Figure 2.

Inhibition of HIF-2α (hypoxia-inducible factor-2α) activation in human lung microvascular endothelial cells (HLMVECs) by treatment with HIF-2α inhibitors. (A) Luciferase assay showing that HIF-2α inhibitors (H2A [HIF-2α antagonist 2] and HIF-2α translational inhibitor compound 76 [C76]) suppressed HRE (hypoxia response element) activity induced by PHD2 (prolyl hydroxylase-2) deficiency in HLMVECs. HLMVECs were cotransfected with HRE-Luc plasmid and pRL-TK plasmid as well as PHD2 siRNA (siPHD2) or control scrambled RNA (siCtl). Six hours after transfection, the cells were treated with either H2A (20 μM), C76 (20 μM), or vehicle (DMSO) for another 14 hours and then lysed for luciferase assay. (B) Representative Western blot demonstrating that C76 treatment reduced HIF-2α protein levels but not HIF-1α protein levels in PHD2-deficient HLMVECs. (C) Quantitative RT-PCR analysis showing inhibited expression of HIF-2α target genes by C76 treatment in PHD2-deficient HLMVECs. (D) A diagram showing the strategy of smooth muscle cell (SMC) treatment with conditioned medium from ECs. (E) 5-Bromo-2′-deoxyuridine (BrdU) immunostaining demonstrating C76 inhibition of idiopathic pulmonary arterial hypertension (IPAH) pulmonary arterial SMC (PASMC) proliferation induced by conditioned medium from PHD2-deficient HLMVECs. Forty-eight hours after transfection with either PHD2 siRNA or control, HLMVECs were treated with DMSO or C76 (20 μM) for 24 hours in serum-free medium. Conditioned medium from these HLMVECs was then added to human PASMCs for 12 hours. After 12 hours of incubation with BrdU (10 μM), SMCs were fixed and immunostained with anti-BrdU (green) for quantification of cell proliferation. Red arrows indicate BrdU-positive cells. Sample size: (A–C) n = 3, (E) n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s. = not significant. (A–C and E) One-way ANOVA with Tukey post hoc analysis. (E) Scale bar: 50 μm. A.U. = arbitrary units.