| Background and purposeHigh perfusion volume and excessive mechanical stress of blood flow on arterial wall in pulmonary circulation system can result in endothelial cell damage, smooth muscle proliferation, pulmonary vascular stenosis, pulmonary vascular remodeling (PVR) and the formation of pulmonary arterial hypertension (PAH) in patient suffered from congenital heart disease (CHD) with left-to-right shunt(LTRS). The treatment and prognosis of CHD with LTRS are largely dependent on the severity of PAH. It is difficult to treat advanced PAH with poor prognosis. PAH secondary to LTRS is still an important topic worthy of study.Recent researches of ischemic cardiomyopathy have indicated that hepatocyte growth factor (HGF) holds a strong effect in facilitating vascular endothelial cell proliferation and vascular endothelial repair. HGF protects vascular endothelium through the promotion of the differentiation and proliferation of vascular endothelial cells. It can also restrain apoptosis of endothelial cell, and inhibit fibrosis and proliferation of smooth muscle cells. Studies have shown that exogenous HGF can promote vascular endothelial cell proliferation in post-ischemic infarct myocardium of rabbits, repair damaged blood vessel intima to prevent the formation of thrombosis, and contribute to the formation of new capillaries. c-Met is by far the only known high affinity HGF receptor. Currently, researches are focused on mechanism of vascular injury and cellular repair. It is still unknown that the expression and impact of HGF/c-Met in lung undergoing PAH secondary to LTRS.In this study, a model of PAH secondary to left-to-right shunt(LTRS)in rat was established. Exogenous HGF was administrated. Pulmonary hemodenamics and pathology were investigated, Expressions of HGF and its receptor, c-Met, as well as Caspase-3 were detected by immunohistochemical and Western blotting in rat with PAH secondary to LTRS. All of them are undertaken to study the impact of HGF/c-Met on PVR in rat with PAH secondary to LTRS. This study will be helpful for exploring the mechanism of PAH secondary to LTRS, and impact of HGF/c-Met on PVR resulting in PAH.MethodsTwo parts were included in this study.Part I: Expression and effect of HGF/c-Met in rat with pulmonary arterial hypertension secondary to left-to-right shuntMale Sprague-Dawley (SD) rats were randomly divided into control group, shunt group and HGF group. Common carotid artery-external jugular vein shunt with conduit connector was performed in rats in shunt group and HGF group to establish the model of PAH, while sham-operated on rats in control group. HGF was given by intraperitoneal injection (2mg/kg/day) since the third day of operation in the HGF group. Ultrasonic examinations was taken at the 4th, 8th, 12th, and 16th week postoperatively to ensure the patency of the shunt, and to detect pulmonary artery pressure and right ventricular hypertrophy index in the groups. The pathological changes of pulmonary medium artery were investigated by routine HE staining, and the percentage of pulmonary artery wall thickness accounted for the artery diameter recorded by computer image analysis. The method also uses immunohistochemistry and Western blotting to identify the expression of HGF and c-Met protein in pulmonary artery was decided by immunohistochemistry and Western blotting to identify the effects of HGF on pulmonary artery pressure, PVR in rats subjected to LTRS.Part II: Impact of HGF/c-Met on apoptosis and proliferation of pulmonary vascular smooth muscle cell in rat with pulmonary arterial hypertension secondary to left-to-right shuntExpression of Caspase-3 protein and PCNA protein in pulmonary medium artery was examinated by immunohistochemistry. Expression of Caspase-3 protein in rat lung was examinated by Western blotting. The apoptosis in pulmonary medium artery checked by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). And the percentage of apoptotic cells (apoptotic index, AI) and proliferation cells (proliferation index, PI) accounted for smooth muscle cells (SMC) in pulmonary medium artery (AI and PI) analyzed by computer image analysis system in the three groups. All of them were used to study the impacts of HGF/c-Met on apoptosis and proliferation of pulmonary medium artery smooth muscle cells and the relationship between PAH and the apoptosis and proliferation of smooth muscle cells in rats with left-to-right shunt.ResultsPart I: Expression and effect of HGF/c-Met in rat with pulmonary arterial hypertension secondary to left-to-right shunt1. All the rats survived. The percentage of shunt patency in shunt group and in HGF group was 100% four weeks after the operation, 95% eight weeks after the operation, 80% twelve weeks after the operation, 75% sixteen weeks after fhe operation. The overall percentage of shunt patency was 87.5%.2. The PASP and mPAP in the shunt group were significantly higher (p<0.01) than those in the control group four weeks after