National Heart, Lung, and Blood Institute program in
Acute Lung Injury
Specialized Centers of Clinically Oriented Research (ALI SCCOR)

Johns Hopkins University, Baltimore

SCCOR in Ventilator-Associated Lung Injury:
Molecular Approaches

Project Listing        Publications        Progress Summary

        Roy G. Brower, MD
           Program Director
           Tel: 410-614-0158
           Email: rbrower@jhmi.edu

 PROGRAM OFFICE ADDRESS

        Johns Hopkins University Department of Medicine
        Pulmonary and Critical Care Medicine
        1830 East Monument Street
        Baltimore, Maryland 21287

 

           

        Paul M. Hassoun, MD
        Program Co-Director
        Tel: 410-550-2606/443-287-3344
        Email: phassoun@jhmi.edu
       
        Johns Hopkins University Department of Medicine
        Pulmonary and Critical Care Medicine
        Johns Hopkins Asthma and Allergy Center
        5501 Hopkins Bayview Circle
        Baltimore, Maryland 21224

 

PROGRAM OBJECTIVE

Acute lung injury is a devastating illness occurring in the context of sepsis and other systemic inflammatory disorders with a clear contribution of mechanical ventilation-mediated stress to adverse patient outcomes.  This SCCOR application is focused on understanding the complex interplay between mechanical ventilation and the increased morbidity and mortality noted in patients with ALI. In concert with the mapping of the Human Genome, high throughput technologies now provide the potential for meaningful translational research to address (i) the molecular basis for rational mechanical ventilation strategies, and (ii) the relationship of mechanical stress to the activation of pathological gene expression in genetically susceptible patients.  Because ALI and ventilator-associated ALI (VALI) are likely manifestations of heterogeneous molecular processes, it is a thematic underpinning of this SCCOR application that advances in genomic technology provide the opportunity to not only characterize pulmonary responses to VALI with increased sensitivity and clarity, but to also identify new molecular targets for potential therapy.   We are conducting comprehensive genomic and proteomic studies of human and animal models of acute lung injury, and characterizing potentially important polymorphisms in a large cohort of well-phenotyped patients with ALI. Importantly, these studies are complemented by innovative studies designed to assess alternate ALI ventilatory strategies and to test novel therapies for VALI-mediated lung edema formation.

This program represents a consortium of investigators with multi-disciplinary expertise, and the common goal to translate basic research discoveries into direct benefit for patients with ALI. Supported by six highly interactive cores (Administration, Data Management and Analysis, Molecular Pathology, Canine Models Core, Genomic/Genotyping, and Biomarkers/ Proteomic), the six human and animal projects utilize state-of-the-art molecular approaches with novel phenotyping instrumentation that are not only likely to provide the deepest understanding of critical pathobiologic processes in VALI to date, but to define key genetic determinants relevant to acute lung injury.  We anticipate our work will facilitate development of new strategies, uncover new therapeutic targets and define new prognostic indicators that will limit the adverse effects of mechanical ventilation on the acutely injured lung.

 SPECIFIC OBJECTS OF THIS PROGRAM ARE:

Our long-term objectives are to improve the care of patients with acute lung injury (ALI) by developing better mechanical ventilation (MV) techniques. MV provides life-sustaining support and is essential for recovery of patients with acute lung injury (ALI). However, MV may itself cause lung injury (ventilation- associated lung injury, VALI), which could prevent recovery from treatable causes of ALI. High frequency oscillatory ventilation (HFOV) is a new mode of MV designed to prevent VALI while ensuring adequate respiratory support. However, several important questions must be answered to optimize HFOV techniques. Moreover, responses of ALI patients who receive HFOV must be compared to those who receive conventional ventilator-based lung protective approaches to MV (CVlp).  This project will use state-of-the-art molecular, imaging, physiologic, and clinical methods to optimize HFOV strategies and to characterize responses of ALI patients to optimized HFOV and conventional ventilator-based lung-protective ventilation (CVlp) approaches. 

Experiments in Specific Aim 1 will characterize effects of different mechanical ventilation strategies on molecular mediators, cytokines, and markers of ALI and VALI and to identify novel molecular pathways in ALI and VALI.   

