Experimental Studies Of Transplantation Of Endothelial Progenitor Cells Transfected With AD.bFGF₂ In Ischemic Myocardium

Background:Ischemic heart disease (IHD) is the primary cause of death in the industrialized world. Traditionally, patients with IHD were treated with medicine, percutaneous transluminal ballon angioplasty, or coronary artery bypass graft. Despite the fact that advances in modern medical, surgical and interventional treatment of the disease have produced significantly improved results, the efficacy of all these methods is limited in patients with diffuse distal coronary artery disease. According to reports, complete revascularization was found in only about 37 percent of patients who had coronary artery bypass graft, however, once the myocardium was revascularized completely, the incidents of angina would be decreased and the incidence of 5 year survive was improved significantly. Therefore, additional therapeutic measures were needed in those patients. Recent advances in vascular biology suggest the possibility of a novel therapeutic approach to treatment of advanced coronary artery disease. The definition of gene therapy in ischemic heart desease is to transfect special angiogenic growth factor genes and enhances the expressing of growth factors into myocardium. This approach seeks to augment normal collateral development by exposing the heart to growth factors capable of stimulating the growth of new blood vessels.During myocardial ischemia, compensatory angiogenesis occurred to adapt to ischemic and anoxaemia. In this procedure, growth factors were essential. Fibroblast growth factor (FGF) is the major regulators of vascular growth. It stimulates proliferation of new vessels, improve the microenvironment of ischemic tissue and benefit for tissular reparation or anagenesis. Fibroblast growth factor was divided into two kinds (aFGF and bFGF). They have the same receptors and biological effect .The effect of bFGF is 10 to 30 times power than aFGF in regulators of vascular proliferation. Experimental and clinical studies have shown that FGF may stimulate vessel growth to the ischemic region. Recently, using FGF treatment in patients with severe ischemic heart disease has been implemented as a medical therapy for coronary artery disease. However, under some circumstances, such as in old and feeble, diabetes or hypercholesteremia patients, it exists not only synthesis decreasing of FGF , but also functional impairment of endothelial cells (ECs) that makes its reaction to FGF decrease. Under this circumstance, only using FGF doesn't improve blood supply of ischemic tissue efficiently, so it was considered that ECs should be provided under using FGF by many oversea scholars.It was found in recently research that there were endothelial progenitor cells (EPCs), the precursor cell of ECs which existed in bone marrow, peripheral blood and cord blood. EPCs may be distributed to ischemic position specially, differentiate to mature ECs and take part in angiogenesis. This approach is called "supply side" strategy that collateral development is augmented by supplying active EPCs. The method of alleviating myocardial remodeling by implanting cells which are transfected with special angiogenic growth factor genes to enhance the angiogenesis may present a new approach to the treatment of ischemic heart disease. However, whether transfecting with special angiogenic growth factor genes reinforce the function of implanting cells or not isn't reported in previous documents.In our study, Firstly, recombinant adenovirus (Ad) vector carrying the basic fibroblast growth factor 2 (bFGF₂) was amplifed, identified, and rat bone marrow endothelial progenitor cells were induced. Then EPCs with Ad. bFGF₂ was transfected in vitro and the efficiency of transfection and protein expression were tested. The proliferation and differentiation effect of Ad. bFGF₂ on EPCs were certified. Finally, the EPCs transfected by Ad. bFGF₂ were transplanted into rat ischemic myocardium and the survival of the transplanted cells was found and the ability to increase the growth of neovascularization in ischemic zone which enhance local blood perfusion and subsequently improve myocardial function was identified. Part 1. Propagation, identification of recombinant adenovirus carrying thebasic fibroblast growth factor 2 gene.In this study, human embroynic kidney 293 cells were transfected with Ad.bFGF₂ and Ad-GFP. Virus clones were isolated and propagated for restriction analysis. After amplified in 293 cells, the obtained adenovirus DNA was detected by PCR. Meanwhile, recombinant adenovirus was further identified by RT-PCR in COS-7 cells. