NADPH Oxidase 5 Is a Pro-Contractile Nox Isoform and a Point of Cross-Talk for Calcium and Redox Signaling-Implications in Vascular Function

Abstract

Background: NADPH Oxidase 5 (Nox5) is a calcium-sensitive superoxide-generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro-contractile signaling and vascular function.

Methods and results: Transgenic mice expressing human Nox5 in a vascular smooth muscle cell-specific manner (Nox5 mice) and Rhodnius prolixus, an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5-expressing mice, agonist-induced vasoconstriction was exaggerated and endothelium-dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N-acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca²⁺]_i, increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro-contractile signaling molecules MLC20 (myosin light chain 20) and MYPT1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild-type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus, gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS2870 (Nox1/2/4 inhibitor).

Conclusions: Nox5 is a pro-contractile Nox isoform important in redox-sensitive contraction. This involves calcium-calmodulin and endoplasmic reticulum-regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro-contractile molecular machinery in vascular smooth muscle cells.

Keywords: cell signaling; contraction; vascular biology.

Figure 1

Phenotypic characterization of Nox5⁺ SM22⁺ mice. (A) Immunolocalization of Nox5 (green fluorescence) in WT, SM22⁺, Nox5⁺, and Nox5⁺/SM22⁺ mice. Blue fluorescence indicates nucleus (DAPI). B, Plasma TBARS levels in WT, SM22⁺, Nox5⁺, and Nox5⁺/SM22⁺ mice. C, Systolic blood pressure assessed by plethysmography for 4 weeks in WT, SM22⁺, Nox5⁺, and Nox5⁺ SM22⁺ mice treated or not with Ang II (600 ng/kg/day). Results are mean±SEM of 5 to 8 mice/group. *P<0.05 vs control WT group or untreated group (BP); **P<0.05 vs nontreated counterpart (TBARS). Ang II indicates angiotensin II; BP, blood pressure; DAPI, 4′,6‐diamidino‐2‐phenylindole; Nox5, NADPH Oxidase 5; TBARS, thiobarbituric acid reactive substances; WT, wild‐type.

Figure 2

Cardiac phenotypic characterization of Nox5⁺ SM22⁺ mice. A, Superoxide anion levels assessed by HPLC in heart tissue from WT (open bars) and SM22⁺Nox5⁺ (closed bars) mice, before and after Ang II treatment. B, Heart weight in WT, SM22⁺, and Nox5⁺ mice, before and after Ang II treatment. C, Representative images of Sirius Red staining of heart tissue from WT and SM22⁺Nox5⁺ mice, treated or not with Ang II, exposed to polarized light microscopy. D, Total collagen deposition in heart tissue from WT and SM22⁺Nox5⁺ mice, before and after Ang II treatment. E, Cardiomyocyte area in heart tissue from WT and SM22⁺Nox5⁺ mice, before and after Ang II treatment. Results are mean±SEM of 5 to 8 mice/group. *P<0.05 vs control WT group; **P<0.05 vs nontreated counterpart. Ang II indicates angiotensin II; HPLC, high‐performance liquid chromatography; Nox5, NADPH Oxidase 5; WT, wild‐type.

Figure 3

Vascular contraction is increased in Nox5⁺/SM22⁺ mice. A, Vascular contractility to U46619 assessed by wire myography in mesenteric arteries from WT (open circle), SM22⁺ (open triangle), Nox5⁺ (open rhombus) and Nox5⁺/SM22⁺ (open square). B, Vascular contractility to U46619 in mesenteric arteries from WT (open circle), WT+Ang II (closed circle), Nox5⁺/SM22⁺ (open square), and Nox5⁺/SM22⁺+Ang II (closed square) mice. C, Vascular contractility to U46619 in mesenteric arteries from WT (open circle) and Nox5⁺/SM22⁺ (open square) mice in the presence of N‐acetylcysteine (NAC; 10 μmol/L) (WT, closed circle; Nox5⁺/SM22⁺, closed square). Vessels were pre‐incubated with NAC for 1 hour. D, Vascular contractility to U46619 in arteries from WT (open circle) and Nox5⁺/SM22⁺ (open square) mice in the presence of GKT137831 (10 μmol/L; WT, closed circle; Nox5⁺/SM22⁺, closed square). Vessels were preincubated with GKT137831 for 1 hour. E, Vascular contractility to U46619 in mesenteric arteries from WT (open circle) and Nox5⁺/SM22⁺ (open square) mice in the presence of calmidazolium (Calmid; 1 μmol/L; WT, closed circle; Nox5⁺/SM22⁺, closed square). Vessels were preincubated with calmidazolium for 1 hour. F, Vascular contractility to U46619 in arteries from WT (open circle) and Nox5⁺/SM22⁺ (open square) mice in the presence of dantrolene (Dant; 10 μmol/L; WT, closed circle; Nox5⁺/SM22⁺, closed square). Vessels were preincubated with dantrolene for 1 hour. Results are mean±SEM of 3 to 8 mice/group. *P<0.05 vs WT; **P<0.05 vs untreated WT or Nox5⁺/SM22⁺ (open symbols). Ang II indicates angiotensin II; Nox5, NADPH Oxidase 5; WT, wild‐type.

