Angiogenesis is required for embryonic development and growth for successful hemochorial placentation (for recent reviews, see [23
]). Distinct vascular processes occur during pregnancy, starting with adaptations of the endometrial vasculature to support blastocyst implantation, followed by expansion and de novo formation of blood vessels to support embryo growth and development. Even at later stages of pregnancy, the placental vasculature continues to be remodeled to enable blood flow to the increasing metabolic demands of the fetus. Sufficient uteroplacental blood flow requires remodeling of the spiral arteries by the extravillous trophoblasts [43
]. Failure of spiral artery remodeling has been associated with complications of pregnancy, such as preeclampsia and fetal growth restriction [44
]. Therefore, a better understanding of the stimuli needed for cell production of angiogenic growth factors during the processes of uteroplacental vascular remodeling and the generation of the placental vascular network is of major clinical interest.
Trophoblasts play an important role in the physiological changes of the spiral arteries as well in other processes during establishment of the placental vasculature, but together with trophoblasts, other cell types clearly are involved in the regulation of angiogenesis during pregnancy. Among them are decidual and placental macrophages as well as dendritic, endothelial, uNK, and mesenchymal cells. Many of these cells secrete or respond to angiogenic factors, such as members of the VEGF family [40
Besides its well known role in angiogenesis, VEGFA has been suggested to regulate trophoblast behavior in an autocrine manner [51
]. Other members of the VEGF family, such as PGF and VEGFC, promote endothelial survival and vascular remodeling [38
]. In addition, PGF was shown to play an important role in successful uNK cell proliferation and/or differentiation [37
] and VEGFC to facilitate immune tolerance and endovascular activity of uNK cells [35
]. Therefore, we decided to investigate whether PSG1 induces the expression of different members of the VEGF family. When exploring the possible induction by PSG1 of VEGFC in HTR-8/SVneo cells and of PGF in BeWo cells, we found that these cells express very high basal levels of these growth factors and that whereas we saw significant induction in some experiments, this induction was not reproducible in others. Therefore, at this time, we are unable to reach a definite conclusion.
Expression of VEGFA by placental macrophages has been documented [52
], and whereas macrophages have been shown to be involved in angiogenesis, they require stimulation by activating factors [53
]. We observed that PSG1 induced significant secretion of VEGFA and TGFB1 in primary monocyte/macrophages, with a small variation in the concentration of PSG1 required depending on the donor. The cells used in our in vitro studies most likely do not exactly replicate the phenotype of placental macrophages, but we propose that PSGs could be one of the placental products, which increases the secretion of VEGFA by these cells. Whereas we found that monocyte-derived dendritic cells secreted TGFB1 in response to PSG1 treatment, VEGFA secretion was not induced. Recent reports indicate that uterine dendritic cells fine-tune decidual angiogenesis by producing TGFB1 and secreted FLT1 and that they have an important role in vascular development [49
]. Interestingly, only alternative activation—not TGFB1 treatment—was reported to lead to VEGFA production by dendritic cells [55
]. Therefore, it remains to be investigated whether, in the decidual microenvironment, PSG1 may induce dendritic cells to secrete VEGFA.
When exploring the potential interaction of PSG1 with nonimmune cells, we found that PSG1 induced the proangiogenic factor TGFB1 in different primary endothelial cells, HEEC, the choriocarcinoma cell line BeWo, and two first-trimester extravillous trophoblast cell lines, HTR-8/SVneo and SGHPL-4. Of the cells listed above, VEGFA induction was only observed in the HTR-8/SVneo and SGHPL-4. Therefore, PSG1 could potentially play a role in trophoblast invasion, migration, and differentiation through its ability to regulate VEGFA secretion in EVT.
Our data show that PSG1 is involved in capillary morphogenesis and that it also influences vascular morphogenetic processes induced by VEGFA. PSG1 induced endothelial cell tube formation in the presence and absence of VEGFA. When VEGFA was added to the cells together with PSG1, we observed that the network was enhanced over the cells in which PSG1 or VEGFA was added alone. Further experiments are required to determine the molecular and signaling mechanisms by which PSG1 induces endothelial tube formation. Tube formation likely is not the result of induction of VEGFA by PSG1, because we did not observe significant PSG1-mediated VEGFA secretion in endothelial cells at PSG1 concentrations employed in the tube assays. Additionally, we did not observe an increase in endothelial cell migration upon PSG1 treatment, which would be expected as a result of up-regulation of VEGFA. Therefore, while PSG1 may increase the availability of VEGFA in the placenta, the effect on endothelial cells, because of its ability to induce VEGFA secretion from other cell types, may be of a paracrine nature or could be mediated by the observed up-regulation of TGFB1. PSGs belong to the CEA family, and it is interesting to note that other members of this family, which are membrane bound, have been implicated in the processes of immunoregulation, human trophoblast invasion, and angiogenesis [27
The PSG concentration increases progressively to reach a plateau in the last 4 wk of pregnancy. At 200 μg/ml, PSGs are the most abundant fetal proteins in the maternal bloodstream during late pregnancy [62
]. At this time, and to our knowledge, no specific antibodies can distinguish the different members of the human PSG family, but splice variants for most of them have been reported to be expressed, with most of these including the N-domain [19
]. Recently, transcripts for PSG1 were found in sperm, and PSG1 protein was detected during zygotic development, suggesting a possible role for this protein in the early stages of embryogenesis and/or implantation [63
]. Within the N-domain, most PSGs have an RGD or an RGD-like sequence. The RGD motif forms the minimal functional binding unit in some integrin ligands [64
], and its presence in one of the solvent-exposed loops of most PSGs was assumed to be required for PSG function [20
]. Within this sequence, the conservation of the aspartic acid (D) at position 95 has been cited as essential for function [18
]. Our results indicate that at least for some functions of PSGs, the G in position 93 and the D at position 95 are not essential. Binding assays showed that although the PSG1gdd→sdl
mutant binds to target cells, such as HTR-8/SVneo, significantly over the control protein, the binding (measured as mean fluorescence intensity) was approximately half that of the wild-type protein. Whether other, as-yet-unknown functions of this protein could be mediated by these amino acids, or how the mutation affects the affinity of the interaction with the receptor, remains to be determined.
Mutations of the two conserved potential N
-linked glycosylation sites and of one of the amino acids (R64) involved in the formation of a salt bridge rendered a protein with no activity. The introduced mutations very likely resulted in major conformational changes in the protein. Preliminary data from our laboratory indicates that enzymatic removal of N
-linked carbohydrates with peptide-N-glycosidase F results in a significant reduction of the activity of the protein. In addition, we have previously established that N
-linked glycosylation is required for interaction of murine PSG17 with CD9 [65
]. It remains to be determined whether the removal of the carbohydrates just in the N-domain is enough to destroy the activity of the protein.
At present, the receptor for human PSGs remains unknown. Because human PSGs share 80% identity at the amino acid level, it is worth exploring whether all 11 members of the family bind to the same receptor to perform these functions. We performed our studies with two recombinant PSG1 proteins, which differ in the nature of the tag at the C-terminus. Both proteins had the same activity in target cells with small variations in the concentration of protein required to observe the effects reported.
In the present study, we show that different cell types can respond to PSG1, secreting growth factors known to regulate vascular development and trophoblast behavior. In addition, the in vitro studies described here indicate that the interaction of PSG1 with endothelial cells can have functional consequences. Therefore, we propose that besides their ability to regulate the immune response, PSGs may have the previously unrecognized ability to contribute to the establishment of the vasculature during pregnancy. The precise processes during angiogenesis in the maternal-fetal unit that could be modulated by this family of proteins require further investigation.