Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus
Anseth KS (2012) Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated
Reduction in Substrate Modulus. PLoS ONE 7(7): e39969. doi:10.1371/journal.pone.0039969
Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus
Huan Wang 0
Sarah M. Haeger 0
April M. Kloxin 0
Leslie A. Leinwand 0
Kristi S. Anseth 0
Elena Aikawa, Brigham and Women's Hospital, Harvard Medical School, United States of America
0 1 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemical and Biomolecular Engineering and Department of Materials Science and Engineering, University of Delaware, Newark, Delaware, United States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Biofrontiers Institute, University of Colorado , Boulder, Colorado , United States of America
Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ,32 kPa, mimicking pre-calcified diseased tissue, to ,7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in a-smooth muscle actin (a-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including a-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-b1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.
Funding: The work was funded by National Institutes of Health (NIH) R01 HL089260, NIH R01 HL50560 and Howard Hughes Medical Institute. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The microenvironment of a cell regulates cellular functions
dynamically [1,2,3]. In particular, substrate elasticity has
recently been shown to direct cell functions, such as stem cell
differentiation  and renewal , independent of soluble
growth factors. Soft matrices that mimic the stiffness of brain
with Youngs modulus (E) around 0.11 kPa promote
neurogenic differentiation of human mesenchymal stem cells, while
rigid matrices that mimic collagenous bone (E, 2540 kPa)
promote osteogenesis . Further, rigid plastic tissue culture
plates have a non-physiological stiffness (10,000| that of soft
tissue) and are known to affect cell phenotypes. For example,
when fibroblasts are cultured on plastic plates, they
spontaneously differentiate into myofibroblasts with fibrogenic properties
[6,7,8]. In contrast, soft substrata derived from
polyacrylamideor poly(ethylene glycol)-based hydrogels with
physiologicallyrelevant moduli (E10kPa) inhibit myofibroblast differentiation,
better preserving the inactivated cellular phenotype [9,10,11].
From this perspective, hydrogels with a tissue-mimicking elastic
modulus provide an important culture system to study and
direct fibroblast functions in vitro.
When fibroblasts are transformed into myofibroblasts over long
time periods in vivo, tissue fibrosis can develop [12,13]. Tissue
fibrosis presents serious health problems affecting multiple organs,
including skin , lung , liver , kidney  and heart
. Resident fibroblasts in these tissues have been shown to play
critical roles in disease progression. Fibroblasts respond to both
chemical cues and the physical stiffness of tissue to become
myofibroblasts [18,19,20]. For example, transforming growth
factor b1 (TGF-b1) is a potent profibrotic cytokine that activates
fibroblasts from valve, skin or liver to become myofibroblasts
[9,18,21]. Myofibroblasts, characterized by increased secretion of
ECM proteins (e.g., collagen and fibronectin) and higher
contractile function mediated through a-smooth muscle actin
SMA) stress fibers, exacerbate fibrosis [20,22]. Strategies to reverse
the myofibroblast phenotype into a native fibroblastic phenotype
could be of significant therapeutic impact in abrogating tissue
Primary fibroblasts isolated from pig aortic valves serve as a
model system to study how the pathogenic myofibroblast
phenotype is regulated by microenvironment modulus
[23,24,25]. These fibroblasts, valvular interstitial cells (VICs), are
the main cell population residing in aortic valves . In a healthy
valve, VICs maintain a quiescent fibroblastic phenotype; however,
in a sclerotic valve, VICs are activated to myofibroblasts, which
secrete excessive ECM degradative enzymes (e.g., MMPs) and
collagen, leading to deterioration of the original valve structure
and tissue thickening [27,28,29]. Persistence of the myofibroblast
phenotype leads to further valve stiffening, which eventually
restricts blood flow from the left ventricle to the aorta. Increased
rigidity of valvular tissue associated with aortic valve (AV) sclerosis
is not only a result of collagen deposition by myofibroblasts, but
also can promote pathology by a positive feedback mechanism,
leading to the accumulation of myofibroblasts and the continual
production of ECM .
Microenvironment stiffness has been shown to regulate the fate
of fibroblasts, including differentiation into myofibroblasts,
apoptosis and proliferation. When valvular, hepatic or lung fibroblasts
are cultured on low modulus substrata (E10 kPa), they maintain
an un-activated phenotype; however, when cultured on higher
modulus substrata, they are activated to myofibroblasts
[10,11,31,32]. Reducing the elastic modulus of the substratum to
a very low level (E1 kPa) promotes apoptosis in various
fibroblasts [11,33,34]. Moreover, NIH3T3 fibroblasts grown on
compliant substrata (E , 5 kPa) show a decrease in proliferation,
compared with cells cultured on stiff substrata (E , 14 kPa) .
