John W Calvert - US grants

[unreadable] DESCRIPTION (provided by applicant): Liver dysfunction or failure, as a result of hepatic ischemia/reperfusion (I/R), is a leading cause of morbidity and mortality for patients who have undergone liver transplantation surgery. Currently, no therapeutic strategy exists and the ever growing gap between supply and demand for donors has forced the consideration of cadaveric or steatotic graphs, which are very susceptible to I/R. Because intervention on more than one level is likely needed to allow for the recovery of cellular and organ failure, the most promising protective strategy against I/R injury explored in the last few years is preconditioning. Therefore, ischemic preconditioning or pharmacological interventions that mimic these effects may have the greatest potential to improve clinical outcome in liver transplantation and liver surgery. Recently, nitrite through the generation of nitric oxide (NO) has been shown to have cytoprotective effects in the setting of hepatic I/R, suggesting that nitrite may serve as a biological storage reserve of NO subsurving a critical function in tissue protection from ischemic injury. Furthermore, preliminary data from our lab indicates that the administration of nitrite 24 hours prior to I/R protects the liver against injury. Moreover, nitrite preconditioning was found to attenuate the l/R-induced suppression of mitochondrial respiration. Therefore, the objective of this proposal is to delve into the role of the mitochondria in nitrite mediated hepatic preconditioning with the central hypothesis being that nitrite preconditioning preserves the structure and function of the mitochondria following hepatic I/R, thereby attenuating hepatocellular injury. The rationale for the proposed research is that identifying the mechanisms of nitrite preconditioning may provide a basis for extending the clinical application to patients facing liver transplantation or liver surgery. So, we plan to test our central hypothesis and accomplish the objective of the proposed study by pursuing two specific aims. Specific aim 1 will further expand on our preliminary data by exploring the-protection of the mitochondria through the evaluation of mitochondrial matrix volume, mitochondrial membrane potential, mitochondiral uptake of calcium, mitochondrial respiration of the different complexes, ATP production, redox potential, ROS production, and finally the release of cytochrome C from the mitochondria into the cytosol. Specific aim 2 will take a step back and explore the signaling mechanisms of nitrite preconditioning by examining the role of mitochondrial K+ channels. We believe that nitrite, through its conversion to NO, protects the mitochondria from a subsequent I/R injury by mediating the opening of the mitochondrial K+ channels, mKATP and mKCa, via the molecular signaling of PKC and PKA, respectively. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Heart failure is the inability of the heart to meet hemodynamic demands and represents the end stage of various forms of cardiovascular disease. In industrialized nations, heart failure represents a major health problem that has been increasing in prevalence and incidence. It is estimated that 5.7 million people in the United States have heart failure resulting in about $37.2 billion being spent a year to cover associated health care related costs. Therefore, drug therapy designed to coincide with the standard means of care are needed to decrease the extent of injury leading to the development of heart failure. Recently, hydrogen sulfide (H2S) has been shown to be cardioprotective in various in vitro and in vivo models of cardiac injury. Although the physiological and cardioprotective effects of H2S in acute models of ischemia- reperfusion injury have previously been documented, the signaling mechanisms that mediate these effects have not been fully studied. Moreover, the signaling mechanisms that have been attributed to H2S have predominantly been studied in in vitro model systems, with very few studies actually exploring the protective effects in in vivo systems. Additionally, the cardioprotective effects of H2S in the setting of heart failure have not been investigated. For this reason, the studies proposed in this application are extremely important and timely. The overall aim of this proposal is to evaluate the signaling mechanisms responsible for the observed cardioprotective effects of H2S therapy in the setting of heart failure. To this end, the transcription factor, Nrf2, has been identified as a possible regulator of these cardioprotective effects. Therefore, the central hypothesis for the proposed studies is that H2S up-regulates endogenous antioxidants, alleviates mitochondrial dysfunction, and reduces hypertrophy in a Nrf2-dependent manner. To test this hypothesis, 3 Specific Aims have been proposed. Specific Aim 1 will evaluate the role of Nrf2 signaling in mediating the antioxidant effects of H2S. Specific Aim 2 will investigate if H2S suppresses apoptosis and cardiac hypertrophy via Nrf2/Trx1- dependent signaling. Specific Aim 3 will investigate if H2S induces mitochondrial biogenesis via Nrf2- dependent signaling. The proposed studies will significantly advance our current understanding of the mechanisms responsible for the development of heart failure and will answer important questions regarding the signaling mechanism responsible for the cardioprotective effects of H2S in the setting of heart failure. Additionally, information gained from these studies will provide the foundation for the development of H2S therapy for the treatment of heart failure. PUBLIC HEALTH RELEVANCE: Despite numerous advances in health care, cardiovascular disease remains the number one killer in the United States and heart failure, as a direct result of cardiovascular disease, affects nearly 5.3 million people in the United States resulting in about $34.8 billion being spent a year to cover associated health care related costs. The proposed studies will evaluate the efficacy of a hydrogen sulfide releasing compound in clinically relevant and highly translational experimental model systems of heart failure. The proposed studies will significantly advance our current understanding of the mechanisms responsible for the development of heart failure and will answer important questions regarding the signaling mechanism responsible for the cardioprotective effects of hydrogen sulfide in the setting of heart failure.

