Effects of heparin and endothelial cell growth supplement on haemostatic functions of vascular endothelium.

Heparin in combination with endothelial cell growth factor (ECGF) affects physiological responses and growth of human umbilical vein endothelial cells (HUVEC). We have examined the effect of heparin, crude ECGF (endothelial cell growth supplement [ECGS]), or both on the basal and thrombin challenged output of metabolites by HUVEC. The supernatant and/or cell lysate was assayed for released prostacyclin, von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor and thrombospondin. Heparin modified release of all these metabolites when in combination with ECGS, and in general these responses were the opposite of those generated by inflammatory mediators such as interleukin-1. It has been postulated that heparin acts by potentiating the effect of ECGF, but heparin inhibited thrombospondin release and enhanced that of von Willebrand factor in the absence of ECGS, while ECGS alone inhibited release of plasminogen activator inhibitor. Thus, under our experimental conditions it would appear that heparin and crude ECGF can affect HUVEC independently of one another.

The effect of heparin and other glycosaminoglycans on levels of tissue plasminogen activator and plasminogen activator inhibitor in cultured human umbilical vein endothelial cells.

Human umbilical vein endothelial cells (HUVEC) were cultured in the presence of various glycosaminoglycans and the intracellular levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) measured by ELISA. 10 IU/ml heparin (90 micrograms/ml) brought about a significant (20-fold) increase in intracellular tPA levels over the 6-day culture period; intracellular PAI-1 levels were significantly decreased (by 60-70%) and culture growth rate promoted. The final cell density of heparin-containing cultures was 1.7 to 2.3 times greater than that of control cultures. Low molecular weight heparin (First International Standard) had similar effects but was less potent than unfractionated heparin. Chondroitin sulphate and heparan sulphate had no effect on tPA and PAI-1 levels but dermatan sulphate reduced PAI-1 significantly. The changes observed following exposure of HUVEC to heparin are consonant with the view that glycosaminoglycans may affect endothelial production of fibrinolytic components.

Fibroblast growth factor and heparin protect endothelial cells from the effects of interleukin 1.

Vascular endothelium is involved in both active and passive processes in haemostasis, but inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF) have been reported to convert the comparatively inert endothelial cell to an inflammatory state. Acidic fibroblast growth factor (aFGF) in the presence of heparin has effects opposite to IL-1 on cultured human umbilical vein endothelial cells (HUVEC); therefore, we have investigated the modulation of IL-1-induced effects by the c combination of aFGF and heparin (aFGF/heparin). First passage HUVEC were cultured for 6 days in the presence of 20% human serum with and without the addition of 625 pM human recombinant aFGF (hr aFGF) and 7 microM heparin. On day 5, recombinant IL-1 beta was included for 24 h. The following day the cells were washed and measurements made of the release of prostacyclin, von Willebrand factor, plasminogen activator inhibitor type 1, and thrombospondin, both in the resting state and following stimulation for 60 min with 1 U/ml thrombin. Tissue-type plasminogen activator was assayed in HUVEC lysates. Similar experiments were performed to assess effects on the expression of vascular adhesion molecule, intracellular adhesion molecule, and E-selectin using an ELISA on cells in situ. This study indicates that aFGF/heparin in the culture medium of HUVEC abrogates the measured responses to IL-1. These data imply that routine endothelial cell culture with aFGF/heparin may cause artefacts, the effects of FGF and Il-1 may involve common pathways, and FGF/heparin may offer an approach to design therapeutics to counter the adverse effects of IL-1.