Mechanisms of Myofibroblast Activity and Phenotypic Modulation
Guido Serini*,† and Giulio Gabbiani‡,1
*Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment, and †Department of Genetics, Biology
and Biochemistry, University of Torino School of Medicine, Str. Prov. 142, Km. 3.95, 10060 Candiolo (TO), Italy;
and ‡Department of Pathology, University of Geneva-CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
The name myofibroblast was proposed several years
agofor fibroblastic cells located within granulation tis-
sue and exhibiting an important cytoplasmic microfila-
mentous apparatus [1, 2]. The suggestion that these
cells are responsible for the phenomenon of wound
contraction was rapidly taken into consideration be-
cause about the same time the concept that any cell,
and not only musclecells, is provided with a contractile
apparatus responsible for the regulation of such phe-
nomena as cell shape modulation or cell motility was
becoming more and more popular (for review see ).
Several laboratories havereported thepresenceof cells
with myofibroblastic features during a variety of
pathological situations  and also in normal tissues,
where they could exert a mechanical function (for re-
view see ). Table 1 lists the locations of myofibro-
blasts in normal tissues. Later, it was observed that
the microfilamentous apparatus of myofibroblasts con-
tains actin and myosin and in particular ?-smooth
muscle (SM)2actin, the actin isoform typical of SM
cells located in the vessel wall . ?-SM actin became
the most reliable marker of myofibroblastic cells. It
was also discovered that myofibroblasts could express
several other SM cell markers according to the patho-
logical situation and/or to the location . Hence, the
proposal was made that a spectrum of phenotypes ex-
ists between fibroblastic and SM cells . The modu-
lations within this spectrum are probably controlled by
a local network, including growth factors, cytokines,
adhesion molecules, and extracellular matrix (ECM)
components. The purpose of this review is to summa-
rize the most recent achievements in the understand-
ing of: (1) the role of contractile proteins, ?-SM actin in
particular, in the production of retractile and fibrotic
phenomena and (2) the local factors controlling the
modulation and activity of myofibroblasts.
MECHANISMS OF FORCE GENERATION
Observations made by several laboratories have in-
dicated that fibroblastic cells in vitro exert mainly iso-
metric traction forces rather than an isotonic contrac-
tile activity . It is possible that a similar mechanism
takes place in vivoduring granulation tissue or fibrotic
tissuecontraction. Indeed, when examined by means of
confocal microscopy, human or animal granulation tis-
sue (Bochaton-Piallat et al., submitted for publication)
exhibit morphological features, including the presence
of stress fibers similar to those of cultured fibroblastic
cells (Fig. 1). It has been shown that myofibroblasts are
connected with the ECM by means of specialized struc-
tures called fibronexi , and it has been postulated
that cytoskeletal elements interconnecting different
cells are organized as a tensegrity structure in which
cytoskeletal components, such as microtubules or in-
termediate filaments, exert a resistance on the tension
produced by contractile elements, such as actin and
myosin . In this context, an interesting aspect of
myofibroblast research has concerned the role of ?-SM
actin in the generation of myofibroblast contractile
forces. In addition to being a marker of myofibroblast
differentiation, ?-SM actin could be functionally im-
portant for myofibroblast contraction if one takes into
account the fact that ?-SM actin is expressed through-
out thephilogenetic treein cells whosemain function is
contraction, e.g., SM cells or myoepithelial cells, and
that the neoexpression of ?-SM actin in pathological
tissues coincides with the appearance of contractile
properties . Actin isoforms are very conserved and
exhibit relatively small differences in amino acid se-
quence, which probably correspond tofunctional differ-
ences. Wehavecharacterized thesequencecorrespond-
1To whom reprint requests should be addressed.
2Abbreviations used: basic fibroblast growth factor, bFGF; cellular
fibronectin, cFN; endothelin, ET; extracellular matrix, ECM; granu-
locyte-macrophage colony-stimulating factor, GM-CSF; fibronectin,
FN; interferon, IFN; large latent complex, LLC; latent associated
protein, LAP; latent TGF? binding protein, LTBP; plasma fibronec-
tin, pFN; platelet-derived growth factor, PDGF; receptor tyrosine
kinases, RTKs; recombinant ED-A, (rED-A); small latent complex,
SLC; SM, smooth muscle; tumor necrosis factor ?, TNF?; transform-
ing growth factor-?, TGF?; thrombospondin-1, TSP-1.
