|
RESEARCH
STUDIES
THE EFFECT OF KGF-2 ON THE PROLIFERATION OF HUMAN KERATINOCYTES
IN VITRO
Y.H. Yang, M.A. Hughes and G.W. Cherry, Wound Healing Institute, The
Churchill Hospital, Oxford, UK
Purpose: In vitro studies have shown that Keratinocyte
Growth Factor-2 (Repifermin, KGF-2) has a proliferative effect on neonatal
foreskin keratinocytes. The purpose of this study was to investigate the
proliferative effect of KGF-2 on keratinocytes from an adult subject.
Methods: Standard medium was Keratinocyte Growth Medium without bovine
pituitary extract, hydrocortisone or Epidermal Growth Factor (EGF). Keratinocytes
cultured from a 58-year old subject were seeded at 2 x 104 in 32 mm dishes
in standard medium with rEGF. After 24 hours, medium was changed to standard
medium with or without KGF-2 at 4, 16, 125 and 500 ng/ml. Standard medium
with EGF was used as a positive control. The growth of keratinocytes was
monitored by the MTT assay and by photographs at day 3, 5 and 7. Each
treatment was performed in triplicate.
Results: KGF-2 concentrations from 4 to 500 ng/ml all showed a significant
proliferative effect on days 3 to 7 compared to the negative control.
By day three the response of keratinocytes in KGF-2 was 1.5 to 2.5-fold,
by day five 3 to 5-fold, and by day seven, 3 to 12-fold, relative to the
negative control. The optimal response was at the 125 ng/ml concentration
on day seven. Cell proliferation was also significantly higher at all
KGF-2 concentrations compared to the EGF positive control.
Conclusion: KGF-2 exerted a significant effect on the proliferation of
adult keratinocytes that was higher than that of the EGF standard. This
supports the positive clinical results in a venous ulcer wound-healing
study presented at the International Wound Healing meeting in 2000.
INDUCTION OF ARGINASE IN WOUND DERIVED FIBROBLASTS-A
NOVEL PHENOTYPIC CHARACTER
Maria Witte, University of Tübingen, Germany
Introduction: Wound fibroblasts (WFB) are induced to synthesize
nitric oxide from L-arginine. Little is known on the alternative pathway
of L-arginine via arginase. Theoretically arginase leads to proline and
polyamine formation, both important for cell proliferation and collagen
synthesis. There are two distinct arginase isoforms (AI and AII). We investigated
this alternative L-arginine metabolism by WFB compared to normal dermal
fibroblasts (NFB) and its regulation by TGF-b1.
Methods: Male Lewis rats were used to extract cells. NFB were harvested
from uninjured skin and wound fibroblasts from subcutaneously implanted
polyvinylalcohol sponges seven and ten days after wounding. Cells were
used between passage 1 and 5. Using arginase I and II specific primers,
cDNA was amplified using semiquantitative RT-PCR. Results were normalised
for GAPDH as housekeeping gene.
Results: WFB from day 7 and 10 post-wounding expressed increased amounts
of AI compared to NFB. AII expression was in general significantly lower
compared to AI, but not different between NFB and WFB. TGF-b1 at 5ng/ml
significantly induced AI expression in WFB, lower concentrations however
(0.1ng and 1ng) decreased AI expression. TGF-b1 did not affect AII expression
in either NFB or WFB.
Conclusion: The WFB phenotype is characterised by a distinct induction
of the Arginase I isoform expression. In view of the importance of the
arginase pathway for wound healing, this phenotype needs further investigation.
Supported by a grant from the DFCG (WI 1499/2-1)
KERATINOCYTE ACTIVATION IN ACUTE AND CHRONIC WOUND
IS CHARACTERISE BY ALTERED EXPRESSION OF KERATINS (K6, K16 AND K17)
G.K. Patel1,2, A.Y. Finlay1, K.G. Harding2 and P.E. Bowden1, 1: Department
of Dermatology, and 2:Wound Healing Research Unit, Cardiff, UK
Re-epithelialisation begins within hours of injury and
persists throughout the proliferative phase. Keratinocytes switch phenotype
and become 'activated' enabling phagocytosis and horizontally migration.
