Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast

Abstract

Objectives: Preeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known.

Study design: Human primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O(2) or 1%O(2) conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA.

Results: Hypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O(2) culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression.

Conclusions: NO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.

Figure 1

Effects of hypoxia on PGF, VEGF and sFlt-1 mRNA expression in syncytiotrophoblast. Cells were cultured under 21%O₂ or 1%O₂ for 24 hrs, RNA was isolated and relative changes in gene expression determined by qRT-PCR. Data were normalized to controls at 21%O₂; set to 100% and plotted; n = 7 cultures, *=p< 0.01.

Figure 2

Nitric oxide generation significantly increases PGF and VEGF mRNA, but decreases sFlt-1 mRNA in hypoxic syncytiotrophoblast. (A.) Primary syncytiotrophoblast were treated with SNP or L-NAME and subjected to 1%O₂ for 24hrs. Relative changes in RNA expression was determined by qRT-PCR, normalized to values at 1%O₂; set to 100% and plotted; n=7, *=p<0.05. (B.) Divergent effects of NO generation under hypoxia on PGF, VEGF and sFlt-1 mRNA in primary syncytiotrophoblast. Relative changes in RNA for each treatment were determined by qRT-PCR, normalized to controls at 21%O₂; set to 100% and plotted, n=7, *=p<0.005.

Figure 3

NO generation decreases sFlt-1/mFlt-1 mRNA expression ratios in hypoxic syncytiotrophoblast. (A.) Relative changes in mFlt-1 mRNA for each culture condition were determined by qRT-PCR and normalized data plotted relative to values at 21% O₂; set =100%, n=7. (B.) Relative sFlt-1/mFlt-1 expression ratios in syncytiotrophoblast under hypoxia and hypoxia with SNP-induced NO-production shows preferential decrease in sFlt-1 mRNA expression with SNP treatment during hypoxia. ANOVA p<0.0001; *=p< 0.01; **=p<0.001, n=7 each.

Figure 4

Nitric oxide generation significantly inhibits sFlt-1 protein release in hypoxic syncytiotrophoblast. Relative changes in sFlt-1 protein were determined by capture ELISA and data were normalized to control values from trophoblast cultured at 1% O₂; set = 100% and plotted, n=3 each, *=p< 0.005.