Construction And Biological Behavior Study Of Angiopoientin 1 Functionalized Surface

Restenosis(RS)and thrombosis formation were the most common complications for the long-term implantation of cardiovascular devices.The major reason caused the complication is that the biocompatibility is not enough such as the shortage of blood compatibility,inflammation compatibility and cytocompatibility.Many researchers tried to introduce bio-active molecule onto the surface of cardiovascular implantations to aim on promoting endothelialization and reducing thrombosis.This kind of surface modification method had made great improvement,while,the current studies mostly focused on improving one aspect of biocompatibility,for instance,meliorating blood compatibility or inhibiting hyperplasia.Recently,the multi-functional surface modification of cardiovascular materials is gradually recognized to achieve both anti-coagulation,anti-hyperplasia and rapid endothelialization However,the researches about vascular inflammation after the materials implantation were barely.And there were less studies about the strategies of establishing cardiovascular materials which could promote vascular healing and regulate vascular immune response in the same time.In addition,the connection between inflammatory response and blood vessel repair was still unclear.In this study,angiopoietin-1(Ang-1),which could keep endothelia alive and regulate inflammation,was firstly employed to modify 316L stainless steel.Based on the two different states of Ang-1 in vivo,two kinds of modification approaches were established.One approach was that Ang-1 molecules were covalent--immobilized on the materials surface,and the other was that Ang-1 were physically loaded on the materials surface.The effects of the free-state Ang-1 on human umbilical vein endothelial cells(HUVECs),smooth muscle cells(SMCs)and endothelial progenitor cells(EPCs)and macrophage were firstly studied.These studies were used to analyze the feasibility of using Ang-1 to regulate vascular inflammation and repair.The studies showed that Ang-1 could effectively promote the proliferation and migration of HUVECs,the proliferation of EPCs and the apoptosis of macrophages in vitro.Moreover,Ang-1 could also inhibit the apoptosis of HUVECs and the proliferation of macrophages,and did not stimulate SMCs over proliferation.These results suggested that the Ang-1 was one promising candidate bio-molecule for the application in cardiovascular devices aimed on promoting endothelialization and adjusting inflammatory response.Based on the above,the poly-dopamine(PDA)coating was employed as a modification platform,and then the Ang-1 molecules were immobilized onto the PDA surface via the Schiff base reaction and Michael addition reaction between the amine groups of Ang-1 and the quinone groups on PDA surface.The introduction of Ang-1 was confirmed by quartz crystal microbalance with dissipation(QCM-D),Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS)and immunofluorescence analysis methods,and the stability and activity of Ang-1 were also investigated.For certain range of concentrations,the higher Ang-1 concentration modified surface had higher surface intensity of Ang-1,,and the activity of immobilized Ang-1 was well retained.And,the introduction of Ang-1 had little influence on the hemocompatibility.Then,the adhesion,proliferation,apoptosis and migration behaviors changes of vascular cells and macrophage growing on the different concentration Ang-1 modified surface were investigated.And,the best optimum concentration of Ang-1 was screened out.The influences of the best optimum Ang-1 modified surface on secretion of inflammatory factors and repairing factors of the vascular cells and macrophages were also evaluated.The results showed that the Ang-1 modified surface could promote the proliferation and migration of HUVECs,stimulate the proliferation of EPCs and inhibit the growth of macrophages.Meanwhile,the inflammatory response of Ang-1 surfaced-modified materials was investigated in vitro.The in vitro experiments results and the mechanism analysis revealed that the involving of Ang-1 could inhibit the inflammatory factors secretion of HUVECs,SMCs and macrophages,such as monocyte chemoattractant protein-1(MCP-1),interleukin-1(IL-1),interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a).In addition,the Ang-1 modified surface could also promote the secretion of interleukin-10(IL-10)and vascular endothelial growth factor(VEGF).In the meantime,the real-time polymerase chain reaction(RT-PCR)results indicated the Ang-1 modified surface could promote the expression of tissue inhibitor of metalloproteinase-1(TIMP-1)of macrophages,and had positive effects on keeping vascular stabilization.Furthermore,the Ang-1 surface-modified materials were implanted under the skin of SD rat and in the abdominal aortic vessel of SD rats.In vivo experiments results revealed that the Ang-1 modified surface had better tissue compatibility.In order to construct the physically-loaded Ang-1 modified surface,the RADA16 peptide was alsochosen to load Ang-1,and a Ang-1 releasing bionic coating was successfully built.This coating could mimic the vascular tissue under the endothelium,which would secrete and release Ang-1 into the vascular endothelium.Based on the results,the continuously releasing of Ang-1 could last more than 14 days.The peptide coating with or without Ang-1 loading had good bio-compatibility.While,the Ang-1 in the coating had obvious promotion effects on HUVECs growth and migration,and inhibited the growth and inflammatory factors secretion of macrophages,such asIL-6 and TNF-a.The RADA16 peptide coating loaded with Ang-1 could promote endothelia growth and regulate inflammatory response in vitro.These two kinds of Ang-1 biomimetic surfaces all had the ability to regulate inflammatory factors secretion,adjust immune response and promote endothelium healing.Thus,this research had certain significance and researching value for the application of Ang-1 in cardiovascular implant devices.This investigation puts forward a novel surface modification strategy of cardiovascular devices,which could regulate vascular immune response and promote endothelium healing in the same time,and this work could provide the basis for the development of novel cardiovascular implantation materials.