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nalysis to quantify the degree of chimerism. 8-mm dermal punchiopsy wounds were made on chimeric mice and injected with a

entivirus containing the SDF-1alpha and LacZ transgenes. Woundsere harvested and stained for GFP and lacZ at days 3 and 7 postounding.

ESULTS: Diabetic mice and their nondiabetic counterparts were4% to 99% chimeric. SDF-1alpha overexpression significantly in-reased the number of bone marrow–derived GFP� progenitor cellsecruited to the wound site in both groups (p�0.021, 11 vs 6 cells/PF), with the majority of these bone marrow–derived progenitor

ells seen at the wound edge on day 3 (p�0.046).

ONCLUSIONS: Chimeric GFP� diabetic mice can be success-ully created to achieve excellent levels of chimerism. The correctionf the wound-healing defect of diabetics with SDF-1alpha overex-ression is due, in part, to increased recruitment of bone marrow–erived progenitor cells. Further studies are needed to characterizehe exact progenitor cell populations involved.

rnithine alpha ketoglutarate enhances woundealingrti Uzgare PhD, Kavalukas Sandra MPA, Qiang Zhang MD,uc Cynober PhD, Adrian Barbul MD, FACSinai Hospital of Baltimore, Baltimore, MD

NTRODUCTION: Ornithine alpha-ketoglutarate (OKG) has beenhown to have strong anti-catabolic, anabolic and immunomodula-ory function in injured rodents and humans. In the current experi-ents, the effects of OKG on wound healing and collagen synthesisere studied.

ETHODS: Twenty male SD rats (325–350 g) underwent a 7-cmorsal skin incision and implantation of sterile polyvinyl alcoholponges into subcutaneous pockets; wounds were closed with surgi-al staples. Immediately postoperatively, groups of 10 rats were fedhow supplemented with 2.5% OKG or with 1.94% nonessentialmino acids (equimolar amounts of glycine, alanine, serine, and his-idine). The 2 supplements were iso-nitrogenous. Food and tap waterere offered ad libitum.Ten days post wounding, rats were sacrificed,nd wound breaking strength and sponge collagen content were de-ermined.

ESULTS: The food and water intake as well as cumulative bodyeight gains (g � SEM) were similar in both groups. Supplementa-

ion with 2.5% OKG significantly increased wound breakingtrength (g � SEM) and wound collagen deposition (micro g/100g sponge � SEM) (t test).

roup Weight Gain WBS Collagen

EAA 35 � 4 414 � 16 8,029 � 706KG 41 � 2 535 � 22 13,076 � 810

NS �0.001 �0.001

ONCLUSIONS: The data show that dietary supplementation withKG has significant positive effects on wound healing. Possible

echanisms include provision of substrate for proline biosynthesis d

or incorporation into collagen and/or improved in postinjury pro-ein metabolism.

ypoxia induces upregulation of VEGF-C andymphatic differentiation via Hif-1a pathwayanjay V Daluvoy MD, Tomer Avraham MD, Jennifer Kasten BA,abak J Mehrara MD, FACSemorial Sloan-Kettering Cancer Center, New York, NY

NTRODUCTION: Recent studies have demonstrated a correlationetween Hif-1a expression and lymphatic metastases. However, theolecular mechanisms by which HIF-1a promotes lymphangiogen-

sis in wound healing remain unknown. Therefore, this study aimedo determine the effects of hypoxia on lymphatic endothelial cellsLECs) and lymphatic regeneration.

ETHODS: Human LECs were incubated under normoxic (21%

2) or hypoxic conditions (1% or 5% O2) for varying time,periodsnd expression of Hif-1a, VEGF-C, and Prox-1 (a marker of lym-hatic differentiation) RNA and protein was assessed. To determinehe impact of Hif-1a on LECs, cells were incubated with Hif-1agonists or antagonists. To evaluate expression of Hif-1a andEGF-C in vivo, alymphatic wounds were created in mice tails har-ested at multiple time points and analyzed using immunohisto-hemistry and PCR.

