Heterozygous deficiency of hypoxia-inducible factor-2alpha protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia

Abstract

Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2alpha is a homologue of HIF-1alpha and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2alpha-deficient (Hif2alpha(+/-)) mice after exposure to 10% O(2) for 4 weeks. In contrast to WT mice, Hif2alpha(+/-) mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2alpha in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2alpha(+/-) mice after hypoxia, suggesting that HIF-2alpha-mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

Figure 1

(a) Western blot analysis showed that pulmonary HIF-2α protein levels were decreased in Hif2α^+/– mice compared with WT mice after normoxia (N) and after 1 week of hypoxia (H). Extracts from PC12 cells, exposed to normoxia or hypoxia (1% oxygen, 4 hours) as previously described (17, 26), were used as controls. (b) Systolic RV pressures in WT and Hif2α^+/– mice under normoxia or after hypoxic exposure. Hemodynamic analysis revealed no differences in RV pressure under normoxia; however, WT mice showed increased RV pressure after 4 weeks of hypoxia, but Hif2α^+/– mice did not. (c) RV hypertrophy analysis in WT and Hif2α^+/– mice revealed no differences under normoxic conditions. After hypoxic exposure for 4 weeks, the ratio of the mass of the right ventricle to the mass of the left ventricle plus septum [RV/(LV+S)] was increased in WT mice but not in Hif2α^+/– mice. (d) Pulmonary ET-1 transcript levels were increased in WT mice after exposure to chronic hypoxia for 6 days and were still elevated after 4 weeks of hypoxia. In contrast, chronic hypoxia failed to induce ET-1 expression in lungs of Hif2α^+/– mice. Data represent means ± SEM of systolic RV pressure (mmHg; n = 4–7) (b), of the ratio of the mass of the right ventricle to the mass of the left ventricle plus septum [RV/(LV+S); n = 6–7] (c), and of the ET-1 mRNA copy number per 100 copies of HPRT (n = 5–6) (d). *Statistically significant (P < 0.05) vs. control (WT, normoxia).

Figure 2

(a–e) Hart’s elastin staining revealed the presence of vessels, located distally to the bronchi, at the level of alveoli and alveolar ducts, that contained only an IEL (or an IEL plus an incomplete EEL) (arrows) in lungs of normoxic (N) WT (a) and Hif2α^+/– mice (b). Lungs of hypoxic (H) WT mice showed the presence of thick-walled vessels containing both an IEL and a complete EEL (arrows) (c and d), whereas no hypoxia-induced vascular remodeling occurred in Hif2α^+/– mice (arrows) (e). (f–j) SMC α-actin staining shows the presence of partially muscularized peripheral vessels (arrows) in lungs of normoxic WT (f) and Hif2α^+/– mice (g). Chronic hypoxia caused pulmonary vascular remodeling in WT mice, as revealed by the presence of fully muscularized vessels (arrows) (h and i), but not in Hif2α^+/– mice (arrows) (j). Bar = 50 μm in all panels.