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FOCUS
MEETING ON TISSUE REPAIR, CONTRACTION AND THE MYOFIBROBLAST
Held at the Beau Rivage Hotel, Nyon, Switzerland, in November 2004
Report by Margaret Hughes, ETRS Business Office
A special
book Tissue repair, Contraction and the Myofibroblast containing
more detailed reviews by the invited speakers at the ETRS Focus meeting
in Nyon will be published shortly by Eureka/Landes Bioscience and Springer
Science.
This Focus meeting was held in Nyon, Switzerland, in November 2004 at
the Beau Rivage Hotel – very aptly named. It was an idyllic setting
on the shore of Lac Leman and the weather was kind. The Meeting was organised
by Giulio Gabbiani and Christine Chaponnier of the University of Geneva
together with Alexis Desmoulière of the Victor Segalen University
in Bordeaux France. Fifty-five participants gathered there, twelve delegates
from the host country, nine from USA, eight each from France and the UK,
four from Japan, three each from Spain, Sweden and The Netherlands, two
from Italy and one each from Belgium and Russia.
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Above:
Giulio Gabbiani and Christine Chaponnier of the University of Geneva
with Alexis Desmoulière (left) of the Victor Segalen University
in Bordeaux, France. |
Industry support was
given by L’Oreal Recherche and Pierre Fabre Dermo-Cosmétique,
who were the main sponsors, and also by Carl Zeiss, Promocell, Roche Pharma
and Urgo. Financial support was given by the Swiss National Science Foundation
and the European Tissue Repair Society, the Main Sponsors of the meeting.
The term ‘myofibroblast’ was coined by Majno and Gabbiani
et al in 1971, so, as participants, we felt tremendously privileged to
be welcomed to the meeting by Professor Gabbiani himself, who said: ‘Thirty-three
years after the first description of the myofibroblast, the number of
publications concerning this cell is very impressive and continuously
expanding, and the work on the myofibroblast involves many laboratories
throughout the world. The myofibroblast has been implicated in developmental
and physiological phenomena, as well as in a variety of pathological situations,
going
from wound healing and fibrotic changes to asthma and cancer invasion.
Many aspect of myofibroblast biology have been clarified, such as the
role of TGF-beta in its differentiation and the role of alpha smooth muscle
actin in tension production by this cell. This focus Meeting, for which
we thank the European Tissue Repair Society and all the Sponsors, as well
as the book that originates from it, will concentrate on the most recent
advances in the biology of the myofibroblast differentiation, the interaction
between myofibroblasts of the stroma and malignant epithelial cells and
the possible practical applications of the experimental finding in these
topics. We hope this assembling in the relaxed atmosphere of Nyon, of
most of the leading scientists in this field will be the occasion for
animated and fruitful discussion and for inspiration of the young audience
of students and postdoctoral fellows.’
Esther Middelkoop, President of the European Tissue Repair Society also
warmly welcomed participants to Nyon for the Focus Meeting.
Introduction
Professor Gabbiani noted that considerable progress in understanding pathologies
has followed the recognition of the existence and functions of the myofibroblast
in the contraction of granulation tissue and the remodelling of connective
tissue in wound healing and in some of the fibrotic diseases. He emphasised
the need for further research pathologies, fibroblast and myofibroblast
heterogeneity and on the mechanisms of apoptosis.
There were four sessions covering the topics: General Aspects of Connective
Tissue Remodelling; The Origin of the Myofibroblast; Interactions of Myofibroblasts
with Other Cells; and Organ Fibrosis.
First Session:
General Aspects of Connective Tissue Remodelling
Connective tissue remodelling is crucial for the strength of repaired
tissue and for its appearance in the case of visible tissue. Failure in
the process can result in too little remodelling or too much, both of
these extremes resulting in impaired function. Research to determine the
mechanisms of the complex processes involved is essential in order to
advance the possibilities of righting or preventing the impaired remodelling.
Invited speakers:
Robert Brown, from London, UK in ‘Cytomechanics
in Connective Tissue Repair and Engineering’ emphasised the role
of mechanical forces in the control of connective issue repair, remodelling
and scarring. Most tissues are anisotropic and tissue mechanics is different
from cytomechanics in the complex interplay of cell and extracellular
matrix. The anisotropic properties of collagen substrates can transmit
important cues to the fibroblasts and the use of rectangular collagen
gels to investigate some of these interactions was reported. Anchoring
the gels at the ends produces a stress-shielded zone and the expression
of metalloproteinases 1, 2 and 3, for example, was shown to be greater
in this zone than in the centre. Disorganised structure such as in scar
tissue can affect the signalling cues and hence the remodelling processes.
