Ravichandran Ramasamy - US grants

Ischemia limits the ability of a myocardium to generate sufficient high energy phosphates to maintain myocyte viability. Although reperfusion, especially early after the onset of occlusion has revolutionized the treatment of acute myocardial ischemia, beneficial effects are limited by the amount of ischemic damage that occurs prior to reperfusion. In this context, the enhancement of myocardial metabolism during ischemia as means of protecting the myocardium assumes greater importance. In recent years, a variety of metabolic therapies that enhance myocardial metabolism during ischemia have been proposed. They focus on (a) increasing myocardial glycolysis by increasing glucose uptake during ischemia or (b) limiting the inhibitory effects of fatty acids to increase glucose metabolism and flux through pyruvate dehydrogenase (PDH) on reperfusion, and (c) inhibiting sodium and calcium influx pathways during ischemia that deplete high energy phosphates. Recent studies from our laboratory demonstrated a novel metabolic approach of protecting ischemic rat hears by inhibiting aldose reductase, a key regulatory enzyme in the substrate flux via the polyol pathway. This novel intervention limited infarct size and improved function and metabolic recovery after ischemia. The aim of this proposal research is to delineate the role of aldose reductase protects ischemic myocardium. Specifically, studies will be performed in isolated rat/mice hearts to investigate if (a) ischemia increases aldose reductase activity, and if the hearts over-expressed for aldose reductase exhibit increased ischemic injury in mice transgenic for aldose reductase, the rate of glycolysis and changes in metabolites, the activity of the Na+, K+-ATPase as well as other sodium transporters, and changes in intracellular sodium and calcium during ischemia in aldose reductase inhibited hearts. Biochemical assays will be used to measure enzyme activities and metabolites, while nuclear magnetic resonance (NMR) spectroscopy will be used to measure time dependent changes in intracellular sodium and calcium. The ability to examine the relationship between metabolism and function using biochemical assays and NMR spectroscopy will enhance our understanding of the mechanism by which aldose reductase inhibition protects the myocardium from ischemic injury. Further, this novel approach of protecting the myocardium from ischemic injury, may become a useful therapeutic intervention in treating myocardial infarction in patient.

DESCRIPTION: (Adapted from the Investigator's Abstract) Accelerated vascular disease, involving both the micro- and macro-vasculature accompanies diabetes mellitus (both types I and II) and syndromes of insulin resistance. Nonenzymatic glycation of proteins and lipids occurs with hyperglycemia and is most marked in macromolecules with long turnover times, ultimately leading to formation of advanced glycation endproducts (AGEs). A principle means by which AGEs affect cellular properties is by interacting with binding proteins, the best characterized of which is receptor for AGEs (RAGE), a member of the immunoglobulin superfamily. Based on the applicant's pilot data, they hypothesize that engagement of RAGE by AGEs underlies changes in those cells critical to atherogenesis, especially endothelial cells and monocytes, affecting cell function in ways that predispose to the development of atherosclerosis and microvascular disease. The major goal of this project is to develop and utilize molecular tools allowing dissection of the contribution of RAGE to diabetic atherosclerosis. Their specific aims are: (1) To develop transgenic (Tg) mice overexpressing sRAGE and RAGE gene knock-out (0) mice, in order to study the role of RAGE in hyperfibrinogenemia, elevated plasminogen activator inhibitor-I (PAI-I) and vascular hyperpermeability associated with diabetes; (2) to develop a diabetic murine model of accelerated atherosclerosis, and, using this model, to determine the contribution of RAGE to lesion formation. These experiments will initially employ apoE 0 mice rendered diabetic with streptozotocin, based on the applicant's pilot data showing more extensive atherosclerosis in these animals than in euglycemic apoE 0 mice, as well as cross-breeding with murine genetic models of diabetes. Tg mice overexpressing apoB, an excellent substrate for nonenzymatic glycation and oxidation, will also be used to develop an atherosclerosis/diabetes model. (3) To assess if AGE engagement of RAGE leading to increased plasma soluble (s) VCAM-1 antigen provides a marker of ongoing endothelial perturbation in diabetes. The impact of RAGE blockade and antioxidant therapy on plasma sVCAM-1 and indices of vascular dysfunction in experimental murine diabetes will be evaluated. Then, using optimal conditions from their murine study, a clinical trial of antioxidants will be undertaken in diabetic patients with microalbuminuria to determine the effect on sVCAM-1 and plasma markers of oxidant stress. The results of these experiments are directed towards our long-term goal, understanding mechanisms important in the pathogenesis of accelerated atherosclerosis in diabetes.

