Myofibroblasts and lung fibrosis induced by carbon nanotube exposure
Dong and Ma Particle and Fibre Toxicology
Myofibroblasts and lung fibrosis induced by carbon nanotube exposure
Jie Dong 0
Qiang Ma 0
0 Receptor Biology Laboratory, Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , 1095 Willowdale Road, Morgantown, WV , USA
Carbon nanotubes (CNTs) are newly developed materials with unique properties and a range of industrial and commercial applications. A rapid expansion in the production of CNT materials may increase the risk of human exposure to CNTs. Studies in rodents have shown that certain forms of CNTs are potent fibrogenic inducers in the lungs to cause interstitial, bronchial, and pleural fibrosis characterized by the excessive deposition of collagen fibers and the scarring of involved tissues. The cellular and molecular basis underlying the fibrotic response to CNT exposure remains poorly understood. Myofibroblasts are a major type of effector cells in organ fibrosis that secrete copious amounts of extracellular matrix proteins and signaling molecules to drive fibrosis. Myofibroblasts also mediate the mechano-regulation of fibrotic matrix remodeling via contraction of their stress fibers. Recent studies reveal that exposure to CNTs induces the differentiation of myofibroblasts from fibroblasts in vitro and stimulates pulmonary accumulation and activation of myofibroblasts in vivo. Moreover, mechanistic analyses provide insights into the molecular underpinnings of myofibroblast differentiation and function induced by CNTs in the lungs. In view of the apparent fibrogenic activity of CNTs and the emerging role of myofibroblasts in the development of organ fibrosis, we discuss recent findings on CNT-induced lung fibrosis with emphasis on the role of myofibroblasts in the pathologic development of lung fibrosis. Particular attention is given to the formation and activation of myofibroblasts upon CNT exposure and the possible mechanisms by which CNTs regulate the function and dynamics of myofibroblasts in the lungs. It is evident that a fundamental understanding of the myofibroblast and its function and regulation in lung fibrosis will have a major influence on the future research on the pulmonary response to nano exposure, particle and fiber-induced pneumoconiosis, and other human lung fibrosing diseases.
Carbon nanotube; Myofibroblast; Lung fibrosis; Animal model; Mechanism; Extracellular matrix
Mammalian lungs are among the most susceptible
organs to fibrosis . As the primary respiratory organ,
the lungs perform gas exchange between the blood and
the inhaled air through a thin layer of alveolar septal
structures. These structures are vulnerable to structural
and functional alterations, such as interstitial
thickening and alveolar destruction, major pathologic features
of lung fibrosis. Respiration also exposes the lungs
constantly to numerous inhaled fibrogenic agents including
toxic chemicals, mycobacteria, and particulate matters
[2, 3]. Some chemicals are preferentially uptaken by
lung epithelial cells and therefore tend to accumulate in
the lungs upon exposure through either respiration or
systemic means [4–6]. These exposures can damage the lung
tissue to give rise to induced fibrosis. Lung fibrosis also
occurs as a common, end stage pathologic development of
existing lung diseases caused by infection, chronic
inflammation, cardiovascular malfunction, autoimmunity, and
idiopathy . In many cases, human lung fibrosis is
progressive and refractory to therapy, causing high rates of
mortality and disability .
The progressive nature and severe outcome of human
lung fibrosing diseases are well illustrated by idiopathic
pulmonary fibrosis (IPF). IPF initiates insidiously with
no known etiology and follows a chronic but progressive
course that is ultimately lethal, indicated by the median
survival of IPF patients within 2–5 years after diagnosis
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[9–11]. IPF exhibits a histopathologic pattern of usual
interstitial pneumonia, including the occurrence of
mild-to-moderate inflammatory infiltration, injury and
hyperplasia of alveolar epithelial cells, excessive
deposition of the extracellular matrix (ECM), thickening of
the alveolar septa, scarring and formation of fibroblastic
foci, and temporally heterogeneous fibrotic remodeling
of the lung structure, such as honeycombing [9, 11]. No
effective therapy is available to the patients with this
destructive lung disease except lung transplantation at
the present time. Several animal models have been
developed and studied for lung fibrosis such as IPF
during the past few decades [12, 13]. Among these, the
bleomycin-induced lung fibrosis is the most commonly
used model. However, bleomycin-induced lung fibrosis
differs from IPF in several pathologic aspects. First,
bleomycin induces severe alveolar epithelial cell death
in the lungs before the onset of fibrosis, whereas extensive
alveolar epithelial cell lesions are rarely seen in IPF.
Second, bleomycin causes diffusive fibrosis throughout the
lung parenchyma with scattered fibrotic foci, while the
fibrosis in IPF is predominantly in the form of fibroblastic
foci. Third, fibrosis in the bleomycin mouse model
resolves spontaneously and thus is reversible, but that in IPF
is progressive and irreversible. Overall, the research using
the bleomycin and other fibrosis models has not yielded
effective translation to the treatment of IPF and other
human lung fibrosing diseases including pneumoconiosis
caused by exposure to fibrogenic particles and fibers, such
as silica and asbestos [4, 14]. As a result, there are
increasing efforts to gain improved fundamental understanding
of the pathogenesis and development of lung fibrosis, as
well as renewed interests in identifying new animal
models that mimic human lung fibrosis, in order to
achieve better treatment and prevention against human
lung fibrosing diseases .
Carbon nanotubes (CNTs) are long and hollow
nanostructures made of a single layer or concentric multiple
layers of one-atom-thick carbon walls, designated as
single-walled CNTs (SWCNTs) and multi-walled CNTs
(MWCNTs), respectively . As new materials, CNTs
have been developed with a variety of industrial and
commercial applications for electronic, biomedical, and
energy-related uses. The annual production of CNTs
has been increased rapidly in recent years . On the
other hand, most CNTs are respirable fibers with
physicochemical features like nano-scaled diameter,
fiberlike shape (high aspect ratio), large surface area, poor
solubility, and excessive biopersistence, properties often
associated with the fibrogenic and tumorigenic
activities of inhaled particles and fibers, thus raising concern
over the potential adverse health effects of human
exposure to CNTs [17, 18]. Indeed, a marked progress in
the understanding of CNT toxicity has been achieved
in experimental animal and cell systems during the past
decade. Importantly, the research has identified CNTs as a
significant fibrogenic inducer in the lungs and the pleural
space to cause interstitial, bronchial, and pleural fibrosis
in animals [18–27]. The pathologic development and
features of CNT-induced pulmonary interstitial fibrosis
overlap with those of IPF and pneumoconiosis
considerably. From the experimental research point of view,
this finding suggests a possible application of
CNTinduced lung fibrosis in the study of the human lung
fibrosing diseases with regard to their pathogenesis,
therapeutic targeting, and biomarkers for exposure and
disease monitoring [18, 24, 28]. Recent field studies on
CNT-exposed populations demonstrate marked
accumulation of inflammatory and fibrotic mediators and
biomarkers in the body fluids of workers manufacturing
MWCNTs, highlighting a need to protect humans from
nano exposure from occupational, environmental, and
commercial sources [29–31].
The mechanisms by which CNTs cause lung fibrosis
remain unclear, but are believed to involve an
exacerbated fibroblastic response 1. During fibrosis
development, fibroblasts and myofibroblasts act as major
effector cells to produce excessive amounts of collagen
fibers and other ECM proteins, and to remodel and
contract the fibrosing tissues [32–40]. The fibroblastic
response in fibrosis bears certain similarity to wound
healing following tissue injury. At the early stage of lung
fibrosis, resident fibroblasts in the lung interstitial space
are activated upon stimulation. Activated fibroblasts
migrate and proliferate to result in the accumulation of
active fibroblastic cells in regions where injury takes place.
At the same time, activated fibroblasts differentiate into
myofibroblasts, which are a group of multi-functional
mesenchymal cells implicated in wound healing, organ
fibrosis, tumorigenesis, and cancer metastasis.
Myofibroblasts are characterized by their simultaneous
presentation of a high capacity of ECM protein production
and their smooth muscle cell-like contractile features
obtained through the de novo synthesis of α-smooth
muscle actin (α-SMA)-containing stress fibers . In
physiologic wound healing, excessive ECM production
and remodeling are avoided, as the majority of the
αSMA-expressing myofibroblasts disappear by way of
apoptosis upon scar formation. However, during
pathologic fibrosis, myofibroblasts become resistant to
apoptosis and thereby persist to continuously synthesize and
remodel the ECM, which ultimately leads to organ
fibrosis and destruction . Understanding the
formation, function, and fate of myofibroblasts in tissue
remodeling may hold a key to differentiating between
physiologic wound healing and the development of organ
fibrosis including IPF, pneumoconiosis, and CNT-induced
Several excellent reviews have been published to sum
up the biological effects, the overall mode of action,
and the interrelations between the physicochemical
properties and the bioactivities of CNTs from a
toxicological point of view [17–19, 42]. However, the cellular
and molecular basis underlying the fibrotic response to
CNTs, which is key to understanding the adverse health
effects from CNT exposure, remains a topic of
considerable challenge. In part, this issue is due to a lack of
significant mechanistic insights into organ fibrosis in
general. Given the emerging and rapidly advancing
research on myofibroblasts and their function in the
pathogenesis of fibrosis, we discuss the current
literature on CNT-induced rodent lung fibrosis with focus
on the formation and role of myofibroblasts in the
development of pulmonary fibrosis. Possible mechanisms by
which CNTs regulate myofibroblast functions and
dynamics during fibrosis development in the lungs will be
discussed. We anticipate that such analysis will have a major
impact on the future research of the pulmonary response
to CNT exposure and therefore will aid in the
fundamental understanding, as well as the risk assessment, of lung
fibrosis caused by exposure to CNTs, other nanomaterials,
and other fibrogenic particles and fibers. The knowledge
obtained will also facilitate the identification of new drug
targets and biomarkers for the treatment and exposure
and disease monitoring of human lung fibrosing diseases.