the operation. They droped a little eight weeks after operation and increased gradually and peaked eventually at 12 and 16 weeks after the operation. The PASP and mPAP in the HGF group postoperatively were significantly (p <0.01) higher than those in the control group at all time points, and markedly lower than those in the shunt group (p<0.01) 12 weeks and 16 weeks postoperatively. ASP and mAP showed no differences in three groups (p>0.05).3. RVHI in the shunt group had been risen significantly since eighth week after operation, while the RVHI with no increased in the HGF group, and with no significantly difference when compared with that in the control group.4. Proliferation, increased nucleus, and changed morphology from flat to high columnar were found in endothelial cell (EC) , as well as proliferation and more nucleus showed in smooth muscle cells four weeks after operation in the moderate pulmonary artery in the shunt group MT% increased significantly higher in the shunt group than that in the control group (p <0.01). In the HGF group, significant proliferation, nucleus increased and more high columnar-shape cell body of EC in pulmonary arterial wall were found with no proliferation of SMC, MT% showed no significant difference in the HGF group when compared that in the control group, but significantly lowerthan that in shunt group four weeks after operation (p<0.05), and the difference was more and more significant thereafter (p<0.01). 5. Expressions of HGF and c-Met in EC and SMC in pulmonary artery were found in the three groups. The expression levels of HGF and c-Met protein in the shunt group reached its peak eight weeks after operation and significantly higher than those in the control group (p <0.01) according to the cumulative optical density (IOD). The expression levels in the shunt group were fallen 12 weeks after operation, but still significantly higher than those in the control group (p <0.01) and then returned to the levels in the control group 16 weeks after operation with no difference (p>0.05). The expressions of HGF and c-Met protein in the HGF group were risen considerably at each time points and peaked eight weeks after operation. They then were decreased slightly but still significantly (P <0.01) higher than those in the HGF group.6. In the control group, the expression of of HGF protein (80kd) and c-Met protein (142kd) in lung were showed weak positive bands at each time points. In the shunt group, they were showed enhanced positive bands, which became more obvious eight and 12 weeks after operation. In the HGF group strong positive bands were showed at each time points. The gray-scale semi-quantitative measurement can analyze the levels of HGF/β-actin and c-Met/β-actin by computer image software. HGF/β-actin in the shunt group was significantly increased four weeks after operation , peaked eight weeks after operation and markedly higher (p<0.01) when compared with that in the control group, and then returned to the levels in the control group 12 and 16 weeks after operation. HGF/β-actin in the HGF group was gradually increased and peaked 16 weeks after operation, and showed significant difference when compared with that in the control group and in the shunt group at each time points(p<0.01). C-Met/β-actin in the shunt group was increased considerably four weeks after operation, and peaked at eightth week after operation with significant higher than that in the control group (p <0.01). C-Met/β-actin in the shunt group fallen 12 weeks after operation, but was still significantly higher than that in control group (p <0.01), then returned to the level in the control group 16 weeks after operation (p >0.05). HGF/β-actin and c-Met/β-actin in the HGF group risen markedly at each time points, and were significantly higher than those in the control group and in shunt group (p <0.01).7. Expression levels of HGF and c-Met protein were positively correlated (r = 0.962, p<0.01) when analyzed by Pearson correlation analysis, whereas expression level of HGF and MT% were negative correlated (r = -0.328, p<0.05), and so did c-Met and MT% (r = -0.402, p =0.01). Expression level of HGF and PASP were negative correlated (r = -0.568, p<0.05), and the same to c-Met and PASP(r = -0.607, p=0.01). Expression level of HGF and mPAP were negative correlated (r = -0.593, p=0.01), and so did c-Met and mPAP were negative correlated (r = -0.634, p<0.01). MT% and PASP were positively correlated (r = 0.767, p<0.01), and also MT% and mPAP were positively correlated (r = 0.789, p<0.01).Part II: Impact of HGF/c-Met on apoptosis and proliferation of pulmonary vascular smooth muscle cell in rat with pulmonary arterial hypertension secondary to left-to-right shunt1. Express of caspase-3 protein was found in EC and SMC in pulmonary artery in the three groups by immunohistochemical. IOD semi-quantitative analysis by Image-Pro Plus 5.1 image analysis software showed that expression of caspase-3 protein in the shunt