The Roleof Fucosylationinthe Promotion Of Endothelial Progenitor Cells In Neovascularization And Bone Repair

Part I. Fucosylation Promotes Human Cord Blood EndothelialProgenitor Cell Homing and Enhances NeovascularizationObjective: Because of the angiogenic and vasculogenic potential, EPCs have beenexploited as a cell therapy to treat ischemic cardiovascular disease, and in which thetherapeutic efficacy largely depends on EPC homing to intended tissues.The first and mostimportant thing is the binding of p-selectin and E-selectin with the P-selectinglycoprotein(PSGL-1), aprotein expressed by the EPCs. The natural EPCs are lack of thesialyl Lewis X (sLeX), an2,3-sialylated and1,3-fucosylated tetrasaccharide on core-2O-glycan.Treating these cells with an exogenous FucT enhanced selectin ligand expression,we test whether it is good for the the homing of EPCs to the injured tissues and the ability ofangiogenesis and vasculogenesis.Methods: Heparin-anticoagulated cord blood was collected from healthy full-termdeliveries.CD34+cells were purified from the mononuclear cells,isolated by density gradientcentrifugation, and cultured in human fibronectin-coated plates with endothelial cellmedium. We analyze cell surface markers by flow cytometry and immunostaining.Dil-AcLDL uptake was examined under a fluorescent microscope and capillary tubeformation onto the Matrigels under a light microscope. FucT VI protein was analyzed byWestern blotting. Flow cytometry was used to measure cell surface sLeXantigen and totestbinding of E-and P-selectins on EPCs.We also tested the differences between FucT VI-and vector-treated EPCs adhesion to HUVECs.At last, we analysed the role of FucT VIexpression in EPC homing and neovascularization in ischemic muscles in this mouse modelby immunostaining and Laser Doppler perfusion imaging (LDPI).Results:EPCs were isolated from human cord blood. In culture, these cells formedcolonies and had cobblestone-like appearance. In flow cytometry, the EPCs were positive for endothelial markers (CD31, CD105, CD144, CD146and VEGFR2) and progenitor markers(CD34and CD133), but negative for hematopoietic markers (CD14and CD45).Immunostaining confirmed CD31, CD144and VWF expression in the EPCs, which werecapable of up-taking Dil-AcLDL and forming capillary-like tubes in Matrigels. By Westernblotting, FucT VI protein was detected in the conditioned medium and cell lysate from FucTVI-transfected, but not vector-transfected, EPCs. In flow cytometry,<5%of the EPCsexpressed sLeXantigen and <10%of the cells bound to E-or P-selectins.89.92.1%ofFucT VI-treated EPCs (EPCs-FucT) were sLeX-positive and>80%of the cells bound to E-and P-selectins. An~2-fold increase in cell adhesion to tumor necrosis factor-(TNF-α)-activated HUVECs was observed with FucT VI-EPCs, compared with controlEPCs. More Dil-AcLDL-positive EPCs were found in mice treated with FucT VI-EPCs thanwith saline or control EPCs (p<0.001). By CD31staining, most capillaries surroundingmuscle fibers were found in mice treated with FucT VI-EPCs (p<0.01). Consistently, bloodflow in the ischemic legs increased by906and423%, respectively, in the FucT VI-EPCand control EPC groups, compared with that in the saline group.Conclusions: Here we show that EPCs from human cord blood expressed low levels ofsLeXantigen and bound poorly to E-and P-selectins. Such a phenotype can be altered byexpressing FucT VI in these cells. In mouse models of hind limb ischemia, the enhancedfucosylation in EPCs markedly improved EPC homing, neovascularization and tissue repair.Thus, our findings support a possible strategy to use fucosylated EPCs as an improvedcellular source to achieve better therapeutic efficacy in ischemic disease. Part II. Roleof fucosylationinthe promotion of endothelial progenitorcells in bone repairObjective:An appropriate blood supply has been recognized as one of the essentialcomponents of normal healing fracture healing, Inadequate blood supply is a major causeofdelayed union or non-union during fracture healing.Because of the angiogenic andvasculogenic potential, EPCs have been exploited as a cell therapy to bone repair.Early studies from our group in aischemicmouse model described that the enhanced fucosylationin EPCs markedly improved EPC homing, neovascularization and tissue repair.So we wantto test the role of fucosylation of EPCs in the homing to the fracture site and the influence inthe bone healing in a fracture mouse model.Methods: A closed diaphyseal fracture was introduced in the femur by a3-pointbending method. Two h after the bone fracture was introduced, EPCs (1×106cells in100μLsaline) or saline were injected via tail vein. At different time points, mice were anesthetizedfor X-ray radiography and blood flow tests, and then euthanized for micro-CT andhistological analyses. The mRNAs of the EPCs homing to the fracture sites was analyzed byRT-PCR. We analysed the role of FucT VI expression in EPCs homing to the fracture sitesand neovascularization in periosteal tissues around the fracture sites in this mouse model byimmunostaining and Laser Doppler perfusion imaging (LDPI). Serial X-ray radiographs andmicro-CT scanning were taken after bone fracture. Reparative callus areas were quantifiedusing Image J software (NIH).Values for bone volume (BV), total volume (TV) and BV/TVratio of the femur were determined. Sagittal sections (5μm) of fractured femurs were madeand stained with Safranin-O/FastGreen.Histology was examined under a light microscope.Results: EPC homing was examined one week pose-surgery. Dil-AcLDL-positive cellswere absent in saline-injected mice, but more in mice treated with FucT VI-EPCs than that inthecontrol EPC group (766vs.274cells/mm2, p<0.001). By RT-PCR, human CD31andVE-cadherin mRNAs were detected in peri-fracture tissues from mice treated with control orFucT VI-EPCs, but not saline-treated mice. Most abundant blood vessels, shown byPECAM-1-staining, were found in mice treated with FucT VI-EPCs, compared with that inthe control EPC and saline groups. The difference between the FucT VI-and control EPCgroups was greater at2w than4w. The blood flow rate, however, was greater in the FucTVI-EPC group than the other two groups (both p values <0.05), and the flow rate in thecontrol EPC group was greater than the saline group (p<0.05). At2w, the callus area waslarger in EPCs-FucT treated than that in the other two groups (both p values <0.05). By4w,the callus area was smaller in the FucT VI-EPC group than the other two groups (both pvalues <0.05). By6w, the difference between the saline group and the other two EPC groupsremained significant (both p values <0.05). By8w, the callus area was similar in all threegroups.As analyzed in reconstructed3D micro-CT images, the total bone volume wasgreater in FucT VI-EPC-treated mice than the other two groups at2and4w post-surgery. FucT VI-EPC-treated mice had the highest BV/TV ratio throughout the healing period,whereas the ratio in the control EPC group also was higher than the saline group. Byhistological analysis, the callus in the FucT VI-EPC group indicated a faster healing processin this group of mice.Conclusions: In mouse models of femoral fracture, the enhanced fucosylation in EPCscan markedly improve EPC homing, neovascularization and bone healing process with ahigher bone densities in the repaired femurs, which has a broad prospect for the treatment ofbone repair.