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The Intrinsic Apoptotic Pathway Is Required for Lipopolysaccharide-Induced Lung Endothelial Cell Death¹

Helena L. Wang^*, I. Ozkan Akinci, Christina M. Baker, Daniela Urich, Amy Bellmeyer, Manu Jain, Navdeep S. Chandel, Gökhan M. Mutlu and G. R. Scott Budinger^2,

^* Division of Pulmonary and Critical Care Medicine, Loyola University, Maywood, Illinois 60153; Department of Anesthesiology and Reanimation, Istanbul University Medical Faculty, Capa-Istanbul, Turkey; and Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X_L were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid^–/–) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (⁰ cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by HL078131, HL071643, GM060472, HL067835, ES015024, ES013995, The American Lung Association, and The Crane Asthma Center.

² Address correspondence and reprint requests to Dr. G. R. Scott Budinger, Northwestern University, Division of Pulmonary and Critical Care Medicine, 240 East Huron, McGaw Pavilion M300, Chicago, IL 60611. E-mail address: s-buding{at}northwestern.edu

³ Abbreviations used in this paper: ROS, reactive oxygen species; HmVEC, human lung microvascular endothelial cell; CHX, cycloheximide; PI, propidium iodine; GPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase; WT, wild type; BAL, bronchoalveolar lavage; BALF, BAL fluid; DISC, death-inducing signaling complex.