Abstract
OBJECTIVE: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.