MECHANISMS

DRUG DISCOVERY

TODAY

DISEASE

Endothelial progenitor cells: Cellularbiomarkers in vascular diseaseMihail Hristov1,2,*, Christian Weber1

1Institut fur Molekulare Herz-Kreislaufforschung (IMCAR), Universitatsklinikum der RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany2IZKF ‘‘BIOMAT’’, Universitatsklinikum der RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

Drug Discovery Today: Disease Mechanisms Vol. 5, No. 3–4 2008

Editors-in-Chief

Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA

Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA

Cardiology

Recently, the research on adult endothelial progenitor

cells (EPCs) in peripheral blood raised great interest.

EPCs were significantly involved in endothelial regen-

eration after arterial injury and in neovascularisation of

ischemic tissue. Moreover, EPC counts might correlate

with clinical outcomes in patients with heart disease or

cancer. This mini-review intends to discuss the validity

of some circulating EPC subsets as surrogate prognos-

tic/diagnostic cellular biomarkers for monitoring vas-

cular homeostasis in atherosclerosis and cancer.

Introduction

Along leukocyte recruitment, lipid accumulation, chronic

inflammation and immune cues, a crucial involvement of

vascular progenitor cells in the pathogenesis of atherosclero-

sis has been newly emerged [1–3]. For instance, endothelial

progenitor cells (EPCs) were identified in adults and these

cells have been shown to play an essential role in arterial

remodelling [4,5]. Adult EPCs were intensively investigated

within the past decade by several groups and numerous

published data revealed their contribution in the pathogen-

esis of transplant arteriosclerosis, neovascularisation of

ischemic tissue and regeneration of the arterial wall after

injury [3–5]. Along atherosclerotic disease EPCs were also

involved in other pathological conditions such as cancer,

diabetes and renal failure, thus highlighting broadly based

and multidisciplinary research on EPC biology within the

medical community [3,6]. Moreover, EPCs have recently

*Corresponding author: M. Hristov (mhristov@ukaachen.de)

1740-6765/$ � 2008 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmec.2008.07.001

Section Editor:Christian Weber – Institute for Molecular CardiovascularResearch (IMCAR), RWTH, Aachen University, Germany

generated great attention as potential novel diagnostic/prog-

nostic biomarkers for vascular integrity and therapeutic clin-

ical approaches using these cells are on the way.

Origin and characterization of circulating adult EPCs

During the embryonic development primitive capillary net-

works were formed by angioblasts, a process known as vas-

culogenesis [7]. Some of these primordial vascular structures

gradually sprout and become stabilised by peri-endothelial

matrix, smooth muscle cells and pericytes, thus transforming

into pressure resistant contracting arteries, a process defined

as arteriogenesis [7]. Parallel to these mechanisms, a common

precursor cell positive for the vascular endothelial growth

factor receptor-2 (VEGFR2) exists, which can give rise to both,

endothelial and smooth muscle lineage during vessel devel-

opment in the embryo [8]. Of note, the above mechanisms

still persist in adults since several recent studies have

described adult vascular progenitor cell subtypes correspond-

ing to embryonic stem cell progeny. These vascular precur-

sors have the ability to promote postnatal vasculogenesis and

neovascularisation of ischemic tissue by assuming for

instance endothelial phenotype and by paracrine delivery

of angiogenic compounds [3–5,9].

As the adult progenitor cell most widely studied, the

putative EPC can be localised in bone marrow and peripheral

blood or reside on ‘stand-by’ within the vascular wall [3–

5,10]. Despite comprehensive amount of published results,

an exact definition of the EPC remains rather controversial

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Drug Discovery Today: Disease Mechanisms | Cardiology Vol. 5, No. 3–4 2008

and inconsistent, based in part on the plasticity of distinct

adult haematopoietic or mesenchymal cell subpopulations

(e.g. CD34+/�, CD14+) in acquiring the endothelial pheno-

type [11,12]. Nevertheless, standard lineage marker combi-

nations commonly established and largely used for

identifying functional circulating EPCs comprise CD133/

CD34/VEGFR2 or CD34/VEGFR2 [3–5]. Some groups have

additionally identified even ‘late’ circulating EPCs with a

CD34+CD144+ phenotype [13,14]. These cells were mainly

located within the CD3� lymphocyte subpopulation [14].

