Objective— Transplant arteriosclerosis is the leading cause of graft failure and death in patients with heart transplantation. Endothelial progenitor cells (EPCs) contribute to endothelial regeneration in allografts. We investigated whether increased HDL cholesterol induced by adenoviral human apoA-I (AdA-I) transfer increases number and function of EPCs, promotes incorporation of EPCs in Balb/c allografts transplanted paratopically in C57BL/6 ApoE −/− mice, and attenuates transplant arteriosclerosis. Methods and Results— EPC number in ApoE −/− mice was increased after AdA-I transfer as evidenced by 1.5-fold ( P <0.01) higher Flk-1 Sca-1–positive cells and 1.4-fold ( P <0.01) higher DiI-acLDL isolectin-positive spleen cells. In addition, HDL enhanced EPC function in vitro. Incorporation of bone marrow–derived EPCs was 5.8-fold ( P <0.01) higher at day 21 after transplantation in AdA-I-treated apoE −/− mice compared with control mice. Enhanced endothelial regeneration in AdA-I-treated apoE −/− mice as evidenced by a 2.6-fold ( P <0.01) increase of CD31-positive endothelial cells resulted in a 1.4-fold ( P =0.059) reduction of neointima and a 3.9-fold ( P <0.01) increase of luminal area. Conclusion— Human apoA-I transfer increases the number of circulating EPCs, enhances their incorporation into allografts, promotes endothelial regeneration, and attenuates neointima formation in a murine model of transplant arteriosclerosis.
Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34 ؉ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 ؋ 10 3 /L). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagenrelated peptide and thrombin receptoractivating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ib␣ (GPIb␣) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo. (Blood. 2009;114:5044-5051) IntroductionHuman platelets are anucleated cells that not only play a crucial role in primary hemostasis and wound repair, but are also particularly important in pathologic conditions such as thrombosis, vascular remodeling, and inflammation. Platelets originate from megakaryocytes (MK) in the bone marrow (BM) by fragmentation of pseudopodial elongations called proplatelets in a process that consumes the entire cytoplasmic content and is tightly regulated by thrombopoietin. 1,2 Human megakaryopoiesis has been studied ex vivo by measuring colony-forming units (CFUs; eg, CFU-MK, CFU-granulocyte, erythrocyte, macrophage, megakaryocyte), 3,4 MK polyploidy state, 5,6 expression of MK markers, 7 and novel genes expressed during MK differentiation. [8][9][10] Unraveling molecular mechanisms involved in megakaryopoiesis and thrombopoiesis is particularly relevant in the light of thrombocytopenia and pancytopenia associated with widespread use of high-dose chemotherapy for treatment of most cancers, also occurring after stem cell transplantation. Therefore, there has been an increasing interest in generating human platelets from MK in culture as well as in developing animal models of human hematopoiesis.Human platelet production has been described from differentiation of CD34 ϩ progenitor cells, isolated from mobilized peripheral blood (PB) or umbilical cord blood (CB), cultured in medium with a cytokine mixture containing thrombopoietin. 5,[11][12][13][14][15] Such produced platelets are functional, as demonstrated in aggregation assays and by expression of P-selectin on the platelet surface or by activation of glycosylphosphatidylinosito...
The dose of cells expressing the surface antigen CD34 (CD34+) has been shown to be a reliable predictor of the time to engraftment following transplantation of PBPC to support high-dose chemotherapy. However, evaluation of rare cells is complicated by a number of factors, including the variability in operator and technical procedures. Recently, Becton Dickinson Immunocytometry Systems introduced a new CD34+ cell analysis system, the ProCOUNT cell enumeration kit, which automates the analysis of CD34+ cells and minimizes the variabilities of this procedure. We have evaluated the ProCOUNT system in comparison to a standard CD34 cell analysis (based on the Milan approach) using leukapheresis products from patients and normal donors mobilized with chemotherapy plus recombinant human G-CSF (rhG-CSF) or with rhG-CSF alone. In addition, we compared these analyses using CD34+ cell-selected mobilized leukapheresis products with purities of 75% or greater. The standard CD34 cell analysis methodology quantitated the frequency of cells identified as CD45+, low side scatter, and CD34+. A high correlation coefficient was obtained between the ProCOUNT methodology and the standard CD34 cell analysis methodology for cells obtained from leukapheresis products mobilized with chemotherapy plus rhG-CSF (r = 0.98), rhG-CSF alone (r = 0.96), and CD34+-selected mobilized leukapheresis products (r = 0.83). A comparison was also made between technicians using both analysis methods. Whereas the correlation coefficient between two technicians using the standard methodology was r = 0.77, the correlation coefficient was much higher when using ProCOUNT (r = 0.99). These data demonstrate that the use of ProCOUNT is associated with less variability between data analyzed by different operators. Also, ProCOUNT is consistent with existing CD34+ cellular analysis methodologies. An additional advantage is the ability to determine the absolute concentration of CD34+ cells, thereby allowing calculation of total CD34+ cell numbers without using WBC counts, which also have inherent errors. The ProCOUNT system provides an automated analysis procedure that minimizes the variables in CD34+ cell analysis and may be useful for standardization of methodology between laboratories.
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