Faculty Bibliography

The coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented challenge and opportunity for translational investigators to rapidly develop safe and effective therapeutic interventions. Greater risk of severe disease in COVID-19 patients with comorbid diabetes mellitus, obesity, and heart disease may be attributable to synergistic activation of vascular inflammation pathways associated with both COVID-19 and cardiometabolic disease. This mechanistic link provides a scientific framework for translational studies of drugs developed for treatment of cardiometabolic disease as novel therapeutic interventions to mitigate inflammation and improve outcomes in patients with COVID-19.

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet-fed Ldlr-/- mice into diabetic Ager-/- (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow-derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

The escalating problem of obesity and its multiple metabolic and cardiovascular complications threatens the health and longevity of humans throughout the world. The cause of obesity and one of its chief complications, insulin resistance, involves the participation of multiple distinct organs and cell types. From the brain to the periphery, cell-intrinsic and intercellular networks converge to stimulate and propagate increases in body mass and adiposity, as well as disturbances of insulin sensitivity. This review focuses on the roles of the cadre of innate immune cells, both those that are resident in metabolic organs and those that are recruited into these organs in response to cues elicited by stressors such as overnutrition and reduced physical activity. Beyond the typical cast of innate immune characters invoked in the mechanisms of metabolic perturbation in these settings, such as neutrophils and monocytes/macrophages, these actors are joined by bone marrow-derived cells, such as eosinophils and mast cells and the intriguing innate lymphoid cells, which are present in the circulation and in metabolic organ depots. Upon high-fat feeding or reduced physical activity, phenotypic modulation of the cast of plastic innate immune cells ensues, leading to the production of mediators that affect inflammation, lipid handling, and metabolic signaling. Furthermore, their consequent interactions with adaptive immune cells, including myriad T-cell and B-cell subsets, compound these complexities. Notably, many of these innate immune cell-elicited signals in overnutrition may be modulated by weight loss, such as that induced by bariatric surgery. Recently, exciting insights into the biology and pathobiology of these cell type-specific niches are being uncovered by state-of-the-art techniques such as single-cell RNA-sequencing. This review considers the evolution of this field of research on innate immunity in obesity and metabolic perturbation, as well as future directions.

During obesity, macrophages infiltrate the visceral adipose tissue and promote inflammation that contributes to type II diabetes. Evidence suggests that the rewiring of cellular metabolism can regulate macrophage function. However, the metabolic programs that characterize adipose tissue macrophages (ATM) in obesity are poorly defined. Here, we demonstrate that ATM from obese mice exhibit metabolic profiles characterized by elevated glycolysis and oxidative phosphorylation, distinct from ATM from lean mice. Increased activation of HIF-1α in ATM of obese visceral adipose tissue resulted in induction of IL-1β and genes in the glycolytic pathway. Using a hypoxia-tracer, we show that HIF-1α nuclear translocation occurred both in hypoxic and non-hypoxic ATM suggesting that both hypoxic and pseudohypoxic stimuli activate HIF-1α and its target genes in ATM during diet-induced obesity. Exposure of macrophages to the saturated fatty acid palmitate increased glycolysis and HIF-1α expression, which culminated in IL-1β induction thereby simulating pseudohypoxia. Using mice with macrophage-specific targeted deletion of HIF-1α, we demonstrate the critical role of HIF-1α-derived from macrophages in regulating ATM accumulation, and local and systemic IL-1β production, but not in modulating systemic metabolic responses. Collectively, our data identify enhanced glycolysis and HIF-1α activation as drivers of low-grade inflammation in obesity.

Background: Diabetic Cardiomyopathy (DbCM) leading to overt heart failure is a common sequalae of both Type 1 and Type 2 Diabetes. Previous attempts to develop treatments for DbCM through inhibition of the enzyme Aldose Reductase (AR) were unsuccessful, due to low AR binding affinity and poor specificity. Off-target inhibition of Aldehyde Reductase (AldR), an enzyme critical for detoxification of aldehydes in the liver and normal hepatocyte physiology, led to common liver-related safety and tolerability issues with first generation ARI compounds. We report on the selectivity and specificity of AT-001, a novel small molecule ARI with optimized affinity and specificity for AR and minimal to zero off-target AldR activity.
Methods and Results: AT-001 was evaluated for AR binding affinity as compared to zopolrestat, a first-generation ARI. AT-001 demonstrated a >300-fold greater affinity for AR versus zopolrestat (IC50 of 30 pmol for AT-001 and ~10 nmol for zopolrestat). In contrast to zopolrestat, AT-001 showed no inhibition of AldR at 50x and 100x EC50 levels in assay medium, while zopolrestat inhibited Aldehyde Reductase by 50% and 60% respectively (spec activity of 2.3 and 1.9 mmol NADPH/min/ mg protein). In addition to these analyses, AT-001 was evaluated in a standard off-target receptor binding analysis of 87 substrates (13 enzyme, 74 binding assays) to characterize pharmacological specificity and selectivity. No significant off-target results (defined as >= 50% inhibitory activity) were observed in this panel.
Conclusion(s): The unique structure and activity of AT-001 provide selectivity for Aldose Reductase and lack of off-target effects. The in-vitro safety of this agent together with the positive safety data from the phase 1/2 program, supports the ongoing pivotal study in DbCM. Keywords: AT-001, Diabetic Cardiomyopathy, Aldol Reductase Inhibitor, Heart Failure Abbreviations: DbCM, CHF, AR, ARI Funding and Conflicts of Interest: Applied Therapeutics Inc (Salary) (Perfetti, Shendelman). Applied Therapeutics Inc (Equity / Shareholder)(Perfetti, Shendelman). Applied Therapeutics Inc (Study Funding) (Ramasamy).
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The role of advanced glycation end products (AGEs) in promoting and/or exacerbating metabolic dysregulation is being increasingly recognized. AGEs are formed when reducing sugars non-enzymatically bind to proteins or lipids, a process that is enhanced by hyperglycemic and hyperlipidemic environments characteristic of numerous metabolic disorders including obesity, diabetes and its complications. In this mini-review, we put forth the notion that AGEs span the spectrum from cause to consequence of insulin resistance (IR) and diabetes, and represent a "common soil" underlying the pathophysiology of these metabolic disorders. Collectively, the surveyed literature suggests that AGEs, both those that form endogenously as well as exogenous AGEs derived from environmental factors such as pollution, smoking and "Western" style diets, contribute to the pathogenesis of obesity and diabetes. Specifically, AGE accumulation in key metabolically-relevant organs induces IR, inflammation and oxidative stress, which in turn provide substrates for excess AGE formation, thus, creating a feed-forward fueled pathological loop mediating metabolic dysfunction.