Recent data advocate to include also circulating CD14+

myeloid cell subtypes (e.g. CD14+CD34low, CD14lowCD16+

Tie-2+, CD14+VEGFR2+CXCR2+/�) which were characterized

as functional endothelial-like cells with significant contri-

bution to endothelial regeneration, ischemic or tumour

angiogenesis [15–17]. Thus, the heterogeneous pool of adult

EPCs includes subsets of multiple origin, phenotype and

differentiation stage, which commonly share expression of

progenitor, myeloid and endothelial differentiation mar-

kers (e.g. CD133, CD34, VEGFR2, CD14, CD31, CD144, Tie-

2, von Willebrand factor) together with clonogenic capa-

city, generation of endothelial outgrowth and formation of

vascular network.

Contribution of EPC subsets in arterial remodelling

and tumour angiogenesis

Studies in animal models have revealed that infused or endo-

genously mobilised EPCs effectively contributed to endothe-

lial regeneration and reduction of neointimal formation after

arterial injury [17–19]. EPCs have also been shown to partially

regenerate damaged endothelial cells in atherosclerosis-

prone ApoE�/� mice and a large percentage of renewed

endothelial cells in vascular grafts originated from circulating

progenitors [20,21]. Translating this knowledge to the clinics

has introduced the application of in vitro expanded autolo-

gous angiogenic mononuclear cells as a cell-based additive

therapy for acute or chronic myocardial ischemia [22]. How-

ever, the outcome on recovery of heart function remains

altogether moderate and controversial as this has been also

revealed during intracoronary application of bone marrow

mononuclear cells after acute myocardial infarction [22].

These modest effects in left ventricular functional recovery

very probably associated with improved neovascularisation

and paracrine action, but not with transdifferentiation of

infused cells into cardiomyocytes [3,22,23]. Current clinical

concepts further claim a protective role of EPCs in athero-

sclerotic disease. Reduced numbers of CD34+VEGFR2+ EPCs

and impaired clonogenic capacity have been shown to cor-

relate with the Framingham risk factor score and to predict

cardiovascular events [24]. By contrast, some trials revealed

that EPC numbers were unaffected by cardiovascular risk

factors but rather associated with the severity of stenotic

coronary artery disease (CAD) [25–27]. These results may also

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be ascribed to ischemia occurring in severe CAD or reflect the

delicate equilibrium between EPC mobilisation and periph-

eral demand. The different diagnostic/therapeutic effects of

EPCs may also be related to the distinct cell models used. For

instance, only the fraction of CD34+ cells has been shown to

independently predict functional improvement at follow-up

after acute myocardial infarction [28]. However, many of the

clinical studies above have therapeutically used the entire

mononuclear cell fraction including also neutrophils and

inflammatory monocytes, thus inducing secondary leukocy-

tosis. Such an elevation in leukocyte counts has been still

demonstrated to associate per se with incidence of CAD [29].

Furthermore, published data from animal trials have shown

that infusion of EPCs increased plaque size and decreased

plaque stability in ApoE�/� mice [30]. This observation could

be explained in part by pro-angiogenic and proteolytic prop-

erties of EPCs. Influx of EPCs may also be involved in cancer

progression due to increased tumour vascularisation and

assessment of circulating EPCs might adequately support

the monitoring of anti-angiogenic therapy [6]. This knowl-

edge introduced EPCs as potential targets for manipulating

tumour growth and metastasis. Conversely, other data

showed that bone marrow-derived EPCs failed to maintain

tumour vascular endothelium [31]. Nevertheless, the level of

EPC contribution to cancer vessels might depend on tumour

type, grade and localisation [6]. For instance, evidence from

research on multiple myeloma has shown that circulating

EPCs are related to the neoplastic clone and this represent one

potential mechanism for up-regulation of tumour neovascu-

larisation [32]. Very recent data have further identified selec-

tive accumulation of pro-angiogenic CD14lowCD16+Tie-2+

monocyte subsets within the neoplastic tissue and their

contribution in promoting tumour angiogenesis and cancer

growth, respectively [15]. This specific, even resident mono-

cyte subpopulation differ from the classical CD14high inflam-

matory monocytes and parallel to elevated expression of Tie-

2 also demonstrate higher levels of the chemokine receptors

CXCR4 and CCR5 [15,33].