Experimental Cell Research 250, 273–283 (1999)
Article ID excr.1999.4543, available online at http://www.idealibrary.com on
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
ing to the epitope of the monoclonal antibody against
?-SM actin, which is Ac-EEED, and shown that this
sequence is important for ?-SM actin polymerization
. Moreover, the microinjection of the epitopic pep-
tide into fibroblastic or SM cells results in the disap-
pearance of ?-SM actin immunostaining in stress fi-
bers. Recent work has shown that inhibition of ?-SM
actin expression in SM cells or in fibroblasts increases
their motility and likely decreases their contractile
activity . All these observations strongly suggest
that ?-SM actin plays an important role in the devel-
opment of myofibroblastic force. It may even be possi-
ble toenvisage the possibility of negatively influencing
myofibroblast contraction by means of the application
of the ?-SM actin N-terminal peptide.
FACTORS INFLUENCING MYOFIBROBLAST
Modulation of cell differentiation and behavior de-
pends on cooperation among cytokines and ECM mol-
ecules, whosereceptors generatecollaborativeintracel-
lular signals responsible for the control of gene
expression, proliferation, apoptosis, and cytoskeletal
dynamics [13–16]. Indeed, cytokine-elicited signals are
integrated by target cells within the context of their
surrounding ECM, so that adhesion receptor-gener-
ated signals intercept and join with the signal trans-
duction pathways triggered by cytokine receptors .
There is now mounting evidence that the mechanisms
leading to the development of the myofibroblastic phe-
notype mirror this paradigm as well.
A common early event in myofibroblast differentia-
tion during embryonic development (for review see
) and wound healing in the adult organism (for
review see ) is represented by the proliferation and
migration of platelet-derived growth factor (PDGF)-
PDGF-expressing target cells and within the PDGF-
containing provisional matrix of the wound clot, re-
spectively. In particular, during development myofi-
broblastsregulate the structural
stability of small-caliber hollow physiological units,
such as the capillary , the glomerulus [21, 22], and
the lung alveolar sack [23, 24]. Three different sets of
myofibroblasts are lost in PDGF-null mice, i.e., capil-
lary pericytes and glomerular mesangial cells in
PDGF-B knockouts and alveolar myofibroblasts in
PDGF-A knockouts. Lack of these myofibroblasts
causes dilatation of the structures to which they be-
long, resulting in capillary microaneurysms, balloon-
ing of the glomerular tufts, and lung emphysema. Re-
cently, a new model for the recruitment of pericytes,
mesangial cells, and alveolar myofibroblasts has been
proposed according to which PDGF receptor-carrying
progenitors, localized where typical SM cells will sub-
sequently develop (e.g., larger vascular and bronchial
walls), spread from proximal to distal sites along
PDGF-secreting epithelial or endothelial tubes and
eventually differentiate intomyofibroblasts . These
data clearly show that PDGF is a potent mitogenic and
chemotactic agent for myofibroblast progenitors in vivo
and unravel new fundamental roles for myofibroblastic
cells during development.
Besides PDGF , other cytokines released within
and around the clot at the wound site may act as
mitogen and/or chemotactic agents for resident dermal
fibroblasts in the neighborhood of the wound (for re-
view see ). Tumor necrosis factor ? (TNF?), pro-
duced by inflammatory cells, and basic fibroblast
growth factor (bFGF), synthesized by macrophages
and endothelial cells, are mitogens for fibroblasts both
in vitro and in vivo [27–29]. Connective tissue growth
factor, a mitogenic and chemotactic agent for SM cells
and fibroblasts, is also actively synthesized and re-
leased by granulation tissuefibroblasts as a transform-
ing growth factor-?1 (TGF?1)-induced immediate-
early gene . However, cytokines implicated in the
early events of myofibroblast precursor proliferation
and migration do not induce the expression of SM
differentiation markers, such as ?-SM actin, either in
vitro or in vivo [28, 29].
