The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5'-iodotubercidin, and by silencing AMPK-α(1/2) and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α(1). AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease.

Objective This study investigated whether arginase contributes to endothelial dysfunction and hypertension in obese rats. Design and Methods Endothelial function and arginase expression were examined in skeletal muscle arterioles from lean and obese Zucker rats (ZR). Arginase activity, arginine bioavailability, and blood pressure were measured in lean and obese animals. Results Arginase activity and expression was increased while global arginine bioavailability decreased in obese ZR. Acetylcholine or luminal flow caused dilation of isolated skeletal muscle arterioles but this was reduced or absent in vessels from obese ZR. Treatment of arterioles with a nitric oxide synthase inhibitor blocked dilation in lean arterioles and eliminated differences among lean and obese vessels. In contrast, arginase inhibitors or L-arginine enhanced vasodilation in obese ZR and abolished differences between lean and obese animals, while D-arginine had no effect. Finally, mean arterial blood pressure was significantly increased in obese ZR. However, administration of L-arginine or arginase inhibitors lowered blood pressure in obese, but not lean animals, and this was associated with an improvement in systemic arginine bioavailability. Conclusions Arginase promotes endothelial dysfunction and hypertension in obesity by reducing arginine bioavailability. Therapeutic approaches targeting arginase represent a promising approach in treating obesity-related vascular disease.

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor-2 (Nrf2). Compound C also stimulated Nrf2 expression and this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-L-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions.

6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine (compound C) is a cell-permeable pyrrazolopyrimidine derivative that acts as a potent inhibitor of AMPactivated protein kinase (AMPK). Although compound C is often used to determine the role of AMPK in various physiological processes, it also evokes AMPK-independent actions. In the present study, we investigated whether compound C influences vascular smooth muscle cell (SMC) function through the AMPK pathway. Treatment of rat aortic SMCs with compound C (0.02-10 M) inhibited vascular SMC proliferation and migration in a concentration-dependent fashion. These actions of compound C were not mimicked or affected by silencing AMPK␣ expression or infecting SMCs with an adenovirus expressing a dominant-negative mutant of AMPK. In contrast, the pharmacological activator of AMPK 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside inhibited the proliferation and migration of SMCs in a manner that was strictly dependent on AMPK activity. Flow cytometry experiments revealed that compound C arrested SMCs in the G 0 /G 1 phase of the cell cycle, and this was associated with a decrease in cyclin D1 and cyclin A protein expression and retinoblastoma protein phosphorylation and an increase in p21 protein expression. Finally, local perivascular delivery of compound C immediately after balloon injury of rat carotid arteries markedly attenuated neointima formation. These studies identify compound C as a novel AMPKindependent regulator of vascular SMC function that exerts inhibitory effects on SMC proliferation and migration and neointima formation after arterial injury. Compound C represents a potentially new therapeutic agent in treating and preventing occlusive vascular disease.