Based on previous findings, we speculated that substrata with E
lower than 10 kPa, but not too low to activate apoptosis, may
provide healthy physical signals to maintain quiescent fibroblast
phenotypes. Specifically in this study, we test the hypothesis that
the valvular myofibroblasts will either undergo apoptosis or
deactivate to a quiescent fibroblast state when the substrate modulus
We utilized a photodegradable poly(ethylene glycol) (PD-PEG)
hydrogel  to study the fate of VICs in response to substrate
modulus reduction. With this unique photo-sensitive material,
we can change E in situ while VICs are adhered to the gels
from ,32 kPa, mimicking collagenous bone (which has been
detected in diseased valves ), to ,7 kPa, mimicking healthy
valve fibrosa . We found that VICs switched their fate from
activated myofibroblasts to fibroblasts with reduced proliferation
when the substrate was changed from stiff to soft. This
deactivation process was not associated with significant apoptosis,
but was characterized by down-regulation of critical
myofibroblast phenotypic markers, including loss of a-SMA stress fibers,
significant reduction in the expression of myofibroblast gene
signatures (a-SMA and CTGF) and a significant decrease in
proliferation. These results provide insight into the potential fate
of valvular myofibroblasts in vivo after tissue repair. Further, we
established that de-activated VICs still maintain the potential to
activate the expression of myofibroblast genes in response to
TGF-b1 and to proliferate in response to growth stimuli,
indicating a reversible fibroblast state of the cells and
contributing to our understanding of how modulus regulates
the myofibroblast-fibroblast transition. This could be useful in
developing novel treatments for tissue fibrosis and could result
in new approaches to direct cell fate and function for tissue
Materials and Methods
The photodegradable crosslinker (PD-PEG) was synthesized as
previously described . PD-PEG (Mn ,4070 g/mol, 8.2 wt%)
was copolymerized with PEG monoacrylate (PEGA, Mn ,400 g/
mol, 6.8 wt%, Monomer-Polymer and Dajac Laboratories, Inc)
and an acrylated adhesion peptide sequence RGDS (5 mM,
described below) via redox-initiated free radical chain
polymerization . The hydrogels were synthesized as thin films
(0.25 mm thick) covalently attached to methacrylated coverglass.
Hydrogels with surface areas (A) of , 250 mm2 and , 370 mm2
were made on coverglass of 18 mm and 22 mm in diameter,
respectively, to enable harvest of appropriate cell numbers for
specific assays. To reduce the crosslinking density and modulus of
the hydrogels, samples were irradiated with long wavelength, low
intensity ultraviolet (UV) light for 5 minutes (365 nm at 10 mW/
cm2). These conditions have been previously demonstrated to be
cytocompatible . Hydrogel moduli were verified with
rheometry and atomic force microscopy as described previously
, where moduli of hydrogels (0.25 mm thick) in phosphate
buffered saline (PBS) were measured as E ,32 kPa and ,7 kPa
after 0 minute and 5 minutes of irradiation (365 nm at 10 mW/
An integrin-binding adhesion peptide was synthesized and
incorporated within the cell culture platform to promote cell
attachment based on established protocols [37,38] Briefly,
OOGRGDSG (diethylene glycol-diethylene
glycol-glycine-arginine-glycine-aspartic acid-serine-glycine) was made on a solid
phase peptide synthesizer (Tribute, Protein Technologies, Inc.)
with HBTU/HOBt amino acid activation and Fmoc chemistry
. After synthesis of the primary sequence, the N-terminus of
the peptide was modified on resin with an acryloyl group by
reaction with HATU-activated acrylic acid in the presence of
diisopropylethylamine (4 molar excess of each relative to
Nterminus amine) . Complete reaction of the amine was verified
using the Kaiser test , and the peptide was cleaved from resin
(5 wt% phenol in 95% trifluoroacetic acid, 2.5%
triisopropylsilane, and 2.5% DI water) stirring for 2 hours at room temperature.
The cleavage solution was precipitated in and washed with ice cold
ethyl ether (3x), dried under vacuum overnight, purified by
highperformance liquid chromatography (HPLC), and characterized
by matrix-assisted laser desorption/ionization mass (MALDI-MS)
. The peptide has a molecular weight of ,892 g/mol,
consistent with its amino acid sequence.
Fresh porcine hearts were obtained from Hormel Foods
Corporation (Austin, MN, USA) within 24 hours of sacrifice and
aortic valve leaflets were excised. Primary VICs were harvested
from porcine aortic valve leaflets based on a sequential collagenase
digestion as previously described . The isolated cells were
cultured in growth medium (Medium 199, 15% fetal bovine serum
(FBS), 50 U/ml penicillin, 50 mg/ml streptomycin, and 0.5 mg/ml
fungizone) and expanded up to passage 3. Passage 3 VICs were
seeded on hydrogels at 35,000 cells/cm2 and were cultured in low
serum medium (Medium 199, 1% FBS, 50 U/ml penicillin,
50 mg/ml streptomycin, and 0.5 mg/ml fungizone) for up to 5
days. Treatment with fibroblast growth factor 2 (FGF2, Sigma
Cat# F0291, 16 ng/ml) in 15% FBS medium or TGF-b1 (R&D
systems, Cat# 101-B1-001, 5 ng/ml) in 1% FBS medium was
applied on day 4 for 24 hours.
Hydrogels (A , 250 mm2) were fixed with 4%
paraformaldehyde (overnight at 4uC), permeabilized in 0.1% TritonX100, and
blocked with 5% bovine serum albumin (BSA). Mouse
anti-aSMA antibody (Abcam, Cat# ab7817) or rabbit anti-vimentin
antibody (Cell Signaling Technology, Cat#5741) was diluted at
1:100 in PBS with 1% BSA and incubated with the samples
overnight at 4uC. Following washes in PBS with 0.05% Tween 20,
samples were labeled with goat-anti-mouse Alexa Fluor 488
secondary antibody (Life technologies (Invitrogen), Cat#
A11001) or goat-anti-rabbit Alexa Fluor 488 secondary antibody
(Life technologies (Invitrogen), Cat# A11070), along with DAPI to
visualize nuclei. Samples were subsequently imaged on a LSM 710
Laser Scanning Microscope with transmitted light detector for
differential interference contrast (DIC) (Carl Zeiss) or inverted
epifluorescent microscope (Nikon), each with a 20X magnification
objective. Images of different fluorescence channels were compiled
with Zen or Metamorph software and analyzed by ImageJ for total
nuclei number (Analyze Particles function, NIH). Myofibroblasts
were counted as cells with a-SMA staining organized into fibrils,
and the percentage of myofibroblasts was calculated as (number of
myofibroblasts/total number of cells) x 100%. To quantify each
sample, 6 random fields of view were imaged, counted, and
Annexin V Staining
VICs were washed in PBS and detached from hydrogels (A
, 370 mm2) using TrypLE (Life technologies, Cat# A1285901).
