Thrombospondin-1 inhibits angiogenesis and promotes follicular atresia in a novel in vitro angiogenesis assay.
Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P < 0.01) and promoted follicular apoptosis (P < 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P < 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.
Garside
Samantha A
SA
Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, Queens Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom.
Harlow
Christopher R
CR
Hillier
Stephen G
SG
Fraser
Hamish M
HM
Thomas
Fiona H
FH
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D005496:Follicular Atresia / Q000502:physiology*; D018919:Neovascularization, Physiologic; D006080:Ovarian Follicle / Q000098:blood supply / Q000254:growth & development*; D051381:Rats; D017208:Rats, Wistar; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1 / Q000502:physiology*; D046509:Tissue Culture Techniques; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology
D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D040262:Receptors, Vascular Endothelial Growth Factor