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Characterization of two populations of human coronary artery endothelial cells
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS P76. Acellular Scaffolding for Vascular Tissue-engineering in a Canine Model. P. J. Schaner, M.D., F. E. Rosato, Jr., M.D., N. A. Tarola, M.D., J. Eisenberg, M.D., N. Martin, M.D., S. Abdul-Misih, M.D., J. Tamburrino, M.D., D. A. Popowich, M.D., P. J. DiMuzio, M.D. Thomas Jefferson University Department of Surgery, Philadelphia, PA. Introduction: We are presently investigating acellular venous tissue as a bioscaffold to be recellularized with the recipient’s own cells prior to implantation. This study tests the biocompatibility and mechanical stability of this construct in terms of graft patency, acute dilatation and rupture, the ability to achieve and maintain hemostasis and resist infection in a canine model. Methods: Twenty-six carotid interposition grafts were constructed using 13 mongrel dogs. Graft material was obtained from the external jugular veins of the same 13 animals (14 autografts, 4 acellular autografts, 4 allografts, and 4 acellular allografts). Decellularization was performed using 0.075% SDS. At two weeks, each graft was evaluated for evidence of gross patency and dilatation, and perfusion fixed for detailed histological and immunohistochemical analyses (H&E, Factor VIII, smooth muscle actin, and inflammatory markers). Morphometric analysis of the cells present in 20 random high-powered fields (hpf) was conducted at the graft’s midpoint. Healing response was further characterized by evaluation of graft incorporation into surrounding tissues and recellularization at the anastamotic sites. Results: All animals survived without neurologic deficit with 100% patency, and no dilatation or rupture. There were no clinically significant infections. The quantified inflammatory reaction was significantly greater in fresh allografts compared to acellular allografts, acellular autografts and fresh autografts: 59 ⫾ 24 vs 19 ⫾ 15, 27 ⫾ 18 or 19 ⫾ 8 cells/hpf, respectively, P ⬍ 0.05. There was no significant difference between fresh autografts and acellular allografts, P ⬎ 0.05. Fresh autografts also retained endothelial and smooth muscle cells, which were absent in decellularized grafts at implantation and at two weeks. Conclusions: We have previously shown sufficient strength, the ability to support endothelial cells, preserved extracellular collagen and elastin, and reduced allosensitization. Acellular venous scaffolding also appears to avoid acute dilatation or rupture, maintain hemostasis and remain free of infectious complications. It is our hope that this may improve the overall utility and functionality of autologous cell seeding and continue to remodel as the recipient’s new blood vessel in vivo. P77. Catechin and Sulfated Polysaccharide Nutriceuticals Inhibit MMP-2 (Gelatinase-A) of Fibroblasts Cultured from Abdominal Aortic Aneurysms (AAAs). S. J. Belsley, M.D., J. Obunike, Ph.D., P. Collin, B.S., M.D., M. D. Tilson, M.D. St. Luke’s–Roosevelt Hospital Center and Columbia University. Background: Matrix MetalloProteinases (MMPs), particularly #’s 2 and 9, have been implicated in the cascade of proteolytic events that lead to aneurysmal dilation of the aorta; and a recent clinical trial of doxycycline has addressed the potential of an MMP inhibitor to retard the expansion of small AAA’s. However, the rate of AAA expansion was 0.63%/month; and several side effects were noted, including photosensitivity. Accordingly, we have commenced screening other compounds for inhibitory activity against MMP-2, which is the predominant gelatinase produced by cultured AAA fibroblasts. We have concentrated on nutriceuticals that are currently in the human food chain and have GRAS (Generally Regarded As Safe) status in the USA. We tested two products, AneuMastat ™ , a green tea polyphenol known to inhibit MMPs #2 and #9, and InflaMastat®, a sea cucumber-derived sulfated polysaccharide commercially available in the USA. Methods: Fibroblasts cultured from AAA specimens were lysed, and the protein content of the supernatant was quantified. Substrate gel enzymography (SGE) was used to test inhibitory properties of candidate compounds against MMP-2 of AAA
