Experimental Study Of EPCs Modified By Lentiviral Vector Containing VEGF To Effect On Angiogenesis In Cerebral Ischemia

Background:In China, there are about 7 million patients with cerebrovascular disease each year, nearly 70% for cerebral ischemia. EPCs have characteristics of stem cells and endothelial cells, which are involved in angiogenesis of ischemic diseases. EPCs participate in the reconstruction of vascular network and secrete growth factors or cytokines, stimulate endothelial cells migration, growth and function to enhance nerve regeneration after cerebral ischemia.In ischemic tissue, VEGF promote angiogenesis, to stimulate endothelial progenitor cells homing the ischemic injure in circulating blood.In MCAO rat models, EPCs take part in angiogenesis by the biological axis of chemokine stromal cell-derived factor-1 (SDF-1) and C-X-C and its receptor Chemokine receptor type 4 (CXCR4). With the hypoxia, HIF-la increases to provide the protection of cells and angiogenesis.Objectives:To explore the effects and mechanisms of EPCs and VEGF to promot angiogenesis in cerebral ischemia via EPCs modified by lentiviral vector containing VEGF.Methods:VEGF 164 was obtained by RT-PCR in blood mononuclear cells from male Wistar rats. pLVX-VEGF164-IRES-ZsGreenl was constructed and identified by restriction enzyme digestion analysis, RT-PCR, the sequencing and western blotting, which could achieve the functionality at mRNA and protein levels. EPCs were separated from the bone marrow in male Wistar rats by gradient separation and cultured to determine EPCs by surface markers CD133, CD34, and KDR in immunofluorescence staining. EPCs modified by Lentiviral vectors containing VEGF (EPCs-VEGF) were obtained. PBS, EPCs and EPCs-VEGF were transplantated into lateral ventricle of MCAO rats model. Through neurological assessment of the mNSS, body mass index, immunohistochemical staining, immunofluorescence double staining, Tunel staining and Western blotting analysis, the indexes of EPCs treatment group were different from which of PBS treatment group on the 3rd,7th,14th days in the pathology and therapeutic effect of MCAO rats model, and the protein expression of HIF-1α, VEGF, MMP-9, CD34, SDF-1 and CXCR4 were analysed and explored in the 7th day of MCAO models among the PBS, and EPCs and EPCs-VEGF treatment groups for cerebral infarction.Results:(1) pLVX-VEGF164-IRES-ZsGreenl was built and packaged successfully in the 293T cells. Lentiviral vector with VEGF 164 could express VEGF stablely at mRNA and protein levels. (2) Using density gradient centrifugation and EGM-2, EPCs were cultured and marked by immunofluorescence identification of CD133,CD34 and KDR. (3) Lentiviral vectors with VEGF 164 infected EPCs. Then,70% of EPCs modified by Lentiviral vectors containing VEGF (EPCs-VEGF) expressed green fluorescent protein, and EPCs-VEGF made VEGF expressed upregulatedly in western blotting (P<0.05). (4) PBS and EPCs were injected into the lateral ventricle of MCAO rats, and mNSS and the weight ratio were different significantly between the two groups in the 7th day (P<0.01). There were results of immunohistochemistry and western blotting showed that VEGF protein expressed in the 7 day at peaked, CD34 gradually increased, apoptosis of penumbra improved, and HIF-la in early ischemic expressed highly, (P<0.05). (5) PBS, EPCs and EPCs-VEGF were injected into lateral ventricles in MCAO rats, and the 7th day of mNSS and body weight ratio among the groups had significant difference (P< 0.05). In immunohistochemistry and western blotting, VEGF, HIF-1, CD34, SDF-1 and CXCR-4 in the EPCs-VEGF treatment group had the highest expression. MMP-9 in the PBS group was the highest (P<0.05).Conclusions:(1) The pLVX-VEGF164-IRES-ZsGreenl plasmid was constructed and packaged successfully. The lentiviral vector carrying VEGF infected cells to express VEGF at RNA and protein level. (2) Bone marrow derived EPCs were obtained and identified, satisfying with the requirements of the experimental cells. (3) EPCs modified by Lentiviral vectors containing VEGF improved the ischemia of MCAO rat models to by HIF-VEGF and SDF-1/CXCR4 signaling pathways to involve in angiogenesis, reduce cerebral infarction volume, and promote nerve functional recovery.