the 2007 Abstract Presentations - Wound Healing Society

Abstracts
106
IMPAIRED NOTCH SIGNALING SIGNIFICANTLY DELAYS
WOUND HEALING
Srinivasulu Chigurupati 1 , Tiruma V. Arumugam 2 , Shafaq Jameel 2 , Mohamed
R. Mughal 2 , Mark P. Mattson 2 , Suresh Poosala 1
1 Comparative Medicine Section, National Institute on Aging Intramural
Research Program, 5600 Nathan Shock Drive, Baltimore, Maryland 21224,
USA,
2 Laboratory of Neurosciences, National Institute on Aging Intramural
Research Program, 5600 Nathan Shock Drive, Baltimore, Maryland 21224,
USA
The evolutionarily conserved Notch-mediated intercellular signaling pathway
is critically involved in cell fate decisions during development of many tissues
and organs. Angiogenesis, the formation of new blood vessels, plays a central
role in number of physiological and pathological conditions including wound
healing which involves a switch in endothelial cell phenotype from quiescence
to proliferation, migration and network formation, and back to quiescence. In
the present study we employed in vivo and cell culture models to elucidate the
role of Notch signaling in wound healing. Inhibition of Notch signaling in
Notch antisense transgenic mice, and in normal mice treated with the g
secretase inhibitor N-[N-(3,5Difluorophenylacetyl)-(S)-alanyl]-(S)-phenylglycine
tert-butyl ester (DAPT), resulted in a significant delay in the healing of
full-thickness dermal wounds. To further examine the role of Notch signaling in
cells involved in wound healing, we evaluated the effects of Notch inhibition on
the behaviors of human umbilical vascular endothelial cells (HUVEC) in a
scratch wound healing test, a cell proliferation assay, cell migration assay and a
tube formation assay in Matrigel. Treatment of HUVEC with DAPT inhibited
scratch wound healing, cell proliferation and cell migration. Tube morphogenesis
was also significantly disrupted by DAPT treatment. These results suggest
that Notch signaling is involved in wound healing and tissue repair, therefore
we conclude that targeting the Notch pathway might provide a novel strategy
for wound repair and treatment.
108
IN SITU TISSUE ENGINEERING USING SELF-ASSEMBLING
PEPTIDE IMPROVES WOUND HEALING AND
NEOVASCULARIZATION IN A DIABETIC MOUSE MODEL
S.S. Vaikunth 1 , S. Balaji 2 , A.R. Maldonado 1 , J. Parvadia 1 , F.Y. Lim 1 , T.M.
Crombleholme 1 , D.A. Narmoneva 2
1 Cincinnnati Childrens’ Hospital Medical Center,
2 University of Cincinnati
Self-assembling RADAII-16 peptide is a novel biomaterial which has been
shown to create an angiogenic microenvironment and promote neovascularization
in murine cardiac muscle. We asked whether this peptide would have a
similar effect in an impaired diabetic wound healing model. We hypothesized
that peptide would increase neovascularization and improve wound healing in
db/db mice. Db/db mice were confirmed to be diabetic with blood sugars
greater than 300 gm/dL. Eight millimeter excisional wounds were made in the
flanks of mice. The wounds were then treated with 50 ml PBS (n = 5) or peptide
(n = 4). Mice were harvested at day 7 and wounds were analyzed by computerbased
morphometric measurements of epithelial gap, epithelial height, and
granulation tissue area. Vessel density was analyzed using lectin staining.
Results are presented as mean standard error, and statistical analysis was
performed using ANOVA. Peptide treatment resulted in larger granulation
tissue area (2.77 0.6 mm 2 vs. 1.45 0.4 mm 2 ,po 0.05, peptide vs. PBS), larger
epithelial height (0.195 0.010 mm vs. 0.148 0.012 mm, p o 0.05, peptide vs.
control), and enhanced vessel density (5.2 0.8 caps/HPF vs. 2.5 0.1 caps/
HPF, p o 0.05, peptide vs. control). No significant difference in epithelial gap
was observed between the two treatment groups (4.8 0.4 mm vs. 4.4 0.4 mm,
peptide vs. PBS). These results suggest that self-assembling peptide scaffold
improves wound microenvironment and healing in diabetic animals and may
offer a novel approach for in situ tissue engineering to augment wound healing.
Acknowledgments: Funded by (TMC) 5RO1-DK072446, (TMC) 5RO1-
DK074055.
