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rhodamine to allow for intracellular visualization. Rhodamine-la-beled expansile nanoparticles (Rho-eNP) were added to the media ofeach treatment group and co-incubated for an additional 4h. Cellswere collected and washed in FACS buffer and the intracellular pres-ence of Rho-eNP within tumor cells was studied using flow cytometry(LSRFortessa, BD Biosciences Bedford, MA). Results: MSTO cellsrapidly demonstrate uptake of Rho-eNP within 4h. in the presenceof inhibitors of macropinocytosis (wortmannin and hexamethyleneamiloride) the uptake of Rho-eNP is reduced by greater than 40%.in contrast, MSTO cell uptake of Rho-eNP were not affected by inhib-itors of general endocytosis, cholesterol dependence, caveolae-medi-ated or clathrin mediated pathways of endocytosis, as Rho-eNPuptake remained greater than 95% of untreated controls. Conclu-sions:Uptake of expansile nanoparticles by tumor cells utilizes path-ways of macropinocytosis. This pathway is non-receptor dependentthus allowing rapid nanoparticle uptake to occur in tumors of variouscell origins; thus explaining similar anti-tumor efficacy against breastand ovarian carcinoma in vivo.

FIG. 1. Flow Cytometry demonstrating decreased uptake ofRho-eNP in the presence of inhibitors of macropinocytosis pathways(hexa methylene amiloride and wortmannin).

37.8. Vascularized Adipose Tissue Flaps Engineered WithMinicircle DNA Provide a Platform for SustainableTransgene Expression. R. C. Rennert, M. Sorkin, S.Morrison, P. Tran, L. H. Evers, M. T. Longaker, G. C.Gurtner; Stanford University School of Medicine, Stanford, CA

Introduction: Several single gene disorders, including factor IX de-ficiency, require gene delivery strategies that would provide a stable,safe and sustainable systemic protein release. Previous techniques re-lying on viral or plasmid gene delivery are clinically limited by risk ofinsertionalmutagenesis or poor efficiency due to gene silencing.Mini-circle (MC) DNA represents an ideal gene delivery system that is epi-somal and resistant to in vivo gene silencing. MCs are engineeredthrough removal of the bacterial backbone of plasmid DNA, prevent-ing immunogenicity and subsequent transgene elimination. Adiposetissue flaps are ideal candidates for MC transfection due to their ex-cellent vascularization, peripheral location and expendability. Wepropose MC transfection of an adipose tissue flap as a safe, reversibleand clinically translatable gene delivery system. Methods: In vitro,murine adipocytes and adipose derived stem cells (ASCs) were iso-lated from inguinal fat pads and transfected with MC DNA encodingfor luciferase or GFP via nucleofection. In vivo, naked MC DNA wasinjected into murine inguinal fat pads at two doses (10ug or 30ug,n¼6), and compared to injection of PBS alone (n¼4). for all experi-ments, successful transgene expression was determined using fluo-rescence microscopy for GFP and bioluminescent in vivo luciferaseimaging (IVIS) for luciferase MCs.Results: In vitro nucleofection re-sulted in efficient transgene expression in both murine ASCs and ad-ipocytes. In vivo luciferase MC DNA injection showed a localized,

initial maximum luminescence of 1.81x106 and 9.44x105 photons/s/sr/cm2 at 30 and 10ug, respectively, compared to 1.02x104 photons/s/sr/cm2 in the negative control (p¼0.02). Although luciferace expres-sion declined initially, it remained localized to the fat pad and wastraceable over a mid-term period of time.Conclusions: Vascularizedadipose tissue flaps are particularly suitable for sustainable gene de-livery approaches since they are easily accessible and expendable. Wesuccessfully show that transfection withMCDNA is a viable and safein vivo gene delivery system that provides efficient mid-term trans-gene expression with minimal adverse effects. This has significantclinical implications, specifically with respect to the treatment of sin-gle gene disorders where a constant systemic release of a factor isrequired.

