Syncytialization of BeWo trophoblasts induces changes in angiogenic signaling and response to hypoxia

Abstract

During pregnancy, the placenta produces significant amounts of pro and anti‐angiogenic factors, notably VEGF, PlGF, and sFlt‐1. The exact cell population(s) most responsible for this production is not definitively known. In the event of placental insufficiency, placental ischemia can cause alterations in the production of these factors, favoring the anti‐angiogenic sFlt‐1 protein. This mechanism is believed to be a major driver of preeclampsia. Within the placenta, the maternal/fetal interface is defined by a layer of specialized syncytiotrophoblasts (ST) which line the maternal side of the underlying fetal chorionic vessels. These large multinucleated cells define the selectivity of nutrient/waste exchange from the maternal to the fetal circulation. In studying trophoblast function, immortalized trophoblast‐derived cell lines are commonly utilized, such as the BeWo choriocarcinoma cell line. These cells however, do not spontaneously syncytialize, and therefore do not likely reflect in vivo ST function. We hypothesized that inducing syncytialization of BeWo cells would alter their production of angiogenic factors in vitro. Syncytialization of BeWo cells was induced with forskolin treatment for 48 hours. At 48 hours, cellular morphology shifted from dense cobblestone patterning into majority large, multinucleated cells. Syncytialization caused ~5 fold increase in both VEGF and PlGF mRNA (p<0.0001). There were significant (p<0.0001) increases in the levels of sFlt‐1 splice variants 2 (~5 fold), 3 (~50 fold), and 4 (~3fold). As a result, there were significant (p,<0.005) ~2 fold increases in secreted VEGF and PlGF protein, while sFlt‐1 went from not detectable to 53 pg/ml as measured by ELISA. Perhaps most interestingly, when cultured in oxygen concentrations mimicking healthy and ischemic placentas (8% and 1% respectively), there was differential regulation of these factors at the transcriptional level. There was no change in VEGF, while sFlt V2 and V3 were significantly upregulated (2‐fold each, p<0.0005), while PlGF was significantly down regulated by 40% (p<0.0001). Taken together, these data suggest that syncytialization significantly alters expression of both pro and anti‐angiogenic factors in vitro. Future studies should take syncytialization state into account when extrapolating from cell culture to in vivo placental function.