This was a mouse model study conducted to assess the role of the COUP-TFI gene in the expression pattern of some major genes involved in the process of placentation. Here we found that VEGF-A and Bax mRNA expression was augmented in COUP-TFI null mice compared to wild-type mice placentas. The positive correlations between the following mRNA pairs observed in normal placental tissue were lost in COUP-TFI null mutants: Bcl-2 and INHA; p21 and ENG; HIF1α and Flt-1. Interestingly, COUP-TFI null mice resulted also to have a significantly lower weight at birth compared to wild-type mice.
We focused our attention on COUP-TFI because this family of proteins carries out vital roles in physiological processes, including proliferation, apoptosis and cell signaling (18). Thanks to the level of conservation of COUP-TFI, understanding the pathological pathway in relation to its expression in mice models could help to focus future studies on genes that are known to be relevant during the process of physiological placental development. In particular, we analyzed the expression of Hif1α, ENG, Flt1, PlGF, VEGF-A, which are main players in angiogenesis and vascular pathfinding, P21 and P53 in cellular proliferation, as well as Bax, Bcl2 and INHA, which are involved in apoptosis and cell survival. All these genes regulate crucial aspects of placental development, and their variation was shown to be associated with impaired development of the ongoing pregnancy (19,20).
When considering the influence of COUP-TFI on the expression of the most common angiogenetic factors, we found that VEGF-A mRNA was consistently up-regulated in Coup-TFI −/− placentas compared to control mice. The isoform A of the vascular endothelial growth factor (VEGF-A), belonging to the VEGF family, is considered the most crucial factor promoting the differentiation of mesenchymal cells in villi into hemangioblastic stem cells. VEGF-A expression is induced by hypoxia, as a potent stimulus, and is mediated via HIF1α expression (21–24). Indeed, VEGF-A is strongly expressed by the cytotrophoblast cells during the first trimester of pregnancy and strong evidence indicates that high VEGF-A expression in fetal growth restriction reflects the hypoxic status of the placenta (25,26).
Supporting this evidence, we found in COUP-TFI null mutants a lost in the positive correlation between mRNA expression of HIF1α and Flt-1, this latter encoding the vascular endothelial growth factor receptor 1 (VEGFR1), one of the receptors for vascular endothelial growth factors (VEGF). In a hypoxic environment, Hif1α could regulate the expression of VEGF-A, Flt-1 and other angiogenic factors, being this a compensatory mechanism aimed to restore normal placental blood flow and rescue the normoxia (27,28). This finding overlap with studies showing that VEGF-A mRNA and protein levels are significantly reduced in patients with growth restriction and that an adenovirus-mediated overexpression of VEGF can improve fetal growth in a sheep model (29,30). Regarding the other angiogenic factors considered in our study, PlGF and ENG, no significant differences were found in COUP-TFI −/− placentas compared to control mice.
Considering the most common genes involved in cell proliferation and survival control, we observed an increase of Bax mRNA in COUP-TFI−/− placentas. An augmented Bax expression is in line with other studies conducted on human placenta (31–33). Bax is a pro-apoptotic protein that exerts, in concert with the anti-apoptotic protein Bcl-2, a crucial role in apoptosis. Both are regulated by the p53 tumor suppressor gene (34). Apoptosis contributes to the turnover of villous trophoblasts and plays a crucial function in the remodeling of spiral arteries in human placenta. Apoptosis in placental villi changes throughout normal pregnancy: it is low in the first trimester, increases in the second, and then reaches the highest levels beyond 40 weeks of gestation (35). Furthermore, the amount of apoptosis is increased in villous trophoblast in placental pathologies, including preeclampsia (36).
In addition, we observed a significant positive correlation of Bcl-2 and INHA in normal mice. This could be due to a regulatory role of INHA on trophoblast growth through inhibition of activin receptor, known to have a fundamental role in trophoblast development and correct placentation (37). This, in turn, could result in reduced placenta proliferation and increased apoptosis characterizing old placentas at the end of gestation (38). According to our study, this correlation seems to be disturbed by the absence of COUP-TFI.
Interestingly, we also observed in COUP-TFI −/− mice a level of p53 expression that was lower than its downstream target p21 and that the expression rate of the two genes positively correlated. This data confirmed previous data on human placentas (20). We could further hypothesize a role of p21 independent from p53. Usually p53, through p21, promotes cell cycle arrest or apoptosis through the augmented expression of Bax (34,39). In the current study, we found a significant positive correlation between p21 and ENG. ENG is part of the transforming growth factor-beta receptor complex. Angiogenesis, apoptosis, and cell cycle arrest could be promoted by the transforming growth factor-beta pathway, reported to be implicated in fetal growth restriction (40). p21 could possibly interact in placental tissue with this cascade triggered by transforming growth factor-beta receptor complex (41). As the correlation between p53 and p21 is lost in COUP-TFI -/- mice, this interaction could be disturbed by the absence of COUP-TFI
Finally, we looked at mice phenotype linker to placental function in terms of weight of pups recorded at birth. Interestingly, our results showed that COUP-TFI −/− mice presented a significant lower weight than WT controls. These data further support the pathological significance of COUP-TFI in placental development, potentially related to fetal growth restriction, a common complication associated with impaired placental function in humans (13,42,43). Our preliminary results support further studies on specific downstream cascades of molecular markers found to be linked to COUP-TFI, both in mouse models and humans.
Limits of the study
This was an explorative study performed to find out potential markers involved in impaired placental function linked to COUP-TFI disruption. Thus, this study lacks a detailed study of mechanisms underlying the downstream regulation of angiogenic, cell regulation and apoptotic factors included in this mouse model. Moreover, in our experiments we have not assessed the function COUP-TFII, a highly redundant to COUP-TFI transcription factor. COUP-TFII, being more expressed in developing organs, shows a high degree of homology at the amino acid level with COUP-TFI. Even though above-mentioned peculiarity, COUP-TFI and COUP-TFII expression patterns overlaps in many areas, possibly resulting in redundant functions (9,44,45). Thus, COUP-TFII may be able to mitigate for the absence of COUP-TFI in COUP-TFI −/− mice. Further experiments on placentas lacking both COUP-TFI and COUP-TFII deriving from matings between COUP-TFI −/− and COUP-TFII −/− mutant mice, could shed new light on the interplay between these two orphan receptors during placenta development.