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated, and the safety of using was tested.Results: Recombinant adenoviral bFGF₂ was propagated successfully, in which the expressing of bFGF₂ was confirmed by PCR and RT-PCR. The Ad. bFGF₂ were amplified in 293 cells for 4 times. The viral particle tite of Ad. bFGF₂ were determined with the TCID50. The viral particle tite of Ad. bFGF₂ was 1.1×10⁹pfu/ml, and viral particle tite of Ad. bFGF₂ was 2.0×10¹²pfu/ml after purified by density gradient ultracentrifuge. Hela cells which were infected with Ad. bFGF₂ for 7 days didn't show up pathosis reaction, so the safety of using was proved.Part 2. Isolation, Culture and identification of endothelial progenitor cells.The number of EPCs is estimated to be too few in bone marrow, so it is important to expand and purify the EPCs for the following experiments. Bone marrow cells suspension of adult rat was prepared by discontinuous gradient centrifugation on Ficoll, mononeuclear cells in the middle layer was collected, cultured for 24 hours, then; suspending cells were put into selective culture medium and cultured in the 10% fetal calf serum coated plate. EPCs were isolated, cultured and expanded. The cells were identified by transmission electron microscope, flow cytometer, immunohistochemical staining with anti-factorâ…§, CD31, CD34, VRGFR-2 antigen, the ability of secreting nitrous oxide and combining BS1-Lectin.Results: Colonies of EPCs formed at 7th day at primary culture. At about 14th day, EPCs got together about 80～90%, and serial subcultivation was made. The passage EPCs proliferated fast, and could be subcultured about every 10 days. EPCs were identified by flow cytometer and immunohistochemical staining (anti-factorâ…§, CD31, CD34, VRGFR-2 antigen) with high purity. Under transmission electron microscope, the cells displayed Weibel-Palade bodies which are characteristic of endothelial cells. The cells could also combine BS-1 lectin and secrete nitrous oxide. So, the cells induced and cultured in vitro have the same characters as endothelial cells.Part 3. Transfection of Ad.bFGF₂ to EPCs and its effects of proliferationand differentiation on EPCs in vitro.To determine the best MOI, transfection efficiency of adenovirus vector to EPCs were identified by infection with various titrations of Ad-GFP. Ad. bFGF₂ was transfected to EPCs with MOI 50:1. After Ad. bFGF₂ transfection, bFGF₂ mRNA transcription was detected by RT-PCR. bFGF₂ protein expression in EPCs and its secretion in culture medium were measured by immunohistochemical staining, Western-blot respectively. The level of NO in culture medium was also measured. The ability of proliferation of Ad. bFGF₂ on EPCs were measured by MTT assay. Finally, the differentiatial ability of Ad. bFGF₂ on EPCs was examinedResults: The transfection efficiency of Ad-GFP to EPCs was increased with MOI. When MOI reached 50, the efficiency was increased to more than 90%. From then on, the transfection efficiency increased limitedly. So 50:1 was choosed as the most suitable MOI. The RT-PCR, immunohistochemical staining and Western-blot showed that bFGF₂ gene could be correctly transfected into EPCs and bFGF₂ protein could express in EPCs. Its secretion in culture medium was detected. After the transfection, the level of NO in the culture medium was also higher than control group. EPCs transfected with Ad. bFGF₂ were revealed a significant increase of cell number which showed by MTT assay. Furthermore, when seeded on Matrigel Matrix, EPCs transfected by Ad. bFGF₂ were induced into capillary-like structures.Part 4.Studies of transplantation of endothelial progenitor cells transfected by bFGF₂ gene into ischemic myocardium.Transplantation of EPCs transfected by Ad. bFGF₂ was carried out in vivo following the above studies. Sixty Wistar rats in which acute myocardial infarction was generated by ligating the left anterior descending coronary artery were divided into four groups (Groupâ… , transplanted with EPCs transfected by Ad. bFGF₂; Groupâ…¡, transplanted with EPCs alone; Groupâ…¢, injected with Ad. bFGF₂ alone; Groupâ…£, injected with PBS). EPCs were labeled with PKH-26 and BrdU before transplantation. Ten minutes after myocardial infarction the animals were used for transplantation. Seven days after transplantation, survival of transplanted cells, expression of bFGF₂ and apoptosis of ischemic myocardium were measured by immunohistochemical staining, Western-blot, tunnel staining respectively. Four weeks later, the cardiac function of all alive animals was assessed by echocardiography. Finaly, the hearts were harvested for the pathological examination.Results: Seven days after transplantation, the transplanted EPCs can survive in the zone of ischemic myocardium. There were more cells survive in group I than that in the other groups. More bFGF protein was expressed and less apoptosis of ischemic myocardium. Four weeks later, the histological study showed differentiation of the transplanted EPCs which formed vascular endothelium. Angiogenesis was more remarkable than that in the other Groups. Echocardiography showed that the systolic and diastolic function in Group I were significantly improved compared with the other groups.Conclusions:The transplantation of EPCs transfected with Ad. bFGF₂ could improve the survival ability of transplanted cells, inhibit apoptosis of ischemic myocardium. With bFGF₂ protein expressed, The transfected EPCs have strong potency to protect cardiac function by stimulating angiogenesis. Such a strategy might be useful in the treatment of patients with ischemic heart disease. This dissertation is just the beginning of the reseach of angiogenesis by gene or cell therapy. There are more unknown questions to be studied and reasearched for us.