the 2007 Abstract Presentations - Wound Healing Society

Abstracts
71
RADIATION IS A KEY REGULATOR OF ANGIOGENIC AND
ANGIOSTATIC CXC CHEMOKINE EXPRESSION IN HUMAN
ENDOTHELIAL CELLS
Chang C.C., Lerman O.Z., Thanik V.D., Scharf C.L., Warren S.M., Levine
J.P.
Institute of Reconstructive Plastic Surgery, NYU School of Medicine
Introduction: Neovascularization in wound healing requires complex signaling
involving angiogenic and angiostatic chemokines in the CXC family. Increasing
evidence suggests that low-dose ionizing radiation (XRT) may be aprovasculogenic
stimulus. However, because radiation is also known to cause tissue
damage, hypovascularity, and impaired wound healing, the mechanism of
angiogenic stimulation must be clarified. Compared to pathologic doses of
radiation, we hypothesize that low dose radiation induces proangiogenicpatterns
of CXC chemokine signaling in human vascular endothelial cells.
Methods: HUVECs were exposed to 0 Gy, 5 Gy, or 20 Gy ionizing radiation.
Cells were grown in basal media for 24–48 hrs. Total cell protein lysate
washarvested and cytokine expression was analyzed by antibody array.
Candidatecytokine upregulation was then confirmed by quantitative real time
RT-PCR and ELISA. Annexin V and CXC chemokine receptor profiles were
analyzed using flow cytometry.
Results: All proangiogenic chemokines CXCL1-CXCL8 demonstrated doseresponse
induction up to 3- and 13-fold when exposed to 5 Gy and 20 Gy
irradiation, respectively. IFNg and TNF-b were upregulated at 20 Gy by 1.5
fold and 3-fold, respectively. 20 Gy XRT also led to increased angiostatic CXC
chemokine production. Annexin-V assay demonstrated decreased apoptosisfrom
0 Gy–5 Gy XRT (16% vs. 4.5%), however 20 Gy XRT showed increased
annexin-V (24%). CXCR4 expression showed a dose-response relationship
tohypoxia, which was independently augmented by 8- and 16-fold 24 and 48 hrs
after radiation.
Conclusion: XRT induces a dose dependent upregulation of angiogenic members
of the CXC family of chemokines in HUVECs. Furthermore, we observed
induction of inflammatory signals and their respective angiostatic CXC
chemokines at 20 Gy. This suggests that XRT induces an angiogenic response
at low doses while proinflammatory and apoptotic effects at highdoses result in
tissue damage. Elucidation of the mechanism by which CXC chemokine
expression is balanced in response to XRT may reveal novel targetsfor
therapeutic intervention.
73
STROMAL PROGENITOR CELLS (SPC) CORRECT THE
WOUND DEFECT IN MMP-9 KNOCKOUT MICE BY
ENHANCED REEPITHELIALIZATION NOT GRANULATION
TISSUE PRODUCTION
A.T. Badillo, S. Chung, R.A. Redden, L. Zhang, E.J. Doolin, and
K.W. Liechty
Children’s Hospital of Philadelphia, Philadelphia, PA
MMP-9 knockout (MMP-9KO) mice exhibit local impairments in wound
healing as well as impaired progenitor cell release from bone marrow. We have
previously shown that application of SPC to murine diabetic wounds can
improve diabetic wound healing through a combination of direct and indirect
mechanisms. Based on these observations we hypothesize that direct application
of SPC to MMP-9KO wounds can both restore local wound deficiencies
and replace the need for mobilization of endogenous progenitor cells to achieve
wound healing.
Methods: 8 mm excisional wounds were created in16-week-old MMP-9KO
mice and immediately treated with either 1 10 6 passage 4 fetal liver derived
SPC expressing green fluorescent protein (GFP) or PBS vehicle control. At 7
days post wounding, wounds were harvested for molecular and histological
analysis of epithelial gap (EG) and granulation tissue (GT) area.
Results: The EG of SPC-treated wounds was significantly smaller than vehicletreated
controls (SPC 2.4 0.4 mm vs PBS 4.7 0.6 mm, p o 0.008). However,
there was no significant difference in GT area between the two treatment
groups. GFP1 cells were present in all SPC-treated wounds at the time of
analysis. No GFP signal was seen in vehicle-treated wounds. Analysis of total
wound cellular mRNA revealed a 2-fold increase in the production of EGF and
VEGF in SPC-treated wounds (p o 0.05).
Conclusions: Direct application of SPC to MMP-9KO wounds significantly
improves reepithelialization and occurs without significant new GT production.
