Lin Shiyu, Xie Jing, Gong Tao, Shi Sirong, Zhang Tao, Fu Na, Ye Ling, Wang Min, Lin Yunfeng
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Cell Prolif. 2015 Dec;48(6):729-37. doi: 10.1111/cpr.12222. Epub 2015 Oct 21.
This study aimed to investigate the role of the TGFβ signalling pathway in angiogenesis in a three-dimensional (3D) collagen gel model, with co-culture between adipose-derived stromal cells (ASCs) and endothelial cells (ECs).
A 3D collagen gel, implanted with green fluorescent protein-labelled mouse ASCs and red fluorescent protein-labelled mouse ECs, was established in vitro. Phenomena of angiogenesis with or without type I TGFβ receptor inhibitor (LY2157299) treatment, were observed 7 days post-implantation, using confocal laser scanning microscopy. To detect expression of angiogenesis-related genes, semi-quantitative PCR and quantitative real-time PCR were conducted. Zymography was performed to explore secretion of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) after treatment with LY2157299 of 5, 10, 20 to 50 μm concentrations, for 24 h.
Angiogenesis was found to be attenuated in co-culture gels after ASC and EC treatment with LY2157299. Genes VEGF-A, VEGF-B, VE-ca, FGF-1, FGF-2, PDGF, HGF, BMP-4 were significantly reduced in the presence of LY2157299 in both mono-cultured and co-cultured ECs. Furthermore, reduction in co-cultured ECs was prominent relative to mono-cultured ECs, while the same results did not occur to ASCs. We further confirmed that gelatinases secreted by ECs were reduced in a dose-dependent manner, after treatment with LY2157299.
These results indicate that in ASC/EC co-culture, the TGFβ signalling pathway regulated angiogenesis via EC activity. Co-cultured ECs were regulated more significantly than mono-cultured ECs suggesting that inhibition of TGFβRI may regulate paracrine secretion of ASCs to further modulate EC angiogenesis.
本研究旨在探讨转化生长因子β（TGFβ）信号通路在三维（3D）胶原凝胶模型血管生成中的作用，该模型采用脂肪来源的基质细胞（ASC）与内皮细胞（EC）共培养。
体外构建植入绿色荧光蛋白标记的小鼠ASC和红色荧光蛋白标记的小鼠EC的3D胶原凝胶。植入后7天，使用共聚焦激光扫描显微镜观察有无I型TGFβ受体抑制剂（LY2157299）处理时的血管生成现象。为检测血管生成相关基因的表达，进行了半定量PCR和定量实时PCR。进行酶谱分析以探究用5、10、20至50μm浓度的LY2157299处理24小时后基质金属蛋白酶2（MMP - 2）和基质金属蛋白酶9（MMP - 9）的分泌情况。
用LY2157299处理ASC和EC后，共培养凝胶中的血管生成减弱。在单培养和共培养的EC中，LY2157299存在时，血管内皮生长因子A（VEGF - A）、血管内皮生长因子B（VEGF - B）、血管内皮钙黏蛋白（VE - ca）、成纤维细胞生长因子1（FGF - 1）、成纤维细胞生长因子2（FGF - 2）、血小板衍生生长因子（PDGF）、肝细胞生长因子（HGF）、骨形态发生蛋白4（BMP - 4）基因显著减少。此外，相对于单培养的EC，共培养的EC中的减少更为显著，而ASC未出现相同结果。我们进一步证实，用LY2157299处理后，EC分泌的明胶酶呈剂量依赖性减少。
这些结果表明，在ASC/EC共培养中，TGFβ信号通路通过EC活性调节血管生成。共培养的EC比单培养的EC受到更显著的调节，提示抑制TGFβRI可能调节ASC的旁分泌分泌，从而进一步调节EC血管生成。
