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
| Identifer | oai:union.ndltd.org:YALE_med/oai:ymtdl.med.yale.edu:etd-06282006-115727 |
| Date | 15 November 2006 |
| Creators | Meade, Eliza |
| Contributors | Seth M Guller |
| Publisher | Yale University |
| Source Sets | Yale Medical student MD Thesis |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://ymtdl.med.yale.edu/theses/available/etd-06282006-115727/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Yale School of Medicine or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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