Transfection Of Human Hepatocyte Growth Factor Gene Suppresses Advancing Pulmonary Arterial Hypertension Induced By Shunt Flow In A Rabbit Model

Objective:To amplify and identify the recombinant adenovirus (Ad) vector carrying the Hepatocyte growth factor (HGF) and green fluorescent protein (GFP) The desired Ad vectors were purified by density gradient ultracentrifuge and titrated. Its biological safety was tested.Methods:Human embroynic kidney293cells were transfected with Ad-HGF and Ad-GFP. Virus clones were isolated and propagated for restriction analysis. After amplified in293cells, the obtained adenovirus DNA was detected by PCR, Meanwhile, recombinant adenovirus was further identified by RT-PCR in293cells. The virus infection titration was titrated with CPE and plaque assay. The desired Ad vectors were purified by density gradient ultracentrifuge, titrated and the safety of using was detected. The virus replication ability was tested by the cytomathy of sensitive cell.Results:Recombinant adenoviral HGF was successfully propagated, which the expressing of HGF was confirmed by PCR and RT-PCR. The Ad-HGF were amplified in293cells for4times.The viral particle tite of Ad-HGF was1.1×109pfu/ml, and viral particle tite of Ad-HGF was2.0×1012pfu/ml after purified by density gradient ultracentrifuge. A549cells were infected with Ad-HGF for7days and did not show any pathologic reaction. This would verify the safety of Ad-HGF.Conclusion:Recombinant adenoviral HGF was propagated and identified successfully. The higher viral particle tite of Ad-HGF were got with propagated in293cells, which made it possible that we could further our research on gene therapy of pulmonary arterial hypertension. Objective:To explore the transfection efficiency of adenovirus vector to HUVECs, the expression of HGF after the transfection of Ad-HGF to HUVECs, and the effects of proliferation and differentiation on HUVECs after gene transfection in vitro.Methods:The best transfection efficiency of adenovirus vector to HUVECs was identified by various infection titrations of Ad-GFP. Then we transfected Ad-HGF to HUVECs with MOI50:1. After Ad-HGF transfection, HGF mRNA transcription was detected by RT-PCR. HGF protein expression in HUVECs was measured by immunohistochemical staining. The level of NO in the culture medium was also measured. The proliferation effect of Ad-HGF on HUVECs was measured by MTT assay. Finally, we also examined the differentiation effect of Ad-HGF on HUVECs in Matrigel Matrix.Results:The transfection efficiency of Ad-GFP to HUVECs increased with the MOI increasing. When MOI reached50, the transfection rate was more than90%. From then on, the transfection efficiency was not increasing any more. So we chosed50:1as the most suitable MOI. RT-PCR and immunohistochemical staining showed that HGF gene could be efficiently transfected into HUVECs and HGF protein could be expressed in HUVECs. The secretion of HGF was also detected in the culture medium. After the transfection of HGF, the level of NO in the culture medium was also higher than that in the control group. HUVECs transfected with Ad-HGF were revealed a significant increase of cell number which showed by the MTT assay. Furthermore, when seeded on Matrigel Matrix, HUVECs transfected with Ad-HGF were induced into capillary-like structures.Conclusion:The most suitable MOI in the research is50:1. HGF gene could be successfully transfected into HUVECs and the HGF protein was successfully produced and persistently secreted in the transfected HUVECs. Ad-HGF not only stimulated the proliferation of HUVECs but also induced the differentiation of HUVECs into the capillary-like structures in vitro. Objective:To investigate the possibility of establishing a model of hyperkinetic pulmonary hypertension by chronic Left internal jugular vein-common carotid artery shunt by end-to-side anastomosis in immature rabbit.Methods:Rabbits (n=60) were randomly separated into shunt group (n=30), sham group (n=15) and normal group (n=15). Left internal jugular vein-common carotid artery shunt by end-to-side anastomosis was performed in rabbit of shunt group, which was anticoagulated by low molecular heparin and aspirin. Four rabbits in each group were killed on the4th and8th weeks after operation, and the residual rabbits were sacrificed on the12th postoperative week. The shunt vessel and thickness of right ventricular anterior wall (RVAW) and left ventricular posterior wall (LVPW) were detected by echocardiogram. Invasive examination included the right heart catheter and thoracotomy for the measurement of pulmonary arterial pressure. Pathomorphology analysis of pulmonary arterioles included vascular wall thickness index (TI), relative vascular wall area index (AI) and the percentage of the muscularized vessels. The weight rate of right ventricle to the left ventricle plus interventricular septum was calculated to assess the hypertrophy of right ventricle.Results:No rabbit died during the operation. The surgery mortality rate was0%. Four rabbits died of other accidents after operation.The postoperative mortality rate was13.3%(4/30).In the26residual living rabbits,6animals were excluded