Heterogeneity of endothelial cells  role in vessel specialization
and cooperation in vasculogenic mimicry
ABSTRACT
Claudine Kieda* mong endothelial cells (ECs), subpopulations are mainly distinguished in terms of
Amaturation, tissue specialization and functions. Heterogeneity is an important char-
acteristic of endothelial cells responsible for organ-specific cell and molecule delivery.
Centre de Biophysique Moléculaire, UPR
Endothelial cell heterogeneity is a key to embryonic development as well as cell recruit-
CNRS 4301, Orleans CEDEX 2, France
ment in adult organism during the immune response; it determines also the pathologic
spreading of diseases, such as cancer invasion and infectious disease progression among
*
Cell recognition and Glycobiology,
species. Heterogeneity is also a feature of intra-organ specialization of endothelial cells.
B2CT Department, Centre de Biophysique
ECs are highly reactive to the microenvironment and their condition reflects healthy vs.
Moléculaire, UPR CNRS 4301, Rue Charles
diseased states. They respond to tissue hypoxia which brings a new parameter to en-
Sadron, 45071 Orleans CEDEX 2, France ; tel.:
dothelial heterogeneity. Hypoxia changes the phenotype and biology of ECs by turning
(00 33) 238 25 55 61, (00 33) 675 68 38 92, fax : (00
on angiogenesis to restore physioxia. Highly responsive to hypoxia are the endothelial
33) 238 25 54 59, e-mail: claudine.kieda@cnrs- precursor cells (EPCs) and the selected cancer stem cell (CSC) populations, the most ag-
orleans.fr
gressive dedifferentiated tumor cells. They cooperate with one another and contribute to
the vascular mimicry process of angiogenesis. This has a most effective impact on tumor
cells escape and spreading.
Received: October 14, 2013
Accepted: November 4, 2013
INTRODUCTION
Key words: angiogenesis, endothelium, hypo-
Endothelium has long been considered as a tissue whose sole function was
xia, endothelial progenitor cells, cancer stem
lining of the vessel walls. The recognition of the variety of biological roles that
cells, vasculogenic mimicry
endothelial cells are able to perform prompted the research on endothelial tissue
selectivity [1].
Abbreviations: CSC  cancer stem cell; EC 
endothelial cell; EPC  endothelial precursor
cell; CK  chemokines; VEGF  vascular en-
Endothelium has been intensively studied since it was recognized that en-
dothelial growth factor
dothelial cells (ECs) mediate the exchanges between the lumen of vessels and
tissues. This action concerns intra/extravasation of cells as well as selection
Acknowledgements: Reported work was
or presentation of the circulating molecules. Endothelial cells display distinct
partly supported by the French-Polish Grant
and characteristic properties depending on the organs they are in and this ap-
INCa/CNRS/MNiSW (347/N-INCA/2008)
for cooperation, ANR  triple sense project , pears to be a key parameter for the behavior and homing of circulating cells.
the LNCC, INCa, CNRS and INSERM.
In invasive disorders these properties become a condition for the propaga-
tion of diseases such as cancer, in which pathological circulating cells are
able to infiltrate into a secondary site by selective adhesion and extravasation
through vessel-lining endothelial cells via recognition and adhesion process-
es. Moreover, endothelial cells favor tumor cell spreading, because tumor
angiogenesis [2] gives rise to non-structured and non-sealed tube walls that
are permissive for cell escape [3].
Pathological angiogenesis is a response to tumor hypoxia that, like in the
embryo, makes the cells produce VEGFs and other proangiogenic factors in
an excessive manner [4]. The tumor angiogenesis process is achieved by sev-
eral mechanisms [5], named: sprouting of the preexisting vessels, intussus-
ception, cooption, and vessel mimicry by tumor cells [6]. The resulting ves-
sels are permissive in several ways. While the non-sealed junctions between
cells are indeed allowing exchanges, the interplay between cancer cells and
endothelial cells generates pseudo vessels and gives rise to pathological ves-
sel walls. Endothelial cells, cancer cells and other recruited cells are able to
interact within the tumor stroma and create pseudo vessels that incorporate
those various cells in a mosaic type of structure [7] which is very favorable
for cancer cell shedding into the vessel lumen. Their subsequent presence
in the circulation permits further homing into secondary sites where they
settle into metastases. Cells cooperating to generate vessels by vascular mim-
icry mechanism are mostly cancer stem cells that combine their action with
endothelial precursors. They cooperate to build a tumor microenvironment,
the composition of which reflects the hypoxic conditions of the tumor, and
contribute efficiently to cancer cells dissemination and aggressiveness [7].
