| Background:Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by pulmonary vascular obstructive lesions and progressively elevated pulmonary vascular resistance. Its main pathological changes are proliferous hypertrophy of tunica media, occlusion and plexus lesions in pulmonary arterioles. Hyperkinetic PAH which is the most common complication of patients with congenital heart disease is mainly caused by the abnormal hemodynamics of systemic-pulmonary shunt, and is a significant contributor to the morbidity and mortality in patients as the disease progresses. At present, the long-term efficacy of drugs for the treatment of PAH is unsatisfactory, but the angiogenesis therapy based on stem cell transplantation has been made new progresses in various research fields. Endothelial progenitor cells (EPCs) exist in peripheral blood and bone marrow and have the ability to differentiate into mature endothelial cells. They are involved in angiogenesis through mobilizing to the ischemia or injury parts of tissues. The long-term efficacy of EPCs transplantation treatment in ischemic disease has been confirmed. Hypoxia-inducible factor (H1F-1) which is a transcription factor widely distributaed in the body and involved in angiogenesis can increase the number of vascular bed to improve ischemia under the condition of hypoxia, and its biological activity mainly depends on HIF-1α subunit. Based on the synergistic effects of EPCs and HIF-1α on ischemia and hypoxia, we hypothesize that transplantation of EPCs combined with high expression of HIF-la through lentivirus mediated transfection can promote angiogenesis, alleviate PAH and reverse pulmonary vascular remodeling. This study could provide the theory basis for the combination of gene and stem cell therapy to congenital heart disease patients with PAH in the future.Objectives: 1. To explore and establish a reliable,.stable and. economic hyperkinetic PAH model and to determine the exact time of successful establishment of a reversible or an irreversible PAH model, which can lay a foundation for the further study of PAH.2. To isolate, purify and identify the bone marrow derived EPCs by density gradient centrifugation, which is the preparation of EPCs transplantation.3. To construct and identify hHIF-1α lentivirus vector, then transfect hHIF-1α to EPCs in vitro and to determine the best MOI through the transfection efficiency and to observe the growth status and hHIF-1α expression of EPCs in hypoxic environment after transfection.4. When hHIF-1α is highly expressed in EPCs, the cells were transplanted into hyperkinetic PAH rabbits, pulmonary vascular hemodynamics and pathologic changes were observed to evaluate the effect of EPCs and hHIF-1α transfected EPCs on PAH and also to confirm its angiogenesis mechanism.Methods:1. We sought to discuss the advantages and disadvantages of common carotid artery-internal jugular vein anastomosis and cuff shunt in1month New Zealand rabbits. After1,2,3,6and12postoperative months, catheterization was used to measure the systolic pulmonary artery pressure (SPAP) and mean pulmonary artery pressure (MPAP). In order to eliminate the influence of shunt to pulmonary artery pressure, we measured the SPAP and MPAP before and after the closure of the shunt respectively. The ratio of the right ventricle to left ventricle plus septum weight was also measured and calculated. The pulmonary vascular pathological changes and Heath-Edwards classifications of each group were evaluated in different postoperative time periods through HE stain to determine the exact time of establishing a reversible or an irreversible PAH model.2. Mononuclear cells were isolated by Ficoll lymphocyte separation medium from the femoral bone marrow of lower limbs. EGM-2medium was used to culture cells which were purified by repeated passage. EPCs were identified by the morphology of cells at different times, cells immunophenotype such as CD34, CD133and VEGFR-2through flow cytometry, and the double phagocytosis of Dil-Ac-LDL and FITC-UEA-1observed by fluorescent staining. Besides, CM-DiL was used to label cells, after that label efficiency and cell growth activity were also observed which provide the basis for subsequent experiments. 3. Primers were designed according to hHIF-la sequence in NCBI database, and hHIF-1α cDNA was used as a template for PCR amplification. hHIF-1α plasmid and blank carrier were recombined and cloned by Nhel and Bam H1enzyme digestion. hHIF-1α lentivirus were packaged according to the ViraPower lentivirus packaging system of Invitrogen corporation, and the virus titer was also tested. After that EPCs which were cultured in hypoxic incubator were transfected with lentivirus according to MOI=0,5,10,20,50,100,200. The best MOI was determined through EPCs growth activity and transfection efficiency, in addition Western blot was used to detect the expression of hHIF-1α.4. Hyperkinetic PAH model was established by common carotid artery-internal jugular vein anastomosis. After detecting the anastomotic patency by ultrasonic cardiogram and pulmonary artery pressure by catheterization, the successful models and sham operation rabbits were randomly divided into6groups, each with10animals:sham group (sham operation group), blank group (without any treatment after establishing PAH model), EPCs group (EPCs were transplanted through left jugular vein to the model rabbits), hHIF-1-EPCs group (hHIF-la transfected EPCs were transplanted through left jugular vein to the model rabbits), control-EPCs group (injection of mock-vehicle transfected EPCs), medium group (injection of EGM-2medium