Fig 2: Effects of FG-4592 on the regulation of AGTR1, AGTR2, eNOS, p-eNOS, and HIF1α in cultured cells.(A) Smooth muscle cells were treated with FG-4592 and HIF1α siRNA. AGTR1, AGTR2, and HIF1α expressions were evaluated by immunoblotting. (B–D) Quantification of immunoblotting analysis from A. (E) Endothelial cells were treated with FG-4592 and HIF1α siRNA. p-eNOS, eNOS, and HIF1α expressions were evaluated by immunoblotting. (F–H) Quantification of immunoblotting analysis from (E). β-Actin was used as loading control. Data are presented as mean ± SD (n = 3 per group). Significance values were determined by 1-way ANOVA. Newman-Keuls multiple-comparison test was used for multiple comparisons. NC, negative control (a control of scrambled siRNA). Vehicle, DMSO in DMEM medium. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Empagliflozin alleviates I/R-induced CMEC damage.In vivo, mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts. The cells were cultured for 24 h and then used for functional analyses. (A–C)In vivo, proteins were isolated from reperfused heart tissues, and Western blots were used to analyze eNOS and ET-1 levels. (D, E)In vivo, immunofluorescence staining of VE-cadherin was performed to observe the changes in endothelial integrity and barrier function. (F, G) Immunohistochemistry was used to assess the expression of ICAM1, an endothelium-specific adhesive factor. (H–J) Proteins were isolated from CMECs, and the levels of Src and Fak were analyzed through Western blots. (K, L)In vitro, FITC-dextran clearance and TER assays were used to analyze the endothelial barrier function and integrity of CMECs. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05.

Fig 4: Transplantation of MSCs or MSC‐Exos influence the nitric oxide synthase (NOS) content in the corpus cavernosum and reduce the organization oxidative stress damage. A, Representative images of western blots for nNOS, eNOS, iNOS and SOD in cavernosum in each group. B‐E, Western blots' data are presented as the relative density of nNOS, eNOS, iNOS and SOD compared with that of GAPDH. F and G, Immunohistochemically semi‐quantitative data of the proportion of SOD positive expression area and 8‐OHdG positive expression endothelial cells. Each bar depicts the means ± standard deviation from n = 6 animals per group. *P < .05 vs the PBS group, **P < .01 vs vs the PBS group, #P < .05 vs the Sham group, ##P < .01 vs the Sham group. H and I, Immunohistochemical expression of SOD and 8‐OHdG, respectively, in corpus cavernosum. The negative control (NC) group used PBS to serve as the primary antibody. Red arrowheads refer to the area of positive tissue expression of SOD. Original magnification, ×200

Fig 5: Effect of the administration of exendin‐4 on eNOS expression. (a) Capillary western blot of total eNOS and phosphorylated eNOS expression in the rat retina. The red box indicates the target protein. (b and c) Fold change in the expression of total eNOS (b) and phosphorylated eNOS (c) in the rat retina (n = 7 in control group; n = 6 in Hp + PBS group; n = 9 in other groups). (d and e) Capillary western blot and fold change in the expression of GLP‐1 receptors (GLP‐1R) (d), PI3K, and Akt (e) in the rat retina. * P < .05, significantly different from CSF group; # P < .05significantly different from noradrenaline without exendin‐4 group. (f) NO content in human retinal microvascular endothelial cells (n = 6, 5, 5, 6, 7, 5, and 6 for group of control, OGD, OGD + Ex‐4, OGD + Ex‐4 + Ex‐9–39‐L, OGD + Ex‐4 + Ex‐9–39‐H, OGD + Ex‐4 + l‐NAME‐L, and OGD + Ex‐4 + l‐NAME‐H, respectively). One‐way ANOVA with LSD or Dunnett's T3 test were performed. C, control group; HP, high pressure injury group; EX‐4, exendin‐4; s.c., subcutaneous injection of exendin‐4; i.v., intravitreal injection of exendin‐4; od, eye drops of exendin‐4; OGD, oxygen glucose deprivation model; EX‐9–39‐L, low concentration of exendin‐9‐39 (10 nM); EX‐9–39‐H, high concentration of exendin‐9‐39 (20 nM); l‐NAME‐l, low concentration of l‐NAME (50 μM); l‐NAME‐H, high concentration of l‐NAME (100 μM)