Shear Stress-Induced SIRT1 Activation: Effects on Mitochondrial Biogenesis and Endothelial Senescence.

Abstract

Vascular aging has been implicated in the etiology of many vascular diseases. A common process linking the vascular aging to the development of vascular complications is the accumulation of senescent endothelial cells (ECs) in the vessel wall. Senescent ECs often exhibit the endothelial activation, a cell interactive pro-inflammatory and pro-coagulant state, which becomes a hallmark of early stage of atherosclerosis. Regular practice of aerobic exercise has been recognized as the single most effective non-pharmacological anti-aging intervention. While multiple factors play a role in bringing about the salutary effect of aerobic exercise, I hypothesized here that unidirectional laminar shear stress (LSS) is a prominent cellular mechanism responsible for the protective effect of aerobic exercise against vascular aging. In chapter 1, the purpose of study was to investigate the protective effects of LSS against EC senescence. In in vivo study, C57BL/6J mice were allowed for 4-week voluntary wheel exercise after a partial ligation surgery on the left carotid artery (LCA) as to induce a local intravascular disturbed flow. Right carotid artery (RCA) was used as an internal control. In in vitro studies, human umbilical vein endothelial cells (HUVECs) were preconditioned by being exposed to LSS for 36 hours (20 dyne/cm2 for 12 hours and 5 dyne/cm2 for 24 hours) using a cone-and-plate shear apparatus. EC senescence was induced by incubating cells in 100 μM H2O2 contained media for 1 hour followed by 72 hours of serial culture in normal growth media. For mechanistic studies, we used resveratrol (RSV) and sirtinol as a SIRT1 activator and a sirtuin deacetylase inhibitor, respectively. EC senescence was determined by the percentage of senescence-associated β-galactosidase (SA-β-gal) positive cells as well as the expression levels of cell senescence markers such as p53, p21, and p16. In in vivo study, en face SA-β-gal staining showed deposition of senescent cells only in the LCA but not in the RCA, suggesting a direct association of disturbed flow with vascular cell senescence. In in vitro studies, the number of SA-β-gal positive ECs and the expressions of p53, p21 and p16 were significantly increased in H2O2-induced senescent ECs. LSS pre-conditioned cells showed significant reduction in the expression levels of cell senescence markers. Furthermore, in senescent ECs, increased reactive oxygen species generation, decreased cell migration and angiogenic dysfunction were observed, which were improved by LSS pre-conditioning. The protective effect of LSS against EC senescence was completely abolished by SIRT1 inhibition. This study suggests that high-flow LSS on endothelial cells has protective effects against H2O2-induced senescence through a SIRT1-dependent mechanism. In chapter 2, the purpose of study was to investigate the effects of LSS on mitochondrial remodeling and endothelial activation. To investigate the effects of exercise on endothelial homeostasis, twenty one pre-hypertensives have performed supervised 6-month aerobic exercise training (AEXT). In in vitro studies, HUVECs were exposed to high-flow LSS using a cone-and-plate shear apparatus. Endothelial activation was determined by measuring released levels of endothelial microparticles (EMP). SIRT1 siRNA was used to determine the underlying mechanism. Circulating levels of microparticles released by activated (CD62E+) and apoptotic (CD31+/CD42a-) endothelial cells were significantly decreased after a 6 month supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced endothelial activation and apoptosis, which was accompanied by an increase in mitochondrial biogenesis through a SIRT1-dependent mechanism. RSV treatment showed similar effects. SIRT1 knockdown using siRNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or PGC-1α siRNA provoked the activated- and apoptotic- statuses in endothelial cells. However, shear stress normalized these impairments. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. In chapter 3, the purpose of study was to investigate the combined effects of RSV and LSS on mitochondrial biogenesis in ECs. HUVECs were exposed to 20 μM RSV and/or LSS at 20 dyne/cm2. The levels of SIRT1, mitochondrial biogenesis factors and mitochondrial content were significantly increased in the treatment of high-flow LSS and the combined treatment of RSV and LSS. However, the additive effect of combined treatment on mitochondrial biogenesis was not found in ECs. Taken together, exercise-induced increased LSS protects against endothelial activation and premature endothelial senescence partly by SIRT1 activation and improving mitochondrial biogenesis in the vascular endothelial cells. These findings suggest that aerobic exercise is an effective strategy to prevent diseases related to vascular aging.