Background-Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. Methods and Results-Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22Ϯ0.02) compared with vehicle-treated rats (intima/media ratio, 0.92Ϯ0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03Ϯ0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27 kip1 in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. Conclusions-We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension. Key Words: atherosclerosis Ⅲ muscle, smooth Ⅲ remodeling Ⅲ signal transduction Ⅲ hypertension E levated vascular tone contributes to the pathogenesis of hypertension. Rho-associated kinase (ROCK), 1 a target of small GTPase Rho, regulates vascular contractility by increasing the level of phosphorylated myosin light chain and thereby elevating the calcium sensitivity of vascular smooth muscle cells (VSMCs). 2 Recently, Uehata et al 3 developed a potent, specific, ROCK inhibitor, Y-27632. The administration of Y-27632 to several hypertensive rat models markedly reduced systolic blood pressure (SBP), implicating ROCK as a key mediator in the pathogenesis of hypertension. 3 We and others have reported that regulators of vascular tone, such as angiotensin II or natriuretic peptides, are also involved in vascular growth. 4 Thus, we postulated that intracellular mechanism(s) should exist that govern both vascular contraction and growth. Using Y-27632 and dominant-negative ROCK, the present study demonstrates that ROCK, the key regulator of vascular contraction, also controls vascular growth in vitro and in vivo. Methods MaterialsY-27632 was obtained from Yoshitomi Pharmaceutical Industries, Osaka, Japan. The pCAG-myc and pCAG-myc-KD-IA plasmids 1 were a gift from T. Ishizaki and S. Narumiya (Kyoto University). The pEXV-myc-N19RhoA was from M. Symons (the Picower Institute for Medical Research), and...

Background-Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.

Abstract-We recently reported that C-type natriuretic peptide (CNP) occurs in vascular endothelial cells and acts as a vascular-type natriuretic peptide. In the present study, we stimulated the cGMP cascade in proliferating smooth muscle cells (SMCs), in which particulate guanylate cyclase-B, the specific receptor for CNP, is predominantly expressed, by use of an adenovirus encoding rat CNP cDNA (Ad.CNP). In the Ad.CNP-treated cultured SMCs, CNP caused the growth inhibition of SMCs at G 1 phase with an early increase of p21 CIP1/WAF1 expression and subsequent upregulation of p16 INK4a . The expression of smooth muscle myosin heavy chain-2, which is the molecular marker of highly differentiated SMCs, was reinduced in the Ad.CNP-treated SMCs. The Ad.CNP-treated SMCs also reexpressed particulate guanylate cyclase-A, which shows high affinity to atrial and brain natriuretic peptide and is exclusively expressed in well-differentiated SMCs. CNP, which was overexpressed in rabbit femoral arteries in vivo at the time of balloon injury, significantly suppressed neointimal formation. Furthermore, an enhancement of the expression of smooth muscle myosin heavy chain-2 occurred in the residual neointima. In addition, early regeneration of endothelial cells was observed in the Ad.CNP-infected group. The natriuretic peptide family consists of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), which act as vasodilators and growth inhibitors of vascular smooth muscle cells (SMCs). 1,2 These peptides elicit their biological effects via the elevation of intracellular cGMP by activating 2 biologically active natriuretic peptide receptors, namely, membrane-bound guanylate cyclase-A (GC-A) and guanylate cyclase-B (GC-B). We and others have demonstrated that ANP and BNP show high affinity to GC-A, whereas CNP selectively binds to 4 Although ANP and BNP are cardiac hormones secreted mainly from the atrium and the ventricle of the heart, respectively, we have demonstrated that CNP is produced in and secreted from vascular endothelial cells (ECs) 5 to act as a local regulator of vascular tone and growth. 6,7 We have also revealed that the endothelial secretion of CNP is stimulated by various cytokines and growth factors that are produced and activated in proliferative vascular lesions, especially transforming growth factor-␤ and tumor necrosis factor-␣. 5,8 In the pathogenesis of proliferative vascular lesions, alteration of differentiation of SMCs (from the "contractile" to "synthetic" phenotype) is considered to be important. 9 We

Recently we have developed new sustained release system of basic fibroblast growth factor (bFGF) using gelatin hydrogel as a carrier. Using this system, we examined the effect of topical sustained release of bFGF on angiogenesis and tissue blood perfusion in a rabbit model of hind limb ischemia. Thirty-two rabbits underwent excision of right femoral artery under general anesthesia. Two weeks later the rabbits were randomized into four groups (n = 8 each): no treatment, intramuscular injection of gelatin hydrogel alone, and intramuscular injection of gelatin hydrogel incorporating 30 microg and 100 microg of bFGF. Four weeks after each treatment, selective angiography, tissue blood flowmetry using laser Doppler perfusion imaging, and histological examination of thigh muscle were performed. In groups treated with bFGF incorporating gelatin hydrogel, tissue blood flow, number of arterioles, and vascular density were significantly increased in a dose-dependent manner 4 weeks after the treatment. Serum concentrations of bFGF and vascular endothelial growth factor were not elevated 4 weeks after the treatment. In conclusion, sustained release of bFGF using gelatin hydrogel augmented angiogenesis and improved tissue blood flow after excision of the femoral artery.