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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 952-958
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology-Oncology, Weill Medical College of
Cornell University, New York, NY; Division of Molecular, Cell Biology
and Immunology, ImClone Systems, New York, NY; Department of
Cardiothoracic Surgery, Columbia-Presbyterian Medical Center, New York,
NY; and Division of Developmental Hematopoiesis, Sloan Kettering Cancer
Center, New York, NY.
Emerging data suggest that a subset of circulating human
CD34+ cells have phenotypic features of endothelial
cells. Whether these cells are sloughed mature endothelial cells or
functional circulating endothelial precursors (CEPs) is not known.
Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that
1.2 ± 0.3% of CD34+ cells isolated from fetal liver
(FL), 2 ± 0.5% from mobilized peripheral blood, and 1.4 ± 0.5%
from cord blood were VEGFR-2+. In addition, most
CD34+VEGFR-2+ cells express hematopoietic
stem cell marker AC133. Because mature endothelial cells do not express
AC133, coexpression of VEGFR-2 and AC133 on CD34+ cells
phenotypically identifies a unique population of CEPs. CD34+VEGFR-2+ cells express
endothelial-specific markers, including VE-cadherin and E-selectin.
Also, virtually all CD34+VEGFR-2+ cells
express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34+ cells that give rise to endothelial
colonies, CD34+ cells derived from FL were incubated with
VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and
plating of nonadherent FL-derived VEGFR-2+ cells with
VEGF and FGF-2 resulted in differentiation of AC133+
VEGFR-2+ cells into adherent
AC133
Wound healing and tumor growth require active
endothelial proliferation, a process referred to as neo-angiogenesis.
Neo-angiogenesis involves the recruitment of endothelial cells to the
site of injury or to the tumor vascular bed. Two possible sources of
endothelialization are (1) endothelial migration and sprouting from
preexisting endothelial cells or (2) recruitment of endothelial
precursor cells from the circulation. There is ample evidence for the
first scenario. However, the existence of circulating endothelial
precursor (CEP) cells in adult humans has only recently been suggested
and is under intensive scrutiny.
Many studies have demonstrated the presence of mature circulating
endothelial cells in the peripheral circulation.1-6 Mature endothelial cells may appear in the circulation randomly by
shedding from the vascular wall. Trauma induced by surgery or increased intravascular turbulence may also result in the introduction of endothelial cells to the peripheral circulation. Patients with sickle cell crisis have been shown to have increased numbers of activated circulating endothelial cells.5 Although
circulating endothelial cells have been suspected to have the capacity
to colonize vascular grafts,1,4,6,7 the contribution of these cells to postnatal angiogenesis or vasculogenesis is not known.
Endothelial precursor cells have properties similar to those of
embryonic angioblasts, which can be defined as migratory endothelial cells with the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but which have not yet acquired characteristic mature endothelial markers and have not yet formed a
lumen.8-10 Although there is plethora of evidence for the
existence of angioblasts during embryonic development, the existence of angioblast-like endothelial precursor cells in adult circulation has
been hampered by the absence of specific phenotypic markers and
functional assays to define this unique cell population.
Both endothelial precursor cells and mature endothelial cells may
express similar endothelial-specific markers, including vascular
endothelial growth factor receptor-2 (VEGFR-2),11
Tie-1,12,13 Tie-2,14 and vascular endothelial
(VE)-cadherin.15,16 Therefore, it may be impractical to use
these markers to differentiate between the 2 populations.
Identification of the differences between endothelial precursor cells
and circulating mature endothelial cells is further complicated by the
fact that hematopoietic stem and progenitor cells express markers
similar to those of endothelial cells, such as VEGFR-1 (Flt-1), CD34,
platelet endothelial cell adhesion molecule (PECAM), Tie-1, Tie-2, and
von Willebrand's factor (vWF), and they also have the capacity to
incorporate acetylated low-density lipoprotein
(Ac-LDL).
One study has shown that CD34+VEGFR-2+ cells
can be detected in the peripheral circulation.17 However,
in this report a polyclonal antibody to the intracellular domain of
VEGFR-2 was used to identify viable
Ac-LDL+CD34+ endothelial progenitor cells. This
may have resulted in the co-isolation and transplantation of
contaminating hematopoietic cells as well as endothelial cells. We have
shown that allogeneic sex-mismatched bone marrow transplantation
results in the transfer of endothelial cells to recipient
dogs.2 Replacement of the aorta of the recipient dogs
months after transplantation with impervious Dacron grafts resulted in graft endothelialization arising exclusively from the
transplanted bone marrow. In humans, similar evidence for endothelial
precursor cells originates from patients implanted with a left
ventricular assist device (LVAD). We demonstrated colonization of the
flow surface of the titanium housing of LVADs with CD34+
endothelial-like cells 6 months after the devices were
removed.1 However, none of these studies has conclusively
demonstrated the existence of a phenotypically and functionally
distinct population of CEPs.
