Adhesion of retrovirally transduced endothelial cells to expanded polytetrafluoroethylene vascular grafts

Author

Kelly Rae Morgan

Date of Award

12-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Engineering Science

Major Professor

Judy L. Cezeaux

Committee Members

John H. Forrester, Jill E. Sackman

Abstract

Seeding small inner diameter vascular grafts with endothelial cells genetically modified to secrete anticoagulant proteins offers a means in which to improve graft patency. The endothelium is an attractive site for gene transfer due to its proximity to circulating blood. Previous in vivo and in vitro animal studies using naive cells seeded onto vascular grafts have demonstrated promising results. However, genetically modified ECs appear impaired in their ability to adhere to synthetic graft materials under in vivo and in vitro flow conditions. This study addresses the issue of retrovirally transduced EC adhesion to expanded polytetrafluoroethylene (ePTFE) vascular grafts. Human umbilical vein endothelial cells (HUVEC) were retrovirally transduced with the neoR gene (LN) or the neoR and tPA genes (LtSN). The cells were also "mock-transduced" (PA317) with empty virus particles. All transduced and packaging cell lines were positive for the neoR gene using polymerase chain reaction amplification of the transduced gene sequence. Enzyme-linked immunosorbant assay (ELISA) yielded tPA production in the LtSN PA317 packaging line but no increased tPA production in the HUVEC LtSN cell line. The cells were seeded onto fibronectin coated ePTFE grafts at a density of 2.25x10⁵ cells/cm² and incubated for 72 hours. The cells were labeled with tritiated thymidine prior to seeding which allowed for quantification of cell retention. The grafts were placed in 37°C, steady flow in vitro flow circuit for 4 hours at a flow rate of 377 ml/min and shear stress of 40 dynes/cm². Cell retention was quantified using liquid scintillation counting. Naive and "mock-transduced" EC retention was significantly higher than both the HUVEC LNcll and HUVEC LtSN cells. These results indicate retrovirally transduced ECs ability to adhere to fibronectin coated surfaces has been impaired.

Recommended Citation

Morgan, Kelly Rae, "Adhesion of retrovirally transduced endothelial cells to expanded polytetrafluoroethylene vascular grafts. " Master's Thesis, University of Tennessee, 1996.
https://trace.tennessee.edu/utk_gradthes/10915