Autophagy maintains vascular function through a novel glycolysis-linked pathway regulating eNOS.

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SUMMARY One report indicates endothelial cell (EC) autophagy is compromised in aged humans. We showed that genetic repression of autophagy in ECs negates shear-stress induced EC nitric oxide (NO) synthase (eNOS) activation and NO generation. It is unknown whether disruption of autophagy specifically in ECs has functional relevance in vivo, and the mechanism whereby repressed EC autophagy compromises NO generation is not known. In Aim 1 we will test the hypothesis that conditional deletion of autophagy in ECs from adult mice (iecAtg3KO mice) phenocopies arterial dysfunction that is present in old mice. Further, based on compelling preliminary data, we will test the hypothesis that repressed EC autophagy in mutant mice and old mice evokes a p53- mediated block in glycolysis, leading to decreased signaling by extracellular ATP via the P2Y1-R and PKCδ to eNOS, resulting in arterial dysfunction. Aim 2 will explore the translational potential of this novel pathway in the context of human aging. In immortalized human arterial endothelial cells (HAECs) we hypothesize that genetic autophagy suppression prevents shear-stress induced purinergic signaling to eNOS. Next, this pathway will be evaluated in primary arterial ECs obtained from old (> 60 y) and adult (18-30 y) subjects before and following rhythmic handgrip exercise that elevates brachial artery shear-rate similarly in both groups. ECs will be used to quantify EC autophagy, eNOS activation, and NO generation. Importantly, pharmacological and genetic approaches that restore purinergic mediated signaling to eNOS will be used in these models of genetic and aging-associated autophagy repression in human ECs. Aim 3 will use adult and aged mutant mice to determine whether exercise-training attenuates the aging-associated decline in EC autophagy, and whether intact autophagy is required for training-induced vascular improvements. To evaluate translational potential, we will discern whether one-limb rhythmic handgrip exercise training by old (> 60 y) human subjects is sufficient to elevate basal and shear-induced EC autophagy initiation, eNOS activation, and NO generation vs. the contralateral sedentary limb. Results from this work have tremendous potential to reveal a new therapeutic target and approach for restoring / maintaining vascular function in the aging population.