alone). After2weeks of transplantation, the hemodynamic parameters were detected by catheterization, and pulmonary vascular pathological changes were evaluated by lung tissue pathological slices with HE stain. The distribution of EPCs in the lung tissues and expression of hHIF-1α were observed by immunofluorescence of frozen section and Western blot, respectively. We also observed the capillary density in each group by detecting the expression of anti VIII factor through immunohistochemistry, which represented impact of transplantation on the pulmonary vascular density.Results:1. The mean operative time in cuff group was1.40±0.12h, while it took1.48±0.21h in anastomosis group. The mortality rate in the cuff group (27.5%) was higher compared with the anastomosis group (12.5%). Compared with the sham-operation group, the cuff and anastomosis groups showed higher SPAP, MPAP and RV/(LV+S) before closure of the shunt (P<0.05). Although, the PAP decreased after closure in the sham-operation group, the SPAP and MPAP maintained at a higher value in the cuff and anastomosis groups (P<0.05). When we compared the cuff group with the anastomosis group, there was no significant difference. Based on the observed pathological changes and the Heath-Edwards classification system, a reversible and an irreversible pulmonary artery hypertension model was established at3and6months following operation, respectively.2. Mononuclear cells which were isolated from bone marrow began the adherence during the first24h culture, the typical colony was formed after7days culture, and cloned growth of EPCs started from the2week. Fluorescence-activated cell sorter analysis showed that the cultured cells were positive for CD34, CD133and VEGFR-2. Dual-positive adherent cells indicated that the cells expressed the scavenger receptor for ac-LDL and the ligand for UEA-1. And95%cultured cells could be labeled by CM-DiL which had no negative effects on the cells growth activity.3.293TN cells which were transfected with hHIF-la lentivirus were observed green fluorescence, and the virus titer of hHIF-la lentivirus was1.5×107TU/ml, while the blank carrier lentivirus was5×108TU/ml. When MOI=100, the transfection efficiency was the highest, the growth of EPCs was active, and the protein was expressed stably. After96h of transfection, there was expression of hHIF-la in the hHIF-la transfected EPCs group, while no expression in blank carrier group which also indicated that hHIF-la was transfected to EPCs successfully.4. After2weeks of transplantation, compared with sham group, SPAP, MPAP and RV/LV+S were significantly increased in blank group and medium group (P<0.05)(sham group:13.3±1.636mmHg,8.97±1.356mmHg and0.247±0.021. blank group:35.4±2.066mmHg,23.1±1.331mmHg and0.436±0.034. medium group:35.9±2.132mmHg,23.4±1.389mmHg and0.433±0.026). There was no significant difference between blank group and medium group. Compared with blank group, SPAP, MPAP and RV/LV+S were significantly decreased in EPCs group and hHIF-1-EPCs group (P<0.05)(EPCs group:24.5±4.301mmHg,17.3±2.908mmHg and O.353±0.O56. hHIF-1-EPCs group:15.4±1.897mmHg,10.3±1.515mmHg and0.278±0.029). However, SPAP, MPAP and RV/LV+S decreased more obviously in hHIF-1-EPCs group than that in EPCs group (P<0.05). There was no significant difference between EPCs group and control-EPCs group. The frozen slice analysis revealed that the engrafted EPCs in the lung almost located in the surroundings of the capillary vessels and alveolar wall. Western blot showed that hHIF-la specific bands were detected in hHIF-1-EPCs group, and no band was observed in other groups. Compared with sham group, blank group and medium group showed a significant increase in the wall thickness (WT) and wall area (WA) of pulmonary arteries (P<0.05) in HE stain of lung tissue slices. There was no significant difference between blank group and medium group. EPCs group and hHIF-1-EPCs group had a significantly decreased WT and WA of pulmonary arteries compared with the blank group (P<0.05). However, WT and WA decreased more obviously in HIF-1-EPCs group than EPCs group (P<0.05). There was no significant difference between EPCs group and control-EPCs group. An immunohistochemical examination of anti ⅤⅢ factor showed that the capillary density of EPCs group, hHIF-1-EPCs group and control-EPCs group were higher than that in blank group and the differences were significant (P<0.05)(EPCs group:4.50±1.354/mm2. hHIF-1-EPCs group:8.20±1.814/mm2. control-EPCs group:4.30±1.337/mm2. blank group:2.30±1.494/mm2). However, hHIF-1-EPCs group had a higher capillary density than EPCs group and control-EPCs group (P<0.05).Conclusions:1. The common carotid artery and jugular vein anastomosis method is a stable hyperkinetic PAH model in rabbits. Reversible and irreversible pulmonary artery hypertension models were established at different times after operation (e.g.3and6months post-surgery).2. Large amounts of EPCs could be isolated and purified by density gradient centrifugation and could be directionally induced into endothelial cells through EGM-2medium, respectively.3. When MOI=100, hHTF-1α and GFP could be transfected into EPCs successfully and efficiently. Meanwhile EPCs were in active growth and expressed hHIF-1a protein stably.4. Combined hHIF-1a and homogeneous EPCs therapy could attenuate shunt flow-induced PAH and reverse pulmonary vascular remodeling. The main mechanism was angiogenesis in lung tissues, thus increased pulmonary vascular bed density and then reduced resistance of the pulmonary circulation. |
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