The Inhibition Effect Of Human Arresten Gene Transfection On The Intimal Hyperplasia Of Autogenous Vein Graft In Rabbits

BACKGROUND For patients with coronary artery disease or limb ischemia, placement of a vein graft as a conduit for a bypass is an important and generally durable strategy among the options for arterial reconstructive surgery. Autogenous Vein is the gold standard for vascular graft conduits. Compared with other types of graft conduits, the most distinctive property of the vein graft is the adaptive response to the arterial environment during the post-surgical process; this adaptation is thought to be responsible for the superior performance of vein grafts compared with prosthetic grafts. However, it is also known that abnormal, or uncontrolled, adaptation may lead to abnormal vessel wall remodeling with excessive neointimal hyperplasia with deposition of smooth muscle cells (SMC) and extracellular matrix (ECM), ultimately vein graft failure and clinical complications. Since1950autogenous vein has been widely used in patients with ischemic disease, but20-50%of vein grafts are ultimately failure because of its remodeling process leading to thrombosis, restenosis et al. Once restenosis occur after revascularization, vascular reconstructive surgery may be taken, which will lead to the formation of restenosis again. There is currently no effective treatment to reduce excessive thickening of the graft wall. The mechanism and prevention measures of graft restenosis have been the focus of clinical research.Numerous physiologic and molecular mechanisms of vein graft adaptation have been discovered. However, this complex process has yet to be characterized well, and we do not yet have the ability to therapeutically control graft wall thickening. Early restenosis is associated with acute thrombosis, intimal proliferation.Long-term restenosis is the pathematology alteration after operation. Vascular smooth muscle cells and extracellular matrix are the essential component of the neointima.The pathophysiology of vein graft failure involved a complex mechanism. Conventional pharmacotherapy had limited impact on intima proliferation of vein graft. Along with the development of gene engining and molecular biology, gene therapy may become the method to solve the problem. DNA/RNA can be encapsulated in nanoprecipitation or adsorbed into the surface of nanoprecipitation, which can be uptakeen into the cell to achieve the purpose of sustained and stable expression of the gene. Biodegradable PLGA nanoprecipitation play an important role in the study of nanoparticles because of sustained-release drug delivery systems.Arresten is the26kDa C-terminal globular non-collagenous(NC1) domain of the al chain of type IV collagen (al[IV]NCl)(Colorado et al.,2000), with molecular weight about26kD. It was initially isolated from human placental basement membrane. In recent years, some study showed that arresten could inhibit the endothelial cells’proliferation and migration, induce endothelial cells’apoptosis and also inhibit tumor growth and metastasis. In early study, arresten showed effective in the inhibition of proliferation of vascular smooth muscle cells and neovascularization. So arresten play an important roll in neintimal hyperplasia and restenosis after vscular recomstructive operation.OBJECTIVE Construction of eukaryotic expression vector of Arresten gene. Construction of Arresten gene loaded nanoparticles biodegradable composed of biodegradable and biocompatible materials PEG-PLGA, to construct rabbit autogenous vein transplantation model, and then the recombinant plasmid was locally transformed into the vein graft; and the influence on neointimal hyperplasia of vein graft was explored.METHODS(1) A synthetic design arresten sequences, according to building requirements at the5’end to add start codon ATG, and add the enhanced expression of kazak sequence GCCACC, remove the termination codon at the3’end. After loading sequence synthesis to pUC57carrier, enzyme digestion and sequencing were conduct. The conduction was inserted into pEGFP-Nl vector.The target gene sequence were affirmed correctly.(2) A modified nanoprecipitation method was established to formulate the target gene loaded PEG-PLGA nanoparticles. Accurately weighted (20mg) PEG-PLGA was dissolved in1mL DMSO containing200μg of plasmid DNA. The resulting clear solution was slowly (30mL/h) injected by a microsyringe pump into20mL magnetic ally stirring0.5%(w/v) Pluronic F127aqueous solution (600rpm) and agitated for5hours at room temperature. The DNA-loaded PEG-PLGA nanoparticles formed instantaneously.(3) Preparation of rabbit autogenous vein graft model, and the recombinant plasmids pEGFP-N1-arresten were locally transfected into vein graft. Detection of arresten expression in vein graft by RT-PCR. Intimal and medial hyperplasia degree was measured by the thickness, which was measured by computer after hematoxylin eosin stain and Masson stain. Smooth muscle alpha-actin (a-SMA)、Proliferating cell antigen (PCNA)、matrix metalloproteinase(MMP-2) and MMP-9of vein graft were analysised by immunohistochemical labeling.RESULTS(1) Sequence analysis showed that, the sequencing results for human Arresten cDNA was cloned in the same prostage sequence. Objective gene into the expression vector is correct. The Arresten gene fragment inserted into the vector between the coincidence. Indicated that arresten gene was cloned into the expression vector comprehensively, accurately to the correct expression of target protein. Plasmid pEGFP-Nl-arresten was successfully constructed.(2) In nano PEG-PLGA nanoparticles modified precipitation method has uniform spherical particles, an average of219.8±2.28nm with narrow size distribution, a negative Zeta potential in pH7.4-24.52±0.644mv. Using the nano modified precipitation method, the obtained PEG-PLGA nanoparticles exhibited greater loading efficiency (96.64±0.202%).(3) To establish the model of autogenous vein graft. RT-PCR results showed that the genome of arresten-transferred tissue contained fragment of arresten gene; about the thickness of the medial and intimal issue, arresten transfection group was less than the control group and blank group, and the difference was statistically significant (P<0.05). Vascular smooth muscle cells consisted in the hyperplasic intima majure; the number and ration of MMP-2、MMP-9and PCNA positive-stained in arresten transfection group was lower than that of the control group and blank group (P<0.05).CONCLUSIONS Using the nano modified precipitation method the obtained PEG-PLGA nanoparticles exhibited greater loading efficiency (>95%). Autogenous vein graft model, by end to end anastomosis between the jugular vein and the ipsilateral carotid artery could lead to restenosis of vein graft. The human Arresten gene transfection locally can inhibit intimal hyperplasia of vein graft, improve the degree of patency. They show a good prospect in clinical application in inhibition and cure of rstenosis after vascular transplantation.