The Division of Pulmonary and Critical Care Medicine published 12 peer-reviewed research papers in 2007-2008. Of these, four studies are especially noteworthy accomplishments in Pulmonary and Critical Care Medicine.

Yacobi NR, DeMaio L, Xie J, Hamm-Alvarez SF, Borok Z, Kim KJ, Crandall ED: Polystyrene nanoparticle trafficking across alveolar epithelium. Nanomedicine 4:139-145, 2008.

We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified [negatively charged] and amidine-modified [positively charged]) across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration ([PNP]) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy. Fluxes increased as charge density became less negative/more positive, with positively charged PNPs trafficking 20-40 times faster than highly negatively charged PNP of comparable size. Trafficking rates decreased with increasing PNP diameter. PNP fluxes tended to level off at high apical [PNP]. Fluxes at 4 degrees C were significantly lower than those at 37 degrees C. Confocal microscopy revealed nanoparticles localized to cell cytoplasm, whereas cell junctions and nuclei appeared free of PNP. These data indicate that (1) trafficking of PNP across RAECM is strongly influenced by charge density, size, and temperature, (2) PNP translocate primarily transcellularly, and (3) PNP translocation requires cellular energy.

Liebler JM, Lutzko C, Banfalvi A, Senadheera D, Aghamohammadi N, Crandall ED, Borok Z: Retention of human bone marrow-derived stem cells in murine lungs following bleomycin-induced lung injury. Am J Physiol, in press, 2008.

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/beta(2) microglobulin (beta(2)M)(null) mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA(+) cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, <0.005% of enzymatically dispersed lung cells from NOD/SCID mice were CMFDA(+), which increased 10- to 100-fold in NOD/SCID/beta(2)M(null) mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA(+) cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.

Qiao R, Yan W, Clavijo C, Mehrian-Shai R, Zhong Q, Kim KJ, Ann D, Crandall ED, Borok Z: Effects of KGF on alveolar epithelial cell transdifferentiation are mediated by JNK signaling. Am J Respir Cell Mol Biol 38:239-246, 2008.

Rat alveolar epithelial cells (AEC) in primary culture transdifferentiate from a type II (AT2) towards a type I (AT1) cell-like phenotype, a process that can be both prevented and reversed by keratinocyte growth factor (KGF). Microarray analysis revealed that these effects of KGF are associated with upregulation of key molecules in the mitogen-activated protein kinase (MAPK) pathway. To further explore the role of three key MAPK (i.e., extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38) in mediating effects of KGF on alveolar epithelial cell (AEC) phenotype, primary rat AEC cultivated in minimal defined serum-free medium (MDSF) were treated with KGF (10 ng/ml) from Day 4 for intervals up to 48 hr. Exposure to KGF activated all three MAPK, JNK, ERK1/2, and p38. Inhibition of JNK, but not of ERK1/2 or p38, abrogated the ability of KGF to maintain the AT2 cell phenotype, as evidenced by loss of expression of lamellar membrane protein (p180) and increased reactivity with the AT1 cell-specific monoclonal antibody VIIIB2 by Day 6 in culture. Overexpression of JNKK2, upstream kinase of JNK, increased activation of endogenous c-Jun in association with increased expression of p180 and abrogation of AQP5, suggesting that activation of c-Jun promotes retention of the AT2 cell phenotype. These results indicate that retention of the AT2 cell phenotype by KGF involves c-Jun and suggest that activation of c-Jun kinase may be an important determinant of maintenance of AT2 cell phenotype.
Zhou B, Ann DK, Li K, Kim KJ, Lin H, Minoo P, Crandall ED, Borok Z: Hypertonic induction of aquaporin 5: novel role of hypoxia inducible factor-1alpha. Am J Physiol 292:C1280-C1290, 2007.

