| ObjectiveTo further elucidate the mechanism involved in the pathogenesis of pulmonary artery hypertension (PAH) secondary to congenital heart disease, an easier approach was made to create an accurate and reliable experimental rat model of PAH with increased pulmonary blood flow.MethodsLeft common carotid artery-external jugular vein shunts were created with cuffs in 4 weeks old Wistar rats. At week 1, 2, 4, 8 of the study, rats were underwent right ventricular systolic pressure (RVSP) measurements with right cardiac catheterization to indicate pulmonary artery systolic pressure (PASP), blood gases were analyzed to calculate Qp/Qs. Morphologic assessments included the measurements of the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+SP) and the percentage of media wall thickness (%MT) in moderate pulmonary arteries. Proliferating smooth muscle cells (PASMCs) were evaluated by proliferating cell nuclear antigen (PCNA) immunohistological staining. Apoptotic PASMCs were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method.ResultsShunted rats failed to thrive. Of all rats in shunt groups, the total survival rate was 85% (51/60), of which the total patency rate was 86.3% (44/51), the mean Qp/Qs was 2.26±0.35. Compared with the control group, RVSP in shunt groups increased significantly both at week 1 and week 8 (23.7±3.5 vs 12.8±2.6 mmHg, t=-8.799; 27.1±6.7 vs 14.3±3.1 mmHg, t=-5.332; respectively, P< 0.01). At week 4, small to moderate size pulmonary arteries in shunt group began to exhibit obvious medial wall thickening, of which PASMCs presented as marked proliferation and derangement, the luminal diameter significantly decreased, MT% significantly increased compared with the control group (28.2±4.8 vs 12.9±2.9, t=-9.192, P < 0.01). At week 8, above morphologic alteration continued to deteriorate, PASMCs predominantly presented as obvious hypertrophy and derangement, endothelial cells presented as slight proliferation, vascular lumens exhibited obvious asymmetry and stenosis, more than half area of the intravascular lumen in most of the pulmonary arterioles was occluded, some non-muscular pulmonary arterioles were muscularized so serious that their lumens were nearly occluded, MT% increased to 3.2 folds more than that of the control group (52.7±9.3 vs 16.4±3.2, t=-12.617, P < 0.01). In contrast, the alteration of RV/( LV+SP) appeared more later. Compared with the control group, no significant increase of RV/( LV+SP) in shunts group was observed until week 8 of the study (36.3±3.2 vs 31.2±4.1, t=-3.157, p < 0.01). Compared with the control group, the percentage of PCNA-positive PASMCs in shunted group increased significantly at week 1 (9.7±2.5 vs 7.5±2.1, t=-2.438, P < 0.05), and reached the maximal level at week 2 (15.8±2.9 vs 8.4±2.5, t=-7.213, P < 0.01), then, it began to decrease at week 4 (9.8±2.5 vs 7.2±1.9, t=-2.711, P < 0.05), and continued to decrease to a level comparable to that of the control at week 8 (5.7±1.6 vs 7.1±1.6, t=1.925, P > 0.05). The percentage of TUNEL-positive PASMCs decreased significantly compared with the control group at week 2 (0.61±0.21 vs 0.84±0.27, t=2.418, P < 0.05), and continued to decrease to a level of which apoptotic PASMCs was scarcely observed at week 8 (0.21±0.17 vs 0.62±0.22, t=4.582, P < 0.01).ConclusionThe connection of common carotid artery and external jugular could result in increased pulmonary blood flow, elevation of PASP and the development of pulmonary artery remodeling characterized with increased proliferation and decreased apoptosis. This is an easier and reliable approach to create a model of pulmonary hypertension with increased pulmonary blood flow in rats. ObjectiveA large body of evidences have now been obtained regarding the important functions of Rho kinase in vascular physiology, particularly in vascular smooth muscle cells. The involvement of RhoA/Rho kinase pathway has been demonstrated in the pathogenesis of hypoxia and monocrotaline induced PAH. 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.MethodsWistar rats aged 4 weeks old in the shunt groups were underwent left common carotid artery-external jugular vein shunt operation, those in control groups received sham-operation. At week 1, 2, 4, 8 of the study, rats were underwent 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. PASMCs were evaluated by PCNA immunohistological staining. Apoptotic PASMCs were detected by TUNEL method. Expression of RhoA/Rho kinase (ROCK2) mRNA expression was detected by using RT-PCR. RhoA activity in pulmonary arteries was detected using Rho activity Assay Reagent. