Increased expression of chloride intracellular channel 4 (CLIC4) is a feature of endothelial dysfunction in pulmonary arterial hypertension (PAH) but its protein targets and therapeutic potential has not been characterized.
To identify CLIC4 target proteins with therapeutic potential using proteomics and to study its mechanism and effects in animal models of pulmonary hypertension (PH).
Methods and Results
Proteomic screening of CLIC4-overexpressing human pulmonary artery endothelial cells (HPAECs) identified pathways associated with endosomal trafficking and lysosomal function as well as NFB and TNF-α signalling, whereas proteomic analysis of CLIC4-interacting proteins identified endosomal regulators such as clathrin, ADP Ribosylation Factor 6 (ARF6) GTPase activating proteins (GIT1 and GIT2), dynein, actin binding proteins and tubulin. CLIC4 overexpression increased clathrin-mediated receptor internalization, increased accumulation of LAMP1-positive late endosome/lysosome fraction and increased lysosome acidification. CLIC4 significantly reduced the levels of gyrating clathrin responsible for rapid receptor recycling of membrane proteins. These changes were accompanied by increased activation of NFB, reduced expression and signalling of BMPRII and enhanced TGF-β-signalling. CLIC4-induced ARF6 but not ARF1 activity and reduced BMPRII expression in HPAECS and in colony-forming blood-derived endothelial cells from idiopathic PAH patients. SecinH3, an ARF6 inhibitor, prevented these CLIC4-induced effects. We assesed the effects of SecinH3 in a preventive strategy using Sugen/hypoxia mice model of PH and in a treatment regimen using the MCT rat model. In both models, SecinH3 reduced pulmonary artery pressure, right ventricular hypertrophy and muscularisation of pulmonary vessels. These effects were accompanied by a significant reduction in Arf6 and NFkB activity and increased expression of BMPRII in the lung.
Targeting ARF6 represents a new treatment strategy in PAH.