Department of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr-University Bochum, Georgstr. 11, D-32545 Bad Oeynhausen, Germany.
Department of Internal Medicine I, Ludwig-Maximilians-University, Campus Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany.
Cells. 2020 Mar 2;9(3):588. doi: 10.3390/cells9030588.
The potential therapeutic role of endothelial progenitor cells (EPCs) in ischemic heart disease for myocardial repair and regeneration is subject to intense investigation. The aim of the study was to investigate the proregenerative potential of human endothelial colony-forming cells (huECFCs), a very homogenous and highly proliferative endothelial progenitor cell subpopulation, in a myocardial infarction (MI) model of severe combined immunodeficiency (SCID) mice.
CD34+ peripheral blood mononuclear cells were isolated from patient blood samples using immunomagnetic beads. For generating ECFCs, CD34 cells were plated on fibronectin-coated dishes and were expanded by culture in endothelial-specific cell medium. Either huECFCs (5 × 10) or control medium were injected into the peri-infarct region after surgical MI induction in SCID/beige mice. Hemodynamic function was assessed invasively by conductance micromanometry 30 days post-MI. Hearts of sacrificed animals were analyzed by immunohistochemistry to assess cell fate, infarct size, and neovascularization (huECFCs = 15 vs. control = 10). Flow-cytometric analysis of enzymatically digested whole heart tissue was used to analyze different subsets of migrated CD34+ /CD45+ peripheral mononuclear cells as well as CD34/CD45 cardiac-resident stem cells two days post-MI (huECFCs = 10 vs. control = 6).
Transplantation of human ECFCs after MI improved left ventricular (LV) function at day 30 post-MI (LVEF: 30.43 ± 1.20% vs. 22.61 ± 1.73%, < 0.001; ΔP/ΔT 5202.28 ± 316.68 mmHg/s vs. 3896.24 ± 534.95 mmHg/s, < 0.05) when compared to controls. In addition, a significantly reduced infarct size (50.3 ± 4.5% vs. 66.1 ± 4.3%, < 0.05) was seen in huECFC treated animals compared to controls. Immunohistochemistry failed to show integration and survival of transplanted cells. However, anti-CD31 immunohistochemistry demonstrated an increased vascular density within the infarct border zone (8.6 ± 0.4 CD31 capillaries per HPF vs. 6.2 ± 0.5 CD31 capillaries per HPF, < 0.001). Flow cytometry at day two post-MI showed a trend towards increased myocardial homing of CD45 /CD34 mononuclear cells (1.1 ± 0.3% vs. 0.7 ± 0.1%, = 0.2). Interestingly, we detected a significant increase in the population of CD34/CD45/Sca1 cardiac resident stem cells (11.7 ± 1.7% vs. 4.7 ± 1.7%, < 0.01). In a subgroup analysis no significant differences were seen in the cardioprotective effects of huECFCs derived from diabetic or nondiabetic patients.
In a murine model of myocardial infarction in SCID mice, transplantation of huECFCs ameliorated myocardial function by attenuation of adverse post-MI remodeling, presumably through paracrine effects. Cardiac repair is enhanced by increasing myocardial neovascularization and the pool of Sca1 cardiac resident stem cells. The use of huECFCs for treating ischemic heart disease warrants further investigation.
内皮祖细胞（EPCs）在缺血性心脏病中的潜在治疗作用是心肌修复和再生的主题，受到了广泛的研究。本研究的目的是研究人内皮集落形成细胞（huECFCs）在严重联合免疫缺陷（SCID）小鼠心肌梗死（MI）模型中的促再生潜能。
使用免疫磁珠从患者的外周血单核细胞中分离 CD34+细胞。为了生成 ECFCs，将 CD34 细胞铺在纤维连接蛋白包被的培养皿上，并在内皮特异性细胞培养基中进行扩增。在 SCID/beige 小鼠手术后诱导 MI 后，将 huECFCs（5×10）或对照培养基注射到梗死区周围。在 MI 后 30 天通过传导微测压术进行侵入性血流动力学功能评估。用免疫组化分析处死动物的心脏，以评估细胞命运、梗死面积和新血管生成（huECFCs = 15 与对照 = 10）。通过酶消化的全心脏组织的流式细胞术分析，分析 MI 后两天不同亚群的迁移 CD34+/CD45+外周单核细胞以及 CD34/CD45 心脏驻留干细胞（huECFCs = 10 与对照 = 6）。
与对照组相比，MI 后移植 huECFCs 可改善 LV 功能（LVEF：30.43±1.20% vs. 22.61±1.73%，<0.001；ΔP/ΔT 5202.28±316.68 mmHg/s vs. 3896.24±534.95 mmHg/s，<0.05）。此外，与对照组相比，huECFC 治疗动物的梗死面积明显减小（50.3±4.5% vs. 66.1±4.3%，<0.05）。免疫组化未能显示移植细胞的整合和存活。然而，抗 CD31 免疫组化显示梗死边界区的血管密度增加（8.6±0.4 CD31 毛细血管/高倍视野 vs. 6.2±0.5 CD31 毛细血管/高倍视野，<0.001）。MI 后两天的流式细胞术显示心肌归巢 CD45/CD34 单核细胞的趋势增加（1.1±0.3% vs. 0.7±0.1%，=0.2）。有趣的是，我们检测到 CD34/CD45/Sca1 心脏驻留干细胞的数量明显增加（11.7±1.7% vs. 4.7±1.7%，<0.01）。亚组分析显示，来自糖尿病或非糖尿病患者的 huECFCs 的心脏保护作用无显著差异。
在 SCID 小鼠心肌梗死模型中，huECFCs 的移植通过减轻不良的 MI 后重塑作用改善了心肌功能，可能是通过旁分泌作用。通过增加心肌新血管生成和 Sca1 心脏驻留干细胞池来增强心脏修复。使用 huECFCs 治疗缺血性心脏病值得进一步研究。
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