GCN2 kinase activation mediates pulmonary vascular remodeling and pulmonary arterial hypertension
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
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Research Article Vascular biology

GCN2 kinase activation mediates pulmonary vascular remodeling and pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding general control nonderepressible 2 kinase, GCN2) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely in patients with PAH. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here, we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and patients with idiopathic PAH. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4–/– mice with genetic disruption of the kinase domain induced neither PVOD nor pulmonary hypertension (PH) but inhibited hypoxia-induced PH. RNA-sequencing analysis suggested endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4–/– mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss-of-function mutations.

Authors

Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao

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Figure 5

Restored expression of endothelial Edn1 in Gcn2-deficient mice partially reverses the reduced PH phenotype.

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Restored expression of endothelial Edn1 in Gcn2-deficient mice partially...
(A) Diagram showing the experimental procedure to restore Edn1 expression selectively in ECs of WT and Gcn2-deficient mice. Mixture of nanoparticles/plasmid DNA expressing Edn1 or GFP (control) under the control of CDH5 promoter was administered retro-orbitally to 11-week-old KO mice and WT mice. After overnight (16 hours), the mice were subjected to hypoxia. Nanoparticles/plasmid DNA mixtures were administered weekly for 3 doses total, and each mouse received 30 μg plasmid DNA each time. (B) Western blotting demonstrating restored Edn1 expression in ECs of KO mice with Edn1 plasmid administration compared with WT and KO mice with GFP plasmid. Lung ECs were isolated for Western blotting. (C) RVSP measurement showing reduced PH in KO mice with GFP plasmid was partially reversed in KO mice with Edn1 plasmid. (D) RV/(LV+S) ratio also showing reduced RV hypertrophy in KO mice with GFP plasmid was partially rescued with restored Edn1 expression. N = 5–8/group. (E) Representative micrographs of anti–α-SMA staining of lung sections showing reduced number of muscularized distal pulmonary vessels in hypoxic KO+GFP lungs was partially reversed in hypoxic KO+Edn1 lungs. Nuclei were counterstained with DAPI (blue). Arrows point to muscularized vessels. (F) Quantification of the number of muscularized distal pulmonary vessels. N = 5/group. (G) Representative micrographs of Russell-Movat pentachrome staining of mouse lung sections. Br, bronchiole; V, vessel. (H) Quantification of pulmonary vessel media wall thickness. N = 5/group. Scale bars, 50 μm. Data are shown as means + SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. One-way ANOVA with Tukey’s multiple comparisons test (C, D, F, and H).