|
ETRS
STUTTGART MEETING, 2005
Young Investigators Awards
Design
of a novel proteolysis resistant VEGF variant
Eming S.1, Roth D.1, Lauer G.1, Paulsson M.2, Davidson
J.3 and Krieg T.1,
1Dermatology, University of Cologne, Germany,
2Center for Biochemistry, University of Cologne, Germany, 3Department
of Pathology, Vanderbilt
University School of Medicine and VA Medical Center, Nashville, Tennessee,
U.S.A.
Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator
in tissue repair. Recently we demonstrated that in non-healing human wounds
a plasmincatalyzed cleavage significantly reduces the bioactivity of the
VEGF-A isoform VEGF165, suggesting that VEGF165 degradation and inactivation
contribute to an impaired healing response. Plasmin digestion results
in loss of the carboxyl-terminal heparin-binding domain. To investigate
the biological relevance of the protease sensitivity of VEGF165 during
cutaneous repair, we assessed the activity of a VEGF165 mutant resistant
to plasmin proteolysis (VEGF165-MutPro111) in a genetic mouse model of
impaired wound healing (db/db mouse). In this mouse model the stability
of the mutant VEGF165 was substantially increased in wound tissue lysates
in comparison to VEGF165 wild type thus indicating a prolonged activity
of the plasmin resistant VEGF165 mutant. The db/db delayed healing phenotype
could be reversed by topical application of VEGF165-MutPro111 and most
interestingly
the resistance of VEGF165 to plasmin cleavage resulted in the increased
stability of vascular structures during the late phase of healing due
to delayed and reduced endothelial cell apoptosis. Our data provide the
first indication that plasmin-catalyzed cleavage modulates VEGF165 mediated
angiogenesis in vivo. Our experiments propose that the ability to stabilize
the heparin-binding capability of VEGF165-A may help to preserve the biological
function of VEGF165 under conditions in which proteases are highly active
such as wound repair and inflammation.
Contact:
Priv.-Doz. Dr med. Sabine Eming, University of Cologne, Joseph-Stelzmann
Str. 9,
50931 Köln, Germany Tel: +49 221 4783196
Role
of Shc family signaling protein in mesenchymal stem cell
Akino K.1, Imaizumi T.2, Mori N.1, Hirano A.2 and Akita S.2
1Nagasaki University, Department of Developmental and Reconstructive Medicine
2Nagasaki University, Department of Plastic Surgery, Nagasaki, Japan
Mesenchymal stem cells are implicated in the neurological disorders and
neuronal differentiation. The neurological
imbalance and abnormality may lead to impaired wound healing such as paraplegic
and diabetic diseases. Mesenchymal stem cell grafting in the cutaneous
wounds successfully improves the rate of the healing, however, the further
mechanisms are unknown. Shc is one of intracellular signaling proteins
related to various cell functions. Detail analysis of Shc in mesenchymal
stem cells may implicate in neuronal and curtaneous regeneration. Shc
recognizes the phosphorylation of various growth factor receptors and
the activated signals are transported to Ras- ERK or JNK via Grb2-SOS.
Therefore, Shc is the cytosolic protein involves in cell proliferation,
cell differentiation, and induction of apoptosis. Among Shc family, ShcC/Neuronal
Shc (N-Shc) is one of the homologues of Shc proteins and demonstrated
the neuron-specific actions. Human mesenchymal stem cells (hMSCs), which
is distinctively sorted by cell surface antigens and the cell properties
and bone marrow stromal cell derived from ShcC deleted mice are further
analyzed in vitro and in vivo. Various neuronal markers such as Shc family
and stathminrelated proteins are investigated. The hMSCs demonstrated
immunoreactivities in SCG10 and OP-18(stathmin) in cytoplasms, whereas
there were only Shc and ShcC cytoplasmic immunoreactivites but not in
Sck, which is specific to membrane depolarization. The immunoreactively
positive cells were all blackout in the nuclei, in which DAPI immunoreactivities
were also observed. The RT-PCR transcript levels normalized by the internal
G3PDH control demonstrated the SCG10 SCLIP transcripts in hMSCs. Since
the stathmin (OP-18) antibody used for immunocytochemistry was not able
to distinguish stathmin antigen from related
antigens such as SCLIP and Rb3, the detail profile of the gene products
first elucidated by RT-PCR. SCG10, SCLIP and stathmin were detectable
but not RB3. The expression levels of SCG10 and SCLIP in hMSCs were cell-cycle
dependent (p<0.01). In the bone marrow derived from ShcC ‘knockout’
mice in three passages, it is demonstrated the biphasic morphology in
size. In cutaneous defects in ShcC ‘knockout’ mice significantly
delayed the wound healing. Therefore, ShcC may involve in cutaneous wound
healing by mesenchymal stem or stromal cells.
