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. 2011 Dec;301(6):L881-91.
doi: 10.1152/ajplung.00195.2011. Epub 2011 Sep 16.

The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo

Bum-Yong Kang  1 , Jennifer M Kleinhenz, Tamara C Murphy, C Michael Hart
Affiliations

The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo

Bum-Yong Kang et al. Am J Physiol Lung Cell Mol Physiol. 2011 Dec.

Abstract

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O(2)) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for 3 wk with or without gavage with RSG (10 mg·kg(-1)·day(-1)) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

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Figures

Fig. 1.
Fig. 1.
Treatment with rosiglitazone (RSG) attenuates hypoxia-induced endothelin (ET)-1 protein levels in vivo and in vitro. A: pulmonary artery endothelial cells were isolated from the lungs of control or idiopathic pulmonary arterial hypertension (IPAH) patients and characterized as reported (35). Quantitative real-time PCR was performed for ET-1. Each bar presents the mean ± SE ET-1 mRNA levels relative to 9S. *P < 0.05 vs. control, n = 3 experiments. B: whole lung homogenates were collected from mice exposed to normoxia (NOR, 21% O2) or hypoxia (HYP, 10% O2) for 3 wk. During the last 10 days of this exposure, selected animals were also treated ± RSG (10 mg·kg−1·day−1 by gavage) as reported (39). Lung ET-1 levels were measured with enzyme-linked immunosorbent assay (ELISA). P < 0.05 vs. NOR (*) and vs. HYP (+); n = 5–6. C: human pulmonary artery endothelial cells (HPAECs) were exposed to NOR (21% O2) or HYP (1% O2) for 72 h. Selected cells were treated during the final 24 h of exposure with RSG (10 μM). At the conclusion of the study, HPAEC media were collected, subjected to ET-1 ELISA assay, and normalized to total cell protein. Each bar represents the average ET-1 level ± SE from 6–9 samples in each treatment group expressed relative to control values. P < 0.05 vs. NOR (*) and vs. HYP (+). D: immunohistochemical detection of ET-1 in lungs from C57BL/6 mice following exposure to NOR or HYP and treatment with vehicle or RSG. Representative 8-μm sections are presented. Immunostaining (brown coloration) was noted in small arterioles and alveolar structures in each treatment group. Scale bar = 50 μm. There was no staining with nonimmune IgG isotype control primary antibody (data not shown). E: HPAEC were exposed to NOR or HYP ± RSG (as described in C), and cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. DMSO, dimethyl sulfoxide. Each bar represents the average HPAEC proliferation level ± SE from 6–9 samples in each treatment group expressed relative to control values. P < 0.05 vs. NOR (*) and vs. HYP (+).
Fig. 2.
Fig. 2.
RSG attenuates HYP-induced increases in ET-1 signaling pathway mRNA levels in mouse lung. C57BL/6 mice were exposed to NOR (21% O2) or HYP (10% O2) ± treatment with RSG as described in Fig. 1. Each bar represents the mean ± SE ET-1 (A), endothelin-converting enzyme (ECE)-1 (B), endothelin receptor A (ETAR, C), and endothelin receptor B (ETBR, D) mRNA relative to ribosomal 9S and expressed relative to control. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 5. Mouse lungs were collected for Western blotting using antibodies directed against ECE-1, ET-1, ETBR, ETAR, and CDK4 (E). Representative blots from 3 separate experiments are presented above average densitometric values ± SE for each protein. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3.
Fig. 3.
Fig. 3.
RSG attenuates HYP-induced increases in HPAEC ET-1 signaling components and proliferation. HPAEC were exposed to NOR (21% O2) or HYP (1% O2) for 72 h. Selected cells were treated during the final 24 h of exposure with RSG (10 μM). Each bar represents the mean ± SE ET-1 (A), ECE-1 (B), and ETBR (C) mRNA levels relative to ribosomal 9S normalized to the control value from 6–9 samples in each treatment group. P < 0.05 vs. NOR (*) and vs. HYP (+). HPAECs were collected for Western blotting using antibodies directed against ECE-1, ET-1, ETBR, and CDK4 (D). Representative blots from 3 separate experiments are presented above average densitometric values ± SE for each protein. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3. The impact of HYP ± RSG on HPAEC proliferation was determined using cell counting and MTT assays (E). Each bar represents the mean ± SE relative to control. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 6.
Fig. 4.
Fig. 4.
RSG attenuates HYP-induced HYP-inducible factor (HIF)-1α and nuclear factor (NF)-κB nuclear binding in HPAEC. HPAEC were exposed to NOR (21% O2) or HYP (1% O2) for 72 h. During the final 24 h of exposure, selected cells were treated with RSG (RSG, 10 μM). Nuclear proteins were extracted from HPAECs and then incubated with radiolabeled HIF-1α (A) and NF-κB (C) oligonucleotides and subjected to electrophoretic mobility shift assay analysis. DNA-protein complexes were separated on a native polyacrylamide gel, and densitometric analysis of bands was performed (B and D). Arrows, probe shift due to HIF-1α and NF-κB protein binding and unbound free probe; P, probe alone; C, control 50× unlabeled probe; Cm, control mutated 50× probe. Each bar represents the mean ± SE. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3. In E, the intracellular localization of the NF-κB subunit p65 was investigated by immunofluorescence by using specific p65 antibody. Images are representative of 3 experiments. Scale bar = 10 μm. There was no staining with nonimmune IgG isotype control primary antibody (data not shown).
Fig. 5.
Fig. 5.
RSG attenuates HYP-induced increases in HIF-1α and NF-κB mRNA levels in mouse lung and in HPAEC. C57BL/6 mice (A and B) or HPAEC (C and D) were exposed to NOR or HYP ± RSG as described in Fig. 1. Each bar represents the mean ± SE HIF-1α or NF-κB mRNA level relative to ribosomal 9S and expressed relative to control. P < 0.05 vs. NOR (*) and 9s. HYP (+); n = 5–9.
Fig. 6.
Fig. 6.
Modulation of HYP-induced ET-1 mRNA and protein levels by inhibitors of HIF-1α or NF-κB in HPAECs. HPAECs were pretreated with DMSO, with the HIF-1α inhibitor chetomin (CTM, 25 nM), or with the NF-κB inhibitor caffeic acid phenethyl ester (CAPE, 20 μM) for 3 h. HPAECs were then exposed to NOR (21% O2) or HYP (1% O2) for 72 h in the presence of absence of CTM or CAPE. During the final 24 h of exposure, selected HPAEC were treated with RSG (10 μM). In A, each bar represents the mean ± SE ET-1 mRNA level relative to 9S and expressed relative to control. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3. In B, representative Western blots for ET-1 and CDK4 are presented. In C, average ET-1 densitometric values ± SE are presented. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3.
Fig. 7.
Fig. 7.
Putative peroxisome proliferator-activated receptor (PPAR) γ-regulated pathways of endothelin signaling in HYP-induced pulmonary hypertension (PH). HYP activates transcription factors, such as HIF-1α or NF-kB, that increase levels of ET-1 and related signaling components (ECE-1, ETAR, and ETBR). RSG attenuates hypoxic stimulation of HIF-1α and NF-kB nuclear binding, thereby reducing hypoxic upregulation of ET-1 and its signaling components that contribute to pathophysiological derangements such as pulmonary vasoconstriction and vascular remodeling. Attenuation of hypoxic activation of ET-1 signaling thereby contributes to the ability of PPARγ ligands to attenuate PH.
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