Stress preconditioning and alveolar epithelial injury. (RO1 GM62188, 2001-2005). Trauma and hemorrhagic shock are one of the major causes of acute lung injury in humans. Twenty to twenty-five percent of patients with severe trauma develop acute lung injury, and trauma is the second most common cause of acute lung injury after sepsis. Upregulation of alveolar epithelial fluid transport by endogenous catecholamines is a major mechanism that prevents alveolar flooding after hemorrhagic shock. However, after severe hemorrhage, this protective mechanism is abolished by the development of an oxidative stress to the alveolar epithelium. The oxidative stress to this lung barrier is caused by the release of a large quantity of nitric oxide (NO) and radical oxygen species that directly alter the function of membrane proteins involved in the beta-adrenergic receptor-cAMP signaling pathway in the alveolar epithelium and by the sequestration of neutrophils in the lung that amplify oxidative stress to the alveolar epithelium.
The heat shock or stress response is a highly conserved cellular defense mechanism characterized by the increased expression of stress proteins that allows the cells to withstand a subsequent lethal insult, a phenomenon referred as "thermotolerance" or "preconditioning". Stress preconditioning with heat has been shown to protect against ischemia-reperfusion injury to the lung, although the mechanisms of protection are poorly understood. Our preliminary data indicated that induction of thermotolerance either with heat or non-thermal stimulus restored normal alveolar epithelial fluid transport after severe hemorrhage in rats. Therefore, we propose to test the hypothesis that the protection against oxidative stress to the alveolar epithelium provided by stress preconditioning with heat is mediated by: (a) a decrease of the inflammatory response in the lung by inhibition of the expression of proinflammatory mediators (NO, peroxynitrite) that directly inhibit ion transport across the alveolar epithelium (aim 1); (b) a decrease in the neutrophil sequestration in lung after severe hemorrhage by decreasing the release of neutrophils from the bone marrow and by increasing the rate of neutrophil apoptosis, as well as by decreasing the expression of adhesion molecules on the surface of the lung endothelium and the airspace release of chemokines CINC-1 and/or MIP-2 (aim 2). In aim 3 of this proposal, we will explore how stress preconditioning using clinically relevant strategies, such as geldanamycin or direct inducers of Hsp 32 expression, protects against oxidative stress to the alveolar epithelium after hemorrhage.

TGF-beta and alveolar epithelial injury. (SCCOR grant P50HL74005, Project 4, 2003-2007). Acute Lung Injury (ALI) is a devastating syndrome with a mortality rate of 30-40 percent. Alveolar epithelial damage is a characteristic morphologic feature in patients with ALI. The loss of epithelial integrity contributes to the alveolar flooding and disrupts normal lung epithelial fluid transport that is important for the removal of pulmonary edema fluid from the airspaces. The molecular steps regulating the development and resolution of alveolar flooding in ALI are poorly understood. The cytokine transforming growth factor ?? (TGF-??) plays a critical role in the resolution of ALI and in the development of lung fibrosis often associated with this syndrome. We previously reported that the expression levels of several TGF-?1-inducible genes are dramatically increased early after the induction of experimental ALI induced with bleomycin. We also found the??v?6 integrin-mediated local activation of TGF-?1 is critical to the development of pulmonary edema in ALI and that the activation of TGF-?1 depends on a change in the conformation of the??v?6 integrin. However, the mechanism of activation of this integrin is still unknown. IL-1? was found to be biologically active and primarily responsible for the inflammatory activity within the airspaces of patients with ALI. Moreover, transient overexpression of IL-1?, but not of TNF-?, in the lung by adenoviral gene transfer was associated with progressive fibrotic changes and an increased expression of TGF-?1. Finally, preliminary experiments from our laboratory indicate that IL-1?, but not TNF-?, causes activation of the ??v?6-mediated TGF-?1–dependent cell signaling pathway in alveolar epithelial cells. Thus, this application will test the hypotheses that (a) the release of IL-1? within the airspaces is responsible for the ?v?6 integrin-mediated activation of TGF-?1 (aim 1); (b) the activation of the focal adhesion kinase (FAK) and/or its downstream cell effectors, phosphoinositol-3-kinase and small GTPases, Rac-1 and RhoA, is required for IL-1?-induced ?v?6 integrin-mediated local activation of TGF-?1 (aim 2); (c) locally activated TGF-?1 decreases basal and c-AMP regulated lung epithelial fluid transport by altering the expression of amiloride-sensitive sodium channel, ENaC, on the cell membrane of lung epithelial cells (aim 3).
Principal investigator, REAC Grant 2000-01

Principal Investigator, NIH RO1 GM62188 2001-05
Stress preconditioning and alveolar epithelial injury

Co-investigator, NIH RO1 HL66410 (PI: TW Taeusch) 2001-04
Polymer Prevention of Surfactant Inactivation

Co-investigator, NIH RO1 HL51854 (PI: MA Matthay) 2001-08
Alveolar Epithelial Barrier in Clinical Lung Injury

Principal investigator, SCCOR grant P50HL74005, Project 4 2003-08
TGF-beta and alveolar epithelial injury
Most Significant Publications
1. PITTET JF, MJD GRIFFITHS, T GEISER, N KAMINSKI, SL DALTON, X HUANG, LAS BROWN, PJ GOTWALS, VE KOTELIANSKY, MA MATTHAY, D SHEPPARD. TGF-beta is a critical mediator of acute lung injury. J Clin Invest 107:1537-1544, 2001

2. PITTET JF, LN LU, DG MORRIS, K MODELSKA, WJ WELCH, HV CAREY, J ROUX, MA MATTHAY. Reactive nitrogen species inhibit alveolar epithelial fluid transport by NF-kB dependent mechanisms after hemorrhagic shock in rats. J Immunol 166:6301-6310, 2001.

3. PITTET JF, LN LU, T GEISER, H LEE, MA MATTHAY, WJ WELCH. Stress preconditioning attenuates oxidative injury to the alveolar epithelium following hemorrhage in rats J. Physiol. London 538.2: 583-597, 2002.

4. NUKTON SL. JA ALONSO, RH KALLET, BM DANIELS, JF PITTET, MD EISNER, MA MATTHAY. Pulmonary dead-space fraction as a risk factor for death in the acute respiratory distress syndrome. N Engl J Med 346: 1281-1286, 2002.
I was at the origin of the study, contributed significantly to the realization of the study at SFGH and helped Dr Matthay to write the paper. Except for Dr Nukton, the next 3 authors are respiratory therapists that did the dead space measurements. Dr Eisner helped us with the statistical analysis.

5. FRANK J, J ROUX, H KAWAKATSU, G SU, A DAGENAIS, Y BERTHIAUME, MB HOWARD, C CANESSA, XH FANG, D SHEPPARD, MA MATTHAY, JF PITTET. TGF-beta1 decreases alpha-ENaC expression and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2 dependent mechanism. J Biol Chem 278:43939-50, 2003.
The experimental plan was designed by me and 90% of the work was completed in my laboratory.