Contact:
Dr Sadanori Akita, Nagasaki University, 1-7-1 Sakamoto,
8528501, Japan Tel: 81 95 849 7327
Chronic
wound fibroblasts: ageing before their time?
Wall I.1, Baird D.2, Price P.3, Harding K.3, Kipling D.2, Thomas
D.W.4 and Stephens P.4
1Wound Biology Group, Cardiff Institute of Tissue Engineering and Repair,
Dept. Oral Surgery, Medicine & Pathology, School of Dentistry; Wound
Healing Research Unit, School of Medicine, Cardiff University, Cardiff,
UK,
2Dept. Pathology, School of Medicine, Cardiff University, Cardiff, UK,
3Wound Healing Researh Unit, School of Medicine, Cardiff University, Cardiff,
UK,
4Wound Biology Group, Cardiff Institute of Tissue Engineering and Repair,
Dept. Oral Surgery, Medicine & Pathology, School of Dentistry, Cardiff
University, Cardiff, UK
INTRODUCTION:
Chronic non-healing wounds are a major health problem and affect 3% of
the population over 65 years of age. Age-related functional changes in
dermal fibroblasts may contribute to this dysfunctional healing phenotype.
The aim of this study was to investigate potential genotypic and phenotypic
differences between patient- matched chronic wound fibroblasts (CWF) and
normal skin fibroblasts (NF). In addition, we sought to investigate the
role played by cellular and molecular ageing as a contributory factor
in these wounds.
METHODS:
CWF and NF were established in culture (n = 4) and RNA was extracted for
transcriptional profile analysis using Affymetrix microarrays after serum-starvation
(to synchronise the cells in G0) and subsequent serum restimulation (to
mimic a wound healing response). Phenotypic characteristics were also
assessed throughout their proliferative lifespans including cellular proliferation
(population doublings), senescence (cell morphology, senescence associated
b-Galactosidase activity [SA b-Gal] and telomere length [using the novel
PCR-based single telomere length analysis; STELA]), extracellular matrix
reorganisation and ability to repopulate an experimental wound.
RESULTS:
Microarray analysis of CWF and NF following serum-stimulation demonstrated
differential expression of 118 genes, including molecules involved in
the protection against oxidative stress (e.g., glutaredoxin, oxidative
stress responsive-1). In long-term culture CWF proliferated more slowly
than NF and senesced at earlier time points (p < 0.01). The onset of
premature senescence within the CWF populations significantly decreased
their abilities to reorganise the surrounding extracellular environment
(p < 0.01) and repopulate a wound. Premature senescence in CWF was
confirmed by increased SA b-Gal staining and their larger, polygonal morphology.
Analysis of telomere lengths in these samples revealed that replicative
senescence was predominantly (3/4 samples) telomere-independent.
CONCLUSIONS:
The results demonstrate that CWF have a genotype and phenotype distinct
from patient-matched NF. The senescent phenotype of fibroblasts from some,
but not all wounds, is independent of telomere shortening. One hypothesis
that supports telomere-dependent and -independent mechanisms of cellular
senescence involves differential levels of oxidative stress within the
wound environment. Elevated levels of stress could drive telomeredependent
senescence by stimulating cell turnover. However, extremely high levels
of oxidative stress bypass the normal methods of telomere-dependent senescence
causing direct DNA damage and subsequent cell senescence.
