This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A pilot study was supported by using SNPRC fund during this period. The study was planned to develop a baboon model to investigate circulating endothelial progenitor cell (CEPC) mobilization and maturation kinetics in baboons. The results of this study will serve as pilot data for an NIH grant application aimed at identifying the factors, particularly the genetic factors, which may influence individual variation in cellular responses which in turn control susceptibility to CVD development progression and recovery. We have completed the following works using this fund. 1. Identify mouse monoclonal antibodies reacting with baboon cell surface antigens. Because most of monoclonal antibodies to identify the CEPC are originally produced against the corresponding human antigens, these antibodies must be tested for cross-reactivity with baboon cells. We have screened a panel of mouse monoclonal antibodies by flow cytometry method; and determined a clone that has immunological specificity to the baboon sample. We have used this antibody to characterize CEPC and validated positive cross-reactivity results by functional analyses of EPCs that show features of endothelial lineage.2. Investigate circulating CEPC kinetics after G-CSF mobilization. Granulocyte colony-stimulating factor (G-CSF) administration is a well-established stimulator for CEPC mobilization. We have quantified CEPC numbers at different times to document the time- and dose-dependent changes of CEPC mobilization in five baboons after G-CSF injection. Our results have indicated the maximal time point and magnitude of mobilized CEPC in peripheral blood as well as bone marrow samples. 3. We hand-picked representative colonies from both groups and spun down by Cytofuge (StatSpin) onto slides. Immunostaining with various antibodies were carried out. Images from these Immunocytochemical analysis revealed that the colonies dominant (11 out of 15 colonies) in G-CSF treatment samples expressed highly a wide range of hematopoietic related proteins (CD45, CD14 and CD235a), as well as endothelial cell markers like CD31 and CD146, but not vWF. 4 out of 15 colonies had positive anti-vWF staining in this group. However, colonies harvested from arterial ligation group (14 out of 15 colonies) have shown to stain strongly for CD31, CD146, and vWF, moderate CD45 positive cells, and negative CD14 and CD235a in these samples. Ultimately, we would like to utilize 500 genetically characterized pedigreed baboons to characterize genes that contribute to baseline and mobilizable CEPCs. To reach this goal, we will need to establish a baboon model to study CEPC kinetics in peripheral blood.
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