Elsevier

Thrombosis Research

Volume 109, Issue 4, 15 February 2003, Pages 175-180
Thrombosis Research

Regular Article
Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

https://doi.org/10.1016/S0049-3848(03)00064-1Get rights and content

Abstract

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-α to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.

Introduction

Endothelial cells (EC) provide a non-thrombogenic and non-adhesive surface but under pathologic conditions they become proadhesive and procoagulant [1], [2]. The expression of surface antigens on resting and stimulated EC has been extensively studied [3]. Upon exposure to proinflammatory cytokines, such as TNF-α or IL-1β, EC synthesize and express on their surface numerous adhesins and other molecules which participate in leukocyte and platelet recruitment, coagulation and inflammation [3], [4]. Apoptosis, on the other hand, elicits distinctive pathways, one prominent manifestation of which is the reversal of the EC membrane to expose anionic phospholipids, thereby providing a procoagulant surface [5], [6]. In addition, apoptotic EC have been shown to shed membrane vesicles (EMP) with procoagulant activity [7].

More recently, it was shown that EC release membrane-derived microparticles (EMP) upon activation or apoptosis [8], [9]. Hamilton et al. first reported the flow cytometric detection of microparticles released by umbilical vein EC (HUVEC) in response to complement C5b9 and calcium ionophore [10]. Subsequently, Combes et al. [8] partially characterized EMP released by HUVEC in response to TNF-α showing that they express membrane antigens PECAM-1 (CD31), vitronectin receptor (CD51), ICAM-1 (CD54) and E-selectin (CD62E).

We previously reported that EC of two different cell lines (renal and brain microvascular EC) release EMP upon activation or apoptosis in vitro [9]. The EMP released expressed antigenic markers CD31 and CD51, and exhibited procoagulant activity as defined by platelet factor 3 activity and tissue factor [8], [9]. In clinical studies, we reported elevated EMP in patients with thrombotic thrombocytopenic purpura (TTP), multiple sclerosis (MS), acute coronary syndromes, preeclampsia, and extreme hypertension [9], [11], [12], [13], [14], [15]. In both TTP and MS, we found that EMP rose in relapses and normalized upon remission [9], [11]. Others have reported elevation of EMP in patients with lupus anticoagulant [8] and coronary ischemia [15], [16].

The present report concerns recent findings on the phenotypic characteristics of EMP released upon activation and apoptosis, defined in terms of antigenic expression. The aim of the study was to determine whether analysis of EMP can discriminate these types of endothelial injury, since such a method could offer new insights to the pathophysiology of thrombotic disorders, and possibly new avenues in their diagnosis and treatment.

Section snippets

EC culture

Renal and brain microvascular EC (MiVEC) and coronary artery (macrovascular) EC were obtained from Cell Systems (Kirkland, WA, USA; Cat. Nos. ACBRI 376, ACBRI 128 and ACBRI 377, respectively) and were cultured as previously described [9]. Upon confluency, cells were detached with a passage reagent group (Cell Systems) following manufacturer's protocol, resuspended in CS-C medium and replated in 12-well tissue culture multi-well clusters (Corning, NY, USA), precoated with attachment factor (Cell

Analysis of EMP and whole EC phenotypes

GFD induced apoptosis in MiVEC as evinced by TUNEL positive results and viability by Trypan blue (65±10% and 10±2% viable EC for RMiVEC, 50±10% and 12±3% viable EC for BMiVEC). Coronary artery (CA) MaVEC were comparatively resistant to GFD at 24 h, showing only modest increase in TUNEL positivity, confirmed by Trypan blue dye uptake (5±3% and 95±8%, respectively). Exposure of MiVEC or MaVEC to TNF-α did not result in apoptosis under our conditions, judged by TUNEL assay and Trypan blue dye

Discussion

In the present study, we investigated a wider spectrum of EMP markers than in previous studies, and report antigenic profiles of the parent whole EC as well as EMP in resting, activated, and apoptotic states in three EC lines. We have demonstrated that EMP are phenotypically distinct in apoptosis vs. activation. This paves the way for further studies of possibly distinctive EMP phenotypes in various kinds of endothelial injury.

In general, EMP expressing constitutive markers, such as CD31 and

Acknowledgements

This work was supported by the Wallace H. Coulter Foundation. We are also grateful for support from the Roz and Cal Kovens Research Fund, the Charles and Jane Bosco Research Fund, and the Mary Beth Weiss Research Fund.

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