Role of endothelial nitric oxide synthase and collagen metabolism in right ventricular remodeling due to pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) causes elevated right ventricular (RV) systolic pressure, RV remodeling and finally RV failure to death. However, the mechanisms of RV remodeling in PH remain to be fully elucidated. METHODS AND RESULTS: RV autopsy samples from 6 PH patients with RV failure against 3 age- and sex-matched controls were first examined. Next, RV remodeling in 2 mouse models of chronic hypoxia-induced PH with endothelial nitric oxide synthase-deficient (eNOS(-/-)) and collagenase-resistant knock-in (Col(R/R)) mice were examined. In humans, RV failure was associated with RV hypertrophy, interstitial and perivascular fibrosis, decreased RV capillary density and increased macrophage recruitment. Furthermore, immunostaining showed that perivascular matrix metalloproteinase-2 was increased in PH patients with RV failure. In animals, both hypoxic eNOS(-/-) and Col(R/R) mice developed a greater extent of RV hypertrophy, perivascular remodeling and macrophage infiltration compared with wild-type mice. Capillary rarefaction was developed in hypoxic eNOS(-/-) mice, while Col(R/R) mice were able to increase their capillary density in the RV in response to chronic hypoxia. Both mouse models showed increased autophagy even under normoxic condition. CONCLUSIONS: These results indicate that RV remodeling occurs early during PH development through fibrosis, perivascular remodeling, capillary rarefaction and autophagy, in which the eNOS pathway and collagen metabolism might be involved.

Combination therapy with atorvastatin and amlodipine suppresses angiotensin II-induced aortic aneurysm formation.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. It is controversial whether statin and calcium channel blockers (CCBs) has an inhibitory effect on the expansion of AAA. Some studies reported that CCBs have an inhibitory effect on Rho-kinase activity. Rho-kinase plays an important role in the pathogenesis of various cardiovascular diseases. However, there is no study reporting of the association between Rho-kinase and human AAAs. METHODS AND RESULTS: Experimental AAA was induced in Apolipoprotein E-deficient (ApoE(-/-)) mice infused with angiotensin II (AngII) for 28 days. They were randomly divided into the following 5 groups; saline infusion alone (sham), AngII infusion alone, AngII infusion plus atorvastatin (10 mg/kg/day), AngII infusion plus amlodipine (1 mg/kg/day), and AngII infusion plus combination therapy with atorvastatin (10 mg/kg/day) and amlodipine (1 mg/kg/day). The combination therapy significantly suppressed AngII-induced increase in maximal aortic diameter as compared with sham, whereas each monotherapy had no inhibitory effects. The combination therapy significantly reduced AngII-induced apoptosis and elastin degradation at the AAA lesion, whereas each monotherapy did not. Moreover, Rho-kinase activity, as evaluated by the extent of phosphorylation of myosin-binding subunit (a substrate of Rho-kinase) and matrix metalloproteinase activity were significantly increased in the AngII-induced AAA lesion as compared with sham, both of which were again significantly suppressed by the combination therapy. In human aortic samples, immunohistochemistory revealed that the activity and expression of Rho-kinase was up-regulated in AAA lesion as compared with abdominal aorta from control subjects. CONCLUSIONS: Rho-kinase is up-regulated in the aortic wall of human AAA. The combination therapy with amlodipine and Atorvastatin, but not each monotherapy, suppresses AngII-induced AAA formation in mice in vivo, for which Rho-kinase inhibition may be involved.

Rho-kinase activation in patients with heart failure.