DESCRIPTION (provided by applicant): Diabetic patients, both Type 1 and 2 display both increased incidence of cardiovascular disease, and complications of myocardial infarction and heart failure. Altered cardiac function, metabolism of glucose and fatty acids, and sodium homeostasis have been demonstrated in both types of diabetes. Altered glucose metabolism, in particular, flux of glucose via the polyol pathway may be responsible, in part, for the enhanced vulnerability of diabetic myocardium to ischemic injury. In polyol pathway, glucose is reduced to sorbitol by aldose reductase (AR) and subsequently oxidized to fructose by sorbitol dehydrogenase (SDH). We have demonstrated that flux via the polyol pathway is partly responsible for impaired myocardial glycolysis and energy production. When diabetic hearts are subjected to ischemia, the ability to generate sufficient high-energy phosphates for maintaining myocyte viability and for sodium homeostasis is severely compromised. Our work, as well as that of others, has shown that enhancement of glycolytic metabolism during ischemia is a feasible approach to maintain myocyte viability, energy metabolism and sodium homeostasis. In this context, our preliminary data demonstrate increased AR and SDH activities in both type 1 and Type 2 diabetic rat hearts and that pharmacological inhibition of AR normalized glycolysis and Na+,K+-ATPase activity via protein kinase C-B Induction of ischemia further increases AR and SDH activity in diabetic hearts and is associated with increased myocardial ischemic injury and poor functional recovery on reperfusion. Inhibition of the polyol pathway reduced ischemic injury, attenuated changes in intracellular sodium homeostasis, and improved functional and metabolic recovery after ischemia in diabetic hearts. These data lead to the hypothesis that in Type 1 and 2 diabetes, increased activity of polyol pathway enzymes AR and SDH increases myocardial vulnerability to ischemic injury, and that this can be attenuated by polyol pathway inhibitors. Mechanisms by which diabetes increases myocardial AR activity, how augmented AR activity in diabetes and ischemia acts to increase myocardial damage, and impairs sodium homeostasis and energy metabolism will be investigated. Type 1 and Type 2 diabetic and non-diabetic littermate rats, as well as transgenic mice, will be used to test our hypothesis and elucidate mechanisms using NMR spectroscopy, biochemical, and molecular techniques. The proposed studies will provide a rationale for the use of polyol pathway inhibitors as an adjunctive therapeutic intervention for treating diabetic patients with myocardial infarction.

The goals of the Biochemistry and Pathology Core, are two-fold: First, the Biochemistry Unit of the Core will serve to perform assays on tissues/plasma of mice and cultured cells studied in the individual projects, including assessment of 4-hydroxynonenal, malondialdehyde, nitric oxide, 3-deoxyglucosone, diacylglycerol, and activities of superoxide dismutase (CuZn SOD and MnSOD), aconitase, aldose reductase, sorbitol dehydrogenase and Protein Kinase C. This unit will be led by Dr. Ravichandran Ramasamy, an experienced biochemist whose laboratory routinely performs these analyses; extensive quality control measures and equipment maintenance/rapair are in place in order to ensure consistency of results across the projects. Second, the Pathology Unit of this Core will serve to perform pathologic analyses of mouse hearts, aortae en face, innominate arteries, and aortic sinus, as well as routine immunohistory with semi-quantitative analysis of extent/intensity of immunostaining. This unit will be led by Dr. Vivette D'Agati, who is a long-time collaborator of the Principle Investigator of the Program Project. The biochemical and pathologic analysis of tissues is best done under the auspices of a core unit in order to ensure consistency of sample preparation, assay performance and quantitative analysis. The Biochemistry and Pathology Core wioll serve all three projects of the Program during each of the five years of the Program Project Grant.

PROJECT SUMMARY (See instructions): Myocardial infarction and its consequences are a leading cause of morbidity and mortality. Earlier studies by us have uncovered key roles for RAGE in myocardial infarction, as global deletion RAGE resulted in decreased myocardial necrosis, increased functional recovery and preservation of ATP compared to wildtype littermates 48 hours after ischemia/reperfusion (l/R). Our studies have uncovered that RAGE contributes to oxidative stress consequent to l/R and influences mitochondrial dysfunction that accompanies injury to the heart. A thorough approach to understanding the basic mechanisms underlying the effects of global RAGE deletion requires cell-specific dissection of the precipitating pathways of injury. Novel findings from our group during the past year enhance the direction of the proposed studies in this application: the RAGE cytoplasmic domain interacts with diaphanous-1 (mDia-1), a member of the formin homology domain protein family and an effector of Rho GTPases. mDial is essential for RAGE ligand-mediated cellular migration and activation of cdc42/rac-1. In this project, we will probe the signaling mechanisms in cardiomyocyte stresses evoked by l/R in the heart using murine models, both in the absence and presence of diabetes. We hypothesize that cardiomyocyte RAGE and mDial, highly upregulated in the murine heart after l/R, signals devastating metabolic consequences in the myocardium, which trigger mitochondrial dysfunction, in part through GSK-3n, ROCK and apoptotic events. These concepts will be explored in depth using novel RAGE and mDial floxed mice in murine models of l/R in the heart. Project 3 is integrally linked within the Program and will study cell-specific RAGE and mDia-1 signaling in myocardial infarction. Project 3 shares findings from Affymetrix gene array studies with Projects 1&2 to create integrated pathways by which RAGE signaling regulates cardiovascular stress. Project 3 uses all three Cores of the Program during all five years.

