Regular ArticleEndothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis
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.
References (29)
- et al.
Leukocyte–endothelial adhesion molecules
Blood
(1994) - et al.
Apoptotic vascular endothelial cells become procoagulant
Blood
(1997) - et al.
Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex
J. Biol. Chem.
(1990) - et al.
TNF signalling in vascular endothelial cells
Exp. Mol. Pathol.
(2001) - et al.
Induction of endothelial cell apoptosis by TNF alpha: modulation by inhibitors of protein synthesis
Exp. Cell. Res.
(1994) Functional heterogeneity of vascular endothelial cells
Biochem. Pharmacol.
(1987)- et al.
Heterogeneity of vascular endothelial cells in normal and disease states
Pharmacol. Ther.
(1998) - et al.
Human cardiac microvascular and macrovascular cells respond differently to exudatively modified LDL
Atherosclerosis
(1998) - et al.
Heterogeneity of vascular endothelial cells: differences in susceptibility to neutrophil-mediated injury
Microvasc. Res.
(1998) - et al.
Anticoagulant properties of the vascular endothelium
Thromb. Haemost.
(1997)
Hemostatic properties of normal and perturbed vascular cells
FASEB J.
Effects of tumor necrosis factor and related cytokines on vascular endothelial cells
Ciba Found. Sympos.
Apoptosis in vascular disease
Thromb. Haemost.
Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus
Proc. Nat. Acad. Sci. U. S. A.
Cited by (469)
Large extracellular vesicles do not mitigate the harmful effect of hyperglycemia on endothelial cell mobility
2022, European Journal of Cell BiologyExtracellular vesicle-induced cyclic AMP signaling
2022, Cellular SignallingNetwork biology and artificial intelligence drive the understanding of the multidrug resistance phenotype in cancer
2022, Drug Resistance Updates