ESULTS: Hypoxia significantly upregulated the expression ofif-1a, VEGF-C, and Prox-1 by LECs in vitro (p�0.05). Upregu-

ation of VEGF-C and Prox-1 by hypoxia was completely blocked byretreatment with YC-1, a HIF-1a antagonist. Cobalt, a Hif-1a ag-nist, caused an increase in VEGF-C (2.5-fold) and Prox-1 (3.5-fold)xpression. Changes in VEGF-C or Prox-1 levels were not due tolterations in mRNA stability. Immunohistochemistry demonstratedhat Hif-1a expression peaked 2 weeks after surgery and correlatedith VEGF-C expression. Hif-1a and VEGF-C expression were co-

ocalized in LECs using confocal microscopy of double immunoflu-rescent stained sections.

ONCLUSIONS: Short-term hypoxia results in a Hif-1a-dependentncrease in VEGF-C production. In addition, Hif-1a promotes lym-hatic differentiation. These findings suggest that hypoxic stimula-ion of Hif-1a contributes to regulation of lymphatic regenerationuring wound repair.

adiation results in persistent subclinicalymphatic dysfunctionomer Avraham MD, Sanjay V Daluvoy MD, Jennifer E Kasten BA,abak J Mehrara MD, FACSemorial Sloan-Kettering Cancer Center, New York, NY

NTRODUCTION: Radiation therapy (XRT) is a known risk factoror developing lymphedema following lymph node dissection. Theechanisms responsible for this increased risk are unknown. The

urpose of this study was therefore to evaluate the effects of XRT onymphatic endothelium and function.

ETHODS: The tails of C57B6 or acid sphingomyelinase–

eficient transgenic (Asmase) mice were irradiated at 0, 15, or 30 Gy.

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S73Vol. 209, No. 3S, September 2009 Surgical Forum Abstracts

he effects of XRT on lymphatic function were evaluated using tailolume measurements, lymphoscintigraphy, histology, and immu-ohistochemistry at various time points ranging from 4 hours to 24eeks. Confirmatory in vitro experiments were performed with XRToses of 4 or 8 Gy.

ESULTS: Both doses of XRT caused mild lymphedema, whichesolved by 12 weeks. Lymphoscintigraphy, however, demon-trated markedly abnormal lymphatic function even after 24eeks (p�0.0001). These abnormalities were associated with tis-

ue fibrosis (p�0.02), lymphatic fibrosis, and decreased numbersf lymphatic capillaries (p�0.006). XRT caused lymphatic endo-helial cell (LEC) apoptosis, which peaked 10 hours followingreatment. Interestingly, in contrast to microvascular endothelialells, Asmase deficiency did not confer protection to LECs frompoptosis. LECs in culture responded to XRT by apoptosing andenescing.

ONCLUSIONS: XRT causes direct injury to LECs resulting inpoptosis independent of the Asmase pathway, decreased density ofymphatic vessels, tissue fibrosis, and impaired lymphatic functionhat persists even months after injury. Persistent subclinical lym-hatic dysfunction noted in our study suggests that XRT acts as aredisposing factor for the development of lymphedema by decreas-ng lymphatic reserve. Thus, lymphedema may result followingeemingly trivial further insult.

GF-beta1 blockade results in improved lymphaticegenerationomer Avraham MD, Sanjay V Daluvoy MD, Jennifer E Kasten BA,ssie Kueberuwa MD, Babak J Mehrara MD, FACSemorial Sloan-Kettering Cancer Center, New York, NY

NTRODUCTION: TGF-beta1 is a growth factor that is also a potentegulator of fibrosis. We have previously shown that wound fibrosisnd increased TGF-beta1 expression are associated with delayed lym-hatic repair. Therefore, the purpose of these studies was to evaluatehe effect of TGF-beta1 blockade on lymphatic regeneration duringound healing.