Experiments in Specific Aim 2 will characterize the interactions between HFOV settings and specific patient characteristics. These studies will reveal magnitudes of HFOV tidal volumes under different physiologic conditions, effects of HFOV pressures on lung recruitment, and effects of HFOV on regional distribution of lung volumes and ventilation. Completion of the experiments for Specific Aims 1 and 2 will provide critical information needed to optimize HFOV and CVlp approaches.

Studies in Specific Aim 3 will utilize this information to design optimal HFOV and CVlp approaches and then conduct an exploratory clinical trial to compare clinical, physiologic, and molecular indicators of efficacy and safety in patients who receive either optimized- HFOV or optimized-CVlp.  Completion of this program of investigation will provide insights regarding pathogenesis of ALI and VALI and will guide future mechanistic and clinical studies designed to improve clinical outcomes in ALI patients.     

 PERFORMANCE SITES 

• Johns Hopkins Hospital, Baltimore, Maryland
• Johns Hopkins Bayview Medical Center, Baltimore, Maryland
• University of Maryland Medical Center, Baltimore, Maryland
• Veterans Administration Medical Center, Baltimore, Maryland
• R. Adams Cowley Shock Trauma Center, Baltimore, Maryland

Project 2:  Long-term outcomes of specific ventilator strategies

Peter J.  Pronovost, MD, PhD

Acute Lung Injury (ALI) is relatively common and associated with significant short-term mortality and long-term morbidity. Despite evidence that a lung-protective conventional ventilator approach (CVLP) is associated with reduced 28-day mortality, it is unknown how widely CVLP is used, and we have a poor understanding of the barriers to its use. Moreover, little is known regarding the long-term mortality and outcomes in ALI patients or whether CVLP improves these long-term outcomes. To improve outcomes for patients with ALI, we must understand the barriers to compliance with CVLP, long-term mortality and other outcomes associated with ALI, and the constellation of factors and other therapies that may be associated with those outcomes. This study will add important new knowledge to our understanding of ways to improve the care of patients with ALI. We will evaluate the following two specific aims:

Specific Aim 1A.  Evaluate the use of CVLP as a care strategy in a cohort of patients with ALI. 

Specific Aim 1B.  Identify patient, provider and organizational barriers to using CVLP.

Specific Aim  2.   Evaluate the association between CVLP ventilation and other aspects of care with long-term mortality in patients with ALI.  We will also undertake a secondary aim: evaluating the association between CVLP and other aspects of care with a broad group of patient outcomes.     To evaluate the aims, we will conduct a cohort study at four hospitals (six intensive care units (ICUs)) in the Baltimore metropolitan area. We will evaluate exposure to specific medical therapies including the use of CVLP, paralysis, steroids, antibiotics and nutrition; and timing of tracheotomy during patient ICU stay.

Outcome variables are: discharge, six months, one year and two year mortality; and disorders of biologic functioning, clinical events and patient reported outcomes. We will evaluate outcomes in the General Clinical Research Center at discharge, one year and two years after diagnosis of ALI.  We will evaluate outcomes at six months via telephone survey. This study is significant as it adds new knowledge about barriers to using CVLP strategies, long-term outcomes of patients with ALI and the impact of CVLP on these outcomes. Moreover, by identifying medical therapies in addition to CVLP that are associated with these outcomes, we will identify opportunities to improve care of patients with ALI.

PERFORMANCE SITES

• Johns Hopkins Hospital, Baltimore, Maryland
• Johns Hopkins Bayview Medical Center, Baltimore, Maryland
• University of Maryland Medical Center, Baltimore, Maryland
• Veterans Administration Medical Center, Baltimore, Maryland

KEY PERSONNEL

Project 3:  Genetic modifiers in acute lung injury

Kathleen C. Barnes, PhD

Whether a clear genetic basis exists for susceptibility to sepsis, acute lung injury, and ventilator-induced acute lung injury remains unresolved.  To address this critical question, we are employing high throughput genomic technologies to examine (i) the patterns of gene expression and (ii) candidate gene polymorphisms which explain susceptibility to ALI. 

Specific Aim 1 will prioritize novel sepsis and sepsis/VALI candidate genes obtained by extensive temporal expression profiling conducted in animal models of sepsis/ALI, and in conjunction with Project #1 personnel, in a small, well phenotyped subset of patients with sepsis and ALI.  These patterns of expression will be analyzed within each clinical and experimental condition, within each species, and then across species.  This approach will allow us to determine both concordantly and discordantly regulated gene clusters, link these gene clusters with physiological measurements of lung function, and determine the gene profiles responsible for the development and maintenance of either resistance or susceptibility to ALI.

