Received: 20 March 2021 Revised: 10 May 2021 Accepted: 17 May 2021

DOI: 10.1111/wrr.12952

PERSPECTIVE ARTICLE

Myofibroblast fate plasticity in tissue repair and fibrosis:

Deactivation, apoptosis, senescence and reprogramming

Wolfgang Merkt MD1,2 | Yan Zhou MSc1,3 | Hongwei Han PhD1 |

David Lagares PhD1

1
Fibrosis Research Center, Center for
Immunology and Inflammatory Diseases, Abstract
Division of Pulmonary and Critical Care In response to tissue injury, fibroblasts differentiate into professional repair cells
Medicine, Massachusetts General Hospital,
Harvard Medical School, Boston, called myofibroblasts, which orchestrate many aspects of the normal tissue
Massachusetts repair programme including synthesis, deposition and contraction of extracellular
2
Department of Hematology, Oncology and
matrix proteins, leading to wound closure. Successful tissue repair responses
Rheumatology, Internal Medicine V, University
Hospital of Heidelberg, Heidelberg, Germany involve termination of myofibroblast activities in order to prevent pathologic
3
Department of Physiology, Xiangya Medical fibrotic scarring. Here, we discuss the cellular and molecular mechanisms
School, Central South University, Changsha,
China limiting myofibroblast activities during physiological tissue repair, including
myofibroblast deactivation, apoptosis, reprogramming and immune clearance of
Correspondence
David Lagares, Fibrosis Research Center, senescent myofibroblasts. In addition, we summarize pathological mechanisms
Center for Immunology and Inflammatory leading to myofibroblast persistence and survival, a hallmark of fibrotic diseases.
Diseases, Division of Pulmonary and Critical
Care Medicine, Massachusetts General Finally, we discuss emerging anti-fibrotic therapies aimed at targeting
Hospital, Harvard Medical School, Boston, MA, myofibroblast fate such as senolytics, gene therapy, cellular immunotherapy and
USA.
Email: dlagares@mgh.harvard.edu CAR-T cells.

KEYWORDS
apoptosis, cellular senescence, fate, fibrosis, myofibroblast, plasticity, reprogramming, tissue
repair

1 | I N T RO DU CT I O N : M YO F I BR O B LA S T pro-repair responses during normal wound healing, ultimately promot-

F A T E S U P O N CO M P L E T I O N OF TI S S U E
REPAIR
During homeostasis, scattered tissue-resident fibroblasts actively
maintain the fitness of connective tissues through processes of syn-
thesis, degradation and remodelling of extracellular matrix (ECM) pro-
teins. These processes are controlled through a series of feedback
mechanisms involving both mechanical and biochemical cues that
instruct fibroblast to produce or degrade the ECM. Upon tissue injury,
ECM breakdown, haemostasis and inflammation unleash fibroblast
ing fibroblast proliferation and differentiation into professional repair
cells named myofibroblasts.1 Activated myofibroblasts are character-
ized by increased synthesis of ECM proteins including type I collagen
and the neo-expression of alpha smooth muscle actin (α-SMA), a pro-
tein that confers these cells a hyper-contractile phenotype.2 These
two cellular activities are essential at promoting wound closure and
preventing blood loss and infections.3 During the late phase of tissue
repair, myofibroblasts orchestrate a series of cellular responses aimed
at fully regenerating or partially restoring parenchyma function.1 Suc-
cessful tissue repair responses involve termination of myofibroblast

Abbreviations: α-SMA, α smooth muscle actin; AAV, adenoassociated virus; BET, bromodomain and extra-terminal; c-Abl, cellular Abelson; CKD, chronic kidney disease; DNMT3A, DNA
methyltransferase 3A; DRs, death receptors; ECM, Extracellular Matrix; ET, endothelin; FAP, fibroblast activation protein; FasL, Fas ligand; FGF1, fibroblast growth factor 1; H3K9, histone 3
lysine 9; H3K9Me3, histone 3 lysine 9 trimethylation; HDAC4, histone deacetylase 4; HGF, hepatic growth factor; IFNγ, Interferon gamma; IL-1β, interleukin-1β; IPF, idiopathic pulmonary
fibrosis; kPa, kilopascal; MeCP2, methyl cap binding protein 2; miRs, microRNAs; MOMP, mitochondrial outer membrane permeabilization; MRTF-A/B, myocardin-related transcription factor-A/
B; NK, natural killer; PDGF, Platelet-derived growth factor; PPARγ, peroxisome proliferator-activated receptor gamma; ROS, reactive oxygen species; SAHA, suberoylanilide hydroxamic acid;
SASP, senescence-associated secretory phenotype; SSc, systemic sclerosis; TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ, transforming growth factor; TRAIL, TNF-related
apoptosis inducing ligand; uPAR, urokinase-type plasminogen activator receptor; YAP, yes-associated protein..

Wound Rep Reg. 2021;1–14. wileyonlinelibrary.com/journal/wrr © 2021 The Wound Healing Society. 1
2 MERKT ET AL.

