Conference: 2008 International PHA Conference and Scientific Sessions
Release Date: 06.20.2008
Presentation Type: Abstracts
Hawkins B.J., Solt L.A., Chowdhury I., Kazi A.S., Abid M.R., Aird W.C., May M.J., Foskett J.K., Madesh M.
Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA
BACKGROUND: Thrombin is increasingly acknowledged as a potent activator of pulmonary endothelial cells and has been implicated in pulmonary arterial hypertension and Acute
Respiratory Distress Syndrome (ARDS). Thrombin acts on pulmonary vascular cells by interacting with protease-activated receptors (PARs) to mobilize intracellular Ca2+ via inositol 1,4,5-trisphosphate (InsP3). During normal signaling, InsP3-mediated Ca2+ transients transmit to the mitochondria, raising mitochondrial matrix Ca2+, and enhancing mitochondrial bioenergetics. Mitochondrial Ca2+ uptake is also correlated with an increase in reactive oxygen species (ROS) production. However, whether thrombin-mediated mitochondrial ROS participates directly in physiologic signaling cascades or occurs simply as a by-product of enhanced mitochondrial respiration is unclear.
METHODS: Primary murine and human pulmonary microvascular endothelial cells (ECs) were activated with thrombin and simultaneously assessed for cytosolic Ca2+ mobilization and mitochondrial function (Ca2+ uptake, reactive oxygen species production, membrane potential alterations) using real-time confocal laser scanning microscopy. Activated ECs were assessed for adhesion molecule expression by western blot. NF-κΒ nuclear translocation and transcriptional activity were determined via electrophoretic mobility shift assay and luciferase activity, respectively. Endothelial/leukocyte adhesion was revealed using fluorescently labeled cells in confocal imaging parallel plate chamber.
RESULTS: Here we demonstrate a previously unknown mechanism in which physiologic Ca2+-evoked mitochondrial ROS production plays a pivotal role in EC activation and leukocyte firm adhesion. PAR-mediated inositol 1,4,5-trisphosphate-dependent mitochondrial Ca2+ uptake resulted in NADPH oxidase-independent mitochondrial ROS production. However, thrombin-linked mitochondrial ROS production did not alter mitochondrial function or trigger cell death, but rather contributed to activation of NF-κΒ and leukocyte cell adhesion via the EC induction of ICAM-1. Dismutation of mROS by manganese superoxide dismutase overexpression and a cell-permeative superoxide dismutase mimetic ablated NF-κΒ transctiptional activity and facilitated leukocyte detachment from the endothelium under simulated circulation following thrombin- but not cytokine-induced activation.
COMMENTS AND CONCLUSIONS: These results demonstrate that mitochondrial ROS is the downstream effector molecule that translates receptor-mediated Ca2+ signals into pro- inflammatory signaling and monocyte/EC firm adhesion. They also offer a mechanistic explanation for thrombin-induced vascular inflammation and implicate the use of antioxidant therapy in pulmonary hypertension patients with elevated circulating thrombin levels.