The Targeted Treatment Study Of Promoting Angiogenesis Of CD151 Gene Delivered Into Rat Ischemic Hearts

Background & ObjectiveThe theraputic angiogenesis provides a novel approach for the treatment of coronary artery disease,that is using exogenous growth factors(protein or gene) to promote angiogenesis and improve the blood supply of the tissue.Among the many signal moleculars,the transmembrane-4 superfamily(TM4SF) proteins have a direct relationship with theraputic angiogenesis.TM4SF located in the membrane are a group of highly conserved proteins in the process of proteins molecular evolvement and are also proteins related with various integrins. CD151 is the most important member of the transmembrane-4 superfamily proteins. Studies show that CD151 involves in many cell functions,such as the cell morphology, migration, adhesion,hemidesmosome structure formation.It also mediates the angiogenesis together with integrins.In recent years, many studies about angiogenesis of CD151demonstrated that CD151 maintains the cell morphology and the ability of angiogenesis in vitro.CD151 transfected into HUMC enhances cell proliferation, migration, and capillary tube formation on Matrigel.Direct injection of an AAV vector carrying CD151 gene increases the number of microvessels in a rat ischemic hind limb and CD151 gene delivery increases microvessel density of ischemia myocardium,reduces the area of myocardial ischemia and improves the function of the heart in a rat myocardial ischemia model and in a pig myocardial ischemia model.Further study on CD151 knockout mice indicated that the CD151 null mice display marked reduction in the angiogenesis.These observations strongly suggest that CD151 can be used for gene therapy on myocardial ischemia by promoting angiogenesis.Upon the progress of gene therapy, there are more concerns regarding the safety of gene therapy and the routes of gene delivery.How to diliver the exogenous gene to the target tissue without trauma to treat the disease,reduce the absorption of other tissues and the possible side-effects at the same time is the next research emphasis.we plan to use CD151 to treat coronary artery disease in the future,that needs not only the safe delivery of CD151 into the ischemia myocardium,but also reduction of the possible side-effects caused by other tissues'absorption.So we still selected AAV to diliver CD151 gene and attempt to reconstruct the ptomoter region of AAV vector to make CD151's treatment on myocardial ischemia disease truly safe,efficient and targeted.Methods1. Construct the plasmids of pAAV-HRE-CD151,pAAV-MLC-CD151 and pAAV-HRE-MLC-CD151,amplify, extract and purify them.2. H9C2 cells in good condition were cultured on 6-well plates to 60%-70% confluence and transfected with pAAV-HRE-CD151, pAAV-CD151, or pAAV-GFP.Non-transfected H9C2 cells were included as control. At 48h after transfection, the infected cells were split into two parts:one part was cultured under normoxic condition (95% air,5% CO2) and the other part under hypoxic condition (94% N2,5% CO2) in serum-free medium for 16 h.Then harvested cells,isolated proteins and detected the expression of CD151 in two groups by using Western blot.3. Eighteen adult male Sprague-Dawley rats with successful heart ischemic model were randomly divided into three groups. The rats in control group (n=6) was injected a single dose of 1 ml of saline solution by the sublingual vein,while the rats in pAAV-CD151 or pAAV-MLC-CD151 group (n=6 per group) received with a single dose of 1 ml saline solution containing 200μg of pAAV-CD151, or pAAV-MLC-CD151 plasmids through the same route. Four weeks after plasmids administration,sacrificed the mice and got the heart,liver and kidney tissue.The expression of CD151 in ischemia heart,liver and kidney were detected by using Western blot,the density of microvessels in ischemia myocardium were detected by immunohistochemistry. 