This study investigated the effects of calcium channel blockers on calcium oscillations in human lung fibroblasts induced by transforming growth factor beta (TGF-β). The researchers found that calcium oscillations were substantially reduced by L-type calcium channel blockers like nifedipine as well as blockers of both L- and T-type channels. In a mouse model of pulmonary fibrosis induced by bleomycin, daily treatment with nifedipine prevented fibrotic changes in the lungs like increased stiffness and collagen deposition without affecting lung inflammation. This suggests that nifedipine may disrupt calcium signaling in fibroblasts and protect against pulmonary fibrosis by altering their profibrotic response rather than through anti-inflammatory effects.

1. ORIGINAL RESEARCH
Disruption of Calcium Signaling in Fibroblasts and Attenuation of
Bleomycin-Induced Fibrosis by Nifedipine
Subhendu Mukherjee*, Ehab A. Ayaub*, James Murphy, Chao Lu, Martin Kolb, Kjetil Ask, and Luke J. Janssen
Firestone Institute for Respiratory Health, St. Joseph’s Hospital, Department of Medicine, McMaster University, Hamilton, Ontario,
Canada
Abstract
Fibrotic lung disease afflicts millions of people; the central problem is
progressive lung destruction and remodeling. We have shown that
external growth factors regulate fibroblast function not only through
canonical signaling pathways but also through propagation of periodic
oscillationsinCa21
.Inthisstudy,wecharacterizedthepharmacological
sensitivity of the Ca21
oscillations and determined whether a blocker of
thoseoscillationscanpreventtheprogressionoffibrosisinvivo.Wefound
Ca21
oscillations evoked by exogenously applied transforming growth
factor b in normal human fibroblasts were substantially reduced by
1 mM nifedipine or 1 mM verapamil (both L-type blockers), by 2.7 mM
mibefradil (a mixed L-/T-type blocker), by 40 mM NiCl2 (selective at this
concentration against T-type current), by 30 mM KCl (which partially
depolarizes the membrane and thereby fully inactivates T-type current
but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and
T-type currents). In our in vivo study in mice, nifedipine prevented
bleomycin-induced fibrotic changes (increased lung stiffness,
overexpression of smooth muscle actin, increased extracellular matrix
deposition, and increased soluble collagen and hydroxyproline content).
Nifedipine had little or no effect on lung inflammation, suggesting its
protective effect on lung fibrosis was not due to an antiinflammatory
effectbutratherwasduetoalteringtheprofibroticresponsetobleomycin.
Collectively, these data show that nifedipine disrupts Ca21
oscillations
in fibroblasts and prevents the impairment of lung function in the
bleomycin model of pulmonary fibrosis. Our results provide compelling
proof-of-principle that interfering with Ca21
signaling may be beneficial
against pulmonary fibrosis.
Keywords: calcium signaling; fibrosis; nifedipine; L-type calcium
channel; bleomycin
Clinical Relevance
Fibroblasts and myofibroblasts are prime targets in fibrosis,
and tyrosine kinase inhibitors have been used to inhibit the
actions of growth factors. However, tyrosine kinase inhibitors
have potential problems due to their diverse and deleterious
side effects (off-target effects), which provide an excellent
rationale to explore other targetable pathways that might be
developed. Ca21
channel blockers like nifedipine are in
common use in clinical practice: they are inexpensive and well
tolerated. The data obtained from these studies may open up
entirely new avenues for the treatment of pulmonary fibrosis.
Fibroproliferative disorders are a leading
cause of morbidity and mortality worldwide.
Extensive uncontrolled scarring (fibrosis)
can occur in a variety of organs. It is
suggested that almost half of all deaths
are attributable to various chronic
fibroproliferative diseases (1). Pulmonary
fibrosis is characterized in large part by the
accumulation of extracellular matrix (ECM)
in the alveolar and interstitial spaces,
severely compromising lung gas exchange
(2). Idiopathic pulmonary fibrosis affects
approximately 5 million persons worldwide
(3). In addition, millions of people
worldwide are affected by chronic
(Received in original form January 7, 2015; accepted in final form February 2, 2015)
*These authors contributed equally to this work.
This work was supported by the Canadian Lung Association and the Ontario Thoracic Society, Toronto-Dominion Grant in Medical Excellence Award, and
St. Joseph’s Healthcare Hamilton.
Author Contribution: S.M. and E.A.A. designed and performed the experiment, analyzed the data, and wrote the paper. J.M. and C.L. performed the
experiment. L.J.J. conceived and designed the experiments, analyzed the data, and edited the manuscript. M.K. and K.A. edited the manuscript, shared their
expertise with fibrosis, and provided cultured cells.