Further understanding of the interplay of cell-matrix signals and forces
could lead to possible improved control of connective tissue remodelling
and dysfunction.
Robert Brown
Galina
Bogatkevich from Charleston, USA presented studies on ‘Scleroderma
Lung Fibroblasts: Contractility and Connective Tissue Growth Factor’.
In pulmonary fibrosis and systemic sclerosis (SSc, Scleroderma) lung fibroblasts
differentiate to a myofibroblast phenotype which contracts the matrix
more than normal lung fibroblasts. The SSc fibroblasts overexpress the
receptors for TGF-beta which is one of the main profibrotic cytokines.
Activated SSc fibroblasts bind more proteins than normal cells and produce
more extracellular matrix proteins. Their team has shown that the main
receptor for thrombin is also overexpressed in Scleroderma lung tissue
and this expression is also observed in myofibroblasts. Thrombin can stimulate
smooth-muscle actin expression in normal lung fibroblasts and it also
induces the expression of connective tissue growth factor (CTGF) which
plays a key role in contraction and is constitutively upregulatd in lung
fibrosis. In a collagen lattice model, they showed that concentrations
of CTGF from 0–1000ng/ml resulted in dose-dependent increase in
lattice contraction.
Galina Bogatkevich
Richard
Phipps, from Rochester, NY, USA, in ‘Functional assessment
of fibroblast heterogeneity by the cell surface glycoprotein Thy1’
stressed that the heterogeneity of fibroblasts is shown by their morphology,
their functional and proliferative characteristics, their synthesising
capabilities and various cell surface markers. Their team studied the
glycoprotein cell surface marker Thy 1 which is expressed by epithelial
and fibroblastic cells. Fibroblasts can differentiate into myofibroblasts
or into adipocyte-like cells and interest in the latter arises in relation
to Graves’ ophthalmopathy in which the accumulation of fat can push
the eye out of orbit. They reported studies of human orbital cells and
various female reproductive fibroblasts which, using magnetic beads, separated
cells into Thy1- and Thy1+ subsets. In cultures of both human orbital
and myometrial fibroblasts the Thy1- cells differentiated into lipofibroblasts
and the Thy1+ cells into myofibroblasts. In Thy1+ fibroblasts Src kinase
activity has been shown to be involved in the assembly of focal adhesions
by thrombospondin. It is suggested that it is important to identify the
function and signalling pathway used by Thy1 because regulation of this
might eventually lead to treatment for diseases such as Graves’
ophthalmopathy and idiopathic pulmonary fibrosis.
Richard Phipps
Short
communications:
In his talk ‘Masters and servants of the force: focal adhesion size
controls recruitment of alpha smooth muscle ac-tin (SMA) to stress fibers’:
Boris Hinz, from Lausanne, Switzerland, emphasised that mechanical stress
is a crucial factor for modulating fibroblasts into myofibroblasts. His
group had investigated the size of focal adhesions (FAs) which are longer
(‘supermature’ FAs) in alpha-SMA positive fibroblasts and
are responsible for communicating the level of extracellular matrix stress
to the cytoskeleton, thus controlling myofibroblast differentiation. They
had determined the minimal tension at individual FAs that was necessary
for alpha-SMA recruitment.
One example of imperfect remodelling is scarring of the cornea which results
in an opaque membrane and leads to visual impairment. Annegret Dahlmann,
from the Institute of Ophthalmology in London, presented ‘PDGF-stimulated
Fibroblast Matrix Contraction is Mediated through Direct Stimulation of
cell protrusion and ruffling activity.’ After corneal injury PDGF
is up-regulated in the tear fluid and Dahlmann’s group used the
fibroblast-populated collage matrix (FPCM) model to investigate the role
of PDGF in matrix remodelling by human corneal fibroblasts (HCF). They
showed similarities between the formation of circular ruffles by HCF in
2D cultures and cell protrusive activity and macroscopic contraction in
the 3D model. Both processes were shown to involve the PDGF-BB receptor,
the tyrosine kinase receptor and certain molecules (Arp2/ 3) complex,
Scar and Wasp proteins) essential for filamentous acting assembly.