Carbon nanotube-induced lung fibrosis
CNT-induced lung interstitial fibrosis initiates with a
prominent acute inflammatory response, exhibited by
recruitment and accumulation of inflammatory cells,
including neutrophils, macrophages, and lymphocytes,
and elevated secretion of pro-inflammatory and
profibrotic cytokines, chemokines, and growth factors,
such as TNF-α, IL-1β, IL-6, MCP-1, TGF-β1 and PDGF-A
(PDGF subunit A) [22, 24, 25, 43–47]. Along with the
acute inflammation, MWCNTs trigger a rapid-onset
fibrotic response, indicated by increased deposition of
collagen fibers in alveolar septa, detectable as early as day 1
post-exposure . The acute inflammatory and fibrotic
responses reach an apex by day 7 post-exposure, after
which the pathologic effects transit to chronic fibrosis.
At the chronic phase, CNT-induced fibrotic lesions are
featured with mild inflammation, thickened alveolar
septa, increased deposition of ECM proteins, enhanced
expression of fibrosis markers, and formation of fibrotic
foci and epithelioid granulomas [27, 48]. Figure 1
depicts the pathologic features and transition of the acute
and chronic lung fibrotic lesions induced by CNTs in
Fig. 1 CNT-induced lung fibrosis. CNTs are respirable fibers with a tendency to deposit, penetrate and accumulate in lung tissues (left box).
Exposure to CNTs induces acute phase responses including an inflammatory response, represented by the recruitment of Mac2-positive macrophages,
and a fibrotic response, shown by Picro-Sirius Red staining for collagens I and III. Acute phase responses start as early as day 1, reach an apex on day 7,
and decline after day 7 to significantly lower levels on day 14 post-exposure. In this scenario, day 7 post-exposure may represent an acute-to-chronic
transition of CNT-induced pathology in mouse lungs (middle box). CNT-induced chronic phase responses are characterized by interstitial fibrosis and
formation of epithelioid granulomas, shown by Masson’s Trichrome staining for collagen fibers on day 28 post-exposure. CNT-induced lung fibrosis
appears to be persistent and irreversible in studies for up to 1 year post-exposure (right box)
rodents. The lung fibrotic response to CNT exposure
resembles the pulmonary response to deposition of
fibrogenic foreign bodies in the lungs. In particular, the
pathologic features and dynamics of the pulmonary
interstitial fibrosis induced by CNTs display a high similarity to
those of pneumoconiosis and IPF, both of which are
progressive, incurable, and poorly understood human fibrotic
lung diseases [24, 28].
Notably, CNT-induced lung fibrosis appears to be
persistent and irreversible, which differs from
bleomycininduced lung fibrosis. The fibrotic lesions induced by a
single dose instillation of bleomycin generally resolve after
28 days post-exposure [12, 13], whereas CNT-induced
lung fibrosis was prolonged and was observed 90 days
after a single dose intratracheal instillation of SWCNTs in
mice, shown by the significant formation of epithelioid
granulomas . Similarly, lung fibrosis was observed
90 days after a single dose intratracheal instillation of
MWCNTs in rats, demonstrated by thickened alveolar
wall and increased collagen deposition . In a more
recent study, the long-term effects of CNTs on lung fibrosis
were specifically investigated. Either a single dose
intratracheal instillation of SWCNTs or an inhalation exposure to
SWCNT aerosol (5 h/day for 4 days) induced lung fibrosis
in mice 1 year post-exposure, demonstrated by increased
levels of collagens in the lungs and the presence of fibrotic
histopathological phenotypes . In a separate study with
inhalation of MWCNT aerosol (5 h/day for 12 days, 4
times/week for 3 weeks), it was found that lung fibrosis in
MWCNT-exposed mice, indicated by increased fibrous
collagen in the alveolar region, displayed a progressive
increase in the thickness of the alveolar septal
connective tissue over time, i.e., 0.17 ± 0.02, 0.22 ± 0.02, 0.26 ±
0.03, 0.25 ± 0.02 and 0.29 ± 0.01 μm on days 1, 14, 84,
168 and 336 post-exposure, which was significantly
higher than that of the clean-air control on days 84 and
336 post-exposure . These observations reveal that
CNTs induce progressive and persistent fibrosis in the
lungs, which is not self-resolved even at 1 year
postexposure. From this prospect, studies on CNT-induced
lung fibrosis are promising, as they would, at the very
least, supplement the findings from the bleomycin
model to better reflect human fibrotic lung diseases,
such as IPF and pneumoconiosis.
Some CNTs are capable of inducing bronchial or pleural
fibrosis. In the former case, inhalation of MWCNTs in
ovalbumin-sensitized mice, a murine asthma model,
induced significant airway fibrosis in addition to asthmatic
phenotypes . In the latter case, direct instillation of long
or short MWCNTs into the pleural cavity induced fiber
length-dependent fibrotic responses like asbestos fibers
. The long MWCNTs elicited acute inflammation
followed by progressive fibrosis on the parietal pleura,
whereas the short MWCNTs were rapidly cleared through
the stomata of the pleura and as a result, failed to induce
pleural fibrosis. In both bronchial and pleural fibrosis, the
development of fibrotic lesions involves acute inflammation
followed by fibrotic progression similarly to that of
interstitial fibrosis described above. The CNT-induced airway and
pleural fibrotic lesions may have implications for the study
of human asthma and pleural thickening/mesothelioma,
respectively, which requires further investigation.
CNTs differ considerably from one another in their
physicochemical properties that may impact CNT
toxicity in vivo by affecting both the intrinsic pathogenicity
and the kinetic behavior of CNTs . Cumulative
evidence reveals that the size, shape, surface area, surface
reactivity, fiber rigidity, and biopersistence of CNTs are
among the important properties to influence their
fibrogenic activity in the lungs. The knowledge obtained from
these structure-activity relationship studies provides
insights into the internal (effective) dose of CNTs in lung
fibrosis and hence their risk assessment. The information
would also suggest new ways of reducing CNT fibrogenic
activity by means of prevention-through-product design.
CNTs exhibit length-dependent activities in causing
lung interstitial fibrosis [51, 52], as well as the fibrosis
and granuloma formation in the parietal mesothelium
. In the latter case, the length-fibrosis correlation
can be explained by the observation that long, but
not short, CNTs (i.e., >15 μm in length) are retained
in the pleural cavity to cause the fibrotic lesions,
because the long CNTs are not efficiently cleared off
through either the stomatal drain (3–10 μm in
diameter) or pleural macrophage phagocytosis (<10–15 μm
in diameter), whereas the short CNTs are rapidly
eliminated from the pleural cavity through both
clearance mechanisms . It is believed that the long
CNTs that exceed the diameter of macrophages and
therefore are not effectively engulfed by macrophages
would trigger the so-called “frustrated phagocytosis”.
In this scenario, macrophages that fail to phagocytize
the fibers are activated to release an array of bioactive
and/or cytotoxic agents, which would cause tissue
damage locally, much like the response to long
asbestos fibers . It is also possible that CNTs interact
with cell surface structures, such as the pattern
recognition receptors, to elicit responses from lung cells.
In this case, the long CNTs may have a higher
capacity of stimulating the cell surface receptors than the
short CNT fibers to account for the differential
fibrotic effects between long and short CNTs.
MWCNTs have been shown to bind to the cell
surface bone morphogenetic protein receptor type II
(BMPR2) to stimulate the differentiation and to inhibit the
apoptosis of mouse myoblast cells . However, evidence
supporting a direct interaction of CNTs with cell surface
receptors to stimulate lung fibrosis is currently lacking.
It is worth noting that many CNTs are shorter than
5–10 μm and thus would be engulfed by macrophages
if not agglomerated or tangled into large masses. Yet
these CNTs may still be fibrogenic both in vitro and in
vivo. Moreover, the lung parenchyma does not appear
to have a “sieve” mechanism like the pleural stomata.
Therefore, the length-dependent activities of CNTs in
causing lung interstitial fibrosis may differ from those
defined in the “fiber length pathogenicity paradigm”
derived from mesothelial fibrosis caused by asbestos fibers
with regard to both their phenotypes and mechanisms.
For instance, the needle-like MWCNTs, i.e., Mitsui XNRI
MWNT-7, have a mean length of 3.86 μm and count
mean diameter of 49 nm, but are potent in inducing lung
fibrosis in vivo, suggesting that properties other than fiber
length, in particular, the fiber rigidity in the case of
XNRI MWNT-7, play an important role in modulating
the fibrogenic activity of nanomaterials [23, 24, 27].
A recent comparison among ten commercial MWCNTs
with different morphology, composition, surface area, and
functionalization reveals that surface area, fiber length,
and surface modifications were better predictors of
pulmonary inflammation in vivo . In other studies, the
thin film coating of MWCNTs with Al2O3 via atomic layer
deposition reduced lung fibrosis in mice ; and the
surface functionalization of MWCNTs with carboxylation or
other covalent modifications demonstrates that strong
cationic modifications induced significant lung fibrosis,
whereas carboxylation significantly reduced the extent
of lung fibrosis, compared with pristine CNTs .