372 www.postepybiochemii.pl
ENDOTHELIAL HETEROGENEITY IN EMBRYOS
Consistently, Nrp2 is expressed in venous and lymphat-
ic endothelial cells, while VEGFR-3 expression is initially
The first level of heterogeneity of the endothelium ap- detected in all blood vessels of the early embryo [12], but
pears early during the embryonic development. Angio- becomes restricted first to venous and then to lymphatic
blasts are subjected to a set of maturation factors that permit endothelial cells, when development proceeds [13]. Con-
the progenitor cells to develop into arterial and venous en- sequently, it should be considered that arterial-venous cell
dothelial cells by specific gene expression pattern and, sub- fate determination and subsequent lymphatic development
sequently, to give rise to arteries and veins [8]. are genetically controlled. They are then regulated in a mul-
ti-step manner through various signaling pathways and ac-
It was believed, until recently, that arteries and veins are tivation of transcription factors.
formed from primitive blood vessels evolving according to
hemodynamic changes and physiological factors. The dis- Moreover, the embryonic stem cell studies have shown that,
tinct expression of ephrinB2 and its receptor EphB4 on arte- by taking advantage of the endothelial plasticity, it is now pos-
rial and venous cells, respectively, before circulation begins sible to reproduce in vitro the maturation and differentiation of
in early embryonic development, has proven that genetic endothelial cells and to condition these cells according to the
mechanisms rule the establishment of the arterial vs. venous organ they are to be part of. The endothelial organ specificity
specification [8]. Indeed, ephrinB2 and EphB4 are distinc- that those cells are capable to adopt, starts from the embryonic
tively detected in the primary vascular plexus before the on- state to end with cells that are phenotypically and function-
set of circulation in the developing embryo. Differential cir- ally different. Clearly distinct from one vessel type to another,
culation by hemodynamic forces stabilizes the phenotypic endothelial cells are also deeply involved in regulating the me-
distinction and influences the arterial-venous plasticity. In tabolism of the organ they belong to. Moreover, by interacting
turn, the lymphatic endothelial cells appear as a subpopula- with the surrounding cells, they regulate their activities both in
tion of the venous endothelial cells which differentiate and the vessel lumen and in the underlying tissue.
acquire lymphatic competence [8,9].
ORGAN SPECIFIC ENDOTHELIAL
As a key factor, VEGF (vascular endothelial growth fac- CELL HETEROGENEITY
tor) induces expression of Notch signaling genes, includ-
ing Notch1 and its ligand Dll4 (Delta-like ligand 4), which Among the main characteristics of endothelial cells, no
start arterial specification. The VEGF/VEGFR-2 (VEGF matter where they are located in the organism, is their abil-
receptor-2)/Nrp1 (neuropilin-1) pathway activates Notch ity to create a selective barrier. This allows the endothelium
signaling early in angioblasts, leading to the specification to fulfill its fundamental role in the blood-to-tissue trans-
of arterial cell fate, while CRLR (calcitonin receptor-like port of nutrients and cells, as well as in recruitment and
receptor) controls VEGF expression. Also Dep1 (Density then selection of cells or molecules that are transported from
enhanced phosphatase-1) acts upstream of the VEGF-acti- blood and lymph to the tissue, and vice versa. At this level,
vated PI3K pathway. On the other hand, Dll4 expression is endothelial cells display the necessary degree of specific-
regulated by Foxc1 and Foxc2 transcription factors, while ity to ensure the harmony of intercellular interactions that
ephrinB2 is a downstream target of Notch. Finally, it has regulate immune and inflammatory responses [14].
been shown that Hey1 and Hey2 genes, the bHLH tran-
scription factors and main transducers of Notch signals in These functional properties of ECs also play a role in
mice, or gridlock gene in zebrafish, further promote arterial the pathological autoimmune reactions, allergic response,
differentiation. The combination of genetic commitment cancer growth, tumor cell spreading, and establishment of
and microenvironmental pressure cooperates to achieve metastases. Thus, endothelium functions in a site-specific
arterial/venous differentiation. It was shown in vitro that manner both in physiological and pathological conditions.
shear stress increases expression of the arterial endothelial Comparably to embryonic differentiation, the endothelial
marker ephrinB2 in murine ES cells via the VEGF-Notch cell organ specificity is the result of both the gene expres-
signaling pathways [10]. sion specialization and the microenvironmental cytokine
and transcription factor signaling.
