Screening Of A Specific Small Peptide Targeting Endothelial Progenitor Cells And Endothelial Cells And Study On Its Endothelialization Function For Vascular Grafts

Background and SignificanceCardiovascular diseases are the leading cause of death in the world.Blood vessel replacement is the common treatment for vascular diseases such as atherosclerosis,restenosis and aneurysm.However,appropriate autologous venous and arterial grafts are limited by their sources,availability,as well as the associated additional surgeries and morbidity.In this context,artificial vascular grafts are used widely in this field,but the grafts with small-diameter(ID)(less than 5 mm)are often clogged due to thrombosis and occlusion,resulting in low patency rate.Therefore,tissue-engineered vascular grafts seeded with functional cells have been constructed to improve the patency.However,constructing cellularized grafts has the disadvantages of extensive manipulation of cells in vitro,high risk,high cost and time consuming,which limit their clinical applications.Therefore,it is highly desirable to develop bioactive vascular grafts to promote the recruitment of endogenous cells and improve in situ endothelialization and patency of grafts.Endothelial progenitor cells(EPCs)and endothelial cells(ECs)play a vital role in vascularizing tissue engineered constructs for neotissue formation,long-term survival,and function.Native vascular wall is composed of extracellular matrix(ECM).which is very important for cell growth and neotissue regeneration.In past years,many functional biomolecules have been tried to coat vascular grafts to enhance in situ endothelialization and long-term patency of the grafts.However,these functional biomolecules are not optimal for in situ endothelialization due to their limited stability,functionality and binding specificity to EPCs and ECs.One-bead one-compound(OBOC)combinatorial library technology is an ultrahigh throughput chemical library synthesis and screening method suitable for ligand discovery against a wide range of biological targets,such as integrins.The OBOC library can be designed based on the main triad motif flanking with random amino acids leading to the discovery of specific ligands with higher affinity and selectivity toward targeted molecules.In addition,non-natural amino acids and disulfide bonds with D-cysteines are chosen to synthesize the ligands with higher stability in vivo.Tnerefore,in this study,we screened OBOC libraries and identified a potent peptide ligand targeting integrin expressed on EPCs and ECs with high binding specificity and affinity,high functionality to EPCs/ECs and high structural stability.We then constructed and modified small-diameter artificial vascular grafts with the peptide ligand and found that the modification vascular grafts with this peptide ligand recruited endogenous EPCs and ECs,resisted the attachment of adverse cells and protein adsorption,promoted in.situ rapid endothelialization and achieved long-term patency of the vacular grafts.Contents,Methods and Results1.Screening and characterization of the specificity and affinity of the small molecular peptide ligand targeting EPCs and ECsMethods:Screening and synthesis of a potent small molecular peptide ligand with high binding specificity and affinity to EPCs and ECs using OBOC combinatorial library technology.Binding Specificity of the ligand to EPCs and ECs were tested using on-bead cell binding assay.The binding specificity and affinity of the ligand and conventional RGD peptide to EPCs and ECs were compared using flow cytometry technology.Surface attachment assay was used to further confirm the binding specificity and affinity of the ligand to EPCs and ECs and it with the conventional RGD peptide.To further confirm that LXW7 binds to EPCs and ECs mainly via ?v?3 integrin,a binding/blocking experiment was performed using a monoclonal anti-?v?3 integrin antibody.Results:We screened and synthesized a small molecular peptide LXW7.LXW7 is a disulfide cyclic octa-peptide(cGRGDdvc)containing unnatural amino acids flanking both sides of the main functional motif.therefore it will be more resistant to proteolysis and more stable in vivo compared to linear peptides and peptides consisting of only natural amino acids.LXW7 showed stronger binding affinity to EPCs and ECs but weaker binding to platelets,and no binding to THP-1 monocytes.The flow cytometry results showed that LXW7 had higher binding affinity with different types of EPCs and ECs than the conventional GRGD peptide.Both ligands did not bind to THP-1 monocytes,and LXW7 had very low binding affinity with platelets,but GRGD showed strong binding to platelets.LXW7 treated surface attracted more ECs than the GRGD modified surface.Additionally,both LXW7 and GRGD treated surfaces did not support THP-1 monocyte attachment similar to the control surface.Furthermore,the LXW7 treated surface showed limited platelet adhesion,whereas the GRGD treated surface allowed a significantly higher number of platelets to attach.The results showed ?av?3 integrin was highly expressed on EPCs and ECs.For the binding/blocking experiment,the results showed LXW7 had high binding efficiencies to EPCs and ECs,and the binding efficiencies of LXW7 to EPCs and ECs that were all significantly blocked by the anti-?av?3 integrin antibody.2.Effect of retention of ECs on LXW7 treated surface under shear stress conditionsMethods:The effects of LXW7 on EPC and EC proliferation were tested using MTS assay.The effect of LXW7 on important signaling pathways related EPC or EC functions were tested using Western-blot assay.The effect of LXW7 on EPC maturation was tested using qPCR assay.The retention of ECs on LXW7 treated surface and fibronectin treated surface under shear stress conditions were compared.Results:The MTS results showed that the LXW7 treated surface significantly promoted EPC and EC proliferation after 48 h in culture compared with the