In vivo application of granulocyte-macrophage col-
ony-stimulating factor (GM-CSF) to the rat subcuta-
neous tissue induces fibroblasts to proliferate and
synthesize high levels of ?-SM actin, leading to the
formation of a granulation tissue rich in ?-SM actin-
containing myofibroblasts . Since in vitro stim-
ulation of fibroblasts with GM-CSF does not induce
?-SM actin, this effect on fibroblastic cells is prob-
ably mediated by another factor(s) . Interestingly,
T ABL E 1
Fibroblastic Cells of Normal Organs Displaying Ultra-
structural and/or Immunochemical Features of SM Differen-
Reticular cells of lymph nodes
Intestinal pericryptal cells
Intestinal villous core
Theca externa of the ovary
Adrenal gland capsule
Hepatic perisinusoidal cells
Bone marrow stroma
Capillary and venular
Glasser and J ulian (1986) 
Toccanier-Pelte et al. (1987) 
Sappino et al. (1989) 
Kaye et al. (1968) 
Skalli et al. (1986) 
Czernobilsky et al. (1989) 
Beertsen et al. (1974) 
Bressler (1973) 
Yokoi et al. (1984) 
Kapanci et al. (1992) 
Charbord et al. (1990) 
Lindahl and Betscholtz (1998) 
SERINI AND GABBIANI
F IG. 1.
membrane from a patient suffering from proliferative vitreoretinopathy. The membrane has been stained in toto by means of immunoflu-
orescence with an antibody against ?-SM actin. The colors change in function of the depth of the section. Myofibroblasts show typical stress
fibers. Bar, 10 ?m.
F IG. 2.A model for fibroblast–myofibroblast modulation. TGF?1 is released from platelets (not shown) and macrophages, probably as a
consequence of their activation by GM-CSF. Released TGF?1 activates resident fibroblastic cells tosynthesize and tridimensionally organize
an ECM scaffold containing ED-A FN, which is necessary but not sufficient for the induction of the myofibroblastic phenotype by TGF?1:
ED-A FN exerts a permissive effect on TGF?1 activity.
Three-dimensional reconstruction by means of laserscan confocal microscopy of myofibroblastic cells present in an epiretinal
MYOFIBROBLAST ACTIVITY AND PHENOTYPIC MODULATION
overexpression of GM-CSF in transgenic mice results
in fibrotic lesions at sites where activated-macroph-
ages accumulate ; moreover, the appearance of
?-SM actin-positive myofibroblasts after local applica-
tion of GM-CSF in rats is preceded by cluster-like
accumulation of macrophages . These results sug-
gest that GM-CSF-activated macrophages could se-
crete a cytokine(s) capable in turn of stimulating ?-SM
actin expression in fibroblasts; TGF?1 (see below) is
the most likely candidate for this activity. In fact, ad-
enoviral vector-mediated gene transfer of GM-CSF in
the alveolus results in local accumulation of TGF?1,
followed by ?-SM actin and collagen synthesis by alve-
olar myofibroblasts, and eventually pulmonary fibrosis
. Moreover, in a well-established experimental
model of rat pulmonary fibrosis, intra-alveolar instil-
lation of bleomycin causes first an influx of inflamma-
tory cells in the alveolus , then TGF?1 expression
by macrophages [33, 35, 36], and eventually differen-
tiation of alveolar fibroblasts in ?-SM actin-expressing
myofibroblasts , which are responsible for collagen
production and fibrosis . Recently, our laboratory
has reported that GM-CSF mRNA is early and tran-
siently expressed both in alveolar macrophages and in
polymorphonuclear neutrophils after bleomycin ad-
ministration and precedes the increase of TGF?1 and
TGF? typeII receptor mRNAs . Thesedata support
the possibility that GM-CSF, secreted by inflammatory
cells invading the alveolus, is an early upstream
regulator of TGF?1 production and signaling during
Among cytokines implicated in myofibroblastic mod-
ulation, TGF?1 can be considered the direct inducer of
the myofibroblastic phenotype, because it is capable of
upregulating fibroblast ?-SM actin [40, 41] and colla-
gen [38, 42, 43] in fibroblasts both in vitro and in vivo.
