Adenovirus-mediated Gene Transfer Of Acidic Fibroblast Growth Factor Induces Angiogenesis In Chronic Ischemic Myocardium

It has been a new hot spot of research to theraPy ischemic heart disease with genetransfer of angiogenic growh factors. Previous stUdies focused mainly on vascularendothelial growth factors(VEGFs) which have proven to induce angiogenesis and improvemyocardial function in chronic ischemic myocardium. However. It has not been reported ifthe gene of acidic fibroblast groWth factor(aFGF FGF-l) could show the same effect. TOverify this hypothesis, Here we first consmicted a replication-deficient adenovirus vectorcoding fOr the gene of aFGF (Ad.aFGF), another angiogenic growth factor, which effects ofthe proliferation and differentiation on endothelial celIs(ECs) and smooth musclecells(SMCs) in vitro would be further examined, and directly intramyocardiallyadministrated Ad.aFGF to evaluate its ability to increase the goWth of collateral vessels andimprove myocardial perfusion and function in vivo with a porcine chronic myocardialischemic model.Mitl1ods:l .The construction of cosmid vector inserting aFGF gene (pAxcAwt/aFGF)The mRNA of human brain tissue was isolated using standard procedures. RT-PCR wasused to synthesize and amplify aFGF DNA which was fiJrther purified by electrophoresisand cleaved with aPpropriate restriction enzymes. The DNA was inserted into the plasmidpBV220. transduced into competent cells of Escherichia coli host strain-TGl fOramp1ification and confirmed to be the gene of interest. After that, aFGF DNA was insertedinto cosmid vector pAxcAwt by the same Strategy and the correct clones containing targetgene in right direction were selected.2. The construction of Replication-deficient recombinan adenovirus coding fOr aFGFgene(Ad.aFGF)Human embroynic kidney 293 cells cultured in a 6-cm dish were transfected withDNA-TPC and pAxcAwt/aFGF by the calcium phosphate method. After 8-l8 days. virusclones were isolated and propagated to for restriction analysis. The desired Ad vectors werepurified by density gradient ultracentrifuge and titrated in 293 cells.3.Transfection of Ad.aFGF to cardiomyocytes and its effects of proliferation anddifferentiation on ECs and SMCs in vitro.Rat fetal cardiomyocytes(FCMs) were isolated and cultured. Transfection efficiency ofAd.aFGF on FCMs were examined at various titfations by immunofluorescence Staining.Gene transcription and protein expression was evaluated by Northern blot and Westem blot,respectively. The concentration of aFGF in culture medium was tested by ELISA. Theproliferation effect of Ad.aFGF on human umbilical venous endothelial cells(HUVECs) andbovine aortic smooth muscle cells(BASMCs) was exaxnined by MTT assay. Finally we alsoexamined the differentiation effect of Ad.aFGF on HUVECs plated ed on reconstitutedbasement membrane(Matrigel Matrix).4.The enhancement of collateral vessel formation induced by Ad.aFGF and the functionimProvement of chronic ischemic myocardium.Swine underwent thoracotomy and placement of an Ameroid constrictor on leficircumflex coronmp artery. Four weeks later, all animals were randomly divided into tlireegroups and injected with Ad.aFGF(n=9), Ad.Null(n=9), or PBS(n=8) intramyocardially at 10sites in the circumflex distribution(l0'pfujsite or 100 ll Usite). At the time of 28-30 daysafter vector administration, myocardial fimction and perfusion were assessed byechocardiograPhy and by single photon emission computed tomograPhy imaging(SPECT)with oomTc-sestaxnibi. After all animals were sacrificed, hearts were harvested for Ex vivocoronary angiograPhy to docurnent the existence and distribution of collateral vessels. Tbeexpression of aFGF and the new blood vessels in myocardium were demonstrated with aFGFinununohistochemistry staining and Factor VllI irTununohistochemistry staining.Resultr:1.The purity and integrity of isolated InRNA were acceptable. The segment of DNAobtained by RT-PCR was identical to that expected. The sequence of DNAenzymatic-cleaved from the cosmid proved to be a...