Editorial
Hard Luck Stories: The Reality of Endothelial Progenitor
Cells Continues to Fall Short of the Promise
Arjun Deb, MD; Cam Patterson, MD, MBA
T
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nature of endothelial progenitor cells against atherosclerotic
lesions was illustrated in an elegant study in which recurrent
treatment with bone marrow– derived progenitors from young
nonatherosclerotic ApoE⫺/⫺ mice retarded progression of
atherosclerosis in older ApoE⫺/⫺ mice, despite persistent
hypercholesterolemia. The authors demonstrated that bone
marrow progenitors localized to atherosclerosis-prone regions of the vessel and concluded that bone marrow– derived
progenitors helped decrease atherosclerotic burden by replacing senescent endothelial cells in plaques and by decreasing
proinflammatory and proatherogenic cytokines.6 However,
the initial enthusiasm associated with these studies has been
tempered by other studies demonstrating the acceleration of
atherosclerosis with transplantation of bone marrow– derived
mononuclear cells.7
In this issue of Circulation, Hagensen et al8 challenge the
existing paradigm that bone marrow– derived progenitors
contribute to the endothelium of plaques after arterial injury.
The authors designed an elegant experimental approach
wherein they determined the contribution of bone marrow
cells to plaque endothelium or endothelial regeneration after
mechanical plaque disruption. In a parallel series of experiments, the authors also investigated the contribution of
circulating cells to endothelium in unruptured plaques or after
plaque disruption. From the results of their experiments, the
authors conclude that bone marrow– derived cells rarely
contribute to the endothelium of developing plaques or
participate in endothelial regeneration after plaque rupture.
Second, using orthotopic arterial transplantation, the authors
convincingly demonstrate that circulating cells rarely contribute to plaque endothelium or regeneration of overlying
endothelium after plaque disruption. The experimental results
strongly suggest that the local vessel is the major source of
plaque endothelium and predominantly contributes to regeneration of overlying endothelium after plaque rupture.
So how do we interpret and reconcile this elegant study
with the smorgasbord of studies previously published that
lead us to very different conclusions?2 If bone marrow and
endothelial progenitors do not contribute to the endothelium
of plaques— either during plaque formation or after plaque
rupture, as shown by Hagensen et al—then why is there a
correlation between endothelial progenitor number, function,
and vascular disease? If endothelial progenitors do not
contribute to plaque endothelium, is there a strong scientific
rationale to use them as therapeutic tools to improve vascular
flow and retard atherosclerotic disease? Can we account for
the major discrepancies in experimental results concerning
the role and function of endothelial progenitors in vascular
disease by technical reasons alone? Do seemingly minor
differences in isolation of endothelial progenitor and culture
he discovery of putative endothelial progenitor cells by
Asahara and colleagues1 engendered a new era of vascular biology, full of the promise of using circulating and
bone marrow– derived progenitors for therapeutic angiogenesis. In this landmark article, Asahara et al demonstrated that
a CD34 or Flk-1 positive fraction of mononuclear cells
isolated from human peripheral blood formed endothelial
tube–like structures in vitro and incorporated into capillaries
in regions of ischemic injury. Thus, a new paradigm of
“vasculogenesis in the adult” was born. Since this observation, more than a decade has gone by, with more than 8000
articles published on endothelial progenitors.2 Despite this
extraordinary productivity, therapeutic vasculogenesis with
endothelial progenitor cells continues to be in its infancy—a
hard luck story, dogged by conflicting studies, lack of
uniform criteria for defining an endothelial progenitor, and
nonrigorous methods of determining progenitor-mediated
vasculogenesis.