activities in order to prevent maladaptive repair responses charac-
terized by pathologic fibrotic scarring.4,5 Given the importance of
myofibroblasts in balancing regeneration and fibrosis, a growing
body of research is currently addressing the mechanisms regulating
myofibroblast activities during the resolution phase of wound
repair and fibrosis.6 In this review, we will summarize the state of
the art regarding cellular and molecular mechanisms limiting
myofibroblast activities during normal tissue repair, including
myofibroblast deactivation, apoptosis, reprogramming and immune
clearance of senescent myofibroblasts (Figure 1). We will also dis-
cuss escape mechanisms leading to myofibroblast persistence and
the development of fibrosis. Finally, we will discuss emerging thera-
peutic strategies aimed at reducing myofibroblast activities to treat
fibrotic diseases.
2 | MYOFIBROBLAST APOPTOSIS DURING
W O U ND H E A L I N G
Successful tissue repair requires timely resolution of the inflammatory
and fibroproliferative responses, ultimately leading to reduction in tis-
sue cellularity. Early reports published in the 1990s proposed that
myofibroblast apoptosis is a major mechanism responsible for
myofibroblast clearance in skin repair, as evidenced by morphological
changes and accumulation of DNA double-strand brakes in α-SMA+
cells.4,5,7 More recent studies have validated this concept in animal
models of tissue injury, further supporting the notion that
programmed cell death plays a central role in wound repair by pro-
moting controlled elimination of myofibroblasts during the resolution
of the healing programme.7–10 Whereas the mechanisms that pro-
mote fibroblast proliferation and activation into myofibroblasts have
been extensively studied and reviewed,11–13 the molecular underpin-
nings that control myofibroblast apoptosis remain poorly understood.
We have recently shown that differentiation of fibroblasts into acti-
vated myofibroblasts associates with augmented predisposition to
apoptosis in these cells,6,14,15 increasing the readiness of myo-
fibroblasts to activate programmed cell death. Thus, activated
myofibroblasts are “primed for death,” a mechanism that enables
rapid elimination of these cells upon completion of the tissue repair
programme. On the molecular level, this process is controlled by
dynamic changes in the “mitochondrial apoptotic priming” of myo-
fibroblasts across the different phases of the tissue repair programme.
2.1 | Mitochondrial priming controls myofibroblast
predisposition to apoptosis
Apoptotic priming is dynamically regulated by a molecular rheostat
involving the BCL-2 family of proteins, which compromises both pro-
apoptotic and anti-apoptotic members that control the mitochondrial
or intrinsic pathway of apoptosis.14,16 This pathway is triggered by
intracellular pro-apoptotic stimuli such as oxidative stress, DNA dam-
age or oncogene activation, ultimately inducing mitochondrial outer
membrane permeabilization (MOMP), cytochrome C release and
activation of executioner caspases. Strong inducers of mitochondrial
apoptosis such as the BCL-2 family member BIM are highly expressed
in activated myofibroblasts compared to resting fibroblasts.6,17 The
mechanisms increasing levels of BIM and other pro-apoptotic mole-
cules in myofibroblasts are poorly understood, although increased
production of mitochondrial reactive oxygen species (ROS) has been
associated with mitochondrial damage and apoptotic signalling in
these cells.15,18,19 Platelet-derived growth factor (PDGF) signalling has
been similarly shown to prime cancer-associated myofibroblasts for
apoptosis.20 Despite being tonically primed for death, myofibroblasts
evade apoptosis by upregulating pro-survival molecules that directly
inhibit pro-death signalling. We and others have shown that pro-
apoptotic activities of BIM are selectively blocked by the anti-
apoptotic BCL-2 family protein BCL-XL, which directly binds to and
sequesters BIM in myofibroblasts' mitochondria.17 Up-regulation of
pro-survival BCL-2 proteins including BCL-XL is induced by growth
factors, cytokines and ECM cues. For instance, the cytokine trans-
forming growth factor beta (TGFβ), the vasoactive peptide
endothelin-1 (ET-1) and PDGF have been shown to promote an
apoptosis-resistant phenotype in myofibroblasts by activating pro-