Keratinocyte activation is characterised ultrastructually by dissolution
of des-mosomes and hemidesmosomes, intracellular alteration of tonofilaments
(keratin intermediate filaments) and formation of both F-actin bundles
and pseudopodia. The aim of this study was to compare keratin expression
in normal skin, psoriasis, and acute and chronic wounds.
Frozen tissue specimens from normal skin (3), psoriasis (3), acute wounds
(5), and chronic venous leg ulcer margin (5) were cryostat sectioned (6
m) and air-dried for 20 minutes. Fluorescein-labelled avidin-biotin standard
immunofluorescence techniques were employed with monoclonal antibodies
(mAB) to K6, K16, K17, K1 and K10. Control sections had no primary antibody.
The sections were evaluated by fluorescence photomicroscopy and confocal
microscopy.
Alteration of normal keratin expression was evident in both psoriatic
epidermis and wound healing. There was reduced expression of keratins
associated with normal terminal differentiation (1, K10). In addition,
keratins not normally constitutively expressed in interfollicular epidermis
(K6, K16 and K17) were up regulated in psoriasis and wound healing. Furthermore,
K17 appeared to undergo the highest level of increased expression at the
wound edge, with K16 the lowest and K6 being intermediate. Preliminary
data suggests that expression of K17, and probably K6b, is stimulated
by interferon- via the JAK-STAT-GAS pathway.
SOX18 IS EXPRESSED DURING ANGIOGENISIS IN WOUND REPAIR
I.A. Darby, T. Bisucci, G.E.O. Muscat and P. Koopman, Microvascular
Biology and Wound Healing Laboratory, RMIT University, PO Box 71, Bundoora,
VIC 3083 and Institute for Molecular Bioscience, University of Queensland,
Australia. Microvascular Biology and Wound Healing Laboratory, RMIT University,
PO Box 71, Bundoora, VIC 3083 and Institute for Molecular Bioscience,
University of Queensland, Australia.
Sox18 is a member of a family of developmental transcription
factors including the high mobility group (HMG) proteins. Sox18 shows
a limited pattern of expression during embryonic development in sites
of vasculogenesis and angiogenesis and mutations in Sox18 have been found
to be responsible for the mouse mutant ragged which shows vascular abnormalities.
Sox18 expression in the adult is low in most tissues. We hypothesise that
Sox18 may be expressed during adult angiogenesis during wound healing.
Full-thickness skin wounds were created on the mid dorsal region of rats
and mice. Wound tissue was taken at various time points after wounding
and expression of Sox18, VEGF, Flk-1 and type IV collagen (Col4a1) were
studied using in situ hybridisation. Sox18 expression was detectable as
early as five days post wounding in the sprouting capillaries of the granulation
tissue and persisted through to 16 days when the wounds were re-epithelialised,
however at 21 days Sox18 expression was undetectable. Flk-1 and Col4a1
were expressed in endothelial cells of capillaries showing an identical
pattern to Sox18 expression. Capillaries in the subcutaneous tissue of
the adjacent unwounded skin did not express Sox18 nor was Sox18 expression
detectable in normal skin biopsies. In endothelial cell cultures, Sox18
expression was associated with formation of tubes when cells were cultured
on collagen I or EHS matrix. Overall, our data suggest that Sox18 represents
a transcription factor that is involved in angiogenesis in wound healing
and tissue repair but not in the maintenance of endothelial cells in normal
tissue.
