Abstract 3426: The Generation Of Biosynthetic Small Caliber Grafts With Improved Patency Using Autologous Genetically Modified Endothelial Cells.

Introduction: Seeding polymer-based synthetic vascular grafts with endothelial cells (EC) improves grafts biocompatibility and consequently grafts patency. EC adhesion and survival on the synthetic polymer remains a considerable challenge. We used pseudo-typed retroviral vectors to modify EC to co-express fibulin-5 and VEGF 165 and created a unique EC phenotype with increased adhesion and enhanced proliferation and used these cells to seed vascular grafts. Methods: Polymer-based grafts seeded with autologous venous EC transduced to co-express fibulin-5 and VEGF 165 were implanted in the carotid arteries of the donor sheep. 18 grafts (8 seeded grafts with EC co-expressing fibulin-5 and VEGF, 6 seeded with control EC and 4 bare grafts) were implanted for two week in nine sheep. Six months studies were performed on 18 grafts (6 study, 6 seeded with control EC and 6 bare) implanted in 18 sheep. Histological studies for cell adhesion, intra-luminal thrombosis and transgene expression were tested in implanted grafts after angiography. Results: At two weeks all 8 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent with no significant thrombosis and 73% coverage by seeded EC, while 3/10 control grafts were thrombosed and only 30% of seeded unmodified EC remained on grafts luminal surface. At 6 months, 5/6 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent while 9/12 control grafts (6 bare, 6 seeded with unmodified EC, p<0.04) were occluded. At 6 months, the genetically modified cells were identified on the graft surface and these grafts had improved EC coverage and reduced intra-luminal neo-intima and thrombosis. No local or systemic side effects were attributed to the implanted grafts. Conclusion : Polymer-based vascular grafts seeded with EC transduced to co-express fibulin-5 and VEGF have lower propensity to thrombosis and improved patency. Use of grafts seeded with EC co-expressing fibulin-5 and VEGF provides a potentially clinically relevant method for small caliber blood vessel engineering.

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Synthetic Vascular Grafts Seeded with Genetically Modified Endothelium in the Dog: Evaluation of the Effect of Seeding Technique and Retroviral Vector on Cell Persistence in Vivo

Cell Transplantation ◽

10.1177/096368979500400206 ◽

1995 ◽

Vol 4 (2) ◽

pp. 219-235 ◽

Cited By ~ 4

Author(s):

Jill E. Sackman ◽

Michael B. Freeman ◽

Mark G. Petersen ◽

Zuhair Allebban ◽

Glenn P. Niemeyer ◽

...

Keyword(s):

Endothelial Cells ◽

Gene Transfer ◽

Gene Sequence ◽

Genetically Modified ◽

Vascular Grafts ◽

Retroviral Vectors ◽

Retroviral Vector ◽

Marker Genes ◽

Growth Hormone Gene

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) pre-clotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 103 to 1.5 × 105 cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

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