Nrf and HO are key factors in the regulation
Nrf2 and HO-1 are key factors in the regulation of oxidative stress in the body. Research has shown that KLF2 can activate Nrf2 and HO-1 . To investigate the mechanisms by which KLF2 regulates eNOS uncoupling and oxidative stress, Nrf2 was inhibited or HO-1 was knocked down in KLF2 overexpressing HUVECs. The results show that the expression levels of Nrf2 and HO-1 were significantly increased in KLF2 overexpressing HUVECs, but the protective effects of KLF2 on H/R injury and eNOS uncoupling almost disappeared in KLF2 overexpressing HUVECs treated with Nrf2 inhibitor (ML385) or HO-1 siRNA. These results suggest that KLF2 plays a protective role in H/R-induced HUVEC injury by regulating eNOS uncoupling via the Nrf2/HO-1 pathway.
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Introduction Atherosclerosis is a progressive disease that is characterized by the accumulation of lipids and fibrous elements in the large arteries. The vascular endothelium is responsible for the regulation of vascular tone and the maintenance of vascular homeostasis [1,2]. Many studies have reported that alterations of endothelial function are early events in atherosclerosis development . During the early stage of atherosclerosis, oxidized low-density lipoprotein (OxLDL) is known to enhance oxidative stress and inflammation, thereby inducing endothelial dysfunction and the formation of atherosclerotic plaques [4,5]. Numerous studies have demonstrated that OxLDL increases endothelial permeability and the expression of adhesion molecules that lead to anabatic adherence and the penetration of monocytes into the vascular endothelium [, , ]. β‑catenin is a key modulator in the Wnt signaling pathway, which is involved in vasculogenesis, angiogenesis, intimal thickening and atherosclerosis [, , ]. β‑catenin associates with transcriptional T-cell factor/lymphocyte enhancing factor (TCF/LEF), thus regulating the coordination of cell-cell adhesion and the expression of numerous target genes, including Heparin sodium D1, c-Myc and c-Jun. In recent years, several studies have reported that β‑catenin plays an important role in OxLDL-induced endothelial dysfunction [12,13]. Nevertheless, how OxLDL regulates the β‑catenin pathway remains unclear. As the first discovered gaseous signaling molecule, nitric oxide (NO) affects a number of cellular processes, including those involving vascular cells . Under physiological conditions, NO is produced mainly from L-arginine by endothelial nitric oxide synthase (eNOS) in the endothelium of blood vessel walls . Recently, eNOS has been demonstrated to interact directly with β‑catenin in endothelial cells, and eNOS activation leads to β‑catenin translocation to the nucleus with resultant effects on gene transcription and downstream functional responses . These results indicate that the β‑catenin signaling pathway might rely on its interaction with eNOS and eNOS activity. Protein S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO) , plays an important role in the progression of cardiovascular diseases [, , ]. eNOS can be S-nitrosylated in endothelial, cells 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 [, , ]. 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 . 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 [, , ]. 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.