Minami Yoshiyasu, Nakajima Toshiaki, Ikutomi Masayasu, Morita Toshihiro, Komuro Issei, Sata Masataka, Sahara Makoto
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Department of Ischemic Circulatory Physiology, 22nd Century Medical and Research Center, University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Department of Cardiovascular Medicine, Teikyo University Chiba Medical Center, 3426-3 Anegasaki, Ichihara 299-0111, Japan.
Int J Cardiol. 2015;186:305-14. doi: 10.1016/j.ijcard.2015.03.166. Epub 2015 Mar 17.
Recent studies have suggested that late-outgrowth endothelial progenitor cells (EPCs) derived from human peripheral blood mononuclear cells (hPBMNCs) might have higher angiogenic potential than classically-defined early-outgrowth EPCs (EOCs). However, it still remains unclear which of "so-called" EPC subpopulations defined in a variety of ways has the highest angiogenic potential.
We classified hPBMNC-derived EPC subpopulations by the time of their emergence in culture. EOCs were defined as attached cells on culture days 3-7. Late-outgrowth EPCs, defined as the cell forming colonies with cobblestone appearance since day 10, were further classified as follows: "moderate"-outgrowth EPCs (MOCs) emerging on days 10-16, "late"-outgrowth EPCs (LOCs) on days 17-23, and "very late"-outgrowth EPCs (VOCs) on days 24-30. Flow cytometry analyses showed the clear differences of hematopoietic/endothelial markers between EOC (CD31(+)VE-cadherin(-)CD34(-)CD14(+)CD45(+)) and LOC (CD31(+)VE-cadherin(+)CD34(+)CD14(-)CD45(-)). We found that LOCs had the highest proliferation and tube formation capabilities in vitro along with the highest expression of angiogenic genes including KDR and eNOS. To investigate the in vivo therapeutic efficacies, each EPC subpopulation was intravenously transplanted into immunocompromised mice (total 4 × 10(5) cells) after unilateral hindlimb ischemia surgery. The LOC-treated mice exhibited significantly-enhanced blood flow recovery (flow ratios of ischemic/non-ischemic leg: 0.99±0.02 [LOC group] versus 0.67 ± 0.07 to 0.78 ± 0.09 [other groups]; P < 0.05) and augmented capillary collateral formation in ischemic leg, which were attributable to their direct engraftment into host angiogenic vessels (approximately 10%) and paracrine effects.
hPBMNC-derived late-outgrowth EPCs emerging on culture days 17-23 are superior to other EPC subpopulations with regard to therapeutic angiogenic potential.
最近的研究表明，源自人外周血单个核细胞（hPBMNCs）的晚期生长内皮祖细胞（EPCs）可能比经典定义的早期生长EPCs（EOCs）具有更高的血管生成潜力。然而，以各种方式定义的“所谓”EPC亚群中，哪一种具有最高的血管生成潜力仍不清楚。
我们根据hPBMNCs来源的EPC亚群在培养中的出现时间进行分类。EOCs定义为培养第3 - 7天贴壁的细胞。晚期生长EPCs定义为自第10天起形成鹅卵石样集落的细胞，并进一步分类如下：第10 - 16天出现的“中度”生长EPCs（MOCs）、第17 - 23天出现的“晚期”生长EPCs（LOCs）以及第24 - 30天出现的“极晚期”生长EPCs（VOCs）。流式细胞术分析显示EOC（CD31(+)VE - 钙黏蛋白(-)CD34(-)CD14(+)CD45(+)）和LOC（CD31(+)VE - 钙黏蛋白(+)CD34(+)CD14(-)CD45(-)）之间造血/内皮标志物存在明显差异。我们发现LOCs在体外具有最高的增殖和管形成能力，同时包括KDR和eNOS在内的血管生成基因表达也最高。为了研究体内治疗效果，在单侧后肢缺血手术后，将每个EPC亚群静脉注射到免疫缺陷小鼠体内（共4×10(5)个细胞）。接受LOC治疗的小鼠表现出显著增强的血流恢复（缺血/非缺血腿的血流比值：0.99±0.02[LOC组]对0.67±0.07至0.78±0.09[其他组]；P<0.05）以及缺血腿毛细血管侧支形成增加，这归因于它们直接植入宿主血管生成血管（约10%）和旁分泌作用。
培养第17 - 23天出现的hPBMNCs来源的晚期生长EPCs在治疗性血管生成潜力方面优于其他EPC亚群。