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blot. The expression of RhoA and ROCK2 were also detected with Western blot. ResultsCompared with the control group, exposure to high blood flow induced significant increase in RVSP at both week 1 and week 8 (23.7±3.5 vs 12.8±2.6 mmHg; 27.1±6.7 vs 14.3±3.1 mmHg; P < 0.01. respectively).At week 4, small to moderate size pulmonary arteries in shunt group began to exhibit obvious medial wall thickening, of which PASMCs presented as marked proliferation and derangement, the luminal diameter significantly decreased, MT% significantly increased compared with the control group (28.2±4.8 vs 12.9±2.9, t=-9.192, P < 0.01). At week 8, above morphologic alteration continued to deteriorate, PASMCs predominantly presented as obvious hypertrophy and derangement, endothelial cells presented as slight proliferation, vascular lumens exhibited obvious asymmetry and stenosis, more than half area of the intravascular lumen in most of the pulmonary arterioles was occluded, some non-muscular pulmonary arterioles were muscularized so serious that their lumens were nearly occluded, MT% increased to 3.2 folds more than that of the control group (52.7±9.3 vs 16.4±3.2, t=-12.617, P < 0.01). In contrast, the alteration of RV/( LV+SP) appeared more later, compared with the control group, no significant increase of RV/( LV+SP) in shunts group was observed until week 8 of the study (36.3±3.2 vs 31.2±4.1, t=-3.157, P < 0.01).Compared with the control group, the percentage of PCNA-positive PASMCs in shunted group increased significantly at week 1 (9.7±2.5 vs 7.5±2.1, t=-2.438, P< 0.05), and reached the maximal level at week 2 (15.8±2.9 vs 8.4±2.5, t=-7.213, P < 0.01), then, it began to decrease at week 4 (9.8±2.5 vs 7.2±1.9, t=-2.711, P < 0.05), and continued to decrease to a level comparable to that of the control at week 8 (5.7±1.6 vs 7.1±1.6, t=1.925, P > 0.05). The percentage of TUNEL-positive PASMCs decreased significantly compared with the control group at week 2 (0.61±0.21 vs 0.84±0.27, t=2.418, P < 0.05), and continued to decrease to a level of which apoptotic PASMCs was scarcely observed at week 8 (0.21±0.17 vs 0.62±0.22, t=4.582, P< 0.01). High flow induced increase of RhoA and ROCK2 mRNA expression were first detected at week 1(1.64±0.27 folds of the control, t=-8.955; 1.83±0.25 folds of the control, t=-12.161; respectively, P < 0.01), reached the maximal level at week 2 (2.69±0.39 folds of the control, t=-16.546; 2.97±0.31 folds of the control, t=-20.876; respectively, P < 0.01), and continued to week 4 (2.43±0.23 folds of the control, t=-19.459; 2.89±0.35, folds of the control, t=-17.100; respectively, P < 0.01), then both of them decreased markedly at week 8, although they were still much higher than those of the control groups(1.58±0.26, folds of the control, t=-6.632, 1.71±0.27 folds of the control, t=-8.050; respectively, P < 0.01).Compared with the control group, the expression of RhoA and ROCK2 protein increased significantly at week 1 (1.51±0.32, folds of the control, t=-6.056; 1.56±0.25, folds of the control, t=-8.411; respectively, P < 0.01), and reached the maximal level at week 2 (2.21±0.49,folds of the control, t=-9.342; 2.37±0.49, folds of the control,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 (1.44±0.28, folds of the control, t=-4.743; 1.43±0.29, folds of the control, 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 (1.94±0.34 folds of the control; t=-l0.246, 2.78±0.35 folds of the control, t=-19.11; respectively, P < 0.01), and reached the maximal level at week 4 (3.19±0.28 folds of the control, t=-24.984; 3.63±0.52 folds of the control, 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 (1.57±0.35 folds of the control,t=-4.934; fold 2.36±0.39 s of the control, t=-10.426; respectively, P < 0.01).ConclusionActivated RhoA/Rho-kinase pathway is associated with both high pulmonary flow induced acute pulmonary vasoconstriction and chronic pulmonary artery remodeling in rats. ObjectiveLiteratures suggested that treatment with Rho-kinase inhibitor improved pulmonary hypertension, right ventricular hypertrophy and pulmonary vascularremodeling, resulting in markedly improved survival of monocrotaline- and hypoxia- induced pulmonary hypertensive rats. Therefore, the aim of this study was to test whether Rho kinase inhibitor-fasudil could attenuate high flow induced PAH in rats.MethodsA total of 168 male Wistar rats aged at 4 weeks old were randomly divided into 4 shunt groups, 4 treated groups and 4 control groups, rats in the shunt groups and treated groups were underwent left common carotid artery-external jugular vein shunt operation, rats in control groups were underwent pseudo-operation. Rats in treated groups received fasudil, the others received same dose of saline. At week 1, 2, 4, 8 of the study, rats were underwent RVSP measurement, blood gases were analyzed to calculate Qp/Qs. Morphologic assessments included the measurements of the weight ratio of RV to (LV+SP) and %MT in moderate size pulmonary arteries. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with western blot. Proliferating cells were evaluated using PCNA immunohistological staining. Apoptotic PASMCs were detected by TUNEL method.ResultsCompared with the control group, exposure to high blood flow induced significant increase in RVSP at both week 1 and week 8 (23.7±3.5 vs 12.8±2.6 mmHg; 27.1±6.7 vsl4.3±3.1 mmHg; P < 0.01, respectively). which were all suppressed by fasudil treatment (13.1±3.2 vs 23.7±3.5; 19.4±3.3 vs 27.1±6.7 mmHg; P < 0.01, respectively), although RVSP in treated group was higher than that of the control group at week 8 (19.4±3.3 vs 14.3±3.1 mmHg, P < 0.05).At week 4, small to moderate size pulmonary arteries in shunt group began to exhibit obvious medial wall thickening, of which PASMCs presented as marked proliferation and derangement, the luminal diameter significantly decreased, MT% significantly increased compared with the control group (28.2±4.8 vs 12.9±2.9, P < 0.01). At week 8. above morphologic alteration continued to deteriorate, PASMCs predominantly presented as obvious hypertrophy and derangement, endothelial cells presented as slight proliferation, vascular lumens exhibited obvious asymmetry and stenosis, more than half area of the intravascular lumen in most of the pulmonary arterioles was occluded, some non-muscular pulmonary arterioles were muscularized so serious that their lumens were nearly occluded, MT% increased to 3.2 folds more than that of the control group (52.7±9.3 vs 16.4±3.2, P < 0.01).Fasudil treatment efficaciously attenuated pulmonary artery medial wall thickning, in which PASMCs only presented as slight proliferation and derangement at week 8, MT% was significantly lower than that of the shunt group both at week 4 and week 8 (14.2±2.7 vs 28.2±4.8; 31.2±3.5 vs 52.7±9.3; P < 0.01, respectively), although pulmonary artery PASMCs showed obvious proliferation and MT% increased significantly compared with the control group at week 8 (31.2±3.5 vs 16.4±3.2, P < 0.01). Long term high blood flow also resulted in right ventricular hypertrophy as indicated by the increase of RV/ (LV+SP). Compared with the control group, RV/( LV+SP) of the shunt group increased significantly at week 8 of the study (0.368±0.038 vs 0.311±0.034, P < 0.01), which was also suppressed by fasudil treatment (0.328±0.035 vs 0.368±0.038, P < 0.05).High blood flow increased the proliferative activity of pulmonary artery PASMCs, which was also significantly suppressed by fasudil therapy. Under the influence of high blood flow, the percentage of PCNA-positive PASMCs in small to moderate size pulmonary arteries of the shunt group increased significantly at week 1 (9.7±2.5 vs 7.5±2.1, P =0.01), and reached the maximal level at week 2 (15.8±2.9 vs8.4±2.5, P < 0.01), compared with the control group. Then, it began to decrease at week 4 (9.8±2.5 vs 7.2±1.9, t=-2.711, P < 0.05), and continued to decrease to a level comparable to that of the control at week 8 (5.7±1.6 vs 7.1±1.6, t=1.925, P > 0.05). In contrast, the percentage of PCNA-positive PASMCs of fasudil treated groups was significant lower than that of the shunt groups at week 1, week 2 and week 4 (7.9±1.5 vs 9.7±2.5, p=0.03; 10.5±2.9 vs 15.8±2.8,p< 0.01; 5.9±1.9 vs 9.8±2.5, P < 0.01; respectively).Under the influence of high pulmonary blood flow, the apoptotic activity of PASMCs decreased, compared with the control, the percentage of TUNEL-positive PASMCs began to decrease at week 2(0.61±0.21 vs 0.84±0.27, P < 0.05), and continued to reach a level that TUNEL-positive PASMCs were scarecely observed at week 8 (0.21±0.17 vs 0.62±0.22, P < 0.01).Treatment with fasudil efficatiousely enhanced the apoptosis of PVPASMCs, the percentage of TUNEL-positive PASMCs weresignificantly higher than that of the shunted group at all time points.Western blot analysis of MYPT1 phophorylation indicated that Rho kinase activation induced by high blood flow was well in line with RhoA activity. After 1 week of exposure to high blood flow, a significant increase of MYPT1 phophorylation in the shunt group was recorded compared with the control group ( 2.78±0.35 fold increase over the control group, P < 0.01). The maximal level of phophorylated MYPT1 was also recorded at week 4 ( 3.63±0.52 fold increase over the control group, P < 0.01). Then, at week 8, the level of phophorylated MYPT1 was down regulated markedly, although it remained higher than that of the control group ( 2.36±0.39 fold increase over the control group, P < 0.01). Fasudil treatment strongly inhibited the activation of Rho kinase induced by high blood flow. The extent of MYPT1 phophorylation in treated group was significantly lower than that of the shunt group at all time points. These results demonstrated that activated Rho kinase participates in the pathogenesis of high flow induced PH; fasudil treatment exerts strong inhibitory effect on Rho kinase activation.ConclusionActivated RhoA/Rho kinase pathway particirates in both the acute pulmonary vasoconstriction and the chronic pulmonary artery remodeling of high flow induced PAH, fasudil treatment could improve PAH by inhibiting Rho kinase activity.
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