Obesity is a growing epidemic in the United States, leading to increases in cases of nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, and type 2 diabetes. A common characteristic of these diseases is aberrant lipid and glucose metabolism. This proposal centers on the nuclear hormone receptor, Liver Receptor Homolog 1 (LRH-1), which acts as an important regulator of lipid metabolism, reverse cholesterol transport, glucose sensing, and homeostasis. As such, LRH-1 represents a novel therapeutic target for metabolic diseases. LRH-1 binds to phospholipids (PLs), but until recently, the role of PLs in receptor activation was unclear. Recent studies identified dilauroylphosphatidylcholine (DLPC) as a specific LRH-1 agonist with potent anti-diabetic effects. Despite this untapped therapeutic potential, mechanisms through which LRH-1 is regulated by ligands remain poorly understood. The discovery that LRH-1 is regulated by PL ligands reveals an exciting potential to tune LRH-1 activity for the treatment of metabolic diseases. However, PLs (such as DLPC) are labile and not suitable for clinical use, necessitating the development of small molecule agonists. This has proved challenging thus far, since very few small molecules are capable of displacing endogenous lipids from the large, lipophilic binding pocket. Recent preliminary studies in our lab have characterized a potent class of small molecules that are capable of this feat. Our crystal structures of LRH-1 bound to a set of these agonists have uncovered novel mechanisms of receptor activation and have provided insights into strategies to improve agonist activity. The overall goal of this proposal is to develop improved small molecule modulators using a structure-based, rational design approach. X-ray crystallography combined with hydrogen deuterium exchange mass spectrometry, cellular assays, and animal studies will be used to address this goal in a series of three Aims. Aim 1. Design and synthesize potent, effective, and selective LRH-1 modulators Aim 2. Determine the molecular basis of allosteric modulation of LRH-1 by RJW100 analogues Aim 3. Evaluate the efficacy of LRH-1 modulators as anti-diabetic agents in obesity.

Project Summary DJ-1 is a cytoprotective protein that is activated by oxidative stress. Due in large part to the association of DJ-1 with Parkinson's Disease, most studies aimed at investigating its role in response to pathological stimuli have been confined to the brain or neuronal cells. However, DJ-1 is expressed in the heart where it also possesses cytoprotective actions. Studies indicate that DJ-1 plays an important role in multiple cellular processes, including oxidative stress response, anti-apoptotic signaling, and transcriptional regulation. However, the cellular mechanisms underlying these reported actions remain largely unknown. Given that DJ-1 is a small, dimeric, single-domain protein, it either possesses multiple functions to account for these actions or there is a single biochemical activity that explains all of them. What is known is that DJ-1 is activated by the removal of a 15-amino acid peptide at its C terminus in response to oxidative stress. We recently provided the first evidence that DJ-1 is activated within hours following the onset of acute myocardial ischemia-reperfusion (I/R). Preliminary studies from our lab demonstrate that cardiac DJ-1 remains active for up to 7 days following the onset of ischemic injury. Furthermore, our studies revealed that DJ-1 deficient mice displayed worse cardiac dysfunction in response to ischemic-induced heart failure compared to wild-type mice. Elevated levels of hypertrophy, fibrosis, and inflammation accompanied the worse cardiac dysfunction in the DJ-1 deficient mice. More importantly, we provide novel evidence that the delivery of the active form of DJ-1 using recombinant adeno-associated virus 9 (AAV9-DJ1WT?C) reduces the development of ischemic-induced heart failure. This is the first and only demonstration that an active form of DJ-1 provides cytoprotection in an in vivo model of injury. In this proposal, we will expand on our previous findings and attempt to fill knowledge gaps in the literature regarding the cellular actions of DJ-1. 3 specific aims have been proposed. Aim 1 will determine if DJ- 1 is a novel cardiac deglycase that opposes glycative stress. Aim 2 will determine if DJ-1 attenuates ischemic- induced heart failure by promoting the cardioprotective actions of thioredoxin. Aim 3 will determine if DJ-1 opposes TGF-? signaling by regulating the cardiac expression of Arkadia.