Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation

Abstract

Uncoupled endothelial nitric oxide synthase (eNOS) produces O instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.

Reconstitution of autophagy ameliorates vascular function and arterial stiffening in spontaneously hypertensive rats

“…Impairment of autophagy results in an inhibitory phosphorylation of endothelial NOS (eNOS) at Thr495 and eNOS uncoupling in human umbilical vein endothelial cells. On the other hand, rapamycin restores autophagy to reduce phospho-eNOS Thr495 and enhances the activation of eNOS at Ser1177 (59). Nonetheless, further understanding of the mechanisms by which autophagy induction can reduce vascular age is still outstanding, especially nitric oxide-independent factors.…”

Novel Contributors and Mechanisms of Cellular Senescence in Hypertension-Associated Premature Vascular Aging

“…127 Right now our understanding is centered on the premise that the accumulation of dysfunctional and decaying organelles and misfolded proteins leads to a state of oxidative stress that subsequently quenches nitric oxide bioavailabilty 121,122 (Figure 3) and also uncouples endothelial nitric oxide synthase. 128 Nonetheless, given the close association of autophagy with metabolism and energy homeostasis, we hypothesize that upregulation of autophagy imparts influence on metabolic sensors (e.g., AKT and AMPK) and thereby can modulate vascular function through these mechanisms.…”

ER–Mitochondria Microdomains in Cardiac Ischemia–Reperfusion Injury: A Fresh Perspective

“…Restoration of Beclin1 attenuates HR-mediated cardiomyocyte death ( Ma et al, 2012 ). At the molecular levels, Beclin1 activation is involved in stress protein degradation ( Fuhrmann and Brune, 2017 ), ROS clearance ( Sun et al, 2017 ), inflammation repression ( Chi et al, 2018 ), and eNOS-dependent vasodilation restoration ( Zhang J.X. et al, 2018 ).…”