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-Despite the mounting clinical burden of heart failure, the biomolecules that control myocardial tissue remodeling are poorly understood. TIMP-3 is an endogenous inhibitor of matrix metalloproteinases (MMPs) that has been found to be deficient in failing human myocardium. We hypothesized that TIMP-3 expression prevents maladaptive tissue remodeling in the heart, and accordingly, its deficiency in mice would alone be sufficient to trigger progressive cardiac remodeling and dysfunction similar to human heart failure. Methods and Results-Mice with a targeted timp-3 deficiency were evaluated with aging and compared with age-matched wild-type littermates. Loss of timp-3 function triggered spontaneous LV dilatation, cardiomyocyte hypertrophy, and contractile dysfunction at 21 months of age consistent with human dilated cardiomyopathy. Its absence also resulted in interstitial matrix disruption with elevated MMP-9 activity, and activation of the proinflammatory tumor necrosis factor-␣ cytokine system, molecular hallmarks of human myocardial remodeling. Conclusions-TIMP-3 deficiency disrupts matrix homeostasis and the balance of inflammatory mediators, eliciting the transition to cardiac dilation and dysfunction. Therapeutic restoration of myocardial TIMP-3 may provide a novel approach to limit cardiac remodeling and the progression to failure in patients with dilated cardiomyopathy.

In the failing heart, an imbalance in matrix metalloproteinases (MMPs) and their biological regulators, the tissue inhibitors of MMPs (TIMPs), may result in cardiac dilatation from matrix degradation. We hypothesized that a reduction of myocardial TIMP-3 is associated with adverse matrix remodeling in both human and experimental heart failure. Cardiomyopathic hamsters at age 15 wk (normal), 25 wk (compensated stage), and 35 wk (overt failure) were compared with age-matched normal controls. MMP activity (gelatinase bioassay) was increased in cardiomyopathic hearts (P = 0.03) and peaked during the transition to overt heart failure. TIMP-3 content (immunoblot) was decreased compared with normal controls (74 +/- 5% at 25 wk, 69 +/- 10% at 35 wk; P = 0.001) and its reduction was associated with increased MMP activity (r = -0.6; P = 0.004). TIMP-1 increased progressively (P = 0.001), whereas TIMP-2, TIMP-4, and MMP protein levels were unchanged. Myocardial collagen (hydroxyproline content) increased with time during the progression to end-stage cardiac failure (P < 0.0001). Collagen synthesis ([(14)C]proline uptake) was elevated in cardiomyopathy at 15 and 25 wk (P < 0.05). The collagen cross-linking ratio (insoluble:soluble collagen) was reduced (P = 0.003) as the left ventricle dilated. By confocal microscopy restricted to viable myocardium, collagen content was reduced (P = 0.04) with fragmentation (P < 0.0001) and thinning (P = 0.003) of perimysial collagen fibers. Similarly, patients with end-stage congestive heart failure (n = 7) compared with nonfailing controls (n = 2) had elevated gelatinase MMP activity (P = 0.02) associated with isolated reductions in TIMP-3 (55 +/- 5% of normal; P = 0.003). Reductions of TIMP-3 parallel adverse matrix remodeling in the cardiomyopathic hamster and the failing human heart. TIMP-3 may contribute to the regulation of myocardial remodeling and its reduction may promote a transition from compensated to end-stage congestive heart failure.