Correspondence and requests for reprints should be addressed to Subhendu Mukherjee, Ph.D., St. Joseph’s Hospital, 50 Charlton Avenue East, T3338,
Hamilton, ON, L8N 4A6 Canada. E-mail: smukher@mcmaster.ca
Am J Respir Cell Mol Biol Vol 53, Iss 4, pp 450–458, Oct 2015
Copyright © 2015 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2015-0009OC on February 9, 2015
Internet address: www.atsjournals.org
450 American Journal of Respiratory Cell and Molecular Biology Volume 53 Number 4 | October 2015

2. obstructive pulmonary disease and asthma,
which also involve a substantial degree of
fibroblast-mediated airway wall destruction
and/or fibrosis. Fibroblasts are key
mediators of the lung structural changes
occurring in these diseases (4). Pulmonary
fibrosis involves a number of diverse
changes in the fibroblasts, but this disease
is mainly characterized by scarring of the
lungs after a destructive inflammatory
response in the small airways and alveoli.
In spite of increased knowledge of the
underlying pathobiology, there still is no
specific treatment targeted to pulmonary
fibrosis. N-acetyl cysteine, corticosteroids,
and cytotoxic agents have been used in
treating fibrosis but without any remarkable
progress (5, 6). Two recent clinical trials
investigating the effectiveness of the
drugs pirfenidone and nintedanib in the
treatment of idiopathic pulmonary fibrosis
(7, 8) showed that these drugs may slow
down disease progression but are not
able to completely stop it. This is very
encouraging but also demonstrates the need
for additional and more effective therapies
for idiopathic pulmonary fibrosis.
The primary function of fibroblasts is to
store and secrete cytokines and connective
tissue proteins (1). Fibroblasts respond to
a variety of growth factors and cytokines,
transforming growth factor (TGF)-b1 being
one among them. However, the intracellular
signaling mechanisms underlying these
responses are not entirely clear, and the
positive effect of blocking one pathway
pharmacologically may be masked or
tempered by the contribution of another
one. It is known that TGF-b1 exerts its
function via different canonical kinase
pathways, including those in which Small
Mothers Against Decapentaplegics (SMAD),
phosphoinositide-3-kinase, and mitogen-
activated protein kinase are central players
(9). In addition to these complex pathways,
several studies suggest that TGF-b1 exerts its
action in part via elevating cellular [Ca21
]i
(10–12). In fact, we were the first to show
that TGF-b1 evokes recurring Ca21
oscillations with frequencies that correlate
with growth factor concentration (11). We
also demonstrated that both the influx of
external Ca21
and release of internally
sequestered Ca21
are involved in TGF-
b1–mediated Ca21
activity. The pathway by
which TGF-b1 triggers the influx of external
Ca21
is not clear.
There are dozens of different types of
ion channels in the plasmalemma that are
involved in influx of external Ca21
(13).
Voltage-dependent calcium channels are
among these (13) and include L and T
subtypes. L- and T-subtype calcium
channels are differentiated in part on the
basis of differing activation and inactivation
properties and pharmacological sensitivity
(14, 15). T-type Ca21
currents play a role
as pacemakers of rhythmic activity in
a diverse array of cell types (16–18). In
many cases, L-type Ca21
currents amplify
the changes in [Ca21
]i triggered by these
T-type currents. Due to the involvement of
T- and L-type Ca21
channels in different
cellular and organ functions, many
studies have been conducted to develop
pharmacological tools that modulate their
function, and many of those tools are now
essential in clinical practice (19–21). More
recently, Ca21
channel blockers, which are
selective for both T- and L-type channels
(e.g., efonidipine) (17, 19–21), have been
used in clinical practice.
For this reason, we tested the effects of
L- and T-type Ca21
channel blockers on
Ca21
oscillations in human pulmonary
fibroblasts. We hypothesized that those
blockers will interfere with Ca21
influx
into the fibroblasts, disrupting the Ca21
oscillations, which are important to their
synthetic function and thereby protect
against pulmonary fibrosis. Primary human
pulmonary fibroblasts were cultured and
treated with TGF-b1 and different blockers
under various conditions. The changes in
Ca21
oscillations in response to a variety of
blockers were monitored by confocal [Ca21
]i
fluorimetry. To test the in vivo effect of
calcium blockage, groups of mice were
exposed to bleomycin and treated with
vehicle or nifedipine daily for 1 or 3 weeks
and assessed for pulmonary inflammatory or
pulmonary fibrotic changes. Our results
suggest that L-type Ca21
currents play a key
role in producing the Ca21
oscillations.
T-type Ca21
currents may also play a
role as pacemakers to set Ca21
oscillation
rhythmicity. Most importantly, we found
that nifedipine prevents the fibrotic changes
and impairment of lung function in the
bleomycin model of pulmonary fibrosis.