Modulation of the fibroblast to myofibroblast and the precise roles of
both these cell types in matrix remodelling have been much discussed.
In ‘Collagen Organisation Contracts Wounds’ Paul Erhlich,
from Hershey, PA, USA, described the use of two variations of an FPCL
model, with gels cast at the same time, lattices released one hour after
casting called free floating (FF) FPCKL, or released four days after casting
called ‘attached delayed release gels (ADR) FPCL. Contraction of
the former is complete in two days without any cell contraction and shows
organisation of collagen, but no alpha-SMA staining. (ADR) FPCL contraction
was completed in two hours (in contrast to two days) and showed alpha-SMA
staining, but no organisation of collagen. Vanadate-treated FF FPCL contract
with collagen organisation, but treatment of ADR FPCL impaired both gel
contraction and collagen organisation. The group also investigated the
contraction process in rat wounds with and without vanadate which closed
at the same rate by day 7 and rat granuloma which is exclusively populated
with myofibroblasts after three weeks. Removal of wound fluid daily for
three days led to the replacement of the myofibroblasts by fibroblasts
in parallel with increased organisation of collagen fibres. They
propose that the functions of the myofibroblast are to counteract transient
changes in tension within granulation tissue and to prepare cells to enter
apoptosis, but the wound contraction advances by fibroblast organisation
of collagen in the absence of any cell contraction.
Second session:
The Origin of the Myofibroblast
An editorial in Laboratory Investigation in 1990 commented that ‘The
mysteries of myofibroblasts are those of both space and time. In addition
to explaining the appearance of myofibroblasts, investigators are also
faced with the equally challenging problem of accounting for their disappearance
(e.g., on the completion of wound healing).’ Speakers in the second
session addressed the problem of the ‘Origin of the Myofibroblasts’.
Invited speakers:
Sabrina Mattoli, from Basel, Switzerland, reported on
‘Tissue Repair in Asthma: the Origin of Airway Subepithelial Fibroblasts
and Myofibroblasts’. In asthmatic patients there is remodelling
of the normal bronchial architecture with overdeposition of collagen leading
to airway obstruction. Under the influence of the chemokine SLC/C CL21
circulating fibrocytes are induced to migrate into the airways. Levels
of some cytokines are raised in the airways of asthmatic patients and
the TGFbeta 1 produced by epithelial and inflammatory cells may stimulate
the differentiation of the fibrocytes into myofibroblasts. Their study
showed that exposure to allergy induces the accumulation of fibrocyte-like
cells that are CD34-positive, express collagen I and alpha SMA. In a mouse
model of allergic asthma they labelled circulating fibrocytes and tracked
them, showing that they did accumulate in the bronchial tissue and differentiated
into myofibroblasts. By six to eight weeks of chronic exposure to low-level
allergen there were structural abnormalities of the airways comparable
to those of human asthma, thus indicating that circulating fibrocytes
may be myofibroblast precursors and contribute to the subepithelial fibrosis
of the airway mucosa.
Sabrina Mattoli
Sem
Phan, from Ann Arbor, MI, USA in his talk ‘FIZZy Alveolar
Epithelial Cells Induce Myofibroblast Differentiation’
described the DNA microanalysis of lung gene expression profiles in a
rat model of pulmonary fibrosis. Many genes were up- or down-regulated
twofold or more, with the FIZZ1 (Found in Inflammatory Zone) gene being
upregulatd 25-fold. FIZZ1 was expressed by alveolar epithelial cells (AEC)
and stimulated the differentiation of fibroblasts into myofibroblasts,
the expression of alphaSMA and type I collagen. In an in vitro model they
cocultured AEC and fibroblasts and found increased collagen expression.
The addition of anti-TGFbeta1 did not inhibit this collagen expression.
IL-4 and IL-13 have a role in the induction of FIZZ1 expression in alveolar
epithelial cells and knockout mice for these two cytokines have
shown reduced FIZZ1 expression and reduced fibrosis. The differentiation
to myofibroblasts acts through a FIZZ1 receptor on fibroblasts, but the
nature of the receptor molecule has not yet been identified.