?-SM actin-expressing myofibroblasts generate granu-
lation tissue contraction during wound closure  and
collagen overproduction during fibrotic diseases ;
thus, TGF? acts as a master switch of tissuerepair and
disregulation of its production generates tissue fibrosis
[19, 42–44]. Three isoforms of TGF? (TGF?1, TGF?2,
and TGF?3) exist in mammals, sharing 70–80% ho-
mology; their functional significance is witnessed by
the degree of evolutionary conservation between spe-
cies. We have shown  that (i) TGF?2, like TGF?1,
induces myofibroblast formation in vivo and in vitro;
(ii) TGF?3 acts like a negative regulator of the myofi-
broblastic phenotype in vivo, but not in vitro; (iii) in
vitro, the three different TGF? isoforms are equally
able to induce ?-SM actin mRNA and protein expres-
sion in fibroblasts. Thus, while in vitrothe three TGF?
isoforms behave similarly, in vivo they play different,
but complementary, roles in myofibroblast modulation.
We hypothesize that the ability of TGF? isoforms to
differently modulate in vivo, but not in vitro, ?-SM
actin expression is due to different microenvironmen-
tal conditions, first of all ECM components [46, 47] and
their spatial organization [48, 49]. For example, as
already shown in endothelial cells , the transition
from thetridimensional environment present in vivoto
the bidimensional in vitro culture conditions could per
seinducea changein theTGF? receptor repertoireand
in the ensuing cellular response to TGF? isoforms.
Endothelin (ET) could represent another candidate
for direct induction of the myofibroblastic phenotype.
ETs constitute a family of potent vasoconstrictive fac-
tors whose effects extend beyond vasoregulation [51–
53]. ET-1 has mitogenic activity on cultured fibroblasts
 and upregulates ?-SM actin expression in cultured
vascular SM cells . After liver injury, hepatic stel-
late cells and endothelial cells actively synthesize ET-1
[56, 57] and circulating levels of ET-1 are elevated .
Moreover, hepatic stellate cells expose abundant ETA
and ETB receptors on their cell surface . Intrigu-
ingly, ET-1 directly stimulates the in vitroactivation of
hepatic stellate cells to ?-SM actin-positive myofibro-
blasts and the mixed ETA/Breceptor antagonist bosen-
tan blocks this in vitro process as well as the hepatic
fibrosis induced by liver injury .
Data from our laboratory  have previously shown
that in cultured fibroblasts ?-interferon (?-IFN), a cy-
tokineproduced by T-helper lymphocytes and liberated
during inflammatory phenomena, decreases ?-SM ac-
tin protein and mRNA expression as well as cell pro-
liferation, pointing to ?-IFN as a potential pharmaco-
logical agent to exert antifibrotic activity in vivo. A
preliminary trial  has shown that direct application
of ?-IFN to lesions: (i) decreases hypertrophic scar and
Dupuytren’s nodule clinical manifestations and size
and (ii) inhibits ?-SM actin synthesis in hypertrophic
scar myofibroblasts. In addition, ?-IFN decreases
?-SM actin expression in cultured hepatic stellate cells
 and reduces liver fibrosis in non-A, non-B hepatitis
. Thus, IFN treatment could be considered a new
promising therapeutical strategy for fibrocontractive
diseases in general. Since bFGF has been shown to
directly inhibit the TGF? induction of ?-SM actin in
myofibroblasts , it would be worthwhile totest this
cytokine as an anti-myofibroblastic agent as well.
Pioneer observations by Singer and colleagues [9, 64]
indicated that both in vitro and in vivo myofibroblast
attachment to ECM takes place at discrete adhesive
sites named fibronexi. In these structures, comparable
to typical focal contacts , actin microfilaments and
extracellular fibronectin (FN) fibrils are organized into
a close one-on-one association, mediated by the inte-
grin family of adhesion receptors . Adhesion to
ECM induces integrin clustering at focal contacts,
where structural molecules and regulatory enzymes
interact with the actin cytoskeleton and trigger signal
SERINI AND GABBIANI
transduction pathways [13, 14]. Signaling pathways
activated by integrins synergize with signaling path-
ways downstream of growth factor receptor tyrosine
kinases (RTKs), resulting in a joined control of cell
shape, migration, proliferation, and differentiation [15,
16, 67]. Moreover, ECM ligands can directly interact
with and activate RTKs [68, 69] and integrin-depen-
dent cellular adhesion can depend on growth factor
receptor tyrosine kinase activation . Hence, coop-
eration between ECM and cytokines is required toreg-
ulate cell behavior and orchestrate functions of cells in
tissue formation and homeostasis.