Materials and Methods
Chemicals
TGF-b1 (PeproTech Inc., Rocky Hill, NJ)
was prepared in 4 mM HCl/0.1% BSA
solution. Oregon Green calcium dye, RPMI
medium, and Hanks’ balanced salt solution
were obtained from Invitrogen (Carlsbad,
CA). Masson’s Trichrome and Picro-
sirius red were made at the McMaster
Immunology Research Centre. All other
chemicals were obtained from Sigma-
Aldrich Chemical Co. (Oakville, ON,
Canada), except for a smooth muscle actin
(a-SMA) monoclonal antibody (Dako,
Burlington, ON, Canada) and bleomycin
(Hospira Healthcare Corp., Saint-Laurent,
PQ, Canada), and were prepared in
absolute ethanol, in DMSO (mibefradil and
nifedipine), or as aqueous solutions.
Pulmonary Fibroblasts
All procedures were approved by St.
Joseph’s Hospital Board of Ethics (RP#
00–1839). Normal human pulmonary
fibroblasts (passages 5–10) were obtained
from five different donors (11).
Ca21
Fluorimetry
Cells were loaded with Oregon Green
(5 mM) for 40 minutes at 378C and then
perfused with Hanks’ balanced salt solution
solution for 15 minutes to allow for
complete dye hydrolysis. Confocal
microscopy was performed at room
temperature (22–258C) using a custom-
built apparatus (11).
Animals
Male C57BL6/J mice aged 10 to 12 weeks
(Charles River, Wilmington, MA) were kept
at the Central Animal Facility of McMaster
University. All animal work was conducted
according to the guidelines from the
Canadian Council on Animal Care and was
approved by the Animal Research Ethics
Board of McMaster University.
Administration of Bleomycin and
Nifedipine
Pulmonary fibrosis was induced using
intratracheal instillation of bleomycin (0.06
U/mouse in a volume of 50 ml) delivered
during gaseous isoflurane anesthesia.
Animals were killed after 0, 7, or 21 days.
Nifedipine (10 mg/kg/d) or vehicle
(DMSO) were given via intraperitoneal
injections starting from the day of
bleomycin administration and continuing
until end of the experiment.
Bronchoalveolar Lavage and
Immunohistochemistry
Mice were anesthetized with isoflurane and
killed. Excised whole lung was washed with
ORIGINAL RESEARCH
Mukherjee, Ayaub, Murphy, et al.: Nifedipine Prevents Fibrosis 451

3. PBS for bronchoalveolar lavage fluid (BALF)
collection, and total and differential
cell counts were performed (22). Left
lungs were fixed (in 10% formalin) for
immunohistochemistry (23). A total of 20
to 30 random images from two transversal
sections of a-SMA–stained slides were
captured on a microscope (203 objective)
(DP70 camera; Olympus Canada,
Richmond Hill, ON, Canada) and
quantified using ImageJ software (Version
1.46r; National Institutes of Health,
Bethesda, MD). All images were threshold
adjusted to differentiate total and stained
areas. Each of the 20 to 30 calculated sets of
percent tissue area stain were averaged to
represent each sample. A semiquantitative
assessment of lung fibrosis was established
using the Ashcroft grading procedure (23)
scored in blinded fashion.
Collagen Content
Soluble collagen in whole lung homogenate
was assessed by Sircol collagen assay
(Biocolor Ltd, Carrickfergus, UK), and
insoluble collagen was measured by a
colorimetric assay as described previously
(24).
ELISA
BALF IL-6 and TGF-b1 were quantified
in duplicate by ELISA (R&D Systems,
Minneapolis, MN).
Measurements of Pulmonary
Function
Lung function parameters (pressure–
volume [P-V] loops, quasistatic elastance,
and K value) were measured (23) using
a flexiVent mechanical respirator (SCIREQ,
Montreal, PQ, Canada).
Systemic Vasopressor Response
Animals were restrained in a plastic
restraining tube at 288C. Systolic, diastolic,
and mean arterial pressures and heart rate
were read using a CODA Blood Pressure
Measurement apparatus (Kent Scientific,
Torrington, CT) and are reported as the
mean of the last 8 to 10 consecutive
readings after 40 cycles of measurement.
Data Analysis
Data are reported as mean 6 SEM; n refers to
the number of donors or of mice. Statistical
comparisons were made using Student’s t test
(unpaired), one-way ANOVA, and Newman-
Keuls multiple comparisons test. P , 0.05
was considered statistically significant.
Results
Baseline Recording and Effect of
Growth Factor on Ca21
Activity
Before investigating the effect of different
interventions, we verified the profile of Ca21
oscillations in normal human fibroblasts.
As we reported previously (11), little or no
spontaneous Ca21
oscillations could be
observed in normal fibroblasts (data not
shown), whereas overnight treatment with
1 nM TGF-b1 evoked recurring Ca21
oscillations (Figure 1A). We showed
previously (11) that those oscillations were
sensitive to the TGF-b receptor tyrosine
kinase inhibitor SD-208 (data not shown).
We also found small mechanical responses
(retraction, shifting of cytosolic contents,
etc.) in a very few cells, but these did not
correlate with the Ca21
oscillations (not
shown).
