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Lung Fibrosis: Links to Aging
and Nox4-Nrf2 Redox Balance
Victor J. Thannickal, M.D.
Professor and Director
Division of Pulmonary, Allergy, and Critical Care
University of Alabama at Birmingham
IPF is a Disease of Aging
IPF may be 5 to 10 times more prevalent than previously
thought, likely due to changing demographics, other risk
factors, and improved guidelines for diagnosing IPF
Raghu G, et al. Am J Respir Crit Care Med. 2006;174:810-816.
Idiopathic Pulmonary Fibrosis:
A Disorder of Lung Regeneration?
AGE
GENETICS*
ENVIRONMENT
(telomerase mutations)
(cigarette smoke, toxins/oxidants)
Telomere shortening
(SP-C mutations)
Senescence/Apoptosis
Impaired Regenerative Capacity
(Epithelial Cells)
*Recent studies implicate
gene variants of MUC5B
N Eng J Med 2011; 364:1503
Fibrosis
(Mesenchymal Cells)
Thannickal VJ, Lloyd JE.
Am J Respir Crit Care Med
2008;178:663-665.
Hallmarks
of Aging
Lopez-Otin et al.
Cell (2013)
153:1194-1217
Nox Enzymes: Diverse Biological
Functions Around Central Themes
• Absent in prokaryotes;
present in all eukaryotes –
7 members in humans
• Conserved functions
- host defense; innate
immunity
- development &
morphogenesis; cellular
signaling
- biosynthesis
Aguirre & Lambeth. Free Rad Biol Med, 2010
TGF-β induces ROS
generation in endothelial cells 1993
(Am J Physiol 1993;265:L622)
1995
Biochemical characterization
of a TGF-β-inducible oxidase
(J Biol Chem 270:30334)
Homologs of NADPH oxidase
(gp91phox - Nox2) 1999
(Nature 1999;401:79-82; J Biol Chem
2001;276:1417; Gene 2001;269)
Nox4 mediates TGF-β-inducible
2005 myofibroblast differentiation
Unique enzymatic characteristics 2006
of Nox4
(Circ Res 2005;97:900)
(Cell Signal 2006;18:69; J Biol Chem 2008;
Mol Cell Biol 2010;30:961; J Biol Chem
2012;287:8737)
First report of an in-vivo function
Nox4 in lung fibrosis
2009 (Nat Med 2009;15:1077; Antioxid Redox
Signal 2011;15:607)
Nox4 Mediates Myofibroblast Activation and Fibrogenic
Responses to Lung Fibrosis
TGF-1
Smad3
Nox4
Contractility
H2O2
Differentiation
ECM synthesis
MYOFIBROBLASTS
Hecker L, et al.,
Nat Med, 2009
Young
Aged
Environmental stress/
lung injury
(in myofibroblasts)
Nox4
Nox4
Nrf2
Nrf2
(changes in
cellular redox)
Self-limited
senescence
Sustained
senescence
(altered
cell fates)
Myofibroblast
apoptosis
Resolving
fibrosis
Bcl-2
Myofibroblast
apoptosis-resistance
Persistent
fibrosis
Impaired Resolution of Fibrosis in Aged Mice
Persistence of fibrosis in aged mice is
associated with myofibroblast senescence
Fibroblasts isolated from lungs of aged
mice demonstrate features of senescence
Young
Ctl
3w
Aged
2m
Ctl
3w
2m
p16
Bcl2
β-actin
Myofibroblasts from aged mice are resistant to
apoptosis during the resolving phase of lung injury
IPF myofibroblasts in fibroblastic foci are apoptosis
resistant and demonstrate features of senescence
Aged mice fail to induce Nrf2
following bleomycin injury
Young
control
3w
Aged
2m
control
3w
2m
Nrf2
β-actin
Human IPF
Senescent fibroblasts are impaired in their
capacity to activate Nrf2
17
Failure to induce Nrf2 in senescent fibroblasts
is associated with apoptosis resistance
NT-siRNA
Time
(min):
0 10
Nrf2-siRNA
30 0 10
30
Nrf2
β-actin
Nox4 confers apoptosis resistance
to lung fibroblasts
WT
Staurosporine:
_
Nox4-KO
+
_
+
PARP
Caspase-3
-Actin
19
Bleomycin
Injury
Time (weeks): 0
1
Intranasal siRNA
treatment
2
3
4
5
6
Persistent fibrosis
RNAi targeting of Nox4 in
vivo in aged mice with
non-resolving fibrosis
In vivo targeting of Nox4 down-regulates
expression of senescence and pro-fibrotic markers
In vivo targeting of Nox4 restores
capacity for fibrosis resolution
Trichrome
NT-siRNA
Nox4-siRNA
A pharmacological Nox4 inhibitor, GKT137831,
attenuates senescence and restores
apoptosis susceptibility
23
Nox4 inhibition reverses
established fibrosis in aged mice
Aged mice 6 weeks post-injury
p21
p16
aSMA
Trichrome
Vehicle
GKT
Young
Aged
Environmental stress/
lung injury
(in myofibroblasts)
Nox4
Nox4
Nrf2
Nrf2
(changes in
cellular redox)
Self-limited
senescence
Sustained
senescence
(altered
cell fates)
Myofibroblast
apoptosis
Resolving
fibrosis
Bcl-2
Myofibroblast
apoptosis-resistance
Persistent
fibrosis
Acknowledgements
Thannickal Lab
Louise Hecker, Ph.D.
Yan Sanders, Ph.D.
Ragini Vittal, Ph.D.
Jeffrey C. Horowitz, M.D.
Tommy Hock, Ph.D.
Karen Bernard, Ph.D.
Collaborators
Yong Zhou, Ph.D.
Guangjie Cheng, Ph.D.
Veena B. Antony, M.D.
Gang Liu, Ph.D.
• Maintenance
• Injury-induced
• Compensatory hyperplasia
• Activation of adult stem cells
• Dedifferentiation
Regeneration
Fibrosis
27
Cellular Homeostasis in the Adult Lung
Alveolar
Lumen (AL)
Cp
Cp
AL
AL
Burns AR, Smith CW, Walker DC.
Physiol Rev 83: 309, 2003
Peripheral distribution
Reticulation/
septal thickening
Honeycombing
Traction bronchiectasis
Raghu G, et al. Am J Resp Crit Car Med, 2011; 183:788-824
honeycombing
Basilar
distribution
Raghu G, et al. Am J Resp Crit Car Med, 2011; 183:788-824
Marked fibrosis
and microscopic
honeycombing
Area of normal lung
subpleural, paraseptal
distribution
Raghu G, et al. Am J Resp Crit Car Med, 2011;183:788-824
AS
FF
Katzenstein AL and Myers JL. Am J Respir Crit Care Med 157:1301, 1998
“The potential for additional “carriers” of electron transport
from NADH (donor) to O2 (acceptor) exists. Moreover, it cannot
be determined conclusively from our current experiments if the
reduction of O2 to H2O2 involves the intermediate formation of
O2.- or if this is a direct 2-electron transfer reaction…”
• TGF-β inducible by a mechanism that involves gene transcription
• Extracellular H2O2 generation
• H2O2 > O2.-
Cytoplasmic-nuclear shuttling of Nrf2 is
defective in senescent fibroblasts