Aquaporin-5 (AQP5) is a water channel protein expressed on the apical surface of alveolar epithelial type I cells in distal rat lung, suggesting a role for AQP5 in regulating alveolar surface liquid tonicity and/or cell volume. We investigated the molecular mechanisms underlying hypertonic induction of AQP5 in primary rat alveolar epithelial cells (AEC). Steady-state levels of AQP5 mRNA and protein were increased by exposure to sorbitol (200 mM in culture fluid) for 24 h. The increase in AQP5 was not accompanied by changes in mRNA half-life. Transduction of mouse lung epithelial (MLE-15) cells and primary rat AEC with lentivirus vectors encoding AQP5-luciferase demonstrated transcriptional activation of the reporter by exposure to hypertonic sorbitol solution. Hybridization of proteins from sorbitol-treated cells to a transcription factor DNA array demonstrated induction of hypoxia-inducible factor-1a (HIF-1a) by hypertonicity, which was confirmed by quantitative RT-PCR. Cotransfections of AQP5-luciferase with HIF-1a and HIF-1ß expression plasmids in MLE-15 cells led to dose-dependent transcriptional enhancement, which was partially abrogated by mutagenesis of putative HIF-1a binding sites in the proximal AQP5 promoter. Importantly, hypertonic induction of AQP5 was significantly inhibited by preventing HIF-1a induction with small interfering RNA. Hypertonicity induced activation of a transiently transfected vascular endothelial growth factor (VEGF) hypoxia response element-driven luciferase construct and increased expression of endogenous VEGF. These results demonstrate that hypertonic induction of both AQP5 and VEGF is transcriptionally regulated and mediated, at least in part, by HIF-1a, suggesting a novel role for HIF-1a in modulating cellular adaptive responses to osmotic stress.

OVERVIEW

Basic and clinical research remains a major focus of the Division’s interests and activities. A number of investigators are studying pulmonary structure and function at the organ, tissue, cellular and molecular levels. Current areas of investigation include study of mechanisms and regulation of water, solute and macromolecule transport across the pulmonary alveolar epithelium; development of cell-type specific markers for alveolar type I and type II cells using monoclonal antibodies and genetic markers; and regulation of lung cell growth, differentiation and gene expression in vitro. In addition, clinical research studies in the fields of sarcoidosis and other granulomatous disorders, acquired immune deficiency syndrome (AIDS), asthma, septic shock, barotrauma, pulmonary edema, acute respiratory distress syndrome (ARDS and tuberculosis) and lung transplantation are underway.

Faculty Research Areas

Kamyar Afshar, DO
Lung Transplant
Cystic Fibrosis

Richard G. Barbers, M.D.
Mechanisms of Remodeling in Near-Fatal Asthma
Mechanisms of Remodeling in Pulmonary Fibrosis
Novel Therapeutic Interventions for BOS in Lung Transplant Recipients
Novel Therapeutic Interventions in Severe Asthma

Ahmet Baydur, M.D.
Respiratory Mechanics
Control of Ventilation
Neuromuscular Disorders
Acute Respiratory Distress Syndrome
Sarcoidosis

Zea Borok, M.D.
Alveolar Epithelial Cell Function and Differentiation
Modulation of Alveolar Epithelial Cell Phenotype and Recovery Following Lung Injury
Pulmonary Alveolar Epithelial Cell Homeostasis
Stem Cell Biology in Lung Injury
Transport Properties of Pulmonary Alveolar Epithelium

C. Thomas Boylen, M.D.
Oxygen Therapy in COPD
Immunological Diseases of the Lung
Pleural Diseases
Clubbing and Its Evaluation in an Asbestos Contact Population
Nocturnal Oxygen Therapy Trial
Acute Effects of Welding
Effects of Formaldehyde Exposure

Ching-Fei Chang, M.D.
Interventional Bronchoscopy
Endobronchial Interventions for COPD
Bronchial Thermoplasty for Asthma
Critical Care Ultrasonography
Medical Pleuroscopy
Medical Education

