| Endothelial progenitor cells (EPC) play critical role in physiogenic and pathologicangiogenesis. The processes that EPC were mobilized from bone marrow to angiogenicsite and participated in angiogenesis were regulated by numerous biochemical factors.Stromal cell derived factor1(SDF-1) is one of the most important factors in regulatingEPC participating in angiogenesis. For a long time, SDF-1was considered to act via itsunique receptor CXCR4. However, recent studies identified an alternative receptor ofSDF-1, CXCR7. CXCR7was widely expressed in hematopoietic System, heart, brain,lung, kidney, testis, voary and many tumor cell lines. CXCR7has significantly higherbinding affinity for SDF-1than CXCR4. The discovery of CXCR7forced people toreconsider the action mechanism of SDF-1. Existing studies found that CXCR7canenhance cell survival, proliferation, adhesion, trans-endothelial migration and play acritical role in tumor development and metastasis, immunocyte maturation and stem cellhoming. Our primary research showed that CXCR7was expressed in EPC, but its rolewas indefinite. Investigating the effect of EPC is meaningful to understand mechanismof angiogenesis. In this study, we chosen EPC derived from human cord blood asresearch object, investigated the role of CXCR7in EPC participating in angiogenesisand tried to uncover partial mechanism. On this basis, we constructed CXCR7high-expressing adenovirus and investigated the feasibility of promoting angiogenesisby elevating CXCR7expression in EPC. Our study mainly includes:①Separation, culture and identification of EPC derived from human cord blood.Mononuclear cells were separated from newborn cord blood by density gradientcentrifugation. Mononuclear cells were induced to differentiate into EPC in EGM-2media, and non-adherent cells were removed by changing media daily for7days. Aftercultured for7days, cells showed round or “cobble-stone” appearance and typical cellclusters appeared. Immunofluorescent staining showed most of mononuclear cells werepositive for CD133and VEGFR2, and more than90%cells were double positive stain.Acetylated low-density lipoprotein (ac-LDL) uptaking and ulex europaeus agglutinin(UEA) binding assay showed that mononuclear cell cultured for7days were positivefor DiI-acLDL and FITC-UEA-1stains, and more than80%cells were double positive.Those cells were defined as EPC. Those results indicated that mononuclear cells can beseparated from newborn cord blood by density gradient centrifugation combining with removing non-adherent cells by changing media daily, and induced to differentiate intoEPC by cultured in EGM-2. EPC that separated using this method has high cell purityand good activity.②The expression and function of CXCR7in EPC. Western Blotting resultsshowed that CXCR7was expressed in human cord blood derived EPC，but itsexpression in EPC is lower when compared with positive control Hela cells. Flowcytometry results indicated that CXCR7was scarcely expressed on cell surface, whileintracellular CXCR7was considerable. Blocking CXCR7by antagonist pretreatment,we investigated the role of CXCR7in SDF-1-inducing EPC migration, proliferation,survival, adhesion to active endothelial cells, trans-endothelial migration, tubeformation, nitric oxide (NO) and MMP-2production. Multiple functional assayssuggested CXCR7had no effect on cell migration, proliferation and NO production butexclusively mediated SDF-1induced cell survival. Besides that, CXCR7can mediateEPC tube formation and MMP-2production along with CXCR4, and blocking CXCR7can interfere with human EPCs adhesion to active endothelial layer andtrans-endothelial migration induced by SDF-1/CXCR4.③Construct CXCR7high-expressing adenovirus with Ad-EASY adenoviralvector system. CDNA was reverse-transcripted from total RNA of G2cell, andamplified CXCR7total length DNA sequence using CXCR7special primers. Then,CXCR7total length DNA sequence was conjuncted with pMD19-T plasmid. Using thisplasmid as template and CXCR7special primers with restriction enzyme cutting site,we amplified CXCR7total length DNA sequence with restriction enzyme cutting siteby PCR. Double restriction enzyme digested CXCR7total length DNA sequence withrestriction enzyme cutting site and pAD Track-CMV shuttle plasmid with correspondingrestriction enzymes. Digested DNA was jointed to pAD Track-CMV shuttle plasmid byT4DNA ligase and transfected into DH5α competent cells. Positive bacterium wasscreened using Kanamycin (Kan). Recombinant shuttle plasmid was extracted andsequenced to confirm that CXCR7total length sequence was correctly amplified.Recombined shuttle plasmid was linearized by Pme I digestion. After that, linearizedplasmid was transformed into BJ5183containing framework plasmid pAd EASY-1tohomologous recombinate by electroporation. Positive bacteriaes were screened by Kan.Plasmid was extracted and recombinant was screeninged by Pac I digestion and furtheridentified by sequencing. Recombinant was linearized with Pac I and transfected intoHEK293cells by liposome method to package virus. Virus was collected and identified by virus PCR. Then, best MOI to transfect EPC was screeninged. Sequencing and PCRidentification showed that CXCR7high-expressing adenovirus was correctlyconstructed and the best MOI for transfecting EPC was100.④Investigate the feasibility of promotion EPC-mediating angiogenesis byelevating CXCR7expression. EPC were transfected with CXCR7high-expressingadenovirus to construct CXCR7high-EPC. The level of CXCR7was investigated byWestern Blotting. To investigate whether elevating CXCR7expression in EPC canpromote angiogenic ability of EPC, we compared cell survival, adhesion to activeHUVEC, trans-endothelial migration and in vitro tube formation betweenCXCR7high-EPC and wt-EPC transfected with control adenovirus. Results displayed:transfecting EPC with CXCR7high expressing adenovirus can significantly elevateCXCR7level by4-5fold. Elevating CXCR7expression in EPC can improve EPCsurvival under serum deprivation, enhance EPC adhesion to active HUVEC andtrans-endothelial migration induced by SDF-1, and promote in vitro tube formation ofEPC.In summary, CXCR7was scarcely expressed on cell surface of human cord bloodderived EPC, while intracellular CXCR7was considerable. Multiple functional assayssuggested CXCR7had no effect on cell migration, proliferation and NO production butexclusively mediated cell survival induced by SDF-1. Besides that, CXCR7can mediateEPC tube formation and MMP-2production along with CXCR4, and blocking CXCR7can interfere with human EPC adhesion to active endothelial layer and trans-endothelialmigration induced by SDF-1/CXCR4. Enhancing CXCR7expression in EPC byadenovirus transfection can promote EPC survival, adhesion to active HUVEC,trans-endothelial migration induced by SDF-1and in vitro tube formation. Thispromotive effect of have synergistic reaction with SDF-1addition. Those resultsindicated that CXCR7is a potential target for regulating angogenesis. Blocking CXCR7can impair angiogenic ability of EPC and elevating CXCR7can enhance it. |
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