Cellular plasticity: the good, the bad, and the ugly? Microenvironmental influences on progenitor cell therapy.
ABSTRACT Progenitor cell based therapies have emerged for the treatment of ischemic cardiovascular diseases where there is insufficient endogenous repair. However, clinical success has been limited, which challenges the original premise that transplanted progenitor cells would orchestrate repair. In this review, we discuss the basics of endothelial progenitor cell therapy and describe how microenvironmental changes (i.e., trophic and mechano-structural factors) in the damaged myocardium influence progenitor cell plasticity and hamper beneficial therapeutic outcome. Further understanding of these microenvironmental clues will enable optimization of cell therapy at all levels. We discuss current concepts and provide future perspectives for the enhancement of progenitor cell therapy, and merge these advances into a combined approach for ischemic tissue repair.
- SourceAvailable from: Guido Krenning[Show abstract] [Hide abstract]
ABSTRACT: Experimental clinical stem cell therapy has been used for more than a decade to alleviate the adverse aftermath of acute myocardial infarction (aMI). The post-infarcted myocardial microenvironment is characterized by cardiomyocyte death, caused by ischemia and inflammation. These conditions may negatively affect administered stem cells. As postnatal cardiomyocytes have a poor proliferation rate, while induction of proliferation seems even more rare. Thus stimulation of their proliferation rate is essential after aMI. In metaplastic disease, the pro-inflammatory cytokine interleukin-6 (IL-6) has been identified as potent mediators of the proliferation rate. We hypothesized that IL-6 could augment the proliferation rate of (slow-)dividing cardiomyocytes. To mimic the behavior of therapeutic cells in the post-infarct cardiac microenvironment, human Adipose Derived Stromal Cells (ADSC) were cultured under hypoxic (2% O2) and pro-inflammatory conditions (IL-1β) for 24h. Serum-free conditioned medium from ADSC primed with hypoxia and/or IL-1β was added to rat neonatal cardiomyocytes and adult cardiomyocytes (HL-1) to assess paracrine-driven changes in cardiomyocyte proliferation rate and induction of myogenic signaling pathways. We demonstrate that ADSC enhance the proliferation rate of rat neonatal cardiomyocytes and adult HL-1 cardiomyocytes in a paracrine fashion. ADSC under hypoxia and inflammation in vitro had increased the interleukin-6 (IL-6) gene and protein expression. Similar to conditioned medium of ADSC, treatment of rat neonatal cardiomyocytes and HL-1 with recombinant IL-6 alone also stimulated their proliferation rate. This was corroborated by a strong decrease of cardiomyocyte proliferation after addition of IL-6 neutralizing antibody to conditioned medium of ADSC. The stimulatory effect of ADSC conditioned media or IL-6 was accomplished through activation of both Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) and Mitogen-Activated Protein (MAP) kinases (MAPK) mitogenic signaling pathways. ADSC are promising therapeutic cells for cardiac stem cell therapy. The inflammatory and hypoxic host post-MI microenvironment enhances the regenerative potential of ADSC to promote the proliferation rate of cardiomyocytes. This was achieved in paracrine manner, which warrants the development of ADSC conditioned medium as an "of-the-shelf" product for treatment of post-myocardial infarction complications.Journal of Translational Medicine 01/2013; 11:39. · 3.46 Impact Factor
REVIEW / SYNTHÈSE
Cellular plasticity: the good, the bad, and the ugly?
Microenvironmental influences on progenitor cell
Jan-Renier A.J. Moonen, Martin C. Harmsen, and Guido Krenning
Abstract: Progenitor-cell-based therapies have emerged for the treatment of ischemic cardiovascular diseases where there is
insufficient endogenous repair. However, clinical success has been limited, which challenges the original premise that trans-
planted progenitor cells would orchestrate repair. In this review, we discuss the basics of endothelial progenitor cell therapy
and describe how microenvironmental changes (i.e., trophic and mechano-structural factors) in the damaged myocardium in-
fluence progenitor-cell plasticity and hamper beneficial therapeutic outcome. Further understanding of these microenviron-
mental clues will enable optimization of cell therapy at all levels. We discuss current concepts and provide future
perspectives for the enhancement of progenitor-cell therapy, and merge these advances into a combined approach for ische-
mic tissue repair.
Key words: cardiovascular disease, plasticity, cell therapy, endothelial progenitor cell, microenvironment, inflammation, en-
dothelial–mesenchymal transition, mechanotransduction, fluid shear stress, cyclic strain.
