Endothelial Progenitor Cells

Functional CharacterizationCarmen Urbich and Stefanie Dimmeler*

Increasing evidence suggests that circulating progenitor cells contrib-

ute to postnatal neovascularization. These cells home to sites of

ischemia, adopt an endothelial phenotype, and contribute to new

blood vessel formation. Hence, the identity of the circulating cells that

contribute to neovascularization is not entirely clear. Bone-marrow-

derived hematopoietic progenitor cells can give rise to endothelial cells

and contribute to endothelial recovery and new capillary formation

after ischemia. However, nonhematopoietic stem cells within the bone

marrow and adipose-tissue-derived cells, as well as cardiac and neural

progenitor cells, also differentiate to endothelial cells. Progenitor cells

from the different sources may be useful to augment therapeutic

vascularization. The present review article summarizes the different

subtypes of (endothelial) progenitor cells that can give rise to

endothelial cells, enhance neovascularization, and may be suitable

for therapeutic neovascularization. (Trends Cardiovasc Med

2004;14:318–322) D 2004, Elsevier Inc. All rights reserved.

The formation of new capillaries to

provide oxygen supply for ischemic

tissues or tumors was believed to be

exclusively mediated by the proliferation

and migration of existing endothe-

lial cells (ECs) in a process termed

bangiogenesis.Q Meanwhile, increasing

evidence suggests that circulating cells

home to sites of ischemia and contribute

to adult neovascularization. Analogous to

embryonic development, this processwas

termed bvasculogenesis.Q Although the

exact contribution of angiogenesis and

vasculogenesis to adult neovasculariza-

tion is not clear, both processes may be of

Carmen Urbich and Stefanie Dimmeler are at

the Division of Molecular Cardiology, Depart-

ment of Internal Medicine IV, University of

Frankfurt, Frankfurt, Germany.

* Address correspondence to: Stefanie

Dimmeler, PhD, Division of Molecular Cardi-

ology, Dept. of Internal Medicine IV, Univer-

sity of Frankfurt, Theodor-Stern-Kai 7, 60590

Frankfurt, Germany. Tel.: (+49) 69-6301-7440;

fax: (+49) 69-6301-7113;

email: dimmeler@em.uni-frankfurt.de.

D 2004, Elsevier Inc. All rights reserved.

1050-1738/04/$-see front matter

318

importance. The identity of the circulat-

ing cells that contribute to new blood

vessel development has been the focus of

intensive research in the last years. Hem-

atopoietic and endothelial progenitor

cells (EPCs) share some markers in

embryonic development and possibly

derive from a common precursor, the

hemangioblast. In addition, hemato-

poietic progenitors are mobilized to the

bloodstream. Therefore, hematopoietic

stem cells (HSCs) were suggested as

candidates of an EPC precursor cell

population. Indeed, Asahara et al. (1997)

and Shi et al. (1998) demonstrated that

bone-marrow-derived hematopoietic

progenitor cells can give rise to ECs and

contribute to endothelial recovery and

new capillary formation after ischemia.

EPCs have been subsequently defined as

cells that express HSC markers such as

CD34 or CD133 and an endothelial

marker protein, the vascular endothelial

growth factor receptor 2 (VEGFR2) (see

below). Stimulated by these pioneering

results, various studies have identified

additional sources of cells that can give

rise to ECs. Thus, bone-marrow-derived

non-HSCs as well as tissue-derived pro-

genitor cells were shown to differentiate

to ECs in vitro and in vivo. With these

novel findings, it is becomingmuchmore

difficult to define EPCs. How can we

define EPCs from different sources?

EPCs may be defined by the capacity of

a non-EC to adapt an endothelial pheno-

type. Second, the clonal expansion

capacity and stemness characteristics

(proliferation, resistance to stress may be

additional useful characteristics inde-

pendent of surface-marker expression

(Table 1). At present, the surface markers

for non-HSCs are only poorly defined,

and it is unclear whether all different pro-

genitor cell sources form intermediates

corresponding to the HSC-derived EPCs,

which co-express hematopoietic and en-

dothelial marker proteins (CD133+/

VEGFR2+). Moreover, the plasticity of

progenitor cells and the artificial in vitro

cultivation systems may limit the use of

standard colony assays because the com-

ponents required for growth of the spe-

cific stem cell population are not defined.

