Differential gene profiling in acute lung injury identifies injury-specific gene expression* Claudia C. dos Santos, MD; Daisuke Okutani, MD, PhD; Pingzhao Hu, MSc; Bing Han, MD, PhD; Ettore Crimi, MD; Xiaolin He, MD, PhD; Shaf Keshavjee, MD; Celia Greenwood, PhD; Author S. Slutsky, MD; Haibo Zhang, MD, PhD; Mingyao Liu, MD Objectives: Acute lung injury can result from distinct insults, ies included real-time polymerase chain reaction, Western such as sepsis, ischemia–reperfusion, and ventilator-induced blots, and immunohistochemistry. Physiologic and morpho- lung injury. Physiologic and morphologic manifestations of dis- logic variables were noncontributory in determining the cause parate forms of injury are often indistinguishable. We sought to of acute lung injury. In contrast, molecular analysis revealed demonstrate that acute lung injury resulting from distinct insults unique gene expression patterns that characterized exposure may lead to different gene expression profiles. to lipopolysaccharide and high-volume ventilation. We used Design: Microarray analysis was used to examine early mo- hypergeometric probability to demonstrate that specific func- lecular events in lungs from three rat models of acute lung injury: tional enrichment groups were regulated by biochemical vs. lipopolysaccharide, hemorrhage shock/resuscitation, and high- biophysical factors. Genes stimulated by lipopolysaccharide volume ventilation. were involved in metabolism, defense response, immune cell Setting: University laboratory. proliferation, differentiation and migration, and cell death. In Subjects: Male Sprague-Dawley rats (body weight, 300–350 g). contrast, high-volume ventilation led to the regulation of genes Interventions: Rats were subjected to hemorrhagic shock or involved primarily in organogenesis, morphogenesis, cell cy- lipopolysaccharide followed by resuscitation or were subjected to cle, proliferation, and differentiation. sham operation. First hit was followed by ventilation with either Conclusions: These results demonstrate the application of low (6 mL/kg) or high (12 mL/kg) tidal volume for 4 hrs. functional genomics to the molecular “fingerprinting” of acute Measurements and Main Results: Physiologic and morpho- lung injury and the potential for decoupling biophysical from logic variables were assessed. Total RNA was hybridized to biochemical injury. (Crit Care Med 2008; 36:855–865) Affymetrix chips. Bioconductor was used to identify signifi- KEY WORDS: transcriptional profiling; acute respiratory distress cantly altered genes. Functional enrichment predictions were syndrome; ventilator-induced lung injury; bioinformatics; sepsis; performed in Gene Ontology Tree Machine. Confirmation stud- shock cute respiratory distress syn- high mortality persists (25–50%) (2, 3). It Regardless of the pathogenesis, the drome (ARDS), the most se- has become apparent that mechanical clinical manifestations of ARDS are in- vere manifestation of acute ventilation itself can be injurious to the distinguishable from the original in- lung injury (ALI), is clinically lung (i.e., ventilator-induced lung injury) sult. Therefore, a common pathway of Adefined as severe dysfunction of gas ex- (4). Thus, elucidating the molecular acute inflammatory responses has been change and chest radiographic abnormal- characteristics of ALI/ventilator-induced proposed to explain how different in- ities after a predisposing injury in the lung injury is vital to the development of sults may lead to lung tissue damage, absence of heart failure (1). Despite ad- novel approaches for diagnosing and deterioration of oxygenation function, vances in life support, an unacceptably managing ARDS. and subsequent dysfunction in multiple organs. Iatrogenic injury associated with the *See also p. 1014. Raw data for this article have been deposited to: supportive care of the ALI/ARDS patient From the Thoracic Surgery Research Laboratory, http://www.ncbi.nlm.nih.gov/projects/geo/. Toronto General Research Institute, University Drs. dos Santos and Okutani contributed equally to and the failure of anti-inflammatory ther- Health Network, Toronto, Ontario, Canada (CCS, DO, this study. apies with specific molecules, such as tu- BH, XH, SK, ML); Critical Care Medicine, Saint Mi- For information regarding this article, E-mail: mor necrosis factor-␣ binding protein, chael’s Hospital, Toronto, Ontario, Canada (CCS, EC, [email protected] or claudia.santos@ have contributed to the pressing need for ASS, HZ); and Genetics and Genomic Biology, Hos- utoronto.ca pital for Sick Children, Toronto, Ontario, Canada (PH, CG). Supplementary tables and figures are available at novel approaches in the management of The authors have not disclosed any potential con- www.ccmjournal.org this syndrome in the