Li Fu-hai, Xia Wei, Sun Ruo-peng
Department of Pediatrics, the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministry of Education and Ministry of Health of China, Shandong University Qilu Hospital, Jinan 250012, China.
Zhonghua Er Ke Za Zhi. 2007 May;45(5):387-92.
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.
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.
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).
Activated RhoA/Rho-kinase pathway is associated with both high pulmonary blood flow induced acute pulmonary vasoconstriction and chronic pulmonary artery remodeling in rats.
高肺血流量诱导的肺动脉高压（PH）常伴有血管收缩增强和肺动脉重塑恶化，其确切机制尚未完全阐明。RhoA/Rho激酶途径已被证明参与缺氧和野百合碱诱导的PH发病机制。因此，本研究旨在探讨RhoA/Rho激酶途径是否参与大鼠高肺血流量诱导的肺动脉重塑过程。
分流组4周龄Wistar大鼠行左颈总动脉-颈外静脉分流术，对照组行假手术。在研究的第1、2、4和8周，测量大鼠右心室收缩压（RVSP）；分析血气以计算Qp/Qs。在光学显微镜下观察肺动脉的形态学改变。还测量了中膜壁厚度的平均百分比（%MT），以评估中等大小肺动脉中膜壁增厚的程度。通过增殖细胞核抗原（PCNA）免疫组织化学染色评估增殖的平滑肌细胞（SMC）。采用TUNEL法检测凋亡的SMC。使用下拉法检测肺动脉中的RhoA活性。通过蛋白质免疫印迹法以MYPT1磷酸化程度定量Rho激酶活性。还通过蛋白质免疫印迹法检测RhoA和Rho激酶（ROCK2）的表达。
颈总动脉-颈外静脉分流导致高肺血流量，分流组所有大鼠的平均Qp/Qs为2.26±0.35，均被认为是大分流。与对照组相比，分流组在第1周和第8周时RVSP均显著升高（分别为t = 8.799，t = 5.332，P < 0.01）。与对照组相比，分流组以SMC增殖和肥大为主的中等肺动脉中膜增厚在第4周首次出现，并在第8周变得更加明显，MT%表明了这一点（分别为t = 9.192，t = 11.185，P < 0.01）。与对照组相比，分流组PCNA阳性SMC的百分比在第1周显著增加（t = 2.438，P < 0.05），并在第2周达到最高水平（t = 7.213，P < 0.01），然后在第4周降至显著低于对照组的水平（t = 4.183，P < 0.01），并继续下降至第8周增殖SMC几乎未观察到的低水平（t = 6.152，P < 0.01）。与对照组相比，TUNEL阳性SMC的百分比在第2周显著降低（t = 2.418，P < 0.05），并继续下降至第8周凋亡SMC几乎未观察到的水平（t = 4.582，P < 0.01）。与对照组相比，RhoA和ROCK2的表达在第1周显著增加（分别为t = 6.056，t = 8.411，P < 0.01），并在第2周达到最高水平（分别为t = 9.342，t = 10.437，P < 0.01），然后在第4周开始下降，然而，在第8周时两者仍显著高于对照组（分别为t = 4.743，t = 4.455，P < 0.01）。与RhoA和ROCK2的表达一致，分流组的RhoA和Rho激酶活性在第1周与对照组相比均显著增加（分别为t = 10.246，t = 19.110），并在第4周达到最高水平（分别为t = 24.984，t = 16.124，P < 0.01），然后下降，然而，在第8周时两者仍高于对照组（分别为t = 4.934，t = 10.426，P < 0.01）。
激活的RhoA/Rho激酶途径与大鼠高肺血流量诱导的急性肺血管收缩和慢性肺动脉重塑均相关。
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