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

Isopropylamine NONOate (IPA/NO) prevents vascular smooth muscle cell (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 personal cell analysis (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 < .005 vs control. ^†P < .05 vs 1000 μmol/L IPA/NO. n = 3/treatment group. Data shown are representative of three separate experiments.

Isopropylamine NONOate (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 personal cell analysis (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 < .005 vs control. n = 3/treatment group. Data shown are representative of three separate experiments.

Isopropylamine NONOate (IPA/NO) causes S-phase cell cycle arrest. A, Flow cytometry analysis of untreated and IPA/NO-treated (1000 μmol/L) vascular smooth muscle cell (VSMC). B, Graphical representation of the flow cytometry results after 24 hours of IPA/NO treatment. *P < .05 vs control. n = 3/treatment group. Data shown are representative of three separate experiments.

Isopropylamine NONOate (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 vascular smooth muscle cell (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.

Isopropylamine NONOate (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 < .001 vs injury alone. Units are arbitrary.

Isopropylamine NONOate (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.

Isopropylamine NONOate (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 < .001 vs injury alone.

Isopropylamine NONOate (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 = .001, n = 3/group.