The goals of Core B, Animal Experimentation Core, are to enhance the reproducibility of surgical procedures; pathological analysis; and functional imaging in a cohesive manner for the Program Project. Dr. Ravi Ramasamy and Dr. Yoshifumi Naka have extensive experience in the isolated perfused heart and rat/mouse microsurgery, respectively, and will lead the experimentation to induce I/R stress in aged vs. young mice and rats in distinct tissues. Dr. Vivette D'Agati and Dr. Matthias Szabolcs will coordinate pathologic analyses of mouse/rat tissues subjected to ischemia/reperfusion (I/R) stress. Measurement of injury; routine immunohistochemistry; and semi-quantitative analysis of extent/intensity of immunostaining will be performed. Dr. Shunichi Homma will lead the imaging functions of the core, in order to quantify the degree of infarction triggered by I/R stress. Core B will serve all three projects of the Program during each of the five years of the Program Project Grant.

Aging human subjects display increased incidence of cardiovascular disease and complications of myocardial infarction and heart failure. We have demonstrated that flux via the polyol pathway is partly responsible for impaired myocardial glycolysis and energy production. When hearts from aged rats are subjected to ischemia, the ability to generate sufficient high energy phosphates for maintaining myocyte viability and sodium homeostasis is severely compromised. Our work, as well as that of others, has shown that enhancement of glycolytic metabolism during ischemia is a feasible approach to maintain myocyte viability, energy metabolism and sodium homeostasis. In this revised application, we show that in human aging, that is, without superimposed cardiovascular disease or diabetes, and in aged Fischer 344 rats, expression and activity of aldose reductase (AR) is increased in the heart. Induction of ischemia further increases AR activity in aged hearts, and is associated with increased myocardial ischemic injury and poor functional recovery on reperfusion. Inhibition of the polyol pathway (AR) or the next enzyme in the pathway, sorbitol dehydrogenase (SDH) reduced ischemic injury, attenuated changes in intracellular sodium homeostasis, and improved functional and metabolic recovery after ischemia in aged hearts. Thus, we hypothesize that in aging, increased activity of the polyol pathway enzyme AR increases myocardial vulnerability to ischemic injury, and that this can be attenuated by polyol pathway inhibitors. The proposed studies will probe the mechanisms by which aging increases myocardial polyol pathway activity, and how this augmented activity in aging and ischemia acts to increase myocardial damage Distinct strategies including pharmacological inhibitors of AR and SDH, Fischer 344 rats, and human AR expressing transgenic mice will be employed to test these concepts. Further, to enhance understanding of SDH in aging in the heart, SDH null mice will be bred into the transgenic mouse background in which human-relevant levels of AR are expressing. We will utilize NMR spectroscopy, biochemical, and molecular techniques in our experiments. Project 1 is closely linked to Projects 2&3, as each studies aging-linked enhanced vulnerability to I/R stress in vascular cells and cardiomyocytes. Project 1 shares mouse/rat models with Projects 2 and 3. Project 1 will utilize all three Cores of the Program Project during all five years of the grant.

DESCRIPTION (provided by applicant): Earlier studies from our laboratory have uncovered key roles for RAGE in myocardial infarction, as global deletion RAGE resulted in decreased myocardial necrosis, increased functional recovery and preservation of ATP compared to wild-type mice after ischemia/reperfusion (I/R) in the isolated perfused heart or after occlusion/reperfusion of the left anterior descending (LAD) coronary artery. RAGE is expressed broadly in multiple cell types that impact on the heart's response to I/R injury, such as monocytes/macrophages, endothelial cells and cardiomyocytes. RAGE contributes to oxidative stress consequent to I/R and influences mitochondrial dysfunction that accompanies injury to the heart. In this application, we will probe the cross- talk between inflammatory, vascular and cardiomyocyte stresses evoked by I/R in the heart using murine models, both in the absence and presence of diabetes. Our overall hypothesis is that RAGE signaling in monocytes/macrophages, endothelial cells and cardiomyocytes is, overall, highly detrimental to the injured heart. We predict that in I/R, monocytes/macrophages produce damaging mediators that disrupt effective healing and that RAGE-dependent endothelial stress, particularly in diabetes, thwarts effective remodeling. Lastly, we predict that cardiomyocyte RAGE, highly upregulated in the murine heart after I/R, signals devastating metabolic consequences in the healing myocardium, which trigger mitochondrial dysfunction, in part through GSK-3b and apoptotic events. Two novel findings in our laboratory suggest roles for mDia-1, a member of the formin homology domain protein family and an effector of RhoGTPases, and the ROCK 1 signaling pathway in RAGE-mediated cardiovascular stress. In this application, we will probe the role of RAGE in the full context of RAGE-dependent mDia-1 and/or ROCK signaling in the heart. Only by a full understanding of potentially adaptive roles for cell-specific RAGE signaling in myocardial infarction, and the proximate signaling cascades stimulated by this receptor in I/R, will the optimal design of RAGE antagonism be achieved.