THE USE OF LOW DOSE 5-FLUROURACIL TO MODULATE COLLAGEN
EXPRESSION IN TISSUE REPAIR
V. Mudera, 1N. Bullstrode, 1A. Cambrey and D.A. McGrouther Tissue Repair
Unit, Centre for Plastic and Reconstructive Surgery, The Royal Free and
University College Medical School, London, UK, 1: The RAFT Institute of
Plastic Surgery, Mount Vernon and Watford Hospitals NHS Trust, Northwood,
UK
The purpose of this work was to investigate use of 5-Flurouracil
(5-FU) to modulate collagen expression in tissue repair. Normal tissue
repair results in a fibropro-liferative state where poor control might
produce excess and non-functional matrix (fibrosis). 5-FU is an antipro-liferative
agent currently used a high doses in clinical practice. Previous work
suggests that low dose topical f-FU might have an antifibrotic action.
Materials and Methods: Using fibroblasts from Dupuytren's contracture
fascia (n=4), a type of fibroproliferative condition causing dysfunctional
ECM contracture as a model, monolayers were treated with single application
5-FU (0.25=50mg/ml, 5min) and incubated for 24 hours in serum free media,
controls were normal human tendon fibro-blasts (HTF, n=4). Collagen production
and secreted TGF-b1(pro-fibrotic agent) were measured. Multiplex RT-PCR:
bactin with TGF-b1, Collagen I and III was performed for 5-FU treated
and untreated cells.
Results: Collagen protein synthesis of HTF and Dupuy-tren's fibroblasts
was specifically and selectively down-regulated (at the post-transcriptional
level by a yet unknown mechanism) by 5-FU (22+/-11% and 32+/-7.45%) compared
to untreated fibroblasts (p<0.05), with no effect on mRNA expression
or non-collagenous protein production. Compared to HTF, Dupuytren's fibroblasts
produced a significantly higher amount of TGF-b1 secretion or total de
novo protein synthesis. There was no significant difference between the
5-FU treated and untreated fibroblasts for TGF-b1, Collagen I and III
mRNA expression in HTF or Dupuytren's fibroblasts. Base on this proof
of concept, clinical trials to decrease post surgical recurrence rates
of Dupuytren's contracture are ongoing.
NOVEL USE OF MINIMALLY LETHAL; PHOTODYNAMIC THERAPY
FOR REGULATION OF FIBROBLAST MEDIATED CONTRACTION
Farida Ali, Alexander MacRobert, Robert Brown and Vivek Mudera, University
College London, Centre for Plastic & Reconstructive Surgery, Tissue
Repair Unit & Tissue Engineering Centre, London, UK
The purpose of this work was to investigate the effect
of minimally lethal Photodynamic therapy (PDT) in controlling fibroblast
mediated contraction. Fibroblast mediated scar contracture is a complication
of wound healing. PDT involves light irradiation of cells treated with
a photosensitising agent to generate reactive oxygen species normally
producing cell death. Previous studies have suggested that PDT treatment
inhibits cellular attachment. We hypothesised that inhibition of cellular
attachment can be used to control fibroblast mediated contraction by this
mechanism.
Materials and Methods: We have used tethered and un-tethered fibroblast
populated collagen gels in combination with varying drug and light does
(PDT). Tethered gels were used to quantitate contractile force generated
over 24 hours, using the Culture Force Monitor (CFM). Cell viability and
contraction profiles were assessed using MTT assay, image analysis and
CFM outputs.
Results: The optimal minimally lethal dose for the photosensitising agents
{aluminium phthalocyanate (A1PcS2) and 5-aminolevulinic acid (ALA)} was
established using MTT assay (<35% cell death). A1PcS2 in combination
with optimal light intensity/time exposure (70% viability) completely
blocked contraction. (Untethered = 0%, control = 76% contraction after
five days and tethered = 0dynes, control = 60dynes at 24hrs. 5-ALA also
strongly inhibited cell-matrix contraction (90%). Having established proof
of concept in vitro we are currently testing the effect in vivo using
an established rat dermal injury model. Early results showed a reduction
in wound contraction up to 50% over three days post treatment as assessed
by image analysis. We have shown that minimally lethal PDT reduces fibroblast
mediated contraction in vitro. Further work is needed to test the longer-term
effects of this clinically applicable technique.
|