From a more therapeutic point of

view, the most important criterion prob-

ably is that the progenitor cells function-

ally integrate into the tissue to promote

neovascularization, leading to the gener-

ation of stable vessels, which maintain

long-term tissue oxygen supply (Table 1).

Possibly, cells without bstem cell charac-

teristics Q may also be useful for thera-

peutic application. The present review

article summarizes the different sources

of cells that can give rise to ECs (Figure 1)

and may be suitable for therapeutic neo-

vascularization. Of note, although for

didactic reasons the term bEPC Q is used

throughout the article, the information

regarding clonal expansion and stemness

characteristics is not available for all

progenitor cell subpopulations described

in the following sections.

� Bone-Marrow-Derived ECs

The best-characterized source of EPCs

are HSCs from the bone marrow

(Figure 1). HSCs are maintained in the

bone marrow within the stem cell niche

and are released upon mobilization with

cytokines such as VEGF or stromal cell-

derived factor-1, which are synthesized

by the ischemic tissue (Askari et al. 2003,

Ceradini et al. 2004, Takahashi et al.

1999). However, the bone marrow con-

TCM Vol. 14, No. 8, 2004

Table 1. Endothelial progenitor cells–definitions and requirements fortherapeutic applications

Definition

1. Nonendothelial cell that can give rise to endothelial cells

2. Clonal expansion

3. Stemness characteristics (not yet clearly defined; may include proliferation andresistance to stress)

Therapeutic requirements

1. Cells, which can be easily isolated and promote neovascularization

2. Functional integration into the tissue (homing, migration, invasion)

3. Resistance to stress (apoptosis)

tains additional other stem cells, which

also can differentiate into ECs.

HSC-Derived EPCs

The first studies supporting the con-

cept that HSCs can give rise to ECs were

performed with isolated cells expressing

the classic HSC marker protein CD34 or

the more immature HSC marker protein

CD133. Both cell populations differenti-

ated to ECs in vitro under appropriate

endothelial differentiation-promoting

conditions (Asahara et al. 1997, Gehling

et al. 2000). Most importantly, injection

of CD34+ or CD133+ cells enhanced

neovascularization in animal models

after ischemia (Asahara et al. 1997,

Kocher et al. 2001). According to the

initial discovery, EPCs were defined as

cells positive for both HSC markers

such as CD34 or CD133 and an endo-

thelial marker protein such as VEGFR2.

Because CD34 is not exclusively

expressed in HSCs but—albeit at a

lower level—also in mature ECs, further

studies used the more immature HSC

marker CD133 to better discriminate

between EPCs and mature ECs (Peichev

et al. 2000).

Figure 1. Summary of various cell types wh

endothelial cells.

TCM Vol. 14, No. 8, 2004

The contribution of HSCs to neo-

vascularization improvement has further

been documented by using bone marrow

transplantation with genetically tagged

cells to replenish bone marrow HSCs

after irradiation. Various experimental

studies showed that genetically tagged

bone-marrow-derived cells home to sites

of ischemia and contribute to new blood

vessel formation in ischemic or tu-

mor tissue (Crosby et al. 2000, Garcia-

Barros et al. 2003, Jackson et al. 2001,

Llevadot et al. 2001, Lyden et al. 2001,

Murayama et al. 2002). The level of

incorporation of bone-marrow-derived

cells in the endothelium of capillaries

and vessels varies in the literature (0% to

50%), with the highest levels detected in

tumors (Crosby et al. 2000, Lyden et al.

2001). The difference in incorporation in

the animal models may be explained by

(a) the efficiency of bone marrow trans-

plantation, (b) the extent of EPC mobi-

lization (which is critically influenced by

the extent of injury), (c) the model used

(different tumors, extent of ischemia,

mice strains [good and poor mobilizers],

genetic tag influencing cell function, and

so forth), and (d) the quality of the

immunostaining.

ich have been shown to differentiate into

Although the contribution of embry-

onic and adult progenitor cells to tumor

angiogenesis has been demonstrated

(Hilbe et al. 2004, Lyden et al. 2001,

Vajkoczy et al. 2003), the mechanism as

well as the extent of contribution is still

not clear. The release of growth factors

triggered by the hypoxic environment of

growing tumors may influence progeni-

tor-mediated tumor vasculogenesis.

