ACTIVATION OF TELOMERASE THROUGH VEGF SIGNALING TRIGGERS ANGIOGENESIS IN RESPONSE TO HIND-LIMB ISCHEMIA

Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 Initial culture conditions resulted in the poor collagen deposition as a result of scaffold porosity and resulting media perfusing through the scaffold into the ablumenal circuit. As flow and pressure were modulated to suit the specific grafting material, adhesion and contraction of the gel was observed. Under optimized conditions histological analysis has shown uniform cell rich collagen sheaths around the grafts ablumenal surface. Efficiently seeding high densities of cells onto the surface of any 3D tubular construct is at best problematic. By using the vascular bioreactor and collagen as a cell delivery vehicle we have been able to achieve such a goal in a one-step method. The simplicity of the reactor/construct design, and the one-step cell adhesion method allows efficient usage of valuable autologous, reduced culture time and lowers the risk of contamination. As such, this method may bring the goal of rapid clinical application one step closer.

P508 MECHANICAL ASSESSMENT OF THE HUMAN UMBILICAL VEIN FOR VASCULAR TISSUE ENGINEERING APPLICATIONS. Joel Daniel, Peter McFetridge. University of Oklahoma, Bioengineering Center, Norman, OK. The human umbilical vein (HUV) as a ‘‘glutaraldehyde tanned’’ and biologically inert graft, has had limited success due to the potential for aneurysm formation. This study describes a novel modification of the HUV to allow biological remodeling while increasing the strength as a scaffold for tissue engineering small diameter blood vessels. HUV segments dissected from human umbilical cords were assessed in four categories: 1. fresh dissected vein (HUV), 2. decellularised HUV (dHUV), 3. decellularised and photo-oxidatively crosslinked (xHUV), and 4. collagen gel/cell coated (ccHUV). ccHUV were prepared by inoculation of collagen gels with primary human smooth muscle and fibroblast cell suspensions (1 – 3  106 cell/ml) into the ablumenal void of specifically designed vascular bioreactors. Burst pressure and compliance was assessed by progressive inflation of the vessels whilst recording diameter variation (range 80/120 mmHg) for compliance, until vessel rupture. Analogue video data was converted to digital media for analysis. HUV segments showed a mean compliance value of 11.7%, compared to pHUVand xHUV with values of 8.5% and 9.5% compliance respectively, no significant difference between data sets ( p > .05). Similarly, there was no significant difference between burst pressure ( p > .05), with 1318 mmHg, 1177 mmHg, and 1279 mmHg for HUV, dHUV and xHUV, respectively. Histologically, the ccHUV segments displayed uniform cell/collagen coverage on the ablumenal surface. Preliminary ccHUV data suggest improved mechanical properties over HUV, dHUV and xHUV preparations, but is dependent on gel/cell concentration, and culture conditions. All HUV treatments exhibit compliances closer to native vessels compared to PTFE, Dacron, and a number of processed biological materials. Whilst burst pressure is significantly above the physiological pressure range, additional support by means of the collagen/cell ‘‘coat’’ is likely required for long-term stability. Acknowledging the HUVs clinical history, we believe the full development of the ccHUV will lead to a mechanically sound, compliant vessel with the ability to fully remodel into autologous vascular tissue.

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P509 IN VIVO, NON-VIRAL LOCAL GENE DELIVERY TO MOUSE FEMORAL ARTERY BY ELECTROPORATION: A PROMISING TOOL TO INVESTIGATE POST-ANGIOPLASTY RESTENOSIS. Daniel Eefting, Jennifer L. Young, Jos M. Grimbergen, J. Hajo van Bockel, David A. Dean, Paul H.A. Quax. Leiden University Medical Center and TNO Prevention and Health, Leiden, The Netherlands, Northwestern University, Chicago, Illinois, USA, TNO Prevention and Health, Leiden, The Netherlands, Leiden University Medical Center, Leiden, The Netherlands. The use of gene transfer techniques in the prevention of post-interventional restenosis has been studied for several years now. Although the results using adenovirus mediated gene transfer aiming at inhibition of smooth muscle cell proliferation and migration look very promising, viral gene transfer has several disadvantages such as immune responses. Therefore we have set up an electroporation based non-viral gene transfer method for transfection of the vessel wall in small animals. We have shown that delivery of pEGFP by electroporation to the rat mesenteric arteries using perivascular gene delivery results in a profound GFP expression throughout all layers of the vessel wall, including the endothelium. To apply this approach in mice models for cuff-induced restenosis in the femoral artery, we have adapted the method for non-viral, electroporation mediated gene transfer to the mouse femoral artery. The cuff-induced mouse restenosis model is a highly reproducible method for neointima formation and can be used in transgenic mice with e.g., a human atherosclerotic phenotype. With the modified electroporation technique for perivascular gene delivery to the mouse femoral artery (using a square wave electroporator, 200 V/cm, 8 pulses of 10 msec duration, 2 mg/ml DNA), we delivered either pCMVluciferase or pEGFP plasmids to the femoral artery. To study injury, we placed a non-constricting polyethylene cuff around the vessel following electroporation. Two days after these experiments, we harvested the arteries and measured expression of GFP as well as luciferase. GFP expression could be observed in the vessel wall of the transfected vessel, and was absent in the arteries which received DNA but were not electroporated. Furthermore pCMVLuciferase transfection resulted in expression of approximately100 pg luciferase per vessel. In contrast, no luciferase expression could be detected in the arteries which received DNA but were not electroporated. In conclusion, local non-viral electroporation mediated gene transfer to the mouse femoral artery in combination with cuff placement to induce neointima formation gives a great opportunity to investigate the mechanism of neointima formation in (transgenic) mice and to validate new therapeutic target genes as an approach for prevention of post-interventional restenosis. This work was financially supported by the Netherlands Heart Foundation (grant M93.001 and De Gelder Fonds)

