EPCs Transplantation For Microvascular Repair In Irradiated Tissue

In spite of the significant advances in surgery, about two-thirds of patientswith head and neck cancer still need radiotherapy or chemoradiotherapyadjuvant to surgery. During the process of radiotherapy, not only the cancer cellsbut also the normal tissues are damaged unavoidably. Endothelial cells areradiosensitive and could be destroyed directly by radiation and indirectly byirradiation-induced reactive oxygen species or free radicals. As a result, vesselespecially the microvessel embolizes or even necroses, finally leading the localischaemia and fibrosis. It’s reported that this stage of poor vascularization wouldlast for a long time or even decades after radiotherapy. This would make thepostoperative reconstruction extremely difficult.Endothelial progenitor cells (EPCs) are the precursors of the endothelialcells. They generally local in the bone marrow, but can be mobilized to thecirculation by ischaemia, injury, cytokines and drugs, and can home to the sitesof ischaemia participating in vascular repair and new vessel formation.Nowadays, EPCs have widely been used for therapy in many kinds of ischemicdiseases, such as myocardial ischemia, brain ischemia, limb ischemia and so on.However, there are very few studies about EPCs used in irradiation-induced ischemia. Therefore, this study tried to study the therapeutic potential of EPCs intreatment of irradiated tissue ischaemia in vitro and in vivo, hoping to provide anew way to improve the ischemic state of the irradiated tissues.Part I：EPCs and microvascular endothelial cells (MVECs) cultureEPCs were separated from the bone marrow of F344inbred rats and thenpurified by colonies selection. Flow cytometry analysis showed the cells werepositive for a panel of markers, including CD34(2.45%), CD144(95.8%)，CD31(80.9%)and VEGFR2(46.6%). Also the cells were positive for vWFimmunofluorescence staining， DiI-AcLDL uptake, FITC-UEA-1binding,Weibel-Palade body containing, and could form tubular-like structures inmatrigel. These results showed that we acquired EPCs successfully.MVECs were separated from gastrocnemius of F344inbred rat and thenpurified by immunomagnetic cell sorting. Flow cytometry analysis showed thatthe purified cells had hight level expression of CD31(96.6%). Also, the cellswere positive for vWF immunofluorescence staining，DiI-AcLDL uptake, andcould form tubular-like structures in matrigel. The results showed that we haveisolated the MVECs with high purity.Part II: The change of MVECs after radiation and its effect on EPCsactivation, chemotaxis and adhesionMVECs were irradiated with a single dose of20Gy X-ray using linearaccelerator. Results showed that the irradiated MVECs displayed no cellularproliferation, weak migration and low tube-forming capacity, but had high levelexpression of SDF-1, ICAM-1, VCAM-1and E-selectin.Further, we investigated the activation effect, chemotactic effect andadhesion effect of irradiated MVECs on EPCs. Results showed that theirradiated MVECs could not only enhance the expression level of CXCR4inEPCs, but also successfully induce the EPCs migration and adhesion. Moreover, the migration and adhesion capacities of the EPCs which were pre-coculturedwith the irradiated MVECs were much higer than those of the normal ones. Thuspre-coculture with irradiated MVECs could be a useful method to activate EPCs.During the processes of EPCs migration and adhesion, SDF-1/CXCR4axis andthe adhesion moleculars ICAM-1, VCAM-1and E-selectin played importantroles.Part III: EPCs transplantation for microvascular repair in irradiated tissueIn order to label the EPCs before transplataion, we constructed thelenti-EmGFP and transduction the EmGFP gene into the EPCs. Results showedthat more than90%EPCs could be successfully labeded when the MOI was50.With the purpose of finding the right time for EPCs transplantion, we usedthe irradiated lower hindlimb in rat for the animal model and investigated thehistological changes and the expression levels of SDF-1, ICAM-1, VCAM-1and E-selectin. Results showed that3weeks after radiation, inflammatoryreaction mainly disappeared, but the expression of SDF-1, ICAM-1, VCAM-1and E-selectin kept in high levels. Thus, this time might be a relative right timefor EPCs transplataion.In the selective time, we transplanted the pre-cocultured EPCs into the ratvia tail vein injection. Results showed that EPCs could successfully home to theirradiated tissue and contribute to microvesscular repair, helping to improve theblood flow in irradiated region.Together, this study proved that EPCs transplantation could enhance themicrovessculars repair in irradiated tissue. It would be a new methed to solvedthe ischaemia problem after irradiation, and worth further investigation.