Project

Arginase-II ; Endothelial aging; eNOS ; S6K1; oxdative stress

Lead
Lay summary
Aging  instead the vasoprotective NO, a situation calledeNOS-uncoupling, is involved in vascular aging. Although studies suggest that enhancedarginase activity (arginase-II in humans and mice) causes eNOS-uncoupling, itis still debatable, whether this is due to depletion of L-arginine for eNOS. Importantly,chronic L-arginine supplementation paradoxically induces endothelial cell agingand increases mortality in patients with myocardial infarction or withperipheral artery disease. In addition, our study also shows that hyperactiveS6K1 emzymatic activity is present in aging endothelial cells and causes eNOS-uncoupling.It is, however, not known, how S6K1 causes eNOS-uncoupling and acceleratesendothelial aging. Based on these results, we would like to investigate theroles and molecular/biochemical mechanisms of S6K1 and Arg-II in promotion of eNOS-uncouplingand endothelial aging in cultured cells and animal models. .-2revious studies showed that the enzyme in vascularendothelial cells called endothelial nitric oxide synthase (eNOS) can produce toxicOis aprominent independent risk factor for cardiovascular disease, the underlyingmechanisms of aging-associated cardiovascular diseases are not fully understood, yet. PThis project will explore noval mechanisms ofage-associated cardiovascular diseases. Particularly, how L-arginine therapy isdetrimental for cardiovascular health will be answered. Expected results shall alsojustify therapeutic rationales of targeting S6K1 and Arg-II foraging-associated vascular disease and beyond, since eNOS-uncoupling, increasedS6K1 and enhanced Arg-II activity are also observed under various pathologicalconditions such as atherosclerosis and obesity.

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Last update: 21.02.2013

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Background: Global population aging is accelerating, which will be accompanied with increase in age-associated diseases including cardiovascular disease in our society. Although aging has been proven to be a prominent independent risk factor for cardiovascular disease, the underlying mechanisms of cardiovascular aging have not been fully understood, yet. It has been well demonstrated that oxidative stress, i.e., enhanced production of reactive oxygen species (ROS) such as O2.- plays a critical role in endothelial aging. Among other sources of ROS, endothelial nitric oxide synthase (eNOS)-uncoupling appears to be an important mechanism of oxidative stress in endothelial aging, whereby eNOS generates large amount of O2.- instead the vasoprotective NO. Recent studies from others and our own showed that enhanced arginase activity (arginase-II in humans and mice) can cause endothelial dysfunction and also eNOS-uncoupling, which is generally believed (not proven) to be due to depletion of L-arginine for eNOS. However, a real absolute L-arginine deficiency does not exist either in cultured cells or in vivo, since L-arginine concentrations in culture medium or in plasma of animals or humans are more than sufficient (in mmol/L range) for eNOS. Suprisingly, chronic L-arginine supplementation paradoxically induces endothelial cell senescence. Patients with myocardial infarction or with peripheral artery disease who received L-arginine supplementation therapy for 6 months have higher mortality or shorter claudication working distance than placebo groups. These results do not support L-arginine deficiency theory on one hand and implicate on the other hand that L-arginine metabolites through Arg-II (urea, L-ornithine, polyamines) may promote endothelial dysfunction or eNOS-uncoupling. It is well established that genetic, dietary, and pharmacological inhibition of mTOR/S6K1 signalling extends lifespan in model organisms. Our recent study provides the first evidence for the causative role of hyperactive S6K1 activity in endothelial aging and eNOS-uncoupling in vitro and in vivo animal model. It is, however, not known, how S6K1 causes eNOS-uncoupling and acelerates endothelial aging. Our additional ongoing experiments demonstrate that S6K1 enhances Arg-II protein expression and activity in cultured human endothelial cells. Based on these results, we would like to investigate the hypothesis below Working hypothesis: S6K1 causes endothelial aging partly through Arg-II. The follwoing major specific questions will be addressed: Specific aims: (1) Does Arg-II promote endothelial cell senescence in culture through eNOS-uncoupling? (2) Does Arg-II promote eNOS-uncoupling and senescence through L-arginine deficiency or through its metabolites catalyzed through Arg-II such as urea, L-ornithine, or polyamines? (3) Does S6K1 induce endothelial senescence through activating Arg-II and what are the underlying molecular mechanisms: up-regulation of mRNA or protein translation or protein posttranslational modification? (4) Does Arg-II knock-out in mouse delay endothelial aging and extends lifespan? (5) Does S6K1 deficiency suppress aging-associated vascular Arg-II up-regulation/activation and endothelial dysfunction/senescence?Experimental design and/or methods: These specific questions will be addressed in two aging models i.e. in vitro senescence model with cultured human endothelial cells and in vivo aging models with mice deficient in Arg-II gene (Arg-II-/-) and S6K1 gene (S6K1-/-). Integrative approaches ranging from genetic, molecular/cellular, organ, and systemic analyses will be utilized. The significance of the project: This project will explore the causal role of S6K1 and Arg-II, the predominant isoform of arginase in human endothelial cells, in acceleration of vascular aging in vitro cultured endothelial cells and in vivo aging animal models. Particularly, the mechanisms of “L-arginine paradox” in relation to eNOS-uncoupling induced by Arg-II will be characterized, i.e. the functions of L-arginine metabolites through Arg-II in eNOS-uncoupling and endothelial senescence will be for the first time investigated. Furthermore, a novel function of S6K1 in regulation of Arg-II gene expression and/or enzymatic activity as well as the underlying molecular mechanisms in context of endothelial aging and dysfunction will be deeply explored. Expected results shall also justify therapeutic rationales of targeting S6K1 and Arg-II for aging-associated vascular disease and beyond, since eNOS-uncoupling, increased S6K1 and enhanced Arg-II activity are also observed under various pathological conditions such as atherosclerosis and obesity. Finally, the aspect of lifespan regulation by Arg-II will also be explored.