Recently, it has been shown that growth

factor-enriched conditioned media iso-

lated from several tumor cell lines indu-

ce s bone mar row s t roma l ce l l

proliferation, migration, and tubulogen-

esis (Annabi et al. 2004). Interestingly,

the specific VEGF-blocking antibody

bevacizumab decreased the number of

viable circulating progenitor cells asso-

ciated with a reduction in tumor perfu-

sion and tumor growth (Willett et al.

2004). Differences in the release of

growth factors to mobilize and attract

circulating progenitor cell populations

may contribute to the varying numbers

and different phenotypes of incorporated

cells described thus far. Of note, several

recent studies detected bone-marrow-

derived cells in the perivascular area

(De Palma et al. 2003). Interestingly, the

induction of a suicide gene in these cells

abrogated tumor angiogenesis, indicat-

ing that these bone-marrow-derived peri-

vascular cells are also contributing essen-

tially to new blood vessel formation.

Myeloid Cells

Myeloid cells are also mobilized from

the bone marrow and derive from HSCs.

A recent study (Camargo et al. 2003),

demonstrated that HSC-derived myeloid

intermediates contribute to skeletal

muscle regeneration, suggesting that

myeloid cells may contribute to tissue

regeneration after injury. Indeed, there

is increasing evidence that myeloid cells

also can give rise to ECs. Specifically,

CD14+/CD34� myeloid cells can coex-

press endothelial markers and form

tube-like structures ex vivo (Schmeisser

et al. 2001). Additionally, ex vivo expan-

sion of purified CD14+ mononuclear

cells under conditions favoring endothe-

lial differentiation yielded cells of an

endothelial character, which incorpo-

rated in newly formed blood vessels in

vivo and improved neovascularization

(Urbich et al. 2003). Moreover, a subset

of human peripheral blood monocytes

319

acts as pluripotent stem cells (Zhao et al.

2003). These data would suggest that

myeloid cells within the peripheral blood

can differentiate (or transdifferentiate)

into the endothelial lineage. A recent

study (Ingram et al. 2004) suggested that

monocyte-derived EPCs appear to have a

lower proliferation capacity than do

HSCs or cord-blood-derived EPCs. How-

ever, the different cell types have a

similar capacity to augment neovascula-

rization in experimental models (Hur

et al. 2004, Kalka et al. 2000, Urbich et

al. 2003), suggesting that either the

proliferation capacity is not of major

importance in vivo or that the monocyte-

derived cells may compensate for the

reduced proliferation by an augmented

release of growth factors. Additional

studies are essential to determine the

differences in incorporation and partic-

ularly the long-term fate of HSC- versus

monocyte-derived cells.

Mesenchymal Stem-Cell-Derived ECs

The bone marrow also contains mes-

enchymal stem cells (MSCs). In 2002,

Verfailliea s group (Reyes et al. 2002)

reported that multipotent adult progeni-

tor cells (MAPCs) that copurify with

MSCs can be isolated from postnatal

human bonemarrow.MAPCs are distinct

from HSCs and differentiate into cells

that express endothelial markers, func-

tion in vitro as mature ECs, and contrib-

ute to neoangiogenesis in vivo during

tumor angiogenesis and wound healing

(Reyes et al. 2002). Likewise, MSCs

differentiate into ECs (Oswald et al.

2004) and improve neovascularization

in vivo (Al-Khaldi et al. 2003). Because

MSCs can release a variety of angiogenic

growth factors, this cocktail of growth

factors may also act in a paracrine man-

ner to support angiogenesis and arterio-

genesis (Kinnaird et al. 2004).

A recent study (Kuznetsov et al. 2001)

showing that MSCs can also be isolated

from the peripheral blood gives rise to

the question of whether bone-marrow-

derived MSCs may also be mobilized in

response to ischemia and contribute to

endogenous repair.

