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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS milieu in CVI ulcers.

P127. LYMPHOTOXIN-ALPHA IMPAIRS ENDOTHELIAL FUNCTIONS IN PORCINE AND HUMAN CORONARY ARTERY ENDOTHELIAL CELLS. J. J. Naoum, H. Chai, P. Lin, A. B. Lumsden, Q. Yao, C. J. Chen; Baylor College of Medicine, Houston, TX Background: Lymphotoxin-alpha (LTA) is a member of the tumor necrosis factor family that mediates inflammation and is associated with the pathogenesis of vascular disease. However, it is not clear whether LTA could affect endothelial functions. The objective of this study was to determine the effects of LTA on vasomotor functions and endothelial nitric oxide synthase (eNOS) expression in porcine coronary arteries (PCAs) and human coronary artery endothelial cells (HCAECs). Methods: For the vasomotor studies, PCAs were cut into 5-mm rings, treated with increasing concentrations of LTA (1, 10, 20, and 40 ng/ml), and tested in a myograph tension system. The eNOS expression in PCAs and HCAECs was measured using real time PCR. The eNOS protein level was assessed by immunohistochemistry staining. Superoxide anion production was detected by the lucigenin-enhanced chemiluminescence method and by DHE staining with flow cytometry analysis. Antioxidant selenomethionine (SeMet) was included. Results: Endothelium-dependent vasorelaxation in response to bradykinin was reduced by 21%, 22%, and 28% for the PCA rings treated with 10, 20, and 40 ng/ml of LTA, respectively, as compared to controls (p⬍ 0.05). In PCA endothelial cells treated with 10 and 20 ng/ml of LTA, eNOS mRNA levels were reduced by 38 % and 54%, respectively, as compared to controls (p⬍ 0.05). Similarly, eNOS mRNA expression in HCAECs was reduced by 14%, 40%, and 49% with 1, 10, and 20 ng/ml of LTA treatment, respectively, as compared to controls (p⬍ 0.05). The antioxidant SeMet effectively reversed the effects of LTA on superoxide anion increase and eNOS downregulation in both PCA endothelial cells and HCAECs (p⬍0.05). Immunoreactivity of eNOS in LTA-treated PCA rings was also reduced. Conclusions: LTA impairs endothelium-dependent vasorelaxation in PCAs. This effect is associated with increased superoxide anion production and decreased eNOS expression in both PCAs and HCAECs. This study suggests that LTA may contribute to vascular lesion formation.

P128. LYSOPHOSPHATIDIC ACID DECREASES ENOS EXPRESSION AND INCREASES SUPEROXIDE ANION PRODUCTION IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS AND PORCINE CORONARY ARTERIES. L. N. Ochoa, H. Chai, N. Hedayati, P. Lin, A. Lumsden, Q. Yao, C. Chen; Baylor College of Medicine, Houston, TX

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Objective: Lysophosphatidic acid (LPA) is a phospholipid is the active component of low-density lipoproteins generated upon oxidation. It accumulates in atherosclerotic plaques, and may play a role in atherogenesis. The objectives of this study were to determine the effects of LPA on endothelial cells and to investigate the potential mechanisms of endothelial dysfunction. Methods: Porcine coronary artery rings and human coronary artery endothelial cells (HCAEC) incubated with increasing concentrations of LPA (2, 10, and 50 ␮M) were compared to controls. Rings were also treated with LPA 10␮M plus the antioxidant selenomethionine (SeMet, 20␮M). A myograph tension system was used to evaluate vasomotor function. Superoxide anion (O - 2) production was analyzed by the lucegenin-enhanced chemiluminescence method. Immunohistochemistry (IHC) for eNOS was used to stain LPA-treated rings. Endothelial nitric oxide synthase (eNOS) mRNA expression in HCAECs was evaluated by realtime PCR. Fluorescent-activated cell sorter (FACS) technology was used to evaluate O - 2 production and mitochondrial potential of HCAECs. Finally, the ATPLite assay system was used to measure ATP content in HCAECs. Results: LPA did not alter maximum contraction nor endothelium-independent relaxation. When challenged with 10 -5 M bradykinin,10 and 50 ␮⌴ LPA-treated rings showed a 32% and 49% reduction in endothelium-dependent relaxation as compared with controls (p⬍0.005). Similarly treated rings had a 42% and 95% increase in O - 2 production. 2␮M LPA produced no significant effect. IHC staining demonstrated decreased eNOS expression in rings treated with LPA. SeMet reversed the vasomotor dysfunction and preserved the expression of eNOS induced by LPA. Expression of eNOS mRNA was reduced in 10 and 50␮M LPAtreated HCAECs by 24% and 40% (p⬍0.05), respectively. An increase in superoxide anion production from 40% to 62% (control vs LPA) paralled a corresponding decrease in mitochondrial potential (70% vs 50%) and ATP content (36.25 nmol/100␮g protein vs 25.74) in LPA treated HCAECs (all, p⬍0.05). SeMet reversed these LPA induced effects in HCAECs. Conclusion: This original data demonstrate that LPA alters endothelial cell regulation of vascular tone by a mechanism associated with oxidative stress and decreased eNOS production in porcine coronary arteries and HCAECs. SeMet may potentially be therapeutic in reversing LPA induced vasomotor dysfunction and potentially abrogate early atherogenesis and its progression.