Venous specification is determined by COUP-TFII (chick-
en ovalbumin upstream promoter-transcription factor). The breakthrough in research that has long been fruit-
This orphan nuclear receptor inhibits expression of Nrp1 fully dealing with EC coming from umbilical cord (human
and Notch arterial specific genes and thus promotes venous umbilical vein endothelial cells, HUVEC) revealed the elab-
cell fate. You et al. have shown that COUP-TFII, which is orated level of organ specificity displayed by endothelial
specifically expressed in venous endothelial cells, is a genet- cells. While clear morphological and functional differences
ic determinant factor of venous specification by acting up- are visible between EC from the large and small vessels, or-
stream of EphB4 in the mouse [11]. Arterial-venous determi- gan specificity appears also between cells derived from vari-
nation is progressively followed by lymphatic specification ous microvascular endothelial beds.
during embryonic development. Among the venous cells, a
population that progressively acquires the Sox18 and Prox1 Indeed, endothelium exerts quite distinct functions, such as
transcription factors, differentiates into lymphatic endothe- maintaining the blood brain barrier, on the one hand, and in-
lial cells. COUPTFII also contributes to lymphatic gene ex- suring the activity and selectivity of post capillary venules for
pression by interacting with Prox1 [12]. blood/lymph to tissue extravasation, on the other hand. Thus,
Postępy Biochemii 59 (4) 2013 373
organ specificity appears to be a key parameter of endotheli- [39,40] have been reported as examples of zone selective
um biology, endothelial cell reactions and mechanisms of ac- properties of endothelial cells. Gene and microenvironment
tion [15-17]. Organ specific migration of lymphocytes during dependency of EC activities can also explain some patholo-
their homing [18] illustrates the selection that endothelial cells gies, such as propagation of infectious diseases, species-
provide due to the adhesion molecules they express [19]. This specific disease occurrence [41] or genetic disorders related
suggested a need for a clear-cut description of the endothelial pathologies, including the mobilization and export of lym-
signature in terms of cell surface molecules. phocytes in the Down syndrome [42].
Our work has shown the presence of membrane lectins In the adult organisms, as opposed to the embryo, en-
on lymphocytes [20]. The membrane lectins influence cell dothelial cells are responding to activation mainly by up
recognition and adhesion [21-24], participating in the con- regulation of genes that are not related to growth. When
trol of organ specificity in lymphocyte migration. This is de- the growth response occurs, endothelial cells usually react
termined by the selective interaction of lymphocytes with to the pathological state by contributing to neovasculariza-
specialized endothelial cells in high endothelial venules, as tion and/or angiogenesis, which are the main responses to
described first by Stamper and Woodruff [25]. By showing endothelial growth factors such as VEGFs, PDGFs (plate-
that the organ-restricted endothelial cell determinants me- let-derived growth factors) or FGFs (fibroblast growth fac-
diate the antigen-independent organ specificity in lympho- tors), and chemoattractants, such as IL-8 (interleukin-8),
cyte migration, Butcher et al. suggested that defined pairs of CXCL12/SDF-1 (chemokine C-X-C motif ligand-12/stromal
complementary receptors/ligands on the lymphocytes and cell derived factor-1) or CCL21/SLC (chemokine C-C motif
endothelial cells, mediate lymphocyte-high endothelial ven- ligand-21/secondary lymphoid tissue chemokine) [43].
ule adherence in Peyer s patches and in lymph nodes [18].