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fibroblast origin. Results: InflaMastat® partially inhibited MMP-2 activity (⬃80 %) at 100 ug/ml against 30 mg lysate; and when combined with the catechin-containing product AneuMastat ™ inhibition was 100% complete under the experimental conditions (no detectable lysis). Further experiments with four additional purified compounds from similar sources suggested that the patterns of inhibition are variable and complex. One agent was 90% effective as an inhibitor at all concentrations studied; while another was inhibitory at low concentrations but exhibited intrinsic gelatinolytic activity at higher concentrations. Conclusion: Although the results of the initial clinical trial of doxycycline as an inhibitor of AAA expansion in man were disappointing, the present experiments suggest that there is a strong rationale for investigation of additional inhibitors of matrix metalloproteinases, which may have greater efficacy and fewer side effects than the recently-trialed doxycycline. P78. Differentiation of Endothelial Proginetor Cells from Canine Peripheral Blood Mononuclear Cells. H. Wu, M.D., Ph.D., Q. Yao, M.D., Ph.D., C. Chen, M.D., Ph.D., Baylor College of Medicine. Background: Endothelial cell differentiation from peripheral blood or bone marrow cells is of great significance in angiogenesis, vascular healing, and tissue engineering. The purpose of this study was to characterize endothelial cell morphology, gene expression, and growth curve of endothelial progenitor cells (PEC) differentiated from canine peripheral blood mononuclear cells (PBMCs). Methods: PBMCs were isolated from fresh dog blood by Hitopaque-1077. Cells were cultured in fibronectin coated plates with endothelial basic medium (EBM-2) for different periods of time. PECs were identified from cell morphology and outgrowth characteristics. Gene expression was studied with flow cytometry analysis. Cell counting technique was used to study cell growth curve of PECs in comparison with primary human coronary artery endothelial cells. Results: At first week of canine PBMC culture, cells were round and many cells died every day. The cell proliferation was very low. At 2 weeks, many cells became spindle shape, showing endothelial morphology. At 3 weeks, many colonies of PECs with cobblestone morphology showed outgrowth. Canine PECs had great growth potential and reached a monolayer in a few days. They formed tight junctions, and showed endothelium like characteristics. Cell markers were studied at 25 days of culture. CD34 was significantly decreased compared to 7 days culture (54% to 6.27%). vWF was significantly increased (from 8.2% to 58.55%). CD31 was not changed. VEGF-R2 was slightly increased. Endothelial nitric oxide synthase was significantly increased (from 0 to 14.97%). Canine PECs showed a significant higher proliferation rate as compared to mature human coronary artery endothelial cells at the same culture condition. Conclusions: These data demonstrate that canine PBMCs are able to differentiate into PECs with characteristics of endothelial morphology and cell specific markers, and canine PECs have a greater growth potential than mature endothelial cells. This study suggests that PBMCs could be a source of PECs, which have potential applications in tissue engineering and vascular therapy. P79. Characterization of Two Populations of Human Coronary Artery Endothelial Cells. H. Wu, M.D., Ph.D., Q. Yao, M.D., Ph.D., C. Chen, M.D., Ph.D., Baylor College of Medicine. Introduction: Heterogeneity of endothelial cells may affect angiogenesis, vascular healing, and cardiovascular disease formation. The objective of this study was to identify and characterize populations of human coronary artery endothelial cells (HCAECs), their gene expression levels, and response to TNF-␣ stimulation. Methods: Commercial HCAECs were cultured with EGM-2 complete medium. Cell population was determined by flow cytometry analysis based on size-scatter distribution. Gene expression for each population with or without TNF-␣ (1 ng/ml) stimulation for 16 hours was
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also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies. Results: Two distinguished populations, HCAEC-I (small) and HCAEC-II (large), were identified through all passages (from 2 to 10) during cultures. Both HCAEC-I and HCAEC-II showed more than 90% positive staining for both vWF and CD31, and were negative for CD34 and CD4 (less than 2%). HCAEC-I had substantially higher expression of Cadherin-5 (71.91% vs 51.07%) than HCAEC-II. HCAEC-I also expressed much higher levels of VEGF-R2 (17.35% vs 0.77%), eNOS (44.64% vs 2.54%), VCAM-1 (6.08% vs 1.18%), E-selectin (18.68% vs 1.42%), CXCR4 (61.05% vs 7.98%), and CCR5 (48.66% vs 1.97%) than HCAEC-II. However, HCAEC-II had substantially higher expression of P1H12 (93.07% vs 58.73%) and ICAM-1 (94.37% vs 58.62%) than HCAEC-I. Following TNF-␣ stimulation, both populations substantially increased ICAM-1, VCAM-1 and E-selectin expressions. However, HACEC-I had much higher expressions of VEGFR-2, eNOS, CXCR4, and CCR-5 than HACEC-II after TNF-␣ stimulation. Conclusions: These data demonstrate that commercial HCAECs exist two distinguished populations with different gene expression patterns and different responses