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

Abstract

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 (3)H-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 micromol/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 < or = 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 < or = .001) and medial area (311 +/- 14 vs 449 +/- 16, P < or = .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 < or = .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.

Figure 1

IPA/NO prevents VSMC proliferation in vitro, but does not cause cell death or affect migration. (A) Proliferation of VSMC was assessed using ³H-thymidine incorporation after 24 hours of treatment with IPA/NO. (B) VSMC death was assessed via Guava PCA after 24 hours of treatment with IPA/NO. (C) The effect of IPA/NO (1000 μmol/L) on VSMC migration was assessed by blinded counting of nuclei migrating into the scraped area. The effects of the parent compound, IPA, and breakdown product of IPA/NO, isopropanol, on proliferation, death and migration were also assessed at 1000 μmol/L. The effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on proliferation and cell death were assessed at 1000 μmol/L. *P<0.005 vs. control. †P<0.05 vs. 1000 μmol/L IPA/NO. n = 3/treatment group. Data shown are representative of three separate experiments.

Figure 2

IPA/NO prevents HUVEC proliferation in vitro, but does not cause cell death or affect migration. (A) Proliferation of HUVEC was assessed using ³H-thymidine incorporation. (B) HUVEC death was assessed via Guava PCA after 24 hours of treatment with IPA/NO. (C) The effect of IPA/NO (1000 μmol/L) on HUVEC migration was assessed by blinded counting of nuclei migrating into the scraped area. The effects of the parent compound, IPA, and breakdown product, isopropanol, of IPA/NO on proliferation, death and migration were also assessed at 1000 μmol/L. *P<0.005 vs. control. n = 3/treatment group. Data shown are representative of three separate experiments.

Figure 3

IPA/NO causes S-phase cell cycle arrest. (A) FACS analysis of untreated and IPA/NO-treated (1000 μmol/L) VSMC. (B) Graphical representation of the flow cytometry results after 24 hours of IPA/NO treatment. *P<0.05 vs. control. n = 3/treatment group. Data shown are representative of three separate experiments.

Figure 4

IPA/NO affects cell cycle protein expression, but not the phosphorylation state of cdks. (A) The expression of cyclins, cdks, and CDKIs were assessed in VSMC by Western blot analysis in the presence or absence of IPA/NO for 24 hours. Data shown are representative of three separate experiments. (B) IPA/NO-treated and untreated VSMC lysates were immunoprecipitated (IP) with the indicated antibody, then analyzed for expression of phosphorylated (P) and non-phosphorylated (non-P) cdks. Graphs show the ratio of phosphorylated to non-phosphorylated protein as determined by densitometry. S: starved, C: control, and I: IPA/NO.

Figure 5

IPA/NO moderates neointimal hyperplasia in vivo. (A) Balloon-injured rat carotid arteries treated with or without exogenous periadventitial administration of IPA/NO powder (10 mg) were harvested 14 days after injury and stained by H&E. Whole mount and close-up sections are shown. (B) Quantitation of intimal area, medial area and arterial circumference using ImageJ software (n = 6/group). *P<0.001 vs. injury alone. Units are arbitrary.

Figure 6

IPA/NO decreases apoptosis in the neointima and media of balloon-injured arteries. Balloon-injured rat carotid arteries treated with or without exogenous periadventitial administration of IPA/NO powder (10 mg) and harvested 14 days after injury (n = 4/group) were subjected to TUNEL staining for apoptosis. Arrows indicate positive staining.

Figure 7

IPA/NO decreases inflammation in balloon-injured arteries. (A) Balloon-injured rat carotid arteries treated with or without exogenous periadventitial administration of IPA/NO powder (10 mg) and harvested 14 days after injury (n = 6/group) were subjected to immunoperoxidase staining using antibodies against leukocytes (CD45) and macrophages (CD68). Arrows indicate positive staining. (B) Blinded quantitation of positive CD45 and CD68 staining in untreated and IPA/NO-treated injured vessels was performed using ImageJ (n = 6/group). *P<0.001 vs. injury alone.

Figure 8

IPA/NO prevents re-endothelialization. (A) Balloon-injured rats were administered Evans blue dye intravenously 7 days post-injury, and the harvested carotid arteries were photographed to assess the extent of endothelial regeneration. Blue staining indicates a lack of endothelium (n = 3/group). (B) Quantitation of photographs from panel (A) using ImageJ software. *P=0.001, n = 3/group.