Peking University China-Japan Friendship School of Clinical Medicine, Beijing, China.
Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing, China.
Metabolism. 2016 Dec;65(12):1755-1767. doi: 10.1016/j.metabol.2016.09.007. Epub 2016 Sep 28.
Recent evidence has suggested that circulating endothelial progenitor cells (EPCs) can repair the arterial endothelium during vascular injury. However, a reliable source of human EPCs is needed for therapeutic applications. In this study, we isolated human fetal aorta (HFA)-derived EPCs and analyzed the capacity of EPCs to differentiate into endothelial cells. In addition, because microvascular dysfunction is considered to be the major cause of diabetic foot (DF), we investigated whether transplantation of HFA-derived EPCs could treat DF in a rat model.
EPCs were isolated from clinically aborted fetal aorta. RT-PCR, fluorescence-activated cell sorting, immunofluorescence, and an enzyme-linked immunosorbent assay were used to examine the expressions of CD133, CD34, CD31, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), von Willebrand Factor (vWF), and Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1). Morphology and Dil-uptake were used to assess function of the EPCs. We then established a DF model by injecting microcarriers into the hind-limb arteries of Goto-Kakizaki rats and then transplanting the cultured EPCs into the ischemic hind limbs. Thermal infrared imaging, oxygen saturation apparatus, and laser Doppler perfusion imaging were used to monitor the progression of the disease. Immunohistochemistry was performed to examine the microvascular tissue formed by HFA-derived EPCs.
We found that CD133, CD34, and VEGFR2 were expressed by HFA-derived EPCs. After VEGF induction, CD133 expression was significantly decreased, but expression levels of vWF and ELAM-1 were markedly increased. Furthermore, tube formation and Dil-uptake were improved after VEGF induction. These observations suggest that EPCs could differentiate into endothelial cells. In the DF model, temperature, blood flow, and oxygen saturation were reduced but recovered to a nearly normal level following injection of the EPCs in the hind limb. Ischemic symptoms also improved. Injected EPCs were preferentially and durably engrafted into the blood vessels. In addition, anti-human CD31+-AMA+-vWF+ microvasculars were detected after transplantation of EPCs.
Early fetal aorta-derived EPCs possess strong self-renewal ability and can differentiate into endothelial cells. We demonstrated for the first time that transplanting HFA-derived EPCs could ameliorate DF prognosis in a rat model. These findings suggest that the transplantation of HFA-derived EPCs could serve as an innovative therapeutic strategy for managing DF.
最近的证据表明，循环内皮祖细胞（EPCs）可以在血管损伤时修复动脉内皮。然而，治疗应用需要可靠的人类 EPCs 来源。在这项研究中，我们分离了人胎儿主动脉（HFA）衍生的 EPCs，并分析了 EPCs 分化为内皮细胞的能力。此外，由于微血管功能障碍被认为是糖尿病足（DF）的主要原因，我们研究了 HFA 衍生的 EPCs 移植是否可以治疗大鼠模型中的 DF。
从临床流产的胎儿主动脉中分离 EPCs。使用 RT-PCR、荧光激活细胞分选、免疫荧光和酶联免疫吸附试验检测 CD133、CD34、CD31、血管内皮生长因子受体 2（VEGFR2）、血管性血友病因子（vWF）和内皮白细胞黏附分子 1（ELAM-1）的表达。形态学和 Dil 摄取用于评估 EPCs 的功能。然后，我们通过将微载体注入 Goto-Kakizaki 大鼠后肢动脉来建立 DF 模型，然后将培养的 EPCs 移植到缺血后肢中。使用热红外成像、血氧饱和度仪和激光多普勒灌注成像监测疾病的进展。进行免疫组织化学检查以检查 HFA 衍生的 EPCs 形成的微血管组织。
我们发现 HFA 衍生的 EPCs 表达 CD133、CD34 和 VEGFR2。在 VEGF 诱导后，CD133 的表达明显降低，但 vWF 和 ELAM-1 的表达水平显著增加。此外，在 VEGF 诱导后，管形成和 Dil 摄取得到改善。这些观察结果表明 EPCs 可以分化为内皮细胞。在 DF 模型中，温度、血流和氧饱和度降低，但在下肢注射 EPC 后恢复到接近正常水平。缺血症状也得到改善。注入的 EPCs 优先且持久地植入血管。此外，在移植 EPCs 后检测到抗人 CD31+ -AMA+-vWF+微血管。
早期胎儿主动脉衍生的 EPCs 具有强大的自我更新能力，并可分化为内皮细胞。我们首次证明，移植 HFA 衍生的 EPCs 可以改善大鼠模型中的 DF 预后。这些发现表明，移植 HFA 衍生的 EPCs 可以作为治疗 DF 的一种创新治疗策略。
