[Expression and activity of RhoA/Rho kinase in remodeling of high blood flow pulmonary vessels]

Abstract

Objective: High pulmonary blood flow induced pulmonary hypertension (PH) is often associated with increased vasoconstriction and deteriorating pulmonary artery remodeling, of which the exact mechanism has not been completely elucidated. The involvement of RhoA/Rho-kinase pathway has been demonstrated in the pathogenesis of hypoxia and monocrotaline induced PH. Thus the purpose of this study was to test whether RhoA/Rho-kinase pathway is involved in the process of high pulmonary flow induced pulmonary artery remodeling in rats.

Methods: Wistar rats aged 4 weeks in the shunt group underwent left common carotid artery-external jugular vein shunt operation, those in control group received sham-operation. At weeks 1, 2, 4 and 8 of the study, rats underwent right ventricular systolic pressure (RVSP) measurement; blood gases were analyzed to calculate Qp/Qs. The morphologic alterations of the pulmonary arteries were observed under optical microscope. The mean percentage of media wall thickness (%MT) was also measured to assess the extent of medial wall thickness of moderate size pulmonary arteries. Proliferating smooth muscle cells (SMCs) were evaluated by proliferating cell nuclear antigen (PCNA) immunohistochemical staining. Apoptotic SMCs were detected by TUNEL method. RhoA activity in pulmonary arteries was detected using pull down assay. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blotting. The expression of RhoA and Rho kinase (ROCK2) was also detected with Western blotting.

Results: Carotid artery-jugular vein shunt resulted in high pulmonary blood flow, of all rats in shunted groups, the mean Qp/Qs was 2.26 +/- 0.35, which were all considered large shunts. Compared with the control group, RVSP in shunt group increased significantly at both week 1 and week 8 (t = 8.799, t = 5.332, respectively, P < 0.01). Compared with the control group, moderate pulmonary artery medial wall thickening characterized by SMCs hyper-proliferation and hypertrophy in shunted group was firstly appeared at week 4 and became more significant at week 8, as indicated by MT% (t = 9.192, t = 11.185, respectively, P < 0.01). Compared with the control group, the percentage of PCNA-positive SMCs in shunted group increased significantly at week 1 (t = 2.438, P < 0.05), and reached the maximal level at week 2 (t = 7.213, P < 0.01), then, it decreased to a level significantly lower than that of the control group at week 4 (t = 4.183, P < 0.01), and continued to decrease to so low a level that proliferative SMCs was scarcely observed at week 8 (t = 6.152, P < 0.01). The percentage of TUNEL-positive SMCs decreased significantly compared with the control group at week 2 (t = 2.418, P < 0.05), and continued to decrease to a level that apoptotic SMCs was scarcely observed at week 8 (t = 4.582, P < 0.01). Compared with the control group, the expression of RhoA and ROCK2 increased significantly at week 1 (t = 6.056, t = 8.411, respectively, P < 0.01), and reached the maximal level at week 2 (t = 9.342, t = 10.437, respectively, P < 0.01), then began to decrease at week 4, however, both of them were still significantly higher than those of the control group at week 8 (t = 4.743, t = 4.455, respectively, P < 0.01). In line with the expression of RhoA and ROCK2, both RhoA and Rho kinase activity of shunted group increased significantly compared with the control group at week 1 (t = 10.246, t = 19.110, respectively, P < 0.01), and reached the maximal level at week 4 (t = 24.984, t = 16.124, respectively, P < 0.01), then decreased, however, both of them were still higher than those of the control group at week 8 (t = 4.934, t = 10.426, respectively, P < 0.01).

Conclusion: Activated RhoA/Rho-kinase pathway is associated with both high pulmonary blood flow induced acute pulmonary vasoconstriction and chronic pulmonary artery remodeling in rats.