Mechanism of oxidative/nitrosative stress and inflammation-induced tissue injury

National Institute On Alcohol Abuse And Alcoholism

Investigators

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Abstract

Interplay of oxidative/nitrative stress, inflammation with the endocannabinoid system (ES) in tissue injury and inflammation. Recent evidence highlights the importance of interactions between various organ systems, which may facilitate cardiovascular disease and/or development of various severe cardiovascular complications in a multitude of diseases (e.g. chronic liver and kidney diseases). We have been very interested in understanding the interactions of the liver with the cardiovascular system and kidneys during chronic hepatic inflammation, fibrosis, and injury. Chronic liver diseases are among the top contributors to mortality worldwide and patients with severe acute and chronic liver disease often develop numerous cardiovascular complications including portal hypertension, peripheral splanchnic vasodilation resistant to vasoconstrictors, vasculopathies, hepatopulmonary syndrome, encephalopathy, and cirrhotic cardiomyopathy, which largely contribute to the increased mortality observed in this patient group. Furthermore, liver disease may also profoundly interact other organ systems. Hepatorenal syndrome (HRS) is a very frequent and potentially lethal complication of acute and chronic liver failure (e.g. induced by alcoholic steatohepatitis, cirrhosis, etc.) and is an important predictor of short-term mortality with poor prognosis. Recently, we described and validated novel mouse models of hepatic cardiomyopathy and hepatorenal syndrome in which interorgan interactions can be studied. Taking the advantage of these animal models and by utilizing multiple molecular biology, genomics, lipidomic and proteomics approaches we aim to understand the role of oxidative stress, inflammation, lipid (.e.g. endocannabinoid and eicosanoid) and cell death signaling in mediating these important organ to organ interactions. Cannabinoid receptor 2 (CB2R), primarily expressed in inflammatory cells, is an attractive target for various diseases associated with tissue injury and inflammation. However, there are no specific antibodies and most of the ligands used in preclinical studies are neither selective nor specific. Because of these issues, the target validation is extremely difficult, which is slowing down the clinical development of this class of drugs. We have recently reported the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. These findings will benefit clinical translatability of CB2R based drugs. We found that pharmacological activation of CB2R with a selective agonist attenuated diabetes-induced cardiac inflammation, oxidative/nitrative stress, fibrosis and cell demise, and consequent cardiac dysfunction without affecting hyperglycemia, while genetic deletion of CB2R aggravated myocardial pathology. Thus, selective activation of CB2R ameliorates diabetes-induced myocardial tissue injury and preserves the functional contractile capacity of the myocardium in the diabetic milieu. This is particularly encouraging, since unlike CB1R agonists, CB2R agonists do not elicit psychoactive activity and cardiovascular side effects and are potential clinical candidates in the treatment of diabetic cardiovascular and other complications. Our ongoing collaborative studies with industry and numerous academic institutions have also been focused on development and characterization of improved CB2R ligands and on understanding the role of cannabinoid 2 receptors (CB2R) in kidney and liver inflammation and fibrosis, Our collaborative studies with Dr. Yuri Persidsky have also been exploring the role of CB2R in AIDS using humanized mouse models of the disease. Our impending studies will also focus on the understanding the mechanisms of the activation of the endocannabinoid system during tissue injury and on the further elucidation of the role of the endocannabinoid system (particularly focusing on the endocannabinoid metabolizing enzymes and CB2R in collaboration with Drs. Cravatt, Van der Stelt, Mechoulam, Cinar and Kunos) in various models of liver disease, cardiomyopathy and nephropathy. Role of oxidative/nitrosative stress, inflammation, metabolic dysregulation in tissue injury In collaboration with Dr. Hasko we explored the role of Adenosine 2a receptors(A2aR) in multiple organ failure after hemorrhagic shock. These data demonstrated that activation of A2AR regulates organ injury and apoptosis in the setting of trauma hemorrhagic shock. In another study we investigated if A2ARs suppress neutrophil aging, which precedes cell death, and N1 to N2 polarization. We found that A2AR stimulation slows neutrophil aging, suppresses cell death, and promotes the polarization of neutrophils from an N1 to N2 phenotype. Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. We found that IMP suppressed tumor necrosis factor (TNF)- production and augmented IL-10 production in endotoxemic mice through metabolism to inosine Our ongoing collaborative studies with Dr. Hasko have also been exploring the role of adenosine and adenosine receptors in tissue injury and inflammation. In collaboration with Drs. Lohoff and Koob we discovered that inhibition of PCSK9 in rats with a therapeutically approved monoclonal antibody for the treatment of atherosclerosis may represent a novel therapeutic approach for alcoholic liver disease by attenuating hepatic inflammation and steatosis. In a small-scale clinical study, we plan to explore the translatability of this approach and plan to include numerous cardiovascular endpoints. In collaboration with Dr. Gao we also found that PCSK9 inhibition in mouse model of NASH attenuated liver injury and inflammation. Our future collaborative studies with Drs. Kunos, Gao, Koob, Cinar and Lohoff will also explore the role of oxidative/nitrosative stress, inflammation, lipid endocannabinoid signaling and cell death in various new rat and mouse models of alcohol use disorder. Our ongoing collaboration with Dr. Steven N Ebert will also explore the effects of maternal binge alcohol consumption on cardiac pathology and gene expression