F I G U R E 1 Myofibroblast fate upon termination of the tissue repair programme. Following tissue injury, extracellular matrix (ECM) breakdown
and inflammatory cytokines such as TGFb promote fibroblast differentiation into activated myofibroblasts, which are characterized by increased
synthesis of ECM and the neo-expression of alpha smooth muscle actin (α-SMA), a protein that confers these cells a hyper-contractile phenotype.
These two cellular activities are essential for promoting wound closure and reepithelialization. Successful tissue repair responses involve
termination of myofibroblast activities via alternative mechanisms including deactivation, apoptosis, immune clearance of senescent
myofibroblasts by T and natural killer (NK) cells and reprograming
MERKT ET AL.
survival signalling pathways such as FAK-PI3K-AKT and ABL.21–27
Similarly, matrix cues including ECM stiffness promote myofibroblast
evasion of apoptosis by activating survival mechanotransduction path-
ways such as integrin-FAK-ROCK-YAP/TAZ signalling.6,12 We and
others have recently shown that mechanical activation of YAP/TAZ
directly regulates expression of the anti-apoptotic protein BCL-XL in
17
primed for death myofibroblasts, ensuring their survival. In addition,
TGFβ also blocks the extrinsic pathway of apoptosis by inhibiting Fas–
Fas ligand (FasL) signalling, a mechanism dependent on blockade of
ceramide generation by sphingomyelinase.28 Taken together,
increased mitochondrial priming in activated myofibroblasts predis-
poses these cells towards apoptosis. Primed for death myofibroblasts
are highly dependent on the activation of survival pathways and
expression of anti-apoptotic proteins of the BCL-2 family members. In
the following section, we discuss cellular and molecular mechanisms
that selectively inhibit these pro-survival mechanisms during the reso-
lution phase of wound healing, ultimately leading to myofibroblast
clearance via apoptosis.
2.2 | Mechanisms inducing apoptosis in “primed
for death” myofibroblasts
2.2.1 | Matrix softening and degradation
Upon injury, the wound is filled with a provisional matrix composed of
fibrin, fibronectin and collagens, which matures into scar tissue during
the remodelling phase of the healing programme. As the tissue repair
programme progresses, the mechanical properties of the granulation
tissue change, in particular by increasing its stiffness and resistance to
deformation. These changes in matrix stiffness have been shown
to directly modulate the biology of fibroblasts during wound repair by
promoting their mechano-activation into myofibroblasts.2,12 Previous
reports have shown that dermal stiffness increases from 1 kPa in
healthy skin tissues up to 20 kPa in 7 days post-wounding and 50 kPa
in mature granulation tissue 14 days post-injury.29 At this point,
wound closure is almost completed due to full activation of hyper-
contractile myofibroblasts.30 Of note, massive degradation of the
ECM is observed once the wound is closed, which associates with
myofibroblast apoptosis.31 Several reports have shown that the
degree of myofibroblasts apoptosis appears to correlate with severity
of ECM stiffness and tension drop. For instance, 15% of activated
myofibroblasts undergo apoptosis following release of anchored colla-
gen gels in vitro from high to low tension,31 whereas 40% of activated
myofibroblasts activate apoptosis when tension drops from very high
to zero.32,33 In vivo, releasing the edges of a previously splinted exci-
sional wound, which leads to tension drop from high to medium, cau-
ses apoptosis in 10% of the cells, albeit the percentage of apoptotic
34
myofibroblasts was not calculated. Mechanistically, ECM degrada-
tion and softening have been shown to promote myofibroblast apo-
ptosis by inhibiting mechanically activated FAK signalling and
reducing levels of pro-survival BCL-XL in primed for the myo-
fibroblasts in vitro and in vivo models.31,34 More recently, de-
3
stiffening of the ECM has been shown to activate the pro-apoptotic
Mdm4-p53 pathway and promote myofibroblast apoptosis.35 The cel-
lular mechanisms promoting ECM degradation and softening remain
poorly understood but may involve reprogramming of injury-
associated macrophages that switch their phenotype from pro-repair
to matrix-degrading phenotype during the resolution of tissue
repair.36–38 Accordingly, molecules involved in the degradation and
uptake of collagen such as uPARP and Mfge8 have shown to promote
ECM degradation and prevent tissue fibrosis in mouse models.39,40
2.2.2 | Growth factor withdrawal
During tissue repair, primed for death myofibroblasts become
addicted to cytokines and growth factors that activate pro-survival
signalling pathways, ensuring their survival. Levels of TGFβ, ET-1
and PDGF, which are released by inflammatory cells and platelets
during the inflammatory phase of the healing programme, are
reduced during the resolution phase,6,41,42 putting forward the
hypothesis that withdrawal of survival signals upon completion of
the tissue repair programme leads to myofibroblast apoptosis. 27,43
While this hypothesis has not been demonstrated in vivo, several
reports have shown that starvation of cultured myofibroblasts
associates with reduced FAK-PI3K-AKT signalling and apoptosis.
Together, growth factor “dependence” may control myofibroblast
clearance during wound healing.
2.2.3 | Pro-apoptotic factors
Another plausible mechanism involves factors that specifically
inhibit survival signalling in myofibroblasts. For instance, fibroblast
growth factor 1 (FGF1), interleukin-1β (IL-1β) and hepatic growth
factor (HGF) have been shown to deactivate the FAK-PI3K-AKT
pathway and induce apoptosis of myofibroblasts from skin granula-
tion tissue, but not in healthy control fibroblasts. Similarly, the
prostaglandin PGE2 blocks AKT signalling and increases the sensi-
tivity of myofibroblasts to Fas-induced apoptosis via the extrinsic
pathway. In this regard, activation of the extrinsic pathway of apo-
ptosis by FasL and TNF-related apoptosis-inducing ligand (TRAIL)
has been similarly reported to promote myofibroblast apoptosis
in vivo.8,44,45 Accordingly, loss of pro-apoptotic Fas signalling in
myofibroblasts impairs homeostatic tissue repair resolution and
promotes pulmonary fibrosis in mice.8 In humans, bronchoalveolar
lavage fluid from patients in the resolution phase of lung repair,
but not from patients with active lung injury, induces myofibroblast
apoptosis, further suggesting that myofibroblasts are actively and
timely eliminated via apoptosis.46
Together, the termination of myofibroblasts' pro-repair activities
emerges as a tightly regulated process in which both intrinsic apopto-
tic priming as well as spatiotemporal regulation of mechanical and
biochemical factors fine-tune the activation of myofibroblast apopto-
sis during the resolution phase of the repair programme.
