Chen Lei, Lu Xiao-Sheng, Li Ya-Lan, Mao Zhou-Fei, Xiao Luan-Juan, Yu Yan-Hong
Department of Anesthesia, First Affiliated Hospital, Jinan University, Guangzhou 510632, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2017 May 20;37(5):614-621. doi: 10.3969/j.issn.1673-4254.2017.05.08.
To analyze the effect of globular adiponectin on angiogenesis of ovarian microvascular endothelial cells (OMECs).
Mouse OMECs were isolated and purified by density gradient centrifugation with Percoll and identified by immunofluorescence analysis of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and endothelial cell marker von Willebrand factor (vWF). The capillary-like tube formation of OMECs was determined by vascular endothelial growth factor A (VEGFA) treatment in Matrigel matrix. OMECs treated with recombinant globular adiponectin protein were examined for cell proliferation with MTS assay and cell migration with scratch wound healing assay, and capillary-like tube formation was tested in Matrigel matrix. Western blotting was performed to detect the effect of globular adiponectin on AMPK phosphorylation.
The signals of LHR and vWF, but not that of FSHR, were detected in the isolated cells. VEGFA treatment of the cells induced capillary-like tube formation, indicating their properties of ovarian-specific endothelial cells. Treatment with 1 and 3 µg/mL of recombinant globular adiponectin significantly increased the number of OMECs by (158.72∓14.50) % and (186.50∓4.20)% (P<0.01) and resulted in scratch wound closure rates of (49.43∓3.43)% (P<0.05) and (69.67∓1.2) % (P<0.01) respectively. The cells treated with 3 µg/mL globular adiponectin formed a capillary-tube length 6.63∓0.66 folds greater than that formed by the control cells (P<0.01). Treatment of the cells with 3 µg/mL globular adiponectin for 15 and 30 min resulted in pAMPK/AMPK ratios of 0.86∓0.08 and 0.66∓0.13, respectively significantly higher than that in the control cells (0.13∓0.12, P<0.01). Compound C obviously suppressed the tube formation and AMPK phosphorylation induced by globular adiponectin.
Globular adiponectin promotes angiogenesis of OMECs through activation of the AMPK signal pathway.
分析球形脂联素对卵巢微血管内皮细胞（OMECs）血管生成的影响。
采用Percoll密度梯度离心法分离纯化小鼠OMECs，并通过卵泡刺激素受体（FSHR）、黄体生成素受体（LHR）和内皮细胞标志物血管性血友病因子（vWF）的免疫荧光分析进行鉴定。在基质胶中用血管内皮生长因子A（VEGFA）处理来测定OMECs的毛细血管样管形成。用重组球形脂联素蛋白处理的OMECs，通过MTS法检测细胞增殖，通过划痕伤口愈合试验检测细胞迁移，并在基质胶中测试毛细血管样管形成。进行蛋白质免疫印迹法检测球形脂联素对AMPK磷酸化的影响。
在分离的细胞中检测到LHR和vWF的信号，但未检测到FSHR的信号。用VEGFA处理细胞可诱导毛细血管样管形成，表明其具有卵巢特异性内皮细胞的特性。用1和3μg/mL重组球形脂联素处理显著增加了OMECs的数量，分别增加了（158.72±14.50）%和（186.50±4.20）%（P<0.01），并导致划痕伤口闭合率分别为（49.43±3.43）%（P<0.05）和（69.67±1.2）%（P<0.01）。用3μg/mL球形脂联素处理的细胞形成的毛细血管管长度比对照细胞形成的长6.63±0.66倍（P<0.01）。用3μg/mL球形脂联素处理细胞15和30分钟后，pAMPK/AMPK比值分别为0.86±0.08和0.66±0.13，明显高于对照细胞（0.13±0.12，P<0.01）。化合物C明显抑制了球形脂联素诱导的管形成和AMPK磷酸化。
球形脂联素通过激活AMPK信号通路促进OMECs的血管生成。