group was significantly higher than that in the control group(p <0.01), and it was peaked at the fourth week after operation, then expression level of caspase-3 protein in the shunt group was decreased, but still significantly higher(p <0.05) than that in the control group eight weeks after operation, but lower than that in the control group 12 and 16 weeks after operation(p <0.01). The express level of caspase-3 protein in the HGF group was lower than that in the control group at all time points(p <0.01), gradually decreased to a minimum level 16 weeks after operation.2. Weak positive bands of caspase-3 protein detected in control group at all time points by Western blotting. In the shunt group, strong positive bands showed four weeks after operation. The positive bands then weakened eight weeks after operation and remained weak positive bands 12 and 16 weeks after operation. In the HGF group, positive bands were showed four after operation and weak positive bands showed at other time points. Semi-quantitative analysis found that the magnitude was significantly higher in the HGF group than in the control group(p<0.01), and it reached its peak at the fourth week after operation. It then gradually fell thereafter and was significantly lower than in the control group 12 and 16 weeks after operation(p<0.01).3. Apoptotic positive cells were found in the middle layer of pulmonary medium artery in the three groups by TUNNL. AI in the shunt group was significantly higher than that in the control group(p<0.01), and it reached its peak four weeks after operation, and then gradually fell but still significantly higher than that in the control group 8 and 12 weeks after operation(p<0.01). AI in the shunt group fell to the level in the control group 16 weeks after operation(p>0.05). The number of apoptotic cells in the HGF group fell considerably, and the AI was significantly lower than that in the shunt group(p<0.01), and lower than that in the control group after eight weeks after operation(p<0.01).4. Express of PCNA protein was found in EC and SMC in pulmonary artery in the three groups by immunohistochemical. Computer image analysis system measures the proliferating cells in the meddle layer of pulmonary medium artery as a percentage of SMC (proliferation index, PI). The proliferating cells of the shunt group increased significantly with its PI was significantly higher than that of the control group and HGF group at each time points(p<0.01). The PI fell 16 weeks after operation, but still significantly higher than that of the control group and the HGF group(p<0.01). The PI of the HGF group, significantly higher than that of the control group four weeks after operation(p<0.01), and fell thereafter. The PI of the HGF group had no significant different compared with the control group 8 and 12 weeks after operation(p>0.05), and was significantly lower than the control group 16 weeks after operation(p<0.01).5. While analyzing the PI/AI of the SMC in pulmonary medium artery of rats, it is found that the PI/AI of the shunt group was significantly higher compared with the control group and the HGF group at each time points(p<0.01), with gradual increase over time. Four weeks after operation, the PI/AI of the HGF group was significantly higher than that of the control group(p<0.01), with a gradual decrease over time. The PI/AI of the HGF group had no significant difference compared with the control group 12 weeks after operation(p>0.05), and was significantly lower than that of the control group 16 weeks after operation(p<0.01).6. Pearson correlation analysis showed that levels of Caspase-3 expression was negatively correlated with MT% (r = -0.890, P <0.01), and so did with PI/AI of the SMC (r =-0.630,p<0.01). The PI/AI of the SMC and MT% were positively correlated(r =0.536,p<0.05).Conclusion1. In this study, the shunt between common carotid artery and external jugular vein with conduit connector can cause pathophysiological changes in rats similar to those in patients suffered from congenital heart disease with left-to-right shunt. These changes include continuous increase of pulmonary artery pressure, thickening of the middle layer of pulmonary artery, and right ventricular hypertrophy. The model of rat with PAH secondary to left-to-right shunt is established in this study.2. Express levels of HGF and c-Met protein in lung of rat with PAH secondary to left-to-right shunt appear to be negative correlation with pulmonary artery pressure and MT%. Exogenous HGF can effectively enhance the expression levels of HGF and c-Met protein in lung of rat with PAH, and decrease pulmonary artery pressure3. Unbalance of proliferation and apoptosis of SMC in middle pulmonary artery could result in thickening of pulmonary arterial wall and then PVR in rat with PAH secondary to left-to-right shunt. Exogenous HGF can balance proliferation and apoptosis of SMC, inhibit excessive proliferation of SMC, and attenuate PVR.
|
PDF Full Text Request