PROJECT SUMMARY (See instructions): Core B, the Biochemistry, Pathology and Imaging Core will continue to serve all three Projects through all five years of the Program. The core is composed of three units: (1) The Biochemistry Unit of Core B will perform assays on tissue/plasma of mice and cultured cells in the individual projects for assessment of oxidative stress, AGEs and other biochemical mediators linked to RAGE and vascular dysfunction. (2) The Pathology Unit of Core B will perform pathological analysis of mouse tissues. This Unit will serve all 3 projects for standardization of immunohistochemistry and semiquantitative analyses. The Unit will perform analysis of atherosclerosis (aortas (atherosclerosis at the aortic root and en face assessment of aorta), angiogenesis (Project 2-3) and myocardial infarction size (Project 3). (3) The newly-formed Imaging Unit will perform novel imaging techniques using state-of-the-art new equipment for molecular imaging studies in atherosclerosis and apoptosis (Project 1) and angiogenesis (Project 2).

DESCRIPTION (provided by applicant): The homozygous RAGE null mouse formed the centerpiece of discoveries during the last cycle of this Program. We demonstrated that RAGE plays critical roles in atheroscierosis in apoE null mice, mediates upregulation of pro-inflammatory and tissue-destructive genes in hypoxia, and mediates loss of cardiac function in the heart upon ischemia/reperfusion (l/R). Multiple novel findings shape the direction of our Program: first, we discovered that the RAGE cytoplasmic domain interacts with diaphanous-1 (mDia-1), a member of the formin homology domain protein family and an effector of RhoGTPases. mDia-1 is essential for RAGE ligand-mediated cellular migration and activation of cdc42/rac-1. New discoveries link mDial to key properties of smooth muscle cells, macrophages and cardiomyocyte signaling. Second, Project 2 has discovered the unanticipated finding that RAGE plays opposing roles in acute vs. chronic hypoxia/ischemia on regulation of Egr-1 in endothelial cells and monocytes/macrophages. Third, Project 1 has discovered that RAGE downregulates ABCG1 and cholesterol efflux to HDL. Fourth, Project 3 has discovered that deletion of mDial is highly protective in the heart in l/R. As a Program, we have shared not merely tools and strategies by virtue of our Core units but, more importantly, we have sought to understand the "big picture" of RAGE signaling. As our data unfold, we recognize that RAGE signaling is not "one size fits all," as new discoveries have uncovered distinct pathways of regulation by the receptor depending on cell type, duration of stress, and specific form of cellular stress. The challenge is to put it together. Toward this end, we have generated novel RAGE- and mDial floxed to probe cell-specific signaling of this axis in atherosclerosis (Project 1), angiogenesis (Project 2) and myocardial infarction (Project 3). Taken together, these discoveries form the basis of a highly innovative and significant set of questions testing RAGE and mDial signaling in vascular dysfunction in diabetic- and non-diabetic cardiovascular pathology. Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program.

DESCRIPTION (provided by applicant): We propose to modify a series of courses into a highly relevant NIDDK educational activity. {Despite promising clinical studies in patients with diabetic complications, such as diabetic nephropathy, retinopathy and cardiovascular disease as examples, pharmaceutical companies have been reluctant to pursue drug development in this important area. NIDDK has duly stepped in with several initiatives to support research and drug development for diabetic complications. As such, our ultimate goal will be to teach a diverse group of targeted learners at the graduate level and beyond} to harness novel bench-to-bedside empirical knowledge in their research {and related endeavors} with a focus on diabetes, {diabetic complications}, and obesity, specific, comorbid, and pervasive public health problems. {We will highlight the importance of designing therapeutic interventions based on target changes during the disease; for example, some targets may be more relevant in initiation, others more in progression, and some more in failure of regression and regeneration.} The programmatic theme that emphasizes translation of research discoveries to patient care will be drug discovery and development. The current yearlong course series reviews Phase I-IV trials, post- marketing trajectories, safety monitoring, drug patents, and economic stimulus reform as well as ethics and cost analysis in the fall semester. In the spring semester, the molecular pathways and scientific basis of successful and unsuccessful drugs are discussed. Our preliminary experience has established that together, the two course segments (and ongoing didactic seminar series) help to disabuse the limiting belief that individuals cannot directly work towards inventing drugs, and the educational activity directs the diverse interests of this wide range of trainees towards the inspirations and rewards of translating basic science discoveries into useful patient care tools. Qualitative and quantitative reporting tools and performance tasks will evaluate the program to assure achievement of educational goals as well as future expansion of the program. Progress in achieving these objectives would mean increased NIDDK-relevant translational research skills for the diverse participants to ultimately improve patient care and public health.

