ARF6 as a Novel Activator of HIF-2α in Pulmonary Arterial Hypertension.

ADP-ribosylation factor 6 (ARF6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wildtype, constitutively active, fast-cycling and dominant negative mutants of ARF6. The analysis revealed a novel link of ARF6 with hypoxia-inducible factor (HIF), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late outgrowth endothelial colony forming cells (ECFCs) and hypoxic mouse lungs in vivo. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH ECFCs showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC, and oral CTC attenuated development of PH in chronically hypoxic mice. We identify epidermal growth factor receptor (EGFR) as a direct interactor of ARF6, and EGFR signalling as a crucial mechanism linking ARF6 and HIF activation. In conclusion, we are first to demonstrate a key role of ARF6 in the regulation of HIF-2α activation in vitro and in vivo and show that HIF-2α, a master-regulator of vascular remodelling in PAH, can be targeted by a clinically approved antibiotic chlortetracycline.