Following centrifugation, cell pellets were re-suspended in
Annexin V-binding buffer (10 mM HEPES, 140 mM NaCl,
2.5 mM CaCl2, pH 7.4) and incubated with Alexa Fluor
594labeled Annexin V (Life technologies, Cat# A13203) for 15
minutes at room temperature. DAPI was applied at 0.5 mg/ml to
distinguish live and dead cell populations. Percent of Annexin V+
cells was quantified using a CyAn ADP flow cytometer (Beckman
Coulter). Early apoptotic cells were identified as those with high
fluorescent emission in the Alexa Fluor 594 channel and low
emission in the DAPI channel. On average, over 10,000 events
were collected per sample. As a positive control, VICs were treated
for 18 hours with 25 mM camptothecin, an apoptosis-inducing
For quantifying basal level proliferation amongst different
substrate moduli, VICs were switched to medium with 5% FBS
after photodegradation on day 3 and were subsequently incubated
with 10 mM EdU for 3 hours on day 5. For examining
proliferative response to growth stimuli after modulus-driven
deactivation, VICs were treated with or without FGF2
(SigmaAldrich, Cat# F0291, 16 ng/ml) and 15% FBS on day 4 for 24
hours and then incubated with 10 mM EdU for 1 hour on day 5.
For each condition, cells from two PD-PEG gels of A , 370 mm2
were harvested by trypsinization. For EdU staining, samples were
fixed and permeabilized using the Fixation and Permeabilization
kit (Life technologies, Cat# GAS003). Following washes in PBS
with 1% BSA, the cells were incubated with Click-iT reaction
cocktail prepared from the Click-iT EdU Alexa Fluor 488 kit (Life
technologies, Cat# C10337) for 30 minutes at room temperature.
DAPI was applied at 5 mg/ml as a measure for DNA content.
Samples were analyzed by a CyAn ADP flow cytometer (Beckman
Coulter). Over 10,000 events were collected per sample.
Proliferative cells were counted based on high fluorescence in the Alexa
Fluor 488 channel.
Quantitative Real-Time PCR
For each condition, two PD-PEG gels (A , 370 mm2) with
attached VICs were harvested and submerged in TRI Reagent
(Sigma, Cat# T9424), rapidly frozen in liquid nitrogen, and stored
at -80uC until processing. Samples were homogenized with
individual DNase and RNase free pestles. Total RNA was purified
according to a modified version of the manufacturers protocol for
TRI Reagent. Briefly, after chloroform extraction and aqueous
phase collection, a second chloroform extraction with equal
volume of chloroform to aqueous phase was performed. cDNA
was synthesized from total RNA with Superscript III reverse
transcriptase (Life technologies, Cat# 18080-051) and random
hexamer primers. Gene expression was determined by SYBR
Green-based quantitative real-time PCR (qRT-PCR) using gene
specific primer sets (Table 1) and an Applied Biosystems 7500
Real-Time PCR machine.
Data are presented as mean 6 standard error of the mean
(SEM). SEM was calculated based on three biological replicates.
Each biological replicate was based on an isolation of VICs from
6090 pooled porcine aortic valves as described in 2.2 Cell
Culture. A Students t-test was used to compare data sets and a p
value less than 0.05 was considered statistically significant.
We synthesized PD-PEG hydrogels as dynamic culture
substrates for VICs  with Youngs modulus (E) ,32 kPa (stiff
gels), E ,7 kPa (soft gels), and E changed from ,32 kPa to
,7 kpa (stiff-to-soft gels). A peptide containing the adhesive
sequence RGDS was incorporated within the gel to facilitate cell
adhesion . VICs were cultured on PD-PEG gels in low serum
(1% FBS) medium for up to 5 days (Fig. 1), where the low serum
culture limits cellular response (i.e., differentiation or proliferation)
to growth factors in the serum . At day 3, half of the stiff gels
were irradiated with low intensity UV light to reduce the
crosslinking density and modulus (stiff-to-soft gels in Fig. 1). We
then investigated the mechanisms of myofibroblast deactivation,
examining apoptosis and reversion to quiescence as potential
pathways (Fig. 1). Specifically, Annexin V staining was used to
examine early apoptosis events. Formation of a-SMA stress fibers,
expression of myofibroblast genes, and relative proliferation rate
were quantified by immunocytochemistry, qRT-PCR and an
EdU-based proliferation assay, respectively.
Primary Valvular Myofibroblasts Deactivate in Response
to Substrate Modulus Reduction
The percent of activated myofibroblasts on different substrates
was examined by a-SMA immunocytochemistry (Fig. 2). Fig. 2A
shows representative staining of VICs for a-SMA on different gel
conditions. Myofibroblasts are defined as cells with a-SMA
organized into stress fibers (Fig. 2A, arrows). There were fewer
myofibroblasts and lower a-SMA fluorescence intensity on soft
and stiff-to-soft gels than on stiff gels (Fig. 2A). Some VICs on
softer substrates also showed diffuse a-SMA staining in the
cytoplasm (Fig. 2A, star); however, these cells were not classified as
myofibroblasts. The percentage of activated myofibroblasts was
quantified (Fig. 2B). Stiff gels activated ,55% of VICs to become
myofibroblasts. In contrast, only ,10% myofibroblasts were
observed when VICs were cultured on soft gels (Fig. 2B). From
day 3 to day 5, the fraction of myofibroblasts remained at a similar
level for both the stiff and the soft gels (Fig. 2B), and the total cell
number was not changed significantly on any substrate (Fig. S1).