Résumé : Les nouvelles thérapies à base de cellules progénitrices permettent de traiter les maladies cardiovasculaires isché-
miques présentant une réparation endogène insuffisante. Toutefois, les réussites cliniques sont limitées, ce qui remet en
cause la prémisse originale selon laquelle les cellules progénitrices transplantées orchestreraient la réparation. Dans la pré-
sente synthèse, nous discutons des fondements de la thérapie à base de cellules progénitrices endothéliales et nous décrivons
comment les modifications microenvironnementales (c.-à-d. facteurs trophiques et mécano-structuraux) dans le myocarde
lésé influent sur la plasticité des cellules progénitrices et retardent les résultats bénéfiques des traitements. Une meilleure
compréhension de ces pistes microenvironnementales permettra d’optimiser la thérapie cellulaire à tous les niveaux. Nous
discutons des concepts courants et présentons les nouvelles perspectives concernant la stimulation de la thérapie cellulaire
progénitrice; nous fusionnons ces éléments dans une approche combinée pour la réparation du tissu ischémique.
Mots‐clés : maladie cardiovasculaire, plasticité, thérapie cellulaire, cellule progénitrice endothéliale, microenvironnement, in-
flammation, transition endothélium–mésenchyme, mécanotransduction, contrainte de cisaillement, déformation cyclique.
[Traduit par la Rédaction]
In its barest form, the cardiovascular system consists of a
pump (the heart) and transport conduits (the vascular tree).
The branched composition of the vascular tree is character-
ized by distinct flow profiles and blood pressures, thus de-
manding high degrees of cellular heterogeneity along the
vessels (Aird 2007). Also within the heart, heterogeneity of
cardiomyocytes is instrumental to the function of both atria
and ventricles. Therefore, the tissues that comprise the cardi-
ovascular system have a high degree of built-in phenotypic
In the cardiovascular system, cells are continuously ex-
posed to microenvironmental stimuli (Di Nardo et al. 2010).
These stimuli firstly consist of trophic factors such as hor-
mones, steroids, cytokines, chemokines, and growth factors.
Secondly, physico-chemical factors such as electrical charge,
oxygen tension, surface energy, temperature, and pH. Thirdly
and least investigated, cells experience mechano-structural
factors that include geometry, force, pressure, strain, topol-
ogy, and velocity. These factors are all spatiotemporally regu-
Received 9 August 2011. Accepted 6 October 2011. Published at www.nrcresearchpress.com/cjpp on .
J.-R.A.J. Moonen, M.C. Harmsen, and G. Krenning. Cardiovascular Regenerative Medicine Research Group (CAVAREM), Dept.
Pathology & Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1 (EA11), 9713GZ Groningen,
Corresponding author: Guido Krenning (e-mail: firstname.lastname@example.org) and Martin C. Harmsen (e-mail: email@example.com).
This review is one of a series of papers published in the Special Issue on Stem Cells in Regenerative Medicine.
Can. J. Physiol. Pharmacol. 90: 1–11 (2012)doi:10.1139/Y11-107
Published by NRC Research Press
lated. The plethora of different signals demands a similarly
diverse set of receptors that receive and transduce this infor-
mation to the nucleus, where it is integrated into a (patho)
During disease, pathophysiological conditions challenge
the homeostasis of the cardiovascular system. Mechanical
stimuli such as high blood pressure, metabolic challenges
such as hyperlipidaemia and hypercholesteraemia, and vascu-
lar inflammation are at the basis of cardiovascular patholo-
gies like atherosclerosis, ischemic heart disease, and cardiac
fibrosis. These pathologies are interconnected, e.g., the rup-
ture of an atherosclerotic plaque in a coronary artery may
cause acute myocardial infarction (aMI), which can result in
cardiac fibrosis and subsequent heart failure. Hence, the prin-
ciples and mechanisms that underlie cardiovascular patholo-
gies are often variations on a similar theme.
In this concise review we will discuss the basics of bone-
marrow-derived endothelial progenitor cell therapy for the
treatment of cardiovascular disease. Furthermore, we describe
how changes in the cardiovascular microenvironment influ-
ence the plasticity of progenitor cells, and influence the ther-
apeutic outcome of progenitor-cell therapy.
Endothelial progenitor cell therapy for
Inflammation and the post-infarct healing response
Obstructions of coronary arteries lead to reduced blood
flow and diminished delivery of oxygen and nutrients, poten-
tially resulting in myocardial infarction. The tissue response
to this state of hypoxia is characterized by increased produc-
tion and secretion of factors that stimulate inflammation and
neovascularization (Vandervelde et al. 2007). This inflamma-
tory response is important for the clearance of cell debris,
and for the formation of scar tissue that preserves tissue in-
tegrity (van Amerongen et al. 2007, 2008).
Since the degree of tissue damage is largely regulated by
tissue perfusion, revascularization of the ischemic heart can
prevent apoptosis of cardiomyocytes (Kocher et al. 2001),
thereby inhibiting the development of cardiac failure (Shio-
jima et al. 2005).