Specific Aim 2 will validate the physiological importance of these genes and “high risk” alleles in a large cohort of well phenotyped patients with sepsis/ALI (obtained from the Hopkins/University of Colorado  DNA consortium (CELEG).  We will perform high throughput genotyping for positional candidate genes generated from a combination of (i) human and animal cDNA microarray expression profiles; (ii) preliminary data; and (iii) literature.  We will rely upon public SNP data and the development of novel SNPs and priority will be given to polymorphisms in coding or regulatory regions.

Specific Aim 3 will determine whether frequencies of functional polymorphisms associated with ALI and sepsis vary significantly in African-Americans compared to Caucasians. For any “high risk” alleles/genotypes/haplotypes identified in Specific Aim 2, frequencies will be obtained from the two population-based samples to test the hypothesis that the prevalence of these allelic variants differs according to ethnicity.  We will utilize standard and novel haplotype analytical tools to specifically identify haplotypes, which address the enhanced morbidity and mortality from sepsis and sepsis/VALI.   We anticipate our work will increase understanding of genetic modifiers affecting Acute Lung Injury.

PERFORMANCE SITES

• Johns Hopkins Hospital, Baltimore, Maryland
• Johns Hopkins Bayview Medical Center, Baltimore, Maryland
• University of Maryland, Baltimore, Maryland
• University of Colorado at Denver, Colorado
• Emory University, Atlanta, Georgia

KEY PERSONNEL

Project 4:  Novel targets/therapies in VALI-induced lung edema

Joe G. N. Garcia, MD

A sustained increase in vascular permeability is a defining feature of ventilator-associated lung injury and multi-organ failure, and has important adverse consequences including prolonged physiologic derangement such as hyperoxemia, and reduced lung compliance. This creates a vicious cycle of lung leak, a requirement for continued mechanical ventilation, causes metabolic derangement (malnutrition), and increases the risk of nosocomial and ventilator-associated pneumonia.  There is, therefore, a desperate need for new strategies to reduce vascular leak in patients on mechanical ventilation.  We will utilize well-established models of murine and canine VALI to identify novel molecular targets as well as validate recently described molecular targets involved in VALI- induced edema formation. 

Specific Aim #1 will test the effect of molecular strategies to reduce the activity of the Ca⁺²/calmodulin-dependent myosin light chain kinase in vascular endothelium (EC MLCK), a critical cytoskeletal regulatory enzyme first cloned by the Garcia laboratory.  EC MLCK is intimately involved in multiple aspects of the inflammatory response and directly participates in EC barrier regulation.  

Specific Aim #2 will examine the efficacy of sphingosine 1-phosphate (S1P), a lipid angiogenic critical factor to the barrier protection produced by platelets, as novel therapy in both mice and canine models of ALI.  Our data indicate that Sph 1-P ligates specific endothelial differentiation gene (Edg) receptors to produce rapid, sustained, and dose-dependent increases in the barrier integrity in vitro and in vivo. 

Specific Aim #3 will utilize the lipid-lowering HMG Co A reductase inhibitor, simvastatin, to reduce VALI-induced edema formation.  Recent reports, including our own data, indicate that the statins directly affect vascular remodeling and vascular leak likely via the modulation of intracellular signaling mediated by Rho GTPases and Rho kinase.  Our data indicate that this pathway is utilized by edemagenic agent (such as thrombin and VALI) to increase vascular leak and can be directly modulated by statin treatment resulting in in vitro protection from high permeability edema formation.  SA #1, #2 and #3 will be accompanied by an in silico drug discovery approach designed to identify new therapeutic agents directed against the molecular targets we have identified. 

Specific Aim #4 will define murine strain differences in responses to VALI- mediated vascular leakage, and utilize experimental progeny back cross strategy with multigenic genetic mapping of the murine genome to identify QTLs linked to susceptibility to VALI. Given the profound physiologic derangements which accompany the vascular leak seen in VALI, we speculate that this project, which will explore novel therapies and targets, will more quickly allow us to bridge the movement of scientific discovery into direct patient benefit.