Article see p 898
Notwithstanding the fundamental problems plaguing this
field, numerous studies during the past few years have
suggested that vascular progenitors may be involved in
maintaining vascular homeostasis and repair. The number of
circulating endothelial progenitors is inversely related to
cardiovascular risk, and patients with healthy vessels have a
greater number of circulating endothelial progenitors.3 Coronary endothelial cells of extracardiac origin were observed in
patients who had undergone gender-mismatched cardiac
transplantation, and a similar robust degree of endothelial
chimerism was seen in coronary vessels of patients with
transplant arteriopathy.4,5 Interestingly, the degree of endothelial cells of extracardiac origin was almost log10-fold
higher in diseased segments of arteriopathic vessels than in
nondiseased segments.4 These studies thus strengthened the
notion that bone marrow and circulating progenitors were
participants in atherosclerosis and arterial injury, although the
precise functional role was unclear. The vasculoprotective
The opinions expressed in this article are not necessarily those of the
editors or of the American Heart Association.
From the Departments of Medicine (A.D., C.P.), Pharmacology (C.P.),
Cell and Developmental Biology (C.P.), and Cell and Molecular Physiology (A.D.), McAllister Heart Institute, University of North Carolina,
Chapel Hill, NC.
Correspondence to Cam Patterson, MD, MBA, McAllister Heart
Institute, University of North Carolina at Chapel Hill, 8200 Medical
Biomolecular Research Bldg, Chapel Hill, NC 27599-7126. E-mail
[email protected]
(Circulation. 2010;121:850-852.)
© 2010 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIR.0b013e3181d4c360
850
Deb and Patterson
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methods used, genetic markers used to identify and trace their
fate in tissues, technical methods of fixation, treatment of
tissues, and microscopic analysis explain such widely different experimental findings?
It is likely that no single factor can be a major underpinning
of such different experimental conclusions reached by so
many different groups during the past decade. Rather, it is
probable that a combination of differences in methodology,
differences in biological properties of the cells being used,
and differences in animal models underlie disparate observations that continue to plague a promising field. One of the
biggest biological hurdles in this field of research has been to
effectively compare, across multiple studies, the cell fraction
being called “endothelial progenitors.”9 Many cell types have
been labeled as endothelial progenitors; to complicate matters
even further, endothelial progenitors are thought to exist in
the local vessel wall as well. The term endothelial progenitor
has been defined in so many different ways2 that the reader
can be confused about the “progenitor properties” of an
endothelial progenitor. Culturing bone marrow or circulating
mononuclear cells to yield an endothelial-like “population”
expressing characteristic endothelial surface markers and
tube-forming capacity does not necessarily reflect isolation of
progenitor cells but likely points to a heterogeneous collection of endothelial-like cells that may behave differently
every time cell culture conditions are slightly modified. Thus,
we have early endothelial progenitors and late endothelial
progenitors, composed respectively of cells that are isolated
within a few days (early) to a few weeks (late) of culture of
circulating mononuclear cells. Early and late endothelial
progenitors exhibit great differences in endothelial properties,
including ability to form tubes, incorporation into vasculature
in vivo, and secretion of growth factors. It is thus evident that
experiments conducted with these 2 biologically different cell
types can lead to completely different outcomes and conclusions. We thus propose that until a combination of cell
surface markers is discovered that unequivocally defines an
endothelial progenitor, we as a scientific community strictly
describe our experimental results with the precise phenotype
of cells used and avoid the term endothelial progenitor. This
will perhaps enable comparison of clearly phenotyped cells
(eg, CD34, CD133) and determine functional benefits observed with different cell types.
The article by Hagensen et al also points to the inherent
biological differences of following the fate of endothelial
progenitor cells. eGFP expression was observed in only 60%
of vWF⫹ endothelial cells in eGFP⫹ ApoE⫺/⫺ mice. It is
thus clear that silencing of a transgenic marker in a random
(or, worse, in a cell-specific marker) may dramatically alter
our conclusions about the fate of endothelial progenitor cells
and make multiple corroborative methods essential for validating experimental results. In this regard, the present study
by Hagensen et al used chimeric analysis with the Y chromosome and arrived at a similar conclusion. It is essential that
in the absence of a clear marker defining an endothelial
progenitor, studies should use multiple genetic markers
driven by specific candidate promoters to better delineate the
fate of endothelial progenitors. The tie2 promoter has been
used extensively to follow the fate of endothelial progenitors,
Table.
