Efficacy Of Human VEGF Plasmid Coated With Magnesium Alloy Degradable Stent In Rabbit Carotid Artery Implantation

BackgroundSince the first case of percutaneous transluminal coronary angioplasty in the world in 1977,new breakthroughs have been made in endovascular treatment.With the development of vascular interventional science and the emergence of bare metal stents(BMS)and drug-coated stents(DES),intravascular interventional therapy has reached a new milestone.However,the long-term retention of metal stent in the blood vessel causes damage to the blood vessel wall,secondary inflammatory response,induced thrombus formation and proliferation of smooth muscle cells under the intima of the blood vessel,eventually leading to the occurrence of intravascular restenosis.Although drug-eluting stents(DES)coated with anti-proliferative drugs can well inhibit the intimal hyperplasia of blood vessels and effectively reduce the incidence of intravascular restenosis,they still lead to the formation of subacute or advanced thrombosis,so 12 Antiplatelet therapy for months or longer.Therefore,the emergence of biodegradable scaffolds is inevitable.Biodegradable scaffolds can be divided into Bioresorbable Polymeric Vascular Scaffold(BPVS)and Biodegradable Metallic Alloy Scaffold(BMAS)according to their main components.BPVS induces intravascular thrombosis in the late stage of degradation due to insufficient radial supporting force.In order to enhance the radial supporting force,BPVS uses a thicker stent bone,resulting in stent fracture.As a new type of degradable stent,BMAS has good radial support and degradation characteristics.Among various materials,the magnesium alloy degradable stent(Mg-BMAS)has excellent degradation characteristics and biological safety,and has been applied to human lower extremity blood vessels,cardiopulmonary circulation and large blood vessels.A number of animal experiments have also verified that the application of magnesium alloy degradable stents in cerebral blood vessels has certain safety and effectiveness.In the related research of cerebral vascular aneurysm,it was found that the magnesium alloy stent can degrade itself after assisting aneurysm embolization due to its degradable characteristics,which greatly reduces the inflammatory response of the aneurysm and promotes the healing of the aneurysm.In ischemic cerebrovascular diseases,the implantation of degradable scaffolds contributes to the perfusion of cerebral blood flow,and gradually degrades until disappearance after vascular endothelial repair is completed.The possibility of complications.With the deepening of research,the shortcomings of excessive degradation rate of magnesium-based alloy stents have been exposed.Before the restoration of vascular function,the stent has lost radial support,which has become the main reason for the poor postoperative expectations of magnesium alloy degradable stents.Vascular endothelial growth factor(VEGF),as the most active vasoactive factor,has the effect of promoting endothelialization.Studies have used VEGF gene to transfect human umbilical vein cells and found that over-expression of VEGF can promote cell proliferation,migration and endothelial cell tube formation.Down-regulation of VEGF will inhibit the response of the above-mentioned endothelial cells.We hypothesized that the recombinant human VEGF gene coated on the magnesium alloy degradable scaffold can be transfected into internal cells,thereby promoting the proliferation of endothelial cells and accelerating the endothelialization of the blood vessel at the stent implantation,thus solving the problem of excessive degradation of the magnesium alloy degradable scaffold.Purpose1.Construction of human VEGF recombinant plasmid coated magnesium alloy degradable scaffold.The effects of human VEGF coated magnesium alloy degradable scaffold on endothelial cell proliferation,migration and tube-like formation were verified by in vitro experiments.2.Construction of human VEGF recombinant plasmid coated magnesium alloy degradable scaffold.The degradation rate and endothelialization of human VEGF recombinant plasmid coated magnesium alloy scaffold were verified by animal experiments.Method1.After constructing the recombinant plasmid coated magnesium alloy degradable scaffold of human VEGF,the scaffolds were divided into three groups:group A:bare magnesium scaffold group,B:liposome+magnesium scaffold group,C:VEGF+liposome+magnesium stent group.They were implanted into human umbilical vein endothelial cells and cultured to extract the culture medium.The proteins were extracted by Western blot analysis,endothelial cell proliferation experiments,endothelial cell adhesion experiments,and endothelial cell migration experiments to study the effects of magnesium alloy degradable scaffolds coating materials on endothelial cells.2.The rabbit common carotid artery was implanted with a magnesium alloy degradable stent and the rabbits were divided into two groups:Control group:Mg stent group,Experimental group:Mg scaffold+PLL+liposome group+ VEGF.Follow-up was performed 1,2,and 4 weeks after the stent implantation,and blood vessel-related imaging datawere collected.The changes in the structure and properties of the vascular lumen were analyzed by image data.Results1.Western blot analysis of VEGF-A and VEGFR-1,2 expressed in human umbilical vein endothelial cells.The expression of VEGF-A in group C(Lipo+VEGF)was higher than that in group A(Con)and group B(Lipo);the expression of VEGFR-1 on the surface of endothelial cells in group C was higher than that in other groups and much higher than VEGFR-2,indicating that VEGF recombinant plasmid can promote the expression of VEGF,which is conducive to binding with VEGFR-1.2.The expression of different coatings in the culture medium of human umbilical vein endothelial cells is also different,resulting in significant differences in endothelial cell proliferation,adhesion and tube formation experiments.Compared with other groups,group C(Lipo+VEGF)can better promote the proliferation,tube formation and adhesion of endothelial cells.3.In animal experiments,the vascular diameter of the scaffold segment increased immediately after the biodegradable stenting of magnesium alloy,but the stenosis of the scaffold segment was found in the control group during the 1-week and 2-week follow-up.Severe intravascular stenosis was observed in the experimental group at week 1 and week 4.In combination with IVUS analysis of the degradation degree of scaffolds,it was found that the degradation rate was too fast in the 1-week follow-up and 4-week follow-up groups of the experimental group,which was the main cause of endovascular stenosis.ConclusionExogenous human VEGF recombinant plasmid promotes the expression of VGEF in endothelial cells,which has the same effect as endogenous VEGF in promoting the proliferation,adhesion and angiogenesis of endothelial cells.The main mechanism is to promote the binding of VEGFA and VEGFR-1.In vitro experiments also found that 1-polylysine and liposomes as carrers of plasmid coated scaffolds did not affect the expression of VEGF and cell destruction,so the human VEGF coating was safe and effective.In vivo experiments,although the bare MgZnYNd scaffold was degraded,it could provide effective radial support for blood vessels in the early stage.Through theoretical analysis,the recombinant VEGF plasmid coated with MgZnYNd scaffold can effectively accelerate the function of endothelial repair after stent implantation.In the process of coating,the oxidation of the support and the good holding degree of the support are still to be explored in this study.