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(Circulation. 2000;102:III-370.)
© 2000 American Heart Association, Inc.

Myocardial Protection and Vascular Biology

Hypercholesterolemia Inhibits Angiogenesis in Response to Hindlimb Ischemia

Nitric Oxide–Dependent Mechanism

Junli Duan, MD; Toyoaki Murohara, MD; Hisao Ikeda, MD; Atsushi Katoh, MD; Satoshi Shintani, MD; Ken-ichiro Sasaki, MD; Hidemichi Kawata, BS; Naoki Yamamoto, BS; Tsutomu Imaizumi, MD

From The Cardiovascular Research Institute and Departments of Internal Medicine III and Radiology, Kurume University School of Medicine, Kurume, Japan.

Correspondence to Toyoaki Murohara, MD, PhD, The Cardiovascular Research Institute, Kurume University School of Medicine, 67 Asahi-machi, Kurume, 830-0011 Japan. E-mail toyom{at}med.kurume-u.ac.jp

Background—Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis.

Methods and Results—Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P<0.01) and capillary densities (P<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO_x and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO_x and cGMP contents.

Conclusions—Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

Key Words: peripheral vascular disease • hypercholesterolemia • angiogenesis • nitric oxide • endothelium