Pravastatin accelerates ischemia-induced angiogenesis through AMP-activated protein kinase

Abstract

Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. In this study, we examined the role of pravastatin in ischemia-induced angiogenesis. Unilateral hindlimb ischemia was surgically induced in C57BL/6J mice. Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and endothelial NO synthase (eNOS) was increased in ischemic tissues. Furthermore, mice treated with pravastatin showed higher increases in phosphorylation than did untreated mice. Laser Doppler analysis has shown that pravastatin treatment accelerates the development of collateral vessels and angiogenesis in response to hindlimb ischemia. Capillary density in the ischemic hindlimb was also increased by pravastatin treatment. An in vitro study on human umbilical vein endothelial cells (HUVECs) revealed that pravastatin increased the phosphorylation of AMPK. Pravastatin-induced phosphorylation of eNOS, one of the downstreams of AMPK, was inhibited by compound C, an AMPK antagonist. The increased migration and tube formation of HUVECs by pravastatin were significantly blocked by compound C treatment. The accelerated angiogenesis by pravastatin after hindlimb ischemia was significantly reduced after treatment with compound C. Thus, ischemia induced AMPK phosphorylation in vivo. Furthermore, pravastatin could also activate AMPK in vivo and in vitro. Such phosphorylation results in eNOS activation and angiogenesis, which provide a novel explanation for one of the pleiotropic effects of statins that is beneficial for angiogenesis. Keywords: angiogenesis; endothelium; ischemia; nitric oxide synthase; statins INTRODUCTIONThe AMP-activated protein kinase (AMPK) is a trimeric enzyme comprising a catalytic a-subunit and regulatory-b, g-subunits. AMPK was identified as an upstream kinase that phosphorylates and hence inactivates 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acetyl-coenzyme A carboxylase (ACC), key enzymes that control choresterol/isoprenoid and fatty acid biosynthesis, respectively. AMPK is considered to be a cellular energy sensor that stimulates ATP-producing catabolic pathways and inhibits ATP-consuming anabolic pathways. 1 Thus, the AMPK pathway is thought to be a regulator of stress responses and cellular energy homeostasis. However, recent studies have shown that AMPK also plays an important role in maintaining endothelial functions. 2 AMPK activation has beneficial effects on endothelial functions and in antiatherogenesis. These effects include the induction of the endothelial nitric oxide synthase (eNOS) pathway to increase NO bioavailability; the suppression of endothelial reactive oxygen species production when stimulated by hyperglycemia or high free fatty acids to improve endothelial free fatty acid oxidation and limit lipid accumulation; the inhibition of apoptosis and inflammation; and the modulation of the vascular tone. 3,4

Apelin-13 stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in myocardial microvascular endothelial cells

“…Notably, although the AMPK inhibitor suppressed the endogenous Akt phosphorylation, the Akt inhibitor had no effect on the endogenous phosphorylation of AMPK in MMVECs. The present data suggest that apelin-13 exerts pro-angiogenic effects in MMVECs through the modulation of AMPK and Akt signaling and the activation of eNOS, which is in accordance with previous studies (28,29,30). A study by Nagata et al (11) demonstrated that AMPK did not exhibit an effect on endothelial cell migration, tube formation and NO production under normoxia, which suggests that further in vitro and in vivo studies are required to gain a greater understanding of the role of endothelial cell AMPK in angiogenesis.…”

Heparin Cofactor II, a Serine Protease Inhibitor, Promotes Angiogenesis via Activation of the AMP-activated Protein Kinase-Endothelial Nitric-oxide Synthase Signaling Pathway

“…AMPK has been shown to be crucial not only for regulation of metabolic homeostasis but also for regulation of homeostasis in the cardiovascular system via activating eNOS (35). Indeed, it has been reported that an HMG-CoA reductase inhibitor (36), fenofibrate (37) and adiponectin (38) enhance angiogenesis in response to hindlimb ischemia through activation of the AMPK-eNOS signaling pathway. Previous experiments on genetic or pharmacological inhibition of AMPK activity showed significant reduction in phosphorylation of eNOS, leading to decrease in NO production (39).…”

Percutaneous Carbon Dioxide Treatment using a Gas Mist Generator Enhances the Collateral Blood Flow in the Ischemic Hindlimb

“…The hindlimb blood flow was assessed under anesthesia with sodium pentobarbital (50 mg/kg, i.p.) using a laser Doppler blood flow (LDBF) analyzer (Moor LDI; Moor Instruments, Devon, UK) before and immediately after surgery and on postoperative days 4, 7, 14, 21 and 28, as previously described 11,12) . The LDBF analysis was performed before each treatment session on days 4, 7, 14 and 21 and 24 hours after the last treatment.…”

“…Unilateral hindlimb ischemia was induced under anesthesia with sodium pentobarbital (50 mg/kg intraperitoneally (i.p. )), as previously described 7,[11][12] . Mixed air dry mist (AM) and CO2 mist (CM) was generated using a dry mist production unit (Advance Biotron Co., Ltd., Tokyo, Japan) 10) .…”