Overwhelming evidence suggests that oxidative-nitrosative/nitrative stress and inflammation are involved in essentially all major pathological processes affecting humans, including those induced by excessive alcohol consumption. The research focus of SOSTI is to understand the cellular and molecular mechanisms underlying oxidative/nitrosative/nitrative stress, inflammation, and their downstream effector pathways using clinically relevant animal models of disease (e.g. ischemia reperfusion injury, cardiomyopathy/heart failure), and to identify novel therapeutic targets against these pathologies. Interplay of oxidative/nitrative stress, inflammation with the endocannabinoid system (ES), and natural cannabinoids in tissue injury During the last couple of years we wanted to understand the interplay of oxidative/nitrosative stress, inflammation with the ES;an emerging very promising therapeutic target against various inflammatory and other diseases). Our in vitro studies suggested that cannabinoid 1 receptor (CB1) activation by endocannabinoid anandamide (AEA) in primary human coronary endothelial cells and cardiomyocytes may amplify the ROSMAPK activationcell death pathway in pathological conditions when the endocannabinoid biosynthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, thereby contributing to the development of endothelial dysfunction and pathophysiology of multiple cardiovascular diseases. In order to test the in vivo relevance of the above findings in endothelial cells, we investigated the effects of genetic or pharmacological inhibition of CB1 in a clinically relevant diabetic retinopathy model. Deletion of CB1 or treatment of diabetic mice with CB1 antagonist SR141716 almost completely prevented retinal cell death. Treatment of diabetic mice or human primary retinal endothelial cells (HREC) exposed to high glucose with CB1 antagonist SR141716 attenuated the oxidative and nitrative stress, reduced nuclear factor kappa B activation, and adhesion molecules expression. In addition, SR141716 attenuated the diabetes- or high glucose-induced proapoptotic activation of MAPKs and retinal vascular cell death. To further explore the potential mechanisms of AEA-induced cardiovascular injury and potential interplay of the endocannabinoid signaling with the oxidative/nitrative stress, we have investigated the role of the AEA-metabolizing enzyme (fatty acid amide hydrolase;FAAH) using cardiomyopathy models induced by Doxorubicin (DOX) in mice. The DOX-induced myocardial oxidative/nitrative stress positively correlated with multiple cell death markers, which were enhanced in FAAH knockout mice exhibiting significantly increased DOX-induced mortality and cardiac dysfunction compared to their wild type littermates. The effects of DOX in FAAH knockouts were partially attenuated by CB1 receptor antagonists. Furthermore, anandamide enhanced cell death in human cardiomyocytes pretreated with FAAH inhibitor and enhanced sensitivity to ROS generation in inflammatory cells of FAAH knockouts. CB2 receptor is a very appealing target for drug development for various inflammatory diseases and ischemia reperfusion injury, in which these receptors on immune cells, attenuate inflammatory response and tissue injury. However, most currently available CB2 agonists have problems related to their long term stability, in vivo efficacy, and their potential modulatory effects on CB1 signaling at higher doses, which often result in opposing cellular consequences on inflammation when the dose is increased (bell-shaped dose-response relationship with many cannabinergic ligands has been reported). We have aimed with Professor Raphael Mechoulam at Hebrew University to develop better leads with improved in vivo efficacy and selectivity. One of the best compounds from this new series, HU910, was recently characterized in detail in vitro, and was proven to be considerably better in its in vivo efficacy in models of hepatic I/R injury compared to other CB2 agonists. During these studies we unexpectedly discovered an important function of CB2 receptors in Kupffer cells in regulating inflammatory responses in the liver. With Professor Pertwee we have discovered that delta(8)-tetrahydrocannabivarin delta(8)-THCV), a stable synthetic analog of the plant-derived cannabinoid delta(9)-tetrahydrocannabivarin, which exerts some anti-inflammatory effects in rodents, is a potent CB2 agonist both in vitro and in vivo with anti-inflammatory effects in hepatic I/R injury. We hope that delta(8)-THCV may serve as a basis for the development of a whole new class of novel compounds with anti-inflammatory activities. We also found that another plant-derived cannabinoid, cannabidiol (CBD), significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death associated with hepatic I/R. CBD also attenuated the bacterial endotoxin-triggered NF-kappaB activation and TNF-alpha;production in isolated Kupffer cells in vitro, likewise the adhesion molecule expression in human liver sinusoidal endothelial cells stimulated with TNF-alpha, and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Our impending studies will also be directed towards the understanding of the mechanisms of the activation of the endocannabinoid system during reperfusion injury and on the further elucidation of the role of endocannabinoid system (particularly focusing on the endocannabinoid metabolizing enzymes in collaboration with Dr. Cravatt) in various models of cardiomyopathy and nephropathy. Our future collaborative studies with Drs. George Kunos, Bin Gao and Byoung-Joon Song will also explore the role of oxidative/nitrosative stress and ES in various other models of liver and metabolic disorders. The above mentioned studies may identify new pharmacological targets in various forms of tissue injury associated with increased inflammation and oxidative stress. Role of oxidative-nitrosative stress and apoptosis in ethanol-induced tissue-damage. Moderate and heavy drinking may significantly influence cardiovascular function and aging in different ways. During the course of the last decade, several research groups have reported that, in animal models of myocardial ischemia/reperfusion ethanol and non-ethanolic components of wine may have a specific protective effect on the myocardium, independent of the classical risk factors implicated in vascular atherosclerosis and thrombosis. Apoptosis is a mechanism of cell death implicated in the pathogenesis of alcohol-induced organ damage. Experimental studies have suggested alcohol-mediated apoptosis in the cardiac muscle and liver, and there is also evidence of skeletal muscle apoptosis in long-term high-dose alcohol consumers. Apoptosis is present to a similar degree in the heart muscle of high-dose alcohol consumers and long-standing hypertensive subjects and is related to structural damage. Some of the protective effects of red wine have been attributed to polyphenolic compounds such as resveratrol. We have recently investigated the mechanisms on how resveratrol might afford myocardial protection during I/R injury and described a very interesting signature of the compound on myocardial microRNA expression. Our future studies will be focused on the understanding of the mechanisms of ethanol-induced oxidative/nitrosative stress, inflammation and cell death in the cardiovascular system and also in the liver. We will use clinically relevant models of aging (Fisher rats developed by National Aging Institute) to address the effects of ethanol on the course of oxidative/nitrosative stress and inflammation associated with aging.
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