Adenovirus-mediated Human Endothelial Nitric Oxide Synthase Gene Transfer Inhibits Vascular Smooth Muscle Cells Proliferation And Its Mechanism

Part 1Construction and identification of recombinant adenovirus carrying the human endothelial nitric oxide synthase geneObject To construct recombinant adenovirus vector carrying the human endothelial nitric oxide synthase gene and LacZ gene, packaged the adenovirus in 293 cells.Methods Human endothelial nitric oxide synthase gene was amplificated by PCR, heNOS cDNA was inserted into adenovirus shuttle plasmid PSUCMV to generate a recombinant plasmid PSUCMV-heNOS, transfected into E.coli DH5., positive clonings were selected and confirmed by restriction endonucleas analysis, and DNA sequencing analysis showed heNOS cDNA is the same as the published data in GeneBank. The plasmid PSUCMV-heNOS was co-transfected with 5 type adenovirus backbone plasmid pBHGE3 into 293 cells to generate replication-defective recombinant adenovirus vector carrying human endothelial nitric oxide synthase gene. The recombinant adenoviruses were purified by cesium chloride density purification,. And virus titer was measured by TCID50 method.Results The recombinant adenovirus vector carrying human endothelial nitric oxide synthase gene and LacZ gene was constructed and its titer was 3.5X10¹⁰ PFU/ml and 3.0X10¹⁰ PFU/ml.Conclusions The successful construction of AdCMV-heNOS may provide an opportunity for the gene therapy of vascular intimal hyperplasia.Part 2Adenovirus-mediated human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cells prolferation and its mechanismObject To investigate the effect of Adenovirus-mediated human endothelial nitric oxide synthase gene transfer on in-vitro cultured human vascular smooth muscle cells(VSMCs) proliferation. To explore the effect of heNOS expression in vascular smooth muscle cells on the expression of the cyclin dependent kinase inhibitors p21, p27 and apoptosis of vascular smooth muscle cells.Methods Human umbilical artery smooth muscle cells(HUASMCs) were in-vitro cultured and purified and identified, vascular smooth muscle cells were transduced with adenovirus vector encoding heNOS at different MOI, the effect of heNOS on HUASMCs proliferation was investigated by MTT assay and cells grow curves were described. At the same time, LacZ transduced and Non transduced cells served as additional controls. heNOS gene expression was sought by NAPDH diaphorase staining, immunohistochemistry and Western Blot. cGMP was measured by radioimmunoassay in HUASMCs. The expression of P21 and p27 were studied by western blot analysis. Cell cycle and apoptosis analysis was performed by flow cytometry.Results MTT value decreased with MOI increased. At 120 hours, compared with control group and groups of MOI 100,150,200 respectively, group of MOI 300 significantly inhibited the HUASMCs proliferation (p<0.01). after 48 hours, heNOS expression was detected by NAPDH diaphorase staining, RT-PCR, immunohistochemistry, and Western Blot in transduced VSMCs with MOI 300. control cells had none. The cGMP levels were increased in heNOS-transduced cells compared to LacZ and Non-tranduced cells(p<0.01). Expression of heNOS in VSMCs resulted in a delay in cell cycle G0/G1 and upregulation of p21 and p27(p<0.05). There was no increase in apoptosis detected in all transduced cells after 24 and 72 hours(p>0.05).Conclusions Adenovirus-mediated human endothelial nitric oxide synthase gene transfer to VSMCs inhibits cell proliferation via upregulation of p21 and p27 resulting in a delay in cell progression but not apoptosisPart 3Adenovirus-mediated human endothelial nitric oxide synthase gene transfer inhibits neointimal hyperplasia after balloon injury in the rat carotid arteryObject To investigate the effect of adenovirus-mediated human endothelial nitric oxide synthase gene local transfer on neointima formation after balloon injury in the rat carotid artery.Methods Sprague-Dawley rats underwent balloon injury of the common carotid artery. After local injury, 100ul of recombinant adenovirus heNOS(3.5X10¹⁰ PFU/ml) and LavZ(3. 0X10¹⁰ PFU/ml) was instilled into the common carotid artery and allowed to dwell for 30 minutes. Single injured artery was control group. The rats were humanely killed after 4,7,14,21 days respectively by perfusion fixation, and the carotid arteries were sectioned. heNOS gene was measured by immunohistochemistry and Western Blot in vascular medial surface area. Sections were stained with hematoxylin-eosin and used to measure the degree of neointimal thickening. The neointimal surface areas, medial surface areas, the intima-media(I/M) ratios were calculated for each of the two sections per artery with computerized planimetry by an investigator blind to the experimental procedure, the average of which was used as an index of neointimal hyperplasia.Results Balloon injury in the rat carotid artery model was successfully established. heNOS protein production was confirmed in vascular medial surface area by immunohistochemistry and Western Blot. The neointima was formed in 7 days and vascular smooth muscle cells migration and proliferation, matrix cells proliferation in 14 days after balloon injury. Neointimal hyperplasia was serious and resulted in lumen stenosis. There was no neointimal hyperplasia in heNOS transduced sections. Morphometric analysis showed that neointimal surface areas, I/M ratios were all significantly reduced in heNOS groups compared to LacZ groups and injuried groups in 14, 21 days respectively after injury(p<0.05). There was no statistical difference between the two control groups.Conclusions adenovirual gene of human endothelial nitric oxide synthase local transfer has an inhibitory effect on neointima formation after balloon injury in the rat carotid artery. adenovirus-mediated human endothelial nitric oxide synthase gene local transfer might reduce arterial restenosis after balloon angioplasty.