Therefore, the surface reactivity and surface charge of
CNTs play critical roles in determining their
fibrogenicity and toxicity.
Role of myofibroblasts and fibroblasts in lung
The fibroblastic response in lung fibrosis is responsible
for the fibrotic matrix built-up, ECM remodeling, and
tissue contraction to result in lung scarring and
destruction. This dynamic process involves both myofibroblasts
and fibroblasts that play distinct, but sometimes
overlapping, functions; moreover, the interplay between these
two types of cells represents an important aspect of
fibrosis development, as illustrated in Fig. 2.
Resident fibroblasts are believed to be activated and
play important roles in the early phase of wound healing
and tissue fibrosis. Activated fibroblasts migrate,
proliferate, and secrete certain ECM proteins, cytokines, and
growth factors, to promote the fibrosis development at
the site of injury. Among these functions, fibroblast
proliferation has received increasing attention in fibrosis
development. In this regard, the fibroblastic foci in IPF
exhibit a prominent accumulation of proliferating
fibroblastic cells that are fibroblasts in nature [37, 57].
Fibroblasts isolated from IPF patients show enhanced
proliferation in vitro. Furthermore, a number of cellular
mechanisms have been identified to account for the
proproliferative state of these cells. There was an aberrant
activation of PI3K/Akt signaling and pathological
proliferation in these fibroblasts, which involved a decreased
level of the plasma membrane integrin β1/caveolin-1/
PTEN complex and a decreased activity of PTEN
phosphatase . Activated Akt phosphorylates and
inactivates transcription factor FoxO3a (forkhead box O3a)
that promotes cell cycle arrest by increasing the
expression of CDK inhibitor p27. In a separate study, IPF
fibroblasts were shown to have a high level of inactive
FoxO3a due to a high Akt activity, which resulted in a
reduced level of p27 and, consequently, increased
proliferation of the cells . A decreased level of integrin
α2β1 and an impaired activity of PP2A phosphatase were
also detected in IPF fibroblasts; these changes elevated
the levels of inactive, phosphorylated GSK-3β and active
nuclear β-catenin, both of which promote fibroblast
proliferation . In addition, the IPF fibroblasts display
reduced apoptosis. The increased proliferation and
reduced apoptosis of fibroblasts expand the fibroblast
population and would therefore boost the
fibroblast-tomyofibroblast transformation, which ultimately leads to
the formation of fibroblastic foci and tissue fibrosis
Fig. 2 Fibroblasts and myofibroblasts in fibrosis. Fibroblasts and myofibroblasts act as the effector cells in organ fibrosis. Upon fibrogenic stimulation,
tissue resident fibroblasts are activated to migrate and proliferate. Activated fibroblasts are the major progenitor cells to differentiate into
myofibroblasts, indicated by the de novo expression of α-SMA. Myofibroblasts possess several characteristics, which distinguish them from
fibroblasts and render them unique and critical functions in organ fibrosis
Myofibroblasts represent a heterogeneous group of
fibroblastic cells found in tissues undergoing wound
healing or organ fibrosis, and within the microenvironment of
primary and metastatic cancers [34, 64]. As shown in
Fig. 2, myofibroblasts exhibit several notable and unique
characteristics that are distinct from those of fibroblasts
and are believed to be critical to wound healing, fibrosis,
tumorigenesis and cancer metastasis [34, 35, 65–67]. First,
myofibroblasts possess a high contractile activity through
their de novo synthesis of α-SMA protein. Newly
synthesized α-SMA incorporates into intracellular stress fibers
that are capable of generating strong force upon
contraction, through which myofibroblasts control the cell shape
and movement, ECM reorganization, and tissue
contraction. Second, myofibroblasts possess a high capacity of
protein synthesis and secretion and are believed to be
responsible for the production of a major portion of the
ECM proteins, such as collagens and fibronectin, for
matrix deposition during fibrosis [38, 68–70]. Third,
myofibroblasts demonstrate high levels of constitutive and
induced expression of cytokines, chemokines, growth
factors, and cell surface receptors, which render the cells
some properties of inflammatory cells to allow them to
respond to a variety of inflammatory, immune, and
mechanical signals [71–75]. Fourth, myofibroblasts produce and
release reactive oxygen and nitrogen species (ROS, RNS)
spontaneously and under stimulation, which contribute to
the up-regulation of ECM production and remodeling
[76–80]. Last but not least, myofibroblasts in fibrotic
tissues show high resistance to apoptosis, possibly due to the
persistent activity of TGF-β1 and ECM deposition, leading
to a prolonged survival and activity of myofibroblasts
during the development of fibrosis [61, 81–83]. Combined,
these features of myofibroblasts enable the cells to
perform unique, direct, and critical functions in a broad range
of physiologic and pathologic processes involving ECM
The formation of myofibroblasts involves a complex
and as yet not well understood process. In both IPF and
bleomycin-induced lung fibrosis, activated interstitial
resident fibroblasts are recognized as a major source of
myofibroblasts. Myofibroblasts may also derive through
trans-differentiation from other types of cells, such as
the bone marrow-derived fibrocytes, the pericytes
surrounding small blood vessel walls, and the epithelial cells
overlying connective tissues. However, the contribution
of these trans-differentiated cells to the myofibroblast
pool in lung fibrosis remains a subject of debate, as
studies examining the role of the trans-differentiated cells in
fibrosis development have yielded inconsistent results in
different experimental systems [41, 66, 84–88].
The differentiation of myofibroblasts from fibroblasts
can be induced by a variety of signals. The
transforming growth factor-β (TGF-β; mainly TGF-β1) has been
recognized as a central player in driving fibroblasts to
differentiate into myofibroblasts in both experimental
and clinical settings, though a panel of other factors
including ED-A fibronectin, Wnt, NOX4, integrins, ROS,
and the stiff ECM have also been shown to promote
myofibroblast differentiation, as have been summarized
by Hinz and colleagues . TGF-β1 stimulates all
characteristics of the differentiated myofibroblasts both
in vitro and in vivo . Exaggerated expression and
activation of TGF-β are commonly observed in lung
fibrotic lesions, such as IPF and bleomycin-induced lung
fibrosis [89–93]. Newly synthesized TGF-β is secreted
into the ECM in the form of a latent complex . The
mechanisms by which TGF-β is induced and activated
during fibrosis are not well understood and remain to
be a topic of intensive research, which will be discussed
in more detail in the section on mediators and
mechanisms for CNT-induced lung fibrosis.
Myofibroblasts in carbon nanotube-induced lung
Given the critical role of myofibroblasts in lung fibrosis
and the predominant pathologic phenotypes of lung
fibrosis induced by CNTs described above, the potential
effects of CNTs on myofibroblast differentiation and
function have drawn attention in recent years.
In vitro studies have provided evidence
demonstrating that CNTs stimulate the formation of
myofibroblasts from fibroblasts and other types of cells.
Treatment of macrophages (RAW264.7) with
MWCNTs (average length 10 μm, average outer
diameter 8.7 nm, surface area 220 m2/g, total metal 0.2 %)
stimulated the cells to produce a range of pro-fibrotic
cytokines and growth factors including TNF-α, IL-1β,
TGF-β1, and PDGF at both mRNA and protein levels
. Induction of the proteins correlated with the
activation of the NF-κB signaling pathway. Moreover, the
cellfree medium from the treated culture was found capable
of stimulating fibroblasts (WI38-VA13) to differentiate
into myofibroblasts, as evidenced by significantly elevated
expression of α-SMA protein. Similarly, SWCNTs (fiber
length 0.5–2 μm, outer diameter 1–2 nm, surface area
480 m2/g, total metal 4.5 %) activated NF-κB and
induced the expression of TGF-β1 in macrophages to
produce a medium that induced the formation of
myofibroblasts from lung fibroblasts . These findings
suggest a molecular mechanism by which CNTs
stimulate a coordinated response between macrophages and
fibroblasts to induce the differentiation of fibroblasts
into myofibroblasts via secreted soluble factors, such as
TGF-β1 and IL-1β.
CNTs induce the epithelial-mesenchymal transition
(EMT), i.e., the trans-differentiation of epithelial cells
to mesenchymal cells, to contribute to myofibroblast
formation. During this process, epithelial cells gradually
acquire a mesenchymal (fibroblast-like) phenotype through
the de novo expression of vimentin, FSP-1 (fibroblast
specific protein-1), Collagen I, fibronectin, and α-SMA. In a
culture of human alveolar epithelial cells (A549), long
MWCNTs (length: 5–15 μm) were shown to
downregulate the epithelial cell marker E-cadherin and
upregulate the expression of α-SMA protein, both of which
indicate EMT . MWCNTs also induced EMT in rat
alveolar type II epithelial cells (RLE-6TN), shown by
decreased E-cadherin expression and increased fibronectin
expression . These findings reveal that myofibroblasts
can derive from epithelial cells through EMT when
exposed to CNTs in vitro.
The involvement of myofibroblasts in CNT-induced
lung fibrosis in vivo was addressed in a few recent studies.
CNTs stimulate the expression of α-SMA mRNA and
protein in the lungs of rodents. One study was focused on the
effect of SWCNTs on EMT in the lungs wherein the
expression of α-SMA and surfactant protein C (SPC, a
marker of alveolar epithelial cells) was used as markers to
trace cell differentiation and trans-differentiation .
