Protein S nitrosylation the covalent modification of

Protein S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO) [17], plays an important role in the progression of cardiovascular diseases [[18], [19], [20]]. eNOS can be S-nitrosylated in endothelial, The picture and this modification reversibly attenuates enzyme activity. These studies have also identified zinc-tetrathiolate cysteine residues as the sites in eNOS that undergo S-nitrosylation in intact endothelial cells [[21], [22], [23]]. To our knowledge, the possibility that S-nitrosylation of cysteine residues on eNOS is involved in the modulation of its interaction with β‑catenin remains unexplored. Inducible nitric oxide synthase (iNOS) is regarded as a principal mediator of NO-dependent S-nitrosylation [24]. However, more investigations are needed to understand the expression of iNOS and the subsequent S-nitrosylation of important proteins in endothelial dysfunction. Several studies have shown that OxLDL elevates iNOS expression in related cardiovascular diseases [[25], [26], [27]]. Therefore, we tested the hypothesis that, in order to cause endothelial dysfunction, OxLDL modulates the β‑catenin signaling pathway via eNOS S-nitrosylation induced by iNOS. We report here that OxLDL induces eNOS S-nitrosylation at Cys94 and Cys99 sites. This modification of eNOS enhances its interaction with β‑catenin, induces β‑catenin nuclear translocation, and promotes the transcriptional activity of β‑catenin, thus contributing to OxLDL-induced endothelial dysfunction. Furthermore, the inhibition of iNOS reduces OxLDL-induced eNOS S-nitrosylation and activation of the β‑catenin pathway, subsequently attenuating endothelial dysfunction. Altogether, these findings revealed a new mechanism for the regulation of endothelial dysfunction in atherosclerosis.
Materials and methods
Results
Discussion Endothelial cells are crucial for both vascular homeostasis and protecting the vasculature against atherogenic insults [3]. OxLDL-mediated injury to endothelial cells is crucial for endothelial dysfunction in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture at advanced stages [6]. We confirmed that the phosphorylation of eNOS (Ser1177) was decreased in OxLDL-treated endothelial cells (Suppl. Fig. 2). However, the precise mechanism of OxLDL on endothelial dysfunction remains to be explored. Under physiological conditions, eNOS can bind to β‑catenin in endothelial cells to regulate the downstream β‑catenin signal pathway [16]; therefore, we wondered whether OxLDL could affect the association and nuclear translocation of the eNOS/β‑catenin complex. Our data showed that OxLDL could increase the association and nuclear translocation of eNOS and β‑catenin in endothelial cells, thereby promoting the transcriptional activity of β‑catenin. Furthermore, the association and nuclear translocation of eNOS and β‑catenin were also enhanced in aortic endothelial cells in an atherosclerosis mouse model (Fig. 1).