following TNF-␣ stimulation. This study indicates that the heterogeneity of HCAECs may have biological or pathological significances in coronary arteries. P80. Covalent Linkage of Heparin Provides a Stable AntiCoagulation Surface of Decellularized Porcine Arteries. C. Chen, M.D., Ph.D., B. Conklin, Ph.D., N. Barber, B.S., E. Richter, B.S., Q. Yao, M.D., Ph.D., P. Lin, M.D., S. Hanson, Ph.D., A. Lumsden, M.D., Baylor College of Medicine. Introduction: Establishing thrombosis resistant surface is crucial to develop successful tissue engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anticoagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Methods: Porcine carotid arteries were obtained from a local abattoir. Cellular components of the arteries were completely removed with a newly developed method of decellularization. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulfate salt and heparin-EDC. Bound heparin contents were measured by a quantitative colorimetric assay of toludine blue staining. Anticoagulation property of bound heparin was determined with a clotting time assay using both human and pig fresh blood. In vivo platelet deposition of the vessel was carried out with a baboon model of the femoral arteriovenous external shunt and 111Indium labeling of platelets. Results: The average content of heparin in treated vessels was 35.6 ⫾ 11.6 mg/g tissue (ranging from 31 to 42 mg/g tissue), which represented 6.21 ⫾ 2.03 UPS heparin/cm 2 tissue (ranging from 5 to 8 UPS/cm 2 tissue). The stability of heparin linkage was tested by incubating the heparin linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Furthermore, several vessels were stored in 70% alcohol for more than 2 years and the contents of bound heparin was not reduced. Standardized small pieces (3.3 ⫻ 3.3 mm 2) of non-heparin bound vessels were clotted in both human and pig fresh blood within 13 min, while all heparin bound vessels did not form clot during 1 hour observation. There were 1.38 ⫾ 0.07 ⫻ 10 9, and 0.64 ⫾ 0.11 ⫻ 10 9 baboon platelets deposited on the control and heparin linked vessels, respectively, at 60 min. Conclusions: These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue engineered biological vascular grafts which could use for human coronary or low extremity artery bypasses. P81. Development of a Biologically Active Dacron Vascular Graft Surface. E. R. Deutsch, M.D., M. D. Phaneuf, B.S.,
D. J. Willis, M.D., M. J. Bide, Ph.D., J. A. Kalish, M.D., F. W. LoGerfo, M.D., W. C. Quist, M.D., Ph.D. Israel Deaconess Medical Center. Introduction: Prosthetic arterial graft failure is, in part, a consequence of incomplete healing at the blood/biomaterial interface. Our previous work demonstrated that human umbilical vein endothelial cell (HUVEC) proliferation was enhanced on the novel bifunctionalized Dacron surface, created by exposure to ethylenediamine (EDA). The goal of this study was to covalently bind basic fibroblast growth factor (bFGF), a mitogen for HUVECs, to our bifunctionalized Dacron surface and examine proliferation of HUVECs on this biologically active Dacron surface. Methods: Mylar discs (1.5 cm diameter), a flattened form of Dacron, were exposed to EDA. Mylar discs were then divided into three groups: untreated (CTRL), controlEDA (C-EDA) and Tr-EDA (EDA treated mylar reacted with Traut’s reagent). bFGF was radiolabeled with 125I and reacted with the heterobifunctioal crosslinker sulfo-SMCC. After incubation with 125IbFGF-SMCC for 3 hours, all segments were then washed and gamma counted. The amount of 125I-bFGF (ng) per Mylar segment weight (mg) was calculated using specific activity. HUVEC growth was evaluated on CTRL, C-EDA and bFGF-EDA (non-radiolabeled) surfaces. Mylar discs were placed in a 24-well culture plate, seeded with 20,000 HUVECs and assessed for cellular proliferation over time using an Alamar blue assay. Results: 125I-bFGF binding, which was either non-specific or covalent, occurred on all of our surfaces. 125IbFGF binding to the Tr-EDA segments (32 ⫾ 2.8 ng/mg) was 8.1 fold greater than the CTRL segments (3.9 ⫾ 0.6 ng/mg, p ⫽ 1.1 ⫻ 10 – 6) and 1.5 fold greater than the C-EDA group (22 ⫾ 2.4 ng/mg, p ⫽ 1.5 ⫻ 10 –3). HUVEC proliferation occurred on all of our surfaces throughout the four-day time course. Segments with covalently bound bFGF demonstrated significantly greater proliferation (27 ⫾ 7.0) as compared to HUVEC proliferation on CTRL surfaces (12 ⫾ 4.1, p ⫽ 2.7 ⫻ 10 -6) and C-EDA surfaces (13 ⫾ 7.3, p ⫽ 2.5 ⫻ 10 – 4). Conclusions: This study demonstrates that growth factors such as bFGF can be covalently bound to Dacron and maintain its biologic activity in vitro. Immobilization of biologically active proteins may potentially aid in avoiding some of the complications that occur following prosthetic arterial grafting.