PERFORMANCE SITES

• The University of Chicago, Illinois

KEY PERSONNEL

Project 5:  Lung-kidney interactions in ventilator-associated lung injury

Hamid Rabb, MD

Acute renal failure (ARF) is associated with a 40% mortality rate in the ICU, while respiratory failure (ARDS) is associated with a 35% mortality rate. ARF and ARDS frequently co-exist, and when this occurs, the mortality rate increases to 80%. Despite this frustrating outcome, little is known about the relationships between ARF and ARDS. The persistent high mortality associated with ARF and ventilator-associated lung injury (VALI) demands that investigators broaden their mechanistic studies beyond simply the pathophysiology of isolated organ injury to the more complex arena of inter-organ failure.  Though ARF has long been established to be associated with ARDS in the multiple organ dysfunction syndrome, recent data supports the notion that ARF contributes directly to lung dysfunction. Recently, we found that lungs in experimental ARF had a marked increase in red blood cells in the interstitium with accompanying edema, alveolar haemorrhage, and sludging of red blood cells in the microvasculature. This was accompanied by an increase in pulmonary vascular permeability, which was in part macrophage-mediated. We also found a sharp down-regulation of the lung epithelial sodium channel (ENaC), aquaporin-5 (but not AQ-1), and Na-K/ATPase, which could contribute to lung fluid retention.  More recently, use of lower tidal volumes has decreased mortality in ARDS.  Preliminary data from our group has demonstrated that injurious mechanical ventilation may influence the systemic inflammatory response by leading to up-regulation of intra-renal cytokines (VEGF, IL-6), and apoptosis, which are established mediators in the development of ARF.  We hypothesize that ARF predisposes one to ventilator induced lung injury, and that in turn, VALI predisposes to ARF. 

Specific Aim 1 will determine the effects of ARF on lung function and susceptibility to ventilator-associated lung injury. Using an established mouse model of ARF, we will measure the effects of high and low tidal volume ventilation on lung histology, function (gas exchange, mechanical response to ventilator) and biochemical markers.  In view of the preliminary data and institutional expertise, experimental design will focus on the inflammatory and fluid/solute transport pathways as potential mediators and effectors of the inter-organ dysfunction.

Specific Aim 2 will evaluate the effects of mechanical ventilatory strategy on the kidney and susceptibility to ARF. Kidney histology, function (glomerular filtration rate, sodium transport, urine output) and biochemical markers will be measured.  In both Aim 1 and Aim 2, genomic techniques will be used to develop mechanistic hypothesis and identify novel markers and therapeutics that can intervene in this deadly loop.  Mouse models will be used because of the genetic tools available in the mouse as well as the availability of established mouse models of both ARF and VALI at Hopkins. 

PERFORMANCE SITES

• Johns Hopkins University, Baltimore, Maryland

KEY PERSONNEL

Project 6:  Anti-oxidant regulators in VALI

Sekhar Reddy, PhD
Paul M. Hassoun, MD

The overall aim of this SCCOR application is to understand the complex events related to ventilator-associated lung injury (VALI).

In Project 6, we will determine the role of oxidative stress in VALI.  Oxidative stress has been generally implicated in acute lung injury through production of reactive oxygen species (ROS).  To detoxify ROS, lung cells express a network of integrated antioxidant enzymes (AOE) consisting of both classical and phase II detoxifying enzymes.  However, if the oxidant stress is overwhelming, ROS may suppress or inactivate the AOE system resulting in cellular damage.  We have recently shown by genetic and functional analysis that NF-E2 related factor 2 (Nrf2) plays a critical role in hyperoxia-induced lung injury. This transcription factor is involved in lung epithelial cell repair and stretch, and regulates the induction of phase II AOEs.  We have also shown that Nrf2-knockout mice are more susceptible to hyperoxia-induced lung injury.  In collaboration with the various Cores of the SCCOR, genomic analysis revealed an up-regulation of Nrf2 and AP-1 family members, such as fos-related protein-1 (Fra-1), as well as specific AOEs in the lungs of mice and dogs exposed to mechanical ventilation (MV).  Thus, we hypothesize that activation of transcription factors such as Nrf2 and Fra-1 and their downstream effectors plays a critical role in the modulation of VALI.   To test this hypothesis, we propose the following aims.

