TGFβ signalling pathway regulates angiogenesis by endothelial cells, in an adipose-derived stromal cell/endothelial cell co-culture 3D gel model.
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).


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.
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Shiyu
S
Lin
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Jing
J
Xie
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Tao
T
Gong
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Sirong
S
Shi
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Tao
T
Zhang
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Na
N
Fu
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Ling
L
Ye
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Min
M
Wang
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
Yunfeng
Y
Lin
D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors*; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D011720:Pyrazoles; D011804:Quinolines; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; C557799:LY-2157299; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9