The importance of circulating inflammatory cells in or-
chestrating myocardial repair following infarction, was exem-
plified by mouse experiments in which monocytes were
depleted prior to acute myocardial infarction (van Ameron-
gen et al. 2007). In these animals, repair processes were ab-
sent or severely hampered: there was no clearance of dead
cells, no scar formation, no remodelling, and reduced neovas-
This illustrates the importance of circulating cells in the
natural healing response to tissue damage. However, during
chronic disease, the natural healing response may be affected
and insufficient to adequately prevent further tissue damage
and maintain proper organ function. In those cases, disrup-
tion of coordinated cardiac remodelling and neovasculariza-
tion contributes to the transition to cardiac failure. Hence,
progenitor-cell-based therapies for induction of neovasculari-
zation are promising for the treatment of cardiovascular dis-
ease (Fig. 1).
Endothelial progenitor cells
The cardiovascular system is directly connected to the
main reservoir of stem cells, i.e., the bone marrow. In the
late 1990s, Asahara et al. (1997) discovered that the bone
marrow not only replenishes the blood, but also sends repair
cells out into the circulation, the so-called endothelial pro-
genitor cells (EPC). Upon cardiovascular damage, EPC are
rapidly recruited from the bone marrow into the circulation
and home-in on the lesion (Abbott et al. 2004).
Circulating EPC express either CD34 or CD14 surface
markers, and serve different functions in cardiovascular repair
(Krenning et al. 2009c). CD34+EPC develop into endothelial
cobble-stone-forming cells (Yoder et al. 2007) in vitro, and
phenotypically are endothelial progenitor cells with full endo-
thelial features (Moonen et al. 2010), while CD14+EPC are
monocytes that upon appropriate stimulation differentiate into
pro-angiogenic macrophages (Krenning et al. 2007, 2009b;
Ploeger et al. 2011) formerly known as early EPC.
Circulating CD34+and CD14+progenitor cells can be
readily isolated from adult human peripheral blood and used
to promote angiogenesis in vitro (Krenning et al. 2009b) as
well as in vivo (Popa et al. 2006; van der Strate et al. 2007;
Fig. 1. Cell therapy for cardiac regenerative medicine. Progenitor
cells are isolated from the adult tissues or peripheral blood by cell
sorting techniques (1 and 2). Thereafter, progenitor cells are im-
planted into the ischemic myocardium using catheters (3). In the
myocardium, progenitor cells may induce neovascularization by
either stimulating sprouting angiogenesis of the pre-existing vascu-
lature (4a) or vasculogenesis, wherein progenitor cells start to form
a new primary vascular plexus, which, in time, fuses to the existing
vascular network (4b). (From Krenning 2009 (B.Sc. thesis), repro-
duced with permission of G. Krenning © 2009 G. Krenning).
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2 Can. J. Physiol. Pharmacol. Vol. 90, 2012
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Krenning et al. 2011). Although in experimental clinical
progenitor-cell therapy, isolated cell populations, such as
CD34+cells, are frequently administered (Fig. 1), the com-
bined effect of CD34+and CD14+progenitor cells is more
potent. The working mechanism of these EPC is through se-
cretion of trophic factors. In fact, conditioned medium of co-
cultured CD34+and CD14+progenitor cells evokes neovas-
cularization to a similar extent as the implanted cells. In
vivo, the presence of IL-8, MCP-1, HGF, VEGF, and FGF-
2, secreted by the EPC, firstly induced an influx of inflam-
matory cells (macrophages), followed by neovascularization
(Krenning et al. 2011).
Influences of the trophic microenvironment
on progenitor-cell function
Although it has long been thought that the delivery of pro-
genitor cells to sites of tissue injury would provide therapeu-
tic benefit (Fig. 1), this view has proven to be rather naive,
and has not yielded widespread clinical benefit. First, reten-
tion of implanted progenitor cells at the damage site has
been disappointing, with reported cell retention fractions
of <10% (Goussetis et al. 2006; Terrovitis et al. 2009). Dis-
ruption of cell–matrix interactions by enzymatic digestion
during progenitor-cell isolation, and placing adherent cells in
suspension for injection in the damaged tissue is a highly dis-
ruptive process that may rapidly trigger anoikis (Thomas et
al. 1999; Robey et al. 2008). Also, ischemic tissue damage
induces local hypoxia-driven and inflammatory responses
(Vandervelde et al. 2007). This natural healing response is
characterized by increased vascular permeability (Zymek et
al. 2006; Xu et al. 2010), which facilitates the influx of in-
flammatory cells, the removal of necrotic cell debris, and
neovascularization. However, this open vessel architecture
also facilitates fast clearance of therapeutic progenitor cells
through venous or lymphatic drainage (Fig. 2).