D-biotin treated surface,and this trend was maintained for the entire period of the experiment.The MTS results also showed that the proliferation of EPCs and ECs cultured on the LXW7 modified surface temporarily were not changed after LXW7 removal.Compared with the control group,the Western-blot results showed LXW7 treated surface significantly increased the phosphorylation of VEGFR2 and phosphorylation of ERK1/2 in ECs,and also significantly increased the phosphorylation of ERK1/2 in EPCs.Compared with the negetive control group,the qPCR results showed no significant difference in the expression of KDR,NOS3,CD144,vWF,JAG1,DLL1 and HEY1 on EPCs cultured on LXW7 surface.Compared with mature ECs,the expression of KDR on EPCs were higher,other genes were lower.Under shear stress conditions,ECs can be well retained on the LXW7 treated surface,and the retained cells were more than the cells retained on fibronectin treated surface.3.Fabrication and function of vascular graftsMethods:The artitical vascular scaffold was fabricated using electrospinning technology.The LXW7-N3 was synthesized for chemistry reaction.An optimal protocol was developed to immobilize LXW7 onto the scaffold surface using 'click chemistry'.Attenuated total reflectance-fourier transform infrared(ATR-FTIR)technology and amino acid analysis(AAA)technology were used to determine the immobilization of LXW7 on the scaffold surface.The attachment,proliferation and spreading of ECs on LXW7 modified surface were evaluated in vitro.Results:Microfibrous tubular grafts were fabricated by electrospinning 19%poly-L-lactide(PLLA)and 5%poly-caprolactone(PCL)polymer blends onto a rotating mandrel(diameter = 1 mm).The structure of the grafts was similar to the native extracellular matrix(ECM).The elastic modulus of the grafts was in the same order of magnitude as the native arteries.The results of ATR-FTIR and AAA showed LXW7 had been successfully immobilized on the scaffold surface.Compared with the control group,the results showed that the function of LXW7 was well maintained after chemical modification,and LXW7 modified scaffold surface supported excellent EC attachment,growth and spreading.4.Evaluation of endothelialization and patency of functionalized vascular graftsMethods:Vascular grafts were implanted into the left common carotid artery of(Sprague-Dawley)SD rats by anastomosis and examined at various time points for up to 6 weeks,with 6 animals per group at each time point.Histological analysis of the cross sections of the grafts was performed to confirm the endothelialization and patency.Immunohistochemistry was performed to evaluate recruitment of endogenous EPCs and ECs,rapid endothelialization and endothelium coverage of the grafts.Results:The results showed a lot of new capillary formation on LXW7 modified grafts,but almost none on untreated grafts.Hematoxylin and eosin(H&E)staining of the grafts showed the luminal surface of LXW7 modified grafts was widely open with only little thrombus formation,but the untreated grafts had significant amount of thrombus which is the major mechanism for clogging and failure in small-diameter grafts.The CD31 staining of LXW7 modified grafts showed the obvious EC coverage on the inner and outer surfaces of grafts and many ECs penetrated into the graft materials.For further test,at 1 week,all 100%(6 of 6)of LXW7 modified grafts were patency after implantation,but only 50%(3 of 6)of untreated grafts were patent.At 2 weeks,83%(5 of 6)of LXW7 modified grafts were patent after implantation,but only 17%(1 of 6)of untreated grafts were patent.At 6 weeks,still 83%(5 of 6)of LXW7 modified grafts were still patency after implantation,but only 17%(1 of 6)of untreated grafts were patent.En face immunofluorescence staining results showed,at 1 week,almost no cells were observed on the untreated grafts.In contrast,on LXW7 modified grafts,a large number of CD31 positive cells migrated from adjacent carotid arteries and few EPCs recruited from the blood flow were found at the proximal and distal ends of the grafts.Many CD34 positive cells recruited from blood flow were found in the middle portion of the grafts.At 2 week,low numbers of CD31 positive cells were found on the untreated grafts.On LXW7 modified grafts,the cell density on the grafts was significantly higher than the grafts at 1 week.Especially,a large number of CD31 positive cells were found in the middle portion of the graft suggesting that EPCs have already differentiated into ECs.At 6 week,the LXW7 modified grafts were almost completely covered by CD31 positive ECs,but the untreated grafts have limited endothelialization.ConclusionsThis project has successfully provided a novel solution to the problems associated with currently available small-diameter vascular grafts and achieved rapid endothelialization and long-term patency.The findings in this study provide a promising way of designing and developing functional small-diameter vascular grafts and improving the clinical applications of vascular grafts in the treatment of vascular diseases in the future.(1)Screening and synthesis of a potent small molecular peptide ligand via OBOC combinatorial library technology with high binding specificity and affinity,high functionality to EPCs and ECs and high structural stability both in vitro and vivo.(2)ECs can retain better on the LXW7 treated surface than on the fibronectin treated surface under shear stress condition.(3)Microfibrous vascular scaffolds were fabricated by 19%PLLA and 5%PCL polymer blends using electrospinning technology.The structure of the scaffold was similar to the native ECM and the elastic modulus of the scaffold was in the same order of magnitude as the native artery.(4)An optimal protocol was developed to immobilize LXW7 onto the scaffold surface via 'click chemistry'.(5)Rapid endothelialization and long-term patency of the small-diameter vascular grafts were successfully achieved in this project.