Aging human subjects display increased incidence of cardiovascular disease and complications of myocardial infarction and heart failure. We have demonstrated that flux via the polyol pathway is partly responsible for impaired myocardial glycolysis and energy production. When hearts from aged rats are subjected to ischemia, the ability to generate sufficient high energy phosphates for maintaining myocyte viability and sodium homeostasis is severely compromised. Our work, as well as that of others, has shown that enhancement of glycolytic metabolism during ischemia is a feasible approach to maintain myocyte viability, energy metabolism and sodium homeostasis. In this revised application, we show that in human aging, that is, without superimposed cardiovascular disease or diabetes, and in aged Fischer 344 rats, expression and activity of aldose reductase (AR) is increased in the heart. Induction of ischemia further increases AR activity in aged hearts, and is associated with increased myocardial ischemic injury and poor functional recovery on reperfusion. Inhibition of the polyol pathway (AR) or the next enzyme in the pathway, sorbitol dehydrogenase (SDH) reduced ischemic injury, attenuated changes in intracellular sodium homeostasis, and improved functional and metabolic recovery after ischemia in aged hearts. Thus, we hypothesize that in aging, increased activity of the polyol pathway enzyme AR increases myocardial vulnerability to ischemic injury, and that this can be attenuated by polyol pathway inhibitors. The proposed studies will probe the mechanisms by which aging increases myocardial polyol pathway activity, and how this augmented activity in aging and ischemia acts to increase myocardial damage Distinct strategies including pharmacological inhibitors of AR and SDH, Fischer 344 rats, and human AR expressing transgenic mice will be employed to test these concepts. Further, to enhance understanding of SDH in aging in the heart, SDH null mice will be bred into the transgenic mouse background in which human-relevant levels of AR are expressing. We will utilize NMR spectroscopy, biochemical, and molecular techniques in our experiments. Project 1 is closely linked to Projects 2&3, as each studies aging-linked enhanced vulnerability to I/R stress in vascular cells and cardiomyocytes. Project 1 shares mouse/rat models with Projects 2 and 3. Project 1 will utilize all three Cores of the Program Project during all five years of the grant.

DESCRIPTION: We are requesting funds for a Seahorse XF automated cellular respiration and glycolysis analyzer. This instrument is the only instrument on the market that can assess cellular respiration and glycolysis in physiologically relevant cellular models, through non-invasive measurements that provide real time kinetic results. Currently, there is no available capability to measure cellular respiration at NYULMC. The Seahorse XF will be placed within the Research Support Core at NYULMC, and compliment an existing fleet of user based instruments aimed at increasing the efficiency of and supporting translational research. This Core is administered by the Office of Collaborative Science and overseen by an experienced staff. The institution will support the instrument and the instrument internal review board will administer instrument oversight. NYUSOM Office of Collaborative Science, will generate the business plan for the Research Support Core, maintain the service contract, and put a system in place for scheduling and billing. The major user group spans 13 academic departments and represents both basic and translational research areas such as; aging, cardiovascular, cancer, immunology and mitochondrial disorders. The 9 NIH funded users combined will use 80% of the instrument capacity. The minor users group will occupy between 10% of instrument capacity. Remaining instrument capacity will be made available to any internal or external researcher through an on-line scheduler. The availability of this instrument in a Core setting will be unique in Lower Manhattan and we expect to serve NIH funded researchers from all surrounding institutions and resolve the cell viability issue that many researchers face when trying to transport assays from Lower Manhattan to the only other externally-available, Core based instrument located at the Albert Einstein College of Medicine (Bronx, NY).

DESCRIPTION (provided by applicant): Types 1 and 2 diabetes and their complications are on the rise. There is a recognized lack in both approved complications-based therapies and established disease-specific biomarkers in diabetes complications, which significantly hinders clinical trials. This application focuses on the role of the receptor for advanced glycation endproducts (RAGE) and its cytoplasmic domain binding partner, mammalian form of diaphanous1, mDia1, which is essential for RAGE signaling as a fundamental therapeutic target for diabetic complications. To transform our discoveries from the bench to the development of therapies for diabetes complications, we performed a library screen and identified two lead series of small molecules that inhibit RAGE tail-mDia1 interaction with nM affinity and demonstrate efficacy in in vitro and in vivo experimental assays. We have developed novel RAGE and mDia1 floxed mice to probe their cell-specific contributions to diabetes complications. Our approach will involve testing the following specific aims: AIM 1 will seek to optimize the two lead compound series which block RAGE/mDia1 signaling by maximizing drug-like properties; Aim 2 will dissect the mechanisms by which RAGE-mDia1 signal transduction contributes to the pathogenesis of diabetic nephropathy; Aim 3 will dissect the mechanisms by which RAGE-mDia1 signal transduction contributes to the pathogenesis of ischemia-reperfusion (I/R) injury in the diabetic heart; and Aim 4 will dissect the mechanisms by which RAGE-mDia1 signal transduction contributes to diabetic complications via impaired resolution of inflammation, to serve as a springboard for the development of target engagement biomarkers. We have assembled a multi-disciplinary team with expertise in RAGE/mDia1 signal transduction and diabetes complications; structural biology and NMR spectroscopy; medicinal and computational chemistry; and bioinformatics/biostatistics to tackle the problem of therapies and target engagement biomarkers for diabetic complications.