Effects of Ca21
Channel Blockers
on Ca21
Oscillations in Cultured
Fibroblasts
We previously demonstrated that TGF-
b1–mediated Ca21
oscillations in normal
human pulmonary fibroblasts are
immediately abrogated by removal of
external Ca21
(11), but the types of
channels involved in that influx of external
Ca21
were not clear.
To examine whether L-type Ca21
channels are involved in this phenomenon,
normal fibroblasts that had been stimulated
with 1 nM TGF-b1 were exposed to
1 mM nifedipine, a dihydropyridine L-
type–selective Ca21
channel blocker (14,
15). We found that nifedipine treatment
significantly reduced the magnitude and
frequency of TGF-b1–mediated Ca21
oscillations (Figure 1B); this blockade was
not instantaneous because the drug was
introduced via an upstream reservoir with
a dead volume of several milliliters at a rate
of approximately 3 ml/min and then
needed to come into equilibrium with the
bathing medium surrounding the cells
(z2 ml). We could not demonstrate
reversibility within the time frame of these
experiments (which we kept to ,30 min to
avoid bleaching of the Ca21
-sensitive dye),
which we attribute to the perfusion-related
delay and to a much slower release of this polar
molecule from the lipid bilayer into the
surrounding aqueous media.
To further test the involvement
of L-type Ca21
channels, we exposed
TGF-b1–stimulated normal fibroblasts to
1 mM verapamil, a structurally unrelated
phenylalkylamine L-type Ca21
channel
blocker (14, 15), or to 1 mM NiCl2 (at this
concentration, Ni21
blocks L- and T-type
Ca21
channels [14, 15]). We found
significant reductions in the frequencies of
TGF-b1–mediated Ca21
oscillations in
both cases (Figures 1C and 1D). These
results strongly suggest the involvement of
L-type Ca21
channels in the influx of
external Ca21
and propagation of Ca21
oscillations in human pulmonary
fibroblasts.
We also examined the possible
involvement of T-type channels in TGF-
b1–mediated external Ca21
influx. We
pretreated a group of normal pulmonary
fibroblasts with 1 nM TGF-b1 overnight
and then perfused these cells with 2.7 mM
mibefradil (a T-type Ca21
-channel blocker
with moderate selectivity [14, 15]), with
40 mM NiCl2 (which is selective at this
concentration for T-type calcium channels
[14, 15]), or with 30 mM KCl (which
depolarizes membrane to 240 mV, which
will not activate L-type currents but will
completely inactivate T-type currents 14,
15]). We found that all these interventions
inhibited the Ca21
oscillations generated by
TGF-b1 (Figures 2A–2C).
Collectively, these data suggest that
both L- and T-type Ca21
channels
contribute to propagation of the Ca21
oscillations. Furthermore, they suggest that
these two contributions are not additive or
complementary because blockage of one
or the other is sufficient to eliminate the
Ca21
oscillations.
Effects of Bleomycin and Nifedipine
on Lung Function and Body Weight
To examine the efficacy of nifedipine against
bleomycin-induced murine pulmonary
fibrosis and inflammation, we treated mice
with bleomycin. Half of these mice were also
treated daily with nifedipine, whereas the
others received vehicle (DMSO); comparisons
were also made against a third group that was
not treated with bleomycin or nifedipine/
DMSO. After 21 days of bleomycin treatment,
we examined lung function using a Flexivent
mechanical respirator: P–V loops were used
to derive the quasistatic elastance and K value
(which indicates the curvature of the P–V
loop). We confirmed that bleomycin flattened
the P–V loop (Figure 3A), decreased the K
value (Figure 3B), and increased quasistatic
elastance (Figure 3C), which measures the
tendency of the lung to return to its normal
ORIGINAL RESEARCH
452 American Journal of Respiratory Cell and Molecular Biology Volume 53 Number 4 | October 2015

4. form after deflation (i.e., its stiffness), but also
found that nifedipine prevented all these
bleomycin-induced changes (Figures 3A–3C).
Overall, our results suggest that nifedipine
largely prevented the deleterious effects on
lung function measured 21 days after
exposure to bleomycin.
We also measured the change in body
weight after bleomycin treatment and
the effect of nifedipine on it. Bleomycin
reduced the body weight by approximately
15% after 7 days of treatment and by
approximately 8% after 21 days of
treatment, whereas nifedipine prevented
this weight loss induced by bleomycin
(Figure 3D).
