The effect of hypoxia on ER-β expression in the lung and cultured pulmonary artery endothelial cells

Indiana University-Purdue University Indianapolis (IUPUI)17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH

Hypoxia Upregulates Estrogen Receptor β in Pulmonary Artery Endothelial Cells in a HIF-1α-Dependent Manner

17β-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERβ expression. Sprague-Dawley rats and ERα or ERβ knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERβ, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERβ mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERβ, whereas HIF-1α knockdown decreased ERβ abundance in hypoxic PAECs. In turn, ERβ knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERβ knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERβ activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERβ regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERβ knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERβ expression and the E2-ERβ axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development

Poor agreement between pulmonary capillary wedge pressure and left ventricular end-diastolic pressure in a veteran population.

BACKGROUND: Accurate determination of left ventricular filling pressure is essential for differentiation of pre-capillary pulmonary hypertension (PH) from pulmonary venous hypertension (PVH). Previous data suggest only a poor correlation between left ventricular end-diastolic pressure (LVEDP) and its commonly used surrogate, the pulmonary capillary wedge pressure (PCWP). However, no data exist on the diagnostic accuracy of PCWP in veterans. Furthermore, the effects of age and comorbidities on the PCWP-LVEDP relationship remain unknown. METHODS: We investigated the PCWP-LVEDP relationship in 101 patients undergoing simultaneous right and left heart catherization at a large VA hospital. PCWP performance was evaluated using correlation and Bland-Altman analyses. Area under Receiver Operating Characteristics curves (AUROC) for PCWP were determined. RESULTS: PCWP-LVEDP correlation was moderate (r = 0.57). PCWP-LVEDP calibration was poor (Bland-Altman limits of agreement -17.2 to 11.4 mmHg; mean bias -2.87 mmHg). 59 patients (58.4%) had pulmonary hypertension; 15 (25.4%) of those met pre-capillary PH criteria based on PCWP. However, if LVEDP was used instead of PCWP, 7/15 patients (46.6%) met criteria for PVH rather than pre-capillary PH. When restricting analysis to patients with a mean pulmonary artery pressure of ≥25 mmHg and pulmonary vascular resistance of >3 Wood units (n = 22), 10 patients (45.4%) were classified as pre-capillary PH based on PCWP ≤15 mmHg. However, if LVEDP was used, 4/10 patients (40%) were reclassified as PVH. Among patients with any type of pulmonary hypertension, PCWP discriminated moderately between high and normal LVEDP (AUROC, 0.81; 95%CI 0.69-0.94). PCWP-LVEDP correlation was particularly poor in patients with COPD or obesity. CONCLUSION: Reliance on PCWP rather than LVEDP results in misclassification of veterans as having pre-capillary PH rather than PVH in almost 50% of cases. This is clinically relevant, as misclassification may lead to inappropriate therapies and adverse events

Patient flow chart.

<p>LHC, left heart catheterization; LVEDP, left ventricular end-diastolic pressure; mPAP, mean pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; RHC, right heart catheterization.</p

Correlation and agreement between PCWP and LVEDP in patients with (A, B) or without (C, D) pulmonary hypertension.

<p>Vertical line in (A) divides patients in patients with PCWP ≤15 mmHg or >15 mmHg; horizontal line divides patients in patients with LVEDP ≤15 mmHg or >15 mmHg. Shaded area in (A) represents the patients with PCWP ≤15 mmHg, but LVEDP >15 mmHg, thus indicating patients that would have been incorrectly classified as pre-capillary PH in absence of LVEDP measurement. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087304#pone-0087304-g003" target="_blank">fig. 3</a> for explanation of Bland-Altman plot labeling.</p

Correlations between PCWP and LEDP in patients with or without diabetes (A, B), hypertension (C, D), COPD (E, F) or CHF (G, H), as well as for patients < or ≥65 years of age (I, J), and for patients with a body mass index (BMI) <25 or ≥30 (K, L).

<p>Note lack of significant correlation between PCWP and LVEDP in COPD patients, and poor correlation in obese patients.</p

Echocardiographic parameters.

<p>Left ventricular ejection fraction, left ventricular posterior wall thickness, and interventricular septal wall thickness are expressed as means±SD. Other values are expressed as absolute numbers with percent of the total study population in parenthesis.</p

Agreement between PCWP and LEDP in the entire study population.

<p>Bland-Altman plot of PCWP and LVEDP pairs for all 101 patients included in the study. Difference indicates difference between PCWP and LVEDP pairs (in mmHg), with positive values indicating that PCWP is higher than corresponding LVEDP for that particular patient, and with negative values indicating that PCWP is lower. Average indicates value of corresponding PCWP and LVEDP pairs divided by 2 ([PCWP+LVEDP/2]). Upper and lower horizontal lines indicate upper and lower borders of 95% limits of agreement, respectively; horizontal line in middle represents mean bias.</p

Hemodynamic Parameters of the 101 study subjects.

<p>If a parameter was not obtained in all 101 patients, the number of patients in which this was measured is indicated in parenthesis in the left column. Values are expressed as means±SD, or as absolute numbers with percent of the total study population in parenthesis.</p

Distribution of patients with LVEDP ≤15 mmHg or LVEDP >15 mmHg in patients with PCWP ≤15 mmHg and PVR >3 WU (upper panel; black), TPG >12 mmHg (middle panel; dark grey), or DPG >7 mmHg (bottom panel; light grey).

<p>DPG, diastolic pressure gradient; PVR, pulmonary vascular resistance; TPG, transpulmonary gradient.</p