Mechanisms of vascular smooth muscle NADPH oxidase 1 (Nox1) contribution to injury-induced neointimal formation

Abstract

Objective: Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models.

Methods and results: Wire injury-induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1(y/-) mice, but there was little difference in Tg(SMCnox1) mice compared with wild-type (WT) mice. Proliferation and migration were reduced in cultured Nox1(y/-) VSMCs and increased in Tg(SMCnox1) cells. Tg(SMCnox1) cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1(y/-) cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1(y/-) cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1(y/-) VSMCs.

Conclusions: These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

Figure 1

Analysis of neointima formation in WT, Nox1^y/- and Tg^SMCnox1 mice. Injury was induced by insertion of a guide wire into the left femoral artery, and arteries were harvested after 21 days. A. Representative images of arterial cross-sections from WT, Nox1^y/- and Tg^SMCnox1 mice. B. Assessment of medial and intimal area, intima/media ratio, and %stenosis. Areas of media and intima were averaged from duplicate cross-sections, from which intima/media ratio and %stenosis were calculated. Values are means±SE of 13 (WT) and 6 (Nox1^y/- and Tg^SMCnox1) animals. *Significantly different from WT control (p < 0.05). Scale bar=50 μm.

Figure 2

Histological analysis of proliferation, apoptosis, and extracellular matrix distribution in arterial cross-sections obtained from WT, Nox1^y/- and Tg^SMCnox1 mice. Arterial sections obtained on day 21 after injury induction were stained for proliferating cell nuclear antigen (PCNA) (A), fibronectin (C, top panels) and collagen (C, bottom panels). TUNEL staining was performed after 2 hrs of injury induction (B). PCNA and TUNEL positive cells were stained red and dark brown, respectively (A and B). Red or orange color indicates fibronectin or collagen, respectively (C). Background images were obtained from WT arteries omitting primary antibody staining (A and C upper panel), and nuclei were counterstained with hematoxylin (C upper panel). Arrowhead indicates positive staining of PCNA (A), and neointimal area analyzed for fibronectin is indicated with a solid line in images (C upper panel). Images for PCNA, TUNEL, collagen and fibronectin staining are representative of sections from at least 3 animals of each genotype. Mean±SE are provided to the right of each image. `M' and `N' denote media and neointima (A). Scale bar=30 (A), 20 (C upper panel), 50 (B and C lower panel) μm.

Figure 3

Characterization of primary-cultured aortic smooth muscle cells from Nox1^y/- and Tg^SMCnox1 mice. A. PCR was conducted with primer pairs that can distinguish cDNA of Nox1^y/- cells from that of WT or Tg^SMCnox1 cells. PCR products were detected on agarose gel electrophoresis. A representative gel image is presented. B. Nox4 protein expression was measured by Western blotting. CDK4 was measured as a loading control. Representative images are shown in upper panels. Values are means±SE from 3 independent experiments. C. Serum-starved VSMCs were treated with 25 ng/ml PDGF for 4 h, which corresponds to maximal activation of NADPH oxidase. Membrane fractions were prepared, and NADPH-dependent, SOD-inhibitable superoxide generation was measured with ESR using CPH. Values shown are means±SE from 5 independent experiments. *Significantly different from corresponding control (p < 0.05).

Figure 4

Effect of Nox1 expression on proliferation, migration and extracellular matrix (ECM) production. A. Growth curves were obtained from WT, Nox1^y/- and OE cells. B. Cell migration was assessed using the modified Boyden chamber assay. Quiescent VSMCs were treated with 10 ng/ml PDGF for 3 h. C. Measurement of collagen I and fibronectin in culture media. Serum-starved VSMCs were maintained in the presence of 50 μg/mL ascorbic acid and β-aminopropionitrile with or without 10 ng/ml PDGF for 72 h. Media was collected and subjected to Western blotting. Representative immunoblots are shown in C. Values are means±SE of 6 (A) and 3 (B and C) independent observations. * Significantly different from corresponding control (p < 0.05).

Figure 5

Alteration of cofilin, mDia and PAK1 expression or activity in WT, Nox1^y/- and Tg^SMCnox1 VSMCs. Lysates were prepared from serum-deprived, untreated cells for total protein measurement. To investigate phosphocofilin levels, serum-starved VSMCs were treated with or without 10 ng/ml PDGF for 15 min, and cell lysates were subjected to Western blotting. Band density of each protein was normalized to that of CDK4 (cofilin and PAK1), cofilin (phospho-cofilin), or β-tubulin (mDia1), and the relative fold-changes over WT groups were calculated. Values are means ± SE of 4 (cofilin) or 3 (phospho-cofilin, mDia1, PAK1) independent experiments. * Significantly different from corresponding control (p < 0.05).

Figure 6

Recovery of impaired migration in Nox1^y/- VSMCs by expression of constitutively active cofilin, S3A. pcDNA3/S3A or pcDNA3 was introduced into Nox1^y/- or WT VSMCs by electroporation. After 12 h, serum was removed for 48 h, and migration induced by PDGF (10 ng/ml) was measured. Values are means±SE of 3 independent observations. *Significantly different from corresponding control, Nox1^y/- cells transfected with pcDNA (p < 0.05).