores will be housed at UCSD under the Direction of Dr. Witztum with assistance of a UCSD expert in liver pathology (Dr. Tatiana Kisseleva), as will the services of the Lipoprotein Core. High- throughput selected proteomic type analyses of cytokines, chemokines and related proteins will be performed at the Salk Institute under the direction of Drs. Ron Evans and Michael Downes. Finally, the Lipid Maps Lipidomic Core, under the direction of Drs. Oswald Quehenberger and Ed Dennis is also located at UCSD in the same building as Dr. Witztum?s laboratories. Thus, all sample collection will be centralized in a central laboratory at UCSD, where they will be logged into an Excel spreadsheet to provide a central organizational pathway for distribution of samples to proper Sub-Cores and in turn, a central site for data collection of results.

PROJECT SUMMARY Project 3. Pivotal Role of Oxidation-specific Epitopes in CVD and NASH Lipid peroxidation, a central event in atherogenesis and NASH, results in formation of oxidation-specific epitopes (OSE) such as oxidized phospholipids (OxPL), malondialdehyde (MDA) and complex MDA adducts termed MAA, which we termed ?oxidation-specific-epitopes? (OSE). They are proinflammatory and promote chronic inflammation. Because OSE are mostly products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them were unavailable until now and their actual roles in vivo in disease states such as atherosclerosis and NASH are unknown. Project 3 is focused on understanding the role of OSE in both atherosclerosis and NASH and the common (or unique) mechanisms by which they contribute to these diseases. This is now feasible based on our recent development of transgenic mice that constitutively express single chain antibodies that target OxPL--the E06-scFv mice?or target MDA/MAA?the IK17-scFv mice. In recently published and new preliminary data we demonstrate that targeting OxPL in mice consuming NASH producing diets decreases both atherosclerosis and NASH. Preliminary studies demonstrate that targeting of MDA/MAA can also reduce the progression of atherosclerosis and hepatic inflammation in a NASH model. Patients with NAFLD are at increased risk for CVD, and share common risk factors. However, NAFLD confers additional risk for CVD above that due to known shared risk factors. This Project will test the hypothesis that OSE are a previously unrecognized common risk factor. Uniquely, this project will simultaneously focus on the role of OSE in the pathogenesis of atherosclerosis and NASH and define common (or distinct) mechanisms by which OSE promote these diseases. Specific Aim 1 will use the E06-scFv transgenic mice to study the Role of OxPL in Atherosclerosis and Hepatic Steatosis/NASH/Fibrosis in mouse models and determine if targeting OxPL can simultaneously reduce disease burden in both the artery and liver. We will use the E06-scFv mice to investigate the specific mechanisms by which neutralization of OxPL impacts atherogenesis and liver disease. Specific Aim 2 will study the Role of MDA/MAA in Atherosclerosis and Hepatic Steatosis/NASH/ Fibrosis in mouse models in parallel studies to Aim 1. Specific Aim 3 are Translational Studies of the Role of OSE in CVD and NAFLD/NASH and will seek to determine if targeting OxPL and/or MDA/MAA can not only prevent progression of disease but cause regression of existing disease. This will be accomplished using newly generated transgenic mice that conditionally express the antibodies targeting OSE so that enhanced titers can be achieved after disease is established. This will allow studies to determine if targeting OSE can be therapeutic and reduce disease burden. These studies should provide an understanding of novel common risk factors connecting NASH and atherogenesis. Because an antibody-mediated approach to neutralize OSE could target both NASH and atherogenesis simultaneously, these studies may lead to innovative translational applications.