Impaired fetoplacental angiogenesis in growth-restricted fetuses with abnormal umbilical artery doppler velocimetry is mediated by aryl hydrocarbon receptor nuclear translocator (ARNT)

Abstract

Context: Fetal growth restriction with abnormal umbilical artery Doppler velocimetry (FGRadv), reflective of elevated fetoplacental vascular resistance, is associated with increased risks of fetal morbidity and mortality even in comparison to those of growth-restricted fetuses with normal placental blood flow. One major cause of this abnormally elevated fetoplacental vascular resistance is the aberrantly formed, thin, elongated villous vessels that are seen in FGRadv placentas.

Objective: The purpose of this study was to determine the role of fetoplacental endothelial cells (ECs) in angiogenesis in normal pregnancies and in those complicated by FGRadv.

Design and participants: Human placental specimens were obtained from FGRadv and gestational age-matched, appropriately grown control pregnancies for EC isolation/culture and for immunohistochemical studies. Additional mechanistic studies were performed on ECs isolated from subjects with term, uncomplicated pregnancies.

Main outcome measures: We evaluated tube formation and differential angiogenic gene expression in FGRadv and control ECs, and we used ECs from uncomplicated pregnancies to further elucidate the molecular mechanisms by which angiogenesis is impaired in FGRadv pregnancies.

Results: Tube formation assays showed that FGRadv ECs demonstrate fewer branch points and total length compared with those from gestational age-matched controls, and this defect was not rescued by exposure to hypoxia. FGRadv ECs also demonstrated lower aryl hydrocarbon receptor nuclear translocator (ARNT) expression. ARNT knockdown resulted in suppression of key angiogenic genes including vascular endothelial growth factor A expression and led to deficient tube formation.

Conclusions: ARNT expression in the placental vasculature mediates key angiogenic expression and fetoplacental EC angiogenesis, and low ARNT expression in FGRadv ECs appears to be a key factor in deficient angiogenesis. This, in turn, results in malformed thin villous vessels that structurally contribute to the abnormally elevated fetoplacental vascular resistance that is associated with high morbidity and mortality in fetal growth restriction.

Figures

Figure 1.

Matrigel tube formation assays in the presence of normoxia and hypoxia. A, Representative tube formation images with ECs isolated from pregnancies complicated by FGRadv demonstrate deficient tube formation compared with that of their gestational age–matched controls. Tube formation is not able to be rescued in the setting of hypoxia. B, Graphical representation shows that the impaired tube formation occurred as a result of fewer branch points and total tube length as calculated by the ImageJ Angiogenesis Analyzer. Overall P < .0001 for one-way ANOVA: a and b, P < .001; c, P < .01; d, P < .001. C, Representative Western blot demonstrates that both control and FGRadv ECs are hypoxia responsive via stabilization of HIF1α and HIF2α protein expression. N, normoxia; H, hypoxia.

Figure 2.

Expression profiles of key angiogenic proteins. A, Western blot and densitometric analysis demonstrate differential ARNT and VEGFA expression within total protein extracted from FGRadv ECs (n = 6) in comparison to controls (n = 6; *, P < .05; ∧, P = .058). B, This difference was also seen via immunofluorescence utilizing a different ARNT antibody with a representative image shown. C, Representative immunohistochemical analyses and modified H-scoring also demonstrate lower ARNT expression in fetoplacental endothelium of FGRadv placentas (n = 8) than that for gestational age-matched controls (n = 8; #, P < .01).

Figure 3.

RNAi of ARNT effect on VEGFA expression. A, Real-time PCR demonstrates that hypoxia exposure results in induction of VEGFA, ANGPTL4, and PTGIS expression under control siRNA transfection. In contrast, this induction is suppressed in the setting of ARNT knockdown. Overall P < .01 for one-way ANOVA: a, P < .001 in comparison to normoxic control siRNA transfection; b, P < .005 in comparison to normoxic control siRNA transfection; c, P < .0001 in comparison to hypoxic control siRNA transfection; d, P < .0001 compared with expression after hypoxic control siRNA transfection. B, Representative Western blot demonstrating ARNT knockdown effects. C, Densitometric analysis of Western blots after ARNT knockdown confirm mRNA results. Overall P < .05 for one-way ANOVA: e and f, P < .001 in comparison to normoxic control siRNA transfection; g, P < .001 in comparison to hypoxic control siRNA conditions; h, P < .05 compared with expression after hypoxic control siRNA transfection. N, normoxia; H, hypoxia.

Figure 4.

Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding of the HIF1α/ARNT heterodimer complex to key HREs within the VEGFA proximal promoter when normalized to IgG. Overall P < .05 for one-way ANOVA: *, P < .0001; ∧, P < .001.

Figure 5.

Tube formation assays after ARNT knockdown. A, Representative images of tube formation after transfection with either control siRNA or ARNT siRNA in the presence and absence of hypoxia. B, ARNT knockdown also results in decreased branch points. Overall P < .01 for one-way ANOVA: a and b, P < .05 under normoxic and hypoxic conditions. Similarly, ARNT knockdown also suppressed tube length under normoxic conditions. Overall P < .05 for one-way ANOVA: c, P < .05, with a trend toward suppressed tube length under hypoxic conditions.

Figure 6.

Schematic diagram of ARNT effects in FGRadv. FGRadv ECs have lower ARNT expression than control ECs. This results in decreased HIF-ARNT heterodimer formation and less binding to key HREs within key angiogenic genes such as the VEGFA promoter, which in turn results in impaired angiogenic potential and response, leading to malformed villous vessels that contribute to abnormal fetoplacental vascular resistance and blood flow as clinically manifested by abnormal umbilical artery Doppler velocimetry.