Endothelial Progenitor Cells
851
Recommendations About Future EPC Studies
Describe clearly and precisely the phenotype of bone marrow cells used,
particularly in studies determining functional effects in vivo. Avoid the term
EPC. Instead, describe the precise phenotype of the cell used.
Recognize and understand that many genetic markers or dyes used for
labeling and tracking EPC fates are often not entirely specific, and that there
is a considerable overlap with cells of the monocyte/macrophage lineage.
Ascribe high priority to studies that take the effort to explain discrepant
findings. Granted, these studies may not be novel or groundbreaking but
they will have a huge impact in moving the field in the right direction.
Assign greater emphasis into the mechanism of EPC action, recognizing that
this may be different for different cell types considered. This is also critical
for fine-tuning EPC therapy. We recommend that even clinical studies of
EPC therapy use sophisticated imaging tools to better determine anatomical/
structural changes in addition to functional changes in affected tissue.
EPC indicates endothelial progenitor cells.
but tie2 is not a specific marker and is expressed by different
nonendothelial cell types, including a monocytic/macrophage
cell fraction. Studies using tie2 to identify bone marrowderived endothelial cells can lead to confounding results,
particularly after injury, inasmuch as many inflammatory
cells at the site of injury may express tie2 without being
committed to the endothelial pathway.10 Similarly, tracking
of cells with endothelial-specific dyes should be interpreted
with caution because many nonendothelial cell types, particularly those of the monocytic-macrophage lineage, also take
up endothelial-specific dyes and can lead to erroneous interpretation of the fate of endothelial progenitors.
Although the article by Hagensen et al questions the role of
bone marrow and circulating cells toward the contribution
and regeneration of the endothelium in plaques, the experimental conclusions do not necessarily imply that endothelial
progenitors or bone marrow– derived cells may not be useful
for retarding atherosclerotic vascular disease. We must be
open to alternative biological explanations such as paracrine
effects rather than regeneration; injected bone marrow cells
may alter the angiogenic milieu in the vessel wall and plaque,
and this could lead to salutary effects on plaque size and
atherosclerotic burden. Hagensen et al suggest that regeneration of plaque endothelium is likely from local endothelial
cells or other progenitor cells residing within the local vessel
wall. It is conceivable that injected endothelial progenitors
could augment this reparative process without directly regenerating new endothelium. Such a salutary paracrine effect has
been observed in myocardial infarction, wherein bone marrow– derived C-kit cells alter the local cardiac milieu to
augment cardiac repair.11 Similarly, bone marrow– derived
mesenchymal cells are known to exert cardioprotective effects through paracrine mechanisms.12
In summary, the elegant experimental approach by Hagensen et al reminds us of the inherent controversies with
which the endothelial progenitor biology has been fraught. It
is time that we pay attention to the precise type of cell
used; the animal model considered; the fallacy of genetic
markers; and alternative biological explanations, methods
of analysis, and measurement of outcomes before we draw
definitive conclusions. It is only by pursuing a rigorous,
uncompromising approach, and adopting uniform criteria
852
Circulation
February 23, 2010
for many of the above variables, that we as a scientific
community can make significant inroads in this stillnascent field brimming with promise but dogged by a
decade of hard luck (Table).
Disclosures
None.
References
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8. Hagensen MK, Shim J, Thim T, Falk E, Bentzon JF. Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine
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KEY WORDS: Editorials
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angiogenesis
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atherosclerosis
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endothelium
Hard Luck Stories: The Reality of Endothelial Progenitor Cells Continues to Fall Short of
the Promise
Arjun Deb and Cam Patterson
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation. 2010;121:850-852; originally published online February 8, 2010;
doi: 10.1161/CIR.0b013e3181d4c360
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
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