Effects of Nifedipine on Bleomycin-
Induced Fibrosis
We examined the fibrotic response to
bleomycin treatment by staining lung tissue
sections for a-SMA immunohistochemistry
(Figures 4A and 4D) or collagen (Picro-sirius
red stain and Masson’s trichrome stain)
(Figures 4B, 4C, and 4E) and by
homogenizing whole lungs to measure
soluble collagen (Sircol assay) (Figure 4F) and
insoluble collagen (hydroxyproline content)
(Figure 4G). We found that bleomycin
treatment substantially increased a-SMA
content and collagen deposition, but
nifedipine treatment reduced a-SMA content
(by 80%) and collagen deposition (Ashcroft
score reduced by 67%, soluble collagen
content reduced by 38%, and insoluble
collagen content reduced by 42%). Taken
together, these results suggest that nifedipine
prevented bleomycin-mediated fibrosis.
Effects of Nifedipine on Bleomycin-
Induced Pulmonary Inflammation
To check whether these protective
properties of nifedipine are secondary to an
effect on bleomycin-induced pulmonary
inflammation, we examined the level
of some inflammatory markers after
bleomycin and nifedipine treatment. We
found that bleomycin increased the total
inflammatory cell count in BALF as well as
specific BALF counts of macrophage and
lymphocytes 7 and 21 days after bleomycin
treatment. Neutrophil counts were
increased after Day 7 but were reduced
dramatically by Day 21. Nifedipine did not
produce any significant change in total
BALF cell counts, macrophage counts, or
lymphocyte numbers but slightly reduced
the neutrophil counts in BALF after 7 days
of bleomycin treatment (Figures 5A–5D).
In addition, no significant effect of
nifedipine was observed in the BALF
level of TGF-b1 (total) and IL-6 at Day 7
(Figures 5E and 5F). Levels of TGF-b1
and IL-6 at Day 21 were undetectable.
Collectively, these results show that
nifedipine did not prevent or ameliorate
pulmonary inflammation in bleomycin-
treated mice.
Measurement of Systemic
Vasopressor Response to Nifedipine
Nifedipine is well known to reduce blood
pressure. To confirm that nifedipine was
100
80
60
40
20
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0 500 1000 1500
F510
Time (sec)
1nM TGFβ (O/N)A
120
100
80
60
40
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250
750
1250
500
1000
1750
1500
F510
Time (sec)
1μM Nifedipine
B 5
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oscillation
peakin10minutes
TGF β TGF β +
nifedipine
***
50
40
30
20
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0 200 600 1000400 800 14001200
F510
Time (sec)
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C 6
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+Verapamil
***
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D
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oscillation
peakin10minutes
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NiCl2
***
Figure 1. Overnight treatment with 1 nM transforming growth factor (TGF)-b1 evoked recurring Ca21
oscillations in normal human pulmonary fibroblasts (A). TGF-b1–evoked Ca21
oscillations in normal
fibroblasts are substantially reduced by blocking L-type calcium current using 1 mM nifedipine (B), 1 mM
verapamil (C), or 1 mM NiCl2 (D). F510: fluorescence measured at 510 nm. Each tracing is representative
of recordings made from batches of cells derived from five donors (at least four cells per batch). Bar
diagram indicates mean (6 SEM) responses to 1 nM TGF-b1 (number of Ca21
oscillations) before and
during perfusion with different blockers. ***P , 0.0001 versus TGF-b1 alone (n = 5).
ORIGINAL RESEARCH
Mukherjee, Ayaub, Murphy, et al.: Nifedipine Prevents Fibrosis 453

5. effective in our own study, we treated
a group of mice (n = 3) with 10 mg/kg
nifedipine (administered intraperitoneally)
daily for 3 days. We measured the blood
pressure of these mice before the
administration of nifedipine on Day 1 and
every day after 4 hours of nifedipine
administration. In two of the animals,
systemic arterial pressures were decreased
from 130/97 to 95/67 mm Hg and from
116/95 to 97/67 mm Hg, respectively. In
the third animal, the prenifedipine blood
pressure was obtained, but the post-
treatment pressure could not be measured.
Nonetheless, these cursory data are
consistent with several previous reports that
this strategy is effective for blocking
systemic (25–30) and pulmonary (31)
vascular L-type Ca21
channels.
Discussion
TGF-b1, a vital multifunctional growth
factor for all mammals, is involved in
a number of cell functions, including
protein synthesis. Excessive extracellular
matrix (ECM) deposition is the main
culprit in the case of pulmonary fibrosis.
TGF-b1 regulates both formation and
degradation of ECM. Eventually TGF-b1
up-regulates the expression of ECM genes
and down-regulates many genes involved in
the degradation of ECM (32, 33).
We were the first to show that growth
factors stimulate a series of recurring
oscillations in [Ca21
]i (11), that the stimulus
strength (agonist concentration) is encoded
within the Ca21
oscillation frequency, and
that disruption of those Ca21
oscillations
suppresses gene transcription (11, 34). We
also showed that the Ca21
oscillations were
abrogated immediately upon removal of
Ca21
from the bathing medium, and they
immediately resumed upon reintroduction
of that Ca21
. This finding suggests the
involvement of Ca21
-permeable ion
channels on the plasmalemma.
