Abstract 3571: Evidence for Rho-Kinase Activation in Patients with Pulmonary Hypertension

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Pulmonary hypertension (PH) still remains a fatal disease characterized by hyperconstriction and remodeling of pulmonary arteries (PA). We and others have previously demonstrated that long-term inhibition of Rho-kinase is useful for treatment of PH in animal models, however, it remains to be examined whether Rho-kinase is actually activated in patients with PH. First, we examined whether Rho-kinase activity is enhanced in circulating neutrophils from 40 healthy age- and gender-matched controls and 40 PH patients with various etiologies, including idiopathic PAH (n=18), and PH associated with connective tissue diseases (n=8), congenital heart diseases (n=7), or chronic thromboembolism (n=7). We measured total and phosphorylated forms of myosin binding subunit (MBS), a substrate of Rho-kinase, by Western blotting, and defined the p-MBS/t-MBS ratio as an index of systemic Rho-kinaes activity. Next, we examined Rho-kinase activity by immunostaining in lung tissues from 5 controls and 5 IPAH obtained during lung surgery and transplantation, respectively. Finally, we examined vascular responses of isolated small PA from those subjects in vitro. Systemic Rho-kinase activity was significantly increased in the PAH patients compared with the controls (P<0.0001). Among the 4 subgroups of PH, Rho-kinase activity was significantly increased in all except for PH with thromboembolism (P<0.05). Significant correlations were noted between Rho-kinase activity and mean PA pressure, pulmonary vascular resistance, and duration of the disorder in the PH patients (all P<0.05). Rho-kinase expression in small PA and Rho-kinase activity in the lung tissue also were significantly increased in the PAH patients compared the controls (both P<0.0001). Endothelium-dependent relaxations were markedly impaired and serotonin-induced contractions were markedly enhanced in the PAH patients compared with the controls, and the hypercontractions were abolished in the presence of hydroxyfasudil, a specific Rho-kinase inhibitor (all P<0.01). These results provide the first direct evidence for Rho-kinase activation in patients with PH, confirming the therapeutic importance of Rho-kinase in the treatment of PH in humans.