Research Of Perlecan/VEGF-165on Enhancing Angiogenesis In Mice

Angiogenesis is essential for wound repair. New formed blood vesselsprovide nutrients and oxygen to support cell growth and tissue repair.Neovascularization is a complex multistep process that is dynamic and tightlyregulated in a spatial and temporal manner by a variety of signals from solublefactors, extracellular matrix, pericytes, and surrounding stromal cells. Recently,endothelial progenitor cells are known to be involved and, in fact, play a criticalrole in neovascularization. Endothelial progenitor cells have multiple ingredients,of which Perlecan and VEGF-165are most important for angiogenesis.Perlecan is a large multi-domain extracellular matrix proteoglycan thatplays a crucial role in tissue development and organogenesis. Perlecan, the mainproteoglycan of basement membranes and pericellular spaces, is one of thelargest single-chain polypeptides of vertebrate animals. The five modules ofPerlecan are related to molecules involved in nutrient metabolism, mitogenesisand adhesion. These structural motifs, when translated into multimeric functionalunits, could be effectively utilized by diverse tissues during development,remodelling or neoplastic growth. Perlecan was subsequently found to beexpressed in nearly all basement membranes as well as mesenchymal organs and connective tissue,which participates in angiogenesis and has the function ofstorage, protection and strengthen blood vessel growth factors. Perlecan has animportant role in regulating blood vessels in biological behaviour and repairingdamages. These characteristics of Perlecan provide opportunities to improvebiological activities of materials used for tissue engineering and wide-range ofclinical therapies. Perlecan has been demonstrated to have a role inangiogenesis.Angiogenesis refers to the formation of new blood vessels viaextension or remodeling of existing blood vessels as opposed to vasculogenesis,where blood vessels develop from progenitor cells that coalesce anddifferentiate to form vessels. Perlecan is detected around newly formed bloodvessels.Perlecans can be decorated with HS, CS or keratan sulfate (KS), however,Perlecan isolated from endothelial cells has so far been shown to containpredominantly HS. Endothelial cell-derived Perlecan has been shown to bind theangiogenic factors FGF1,2and FGF receptor1or3/FGF2complexes via theHS chains, as well as to promote FGF signaling in vitro.VEGF is essential for growth plate angiogenesis and subsequent boneformation. Proteolysis of the VEGF-165/CTGF complex by MMP-1/-3,-7or-13, which are upregulated during cartilage remodeling, was shown to degradeCTGF and result in release of active VEGF. This is thought to be due todecreased availability of VEGF to interact with its receptors on endothelial cells.This mechanism is also supported by the finding that infusion of dimerizedsoluble receptor Flt-1(VEGF receptor1) reversibly delayed bone formation inthe growth plate.Poly lactic acid (polylactic acid, PLA) is the most usually used to constructthe blood vessels of the tissue engineering scaffolds materials. OPLA isconsisted of D, D-L, L polylactic acid, which has easy biodegradability,plasticity and good biocompatibility, so we used this kind of scaffolds tocombine with Perlecan／VEGF-165to promote angiogenesis. Understandingthe molecular mechanisms that regulate wound angiogenesis may provide novel approaches for treating chronic wounds. The ischemia caused by blood vesseldamage causes of morbidity and mortality are higher, Angiogenesis to tissuerepair plays an important role in injury tissue, biological materials promoteangiogenesis will greatly improve the success rate of injury repair.The aim of this research is to combine scaffolds with purified Perlecan andVEGF-165implanted invivo to abserve angiogenesis process. Angiogenesis isan essential signal of tissue remodeling and regeneration and wound healing，and has long been a desired therapeutic access to improve clinical outcomes ofsituations typified by ischemia. VEGF-165has demonstrated ability to generatenew blood vessels，and continuous delivery of VEGF-165has been provednecessary to generate blood vessels as demonstrated by implanted, controlledrelease devices.The preparation and purification process of Perlecan has been confirmed byWuDan and Perlecan combined with VEGF-165has good reliability andstability and specificity. So based on researches has been done bu WuDan, myexperiment is arranged as follows:1. The effect of enchancing angiogenesis by qualitative analysis in miceOPLA is the complication of D, D-L, L polylactic acid. OPLA has poroussmall plane structur,so it is specialy suitable for all kinds of ingredientsabsorption. There were two groups in my experiments, which were treatmentgroups and control group. There were three subgroups under treatment groups:OPLA combined with Perlecan, OPLA combined with VEGF-165and OPLAcombined with both Perlecan and VEGF-165. Control group was OPLA has notreatment.My study was performed under the aggreement and applying to theguidelines of the Institutional of Animal Care and obey Committee of FourthMilitary Medical University. Scaffolds were implanted into male mice andharvested at different timepoints. HE was employed to investagatedangiogenesis and restructuring effect by qualitative analysis. We found that scaffolds degraded with time increasing and combined closely with surroundingtissues and there were visuable microvessels extended into the scaffolds.Microvessels surrounding scaffolds examplified with time increasing whichwas the most obvious at8weeks. At the same time, tissues with microvesselsgrown into the scaffolds and divided the scaffolds into pieces, each group can beobserved angiogenesis, especially the OPLA combined with both Perlecan andVEGF-165group.2. The effect of enchancing angiogenesis by quantitative analysis in miceCD31antigen widely distributed in various endothelium blood vessel cellsand can be used as a symbol of blood vessel formation. Through the quantitativeanalysis of the CD31expression of restructuring microvessels, we found thatthe OPLA+VEGF-165group was almost twice as much as the control group at2weeks; microvessel density (MVD) of OPLA+VEGF-165group showed nostatistically significant difference with the control group, but showed statisticallysignificant difference with OPLA+Perlecan group and OPLA+Perlecan+VEGF-165group. That demonstrated that VEGF-165was rapid released at2weeks and promoting the angiogenesis was the most obvious at2weeks, Until8weeks, MVD of OPLA+VEGF-165+Perlecan group remained keeping a highspeed explained Perlecan regulated the release of the VEGF-165.At1week,MVD of OPLA+Perlecan group and OPLA+VEGF-165+Perlecan group hadno statistically significant difference; but at2weeks, there was statisticallysignificant difference; at3weeks, though the difference became narrow, MVDwere higher than control group. All those helped us to undstand Perlecan wasgood at enhancing angiogenesis, Perlecan combined with VEGF-165was betterat enhancing angiogenesis. MVD of OPLA+Perlecan+VEGF-165group wasthe highest at any time point, so we saw that OPLA combined with VEGF-165and Perlecan had a synergy effect at enhancing angiogenesis precess.