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.

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) preclotted 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 x 10(3) to 1.5 x 10(5) 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 micrograms/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.

The use of radionuclide angiography to study blood flow through endothelial cell seeded extrathoracic bypass grafts in the dog.

Endothelial seeding of vascular grafts has been shown to decrease graft thrombogenicity and prolong longevity when implanted in vivo. Previous studies have utilized anatomic grafts to study endothelialization and healing. Anatomic thoracoabdominal grafts do not allow for sequential biopsy for evaluation of individual grafts nor do they approximate the environment for long bypass grafts used in limb salvage. This study evaluated the use of an extra-anatomic aortic bypass graft to assess the healing of endothelial cell seeded expanded polytetrafluoroethylene (ePTFE). Radionuclide angiography was used to evaluate graft patency and quantify blood flow through the graft. Dogs underwent placement of an extra-anatomic 60 cm long, 8 mm internal diameter, graft seeded with autologous endothelium. Grafts were biopsied from 2 weeks up to 1 year. Radionuclide studies were performed postimplantation and following each graft biopsy. Graft placement and biopsies were well tolerated in all dogs. Biopsied segments of graft allowed for sequential studies of the healing of implanted grafts by scanning electron and light microscopy. Flow through the implanted graft was close to 50% of the total caudal abdominal aortic flow. No significant difference in graft flow was noted either between animals or over time.

Healing characteristics of intraarterial stent grafts in an injured artery model.

Previous investigations reveal in the absence of endothelial cell (EC) injury, intraarterial polytetrafluoroethylene (PTFE) stent graft (SG) exhibit greater EC repaving than PTFE interposition grafts (CG). The investigation evaluated EC repaving of SG compared to CG after balloon injury. Twenty adult dogs underwent SG (n = 10) or CG (n = 10) placement in the infrarenal aorta after balloon injury with harvest at 1 and 6 weeks. Endothelial repaving, intima-to-media height ratios (IMHR), and inflammatory stains were performed. Endothelial repaving was greater in 6-week SG compared to CG (51% +/- 5.0 versus 10% +/- 5.0, p < or = 0.05). IMHR was less in 6-week SG compared to CG at the proximal (1.22 +/- 0.16 versus 1.82 +/- 0.16, p < or = 0.05) and distal anastomosis (0.81 +/- 0.25 versus 1.33 +/- 0.25, p < or = 0.05). Smooth muscle cell (SMC) alpha-actin was greater in 1-week SG compared to CG at the distal anastomosis (121.5 +/- 7.2 versus 94.0 +/- 7.2, p < or = 0.05). Proliferating cell nuclear antigen (PCNA) was less in 6-week SG compared to CG at the proximal (5.6 +/- 1.4 versus 9.4 +/- 1.1, p < or = 0.05) and distal anastomosis (3.8 +/- 0.6 versus 11.2 +/- 1.1, p < or = 0.05). Macrophage CD-44 was less in 6-week SG compared to CG at the proximal (10.4 +/- 1.6 versus 32.6 +/- 3.6, p < or = 0.05) and distal anastomosis (8.6 +/- 0.9 versus 35.6 +/- 3.6, p < or = 0.05). Intraarterial SG placed after balloon injury exhibited significantly greater endothelialization and less intimal hyperplasia when compared to CG.