Pulmonary arterial hypertension (PAH) is a severe and incurable disease characterised by progressive pulmonary vascular remodelling and right heart hypertrophy. PAH bears many hallmarks of cancer, including apoptosis resistance, increased cell proliferation and mitochondrial dysfunction characterised by membrane hyperpolarization, mitochondrial fission and metabolic switch from mitochondrial oxidative phosphorylation to glycolysis. Chloride intracellular channel proteins CLIC1 and CLIC4 are highly expressed in the remodelled pulmonary vasculature in PAH. These redox-sensitive proteins regulate cell proliferation and angiogenesis. CLIC1, CLIC4 and CLIC5 are also structural components of mitochondrial membranes but their role has not been investigated.
We hypothesized that increased expression of CLIC proteins induces mitochondrial dysfunction in human pulmonary vascular endothelial cells (HPAECs) in vitro, consistent with pathological changes seen in PAH.
CLIC1, CLIC4 and CLIC5 were overexpressed via adenoviral gene transfer and their effects on mitochondrial fission/fusion, mitochondrial membrane potential, reactive oxygen species (ROS) generation, expression of mitochondrial proteins, glycolysis and mitochondrial respiration, were studied.
Physiological levels of CLIC1, CLIC4 and CLIC5 overexpression induced mitochondrial fission, significantly increased mitochondrial membrane potential, mitochondrial reactive oxygen species (ROS) generation and extracellular acidification rate (ECAR) in HPAECs. Mitochondria isolated from CLIC1- and CLIC4-overexpressing cells showed a significant decrease in the expression of MFN2 and p-Ser637 DRP1, while overexpression of CLIC5 increased expression of FIS1. Mitochondrial fragmentation was accompanied by the reorganization of microtubule cytoskeleton and reduced phosphorylation of pyruvate dehydrogenase at Serine 293. Increased expression of CLIC proteins and corresponding changes in mitochondrial dynamics were also seen in blood-derived endothelial cells from PAH patients, compared with healthy controls.
Our results suggest that CLIC proteins are likely to play a contributory role in mitochondrial dysfunction in PAH.