Hypothesis: The vast majority of vascular graft endothelialization strategies examined to date have employed integrin-dependent ligand treatments to improve lumenal EC adhesion and retention. However, none of these strategies have been successful in small diameter synthetic grafts because they do not rapidly establish the firm initial EC attachments needed to produce stable, adherent and quiescent endothelium in the graft lumen. The hypothesis governing this project is -- any successful ligand-based endothelialization strategy must employ a straightforward and benign mechanism that (1) brings the EC membrane in rapid and stable apposition to the graft surface, and (2) that in turn nucleates the integrin mediated cell adhesion events leading to focal contact formation, cell spreading and the production of the cell cytoskeleton. Four additional, research-specific hypotheses that are tested herein emanate from this guiding principle. Innovation & Approach: Through this NHLBI grant we have developed a unique """"""""dual ligand"""""""" EC adhesion treatment that synergistically combines integrin-independent, high affinity streptavidin-biotin binding with lower affinity fibronectin-integrin ligand binding. Streptavidin-biotin is the """"""""anchor"""""""" that stabilizes the initial EC-substrate contact, bringing the cell membrane close to adsorbed fibronectin, that in turn nucleates the formation of integrin-mediated focal adhesions. We have further refined this technique by employing an RGD-streptavidin mutant that contains both integrin-independent and integrin-dependent binding functions in a single cell-substrate bridging protein. We combine the dual ligand EC adhesion treatment with subsequent flow preconditioning to further augment cytoskeletal rearrangement, EC retention under flow, and adaptation of seeded EC to the quiescent and anti-thrombotic status. Objective: This competitive renewal proposes to advance this technology from protein treated glass surfaces to the precipice of clinical application, by completing two primary experimental goals that are comprised of six Specific Aims. Goal 1: in vitro optimization of the dual ligand treatment for adhesion of rat EC on Dacron and Goretex small diameter grafts. Goal 2: in vivo validation of dual ligand treated small diameter grafts in non-survival and survival animal studies.
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