Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor.

R R Schleef,M P Bevilacqua,M Sawdey,M A Gimbrone,D J Loskutoff

doi:10.1016/s0021-9258(18)60636-2

Abstract

Regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (HUVECs) by recombinant interleukin 1 beta (rIL-1 beta) and tumor necrosis factor alpha (rTNF alpha) was investigated. Functional and immunologic assays indicated that both cytokines decreased HUVEC tissue-type plasminogen activator (tPA) and increased type 1 plasminogen activator inhibitor (PAI-1) in a dose- and time-dependent manner. Maximal effects (50% decrease in tPA antigen; 300-400% increase in PAI-1 activity) were achieved with 2.5 units/ml rIL-1 beta and 200 units/ml rTNF alpha. Combinations of rIL-1 beta and rTNF alpha were not additive at these maximal concentrations. After a 24-h pretreatment with rIL-1 beta, HUVECs secreted tPA at one-quarter of the rate of control cells and released PAI-1 at a rate that was 5-fold higher than controls. Neither the basal rate of PAI-1 release nor the increased rate of release of PAI-1 in response to rIL-1 beta was affected by subsequently treating the cells with secretagogues (e.g. phorbol myristate acetate) suggesting that PAI-1 is not contained within a rapidly releasable, intracellular storage pool. Northern blot analysis using a PAI-1 cDNA probe indicated that the cytokines increased the steady-state levels of the 3.2- and 2.3-kb PAI-1 mRNA species, but with a preferential increase in the larger mRNA form. The fact that both rIL-1 beta and rTNF alpha act in a similar manner strengthens the hypothesis that the local development of inflammatory/immune processes could reduce endothelial fibrinolytic activity.

Highlights

Regulation of the fibrinolytic systemof cultured hu-’ [1,2]
We recently demonstrated that treatmenotf HUVECs with human monocyte-derived interleukin 1 (IL-1), rIL-la or rIL-lP results ina decrease in the secretion of tPA antigen and an increase in PAI-1 activity[16]
To determineif T N F alters the release of these fibrinolytic components, conditionedmedium was collected from HUVECs incubated continuouslyfor 24 h in thepresence of recombinant TNFa (rTNFa) (200 units/ml) or rlL-1P(2.5 units/

Summary

Introduction

Cultured endothelial cells synthesize and man umbilical vein endothelialcells (HUVECs) by re- secrete type 1plasminogen activator inhibitor (PAI-1) [2], a combinant interleukin 18 (rIL-18) and tumor necrosis fast-actinginhibitor of tPAandurokinase[3,4]. Functional animd - regulation of these fibrinolytic proteins is critical for effective munologic assaysindicatedthatbothcytokines de- hemostasis since alterations in the production of either tPA creased HUVEC tissue-typeplasminogenactivator or PAI-1 havebeen detected duringdeep vein thrombosis IL-1 rate of PAI-1 release nor the increased raotfe release suppresses thefibrinolytic system of endothelial cells, increasof PAI-1 in response to rIL-18 wasaffected by subse- ingPAI-1activity severalfold [13,14,15,16] and decreasing tPA quentlytreatingthe cells withsecretagogues (e.g. antigen by 50% [16].Systemic injection of IL-1intorats phorbol myristate acetate) suggesting that P1AisI-not increases blood PAI-1 activity, supporting an in vivo role for containedwithin a rapidlyreleasable,intracellular this cytokine in modulating the vascular fibrinolytic system storage pool. Northern blot analysisusing a PAI-1 [14]

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Results

Conclusion