Contact:
Ivan Wall, Cardiff University, 4th Floor Dental School, CF14 4XY, United
Kingdom Tel: 02920 744252
Wound-healing
defect of CD18-/- mice due to a decrease in TGF-b1 and myofibroblast differentiation
Peters T.1, Sindrilaru A.1, Hinz B.2, Al-Azzeh E.1,
Menke A.3, Hinrichs R.1, Oreshkova T.1, Kess D.1, Wang H.1, Holzwarth
K.4, Renkl A.1, Wlaschek M.1, Sunderkötter C.1, Krieg T.4, Scharffetter-Kochanek
K.1
1University of Ulm, Dept. of Dermatology and Allergic Diseases, Ulm, Germany,
2Swiss Federal Institute of Technology, Laboratory of Cell Biophysics,
Lausanne, Switzerland,
3University of Ulm, Dept. I of Internal Medicine, Ulm, Germany,
4University of Cologne, Dept. of Dermatology and Venerology, Cologne,
Germany
The b2 leukocyte integrins are heterodimers, composed of a common b chain
(CD18) and one out of four distinct a chains (CD11). They are pivotal
for migration and signaling of hematopoietic cells during inflammatory
processes and immune responses. Lack of functional b2 integrin causes
leukocyte-adhesion deficiency type 1 (LAD1), a life-threatening primary
immunodeficiency syndrome with severe recurrent microbial infections,
leukocytosis and impaired wound healing. Initial data revealed significantly
increased wound sizes in a murine model for LAD1 (CD18-/-) from day 5
to 14 after application of full thickness wounds on mouse backs. We now
addressed the question whether this impairment in wound healing was a
consequence of reduced wound contraction potentially caused by disturbed
myofibroblast recruitment. Therefore, we analyzed expression of markers
critical for myofibroblast differentiation by immunohistochemistry and
Western blotting. We found that both splice variant ED-A of fibronectin
and a-smooth muscle actin were substantially reduced in CD18-/- mice at
day 5 and 7 after wounding, respectively, indicating impaired myofibroblast
differentiation. Interestingly, TGFb1 and its receptor TGF-bRII were also
largely decreased. Since TGF-b1 is a key factor for granulation tissue
formation and promotes wound contraction, we supplemented TGF-b1 by subcutaneously
injecting two different doses (0.45 mg, 0.1 mg) into the wound margins
at day 1, 3 and 5 after wounding of CD18-/- and wild-type mice. As a result,
we observed a rescued wound closure in CD18-/-, similar to wild-type mice.
Since in wounds of CD18-/- mice, defective migration leads to a severe
reduction of neutrophils, we envisioned that infiltrating macrophages
may not be able to phagocytose apoptotic CD18-/- neutrophils, thus, lacking
their main stimulus to secrete TGF-b1. We demonstrated that in the absence
of neutrophils, or in cocultures with CD18-/- neutrophils, TGF b1 release
by macrophages was dramatically reduced due to defective phagocytic clearance
of CD18-/- neutrophils, whereas proinflammatory cytokines were increased.
Deviant from former views, our data demonstrate that growth factors released
by neutrophils in a paracrine fashion are important for wound contraction
and healing. It remains to be seen whether CD18 is also involved in migration
of myofibroblast precursors.