� Non-Bone-Marrow-Derived EPCs

Several studies suggested that other cell

populations in addition to bone-marrow-

320

derived cells can give rise to ECs

(Figure 1). The first evidence came from

a study using transplanted grafts, where

non-bone-marrow-derived cells replaced

the ECs (Hillebrands et al. 2002). Mean-

while, other tissues such as fat tissue or

the heart itself were shown to contain

cells that are capable of differentiating

into the endothelial lineage (Beltrami

et al. 2003). Although it is unclear at

present whether these cells derive

entirely from repopulated bone-marrow-

derived (hematopoietic) stem cells or are

tissue-residing remnants from embry-

onic development, it is of major scien-

tific and possible therapeutic interest to

determine whether these tissue-derived

cells may have superior capacities com-

pared with bone-marrow-derived or

peripheral-blood-derived cells.

Fat Tissue

Adipose tissue is an alternative source

of autologous adult stem cells that can

be obtained in large quantities under

local anesthesia and with minimal dis-

comfort. Human lipoaspirate contains

multipotent cells that can differentiate

into different lineages (Zuk et al. 2001).

Additionally, other groups isolated adi-

pose-tissue-derived, cultured, stromal-

vascular fractions lacking the HSC

marker CD34 and the endothelial marker

CD31 (thus resembling mesenchymal/

stromal cells), which differentiated into

ECs and promoted angiogenesis (Planat-

Benard et al. 2004). Stromal-vascular

fractions obtained from adipose tissue

also contained hematopoietic-marker-

expressing cells (CD34+) that were neg-

ative for the endothelial protein CD31.

These isolated CD34+/CD31� cells also

were capable of differentiating into ECs

and potently promoted neovasculariza-

tion (Miranville et al. 2004).

Tissue-Resident Stem Cells

Tissue-specific stem cells reside in

certain adult tissues, yet their specific

properties often are difficult to assess

because of their heterogeneity and tech-

nical difficulties in identifying them and

tracing their progeny. Adult tissue stem

cells are responsible for regenerating

damaged tissue and maintaining tissue

homeostasis—for example, physiologic

replenishment of skin and gut. Recently,

tissue-resident c-kit+ stem cells have

been isolated from the heart, which are

capable of differentiating into the endo-

thelial lineage (Beltrami et al. 2003).

Similarly, neural stem cells differenti-

ated into the endothelial lineage in vitro

and in vivo (Wurmser et al. 2004),

suggesting that tissue-resident stem/pro-

genitor cells may contribute to vascular

growth and tissue oxygenation.

� Cord Blood

A rich source of EPCs is the cord blood.

Cord blood contains higher numbers of

CD133+ and CD34+ HSCs compared with

peripheral blood from adults (Ingram et

al. 2004). CD133+ and CD34+ cells iso-

lated from cord blood were ex vivo

cultivated and differentiated to ECs

(Murohara et al. 2000). Cord-blood-

derived EPCs showed a higher prolifer-

ation capacity and express telomerase, a

functional characteristic of stem cells,

which is very low or absent in other

progenitor cell populations (Ingram

et al. 2004).

� Open Questions

Various distinct cell populations within

the adult organisms have the ability to

contribute to and enhance vessel

growth. The characterization of the

precursor of the ECs derived from differ-

ent sources is not clear. Current studies

are predominantly aimed at detecting

the expression of surface markers such

as CD133 and VEGFR2 expression for

HSC-derived EPCs. Possibly, transcrip-

tional profiling will yield better func-

tional markers— for example, by

identifying transcription factors that

are necessary to mediate the switch

toward the endothelial lineage. More-

over, functional assays (e.g., colony

assays) are of utmost importance. Be-

cause culture conditions affect the out-

come greatly, there is an urgent need for

development of serum-free ubiquitously

usable colony assays. For that purpose,

the growth factors—which are necessary

for colony formation and proliferation

have to be defined. Despite these chal-

lenges, EPCs from different sources

appear to be promising options for neo-

vascularization improvement in patients

with peripheral vascular disease or acute

myocardial infarction (Assmus et al.

2002, Tateishi-Yuyama et al. 2002). By

TCM Vol. 14, No. 8, 2004

physically forming vessels and/or provid-

ing growth factors, EPCs are powerful

tools for themodulation of vessel growth.

It is hoped that better understanding of

thebiology ofEPCswill provide strategies

to additionally enhance neovasculariza-

tion improvement.

� Acknowledgments

The authors apologize that many of the

important publications could not be

cited due to space limitations. The

authors are supported by the Dutch

Forschungsgemeinschaft (FOR 501,

Di600/4-1 and 6-1).

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