107
IN VIVO EXPRESSION OF VEGF-R2 DURING EARLY
SKELETAL ISCHEMIA-REPERFUSION INJURY
M. Hofmann, R. Mittermayr, T. Morton, S. Pfeifer, M. van Griensven, H. Redl
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology –
Research Center of the AUVA, Vienna, Austria
Introduction: Different strategies were developed to reduce ischemia/reperfusion
injury. One of the concepts includes therapeutic angiogenesis with VEGF
and its receptor as pivotal players. This study investigates the in vivo expression
of VEGF-R2 during early murine hindlimb ischemia/reperfusion and its
alteration via VEGF165 released from a fibrin biomatrix (FS).
Material and Methods: Male/female transgenic FVB/N-Tg(Vegfr2luc)Xen
mice (n = 12/group) were used for non-invasive, real-time assessment of the
VEGF-R2 (Flk-1/KDR) induction. Ischemia was induced by tension controlled
(250 g) tourniquet to the hindlimb and verified by laser Doppler imaging
(LDI, Moor Instruments Inc.) technique. Ischemia was maintained for 2 hours
with subsequent reperfusion for 4 or 24 hours. Control animals received no
treatment whereas the animals of the FS/VEGF groups received 0 or 20 ng
VEGF/ final FS clot (0.2 ml in total) in their hindlimb subcutaneously, to
distribute evenly around the muscle compartment, 15 min prior to reperfusion.
The contralateral leg was used as an internal control. At different time points
LDI was done to show hindlimb perfusion and bioluminescence detection was
done to observe VEGF-R2 expression (VivoVision s IVIS s , Xenogen).
Results: Applying the tourniquet resulted in ischemia as verified by a reduction
of leg perfusion to approximately 10% in all groups. Ischemia was maintained
for the entire 2 hours. Furthermore, restoration of blood flow was seen to 83%
of baseline in the control group after 24 hours reperfusion. Similar reperfusion
was observed in the vehicle and 20 ng FS/VEGF groups (112% and 95% of
baseline). Edema was found in all groups in the injured hindlimb. The VEGF-
R2 was slightly up-regulated in the control group after 24 hours of reperfusion
in the ischemic, but not in the contralateral hindlimb. With VEGF in the fibrin
biomatrix a significant increase in receptor expression was observed at 24 h,
compared to baseline as well as to the control group while after 4 hours no
receptor alterations were seen.
Conclusion: This ischemia/reperfusion model in transgenic mice enables in vivo
observation of the VEGF-R2 expression, a key receptor in angiogenesis.
VEGF-R2 is only marginally up-regulated in the reperfusion period after severe
ischemic conditions. However following the concept of therapeutic angiogenesis,
VEGF application from a fibrin biomatrix causes an increase in VEGF
receptor expression at 24 h.
109
LASER CAPTURE MICRODISSECTION AND TISSUE
ANALYSIS: EMERGENT TECHNOLOGIES IN WOUND
RESEARCH
Chandan K. Sen and Sashwati Roy
The Ohio State University Comprehensive Wound Center, Columbus, OH
43210
Spatially-resolved cell-specific molecular analysis of tissue affected by focal
injury is now possible because of current laser capture microdissection (LCM)
technology. A variant of LCM is laser microdissection and pressure catapulting
(LMPC). In LMPC, thebiological material is placed directly on top of a
thermoplastic polyethylene napthalate membrane. The membrane acts as a
scaffold to allow for catapulting relatively large amounts of intact material. A
focused laser beam cuts out an area of the membrane and corresponding
biological material. Next, the beam is defocused and the energy used to catapult
the membrane and material from the slide. A motorized robotic stage moves the
sample through the laser beam path to allow the user to control the size and
shape of the area to be cut. The catapulted sample is generally captured in an
aqueous media (e.g. RNA or protein stabilizing solution). Under direct
microscopic visualization, LMPC permits rapid procurement of histologically
defined tissue samples from spatially-resolved (e.g. infarct vs peri-infarct;
wound core vs leading edge) regions of the tissue. Recently, our laboratory has
developed novel methods that enable the molecular study of specific regions of
the infarcted heart with single cell resolution (Roy et al. Physiol. Genom 2006;
Kuhn et al. AJP-Heart & Circ 2006 & 2007). Even in frozen tissues, RNA
degradation occurs rapidly. Thus, most standard immunohistochemical staining
techniques are not suitable if the measurement of gene expression is desired.
Rapid techniques for accurate histological identification are needed. We have
developed an approach to identify and capture blood vessels from wound-edge
tissue biopsies collected from patients with chronic wounds. Subsequent
techniques to perform real-time PCR aswell as transcriptome analysis have
been optimized. These developments and the potential of LCM/LMPC technology
to illuminate new aspects ofwound biology from a novel vantage point
will be presented. Supported by NIH GM069589 and HL073087.
Wound Rep Reg (2007) 15 A14–A54 c 2007 by the Wound Healing Society
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