Specific Aim 1 will determine the molecular responses of the lung in response to MV and hyperoxia in the development of VALI.  We will compare gene and protein expression profiles and markers of injury in the lungs of mice and dogs exposed to MV and hyperoxia.

Specific Aim 2 will examine the role of Nrf2 and identify its downstream effectors in VALI. 

Specific Aim 3 will investigate the role of Fra-1 and identify its downstream effectors in VALI.

For these studies, we will use respective knockout and/or transgenic murine models with specific lung over-expression of mutant or wild type proteins of Nrf2 and Fra-1, as well as genomic and proteomic techniques in collaboration with Cores E and F.  Cross species comparison and validation of these results in human models will be done through collaboration with other Basic and Clinical Research Projects, respectively.  Results of these studies will provide further insight into the role of oxidative stress as well as the molecular and genetic basis of VALI.  The identification of molecular targets will aid in the development of new strategies aimed at minimizing the potential hazardous effects of mechanical ventilation and hyperoxia.   

PERFORMANCE SITES

• Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland
• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE A - ADMINISTRATIVE

Roy G. Brower, MD
Paul M. Hassoun, MD

Core A (Administrative Core), led by the Program's Co-Principal Investigators, Roy G. Brower, MD and Paul M. Hassoun will provide essential administrative and secretarial support and ensure overall direction and organization of the entire Program.  In addition, this Core will provide accounting support that will ensure appropriate fiscal and scientific oversight, monitoring and compliance with federal and institutional grant management regulations, the latter through several formal mechanisms. 

The objectives of the Administrative core are (i) centralization of all administrative actions and financial recording keeping, (ii) to provide statistical and data processing support for the projects (iii) to prepare scientific and financial reports as required by the university and the NHLBI, (iv) to ensure that the SCCOR research meets the highest standards through periodic review by the internal and external review panels, (v) to facilitate the use of common resources, (vi) to foster exchange of scientific information and ideas, (vii) maintain the various consortium agreements such as with the University of Colorado concerning Projects 1 and 3, and the University of Chicago concerning Project 4, and (viii) provide the projects and cores with a review of all expenditures on a monthly basis and deal with University Accounting and Grants offices concerning grant budgets. 

Core A will coordinate the inter-project, inter-departmental, and inter-institutional collaborative arrangements and evolve new arrangements as deemed necessary for the scientific progress of the SCCOR program as a whole.  Core personnel will orchestrate monthly meetings of the project leaders that will be held to discuss scientific and administrative matters.  Core A will organize regular research seminars, which will allow SCCOR investigators to present their work in progress to other researchers.  Coordinated administrative services will ensure optimal purchasing practices, facilitate communications, and promote scientific interaction.

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE B – DATA MANAGEMENT/ANALYSIS

Gregory B. Diette, MD, MHS

The data management and analysis core (DMAC) is an assembly of outstanding investigators, staff and material resources that will provide key infrastructure support to the ALI SCCOR application. 

The DMAC, headed by Gregory B. Diette, MD, MHS who is both a critical care physician and a rigorously trained epidemiologist, will avail itself of extensive resources readily available on the Johns Hopkins campus, including those of the Bloomberg School of Public Health and the School of Medicine. Performance of high quality data management and analysis functions are essential for assuring that these studies are able to provide accurate detailed documentation of their findings and timely presentation of results. 

This core will be operational at the outset of the project and will include assistance with planning studies, including advice on research design and methods as well as development of study manuals and documents that are essential for conduct of clinical studies. The DMAC will assume responsibility for management of data once collected by the primary investigators and development of databases that facilitate scientific queries initiated by the study investigators. 

The core will also function to provide expert advice on the approach to data analysis as well as execute analysis plans that are developed with the clinical study investigators.  Collectively, these functions will assure that data are stored in a safe and secure manner while also supporting the rapid transmission of results via reports, abstracts, manuscripts and postings to relevant web sites. 

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE C – MOLECULAR PATHOLOGY

Rubin M. Tuder, MD

The main objective of the Molecular Pathology Core is to provide the investigators in this SCCOR program group with a complete range of expertise, training, reagents, and equipment to accomplish the imaging of histologic, molecular, and mRNA targets in fixed and living cells and tissues.