GGCATTGATGAAGAACCCTTG AGGAAGAGATGGCCCGTCA doi:10.1371/journal.pone.0039969.t001
When E was reduced from 32 kPa to 7 kPa (stiff-to-soft gels,
Fig. 2B), the percentage of myofibroblasts decreased from
56.7+5.2% to 24.7+3.2% over the course of 2 days.
The Decrease in the Proportion of Valvular
Myofibroblasts is not due to Apoptosis
To investigate apoptosis as a potential mechanism of the
reduced proportion of myofibroblasts on stiff-to-soft gels,
expression of the early apoptotic marker phosphatidylserine on the outer
plasma membrane was examined. Annexin V, which binds
phosphatidylserine, and DAPI staining coupled with flow
cytometry was used to quantify the percentage of apoptotic cells. Live
cells at an early stage of apoptosis stain positive for Annexin V and
minimally for DAPI (Fig. 3A, red box). Fig. 3A shows a
representative scatter plot of apoptosis staining for VICs cultured
on stiff gels harvested on day 3. During 5 days of VIC culture, no
significant change in cell number was observed, and on average,
minimal dead cells were detected across all culture conditions
based on low DAPI staining (Fig. 3A as an example). On day 3,
similar percentages of apoptotic cells in VIC culture on stiff gels
(3.58+0.80%) and on soft gels (4.41+0.09%) were detected.
Interestingly on day 5, we observed a slight but significant increase
in apoptosis on stiff-to-soft gels (5.11+0.62%) as compared to stiff
gels (2.97+0.67%) (Fig. 3C). However, the level of apoptosis was
statistically the same between VICs on soft gels (5.22+1.90%) and
those on stiff-to-soft gels on day 5. As a positive control, VICs
cultured on plastic plates were treated with camptothecin, an
apoptosis-inducing reagent. The percentage of apoptotic cells
increased from 3.72% to 32.10% (Fig. 3C) with camptothecin
treatment. Additionally, we examined the morphology of the
Annexin V stained cells to confirm our observations. Cells that
stained positive for Annexin V expressed a rounded morphology,
whereas those that did not express Annexin V maintained a
spindle-shaped morphology (Fig. 3B).
Valvular Myofibroblasts Transform into a Less
Proliferative Fibroblast Phenotype on Softer Substrate
We hypothesized that, when the modulus of the
microenvironment decreases, valvular myofibroblasts will revert to a quiescent
fibroblast state. Expression of myofibroblast gene markers,
Figure 1. Cell fate in response to substrate modulus reduction. Valvular interstitial cells (VICs) were seeded on photodegradable
poly(ethylene glycol) (PD-PEG) gels on day 0. At day 3, a portion of the stiff gels was softened with light (365 nm at 10 mW/cm2). The fate of VICs on
continuously stiff, continuously soft and stiff-to-soft gels was subsequently examined on day 3 and/or day 5 based on immunocytochemistry,
apoptosis staining, proliferative assay and mRNA expression.
Figure 2. Reduced myofibroblast activation in response to lowering substrate modulus. VICs cultured on stiff, soft or stiff-to-soft gels as
shown in Fig.1 were fixed on day 3 and day 5, and stained for a-smooth muscle actin (a-SMA). (A) Representative staining of the myofibroblast
phenotype for VICs cultured on substrates with different stiffnesses on day 3 and day 5. Green: a-SMA. Blue: nuclei. Arrows: myofibroblasts
characterized by organized a-SMA+ stress fibers. Star: a cell stained with diffusive a-SMA. Scale bar: 100 mm. (B) Quantification of the percent of
myofibroblasts on the substrates based on staining in (A). The percentage of myofibroblasts on stiff-to-soft gels or soft gels was significantly lower
than that on stiff gels. * indicates p,0.05.
including a-SMA [20,44] and CTGF , and a fibroblast gene
marker, vimentin , were measured by qRT-PCR at 6 hours
after softening substrates on day 3. Fig. 4A shows that VICs on
stiff-to-soft gels expressed 49% less a-SMA mRNA and 83% less
CTGF mRNA than those cultured on stiff gels. VICs cultured
continuously on a soft substrate expressed a similarly low level of
a-SMA and CTGF as those cultured on stiff-to-soft gels. However,
the expression of vimentin mRNA was not changed significantly
for cells cultured on any substrates (Fig. 4A). Consistently, cells on
gels with different moduli have the characteristic staining for
vimentin, similar to those fibroblasts cultured on plastic plates
(Fig. 4D). Next, we examined the proliferative ability of VICs
grown on PD-PEG gels by quantifying the incorporation of EdU,
an analogue of thymidine, during DNA synthesis. One hour after
irradiating stiff gels to soften them on day 3, VICs were treated
with medium containing 5% FBS for all gel conditions. This was
done to induce measurable proliferation with EdU treatment for 3
hours on day 5 followed by flow cytometry. As shown in Fig. 4B,
the percent of EdU+ cells on stiff-to-soft gels and soft gels was
,30% less than those cultured on stiff gels. We also observed that
more cells stalled in the G2 or mitosis (M) phase of the cell cycle on
soft or stiff-to-soft gels than on stiff gels (Fig. S2). When VICs were
cultured on a plastic tissue culture plate and assayed for
proliferation using the same experimental conditions, there were
2 fold more EdU+ cells on the plastic tissue culture plate than on
the stiff gels (data not shown). Confocal fluorescence images of
samples prepared for flow cytometry confirmed that EdU staining
was present in the nuclei of cells (Fig. 4C).