Combined, these factors greatly reduce the long-term re-
tention of transplanted therapeutic cells to <1% at 4 weeks
post implantation (Zeng et al. 2007). As therapeutic benefit
is strongly correlated to the number of engrafted progenitor
cells (Terrovitis et al. 2009; Cheng et al. 2010), the need to
develop new and more effective cell delivery and retention
methods is evident, and would significantly increase the po-
tential of cell therapy.
Progenitor-cell apoptosis, activation, and adverse
Second, even if therapeutic progenitor cells are retained at
the site of tissue damage, they are faced with a rather hostile
microenvironment (Fig. 2). The natural healing response,
which directly follows tissue damage, consists of many in-
structive factors that may influence the behavior of therapeu-
tic cells (Vandervelde et al. 2005, 2006), challenging the
primitive idea that therapeutic cells instruct tissue repair. In
fact, recent evidence suggests that implanted therapeutic cells
may be adversely affected by this local environment, impair-
ing their therapeutic behavior (Verma et al. 2004). For in-
stance, high expression of inflammatory mediators, such as
interleukin (IL)-1b or C-reactive protein (CRP), commonly
present at sites of tissue damage, may directly cause apopto-
sis of transplanted therapeutic cells (Suzuki et al. 2004;
Verma et al. 2004; Desouza et al. 2011) in a NFkB-depend-
ent manner. Furthermore, the mere presence of such apop-
totic progenitor cells amongst a population of viable
progenitor cells may drastically hamper the survival, and
thus the therapeutic efficacy, of the whole population (Mou-
quet et al. 2011). In contrast, Thum et al. (2005) put forward
the “dying stem cell hypothesis”, which states that the in-
creased inflammatory response towards transplanted apop-
totic progenitor cells would improve cardiac outcome. Yet, it
seems counter-intuitive to develop therapies that are based on
the deliberate administration of apoptotic cells. The contra-
diction, however, does show the importance of timed immu-
nomodulation during cardiovascular repair.
Third, chronic exposure to inflammatory mediators may
also functionally impair progenitor cells (Werner and Nicke-
nig 2006; Moonen et al. 2007). Indeed, numerous studies
now show that the therapeutic capacity of progenitor cells is
impaired in situations where chronic inflammatory stimula-
Fig. 2. Progenitor cell and microenvironmental influences during
cardiac regeneration. Therapeutic progenitor cells are transplanted
into the ischemic heart to provide therapeutic benefit. Progenitor
cells supposedly provide pivotal clues that govern processes such as
myocardial survival, tissue remodeling, and neovascularization.
However, the microenvironment of a diseased heart has a major in-
fluence on the retention and behavior of progenitor cells, caused by
the presence of ischemia, inflammatory mediators, profibrotic mole-
cules, and mechano-structural changes. The interaction between car-
diac microenvironment and progenitor cells represents a continuum
of which the therapeutic outcome relies on the extent of initial da-
mage and the progenitor-cell susceptibility to these clues.
Moonen et al.3
Published by NRC Research Press
tion is present, such as organ failure (Heeschen et al. 2004;
Krenning et al. 2009a). Inflammatory mediators, such as
CRP, reduce the secretion of pro-angiogenic factors by pro-
genitor cells. In this respect, Nan et al. (2009) showed that
CRP was able to completely inhibit the secretion of IL-8 by
endothelial progenitors in a p38-MAPK dependent manner.
This inhibition may drastically affect EPC-induced neovascu-
larization, as IL-8 is a key mediator and its neutralization was
shown sufficient to block differentiation and neovasculariza-
tion by EPC, in vitro and in vivo (Krenning et al. 2009b,
Also, the anti-thrombogenic function of EPC is negatively
affected by chronic inflammatory stimulation. Exposure of
progenitor cells to inflammatory cytokines causes increased
expression of tissue factor (TF) and Factor VIIa-dependent
generation of thrombin (Krenning et al. 2009a; Cuccuini et
al. 2010). In fact, this associates EPC therapy with increased
thrombogenic risk, and may hamper clinical application.
Most disturbing is the fact that inflammatory mediators
stimulate the secretion of pro-inflammatory molecules by
progenitor cells (Zhang et al. 2009), providing a feed-forward
mechanism for inflammation at sites of tissue damage. Pro-
genitor cells that are stimulated with the pro-inflammatory
cytokine tumor necrosis factor (TNF)-a start to secrete high
amounts of monocyte chemoattractant protein (MCP)-1
(Zhang et al. 2009). Although MCP-1 may aid in the recruit-
ment of monocytes/macrophages that are necessary for neo-
vascularization and healing of damaged tissues (Dewald et
al. 2005; van Amerongen et al. 2007, 2008), overproduction
of MCP-1 may contribute to additional cardiomyocyte death
(Zhou et al. 2006; Younce et al. 2010; Younce and Kolat-
From the above, one can conclude that the inflammatory
microenvironment that is present at sites of tissue damage,
poses a huge hurdle for the successful clinical application of
progenitor-cell transplantation. Therefore, reducing the sensi-
tivity of progenitor cells to inflammatory mediators may pose
a viable option to increase therapeutic outcome of progenitor-
cell therapy. Proof-of-concept for this idea was delivered by
Liu et al. (2010). In their studies they used genetically modi-
fied progenitor cells that express the anti-inflammatory pro-
tein A20, and showed reduced sensitivity of these progenitor
cells towards pro-inflammatory cytokines TNFa and IL1b.