Accordingly, the ambiguous contribution of circulating

CD34+VEGFR2+ EPCs and myeloid CD14low/+ angiogenic cells

to vascular remodelling during arterial injury, advanced

atherosclerosis and cancer disease requires a meticulous re-

evaluation.

Effect of various conditions, cytokines and

pharmacological compounds on the number of EPCs

Published data from animal studies and clinical trials have

reported effects of some conditions, cytokines and therapeu-

tics on the number of EPCs (Fig. 1). For instance, ischemia and

exercise training associated with mobilisation and improved

homing of EPCs in patients with peripheral arterial occlusion

[34]. Furthermore, VEGF, erythropoietin and granulocyte

colony-stimulating factor have been shown to mobilise EPCs

Vol. 5, No. 3–4 2008 Drug Discovery Today: Disease Mechanisms | Cardiology

Figure 1. Circulating putative progenitors for endothelial cells as fundamental players in vascular homeostasis. The heterogeneous pool of adult

endothelial progenitor cells in peripheral blood includes subsets of multiple origin, phenotype and differentiation stage, which commonly share expression

of progenitor (CD133, CD34), myeloid (CD14) and endothelial (VEGFR2, CD144, Tie-2) differentiation markers. These cells obviously participate in

endothelial repair and angiogenesis, and their number is differentially affected by several conditions, cytokines and therapeutic compounds. A routine

quantification of circulating endothelial progenitor cells by using multi-parametric flow cytometry analysis of peripheral blood may strengthen their

significance as novel cellular biomarkers for monitoring drug activity and endothelial maintenance during atherosclerosis and cancer disease. ACE,

angiotensin-converting enzyme; G-CSF, granulocyte colony-stimulating factor; VEGF(R), vascular endothelial growth factor (receptor).

[3]. Therapeutically, the HMG-CoA reductase inhibitors

(statins) induce a transient mobilisation of circulating

CD34+VEGFR2+ EPCs and stimulate their neo-endothelial

incorporation which may also assist in preventing neointimal

hyperplasia [3,18]. Interestingly, long-term treatment with

statins dose-dependently decreased the number of circulating

CD34+VEGFR2+ EPCs but increased the number of the even

more mature CD34+CD144+ EPCs in peripheral blood of

patients with CAD [13,27]. Further data have revealed a rise

in circulating EPCs after application of peroxisome prolifera-

tor-activated receptor-gamma agonists (thiazolidinediones)

or the angiotensin-converting enzyme inhibitor enalapril

[35,36]. Conversely, the number of circulating EPCs has been

reduced after treatment with bevacizumab (a specific mono-

clonal antibody against VEGF) in cancer patients [37]. Simi-

larly, application of small molecule VEGFR2-antagonist was

reported to inhibit the mobilisation of EPCs in mice [6].

EPCs: cellular biomarkers in vascular disease

From the diagnostic/prognostic clinical point of view quan-

tification and functional assessment of circulating EPCs have

been reported to reflect endothelial complexity and main-

tenance [3,6,24]. Thus, such a procedure could be routinely

introduced as an alternative predictive and/or diagnostic

biomarker of several vascular disorders (e.g. acute coronary

syndrome, restenosis, transplant vasculopathy, diabetic

angiopathy, etc.) but also in cancer [6,37,38]. To date, direct

ex vivo flow cytometry from anti-coagulated peripheral blood

and in vitro enumeration of colony forming units (CFUs; a

marker for clonal expansion capacity) are the most com-

monly used methods for evaluation of adult EPCs [3,38].

Flow cytometry from freshly drawn peripheral venous

blood offers the advantage of high sensitive and reproducible

multi-parameter approach allowing direct quantification of

the target population. This is a quickly done and minimally

invasive procedure which requires very small blood volume

parallel to convenient laboratory equipment, expenses and

efforts. However, a general agreement for enumeration of

circulating EPCs is still absent. This may in part explain some

controversial results in published clinical trials [24–27]. Basi-

cally, the direct quantification of circulating CD34+ haema-

topoietic progenitor subsets by flow cytometry is well

established in haematology and haematooncology. Of note,

analysis of CD34+ cells in peripheral blood may also reflect

endogenous vascular homeostasis and endothelial regenera-

tive potential. For example, an increase in circulating CD34+

cells after coronary stent implantation independently pre-

dicted in-stent stenosis and may be suggestive for the invol-

vement of CD34+ subpopulations in the pathogenesis of

neointimal hyperplasia [39]. Other clinical trials revealed a

significant increase of circulating CD34+ cells in patients with

acute myocardial infarction and a biphasic response during

the early and advanced phases of chronic heart failure,

respectively [3]. Despite variations in the methodology for

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Drug Discovery Today: Disease Mechanisms | Cardiology Vol. 5, No. 3–4 2008

