This work is the first systematic study to comprehensively illustrate the effect of the APELA signal pathway on the luteal angiogenesis. In this study, we found that APELA/APLNR as a factor that helps to regulate luteal angiogenesis, suppression of which would result in luteal dysfunction and angiogenic defects. Regarding the mechanism, APELA/APLNR upregulates TGFβ1 and TGFβR1, and activates SMAD3 phosphorylation, and ultimately sensitizes the luteal angiogenesis.
APLNR is expressed on reproductive system, such as ovary, uterus and CL [25, 26], whereas the expression of APELA is still unclear in reproductive system. Our study found that luteal cells express high levels of APELA and APLNR, especially luteal granulosa cells. This result is partially similar to Mercatil’s result, in which APELA and APLN (another ligand of APLNR), are high expressions in luteal cells [27]. However, whether APELA has an effect on the luteal function is still unknown, so we conducted further investigation through relevant experiments.
The main function of CL is to produce progesterone [1]. The progesterone production depends on the expression of ovarian steroidogenic enzymes. Here we found that APELA/APLNR has been demonstrated to enhance progesterone secretion and affect endocrine functional enzymes expression, such as Star, Hsd3b1, Cyp11a1 and Cyp19a1. This is similar to the report by Roche et al., in which they revealed that APLN promoted steroidogenesis in human luteinized granulosa cells [28]. These results suggest that APELA/APLNR is a promoting factor, which can trigger endocrine function.
Luteal angiogenesis is the guarantee for growth and development of CL [29]. It forms a dense capillary network to supply nutrients efficiently to the luteal cells and provides low density lipoprotein cholesterol acts as a substrate for steroid formation [30]. CD31 is the marker of angiogenesis, and Vegfa, Ang2, Tie2, Fgf2, and hif1 are the biomarker of the luteal angiogenesis [31]. The results displayed that the inhibition of APELA/APLNR expression led to a decreased expression of CD31 [32], suggests that APELA/APLNR has effect on angiogenesis. And, our results found that APELA/APLNR enhance the mRNA levels of pro-angiogenesis factor, such as Vegfa, Ang2, Tie2, and hif1. Interestingly, because of the suppressive effect of FGF2 by APLNR [33], the expression levels of Fgf2 mRNA is shown to be opposite performance. These results reveal that the role of APELA/APLNR for luteal angiogenesis is benefit.
Further, we investigated whether it is possible to accelerate angiogenesis in vitro culture by adding exogenous APELA to the culture media. Our results showed that APELA is a favorable element in the multiple stages of angiogenesis, including endothelial cell proliferation, migration, endothelial cell tip formation, and lumen formation, those of which are the very important processes in the angiogenesis [34]. In addition, APELA promotes the mRNA levels of pro-angiogenesis factors, such as Vegfa, Ang2, Tie2, and hif1. And, the protein level of VEGFA is increasing. These results reveal that APELA/APLNR is a proangiogenesis factor and may be beneficial to angiogenesis. Nevertheless, the potential mechanism of its action was still unrevealed.
To determine the mechanism of APELA/APLNR in luteal angiogenesis, we detected the expression levels of TGFβ pathway, which plays an important role in the reproductive system [35, 36] and ovarian angiogenesis [37]. The results showed that ML221 inhibited VEGFA expression along with decreased expression levels of TGFβ1, TGFβR1 and SMAD3 phosphorylation. We also demonstrated in vitro that APELA/APLNR is involved in angiogenesis by regulating the TGFβ pathway. These results provide a supplementary explanation for the mechanism of APELA/APLNR regulating luteal angiogenesis.
Our present study is limited to the effect of APELA/APLNR in luteal angiogenesis. These results require further deepening of the role of APELA/APLNR in luteal disease. According to earlier studies, luteal phase defect (LPD) are closely related to luteal angiogenesis, and the dysregulation of luteal blood flow can lead to luteal dysfunction [38], while impaired angiogenesis can lead to LPD [39, 40]. This suggests that APELA/APLNR could be a potential therapeutic target for LPD.
In conclusion, in this study, we demonstrated that APELA/APLNR is required for the development and function of CL and is involved in luteal angiogenesis through TGFβ pathway. Therefore, APELA/APLNR could be further studied as a therapeutic target for LPD.