However, whether such progenitor-cell adaptations result in
functional maintenance and increased survival in vivo, re-
mains to be addressed.
Lastly, it is not only pro-inflammatory cytokines that ren-
der the microenvironment of damaged tissues hostile to ther-
apeutic progenitor cells (Fig. 2). Following tissue damage,
which limits the bioavailability of NO, by uncoupling of ni-
tric oxide synthases (i.e., endothelial (e)NOS, inducible (i)
NOS and neuronal (n)NOS), leading to the formation of reac-
tive oxygen species (ROS) (Umar and van der Laarse 2010).
Consequently, ROS promote the expression and secretion of
transforming growth factor (TGF)-b by inhibition of cGMP-
signaling and stimulation of the angiotensin II type 1 recep-
tors (Tomita et al. 1998; Saura et al. 2005). The TGFbs are
pleiotropic molecules, which may dampen the inflammatory
reaction present during the healing response (Frangogiannis
2004), they may also hamper
(Coomes and Moore 2010).
Endothelial-to-mesenchymal transition (EndMT) is a proc-
ess in which mature and progenitor endothelial cells lose
their endothelial phenotype and acquire a mesenchymal phe-
notype (Krenning et al. 2008; Moonen et al. 2010; Medici et
al. 2011). Binding of TGFb to its receptor ALK5 on endothe-
lial (progenitor) cells stimulates the activation of Smad3 and
evokes downstream repression of endothelial specific genes
(Moonen et al. 2010; Medici et al. 2011). Consequently,
EPC lose their ability to induce neovascularization and start
to produce collagens, contributing to the development of or-
gan fibrosis (Krenning et al. 2010; Moonen et al. 2010). In-
terestingly, Medici et al. (2010) recently described the
generation of bone-forming multipotent cells through EndMT
of mature endothelial cells. This suggests that the risk for
cardiac calcification or terratoma formation may be increased
following progenitor-cell therapy. Indeed, multiple reports
have described the occurrence of these adverse effects of
progenitor-cell therapy in animal experiments as well as in
humans (Yoon et al. 2004; Iwatani et al. 2009; Jeong et al.
In summary, progenitor cells are highly plastic cells that
may provide direct therapeutic benefit in many pathologies.
Hence, progenitor-cell therapy has rapidly moved into clinical
trials where these cells where expected to contribute to tissue
repair and do away with potential damaging factors. How-
ever, the trophic microenvironment in which progenitor cells
must function has a major influence on their behavior, poten-
tially limiting their therapeutic ability, or worse, causing ad-
verse differentiation into pathogenic cells. Careful analysis
and in-depth understanding of these microenvironments is
warranted to overcome these difficulties in progenitor-cell
therapy and increase therapeutic benefits.
Influences of the mechano-structural
microenvironment on progenitor-cell function
Mechano-transduction: relaying extracellular and
Not only trophic factors affect progenitor-cell function; the
mechano-structural microenvironment in which progenitor
cells function also plays an important role in many physio-
logical and pathological processes, and may be a prime deter-
minant of cell-therapy outcome (Fig. 2).
The first notion that mechanical forces are involved in the
general behavior of cells was derived from the observation
that many growth factors, cytokines, and integrins activate
myosin light chain kinase (MLCK) and RhoA, 2 major path-
ways involved in myosin activity, thereby regulating cell
shape and migratory activity (Klemke et al. 1997; Fukata et
al. 2001). Furthermore, inhibition of either MLCK or RhoA
activity not only blocks cytoskeletal functions, such as cell
migration, but also blocks cell proliferation (Olson et al.
1995; Zhou et al. 2008) and influences the differentiation of
progenitor cells (McBeath et al. 2004), suggesting that the
physical microenvironment of transplanted progenitor cells
may be a decisive factor for clinical outcome.