37.9. Manipulation of the Intrinsic Apoptotic Pathway to En-hance Stem Cell Survival Towards an Osteogenic Line-age. D. Lo, J. Hyun, D. Montoro, M. Grova, D. C. Wan, M. T.Longaker; Stanford University School of Medicine, Stanford,CA

Introduction: There are significant hurdles to overcome for the ulti-mate widespread clinical application of stem cells. the exact fate ofthese cells when implanted in vivo remains a challenging and impor-tant question. the clinical application of these cells often involvesplacement into a wound environment that is hypoxic with an upregu-lation of inflammatory mediators. Within this hostile environment,a large percentage of the implanted progenitor cells undergo apopto-sis. the Bcl-2 family is a group of proteins that function as one of themain mediators of cellular apoptosis and survival. These proteinsare divided into pro-survival and pro-apoptosis groups and the bal-ance of expression between the two groups is a critical component indetermining cellular fate. in this study, we overexpressed Bcl-2,a pro-survival protein, in order to evaluate whether it would increasecellular survival during osteogenic differentiation.Methods:Humanadipose-derived stromal cells (hASC) were obtained from lipoaspirateaccording to Stanford IRB protocols. Human embryonic stem cells

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(hESC) were obtained through existing federally mandated cell lines.the intrinsic apoptosis pathway was manipulated through the over-expression of the pro-survival protein, Bcl-2 in hASC and hESC. theability of hASC and hESC with Bcl-2 over-expression to undergoin vitro osteogenic differentiation was tested using qPCR, westernblotting, immunocytochemistry and staining for alkaline phospha-tase and Alizarin Red after placement in osteogenic medium. En-hanced cellular survival with Bcl-2 overexpression in hASC andhESC was evaluated by placement of these cells under hypoxic condi-tions and through the induction of apoptosis using a pro-apoptoticchemotherapeutic agent.Western blotting and ELISAwere employedto evaluate levels of pro-apoptotic mediators cytochrome c and Cas-pase 3, 7, and 9. Results: Bcl-2 over-expression in hASC and hESCdid not compromise the ability for these stem cells to differentiatedown an osteogenic lineage in vitro as shown by comparable expres-sion of early osteogenic genes, alkaline phosphatase and Runx2.Bcl-2 overexpression also showed comparable mineral deposition asshown by Alizarin Red staining. However, Bcl-2 over-expression sig-nificantly decreased mediators of apoptosis in vitro under hypoxicconditions and during the introduction of a pro-apoptotic chemother-apeutic agent. Specifically, Bcl-2 over-expression inhibited the apo-ptotic mediators cytochrome c and Caspase 3, 7, and 9 (*p<0.05).Conclusions: These data show that overexpression of Bcl-2 canmod-ulate effectors of apoptosis leading to increased survival of stem cellsdirected towards an osteogenic lineage. the ability to increase survivalof implanted progenitor cells will be a critical step for the widespreaduse of these cells in regenerative medicine.

37.10. Alginate Gels Treated With Nonthermal Plasma: ANovel Wound Dressing. A. E. Poor,1 U. K. Ercan,1 S. G.Joshi,1 A. Fridman,2 G. Fridman,2 G. Friedman,2 A. D.Brooks1; 1Drexel University College of Medicine,Philadelphia, PA; 2A.J. Drexel Plasma Institute, Camden, NJ

Introduction: Treatment and prevention of wound infection re-mains a challenge in medicine. Currently, many dressings are usedwith varying efficacy as antimicrobials and barriers to contamination.Alginate hydrogels are often used because they are soft, inert, havehemostatic and bacteristatic properties, and can absorb largeamounts of exudate relative to their size. Alginates are often modifiedwith bactericidal materials (chlorhexidine or silver ion) to improvetheir efficacy. Generally, the more effective antimicrobial agents aremore irritating to tissue, thus the most effective antimicrobial dress-ings cannot be placed on open wounds. Methods: We used non-ther-mal plasma discharge to confer antimicrobial properties to analginate hydrogel. Non-thermal plasma energizes electrons, whichthen produce excited species, free radicals, and ions that have biocidaleffect. We used colony count assays to determine bacterial killing andViaCount assays to determine cellular toxicity. Results: Alginate

