Doctoral Dissertations

Date of Award

5-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Clinton D. Lothrop

Committee Members

Roger C. Carroll, Michael B. Freeman, D.J. Krahwinkel, David O. Slauson

Abstract

Unique characteristics of endothelium make it an attractive target for gene transfer. Vascular prostheses seeded with autologous canine endothelial cells transduced with the B-galactosidase reporter gene have retained evidence of gene expression for up to 5 weeks post-implantation. The purpose of the first part of this study was to evaluate the persistence of a non-inducible reporter gene (neoR) in endothelial cells seeded on Dacron vascular grafts for up to 360 days. The second phase evaluated recombinant endothelial cell persistence on polytetrafluoroethylene (PTFE) vascular grafts with 3 different reporter genes. PTFE vascular grafts were evaluated by multiple biopsies over a 5 to 7 month period. Canine endothelial cells were cultured in vitro and verified as endothelial in origin by immunoperoxidase staining for von Willebrand's factor. Cultured endothelium was transduced with either the N2, G2N, SFF-ghGH or LXSN retroviral vector. Cells transduced with the neoR gene (N2, G2N or LXSN vectors) were selected in G418 sulfate. Cells selected in G418 were positive for the neoR gene sequence by polymerase chain reaction (PGR) and neomycin phosphotransferase (NPT) enzyme activity. Cells transduced with the SFF-ghGH vector were evaluated for human growth hormone production via radioimmunoassay and PCR amplification of a human growth hormone sequence. Autologous transduced cells in the first group of dogs (n=3) were seeded on Dacron vascular prostheses and implanted in vivo as intra-abdominal aortic bypass grafts. The next series of dogs (n=3) had autologous transduced endothelium seeded and cultured for 5 days on PTFE grafts prior to implantation. The following 2 dogs had harvested endothelium immediately transduced and seeded on fibronectin coated PTFE grafts. The last two animals had in vitro cultured autologous endothelium seeded at high density on fibronectin coated PTFE. All PTFE grafts were implanted as subcutaneously tunneled extra-anatomic aortic bypass grafts. Dacron grafts were harvested 250 to 360 days post-implantation. Patent grafts had complete endothelialization of the luminal surface at the time of harvest. Endothelial cells subcultured from graft explants failed to survive in G418. DNA analysis by PGR and Southern blotting for the neoR gene sequence was negative in all recovered grafts. All of the in vitro cultured PTFE grafts were coated with a confluent layer of endothelium at implantation. Two of the dogs with in vitro culturing of PTFE grafts died. One dog died immediately postoperatively, the other 6 weeks following implantation secondary to the rupture of a false aneurysm. The third dog in this group had no evidence of graft endothelialization on biopsies up to 19 weeks. All the remaining PTFE grafts were biopsied at 2, 6 and 20 weeks. Grafts with immediate cell seeding were partially endothelialized and remained patent at all biopsies. DNA analysis by PGR and Southern blotting was negative for the neo^ sequence in all samples from these dogs.. The final two animals with in vitro cultured endothelium and high density seeding had significantly improved graft endothelialization. The graft seeded with human growth hormone (hGH) transduced cells was negative by PGR and Southern blotting for the growth hormone sequence at all biopsy times. No hGH levels were ever detected in the serum from this animal. The dog seeded with LXSN transduced cells exhibited complete graft endothelialization and positive DNA by PGR for the neoR sequence at 3 weeks. Although endothelial cells are readily transduced in vitro and seeded on vascular grafts, seeded cells appear to be replaced by ingrowth of endogenous endothelium when evaluated on knitted Dacron at 250 to 360 days post-implantation. Endothelial cells seeded and cultured in vitro on PTFE grafts for 5 days appear to be unable to maintain graft colonization after implantation in vivo. Endothelial cells seeded at low density immediately after harvest appear to partially colonize fibronectin coated PTFE grafts; however, brief exposure to low titer SFF-ghGH retroviral supemate in suspension is an inefficient way to transduce cells being seeded. By optimizing the percentage of cells transduced, the number of transduced cells seeded through use of a selectable marker gene, and improving the attachment substrate, genetically marked endothelium is capable of colonizing PTFE for at least 3 weeks.

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