Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

One driving factor for developing preeclampsia—a pregnancy disorder, often associated with poor spiral artery (SpA)-remodeling and fetal growth restriction—is the anti-angiogenic sFLT1 (soluble fms-like tyrosine kinase-1), which is found to be highly upregulated in preeclampsia patients. The sFLT1-mediated endothelial dysfunction is a common theory for the manifestation of maternal preeclampsia symptoms. However, the influence of sFLT1 on SpA-remodeling and the link between placental and maternal preeclampsia symptoms is less understood. To dissect the hsFLT1 (human sFLT1) effects on maternal and/or fetoplacental physiology in preeclampsia, sFLT1-transgenic mice with systemic hsFLT1 overexpression from midgestation onwards were used. SpA-remodeling was analyzed on histological and molecular level in placental/mesometrial triangle tissues. Maternal kidney and aorta morphology was investigated, combined with blood pressure measurements via telemetry. hsFLT1 overexpression resulted in maternal hypertension, aortic wall thickening, and elastin breakdown. Furthermore, maternal kidneys showed glomerular endotheliosis, podocyte damage, and proteinuria. preeclampsia symptoms were combined with fetal growth restriction already at the end of the second trimester and SpA-remodeling was strongly impaired as shown by persisted vascular smooth muscle cells. This phenotype was associated with shallow trophoblast invasion, delayed presence of uterine natural killer cells, and altered lymphatic angiogenesis. Overall, this study showed that circulating maternal hsFLT1 is sufficient to induce typical maternal preeclampsia-like symptoms in mice and impair the SpA-remodeling independent from the fetoplacental compartment, revealing new insights into the interaction between the placental and maternal contribution of preeclampsia.

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1–mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.