SFRBM 2006 Mantell 1

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Oxygen, Redox Signaling and Lung Injury

• Lin L. Mantell, MD/PhD
• Department of Pharmaceutical Sciences
• St. Johns University College of Pharmacy
• The Feinstein Institute for Medical
  Research/Department of Surgery, North Shore
  University Hospital, North Shore-LIJ Health
  SystemNew York University School of Medicine

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Acknowledgements

• Mantell Laboratory
• Current Members
• Tahereh E. Zaher, PhD
• Binh Phan, MD
• Mark Caryl, MD
• Ashwini Pathak
• Nicole Palma
• Maitreyi G. Muralidhar
• Swapna Morisetti

• Collaborators
• Stuart Horowitz, PhD
• Ed Miller, PhD
• Haichao Wang, PhD
• Kevin J. Tracey, MD
• Saul Powell, PhD
• Luis Ulloa, PhD

• Former Members
• Mohammad Javdan, PhD
• Dympna Morrow, PhD
• William A. Franek, MS
• John Romashko III, MS
• Hong Zhu, MD
• Leah Stansberry, MS

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9/11
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pulmonary Sarcoidosis
Felicia Dunn-Jones, whose death was deemed a
result of 9/11 dust.
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(No Transcript)
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Physiology of the Lung

• The primary entry site

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Facts About Oxygen

• Discovered in the late 18th century - Joseph
  Priestley
• Derives from ???? (oxys) and -?e??? (-genes)
• The most prevalent element in the earth crust --
  49.2 by weight
• A significant component of earth atmosphere --
  20.95
• The most abundant element in the human body --
  65
• Essential for aerobic respiration
• The most important source of cellular energy in
  aerobic life

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Anatomy of the Lung

www.bme.arizona.edu
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Structure of an Alveolus
Respiratory Membrane
Diffusion of O2
Diffusion of CO2
John Wiley Sons
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Physiology of the Lung
Edmund Miller

• Anatomic location
• Easy diffusion
• Large surface area
• Greater oxygen tension
• The major target of air pollution
• 3 x 108 liters

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Most oxygen is reduced by electrons to water,
however..
HOONO

NO2.
OONO-
1O2
Bauer et al.
HOCl
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Antioxidants in the Lung

• Enzymatic
• SOD (superoxide dismutase)
• Catalase
• GPx (glutathione peroxidase)
• Thioredoxin
• Non-Enzymatic
• Vitamin A, C E
• Thios Cysteine, glutathione, methionine
• Bioflavines

Under physiological conditions

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Oxidative Stress in Lung Injury
AO
ROS O2??, H2O2 , 1O2, ?OH, HOCl, NO, NxOy
Experimental evidence Increased H2O2 in the
exhaled breath condensate ARDS patients.
Baldwin et al. 1986. Lancet Relative deficient
levels of glutathione in patients with sepsis and
ARDS. Pacht et al. 1991. Chest

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What are the sources of ROS/RNS in the lung?
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Oxidative Stress in the Lung

• Endogenous
• NADPH oxidase
• nitric oxide synthases (NOS)
• mitochondrial electron transport chain
• cytochrome P450
• xanthine oxidase

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Oxidative Stress in the Lung

• Exogenous
• Mechanical ventilation
• Air pollutants, ozone, asbestos, nitrogen
  dioxide.
• Cigarette smoke
• Infection

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Mechanisms of lung injury during bacterial
infection
Chow et al. Am. J. Respir. Cell Mol. Biol.
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Pathological Effects of ROS

• At the molecular level
• DNA
• Lipid
• Proteins (methionine, tyrosine, cysteine)
• At the cellular level
• Cell injury/cell death
• Proliferation
• At the tissue level
• Acute inflammatory lung injury
• Chronic lung injury emphysema

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The Clinical Problems with Lung Injury

• Acute lung injury (acute respiratory distress
  syndrome)
• a common, devastating clinical problem that
  affects both medical and surgical patients
• a major cause of acute respiratory failure with
  high morbidity and mortality in these patients
• Resolution following lung injury plays an
  essential role to the survival of these patients
• Pulmonary fibrosis is a common response to
  various lung injury

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The Clinical Problems with Lung Injury

