Introduction and Objectives CLIC-4 belongs to the family of chloride intracellular channels, proteins structurally homologous to glutathione transferases. CLIC4 has been implicated in tumour angiogenesis and signalling pathways important in the pathogenesis of pulmonary arterial hypertension (PAH). We have previously demonstrated increased CLIC4 protein expression in whole lung samples from patients with PAH and animals with chronic hypoxia- and monocrotaline-induced PH. CLIC4 is particularly abundant in pulmonary endothelium of patients with PAH. In this project we aimed to establish a role of CLIC4 plays in the regulation of pulmonary endothelial cell responses to hypoxia.
Methods Human pulmonary artery endothelial cells (HPAECs) were cultured in normoxic conditions or were exposed to hypoxia (2% O2, 5% CO2, 93% N2) for 1–24 h. CLIC4 expression and localisation was studied by Western blotting, immunofluorescence and confocal microscopy. The wildtype and nuclear-targetted CLIC4 were overexpressed via adenoviral gene transfer while chloride channel inhibitor NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid) was used to inhibit CLIC4 activity. We studied the effects of CLIC4 on endothelial permeability, angiogenesis and proliferation.
Results Overexpression of the wildtype CLIC4 in HPAECs increased pulmonary endothelial proliferation, compromised barrier function and increased angiogenic responses to chronic hypoxia in vitro (Abstract S156 Figure 1). These effects were prevented by chloride channel inhibitor NPPB. In normoxic HPAECs CLIC4 was localised predominantly to the cell nucleus and cytoplasm. Upon stimulation with hypoxia, CLIC-4 translocated to the cell periphery, localising in particular to membrane protrusions (filopodia and lamellipodia), the effect mimicked by overexpression of CLIC4.
Conclusions Hypoxia induces translocation of CLIC-4 to the membrane of HPAECs. This behaviour has been linked to increased motility and malignant phenotype in other cell types. CLIC-4 overexpression in HPAECs also increases endothelial cell responses to hypoxia in vitro. These findings suggest increased CLIC-4 expression in PAECs in PAH may play a role in pathogenesis of PAH and provide novel insights in to disease pathogenesis and treatment strategies.
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