Edward D. Crandall, Ph.D., M.D.
Markers of Rat Alveolar Epithelial Cell Development and Differentiation
Regulation of Pulmonary Epithelial Cell Differentiation
Acute and Chronic Lung Injury and the Factors that Influence Recovery
Transport Properties of Pulmonary Alveolar Epithelium
Cell Pathophysiology of Alveolar Epithelium
Nanoparticle Interactions with Lung

Sivagini Ganesh, M.D.
Lung Transplant
Pulmonary Hypertension

Ricardo H. Juarez, M.D.
Mechanical Ventilation in Respiratory Failure
Preoperative Assessment of Obese Patients

Kwang-Jin Kim, Ph.D.
Drug Delivery through the Lung
Tight Junctions in Alveolar Epithelium

Janice M. Liebler, M.D.
Cell-Based Treatment of Lung Diseases
Lung Epithelial Cell Biology
Lung Injury and Repair
Lung Transplantation

Richard L. Lubman, M.D.
Cell-Matrix Interactions by Human Embryonic Stem Cells
Cell-Matrix Interaction by Alveolar Epithelium in Repair of Lung Injury
Regulation of Intracellular pH and CO2 Transport by Alveolar Epithelium

Albert H. Niden, M.D.
Pathogenesis, Diagnostic Techniques and Treatment of Diffuse Interstitial Lung Disease

Renli Qiao, M.D., Ph.D.
Mechanisms of Alveolar Homeostasis

Adupa P. Rao, M.D.
Cystic Fibrosis
Sepsis

Bertrand J. Shapiro, M.D.
Cystic Fibrosis

Om P. Sharma, M.D.
Predictors of Survival in Patients with Sarcoidosis
Long Term Prognosis of Myocardial Sarcoidosis
Neurological Sarcoidosis

Hidenobu Shigemitsu, M.D.
Interstitial Lung Disease

Robert S. Swinney, M.D.
Clinical Applications of Computers in Critical Care Medicine

SPECIAL BASIC TRANSLATIONAL RESEARCH ACTIVITIES

Zea Borok, M.D.
Associate Director, Will Rogers Institute Pulmonary Research Center
Regulation of Gene Expression in Lung Growth and Differentiation. The alveolar epithelium lining the gas exchange surface of the adult lung consists of two highly specialized cell types, type II and type I cells. These cell types are distinguished from each other by their characteristic morphologic appearances and by expression of unique cell-specific phenotypic markers. Type II cells have been well-characterized with regard to their role in surfactant production. In contrast, despite the fact that type I cells cover ~90% of the gas exchange surface, little is known of their functional properties or of the mechanisms that regulate gene expression specifically in type I cells. Aquaporin-5 (AQP5) is a member of a family of water channel proteins that is expressed in type I, but not type II, cells of the alveolar epithelium. Dr. Borok is studying the mechanisms underlying cell-specific expression of AQP5 in order to elucidate how gene expression is regulated in type I cells. She and her associates have isolated the regulatory (promoter) region of AQP5 from rat genomic DNA. They have mapped the site at which transcription is initiated and, using transient transfections into a lung cell line, have delineated regions of the promoter that appear to be important for high levels of expression in lung. Dr. Borok is also using in vivo approaches to evaluate the ability of the AQP5 promoter to regulate expression of a reporter gene in type I cells within the lungs of transgenic mice. These studies will provide important new insights into the mechanisms that regulate gene expression in type I cells. Identification of a promoter that is able to direct gene expression in a cell-specific fashion in type I cells should prove useful for targeted delivery to type I cells of potentially therapeutic genes in order to modulate type I cell function and accelerate restoration of normal alveolar architecture following injury.