SPC have the potential to enhance epithelialization through production of
trophic factors and restoration of MMP-9 activity. Furthermore, direct
application of SPC may re-establish physiologic tissue repair processes normally
orchestrated by absent endogeneous progenitor cells. This data has
significant implications for the development of SPC based therapeutic strategies
for a broad range of wound healing deficits and also provides a rationale for
further study of the role of progenitor cells in tissue repair.
72
APPLICATION OF AAV2-MEDIATED BFGF GENE THERAPY
ON SURVIVAL OF ISCHEMIC FLAP: EFFECTS OF TIMING
OF GENE TRANSFER
Xiao Tian Wang, MD, Paul Y. Liu, MD, Jin Bo Tang MD
Department of Surgery, Roger Williams Hospital, Providence, RI, USA
Purpose: Necrosis of surgically transferred flaps is a major problem in
reconstructive surgery. Enhancement of survival of ischemic flap has been a
focus of investigations for a long period of time and remains a challenge to both
researchers and surgeons. The objective of this study was to investigate efficacy
of a new vector system—adeno-associated viral 2 (AAV2) vector mediated gene
transfer to enhance survival of the ischemic skin in a rat model.
Materials and Methods: Thirty-eight Sprague-Dawley rats were divided into 3
gene therapy groups and one non-treated control of 9 or 10 each.
7.5 10 10 AAV2 particles harboring rat bFGF genes were injected to the
dorsum of each of the 29 rats; these rats were then divided into 3 groups
according to the timing of flap elevation. At the time of surgery, one week, and
two weeks after surgery, random skin flaps of 3 7 cm were raised on the dorsal
aspect of rats. One week after flap elevation, flap viability was determined by
measurement of percentage area of survival. The efficacy of gene therapy was
evaluated by flap survival and vascularization of histologic sections.
Results: The viability of the flap was significantly improved after injection of
the AAV2-bFGF at the time of surgery (57.6 3.3) compared with non-treated
controls (50.9 5.2%)(p = 0.0033). However, the surviving area of the flap was
significantly smaller after delivery of the AAV2-bFGF one week before surgery
(42.7 8.4%) compared with non-treated controls (p = 0.025). When the flap
was raised two weeks after injection of the AAV2-bFGF, the average flap
survival area was increased by about 10% over the controls, but greater
deviations were seen. Statistically, the changes did not reach significance.
Histologically, vascular counts were significantly increased in the groups with
AAV2-bFGF injection.
Discussion: The novel approach of using AAV2-bFGF gene therapy at appropriate
time shows encouraging manifestations in improving survival of ischemic
flaps. However, further optimization of doses and timing of delivery is needed to
determine the doses and timing consistently yielding improvement of flap survival.
74
STEM CELL THERAPY FOR DIABETIC WOUNDS
C.D. Lin 1 , O.Z. Lerman 1 , D. Brown 1 , O.T. Tepper 1 , P.B. Saadeh 1 ,
S.M. Warren 1 , J.P. Levine 1
1 New York University School of Medicine, NY USA
Purpose: In recent years the critical role that bone marrow (BM)-derived stem
cells play in ischemic tissue repair and wound healing has become evident.
Adult stem cell technology poses therapeutic potential in diseases such as
diabetes, where progenitor cell function is known to be impaired. The following
study set out to determine if BM-derived stem cells delivered locally to a wound
bed can accelerate diabetic wound healing via incorporation into the wound
bed, improved neovascularization, matrix deposition and restoration of cellular
architecture.
Methods: Uncommitted lineage negative progenitor cells from the BM of wild
type mice were magnetically separated. These cells were tagged with DiI, and
were applied topically within a collagen gel to excisional wounds on diabetic
mice. The established wound model uses full-thickness dorsal wounds that are
splinted open in order to more closely mimic human wound healing. Wounds
treated with lineage positive cells served as a control. Wounds were analyzed for
time to closure, epithelial gap, granulation tissue formation, cell proliferation,
and vascularity.
Results: Lineage negative stem cell treated wounds showed a statistically
significant decrease in time to closure as well as a decrease in epithelial gap
relative to controls (80% vs 60% closure at day 18; p o 0.05; 100% vs 73% at
day 21; p,0.05). Viable DiI-positive cells were noted in the tissue as far as 21
days, and co-staining for CD31 demonstrated the formation of new blood
vessels by transplanted cells. Stem-cell treated wounds exhibited increased
vascularity with concomitent cell proliferation relative to controls.
Conclusions: We conclude that topical delivery of cells represents a suitable
approach to adult stem cell therapy for wound healing. Transplanted cell have
the potential to survive and incorporate into the underlying wound bed.
Delivery of BM-lineage negative cells in this manner may provide a therapeutic
modality to improving wound healing in diabetes.
Wound Rep Reg (2007) 15 A14–A54 c 2007 by the Wound Healing Society
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