Immunofluorescence staining showed that the α-SMA
expression occurred in a portion of hyperplastic SPC
positive epithelial cells in the lungs on days 28 and 42
postexposure to SWCNTs; whereas, flow cytometry analysis
revealed that about 17, 30, 33, and 29 % of the α-SMA
positive cells were SPC positive on days 14, 28, 42 and 56
post-exposure to SWCNTs, respectively. These data
demonstrate that a subset of α-SMA positive cells were
differentiated from the epithelium-derived fibroblasts
under SWCNT exposure. Although the overall effect of
SWCNTs on α-SMA expression and myofibroblast
differentiation in the lungs was not assessed, this study was the
first to report α-SMA induction in CNT-exposed lungs. In
a separate study, the toxicity of functionalized SWCNTs
in mouse lungs was analyzed . The protein level of
αSMA was significantly increased in lung tissues on day 14
post-exposure to certain surface-modified and
functionalized SWCNTs. Therefore, SWCNTs are capable of
inducing myofibroblast transformation in the lungs, as shown
by increased number of α-SMA positive cells in lung
tissues. In another report, the induction of EMT by short
(length: 350–700 nm) and long (length: 5–15 μm)
MWCNTs was investigated in mouse lungs with α-SMA
as a marker of the mesenchymal cells . The percentage
of SPC positive cells stained for α-SMA in the lungs was
significantly elevated by long, but not short, MWCNTs on
days 28 and 56 post-exposure. Thus, MWCNTs induce
αSMA expression in SPC positive cells in the lungs through
EMT in a fiber length-dependent manner.
In an attempt to assess the effect of CNTs on
myofibroblast transformation during lung fibrosis directly, we
conducted a study in mouse lungs exposed to MWCNTs
(XNRI MWNT-7, median length 3.86 μm, mean
diameter 49 nm, average surface area 26 m2/g, total metal
0.78 %), a potent fibrogenic agent that induces
rapidonset lung fibrosis in mice [23, 24, 43]. We found that a
single dose pharyngeal aspiration of MWCNTs (40 μg)
significantly induced the expression of α-SMA protein
and increased the number of α-SMA positive cells in
mouse lungs on days 1, 3, 7 and 14 post-exposure. The
increases were especially apparent in the interstitial
fibrotic foci where MWCNTs deposited, as revealed by
immunohistochemistry and immunofluorescence1. Rep
resentative images showing the induced expression of
αSMA on day 7 post-exposure are presented in Fig. 3,
which demonstrate, for the first time, that MWCNTs,
exemplified by XNRI MWNT-7, can remarkably
stimulate myofibroblast transformation for the formation of
fibrotic foci during lung fibrosis in vivo.
Several ECM proteins other than α-SMA typically
expressed and produced by myofibroblasts during organ
fibrosis display increased expression in CNT-exposed
lungs, indicating that the myofibroblasts under CNT
exposure are active and functional with regard to fibrosis
development. A number of studies using traditional
methods of collagen analysis demonstrate remarkably
increased amounts of collagen fibers in CNT-exposed
lungs, exemplified by the investigations listed in Table 1
with references included therein. Elevated expression
and deposition of ECM proteins in the interstitial tissues
of CNT-exposed lungs, especially the fibrotic foci where
Fig. 3 α-SMA expression induced by MWCNTs in mouse lungs.
Pulmonary exposure to MWCNTs (XNRI MWNT-7, 40 μg) for 7 days
strongly induces α-SMA expression. α-SMA expression in
wellformed fibrotic foci is shown by immunohistochemistry (upper
panel) and in less well-formed, early stage fibrotic foci by
immunofluorescence (lower panel), respectively (scale bar: 20 µm; for
immunofluorescence, red: α-SMA staining, blue: nuclear DAPI staining)
Table 1 Increased pulmonary collagen deposition by CNTs as revealed by traditional assays
SWCNT intratracheal injection
SWCNT pharyngeal aspiration
MWCNT intratracheal injection
MWCNT pharyngeal aspiration
Sircol soluble collagen assay
Masson’s Trichrome staining
Sircol soluble collagen assay
Masson’s Trichrome staining
Sircol soluble collagen assay
Masson’s Trichrome staining
Masson’s Trichrome staining
Masson’s Trichrome staining
Masson’s Trichrome staining
Masson’s Trichrome staining
Masson’s Trichrome staining
CNTs deposited, were also observed by molecular
biology techniques. In one study, long MWCNTs (length:
20–50 μm), but not short MWCNTs (length: 0.5–
2 μm), induced the expression of Collagen I mRNA on
day 30 and Collagen III mRNA on day 7 and day 30
post-exposure in rat lungs . Certain forms of
functionalized SWCNTs significantly increased the levels of
Collagen I and Collagen III in mouse lung tissues on
day 14 post-exposure . We recently demonstrated
that MWCNTs (XNRI MWNT-7, 40 μg) caused
significantly elevated mRNA expression of Col1a1 and Col1a2,
the genes encoding the pro-alpha1 and pro-alpha2 chains
of Collagen I, on days 3 and 7 post-exposure in mouse
lungs [24, 101]. The treatment also dramatically increased
the production and accumulation of Collagen I in
interstitial lung tissues on days 1, 3, 7 and 14 post-exposure. A
dose-dependence study showed that the induction of
Collagen I accumulation by MWCNTs occurred at low
doses, i.e., 5 and 20 μg, on day 7 post-exposure .
Additionally, it was detected that the expression of
fibronectin mRNA was significantly increased on day 3 and
day 7, and the level of fibronectin protein was markedly
elevated on day 7, in mouse lungs exposed to MWCNTs
(XNRI MWNT-7, 40 μg) .
Induction of the platelet-derived growth factor
receptorβ (PDGFR-β) expression is another feature of
myofibroblast activation during fibrosis in several organs
including the liver, kidney and lung [102–105]. By using a
double-fluorescent Pdgfrb-Cre reporter mouse strain, it
was demonstrated that, in the lungs following
bleomycin treatment for 28 days, a marked expansion of
the reporter cells occurred in fibrotic regions, in which
almost all the reporter cells expressed PDGFR-β and
most of the cells expressed α-SMA, indicating that a
high percentage of PDGFR-β-expressing cells are lung
myofibroblasts . Therefore, an induced expression
of PDGFR-β is a marker for myofibroblast activation
during fibrogenesis. We examined the level of
PDGFRβ in MWCNT-induced lung fibrosis and demonstrated
that MWCNTs (XNRI MWNT-7, 40 μg) remarkably
increased the PDGFR-β expression and the number of
PDGFR-β positive cells in mouse lungs on day 7
postexposure, especially in interstitial fibrotic foci1. These
findings further support the activation of myofibroblasts
by CNTs in the lungs in vivo.
Candidate mediators and mechanisms for
CNTinduced myofibroblast differentiation and
A number of signaling molecules and cellular processes
induced by CNTs have been suggested to play important
roles in myofibroblast differentiation and activation. In
this section, we discuss evidence supporting their
involvement and their potential mechanisms in the regulation of
myofibroblast formation and behaviors in CNT-induced
lung fibrosis with the goal to gain insights into the
molecular underpinnings of induced lung fibrosis.
TGF is a superfamily of more than sixty structurally
related growth factors that regulate many different
physiological and disease processes across species.
TGFβ1, along with its isoforms TGF-β2 and 3, is a prototype
of the TGF superfamily. TGF-β1 inhibits the
proliferation of most types of cells and induces the apoptosis of
epithelial cells; conversely, it stimulates mesenchymal
cells to proliferate and differentiate into myofibroblasts,
which prompts wound healing or tissue fibrosis in various
organs . Evidence supports the notion that TGF-β1 is
arguably the most predominant pro-fibrotic growth factor
in vivo. For instance, TGF-β1 expression is elevated in
lung fibrotic lesions, such as IPF and bleomycin-induced
lung fibrosis [90–92]; TGF-β1 induces fibroblast activation
in vitro and overexpression of active TGF-β1 leads to
persisting lung fibrosis in vivo [107, 108]; knockout of the
TGF-β1 gene in mice causes severely impaired wound
repair alongside severe wasting, generalized inflammation,
and tissue necrosis leading to organ failure and death,
whereas the epithelium-specific deletion of TGF-β
receptor type II protects mice from bleomycin-induced lung
fibrosis [109, 110]; lastly, blocking TGF-β1 signaling
ameliorates lung fibrosis in animal models [111, 112]. Relevant
to this review, TGF-β1 has been recognized as one of the
most important endogenous regulator to drive
myofibroblast differentiation and activation, because it directly
controls the de novo expression of α-SMA and the
induced expression of ECM proteins including collagens
and fibronectin; moreover many fibrogenic signals
activate myofibroblast functions by modulating the
expression and/or activation of TGF-β1, or by
crossinteracting with TGF-β1 signaling in and outside of the
cell (Fig. 4a) [34, 41].
As a pro-fibrotic growth factor, TGF-β1 can be
induced by injury and other fibrosis-stimulating signals
from several types of cells including bone
marrowderived cell lineages, such as macrophages, neutrophils,
and T lymphocytes, as well as structural cells, such as
airway epithelial cells, endothelial cells, and mesenchymal
cells, i.e., fibroblasts and myofibroblasts [34, 90, 113–115].
