Date of Award
Open Access Thesis
Medical Doctor (MD)
Seth M Guller
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.
Meade, Eliza, "Hypoxic Regulation of VEGF and PAI-1 Expression by HIF-1α and HIF-2α in First Trimester Trophoblasts" (2006). Yale Medicine Thesis Digital Library. 271.