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Hypoxia Increases Proliferation and Adhesion of Human Umbilical Vein Endothelial Cells on Synthetic Graft Membranes

Johnnal Stroud, Feng Zhao, and Teng Ma. Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, 2525 Pottsdamer St., Tallahassee, FL 32310

One treatment option for patients with occluded arteries is the bypass graft, which directs blood away from the occluded vessel and through the graft. Many grafts provided for patients are autologous veins; however, in some patients these are not available. Therefore, synthetic grafts are used as well. In large diameter grafts (>6 mm), synthetic grafts show great patency, with ePTFE and Dacron being the prevalent types used in large diameter synthetic grafts. However, synthetic grafts have displayed poor patency results for small diameter grafts (<6 mm), due to a lack of compliance by the synthetic material and the thrombogenicity of the synthetic material to blood, among other things. The endothelium is the non-thrombogenic layer found in vivo that comes into contact with blood and plays a significant role in providing the non-thrombogenic surface on the synthetic vascular grafts. In vitro endothelialization, the process in which endothelial cells are seeded onto synthetic surfaces, plays an important role in the success of the vascular grafts. However, many endothelial cells seeded onto synthetic grafts are lost when grafts are placed under fluid flow, as many of the endothelial cells are detached from the graft surface by shear stress. Many methods have been sought to increase the retention and adhesion of endothelial cells to synthetic graft surfaces, which include chemical modification, protein adsorption, and seeding under force conditions. As a strategic barrier between the intravascular compartment and underlying tissues, endothelial cells are exposed to low ambient oxygen tension, i.e. hypoxia, during their development. In this study, we hypothesize that hypoxia will stimulate endothelial cell proliferation and adhesion on synthetic graft surfaces. We investigated the effects of oxygen tension on human umbilical vein endothelial cells (HUVEC) adherence and growth on treated poly(ethylene terethphthalate) (PET) and ePTFE surfaces under physiologically relevant oxygen environment (5% O2) in an attempt to improve endothelial cell adhesion and proliferation onto these surfaces. Cell counts were significantly higher under hypoxic conditions than when cells were grown at standard cells culture conditions (20% O2) on PET surfaces. Ki67 staining and BrdU incorporation experiments showed that cells exposed to hypoxia were much more active in proliferation as compared to normoxic-exposed cells. We are currently investigating the effects of oxygen tension on endothelial cell proliferation and adhesion on ePTFE surfaces. We have also investigated endothelial cell retention on both PET and ePTFE surfaces under shear stress. Preliminary data suggest that hypoxia improves endothelial cell retention on both surfaces. For PET surfaces, at a shear stress of 10 dynes/cm2 for 6 hours and a seeding density of 4,000 cells/cm2, the seeding density was 4,000 cells/cm2 and 5,500 cells/cm2 for normoxia and hypoxia, respectively. At a shear stress of 5 dynes/cm2 for 6 hours and seeding density of 4000 cells/cm2, cell retention for endothelial cells on ePTFE surfaces under normoxic and hypoxic conditions was 7,000 and 12,500 total cells, respectively. These results strongly indicate that low oxygen tension can promote endothelial cell proliferation on PET and ePFTE surfaces. We are currently investigating the effects of hypoxia on ECM secretion and expression of endothelial specific markers. The expression of fibronectin, laminin, collagen I, collagen IV, CD 31, CD 105 will be tested in cells cultured under hypoxia condition. Our overall objective is to determine the optimized physiological parameters to facilitate the endothelialization of the vascular grafts.