Production of plasminogen activator inhibitor-1 by human saphenous vein endothelial cells seeded onto endarterectomy surfaces in vitro.

PURPOSE: Endothelial cells produce many biologically important factors that may be used as functional markers, including plasminogen activators and their inhibitors (PAI). PAI-1 is a common peptide with a central role in the balance of thrombosis and fibrinolysis in vivo, and its production by vascular endothelial cells has been demonstrated for many in vitro cell lines. METHODS: The basal rate of PAI-1 release from cultured human adult endothelial cells was studied in both a well-plate-seeding model and after seeding onto human endarterectomy specimens. The effect of nonspecific stimulation with thrombin on PAI-1 release was examined in well-plate cultures. PAI-1 was measured by enzyme-linked immunosorbent assay. RESULTS: Cultured human saphenous endothelial cells release PAI-1 constitutively at a steady rate, which can be increased in the short term by the addition of thrombin. CONCLUSION: After seeding onto endarterectomy specimens, seeded endothelial cells release significant amounts of PAI-1, which suggests that they retain functional integrity and may potentially influence thrombosis in vivo after seeding.

Dialysis fluid cytotoxicity and inhibition of host defence in cultured human mesothelial cells are neutralized rapidly with incubation in the peritoneum.

BACKGROUND: A recent survey puts the global dialysis population at 535100; of those on peritoneal dialysis, 85% are on continuous ambulatory peritoneal dialysis. Current hyperosmolar dialysis fluids are toxic to peritoneal cells and inhibit certain host-defence functions. An alternative preparation, glucose polymer, has recently been developed. METHODS: Mesothelial cell viability, interleukin-6 and prostacyclin synthesis, after exposure to 7.5% glucose polymer, 1.36% glucose or 3.86% glucose peritoneal dialysis effluent solution was assessed. RESULTS: In its neat form, at an original pH of 5.4, glucose polymer was as toxic as hyperosmolar solutions (P < 0.01). Synthesis of interleukin-6 and prostacyclin were significantly inhibited by neat dialysis fluid, (P < 0.01). However, after an in vivo intraperitoneal incubation of only 15 min the toxicity of all solutions tested in vitro was lost. CONCLUSIONS: Despite rapid in situ neutralization of dialysis fluid toxicity, mesothelial injury and inhibition of host-defence function, early in the dialysis cycle, may affect peritoneal physiology given the complex network of pathways to which these cells contribute. Although recent trials indicate improved ultrafiltration is achievable with glucose polymer, it is not a biocompatible dialysis fluid in its current manufactured form.

Endothelial cell seeding kinetics under chronic flow in prosthetic grafts.

Improved patency of endothelial cell seeded grafts relies on good initial adherence and cell retention when the circulation is restored. In this study human adult endothelial cells (HAECs) were used to evaluate the suitability of commercially available prostheses for seeding. Acutely seeded indium-111 oxine labeled HAECs were used to measure cell adherence to plain and fibronectin (FN)-coated expanded polytetrafluoroethylene (ePTFE), gelatin-impregnated Dacron (Gelseal), and collagen-impregnated Dacron (Hemashield) grafts. Cell loss from FN-coated prostheses, when exposed to a simulated human arterial blood flow of 200 ml/min in an artificial pulsatile circulation, was quantified from the loss of gamma activity from the graft over 24 hours, pressure in the circulation being reduced to 15 mm Hg to reduce fluid loss. Initial HAEC adherence (mean [SD]) to plain grafts was 3(1)%, 47(9)%, and 53(9)% for ePTFE, Gelseal, and Hemashield, respectively. This improved significantly with FN coating (78[6]%, 60[8]%, and 76[4]%). Cell retention after 24 hours of flow to FN-coated grafts was 16(10)%, 25(5)%, and 65(4)% and was confirmed qualitatively by scanning electron microscopy and environmental scanning electron microscopy. FN significantly improved initial cell adherence with Dacron grafts showing the better adherence. Cell retention after 24 hours of flow was better with FN-coated Dacron than with ePTFE but was best with Hemashield grafts.