Deactivated Fibroblasts on Stiff-to-soft Gels Maintain
Responsiveness to a Proliferative Stimulus or TGF-b1
To assess whether the de-activated myofibroblast phenotype
was reversible, the ability of deactivated cells to proliferate and
reactivate in response to chemical cues was examined. VICs cultured
on stiff-to-soft gels were treated for 24 hours with (i) fibroblast
growth factor-2 (FGF-2) and 15% FBS to induce proliferation, or
(ii) TGF-b1 to induce myofibroblast differentiation. As shown in
Fig. 5A, deactivated VICs responded to growth stimuli and
exhibited increased proliferation by ,4 fold. These cells also
exhibited up-regulated myofibroblast gene markers in response to
TGF-b1 (Fig. 5B). CTGF mRNA expression was increased by 3.6
fold (Fig. 5B), and ECM genes, such as collagen 1A1 (Col1A1) and
fibronectin 1 (FN1), were also significantly up-regulated, by 2.9
fold and 2.3 fold respectively, with TGF-b1 treatment (Fig. 5B).
Interestingly, a-SMA mRNA level was not significantly changed,
and the number of mature myofibroblasts with a-SMA stress fibers
was not increased on stiff-to-soft gels with TGF-b1 treatment,
(Fig. 5C). Similarly, cells cultured continuously on soft gels did not
activate to a-SMA+ myofibroblasts in response to TGF-b1 (Fig.
Here, we have begun to explore how substrate modulus serves
as a mechanical cue to regulate the fate of activated valvular
myofibroblasts. Our studies revealed striking phenotypic changes
from activated myofibroblasts to less proliferative, quiescent-like
fibroblasts when the culture substrates elastic modulus was
reduced from 32 kPa to 7 kPa. Apoptosis was minimally
associated with this de-differentiation process. Within 6 hours of
in situ substrate elasticity reduction, gene signatures of
myofibroblasts (a-SMA and CTGF) were down-regulated, while a fibroblast
gene (vimentin) stayed at a similar level, confirming myofibroblast
de-activation and suggesting potential signaling cascade
mechanisms. Mechanically-reprogrammed VICs on stiff-to-soft gels were
able to proliferate and re-initiate expression of myofibroblast genes
in response to chemical cues. Considering the extensive health
effects of tissue fibrosis, our study provides insight into possibly
reducing fibrosis through preventing myofibroblastic activation
and will assist with strategic in vitro tissue engineering to replace or
re-organize severely fibrotic or calcified tissue.
Human tissues have stiffnesses ranging from ,0.1 kPa to
,20 GPa [4,47]. To recapitulate native stiffness in vitro, it is critical
to culture cells on substrata with a physiologically relevant stiffness
for understanding their functions. Previous studies indicated that
normal valve fibrosa have a bulk elastic modulus from 0.88 kPa
. When healthy valves become stenotic, osteoid, which is
crosslinked collagen matrix as precursor to bone, has been
detected in the valve . While the stiffness of calcified valves
has not been measured to our knowledge, Engler et al. have shown
that osteoid matrix secreted by human mesenchymal stem cells has
E ,27+10 kPa . To mimic these microenvironments, we
synthesized hydrogels with either a normal mesenchyme-like
modulus (,7 kPa, soft gels) or a pathological osteoid-like modulus
Figure 5. Deactivated VICs on stiff-to-soft gels enter the cell cycle with proliferative stimulus and activate myofibroblast the gene
program in response to TGF-b1. VICs cultured on stiff-to-soft gels were treated with either proliferative media with 15% FBS and fibroblast
growth factor 2 (FGF-2) or fibrogenic chemokine (TGF-b1) on day 4 for 24 hours. (A) Proliferation was measured by EdU chase for 1 hour on day 5.
VICs treated with growth stimulus had ,4 fold more proliferating cells than those in control medium. (B) Myofibroblast gene markers, including
CTGF, collagen 1a1 (Col1a1) and fibronectin 1 (FN1), were significantly up-regulated in deactivated VICs treated with TGF-b1. The mRNA level of
aSMA was not changed significantly by TGF-b1 treatment. (C) Immunocytochemistry of a-SMA showed similar levels of myofibroblasts on stiff-to-soft
gels treated with or without TGF-b1. Green: a-SMA. Blue: nuclei. These results show that the de-activated fibroblasts have the potential to proliferate
and to activate fibrogenic associated genes in response to chemical cues, but a stiffer substratum is likely required for a-SMA stress fiber formation.
Scale bar: 100 mm. * indicates p,0.05.
(,32 kPa, stiff gels) to probe the cell fate of VICs. Kloxin et al.
previously demonstrated that valvular myofibroblast
differentiation was promoted on stiff gels, but inhibited on soft gels .