4 MERKT ET AL.
2.2.4 | Myofibroblast senescence and immune
clearance
In addition to self-clearance by apoptosis, acquisition of a senescent
phenotype has been shown to reduce myofibroblast activities and
promote their clearance by cytotoxic immune cells. The role of cellular
senescence in normal tissue repair remains poorly understood,
although it appears to be a mechanism to arrest myofibroblasts and to
limit their pro-repair activities47,48 during the resolution phase of the
tissue repair programme in the liver,49 lungs,50,51 skin,52,53 kidney,54
oral submucosa55 and pancreas.56 Mechanistically, the matricellular
protein CCN1 has been shown to promote myofibroblast senescence
in vivo in the context of liver and skin injury, promoting liver regenera-
tion and limiting fibrosis in mouse models.52,57 In addition, the
senescent phenotype appears to target myofibroblast for recognition
and clearance by immune cells via not yet fully understood mechanisms.
A growing body of evidence has reported that the senescence-
associated secretory phenotype (SASP) of senescent myofibroblasts
includes pro-inflammatory chemokines and cytokines that lead to the
recruitment and activation of cytotoxic immune cells. Thus, senescent
myofibroblasts appear to be programmed to be eliminated by the
immune system. This concept has been demonstrated in multiple studies
confirming the role of immune cells in eliminating senescent myo-
fibroblasts during tissue repair, and that impaired immune clearance of
49,58–60
senescent cells leads to overwhelming fibrosis. Several immune
cell types have shown the capacity to eliminate senescent cells in vitro
and in vivo, including innate immune cells such as natural killer (NK) cells,
61 59,62,63
macrophages and neutrophils, but also T cells. In the context of
wound healing, NK cells expressing TRAIL have been involved in the res-
olution of the tissue repair programme by promoting apoptosis of senes-
44,49
cent myofibroblasts and other cell types. Similarly, cytotoxic CD4+
T cells expressing pro-apoptotic FasL have been shown to eliminate
myofibroblasts in the context of lung injury.45,64 In addition, senescent
dermal myofibroblasts can be cleared out by CD8+ T cells and NK cells
65
ex vivo. Mechanistically, the acquisition of a senescent phenotype
leads to up-regulation of stress ligands in myofibroblasts and other cell
types, which can be recognized by specific receptors expressed in cyto-
toxic lymphocytes.66 Stress signals upregulated in senescent myo-
fibroblasts, but not healthy cells, selectively target them for apoptosis by
67–69
immune cells. For instance, up-regulation of MIC-A/B and ULBPs in
senescent myofibroblasts have been shown to activate both NK cells
and a subset of CD8+ T cells via the NKG2D receptor.44,49,65,70 NKG2D
activation leads to NK and CD8+ T activation and the release of cyto-
toxic granules containing perforin and granzymes that induce
myofibroblast apoptosis. Accordingly, both NKG2D- and perforin-
deficient mice show accumulation of senescent liver myofibroblasts and
increased fibrosis in mouse models of liver injury.64,70 More recent
reports have shown that senescent myofibroblasts may escape immune
clearance in pathological settings by co-expressing inhibitory ligands or
decoy receptors that block immune cell function. For instance, up-
64
regulation of the decoy death receptor Dcr2 or the inhibitory molecule
HLA-E65 in senescent myofibroblasts have been associated with resis-
tance to immune cell killing. In addition, leukocytes have been involved
in myofibroblast apoptosis by secreting interferon gamma (IFNγ), which
facilitates myofibroblast apoptosis by NK cells in the context of liver
injury.71–73 Finally, myofibroblast apoptotic bodies are subsequently
phagocytosed by macrophages via efferocytosis, which associates with
anti-inflammatory and wound healing resolution responses.74,75
3 | MYOFIBROBLAST DEACTIVATION
A ND R E P R O G RA M M I N G D U R I N G W O U N D
HE AL IN G
Genetic lineage-tracing studies in mice have recently demonstrated
that myofibroblasts undergo dynamic phenotypic transitions through
different activation states in vivo.10,76–78 These studies show that myo-
fibroblasts get deactivated upon termination of the normal tissue repair
in vivo, a phenotype characterized by reduction in α-SMA levels, con-
tractile activities and ECM synthesis.79,80 The mechanisms promoting
myofibroblast deactivation remain poorly defined, although several
reports have demonstrated a major role for peroxisome proliferator-
activated receptor gamma (PPARγ) at promoting myofibroblast deacti-
vation by blocking the TGF-β pathway. In the liver,
50% of total
myofibroblasts get deactivated during the resolution of murine liver
fibrosis via up-regulation of PPARγ, albeit these reprogrammed cells
acquire a phenotype distinct from quiescent hepatic stellate cells.10,76
In the lungs, lineage-tracing studies show that myofibroblasts get simi-
larly deactivated by turning into lipofibroblast-like cells during lung
fibrosis resolution, a process associated with PPARγ up-regulation.81 In
the heart, periostin-traced myofibroblasts get deactivated to resting
fibroblasts upon injury resolution in myocardial infarction models, a
mechanism associated with re-gaining expression of the transcription
factor Tcf21.77 Accordingly, overexpression of Tcf21 deactivates
hepatic myofibroblasts in vitro and protects mice from CCl4-induced
liver fibrosis and diet-induced steatohepatitis.82 In the skin, lineage-
tracing studies have also demonstrated that dermal myofibroblasts get
deactivated via reprogramming to adipocytes through activation of the
BMP-ZFP423 pathway upon termination of the wound healing pro-
gramme.78 Further studies have shown that the transcription factor
MyoD acts as a critical switch controlling myofibroblasts activation and
deactivation in vivo by modulating the TGF-β pathway.83 Together,
these studies elegantly demonstrate the capacity of wound myo-
fibroblasts to get deactivated in vivo via reprogramming into resting
cells or converting to another cell type. The molecular mediators
controlling these processes remain poorly understood, although
soluble factors such as PGE2, BMP4, PDGFB, HRAS, EGR4 and FGF-1
have been shown to block the TGF-β pathway in cultured myo-
fibroblasts.80,84,85 In addition, matrix softening has been also reported
to induce myofibroblast deactivation. Several studies have shown that
stiffness-activated myofibroblasts get deactivated when plated on soft
matrices.86–89 Similarly, desplinting and releasing the wound edge has
been shown to induce myofibroblast deactivation in vivo.29,90 In addition
to these cell-intrinsic mechanisms, bystander cells including immune cells
and adipocytes have been shown to promote myofibroblast deactivation.
For instance, myofibroblasts in aortic valve stenosis get deactivated after
MERKT ET AL. 5