Endothelial precursor cells may share similar characteristics with
hematopoietic stem cells. CD34+ hematopoietic stem cells
can be detected at low numbers in bone marrow, fetal liver, and
umbilical cord blood. Although the number of circulating hematopoietic
stem cells in the peripheral circulation is very low, cytokines such as
granulocyte colony-stimulating factor (G-CSF) promote mobilization of
stem cells from bone marrow to the peripheral blood (PB). In addition,
hematopoietic stem and progenitor cells express unique surface markers,
such as CD34 and the newly discovered early hematopoietic stem cell
marker AC133. Expression of CD34 and AC133 diminish with maturation and differentiation.18,19
AC133 is a novel 120-kd glycosylated polypeptide that contains
5-transmembrane domains with an extracellular N-terminus and a
cytoplasmic C-residue.18,19 The function of AC133, which does not share homology with any previously described hematopoietic cell surface antigen, is not known. However, isolation of a
subpopulation of CD34+ cells using
monoclonal antibody (MoAb) to human AC133 has resulted in the
identification of functional CD34+ population of
hematopoietic stem cells. Human-derived AC133+ cells can
repopulate sheep bone marrow19 and, therefore, can be
considered pluripotent hematopoietic stem cells. Expression of AC133 is
rapidly downregulated as hematopoietic progenitors and stem cells
differentiate into more mature postmitotic cells. In fact, virtually
all mature hematopoietic cells, including mature myeloid,
megakaryocytes, erythroid, and lymphoid cells and terminally differentiated hematopoietic cells, fail to express
AC133.18,19 Therefore, subsets of CD34+ cells
that express AC133 are truly a phenotypic and functional marker of an
immature population of hematopoietic stem and progenitor cells.
In search of unique endothelial markers to facilitate the isolation and
characterization of endothelial precursor cells, we have found that
AC133 is expressed on subset of CEPs but not on mature differentiated
endothelial cells. To examine the possibility that the
endothelial-specific marker VEGFR-2 may be expressed on subsets of
circulating AC133+ cells, we have used a combination of
high affinity to the extracellular domain of VEGFR-2 to identify CEPs.
In this paper, we demonstrate that a small subset of CD34+
cells derived from different hematopoietic sources express AC133 and
VEGFR-2. In addition, these cells express endothelial-specific antigens, including E-selectin and VE-cadherin. Almost all circulating VEGFR-2+ cells express the chemokine receptor CXCR-4 and
migrate in response to the CXCR-4 ligand, stromal-derived factor
(SDF-1), as well as VEGF. Incubation of nonadherent CD34+
cells coexpressing AC133 and VEGFR-2 cells with VEGF, fibroblast growth
factor (FGF)-2, and collagen results in their proliferation and
differentiation into adherent AC133 Flow cytometry studies
Migration studies
Endothelial colony assay Freshly isolated CD34+ cells were analyzed for the presence of AC133+VEGFR-2+ cells. Subsequently, 106 CD34+ cells were incubated in EC media containing medium 199 (M199; GIBCO-BRL, Gaithersburg, MD) supplemented with 20% fetal bovine serum (HY CLONE, UT), FGF-2 5 ng/mL (human recombinant fibroblast growth factor-basic; SIGMA, St. Louis, MO), and heparin 5 units/mL. After 3 days, nonadherent cells were transferred to collagen-coated (1 µg/mL) plastic dishes and grown in the same media for 2 weeks. This incubation resulted in attachment and proliferation of CD34+ cells expressing VEGFR-2 into monolayers of mature endothelial cells. Cells were stained for endothelial-specific antigens, including vWF, VE-cadherin and, after activation with interleukin (IL)-1 (10 units/mL), for
E-selectin expression. AC133 staining of the newly formed endothelium
was done using the protocol as described above.
Identification of VEGFR-2+ cells from LVAD neo-intima Mononuclear cells from LVAD neo-intimal surfaces were obtained as previously described.1 Briefly, after explantation of LVADs, the neo-intima covering the textured surface was removed, washed, and digested with 0.1% collagenase. Mononuclear cells were recovered and washed, and the number of CD34+VEGFR-2+ cells and AC133+VEGFR-2+ cells was determined by dual-color flow cytometry. A total of 6 LVADs were processed and extracted cells analyzed for the presence of AC133+ VEGFR+ cells. Depending on the clinical circumstances, the LVADs were explanted at different times, ranging from 28 days to 6 months.Statistical analysis Data are expressed as mean ± SEM of at least 3 independent experiments. To detect differences between migrating and nonmigrating cells, the t test for paired samples was applied. A P value of <.05 was considered statistically significant.
MoAbs to the VEGFR-2 identify a small subpopulation of circulating CD34+ endothelial cells To quantify the number of CD34+VEGFR-2+ cells within different hematopoietic sites, CD34+ cells isolated from cytokine-mobilized PB, CB, and FL were analyzed for the presence of CD34+ VEGFR-2+ cells by 2-color flow cytometry. Using FITC-labeled MoAbs to the extracellular domain of VEGFR-2, we found that 2.0 ± 0.5% (n = 5) of CD34+ cells derived from G-CSF mobilized PB, and 1.4 ± 0.5% (n = 3) of CB-derived CD34+ cells, and 1.2 ± 0.3% of CD34+ isolated from FL (n = 3) were VEGFR-2+ (Figure 1). Similar analysis of the frequency of CD34+VEGFR-2+ cells in nonmobilized PB showed a very small number of these cells (data not shown). These results demonstrate the presence of small but distinctive population of VEGFR-2+ cells within CD34+ hematopoietic cells.