The combined differential gene expression and microen-
To demonstrate this selectivity by a simple and precise vironmental conditioning of EC, resulting in endothelial cell
method we based our approach on the isolation of murine en- heterogeneity, is also true inside the tumor vasculature [44].
dothelial cells from different organs with regard to their distinct This finding allowed proposing a new approach to treating
biochemical and biological behavior in terms of lymphocyte tumors [45], based on the differences in endothelial cells lo-
adhesion and cell selection [26,27]. This model allowed us to cated in the tumor as compared to normal tissues.
show that subtle post translational modifications of addressins
[29] facilitate efficient selection of distinct lymphocyte popula- HETEROGENEITY OF ENDOTHELIAL
CELLS IN TUMOR ANGIOGENESIS
tions [19,30,31] in the high endothelial venules from peripheral
lymph nodes compared to the Peyer s patches [28]. Denis et
al. [28] demonstrated microenvironmental modulation of the In tumors, the mechanisms of neovascularization also re-
composition of endothelial addressins upon conditioning by flect endothelial heterogeneity. The best known mechanism
properly chosen soluble factors [28]. of tumor angiogenesis is the sprouting of capillaries from
preexisting vessels (Fig. 1), long understood as the only
Some adhesion molecules, such as selectins, are lectins that means of tumor vascularization. The hypoxia regulated
interact with glycanic ligands such as, for example, addressins, VEGF and other proangiogenic factors recruit endothelial
and dictate the first level of interactions that govern the rolling cells from the vicinal vessels depending on the type of tu-
and arrest of leukocytes on the endothelium of a given organ mor and its degree of development [46].
[31] in a highly sophisticated way. This type of intercellular
recognition relies on endothelial cell expressed selectins and During the sprouting process differential expression of
leukocyte expressed ligands, leading to the so called double genes directs endothelial cells mobilization and differentia-
sugar-protein recognition, first described in the case of cross- tion [46]. Comparably to the mechanisms observed during
talk between tumor and endothelial cells [23]. Promoting or embryonic development, in the adult organism the long-
inhibiting leukocyte recruitment requires knowledge on the term quiescent endothelial cells can be activated to adopt an
fine tuning of molecular signal transduction cascades which angiogenic phenotype. Migrating cells guide the proliferat-
underlie cell interactions, with special regard to the role of ing stalk cells that elongate the vessels. This process needs
chemokines and protein glycosylation [32]. This brings a sec- to be strongly coordinated by expression of VEGF respond-
ond level of sophistication to the differential gene expression ing genes in the tip cells and stimulation of the stalk cells by
patterns among the heterogeneous organ-specific EC popula- interaction of Dll4/JAG-1 (Jagged-1)/Notch proteins.
tions (as reviewed by Ribatti et al. [17]). Using gene differential
display [15] and gene expression profiling, it was demonstrat- Endothelial tip, stalk and phalanx cells coordinate the ini-
ed that microvascular endothelial cells differ significantly in tiation of sprouting. Different morphologies and functional
terms of phenotype and gene expression profile [33] accord- properties of tip and stalk endothelial cells reflect their spe-
ing to their organ of origin. These differences are functionally cific features. Endothelial tip cells are migrating cells with
important for cells during their specific chemokine-directed a low proliferation rate, while endothelial stalk cells are ac-
homing [34] and during recruitment of lymphocytes in normal tively proliferating. In 2003, the concept of tip and stalk cell
responses and in pathologies, such as asthma [35]. phenotype was described for emerging sprouts [47]. Thus,
the tip cell is migratory and polarized, whereas the stalk cell
Intra organ specialization further illustrates endothelial proliferates during sprout extension and forms the vascular
heterogeneity. Different specialized functions performed by lumen cell. This clear specialization of endothelial cells is
EC in distinct zones of the lungs [36,37], liver [38] and brain very transient and reversible. It is the effect of the balance be-
374 www.postepybiochemii.pl
ited the recruitment of multiple
cell types from the bone marrow
and their incorporation into the
tumor vascular compartment.
Endothelial cells can actively re-
cruit the bone marrow-derived
cells in an angiocrine manner,
such as Notch ligands secretion
that attracts circulating stem
and progenitor cells.
