• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • OxLDL down regulates eNOS and up regulates iNOS


    OxLDL down-regulates eNOS and up-regulates iNOS, thereby augmenting the formation of NO and protein S-nitrosylation in human endothelial Phosphatase Inhibitor Cocktail 3 (100X in DMSO) [26]. Importantly, iNOS-mediated S-nitrosylation plays an increasingly significant role in cardiovascular diseases [34]. For example, iNOS-mediated IRE1α S-nitrosylation links obesity-associated inflammation to endoplasmic reticulum dysfunction [34]. Additionally, S-nitrosylation of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma [35]. Recent studies have discovered that innate immunity is necessary for the transdifferentiation of fibroblasts to endothelial cells. Innate immune activation increases the iNOS generation of NO to S-nitrosylate RING1A, thus releasing epigenetic repression to achieve effective transdifferentiation [36]. We also confirmed that OxLDL significantly increased iNOS expression and NO release in endothelial cells (Fig. 5 and Suppl. Fig. 3), but without change in GSNOR and Trx expression (Suppl. Fig. 4). To explore whether eNOS S-nitrosylation induced by OxLDL was ascribed to iNOS-derived NO, 1400 W, which is a specific inhibitor of iNOS, was used in our study. As our results demonstrated, the iNOS inhibitor suppressed OxLDL-induced eNOS S-nitrosylation, cell migration and adhesion molecule expression (Fig. 5). Mechanistically, the iNOS inhibitor also reduced the association and nuclear translocation of eNOS and β‑catenin in OxLDL-treated endothelial cells (Fig. 6). These results suggested that iNOS activation contributes to OxLDL-induced eNOS S-nitrosylation and endothelial dysfunction. In conclusion, this study provides evidence that OxLDL increases iNOS-mediated S-nitrosylation of eNOS at Cys94 and Cys99 to regulate the interaction of eNOS and β‑catenin to induce endothelial dysfunction (Fig. 7). These data highlight a novel insight into the mechanism of atherosclerosis. The following are the supplementary data related to this article.
    Conflict of interest
    Transparency document
    Introduction O2 concentration directly affects cells and tissues is thereby damaging to all aerobic organisms. In particular, vascular endothelial cells, which are constantly in contact with flowing blood, are highly susceptible to hypoxia. Under conditions of low oxygen, nitric oxide (NO) regulates oxygen (O2) delivery by controlling blood vessel relaxation locally [1]. NO is also produced by various NO synthase (NOS) enzymes via the L-arginine-nitric oxide pathway [2], and three distinct isoforms of NOS include neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Among these, eNOS expression is largely restricted to vascular endothelial cells, primarily to those in medium- and large-sized arteries and arterioles [1]. In quiescent cells, eNOS is inactivated by binding to the caveolin-constituting protein Caveolin-1 in cell membrane caveolae [3]. eNOS is activated by Ca2+-dependent and -independent mechanisms. Increasing intracellular Ca2+ concentrations lead to assembly of Ca2+/calmodulin complexes, which binds eNOS instead caveolin-1 [3]. Alternatively, Ca2+-independent phosphorylation and de-phosphorylation networks strongly influence eNOS activities via the phosphorylation of serine and threonine residues. Known activating phosphorylation sites of eNOS include Ser1179, Ser635, and Ser617, and inactivating sites include Ser116 and Thr497 (based on bovine eNOS sequence and equivalent to human eNOS-Ser1177, Ser633, Ser615, Ser114, and Thr495, respectively) [[3], [4], [5]]. Previous studies show that eNOS is phosphorylated at Ser1179 and Ser635 by drug-mediated stimulation of bradykinin and vascular endothelial growth factor (VEGF) and by physical stimuli, such as fluid shear stress [4,6,7]. Specifically, shear stress of 25 dyn/cm2 for 60 min increased NO production and eNOS phosphorylation in bovine aortic endothelial cells (BAECs) [8]. Moreover, phosphorylation of eNOS increased in porcine aortic endothelial cells by up to 1.80 times after 5-min exposures to fluid shear stress of 12 dyn/cm2, and eNOS serine phosphorylation remained detectable for up to 60 min [9]. Other studies also show increased phosphorylation of eNOS at Ser1179 and Ser635 following fluid shear stress of BAECs [5,10,11]. In addition to fluid shear stress, the effects of O2 concentration have been investigated in various ECs [[12], [13], [14], [15]]. Increases in eNOS phosphorylation have been observed under conditions of hypoxia, and exposures of porcine coronary endothelial cells to low oxygen environments (pO2 = 10 mmHg) led to early (15 min) and sustained increases in eNOS phosphorylation at Ser1179 [12]. Simultaneous applications of oxygen tension (medium O2 concentrations = 5˜21%) and shear stress of 10 dyn/cm2 also led to the accumulation of NO metabolites in BAECs, and these were highest under a combination of hyperoxia and shear stress [16]. However, ratios of phosphorylation at Ser1179 to total eNOS did not differ significantly among O2 concentrations, suggesting the involvement of other phosphorylation sites, such as Ser635 [16]. The effect of phosphorylation of eNOS is already studied widely and several mechanisms have been proposed. However, endothelial cells (ECs) are exposed constantly by various physiological stimuli, such as shear stress, O2, and hormone, and the combinatory effects of these mechanisms on eNOS phosphorylation is not known well. Thus, in this study, we investigated the effects of fluid shear stress and O2 concentrations on the phosphorylation of eNOS at Ser635 in cultured BAECs.