This Core supports the SCCOR’s six research projects with services related to optical detection, visualization, and quantitative microscopy.  It offers access to experienced use of the complete resources of the Johns Hopkins University School of Medicine Microscope Facility (which includes an excellent electron microscope facility) and the Department of Medicine's Multiphoton Laser Scanning Microscope facility.  Dr. Tuder has assembled the personnel and laboratory facilities to satisfy a wide range of experimental imaging needs.  Molecular Pathology Core personnel have professional experience spanning the fields of tissue preparation, cell culture, differential detergent extraction and cytoskeletal preservation, cell component-specific staining, microscopy and digital image analysis.  The technical staff of the Molecular Pathology Core provides essential support to this SCCOR application. 

The core facility features access to multiple microscopes that utilize both Windows PCs and MacIntosh work stations, extensive software for image analysis and visualization, and software and hardware for producing manuscript-ready images and figures in hard copy.  Specific competencies provided by this Core include:  consultation on experimental design, reagents for immunolabeling and fluorescence protein chimera expression, specimen preparation, biopsy processing, in situ hybridization, auto-radiographic studies of radio-labeled material in tissues, immunocytochemical labeling for light, epiflurorescence, multiphoton, or electron microscopic examination, transmitted light and spinning disc confocal implementation of two- and three-dimensional image processing, analysis algorithms and quantitative image analyses, three-dimensional reconstruction and stereoscopic visualization, and graphics services involving digital imaging. 

This core will prepare and analyze specimens from a broad range of research endeavors.  Importantly, during the preparation of preliminary data for this proposal, this Molecular Pathology Core has demonstrated that its flexibility, expertise, and resources are available and essential to all of projects in this SCCOR.

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE D – CANINE MODEL

Brett A. Simon, MD, PhD

This SCCOR proposal encompasses and interrelates approaches to ventilator-associated lung injury (VALI) ranging from genetic and molecular mechanisms, through cellular responses and animal models, and finally to cohort human studies.  Large animal models of VALI are a crucial component of this continuum because they capture the heterogeneity of lung mechanical dysfunction found in human acute lung injury (ALI). 

The function of the Canine Model Core (D) is to provide the project leaders in this SCCOR with large animal models of acute lung injury, with state-of-the-art evaluation of cardio-pulmonary pathophysiology, interventions, data analysis, and interpretation, in order to provide insight into the efficacy and mechanisms of clinically relevant management approaches and to facilitate the translation of basic research to the clinical arena. 

The Core will have the capability to implement several lung injury models, manage mechanical ventilation with conventional and novel techniques, and monitor gas exchange, lung mechanics, fluid status, and hemodynamic function.  A unique feature of the Canine Model Core is its expertise with functional lung imaging using high-speed multi-slice computed tomography (CT) permitting the non-invasive measurement of regional lung mechanics, aeration, lung water, and ventilation and perfusion distributions.  Additionally, in conjunction with the Genomics (E) and Biomarkers (F) Cores, we will develop and validate a new methodology for canine genomic analysis in acute lung injury.  These results will be used to develop novel genomic and proteomic biomarkers, which will be available to all projects.  The combination of these methods will allow the SCCOR investigators to characterize the effects of ventilator management protocols and pharmacologic interventions on regional lung function and cellular responses, and to relate these changes to the local mechanical stresses and regional physiology measured with functional CT imaging. 

By providing large animal genomic and proteomic tools previously available only in rodent models, it will be possible to evaluate promising discoveries and test hypotheses in disease models with clinically relevant mechanical heterogeneity and scale, a necessary step in the translation of basic science to clinical study and practice.

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE E – GENOMICS/GENOTYPING

Kathleen C. Barnes, PhD
Aravinda Chakravarti, PhD

Core E, the Genomics and Genotyping Core, will support the extensive gene expression profiling studies proposed throughout this SCCOR application.   Key to the success of this Core is the availability of a dedicated high-throughput microarray facility, which is based in the Center for Translational Respiratory Medicine.  This array facility includes highly experienced and proficient personnel who utilize an Affymetrix platform to conduct gene array analysis of lung and kidney tissues derived from murine and canine models of ALI as well as human blood and bronchoalveolar lavage cellular elements.

