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Protease Activated Receptor Ca2+-Linked Mitochondrial Reactive Oxygen Species are Essential for Endothelial/Leukocyte Adherence

Brian Hawkins

L. A. Solt

I. Chowdhury

A. S. Kazi

M. R. Abid

W. C. Aird

M. J. May

J. K. Foskett

M. Madesh


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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.