intensive care unit flicts of interest. Copyright © 2008 by the Society of Critical Care (5, 6). There is increasing awareness that Supported, in part, by operating grants MOP- Medicine and Lippincott Williams & Wilkins injury-specific mechanisms and individu- 13270 and MOP-42546 and by fellowship 6LA-54707 DOI: 10.1097/CCM.0B013E3181659333 from the Canadian Institutes of Health Research, Ot- alized therapies for ARDS patients may tawa, Ontario, Canada. become attractive therapeutic options in Crit Care Med 2008 Vol. 36, No. 3 855 the future (7). To this end, specific mech- signatures” for clinical diagnosis and METHODS anisms of injury in the lung must be prognosis (7). For example, multiple CXC Animal Experiments. Male Sprague-Daw- elucidated. chemokines up-regulated by tumor ne- ley rats (n ϭ 24, 300 to 350 g; Charles River, In the present study, we explored the ␣ crosis factor- in human lung epithelial Montreal, Quebec, Canada) were anesthetized hypothesis that ALI is not a stereotyped cells are located closely on the same with ketamine hydrochloride (80 mg/kg; Ay- response of the lung to injury but rather a chromosome (8). A group of transform- erst Veterinary Laboratories, Guelph, Canada) composite of specific responses to different ing growth factor-␤–inducible genes is and xylazine (8 mg/kg; Bayer, Toronto, Can- coexisting injury mechanisms. We postu- up-regulated together in bleomycin- ada) administered intraperitoneally. After tra- lated that exploring the global response to treated animals (9). Using genetic linkage cheostomy, a 14-gauge catheter was inserted injury using microarray technology would analysis, a pulmonary irritant-sensitive into the trachea. The right carotid artery was reveal the presence of injury-specific differ- cannulated with a 24-gauge Angiocath (Bec- locus on murine chromosome 6 was ton Dickinson, Franklin Lakes, NJ) for mea- ential gene expression patterns in com- identified (10). Inspired by these investi- parable lung injury models. We also pro- suring mean arterial blood pressure (MAP), gations, the objective of the present study posed that such patterns would contain blood withdrawal, and resuscitation. The tail was to explore whether injury-specific vein was catheterized with a 22-gauge Angiocath genes that are biologically plausible and differential gene expression patterns (Becton Dickinson) for continuing sedatives of functionally related and thus can be ex- Ϫ1 Ϫ1 could be recognized and separated from ketamine hydrochloride (20 mg·kg ·hr ), xy- ploited for future mechanistic studies Ϫ1 Ϫ1 genes that are commonly involved in ALI lazine (4 mg·kg ·hr ), and pancuronium and, more importantly, may serve as a (0.3 mg·kgϪ1·hrϪ1). Hemodynamics, lung from comparable and otherwise indistin- template for novel molecular diagnostics elastance, and arterial blood gas measure- of ALI. guishable animal models of lung injury. ments were performed (11). Animals in all Whole-genome approaches have been In the present study, we used adult rats groups underwent the same initial instrumen- proposed as feasible and efficient ways of treated with lipopolysaccharide (LPS; tation and anesthesia. Rats were randomized dissecting the molecular response to in- model of septic shock) or hemorrhagic to receive HS (volume reduction to MAP of 40 jury. Stimulus-specific co-expression pat- shock/resuscitation (HS; model of isch- mm Hg for 30 mins) or systemic administra- tion of LPS (5 mg/kg intravenously) followed terns describing the transcriptional be- emia/reperfusion) as the “first hit,” fol- lowed by mechanical ventilation as the by resuscitation or to undergo sham operation havior of multiple genes simultaneously (without HS or LPS). Animals were subse- in response to an insult may provide “second hit” to test our hypothesis that ALI quently randomized again to receive mechan- clues to the underlying molecular mech- from different causes can lead to specific ical ventilation with either a low tidal volume anisms of injury; these injury-specific molecular profiles based on injury-specific (LV) of 6 mL/kg and 5 cm H2O of positive patterns could also be used as “molecular differential gene expressions. end-expiratory pressure or a high tidal volume (a) (b) 120 200 Ventilation (e) LV HV 100 150 80 * * * LPS * * * 60 HSHV 100 * *+ HSLV (mmHg) * Sham 2 MAP (mmHg) 40 LPSHV Hemorrhage *^+ LPSLV PaO 50 20 ShamHV Resuscitation ShamLV 0 0 BL -1 -0.5 0 0 1 2 3 4 (h) BL 0 1 2 3 4 (h) (c) (d) *^ +^ 8 *+ ** + LPS 160 ^* **+ * +* * 7 ** * 140 * * ** * 6 120 Elastance (%) S 5 Lung Wet/Dry Ratios H 100 4 01234(h) LVHV LV HV LV HV Sham LPS HS Figure 1. Physiologic variables of acute lung injury. Animals received lipopolysaccharide (LPS; 5 mg/kg intravenously) or hemorrhagic shock (HS) followed by resuscitation, or received sham