P510 ACTIVATION OF TELOMERASE THROUGH VEGF SIGNALING TRIGGERS ANGIOGENESIS IN RESPONSE TO HIND-LIMB ISCHEMIA. Carlo Gaetano, Germana Zaccagnini, Linda Della Pietra, Simona Nanni, Maurizio C. Capogrossi, Antonella Farsetti. Istituto Dermopatico dell’Immacolata, Roma, Italy, Istituto Cardiologico Monzino, Milano, Italy, Istituto Regina Elena, Roma, Italy, Istituto Dermopatico dell’ Immacolata, Roma Italy, Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italy. In mammals, age-dependent telomere dysfunction may contribute to a reduction in cell viability, altered differentiation functions and impaired regenerative/proliferative responses. Recent advances indicate that sustained level of telomerase activity in endothelial cells and precursors may confer a pro-angiogenic phenotype to these cells. The present study was aimed at investigating whether telomerase contributes in vivo to the process of tissue

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Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200

regeneration following hind-limb ischemia and adenoviral-mediated VEGF165 treatment in young and aged rats. Unilateral hind-limb ischemia was induced by surgical dissection of the femoral artery in young (3 months) and aged (22 months) male Fisher rats. Coincidentally, animals were injected i.m. with saline solution, AdCMV.Null or AdCMV.VEGF165 (5X10^7 pfu). Evaluation of angiogenesis by capillary count and of telomerase by RT-PCR, immunoistochemistry and TRAP assays were performed on adductor and quadriceps muscles at 3, 8 and 14 days upon treatment (n = 6 animals/point). VEGF delivery to ischemic tissues induces angiogenesis and a significant increase of TERTmRNA, TERT protein and telomerase activity in skeletal muscles, vascular endothelial, smooth muscle and satellite skeletal muscle cells in young and, to a lower extent, older animals, presumably as a consequence of tissue remodeling. To explore the molecular mechanisms underlying this phenomenon, VEGF-dependent induction of TERT expression and activity was reproduced in vitro in differentiated murine myoblasts C2C12. Such increase was abrogated by addition of 7-Nitroindazole, a Nitric Oxide Synthases inhibitor, indicating that VEGF-mediated synthesis of nitric oxide is involved in this process. In addition, adenoviral-mediated hTERT gene transfer in young rats induced angiogenesis 3 and 8 days after ischemia with an efficiency similar to that elicited by VEGF165, as assessed by measurement of capillaries density. Thus TERT appears to directly contribute to angiogenesis in vivo, suggesting an extracurricular telomerase function.