4. Twenty-four adult male Sprague-Dawley rats with successful heart ischemic model were randomly divided into four groups. The rats in control group (n=6) was injected with a single dose of 1 ml of saline solution by the sublingual vein,while the rats in pAAV-CD 151 pAAV-MLC-CD151 or pAAV-HRE-MLC-CD151 group (n=6 per group) received a single dose of 1 ml saline solution containing 200μg of pAAV-CD151, pAAV-MLC-CD151 or pAAV-HRE-MLC-CD151 plasmids through the same route. Four weeks after plasmids administration,sacrificed the mice and got the heart tissue.The expression of CD151 protein in ischemia heart were detected by using Western blot,the mRNA level of CD151 were detected by Real-time PCR,the density of microvessels in ischemia myocardium were detected by immunohistochemistry.Results:1. The plasmids of pAAV-HRE-CD151,pAAV-MLC-CD15 and pAAV-HRE-MLC-CD151 were constructed and confirmed by the nucleotide sequencing.2. HRE-regulated CD151 expression in cardiomyocytes in vitro. We transfected the plasmids of pAAV-HRE-CD151, pAAV-CD151 or pAAV-GFP into H9C2 cardiomyocytes, respectively. We detected CD151 protein expression in every group cells by western blotting.Under the hypoxic condition, the expression of CD151 proteins was significantly increased in the pAAV-HRE-CD151 group compared to the pAAV-CD151, pAAV-GFP, and non-tranfected cardiomyocyte group (P values< 0.01). Under the normoxic condition, the expression of CD151 protein was increased significantly in the pAAV-HRE-CD151 group compared to the pAAV-GFP and non-tranfected cardiomyocyte groups (P<0.01), but there was no significant difference in CD151 expression between the pAAV-HRE-CD151 and pAAV-CD 151 groups.3. MLC-induced cardiac-specific CD151 gene expression in vivo.we delivered the plasmids of pAAV-CD151,pAAV-MLC-CD151 into the heart ischemic model rats by intravenous injection.We used western blot to detect CD151 protein expression in the hearts.We observed in our experiment that MLC-mediated CD151 expression in ischemic hearts increased significantly in pAAV-MLC-CD151 group compared with control group and pAAV-CD151 group (P<0.01).The CD151 expression was also detected in the livers and kidneys.The CD151 expression in livers or kidneys decreased significantly in pAAV-MLC-CD151 group compared with the pAAV-CD151 group respectively (P<0.01). The CD151 expression in livers or kidneys between the control and pAAV-MLC-CD151 group have no significant difference(P>0.05).The immunohistochemistry results showed higher microvessel densities in ischemic hearts of the pAAV-MLC-CD151 group and less neovascularization in the control group,the pAAV-CD151 group.the difference is significant(P<0.05)4. HRE and MLC-regulated CD151 gene expression in vivo. we delivered the plasmids of pAAV-HRE-MLC-CD151,pAAV-MLC-CD 151 or pAAV-CD 151 into the heart ischemic model rats by intravenous injection.We detected CD151 expression in the hearts by western blotting analysis and detected the mRNA level of CD151 by Real-time PCR.We observed in our experiment that CD151 protein expression and mRNA levels in ischemic hearts increased significantly in the pAAV-HRE-MLC-CD151 group compared with control group,pAAV-CD151 group and pAAV-MLC-CD151 group (P<0.01).The immunohisto-chemitry results showed higher microvessel and arterioles densities in ischemic hearts of the pAAV-HRE-MLC-CD151 group and less angiogenesis in the control group,the pAAV-CD151 group and pAAV-MLC-CD151group,the difference is significant(P<0.05).Conclusions1. Hypoxic response elements(HRE) can increase CD 151 expression in cardiomyocytes under the hypoxic condition.2. Cardiac-specific promoter(myosin light chain,MLC) can induce cardiac-specific CD151 gene expression in ischemic heart rats.3. Adeno associated viral vector-delivered, hypoxia response element and cardiac speci-fic promoter-regulated CD151 expressed high and improved the transfection efficiency of gene therapy.The higher expression of CD151 can promote the formation of microvessels and arterioles of ischemic myocardium.