• Conventional therapy using glucocorticoids or
  immunosuppressive drugs is usually ineffective in
  preventing progression of fibrosis

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Alveolar fluid lining with pulmonary surfactant

Ware Matthay N Engl J Med 2000
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Resolution
Fibrosis
Epithelial Integrity
Cytokine/chemokines
Chapman, 2004, JCI
Ware Matthay N Engl J Med 2000
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Pulmonary Epithelium in Lung Injury and Repair

• Essential for pulmonary respiratory functions and
  inflammatory responses
• crucial for the restoration of the alveolar
  epithelial barrier and recovery from lung injury
• disorganized or insufficient epithelial repair
  may lead to fibrosis

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The cellular mechanisms of lung injury
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ARDS BAL fluid caused apoptosis and disruption
of the monolayer of human distal lung epithelial
cells
normal BAL fluid
ARDS BAL fluid
ARDS BAL fluid
ARDS BAL fluid
Matute-Bello, Martin, 1999, J Immunol
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Apoptosis of alveolar epithelium in ARDS
More positive signals of TUNEL-labeled nuclei,
caspase-3-labeled cytoplasm, Bax-labeled
cytoplasm in the tissue sections from the
patients who died with ALI or ARDS compared to
those who died without pulmonary disease
Albertine, et al. 2002
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Apoptosis of Lung Epithelium in Patients with
idiopathic pulmonary fibrosis
The TUNEL method stained red the nucleus of
hyperplastic alveolar epithelial cells (arrows).
TUNEL
The expression of caspase-3 was detected both in
the cytoplasm (bottom, C) and the nucleus (bottom
inset, D) of alveolar epithelial cells (arrows).
caspase-3
Plataki et al, Chest 2005
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Apoptosis may be responsible for the resolution
of type II pneumocytes in acute lung injury
TUNEL
HE

• During the resolution phase of acute lung injury,
  extensive apoptosis of type II pneumocytes is the
  main cellular mechanism that accounts for the
  disappearance of these cells
• Fas/Fas ligand is involved in the resolution of
  type II pneumocytes

control rat lung
control rat lung
1 days
Wang HC, et al., Crit Care Med. 2002
3 days
7 days
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The role of ROS/RNS in the programmed cell death
of pulmonary epithelial cells
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Apoptosis of Lung Epithelium in Hyperoxic Lungs
100 Oxygen
Room Air Control
TUNEL
DAPI

• Mantell, LL et al, 1997. Cell Death Differ
• Mantell et al, 2002, Am J Physiol. Lung
• Petrache et al, 1999, Am J Physiol. Lung

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RNS mediates apoptosis of Lung Epithelial Cells
Janssen YM, et al., 1997, Am J Physiol. Lung
1997 Persinger et al., 2001.Am J Respir Cell Mol
Biol.
TUNEL-positive
Nitrotyrosine reactivity
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H2O2 inhibits alveolar epithelial repair
H2O2 inhibited alveolar epithelial wound repair
in vitro in a concentration-dependent manner
Geiser et al., 2004
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Alveolar epithelial cell apoptosis is an
important mechanism of H2O2-induced inhibition of
alveolar epithelial repair
Annexin V-positive
phase-contrast
Geiser et al., 2004
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ROS/RNS-induced lung cell death may play critical
roles in the pathogenesis of lung injury and the
following repair process
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Cell Signaling in ROS/RNS-induced Lung Cell Death

• Transcription factors (AP1, NF-kB)
• Protein kinases (MAPK, SATA, PI-3K/AKT)
• Mitochondria-mediated events
• Cell surface receptor (epidermal growth factor
  receptor)
• DNA repair/Cell cycle arrest (p53, p21, GADD45)