Edward D. Crandall, Ph.D., M.D.
Director, Will Rogers Institute Pulmonary Research Center
Gene Regulation in Lung Injury and Repair. Dr. Crandall’s research group focuses on the lung’s primary barrier between the external environment and the internal milieu, the alveolar epithelium (lining cells of the air sacs). In addition to several specific projects described below that are currently in progress under the supervision of Division faculty, Dr. Crandall and his group are comprehensively studying many additional aspects of alveolar lung cell biology. Current projects include studies on the effects of injury from excess oxygen exposure (hyperoxia) and lack of oxygen (hypoxia) on the alveolar epithelial barrier, on pharmacological agents that can induce the alveolar epithelium to increase water clearance from the lungs, and on the process of alveolar epithelial cell differentiation after lung injury. The ultimate goal of these studies is to develop a better understanding of the pathways of salt and water absorption by the lungs, and to develop new therapeutic approaches for diseases that cause respiratory failure due to excess alveolar fluid (i.e., pulmonary edema). Conditions such as congestive heart failure (CHF) and the adult respiratory distress syndrome (ARDS), two major causes of morbidity and mortality in the U.S., could potentially be ameliorated by manipulation of alveolar fluid balance and modulation of the process of alveolar epithelial cell differentiation.

Kwang-Jin Kim, Ph.D.
Senior Investigator, Will Rogers Institute Pulmonary Research Center
Drug Delivery Through the Lung. The alveolar and airway epithelium (lining cells) form the primary barrier between the relatively dry alveolar air space and aqueous internal milieu. Dr. Kim and associates are currently studying transport of water, ions, peptides, and proteins across the alveolar epithelial barrier using in vitro models of the alveolar epithelium, including air-interfaced monolayer cultures of alveolar pneumocytes. Trafficking of white blood cells (leukocytes, lymphocytes, and macrophages) and tuberculous bacteria (mycobacteria) across the alveolar epithelium are also being studied, as are the effects of injurious agents (H2O2, NO2 acrolein, acetaldehyde, and cigarette smoke) on alveolar epithelial barrier properties. These studies are expected to yield important information concerning the mechanisms involved in maintenance of normal alveolar fluid balance, and will help to characterize drug absorption by the alveolar epithelium as an alternative systemic drug delivery route.

Janice M. Liebler, M.D.
Investigator, Will Rogers Institute Pulmonary Research Center
Cell-Based Therapy for Lung Diseases. Although most lung injury is repaired by locally derived progenitor cells, recent information suggests that cells that originate outside the injured organ, presumably derived from the bone marrow, may also repopulate the lung. Dr. Liebler is interested in learning whether human bone marrow-derived cells, in contrast to murine bone marrow-derived cells, are able to promote repair of injured lung tissue. Dr. Liebler is using well-characterized mouse xenograft models to determine the potential of adult human bone marrow-derived cells to migrate to the lungs of immunodeficient mice following a single intravenous infusion. Since previous studies have established the need for lung injury to be present to show significant levels of engraftment, mice are studied with and without bleomycin (bleo)-induced lung injury. These studies may provide important insights into the potential of cell-based therapy in the treatment of lung diseases.

Renli Qiao, M.D., Ph.D.
Investigator, Will Rogers Institute Pulmonary Research Center
Gene Therapy for Acute Lung Injury. Pulmonary edema is a common and severe condition resulting from acute lung injury (ALI) of various causes. In its most severe form, edema fluid fills the alveolar air spaces resulting in hypoxemia and respiratory failure. The resolution of alveolar edema depends on active ion transport (accompanied by water) across the alveolar epithelium driven by basolaterally located Na pumps. Augmentation of active Na transport via upregulation of Na pump activity through gene transfer of Na pump subunit(s) in the alveolar epithelial cells (AEC) is a potential strategy for enhancing alveolar fluid clearance following ALI and is the focus of Dr. Qiao’s research. Currently, he is trying to 1) develop a lentivirus vector that can efficiently infect AEC, and 2) investigate the best strategy of gene delivery to AEC for upregulating functional Na pumps. Since functional Na pumps consist of two structurally and functionally distinct subunits, to achieve augmentation of Na pump activity, genes of both subunits have to be delivered simultaneously into each target cell. Dr. Qiao has successfully incorporated two different genes into the backbone plasmid of a lentivirus vector and in pilot experiments has demonstrated that both genes are efficiently made by this construct. He has determined that the best route of administration of lentivirus to transfect AEC is from the alveolar side (instead of basolateral side) which is encouraging for the development of inhalational strategies for gene delivery. He is currently evaluating lentivirus vectors generated with these plasmids to overexpress functional Na pump in cultured AEC and in vivo.