TGF? possesses the unique capability to control cell
adhesion and migration by modulating the integrin
expression pattern [71, 72] and the synthesis of ECM
ligands, such as FN and collagen [73, 74]. FN is a
440-kDa disulfide-bonded dimeric protein, implicated
in a wide variety of cellular properties, such as cell
adhesion, morphology, cytoskeletal organization, mi-
phagocytosis, and hemostasis . FN exists as a sol-
uble plasma FN (pFN) and as an insoluble cross-linked
multimeric cellular FN (cFN) which is deposited as
ECM fibrils . Each FN subunit is composed of re-
peating homologous modules containing binding sites
for integrins and for other ECM components. Alterna-
tive splicing of the type III segments ED-A, ED-B, and
IIICS generates FN polymorphism, with ED-A and
ED-B alternative spliced segments included in cFN,
but not in pFN [65, 75, 76]. FN is expressed at high
levels in healing wounds ; here both granulation
tissue fibroblasts and macrophages express FN tran-
scripts, including ED-A and ED-B domains [78, 79].
Interestingly, TGF?1 dramatically increases the rela-
tive amount of mRNA for ED-A- and ED-B-containing
FN isoforms [80–82] and ED-A FN modulates hepatic
stellate cells to ?-SM actin-expressing myofibroblast
. We have recently shown that the induction of the
twomain myofibroblast markers ?-SM actin and colla-
gen type I by TGF? depends on an ED-A FN-derived
permissive outside-in signaling, under the control of
the TGF? itself . Indeed, ED-A FN deposition pre-
cedes ?-SM actin expression both in vivo, during gran-
ulation tissue evolution, and in vitro, after TGF?1
stimulation. Importantly, while seeding fibroblasts on
ED-A FN does not elicit per se ?-SM actin expression,
selectively blocking the ED-A domain of cFN by IST-9
monoclonal antibody inhibits ?-SM actin and collagen
type I mRNA induction by TGF?1. In addition, treat-
ment of fibroblasts with soluble recombinant ED-A do-
main (rED-A) inhibits TGF?1 induction of the myofi-
broblastic phenotype as well, but neither IST-9 nor
rED-A alter FN matrix assembly. Our data show that
ED-A-containing polymerized FN is necessary but not
sufficient for the induction of the myofibroblastic phe-
notype by TGF?1 and that it exerts a permissive effect
on TGF?1 activity. The fact that the myofibroblast
inhibitor ?-IFN inhibits dose-dependently the synthe-
sis of ED-A FN at the transcription level  further
points to ED-A FN as an indispensable ECM molecule
for the modulation of myofibroblastic differentiation.
Our observation, that the addition of isolated rED-A
domain inhibits TGF?1 activity similarly to the addi-
tion of IST-9, indicates that binding of the ED-A do-
main to either a second ECM component or a cell
surface receptor is mandatory to elicit its permissive
signaling. The latter hypothesis is supported by three
lines of evidence: (i) the isolated rED-A domain pro-
motes cell adhesion ; (ii) activated ?1 integrins can
mediate cell adhesion and spreading on recombinant
noncanonical FN type III repeats lacking the RGD
motif [87–89]; (iii) the ?5?1 integrin transduces dis-
tinct signals upon interacting with either the N-termi-
nal or RGD region of FN . The increased accessi-
bility of the RGD motif to integrin ?5?1 because of an
ED-A-induced conformational change of FN described
by Manabe et al.  could represent a second inde-
pendent and indirect function of the ED-A domain.
According to the dominant negative function exerted
by the isolated rED-A domain, the ED-A receptor
should beabletointeract with its ligand independently
of the molecular context and to generate intracellular
signals in a conformationally sensitive manner. In-
triguingly, this is somehow reminiscent of the proper-
ties of the newly described collagen receptor tyrosine
kinases DDR1 and DDR2, which bind to and are acti-
vated by collagen in a conformation-dependent way
Inhibiting TGF? induction of myofibroblasts may be
therapeutically useful in many pathological settings,
such as Dupuytren’s disease , organ fibrosis ,
arterial intimal thickening , and stroma reaction to
epithelial cancers . However, direct blocking of the
TGF?/TGF? receptor system exposes to a high risk of
side effects, such as autoimmune diseases [96, 97] and
malignant transformation [98, 99]. The ED-A domain
of cFN could be considered an alternative and safe
therapeutical target for different reasons: (i) in con-
trast to TGF?1, ED-A FN drops to undetectable levels
in adult normal tissues ; (ii) ED-A FN is actively
synthesized by myofibroblasts in all the pathological
situations described above; (iii) in animal models
where TGF? inhibition has been shown tobe therapeu-
tic , a concomitant dramatic reduction of ED-A FN
deposition was reported [101, 102]; (iv) ED-A FN is an
easily reachablemoleculeand in vivotargeting of other
alternatively spliced FN type III domains in patholog-
ical tissues has been shown to be feasible [103–105].