Similarly, fibroblasts isolated from different tissues, including lung
 and liver , have been shown to activate with E .15 kPa
and maintain the a-SMA negative fibroblast phenotype when the
microenvironment had E10 kPa. We observed consistent results
for VICs cultured on either stiff or soft gels (Fig. 2A). In addition,
when we irradiated stiff gels with UV light to reduce the substrate
modulus, valvular myofibroblasts were de-activated and lost
previously formed a-SMA stress fibers (Fig. 2A). Similar behavior
was observed for rat pulmonary fibroblasts (Fig. S4 and Text S1),
indicating a general role of substrate modulus in regulating the
differentiation of myofibroblasts.
In these experiments, VICs were cultured on a 2-dimensional
surface. While this is different from the 3-dimensional (3D) valve
tissue in which the endogenous cells reside, a 2D culture approach
has several advantages in understanding basic biological systems.
2D surfaces of functionalized biomaterials have served as unique
tools for understanding how cells collectively migrate and how
they differentiate in response to stiffness or shape [4,48,49].
Additionally, 2D culture enables one to readily monitor and image
cells over time using real time microscopy tracking tools and to
collect intracellular proteins or RNA more easily compared with
3D cultures. Currently, two types of scaffolds have been used for
the 3D culture of VICs, enzymatically degradable synthetic gels
 and natural matrices comprised of collagen  or hyaluronic
acid . Both of these materials have the complication that cells
are changing their mechano-environment by degrading the
matrix, so it becomes difficult to know the mechanical properties
of the matrix in the pericellular region. In contrast, if VICs are
encapsulated in non-degradable matrices with precisely defined
mechanical properties, they remain in a rounded and un-natural
morphology. Thus, it is difficult to de-couple the effect of modulus
and cell spreading on the cell fate within a 3D highly cross-linked
matrix. For these reasons, our studies focus on isolating and
understanding the effect of modulus on cell fate in 2D and believe
that this knowledge will be helpful in better understanding VIC
function in more complex, 3D matrices in future studies.
The fate of myofibroblasts after normal tissue repair has been an
ongoing debate. In granulation and scar tissue, massive apoptosis
has been observed . Induction of apoptosis has been associated
with matrix tension. For example, sudden release of collagen gels
from their anchor causes programmed cell death in human dermal
fibroblasts . Additionally, compliant substrata with E ,1 kPa
have been shown to induce significantly higher caspase 3 activity
than stiff substrata in lung fibroblasts . Consistently, we
observed a small but significant increase of apoptosis on stiff-to-soft
gels in comparison to stiff gels on day 5 (Fig. 3C), indicating that
some valvular myofibroblasts underwent apoptosis in response to
reduction in modulus. However, this level of apoptosis on
stiff-tosoft gels was similar to that observed for cells cultured on statically
soft substrates. Additionally, the average level of apoptosis in VICs
was ,5% on stiff-to-soft gels, which was too small to account for a
nearly 35% decrease in the myofibroblast population. Therefore,
most myofibroblasts did not undergo apoptosis in response to
substrate modulus reduction. These findings within the context of
the literature suggest that there may be different thresholds of
substrate modulus for regulating myofibroblast activation and
apoptosis. While E ,7 kPa is sufficient to de-activate valvular
myofibroblasts without inducing significant apoptosis, we speculate
that further reduction of E below or around 1 kPa would induce
most cells to undergo apoptosis. Additionally, softening the
substrate did not select for specific populations of cells, as the
cell number counted as described in the Text S1 was not changed
significantly from day 3 to day 5 across all gel moduli (Fig. S1), and
cells did not proliferate or undergo apoptosis significantly over
time. There were slightly fewer cells attached on soft gels at day 1
than stiff gels, so we observed fewer cells on soft gels than on stiff
gels from day 3 to day 5 (Fig. S1).
Since valvular myofibroblasts did not undergo significant
programmed cell death, we hypothesized that these cells
dedifferentiated into a dormant, or quiescent-like, fibroblast state.
Myofibroblasts are differentiated from fibroblasts through
increased a-SMA expression and its organization into stress fibers,
which is regulated by mechanical stress [20,54]. When cells adhere
to surfaces, traction forces are generated based on the resistance of
the matrix to cellular adhesion and movement . Cells on
substrates with higher moduli have been shown to exert higher
traction forces as measured by deformation of embedded
fluorescent beads . Mechanical strain generated on higher
substrate moduli activate intracellular signaling through p38
MAPK, Rho kinase and focal adhesion kinase to up-regulate
transcription of a-SMA and subsequently incorporation of a-SMA
into stress fibers [56,57]. Our results confirmed that a-SMA stress
fibers in VICs are dependent on substrate modulus. Based on
Fig. 2A, a-SMA stress fibers in VICs were disassembled after 2
days of lowering substrate elasticity. Myofibroblast activation on
substrates with varying moduli was independent of their time in
culture (Fig. 2B), indicating minimal influence from soluble factors
in the medium. On stiff-to-soft gels, we observed a higher
percentage of activated myofibroblasts (,25%) than that on soft
gels (,10%). This indicates that not every myofibroblast can be
efficiently de-activated by modulus reduction on stiff-to-soft gels.
Myofibroblasts not only differ from fibroblasts in the formation
of a-SMA stress fibers, but also have a distinct gene expression
profile [58,59,60,61]. Through previous research, gene signatures
to distinguish myofibroblasts from fibroblasts have been revealed,
such as a-SMA and CTGF. a-SMA is highly regulated at the
transcriptional level with multiple serum response elements and
CArG motifs in the promoter region of the gene . CTGF
expression is involved in the pathogenesis of fibrosis for various
tissues and is tightly associated with the myofibroblast phenotype
. Both genes are more highly expressed by myofibroblasts than
fibroblasts. As shown in Fig. 4A, these myofibroblast genes,
aSMA and CTGF, were significantly down-regulated with substrate
modulus reduction, and the expression level of these genes was
similar on stiff-to-soft gels compared with soft gels, suggesting
reversion of activated VICs to a fibroblast-like phenotype. The
reduction of these mRNAs was observed 6 hours after irradiating
stiff gels to make them soft, indicating that cells change their
molecular phenotype quickly in response to the mechanical cues.
