• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • Inducible nitric oxide synthase iNOS is regarded as a


    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
    Discussion Endothelial Omadacycline inhibitor 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). Previous reports demonstrated that eNOS is S-nitrosylated at Cys94 and Cys99 in endothelial cells, and eNOS S-nitrosylation is inversely related to eNOS activation (phosphorylation at Ser1179) [21,22]. S-nitrosylation is a dynamic post-translational modification for the regulation of protein function [31]. Cys94 and Cys99 are widely investigated sites that have been shown to be S-nitrosylation cysteine sites of eNOS, and they form a zinc-tetrathiolate cluster at the eNOS homodimer interface, which is responsible for dimer formation of the active enzyme [21,22]. Cys441 is the last residue near the C-terminus of the oxygenase domain in the dimer interface and is located between acidic (glutamine) and basic (arginine) amino acids of eNOS [32]. Collectively, these three cysteine sites play a crucial role in maintaining the normal function and activation of eNOS. To examine the correlation between OxLDL-induced eNOS S-nitrosylation and endothelial dysfunction, Cys94, Cys99 and Cys441 mutants were used in our experiments. Our findings demonstrated that the Cys94 and Cys99 sites participated in OxLDL-induced eNOS S-nitrosylation, whereas Cys441 rarely influenced OxLDL-induced eNOS S-nitrosylation (Fig. 2). The S-nitrosylation of eNOS enhanced cell migration and adhesion molecule expression in endothelial cells after treatment with OxLDL, but this effect was abolished by mutation of Cys94 and Cys99 in eNOS (Fig. 3). These results provide the first evidence that eNOS S-nitrosylation at Cys94 and Cys99 is involved in OxLDL-induced endothelial dysfunction.