Newly synthesized TGF-β1 is confined within the
latencyassociated peptide (LAP) and is further associated with
the latent TGF-β binding protein (LTBP). This large
latency complex (LLC) is secreted into the ECM where it
anchors to the ECM by binding to fibronectin and Fibrillin
1 through LTBP. This configuration keeps TGF-β1 in a
latent state that needs to be released from the complex to
be activated. Indeed, cumulative evidence reveals that, in
addition to induced expression of TGF-β1 mRNA and
protein, much of the regulation of TGF-β1 in physiologic
and pathologic processes centers on the activation of
the latent TGF-β1 [92, 94]. Upon activation, TGF-β1
binds to its receptors to form a complex consisting of a
TGF-β1 homodimer, two TGF-β type I receptors, and
two TGF-β type II receptors on the cell surface. The
formation of the TGF-β1-receptor complex leads to the
activation of the Smad-dependent transcription of
fibrotic genes encoding α-SMA, collagens, fibronectin,
etc., as well as Smad-independent signaling, to drive
myofibroblast differentiation from fibroblasts (Fig. 4a).
CNTs have been shown to increase the level of
TGFβ1 protein in vitro and in vivo in several types of cells
(Fig. 4b). Treatment of mouse leukemic
monocytemacrophage RAW264.7 cells with either MWCNTs or
SWCNTs significantly induced the expression of
TGFβ1 mRNA and protein; moreover, the cell-free and
CNT-free supernatant of a conditioned medium from
the treatment stimulated human normal lung
fibroblasts (WI38-VA13) to differentiate into myofibroblasts,
as indicated by the induced expression of α-SMA [95, 96].
In separate studies, RAW264.7 macrophages exposed to
SWCNTs displayed a potent induction of secreted
TGFβ1 in the culture medium , and RAW264.7
macrophages exposed to long MWCNTs (length: 20–50 μm)
had a remarkably increased expression of TGF-β1 mRNA
as well as an elevated level of secreted TGF-β1 in the
culture medium . Induced expression of TGF-β1 mRNA
and protein was also observed in human normal bronchial
epithelial cells (BEAS-2B) treated with either MWCNTs
or SWCNTs; and the level of secreted TGF-β1 in the
culture medium of BEAS-2B cells was increased by
SWCNTs [95, 96, 116]. In the above cases, TGF-β1
produced from macrophages and epithelial cells stimulates
the differentiation of fibroblasts to myofibroblasts as a
CNTs also induce the production and secretion of
TGFβ1 from fibroblasts directly. SWCNTs induced TGF-β1
expression and promoted rat vascular adventitial
fibroblasts to transform to myofibroblasts, indicated by the
gained expression of SM22-α (smooth muscle protein
22α), which is a smooth muscle cell-specific protein and a
marker of myofibroblast differentiation . SWCNTs
induced TGF-β1 secretion and activation in a
dosedependent manner in human lung fibroblasts (CRL-1490)
. Long and short SWCNTs with median lengths of
12.31 and 1.13 μm, respectively, induced TGF-β1
expression and secretion in normal human lung fibroblasts
(NHLF); but the induction by the long SWCNTs was
significantly more pronounced than that by the short ones
. SWCNTs and MWCNTs increased the protein
expression of TGF-β1 and Collagen I in CRL-1490 cells as
well as the level of secreted TGF-β1 in the culture medium
. MWCNTs also induced the expression of α-SMA
mRNA and protein in mouse embryonic fibroblasts (NIH
Fig. 4 Regulation of myofibroblast formation by TGF-β1. a Schematic presentation of TGF-β1 signaling in myofibroblast formation. Upon stimulation,
latent TGF-β1 is activated and active TGF-β1 is released to bind to its receptors on the cell surface to drive the Smad-dependent pathway,
which directly up-regulates the transcription of fibrotic genes encoding α-SMA, collagens, and fibronectin. Binding of active TGF-β1 to its
receptors also elicits a number of Smad-independent pathways, such as the PI3K-AKT signaling, which may promote myofibroblast differentiation
and function. b Role of TGF-β1 in CNT-stimulated myofibroblast differentiation. CNTs induce the production and secretion of TGF-β1 by
macrophages and epithelial cells, which serves as a paracrine factor to stimulate fibroblast-to-myofibroblast differentiation. CNTs also directly induce
fibroblasts to produce and secrete TGF-β1, which functions as an autocrine factor for fibroblasts to differentiate into myofibroblasts. CNTs may directly
promote fibroblast-to-myofibroblast differentiation by mimicking the ECM or intracellular collagen fibers to generate mechanical stress. In addition,
CNTs stimulate epithelial cells to produce and secrete TGF-β1, which may induce the trans-differentiation of epithelial cells to myofibroblasts via EMT
3T3) . In the above cases, CNT exposure activated
fibroblasts to produce TGF-β1, which functions as an
autocrine factor to induce α-SMA expression and promote the
Elevation of the TGF-β1 protein level in the
bronchoalveolar lavage (BAL) fluid provides a measurement of
TGF-β1 induction in vivo. SWCNTs remarkably increased
the level of TGF-β1 in the BAL on day 7 post-exposure in
mice . SWCNTs and MWCNTs elevated the level
of TGF-β1 in the BAL on day 21 post-exposure in mice
[56, 116, 121, 122]. Long MWCNTs (length: 20–50 μm)
induced an elevated level of TGF-β1 in the BAL on day
1 and in alveolar macrophages on day 7, as well as an
increased level of Smad2 phosphorylation, a marker of
the activation of TGF-β1 signaling, on day 7
postexposure in rat lungs . These findings implicate
TGF-β1 signaling in CNT-induced lung fibrogenesis. In
a separate study, long MWCNTs (length: 5–15 μm),
but not short MWCNTs (length: 350–700 nm),
increased the level of TGF-β1 in the BAL on days 7 and
28 post-exposure; moreover, the percentage of SPC and
α-SMA double positive cells in mouse lungs was
significantly elevated by the long MWCNTs, but not the short
MWCNTs, on days 28 and 56 post-exposure,
suggesting that the induction of TGF-β1 by MWCNTs leads to
α-SMA expression in SPC positive cells in the lungs,
possibly through the trans-differentiation of alveolar
epithelial cells to myofibroblasts .
The widely used and well characterized MWCNTs in
the study of CNT-induced lung fibrosis, XNRI
MWNT7, are intermediate in length (median length: 3.86 μm)
compared with the long and short MWCNTs discussed
above. MWNT-7 CNTs have been shown to significantly
increase the levels of TGF-β1 protein in mouse BAL on
days 3, 7 and 14 post-exposure, and in mouse lung tissues,
especially in the regions where fibrosis occurs, on day 7
post-exposure . These findings demonstrate a
remarkable induction of TGF-β1 as an acute response to
MWCNTs, which might control myofibroblast
differentiation and activation, indicated by highly induced
expression of α-SMA, Collagen I, and fibronectin during the
acute phase response, in MWCNT-exposed lungs 1.
Taken together, these studies support that TGF-β1 is
significantly induced and may play a critical role in
promoting myofibroblast differentiation and activation in
CNT-triggered lung fibrosis.
A number of mechanisms have been described to
account for the activation of the latent TGF-β1 stored in
LLC, some of which have implications for CNT-induced
lung fibrosis. Activation of the latent TGF-β1 can occur
(a) by way of cellular acidification that denatures LAP to
release TGF-β1, (b) via ROS that oxidize LAP to perturb
the interaction between LAP and TGF-β1, (c) through
thrombospondin-1 (TSP-1) that directly interacts with
the latent complex to prevent it from binding matured
TGF-β1, (d) by proteases including plasmin, MMP-2
and MMP-9, tryptase, elastase, and thrombin that
activate TGF-β1 by proteolytic degradation of LLC and then
LAP of the latent TGF-β1 complex, and (e) through
integrins, such as the epithelial cell-specific αvβ6, which
mediates matrix contraction to release active TGF-β1
close to the cell surface, and the fibroblast αvβ8, which
presents latent TGF-β1 to a membrane-bound protease
(i.e., MT1-MMP) to activate TGF-β1 [123, 124]. It is
known that both MWCNTs and SWCNTs stimulate the
production of ROS in vitro and in the lungs [95, 96, 101,
125], which would induce the activation of TGF-β1 in a
manner analogous to that of asbestos [126, 127]. In
recent studies, we have shown that the lung expression of
TSP-1 was significantly induced by MWCNTs in vivo
, whereas that of MMP-2 was induced during lung
fibrosis from exposure to silica, paraquat, or bleomycin
. Direct evidence supporting a role of these
mediators in the activation of latent TGF-β1 in CNT-induced
lung fibrosis awaits further investigation.
Certain SWCNTs and MWCNTs have been shown to
induce EMT to generate myofibroblasts in the lungs,
which is accompanied by the activation of TGF-β1
signaling [97, 99]. These studies suggest that TGF-β1
may induce myofibroblast formation through EMT in
CNT-exposed lungs. However, studies on the
contribution of myofibroblasts derived via EMT in IPF and in
bleomycin-induced lung fibrosis, as well as in fibrosis
occurring in other organs such as kidney and liver, are
controversial; thus, the role of EMT in fibrosis remains
uncertain currently [88, 128]. Evaluation on whether
and, if so, how much the TGF-β1-regulated
myofibroblast trans-differentiation through EMT contribute to
CNT-induced lung fibrosis might provide an answer to
this question with regard to the phenotype, mechanism,
and function of EMT.
