Fig 2: Nox4 deficiency in cultured endothelial cells improves vascular function in D-gal-induced endothelial senescence. (A) HUVECs were transiently transfected with Nox4 siRNA, followed by transfection with pcDNA-Nox4 or empty vector. (B) Representative senescence-associated ß-galactosidase (SA-ß-gal) staining in HUVECs. (C) Western blots analysis of anti-phospho-eNOS Ser1177 or total-eNOS antibody. (D) Representative fluorescent images of NO formation in HUVECs were shown using NO probe DAF-2DA. (E) Representative cells were stained for DHE. (F) Images showing cell transfected with ER-targeted HyPer-Red (HyPer Red ER). ER NADPH oxidase activity (G) and hydrogen peroxide level (H) were measured in HUVECs. (I) Irreversible sulfonation of IRE1a was analyzed with transiently transfected with non-specific-siRNA or Nox4 siRNA. Scale bars, 20 µm. Data are mean ± standard deviation. #, p < 0.05; *, p = 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Schematic illustrating the regulation of NO bioavailability and EDD function by mTOR signaling.Inhibition of mTORC1 suppresses eNOS gene expression, by impairing p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, targeting mTORC2 does not affect eNOS expression. Rather, disruption of mTORC2 results in activation of MAPK (p38 and JNK), which are normally suppressed. A positive-feedback loop with Nox2 upregulation is enabled that contributes to the excessive generation of reactive oxygen species (ROS), leading to eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC.

Fig 4: (a) eNOS and (b) TRPV1 expression in aortic tissue. Control (C) and CS, CZ, and CZ + CS treatments. The bars represent mean ± SEM of 4 animals per group. #p < 0.05; *p < 0.05 vs. control group. Representative western blot analysis is shown.

Fig 5: Treatment of melatonin in the bone marrow transplantation model. (A) Concomitant expression of enhanced green fluorescent protein (eGFP, marker of transplantation donor) and CD31 (red, endothelial marker) in EPC-derived endothelial cells show yellow in color. A linear scale, 50 µm. (B) Density of EPC, calculated by ratios of eGFP+/CD31+ cell counting in a high-power field (HPF, ×100). The results are shown as means ± standard deviations. # p < 0.05 vs. diabetes. (C) Melatonin prevented EPC dysfunction and repaired blood flow recovery by increasing the expression of eNOS, AMPK, and HO-1 as well as by lowering oxidative stress. eGFP, enhanced green fluorescent protein; CD31, cluster of differentiation 31, EPC, endothelial progenitor cell.