Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome

Abstract

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.

Fig. 1.

Nitric oxide (NO) inhibits [³H]thymidine incorporation to a greater extent in type II diabetes mellitus (DM) and metabolic syndrome compared with control. A: rat aortic vascular smooth muscle cells (VSMC) harvested from lean Zucker (LZ; control), obese Zucker (OZ; metabolic syndrome), and Zucker diabetic fatty (ZDF; type II DM) rodents were exposed to starvation media [0% fetal bovine serum (FBS)], control media (10% FBS), or varying concentrations of NO donor S-nitroso-N-acetylpenicillamine (SNAP; 0–1,000 μM) for 24 h and [³H]thymidine incorporation was assessed. *P < 0.05 compared with 10% FBS for each group. B: LZ cells were exposed to control media or hyperglycemic (G, 25 mM) or hyperinsulinemic (I, 200 nM) environments and then treated with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO). [³H]thymidine incorporation was assessed at 24 h. *P < 0.001 vs. control; **P < 0.007 vs. DETA/NO, 0.5–1.0 mM. Each group was assessed in triplicate. Values are representative of 5 independent experiments.

Fig. 2.

NO causes a greater G₀/G₁ cell cycle arrest in type II DM and metabolic syndrome compared with controls. Rat aortic smooth muscle cells harvested from LZ (control), OZ (metabolic syndrome), and ZDF (type II DM) rats were exposed to control media (10% FBS) or NO donor DETA/NO (1,000 μM) for 24 h. From left to right: representative flow cytometry analysis of control cells, then DETA/NO treated cells, followed by Modfit quantification of the percentage of cells in each stage of the cell cycle for LZ (control) (A), OZ (metabolic syndrome) (B), and ZDF (type II DM) (C) cells. *P < 0.05 compared with uninjured. Each group was assessed in triplicate. Values are representative of 4 independent experiments.

Fig. 3.

NO increases expression of the cyclin-dependent kinase inhibitor p21 more in type II DM vs. controls. Western blot analysis is shown of the cyclin-dependent kinase inhibitors p21 and p27 for LZ (control), OZ (metabolic syndrome), and ZDF (type II DM) VSMC. The p21 Western blot is representative of 7 independent experiments, whereas the p27 Western blot is representative of 5 independent experiments. The graphs represent densitometry of the Western blots shown, adjusted according to the control within each cell type.

Fig. 4.

NO inhibits neointima formation following arterial injury more effectively in type II DM and metabolic syndrome. NO treatment groups received a topical application of NO donor disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO); arteries were harvested 2 wk after injury and analyzed morphometrically. A: representative hematoxylin-eosin-stained sections from LZ (control, n = 11), OZ (metabolic syndrome, n = 12), and ZDF (type II DM, n = 13) rats: uninjured, injury alone, or injury plus application of PROLI/NO gel. Intimal area (B), medial area (C), and intima-to-media area ratios (I/M) (D) are shown for rodents subjected to vascular injury alone and vascular injury followed by application of PROLI/NO gel. *P < 0.05 compared with injury alone; **P < 0.05 compared with LZ injury alone.

Fig. 5.

NO release from the PROLI/NO gel. NO release from the PROLI/NO and control gels was assessed using the Greiss reaction, which measures nitrite. The PROLI/NO gel released a large amount of nitrite within the first 24 h and continued to release nitrite for 6 days.

Fig. 6.

NO causes a greater inhibition of reactive oxygen species following injury in type II DM and metabolic syndrome. A: representative dihydroethidium-stained rat carotid artery sections from LZ (control), OZ (metabolic syndrome), and ZDF (type II diabetes) rats: uninjured, injury alone, or injury plus application of PROLI/NO gel. B: quantification of dihydroethidium stain throughout all layers of the arterial wall for LZ (control), OZ (metabolic syndrome), and ZDF (type II diabetes) rats subjected to no injury, vascular injury alone, and vascular injury followed by application of PROLI/NO gel. Blinded grading was performed on a 0–3 scale. Positive control consisted of sections exposed to hydrogen peroxide (200 μM). *P < 0.05 compared with uninjured; **P < 0.05 compared with injury alone.

Fig. 7.

NO increases nitrotyrosine expression. Representative immunohistochemical staining is shown for nitrotyrosine expression of rat carotid artery sections from LZ (control), OZ (metabolic syndrome), and ZDF (type II diabetes) rats: uninjured, injury alone, or injury plus application of PROLI/NO gel.