P129. IN VIVO STUDY OF A TISSUE ENGINEERED GRAFT SEEDED WITH AUTOLOGOUS STEM CELLS. N. Golesorkhi, C. Dimatteo, D. Grabo, L. Fischer, N. Tarola, I. Shapiro, T. Tulenko, R. A. Carabasi, III, R. Larson, J. Lombardi, P. J. Dimuzio; Thomas Jefferson University, Philadelphia, PA Introduction: Autologous human adult stem cells are an attractive source of cells for vascular tissue engineering. Recent work suggests that stem cells derived from adipose tissue can acquire endothelial phenotype. In this study, we examined the heeling of a natural vascular tissue scaffold (decellularized vein allograft) seeded with autologous stem cells isolated from adipose tissue, hypothesizing that the seeded cells would provide the grafts with a non-thrombogenic lumen. Method: The tissueengineered grafts were evaluated in a canine model over two weeks. External jugular vein was harvested from donor animals and decellularized with sodium dodecyl sulfate. Autologous stem cells (ASC) and microvessel endothelial cells (EC) were isolated from adipose tissue removed from the falciform ligament of recipient animals. Bilateral carotid interposition grafts created using decellularized vein allografts were implanted as follows: 1) unseeded controls (n⫽6), 2) seeded with undifferentiated ASC (n⫽6), and 3) seeded with microvessel EC (n⫽4). All animals were maintained on aspirin (81mg) daily. Prior to harvest, graft patency was evaluated by duplex ultrasound. Perfusion-fixed grafts were then submitted for histologic analysis. Results: All animals (n⫽8) survived to harvest without any neurological deficit. No aneuris-

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

mal dilatation or graft rupture occurred. Graft occlusion was observed in 2/6 ASC grafts, 1/4 EC grafts, and 0/6 unseeded grafts. Upon harvest, significant perivascular inflammation was noted surrounding the ASC grafts. Macroscopic examination of the patent grafts revealed fibrin deposition on both the ASC and EC seeded grafts. Histologic analysis revealed significant transmural inflammation within the ASC grafts compared to EC grafts and unseeded controls. Conclusion: Tissue-engineered vascular grafts seeded with undifferentiated stem cells derived from adipose tissue exhibit inflammation and thrombogenicity. These findings may in part be due to the undifferentiated state of these cells, as grafts seeded with microvessel endothelial cells from the same tissue source showed less inflammation. These results suggest that ex vivo differentiation of ASC will likely be required prior to implantation if they are to be used for vascular graft creation.

P130. ARGINASE BLOCKADE LESSENS ENDOTHELIAL DYSFUNCTION AFTER THROMBOSIS. C. Lewis, M. L. Pavkov, C. M. Kinney, P. E. Dicorleto, V. S. Kashyap; The Cleveland Clinic Foundation, Cleveland, OH Introduction: Acute arterial thrombosis causes endothelial dysfunction in multiple animal models. This thrombus-induced endothelial dysfunction is attributed to decreased nitric oxide (NO) levels and is ameliorated via L-arginine supplementation. Substrate competition by arginase and NO synthase may be important in modulating intracellular L-arginine levels. The purpose of this study was to identify the role of arginase in the endothelial dysfunction and to study the effect of arginase inhibition on the vasomotor response in arteries exposed to thrombus. Methods: Adult male Sprague-Dawley rats underwent aortic occlusion via clip ligature to induce arterial thrombosis. After 1 hour, ring segments from the infrarenal aorta (IRA) were harvested and placed into physiologic buffered baths to measure endothelial-dependent relaxation (EDR) and endothelial independent relaxation (EIR) with the use of a force transducer (Radnoti Systems, Inc). Arginase inhibition was performed by incubating IRA with arginase inhibitors for 1 hour before measuring EDR. Animals with non-thrombosed normal aortic segments served as controls. ANOVA analyses were used to compare EDR and EIR curves. A P value ⬍0.05 was considered significant. Results: Arterial thrombosis causes a decrease in EDR. The IRA exposed to thrombosis for 1 hour (n⫽8) had diminished EDR curves when compared to control IRA (n⫽14). The max EDR (acetylcholine 10 -5 M) in control IRA was 108%⫾ 4.3 (⫾ SEM) as compared to 63%⫾ 6.2 for IRA exposed to thrombus ( p⬍0.001). Pre-incubation of the IRA (n⫽9) with a non-specific arginase inhibitor (DFMO) for 1 hour significantly increased the max EDR as compared to untreated thrombosed IRA (n⫽7) (104⫾ 5.2 vs 53%⫾ 7.0, p⬍0.001). Pre-incubation with a specific arginase inhibitor (BEC) (n⫽6) also significantly increased max EDR compared to untreated thrombosed IRA (108⫾ 7.6 vs 53%⫾ 7.0, p⬍0.001). The EIR of control, untreated thrombosed IRA, and IRA pre-incubated with DFMO and BEC were similar (105⫾ 4.8, 104⫾1.3, 105⫾2.3, and 102 ⫾0.4 % respectively, sodium nitroprusside 10 -7 M, P ⫽ N.S.) indicating normal smooth muscle function. Norepinephrine-induced vasoconstriction in control IRA (1.0g⫾0.7), in untreated thrombosed IRA (0.96g⫾ 0.1), and in DFMO (0.72g⫾ 0.04) and BEC (0.92g⫾ 0.2) pre-incubated IRA were similar (P ⫽ N.S.). Immuno-blot analyses of rat aortic specimens indicated arginase I, but not arginase II, protein concentration was increased following arterial thrombosis. Conclusions: Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Endothelial dysfunction caused by acute arterial thrombosis is due to arginase I and is reversed by both specific and non-specific arginase inhibitors. These results indicate that arginase plays a critical role in endothelial dysfunction and its blockade can lead to normalization of arterial vasomotor function.

