Review
Circulating endothelial cells: A novel marker of endothelial damage

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Abstract

Circulating endothelial cells (CECs) were first described over 30 years ago in smears of peripheral blood. Since then, more sophisticated techniques for CEC isolation have become available. In particular, immunomagnetic isolation and fluorescence-activated cell sorting (FACS) have been employed with success. We provide a short historical perspective and a comprehensive review on the subject. We review isolation and enumeration of CECs with an emphasis on CD146-driven immunomagnetic isolation and FACS. We describe, in great detail, advantages and pitfalls of both approaches and compare their specificity. Moreover, we provide a comprehensive list of clinical studies in this field and describe the possible clinical use of CECs. We also describe the phenotype of these cells and list typical surface markers. In addition, we review the phenotype of CECs and discuss mechanisms of detachment. We speculate about potential interactions between CECs and other cell subsets. We also describe other serum markers of endothelial damage and compare CECs with these markers. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CECs must now be regarded as a sensitive and specific marker of endothelial damage. We emphasize that use of CECs in a clinical setting is on the horizon and pathogenetic clues may also be obtained.

Section snippets

Circulating endothelial cells — a historical perspective

Circulating endothelial cells (CECs) were first described over 30 years ago by Bouvier and Hladovec. Their techniques included vital light microscopy, Giemsa staining and separation by density centrifugation ([1], [2], [3], [4]). Increased numbers of circulating endothelial cells were also detected in animal models of shock, such as treatment with endotoxin ([3]). Between 1970 and 1980 studies in humans followed and these cells were identified in various conditions, such as smoking, acute

Immunomagnetic isolation of CECs

In healthy individuals the endothelial layer is continuously renewed at only a low replication rate of 0–1% per day. Endothelial proliferation is clustered at sites of branching [19] while laminar flow has been reported to suppress endothelial apoptosis [20]. The turnover of endothelial cells varies greatly in different organs. In accordance with these data, detection of circulating endothelial cells in a healthy adult is a rare event and immunomagnetic isolation has consistently yielded as few

Isolation of CECs by fluorescence-activated cell sorting (FACS)

Flow cytometry represents another attractive approach to isolate and enumerate CECs [29], [30], [31], [32], [33]. In general, multi-parametric flow cytometry is used to detect endothelial cells and discriminates them from cells with overlapping expression of antigens. For example, CD 146 expression on activated T cells can be distinguished from CD 146 on endothelial cells by co-staining with CD45 or CD3 (or both). CD 133 may help to identify EPCs because it is not present on CECs or any mature

CECs in vascular disorders

Numbers of CECs also reflect the extent of the endothelial lesion. High cell counts have been observed in diseases with widespread vascular damage, such as rickettsial infection, sickle cell disease or vasculitis [22]. In contrast, localized damage may be seen in patients undergoing coronary angioplasty and low cell numbers have been observed in this setting. In healthy subjects, renewal of the endothelial layer takes place at a low replication rate of 0–1% per day. Therefore, detection of

Phenotype and mechanisms of detachment of endothelial cells

The vascular network has an estimated surface area of more than 1000 m2 and maintains an anticoagulant, anti-thrombotic and anti-inflammatory state [78]. Endothelial cells can be activated by various stimuli, such as pro-inflammatory cytokines, growth factors, infectious agents, lipoproteins, or oxidative stress. Irreversible loss of integrity of the endothelial layer eventually leads to cell detachment. It must be noted that detached cells could be apoptotic or necrotic [79]. Unfortunately,

Interactions of circulating endothelial cells with other cell subsets

It is unknown whether circulating endothelial cells are pro-inflammatory [79]. Several products released by necrotic cells have been found to initiate an inflammatory response. These products include high mobility group 1 protein (HMGB1) [94], cytochromes, plasma DNA or heat shock proteins. Other studies have observed that necrotic but not apoptotic cells initiate a Toll-like-receptor-2/NF κB-dependent reaction in monocytes and fibroblasts [95]. The uptake of necrotic cellular material

CECs and other circulating markers of vascular damage

Endothelial cells express a broad variety of proteins [97] but only few of these have been studied in serum or plasma in vascular disease. Currently, von Willebrand factor (vWF), thrombomodulin and soluble E-selectin are best described [98], [99], [100], [101]. It must be noted that several factors may influence the levels of these circulating proteins. For example, thrombomodulin undergoes renal excretion. Hence, serum levels are influenced by renal function. Other confounding factors, such as

CECs and EPCs

CECs and EPCs represent two groups of non-hematopoietic cells in the blood. Agreement on the phenotypic differentiation of EPC and CEC is still lacking, not least because several markers occur on both. It is believed that CECs and EPCs have different origins. CECs derive from mature endothelium while EPCs derive from the bone marrow. Accordingly, EPCs are characterized by the expression of CD 34 and the receptor for vascular endothelial growth factor (VEGFR-2 or KDR/Flk-1) [108]. Many

Conclusion

Circulating endothelial cells are a novel marker of vascular damage. Cell numbers correlate with disease activity across a variety of diseases. Their use in a clinical setting is on the horizon and pathogenetic clues may also be obtained. The phenotype and functional capacity of these cells as well as interactions with other cell subsets need to be further elucidated. Another crucial issue is standardization of the technique, a task that has been addressed by a European multi-center effort. In

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