Interaction between human placental microvascular endothelial cells and a model of human trophoblasts: effects on growth cycle and angiogenic profile

Abstract

Intrauterine growth restriction (IUGR) is a leading cause of perinatal complications, and is commonly associated with reduced placental vasculature. Recent studies demonstrated over‐expression of IGF‐1 in IUGR animal models maintains placental vasculature. However, the cellular environment of the placental chorionic villous is unknown. The close proximity of trophoblasts and microvascular endothelial cells in vivo alludes to autocrine/paracrine regulation following Ad‐HuIGF‐1 treatment. We investigated the co‐culturing of BeWo Choriocarcinoma and Human Placental Microvascular Endothelial Cells (HPMVECs) on the endothelial angiogenic profile and the effect Ad‐HuIGF‐1 treatment of one cell has on the other. HPMVECs were isolated from human term placentas and cultured in EGM‐2 at 37°C with 5% CO2. BeWo cells were maintained in Ham's F12 nutrient mix with 10% FBS and 1% pen/strep. Co‐cultured HPMVECS+BeWo cells were incubated in serum‐free control media, Ad‐HuIGF‐1, or Ad‐LacZ at MOI 0 and MOI 100:1 for 48 h. Non‐treated cells and mono‐cultured cells were compared to co‐cultured cells. Angiogenic gene expression and proliferative and apoptotic protein expression were analysed by RT‐qPCR and immunocytochemistry, respectively. Statistical analyses was performed using student's t‐test with P <0.05 considered significant. Direct Ad‐HuIGF‐1 treatment increased HPMVEC proliferation (n =4) and reduced apoptosis (n =3). Co‐culturing HPMVECs+BeWo cells significantly altered RNA expression of the angiogenic profile compared to mono‐cultured HPMVECs (n =8). Direct Ad‐HuIGF‐1 treatment significantly increased Ang‐1 (n =4) in BeWo cells. Ad‐HuIGF‐1 treatment of HPMVECs did not alter the RNA expression of angiogenic factors. Trophoblastic factors may play a key role in placental vascular development and IGF‐1 may have an important role in HPMVEC growth.

Hypoplastic left heart syndrome is associated with structural and vascular placental abnormalities and leptin dysregulation

“…RNA yield and integrity were quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA expression of Leptin, VEGF-A and FLK-1 was analyzed by real-time PCR as previously described [37]. Briefly, 1 µg of purified RNA was reverse transcribed into cDNA using Hi-Cap cDNA Conversion Reaction on MJ Research PTC 200 Thermal Cycler (MJ Research Inc., St. Bruno, Canada) following manufacturer’s protocol.…”

Decidual vascular endothelial cells promote maternal–fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling

“…Mouse primary DVECs were isolated and cultured based on previous literatures 43 , 44 , 45 , 46 . Briefly, deciduas were collected and minced as described above, followed by digestion for 2 h in a shaking incubator at 37 °C in 0.28% (W/V) collagenase II, 0.25% dispase II and 2.5 mg DNase I (all from Roche) in Hepes‐buffered Hanks' balanced salt solution.…”

Single-cell transcriptomics of the human placenta: inferring the cell communication network of the maternal-fetal interface

“…Tissue underwent serial trypsin and collagenase digests as previously described (Troja et al 2014), and the resulting cell suspension was layered on a continuous Percoll gradient. Cells were collected from the 20%-60% fractions.…”