Aging Enhances the Sensitivity of Endothelial Cells Toward Apoptotic Stimuli

Abstract

Abstract-Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-␣-induced apoptosis and caspase-3-like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35Ϯ10%) and a decrease in the overall S-NO content (33Ϯ3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3-like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

“…19,23,26 Our results show that staurosporine-induced reduction in eNOS activity was associated with caspasemediated cleavage of eNOS. Long-term (18-24 h) exposure of either eNOS-transfected COS-7 cells or BAECs to staurosporine resulted in a significant loss of eNOS protein and activity, an effect that was significantly reduced by caspase inhibitors.…”

“…[20][21][22][23][24][25] Diminished eNOS-catalyzed production of NO or its bioavailability leads to endothelial apoptosis, and it has been speculated that this is associated with enhanced susceptibility to endothelial dysfunction and atherosclerosis. [26][27][28] As NO inhibits apoptosis in endothelial cells, it is intriguing to speculate that caspase-mediated loss of eNOS-derived NO further activates the caspase cascade, thereby promoting the apoptotic process by removing an impediment to cell death.…”

“…[20][21][22][23][24] Thus, eNOS-synthesized NO inhibited caspase-mediated apoptosis in endothelial cells; 23 it has been speculated that diminished production or availability of NO and enhanced apoptosis is related to susceptibility to atherosclerosis or age-related endothelial dysfunction. [25][26][27][28] In view of a role for eNOS in endothelial cell apoptosis, the effect of staurosporine-induced apoptosis on levels of eNOS protein and activity was studied in COS-7 cells, overexpressing eNOS, and in bovine aortic endothelial cells (BAEC). We hoped to determine whether eNOS, having a role as an inhibitor of apoptosis, may itself be a target of apoptosismediated events.…”

Tumor Necrosis Factor-α Receptor p75 Is Required in Ischemia-Induced Neovascularization

“…12,33,39 The reduced expression of TNFR2/p75 associated with increasing age, coupled with postischemic increases in the systemic levels of TNF-␣, favors apoptosis in adult ECs, 39 which could subsequently lead to inhibition of angiogenesis. Suppression of TNF with soluble TNFR1/p55 was reported to accelerate angiogenesis via upregulation of VEGF receptor KDR/Flk-1.…”

Cellular senescence: from growth arrest to immunogenic conversion

“…For example, early work by one of us (RGAF) failed to show any elevation in spontaneous apoptosis rates in HUVECs cultured to senescence (although baseline apoptosis rates as measured by TUNEL were significantly higher than those seen in fibroblasts) (Kalashnik et al 2000). Later studies (Hoffmann et al 2001) demonstrated that late passage HUVECs were more sensitive to apoptosis induced by oxidized LDL or TNFα compared to early passage cells. Jeon and Boo (2013) have recently shown that upregulation of the Fas receptor at both the mRNA and protein level in senescent HUVECs probably underlies their enhanced potential to undergo programmed cell death.…”