Isopropylamine NONOate (IPA/NO) moderates neointimal hyperplasia following vascular injury

Objective

Isopropylamine NONOate (IPA/NO) is a nitroxyl (HNO) donor at physiologic pH. HNO is a positive inotrope and vasodilator, but little is known about its effect on neointimal hyperplasia. The aims of this study are to determine the effect of IPA/NO on endothelial and vascular smooth muscle cells (VSMC) in vitro and to determine if IPA/NO inhibits neointimal hyperplasia in vivo.

Methods

VSMC were harvested from the abdominal aortas of male Sprague Dawley rats, and human umbilical vein endothelial cells were purchased from ATCC. In vitro, cellular proliferation was assessed by ³H-thymidine incorporation, cell migration was assessed using the scrape assay, and cell death was assessed using Guava personal cell analysis (PCA). Cell cycle analysis was performed using propidium iodide staining and flow cytometry analysis. Protein expression was assessed using Western blot analysis. Phosphorylated proteins were assessed using immunoprecipitation and Western blot analysis. In vivo, the carotid artery injury model was performed on male Sprague Dawley rats treated with (n = 12) or without (n = 6) periadventitial IPA/NO (10 mg). Arteries harvested at 2 weeks were assessed for morphometrics using ImageJ. Inflammation was assessed using immunohistochemistry. Endothelialization was assessed by Evans blue staining of carotid arteries harvested 7 days after balloon injury from rats treated with (n = 6) or without (n = 3) periadventitial IPA/NO (10 mg).

Results

In vitro, 1000 μmol/L IPA/NO inhibited both VSMC (38.7 ± 4.5% inhibition vs control, P = .003) and endothelial cell proliferation (54.0 ± 2.9% inhibition vs control, P ≤ 0.001) without inducing cell death or inhibiting migration. In VSMC, this inhibition was associated with an S-phase cell cycle arrest and increased expression of cyclin A, cyclin D1, and the cyclin-dependent kinase inhibitor p21. No change was noted in the phosphorylation status of cdk2, cdk4, or cdk6 by IPA/NO. In rodents subjected to the carotid artery balloon injury model, IPA/NO caused significant reductions in neointimal area (298 ± 20 vs 422 ± 30, P ≤ .001) and medial area (311 ± 14 vs 449 ± 16, P ≤ .001) compared with injury alone, and reduced macrophage infiltration to 1.7 ± 0.8 from 16.1 ± 3.5 cells per high power field (P ≤ .001). IPA/NO also prevented re-endothelialization compared with injury alone (55.9 ± 0.5% nonendothelialized vs 21 ± 4.4%, respectively, P = .001). Lastly, a 50% mortality rate was observed in the IPA/NO-treated groups.

Conclusions

In summary, while IPA/NO modestly inhibited neointimal hyperplasia by inhibiting VSMC proliferation and macrophage infiltration, it also inhibited endothelial cell proliferation and induced significant mortality in our animal model. Since HNO is being investigated as a treatment for congestive heart failure, our results raise some concerns about the use of IPA/NO in the vasculature and suggest that further studies be conducted on the safety of HNO donors in the cardiovascular system.

Clinical Relevance

Isopropylamine NONOate (IPA/NO) is a predominately nitroxyl (HNO) releasing compound that is currently being investigated as a pro-inotropic drug for the cardiovascular system. The effects of IPA/NO on the development of neointimal hyperplasia have not been investigated. Here, we demonstrate that IPA/NO has a modest beneficial effect on preventing the development of neointimal hyperplasia, but IPA/NO also inhibits endothelial cell growth. Given that the latter is less ideal for a therapy targeted toward promoting vascular health, we caution its use as an antiproliferative agent in the vasculature. However, we note that it may be a good antiangiogenic agent for cancer therapeutics.