from the study because the occlusion of the shunt was detected by echocardiogram. The patency rate was76.9%(20/26).At the12th postoperative week, the thickness rate of RVAW to LVPW in the shunt animals increased (45.02±8.36) compared with that in the normal control animals (26.08±2.54) and sham group(26.99±2.63) or that in the shunt4th week (25.46±2.41) and the shunt8th week (30.24±2.27) animals, pulmonaryarterial pressure was higher in the shunt rabbits(PASP40.42±3.48mmHg、 PADP31.34±2.72mmHg、mPAP35.33±2.67mmHg)than that in the sham animals(PASP19.32±2.45mmHg、PADP10.35±2.19mmHg、mPAP14.78±2.23mmHg),the normal control animals(PASP18.59±4.51mmHg、PADP11.32±2.41mmHg, mPAP13.62±1.96mmHg), in the shunt4th week (PASP17.94±3.27mmHg, PADP11.31±2.38mmHg、mPAP13.54±3.42mmHg),in the8th week (PASP27.63±3.90mmHg、PADP12.31±3.51mmHg、mPAP17.59±5.04mmHg) which was confirmed by the right heart catheter and the thoracotomy for the pressure measurement. At the12th postoperative week, the thickness rate of RV/LV+S in the shunt animals increased (0.49±0.02)compared with that in the normal control animals (0.27±0.01),sham rabbits (0.25±0.02), in the shunt4th week (0.31±0.04),in the shunt8th week (0.35±0.04)Pathological examination for the pulmonary arterioles showed that the TI、AI and the percentage of the muscularized vessels were all increased in the shunt animals (42.35±5.11%,73.72±6.19%and56.59±7.21%, respectively) than that in the normal rabbits (16.71±2.99%、29.05±3.79%and22.09±3.77%, respectively), the sham animals(18.12±2.68%,31.92±3.01%and24.26±3.13%, respectively),the shunt4th week (17.01±3.08%、30.61±2.44%and25.12±2.99%, respectively),in the shunt8th week (20.09±3.57%、35.24±4.01%and30.54±3.71%, respectively)(P<0.05)Conclusion:A hyperkinetic pulmonary arterial hypertension model could be established by left internal jugular vein-common carotid artery shunt by end-to-side anastomosis in rabbit after12weeks if the shunt was patent. The pathological alteration conforms to the hyperkinetic pulmonary hypertension. Objective:To transfect exogenous HGF gene with adenovirus through the internal jugular vein injection into the rabbit models of advancing hyperkinetic pulmonary hypertension; To explore the feasibility of suppression pulmonary artery pressure by therapeutic angiogenesis and amelioration of endothelial cell injury induced by HGF gene.Material and Methods:Rabbit models of hyperkinetic pulmonary hypertension was established by the left internal jugular vein-common arotid artery shunt with end-to-side anastomosis. The models animals were randomly separated into four groups:the PAH group, HGF gene group (six weeks after operating,2×109Ad-HGF in0.5mL DMEM was injected per rabbit through right internal jugular vein), Empty group (six weeks after operating,2×109adenovirus in0.5mL DMEM was injected per rabbit through right internal jugular vein) as blank control and the sham group were taken as negative control. Two weeks after transfection, Immunohistochemistry, ELISA, Real time PCR and Western blot analysis were done to demonstrate the expressions of mRNA and protein of HGF, eNOS and ET-1. Six weeks after transfection, echocardiogram, and right heart catheter were performed, by which the thickness rate of right ventricular anterior wall to left ventricular posterior wall (RVAW/LVPW) and pressure ratio of right ventricular to left ventricular were assessed. Immunohistochemistry of VIII factor was also performed to compare the vascular number in the lung tissue, meanwhile, right ventricular hypertrophy RV/(LV+S) was evaluated. Results:Rabbit models of hyperkinetic pulmonary hypertension were established by left internal jugular vein to common carotid artery shunt with end-to-side anastomosis after12weeks. Two weeks after HGF transfection, the HGF transfection augments the HGF expression in lung which was confirmed by Immunohistochemistry, RT-PCR, ELISA and Western blot analysis. In addition, the expression of eNOS in lung was also increased after transfection, in accordance with the increasing of the HGF. But the expression of ET-1was decreased following the increasing of the HGF and eNOS. Six weeks after HGF transfection, the thickness rate of right ventricular anterior wall to left ventricular posterior wall (RVAW/LVPW) was also significantly lower compared with the PAH and Empty groups. Mean pulmonary arterial pressure in the transfection group (14.62±3.23mmHg) was decreased than that in the PAH group (36.35±4.63mmHg) and Empty group (34.02±4.93mmHg), P<0.05). RV/(LV+S), as an index for right ventricular hypertrophy was (0.28±0.09) lower than those of the PAH group (0.48±0.06) and Empty group (0.49±0.11). Finally, VIII factor immunohistochemistry stain showed that the vessels in certain area of the lung tissues were much more than that in the PAH and Empty group (P<0.05).Conclusion:HGF transfection suppressed pulmonary hypertension induced by shunt flow, reversed the deteriorative hemodynamics caused by PAH, and inhibited the pathological process of the pulmonary arterial. All the results mentioned above might due to the increased expression of HGF, which improved the angiogenesis and ameliorated injury endothelial cells.