ETHODS: Circumferential full-thickness skin excisions and mi-rosurgical lymphatic ligation were performed on the tails of mice.he excision site was covered with a collagen gel dressing and treatedith vehicle, recombinant TGF-beta1, dominant negative TGF-beta

eceptor II adenovirus (DN-TBRII), or control LacZ virus. Lym-hatic regeneration and function were evaluated using tail volumeeasurements, lymphoscintigraphy, and immunohistochemistry at

arious time points.

ESULTS: When compared with vehicle controls, exogenousTGF-eta1 resulted in nearly 40% greater increase in tail volumes fromaseline 6 weeks postoperatively (p�0.05), impaired lymphaticransport (p�0.005), increased lymphatic fibrosis, impaired lym-hatic capillary regeneration, decreased LEC proliferation (p�0.01),nd migration. Blockade of TGF-beta1 function using the TBRIIirus resulted in 40% less increase in tail volumes from baseline

p�0.03), improved lymphatic transport (p�0.05), and increased b

EC proliferation and recruitment. The LacZ virus group demon-trated effective transfection, but no significant differences from ve-icle control.

ONCLUSIONS: We have shown that blockade of TGF-beta1, aotent inhibitor of lymphangiogenesis, results in improved LECigration, proliferation, and function. These studies demonstrate

or the first time that inhibition of fibrosis and the direct anti-ymphangiogenic effects of TGF-beta1 can significantly acceler-te and improve lymphatic regeneration and may, therefore, be alinically relevant means of preventing the development ofymphedema.

echanisms of improved diabetic wound healingchieved with topical silencing of p53huong D Nguyen MD, John P Tutela MD, Vishal D Thanik MD,obert J Allen Jr MD, Oriana D Cohen BA, I Janelle Wagner MD,

amie P Levine MD, Stephen M Warren MD,ierre B Saadeh MD, FACSew York University Langone Medical Center, New York, NY

NTRODUCTION: Impaired diabetic wound healing is multifacto-ial and incompletely understood. p53, a master gene regulator, ispregulated in diabetic wounds. We have demonstrated improvediabetic wound healing through a novel topical p53 silencing system.e hypothesized that topical silencing of p53 improves diabeticound healing by promoting vasculogenesis and abrogating apopto-

is.

ETHODS: In vitro, p53 was silenced in Leprdb/db diabetic mouseermal fibroblasts in normoxia (21% O2) and hypoxia (�1% O2) toimulate injury. In vivo, p53 was silenced in 4-mm wounds oneprdb/db mice (control � nonsense siRNA). Wounds were photo-etrically assessed and harvested for histology. In both in vitro and in

ivo groups, vasculogenic and apoptotic cytokine expression werevaluated (Western, RT-PCR, and ELISA).

ESULTS: RT-PCR demonstrated a 766% increase in proapop-otic Bax expression in diabetic fibroblasts exposed to hypoxiaersus an 11.97-fold decrease after silencing p53. Wounds closedaster with p53 silencing (18 � 1.3 d) versus controls (28 � 1 d)p�0.05). Corresponding histology showed near-complete p53nockdown and increased granulation tissue compared withonsense-treated wounds. Treated wounds showed a 7.63-fold

ncrease in CD31 endothelial cell staining and decreasedytochrome-c staining, an apoptosis marker, over controls. At day0, VEGF was significantly increased in treated wounds (109.3 �3.9 pg/mL) versus controls (33.0 � 3.8 pg/mL) (p�0.05). RT-CR demonstrated a 1.86-fold increase in SDF-1 expression inreated wounds.

ONCLUSIONS: Our data suggest that p53 silencing improves di-betic wound healing by abrogating apoptosis and augmenting vas-ulogenesis. These results aid in elucidating the mechanisms of dia-

etic wound healing and in developing targeted therapies.