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  • DL-AP5 Sodium salt In vasculature NADPH oxidases NOXs


    In vasculature, NADPH oxidases (NOXs) are the main sources of O2− generation that involve in the transfer of electrons to molecular oxygen. Endothelin-1 [25] and thromboxane A2 [26] were reported to induce eNOS uncoupling via upregulation of NOXs. Additionally, inhibition of p47 phox, a subunit of NOXs, could reduce oscillatory SS induced O2− release [21]. Thus, O2− increasing might initiate eNOS uncoupling in the context of low SS exposure. In current work, we discovered low SS could elicit eNOS uncoupling. However, this process is likely to be O2− independent. To elucidate the correlation of eNOS uncoupling and O2− production, we first obligated O2− beforehand and found these maneuvers could not reverse NO decreasing in sheared ECs. It is proposed that in circumstances of redox stress, such as hydrogen peroxide [27] or ox-LDL treatment [28], dephosphorylated eNOS Thr 495 increases in ECs. We next repeated the experiment of ox-LDL treatment in order to use it as a control to observe the DL-AP5 Sodium salt of eNOS Thr 495 during low SS exposure. Phosphorylation of eNOS Thr 495 was conversely regulated by ox-LDL vs. low SS. This controversy supported the notion that other signals apart from redox regulation might participate in low SS induced eNOS uncoupling. Given the evidence that autophagy agonist rapamycin can appease low SS induced oxidative stress [19], rationale for investigating autophagy in low SS condition is strong. Autophagy is a survival pathway to adapt to environmental stress. Physiological SS was reported to induce endothelial autophagy [29,30], while limited information exists about the influences of low SS on autophagy. Ding and his colleagues [31] discovered that low SS of 3 dyne/cm2 could activate LC3-II formation. Consistently, lipidation of LC3-II and amount of Beclin1 were boosted by low SS in our study. Nevertheless, increased lipidation of LC3-II may reflect either an increase of autophagosome or inefficient clearance of sequestered cargos. Hence, a question arose from it was the increasing of LC3-II formation should be ascribed to enhanced autophagy or to impaired autophagic flux in response to low SS. This prompted us to observe the late stage of autophagy and autophagic flux in our investigation. During the late stage of autophagy, p62, an adaptor protein to carry autophagic cargos [32,33], is itself degraded mainly in the autophagic process [34]. Therefore, it can be used as an indicator for late stage of autophagic flux. We found that p62 accumulation and autophagic flux was impaired during low SS exposure, which is in accordance with an in vivo study that detected stronger p62 staining in low SS field of rabbit aorta [35]. ULK1/Atg1 kinase complex functions as the most upstream target during the assembly of pre-autophagosomal structure [36]. Likewise, results from our study observed that ULK1/Atg1 signal was increased in low SS exposed ECs, suggesting autophagic flux was positively regulated at first even though it was impaired later. Our third finding is the interconnection with eNOS uncoupling and autophagic flux impairment in low SS exposed ECs. We observed that improvement of eNOS uncoupling did not restore autophagic flux, which was validated by the result that low DL-AP5 Sodium salt SS induced eNOS uncoupling was nonetheless decreased by L-NAME in condition of pharmacological autophagic flux blockade. In contrast, we noticed that restoration of low SS- impaired autophagic flux by rapamycin and WYE-354 increased NO release in vitro. And this was substantiated In vivo that rapamycin attenuated O2− concentration in low SS area of mice carotid arteries. Taken together, we conclude low SS-induced eNOS uncoupling is secondary to impaired autophagic flux. Phosphorylation is a crucial posttranslational modification to regulate eNOS enzymatic activity. Even though it was argued that physiological SS activated Ser 1177 and Ser 633 residues to produce NO [37], the details responsible for influence of low SS, <10 dyne/cm2, on eNOS activity remains largely unexplored. Phosphorylation of eNOS Ser 114 and Thr 495 is commonly assumed to inhibit eNOS activity. In recombinant BH(4)-free eNOS, eNOS uncoupling were tuned by phosphorylation at Thr495/Ser1177 sites [38]. Given that low SS induced eNOS uncoupling was not in reliance on O2− increasing, and low SS phosphorylated the negative regulating residue Thr 495, we inferred the mechanism of low SS induced eNOS uncoupling was similar to that of BH(4)-free eNOS, through eNOS phosphorylation other than BH(4) oxidation.