Uniquely, in comparison to substrates fabricated with discrete
stiffness, changing substrate modulus in situ using PD-PEG gels
enabled us to track dynamic transcriptional changes during
myofibroblast de-activation and further reveal the molecular
mechanisms regulating this process. As CTGF has been shown to
be down-regulated through the YAP/TAZ pathway on soft
substrata , it is possible that this signaling is involved in the
early phase of myofibroblast deactivation on stiff-to-soft gels. Hinz
et al. have discovered that latent TGF-b1 from the ECM is
activated by contraction of a-SMA stress fibers in myofibroblasts
. Given that VICs rarely form a-SMA stress fibers on soft or
stiff-to-soft substrata, this result indicates a limited ability to
activate TGF-b1 from their microenvironment. This mechanism
may act at a later phase to reinforce the un-activated fibroblast
phenotype on stiff-to-soft gels.
Another functionally significant characteristic of myofibroblasts
is their high rate of proliferation. Lung fibroblasts cultured on
substrates with high modulus (E ,100 kPa) exhibited increased
myofibroblast activation and more proliferation . In fibrotic
lesions, a large number of myofibroblasts, generated through cell
proliferation, exacerbates the inflammatory response and collagen
deposition . In contrast, VICs residing in healthy compliant
valve matrices are mostly quiescent . We found that the
number of proliferating VICs was decreased by ,30% on
stiff-tosoft gels in comparison to stiff gels. This result indicates that
lowering substrate modulus inhibits cell cycle progression and
directs cells to a more quiescent-like phenotype. In particular, a
higher fraction of the cell population stalled in the G2 or mitosis
(M) phase of the cell cycle on soft or stiff-to-soft gels than on stiff
gels (Fig. S2), suggesting that mechanical tension conferred by
substrate modulus is an important regulator for the G2/M phase
of the cell cycle. From both Fig. 4A and 4B, reducing substrate
modulus not only down-regulated myofibroblast differentiation,
but also controlled the proliferative response of these cells.
The myofibroblast phenotype has been suggested to be plastic,
where myofibroblasts can be inhibited through different means
including TGF-b1 antagonist treatment  and low substrate
modulus . If the valvular myofibroblasts were reprogrammed
to quiescent fibroblasts on stiff-to-soft gels, then these cells should
maintain the fibroblast gene expression and the potential to
proliferate and differentiate into myofibroblasts. Vimentin is an
intermediate filament protein expressed in mesenchymal cells,
including fibroblasts . VICs expanded on plastic plates are all
positive for vimentin staining (Fig. 4D). This fibroblast property
was preserved when substrate modulus was decreased. Based on
Fig. 4A and 4D, mRNA and protein expression of vimentin was
present at a similar level in the de-activated cells on stiff-to-soft
gels, compared with cells on either stiff or soft gels, indicating that
the de-activated cells were still fibroblasts. Our results also suggest
that these deactivated cells are in a reversible state and respond to
FGF2 and increased serum by entering the cell cycle and respond
to TGF-b1 by expressing myofibroblast gene markers (Fig. 5A and
5B). Cell plasticity has become a blooming field of research with
the paradigm-shifting discovery of reprogramming adult somatic
fibroblasts into pluripotent stem cells by activating four
transcription factors [67,68]. A culture substratum with appropriate elastic
modulus and binding epitopes shows promise as a complementary
approach to reprogram the cells into a developmental stage of
interest and to dynamically dictate cell phenotype and fate in a
The timing and duration of matrix signaling events are
emerging as important factors in myofibroblastic differentiation
plasticity and ultimate cell fate. We observe myofibroblastic
deactivation of VICs with substrate modulus changes at short culture
times. In complementary studies, Balestrini et al. have observed
that lung myofibroblasts memorized the stiff or soft substrates
on which they were propagated for 3 weeks and stayed activated
or un-activated even after they had been transferred to substrates
with opposite stiffness . To compare, our cells have been
cultured 7 days on stiff plastic plates before seeding on soft
hydrogels for subsequent modulus tuning of 6 days in culture.
Further, we observed similar levels of activation for freshly isolated
VICs on stiff gels and stiff-to-soft gels as VICs at passage 3 (Fig.