Moreover, this mechanism
contributes to the recruitment of
monocytes into the tumor site,
giving rise to tumor-associated
macrophages (TAM). Through
the release of angiopoietin-2
the endothelial cells mobilize
Tie-2+ monocytes. These cells
effectively support the tumor
blood vessel formation through
production of proangiogenic
growth factors, and consequent-
Figure 1. Schematic representation of the sprouting angiogenesis compared to the vasculogenic mimicry.
ly enhance the speed of tumor
progression. New research
strategies aiming at reduction or
tween pro-angiogenic factors, such as VEGF and JAG-1, and
inversion of the cell populations that are recruited to the tu-
suppressors of endothelial cell proliferation, as Dll4-Notch
activity [48,49]. While tip cells express Dll-4, PDGF-B, unc- mor by the tumor stroma microenvironment are intensively
investigated. As endothelial precursors appear to be decisive
5 homolog-b (UNC5b), VEGFR-2 and VEGFR-3/Flt-4, they
in supporting tumor growth their interactions with the tumor
poorly express Notch [50-52]. Stalk cells proliferate, make
cells are critical for tumor development [63]. Suppression of
tubes, branches, vascular lumen [53], and junctions [54]. The
the recruitment of circulating EPCs to a tumor may reduce tu-
phalanx type of EC appears transiently also as a response
to VEGF balance [55,56], similarly the tip cell migration re- mor resistance and enhance the process of tissue regeneration
following anticancer therapy [64].
sponds to VEGF gradient while proliferation of stalk cells is
dependent on VEGF concentration [50]. It is the VEGFR-2
VASCULOGENIC MIMICRY
expression on all endothelial cells that dictates the prolifera-
tive and chemotactic responses of endothelial cells to VEGF.
Among the lines of evidence that led to the idea of vasculo-
On the other hand, VEGFR-1 expression is induced by Notch.
genic mimicry, as proposed by Hendrix [65], is the observation
Higher VEGFR-1 expression lowers VEGF availability and,
that tumor cells differentiate into vessel-like structures and
consequently, the tip cell migration. VEGFR-1 is present
participate in endothelial independent angiogenesis (Fig. 1).
mainly in stalk cells and thus controls tip cell activity [57].
Although a number of recent studies have proposed the in-
Hence, the angiogenic mechanisms of sprouting confirm the
volvement of cancer stem cells in this process, the existence
importance of endothelial cell heterogeneity.
and functional importance of VM in growing tumors remains
Despite the fact that for years the sprouting angiogenesis the subject of debate [66,67]. The plasticity of tumor cells is of-
has been admitted as the exclusive mechanism of tumor vas- ten evoked to explain the vasculogenic mimicry. It is hypoth-
cularization [58], it is now commonly accepted that new blood esized that, among tumor cells, the cancer stem cells have a
vessel formation within tumors may result from several others higher propensity to mimic endothelial cells. The phenomenon
mechanisms as well. They were identified as vessel-cooption, of vascular mimicry may occur when tumor cells relocate to
intussusception, recruitment of EPCs [59]. Another mecha- form vascular structures similar to endothelial tubes. During
nism, called vasculogenic mimicry (VM), was considered such adaptation to external signals some malignant tumor cells
as independent of endothelial cells [60]. This diversity in the can become endothelial-like cells. This has been shown for can-
mechanisms of tumor vessel formation was revealed by Rafii cer stem cells differentiating into vascular cells mainly in ma-
et al. [61]. The authors have shown that impairment of bone lignant glioblastoma, where neural cells, the stem-like cancer
marrow-derived endothelial and hematopoietic precursor cells, give rise to endothelial-like cells [68,69]. This has been
cells recruitment led to inhibition of tumor angiogenesis. repeatedly reported in other cancer types such as breast [70],
kidney [71], and ovary [72] tumors, as well as in leukemia [73].
Endothelial precursor recruitment to vasculogenesis (forma-
tion of blood vessels de novo) occurs in adults, when the bone The search for cancer stem cell population, that is able to
marrow-derived EPCs move to an angiogenic site, forming give rise to endothelial-like cells, indicated that this property is
new vessels (Fig. 1) in response to hypoxia and during injury restricted to a certain stem cell set, mainly the CD133+ cells. In
repair [62]. The dual blockade of VEGFR-1 and VEGFR-2 lim- mouse models, ablation of the stem cell subpopulation positive
Postępy Biochemii 59 (4) 2013 375
for the CD133 glycoprotein limits angiogenesis and induces tu- the diverse endothelial activities. This feature has been neglect-
mor regression [74]. As CD133 is dependent on Notch expres- ed for a long time, but now the pathologies linked to EC func-
sion, the switch of the cancer stem-like cells into endothelium- tion are the subject of strong interest.
