Immunomodulation of cultured vascular endothelial cells by serial cell passage.

UNLABELLED: Endothelial cell seeding has been successful in reducing the thrombogenecity of prosthetic vascular grafts in animal models, but results from clinical trials have been largely disappointing. These poor results have been associated with poor graft coverage in immediate seeding trials, and failure of cell culture in staged procedures. These problems could be largely overcome by utilising a bank of allogeneic endothelial cells, providing an ever ready supply. However, one potential pitfall with this technique would be the possibility of a rejection response following transplantation. AIM: To study the effects of prolonged tissue culture, on the ability of endothelial cells to generate an immune response. METHODS AND RESULTS: The immunogenecity of human umbilical vein endothelial cells was measured using the mixed lymphocyte endothelial reaction. It was demonstrated that prolonged tissue culture significantly reduced the immunogenecity of the cells, from a mean of 7261 cpm (S.E. +/- 243, n = 3) for cells of subculture 3, to 5478 cpm (+/- 156, p = 0.04) for cells of subculture 7 (p = 0.04, Wilcoxon paired rank test), but did not significantly impair morphology or antithrombotic function. CONCLUSION: This study provides evidence that prolonged tissue culture provides morphologically and functionally intact, immunomodified endothelial cells which may potentially be used in seeding prosthetic vascular grafts.

Attachment, replication and thrombogenicity of genetically modified endothelial cells.

UNLABELLED: Endothelial cell seeding of prosthetic surfaces has been proposed as a technique to improve the patency of vascular grafts following arterial reconstruction. The introduction of specific recombinant DNA into seeded endothelial cells may enhance the anti-thrombogenic nature of the endothelial-blood interface with a consequent reduction in graft thrombosis. However, the successful use of genetically modified endothelial cells in the seeding process relies on the cells retaining normal function in terms of cellular replication, attachment and secretion of anti-thrombotic mediators. Successful genetic manipulation of human endothelial cells has been accomplished by viral and chemical methods. AIM: To study the functional characteristics of electrontransfected endothelial cells. METHODS AND RESULTS: Endothelial cells were electro-transfected with the test plasmid pTCF at a transfection efficiency of 10% utilising a single electric pulse with an electric field of 1000 volts/cm and a time constant of 12.8ms. The functional status of transfected endothelial cells was then compared with a control endothelial cell population. There were no significant differences in replication (p = 0.76), attachment (p = 0.43), basal (p = 0.89) or stimulated (p = 0.11) prostacyclin release between transfected cells as compared with control endothelial cells. CONCLUSIONS: Genetically modified cells are functionally normal, and may be used in endothelial cell seeding of prosthetic vascular surfaces.

Optimisation of gene transfer into vascular endothelial cells using electroporation.

OBJECTIVES: We have examined the conditions required to obtain optimum transfection efficiencies for human umbilical vein endothelial cells by transduction with a plasmid conferring neomycin resistance. MATERIALS AND METHODS: Preliminary studies examined the effects of electric discharges using the Biorad Gene Pulser on endothelial cells. Post-electroporation, there was a significant decrease in cell survival with increasing voltages (100-400 volts; p = 0.03), capacitances [125-960 microFarads (microF); p = 0.02], number of electric pulses (1-2; p = 0.03) and decreasing cell concentrations (p = 0.01). The optimal cell concentration was 3 x 10(6) cells/ml. Transfection studies utilised the neomycin resistance expressing plasmid, pTCF; transfectants were selected with the neomycin analogue G-148. RESULTS: Electro-transfection was optimised with increasing voltages (p = 0.02) and capacitances (p = 0.01) using a single pulse. Optimal transfection was obtained using 400 volts with a capacitance of 960 microF using a single pulse; the median transfection efficiency was 10%. Transduced endothelial cells stably expressed the plasmid for 12 days and at least two cell passages. CONCLUSIONS: The results indicate that endothelial cells can be efficiently transduced by electroporation to stably express an introduced gene. This may have important implications in vascular surgery.

Effect of pulsatile shear stress on endothelial attachment to native vascular surfaces.

An in vitro model of vascular damage was used to investigate the ability of seeded endothelial cells to resist shear stresses generated in a perfusion circuit. At perfusion rates of 100 ml/min the maximum shear stress reached 16.5 dyn/cm2. At this level the rate of cell detachment from the damaged vascular surface was 88 per cent per h for the first 20 min of flow but gradually decreased to 5 per cent per h after 90 min. These findings suggest that endothelial cells may be retained on damaged vascular surfaces in conditions that approximate to arterial flow.

The effect of transluminal endothelial seeding on myointimal hyperplasia following angioplasty.