AC133 is expressed on CEPs but not on mature endothelial cells. VEGFR-2 is expected to be expressed on both mature circulating endothelial cells and CEPs. Therefore, the CD34+VEGFR-2+ cells identified in Figure 1 may represent a heterogeneous population of mature and immature endothelial cells. In this respect, we have searched for a specific marker that is expressed on CEP but not on mature endothelial cells. We have found that the most CD34+VEGFR-2+ cells express the newly discovered hematopoietic stem cell marker AC133, which is also present on immature hematopoietic cells but is absent on mature endothelial or differentiated hematopoietic cells.
Phenotypic characteristics of putative circulating CEPs.
CD34+ cells isolated from various hematopoietic
microenvironments were analyzed for the presence of other hematopoietic
and endothelial markers. Using dual-color flow cytometry, we showed that human CD34+VEGFR-2+ cells express
endothelial-specific markers, including E-selectin and VE-cadherin
(Figure 3C). In addition, they also
coexpressed common hematopoietic and endothelial markers, including
C-kit (Figure 3A), PECAM, and CD13.
CD34+VEGFR-2+ cells also expressed CXCR-4, the
chemokine receptor for SDF-1 (Figure 3B). Among all of these markers,
AC133 was the only specific marker that was expressed on
CD34+ VEGFR-2+ cells but was absent on both
resting and activated mature HUVECs. VEGFR-2+ cells did not
express myelomonocytic markers, including CD15 and CD14.
SDF-1 and VEGF induce migration of
CD34+VEGFR-2+
cells.
Although mature endothelial cells grow in an anchorage-dependent
manner, their capacity to respond to chemotactic factors can be
assessed using a Boyden chamber transmigration assay. In these studies,
adherent populations of mature endothelial cells are removed by
collagenase digestion, and their capacity for migration is examined in
collagen-coated Boyden chambers.
LVAD neo-intimal surfaces are colonized with
AC133+VEGFR-2+
cells.
We have previously shown that surfaces of LVADs in contact with
circulating blood are colonized with CD34+ cells with high
proliferative capacity, giving rise to a biologically nonthrombogenic
neo-intima. Close scrutiny of the LVAD surfaces shows that
4 ± 1% of the mononuclear cells express AC133+ and
VEGFR-2+ cells, suggesting that circulating CEPs have the
capacity to colonize neo-intimal surfaces (Figure 6). The presence of
AC133+VEGFR-2+ cells detected on LVADs was more
prominent in formed neo-intima explanted early after operation (28 days).
Isolation, identification, and characterization of CEP cells have
been hampered by the absence of (1) specific endothelial markers to
differentiate between CEPs and contaminating sloughed endothelial cells
and hematopoietic progenitor/stem cells and (2) functional assays to
differentiate between mature endothelial cells and CEPs. In this
report, we have found that AC133, an early hematopoietic stem cell
marker, is expressed on a large subset of CEPs but not on the mature
endothelium. The percentage of CD34+ cells expressing
AC133+ and VEFGR2+ cells is only 2% of
circulating CD34+ cells. This figure is much less than the
percentage of CD34+ VEGFR-2+ cells previously
reported by other groups.17 Using a polyclonal antibody to
the intracellular domain of VEGFR-2, another group showed that 27% of
CD34+ cells were also VEGFR-2+. However, we
have used a specific MoAb to the extracellular domain of VEGFR-2,
allowing a more accurate estimation of the circulating CD34+VEGFR-2+ cells. In addition, because AC133
is expressed on putative CEPs, the remaining circulating
AC133 Submitted May 14, 1999; accepted October 1, 1999.
S.R. supported by the American Heart Association Grant-In-Aid; NHLBI
grants R01-HL-58707 and R01-HL-61849; the Dorothy Rodbell Foundation
for Sarcoma Research; and the Rich Foundation. M.P. supported by a
grant from the National Childhood Cancer Foundation. M.A.S.M. supported
by NHLBI grant R01-HL-61401 and the GAR Reichman Fund of the Cancer
Center support grant CA-08748.
Reprints: Shahin Rafii, Weill Medical College of Cornell
University, Hematology-Oncology Division, 1300 York Ave, Room C-606,
New York, NY 10021; e-mail:srafii{at}mail.med.cornell.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
VEGFR-2+Ac-LDL+
(acetylated low-density lipoprotein) colonies (plating
efficiency of 3%). In an in vivo human model, we have found that the
neo-intima formed on the surface of left ventricular assist devices
is colonized with AC133+VEGFR-2+ cells.
These data suggest that circulating CD34+ cells
expressing VEGFR-2 and AC133 constitute a phenotypically and
functionally distinct population of circulating endothelial cells that
may play a role in neo-angiogenesis.
(Blood. 2000;95:952-958)