Most cell types secrete TGF? in a biologically inac-
tive latent form  resulting from the noncovalent
interaction of TGF? with the latent associated protein
(LAP), a homodimer of the N-terminal proteolytic frag-
ment of the large TGF? precursor protein . TGF?
associated with the LAP homodimer is called the small
MYOFIBROBLAST ACTIVITY AND PHENOTYPIC MODULATION
latent complex (SLC) and latent TGF? is mainly se-
creted as a part of a large latent complex (LLC), in
which a second protein, the latent TGF? binding pro-
tein (LTBP), is disulfide linked to LAP . Several
ECM components and their receptors act as direct reg-
ulators of latent TGF? activation. The large homotri-
meric ECM protein thrombospondin-1 (TSP-1) has
been recently identified as an important natural acti-
vator of TGF?1 in vivo. PDGF, bFGF, and TGF?1
itself can induce TSP-1 in cultured cells [109–112].
TSP-1 binds to and activates both SLC and LLC forms
of TGF?1 as a result of an interaction with the LSKL
sequence of LAP and the ensuing conformational
change which makes TGF?1 accessible to its receptors
[108, 113–116]. TSP-1 expression seems also to be in-
timately linked to myofibroblast differentiation. In-
deed, TSP-1 stimulates theexpression of ?-SM actin by
fibroblasts in aortic explants . In several animal
models of glomerulonephritis, the majority of TSP-1-
positive interstitial cells in areas of tubulointerstitial
injury has been shown to be ?-SM actin-positive myo-
fibroblasts . Recent data, showing that adminis-
tration of the LAP-derived LSKL peptide blocks TSP-1
activation of TGF?1 in vivo , suggest new ap-
proaches to locally interfering with pathological devel-
opment of myofibroblasts. However, since the inflam-
matory phenotype displayed by TSP-1-null mice is
clearly less severe than that showed by TGF?1 knock-
out mice, additional mechanisms of TGF? activation
should be taken in account. An intriguing novel mech-
anism has been recently described where the RGD-
dependent adhesion of ?v?6 integrin to LAP-?1 is suf-
ficient to locally activate TGF?1 . Interestingly,
?6–null mice are protected against bleomycin-induced
pulmonary fibrosis and ?v?6 expression is strongly
increased in the lungs of wild-type mice in response to
bleomycin . In addition, binding to ?v?6 does not
activate per se latent TGF?1 and ?v?6 association
with the actin cytoskeleton is required to activate
bound latent TGF?1 . Up to now it has been
shown that LAP isoform 1 is an RGD-dependent ligand
for integrins ?v?1, ?v?5, and ?v?6 [119, 120] and
probably for ?IIb?3 platelet integrin as well .
Because TGF? expression can be maintained by an
autocrine loop [122–124], it could be interesting totest
whether specifically blocking ?v integrins could inhibit
TGF?-induced myofibroblastic differentiation. More-
over, since LAP-containing latent TGF?1 induces ty-
rosine phosphorylation of integrin signal transducers
, the LAP-driven outside-in signaling could repre-
sent an early event during TGF?1 induction of myofi-
broblastic phenotype as well.
Our laboratory has previously reported that a pro-
gressive apoptotic wave is responsible for the gradual
disappearance of granulation tissue myofibroblasts af-
ter wound closure . Little is known about the
stimuli leading to myofibroblast programmed cell
death. Differently from epithelial and endothelial cells,
cultured primary fibroblasts do not undergo apoptosis
following serum withdrawal, irradiation, or loss of ad-
hesion to the ECM [126–128]. However, embedding
primary fibroblasts in contractile collagen gels elicits
their apoptosis, while seeding cells in either anchored
collagen gels or contractile fibrin gels does not .
