Biochemical and Biophysical Research Communications
SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol
Introduction
Reactive oxygen species (ROS) either generated endogenously or by exposure to cigarette smoke (CS) play a major role in the progression of several diseases including cardiovascular and pulmonary diseases [1], [2]. CS is known to cause endothelial dysfunction which is characterized by reduced cell migration, angiogenesis, vasodilation, proinflammatory and prothrombic properties associated with the development of cardiovascular diseases (CVD) [1], [2]. Endothelial nitric oxide synthase (eNOS), a vital signaling molecule, plays a crucial role in endothelial cell function. eNOS regulates vascular tone, blood flow, platelet aggregation, cell adhesion and leukocyte–endothelial cell interactions which are characteristics of endothelial function [3]. It has been shown that accelerated ROS production leads to decreased eNOS levels, which play a central role in CS-mediated endothelial dysfunction [4]. We and others have recently shown that CS-mediated oxidative stress downregulates eNOS levels leading to reduced NO production and endothelium-dependent vasodilatation in endothelial cells [5], [6], [7], [8]. However, it is not known whether eNOS is subjected to post-translational modifications, in particular acetylation, rendering it inactive in response to CS-mediated oxidative stress in endothelial cells.
SIRT1, which is homologous to yeast (S. cerevisiae) silent information regulator protein 2 (Sir 2), plays a critical role in aging, cell cycle regulation, apoptosis and inflammation [9], [10]. It is highly expressed in vascular endothelial cells and plays a key role in regulating the endothelial function [11], [12], [13], [14], [15]. Although SIRT1 has been shown to play a critical role in endothelial vascular biology, it is not known whether SIRT1 regulates endothelial function via acetylation/deacetylation of eNOS in response to CS and oxidants. We therefore hypothesized that CS-mediated oxidative stress downregulates SIRT1 by phosphorylation and degradation leading to eNOS acetylation in endothelial cells. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were exposed to cigarette smoke extract (CSE) and H2O2, and the role of SIRT1 in regulation of eNOS acetylation/deacetylation was determined. We also studied whether pharmacological activation and inhibition of SIRT1 can regulate eNOS acetylation in these cells.
Section snippets
Materials and methods
Human umbilical vein endothelial cell culture. HUVECs culture was established as described previously [7], [8] by using human umbilical cords collected within 48 h of delivery and were grown in EGM-2 (Lonza, Walkersville, MD, USA, previously known as Cambrex) media containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in 75 mm2 flask coated with 0.1% gelatin, and treatments were performed in 0.1% gelatin-coated 6-well plates [7], [8].
CSE
CSE and H2O2 decreased SIRT1 level in HUVECs
SIRT1 plays important role in vascular inflammation and endothelial dysfunction [15]. However, it is not known that whether SIRT1 level is altered in response to CS-mediated oxidative stress in endothelial cells. Therefore, we determined the effects of CSE on SIRT1 protein levels in HUVECs. Treatment of endothelial cells with CSE (0.5% and 1.0%) for 1 and 4 h resulted in significant dose- and time-dependent decrease in SIRT1 levels when compared to control treatments (P < 0.001, Fig. 1A–C).
Discussion
Oxidative stress-mediated chronic inflammation in smokers renders damage to endothelial cells thus increases response to vascular injury [6]. Previously, we have shown that CS-induced oxidative stress impaired the angiogenic function of endothelial cells by downregulating phosphorylation of Akt and eNOS in endothelial cells [7], [8]. Recently, it has been shown that activation of SIRT1 protects endothelial cells against CS-induced oxidative stress [18]. However, the downstream target of SIRT1
Acknowledgments
This study was supported by the NIH R01-HL085613, 1R01HL097751-01 and NIEHS Environmental Health Science Center grant ES-01247.
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