P511 IN VIVO BEHAVIOUR OF DECELLULARISED AORTIC VALVE ALLOGRAFTS IN A RAT IMPLANTATION MODEL: FEASIBILITY OF ALLOGENEIC MATRIX SCAFFOLDS IN REGENERATIVE MEDICINE. Robert W Grauss, Debora R Doorn, Beerend P Hierck, Conny J van Munsteren, Henry Beekhuizen, Mark G Hazekamp, Adriana C Gittenberger-de Groot, Marco C DeRuiter. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands, Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands, Department of Cardiothoracic Surgery, Leiden University Medical Center, Leiden, The Netherlands. Immunological deterioration has been considered as one of the major causes of early aortic valve graft failure. Removal of allogeneic valve cells is therefore one of the most promising approaches in overcoming valve failure. Hardly any attempt has been made to study in detail the effect of cell removal on extracellular matrix (ECM) integrity and the in vivo remodeling of decellularised aortic valve scaffolds. Furthermore, the absence of endothelial cells in these scaffolds would result in a thrombotic surface. The most suitable endothelial source for reseeding of these grafts has to be determined. A two-step detergent-enzymatic extraction method involving sodium dodecyl sulfate as well as DNase and RNase was used to decellularise rat aortic valve conduits. Cellular (N = 4) or acellular (N = 4) heparinised allogeneic conduits were grafted infrarenally into the descending aorta. Heparinised syngeneic transplants (cellular; N = 3, acellular; N = 3) served as controls. After 21 days the valves were explanted and immunohistochemical techniques were used to study ECM constitution (elastin, collagen, GAG, fibronectin and chondroitin sulphate). For reseeding of the valve conduits we isolated and cultured arterial, venous and microvascular endothelial cells (EC) derived from descending aorta, caval vein and fat, respectively. Transplanted cellular allografts lost their normal leaflet structure and became acellular with major changes in the ECM. The decellularisation method resulted in a complete removal of cellular structures from the entire valve conduit with minimal ECM remodeling. Acellular allografts retained normal leaflet structure comparable to transplanted cellular and acellular syngeneic grafts. All acellular grafts showed retrovalvular thrombus formation caused by the absence of (EC). Preliminary data show that both rat arterial and venous EC have to be seeded onto the graft at very high densities and cultured very shortly (maximally 5 days) to prevent phenotype

changes as determined by RT-PCR and immunohistochemistry. The highest proliferation rate was seen for the aortic EC. Our two-step detergent-enzymatic extraction method preserves most of the major valve ECM components. Furthermore, decellularisation appears to reduce ECM damage and valve remodeling demonstrating the feasibility of allogeneic matrix scaffolds in regenerative medicine. The rat implantation model is suitable for future studies on the anti-thrombotic effects of reseeding with various syngeneic EC sources.

P512 INTRAMYOCARDIAL NONVIRAL GENE DELIVERY: THE IN VIVO EVALUATION OF TAT BASED PEPTIDES AS VECTORS FOR PLASMID DNA. Irina Hellgren, Anwar J. Siddiqui, Beston Nore, Karl Henrik Grinnemo, Edvard Smith, Christer Sylve´n. Karolinska Institute at Department of Cardiology Huddinge University Hospital, Stockholm, Sweden, Karolinska Institute at Department of Thoraxical Surgery and Huddinge University Hospital, Stockholm, Sweden., The Clinical Research Centre, Huddinge University Hospital, Stockholm, Sweden, Karolinska Institute at Department of Cardiology Huddinge University Hospital, Stockholm, Sweden. Background: The tat is positively charged peptide that binds DNA electrostaticly. We have evaluated the in vivo use of two tat-based peptides, branched 8tat (8tat) and tat-protamine hybrid (TP), as plasmid vehicles and investigated the factors controlling the efficiency of transfection. Methods and results: To study the degree of the plasmid protection from DNaseI degradation, the plasmid was mixed with either 8tat or TP peptide in different charge ratios in salt and isotonic solutions. The plasmid extensively protected in complex with peptides compared to naked DNA alone when ratio 1:8 (8-tat) or 1:5 (TP) was used. Complexes formed in NaCl and dextrose provided greater protection than compared with HEPES. The size of DNA/peptide particles was established by gel-retardation assay. TP and 8-Tat peptide were used to investigate their ability to facilitate gene transfection in vivo. Mice were injected into the myocardium with the complex of plasmid and peptide at different /+ charge ratios and luciferase gene expression was measured after 24 h. Highest luciferase activity was observed for ratio of 1:1 (8tat) and 1:1/10 (TP). Optimisation of DNA/peptide complex formation conditions resulted in further augmentation of luciferase level. Compared to plasmid DNA alone, transgene expression was improved several times with the use of TP but not 8 tat peptide. Conclusions. These observations suggest that TP and branched 8tat peptides extensively protect the plasmid from DNase I degradation at high DNA/peptide ratios. However, the in vivo reporter gene expression was highest at low DNA/peptide ratios. In summary, TP peptide may be used as an enhancer of intramyocardial gene delivery.

P513 NATURALLY OCCURRING FIBRINOGEN VARIANTS DETERMINE THE EXTENT OF ANGIOGENESIS IN A FIBRIN MATRIX IN VITRO AND IN VIVO. Eric Kaijzel, Monique van Erck, Moniek de Maat, Victor van Hinsbergh, Pieter Koolwijk. Gaubius Laboratory TNO-PG, Division Biomedical Research, Leiden, The Netherlands, Gaubius Laboratory TNO-PG, Division Biomedical Research, Leiden, and Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Introduction: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and growth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. If angiogenesis proceeds too rapidly, inadequate wound healing will occur; if the process proceeds too slowly scar tissue will be formed. The composition of the fibrinous matrix is an important determinant of the invasion of endothelial cells and capillary-tube formation.