Sem Phan
Treatment
of end-stage renal disease involves frequent haemodialysis. An alternative
treatment, which the patient can easily carry out at home, is peritoneal
dialysis (PD). However, one of the complications that may arise is peritoneal
membrane fibrosis. Manuel López-Cabrera from Madrid,
Spain, reported on ‘Epithelial to Mesenchymal Transition of Mesothelial
Cells as a Mechanism Responsible for Peritoneal Membrane Failure in Peritoneal
Dialysis Patients’. Long-term use of PD may be associated with extracellular
matrix deposition and fibrosis of the peritoneal membrane, an increased
number of peritoneal capillaries and the loss of the mesothelial layer.
The group isolated mesothelial cells from the PD effluents of 54 patients
and, using an in vitro scratch model, showed a change in morphology of
the cells to a spindle shape at the edge of the scratch. The effluents
also stimulate the production of VEGF and TGF-beta by the mesothelial
cells. The latter induces the transition of epithelial to mesenchymal
cells which are responsible for the overproduction of collagen I and fibronectin,
leading to fibrosis. How to prevent or reverse the transition should be
a target for potential therapies.
Manuel López-Cabrera
Short
communications
Perfect wound healing would result in regeneration rather than repair
and this is known to occur for cutaneous wounds in early gestational foetuses.
Kerstin Rolfe from Northwood, UK presented work on ‘Fetal cutaneous
myofibroblasts – fact of fallacy?’ and looked at the rôle
of TGF-beta1 which is a pro-fibrotic factor. Recent data has shown that
this growth factor is present in foetal wounds, but for shorter periods,
and they compared early foetal fibroblasts (<14 week EGA) with postnatal
dermal fibroblasts.
Stimulation with 5 ng/ml TGF-beta 1 induced 40% of human foetal fibroblasts
to differentiate into myofibroblasts. Timing for the appearance and disappearance
of myofibroblasts in response to TGF-beta 1 was different in the two cell
strains, with appearance earlier (peak day 2– 3) in foetal cells
than in postnatal cells (peak day 6–8). The induction seen in foetal
cells was blocked by a p-JNK inhibitor, but this did not happen in postnatal
cells. With respect to collagen synthesis in response to TGF-beta1, foetal
fibroblasts produced about 50% of the collagen synthesised by postnatal
fibroblasts. They suggested that a prolonged response to this cytokine
might be responsible for the more fibrotic nature of postnatal wound healing.
More uncontrolled remodelling of extracellular matrix occurs in cases
of ‘overscarring’ such as hypertrophic scars and keloids.
Magda Ulrich, from the Association of Dutch Burn centres, addressed this
problem in ‘Myofibroblasts in Hypertrophic Scars, Where do they
come from?’ The appearance of myofibroblasts in the wound is triggered
by the entry of TGF-beta released from platelets and inflammatory cells.
In partial-thickness wounds the myofibroblasts disappear after the wound
is closed, probably by apoptosis, but in full-thickness wounds they remain
present and healing is often accompanied by hypertrophic scars. Their
group hypothesised the presence of a myofibroblast sub-type which is less
susceptible to apoptotic triggers, and thus sustains the process. In the
case of full-thickness wounds, fibroblasts are recruited from the subcutaneous
fat as well as from the dermis. The group compared
the expression levels of proteins which are thought to play a role in
hypertrophic scar formation in fat fibroblasts (fib), dermal fibroblasts
(dab) and scar-derived fibroblasts (sib). Cultured fFB showed increased
expression of alpha-SMA, collagens I and III and TIMPs and decreased expression
of MMPs in comparison with dFBs. The fFB also showed a collagen cross-linking
profile similar to that of bone collagen where the collagen fibres are
less accessible to proteases. The expression pattern of fFB resembles
that of sFB and suggests that the myofibroblasts from the subcutaneous
fat may play a rôle in the formation of hypertrophic scars. The
remodelling process in the lungs of asthmatic patients leads to sub-epithelial
fibrosis and cells with the phenotypic appearance of myofibroblasts are
thought to be responsible. Westergren-Thorsson and colleagues from Lund,
Sweden reported on this cell type in the ‘Presence of activated
mobile fibroblasts in bronchial alveolar lavage from mild asthmatic patients.’
They detected this phenotype in bronchoalveolar lavage fluid (BALF) from
30% of the asthmatic patients (n = 13), but found no cells in the fluid
from control subjects (n = 17). These myofibroblasts showed increased
migratory ability and an increase in the production of several types of
proteoglycans. By adding biglycan to normal fibroblasts it was possible
to mimic the migratory type of myofibroblasts they had found in BALF.