Contact:
Dr Thorsten Peters, University of Ulm, Maienweg 12,
D-89081, Germany Tel: +49 731 5002 1823
The
fate of human mesenchymal stem cells bone regeneration
Imaizumi T.1, Akino K.2, Hirano A.1, Akita S.1
1Nagasaki University, Department of Plastic Surgery,
2Nagasaki University, Department of Developmental and Reconstructive Medicine,
Nagasaki, Japan
Bone marrow derived cells such as the human mesenchymal stem cells (hMSCs)
successfully bone regenerated, however, there is little data on how and
when the cells contribute to this event. As the hMSCs may be able to differentiate
to mesenchymal lineage in each specific condition, further in vivo analysis
is still lacking and the mechanisms are ambiguous. In order to elucidate
the cell kinetics, proliferation and differentiation, the hMSCstransfected
with Green Fluorescent Protein (GFP) DNA plasmid, further kinetics were
investigated. GFP-transfected hMSCs were grafted with gelatin sponge (Gelform)
in the 5-mm full-thickness cranial bone defects of nude rats. The defects
were healed in the grafted GFP-transfected hMSCs by four weeks post-operatively
in accordance to the previous experiment, which was identical to that
of without GFP-transfection but hMSCs grafting. Superficial layer, independent
of the adjacent rat bone edge, at day 7 demonstrated the strong immunoreactivities
of both GFP and osteocalcin, which indicates the matured bone, with the
cell nuclear antigens of 6-Diamidino-phenylindole, Dihydrochloride (DAPI).
In the same layer, polyomavirus enhancer binding protein 2a (PEBP 2a)
was also depicted as a core binding factor1 (cbfa1), an osteogenic transcription
factor expressed in some cells among the interstitial fibroblast-like
cells along with GFP immunoreactivities. Taken together, PEBP 2a transcription
was co-localized with osteocalcin expressions and these expressions were
all strongly GFP immunoreactive. Therefore, the grafted hMSCs were located
in independent of the pre-existing rat bones and osteogenesis is regulated
transcriptionally via PEBP 2a and osteocalcin by autogeneic regulation
and the osteogenesis was enhanced with presence of osteogenic cytokines
such as bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth
factor (bFGF).
Contact:
Dr Sadanori Akita, Nagasaki University, 1-7-1
Sakamoto, 8528501, Japan Tel: 81 95 849 7327
Upregulation
of lysyl hydroxylase 2 b in wound healing leads to altered collagen cross-linking
Bogaerdt, van den A.J., Ulrich M.M.W., Verkerk M., Reijnen
L., Vlig M. and Middelkoop E. Association of
Dutch Burn Centres, Beverwijk, The Netherlands
Hypertrophic scar formation in deep dermal wounds is vassociated with
excessive collagen deposition and contraction. Besides increase collagen
synthesis and decreased collagen degradation, the appearance of a different
type of cross-linking, has been described in the literature in fibrosis
of the skin. It was shown that collagen cross-linked by the hydroxyl-allysine
route, the fibrotic type, is less assessable for degradation by MMP-1
than cross-links of the allysine route, the normal type of cross-linking
in the skin. This type of cross-linking could therefore contribute to
the process of collagen accumulation. Recently the key enzyme responsible
for this type of cross-linking has been identified as lysyl hydroxylase
type 2 (LH2 or PLOD2). This enzyme also influences fibril formation, over-expression
of LH2 results in a decrease in fibril diameter. In this study we investigated
the expression profile of LH2, collagen type I and III in time after wounding
and determined the collagen cross-linking type of the scars in the experimental
pig model. In pig full thickness wounds were created, the wounds were
transplanted with a meshed (1:3) split skin autograft. At different time
points after wounding biopsies were taken for evaluation. RNA levels were
determined with real time RT-PCR. The type of cross-linking was determined
in hydrolyzed samples by reverse-phase high performance liquid. The hydroxyl-allysine
type
crosslink in tissue samples from scars is significantly higher than in
normal pig skin. The expression level of LH2 is increased in the wound
during the first three weeks after wounding. Collagen type I and III expression
are increased as well during this period. Pico Sirius red staining of
the scar shows an altered architecture of the collagen fibres. In normal
skin the collagen bundles are thicker and randomly orientated (basket
weave pattern) whereas in the scar the bundles are thinner and aligned
parallel to each other. The spatial and temporal increase of LH2 expression
coincides with the elevated collagen type I and III expression. This results
in increased deposition of collagen fibers of the fibrotic type, which
is less susceptible for degradation by collagenase.
Kontakt:
Dr Magda Ulrich, Association of Dutch Burn Centres, PO Box 1015,
1940 EA, The Netherlands, Tel: +31 251 27 55 06
|
