Jill E. Sackman

Vascular endothelial growth factor and heparin in a biologic glue promotes human aortic endothelial cell proliferation with aortic smooth muscle cell inhibition.

BACKGROUND Incomplete luminal endothelialization may contribute to small diameter vascular graft failure. Vascular endothelial growth factor (VEGF) can be used to stimulate endothelialization without provoking smooth muscle cell (SMC) proliferation. Heparin and VEGF in a fibrin glue (FG) were investigated for their ability to promote selective human aortic endothelial cell (HAEC) proliferation and human aortic smooth muscle cell (HASMC) inhibition. METHODS HAECs and HASMCs were seeded on FG containing VEGF (2.5, 10, 30, 100 ng/ml) or VEGF and heparin (5, 50, 500 units/ml). Proliferation assays were performed with tritiated thymidine on days 1 and 3. Results were analyzed by ANOVA, with p < or = 0.05 significant. RESULTS HAEC proliferation on FG with 10, 30, and 100 ng/ml VEGF was significantly greater than FG alone at days 1 and 3. The addition of 50 units/ml heparin to VEGF significantly increased HAEC proliferation to greater than FG with VEGF alone at day 1. Human aortic SMC proliferation was not stimulated by the addition of VEGF. The addition of 5, 50, and 500 units/ml heparin significantly inhibited HASMC proliferation regardless of VEGF concentration. DISCUSSION VEGF at 10 ng/ml combined with heparin at 50 units/ml exhibited maximal stimulation of HAECs with inhibition of HASMCs. VEGF and heparin in a biologic glue may improve patency by selectively promoting HAEC proliferation without HASMC growth on synthetic vascular bypass grafts.

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 ECs, 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.

Mechanical characteristics of lyophilized human saphenous vein valves

PURPOSE We investigated the mechanical characteristics of lyophilized human saphenous vein valves to determine their suitability for use as allogeneic transplants to treat chronic venous insufficiency. METHODS Fresh cadaveric veins were lyophilized in vacuum bottles within 24 hours of harvest and were stored at room temperature. The veins were reconstituted in saline solution and then were placed in an in vitro flow circuit for evaluation. At varied flow rates, pressures proximal and distal to valves during prograde and retrograde flow were measured. Valve closure times were determined with Doppler examination and spectral analysis. The valves were also stressed to 350 mm Hg on a separate apparatus. RESULTS All pressures proximal and distal to the valves remained less than 10 mm Hg during prograde flow. A pressure gradient developed immediately on the reversal of flow. Pressure as high as 200 mm Hg applied against the closed valves was not transmitted beyond the valve. Valve closure times had a mean of 0.31 +/- 0.03 seconds and 0.21 +/- 0.01 seconds for the Doppler examination and spectral analysis, respectively. All valves withstood stress pressures to 350 mm Hg. CONCLUSIONS The in vitro mechanical characteristics of the valves of lyophilized veins are similar to known values for normal in vivo valves.