The BALF myofibroblasts also showed an increase in heat-shock protein
20. They propose that biglycan and mesenchymal progenitor cells could
be potential biomarkers for sub-epithelial fibrosis formation.
Third session:
Interactions of Myofibroblasts with Other Cells
The complex process of wound healing involves several types of cells which,
in normal wound healing, are drawn
into the wound site in an orderly sequence, although several types may
coexist in certain phases of healing, each fulfilling their particular
functions. The third session of the meeting focused on the interactions
of myofibroblasts with other cells.
Invited speakers:
Raghu Kalluri,
from Boston, MA, USA spoke on ‘Experimental Models to Study the
Origin and Role of myofibroblasts in Renal Fibrosis’. There are
several diseases that cause end-stage renal failure and lead to a scarred
fibrotic kidney. Changes in the kidney are similar to those reported in
earlier sessions of this meeting. The first step in the transition of
an epithelial cell to a fibroblast type appears to be the destruction
of the basement membrane, and TGFbeta is the main factor in the differentiation
to a myofibroblast type. There are several endogenous molecules that can
counteract the action of TGF-beta and these include BMP-7 (bone morphogenetic
protein-7). Renal BMP-7, which decreases after acute ischaemic attacks,
is antiapoptotic to epithelial cells, anti-inflammatory and regulates
the cellular transition of epithelial to mesothelial cells (EM transition)
and can even reverse this in adult cells inducing M-E transition, e.g.,
restoring cadherin expression and decreasing the production of collagen.
It does this by counteracting TGF-beta-induced Smad-dependent signalling.
The group did in vivo experiments on mouse kidney and showed reversal
of lesions with BMP-7 as they also did for carbon tetrachloride-induced
liver injury and urethral obstruction. BMP-7 is not expressed in the liver,
but hepatocytes have receptors for it. Recombinant BMP-7 is currently
being developed for clinical use.
Raghu Kalluri
Marc
Mareel,
from Ghent, Belgium reported on the ‘Pro-invasive Molecular Cross-signalling
between Cancer
Cells and Myofibroblasts’. They studied the invasion of cells into
a collagen Type I gel and showed that myofibroblasts derived from colon
cancer tissue stroma stimulate the invasion of colon epithelial cancer
cells into the matrix. Two molecules secreted by the myofibroblasts, scatter
factor/hepatocyte growth factor and the extracellular matrix molecule
tenascin-C, were determined to be the pro-invasive agents. Tenascin-C
is expressed in myofibroblasts, but not in normal fibroblasts. When Sf/HGF
plus tenascin C were added they stimulated colon cancer cells to invade
similarly to Conditioned Medium from the myofibroblasts, producing similar
changes in morphology. The group also investigated the roles of RAC, Rho
and Ncadherin in these processes.
Marc Mareel
The tumour
microenvironment was also investigated by Fernando Vidal-Vanachlocha’s
group (Leioa, Spain) who reported on the ‘Pro-angiogenic Implications
of Hepatic Stellate Cell Transdifferentiation into Myofibroblasts Induced
by Tumour Microenvironment’. He commented that most tumours contain
between 50 and 90% of normal cells. Liver sinusoids are a common site
for metastasis and the peri-sinusoidal hepatic stellate cells can be activated
by invasion- and proliferative-factors released by cancer cells and differentiate
into myofibroblasts. Cancer cells release angiogenic factors and inhibit
angiogenic inhibitors. The myofibroblasts which are present in the avascular
micro-metastases provide a supporting stroma for the recruitment of endothelial
cells present in the tumour microenvironment to proceed to neoangiogenesis.
Fernando Vidal-Vanachlocha
Short
communications
In some cases parallels have been drawn between the processes of wound
healing and the formation of tumours, Bert Nederbragt from Utrecht, Netherlands
presented ‘Cartilage formation as a differentiation route of myoepithelial
cells in canine mammary tumours: involvement of myofibroblasts?’
They investigated the composition of the extracellular matrix (ECM) in
the stroma of these tumours to obtain an indication of the types of different
cells involved. They carried out semiquantitative PCR analyses of different
types of normal tissue and tumours and showed expression of aggrecan and
collagen II in complex and mixed tumours and in epitheliomas, but also
of collagen II in simple tumours. In the spindle cells of complex tumours
there was expression of cytokeratin 14, alpha SMA and vimentin and the
group suggest that this may indicate that the cells are modified myofibroblasts.
