|
HEALING
IN SPECIFIC TISSUES
CARDIAC TISSUE REPAIR USING SKELETAL MUSCLE CELLS
Ch. Rajnoch1, P. Bruneval2 and A.Carpentier2,
1: Laboratory of Cardiac Grafts & Prosthesis, Broussais Hospital,
Paris, France, 2: European Hospital Georges Pompidou, Paris, France.
Introduction: Cardiac muscle in vertebrates is not capable
of regeneration, when destroyed by injury or disease. Cell therapy is,
therefore, a promising technique to treat end-stage heart failure. The
aim of this study was to find the optimal time for cell implantation after
myocardial infarction.
Methods: A myocardial lesion was induced by cardiotoxin injection through
a left thoracotomy in 32 rats. Histological analysis of frozen heart sections
was carried out at 24 hours, 3 days, 1 week, 2, 3 and 4 weeks. The same
lesion was then created in 40 syngeneic Lewis rats, followed by implantation
of one million skeletal muscle cells at one week (n=19) or at two weeks
(n=19) post-myocardial injury. Frozen heart sections were analysed with
a skeletal muscle specific myosin heavy chain antibody, after three weeks
of cell therapy.
Results: The cardiotoxin injection produced a well-delineated transmural
myocardial infarction. Cell implantation at two different time points
corresponded to mild inflammation in the first group and predominant fibrotic
lesions in the second group. Lewis rats, which survived the double thoracotomy
(n=13), showed that one-week-old lesions contained large areas of myocytes
and myotubes, while few single skeletal muscle cells were found in two-week
old lesions.
Conclusion: Implantation of skeletal muscle cells into the myocardium
after infarction is dependent on the state of tissue repair and should
be performed early after injury before significant fibrosis has developed.
LIVER FIBROSIS IS DRAMATICALLY REDUCED IN CARBON TETRACHLORIDE
INJURED JunD KNOCKOUT MICE
David E. Smart, Michael J.P. Arthur, Jonathan Weitzman*, Moshe Yaniv*
and Derek A. Mann. Liver Group, University of Southampton, Southampton,
UK and *Institute Pasteur, Paris, France.
Activation of hepatic stellate cells (HSC) to myofibroblast-like
phenotype is the pivotal event in hepatic wound healing and fibrosis.
There is considerable interest in determining the molecular events that
regulate HSC activation. We have recently identified JunD as an important
regulator of the phenotype of the activated HSC. The Jun family (c-Jun,
JunB and JunD) are key components of the dimeric transcription factor
AP-1. The DNA binding activity of AP-1 is induced during HSC activation
and serves to regulate the expression of at least two pro-fibrogenic genes,
TIMP-1 and IL-6. JunD is the predominant AP-1 factor expressed in activated
HSC and is responsible for regulation of TIMP-1 and IL-6 gene transcription.
To further investigate a role for JunD in hepatic fibrosis we have studied
the fibrogenic response of JunD-/- mice to liver injury. JunD-/- and wild
type control mice were treated with carbon tetrachloride for eight weeks
in order to induce a chronic liver injury. Liver sections from culled
mice were analysed histochemically for the extent of fibrosis and collagen
deposition. JunD-/- mice displayed a dramatically attenuated phenotype,
with substantially reduced fibrosis relative to that obvserved in wild
type mice. Reduced levels of collagen deposition and numbers of activated
hepatic stellate cells relative controls was observed in all JunD-/- mice.
We conclude that JunD is a regulator of the expression of profibrogenic
genes in activated HSC and plays a critical role in the fibrogenic process
in vivo. JunD should therefore now be considered as an important target
for drug design.
ANTI TGF-B2 ANTIBODY (CAT-152) PREVENTS SCARRING AFTER
EXPERIMENTAL GLAUCOMA FILTRATION SURGERY
A.L.Mead1,2, M.F.Cordeiro1,2, I.K.Anderson3 and P.T. Khaw1,2, 1:Wound
Healing Unit, Institute of Ophthalomology, London, UK, 2: Department of
Glaucoma, Moorfields Eye Hospital, London, UK, 3: Cambridge Antibody Technology,
The Science Park, Melbourn, Cambs, UK
Purpose: We have recently demonstrated that a novel human
monoclonal antibody to Transforming Growth Factor b2, CAT-152* improves
glaucoma filtration surgery outcome by inhibiting subconjunctival fibrosis.
This was first shown in our animal model of aggressive scarring, using
a regimen of both intra- and post-operative subconjunctival injections
of CAT-152, and is now in clinical trial. The purpose of this study was
to compare CAT-152 with a current gold standard anti-scarring agent, the
antimetabolite 5-fluorouracil (5-FU) and to determine if the timing of
CAT-152 application affected its efficacy.
Methods: In our model of glaucoma filtration surgery (i) 30 rabbits were
randomly allocated to receive intra-operative subconjunctival injections
(100µl) of CAT-152 (1mg/ml), PBS or no treatment (ii) 24 rabbits
received CAT-152, 5FU (50mg/ml) given as a post-operative course ( 7 injections
between days 2-14) or no treatment. Bleb characteristics, successful drainage
and local reaction to treatment were assessed.
Results: Post-operative CAT-152 significantly improved surgical outcome
compared to 5-FU and control (log rank p= 0.009). Mean bleb survival (days):post-operative
CAT-152 24.6, 5-FU 19.5 and control 16.8. Bleb area and height were significantly
increased by post-operative CAT-152 (p<0.05). Intra-operative treatment
alone did not improve outcome. Treatments were well tolerated.
Conclusions: Post-operative administration of CAT-152 significantly improves
surgical outcome compared to the gold standard anti-scarring agent 5-FU.
