Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall

Highlights

• Nitric oxide increases SOD-1 protein levels in vascular smooth muscle cells, but not in endothelial cells or adventitial fibroblasts.

• The ^•NO-dependent increase in the SOD-1 levels is preceded by an increase in gene expression; it also correlates with an increase in SOD-1 activity and a decrease in superoxide levels.

• Nitric oxide is more effective at inhibiting the development of neointimal hyperplasia after wire femoral injury in wild-type mice than in SOD-1 knockout mice.

• Nitric oxide regulates vascular smooth muscle cell proliferation by differentially regulating the redox environment in these cells.

Abstract

Superoxide (O₂^•−) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide (^•NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether ^•NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether ^•NO exerts cell-specific, differential modulation of O₂^•− levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates ^•NO-dependent inhibition of neointimal hyperplasia. In vivo, ^•NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, ^•NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O₂^•− levels. Lastly, to determine the role of SOD-1 in ^•NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without ^•NO. Interestingly, ^•NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O₂^•− by ^•NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of ^•NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

Introduction

Atherosclerosis remains the leading cause of death and disability in the United States. Cardiovascular disease claims the lives of more than 2150 Americans each day [1]. Current interventions for the treatment of severe atherosclerosis include: balloon angioplasty with or without stenting, endartectomy, or bypass grafting. However, vascular interventions continue to fail from restenosis secondary to neointimal hyperplasia. Neointimal hyperplasia is predominantly characterized by proliferation and migration of vascular smooth muscle cells (VSMC) and adventitial fibroblasts to the neointima [2], [3], [4], [5]. We have demonstrated that local delivery of nitric oxide (^•NO) to the site of arterial injury effectively inhibits neointimal hyperplasia in different animal models [6], [7], [8]. The mechanisms by which ^•NO accomplishes such inhibition are diverse, but include inhibition of VSMC proliferation and migration and promotion of endothelial proliferation [9], [10], [11], [12], [13]. We have recently focused our attention on the cell-specific effect of ^•NO in the vascular wall as it is known that ^•NO affects neighboring cell populations differently. In particular, we have focused on the effect of ^•NO on superoxide (O₂^•−) metabolism, since O₂^•− is integral to many of the cellular processes involved in the arterial injury response.

The role of reactive oxygen species (ROS) following vascular injury has been well-described. ROS, including O₂^•− and H₂O₂, increase following arterial injury and stimulate proliferation and migration of VSMC and adventitial fibroblasts, resulting in neointimal hyperplasia [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Reducing ROS following arterial injury with different antioxidants, NOX inhibitors, genetic NOX deletions, or xanthine oxidase inhibitors has been shown to decrease the formation of neointimal hyperplasia [15], [17], [24], [25], [26], [27]. Specifically demonstrating the importance of SOD to neointimal hyperplasia, overexpression of SOD or treatment with a SOD mimetic attenuated the formation of neointimal hyperplasia [28], [29]. However, the data that exist on the effects of ^•NO on O₂^•− and SOD-1 are conflicting. While ^•NO can inhibit the mitochondrial respiratory chain and increase O₂^•−, ^•NO can both stimulate and inhibit different NOX subunits, thereby increasing and decreasing O₂^•− levels, in different cell types [30], [31], [32]. In particular, in pulmonary smooth muscle cells, Wedgwood et al. showed that ^•NO decreases O₂^•− levels [33]. Furthermore, ^•NO has been shown to increase SOD-1 in some cell types but inhibit SOD-1 in others [34], [35], [36]. Thus, while it is clear that both O₂^•− and SOD are intimately involved in regulating the arterial response to injury, little is known about how ^•NO modulates SOD-1 and O₂^•− levels in the vasculature.

Here we explore the effect of ^•NO on redox homeostasis in the cells that comprise the vascular wall in vitro and in vivo. We hypothesize that cell-specific, differential ^•NO-mediated regulation of SOD-1 levels throughout the vascular wall mediates the ability of ^•NO to prevent neointimal hyperplasia. Cell-specific modulation of SOD-1 levels by ^•NO would represent a novel mechanism of ^•NO-mediated redox modulation. Moreover, these findings could open the door for translational research focusing on cell-specific targeting of antioxidant therapies with much promise to improve the outcomes of patients with vascular disease.