SPECIAL CLINICAL RESEARCH ACTIVITIES

Richard G. Barbers, M.D.
Chronic Inflammation and Remodeling in Asthmatics. Remodeling may occur in mild, moderate and severe asthmatics and may be a reason for persistent and refractory asthma episodes. However, not all asthma patients manifest remodeling. There may be differences in the inflammatory and immune responses. In order to define these processes in the airways, severe asthma subjects will undergo bronchoscopy, bronchoalveolar lavage (BAL) and proximal airway biopsies. The cellular and protein material retrieved by BAL as well as airway biopsies are studied in the laboratory. Our research will attempt to show that abnormal repair processes and growth factors eventually lead to airway fibrosis (remodeling). The information obtained will provide insight into pathogenesis as well as potential therapeutic interventions for severe asthmatics. In addition, with researchers at the University of Washington, Dr. Barbers is exploring similar mechanisms of remodeling in pulmonary fibrosis. This collaborative effort will examine the effect of inhibitors in the fibrotic process.

Ahmet Baydur, M.D.
A New Noninvasive Method for Evaluating Upper Airway Collapse in Sleep Apnea. The major pathophysiologic factor contributing to the generation of sleep disordered breathing is increased upper airway collapsibility. Expiratory compliance of the upper airway is higher than inspiratory compliance and is higher in obstructive sleep apnea syndrome (OSAS) than in normal subjects. Assessment of flow dynamics during expiration, therefore, should provide information about the degree of airway collapse or occlusion. Demonstration of expiratory flow limitation (EFL) has been facilitated by the introduction of the negative expiratory pressure (NEP) technique. In this approach, a small negative pressure (-3 to -5 cm H2O) is applied at the start of expiration during tidal breathing. In normal subjects, an increase in expiratory flow is observed. In subjects with EFL the flow measured during the application of NEP will not exceed spontaneous flow. This simple, noninvasive, effort independent and fast technique can be applied in any body position. EFL has been found to correlate with the desaturation index in OSAS and the severity of OSAS, suggesting that the greater the EFL over tidal expiration, the higher is the collapsibility of the upper airway in apneic/hypopneic patients. This phenomenon has been observed more commonly in the supine position. There remain, however, problems in interpreting the presence of EFL in patients with OSAS and those with intrathoracic obstructive airway disease (COPD and asthma). Patients with the latter condition also exhibit EFL, although their expiratory flow pattern can sometimes be distinguished from those with OSAS. Because of these interpretive difficulties, the potential usefulness of the NEP technique as a diagnostic tool in daily clinical practice is unknown. The purpose of this study, therefore, is to determine the operating characteristics of the NEP technique in detecting OSAS in snoring patients, and to determine differences in the expiratory flow pattern during tidal breathing between patients with intrathoracic airway obstruction and those with extrathoracic airway obstruction. The measurements are recorded in seated and supine postures.

Edward D. Crandall, Ph.D., M.D.
Gene Therapy. Cystic fibrosis (CF) is an inherited disease that causes severe lung disease in children and young adults. Abnormalities in the transport of salt and water within the patient’s airways result in the production of thickened bronchial secretions and respiratory infection. Repeated lung infections cause destruction of lung tissue, resulting in progressive breathlessness, disability and mortality. We are developing programs for treating salt and water transport defects in patients using gene therapy. One object of this therapy is to introduce the normal form of a defective transport channel into the cell membranes of CF airway cells. The DNA sequence of the gene for this channel, known as the Cystic Fibrosis Transmembrane Regulator (CFTR), has been described. Following insertion of copies of this gene into a non-infectious carrier virus and introduction of the vector into airway cells having abnormal CFTR, the abnormal cell will produce normal CFTR. It is hoped that production of the normal channel will correct the transport defect and eliminate the pulmonary abnormalities seen in cystic fibrosis. Other publications of gene therapy are aimed at introducing genes into alveolar epithelial cells that will enhance clearance of pulmonary edema.