transcatheter aortic valve replacement and such deactivation is triggered
by IL-1β and TNF-α released from M1 macrophages in post-surgery
serum.91 Similarly, adipocyte-conditioned medium significantly decreases
the expression of α-SMA and ECM components in skin myofibroblasts
through BMP4 mediated activation of PPARγ signalling.85 More recent
studies have shown that activated myofibroblast can get directly rep-
rogrammed into adipocytes via BMP signalling during wound healing,
contributing to tissue repair and regeneration.78
4 | M Y O F I B RO B L A S T P E RS I S T E N C E L E A D S
TO TISS UE FIBROSI S
Human fibrotic diseases including idiopathic pulmonary fibrosis (IPF),
systemic sclerosis (SSc, scleroderma), liver cirrhosis, cardiac fibrosis
and chronic kidney disease (CKD) are associated with high morbidity
and mortality.92 Persistent myofibroblast activation is a hallmark of
these fibrotic diseases and associates with progressive tissue scarring
6,41
and organ failure. The molecular pathways that promote
myofibroblast persistence beyond the resolution phase of tissue repair
remain poorly understood, although recent studies have identified
mechanisms that keep myofibroblasts in an activated state by promot-
ing evasion of apoptosis, cellular senescence as well as metabolic and
epigenetic reprogramming (Figure 2).
4.1 | Evasion of apoptosis: Addiction to TGF-β1
and mechanical signalling
Persistent growth factor signalling has been shown to promote
myofibroblast evasion of apoptosis by activating pro-survival
pathways. The pro-fibrotic cytokine TGF-β promotes an apoptosis-
resistant phenotype in myofibroblasts by activating the pro-survival
FAK-PI3K-AKT and p38 MAPK pathways.21,25,93 In addition, TGF-β
blocks mitochondrial apoptosis by modulating levels of the BCL-2
family of proteins. For instance, TGF-β inhibits the pro-apoptotic sen-
sitizer protein BAD, upregulates pro-survival proteins such as BCL-2
and BCL-XL and decreases BAX to BCL-2 ratio via cellular Abelson (c-
Abl) signalling.27,94–97 In addition to blocking the intrinsic apoptotic
pathway, TGF-β has been also shown to inhibit FasL-mediated activa-
tion of the extrinsic apoptotic pathways in scleroderma myo-
fibroblasts by blocking ceramide production.94,98 Restoration of Fas
signalling upon treatment with PGE2, a lipid mediator which is found
deficient in IPF, increases Fas receptor expression and sensitizes myo-
fibroblasts to apoptosis by inhibiting AKT signalling.99,100
In addition to TGF-β signalling, increased matrix stiffness in fibrotic
conditions has been shown to block apoptotic signalling in myofibroblasts
via activation of integrin-mediated mechanotransduction pathways.6,17 In
fibrotic diseases, myofibroblasts promote progressive matrix stiffening due
to excessive deposition and crosslinking of ECM proteins, locking them-
selves in a mechanical positive feedback loop. This vicious cycle amplifies
fibrotic responses by enhancing myofibroblast activation and persis-
tence.101 We and others have shown that stiff matrices directly activate
FAK, a pro-survival mechanosensitive protein activated by integrin signal-
ling.22,102 FAK mediates Rho/Rho-associated protein kinase (ROCK) acti-
vation and increases acto-myosin contractility, which leads to activation of
transcriptional coactivators yes-associated protein (YAP)/transcriptional
coactivator with PDZ-binding motif (TAZ) and myocardin-related tran-
scription factor-A (MRTF-A)/MRTF-B, widely recognized as major
mechanotransducers.103,104 We and others have shown that stiffness-
activated YAP/TAZ promotes up-regulation of BCL-XL in myofibroblasts
and that FAK–YAP–TAZ–BCL–XL pathway is hyperactivated in dermal

