Date of Award

11-15-2006

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Seth M Guller

Abstract

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-1β and either HIF-1α or -2α 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/μg protein. Results from 3 to 8 independent experiments were analyzed using unpaired t-tests. Under hypoxic conditions treatment of cells with HIF-1α, HIF-2α or HIF -1α + -2α siRNA resulted in >90% HIF-α 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 ± 0.46 ng/μg protein and VEGF levels were 7.27 ±1.08 pg/μg protein. Treatment with siRNA to HIF-1α, HIF-2α and HIF-1α + -2α significantly reduced PAI-1 levels to 3.3 ± 0.35 (p<0.02), 3.1 ± 0.38 (p<0.03) and 2.4 ± 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-1α (5.79 ± 0.55), HIF-2α (5.50 ± 1.24) and HIF-1α + -2α (4.24 ± 0.93) were reduced compared to the hypoxic control (7.27 ± 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 ± .98), all HIF siRNA treatments promoted a significant reduction in VEGF expression (p<0.003, p<0.02 and p<0.003 for HIF-1α, HIF-2α and HIF-1α+ -2α, respectively). In conclusion, these results indicate that hypoxia-mediated changes in PAI-1 and VEGF expression in trophoblasts are regulated similarly by both HIF-1α and HIF-2α. This provides important insight into the molecular mechanisms regulating hemostasis and trophoblast invasion as well as their potential dysfunction in pregnancies complicated by preeclampsia.

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