Figure 4

Maximum contraction to KCl and ET‐1–induced contraction are increased in arteries from Nox5⁺/SM22⁺ mice. A, Vascular contraction to a single concentration of KCl (62.5 mmol/L) in mesenteric arteries from WT, Nox5⁺/SM22⁺, SM22⁺, and Nox5⁺ mice, before and after treatment with Ang II. B, Vascular contraction to KCl in mesenteric arteries from WT and Nox5⁺/SM22⁺ mice, before and after preincubation (1 hour) with diltiazem (10 μmol/L) or calmidazolium (10 μmol/L). C, Vascular contraction to KCl in mesenteric arteries from WT and Nox5⁺/SM22⁺ mice, before and after preincubation (1 hour) with NAC (10 μmol/L) or GKT137831 (10 μmol/L). D, Vascular contraction to a single concentration of ET‐1 (0.1 mmol/L) in mesenteric arteries from WT and Nox5⁺/SM22⁺ mice. Vascular contraction to ET‐1 in mesenteric arteries from WT and Nox5⁺/SM22⁺ mice, before and after preincubation (1 hour) with NAC (10 μmol/L) or GKT137831 (10 μmol/L; E) or calmidazolium (10 μmol/L; F). Results are mean±SEM of 3 to 8 mice/group. *P<0.05 vs WT; **P<0.05 vs untreated mice. Ang II indicates angiotensin II; ET‐1, endothelin‐1; NAC, N‐acetylcysteine; Nox5, NADPH Oxidase 5; WT, wild‐type.

Figure 5

Vascular expression of Nox5 induces endothelial dysfunction. A, Vascular relaxation to ACh assessed by wire myography in mesenteric arteries from WT (open circle), SM22⁺ (open triangle), Nox5⁺ (open rhombus), and Nox5⁺ SM22⁺ (open square). B, Vascular relaxation to ACh in arteries from WT (open circle), WT+Ang II (closed circle), Nox5⁺/SM22⁺ (open square), and Nox5⁺/SM22⁺+Ang II (closed square) mice. C, Vascular relaxation to ACh assessed by wire myography in mesenteric arteries from WT (open circle), SM22⁺ (open triangle), and Nox5⁺ (open rhombus) before and after Ang II treatment (closed symbols). Results are mean±SEM of 5 to 8 mice/group. *P<0.05 vs WT; **P<0.05 vs untreated WT or Nox5⁺/SM22⁺ (open symbols). ACh indicates acetylcholine; Ang II, angiotensin II; Nox5, NADPH Oxidase 5; WT, wild‐type.

Figure 6

Cross‐sectional area (CSA) in resistance arteries from Nox5⁺/SM22⁺ mice. A, Vascular CSA assessed by pressure myography in mesenteric arteries from WT (open circle), SM22⁺ (open triangle), Nox5⁺ (open rhombus), and Nox5⁺/SM22⁺ (open square). B, Vascular CSA assessed by pressure myography in mesenteric arteries from SM22⁺ (open triangle) and Nox5⁺ (open rhombus), before and after Ang II treatment (closed symbols). C, Vascular CSA assessed by pressure myography in mesenteric arteries from WT (open circle) and Nox5⁺/SM22⁺ (open square), before and after Ang II treatment (closed symbols). Results are mean±SEM of 5 to 8 animals per group. *P<0.05 vs untreated groups. Ang II indicates angiotensin II; Nox5, NADPH Oxidase 5; WT, wild‐type.

Figure 7

ROS production in arteries and VSMCs from Nox5⁺ SM22⁺ mice. A, Nitrotyrosine levels, a marker of ONOO⁻, in aorta from WT and Nox5⁺/SM22⁺ mice measured by ELISA. B, ROS generation in VSMCs from WT and Nox5⁺ SM22⁺ mice assessed by lucigenin chemiluminescence. Relative luminescence units (RLU) were corrected by protein concentration of each sample. C, Basal ROS production in VSMCs from mesenteric arteries from Nox5⁺ SM22⁺ mice after incubation with verapamil (10 μmol/L), calmidazolium (1 μmol/L), and dantrolene (10 μmol/L). D, U46619‐stimulated ROS generation in VSMCs from mesenteric arteries from WT and Nox5⁺ SM22⁺ mice. U46619‐induced ROS production in VSMCs from mesenteric arteries from WT (E) and Nox5⁺ SM22⁺ (F) after preincubation with verapamil and dantrolene. Results are mean±SEM of 3 to 6 experiments. *P<0.05 vs control WT; **P<0.05 vs control nonstimulated WT or Nox5⁺ SM22⁺. W in (F) represents basal ROS generation in WT VSMCs. Nox5 indicates NADPH Oxidase 5; ONOO⁻, peroxynitrite; ROS, reactive oxygen species; VSMCs, vascular smooth muscle cells; WT, wild‐type.

Figure 8

Molecular mechanisms of vascular dysfunction in Nox5⁺ SM22⁺ mice. A, Ca²⁺ influx induced by ionomycin in VSMCs from WT and Nox5⁺/SM22⁺ mice measured by live cell microscopy using the fluorescent probe, CAL520‐AM. B, Representative immunoblotting images of phosphorylation of MYPT1 and MLC20, corrected to total GAPDH expression. C, MYPT1 phosphorylation in VSMCs from WT and Nox5⁺/SM22⁺ mice. D, MLC20 phosphorylation in VSMCs from WT and Nox5⁺/SM22⁺ mice. Results are mean±SEM of 3 to 5 experiments. *P<0.05 vs WT. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; MLC20, myosin light chain 20; MYPT1, myosin phosphatase target subunit 1; Nox5, NADPH Oxidase 5; VSMCs, vascular smooth muscle cells; WT, wild‐type.