Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo

Abstract

Abstract-Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominantnegative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1␤, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of ␤-galactosidase, into the interleukin-1␤-treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRhoK. Recent studies in vitro have suggested that the small GTP-binding protein Rho and its target protein Rho-kinase/ ROK/ROCK 2-4 play an important role in various cellular functions, including smooth muscle contraction, 5,6 actin cytoskeleton organization, 7,8 cell adhesion and motility, 9 cytokinesis, 10 and gene expression, 11 all of which may be involved in the pathogenesis of arteriosclerosis. We have recently demonstrated that Rho-kinase is functionally upregulated at the inflammatory coronary lesions and plays an important role in the pathogenesis of coronary hyperconstriction in our porcine model with interleukin (IL)-1␤. 12 The IL-1␤-treated coronary segment is characterized by luminal reduction caused by neointimal formation and constrictive remodeling. 13 Thus, our porcine model may be useful in the examination of the molecular mechanism of coronary arteriosclerosis in humans.We have recently found that hydroxyfasudil, an active metabolite of fasudil after oral absorption, is a potent and specific inhibitor of Rho-kinase and markedly inhibits coronary hyperconstriction and macrophage migration. 14,15 Thus, it is conceivable that Rho-kinase is involved not only in the pathogenesis of coronary artery spasm but also in that of coronary arteriosclerosis.To test this hypothesis, we examined in the present study whether selective inhibition of Rho-kinase by the local adenovirus-mediated transfer of dominant-negative Rhokinase (DNRhoK) can effectively induce a regression of coronary arteriosclerotic lesions in our porcine model in vivo. MethodsThis experiment was reviewed by the Committee of the Ethics on Animal Experiments at Kyushu University and was carried out under the control of the Guidelines for Animal Experiments at Kyushu University. Animal PreparationDomestic male pigs (2 to 3 months old, weighing 25 to 30 kg) were sedated with intramuscular ketamine hydrochloride...

Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis

“…We used the extent of ERM phosphorylation as a marker of Rho-kinase activity. [42][43][44] Determination of Akt Binding to eNOS. Akt was immunoprecipitated from liver tissue lysates (800 mg) using 5 mL rabbit polyclonal anti-Akt antibody conjugated with protein G-Sepharose (30 mL; GE Healthcare, Uppsala, Sweden).…”

Rho-Kinase Mediates Hypoxia-Induced Downregulation of Endothelial Nitric Oxide Synthase

“…13 Bovine pulmonary endothelial cells were infected at 80% confluence with the indicated recombinant adenoviruses (Ad-LacZ or Ad-DN-Rho-kinase) at a multiplicity of infection of 200. Approximately 24 hours later, cells were either exposed to normoxia (20% O 2 ) or hypoxia (3% O 2 ) for 24 hours before…”

“…The rabbit polyclonal antibody recognized Thr(558) phosphorylation of ezrin and radixin and Thr(564) phosphorylation of moesin. 13 The specificity of the Rho-kinase antibody has been described. 14 The antibody detection kit (enhanced chemiluminescence) and the nylon and polyvinylidene difluoride transfer membranes were purchased from Amersham Corp.…”

Rho-kinase as a novel gene therapeutic target in treatment of cold ischemia/reperfusion-induced acute lethal liver injury: effect on hepatocellular NADPH oxidase system

“…To evaluate the intracellular enzymatic sources responsible for the I/R-induced production of ROS, the measurement was also performed in the presence of the following drugs: (1) indomethacin (100 mM, cyclooxygenase inhibitor), 44 (2) rotenone (5 mM, mitochondrial complex I inhibitor) 45 and (3) apocynin (100 and 200 mM, NADPH oxidase inhibitor). 23,46 Adenovirus-mediated in vivo gene transfer of dominant-negative Rho-kinase into hepatocyte Adenovirus vectors with a mutant (NK1036-TT) Rhobinding (RB) domain of Rho-kinase with a pleckstrin homology domain [RB/PH (TT)] (AdDNRhoK[RB/ PH(TT)]), 20 which are DNRhoK mutants driven by the cytomegalovirus promoter with a c-myc tag, were prepared through homologous recombination between co-transfected pJM17 and the shuttle plasmid in 293 cells. Integration of the transgene into the adenoviral genome was determined by a polymerase chain reaction and restriction analysis.…”

“…17 We have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases in both animals and humans. [18][19][20][21] Rho-kinase is also known to regulate the mechanism of ROS production. 17,22,23 Indeed, we have also recently demonstrated that Rhokinase is involved in the ROS production in the hyperacute phase of the reperfusion and pro-inflammatory cytokine production in hepatic I/R injury in rats in vivo.…”