Data_Sheet_3_Endothelial GABBR2 Regulates Post-ischemic Angiogenesis by Inhibiting the Glycolysis Pathway.ZIP

Purpose: Angiogenesis post-ischemia plays an essential role in preventing ischemic damage to tissue by improving the blood recovery. Determining the regulatory mechanism of ischemic angiogenesis, therefore, could provide effective therapeutics for ischemic injury.
Materials and Methods: The RNA sequencing (RNA-seq) database was used to predict the association of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) with endothelial-specific expression. The role of GABBR2 in angiogenesis was verified in vitro by downregulating GABBR2 in human umbilical vein endothelial cells (HUVECs) with lentiviral vectors. Besides, the in vivo effect of GABBR2 on the blood recovery of an ischemic hindlimb was demonstrated by establishing a hindlimb ischemia model in normal and GABBR2 adenoviral vector-infected mice. Then, the mobilization of endothelial progenitor cells (EPCs) in peripheral blood post-ischemia was determined by flow cytometry. Finally, the XF analyzer and Western blot were used to determine the effect of GABBR2 on endothelial metabolism.
Results: The RNA-seq results indicated a strong association between GABBR2 and endothelial revascularization, and the upregulation of GABBR2 was detected in both hypoxia-treated HUVECs and ischemic mouse hindlimb. Hypoxia treatment for 6 h increased the proliferation, migration, and tube formation of HUVECs, which were inhibited by GABBR2 knockdown. Additionally, GABBR2 downregulation significantly decreased the blood flow recovery of mouse ischemic hindlimb. The expressions of the EPC markers CD34+ and CD133+ significantly decreased in the peripheral blood in hindlimb post-ischemia. Mechanically, glycolysis-dominated metabolism of HUVECs was compromised by GABBR2 knockdown. Evidences of the decreased expressions of HKII, PFKFB3, and PKM1 also supported the compromised glycolysis induced by GABBR2 downregulation.
Conclusion: Our study demonstrated that GABBR2 regulated angiogenesis post-ischemia by inhibiting the glycolysis pathway.

研究目的：缺血后血管生成可通过改善血液灌注恢复，在预防组织缺血性损伤中发挥核心作用。因此，解析缺血性血管生成的调控机制，能够为缺血性损伤的临床治疗提供有效策略。
材料与方法：本研究利用RNA测序（RNA-seq）数据库，预测γ-氨基丁酸B型受体亚基2（gamma-aminobutyric acid type B receptor subunit 2, GABBR2）与内皮特异性表达的关联。通过慢病毒载体下调人类脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）中GABBR2的表达，在体外验证GABBR2对血管生成的调控作用。此外，通过在正常小鼠及GABBR2腺病毒载体感染小鼠中构建缺血后肢模型，验证GABBR2对缺血后肢血液灌注恢复的体内调控效果。采用流式细胞术（flow cytometry）检测缺血后外周血中内皮祖细胞（endothelial progenitor cells, EPCs）的动员情况。最后，利用XF分析仪及蛋白质印迹（Western blot）技术，分析GABBR2对内皮细胞代谢的影响。
研究结果：RNA-seq分析结果显示，GABBR2与内皮血管重建存在显著关联；且在缺氧处理的HUVECs及缺血小鼠后肢组织中，均检测到GABBR2的表达上调。缺氧处理6小时可增强HUVECs的增殖、迁移及管腔形成能力，而GABBR2敲低可显著抑制上述效应。此外，GABBR2表达下调可显著降低小鼠缺血后肢的血流灌注恢复水平。缺血后肢模型小鼠外周血中，内皮祖细胞标志物CD34+及CD133+的表达水平显著降低。机制研究显示，GABBR2敲低可损害以糖酵解为主的内皮细胞代谢；糖酵解关键酶HKII（己糖激酶II）、PFKFB3（6-磷酸果糖-2-激酶/果糖-2-磷酸酶3）及PKM1（丙酮酸激酶M1）的表达下调，进一步证实了GABBR2表达下调可抑制糖酵解通路。
研究结论：本研究证实，GABBR2可通过抑制糖酵解通路调控缺血后血管生成。