treated with non-thermal plasma for 1 minute sterilized 10^9CFU/mL E. coli. By placing a time interval between exposure to plasmaand bacteria, we found that the antimicrobial properties after a 3-minute plasma treatment remain undiminished for 21 days. Wealso found a wide spectrum of activity: treated hydrogels inactivatedevery antibiotic resistant organism tested. Finally, we exposed epi-thelial cells and fibroblasts to treated hydrogels, chlorhexidine-con-taining dressings, and silver-containing dressings. the hydrogelswere less toxic than chlorhexidine-containing dressings and compara-ble to silver-containing dressings. Conclusions: These results sug-gest that this novel product has potential for clinical use. Futureresearch will assess the toxicity of plasma-treated gels in vivo aswell as the efficacy in treating clinically infected wounds.

GASTROINTESTINAL AND NUTRITION 2:CYTOKINES, GROWTH FACTORS &

INFLAMMATION

38.1. Vodka and Wine Consumption in a Swine Model of Met-abolic Syndrome Alters Insulin Signaling Pathways inthe Liver and Skeletal Muscle. N. Y. Elmadhun, A. D.Lassaletta, L. M. Chu, C. Bianchi, F. W. Sellke; Division ofCardiothoracic Surgery, Cardiovascular Research Center,Warren Alpert School of Medicine, Brown University,Providence, RI

Introduction:The purpose of this studywas to examine the effects ofvodka and wine consumption in the context of metabolic syndrome oninsulin signaling pathways in the liver and skeletal muscle.Methods: Male Yorkshire swine developed metabolic syndrome byconsuming a high calorie, high-fat/cholesterol diet for 4 weeks. Pigswere then split into three different groups according to diet supple-mentation for an additional 7 weeks. the control group was continuedon a hypercholesterolemic diet alone (HCC) (n¼9). the hypercholes-terolemic vodka (HCVOD) and hypercholesterolemic wine (HCW)groups were supplemented with 112 mL of vodka (40% EtOH/V)(n¼9) and 375 mL of red wine daily (12.5% EtOH/V) (n¼8) respec-tively. Animals underwent intravenous dextrose challenge prior toeuthanasia and tissue collection. Protein expression in the liver andskeletal muscle was analyzed by western blot. Results: At baseline,HCC, HCVOD and HCW groups had similar blood glucose levels(67.00, 66.13, 62.78 mg/dl respectively). the mean BMI of the HCC,HCVOD and HCW groups were also not statistically different(35.02kg/m2, 32.44kg/m2and 33.17kg/m2respectively). the dextrosechallenge showed that HCVOD and HCW groups had significantlyhigher blood glucose levels at 30 minutes (210 and 206.89mg/dl) and60 minutes (147.13 and 125.89mg/dl) compared to HCC (166.29mg/dl and 94.57mg/dl). Western blot analysis in skeletal muscle demon-strated a marked increase in the markers involved in the insulin sig-naling pathway including phosphorylated insulin receptor substrate1 (p-IRS1) (Ser612), IRS2, AKT, AMP-activated protein kinase(AMPKa), peroxisome proliferating activated receptor a (PPARa),Fox01, and glucose transporter type 4 (GLUT4). There was a decreasein the p-AKT (Thr 308) level in skeletal muscle. in the liver, there wasonly a moderate increase in the markers of insulin signaling in HCWincluding AKT, AMPKa, and GLUT4 (see table). Conclusions:Vodka andwine consumption in a swinemodel of metabolic syndromeincreases glucose intolerance compared to the control. the westernblot data demonstrates an altered activation of the insulin signalingpathway in the liver and skeletal muscle. Whereas an inhibitor ofthe insulin signaling pathway p-IRS-1 is elevated and an activatorof the pathway p-AKT is decreased, there are also elevated levels ofother markers involved in propagating insulin signaling. the mixedsignals may result in the observed glucose intolerance. Alcohol seemsto have a complex and negative effect on the insulin signal transduc-tion pathway.