F I G U R E 2 Targeting myofibroblast fate for the treatment of established fibrosis. Myofibroblast persistence is a hallmark of fibrotic diseases
characterized by progressive tissue scarring. The molecular mechanisms that promote myofibroblast persistence include pathological growth
factor signalling (e.g. TGF-β pathway), mechanical signalling induced by matrix stiffness, cellular senescence and the senescence-associated
secretory phenotype (SASP: TGF-β, IL6, IL8) as well as metabolic and epigenetic reprogramming. This knowledge has paved the way for new
therapeutic strategies aiming at reversing fibrosis by targeting myofibroblast fate and promoting myofibroblast apoptosis and immune clearance,
deactivation and reprogramming. These novel treatments include senolytics, gene therapy, cellular immunotherapy and CAR-T cells, and may not
only reverse fibrosis but also regenerate fibrotic tissues
6 MERKT ET AL.
myofibroblasts from patients with scleroderma.15,105 Similarly, the
mechanosensitive pathway FAK–ROCK–MRTF–BCL-2 promotes sur-
106
vival and persistence of IPF fibroblasts. In addition, mechanical sig-
nalling inhibits the extrinsic apoptotic pathways by blocking acid
sphingomyelinase and reducing Fas expression in myofibroblasts.28,94,98
4.2 | Metabolic reprogramming
Myofibroblasts undergo metabolic reprogramming in order to sustain
the high-energy demand associated with their ECM hyper-synthetic
and contractile phenotype. A growing body of evidence suggests that
metabolic reprogramming, characterized by increased aerobic glycoly-
sis, mitochondrial oxygen consumption and ROS production, pro-
19,107–112
motes myofibroblast persistence and survival. This notion
that myofibroblast bioenergetics are linked to their persistent activa-
tion state has been demonstrated by studies showing that inhibition
of glycolysis in myofibroblasts leads to rapid down-regulation of
α-SMA. 19,113
In addition, metabolic reprogramming and
NOX4-mediated ROS production in myofibroblasts have been associ-
ated with mitochondrial dysfunction characterized by fragmented
mitochondria and impaired activation of mitochondrial apoptosis in
19,107,111
these cells. Accordingly, pharmacological activation of the
energy-sensor with metformin promotes mitochondrial biogenesis,
restores myofibroblast sensitivity to apoptosis and reverses
established lung fibrosis in mouse models.18 Moreover, metabolic
reprogramming associates with enhanced glutamine metabolism in
IPF-derived fibroblasts, leading to increased expression of the apopto-
sis inhibitors XIAP and surviving.114 More recently, increased
glutaminolysis and glycolysis have been linked to Ca2+-dependent
metabolic reprogramming and myofibroblast persistence via epige-
115
netic mechanisms.
4.3 | Epigenetic mechanisms
Epigenetic modifications including DNA methylation, histone modifi-
cation and regulation by non-coding RNA such as microRNAs (miRs),
have been recently associated with myofibroblast activation and per-
sistence.111,116 Difference in DNA methylation has been described in
healthy control fibroblasts compared to fibroblasts isolated from
patients with IPF, CKD and scleroderma.117–122 Mechanistically, TGF-
β signalling induces expression of DNA methyltransferase 3A
(DNMT3A) and DNMT1 in myofibroblasts, leading to increased DNA
123
methylation and modulation of pro-fibrotic gene expression. In this
regard, DNA hypermethylation silences endogenous anti-fibrotic
genes such as PPARγ, a mechanism controlled by methyl cap binding
protein 2 (MeCP2), a transcriptional repressor that binds to methyl-
ated DNA.124 Accordingly, MeCP2-deficient mice showed protection
from fibrosis in the lungs and liver in mouse models.124 Similarly,
hypermethylation of anti-fibrotic genes such as THY-1 and FLI1 leads
to down-regulation of these molecules and increased type I collagen
synthesis in fibrotic fibroblasts isolated from patients with IPF and
scleroderma.118,125 Moreover, hypermethylation and subsequent
silencing of the proapoptotic protein P14ARF promotes an apoptosis-
resistant phenotype in IPF fibroblasts.126 Noteworthy, selective meth-
ylation of three CpG islands in ACTA2 gene (the encoding gene of
α-SMA) has been also shown to reduce α-SMA expression in myo-
fibroblasts.127 On the contrary, DNA hypomethylation and conse-
quent transcription up-regulation of pro-fibrotic genes including
COLIVA1, TGFBR3, SMAD3, SMAD6 has been described in kidney
fibrosis.121 Acetylation and methylation of histone proteins have been
similarly associated with changes in chromatin structure and increased
gene transcription in myofibroblasts. For instance, hyperacetylation of
histone H4 by P300 has been shown to be required for TGF-
β-induced type I collagen expression in myofibroblasts.128 Similarly,
histone 3 lysine 9 (H3K9) hypermethylation of the PPARGC1A gene
promoter suppresses expression of PGC1α via the CBX5/G9a path-
way and promotes sustained activation of IPF fibroblasts.129 More-
over, decreased histone acetylation and increased histone 3 lysine
9 trimethylation (H3K9Me3) of the Fas promoter reduces Fas expres-
sion and promotes resistance to Fas-mediated apoptosis.130 Accord-
ingly, pharmacological inhibition of histone deacetylases with
suberoylanilide hydroxamic acid (SAHA) reverses established lung fibro-
sis by inducing myofibroblast apoptosis via BCL-XL down-regulation.131
On the contrary, histone deacetylation by the histone deacetylase
4 (HDAC4) has been reported to suppress the activated myofibroblast
phenotype by inhibiting α-SMA expression.132 miRs suppress gene
expression via direct binding to 30 UTR of target. In myofibroblasts,
down-regulation of miR-29a and miR-133a has been associated with
myofibroblast persistence and survival, and that increase in miR-29a
promotes myofibroblast apoptosis.133 On the contrary, up-regulation of
miR-21 has been shown to promote myofibroblast survival by reducing
levels of pro-apoptotic BAX.134 Moreover, up-regulation of miR-22 has
been shown to promote myofibroblast persistence and survival by driv-
ing cellular senescence.135
4.4 | Cellular senescence
Myofibroblast senescence has been traditionally associated with the
resolution of the normal tissue repair programme and that elimination
of senescent myofibroblasts prevents the development of fibro-
sis.52,57 However, a growing body of evidence suggests that impaired
clearance of senescent myofibroblasts shifts their pro-repair activities
to pro-fibrotic via persistent secretion of pro-inflammatory and pro-
fibrotic mediators50,136–141 . In this regard, the SASP of pro-fibrotic
senescent myofibroblast has been shown to contain pro-inflammatory
cytokines (IL-1α, IL-1β, IL-6 and IL-8), pro-fibrotic cytokines (TGF-β,
PDGF) and ECM proteins (fibronectin, collagens).50,142 Human fibrotic