? DESCRIPTION (provided by applicant): The problems of obesity and insulin resistance have been linked to type 2 diabetes. Diabetes is on the rise; fully effective treatments are lacking. In the spectrum from obesity, insulin resistance, to diabetes, profound metabolic dysfunction is linked to increased risk for cardiovascular disease. Our goal is to uncover fundamental mechanisms by which macrophage inflammation impacts metabolic dysfunction in high fat feeding and obesity. Our preliminary data reveal that in high fat feeding in mice, ligands of the receptor for AGE (RAGE) are increased in key metabolic tissues even before the development of frank diabetes. Our data reveal that genetic deletion of RAGE results in significant protection against high fat feeding induced obesity and insulin resistance. Importantly, the accumulation, inflammatory polarization, and metabolic properties of adipose tissue macrophages are greatly reduced by deletion of RAGE. We will address the hypothesis that macrophage RAGE regulates obesity, adiposity and metabolic dysfunction in high fat feeding, both inherently and via cross-talk with the adipocyte. Our Project will explore four key properties of macrophage inflammation to discover RAGE-dependent mechanisms in high fat feeding: monocyte recruitment; macrophage retention and stasis; polarization; and metabolic regulation. Finally, we explore how the binding of the RAGE cytoplasmic domain to the formin, mDia1, which is required for RAGE signaling, contributes to macrophage dysfunction in high fat feeding. Translational studies relevant to human subjects will include examination of human adipose tissue macrophages in obesity and, in preclinical models, the testing of novel small molecule antagonists of RAGE signal transduction.

The heart reduces fatty acid (FAs) oxidation and switches to greater glucose utilization with ischemia. While this process allows more ATP production with less oxygen use, it occurs at the expense of limiting the use of FAs, the major substrates for cardiac energy. We hypothesize that this leads to fuel deprivation, especially as blood flow reduction decreases energetic substrate availability. If this hypothesis is correct, then storage of more triglyceride in the heart would lead to reduced ischemic damage; this we speculate is the reason for the ?obesity paradox? in which obese humans who have more ischemic disease also have increased post myocardial infarction survival. This proposal includes experiments to study basic and clinically relevant relationships between heart FA metabolism and heart function. Specifically, we will use mice created by the PI to study how changes in triglyceride stores and lipid uptake alter cardiac response to ischemia/reperfusion. In addition, we will test whether deletion of genes required for normal uptake of FAs by myeloid cells affects their conversion to an alternatively activated and reparative phenotype. These studies will include assessment of cardiac gene changes and lipidomics and will utilize tracer kinetics to assess uptake and oxidation of glucose and lipids, and to determine their downstream products. The experiments will require the collaboration of two laboratories: one with expertise in heart lipid metabolism and the second with expertise in ischemic/reperfusion models and analysis of substrate metabolism. The three aims of this application are the following: Aim 1. To determine whether increased cardiomyocyte storage of triglyceride improves response to ischemia. Aim 2. To assess whether mice with reduced heart lipid uptake have altered response to ischemia. Aim 3. To assess ischemic injury and repair in mice with macrophage-specific deletions of lipoprotein lipase and cluster of differentiation (CD)36. The experiments will use several lines greater cardiac triglyceride stores due to transgenic expression of diacylglycerol acyl transferase 1 and peroxisomal proliferator activated receptor ?, and mice with reduced uptake of FAs due to tissue specific deletions of lipoprotein lipase and CD36. These studies will illustrate possible metabolic approaches to reducing ischemic injury and improving repair of damaged myocardium.

Summary Cardiovascular disease (CVD) represents the major cause of morbidity and mortality in patients with diabetes. Despite aggressive management of levels of lipids and glucose, both types 1 and 2 diabetic patients exhibit earlier onset and more extensive atherosclerotic lesions than non-diabetic subjects, and the response to lipid- lowering strategies is significantly less robust than the beneficial effects noted in non-diabetic subjects. Preclinical studies in diabetic animals have shown accelerated progression and impaired regression of atherosclerotic plaques, as well as increased retention of macrophages (MØs) in plaques. We will focus on diabetes-driven mechanisms linked to impaired atherosclerosis regression and the specific role of MØ perturbation. Our proposed studies are built on the discovery that transplantation of aortic arches from Ldlr-/- mice into diabetic mice deficient in RAGE or its cytoplasmic domain binding partner, DIAPH1, required for RAGE signaling, display significantly improved plaque regression, independent of changes in plasma glucose or lipid levels, compared to wild-type diabetic mice. Our earlier studies demonstrated that activation of RAGE is a critical component of the vessel wall response to hyperglycemia and that RAGE-driven mechanisms accounted for the observed acceleration and progression of atherosclerosis. In this application, using models of hyperglycemia and insulin resistance (IR), we will test the hypothesis that the consequences of hyperglycemia and RAGE/DIAPH1-dependent mechanisms in MØs modulate MØ trafficking (recruitment & retentions/stasis), MØ inflammatory polarization and MØ metabolism and oxidative stress, mechanisms which converge to suppress regression of established atherosclerotic plaques in diabetes. We will test novel pharmacological antagonists of RAGE/DIAPH1 in our murine model of diabetic atherosclerosis regression. These studies will provide novel insights into diabetes specific mechanisms that promote MØ accumulation and impair regression, and foster the development of novel therapeutic adjuncts for diabetic atherosclerosis.

