Understanding mechanisms by which endothelial cells detach from polymer surfaces is central to the design of endothelialized, synthetic vascular grafts. The research objectives of this competitive renewal are to determine the sequence of events involved in cell detachment from polymeric surfaces and to utilize heterogeneous surfaces to manipulate parameters associated with stable adhesion. This work will evaluate three hypotheses: 1) cytoskeleton-integrin interactions control whether cells detach by breaking receptor-ligand bonds (adhesive failure) or local rupture of the membrane (cohesive failure); 2) the mechanism of failure can be controlled by manipulating cytoskeleton-integrin interactions and cytoskeleton organization; and 3) cell adhesion to polymers can be enhanced by integrin-independent cell stabilization during the critical early stages of cell attachment and spreading. A newly developed experimental tool, """"""""real time TIRFM,"""""""" will be used in conjunction with immunofluorescence, immunoprecipitation and immunoblotting, and surface modification to examine in situ contact morphology of human umbilical vein endothelial cells. The strength of adhesion will be determined by a combination of flow studies and computational fluid dynamics. Analysis of separation contours of the basal surface will allow us to evaluate whether the dynamics of membrane movement differ for cells which detach by receptor-ligand dissociation and membrane rupture. Immunofluorescence will be used to determine whether membrane rupture results from the absence of connecting proteins which may reduce the strength of the adhesive contact. Alterations in the phosphorylation of focal contact proteins which could affect contact assembly and disassembly will be assessed by immunoblotting. Heterogeneous surfaces with a hierarchy of binding affinities will be used to promote cell adhesion by a combination of integrin-independent cell attachment and integrin-dependent cell spreading. This novel approach provides a practical stabilization methodology that addresses two important cell seeding problems: 1) early detachment of the loosely bound endothelial cells from polymer surfaces, and 2) the hastening of slower to develop integrin-mediated receptor-ligand bonds for implantation in the clinical environment. The system will be optimized in vitro on glass and polymer substrates using TIRFM, cell morphology assessment and detachment strength measurements. The conditions that produce the best combination of early cell stabilization and rapid cell spreading will be tested in vivo using microvascular grafts in the femoral arteries of the rat.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL044972-08
Application #
2685358
Study Section
Surgery and Bioengineering Study Section (SB)
Project Start
1990-07-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Duke University
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Fernandez, C E; Yen, R W; Perez, S M et al. (2016) Human Vascular Microphysiological System for in vitro Drug Screening. Sci Rep 6:21579
Fernandez, Cristina E; Obi-onuoha, Izundu C; Wallace, Charles S et al. (2014) Late-outgrowth endothelial progenitors from patients with coronary artery disease: endothelialization of confluent stromal cell layers. Acta Biomater 10:893-900
Reichert, William M (2013) Diversity and the Duke BME PhD program: then, now and moving forward. Ann Biomed Eng 41:2019-26
Nichols, Michael D; Choudhary, Rewa; Kodali, Santhisri et al. (2013) Coagulation-induced resistance to fluid flow in small-diameter vascular grafts and graft mimics measured by purging pressure. J Biomed Mater Res B Appl Biomater :
Nichols, Michael D; Choudhary, Rewa; Kodali, Santhisri et al. (2013) Coagulation-induced resistance to fluid flow in small-diameter vascular grafts and graft mimics measured by purging pressure. J Biomed Mater Res B Appl Biomater 101:1367-76
Stroncek, J D; Ren, L C; Klitzman, B et al. (2012) Patient-derived endothelial progenitor cells improve vascular graft patency in a rodent model. Acta Biomater 8:201-8
Brochu, Alice B W; Craig, Stephen L; Reichert, William M (2011) Self-healing biomaterials. J Biomed Mater Res A 96:492-506
Stroncek, John D; Xue, Yujing; Haque, Nabila et al. (2011) In vitro functional testing of endothelial progenitor cells that overexpress thrombomodulin. Tissue Eng Part A 17:2091-100
Angelos, Mathew G; Brown, Melissa A; Satterwhite, Lisa L et al. (2010) Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress. Biophys J 99:3545-54
Brown, Melissa A; Zhang, Lisheng; Levering, Vrad W et al. (2010) Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis. Arterioscler Thromb Vasc Biol 30:2150-5

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