A delayed delivery regimen when scarring is occurring may still be effective.
These findings suggest that CAT-152 may have a potential therapeutic role
as a post-operative agent to prevent subconjunctival scarring after glaucoma
filtration surgery.
INTEGRIN anb3 REGULATES HEPATIC STELLATE CELLS
X. Zhou, J.P. Iredale and R.C. Benyon, Southampton University, UK
Integrins regulate a variety of cellular functions. Following
liver injury, hepatic stellate cells (HSC) become activated to transform
to proliferative myofibroblasts which overproduce extracellular matrix
resulting in liver fibrosis. We have examined the role of anb3 integrin
in regulating rat HSC proliferation and fibrogenic activity.
Western blotting and Taqman RT-PCR showed that HSC increasingly express
integrin anb3 as they transform to myofibroblasts. A 24 hr treatment with
the integrin anb3 antagonist echistatin, 100nM, reduced the proliferation
rate of HSC plated on plastic by 73.5%, by 3H-thymidine incorporation
assay. Western blotting showed reduced activation of MAP kinase p42 and
p44 isoforms. Echistatin treatment also inhibited HSC expression of mRNA
for a variety of profibrogenic molecules: TIMP-1 was reduced by 97.1%;
procollagen-1 by 72.0%; CTGF by 97.4% and TGFb1 by 93.0% when examined
by Taqman RT-PCR. This was paralleled by an increase in the collagenolytic
activity present in pooled cell lysates and supernatants. Secretion of
the gelatinase MMP-9 was also increased, but MMP-2 and MT1-MMP were unchanged
by echistatin treatment. CAT reporter assays showed TIMP-1 promoter expression
was decreased by 68.7% after 56 hours treatment with echistatin.
These findings suggest that integrin anb3 plays an important role in the
interactions between HSC and extracellular matrix which are required for
HSC activation and proliferation. Integrin regulation of HSC might be
important under conditions of fibrogenesis in the liver where there are
major qualitative and quantitative changes in the extracellular matrix
including enhanced deposition of anb3 ligands such as fibronectin, vitronectin
and thrombos-pondin.
HUMAN OCULAR MYOFIBROBLASTS ARE ACTIVE PRODUCERS OF
MMP-1 AND MMP-2: A ROLE IN CONJUNCTIVAL WOUND HEALING
Tina T.L. Wong1, Julie T. Daniels1, Gillian Murphy2 and Peng T. Khaw1,
1: Wound Healing Research Unit, Department of Pathology, Institute of
Ophthalmology and Moorfields Eye Hospital, London, U.K, and 2: School
of Biological Sciences, University of East Anglia, Norwich, UK
Background: The myofibroblast is responsible for the generation
of contractile forces required for wound closure. Continual presence of
myofibroblasts may result in aberrant wound healing. Matirx metalloproteinases
(MMPs), are a group of proteolytic enzymes that degrade and remodel collagen
and matrix components.
Methods: We used the stress-relaxed collagen lattice contraction assay
to investigate the role of MMP production by myofibroblasts. The effect
of broad-spectrum MMP inhibitor Galardin, on MMP activity was also investigated.
Gel Diameters were recorded at specific time points, culture supernatants
were analysed for MMP activity using gelatin zymography and the protein
with Western blot. MMP mRNA expression was quantitated using real-time
Taqman PCR. Colocalisation of MMPs with ASMA was determined by immunofluorescent
staining. Electron microscopy of the cell ultrastructure was determined.
Results: Western blotting confirmed the presence of MMP-1 and MMP-2 and
zymography demonstrated decreasing activity in a dose-dependent manner.
Galardin significantly decreased gel contraction compared with controls
(p< 0.001). Confocal microscopy revealed cells co-staining for ASMA
and MMPs-1 and MMP-2. mRNA expression of MMP-1 and MMP-2 was significantly
reduced in the presence of Galardin.
Conclusions: This study indicates that myofibroblasts are active producers
of MMPs in vitro. MMPs-1 and 2 may initiate and maintain remodelling respectively.
Production of MMP-2 by myofibroblasts may play an important role in the
remodelling of a conjunctival wound and mechanical stress may be an important
component of this cell behaviour.
HEPATOCYTE GROWTH FACTOR (HGF) DELAYS EPITHELIAL WOUND
HEALING AND INCREASES THE PROLIFERATION OF KERATOCYTES
L.M. Carrington and M.E. Boulton, Department of Optometry and Vision
Sciences, Cardiff University, Wales, UK
Aim: To investigate the effect of exogenous HGF on corneal
re-epithelialisation and early stromal wound healing events using serum-free
organ and cell culture.
Method: hrHGF was added at 0.1-100ng/ml to primary keratocytes cultures
and wounded bovine corneas in organ culture. Cell and organ cultures were
maintained for up to five days, proliferation and phenotype was assessed
in the keratocyte cultures while re-epithelialisation, epithelial cell
morphology, keratocyte proliferation and stromal cell phenotype were examined
in organ culture.
Results: HGF significantly delayed epithelial wound healing; 73±6%
healed at 48 hr for controls compared to 25±12% at 100ng/ml HGF
(p<0.01). This correlated with the degree of proliferation seen at
the leading edge. By contrast, HGF stimulated keratocyte proliferation
to double that seen in control cell cultures by day 5 (p<0.01). HGF
(100ng/ml) was seen to effect the spacial location of keratocytes, increasing
numbers immediately below the wound at 24hr (a 21% increase compared to
wounded controls p<0.01). No effect on myofibroblast differentiation
was noted in HGF-treated corneas.
Conclusion: This is the first study to show delayed re-epithelialisation
and increased proliferation of keratocytes in the presence of HGF.
|
