Fig 1: Nox1, Nox4 and Nox5 are involved in Ang II-induced redox signalling in human VSMC. Cells were stimulated with Ang II, (100 nmol/L) for 5 and 15 min in the presence and absence of NoxA1ds (Nox1 inhibitor, 10 µmol/L), GKT137831 (Nox1 and Nox4 inhibitor, 10 µmol/L) and melittin (Nox5 inhibitor, 10 nmol/L). Phosphorylation of MLC (A), Ezrin-Radixin-Moesin (B), p53 (C) and ERK1/2 (D) was detected by western blotting in hVSMC. a-Tubulin and total ERK1/2 were used as loading control. Results are expressed as mean ± SEM of 6–8 separate experiments. *P < 0.05 vs. control (Ctl), +P < 0.05 vs. respective Ang II group. Ang II angiotensin II, Ctl control, p-MLC20 phosphorylated MLC20, p-ERM phosphorylated Ezrin–Radixin–Moesin, p-p53 phosphorylated p53, p-ERK1/2 phosphorylated ERK1/2.
Fig 2: Nox1 and Nox5 but not Nox4 are present in lipid-rafts/caveolae in human VSMCs. VSMCs isolated from small arteries were stimulated with Ang II and subjected to discontinuous sucrose density gradient. (A) Representative immunoblots of Nox5 and Cav-1. Nox5 is present in isolated lipid-rafts fractions 3–4 in human VSMCs. (B) Representative images of Nox5 and Nox1 in lipid rafts (LRs) vs. non-lipid rafts (NLRs) in VSMCs stimulated with Ang II (100 nmol/L) for 5, 15 or 30 min. Isolated LR (fractions 3–4) or high density NLR (fractions 7–12) were pooled together and the percentage ratio of LRs vs. NLRs for Nox5 (C) and Nox1 (D) was determined. (E) Representative images of Nox4 in LRs vs. NLRs. Bar graphs are means ± SEM from 4–5 experiments. Control was taken as 100% and data are presented as the percentage changes relative to control conditions. *P < 0.05 vs. Ctl. #P < 0.05 vs. Ang II, 5 min. Ang II angiotensin II, Ctl control, Cav-1 caveolin-1, LR lipid rafts, NLR non-lipid rafts, M marker, THP1 human acute monocytic leukemia cells, VSMCs vascular smooth muscle cells.
Fig 3: Schematic showing putative interactions between lipid rafts/caveolae, Nox isoforms, DJ-1 and vascular signaling in conditions where lipid rafts/caveolae are intact and when they are discrupted. (i) When cholesterol-rich microdomains are intact, likely in physiological conditions, Nox1 and Nox5 are regulated and ROS production is controlled. Nox4 which is constitutively active, localizes in the cytoplasm where it generates mainly H2O2, which may be vasoprotective. (ii) During lipid-raft disruption, which may occur in pathological conditions or when the Ang II system is upregulated, Nox1 and Nox5 are activated leading to excessive ROS production and oxidative stress. This promotes increased redox-dependent signaling through multiple downstream pathways that influence VSMC function. P phosphorylation, Ox oxidation, MLC20 myosin light chain, ERM Ezrin–Radixin–Moesin.
Fig 4: Protein expressions of oxidative stress in lung parenchyma by day 5 after ARDS induction. (a) Protein expressions of NOX-1, * versus other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (b) Protein expression of NOX-2, * versus other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (c) Expressions of oxidized protein, * versus other groups with different symbols (†, ‡, §, ¶), p < 0.001. MW = molecular weight; DNP = 1,3-dinitrophenylhydrazone. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC = sham control; ARDS = acute respiratory distress syndrome; Mito = mitochondria; SW = shock wave.
Fig 5: Nox isoforms differentially regulate Ang II-induced ROS production in human VSMC. Cells were stimulated with Ang II (100 nmol/L) for 5 and 15 min in the presence and absence of NoxA1ds (Nox1 inhibitor, 10 µmol/L), GKT137831 (Nox1/Nox4 inhibitor, 10 µmol/L) and melittin (Nox5 inhibitor, 100 nmol/L). NADPH dependent O2- production (A) and H2O2 levels (B) were measured by lucigenin-derived chemiluminescence and amplex red, respectively. Results are expressed as mean ± SEM of 6–8 separate experiments. *P < 0.05 vs. Ctl, +P < 0.05 vs. respective Ang II group. Ang II angiotensin II, Ctl control, H2O2 hydrogen peroxide, RLU relative light units.
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