fibroblasts derived from patients with various fibrotic diseases display
a senescent phenotype.143–146 Noteworthy, senescent myofibroblasts
activate pro-survival mechanisms including up-regulation of BCL-XL,
which promotes their persistence within fibrotic tissues. In humans,
senescent myofibroblasts from patients with IPF show elevation of
p16 and p21.50,51,55,136 Mechanistically, acquisition of a senescent
MERKT ET AL.
phenotype appears to be regulated by the DNA damage response,55
independently from TGF-β signalling.
5 | TA RG ETING M YO FIBROB LAS T
P E R S I S T E N CE F O R A NT I - F I B RO TI C TH E R A P Y
Organ fibrosis has been traditionally thought to be irreversible. How-
ever, fibrosis has been shown to regress in humans in response to
pharmacological treatment of the underlying pro-fibrotic process. For
instance, liver fibrosis can regress after treatment of viral hepatitis,
pancreas transplantation can ameliorate diabetic nephropathy, hypo-
tensive drugs can treat hypertensive cardiac fibrosis, and lung fibrosis
can resolve substantially in patients with acute respiratory syndrome.
These examples have in common that defined fibrogenic injuries exist,
and that haltering of these pro-fibrotic insults associates with fibrosis
regression. However, the exact aetiology of most human fibrotic dis-
ease remains unknown such as in IPF or SSc. In these fibrotic diseases,
it is thought that the establishment of self-amplification pro-fibrotic
loops leads to progressive fibrotic responses independently from the
initial insult. Up to date, only nintedanib and pirfenidone have been
approved for the treatment of IPF and scleroderma-associated inter-
stitial lung disease.147–149 Although these drugs slow down the rate
of progression of fibrosis, they do not halt or reverse established
fibrosis.150 The mode of action of these drugs remains poorly under-
stood, but they likely inhibit mechanisms involved in myofibroblast
formation and collagen synthesis rather than mechanisms promoting
fibrosis resolution. Recent insights into the mechanisms promoting
myofibroblast persistence and survival have paved the way for new
therapeutic strategies aiming at reversing fibrosis by targeting
myofibroblast fate. These novel treatments include senolytics, gene
therapy, cellular immunotherapy and CAR-T cells, and may not only
reverse fibrosis but also regenerate fibrotic tissues (Figure 2).
5.1 | Targeting myofibroblast apoptosis
Substantial evidence has demonstrated that activated (senescent)
myofibroblasts in fibrotic tissues are prone to undergo apoptosis due
to increased mitochondrial priming, as described above. This knowl-
edge of apoptotic priming has led to the development of novel anti-
fibrotic strategies aimed at blocking pro-survival mechanisms acti-
vated in senescent myofibroblasts, ultimately inducing activation of
programmed cell death.
5.1.1 | Senolytic drugs unleash the intrinsic
pathway of apoptosis
The so-called BH3 mimetic drugs are small molecules that selectively
antagonize anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, BCL-W,
MCL-1 and A1) by directly binding to specific shallow hydrophobic
grooves.14 This knowledge has led to the discovery and development
7
of ABT-737 and ABT-263 (navitoclax, which blocks BCL-2, BCL-XL,
BCL-W), ABT-199 (venetoclax, a BCL-2 selective inhibitor) and sev-
eral BCL-XL and MCL-1 mono-selective inhibitors.14,151–154 We have
recently shown that ABT-263 reverses established dermal fibrosis in a
mouse model of scleroderma by targeting myofibroblast for
apoptosis.17 Mechanistically, ABT-263 directly blocks the pro-survival
protein BCL-XL, releasing pro-apoptotic proteins such as BIM that
ultimately triggers myofibroblast apoptosis. In vitro, ABT-263 induces
apoptosis of fibrotic fibroblasts isolated from patients with sclero-
derma.17 In addition, ABT-263 has been recently identified as a potent
senolytic agent by targeting senescent cells for apoptosis and revers-
ing lung fibrosis induced by ionizing radiation in mice.155 These data
validate genetic and pharmacological studies showing that elimination
of senescent cells protects mice from lung and kidney fibrosis.51,156–
158
In the heart, ABT-263 has been shown to eliminate senescent
cardiomyocytes and improve cardiac function in mouse models of car-
diac ischemia–reperfusion injury159 and angiotensin II-induced cardiac
hypertrophy and fibrosis.160 Importantly, clearance of senescent cells
improved myocardial remodelling and diastolic activity along with a
higher rate of survival in the event of myocardial infarction.161 In the
liver, the BCL-XL selective inhibitor A-1331852 treats biliary liver
fibrosis in a mouse model of primary sclerosing cholangitis by
targeting senescent cholangiocytes and fibroblasts for apoptosis.162
More recently, the MCL-1 inhibitor S63845 induces apoptosis of pro-
liferative ductular reactive cells, reducing hepatic fibrosis in a mouse
model of cholestasis.163
5.1.2 | TRAIL ligands activate the extrinsic pathway
of apoptosis
As described above, several mechanisms contribute to reduced sensi-
tivity of myofibroblasts to FasL-induced apoptosis.164 In vitro, activa-
tion of the cell-surface death receptors (DRs) of the extrinsic pathway
of apoptosis has been shown to sensitize myofibroblasts to FasL-
induced apoptosis.165 Accordingly, selective agonism of DR4 and DR5
with the TRAIL-TLY012 induces myofibroblast apoptosis and reverses
established skin fibrosis in a mouse model of scleroderma,166
highlighting the therapeutic potential of targeting myofibroblasts for
apoptosis via activation of the extrinsic apoptosis pathway. Moreover,
PEGylated TRAIL ameliorates liver cirrhosis in rats by inducing apo-
ptosis of activated hepatic stellate cells.167
5.2 | Cellular immunotherapy
5.2.1 | Harnessing macrophage and T cell anti-
fibrotic potential
Harnessing the immune system to treat fibrosis has recently emerged
as a promising therapeutic strategy. Recent reports have demonstrated
that CD47, a “don't-eat-me-signal,” is upregulated in scleroderma myo-
fibroblasts and prevents phagocytosis by macrophages.168 Accordingly,
8 MERKT ET AL.
pharmacological blockade of CD47 leads to myofibroblast clearance by
macrophages and reversion of fibrosis in an adaptive transfer model.169
Cytotoxic T cells have been similarly involved in myofibroblast clear-
ance by activating the extrinsic pathway of apoptosis. In vivo,
myofibroblast persist in FasL-deficient mice subjected to lung fibrosis
models, and that reconstitution with FasL+ cytotoxic cells promote
45
myofibroblast apoptosis and fibrosis resolution in mice.
5.2.2 | CAR-T cells
CAR-T cells are T-lymphocytes engineered to express a chimeric
receptor targeting a specific antigen, thereby triggering T-cell activa-
