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  • br Materials and methods br Results br Discussion

    2020-09-09


    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). 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) Dextran sulfate sodium salt 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. In the vasculature, the interaction of VE-cadherin and β‑catenin at endothelial cell-cell junctions controls vascular integrity [33]. However, OxLDL induces the activation of β‑catenin in human aortic smooth muscle cells, and the active β‑catenin associates with TCF4 and translocates into the nucleus, thus playing an important role in atherosclerosis [12]. Despite research on the interaction between eNOS and β‑catenin in endothelial cells, the role of eNOS S-nitrosylation in regulating the eNOS/β‑catenin complex and the subsequent signaling pathway in the context of endothelial dysfunction is not clearly defined [16]. Hence, we examined the involvement of eNOS S-nitrosylation in β‑catenin signaling using a NOS inhibitor and mutations of eNOS. L-NAME, a dual NOS inhibitor, not only diminished eNOS S-nitrosylation (Fig. 3) but also inhibited the binding of β‑catenin to eNOS induced by OxLDL in endothelial cells (Fig. 4). Furthermore, the interaction of β‑catenin and eNOS, nuclear translocation and transcriptional activity of β‑catenin were decreased in eNOS mutation (C94S and C99S)-transfected endothelial cells (Fig. 4). Together, our study suggested for the first time that the increased interaction of eNOS and β‑catenin induced by OxLDL is dependent on the S-nitrosylation of eNOS.