Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF

Abstract

Small caliber synthetic vascular grafts used for dialysis access sites have high failure rates due to neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. We tested the use of ePTFE grafts seeded with autologous ECs expressing fibulin-5 and vascular endothelial growth factor (VEGF), as a dialysis access site in a porcine model. We connected the carotid arteries and jugular veins of 12 miniature pigs using 7-mm ePTFE grafts; five grafts were seeded with autologous venous ECs modified to express fibulin-5 and VEGF, and seven unseeded grafts were implanted at the same location and served as controls. At 6 months, after completion of angiography, the carotid arteries and jugular veins with the connecting grafts were excised and fixed. Autologous EC isolation and transduction and graft seeding were successful in all animals. At 3 months, 4 of 5 seeded grafts and 3 of 7 control grafts were patent. At 6 months, 4 of 5 (80%) seeded grafts and only 2 of 7 (29%) control grafts were patent. Seeding ePTFE vascular grafts with genetically modified ECs improved long term small caliber graft patency. The biosynthetic grafts offer a novel therapeutic modality for vascular access in hemodialysis.

Keywords: dialysis access; gene therapy; tissue engineering.

Figure 1

Graft Implantation and Angiography (A) Immediately after implantation of an end-to-side carotid artery-jugular vein ePTFE graft. (B) Representative angiograms of grafts seeded with endothelial cells (ECs) expressing fibulin-5 and VEGF showed patency at vein and arterial anastomoses after 24 weeks. (C) An un-seeded graft (control) occluded after 12 weeks. White arrow, artery; blue arrow, graft; black arrow, vein. (D) Representative graft segments. Left: segments from arterial proximal, middle, and venous areas of an un-seeded control graft; the lumen of all three sections is fully occluded. Right: same graft segments from EC-seeded grafts. The lumen of all graft segments is patent.

Figure 2

Graft Patency up to 24 Weeks after Implantation Seeded grafts (n = 5) were compared to bare grafts (n = 7).

Figure 3

Histology of an Endothelial Cell Seeded Graft and a Control Unseeded Graft Paraffin-embedded sections of an explanted seeded graft patent 24 weeks after implantation. (A) Low magnification of a transverse section of the graft from an area near the artery. The lumen is fully patent. The circled area is shown in (B). (B) Medium magnification of the graft wall. From the left side to the arrow, fibrin is present on the inner aspect of the graft. (C) Low magnification of the artery. The lumen is fully patent. The segment between the two arrows is composed of fibrous tissue. Arrowheads point to residual graft material and black staining elastic fibers from the arterial wall. (D) Low magnification of a level near the graft-vein junction. The graft lumen is widely patent. The lumen of the vein, located on the right half of the field, is fully patent. There is mild multifocal intimal hyperplasia (arrowheads). (E) and (F) are from paraffin-embedded sections of explanted bare, control graft, which occluded 4 weeks after implantation. (E) Low magnification of a transverse section of the graft near its junction with the artery. The graft lumen is filled with fibrin. In the central area (circled), the fibrin is not organized. Near the inner aspect of the graft, it is laminated (asterisks). The graft is surrounded by mature fibrous tissue (FT).

Figure 4

In Vitro Seeding of Dually Transduced Endothelial Cells on ePTFE Miniature pig endothelial cells (EC) seeded and cultured to near-confluence on tissue culture plates and ePTFE discs. ECs were cultured in 6-well tissue culture plates and on ePTFE 8-mm discs for 72 hr to near-confluence. Cells were then fixed with 4% PFA and stained with DAPI. Cells were counted in nine randomly selected fields using ImageJ software. (A) Bright field image of ECs seeded on a tissue culture plate (magnification ×270). (B) DAPI staining of ECs seeded on a tissue culture plate (magnification ×270). (C) DAPI staining of EC seeded on an ePTFE disc (magnification ×270). Scale bars represent 25 μm.

Figure 5

Graft Seeding Homogeneity Five random fields of a 1-cm seeded graft segment were captured at a magnification ×270. Each field was divided into 12 subfields. The mean and SD of each field was calculated, and the mean and SD of all five fields were calculated and normalized. The upper panel represents a homogeneous graft (normalized SD = 1.7), while the lower panel represents a non-homogeneous graft (normalized SD = 3.36).