The Unexpected Role of GCN2 Kinase Activation in Mediating Pulmonary Arterial Hypertension

Background Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart hypertrophy and failure. Published studies show that recessive mutations of EIF2AK4 gene (encoding GCN2, General control nonderepressibe 2 kinase) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients and EIF2AK4 mutations were also found in PAH patients although very rare. However, the role of GCN2 kinase in the pathogenesis of PAH remains unclear. Methods Eif2ak4-/- mice with genetic disruption of the kinase domain and GCN2 kinase inhibitor A-92 were employed in animal models of PH including chronic hypoxia-exposed mice and monocrotaline-challenged rats. Human lung endothelial cells (HLMVECs) were used for mechanistic studies. Endothelium-targeted nanoparticles were employed to deliver plasmid DNA to adult mice to knockout Eif2ak4 or overexpress Endothelin-1 (Edn1) selectively in ECs. Results Here we show that loss of GCN2 induced neither spontaneous PVOD nor PH in Eif2ak4-/- mice but inhibited hypoxia-induced PH evident by reduced right ventricular systolic pressure, right ventricle hypertrophy and pulmonary vascular remodeling. RNA sequencing analysis suggested Edn1 as the downstream target of GCN2. In cultured HLMVECs, GCN2 was phosphorylated and activated in response to hypoxia, mediating hypoxia-induced Edn1 expression via HIF-2α. Restored Edn1 expression in ECs in Gcn2 -deficient mice reversed the reduced phenotype of hypoxia-induced PH. Furthermore, loss of endothelial Eif2ak4 in mice attenuated hypoxia-induced PH. Monocrotaline-induced PH and pulmonary vascular remodeling in rats were inhibited by GCN2 inhibitor A-92 treatment. The clinical relevance of the observation was validated by GCN2 hyperphosphorylation indicative of activation in ECs of pulmonary vascular lesions of PAH patients. Conclusion These studies demonstrate that GCN2 activation by hypoxia mediates pulmonary vascular remodeling and PAH through Edn1. Thus, targeting GCN2 signaling is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss of function mutations. ### Competing Interest Statement Y.Y.Z is the founder and chief scientific officer of Mountview Therapeutics LLC. This project utilizes technologies subject to the following pending patent PCT/US2019/055787 ?PLGA-PEG nanoparticles and methods of uses? by Zhao, Y.Y. The other authors declare no competing interests. * ECs : endothelial cells CRISPR : Clustered regularly interspaced short repeats, gRNA, guide RNA HLMVECs : Human lung microvascular endothelial cells LV+S : left ventricle+septum MCT : monocrotaline (I)PAH : (idiopathic) pulmonary arterial hypertension PH : pulmonary hypertension PVOD : pulmonary veno-occlusive disease RV : right ventricle; RVSP, right ventricle systolic pressure SMC : smooth muscle cells