In selected experiments, we will utilize custom canine arrays generated here at Hopkins via an NIH funded canine array initiative. Gene expression will be validated by several complementary approaches. Analyses of these extensive data sets will be facilitated by our bioinformatics unit, which includes key personnel from the Data Management and Analysis Core).  This analysis unit is intimately familiar with the complex issues surrounding normalization and analysis of these gene expression data sets and is supported by the extensive resources and data analytical software available within the HopGene Program in Genomics Application (PGA). 

A second major thrust of the Genomics/Genotyping Core is the performance of high-throughput genotyping via Illumina and Taqman technologies to assess the contributions of candidate gene single nucleotide polymorphisms to the dramatic differences in disease severity characteristic of Acute Lung Injury.  The frequencies of ”high risk “ alleles/genotypes will be evaluated with a particular focus on racial-specific variants as Project 3 preliminary data have identified a dramatic segregation of candidate gene SNPs in African-American patients with ALI.  This Core will utilize standard as well as highly novel haplotype tools to address the enhanced morbidity and mortality from sepsis and ALI. 

The feasibility of this approach is assured by the ready availability of an extensive ALI DNA bank derived from the Hopkins-driven Consortium for the Evaluation of Lung Edema Genetics (CELEG).   In summary, this Core will provide critical comprehensive high throughput gene expression profiling and genotyping for SCCOR personnel, which should generate novel insights into the genetic basis of Acute Lung Injury.

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

CORE F – BIOMARKERS/PROTEOMICS

Michael T. Crow, PhD

The major goal of the Biomarkers/Proteomics Core is to provide the principal investigators in this SCCOR program with a comprehensive measurement of biological markers to assess and correlate ventilator associated lung injury (VALI) in human and animal models.  Additionally, this Core will utilize the newly emerging, state of the art techniques of proteomics and genomics to integrate clinical and basic research pertaining to VALI.  This Core supports the SCCOR's six projects with services related to three key areas. 

First the Core will provide a centralized facility to analyze, compile and correlate levels of biomarkers such as interleukin-8 nitrotyrosinated proteins, von Wilebrand Factor, surfactant proteins B, C, and D, protein carbonlys and lipid peroxidation products, isoprostanes and sphingosine-1-phosphate to endothelial and epithelial injury/leakiness in VALI.  This classical approach will utilize plasma, serum and bronchial alveolar lavage (BAL) from HFV versus conventional as well as from clinical patients with sepsis presenting either no acute lung injury or acute lung injury with the ventilator.  The human studies will be

Second, the NIH funded Proteomics Center at the Johns Hopkins University School of Medicine will provide the necessary infrastructure and expertise to initiate and develop a proteomics approach to the Biomarkers Core.  Our investigators have assembled the personnel and state of the art proteomics technology to meet the immediate and long-term needs of the scientific community at Hopkins.  Specific competencies provided by the proteomics facility include:  consultation and training of research faculty and technical personnel in differential imaging gel electrophoresis analysis, isotope-coded affinity tag analysis and utilization of mass spectrometry technology in obtaining structural information, identification and expression of new proteins, determining modifications, map cleavage sites and identifying binding partners. 

Third, the Genomics/Genotyping Core will provide the needed infrastructure and trained personnel to compare the phenotypic changes to gene expression in the endothelial and epithelial cells from human and animal models of VALI.

PERFORMANCE SITES

• Johns Hopkins University School of Medicine, Baltimore, Maryland

KEY PERSONNEL

Johns Hopkins University, Baltimore

SCCOR in Ventilator-Associated Lung Injury: Molecular Approaches

PUBLICATIONS TO DATE

PROJECT 1

Fessler HE, Brower R.  Protocols for lung protective ventilation.  Crit Care Med. 2005;33:S223-S227.

Hager DN, Krishnan JA, Hayden DL, Brower RG. Tidal Volume Reduction in ARDS Patients with Plateau Pressures Less Than 30 to 35 cm H₂O. Am J Respir Crit Care Med 2005 (in press).

Hager DN, Fuld M, Kaczka, DW, Fessler HE, Brower RG, Simon BA. Four methods of measuring tidal volume during high frequency oscillatory ventilation. Crit Care Med 2005 (in press)

Simon BA, Easley RB, Ye SQ, Ma SF, Lavoie T, Grigoryev D, Garcia, JG.  Microarray analysis of regional cellular responses to l