Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-ß-cyclodextrin (MßCD) to disrupt caveolae. We found that after MßCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MßCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Oxygen regulation of aquaporin-4 in human placenta.

RESEARCH QUESTION: We recently reported that blocking of placental aquaporins (AQP) abrogates the apoptotic response of the trophoblast. As trophoblast apoptosis is exacerbated in pre-eclampsia, we hypothesized that changes in AQP in these placentae may trigger programmed cell death. We analysed AQP4 expression in pre-eclamptic placentae and its regulation by oxygen tension. DESIGN: AQP4 expression was studied in placentae from non-pathological and pre-eclamptic pregnancies by reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence and immunohistochemistry. Explants from non-pathological placentae were cultured in normoxia, hypoxia, hypoxia-reoxygenation and CoCl2. AQP4 expression was investigated by RT-PCR and Western blot. Hypoxia responsive elements sites on AQP4 promotor were investigated by in-silico analysis. AQP4 degradation was studied in the presence of proteosomal and lysosomal inhibitors. RESULTS: AQP4 protein expression was weakly detectable in pre-eclamptic placentae, but its mRNA was elevated compared with non-pathological placentae. In non-pathological explants cultured in hypoxia, AQP4 mRNA and protein were increased compared with placentae cultured in ambient oxygen but decreased after reoxygenation. Incubation with CoCl2, that stabilizes hypoxia inducible factor (HIF)-1α, also increased AQP4 levels. In-silico analysis showed three putative binding sites for HIF-1α in AQP4 promotor. CONCLUSIONS: Oxygen may regulate AQP4 expression in human placenta, possibly through HIF-1α. Therefore, the decrease in AQP4 throughout pregnancy, previously reported, is consistent with changes in HIF-1α, and suggests that AQP4 might have a crucial role during placentation. Therefore, the abnormal expression of AQP4 may be involved in the cause of pre-eclampsia, but it does not seem to take part in the apoptotic events.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia.

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

New Insights Into the Role of Placental Aquaporins and the Pathogenesis of Preeclampsia.

Accumulated evidence suggests that an abnormal placentation and an altered expression of a variety of trophoblast transporters are associated to preeclampsia. In this regard, an abnormal expression of AQP3 and AQP9 was reported in these placentas. Recent data suggests that placental AQPs are not only water channel proteins and that may participate in relevant processes required for a normal placental development, such as cell migration and apoptosis. Recently we reported that a normal expression of AQP3 is required for the migration of extravillous trophoblast (EVT) cells. Thus, alterations in this protein might lead to an insufficient transformation of the maternal spiral arteries resulting in fluctuations of oxygen tension, a potent stimulus for oxidative damage and trophoblast apoptosis. In this context, the increase of oxygen and nitrogen reactive species could nitrate AQP9, producing the accumulation of a non-functional protein affecting the survival of the villous trophoblast (VT). This may trigger the exacerbated release of apoptotic VT fragments into maternal circulation producing the systemic endothelial dysfunction underlying the maternal syndrome. Therefore, our hypothesis is that the alteration in the expression of placental AQPs observed at the end of gestation may take place during the trophoblast stem cell differentiation, disturbing both EVT and VT cells development, or during the VT differentiation and turnover. In both situations, VT is affected and at last the maternal vascular system is activated leading to the clinical manifestations of preeclampsia.

Expression of aquaporin-3 (AQP3) in placentas from pregnancies complicated by preeclampsia.

Recently, we have reported that the blocking of AQP3 abrogates the apoptotic response of the trophoblast. Since trophoblast apoptosis is exacerbated in preeclampsia, we hypothesized that placental AQP3 is increased in these placentas in order to trigger the programmed cell death. Here, we examined mRNA levels, protein expression and localization of AQP3 in placentas from pregnancies complicated by preeclampsia and against what we expected, we found that AQP3 expression was significantly reduced, both at protein and mRNA levels, compared to normal placentas. In the light of our results, further studies are required to evaluate whether the decreased expression of AQP3 might be an adaptive response of the placenta to reduce the trophoblast apoptosis, which is related to the clinical manifestations of preeclampsia.