P131. GROWTH-FACTOR RICH ENDOTHELIAL CELL MEDIA INDUCES ADIPOSE-DERIVED STEM CELLS TO ACQUIRE ENDOTHELIAL CELL PHENOTYPE. A. Mericli, N. Tarola, L. Fischer, C. Dimatteo, N. Golesorkhi, D. Grabo, S. McIlhenny, K. Scully, T. Tulenko, I. Shapiro, P. Dimuzio; Jefferson Medical College, Philadelphia, PA Background: This study evaluates the effect of endothelial growth factors and shear stress on the differentiation of adiposederived stem cells (ASC) into endothelial-like cells. We hypothesize that culture in a commercially-available endothelial cell media enriched with growth factors promotes ASC to acquire endothelial phenotypes, and that exposure of these cells to physiologic shear stress aids in differentiation. Methods: Human ASC (CD13 ⫹ 29 ⫹90 ⫹31 -45 -; passage 3-6) isolated from lipoaspirated fat (n⫽4 different donors) were cultured in EGM-2 endothelial cell media for up to 21 days. Cultured cells were additionally exposed to shear stress in vitro (10 dynes/cm 2) for up to 96h using an orbital shaker. Endothelial phenotype was defined as re-alignment with flow and expression of endothelial-specific markers (eNOS, vWF, CD31) as measured by RT-PCR and immunohistochemistry. Differentiated human microvessel endothelial cells and smooth muscle cells were used for positive and negative controls, respectively. Results: At baseline, ASC did not re-align with flow or express any of the noted endothelial markers. After 14 and 21 days of culture in EGM-2 media, no gross morphologic changes were noted; however, ASC expressed eNOS and vWF, but not CD31. Upon exposure to shear stress for 48 and 96h, the cells re-aligned in the direction of flow and expressed CD31, in addition to both eNOS and vWF. Conclusions: ASC cells cultured in media enriched with endothelial growth factors demonstrate endothelial features in as early as 14 days. Exposure to physiologic shear enhances differentiation. Based on the ability to acquire endothelial phenotype, ASC may be a useful source of autologous cells for cardiovascular tissue engineering strategies.

P132. THE EFFECTS OF FLUID RESUSCITATION ON LIMB ISCHEMIA-REPERFUSION INJURY. V. I. Patel, H. Albadawi, T. Goode, R. Crawford, T. Abbruzzese, J. Kang, H. Yoo, W. Austen, Jr., M. T. Watkins; Massachusetts General Hospital, Boston, MA Background: Acute limb ischemia with associated reperfusion injury (IR) is a common clinical problem in vascular surgery. Hydration is a routine component of peri-operative care. These experiments were undertaken to test the local and systemic effects of different hydration fluids on reperfusion injury using a murine model of hind limb IR. Study Design: C57BL6/J male mice (8-10 weeks old) were subjected to 1.5 hours of unilateral hind limb tourniquet ischemia followed by 24 hours of reperfusion. Sham controls (Sham n⫽6) were subjected to anesthesia only. Four groups of mice were treated using the following resuscitation protocols: no resuscitation fluid (NR n⫽7), normal saline (NS n⫽8), Lactated Ringer’s (LR n⫽12), and 5% Dextrose with 150mEq/l sodium bicarbonate (SB n⫽9). Each animal received intraperitoneal injections of 0.4 ml of corresponding fluid at 15 min prior to ischemia, 10 min pre-reperfusion, 5 min and 2 hours post reperfusion, and 0.2 ml at 4, 6, 12, and 22 hours post reperfusion. At 24 hours, serum was collected for measurement of systemic levels of the pro-inflammatory cytokine Interleukin-6 (IL-6) using ELISA. Hind limb muscles were harvested and tissue ATP levels as a measure of metabolic activity were assessed by chemiluminescence. ELISA was used to measure the local levels of the pro-inflammatory cytokine (KC) and the marker of neutrophil infiltration Myeloperoxidase (MPO). Serum lactate levels were measured using spectrophotometry. Comparison between the groups was performed using an analysis of variance (ANOVA) with a p’0.05 considered significant. Results: During reperfusion,