P82. Taurine Supplementation Reverses Endothelial Cell Dysfunction and Promotes Mobilisation of Endothelial Progenitor Cells. M. A. Moloney, M.D., J. J. Kelly, M.D., C. M. Condron, B.Sc., G. Roche-Nagle, M.D., D. H. O’Donnell, M.D., F. M. Fennessy, Ph.D., D. J. Bouchier-Hayes, M.D. Department of Surgery, Royal College of Surgeons in Ireland. Introduction: Cigarette smoking is the main, modifiable risk factor for atherosclerosis. We have previously reported reversal of endothelial dysfunction in young, healthy smokers with taurine supplementation. A heterogeneous population of endothelial cells (shed circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs)) is found in circulation. These correlate with the magnitude of injury and the reparative response respectively. We hypothesise, that taurine decreases structural endothelial injury and restores the reparative mobilisation of EPCs from the bone marrow in young smokers. Methods: We tested this hypothesis with a model of brief upper limb ischaemia, by characterising the response of CECs and EPCs to ischaemia in matched groups of non-smoking (n ⫽ 15) and healthy smoking young males (n ⫽ 15). Smokers were then treated for five days with Taurine, and the test repeated. Endothelial cell populations were determined by flow cytometry using CD45, CD133 and CD145.
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fibroblast origin. Results: InflaMastat® partially inhibited MMP-2 activity (⬃80 %) at 100 ug/ml against 30 mg lysate; and when combined with the catechin-containing product AneuMastat ™ inhibition was 100% complete under the experimental conditions (no detectable lysis). Further experiments with four additional purified compounds from similar sources suggested that the patterns of inhibition are variable and complex. One agent was 90% effective as an inhibitor at all concentrations studied; while another was inhibitory at low concentrations but exhibited intrinsic gelatinolytic activity at higher concentrations. Conclusion: Although the results of the initial clinical trial of doxycycline as an inhibitor of AAA expansion in man were disappointing, the present experiments suggest that there is a strong rationale for investigation of additional inhibitors of matrix metalloproteinases, which may have greater efficacy and fewer side effects than the recently-trialed doxycycline. P78. Differentiation of Endothelial Proginetor Cells from Canine Peripheral Blood Mononuclear Cells. H. Wu, M.D., Ph.D., Q. Yao, M.D., Ph.D., C. Chen, M.D., Ph.D., Baylor College of Medicine. Background: Endothelial cell differentiation from peripheral blood or bone marrow cells is of great significance in angiogenesis, vascular healing, and tissue engineering. The purpose of this study was to characterize endothelial cell morphology, gene expression, and growth curve of endothelial progenitor cells (PEC) differentiated from canine peripheral blood mononuclear cells (PBMCs). Methods: PBMCs were isolated from fresh dog blood by Hitopaque-1077. Cells were cultured in fibronectin coated plates with endothelial basic medium (EBM-2) for different periods of time. PECs were identified from cell morphology and outgrowth characteristics. Gene expression was studied with flow cytometry analysis. Cell counting technique was used to study cell growth curve of PECs in comparison with primary human coronary artery endothelial cells. Results: At first week of canine PBMC culture, cells were round and many cells died every day. The cell proliferation was very low. At 2 weeks, many cells became spindle shape, showing endothelial morphology. At 3 weeks, many colonies of PECs with cobblestone morphology showed outgrowth. Canine PECs had great growth potential and reached a monolayer in a few days. They formed tight junctions, and showed endothelium like characteristics. Cell markers were studied at 25 days of culture. CD34 was significantly decreased compared to 7 days culture (54% to 6.27%). vWF was significantly increased (from 8.2% to 58.55%). CD31 was not changed. VEGF-R2 was slightly increased. Endothelial nitric oxide synthase was significantly increased (from 0 to 14.97%). Canine PECs showed a significant higher proliferation rate as compared to mature human coronary artery endothelial