Role of Rho-Associated Kinase in Neointima Formation After Vascular Injury

Abstract

Background —The Rho/Rho-associated kinase (Rho-kinase) system is implicated in various cellular functions, including migration, proliferation, and apoptosis. Because a possible role of the system is suggested in neointima formation after vascular injury, we sought to examine whether a new specific Rho-kinase inhibitor, Y27632, prevents neointima formation of the balloon-injured rat carotid artery, and if so, to investigate the effects of Y27632 on migration, proliferation, and apoptosis of smooth muscle cells (SMCs) in the injured artery. Methods and Results —Y27632 was administered intraperitoneally from 1 day before to 14 days after vascular injury. Treatment with Y27632 inhibited phenylephrine-induced Rho-kinase activation in the carotid artery on the basis of immunoblotting against the phosphorylated myosin-binding subunit of myosin phosphatase. Y27632 markedly prevented neointima formation at days 7 and 14. In controls, BrdU + proliferating and TUNEL + apoptotic SMCs were transiently and coincidentally increased in the neointima, with a peak at day 7. Y27632 significantly increased the neointimal TUNEL + SMCs at days 7 and 14, but not BrdU + SMCs. Y27642 significantly decreased the number of intimal SMCs at day 4, while not affecting the number of BrdU + or TUNEL + SMCs. Reendothelialization after balloon injury was not significantly affected by Y27632 at days 7 and 14. Conclusions —Y27632 inhibited neointima formation by enhancing SMC apoptosis and probably by suppressing early SMC migration. Therefore, a role of Rho-kinase is suggested in neointima formation after vascular injury.

Dominant negative c-Jun gene transfer inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in rats

“…15 There was no significant difference between vehicle-transfected and DN-c-Jun-transfected groups in TUNEL index of intima (35 ± 4% versus 32 ± 4%, respectively, n = 6), at 7 days after balloon injury. TUNEL index of media was negligible in either the vehicle-transfected or the DN-c-Jun-transfected group.…”

“…15 The counterstain was done with 0.5% methyl green. A negative control was incubated without the TdT.…”

Rho kinase in the regulation of cell death and survival

“…Moreover, inhibition of ROCK with fasudil significantly enhances smooth muscle cell apoptosis in autologous vein grafts in rabbits [40] and in pulmonary hypertension in rats [1], further supporting a pro-survival role of ROCK in smooth muscle cells. Increased smooth muscle cell apoptosis along with inhibition of smooth muscle cell proliferation, inflammatory cell infiltration and/or extracellular matrix protein production contribute to the inhibition of neointima formation via ROCK inhibition [40,84,120]. Therefore, these studies suggest that ROCK plays a pro-survival role in neointimal smooth muscle cells.…”

“…Y27632 treatment significantly increases the neointimal TUNEL-positive smooth muscle cells at 7 and 14 day after vascular injury [120], and this increased apoptosis is associated with significantly up-regulation of the proapoptotic Bax, suggesting that ROCK inhibition induces neointimal smooth muscle cell apoptosis through Bax up-regulation [121]. Consistent with this finding, the inhibition of ROCK with fasudil enhances smooth muscle cell apoptosis in the neointima after stent implantation in the porcine coronary artery, and this increased apoptosis is associated with down-regulation of the anti-apoptotic protein Bcl-2 [84].…”

“…A pro-survival role of ROCK in vascular smooth muscle cells was first observed in neointima formation after balloon injury in the rat carotid artery [120]. Y27632 treatment significantly increases the neointimal TUNEL-positive smooth muscle cells at 7 and 14 day after vascular injury [120], and this increased apoptosis is associated with significantly up-regulation of the proapoptotic Bax, suggesting that ROCK inhibition induces neointimal smooth muscle cell apoptosis through Bax up-regulation [121].…”