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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:827.)
© 2008 American Heart Association, Inc.

Integrated Physiology/Experimental Medicine

Angiopoietin-Related Growth Factor Enhances Blood Flow Via Activation of the ERK1/2-eNOS-NO Pathway in a Mouse Hind-Limb Ischemia Model

Takashi Urano; Yasuhiro Ito; Masaki Akao; Tomohiro Sawa; Keishi Miyata; Mitsuhisa Tabata; Tohru Morisada; Tai Hato; Masato Yano; Tsuyoshi Kadomatsu; Kunio Yasunaga; Rei Shibata; Toyoaki Murohara; Takaaki Akaike; Hidenobu Tanihara; Toshio Suda; Yuichi Oike

From the Laboratory of Vascular Biology and Metabolism (T.U., M.A., M.T., T.M., T.H., Y.O.), Center for Integrated Medical Research, and the Department of Cell Differentiation (T.U., Y.I., M.A., T.M., T.H., T.S., Y.O.), School of Medicine, Keio University, Tokyo, Japan; the Department of Ophthalmology and Visual Science (T.U., Y.I., H.T.), the Department of Microbiology (T.S., T.A.), and the Department of Molecular Genetics (T.U., K.M., M.T., M.Y., T.K., Y.O.), Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; the Molecular Medicine Laboratories (K.Y.), Astellas Pharmaceutical Co Ltd, Tsukuba, Japan; the Department of Cardiology (R.S., T.M.), Nagoya University Graduate School of Medicine, Nagoya, Japan; and PRESTO (Y.O.), Japan Science Technology Agency (JST), Kawaguchi, Saitama, Japan.

Correspondence to Yuichi Oike, MD, PhD, Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail oike{at}gpo.kumomoto-u.ac.jp

Abstract

Objective— Transgenic mice overexpressing angiopoietin-related growth factor (AGF) exhibit enhanced angiogenesis, suggesting that AGF may be a useful drug target in ischemic disease. Our goal was to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define molecular mechanisms underlying AGF signaling in endothelial cells.

Methods and Results— Intramuscular injection of adenovirus harboring AGF into the ischemic limb increased AGF production, which increased blood flow through induction of angiogenesis and arteriogenesis, thereby reducing the necessity for limb amputation. In vitro analysis showed that exposing human umbilical venous endothelial cells to AGF increased nitric oxide (NO) production through activation of an ERK1/2-endothelial NO synthetase (eNOS) signaling pathway. AGF-stimulated eNOS phosphorylation, NO production, and endothelial cell migration were all abolished by specific MEK1/2 inhibitors. Moreover, AGF did not restore blood flow to ischemic hind-limbs of either mice receiving NOS inhibitor L-NAME or eNOS knockout mice.

Conclusion— Activation of an ERK1/2-eNOS-NO pathway is a crucial signaling mechanism by which AGF increases blood flow through induction of angiogenesis and arteriogenesis. Further investigation of the regulation underlying AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.

We sought to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define its molecular mechanisms. Activation of an ERK1/2-eNOS-NO pathway is crucial for AGF-induced angiogenesis and arteriogenesis. AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.

Key Words: angiopoietin-related growth factor • therapeutic angiogenesis • nitric oxide • endothelial nitric oxide synthetase • extracellular signal-regulated kinase 1/2