cells at the same culture condition. Conclusions: These data demonstrate that canine PBMCs are able to differentiate into PECs with characteristics of endothelial morphology and cell specific markers, and canine PECs have a greater growth potential than mature endothelial cells. This study suggests that PBMCs could be a source of PECs, which have potential applications in tissue engineering and vascular therapy. P79. Characterization of Two Populations of Human Coronary Artery Endothelial Cells. H. Wu, M.D., Ph.D., Q. Yao, M.D., Ph.D., C. Chen, M.D., Ph.D., Baylor College of Medicine. Introduction: Heterogeneity of endothelial cells may affect angiogenesis, vascular healing, and cardiovascular disease formation. The objective of this study was to identify and characterize populations of human coronary artery endothelial cells (HCAECs), their gene expression levels, and response to TNF-␣ stimulation. Methods: Commercial HCAECs were cultured with EGM-2 complete medium. Cell population was determined by flow cytometry analysis based on size-scatter distribution. Gene expression for each population with or without TNF-␣ (1 ng/ml) stimulation for 16 hours was
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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies. Results: Two distinguished populations, HCAEC-I (small) and HCAEC-II (large), were identified through all passages (from 2 to 10) during cultures. Both HCAEC-I and HCAEC-II showed more than 90% positive staining for both vWF and CD31, and were negative for CD34 and CD4 (less than 2%). HCAEC-I had substantially higher expression of Cadherin-5 (71.91% vs 51.07%) than HCAEC-II. HCAEC-I also expressed much higher levels of VEGF-R2 (17.35% vs 0.77%), eNOS (44.64% vs 2.54%), VCAM-1 (6.08% vs 1.18%), E-selectin (18.68% vs 1.42%), CXCR4 (61.05% vs 7.98%), and CCR5 (48.66% vs 1.97%) than HCAEC-II. However, HCAEC-II had substantially higher expression of P1H12 (93.07% vs 58.73%) and ICAM-1 (94.37% vs 58.62%) than HCAEC-I. Following TNF-␣ stimulation, both populations substantially increased ICAM-1, VCAM-1 and E-selectin expressions. However, HACEC-I had much higher expressions of VEGFR-2, eNOS, CXCR4, and CCR-5 than HACEC-II after TNF-␣ stimulation. Conclusions: These data demonstrate that commercial HCAECs exist two distinguished populations with different gene expression patterns and different responses following TNF-␣ stimulation. This study indicates that the heterogeneity of HCAECs may have biological or pathological significances in coronary arteries. P80. Covalent Linkage of Heparin Provides a Stable AntiCoagulation Surface of Decellularized Porcine Arteries. C. Chen, M.D., Ph.D., B. Conklin, Ph.D., N. Barber, B.S., E. Richter, B.S., Q. Yao, M.D., Ph.D., P. Lin, M.D., S. Hanson, Ph.D., A. Lumsden, M.D., Baylor College of Medicine. Introduction: Establishing thrombosis resistant surface is crucial to develop successful tissue engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anticoagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Methods: Porcine carotid arteries were obtained from a local abattoir. Cellular components of the arteries were completely removed with a newly developed method of decellularization. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulfate salt and heparin-EDC. Bound heparin contents were measured by a quantitative colorimetric assay of toludine blue staining. Anticoagulation property of bound heparin was determined with a clotting time assay using both human and pig fresh blood. In vivo platelet deposition of the vessel was carried out with a baboon model of the femoral arteriovenous external shunt and 111Indium labeling of platelets. Results: The average content of heparin in treated vessels was 35.6 ⫾ 11.6 mg/g tissue (ranging from 31 to 42 mg/g tissue), which represented 6.21 ⫾ 2.03 UPS heparin/cm 2 tissue (ranging from 5 to 8 UPS/cm 2 tissue). The stability of heparin linkage was tested by incubating the heparin linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Furthermore, several vessels were stored in 70% alcohol for more than 2 years and the contents of bound heparin was not reduced. Standardized small pieces (3.3 ⫻ 3.3 mm 2) of non-heparin bound vessels were clotted in both human and pig fresh blood within 13 min, while all heparin bound vessels did not form clot during 1 hour observation. There were 1.38 ⫾ 0.07 ⫻ 10 9, and 0.64 ⫾ 0.11 ⫻ 10 9 baboon platelets deposited on the control and heparin linked vessels, respectively, at 60 min. Conclusions: These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue engineered biological vascular grafts which could use for human coronary or low extremity artery bypasses. P81. Development of a Biologically Active Dacron Vascular Graft Surface. E. R. Deutsch, M.D., M. D. Phaneuf, B.S.,