The platelet-derived growth factor (PDGF) represents
another important pro-fibrotic growth factor that confers
multiple functions in organ fibrosis including human and
experimental lung fibrosis [128–130]. For instance,
overexpression of PDGF-B (PDGF subunit B) in mouse lungs
induced severe fibrosis . Instillation of rats with
bleomycin caused elevated levels of PDGF-AA (homodimer of
PDGF subunit A) and PDGF-BB (homodimer of PDGF
subunit B) in the BAL fluid; moreover, the concentrated
BAL showed a growth-promoting activity toward lung
fibroblasts that can be partially blocked with anti-PDGF-BB
(64 %) or anti-PDGF-AA (15 %) antibodies . PDGF is
a potent mitogen for cells of a mesenchymal origin, such
as fibroblasts, both in vitro and in vivo; and it boosts the
recruitment and proliferation of fibroblasts, promotes the
differentiation of myofibroblasts from fibroblasts and
other types of cells, and increases the production of ECM
proteins from myofibroblasts during the fibrosis of various
organs [104, 128, 129, 133–135].
PDGF has been shown to be induced by CNTs in the
lungs in a number of recent studies. PDGF-AA was
significantly increased in mouse BAL on day 21
postexposure to MWCNTs in a dose-dependent manner
[56, 121]. During the early phase response to
MWCNTs, PDGF-AA was shown to be significantly
increased in the BAL on days 1, 3 and 7, and in lung
tissues on day 7, post-exposure to XNRI MWNT-7 by
aspiration in mice . In a separate study, PDGF-AA
was induced in the BAL on day 1 post-inhalation
exposure to MWCNTs in mice . Also, a study in rats
revealed that PDGF-AA was induced in the BAL on day
1, and in lung tissues on day 1 and day 21,
postexposure to MWCNTs, which was boosted by
costimulation with bacterial lipopolysaccharides .
These findings suggest the possibility for PDGF to play
a role in promoting myofibroblast differentiation and
activation in the lungs exposed to CNTs. However,
further detailed studies are needed to ascertain this posit.
Th2 cytokines IL-4 and IL-13
The T helper 2 (Th2)-type cytokines IL-4 and IL-13 have
been studied intensively in a variety of fibrotic diseases
and animal models, and have been demonstrated to
function as potent pro-fibrotic mediators to drive fibrosis
development [8, 128, 137, 138]. For instance, increased levels
of IL-4 were detected in patients with IPF or cryptogenic
fibrosing alveolitis [139, 140]; and inhibition of IL-4 by
neutralizing antibodies or inhibitors reduced liver fibrosis
and dermal fibrosis in mice [141, 142]. IL-13 levels were
significantly higher in IPF patients than in normal controls
; overexpression of IL-13 in mouse lungs induced
subepithelial airway fibrosis ; and inhibition of IL-13
by neutralizing antibodies decreased collagen deposition
in mouse lungs exposed to bleomycin .
IL-4 and IL-13 receptors are located on the cell
surface of a number of mouse and human fibroblast
subpopulations [146, 147]. In multiple in vitro studies, it
was shown that, under the stimulation of IL-4 or IL-13,
fibroblasts displayed enhanced proliferation and
differentiation, and increased production of α-SMA and ECM
proteins, such as type I and type III collagens and
fibronectin [146–152], which indicates that IL-4 and IL-13
signaling may promote fibrosis by stimulating
fibroblastto-myofibroblast differentiation and by enhancing tissue
remodeling. The Th2-type response may also promote
myofibroblast differentiation by activating TGF-β1. It
has been reported that IL-13 activates TGF-β1 in two
ways: first, IL-13 induces the production of latent
TGFβ1 from macrophages ; second, IL-13 activates
TGF-β1 by increasing the expression of proteins that
function in the cleavage of LAP, which keeps TGF-β1 as
an inactive form, such as matrix metalloproteinases
(MMPs) and cathepsins [153–155]. Taken together,
cumulative evidence reveals that IL-4 and IL-13 play
critical roles in the initiation and development of fibrosis,
which are in part mediated by inducing myofibroblast
We recently demonstrated that IL-4 and IL-13
expression and signaling were significantly induced by
MWCNTs (XNRI MWNT-7) in mouse lungs . In
a genome-wide microarray gene expression study of
mouse lung tissues, Th2-driven immune responses were
preferentially enriched. In particular, the activation of
IL-4 and IL-13 signaling, the center of Th2-type
responses, was a dominant effect induced by MWCNTs
on day 7 post-exposure. Time-course studies detected
that IL-4 was significantly induced by MWCNTs at the
mRNA level on days 1, 3, 7 and 14, and at the protein
level on days 3, 7 and 14, post-exposure. IL-13 was
significantly induced at the mRNA and protein levels on
days 3, 7 and 14 post-exposure. In addition, a panel of
signature downstream target genes of IL-4/IL-13
signaling, such as Il4i1, Chia, and Ccl11/Eotaxin, were
remarkably induced by MWCNTs at both the mRNA and
protein levels, further supporting the activation of IL-4/
IL-13 signaling. The increased expression of IL-4 and
IL-13 during the early phase fibrotic response, i.e., days
1 to 14 post-exposure, strongly suggests the potential
for IL-4 and/or IL-13 to play a role in promoting
myofibroblast differentiation in MWCNT-exposed lungs to
drive fibrosis development.
Oxidative stress reflects a cellular stress state that occurs
when the production of ROS and antioxidant defense
are out of balance, which causes multiple damages to
the cell, such as DNA strand breaks and DNA mutation,
protein peptide chain breaks, and lipid peroxidation,
leading to cell death in the severe case [157–159]. ROS
have been implicated in promoting fibrosis in multiple
organs, such as the lung, liver, and kidney, through a
number of mechanisms, as have been discussed in
several recent reviews [160–167]. Significantly, it has been
established that ROS promote the transformation of
fibroblasts to myofibroblasts by interacting with the
TGFβ1 signaling pathway [168–172]. ROS can augment the
expression and secretion of TGF-β1 and activate the
latent TGF-β1 to become active and functional. In a
reciprocal manner, TGF-β1 increases ROS production, mainly
through the induction of NOX4 expression. The
NOX4dependent production of hydrogen peroxide (H2O2) is
essential for TGF-β1-mediated myofibroblast
differentiation and ECM production. These findings clearly
demonstrate a necessary role of ROS in myofibroblast
differentiation and activation.
In the studies on CNT-induced toxicity, numerous
observations consistently demonstrate oxidative stress
as a predominant mechanism to link CNT exposures to
their toxicological and pathological outcomes. A large
number of in vitro cell culture studies support that
both SWCNTs and MWCNTs directly stimulate ROS
production in various types of cells, such as macrophages,
fibroblasts, and bronchial and alveolar epithelial cells, as
summarized in two recent reviews [18, 173]. For instance,
MWCNTs were shown to stimulate the production of
ROS in macrophages to activate NF-κB signaling ,
whereas SWCNTs were found to stimulate both fibroblast
proliferation and angiogenesis via the induction of ROS
production . Importantly, a few in vivo studies have
confirmed that CNT exposure results in oxidative stress
in tissues. Exposure of mice to SWCNTs led to a
dosedependent accumulation of 4-hydroxy-2-nonenal (4-HNE,
a lipid peroxidation biomarker) in the BAL as early as
1 day post-exposure, and a dose- and time-dependent
depletion of glutathione (GSH, a major antioxidant) in the
lungs, demonstrating the presence of oxidative stress upon
exposure to SWCNTs . Another indicator of oxidative
stress, heme oxygenase 1 (HO-1), has also been shown to
have an increased level in mouse lungs, aorta, and heart
on day 7 post-exposure to SWCNTs . In the NADPH
oxidase knockout mice that lack the gp91phox (Nox2)
subunit of a NOX enzymatic complex and are deficient in
ROS production, lung fibrosis induced by SWCNTs or
MWCNTs was remarkably attenuated, compared with the
wild-type control mice [122, 125, 175, 176].
We analyzed the roles of ROS in MWCNT (XNRI
MWNT-7)-induced pathologic effects on the lungs, by
using the nuclear factor erythroid 2-related factor 2
(Nrf2)deficient mice, which have an elevated oxidative stress due
to the lack of the defense against oxidative stress mediated
by Nrf2 [177, 178]. Under exposure to MWCNTs, several
markers indicative of oxidative stress, including ROS
production in alveolar macrophages, the levels of DNA
oxidation indicators 8-OHdG (8-hydroxy-2′-deoxyguanosine)
and γH2AX (phospho-Histone H2A.X (Ser139)) and the
level of 4-HNE in lung tissues, were remarkably increased
in the lungs; and the increases were markedly more
pronounced in Nrf2 knockout lungs than in wild-type lungs.
There was also a remarkably higher level of
MWCNTinduced lung fibrosis in Nrf2 knockout lungs than in
wildtype lungs . Taken together, these in vivo studies
strongly support that CNTs induce ROS production in the
lungs, and ROS play an important role in the initiation and
progression of CNT-induced lung fibrosis, suggesting that
CNT-induced ROS can serve as an enhancer to promote
myofibroblast differentiation in the lungs.
Role of inflammation and pro-inflammatory
The role of inflammation in organ fibrogenesis is
complex . Acute inflammation precedes and sometimes
accompanies fibrosis in most, if not all, lung fibrosis
induced by exposure to fibrogenic and cytotoxic agents
including chemicals, microbes, and particles and fibers.