Sayuri Anan from Tokyo, Japan used a newly established
ICR-derived mouse strain with glomerulonephritis (ICGN mice) to study,
‘Increased Smad4 mediated acceleration of TGF beta1 signalling and
myofibroblastic differentiation in tubulointerstitial cells in kidneys
of hereditary nephrotic mice.’ As the hyper-accumulation of ECM
components leads to fibrosis the group investigated the expression and
localisation of TGF-beta1, TGF-B1 type 1 receptor (TGFR1), and bone morphogenetic
protein (BMP)-7 which has a negative role in the regulation of TGF-beta,
using immunohistochemical staining and Western blotting. In tubular epithelial
cells high expression levels of TGF-beta1 and TGFR1 were detected compared
with those in control mice. BMP-7 decreased with the progression of renal
disease and this could lead to uncontrolled levels of TGF-beta 1. These
findings may give a pointer to pathogenic mechanisms involved in human
idiopathic nephrotic syndrome.
Anders Malmström from Lund, Sweden presented studies showing that
‘Activated fibroblasts are required for extracellular matrix remodeling
in the human cervix.’ They hypothesised that fibroblasts from cervices
at different stages of remodelling are of different phenotypes. They studied
cervical fibroblasts cultured from non-pregnant women, women at 36 weeks
of pregnancy and postpartal women, investigating levels of cytokine and
metalloprotease expression and production. Levels of interleukin 6 and
interleukin 8 were three to six times higher and metalloproteases 1 and
3 were three times higher in postpartal fibroblast cultures than in normal
cervical fibroblast cultures, while intermediate levels of these molecules
were seen in fibroblasts from 36-week pregnancy. This suggests that remodelling
of the cervix is carried out by transient activation or differentiation
into fibroblast phenotypes usually found in inflammatory conditions.
Fourth session: Organ Fibrosis
Invited speakers
John Iredale, from Southampton, UK, presented ‘Matrix Metalloproteinases,
Tissue Inhibitors of Metalloproteinase and Matrix Turnover and the fate
of Hepatic Stellate Cells’. The hepatic stellate cell (HSC) produces
both Metalloproteinases, Collagens I and III, and a Tissue Inhibitor of
Metalloproteinase (TIMP1). The balance of TIMP and MMPs determines the
resolution of liver fibrosis, although this may be limited by the degree
of matrix cross-linking. Recovery from fibrosis is related to the apoptosis
of the HSC-expressing TIMP which allows the degradation of the matrix.
Survival factors which may be derived from inflammatory cells and injured
hepatocytes allow the hepatic stellate cells to persist for longer without
going into apoptosis and this results in more prolonged fibrosis. The
group used rat models with carbon tetrachloride-induced injury and showed
that when the administration of CCl4 was stopped after six weeks, the
rats recovered within 28 days. With more prolonged administration for
twelve weeks, the levels of TIMP still decreased, but not to the same
degree. They also investigated the rôle of macrophages which are
involved both in injury and in repair.
Depletion of macrophages in advanced fibrosis led to less scarring and
reduced numbers of myofibroblasts. When
depleted during the recovery stage there was less degradation of the matrix.
Increased understanding of the fate or
regulation of the hepatic stellate cells may point the way to the development
of antifibrotic agents.
Paul Noble, New Haven, USA, presented an account on ‘Innate Immune
Regulation of Lung Injury and Repair’.
There is less understanding of the rôle of the immune response in
non-infectious lung injury than there is for response to infectious agents.
The group identified a rôle in the regulation of lung injury and
fibrosis for the hyaluronic acid (HA) receptor CD44. In irreversible lung
fibrosis there is accumulation of HA. Fragmentation of HA induces the
expression of a number of genes in inflammatory macrophages. In CD44-deficient
mice there is persistent inflammation and failure to clear the accumulation
of HA fragments and other ECM degradation products at the injury site
and the mice tended to die due to overwhelming inflammation. The group
also investigated another surface receptor CXC chemokine receptor 3 (CXCR3)
which is the receptor for a number of IFN-gamma inducible chemokines.
CXCR3-deficient mice suffered increased fibrosis and impaired production
of IFN-gamma which has a protective effect against fibrosis and they had
a greater mortality rate than wild mice. Administration of exogenous IFN-gamma
partly resolved the fibrosis.