tion and killing of target cells.170 Recently, CAR-T cells designed to
selectivity eliminate cardiac fibroblasts expressing fibroblast activation
protein (FAP) have been shown to reduce cardiac fibrosis and restora-
171
tion of cardiac function after injury in mice. Similarly, CAR-T cells
selectively targeting senescent cells expressing the urokinase-type
plasminogen activator receptor (uPAR) has been shown to treat liver
fibrosis in mouse models.172
5.3 | Targeting myofibroblast deactivation
Over the last 20 years, multiple studies have demonstrated the ability of
several agents at treating established fibrosis in preclinical models by pro-
moting myofibroblast deactivation to a low activity state. FDA-approved
anti-fibrotic drugs, pirfenidone and nintedanib, have been shown to
reduce ECM synthesis and myofibroblast numbers by antagonizing the
TGF-β pathway.173,174 Accordingly, direct inhibition of TGF-β ligand,
TGF-β receptors or integrin-mediated TGF-β activation have been simi-
larly shown to attenuate established fibrosis in mouse models.175 More-
over, targeting secondary pro-fibrotic mediators induced by TGF-β such
as ephrin-B2, CTGF or ET-1 reduces ECM synthesis in myo-
15,176,177
fibroblasts. Similarly, antagonizing TGF-β signalling in fibroblasts
with PPARγ agonists reduces matrix deposition both in vitro and
in vivo.175 In addition, inhibitors of mechanotransduction pathways
including FAK, ROCK, YAP/TAZ and MRTFs have been shown to treat
12,178
established fibrosis by reducing myofibroblast pro-fibrotic activities.
Targeting epigenetic modulators such as the histone methylase SET8
with the UNC0379 inhibitor or the bromodomain and extra-terminal
(BET) with JQ1 have been shown to deactivate myofibroblasts and treat
179,180
pre-clinical fibrosis. The list of anti-fibrotic targets aimed at reduc-
ing myofibroblast activities is evergrowing and extensible reviewed else-
where. More recent studies have identified functionally and spatially
distinct fibroblast lineages involved in skin repair and fibrosis, supporting
the notion that specific pro-fibrotic fibroblasts populations can be
targeted for therapeutic treatment.181–183 Specifically, the Engrailed-1
fibroblast lineage is endowed with fibrogenic potential and is the primary
contributor to ECM synthesis during embryonic development, wounding
181
and fibrosis. Accordingly, selective targeting of Engrailed-1+ fibro-
blasts with the CD26 pharmacological inhibitor diprotin A reduced skin
scarring in wounding models.181
5.4 | Targeting myofibroblast reprogramming
Conversion of activated myofibroblasts into epithelial-like cells holds
potential for the treatment and regeneration of fibrotic tissues. For
instance, fibrogenic injury in the liver leads to loss of hepatocytes
and accumulation of activated myofibroblasts. In this regard, recent
studies have demonstrated that direct reprogramming of liver myo-
fibroblasts into hepatocytes reverses established liver fibrosis and
repopulates the hepatocyte pool in mice. In these studies, lentiviral-
mediated ectopic overexpression of the transcription factors
FOXA3, GATA4, HNF1A and HNF4A converts mouse myo-
fibroblasts into hepatocyte-like cells and attenuates liver fibrosis.184
Similarly, liver myofibroblast reprogramming into hepatocytes using
adeno-associated virus (AAV) vectors expressing six hepatic tran-
scription factors including Foxa1, Foxa2, Foxa3, Gata4, Hnf1a or
Hnf4a reverses liver fibrosis in vivo.185 This novel therapeutic
approach may open new avenues for the treatment of chronic liver
fibrosis using gene therapy.
6 | CONC LU SIONS
Spatiotemporal control of myofibroblast functions is regulated by
alternative fates during normal tissue repair and fibrosis.
Myofibroblast deactivation, apoptosis, senescence and repro-
gramming appear to limit pro-repair functions and prevent the
development of fibrosis: Whether these alternative myofibroblast
fates represent specific or redundant fail-safe mechanisms for lim-
iting myofibroblast functions remains to be investigated. Thus, fur-
ther research is needed to understand the mechanisms and
signalling pathways that determine these specific myofibroblast
fates and their functions during normal tissue repair. In this line,
recent studies using single-cell RNA sequencing have shown that
heterogeneous myofibroblast subpopulations undergo dynamic
phenotypic changes during tissue repair and fibrosis, further
suggesting that myofibroblast fate plasticity is essential for our
body to respond to multiple injury scenarios. Despite these
advances, it remains unknown how these different fates are inter-
connected as well as their temporal and local wiring. A growing
body of evidence suggests that persistent myofibroblast activities
lead to fibrosis, a manifestation of a pro-fibrotic phenotype associ-
ated with impaired or chronic myofibroblast fate. Therapeutic
targeting of myofibroblast fates such as induction of apoptosis of
(senescent) myofibroblasts, deactivation or reprograming could
reverse established fibrosis and potentially regenerate chronically
injured tissues. Given that the origin of myofibroblasts depends on
specific progenitors present in each tissue (e.g. resident fibroblasts,
epithelial and endothelial cells, pericytes and adipocytes), it will be
important to understand the contribution and function of specific
myofibroblast fates in different organs and systems, which might
inform about organ-specific anti-fibrotic strategies. Further
research is needed to translate these innovative and promising
therapeutic strategies from the bench to the bedside.
MERKT ET AL.
ACKNOWLEDGMENTS
D.L. gratefully acknowledges funding support from the NIH (grant
R01 HL147059-01) and Sponsored Research Grants from Boehringer
Ingelheim and Merck & Co.
CONF LICT OF IN TR ES T
D.L. declares that he has received research funding from Boehringer
Ingelheim, Indalo Therapeutics, Merck & Co and Unity Biotechnology.
Dr. Lagares has a financial interest in Mediar Therapeutics and Zenon
Biotech. The companies are developing treatments for organ fibrosis.
Dr. Lagares's interests were reviewed and are managed by MGH and
Partners HealthCare in accordance with their conflict of interest poli-
cies. WM, YZ and HH declare no conflict of interest.
DATA AVAI LAB ILITY S TATEMENT
The data that support the concepts described in this manuscript is
derived from resources available in the public domain.
ORCID
David Lagares https://orcid.org/0000-0002-7317-8796
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How to cite this article: Merkt W, Zhou Y, Han H, Lagares D.
Myofibroblast fate plasticity in tissue repair and fibrosis:
Deactivation, apoptosis, senescence and reprogramming.
Wound Rep Reg. 2021;1–14. https://doi.org/10.1111/wrr.
12952