Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways Gabriella Aschkenasy 1. , Zohar Bromberg 1. , Nichelle Raj 2 , Clifford S. Deutschman 2 , Yoram G. Weiss 1,2 * 1 Department of Anesthesiology and Critical Care Medicine and the Goldyne Savad Institute of Gene Therapy, Hadassah-Hebrew University School of Medicine, Jerusalem, Israel, 2 Department of Anesthesiology and Critical Care and the Stavropoulos Sepsis Research Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America Abstract The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis- induced lung injury. Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFa. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co- immunoprecipitation and a caspase-3 activity assay. TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes. We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery. Citation: Aschkenasy G, Bromberg Z, Raj N, Deutschman CS, Weiss YG (2011) Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways. PLoS ONE 6(11): e26956. doi:10.1371/journal.pone.0026956 Editor: Marcelo G. Bonini, University of Illinois at Chicago, United States of America Received October 17, 2010; Accepted October 7, 2011; Published November 23, 2011 Copyright: ß 2011 Aschkenasy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Israel Science Foundation (#586/03 and # 529_/08 - YGW), the Israel Ministry of Health Chief Scientist (#5304 - YGW), the NIH/NIGMS (GM059930-07 - CSD) and the Stavropoulos Sepsis Research Program (CSD). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weiss@hadassah.org.il . These authors contributed equally to this work. Introduction The Acute Respiratory Distress Syndrome (ARDS) is a lethal, incompletely understood syndrome that frequently accompanies sepsis [1,2]. The initial phase of the disorder involves unchecked inflammation that damages and may destroy type I alveolar epithelial cells [2,3,4]. While the exact mechanisms that lead to pulmonary cell death are unknown, it is likely that apoptosis plays an important pathogenic role [5,6]. Normally, apoptosis is a homeostatic response to eliminate damaged or senescent cells [7,8,9]. This form of regulated cell death may be induced by a range of environmental, physical or chemical stresses [10]. Activation of apoptosis depends on a pro- teolytic system that involves some members of a family of intra- cellular enzymes called caspases. Several, such as caspase-3, -8 and -9, initiate and execute the cell death process while others have been implicated in inflammation [11,12,13]. Caspases are present in the cell in a precursor state. Following a pro-apoptotic signal, these pro-caspases are cleaved to yield the activated enzymes. Activation and progression of apoptosis is tightly controlled. Part of the regulatory system involves Heat Shock Proteins (HSPs). These molecular chaperones are expressed both constitutively and in response to cellular and extracellular perturbations [14,15]. As a result, they are involved in a myriad of normal homeostatic processes and can mediate cellular protection and recovery [16]. The involvement of HSPs in such a wide range of cellular activities reflects the ability of these molecules to interact with hydrophobic regions of nearly all proteins or polypeptides. Among HSPs, members of the 70 kDa subfamily, collectively referred to as Hsp70, are phylogenetically conserved, highly inducible and believed to play an essential role in normal cell processes and in the response to noxious stimuli [17]. Previously, we have demonstrated that intra-tracheal adminis- tration of an adenoviral vector that expresses Hsp70 (AdHSP) attenuates lung pathology and improves outcome in lung injury induced by cecal ligation and double puncture (2CLP) in rats [18,19,20]. In this model, AdHSP limited histologic lung injury, attenuated acute inflammation and neutrophil recruitment by suppression of NF-kB activation [21], limited over-proliferation of type II pneumocytes [22] and preserved type I alveolar epithelial cells [20]. This last finding may be related to altered apop- tosis. Indeed, others have established that Hsp70 can attenuate PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26956