quantifying EPCs by flow cytometry from peripheral blood,

the most commonly used combination of surface marker

currently encompasses analysis of CD34+ subpopulations

defined as (CD133+)CD34+VEGFR2+ cells [38]. Principally,

this approach is performed within the mononuclear (lym-

pho/monocyte) population of lysed whole blood by addi-

tionally sub-gating on CD45low/� events [37]. Such a double-

gating strategy allows more exact identification of the very

low number of circulating EPCs (0.01–0.2% of the peripheral

blood mononuclear cells) [37]. Because of these very low

numbers of positive cells additional efforts are necessary to

increase analytical sensitivity, for example, the use of bright

fluorochrome for dimly expressed marker, automatic com-

pensation in the multicolour experiments, minimizing back-

ground noise, smooth fixation and recording more events

(e.g. at least 50,000) within the target gate [6,14,37,38].

The second approach for quantifying EPCs by in vitro

cultivation to obtain CFUs has also been used several times

[24–26]. The number of EPCs (measured by CFU assay) has

been shown to significantly increase in patients with unstable

angina, although their function (determined as adhesion to

fibronectin) did not differ as compared to patients with stable

CAD [40]. These results imply that enhanced number of EPC-

CFU do not necessarily associate with improved function.

However, recent data revealed that the CFU count did not

correlate with the number of circulating CD34+VEGFR2+ cells

[38]. Thus, the ability of separated mononuclear cells to form

CFUs in vitro may rather represent a functional assay for

clonogenic capacity but the enumeration of CFUs is not

the method of choice for direct ex vivo quantification of

circulating EPCs.

Taken together, some controversial findings in the clinical

studies above may also reflect the different methods and

definitions used to assess EPCs in peripheral blood as well

as interpretation of rather functional in vitro parameters (e.g.

CFU count) as quantitative ex vivo marker. Hence, consider-

ing the growing evidence in support of circulating vascular

progenitor cells as predictive biomarkers (positive or nega-

tive) of endothelial maintenance during atherosclerosis and

cancer, it would be crucial for the clinic to establish further

definitive standards for routinely monitoring endothelial-

like cells in peripheral blood. To adequately address the

multiple EPC phenotypes, a simultaneous ex vivo measure-

ment by flow cytometry of ‘classical’ CD34+VEGFR2+C-

D45low/� EPCs in conjunction with CD14low/+ myeloid cells

(e.g. CD14lowCD16+Tie-2+ monocyte subsets) may be more

meaningful in terms of potential prognostic/diagnostic target

(Fig. 1).

Summary and conclusions

Diverse subpopulations of circulating or tissue resident adult

progenitor cells may acquire endothelial phenotype and

functional characteristics of mature endothelial cells, thus

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obviously participating in vascular repair and angiogenesis.

Parallel to inflammatory and immunological cues, these cells

are recognised as fundamental players in the pathology of

atherosclerosis, arterial remodelling after injury but also in

cancer disease. Recent basic and clinical research intensively

discusses the relevance of EPCs as surrogate cellular biomar-

kers in vascular disorder. Standardisation of the multi-para-

metric flow cytometry protocols currently used is crucial for

the direct quantification of circulating angiogenic progenitor

cells as vascular biomarkers. In terms of clinical prognosis and

diagnosis this will strengthen the significance of EPCs in

routinely monitoring drug activity and endothelial home-

ostasis. Last but not least, simultaneous express analysis of

more than one circulating progenitor cell subpopulation (e.g.

as a biomarker package) may substantially contribute to a

more complex prognostic and diagnostic statement.

Acknowledgement

This work was supported by a grant from the Interdisciplinary

Centre for Clinical Research ‘‘BIOMAT’’ within the Faculty of

Medicine at the RWTH Aachen University (NTV B113-a).

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