DataSheet_1_FGA Controls VEGFA Secretion to Promote Angiogenesis by Activating the VEGFR2-FAK Signalling Pathway.docx

BackgroundOur previous work revealed the high expression of fibrinogen alpha chain (FGA) in patients with endometriosis (EM) and that it could promote the migration and invasion of endometrial stromal cells. Angiogenesis is the key condition for the development of EM. This study was aimed to elucidate the role of FGA in endometrial stromal cells involved in angiogenesis in EM.
MethodsImmunohistochemistry was used to detect the microvessel density (MVD) and VEGF expression in the eutopic endometrium samples from EM and non-EM. The conditioned medium (CM) of human primary eutopic endometrial stromal cells (EuESC) and immortalized endometrial stromal cell line hEM15A with FGA knockdown were collected and used to treat human umbilical vein endothelial cells (HUVECs). Then, tube formation assay, EdU assay, wound assay, transwell assay and flow cytometry assays were performed to assess the function of HUEVCs in vitro. The angiogenic capability of HUVECs was further measured using a matrigel plug assay with BALB/c nude mice in vivo. Immunofluorescence was used to detect the expression of F-actin and VE-cadherin. RT-PCR and western blotting were used to detect the expression of angiogenesis-related factors in endometrial stromal cells and downstream signalling pathways in HUVECs.
ResultsMVD and VEGF expression in the eutopic endometrium of EM patients were significantly higher than those in the normal endometrium of non-EM patients, and the increased MVD in EM indicates an increased risk of recurrence. Functionally, we found that CM of endometrial stromal cells with FGA knockdown could inhibit HUEVCs migration and tube formation in vitro and in vivo, while having no significant effect on HUVECs proliferation, apoptosis and cell cycle. Mechanically, the expression of VEGFA, PDGF, FGF-B, VEGF, MMP-2 and MMP-9 was reduced in hEM15A cells with FGA knockdown. CM of hEM15A cells with FGA knockdown reduced the number of microfilaments and pseudopodia, as well as the expression of VE-cadherin, and inhibited the activity of VEGFR2 and the FAK signalling pathway in HUVECs.
ConclusionOur study demonstrated FGA could enhance the interaction between endometrial stromal cells and HUVECs via the potential VEGA-VEGFR-FAK signalling axis and promote EM angiogenesis, revealing a promising therapeutic approach for EM.

研究背景：我们既往的研究揭示了纤维蛋白原α链（fibrinogen alpha chain, FGA）在子宫内膜异位症（endometriosis, EM）患者中呈高表达，且其可促进子宫内膜间质细胞的迁移与侵袭。血管生成是子宫内膜异位症发生发展的关键环节。本研究旨在阐明FGA在子宫内膜异位症患者子宫内膜间质细胞参与血管生成过程中的作用。

研究方法：本研究采用免疫组化（immunohistochemistry）法检测子宫内膜异位症患者与非子宫内膜异位症患者的在位子宫内膜样本中的微血管密度（microvessel density, MVD）与血管内皮生长因子（vascular endothelial growth factor, VEGF）的表达水平。收集敲低FGA的人原代在位子宫内膜间质细胞（human primary eutopic endometrial stromal cells, EuESC）以及永生化子宫内膜间质细胞系hEM15A的条件培养基（conditioned medium, CM），用以处理人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）。随后通过管形成实验、EdU实验、划痕实验、Transwell实验以及流式细胞术等体外实验评估HUVECs的功能。进一步通过BALB/c裸鼠基质胶栓实验体内检测HUVECs的血管生成能力。采用免疫荧光法检测F-肌动蛋白（F-actin）与血管内皮钙粘蛋白（VE-cadherin）的表达。通过RT-PCR与蛋白质印迹法检测子宫内膜间质细胞中血管生成相关因子的表达，以及HUVECs中的下游信号通路活性。

研究结果：子宫内膜异位症患者在位子宫内膜中的MVD与VEGF表达水平显著高于非子宫内膜异位症患者的正常子宫内膜，且子宫内膜异位症患者升高的MVD提示复发风险增加。功能实验显示，敲低FGA的子宫内膜间质细胞条件培养基可在体内外抑制HUVECs的迁移与管形成能力，但对HUVECs的增殖、凋亡及细胞周期无显著影响。机制层面，敲低FGA的hEM15A细胞中VEGFA、血小板衍生生长因子（PDGF）、成纤维细胞生长因子-B（FGF-B）、VEGF、基质金属蛋白酶-2（MMP-2）以及基质金属蛋白酶-9（MMP-9）的表达均下调。敲低FGA的hEM15A细胞条件培养基可减少HUVECs的微丝与伪足数量，降低VE-钙粘蛋白的表达，并抑制HUVECs中血管内皮生长因子受体2（VEGFR2）及FAK信号通路（FAK signalling pathway）的活性。

研究结论：本研究证实，FGA可通过潜在的VEGFA-VEGFR2-FAK信号轴增强子宫内膜间质细胞与HUVECs之间的相互作用，促进子宫内膜异位症的血管生成，为子宫内膜异位症的治疗提供了极具潜力的新策略。