Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis

doi:10.1016/S0039-6060(97)90016-7

Surgery

Volume 122, Issue 2, August 1997, Pages 255-263

https://doi.org/10.1016/S0039-6060(97)90016-7 Get rights and content

Abstract

Background. We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability.

Methods. An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated.

Results. AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3—like protease activity.

Conclusions. Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

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We have shown that periadventitial delivery of the short-acting NO donor PROLI/NO (t1/2=2 s at pH 7.4 and 37 ºC) strongly inhibits neointimal hyperplasia [19,20]. The mechanisms by which NO inhibits neointimal growth are diverse: NO inhibits platelet adherence and aggregation [21], mitigates leukocyte adhesion [22], and prevents VSMC proliferation and migration [23,24], while simultaneously promoting endothelial cell growth [23,25]. However, the effect of NO-based therapies on the temporal profile of inflammation, cellular proliferation, phenotypic marker expression, and inflammation in each layer of the arterial wall has not been established.
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Our results provide evidence that regulation of the redox environment at baseline and following exposure to ^•NO is sex-dependent in the vasculature. These data suggest that sex-based differential redox regulation may be one mechanism by which ^•NO is more effective at inhibiting neointimal hyperplasia in male versus female rodents.
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We have demonstrated that local delivery of nitric oxide (•NO) to the site of arterial injury effectively inhibits neointimal hyperplasia in different animal models [6–8]. The mechanisms by which •NO accomplishes such inhibition are diverse, but include inhibition of VSMC proliferation and migration and promotion of endothelial proliferation [9–13]. 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.
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.
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Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.

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^☆: Supported by National Institutes of Health grants GM-44100, GM-37753, and AI-16869.

^☆☆: Presented at the Fifty-eighth Annual Meeting of the Society of University Surgeons, Tampa, Fla., Feb. 13–15, 1997.

¹: E. T. is supported by a National Research Service Award F32-GM-16645.

²: T. R. B. is the recipient of the George H.A. Clowes Jr., MD, FACS, Memorial Research Career Development Award of the American College of Surgeons.

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Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis☆,☆☆

Abstract

Cell

Cell

Surgery

Biochem Biophys Res Commun

J Biol Chem

Cell

The pathogenesis of atherosclerosis: a perspective for the 1990's

Nature

Molecular therapies for vascular disease

Science

Beneficial effect of SPM-5185, a cysteine-containing nitric oxide donor, in rat carotid artery intimal injury

Circ Res

Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide

J Clin Invest

Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene

Vascular gene transfer of the human inducible nitric oxide synthase: characterization of activity and effects on myointimal hyperplasia

Molec Med