A previous publication has given
evidence that those Ca21
-permeable
channels include members of the transient
receptor potential (TRP) family of channels
referred to as TRPV4 channels (35). TRP
channels are often involved in refilling of
the internal Ca21
store. In the present
study, we present considerable evidence
that Ca21
oscillations also involve L- and
T-type voltage-gated Ca21
channels, using
a variety of pharmacological tools that have
been developed to selectively manipulate
these currents (14, 15).
First, we found that nifedipine
eliminated the Ca21
oscillations triggered
by TGF-b1 in normal pulmonary
fibroblasts. Nifedipine is widely recognized
as being highly selective for L-type Ca21
currents. Nonetheless, there have been
reports of its nonselective inhibitory action
against certain other conductances,
including TRP channels. However, we went
on to show that the Ca21
oscillations were
also blocked by verapamil, a member of
another structurally unrelated class of
blockers that are also recognized as being
highly L-type selective. Verapamil and
other related phenylalkylamines bind to
a different site on those channels; as such,
they do not share the same spectrum
of nonselective actions as do the
dihydropyridines, which include nifedipine.
We also showed that these Ca21
oscillations are sensitive to mibefradil or to
40 mM NiCl2, which at the concentrations
used here are selective for T-type channels
over L-type ones (14, 15). Likewise,
knowing that T-type currents are fully
inactivated at a membrane potential of
240 mV, whereas L-type currents are only
moderately inactivated at that potential
(14, 15), we went on to show that the
Ca21
oscillations are also eliminated by
increasing the bath concentration of [K1
]
to 30 mM, which the Nernst potential
predicts will depolarize the membrane to
240 mV. We are not aware of any reports
that TRPV4 channels are sensitive to
mibefradil or 40 mM NiCl2.
Altogether, our data suggest strongly
that both T- and L-type channels are also
100
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500
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Time (sec)
2.7 μM Mibefradil
A
1250250 10000 750 1500
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NumberCa2+
oscillation
peakin10minutes
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mibefradil
***
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40
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B
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peakin10minutes
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***
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60
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Time (sec)
30 mM KCl
C
0 400 800 1200
6
4
2
0
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oscillation
peakin10minutes
TGF β TGF β + KCl
***
Figure 2. TGF-b1–evoked Ca21
oscillations in normal human pulmonary fibroblasts are substantially
reduced by blocking T-type Ca21
current using 2.7 mM mibefradil (A), by 40 mM NiCl2 (B), or by
30 mM KCl (C). Each tracing is representative of recordings made from batches of cells derived from
five donors (at least four cells per batch). The bar diagram indicates mean (6 SEM) responses to
1 nM TGF-b1 (number of Ca21
oscillations) before and during perfusion with different blockers.
***P , 0.0001 versus TGF-b1 (n = 5).
ORIGINAL RESEARCH
454 American Journal of Respiratory Cell and Molecular Biology Volume 53 Number 4 | October 2015

6. involved in producing the Ca21
oscillations,
together with TRPV4 channels (35).
Furthermore, our data suggest that the
contributions of these three distinct Ca21
conductances to the Ca21
oscillations are
not additive or complementary because
blocking any one of the three selectively is
fully sufficient to abrogate the oscillations.
This speaks to the serial nature of their
interaction in other cell types. That is, we
would propose that the TRPV4 channels
play a role in Ca21
handling by the internal
Ca21
store; that the latter regulates T-type
currents, which in turn play a pacemaker
function; and that the L-type channels are
the primary source of Ca21
for the
repetitive Ca21
oscillations, as follows.
In our previous publications (11, 34),
we documented the disruption of Ca21
oscillations by agents that target the storage
and release of Ca21
by the endoplasmic
reticulum (i.e., cyclopiazonic acid,
ryanodine, U73122, and xestospongin). Our
interpretation is that Ca21
release from the
internal Ca21
pool triggers some kind
of pacemaker current. The latter may
comprise the TRPV4 channels, which
elsewhere are important for refilling of the
internal Ca21
pool (a phenomenon referred
to as store-operated Ca21
entry). Given that
TRPV4 channels conduct both Ca21
and
Na1
, their opening would depolarize the
membrane and would allow Ca21
entry
for refilling of the store. The pacemaker
current may also include Ca21
-dependent
chloride channels activated by the store-
mediated release, which also cause
membrane depolarization.
The depolarization produced by the
pacemaker current would activate T-type
channels, which in turn produces further
20
15
5
Est(cmH2O/mL)
Control
25
10
0
Bleo Bleo +
Nifedipine
**
#C
120
110
100
90
80
70
Weights(%)
D
0 5 10 15
Time (days)
20 25
Control
Bleomycin + Nifedipine
Bleomycin
1.0
0.8
0.4
0.2
0.0
Volume(ml)
Pressure (cm H2O)
A
10
0.16
0.12
0.08
Kvalue(1/cmH2O)
Control
0.6
–0.2
20 30 40 Bleo Bleo +
Nifedipine
*
#B
1
2
3
Bleo (3)
Bleo+Nifedipine (2)
Control (1)
Figure 3. All animals were subjected to lung function measurements 21 days after bleomycin (Bleo)
treatment: mean pressure–volume loops (A), K value (B), and elastance (Est) (C). Bleo treatment
dramatically flattened the pressure–volume loop curves, decreased the K value, and increased Est.