Culture of human adult endothelial cells on endarterectomy surfaces.

OBJECTIVES: Endothelial cell seeding of prosthetic grafts has not been as successful as initially hoped and the application of seeding technology to alternative reconstructive procedures such as endarterectomy and angioplasty has been increasingly considered. The success of such seeding depends on the ability of the seeded cells to attach to, and form a monolayer on the endarterectomised vessel wall which was the aim of this study. METHODS: Using a seeding chamber model, heterologous human adult endothelial cells were seeded onto fresh human endarterectomy specimens and cultured. Studies of endothelial call adherence to endarterectomy specimens were performed using 111-Indium oxine labelled cells using methodology analogous to graft seeding. RESULTS: Mean endothelial cell adherence of 70% (S.D. 10%) after 1 h incubation was achieved and the successful development of a monolayer of human adult venous endothelial cells on endarterectomised arteries was demonstrated in vitro. CONCLUSIONS: These results indicate that closed endarterectomy appears to offer a surface with cell attachment that is superior to prosthetic grafts. Where femoral endarterectomy is appropriate, endothelial seeding potentially offers a method of reducing thrombogenicity and intimal hyperplasia, improving patency and avoiding a prosthetic graft whilst preserving collateral circulation and autologous vein.

Limitations of in-vitro labeling of endothelial cells with indium-111 oxine.

Indium-111 oxine labeling is widely used as a marker of endothelial cell attachment to vascular prostheses. The long term effect of labeling human adult endothelial cells (HAECs) with this isotope has not been determined. In this study the viability of labeled HAECs, leakage of isotope from labeled cells and adherence of circulating isotope to fibronectin coated prostheses were investigated over 24 h. The effect of incubation time on labeling efficiency was also assessed. There were significant differences in cell viability between the labeled and unlabeled groups beyond 4 h (p < 0.005, 2-tailed, unpaired t-test). In the control group cell numbers increased by 42% while in the labeled group this had decreased by 20% at 24 h. Spontaneous leakage increased with time but was maximal in the first 2 h. Adherence of circulating isotope to fibronectin coated expanded polytetrafluoroethylene (ePTFE) grafts was minimal but was significantly greater to gelatin impregnated Dacron (GEL-SEAL) beyond 1 hour (p < 0.05). Incubation times greater than 5 minutes during labeling do not significantly improve labeling efficiency, and may contribute to toxicity by prolonging exposure to oxine. Indium-111 oxine labeling of HAECs is a suitable technique for acute studies of endothelial cell kinetics up to 4 h, but its use in chronic studies may lead to significant underestimations of cell retention.

Endothelial cell seeding: a review.

The concept of endothelial cell seeding, designed to provide vascular grafts with a nonthrombogenic lining, has progressed from crude animal experiments during the past two decades to detailed in vitro functional studies using human cells. Although favorable results have been obtained in animal studies this has yet to be translated to humans, where current application of these techniques has been limited to a very few clinical trials. The history, current status and future directions are reviewed herein.

Fibronectin binding to gelatin-impregnated Dacron (Gelseal) prostheses.

Endothelial cell adherence to uncoated gelatin-impregnated Dacron (Gelseal) is poor but can be significantly improved by precoating with a suitable basement membrane such as fibronectin. To assess the suitability of fibronectin-coated Gelseal for endothelial cell seeding, fibronectin binding to Gelseal and its dissociation kinetics were investigated. Fibronectin binding was quantified by radiolabeling human fibronectin with iodine 125, concentrations of 10, 25, 50, 150, and 250 micrograms/ml being used to coat Gelseal at 30, 60, and 90 min of incubation. The amount of fibronectin bound was directly proportional to the concentration used and increased with time of incubation (p < 0.05). However, the percentage attachment decreased with increasing concentration (p < 0.001). The number of molecules bound per centimeter squared of graft was calculated. In the first 30 min, 75% of bound fibronectin was lost after exposure to a flow rate of 200 ml/min in a pulsatile artificial circulation; thereafter, the fibronectin-Gelseal bond was stable for up to 2 h.