like cells seems to depend on Notch activity, which moreover
Cancer development is particularly dependent on patho-
governs the VEGF/VEGFR-2 interactions [75]. In aggressive
logical tumor angiogenesis. The formation of new tumor
glioblastoma, lineage plasticity of the putative cancer stem
blood vessels is possible due to endothelial cell specialization.
cells suggests their capacity to generate vasculature, providing
As hypoxia is a feature common to many diseased tissues, the
a clue to the definition of steaminess in relation to neovessel
knowledge of the fine tuning of endothelial cells opens oppor-
formation.
tunities to new treatment strategies which would restore the
It was additionally demonstrated that the stem cell-like non-pathological endothelial features. External conditioning of
CD133+ cell subset contains a subpopulation that expresses the highly plastic endothelial precursor cells orchestrates speciali-
vascular endothelial cadherin (VE-Cad, CD144) and displays zation and adaptation to cooperate with other cell types. This
characteristics of endothelial progenitors capable of maturing conditioning might occur in a reciprocal manner between en-
into endothelial cells. Lineage analyses and cloning confirmed dothelial precursors and cancer stem cells in a hypoxic milieu.
that a subpopulation of the CD133+ stem-like cell fraction is Such interconnections might play a role in the tumor cell medi-
multipotent and capable of differentiating along tumor and ated angiogenesis-like process, which is commonly designated
endothelial lineages, possibly via the intermediate CD133+/ as endothelial independent vasculogenic mimicry, with cancer
CD144+ progenitors. stem cells as a main player. In the future, successful treatment
of aggressive cancers could involve the targeting of both vas-
The hypoxia mediated regulatory mechanism determining culogenic mimicry and angiogenesis.
cell phenotype appears to be a common denominator. Indeed,
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Heterogenność komórek śródbłonka  rola w specjalizacji
funkcjonalnej naczyń i mimikrze naczyniowej
Claudine Kieda*
Centre de Biophysique Moléculaire, UPR CNRS 4301, Orleans CEDEX 2, France
*
Cell recognition and Glycobiology, B2CT Department, Centre de Biophysique Moléculaire, UPR CNRS 4301, Rue Charles Sadron, 45071
Orleans CEDEX 2, France; tel.: [phone], fax : [phone], e-mail: [e-mail]
Słowa kluczowe: angiogeneza, śródbłonek, hipoksja, komórki progenitorowe śródbłonka, komórki macierzyste nowotworów, mimikra
naczyniowa.
STRESZCZENIE
Komórki śródbłonka różnią się w zależności od stopnia dojrzałości i tkanki z której pochodzą naczynia. Heterogenność komórek śródbłonkowych
pozwala lepiej zrozumieć różnorodność ich funkcji w poszczególnych narządach, może również ułatwić projektowanie terapii celowanych. Hete-
rogenność jest kluczową cechą w powstawaniu unaczynienia w rozwoju zarodkowym oraz w regulacji interakcji komórek śródbłonka i komórek
układu odpornościowego w organizmie dorosłym; wpływa także na przebieg wielu chorób, w tym przerzutowania nowotworów i międzygatunko-
we rozprzestrzenianie się niektórych chorób zakaz
nych. Komórki śródbłonka silnie reagują na sygnały z mikrośrodowiska, zarówno prawidłowego
jak i zmienionego w wyniku choroby. Odpowiadają też na tkankową dostępność tlenu (ang. physoxia), która jest jednym z czynników determinu-
jących heterogenność śródbłonka. Jeśli dostępność tlenu obniży się, komórki śródbłonkowe ulegają aktywacji i indukują angiogenezę, prowadzącą
do przywrócenia równowagi. Na hipoksję odpowiadają również szpikowe komórki prekursorowe śródbłonka oraz komórki macierzyste nowo-
tworów, obejmujące najbardziej agresywne, oporne na terapię i odróżnicowane komórki nowotworowe. Komórki te współdziałają z komórkami
śródbłonka i ich prekursorami, uczestnicząc w procesie mimikry naczyniowej, a tym samym wpływają na tworzenie odległych przerzutów.
378 www.postepybiochemii.pl