The biochemical response of cells to force is known as
mechano-transduction. It has long been known that externally
applied forces such as gravity and exercise-induced stress
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4Can. J. Physiol. Pharmacol. Vol. 90, 2012
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play important roles in musculo-skeletal development (Dun-
can and Turner 1995; Burkholder 2007); however, recently,
mechano-transduction has received increased attention due to
its potent and pleiotropic effects on progenitor-cell differen-
tiation (McBeath et al. 2004). Cells are physically attached
to their surrounding matrix and adjacent cells through integ-
rins and adhesion molecules, such as cadherins and selectins,
and their cytoskeleton machinery consisting of microfila-
ments, intermediate filaments, and microtubules. The integra-
tion of cytoskeletal proteins, extracellular microenvironment,
and the exciting tensile forces is known as tensegrity (Ingber
1997). Tensegrity suggests that to transduce a mechanical
signal into a biochemical signal, many factors related to cell
behavior must be associated with the cytoskeleton. Indeed,
focal adhesion complexes act as integrators that focus and
transduce mechanical stimuli to a plethora of associated sig-
nal transduction molecules (Ingber 2002) to tune cell re-
sponses to their environment, as discussed below. For blood
vessels in particular, the dynamic microenvironment dictates
the responses of endothelial cells (Hoffman et al. 2011),
which involves a mechanosensory complex of the typical en-
dothelial molecules PECAM-1, VE-Cadherin, and VEGFR2
(Tzima et al. 2005).
Fibrogenic cardiovascular diseases are the major target of
progenitor-cell therapy, especially the treatment of cardiac
failure. A common cause of cardiac failure is aMI. The me-
chanical post-aMI microenvironment is characterized by fi-
brotic scar formation (van Amerongen et al. 2007, 2008).
Increased deposition of extracellular matrix (ECM) in the in-
farcted areas results in increased stiffness of the cardiac tis-
sue, which may increase over 3-fold, reaching stiffness
values that are comparable with cartilage and bone (Berry et
Our understanding of how altered ECM stiffness may alter
progenitor-cell behavior has been greatly improved by study-
ing progenitor-cell behavior on ECM-functionalized polya-
crylamide gels. Polyacrylamide gels have a tunable stiffness,
and thus enable investigation of matrix stiffness without
changing the actual matrix content or architecture. Cells cul-
tured on stiff substrates, which are less prone to deformation,
show increased mechanical stress and increased activation of
focal adhesion kinases, compared with cells cultured on a
similar, but softer substrate with little resistance to deforma-
tion (Pelham and Wang 1997).
In an elegant series of experiments, Engler et al. (2006)
showed that matrix stiffness directs progenitor cell lineage
specification. ECM matrices possess a stiffness comparable
with brain-tissue-induced neurogenic differentiation, whereas
stiffer matrices induced myogenic differentiation, and matri-
ces with a stiffness comparable with bone proved to be osteo-
Matrix stiffness thus has a major influence on progenitor-
cell behavior, which would dictate that progenitor-cell func-
tion may be hampered in a fibrogenic (i.e., stiff) microenvir-
onment (Fig. 2), by the induction of adverse functioning or
differentiation. In line with this reasoning, Engler et al.
(2008) revealed that embryonic cardiomyocytes in culture
continue to contract on matrices with a stiffness comparable
with the cardiac tissue, but stopped contracting on matrices
with a stiffness that mimics fibrotic scar tissue. Furthermore,
Yoon et al. (2004) noted intramyocardial bone formation fol-
lowing cardiac cell therapy, using bone-marrow-derived pro-
genitor cells into a fibrosed heart. These studies exemplify
the influence a mechanical microenvironment has on cellular
function, and improving our understanding of these processes
may facilitate their modulation, thereby providing new oppor-
tunities for cell therapy.
Fluid shear stress and cyclic strain
The mechanical properties of matrices are not the only de-
terminants of a cells mechanical microenvironment. Beating
of the heart causes flow of the blood through the vasculature,
resulting in fluid shear stress (FSS) as well as intermittent ex-
pansion of the vessels (resulting in cyclic strain). Logically,
the endothelial cells that line blood-vessel walls mainly re-
spond to FSS, while smooth muscle cells that are in the cir-
cumference of the vessel respond to cyclic strain. In
particular, the endothelial cell’s responses to FSS play an im-
portant role in the homeostasis of the circulatory system.
Clinical observations indicate that atherosclerotic lesions
preferentially develop in predisposed (i.e., atheroprone) re-
gions (Ku et al. 1985). These regions are characterized by
low levels of nonlaminar shear stress, typically encountered
at vascular bifurcations and at the inner wall of vascular cur-
vatures. This is in contrast to regions with high levels of lam-
inar shear stress, where atherosclerotic lesions are less likely
to develop (Glagov et al. 1988). High levels of laminar shear
stress induce quiescence in endothelial cells, and evoke the
expression of anti-inflammatory and anti-thrombogenic pro-
teins. In contrast, disturbed flow, or nonlinear shear stress
evokes the expression of inflammatory mediators and throm-
bogenic proteins (Traub and Berk 1998).
