APELA promotes luteal angiogenesis through the regulation of TGF-β/SMAD3 signaling pathway

Luteal angiogenesis is absolutely required for the development and function of corpus luteum. Recently, APELA has been shown to be essential to embryonic development and angiogenesis. However, whether APELA is involved in the luteal development and angiogenesis remains unknown. In this study, we established a PMSG/hCG superovulating model and spheroid-based angiogenesis assay of HUVECs to analyze the roles of APELA through APLNR in the luteal development and angiogenesis. Our immunohistochemical results showed that APELA and APLNR were highly expressed in the ovarian corpus luteum, but their expression levels significantly decreased in the PGF2α-induced luteolysis model. ML221 treatment of APLNR antagonist could inhibit the development and endocrine function of corpus luteum as evidenced by the downregulation of luteal area and serum progesterone and estradiol and ovarian steroidogenic enzymes mRNA. Furthermore, ML221 significantly suppressed ovarian CD31 staining and expression levels of Ang2 , Tie2 , Vegfa and Hif1 mRNA, followed by decreased expression of ovarian P-SMAD3/SMAD3, TGFβR1 and TGFβ1 proteins. In vitro experiments demonstrated that recombinant APELA significantly increased the rate of sprouts/spheroid and sprout length and tip-cells at the extremity of sprouts, enhanced expression levels of related angiogenic factors and molecules of TGFβ signaling pathway of HUVECs. Nevertheless, ML221 cotreatment could abrogated the promotional effect of APELA. In summary, we conclude that APELA acting through APLNR has an obligatory role in the luteal angiogenesis and corpus luteum formation.