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Hypoxia Increases Estrogen Receptor Beta Expression in Cultured Pulmonary Artery Cells

M Selej

A Lockett

Marjorie Albrecht

I Petrache

Tim Lahm


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Conference: 2012 International PHA Conference and Scientific Sessions

Release Date: 06.22.2012

Presentation Type: Abstracts

BACKGROUND: 17beta estradiol (E2) exerts protective effects in hypoxic pulmonary hypertension (HPH). These effects occur in an endothelial cell (EC)-dependent and estrogen receptor (ER)-mediated manner, with selective activation of ER subtype beta (ER-beta) attenuating acute hypoxic pulmonary vasoconstriction (HPV). However, it remains unknown if pulmonary artery EC (PAEC) ER subtype expression is altered by hypoxia exposure. We hypothesized that moderate hypoxia primarily affects ER-beta expression without altering ER-alpha. To test our hypothesis, we investigated ER-alpha and ER-beta expression and transcript in isolated primary rat PAECs exposed to acute and subacute hypoxia.

METHODS: Cultured primary rat PAECs were serum-starved for 2h and then exposed to room air (21% O2) or hypoxia (1% O2) at 4, 24 and 72h time intervals. Immunofluorescence for ER-alpha and ER-beta was performed. ER-alpha and ER-beta expression was quantified as megapixel intensity normalized for number of nuclei. In identical experiments, total mRNA was extracted from cell lysates and used for real-time PCR analysis. Additional experiments were performed in hypoxic primary rat PA smooth muscle cells (PASMCs) and primary human pulmonary artery endothelial cells (HPAECs). Additionally, rat PAECs were exposed to chemical hypoxia with the prolyl hydroxylase inhibitor dimethyloxallyl glycine (DMOG; 500uM) for 24 hours. P<0.05 was considered statistically significant.

RESULTS: Hypoxia did not affect ER-alpha expression, but significantly up-regulated ER-beta expression at 24 and 72h. Increased ER-beta expression was associated with a pattern suggestive of increased nuclear translocation. No increase in ER-beta was noted at 4h, suggesting a genomic mechanism of hypoxia-induced ER-beta up-regulation. ER-beta mRNA was significantly increased at 72h. In preliminary experiments, ER-beta expression was also increased in hypoxic primary rat PASMCs and in human PAECs. Chemical hypoxia with DMOG at 24 hours did not alter ER-beta expression, suggesting that hypoxia inducible factor (HIF) activation is not sufficient to increase ER-beta at that time point. Dose response and additional time curve experiments are currently underway.

CONCLUSIONS: Hypoxia time-dependently increases ER-beta, but not ER-alpha expression in isolated primary pulmonary artery cells. Hypoxia effects on ER-beta are not cell type nor species-specific. Interpreted in the context of previous experiments demonstrating ER-beta-mediated attenuation of acute HPV, our data suggests a potential protective role for endothelial and/or smooth muscle ER-beta in the hypoxic pulmonary vasculature. However, our DMOG data suggest that these effects may occur in a HIF-independent fashion. Elucidating ER-beta signaling pathways may reveal potential therapeutic targets in HPH.

FUNDING: ATS/PHA/Pfizer Fellowship in PAH Research (TL), Reba &Floyd Smith Chair in Respiratory Diseases (IP)