So, do FSS and cyclic strain influence progenitor-cell be-
havior? FSS induces endothelial differentiation of embryonic
stem cells in an Flk-1-dependent manner (Yamamoto et al.
2005). Simultaneously, high levels of FSS inhibit muscular
differentiation, possibly through repression of PDGF-signal-
ing (Palumbo et al. 2000), whereas low levels of FSS in-
crease expression of PDGF and TGFb, stimulating muscle
cell differentiation (Qi et al. 2011). Furthermore, FSS induces
the maturation of endothelial progenitor cells by increasing
the expression of endothelial lineage genes VEGFR2, Flt-1,
and VE-Cadherin (Yamamoto et al. 2003). Depending on
FSS levels, arterial specialization by progenitor cells can be
induced (Masumura et al. 2009; Obi et al. 2009).
In contrast, cyclic strain induces the differentiation of
smooth muscle cells from embryonic stem cells by activation
of PDGF-signaling (Shimizu et al. 2008; Ghazanfari et al.
2009). Furthermore, cyclic strain induces expression of
smooth muscle cell lineage markers in bone marrow progeni-
tor and mesenchymal stem cells (Hamilton et al. 2004; Park
et al. 2004). In an ex-vivo model of vein arterialization,
Campos et al. 2009) showed that cyclic strain is the main
stimulus for smooth-muscle differentiation through increased
cysteine- and glycine-rich protein 3/ muscle LIM-domain
protein (CRP3/MLP) expression.
Shear stress and cyclic strain profiles are generally not
modifiable in vivo. However, one can speculate that pre-ex-
posing progenitor cells to mechanical stimuli ex vivo prior
to transplantation may prove advantageous; e.g., by inducing
Moonen et al.5
Published by NRC Research Press
angiogenic phenotypes more resistant to inflammatory and
thrombotic stimuli, thereby increasing neovascularization po-
tential. This rationale was recently evidenced by Boon et al.
(2011), who showed that increased expression of Krüppel-
like factor 2, a major FSS responsive gene, increased the
neovascularization potential of bone marrow-derived progeni-
tor cells. Hence, future research on mechano-transduction
pathways offers great potential to regenerative medicine.
Since the discovery of endothelial progenitor cells, numer-
ous experimental and clinical studies have been performed
wherein therapeutic cells are transplanted to elicit tissue re-
generation. However, the more we learn about tissue regener-
ation, the more apparent it becomes how complex this
process is. Besides the spatiotemporal regulation of growth
factor signaling and extracellular matrix interactions we are
just beginning to understand the regulation of mechano-struc-
tural factors and the role they play in tissue regeneration.
In this review, we focused on the application of bone-mar-
row-derived EPC for cardiovascular regeneration, and how
changes in the cardiovascular microenvironment influence
their plasticity. Besides EPC, other progenitor-cell types are
considered for use in cardiovascular regenerative medicine,
including cardiac progenitor cells (Smits et al. 2009),
epicardium-derived cells (EPDC) (Weeke-Klimp et al. 2010;
Smart et al. 2011), and adipose-derived stem cells (ADSC)
(Gimble et al. 2007; Danoviz et al. 2010). However, which-
ever progenitor cell type used, the local microenvironment
dictates their therapeutic potential. Increasing our understand-
ing of the complex interplay between progenitor cells and the
trophic and mechano-structural microenvironment of the
damaged tissue will help to predict and secure the beneficial
outcome of cell therapy.
Preconditioning the damaged tissue (Fig. 3), both to mobi-
lize and to increase the retention of therapeutic progenitor
cells has evolved from (pre-)clinical experience, and may im-
prove the therapeutic benefit of cell therapy. Introducing
exogenous factors to promote endogenous regeneration and
reduce inflammation (e.g., growth factors or anti-inflamma-
tory cytokines) is a well established method, and has demon-
strated beneficial effects on the cardiac tissue (Okada et al.
2009; Hiesinger et al. 2010; Rastogi et al. 2011). However,
the clinical benefit of these strategies is challenged by the
lack of specificity, transient effects, and the requisite intro-
duction of potentially tumorigenic factors.
The concept to prime progenitor cells before implantation
(Fig. 3), and thereby increase their ability to withstand the
microenvironmental influences and enhance their therapeutic
ability, has received increasing attention (Zemani et al. 2008;
Kim et al. 2009; Stratman et al. 2011). Priming progenitor
cells with cytokines or growth factors increases their reten-
tion and neovascularization potential in animal models. Simi-
larly, alterations in cell culture conditions may prime
progenitor cells. Exposure to hypoxic conditions, such as en-
countered in infarcted hearts, has proven successful in im-
proving the retention and survival of progenitor cells post-
implantation (Akita et al. 2003; Ong et al. 2010). These pri-
ming strategies highlight the capacity of altered culture con-
ditions to increase therapeutic efficacy. Such priming has the
advantage of simplicity, but may lack durability of its effects,
and clinical investigations have not been reported to date.