Project Summary: Project 1 Cardiovascular disease (CVD), that include increased sensitivity of diabetic (DM) myocardium to ischemic injury, represents the major cause of morbidity and mortality in patients with diabetes. We have uncovered key roles for the receptor for advanced glycation end products (RAGE -gene symbol Ager) in DM and myocardial ischemia/reperfusion (I/R), as global deletion of Ager attenuated myocardial injury, reduced oxidative stress, increased functional recovery and preservation of ATP compared to wild-type (WT) littermates. Mechanisms by which Ager deletion confers metabolic and functional protection in DM and non-DM (NDM) I/R hearts will be the focus of this project. Our program discovered that the RAGE cytoplasmic domain interacts with diaphanous-1 (DIAPH1), an effector of RhoGTPases, is essential for RAGE ligand-mediated cellular migration and activation of cdc42/rac-1. Our recent studies demonstrated that DIAPH1 is expressed in cardiomyocytes (CMs) and that I/R increases expression of Diaph1. Key preliminary data reveal reduced infarct size and improved functional recovery after I/R in (a) WT mice transplanted with bone marrow derived cells from Ager null mice,(b) mice with CM specific deletion of Ager/Diaph1, and (c) WT mice hearts perfused with conditioned media from Ager null or Diaph1 null macrophages (M?s). Novel findings in CMs and M?s reveal that DIAPH1 interacts with mitochondrial GTPase mitofusin2 (MFN2) and augments MFN2's tethering of sarcoplasmic reticulum (SR) to mitochondria (mito) and consequently mito calcium regulation. Importantly, our data shows that high glucose, ligands of RAGE, and RAGE-DIAPH1 interaction further augments DIAPH1-MFN2 driven mito-SR tethering. Taken together, these data led us to hypothesize that RAGE/DIAPH1 mediates DIAPH1-MFN2 driven Mito- SR interactions, altered calcium regulation, cell death signaling and metabolic dysfunction in I/R hearts by cell intrinsic mechanisms in M?s, and via M?-CM cross-talk. We will probe comprehensive mechanisms in cardiac stresses evoked by I/R using murine models, both in the absence and presence of DM. We will employ novel Ager and Diaph1 floxed mice, small molecule antagonists of RAGE-DIAPH1 interaction, state-of-the-art molecular techniques, proteomics, and magnetic resonance spectroscopy to uncover mechanisms of I/R injury in DM hearts.Proposed studies in this project will identify novel mechanism M? specific mechanism, as well as M?-CM cross talk mechanisms by which RAGE-DIAPH1 modulates I/R injury in hearts, particularly in DM hearts. Identification of these mechanisms, along with testing of novel small molecules that block RAGE/DIAPH1 interaction, will pave the way for therapeutic interventions to protect DM hearts from I/R injury. Project 1 will work closely with Projects 2 and 3 and the two Cores to achieve these goals.

Project Summary: Scientific Core The overall goal of Core 2 is to provide the key resources, including mice bred-in-house; primary cells from mice (endothelial cells, cardiomyocytes and bone marrow derived macrophages); and surgical procedures on mice required for two of the three Projects. Further, this Core provides proteins/mutant proteins of RAGE, DIAPH1 and MFN2 that are required for the three Projects. In addition, the entire Program Project is served by the data management and biostatistical component of this Core. The rationale to centralize these key activities is to ensure rigorous data management and biostatistical support, resource and procedural consistency, and maximal and most efficient use of resources throughout the Program Project. All three Projects will use this Core 2 over all five years of the Program Project. The five Specific Aims of this core are as follows: Specific Aim 1 will provide data management and biostatistical support; Specific Aim 2 will perform the breeding of the mice to be employed in this Program project; Specific Aim 3 will perform the surgical procedures to induce ischemia and reperfusion injuries to mice in this Program Project; Specific Aim 4 will isolate and characterize the primary macrophages, cardiomyocytes and endothelial cells to be employed in this Program Project ; Specific Aim 5 will prepare and characterize purified proteins and site-directed mutants of RAGE, DIAPH1 and MFN2 to be employed in this Program Project. AIMS 1-4 are based at the NYU School of Medicine/NYU Langone Health, while AIM 5 takes place at the SUNY at Albany. All of the necessary facilities and equipment are available at NYU School of Medicine/NYU Langone Health and at the SUNY at Albany to successfully carry out the proposed studies. The Core leader and Core Co-Investigator have all of the necessary expertise and experience to oversee these core functions. Letters are provided and appended to this Core indicating that there is NO OVERLAP of the five specific aims of this Core with NYU- or Albany-institutionally funded-Cores. This core is in full compliance with the instructions for scientific cores within NHLBI Program Projects. Core 2 serves all three projects of the Program over all 5 years of the Program Project.