D. J. Willis, M.D., M. J. Bide, Ph.D., J. A. Kalish, M.D., F. W. LoGerfo, M.D., W. C. Quist, M.D., Ph.D. Israel Deaconess Medical Center. Introduction: Prosthetic arterial graft failure is, in part, a consequence of incomplete healing at the blood/biomaterial interface. Our previous work demonstrated that human umbilical vein endothelial cell (HUVEC) proliferation was enhanced on the novel bifunctionalized Dacron surface, created by exposure to ethylenediamine (EDA). The goal of this study was to covalently bind basic fibroblast growth factor (bFGF), a mitogen for HUVECs, to our bifunctionalized Dacron surface and examine proliferation of HUVECs on this biologically active Dacron surface. Methods: Mylar discs (1.5 cm diameter), a flattened form of Dacron, were exposed to EDA. Mylar discs were then divided into three groups: untreated (CTRL), controlEDA (C-EDA) and Tr-EDA (EDA treated mylar reacted with Traut’s reagent). bFGF was radiolabeled with 125I and reacted with the heterobifunctioal crosslinker sulfo-SMCC. After incubation with 125IbFGF-SMCC for 3 hours, all segments were then washed and gamma counted. The amount of 125I-bFGF (ng) per Mylar segment weight (mg) was calculated using specific activity. HUVEC growth was evaluated on CTRL, C-EDA and bFGF-EDA (non-radiolabeled) surfaces. Mylar discs were placed in a 24-well culture plate, seeded with 20,000 HUVECs and assessed for cellular proliferation over time using an Alamar blue assay. Results: 125I-bFGF binding, which was either non-specific or covalent, occurred on all of our surfaces. 125IbFGF binding to the Tr-EDA segments (32 ⫾ 2.8 ng/mg) was 8.1 fold greater than the CTRL segments (3.9 ⫾ 0.6 ng/mg, p ⫽ 1.1 ⫻ 10 – 6) and 1.5 fold greater than the C-EDA group (22 ⫾ 2.4 ng/mg, p ⫽ 1.5 ⫻ 10 –3). HUVEC proliferation occurred on all of our surfaces throughout the four-day time course. Segments with covalently bound bFGF demonstrated significantly greater proliferation (27 ⫾ 7.0) as compared to HUVEC proliferation on CTRL surfaces (12 ⫾ 4.1, p ⫽ 2.7 ⫻ 10 -6) and C-EDA surfaces (13 ⫾ 7.3, p ⫽ 2.5 ⫻ 10 – 4). Conclusions: This study demonstrates that growth factors such as bFGF can be covalently bound to Dacron and maintain its biologic activity in vitro. Immobilization of biologically active proteins may potentially aid in avoiding some of the complications that occur following prosthetic arterial grafting.
P82. Taurine Supplementation Reverses Endothelial Cell Dysfunction and Promotes Mobilisation of Endothelial Progenitor Cells. M. A. Moloney, M.D., J. J. Kelly, M.D., C. M. Condron, B.Sc., G. Roche-Nagle, M.D., D. H. O’Donnell, M.D., F. M. Fennessy, Ph.D., D. J. Bouchier-Hayes, M.D. Department of Surgery, Royal College of Surgeons in Ireland. Introduction: Cigarette smoking is the main, modifiable risk factor for atherosclerosis. We have previously reported reversal of endothelial dysfunction in young, healthy smokers with taurine supplementation. A heterogeneous population of endothelial cells (shed circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs)) is found in circulation. These correlate with the magnitude of injury and the reparative response respectively. We hypothesise, that taurine decreases structural endothelial injury and restores the reparative mobilisation of EPCs from the bone marrow in young smokers. Methods: We tested this hypothesis with a model of brief upper limb ischaemia, by characterising the response of CECs and EPCs to ischaemia in matched groups of non-smoking (n ⫽ 15) and healthy smoking young males (n ⫽ 15). Smokers were then treated for five days with Taurine, and the test repeated. Endothelial cell populations were determined by flow cytometry using CD45, CD133 and CD145.