S5). This indicates that our culture of VICs on plastic plates for
about a week did not change the cellular response to substrate
modulus. While there could be inherent differences between
valvular fibroblasts and lung fibroblasts, our results and Balestrini
et al. collectively indicate that as the myofibrobalsts mature over
time, there may be a time limit on their ability to revert back to
Differentiation of myofibroblasts is regulated by multiple
factors, including cell-cell contact , adhesive epitopes ,
TGF-b1 [18,21,71], and substrate elasticity [10,32]. However,
cells in vivo encounter numerous signals and integrate different
types and magnitudes of signals in choosing their fate. For
example, cell-cell contact prevents TGF-b1 from inducing
epithelial-to-myofibroblast differentiation . The ECM protein
fibronectin with ED-A domain is required for TGF-b1 mediated
myofibroblast differentiation . The Wells group has found
that portal fibroblasts need both a stiff substrate and TGF-b1 to
become myofibroblasts . Similarly, we observed that the
myofibroblastic differentiation of VICs is regulated by both
substrate stiffness and TGF-b1. When VICs were cultured on
stiff-to-soft substrata (E, from ,32 kPa to ,7 kPa), they can still
activate fibrogenic genes (CTGF, FN1 and Col1A1) in response
to TGF-b1 (Fig. 5B). However, these cells fail to develop a-SMA
stress fibers on the soft substrata even with TGF-b1 treatment
(Fig. 5B and Fig. S3). We speculate that a stiffer substrate is
required for mature valvular myofibroblast formation and the
deactivated VICs on stiff-to-soft gels were likely in a
protomyofibroblast state when treated with TGF-b1 . The
PDPEG gel system provides a powerful tool in studying cellular
responses to competing signals in vitro, for example reduced
substrate modulus while simultaneously increasing pro-fibrotic
cytokines. This may provide insight into how microenvironment
modulus in combination with other chemical or biological cues
directs cell fate.
In summary, valvular myofibroblasts were reprogrammed to
fibroblast-like cells when substrate modulus was reduced with light
in situ from E ,32 kPa to E ,7 kPa. This de-differentiation
process is characterized by low occurrences of apoptosis,
dissolution of a-SMA stress fibers, down-regulation of
differentiation associated genes (a-SMA and CTGF), and a decline in cell
proliferation. The de-activated fibroblasts on stiff-to-soft gels can
be re-activated by FGF2 and serum to enter the cell cycle and by
TGF-b1 to express fibrogenic genes, such as CTGF, Col1A1 and
FN1. Our data suggest that the fate of valvular myofibroblasts is
regulated by substrate elasticity independent of soluble factors.
This can potentially be applied to equivalent myofibroblasts from
other tissues and presents a promising approach in tempering
tissue fibrosis by de-differentiating activated myofibroblasts. Our
study also provides an example of dynamically reprogramming
differentiated cells through substrate modulus reduction and
shapes the conception of designing user-defined 2-dimensional,
or even 3-dimensional, platforms for controlling the
developmental stage of cells.
Figure S1 Cell number was not changed after gel
irradiation from day 3 to day 5 in culture. Cell number
was counted per field of view for both day 3 and day 5 samples.
Over time, no significant change in cell number was observed for
cells cultured on any of the gel moduli. There were slightly fewer
cells on soft gels than on stiff gels.
Figure S2 More cells reside at the G2/M phase of cell
cycle on softer substrates. VICs cultured on stiff, soft or
stiffto-soft gels were chased with EdU for 3 hours on day 5. Cell cycle
profile was quantified by simultaneously labeling proliferative cells
with EdU-Alexa Fluor 488 and labeling DNA content with DAPI.
Fold change in percent of cells residing in the G2/M phase of the
cell cycle was normalized to the stiff condition. There are 1.8 and
2.1 fold more cells in G2/M phase of the cell cycle on soft and
stiffto-soft gels respectively than those on stiff gels, indicating that
substrate modulus is a regulator for cell mitosis. * indicates
Figure S3 VICs cultured on soft gels did not respond to
TGF-b1 with more a-SMA stress fiber formation. VICs
cultured on soft gels were treated with TGF-b1 on day 4 for 24
hours to induce myofibroblast differentiation. a-SMA organization
was examined by immunocytochemistry. Green: a-SMA. Blue:
nuclei. Few myofibroblasts with a-SMA stress fibers were observed
on soft gels, and TGF-b1 did not induce further cell activation.
Scale bar: 100 mm.
Figure S4 Rat pulmonary myofibroblasts de-activated
with reduction in substrate modulus. Rat pulmonary
myofibroblasts were stained for a-smooth muscle actin (a-SMA)
after culture on stiff, soft or stiff-to-soft gels until day 5. (A)
Representative staining of a-SMA to denote the myofibroblast
phenotype. These cells lost a-SMA stress fibers on softer
substrates. Green: a-SMA. Blue: nuclei. Scale bar: 100 mm. (B)
Quantification of the percent of myofibroblasts on the substrates
based on staining in (A). The percentage of myofibroblasts on
stiffto-soft gels or soft gels was significantly lower than that on stiff gels.
This is consistent with the observation on valvular fibroblasts in
Figure 2, indicating a general role of substrate modulus in
regulating the differentiation of myofibroblasts. * indicates
Figure S5 Freshly isolated VICs (P0 VICs) are activated
on stiff gels and de-activated with reduction in substrate
modulus. Freshly isolated VICs that have not been sub-cultured
on plastic plates were seeded on stiff gels. Cell activation was
examined on day 5 by a-SMA immunocytochemistry. Green:
aSMA. Blue: nuclei. A similar percentage of myofibroblasts was
observed for P0 VICs on stiff gels compared with P3 VICs that
have been expanded on plastic plate. After gel softening with light
(stiff-to-soft gel), activated P0 VICs were de-activated with
significant reduction in the number of myofibroblasts. Scale bar:
The authors would like to thank Mark Tibbitt for generously sharing
materials, Kelly Pollock for assistance with some experiments, and Dr.
Chien-Chi Lin, Dr. Alex Aimetti, and Dr. Daniel Alge for discussions
regarding peptide synthesis.
Conceived and designed the experiments: HW AMK SMK LAL KSA.
Performed the experiments: HW SMH AMK. Analyzed the data: HW
SMH AMK LAL KSA. Contributed reagents/materials/analysis tools:
HW SMH AMK. Wrote the paper: HW SMH AMK LAL KSA.
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