In an aging condition the decreased

In an aging condition, the decreased pattern of eNOS Ser1177 was observed, and its activity decreased with passage progression in the endothelial cells. Phosphorylation of eNOS includes post-translational modifications and is responsible and somehow representative of its activity. eNOS has various sites for phosphorylation, but the serine (Ser) residues are considered predominant over threonine (Thr) and tyrosine (Tyr) [25], [26]. In the aging cells, NO production decreased, whereas superoxide increased with decreased eNOS Ser1177 (Fig. 1B, C, D), suggesting interrelation between superoxide and eNOS to explain the eNOS uncoupling phenomenon. The NADPH-based enzyme Nox4, which is a main superoxide-generating enzyme, was highly increased in aging endothelial Mepivacaine HCl synthesis showing O2- accumulation and eNOS uncoupling. In mitochondria aging theory, there is deprivation of NADPH by glycolysis pathway inhibition, decreased energy production, and an increase in O2- during aging [27]. These factors together inhibit glycolysis, and the resultant glucose-6-phosphate follows the pentose phosphate pathway (PPP), which acts as a source of NADPH and its linked enzymes, e.g., NADPH-dependent oxidase [11], [28]. The mitochondria and ER membrane-localized NADPH-linked system Nox4 is increased in response to the accumulated NADPH through an exposed cytoplasmic signaling motif [29]. In addition, considering that the close contact between mitochondria and ER has been frequently observed in aging (Fig. 2A, D), the phospholipid membrane permeable O2- between the two organelles might also be a contributing mechanism to intra-ER O2- accumulation. Silencing of Nox4 can normalize the O2- level and eNOS activity in mesangial cells [30]. In this study, the ER-localized Nox4 was closely associated with the PDI chaperone, showing that highly increased Nox4 leads to PDI hyperactivation. Carbonylated proteins were highly associated with PDI, especially in the high-passage cells (Fig. 4F), suggesting that the majority of oxidized PDI resides within the heavy molecular weight complex fraction in association with client proteins. If PDI is working effectively, it would not reside within the heavy molecular weight complex fraction in association with Nox4, as shown with low passage cells. For ER homeostasis, ER stress triggers UPR (survival pathway), which is connected with upregulation of ER-derived chaperones and folding enzymes for degradation of unfolded proteins [31], [32]. This study shows reduced/altered function of protein folding capacity, especially in the aging condition. As PDI is unable to finish its protein folding task in aging cells, it remains attached to a client protein, such as Nox4 (Fig. 4C). Nox4 is an ER trans-membrane protein and is functional in both the cytoplasm and ER lumen [11]. In HUVEC, Nox4 expression is high compared with that of other Nox family proteins and is responsible for O2- generation. The expression of Nox4 was observed to be 100-fold greater than that of Nox2 [15]. Consistently, in aged ER lumen, Nox4 causes higher O2- generation [11], [33] and redox imbalance, leading to ER stress and disturbance of PD1 chaperonic activity [15]. Our study also suggests that this redox imbalance significantly affects eNOS coupling.