More importantly, nifedipine reversed all these changes to nearly normal levels. Bar diagrams indicate
mean (6 SEM) responses to different treatments. Bleo administration reduced body weight by 15%
after 7 days of treatment and by 8% after 21 days treatment. Nifedipine markedly ameliorated the
Bleo-induced weight loss (D). *P , 0.05 and **P , 0.002 versus control; #
P , 0.05 versus Bleo.
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
1
2
αSMApositivearea(%)
3
4
***
###
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
500
Solublecollagen
(μgsperrightlung)
1000
1500 **
##
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
2
4
Gradeoffibrosis
6
8
***
###
Control Bleo + Vehicle Bleo + Nifedipine
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
50
Hydroxyproline
(μgsperrightlung)
100
150
200
***
*
#
D F
E
C
B
A
G
Figure 4. Nifedipine prevented Bleo-induced myofibroblast proliferation. Staining for a-smooth muscle actin (a-SMA) (A), Picro-sirius red (B), and Masson’s
trichrome (C) measured 21 days after Bleo administration. Bleo increased a-SMA–positive cells and collagen deposition, whereas nifedipine prevented both
changes (D and E). Ashcroft score (E) was calculated from Picro-sirius red–stained slides. Bleo also increased soluble (F) and insoluble (G) collagen in lung
homogenates, and nifedipine significantly reduced the level of both. Scale bars indicate the size (200 mm, except Bleo 1 vehicle slice, which is 1 mm [C, middle])
of the lung slices. *P , 0.05, **P , 0.002, and ***P , 0.0001 versus control; #
P , 0.05, ##
P , 0.002, and ###
P , 0.0001 versus Bleo 1 vehicle (n = 5).
ORIGINAL RESEARCH
Mukherjee, Ayaub, Murphy, et al.: Nifedipine Prevents Fibrosis 455

7. membrane depolarization (and a small
and brief Ca21
influx). T-type currents
play a role as pacemakers of rhythmic
activity in a diverse array of cell types
(16–18, 36, 37), whereas the larger and
longer-lasting L-type Ca21
currents
amplify the changes in [Ca21
]i triggered
by the T-type currents (or, in the case
of cardiac muscle, triggered by a mixed
sodium/potassium pacemaker current).
Both T- and L-type currents also exhibit
voltage-dependent inactivation, which
then allows [Ca21
]i to return to the
resting level, setting the stage for another
Ca21
oscillation.
Patch-clamp electrophysiological
experiments are needed to fully characterize
the complement of ion conductances found
in the plasmalemma of pulmonary
fibroblasts, including those that set the
membrane potential, any putative
pacemaker current(s), and the voltage-
dependent Ca21
currents.
We have previously shown that the
Ca21
oscillations are critical to the growth
factor–stimulated synthetic function of
the pulmonary fibroblasts (11, 34). We
therefore next sought to test the effects of
pharmacologically interfering with the
Ca21
oscillations in an animal model of
pulmonary fibrosis. Bleomycin is widely
used in studies of experimentally induced
pulmonary fibrosis (38) as well as
in the clinical setting as a cancer
chemotherapeutic tool (29, 39–41). In the
animal model, it induces a profound
inflammatory response within days, which
then progresses to a full pulmonary fibrotic
response over the ensuing 2 to 3 weeks.
We chose to use the dihydropyridine
nifedipine in this study for several reasons.
First, L-type current appears to be the
final player in the sequence of ionic
conductance changes that produce the Ca21
oscillations. Also, this L-type blocker has
already received FDA approval for use in
the clinic for treatment of a wide variety
of cardiovascular problems and is well
tolerated, well characterized, and relatively
inexpensive. Numerous groups have
previously shown that a dose of 5 to
10 mg/kg/d given subcutaneously (30) or
orally (25–28, 42) is sufficient to exert
a powerful systemic vasodilator response
in rodents. Another study showed that
4 mg/kg given by intraperitoneal injection
in mice was sufficient to normalize right
ventricular pressures in a genetic model of
pulmonary hypertension (31). To confirm
that nifedipine was effective in our study,
we showed that systemic arterial pressures
in mice were decreased by approximately
20 to 30 mm Hg after treatment with
nifedipine (10 mg/kg, intraperitoneally).
