CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis (original) (raw)

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-NЈ-(4-butyl-2methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle. vessel growth; vascular endothelial growth factor; electrical stimulation PHYSIOLOGICAL ANGIOGENESIS is a complex process involving an interplay between cells, extracellular matrix molecules, and soluble factors that culminates in cell migration, proliferation, and tube differentiation of endothelial cells. The process of angiogenesis involves preexisting vessels, which send out capillary sprouts to produce new vessels (14). Several cytokines and growth factors have been established to modulate angiogenesis in vitro and in vivo, and, among these factors, vascular endothelial growth factor (VEGF) has been considered the most potent angiogenic inducer (9). Recent studies from our laboratory have further supported the role of VEGF as an important regulator of angiogenesis in skeletal muscle, because treatment with a VEGF-neutralizing antibody blocked the angiogenic response to electrical stimulation and exercise (3, 4). Arachidonic acid (AA) metabolites have been implicated in endothelial cell migration, tube formation, and angiogenesis (29, 30, 34). A recent study provided evidence for the expression of cytochrome P-450A (CYP4A)-hydroxylase in skeletal muscle cells and arterioles of rat cremaster muscle (18). This enzyme is responsible for the formation of both a vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), and a vasodilator, epoxyeicosatrienoic acid (EET). It has been shown that 20-HETE, which can be formed by the actions of enzymes in either the CYP4A and CYP4F families, plays a role in myogenic activation of small arterioles of the cerebral (15, 16) and renal (15, 22) circulations. More recently, Frisbee et al. (10) and Kunert et al. (19) have demonstrated that 20-HETE contributes to the vasoconstrictor responses to elevations in transmural pressure and PO 2 in skeletal muscle resistance arterioles. Much less is known about the role of 20-HETE in the control of growth of blood vessels. Recent studies have suggested that norepinephrine and angiotensin II (ANG II) stimulate the synthesis and release of 20-HETE in vascular smooth muscle cells (25) and that cytochrome P-450 inhibitors block activation of the MAPK system and the mitogenic effects of norepinephrine and ANG II on cultured vascular smooth muscle (VSM) cells. Because 20-HETE serves as a second messenger for the vasoactive and mitogenic actions of ANG II (2) and the local renin-angiotensin system plays a critical role in angiogenesis induced by electrical stimulation (3), the present study examined the role of 20-HETE in this response. MATERIALS AND METHODS Animal surgery. All protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. The rats were housed in the Animal Resource Center of the Medical College of Wisconsin and were given food and water ad libitum. Thirty-two male Sprague-Dawley rats, 7-8 wk old, were anesthetized with an