Search

Finigan JH, Boueiz A, Wilkinson E, Damico R, Skirball J, Pae HH, Damarla M, Hasan E, Pearse DB, Reddy SP, Grigoryev DN, Cheadle C, Esmon CT, Garcia JG, Hassoun PM. Activated protein C protects against ventilator-induced pulmonary capillary leak. Am J Physiol Lung Cell Mol Physiol 296: L1002-L101 1, 2009. First published April 10, 2009; doi: 10.1 152/ajplung.90555.2008.-The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HV[subT]; 20 mI/kg) or low (LV[subT]; 7 mI/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 mm after starting MV. Indices of capillary leakage included bronchoalveolas lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rhoassociated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HVT induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HVT also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from HV[subT]-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-β[sub6]) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection. [ABSTRACT FROM AUTHOR]

Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALl), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and DahI salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 mI/kg, 85 breaths/mm, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as "transcription" and "chemotaxis/cell motility." We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is associated with significant mortality, which increases further when combined with acute lung injury. Experiments in rodents have shown that kidney ischemia-reperfusion injury (IRI) facilitates lung injury and inflammation. To identify potential ischemia-specific lung molecular pathways involved, we conducted global gene expression profiling of lung 6 or 36 h following 1) bilateral kidney IRI, 2) bilateral nephrectomy (BNx), and 3) sham laparotomy in C57BL/6J mice. Bronchoalveolar lavage fluid analysis revealed increased total protein, and lung histology revealed increased cellular inflammation following IRI, but not BNx, compared with sham controls. Total RNA from whole lung was isolated and hybridized to 430MOEA (22,626 genes) GeneChips (n = 3/group), which were analyzed by robust multichip average and significance analysis of microarrays and linked to gene ontology (GO) terms using MAPPFinder. The microarray power analysis predicted that the false discovery rate (q < 1%) and ≥50%-fold change compared with sham would represent significant changes in gene expression. Analysis identified 266 and 455 ischemia-specific, AKI-associated lung genes with increased expression and 615 and 204 with decreased expression at 6 and 36 h, respectively, compared with sham controls. Real-time PCR analysis validated select array changes in lung serum amyloid A3 and endothelin-I GO analysis revealed significant activation (Z > 1.95) of several proinflammatory and proapoptotic biological processes. Ischemic AM induces functional and transcriptional changes in the lung distinct from those induced by uremia alone. Further investigation using this lung molecular signature induced by kidney IRT will provide mechanistic insights and new therapies for critically ill patients with AKI. [ABSTRACT FROM AUTHOR]

Acute lung injury is a complex illness with a high mortality rate (>30%) and often requires the use of mechanical ventilatory support for respiratory failure. Mechanical ventilation can lead to clinical deterioration due to augmented lung injury in certain patients, suggesting the potential existence of genetic susceptibility to mechanical stretch (6, 48), the nature of which remains unclear. To identify genes affected by ventilator-induced lung injury (VILI), we utilized a bioinformatic-intense candidate gene approach and examined gene expression profiles from rodent VILI models (mouse and rat) using the oligonucleotide microarray platform. To increase statistical power of gene expression analysis, 2,769 mouse/rat orthologous genes identified on RG̱U34A and MG̱U74Av2 arrays were simultaneously analyzed by significance analysis of microarrays (SAM). This combined ortholog/SAM approach identified 41 up- and 7 downregulated VILI-related candidate genes, results validated by comparable expression levels obtained by either real-time or relative RT-PCR for 15 randomly selected genes. K-mean clustering of 48 VILI-related genes clustered several well-known VILI-associated genes (IL-6, plasminogen activator inhibitor type 1, CCL-2, cyclooxygenase-2) with a number of stress-related genes (Myc, Cyr61, Socs3). The only unannotated member of this cluster (n = 14) was RIKEṈ1300002F13 EST, an ortholog of the stress-related Gene33/Mig-6 gene. The further evaluation of this candidate strongly suggested its involvement in development of VILI. We speculate that the ortholog-SAM approach is a useful, time- and resource-efficient tool for identification of candidate genes in a variety of complex disease models such as VILI. [ABSTRACT FROM AUTHOR]

Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALl) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell bather protection in vitro, in a murine inflammatory model of ALL C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 µg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; -35-60% reduction). Simvastatin also decreased BAL albumin (-50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with bather protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-KB regulation) and with respect to individual genes implicated in the development or severity of ALT (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI. [ABSTRACT FROM AUTHOR]