Bleomycin markedly decreased lung
compliance, as indicated by a flatter P-V
relationship. Importantly, this increased
stiffness was largely prevented by nifedipine
pretreatment. Analysis of these P-V loops
showed that nifedipine abrogated the
bleomycin-induced deficit in pulmonary
function, as reflected in a normalized K
value and significantly decreased elastance.
Immunostaining also revealed that
nifedipine abrogated the bleomycin-induced
increase in a-SMA, collagen fiber
deposition, and content of soluble and
insoluble collagen. Finally, nifedipine
significantly prevented the weight loss due
to bleomycin treatment.
Bleomycin increased the counts
of several inflammatory cells (e.g.,
macrophages, lymphocytes, and neutrophils)
as well as BALF levels of IL-6 and TGF-b.
Nifedipine had no statistically significant
effect on most of these markers of
inflammation after bleomycin treatment.
The only change that could be considered to
be antiinflammatory in nature was a very
0
Control
Control
Bleo + Nifedipine
Bleo + Vehicle
Day 7 Day 21
2
Cellsperml(×105
)
4
6
**
***
#
***
***
0.0
Control Day 7 Day 21
0.2
0.1
Cellsperml(×105
)
0.3
0.4
**
**
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
40
20
TGFβ1(pg/ml)
60
0
Control Day 7 Day 21
2
1
Cellsperml(×105
)
3
4
**
#
*
***
**
0.0
Control Day 7 Day 21
1.0
0.5
Cellsperml(×105
)
1.5
2.0
**
**
0
Control Bleo +
Vehicle
Bleo +
Nifedipine
40
60
20
IL-6(pg/ml)
80
A
C
E
B
D
F
Figure 5. Nifedipine had little effect on Bleo-induced pulmonary inflammation in mice. Bleo increased
total cell counts in bronchoalveolar lavage fluid (A) and increased differential counts for macrophages (B),
neutrophils (C), and lymphocytes (D) after 7 and 21 days of administration, except the neutrophil counts
were reduced after 21 days. Nifedipine did not significantly alter any of these changes except for
neutrophil counts. Similarly, nifedipine had little effect on Bleo-elevated serum levels of TGF-b (total)
(E) or IL-6 (F) measured 7 days after Bleo treatment. Levels of TGF-b and IL-6 were undetectable
after 21 days. Bar diagrams indicate mean (6 SEM) responses to different treatments. *P , 0.05,
**P , 0.002, and ***P , 0.0001 versus control; #
P , 0.05 versus Bleo 1 vehicle (n = 5).
ORIGINAL RESEARCH
456 American Journal of Respiratory Cell and Molecular Biology Volume 53 Number 4 | October 2015

8. modest decrease in neutrophil counts at
Day 7. Otherwise, the only other nifedipine-
induced change that was statistically
significant was in fact in a proinflammatory
direction: a doubling of lymphocyte counts
at Day 21. There were small changes in
BALF TGF-b and IL-6 levels in nifedipine
group compared with the bleomycin-only
group (increased TGF-b level and
decreased IL-6 level at Day 7), but these
changes were not significant. None of
these nifedipine-induced changes on
inflammation can realistically account for
the profound protective effect of nifedipine
against the subsequent fibrotic response to
bleomycin.
We cannot rule out off-target effects of
nifedipine on other cell types because the
lung may be composed of up to 40 to 50
different cell types, many of which may
express voltage-dependent Ca21
channels;
nifedipine also blocks pH-dependent [Ca21
]i
changes due to its carbonic anhydrase
activity. Irrespective of how or where
nifedipine may be acting, however, our data
clearly show a promising beneficial effect
of nifedipine in protecting against the
progression of fibrotic changes in the lung.
Our finding that several different forms
of L-type channel blockade disrupt Ca21
oscillations in isolated fibroblasts, coupled
with our previous report that disruption
of Ca21
oscillations in isolated fibroblasts
interferes with their synthetic/secretory
response to TGF-b or PDGF, strongly
suggests that nifedipine’s effect was
largely upon the postinflammation
profibrotic response of the fibroblasts.
The transduction pathway by which the
Ca21
oscillations are decoded is unclear
but appears to operate in parallel with
other well-characterized canonical pathways
(e.g., Smad proteins, RhoA, PI-3-kinase, p38,
JNK, and PKC).
Fibroblasts and myofibroblasts are
prime targets in fibrosis, and tyrosine kinase
inhibitors have been used to inhibit the
actions of growth factors. However, tyrosine
kinase inhibitors have potential problems
due to their diverse and deleterious side
effects (off-target effects), which provide
an excellent rationale to explore other
targetable pathways that might be
developed. Ca21
channel blockers like
nifedipine are in common use in clinical
practice: they are inexpensive and well
tolerated. The data obtained from these
studies may open up entirely new avenues
for the treatment of pulmonary fibrosis. n
Author disclosures are available with the text
of this article at www.atsjournals.org.
Acknowledgments: The authors thank
Mrs. Fuqin Duan and Jane Ann Smith for
technical support.
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