A Thrombospondin-Mimetic Peptide, ABT-898, Suppresses Angiogenesis and Promotes Follicular Atresia In Vivo in the Monkey.

Abstract

Ovarian folliculogenesis is regulated by many endocrine, paracrine and autocrine factors, and as follicular development progresses, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Angiogenesis is tightly regulated by a variety of pro- and anti-angiogenic factors, including vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aberrant angiogenesis occurs in a number of pathological conditions, such as polycystic ovary syndrome (PCOS) and endometriosis, and novel ways of targeting this could lead to new therapeutics. TSP-1 is an anti-angiogenic factor in many physiological systems and has been shown to have anti-angiogenic effects on follicles in vitro. ABT-428898 is an octpeptide mimetic of TSP-1 and we investigated the effect of ABT-898 on follicular angiogenesis using an in vitro angiogenesis assay, prior to studying its effects in vivo in the common marmoset (Callithrix jacchus). Intact preantral/early antral follicles from 21-day-old rat ovaries were cultured for six days in the presence of ABT-898 (0, 0.1, 1, 10, 100 and 1000nM). At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. In a subsequent experiment, the potential of this peptide to inhibit follicular angiogenesis was investigated in vivo, in marmoset monkeys. The marmosets (n=5) were treated with 2.5mg/kg ABT-898 twice daily throughout the follicular phase of the cycle and control marmosets (n=5) were treated with vehicle. On d10 of the follicular phase, which corresponds to the peri-ovulatory period in controls, all animals were injected with 20mg bromodeoxyuridine (BrdU) one hour before removal of ovaries which were fixed, sectioned and stained for haematoxylin and eosin, CD31, BrdU, CD31/BrdU and caspase-3. Treatment with ABT-898 resulted in the suppression of follicular angiogenesis, a reduction in endothelial cell proliferation at the pre- and early antral stages of follicular development and the suppression of granulosa cell proliferation at early preantral, preantral and early antral stages of follicular development. In addition to the inhibition of angiogenesis, ABT-898 treatment also resulted in a significant increase in the number of large preantral and early antral follicles undergoing apoptosis. However, follicle selection and ovulation were not inhibited by treatment, suggesting that the effects of ABT-898 are stage-specific. Taken together, these results indicate that the TSP peptide is capable of having a dual effect by inhibiting follicular angiogenesis and promoting atresia of antral follicles in vivo, but does not prevent ovulation, as has been observed with direct VEGF inhibitors. These results suggest that the TSP peptide could be a novel therapeutic to inhibit abnormal angiogenesis and induce atresia of accumulated follicles in polycystic ovary syndrome. This research was supported by a Medical Research Council Ph.D. studentship and a core grant to Medical Research Council Reproductive Sciences Unit (U.1276.00.002.00006.01). (poster)