Hypoxic Regulation of VEGF and PAI-1 Expression by HIF-1[alpha] and HIF-2[alpha] in First Trimester Trophoblasts

by Meade, Eliza

Abstract (Summary)
Preeclampsia results from incomplete trophoblast invasion of the spiral arteries during early pregnancy. Vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) are critical factors involved in angiogenesis, invasion and hemostasis at the maternal-fetal interface. Both factors are transcriptionally regulated by hypoxia inducible factor (HIF), a heterodimeric complex consisting of HIF-1[beta] and either HIF-1[alpha] or -2[alpha] whose specificity or redundancy in gene regulation is cell-type specific. This study uses siRNA technology to dissect the mechanisms of hypoxia-mediated regulation of PAI-1 and VEGF expression in first trimester trophoblasts. Immortalized first trimester human extravillous trophoblasts (HTR8/SVneo cells) were maintained in serum-free and serum-containing media for 4h (n=3-4), 8h (n=6), 24h (n=5) and 48h (n=5) under normoxic (21% O2) and hypoxic (1-2% O2) conditions to determine a time of maximum induction of both VEGF and PAI-1. Subsequently, cells were maintained for 48h in the presence or absence of siRNA for HIF-1[alpha], HIF-2[alpha], HIF-1[alpha] + -2[alpha], a non-targeting (NT) sequence or Cyclophilin B (CB). Media were then removed, cells lysed, and Western blotting used to assess HIF-[alpha] knockdown. VEGF and PAI-1 levels in the media were quantified by ELISA and results expressed as pg or ng/[micro]g protein. Results from 3 to 8 independent experiments were analyzed using unpaired t-tests. Under hypoxic conditions treatment of cells with HIF-1[alpha], HIF-2[alpha] or HIF -1[alpha] + -2[alpha] siRNA resulted in >90% HIF-Ñ protein knockdown as determined by Western blotting. 48h of hypoxic treatment caused a statistically significant increase in PAI-1 levels (p<0.01) and VEGF levels (p<0.001) compared to normoxic controls. Under hypoxic conditions, PAI-1 levels were 4.75 [plus-minus] 0.46 ng/[micro]g protein and VEGF levels were 7.27 [plus-minus] 1.08 pg/[micro]g protein. Treatment with siRNA to HIF-1[alpha], HIF-2[alpha] and HIF-1[alpha] + -2[alpha] significantly reduced PAI-1 levels to 3.3 [plus-minus] 0.35 (p<0.02), 3.1 [plus-minus] 0.38 (p<0.03) and 2.4 [plus-minus] 0.19 (p<0.003), respectively. No significant difference in PAI-1 reduction was noted between the three HIF siRNA conditions. Under hypoxic conditions, levels of VEGF in cells treated with siRNA to HIF-1[alpha] (5.79 [plus-minus] 0.55), HIF-2[alpha] (5.50 [plus-minus] 1.24) and HIF-1[alpha] + -2[alpha] (4.24 [plus-minus] 0.93) were reduced compared to the hypoxic control (7.27 [plus-minus] 1.08), yet these effects did not reach statistical significance. However, when compared with the levels observed in cells treated with NT siRNA (9.90 [plus-minus] .98), all HIF siRNA treatments promoted a significant reduction in VEGF expression (p<0.003, p<0.02 and p<0.003 for HIF-1[alpha], HIF-2[alpha] and HIF-1[alpha]+ -2[alpha], respectively). In conclusion, these results indicate that hypoxia-mediated changes in PAI-1 and VEGF expression in trophoblasts are regulated similarly by both HIF-1[alpha] and HIF-2[alpha]. This provides important insight into the molecular mechanisms regulating hemostasis and trophoblast invasion as well as their potential dysfunction in pregnancies complicated by preeclampsia
Bibliographical Information:

Advisor:Seth M Guller

School:Yale University

School Location:USA - Connecticut

Source Type:Master's Thesis

Keywords:vascular endothelial growth factor a vegf pre eclampsia pregnancy complications pai 1 plasminogen activator inhibitor maternal fetal exchange hemostasis trophoblasts


Date of Publication:11/15/2006

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