Thus, ECM-mediated tensile forces, which act on and
are exerted by myofibroblasts, could represent major
environmental regulators of apoptosis. Interestingly,
soluble RGD peptides, already known to cause apopto-
sis by inhibiting integrin–ECM interactions ,
have been recently shown to enter cells and induce
apoptosis by direct cytoplasmic activation of caspase-3
in fibroblasts cultured on collagen type I, where cell
adhesion is RGD-independent . It would be inter-
esting to investigate if generation of RGD peptides by
local proteolytic degradation of ECM components could
bea relevant in vivomechanism in triggering apoptosis
of granulation tissue myofibroblasts. Indeed, protein-
ase-induced ECM degradation leads to apoptosis in
several systems, such as development, neuron death,
and mammary gland involution (for review see ).
Moreover, Okada et al.  have shown that matrix
metalloproteinase genes are highly expressed during
rat skin wound healing and transcripts for gelatinase-
A/metalloproteinase 2 and its activator membrane
type-1 metalloproteinase are specifically detected in
wound stromal cells during the late phases of granu-
lation tissue evolution, i.e., when RGD proteolytic
products are expected to be generated.
CONCLUSIONS AND PERSPECTIVES
More than 25 years after the suggestions that myo-
fibroblasts are responsible for granulation tissue con-
traction , important progress has been made in the
understanding of the processes regulating connective
tissue retractile activity and of the factors controlling
the phenotypic modulation of fibroblasts intomyofibro-
It appears clear that fibroblastic cells can more or
less temporarily acquire contractile properties which
are generally coupled to the capacity for synthesizing
important amounts of ECM components, among which
collagen type I predominates. It remains to be seen
whether all fibroblastic cells contribute equally to the
appearance of myofibroblasts or whether a special sub-
set of these cells is particularly prone to undergo myo-
fibroblastic modulation. In this respect, more and more
evidence is accumulating in the literature suggesting
that fibroblastic cell phenotype is heterogeneous (for
discussion of this point see ).
The participation of neoexpressed specific contractile
proteins, e.g., ?-SM actin, in force generation by myo-
fibroblastic cells is now well accepted, although the
molecular mechanisms of this action are not yet eluci-
SERINI AND GABBIANI
dated. Further work in this direction may result in the
possibility of influencing clinical situations character-
ized by connective tissue retraction.
Myofibroblastic modulation appears to depend on a
complex microenvironmental network in which growth
factors, cytokines, adhesion molecules, and extracellu-
lar matrix components are actively involved. Figure 2
schematizes what we presently think are the main
mechanisms involved in myofibroblastic differentia-
tion, at least during pathological situations. The stim-
ulating activity of TGF? on ?-SM actin and collagen
production is well accepted. TGF? induction by GM-
CSF may represent a promising target for influencing
the development of fibrotic changes at early steps.
Clearly several events in the process conducing to
fibrosis establishment remain unexplained, such as the
mechanisms of early myofibroblast activation, reflected
by the organization of cytoplasmic actin microfila-
ments into stress fibers , the stimuli inducing an
early expression of cellular FN, the factors influencing
the local production and activity of GM-CSF, and the
mechanisms of the permissive action of ED-A FN. In
addition, the fact that integrins have been recently
shown toplay a rolein TGF? activation  opens the
fascinating possibility that they could play such a role
in myofibroblast phenotypic modulation during granu-
lation tissue evolution and fibrosis as well. We are sure
that these and other important points will be eluci-
dated during the next years, but the knowledge accu-
mulated up tonow already allows us todesignate myo-
fibroblastic modulation as an essential step during
wound healing and fibrosis formation and to envisage
research strategies aimed toward the control of myofi-
broblast force generation and of myofibroblast modula-
tion, with a view to influencing the onset and the
evolution of fibrocontractive diseases.
This work was supported by the Swiss National Science Founda-
tion (Grant 31.50568.97). We thank Dr. M-L. Bochaton-Piallat for
the picture presented in Fig. 1, J -C. Rumbeli and E. Denkinger for
photographic work, and M-H. J aspers-Thurre for help in typing the
1.Gabbiani, G., Hirschel, B. J ., Ryan, G. B., Statkov, P. R., and
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Received March 3, 1999
Revised version received April 22, 1999
MYOFIBROBLAST ACTIVITY AND PHENOTYPIC MODULATION