Mechanical approaches to enhance retention and function
of progenitor cells have emerged from the field of biomate-
rial science (Fig. 3). As described above, loss of tissue archi-
tecture and matrix support can have detrimental effects on
progenitor-cell function. However, biomaterials can be used
to support therapeutic cells by mimicking the native tissue ar-
chitecture of extracellular matrix, and relay essential survival
signals to the transplanted cells (von der Mark et al. 2010).
Moreover, bioactive signals can be incorporated into some
biomaterials to instruct progenitor-cell behavior (Hung et al.
2011). Interestingly, Silva et al. 2008) used these so-called
bioactive materials for the delivery of endothelial progenitors
in a murine hindlimb ischemia model, and reported a 3-fold
increase in neovascularization, compared with control cells in
suspension. Although these results are highly encouraging in
animal models, human precedence has not been provided yet.
Transplantation of a single progenitor-cell type will likely
not be sufficient to regenerate a damaged heart. Aiming at
synergism between progenitor cells, co-transplantation of
cells that improve the damaged microenvironment by the se-
cretion of trophic factors with cells that are capable of form-
ing functional cardiac tissue cells, may prove highly effective
Fig. 3. Past and future advances in progenitor-cell therapy.
Progenitor-cell therapy has been around for over a decade, but with
limited clinical success, owing to the hostile microenvironment these
cells must function in. Efforts made to improve clinical outcome,
such as preconditioning of the target tissue, priming of progenitor
cells, biomaterial-aided delivery of progenitor cells, and transplanta-
tion of synergistic progenitor cell types show promising results in
animal models. However, to really regenerate the cardiac tissue, all
of these advancements have to be combined into a “fluid organ”,
which contains all of the instructive and constructive elements of a
Pagination not final/Pagination non finale
6 Can. J. Physiol. Pharmacol. Vol. 90, 2012
Published by NRC Research Press
for cardiac regeneration (Fig. 3). In fact, co-transplantation of
EPC and skeletal myoblasts after myocardial infarction
showed superior neovascularization compared with either
cell type alone (Bonaros et al. 2008). Similarly, Winter et al.
(2009) co-transplanted epicardial-derived cells and cardiac
progenitor cells, and showed improved post-infarct cardiac
Although the recent advancements described above facili-
tate drastic improvements in the efficacy of progenitor-cell
therapy, future therapies must incorporate all of these facets
to achieve true regeneration of cardiac tissue. An increase in
our understanding of the complex interplay between the local
microenvironment, the method of cell delivery, and the syner-
gism between multiple progenitor-cell types is needed to ad-
vance this field to the next level. A systems biology approach
to regenerative medicine will likely provide us with many of
these insights. Systems biology is an emerging field that ap-
preciates the high complexity of biological systems by inte-
grating life sciences with chemistry, physics, mathematics,
informatics, and engineering. From a more practical point of
view, much progress can be made by implementing organo-
typic culture models which mimic the local environment in
which therapeutic cells are to exert their function.
Reverting back to our initial question (the good, the bad,
and the ugly?), one can conclude that progenitor-cell plasti-
city offers a large number of clinical benefits. The ease with
which progenitor cells can be isolated, culture expanded, and
differentiated in therapeutic cells offers huge clinical potential
(the good). However, it is this same plasticity that threatens
therapeutic benefit. Trophic and mechanistic microenviron-
ments may adversely influence progenitor cells, which subse-
quently lose their therapeutic value, or worse, start to
contribute to disease progression (the bad). If we want to pre-
vent cell therapy from becoming ugly, we need to further our
understanding of the microenvironment we expect progenitor
cells to function in, and use this knowledge to improve cur-
rent cell therapy concepts. Herein, we envision the transplan-
tation of “fluid organs” (Fig. 3), in a formulation that
contains all instructive and constructive elements of a healthy
myocardium. Such a formulation would consist of a degrad-
able biomaterial that provides structural support and transfers
biomechanical cues while it releases growth factors and cyto-
kines to modulate the hostile microenvironment, and instruct
the multiple progenitor cell types that, in synergy, can differ-
entiate into all the cardiac cell lineages.
The authors report that there is no conflict of interest asso-
ciated with this paper.
J.R.M., M.C.H., and G.K. are supported by the Groningen
University Institute Drug Exploration (GUIDE) of the Uni-
versity of Groningen and the University Medical Center Gro-
ningen. M.C.H. is supported by the W.J. Kolff Institute for
Biomedical Materials Science and Application of the Univer-
sity of Groningen. G.K. is supported by the Netherlands Or-
ganization for Scientific Research and the Netherlands
Organization for Health Research and Development (NWO/
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