On the other hand, fibrosis in the absence of apparent
tissue injury may occur without a prominent
inflammatory phenotype; moreover, anti-inflammation alone does
not effectively prevent or block the development of
fibrosis. It is believed that the role of inflammation in
fibrosis development varies among fibrosing diseases;
but once present, increased inflammatory infiltration
and secretion create a milieu rich in pro-fibrotic growth
factors, cytokines, and chemokines that foster the
development of fibrosis, in part mediated by priming or
promoting fibroblasts to differentiate into myofibroblasts. In
this context, a panel of pro-inflammatory cytokines
including TNF-α, IL-1α, IL-1β, and IL-6 have been shown
to be pro-fibrotic factors in both mouse and human lung
fibrosis models [18, 33].
TNF-α and IL-1β are among the earliest cytokines
recognized as pro-fibrotic factors. Overexpression of TNF-α
in mouse lungs resulted in spontaneous lung fibrosis
. TNF-α appears to play important roles in various
fibrosis animal models, such as bleomycin- or
silicainduced lung fibrosis and CCl4-induced liver fibrosis, as
well as a number of human fibrotic diseases, such as IPF
and asbestosis [180–183]. IL-1β and its receptors have
been shown to promote fibrosis in different types of
organ fibrosis, whereas inhibition of IL-1β signaling
reduces the fibrosis development, illustrating a critical
role of IL-1β signaling in organ fibrosis [184–191]. The
IL-1α-deficient mice exhibited reduced collagen
deposition in lung tissues in response to bleomycin treatment
. Enhanced IL-6 level was detected in the BAL of
IPF patients , and IL-6 signaling was found to be
key to driving fibrosis in a mouse model of acute
peritoneal inflammation . Moreover, a number of
studies demonstrate that these cytokines can stimulate
mesenchymal cells from multiple organs to express
αSMA [128, 195–199]. How these pro-inflammatory and
pro-fibrotic cytokines induce myofibroblast
differentiation and activation at the molecular level remains to
Upon exposure to CNTs, the lungs elicit an acute
inflammatory response as a well-characterized feature of
CNT-induced lung toxicity. In many cases, acute
inflammation precedes and accompanies CNT-induced lung
fibrosis. A number of studies have detected increased
expression and production of these pro-inflammatory
cytokines in CNT-exposed lungs. The level of TNF-α
in the BAL was significantly increased by SWCNTs on
day 1 post-exposure in mice , by MWCNTs at 12 h
or on days 1, 3 and 7 post-exposure in mice [24, 55,
200–202], and by MWCNTs on day 3 post-exposure in
rats . The level of IL-1α in the BAL was
significantly increased by MWCNTs (XNRI MWNT-7) on
days 1, 3, 7 and 14 post-exposure in mice . The
level of IL-1β in the BAL was significantly increased by
SWCNTs at 40 h or on days 1, 3, 7 and 28 post-exposure
in mice [22, 116], and by MWCNTs on days 1, 3 and 21
post-exposure in mice [24, 55, 122, 201]. The level of IL-6
in the BAL was significantly increased by MWCNTs at
12 h or on days 1, 3, 7, 14 and 28 post-exposure in mice
[24, 55, 200, 202]. These pro-inflammatory cytokines with
increased production from the acute innate immune
response may contribute to the induction of myofibroblast
differentiation in CNT-exposed lungs.
While induced expression of pro-inflammatory
cytokines remains a major mechanism of up-regulation of
their signaling, the activation of inflammasome
processing of cytokines IL-1β and IL-18 has been increasingly
recognized as a critical process for a variety of host
responses and diseases including fibrosis [203–205].
Inflammasomes are large protein complexes in the
cytoplasm that sense extracellular and intracellular signals to
initiate innate immune responses to microbe exposure
and tissue injury. Particulate and fibrous materials, such
as silica, asbestos, cholesterol crystals, and CNTs, have
been shown to activate inflammasomes, mainly the
NLRP3 inflammasome, to mediate the proteolytic
maturation of IL-1β and IL-18 [188, 205–207]. Activation of
the NLRP3 inflammasome increases the formation of
myofibroblasts in bleomycin-induced skin fibrosis .
Given the broad and critical roles of IL-1β, it is believed
that inflammasome activation plays an important role in
myofibroblast formation and function in CNT-induced
lung fibrosis. However, direct evidence supporting this
notion awaits further investigation.
Role of proliferation
During lung fibrosis, fibroblasts undergo an elevated
proliferation through multiple mechanisms, which
would promote the formation of fibroblastic foci and
the destruction of lung tissues. As fibroblasts are the
major progenitor cells of myofibroblasts, it is rational
to posit that an increase in the number of fibroblasts
leads to a higher number of myofibroblasts and the
accumulation of myofibroblasts in fibrotic foci.
The induction of fibroblast proliferation by CNTs has
been observed in a number of in vitro studies. SWCNTs
induced the proliferation of human lung fibroblasts
(CRL1490) in a dose- and time-dependent manner, which was
mediated by the ROS-regulated activation of p38 MAPK
(mitogen-activated protein kinase) and the induction of
TGF-β1 and VEGF (vascular endothelial growth factor)
[118, 208]. MWCNTs stimulated the proliferation of
multiple types of fibroblasts in tissue culture in a dose- and
physicochemical property-dependent manner [51, 209].
Furthermore, MWCNTs directly promoted the
proliferation of mouse lung fibroblasts (MLg cells) primed with a
low concentration of growth factor TGF-β1 or PDGF, by
prolonging the phosphorylation of the extracellular
signalregulated kinase (Erk) 1/2 . CNTs may stimulate
fibroblast proliferation by inducing the secretion of
soluble factors from epithelial cells, which is NLRP3
inflammasome-dependent, but TGF-β1-independent
. These studies demonstrate that certain types of
CNTs are capable of stimulating fibroblast
proliferation directly and thereby contribute to CNT-induced
The tissue inhibitor of metalloproteinase 1 (TIMP1)
is highly induced and is secreted into the ECM from
macrophages and mesenchymal cells during lung
fibrosis. Using the Timp1-deficient mice, we demonstrated
that TIMP1 plays a critical role in the development of
MWCNT-induced lung fibrosis1. In the lungs of
wildtype mice, MWCNTs (XNRI MWNT-7) remarkably
increased the proliferation of fibroblasts, indicated by the
expression of cell proliferation markers Ki-67 (marker
of proliferation Ki-67) and PCNA (proliferating cell
nuclear antigen). However, this induction was significantly
attenuated in the lungs of Timp1-deficient mice.
Accordingly, the MWCNT-induced fibrotic responses,
including the formation of fibrotic foci, the
differentiation of myofibroblasts, and the production
and deposition of ECM proteins, such as Collagen I
and fibronectin, were significantly higher in wild-type
mice than in Timp1-deficient mice. These findings
strongly suggest that MWCNTs stimulate fibroblast
proliferation in the lungs and thereby promote
myofibroblast differentiation from the enriched fibroblast
pool to boost fibrosis. Mechanistic analysis
demonstrated that MWCNT-induced fibroblast proliferation
might be mediated by the formation of a TIMP1/CD63/
integrin β1 complex on the surface of fibroblasts, which
promotes the Erk1/2 phosphorylation and activation in
fibroblasts in the lungs. These findings establish a
direct mechanistic link among MWCNT exposure,
fibroblast proliferation, myofibroblast differentiation, and
lung fibrosis in vivo.
Role of tissue stiffness, mechanical force, and
In the model of wound healing, the formation of
myofibroblasts generally becomes a major event at 1 week after
tissue injury, which correlates with a significantly
increased tissue tension or stiffness . In fact, it has been
shown that the threshold stiffness for the de novo
expression of α-SMA in stress fibers ranges around 20,000 Pa,
which is about 200 to 2000-fold higher than that of the
early wound provisional ECM (i.e., 10–100 Pa) . This
increase in tissue stiffness and myofibroblast formation
correlates with the increased activation of TGF-β1 protein
within the tissue matrix. Mechanistic studies demonstrate
that, indeed, the increased stiffness is necessary for the
activation of the latent TGF-β1 and the release of activated
TGF-β1 from the large latency complex in the vicinity of
the site of wound healing or fibrosis . In particular, the
mechanical tension generated through the contraction of
myofibroblasts is a well-recognized mechanism for the
activation of the latent TGF-β1 in the matrix . Based
on these findings, it is believed that the CNT-induced
tissue injury and fibrotic changes stimulate myofibroblast
contraction to increase tissue stiffness, which in turn
boosts the differentiation of fibroblasts into myofibroblasts
by activating the latent TGF-β1 locally. This chain of
events sets in motion a positive feedforward response
among myofibroblast formation, contraction, tissue
tension, and TGF-β1 activation to drive organ fibrosis.
However, a direct measurement of tissue stiffness of the lungs
exposed to CNTs in relation to fibrosis and myofibroblast
activation is needed to prove this notion.
At the molecular level, CNTs may modulate cellular
and ECM mechanical properties through several means.
In addition to stimulating the tissue to release soluble
factors, such as TGF-β1, discussed above, CNTs may
induce the contraction and increase the cellular tension of
cells by interfering with the intracellular contractile
structures once inside the cell, as some CNT fibers
resemble the cytoskeletal or contractile filaments in size
and shape. Alternatively, CNT fibers may directly
interact with cell surface receptors, such as the pattern
recognition receptors. SWCNTs have been observed inside
rat vascular adventitial fibroblasts after exposure for 24
and 48 h . MWCNTs were found to be
accumulated on the surface of NIH 3T3 fibroblasts 3 h
postexposure, and some MWCNTs entered the cell by way
of endocytosis 24 h post-exposure . Moreover,
MWCNTs were shown to bind to BMPR2 on the surface
of myoblasts to modulate their differentiation .