Myointimal hyperplasia develops as a generalised response to vascular injury, and may cause stenoses in 40% of all peripheral arterial reconstructions. Disruption of the endothelial monolayer is a prerequisite for the development of intimal hyperplasia, and may be the initiating event in this process. This study examined the hypothesis that restenosis following balloon angioplasty may be reduced by rapid restoration of the endothelial monolayer, achieved by endothelial seeding. Bilateral iliac angioplasties were performed in 11 rabbits. A double balloon catheter was used to seed one angioplasty site with autogenous endothelial cells; the contralateral site was sham seeded with culture medium and acted as a control. Arterial patency rates, the degree of intimal hyperplasia (IH/IEL), and the extent of endothelialisation were quantified at 1 (n = 5) and 3 (n = 6) weeks following balloon dilatation. The results suggest that transluminal endothelial seeding may be a therapeutically applicable technique as it decreases myointimal hyperplasia, and increases patency following angioplasty. This study also illustrates the protective effect of the vascular endothelium following arterial injury, and indicates that intensive efforts should be made to preserve the endothelium during vascular reconstruction.

Platelet deposition after angioplasty is abolished by restoration of the endothelial cell monolayer.

PURPOSE: Percutaneous transluminal angioplasty of an atheromatous plaque causes endothelial desquamation and intimal dissection with the consequent formation of a thrombogenic flow surface. In this study we investigated the hypothesis that platelet deposition after balloon angioplasty may be decreased by rapid restoration of the endothelial cell monolayer, achieved by transluminally seeding angioplasty sites with endothelial cells. METHODS: Bilateral external iliac angioplasty was performed in eight New Zealand white rabbits. One angioplasty site was isolated from the circulation and incubated with a supraconfluent endothelial cell suspension with a double balloon catheter; the contralateral angioplasty site was sham seeded with culture medium. The deposition of autologous indium 111-labeled platelets on the angioplasty sites was quantified 30 minutes after restoration of flow and was referenced to an undamaged segment of aorta that acted as a negative control. RESULTS: Platelet deposition on the nonseeded angioplasty site (13.1 x 10(4) platelets/mm2) was significantly higher than on nondilated segments (3.4 x 10(4) platelets/mm2; p = 0.014). Restorationof endothelial cell coverage by endothelial seeding significantly reduced platelet deposition on dilated arterial segments to levels not significantly higher than in controls (3.6 x 10(4) platelets/mm2; p = 0.014). CONCLUSIONS: These results illustrate that rapid reendothelialization of angioplasty sites decreases subsequent platelet deposition and may reduce the rate of acute arterial reocclusion complicating endovascular techniques.

A method to transluminally seed angioplasty sites with endothelial cells using a double balloon catheter.

Acute arterial reocclusion and late restenosis following angioplasty may be partially caused by loss of the endothelial cell monolayer during balloon dilatation. Rapid restoration of the endothelial cell monolayer by endothelial cell transplantation has the potential to increase the antithrombotic nature of the angioplasty site and also to prevent myointimal hyperplasia which is the cause of late restenosis. We have investigated a method to transluminally deliver endothelial cells to angioplasty sites using a double balloon catheter with a central instillation port. Inflation of the balloons allows a segment of artery to be isolated from the circulation which may then be incubated with infused endothelial cells. The external iliac arteries of New Zealand white rabbits were dilated for 30 s at 8 atm pressure using a 3 mm balloon angioplasty catheter. Indium-111 labelled allogeneic rabbit endothelial cells were seeded onto the angioplasty site and retained in contact with the vascular surface for 30 min by continued inflation of the isolation balloons. After 30 min, the balloons were deflated and flow restored. Percentage cell retention was calculated by determining the gamma activity of the seeded angioplasty site, and referencing this to the contralateral sham seeded site. Results are expressed as a median with 95% confidence intervals and suggest that endothelial cells may be successfully delivered and retained on damaged vascular surfaces [table: see text].

Effect of seeding time and density on endothelial cell attachment to damaged vascular surfaces.

An in vitro model to facilitate the study of endothelial cell seeding of damaged vascular surfaces has been developed. This may have applications in the study of endothelial seeding of angioplasty and endarterectomy sites. Using this model, the optimum endothelial seeding time for attachment to damaged vascular surfaces should not exceed 30 min and, to achieve confluent cell attachment, a seeding density > 5 x 10(5) cells/cm2 should be used.

Endothelial cell seeding of damaged native vascular surfaces: prostacyclin production.

Endothelial cell seeding has been successful in reducing the thrombogenicity of prosthetic vascular grafts in animal and clinical studies. The reduction in thrombogenicity may be attributed to the intrinsic properties of endothelial cells themselves, and their ability to produce anti-thrombogenic mediators such as prostacyclin, and endothelium-derived relaxing factor. Endothelial seeding of damaged vascular surfaces produced during percutaneous transluminal angioplasty and endarterectomy is an attractive possibility due to the excellent attachment characteristics of the sub-endothelial tissue exposed during these procedures. The ability of endothelial seeded damaged vascular surfaces to produce prostacyclin was measured in an in vitro model of vascular injury. Endothelial-seeded damaged surfaces produced significantly higher prostacyclin release than did vessels damaged by balloon dilatation (265.5 pg cm-2 min-1 and 87.5 pg cm-2 min-1 respectively). This study provides evidence that endothelial seeding of damaged native vascular surfaces is technically feasible and that seeding may reduce the thrombogenicity of vascular surfaces following balloon dilatation.