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

Abstract

Objective Isopropylamine NONOate (IPA/NO) is a nitroxyl (HNO) donor at physiological 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 FACS 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=0.003) 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=<0.001) and medial area (311±14 vs. 449±16, P=<0.001) compared to injury alone, and reduced macrophage infiltration to 1.7±0.8 from 16.1±3.5 cells per high power field (P=<0.001). IPA/NO also prevented re-endothelialization compared to injury alone (55.9±0.5% non-endothelialized vs. 21±4.4%, respectively, P=0.001). 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.

Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats

“…Once anesthetized, as evident by absence of response to toe pinch, the animals were euthanized via a bilateral thoracotomy. The aortas were removed, and the cells were isolated and maintained as previously described (30). Briefly, after the aorta is removed, the endothelial layer is gently removed by scraping.…”

“…All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85- 23,1996) and approved by the Northwestern University Animal Care and Use Committee. Thirty 11-wk-old male Sprague Dawley rats weighing between 350 and 400 g underwent the carotid artery balloon injury model, as previously described (17,28,30). Animals were anesthetized with isoflurane (1.5-5%) and treated preoperatively with carprofen subcutaneously (0.15 mg/kg) for pain control.…”

Vasorelaxant and antiaggregatory actions of the nitroxyl donor isopropylamine NONOate are maintained in hypercholesterolemia

“…Studies in support of preserved vascular actions of HNO under pathophysiological conditions include the finding that Angeli's salt-mediated inhibition of ADP-stimulated platelet aggregation was sustained in patients with sickle cell disease (21). IPA/NO has also been shown to limit neointimal hyperplasia in rats (29), and Paolocci and colleagues (22) have indirectly shown that the hemodynamic actions of HNO are sustained in acute experimental heart failure. However, it remains unclear whether HNO donors offer advantages over traditional nitrovasodilators, such as GTN, in the treatment of vascular diseases where there is increased generation of ROS such as O 2 ·Ϫ and reduced NO • signaling.…”

“…Thus, Angeli's salt has been shown to lower blood pressure in the setting of acute experimental heart failure (22) and inhibit the aggregation of platelets from patients with sickle cell disease (21). In addition, IPA/NO has been reported to limit neointimal hyperplasia in rat carotid arteries after balloon injury (29).…”

Nitroxyl donors retain their depressor effects in hypertension

“…Importantly, we have shown that, unlike clinically used nitrovasodilators such as GTN, vascular tolerance to HNO donors does not develop either in vitro (5,11) or in vivo (12) with continued use. In addition, the vasodilatory capacity of HNO is accompanied by an ability of this nitrogen oxide to inhibit platelet aggregation (5, 21) and limit vascular smooth muscle proliferation (30).…”