Role of the Aryl Hydrocarbon Receptor in Sugen 5416-induced Experimental Pulmonary Hypertension

Abstract

Rats dosed with the vascular endothelial growth factor inhibitor Sugen 5416 (Su), subjected to hypoxia, and then restored to normoxia have become a widely used model of pulmonary arterial hypertension (PAH). However, the mechanism by which Su exacerbates pulmonary hypertension is unclear. We investigated Su activation of the aryl hydrocarbon receptor (AhR) in human pulmonary artery smooth muscle cells (hPASMCs) and blood outgrowth endothelial cells (BOECs) from female patients with PAH. We also examined the effect of AhR on aromatase and estrogen levels in the lung. Protein and mRNA analyses demonstrated that CYP1A1 was very highly induced in the lungs of Su/hypoxic (Su/Hx) rats. The AhR antagonist CH223191 (8 mg/kg/day) reversed the development of PAH in this model in vivo and normalized lung CYP1A1 expression. Increased lung aromatase and estrogen levels in Su/Hx rats were also normalized by CH223191, as was AhR nuclear translocator (ARNT [HIF-1β]), which is shared by HIF-1α and AhR. Su reduced HIF-1α expression in hPASMCs. Su induced proliferation in BOECs and increased apoptosis in human pulmonary microvascular ECs and also induced translocation of AhR to the nucleus in hPASMCs. Under normoxic conditions, hPASMCs did not proliferate to Su. However, when grown in hypoxia (1%), Su induced hPASMC proliferation. In combination with hypoxia, Su is proliferative in hPASMCs and BOECs from patients with PAH, and Su/Hx-induced PAH in rats may be facilitated by AhR-induced CYP1A1, ARNT, and aromatase. Inhibition of AhR may be a novel approach to the treatment of pulmonary hypertension.

Keywords: Sugen; VEGF; aryl hydrocarbon receptor; estrogen; pulmonary hypertension.

Figure 1.

Expression of aryl hydrocarbon receptor (AhR) and CYP1A1 in lung tissue from the Sugen/hypoxic (Su/Hx) animal model. Expression of AhR mRNA (A) and protein (B), and expression of CYP1A1 mRNA (C) and protein (D) in whole lungs from male and female Su/Hx rats and their normoxic littermates (n = 3–4 per group, repeated in triplicate) are shown. (E) Representative immunoblots of AhR and CYP1A1 protein expression in whole lungs from male and female Su/Hx rats and their normoxic littermates. Vertical lines have been added to clarify the experimental groups. Data are displayed as mean ± SEM. ***P < 0.001, ****P < 0.0001 determined by one-way ANOVA with Tukey’s post hoc test. CYP1A1 = cytochrome P450 enzyme 1A1; RQ = relative quantity.

Figure 2.

Effect of the AhR antagonist CH223191 on Su/Hx pulmonary hypertension in female rats. (A) Right ventricular systolic pressure (RVSP; n = 5–6 per group), (B) right ventricular hypertrophy (RV/[LV+S]; n = 8 per group), and (C) the percentage of remodeled arteries in lungs without treatment (control) or with CH223191 alone, Su/Hx treatment with vehicle, or CH223191; n = 5–6. (D) Representative images showing elastic laminae stained with elastin/Picrosirius red. Scale bar: 20 μm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 3.

Effect of the AhR antagonist CH223191 on the protein expression of AhR, CYP1A1, and AhR nuclear translocator (ARNT) in female rat lungs. (A) AhR expression (n = 4), (B) CYP1A1 expression, and (C) ARNT expression, with (D) representative immunoblots (n = 4–6). (E) Representative CYP1A1 immunostaining in pulmonary arteries from rats. Scale bar: 50 μm. (F) Representative AhR immunostaining in pulmonary arteries from rats. Scale bar: 50 μm. Data are shown as mean ± SEM. *P < 0.05 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. HIF = hypoxia-inducible factor.

Figure 4.

Effect of an AhR antagonist on aromatase expression and estrogen in Su/Hx rat lungs. (A) Aromatase expression in female rat lungs (n = 3) and (B) representative immunoblots. (C) Local estrogen levels in female rat lungs (n = 4–5). Data are shown as mean ± SEM. *P < 0.05 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. veh = vehicle.

Figure 5.

Effect of Sugen on AhR and CYP1A1 expression in hPASMCs. (A and B) AhR and (C and D) CYP1A1 protein levels in PASMCs from female patients with PAH stimulated with 1 or 5 μM of Sugen 5416 for 24 hours (n = 3–4 different cell lines). (B and D) Representative western blots, with irrelevant lanes removed on the right-hand side. (E) Sugen caused nuclear translocation of AhR after 30, 60, and 90 minutes (n = 3 for all groups, *P < 0.05 as indicated, determined by area under the curve). AhR protein expression was normalized to α-tubulin and nucleoporin as markers for cytosolic and nuclear enrichment, respectively. Data are displayed as mean ± SEM. *P < 0.05 in C as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. hPASMCs = human pulmonary artery smooth muscle cells; PAH = pulmonary arterial hypertension.

Figure 6.

Sugen stimulates the proliferation of PASMCs from female patients with PAH under hypoxia but not under normoxia. Stimulation with Sugen had no effect on proliferation in female hPASMCs in normoxia (A); however, in hypoxia, 1 μM of Sugen 5416 induced cell proliferation (B); n = 4, repeated three times. (C) Stimulation with 1 μM of Sugen 5416 and the AhR agonist FICZ induced a decrease in the number of pulmonary microvascular endothelial cells. Resveratrol (100 μM) was used as the positive control, and endothelial cell growth media served as the negative control for apoptosis; n = 4, repeated three times. Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01 ***P < 0.001 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. CSS = charcoal stripped serum; EC = endothelial cell; FICZ = 5,11-dihydro-indolo[3,2-b]carbazole-6-carboxaldehyde, 6-formylindolo[3,2-b]carbazole.

Figure 7.

Sugen stimulates the proliferation of blood outgrowth endothelial cells (BOECs) from female patients with PAH. Stimulation with Sugen increased proliferation in BOECs (A); however, both the AhR antagonist CH223191 (1 μM) and Sugen 5416 (1 μM) reduced cell viability in BOECs by Trypan blue exclusion (B), n = 3, repeated three times. Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. (C) Sugen caused nuclear translocation of AhR after 60 minutes. AhR protein expression was normalized to α-tubulin and nucleoporin as markers for cytosolic and nuclear enrichment, respectively. The data are displayed as mean ± SEM. *P < 0.05 as indicated, determined by area under the curve. (D) Our data suggest that Su may activate AhR nuclear translocation and subsequent activation of CYP1A1, apoptosis, and aromatase expression. The resulting increase in E2 synthesis and metabolism may contribute to experimental PH. We also demonstrate directly that Su and hypoxia synergize, perhaps via ARNT, to cause hPASMC proliferation, suggesting that inhibition of AhR is a potential approach to the treatment of PAH.