These findings raise the possibility of a direct effect of
CNTs on fibroblasts and myofibroblasts by way of
mechanical activation of the matrix latent TGF-β1 or
biochemical activation of cell surface receptor-mediated
intracellular signaling, to stimulate the activation and
differentiation of fibroblasts into myofibroblasts. In both
scenarios, the physicochemical properties of CNTs, such
as the nano-scaled diameter, fiber length, surface area
and reactivity, and biopersistence, would be critical
parameters to influence their stimulatory activities.
Although attractive, these possibilities remain to be proven
by direct evidence from studies using multiple cellular,
molecular, and biophysical means in the future.
The expanding knowledge on the pathological features
and molecular mechanisms of CNT-induced lung
fibrosis is in agreement with the overall understanding of
lung fibrosis derived from certain human fibrotic lung
diseases and experimental animal models in a number of
ways. This correlation suggests that CNT-induced lung
fibrosis can be used as a new animal disease model for
studying the molecular mechanisms underlying human
fibrotic lung diseases, such as IPF and pneumoconiosis.
Emerging evidence reveals that CNTs potently induce
and activate myofibroblasts both in vitro and in vivo.
Moreover, CNTs are found capable of inducing and
activating a number of critical mediators and cell signaling
pathways that have been implicated in myofibroblast
function and regulation during fibrosis development, as
summarized in Fig. 5. It is clear that the activation of
myofibroblasts likely represents a critical and common
molecular step toward the development of organ
fibrosis, which now includes CNT-induced lung fibrosis.
Although myofibroblasts have long been recognized as
an important group of mesenchymal cells in the
development of fibrosis, the contribution of these cells to the
fibrotic response induced by fibrogenic particles and
fibers, and new materials, such as nanomaterials, has
received attention in the field of toxicology only in recent
few years. In part, this delayed recognition is due to the
difficulty of separating myofibroblasts from fibroblasts
during the development of fibrosis with regard to the
phenotypes and the available techniques and approaches
for analyzing the cells. Indeed, there is a lack of specific
markers for identification of myofibroblasts in tissues.
Moreover, certain mesenchymal functions during fibrosis
are shared between fibroblasts and myofibroblasts, such
as the secretion of some ECM proteins. Therefore, it is
necessary to use multiple markers, including α-SMA
expression, to analyze myofibroblasts and their functions
in a specific tissue, time point, and context in which
fibrosis takes place. In this respect, the study on
CNTinduced lung fibrosis is perhaps advantageous over other
lung fibrosis models for the analysis of myofibroblast
functions, because the formation of myofibroblasts is
exposure-dependent and possibly inducer-specific with
respect to the pathological features, mechanisms, and
consequences, which can now be readily demonstrated
in cultured cells and animals exposed to CNTs.
The fibroblastic response responsible for matrix built-up
and scarring is a rather complex and dynamic process,
which remains poorly understood to date. From the
experimental point of view, the study on myofibroblasts
provides an opportunity to unravel the mechanisms
underlying the fibroblastic response for the initiation and
development of lung fibrosis induced by CNTs, other
nanomaterials, and particles and fibers at molecular and
cellular levels. From this perspective, the list of mediators
and cell signaling pathways summarized above can
serve as a reasonable starting point for the mechanistic
analysis of myofibroblast regulation and function in
fibrosis. Needless to say, further pathological and
molecular studies with the aid of genetically engineered
mouse strains are required to ascertain the contributions
Fig. 5 Mediators of CNT-induced myofibroblast differentiation in the lungs. CNTs stimulate multiple mechanisms and mediators capable of
promoting myofibroblast formation and function, including a pro-fibrotic growth factors TGF-β1 and PDGF, b Th2 cytokines IL-4 and IL-13,
c ROS, d pro-fibrotic cytokines TNF-α, IL-6, IL-1α and IL-1β, e TIMP1, and f tissue stiffness. Activation of these signaling cascades may induce
myofibroblast differentiation and activation directly or by boosting fibroblast proliferation to increase the myofibroblast precursor pool, leading to
fibrosis in the lungs
of these candidate mediators and signaling cascades to the
onset and pathological outcomes of CNT-induced lung
fibrosis in vivo. One caveat to note on this line of research
is that these mediators and pathways are not likely to act
alone, but work in concert in a highly regulated and
timeand context-dependent fashion, to drive the formation
and functioning of myofibroblasts leading, ultimately, to
fibrosis of the lungs. As such, multiple targets from this
myofibroblast-predominant fibroblastic response should
be sought after in order to achieve better intervention
against lung fibrotic diseases.
Among the identified mediators of myofibroblast
activation, TGF-β1 stands out as the most relevant
endogenous factor to drive myofibroblast differentiation
and function. However, many gaps exist in the
understanding of TGF-β1 function and mode of action in the
regulation of myofibroblasts and fibrosis induced by
CNT exposure. In particular, most toxicological studies
on CNT lung fibrosis examined the induction of TGF-β1
mRNA and/or protein expression in cultured cells or in
the lungs, which is necessary to establish the involvement
of TGF-β1 in CNT toxicity. Several critical questions
remain unaddressed. For instance, which signaling
pathways and factors mediate the induction of TGF-β1
by CNTs; whether and, if so, how CNTs activate the
latent form of TGF-β1 stored in the ECM; and how the
activated TGF-β1 controls myofibroblast activation and
function upon CNT exposure? The morphologically
apparent contractive features of myofibroblasts fittingly
explain, at least in part, the inevitable contraction and
ultimate scarring of fibrotic tissues in the lungs and
other organs. Moreover, the mechano-regulation of
matrix remodeling by myofibroblasts appears to be
closely correlated with the activation of latent TGF-β1.
How this interplay among myofibroblast contraction,
tissue stiffness, and TGF-β1 activation occurs to propel
CNT-induced lung fibrosis is currently unclear.
Apparently, a combination of molecular, biophysical, and
genetic approaches is needed to address these questions in
One implication of the findings from the research on
myofibroblasts derives from the notion that organ fibrosis
might arise from a failure to suppress the normal repair
process of tissue injury to result in the persistent presence
and over-functioning of myofibroblasts in fibrotic tissues.
It is therefore rational to expect that the information
obtained from the study of myofibroblasts and their
associated mediators and signaling pathways involved in the
pathogenesis of lung fibrosis is likely to generate new
insights into both the molecular understanding and the
clinical treatment of human fibrotic lung diseases that include
IPF, pneumoconiosis, and nanomaterial-induced lung
1Dong J and Ma Q. TIMP1 promotes multi-walled
carbon nanotube-induced lung fibrosis by stimulating
fibroblast activation and p roliferation. 2016; submitted.
4-HNE: 4-hydroxy-2-nonenal; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; Akt:
vakt murine thymoma viral oncogene homolog; BAL: Bronchoalveolar lavage;
BMPR2: Bone morphogenetic protein receptor type II; Ccl11: Chemokine (C-C
Motif) ligand 11, or eotaxin; CDK: Cyclin-dependent kinase; Chia: Chitinase,
acidic, or AMCase; CNT: Carbon nanotube; ECM: Extracellular matrix;
EDA: Extra domain A; EMT: Epithelial-mesenchymal transition; Erk: Extracellular
signal-regulated kinase; FoxO3a: Forkhead box O3a; FSP-1: Fibroblast specific
protein-1; GSH: Glutathione; GSK-3β: Glycogen synthase kinase-3β;
HO1: Heme oxygenase 1; IL: Interleukin; Il4i1: Interleukin 4 induced 1, or Fig1;
IPF: Idiopathic pulmonary fibrosis; JNK: c-Jun N-terminal kinase; Ki-67: marker
of proliferation Ki-67; LAP: Latency-associated protein; LLC: Large latency
complex; LTBP: Latent TGF-β binding protein; MAPK: Mitogen-activated
protein kinase; MEK: Mitogen-activated protein kinase kinase; MMP: Matrix
metalloproteinase; MWCNT: Multi-walled carbon nanotube;
NADPH: Nicotinamide adenine dinucleotide phosphate; NF-κB: Nuclear
factor-κB; NLRP3: Nucleotide-binding oligomerization domain-like receptor:
pyrin domain-containing 3; NOX: NADPH oxidase; Nrf2: Nuclear factor
erythroid 2-related factor 2; PCNA: Proliferating cell nuclear antigen;
PDGF: Platelet-derived growth factor; PDGFR-β: Platelet-derived growth
factor receptor-β; PI3K: Phosphoinositide 3-kinase; PP2A: Protein phosphatase
2A; PTEN: Phosphatase and tensin homolog; RNS: Reactive nitrogen species;
ROS: Reactive oxygen species; SM22-α: Smooth muscle protein 22-α;
Smad: Sma and Mad related family; SPC: Surfactant protein C; SWCNT:
Singlewalled carbon nanotube; TAK1: TGF-β-activated kinase 1;
TGFβ1: Transforming growth factor-β1; Th2: T helper 2; Timp1: Tissue inhibitor of
metalloproteinase 1; TNF-α: Tumor necrosis factor-α; TSP-1:
Thrombospondin1, or Thbs1; VEGF: Vascular endothelial growth factor; Wnt: Wingless-type
MMTV integration site family; α-SMA: α-smooth muscle actin;
γH2AX: phospho-Histone H2A.X (Ser139)
Availability of data and materials
All data and materials are included in this article.
JD designed and drafted the manuscript. QM revised and finalized the
article. Both authors read and approved the final manuscript.
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