Project Summary/Abstract The proposed T35 program will provide financial support for up to 24 medical students from the NYU School of Medicine (NYUSOM), as well as outside medical schools, to spend 9 weeks during the first summer of medical school working on NIDDK-related research. In the current funding period, our T35 program has steadily grown and attracted committed and passionate students to research. For example, in 2018, 26 students applied to the program and were accepted; of which 24 participated and 2 declined. Since 2003, approximately 40% of students participating in the T35 Program pursued additional high quality research opportunities that included a research year, graduating with research Honors or gaining entrance to our highly competitive MSTP program. The core operational objectives of this renewal application are to: (1) provide students with a pre-selected list of excellent mentors and projects, (2) create a selection process to identify highly qualified students and optimize the probability that their work will lead to publication, (3) create cohesion among the trainees and the participating mentors, (4) acquire data that can be used to judge the success of the program and to implement changes to make it more successful. To accomplish these objectives an internal advisory board (IAB) will advise the Program Director (PD) on selection of mentors, projects and students. An external advisory board (EAB) will review the program each year and make suggestions that assure it meets our core objectives. Each year the IAB and PD will select experienced and successful mentors and solicit from them NIDDK-related projects to be offered to students. Selected mentors must have an excellent mentoring record and be doing work relevant to the NIDDK mission. Mentors will interview applicants and select a student for each project. With the mentor?s guidance, selected students will write a proposal to submit to the PD and program staff for final approval. To further enhance the probability that selected projects will lead to publication, students will submit an updated research plan just prior to starting their summer project. Cohesion among the trainees and mentors will be created through weekly meetings of students and faculty. Selected faculty will discuss their research and careers and, at subsequent meetings, students will present either journal articles or work-in-progress seminars. At the conclusion of their summer project students will be queried about their plans for publication and will be asked to submit an abstract for the annual fall research day to be held at the NYUSOM. To track the success of the program, students and mentors will be queried at the end of the summer regarding progress in the key research competencies and students will be asked to comment on their plans for further research and academic careers. Follow up questionnaires will be sent to students at the end of the clerkship year, at graduation, and post- graduation with the intent to track their publication record, research plans, and their evolving impression of the T35?s influence on their professional careers.

Abstract The complications of diabetes, particularly impaired wound healing and diabetes-associated nephropathy, are major causes of morbidity and mortality in patients with types 1 and 2 diabetes. The RAGE extracellular domains are heterogeneous and RAGE ligands may bind at spatially-distinct sites on these domains, thereby indicating that the use of small molecules or antibodies targeted to the extracellular domains, may be ineffective. We have discovered that the interaction of the cytoplasmic tail of RAGE (ctRAGE) with the intracellular formin molecule, DIAPH1, is essential for RAGE-mediated signal transduction. We previously performed a high-throughput screen of >59,000 compounds with the goal to block the interaction of ctRAGE with DIAPH1. We identified two lead series (LS) that fulfill key criteria for drug-like properties; from one of the series, LSII, RAGE229 emerged as a plausible lead for clinical development because of efficacy, favorable pharmacokinetic profile and promising early-stage off-target and toxicity testing. In vivo efficacy was also demonstrated, as administration of RAGE229 attenuated inflammation in a delayed type hypersensitivity experiment in mice, reduced myocardial infarct size upon ligation and reperfusion of the left anterior descending coronary artery in diabetic mice, and reduced multiple parameters of diabetes-associated kidney pathology and impaired wound healing in diabetic mice, all versus vehicle, in both male and female mice. However, in late-stage testing, RAGE229 tested positive in the Mini-AMES test. To address this liability, we have already prepared new analogs of RAGE229; these analogs retain potency but are negative in the Mini-AMES test. In addition, we have made significant progress in developing back-up candidates in the LS I. Importantly, a lead benchmark compound within LSI tested negative in the Mini-AMES test as well. Our established, multi-disciplinary team is now well-poised to move forward aggressively at this critical juncture to identify lead candidate molecules for LSII and the back-up LSI for ultimate clinical development for diabetic complications. Our drug discovery goals, supported by extensive preliminary data are: 1) to identify potent, selective, and safe candidate analogs of LSII; and 2) to develop LSI backup candidates with a different scaffold to mitigate risk to our RAGE program. If successful, our work will set the stage for the development and clinical testing of a novel class of disease-modifying agents for diabetic complications.