BACKGROUND: Heart failure (HF) is a complex clinical syndrome, resulting from structural and/or functional cardiac disease. The aim of this study was to determine whether the activity of Rho-kinase, which has been identified as an important therapeutic target of cardiovascular disease, is enhanced in HF patients. METHODS AND RESULTS: Total and phosphorylated forms of myosin binding subunit (t-MBS and p-MBS), a substrate of Rho-kinase, were measured on western blotting in circulating leukocytes, and the p-MBS/t-MBS ratio was defined as an index of systemic Rho-kinase activity. First, during the time-course of acute HF (n=12), Rho-kinase activity was significantly elevated in the acute phase compared to the chronic phase (1.19 ± 0.06 vs. 0.97 ± 0.04, P<0.05). Next, Rho-kinase activity was examined in 30 controls and 130 chronic HF patients (cardiomyopathy, n=57; valvular heart disease, n=35; ischemic heart disease [IHD], n=33; and others, n=5). As compared with the controls, Rho-kinase activity was significantly elevated in the total HF group (1.14 ± 0.02 vs. 0.77 ± 0.05, P<0.0001) and in each underlying heart disease (P<0.05 each). Importantly, in the high-risk non-IHD group, Rho-kinase activity was significantly associated with plasma brain nutriuretic peptide level. Finally, p-MBS was expressed in myocardial biopsy samples (immunohistochemistry) in chronic HF patients (n=36), independent of Rho-kinase activity in leukocytes. CONCLUSIONS: Rho-kinase is activated in HF patients, suggesting that it could be a new therapeutic target of the disorder.

Intensive immunosuppressive therapy improves pulmonary hemodynamics and long-term prognosis in patients with pulmonary arterial hypertension associated with connective tissue disease.

BACKGROUND: Pulmonary arterial hypertension (PAH) remains a serious disease characterized by elevated pulmonary artery pressure (PAP) and increased pulmonary vascular resistance (PVR). Among its subtypes, PAH associated with connective tissue disease (CPAH) has the worse prognosis, because of resistance to conventional vasodilator therapy. We hypothesized that intensive immunosuppressive therapy (IIT) could improve the pulmonary hemodynamics in CPAH. METHODS AND RESULTS: In our pulmonary hypertension (PH) cohort of 182 patients, we evaluated 13 consecutive patients with CPAH who received IIT combined with cyclophosphamide and glucocorticosteroids (IIT group, mean age 45 ± 8 years, 12 females and 1 male). We compared them with 8 historical controls (control group: mean age 52 ± 18 years, 8 females) for pulmonary hemodynamics and prognosis. Both groups were treated with conventional vasodilator therapy. Although the mean PAP (mPAP) remained unchanged in the control group, IIT significantly decreased mPAP (40 ± 9 to 29 ± 11 mmHg, P < 0.01) and tended to decrease PVR (700 ± 434 to 481 ± 418 dyne·s·cm⁻5, P=0.07). Importantly, in 6 of the 13 patients in the IIT group, mPAP was almost normalized (< 25 mmHg) and remained stabilized for more than 1 year. Furthermore, the IIT group showed significantly better prognosis compared with the control group (P<0.01). CONCLUSIONS: These results suggest that IIT as well as conventional vasodilator therapy improves the pulmonary hemodynamics and long-term prognosis of patients with CPAH.

Evidence for Rho-kinase activation in patients with pulmonary arterial hypertension.

BACKGROUND: Direct evidence for Rho-kinase activation in patients with pulmonary hypertension (PH) is still lacking. METHODS AND RESULTS: Rho-kinase activity in circulating neutrophils was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit, a substrate of Rho-kinase, in 40 consecutive PH patients and 40 healthy controls. Next, Rho-kinase expression and activity was examined in isolated human lung tissues (5 patients with idiopathic pulmonary arterial hypertension [IPAH], 5 controls) and vascular reactivity of isolated small human pulmonary arteries in vitro (4 IPAH, 4 controls). Rho-kinase activity in circulating neutrophils was significantly increased in the PH patients overall compared with controls (P<0.0001). Significant correlations were noted between Rho-kinase activity and the severity and duration of PAH (all P<0.05). Rho-kinase expression and activity in isolated lung tissues also were significantly increased in the IPAH patients compared with the controls (both P<0.0001). Endothelium-dependent relaxation was markedly impaired and serotonin-induced contraction (in the absence of the endothelium) markedly enhanced in the PAH patients compared with the controls, and the hypercontraction to serotonin was abolished by hydroxyfasudil, a specific Rho-kinase inhibitor. CONCLUSIONS: These results provide the first direct evidence for Rho-kinase activation in patients with PAH, suggesting the therapeutic importance of Rho-kinase in the disorder.