Injury to the eye resulting in scarring leads to the loss of visual acuity
due to the opacity of the corneal scar. James Jester
(Dallas USA) spoke on ‘An eye on Repair: Myofibroblasts in Corneal
Wounds’. Methods, such as radial keratotomy and excimer laser keratectomy,
to improve refractive errors in the eye can lead to a corneal haze. Repair
is
dependent on myofibroblasts migrating into the wound site to form a contractile
network, but they can also be a source
of light scattering that causes a corneal haze. The group studied different
types of wounds such as scrape injury, excimer laser and incisional wounds
to investigate the factors involved in fibrosis. The differentiation of
keratocytes to the activated fibroblast phenotype depends in part on TGF-beta.
Production of this factor can be upregulated by the cell’s own secretion
of PDGF and the signalling cascade involves integrins along with these
two growth factors. Treatment of corneal wounds with anti-TGF-beta inhibited
the fibrosis and the resulting corneal haze.
James Jester
Short
communications
In her presentation ‘Contribution of interstitial fluid flow to
fibroblast alignment and differentiation’ Melody Swartz from Lausanne,
Switzerland described the use of a tissue culture model of interstitial
flow in 3D cultures to examine the influence of biophysical changes on
fibroblast behaviour. Even very low interstitial flows (1–10 microns/
sec) caused both dermal and lung fibroblasts to align themselves perpendicularly
to the direction of flow and to align the collagen fibres, whereas fibroblasts
in fibrin gels did not align. Increased expression of alpha-smooth muscle
actin indicated cells were differentiating to a myofibroblast phenotype.
As interstitial flow in vivo increases in wound healing and in some pulmonary
disorders these studies indicated that the flow may contribute to interstitial
fibrosis. Margaret Hughes from Oxford, England presented the Oxford experience
of ‘Pharmacological and Mechanical Effects on Collagen Lattice Contraction’.
High concentrations of minoxidil at 0.5 to 4 mM inhibited contraction
of dermal fibroblasts from a 74-year old subject, but had no effect on
contraction by neo-natal foreskin fibroblasts. The major metabolite minoxidil
glucuronide did not affect contraction, but the metabolite minoxidil sulphate
was a stronger inhibitor than minoxidil. Tamoxifen inhibited
contraction at 10–100 micromolar, and an extract from Chromolaena
odorata, used for soft-tissue wounds in Vietnam inhibited contraction
at doses from 50–200 microgram/ ml. A regime of intermittent radiant
warming for an hour three times a day, as used to treat ulcers in the
clinic, caused a rise in the temperature of the medium from 33.2 to 36.2°C
after one hour. After one week this led to significantly faster contraction
and increased proliferation in the 3D collagen matrix.
Transdifferentiation of hepatic stellate cells into myofibroblasts leads
to liver fibrosis. Rebecca Wells, from Philadelphia, USA, presented work
on ‘The rôle of matrix stiffness in hepatic stellate cell
(HSC) activation and liver fibrosis’. Hypothesising that the mechanical
properties of the surrounding matrix influenced the transdifferentiation
and that an increased elastic modulus may perpetuate the transdifferentiation,
they used an in vitro system of primary rat HSC on supports with different
elastic moduli from 100 to 12K Pa to test this. Supports were coated with
very thin layers of Type I collagen, fibronectin or MatrigelTM. Irrespective
of the type of coating, as substrate stiffness (elastic modulus) increased
there was an increasing degree of transdifferentiation as determined by
morphology and alpha-smooth muscle actin expression. In vivo they showed
that the elastic modulus of CCl4-treated rats increased progressively
more than threefold over ten weeks, and suggest that alterations in liver
stiffness are key factors driving fibrosis.
Esther Middelkoop,
President of the European
Tissue Repair Society, who welcomed participants to
Nyon for the Focus Meeting.
Posters
Twenty-four posters were exhibited, with a designated session for discussion
with authors on the first day and considerable interest and discussion
during coffee and lunch breaks.
Social Event
A very relaxed reception was hosted by the Mayor of Nyon at the magnificent
historic Chateau de Nyon followed by a banquet at the Hotel Beau-Rivage.
Reference:
Majno G, Gabbiani G, Hirschel BJ, Ryan GB and Statkove PR: Contraction
of granulation tissue in vitro: similarity to smooth muscle.
Science 1971; 173: 548–550.
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