Obesity and diabetes are leading causes of cardiovascular morbidity and mortality. Although extensive strides have been made in the treatments for non-diabetic atherosclerosis and its complications, for patients with diabetes, these therapies provide less benefit for protection from cardiovascular disease (CVD). These considerations spur the concept that diabetes-specific, disease-modifying therapies are essential to identify, especially as the epidemics of obesity and diabetes continue to expand. Hence, as hyperglycemia is a defining feature of diabetes, it is logical to probe the impact of the specific consequences of hyperglycemia on the vessel wall, immune cell perturbation, and endothelial dysfunction-all harbingers to the development of CVD. In this context, high levels of blood glucose stimulate the formation of the irreversible advanced glycation end products, the products of non-enzymatic glycation and oxidation of proteins and lipids. AGEs accumulate in diabetic circulation and tissues and the interaction of AGEs with their chief cellular receptor, receptor for AGE or RAGE, contributes to vascular and immune cell perturbation. The cytoplasmic domain of RAGE lacks endogenous kinase activity; the discovery that this intracellular domain of RAGE binds to the formin, DIAPH1, and that DIAPH1 is essential for RAGE ligand-mediated signal transduction, identifies the specific cellular means by which RAGE functions and highlights a new target for therapeutic interruption of RAGE signaling. In human subjects, prominent signals for RAGE activity include the presence and levels of two forms of soluble RAGE, sRAGE, and endogenous secretory (es) RAGE. Further, genetic studies have revealed single nucleotide polymorphisms (SNPs) of the AGER gene (AGER is the gene encoding RAGE) and DIAPH1, which display associations with CVD. This Review presents current knowledge regarding the roles for RAGE and DIAPH1 in the causes and consequences of diabetes, from obesity to CVD. Studies both from human subjects and animal models are presented to highlight the breadth of evidence linking RAGE and DIAPH1 to the cardiovascular consequences of these metabolic disorders.

Introduction: Hyperactivation of the polyol pathway (PP) contributes to development of diabetic complications such as diabetic cardiomyopathy (DbCM). Aldose reductase (AR), the rate-controlling enzyme in the PP, catalyzes NADPH-dependent reduction of glucose to sorbitol. Under hyperglycemic and ischemic conditions, PP activation causes intracellular sorbitol accumulation leading to osmotic stress, cell death and diabetic complications. Previous AR inhibitors (ARI) failed due to safety or lack of efficacy. AT-001 is a novel oral ARI with optimized specificity and affinity for AR.
Objective(s): To assess cardioprotective activity of AT-001 in an animal model of DbCM and safety, tolerability and target engagement in a phase 1/2 clinical study. Preclinical
Methods and Results: The cardioprotective effect of AT-100 was studied in transgenic mice expressing human levels of AR (Tg hAR) and rendered diabetic with streptozotocin (55 mg/kg for 5d). Mice were treated with AT-001 40 mg/kg, n=6 or vehicle (V, n=6) for 3d prior to cardiac ischemia reperfusion (IR) injury induced by ligation and reperfusion of the left anterior descending (LAD) coronary artery. Control non-DM Tg hAR mice (non-DM, n=9) were also subjected to IR injury. After 48h recovery mice were euthanized and infarct area and fractional shortening (FS) assessed. Mean (+/-SD) infarct area (ratio of infarcted to total left ventricular area) was significantly lower in AT-001 (35.3+/-2.7) vs. non-DM (43+/-2) and V (59.1+/-0.8); P<0.05 vs non-DM and V). FS 48h post reperfusion, a measure of functional recovery, was greater in AT-001 vs non-DM and V: 38.2+/-2, 24+/-3, 21+/-1.5, respectively; P<0.05 vs. non-DM and V. Clinical methods and Results: The phase 1/2 study consisted of single and multiple oral ascending doses (SAD, n=8 per group) of 5, 10, 20 and 40 mg/kg once daily (QD) and multiple doses (MAD) of placebo (Pbo) or AT-001 5, 20, 40 mg/kg QD or 20 mg/kg twice daily for 7d. Subjects with type 2 diabetes (T2DM), age 18-75 and HbA1c 5.0-8.5% were allocated to AT-001 (n=8) or Pbo (n=2) in each dose cohort. AT-001 was well-tolerated with no drug-related AEs. AR inhibition and target engagement were demonstrated by dose-dependent decreases in sorbitol in AT-001 (up to -50 % change from baseline) vs. Pbo subjects (-3 % change). Maximum inhibition was between 2-4 h and lasted several hours.
Conclusion(s): AT-001 significantly attenuated or prevented cardiac damage in a relevant animal model and was safe, well-tolerated and reduced sorbitol levels in T2DM subjects. These findings support clinical development in DbCM.
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