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ENOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

ABSTRACT: Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs to high glucose (22 mM) for 3 days increased senescence-associated- ?-galactosidase (SA-?-gal) activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. The calcium channel blocker nifedipine, but not the ?1-adrenergic blocking agent atenolol or the angiotensin-converting enzyme inhibitor perindopril, reduced SA-?-gal positive cells and prevented a decrease in telomerase activity in a high-glucose environment. This beneficial effect of nifedipine was associated with reduced reactive oxygen species (ROS) and increased endothelial nitric oxide synthase (eNOS) activity. Thus, nifedipine prevented high glucose-induced ROS generation and increased basal eNOS phosphorylation level at Ser-1177. Treatment with N (G)-nitro-L-arginine (L-NAME) and transfection of small interfering RNA (siRNA) targeting eNOS eliminated the anti-senscence effect of nifedipine. These results demonstrate that nifedipine can prevent endothelial cell senescence in an eNOS-dependent manner. The anti-senescence action of nifedipine may represent a novel mechanism by which it protects against atherosclerosis.

SUBMITTER: Hayashi T

PROVIDER: S-EPMC3919771 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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eNOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

Hayashi Toshio T Yamaguchi Tomoe T Sakakibara Yasufumi Y Taguchi Kumiko K Maeda Morihiko M Kuzuya Masafumi M Hattori Yuichi Y

PloS one 20140210 2

Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs to high glucose (22 mM) for 3 days increased senescence-associated- β-galactosidase (SA-β-gal) activity, a senescence marker, and decreased telomerase activity, a replicative se ...[more]

PMID: 24520379

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Project description:The cardiovascular effects of estrogen are mediated in part by augmenting the function of endothelial nitric oxide synthase. Endothelial nitric oxide synthase activity is dependent on many cofactors including Ca(2+). Hence, we investigated the effect of chronic 17 beta-estradiol treatment on the intracellular Ca(2+) concentration and endothelial nitric oxide synthase protein expression in the human endothelial cell line, EA.hy926, using spectrofluorometry and Western blot, respectively. Inhibiting the sarco(endo)plasmic reticulum Ca(2+) ATPase with thapsigargin caused an increase in the intracellular Ca(2+) concentration, which was higher in chronically 17 beta-estradiol-treated (1muM, 24h) cells loaded with Fura-2-acetoxymethyl ester compared to vehicle-treated cells, suggesting a higher endoplasmic reticulum Ca(2+) content in 17 beta-estradiol-treated cells. An enhanced Ca(2+) influx pathway in chronically 17 beta-estradiol-treated cells was also observed. In addition, 17 beta-estradiol-treated cells expressed higher levels of endothelial nitric oxide synthase protein in comparison to vehicle-treated cells. The chronic effect of 17 beta-estradiol on Ca(2+) homeostasis and endothelial nitric oxide synthase expression was attenuated with the nonselective estrogen receptor inhibitor, ICI 182,780 (10muM, 7alpha, 17beta-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl] estra-1,3,5(10)-triene-3,17-diol). Furthermore, analysis of the thapsigargin-evoked Ca(2+) response in chronically 17 beta-estradiol-treated estrogen receptor alpha-knockdown cells showed no significant difference in Ca(2+) response compared to vehicle-treated estrogen receptor alpha-knockdown cells, indicating that the regulation of Ca(2+) homeostasis by 17 beta-estradiol is mediated through an estrogen receptor alpha-dependent pathway. These data revealed an estrogen receptor alpha-dependent modulation of Ca(2+) homeostasis accompanying the enhancement of endothelial nitric oxide synthase expression in 17 beta-estradiol-treated human endothelial cells.

Project description:AimsArginase II (ArgII) plays a key role in the regulation of Ca2+ between the cytosol and mitochondria in a p32-dependent manner. p32 contributes to endothelial nitric oxide synthase (eNOS) activation through the Ca2+/CaMKII/AMPK/p38MAPK/Akt signalling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability.Methods and resultsmRNA levels were measured by quantitative reverse transcription-PCR, and protein levels and activation were confirmed by western blot analysis. Ca2+ concentrations were measured by FACS analysis and a vascular tension assay was performed. ArgII bound to p32, and ArgII protein knockdown using siArgII facilitated the ubiquitin-dependent proteasomal degradation of p32. β-lactone, a proteasome inhibitor, inhibited the p32 degradation associated with endothelial dysfunction in a Ca2+-dependent manner. The amino acids Lys154, Lys 180, and Lys220 of the p32 protein were identified as putative ubiquitination sites. When these sites were mutated, p32 was resistant to degradation in the presence of siArgII, and endothelial function was impaired. Knockdown of Pink/Parkin as an E3-ubiquitin ligase with siRNAs resulted in increased p32, decreased [Ca2+]c, and attenuated CaMKII-dependent eNOS activation by siArgII. siArgII-dependent Parkin activation was attenuated by KN93, a CaMKII inhibitor. Knockdown of ArgII mRNA and its gene, but not inhibition of its activity, accelerated the interaction between p32 and Parkin and reduced p32 levels. In aortas of ArgII-/- mice, p32 levels were reduced by activated Parkin and inhibition of CaMKII attenuated Parkin-dependent p32 lysis. siParkin blunted the phosphorylation of the activated CaMKII/AMPK/p38MAPK/Akt/eNOS signalling cascade. However, ApoE-/- mice fed a high-cholesterol diet had greater ArgII activity, significantly attenuated phosphorylation of Parkin, and increased p32 levels. Incubation with siArgII augmented p32 ubiquitination through Parkin activation, and induced signalling cascade activation.ConclusionThe results suggest a novel function for ArgII protein in Parkin-dependent ubiquitination of p32 that is associated with Ca2+-mediated eNOS activation in endothelial cells.

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ENOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

Publications

eNOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

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