Albert H. Niden, M.D.
Idiopathic Pulmonary Fibrosis Research Study. Idiopathic Pulmonary Fibrosis (IPF) or Usual Interstitial Pneumonia (UIP) is a not uncommon, slowly progressive disease of unknown etiology, which responds poorly to current available therapy. It is also a disease that is difficult to diagnose short of an open lung or thoracoscopic lung biopsy. Tumor Necrosis Factor (TNF), an inflammatory cytokine, has been shown to be associated with inflammation and fibrosis in patients with IPF. TNFR:Fc (Etanercept, Enbrel ®) blocks TNF and has been shown to be safe and effective in treating patients with refractory rheumatoid arthritis. Dr. Niden and co-workers are utilizing a transthoracic core needle biopsy of the lung as an outpatient procedure to pathologically establish the diagnosis of IPF in patients with diffuse interstitial lung disease who have failed to respond to steroid therapy. Patients with TNFR are being treated and their response to therapy with serial pulmonary function and arterial blood gases are being objectively monitored. The study also correlates pathologic changes with lung HRCT and PET scan finings and assesses their ability to monitor response to therapy. Dr. Niden’s group is also initiating a study to identify the cytokines, which promote inflammation and or fibrosis and are present in lung tissue from patients with various diffuse interstitial lung diseases. The researchers hope to identify patterns of cytokine deposition that may be specific for the various disease entities. This would lead to future studies with treatment targeted against specific cytokines to more effectively treat these diseases.

Om P. Sharma, M.D.
Predictors of Survival in Patients with Sarcoidosis. Sarcoidosis is a multi-organ system disease of unknown etiology that primarily affects the lungs, causing severe respiratory impairment in many individuals. Dr. Sharma and colleagues are currently analyzing 100 patients with sarcoidosis who have been closely observed for the last 15 years, with only those patients who have had at least once-a-year lung function evaluation included in the study. Changes in FVC (forced vital capacity), FEV1 (forced expiratory volume at one second)/FVC ratio, DLCO (diffusing capacity), and P(A-a)O2 (alveolar-arterial O2 difference) are being evaluated as predictors of survival in patients with sarcoidosis. A 10% increase in FVC, 20% increase in DLCO (single breath), 10% increase in FEV1/FVC ratio, and <5 mm Hg decrease in P(A-a)O2 will define improvement. Kaplan-Meier survival plots and Cox proportional hazard regression model will be used to analyze survival time after one, three, and five years with and without therapy. This study will provide a rationale for the need and effectiveness of therapeutic regimens for individual patients, and the basis for future comparative therapy trials. Long term follow-up studies are in progress related to diagnosis and effects of new therapies in myocardial and neurological sarcoidosis.

Hidenobu Shigemitsu, M.D.
Pathogenesis of Sarcoidosis. Sarcoidosis is still a disease of unclear etiology with protean clinical manifestations. One of the hypotheses for pathogenesis is an imbalance of T cell subtypes. We are actively investigating this link by collecting gammaglobulin levels prospectively in a large cohort of sarcoidosis patients in order to elucidate a pattern of sarcoidosis associated with abnormalities in gammaglobulin. In a prospective observational study, patients with sarcoidosis will be screened for pulmonary hypertension (PAH) by echocardiogram using the systolic pulmonary arterial pressure (PAP) and Tei-index. In patients with systolic PAP > 35mmHg, right heart catheterization (RHC) will be performed for confirmation. It is anticipated that PAH will be prevalent among patients with advanced radiographic findings, pertinent physical findings, and pulmonary function impairments. In these patients, measurement of systolic PAP in conjunction with Tei-index by echocardiography will accurately reflect the measurements by RHC.