• AP-1, EGFR, PKC, ERK, Brooke T. Mossman,
  University of Vermont
• AP-1, MAPK, IKK/NF-kB, Yvonne M. W.
  Janssen-Heininger , University of Vermont, Irfan
  Rahman, University of Edinburgh R. Maselli,
  University of Catanzaro
• MAPK, AP-1, Augustine Choi, University of
  Pittsburgh Phyllis A. Dennery, Stanford
  University Patty Lee, Yale University
  Barazzone-Argiroffo, University of Geneva L
  Mantell
• P53, AP-1, NF-kB, JNK, XiangLin Shi, National
  Inst. for Occupational Safety and Health
• ERK, telomerase, David Warburton, Childrens
  Hospital Los Angeles Research Institute
• AKT, Prabir Ray, University of Pittsburgh,
• PI-3K, Mitochondria-related, Carl W. White,
  National Jewish Medical and Research Center
• DNA repair/p21, Michael A. O'Reilly, University
  of Rochester
• Bcl-2, Aaron B. Waxman/Jack A. Elias,
  Massachusetts General Hospital/Yale University
• Ceramide, Tzipora Goldkorn, UC Davis

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Oxidative Signaling Pathways
ROS/RNS

• Stimuli
• Concentration
• Cell type
• Time

R
ROS/RNS
ROS
?
Plasma Membrane
Cell Damage
PKC
Cell Cycle Arrest (p21)
ROS/RNS
ROS/RNS
p38
JNK
Cytoplasm
ERK
MAP3K
ROS
Ceramide
Survival
Caspase 9 cascade
JNK
NF-kB
p38
ERK
ROS/RNS
ROS
NF-kB
Caspase 3 Cascade
AP-1
ATF2
Elk1
c-Jun
c-Fos
Nucleus

ROS/RNS
ROS
Survival Gene (AOE, Stress genes)
Inflammation
Non-apoptotic Cell Death
Apoptosis
ROS
Apoptosis
Proliferation/Differentiation
Stress response
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Serial Review Redox signaling in immune function
and cellular responses in lung injury and
diseases Serial Review Editors Victor
Darley-Usmar, Lin Mantell Redox signaling in
immune function and cellular responses in lung
injury and diseases Lin L. MantellRadical
Biology and Medicine Volume 41, Issue 1 , 1 July
2006 

• How do cells respond to oxygen?
• How do lung epithelial cells respond to ROS/RNS
  and viral infection?
• What are the roles of purinergic signaling, heat
  shock proteins, cytokines in cellular adaptation
  to ROS-induced cellular damage?

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NFkB in ROS/RNS-induced stress
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Lung NFkB in the Tolerance to Hyperoxic Lung
Injury
Yang et al, 2004, JCI
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Hyperoxia-induced NF-kB Activation Protects Lung
Epithelium Against Oxidative Apoptosis
Franek et al, 2001, JBC
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Inhibition of NFkB activation by ROS/RNS

• Franek et al, 2002, JBC

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Oxidation of NFkB activation by ROS/RNS

• Franek et al, 2002, JBC

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H2O2 inhibits TNF-induced NFkB activation by
oxidizing IKK
Korn et al, 2001
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H2O2 Induces S-glutathionylation of IKKb
Reynaert et al. 2006, PNAS
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IKKb is a target for S-nitrosylation
Reynaert et al. 2004, PNAS
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NFkB Regulates Cell Survival Under Oxidative
Stress by Affecting the Levels of MnSOD

• Franek et al, 2004, FRBM

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Antioxidants Affect Cell Death Signaling Pathways
Franek et al,

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Signaling pathways in epithelial apoptosis
Pro- Cell Death
Anti- Cell Death
TNF p53, Bax, JNK p38, FasL, TGF-b, ROS IFN-?
IAP, Bcl-2, NF-kB FLIP Bcl-x IL-6 CO, AOE
Kuwano et al, 2004
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Pulmonary epithelial Death in Lung Injury
Resolution
Fibrosis
Kuwano et al, 2004
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Summary

• Cell death of pulmonary epithelium
  during/following lung injury could be envisioned
  as either detrimental or beneficial, depending on
  the cell type, the circumstances, and the timing
• Apoptosis of pulmonary epithelium is important
  for the resolution of lung injury, excessive
  death of alveolar epithelium could lead to lung
  fibrosis, or even death
• ROS and RNS can cause excessive cell death of
  pulmonary epithelium by activating pro-death
  pathways and/or suppressing cell survival
  pathways, leading to more severe injury and/or
  less effective alveolar repair

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''Everything should be made as simple as
possible, but not simpler.''  
Albert Einstein (1879-1955)