JPT-06665; No of Pages 16

Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Mesenchymal stem/stromal cells as a pharmacological and therapeutic

approach to accelerate angiogenesis
Annelies Bronckaers ⁎, Petra Hilkens, Wendy Martens, Pascal Gervois, Jessica Ratajczak,
Tom Struys, Ivo Lambrichts
Group of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek, Belgium

a r t i c l e i n f o a b s t r a c t

Keywords: Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific
Mesenchymal stem/stromal cells world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible
Angiogenesis transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothe-
Endothelial differentiation sized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in
Secretome clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are pre-
Gene therapy
dominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angio-
genic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been
demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence,
MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogen-
esis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the
angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by
limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)
angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial
cells will be evaluated critically.
© 2014 Elsevier Inc. All rights reserved.

Contents

1. Identification and characterization of mesenchymal stromal/stem cells . . . . . . . . . . . . . . . . . . . . . 0

2. Current concepts of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Role of MSCs in angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. MSC as vehicles for targeted (anti-)angiogenic drug and gene delivery . . . . . . . . . . . . . . . . . . . . . 0
5. Therapeutic expectations and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Abbreviations: AD-MSCs, adipose-derived mesenchymal stem cells; Ang-1, angiopoietin-1; BM, bone marrow; BM-MSC, bone marrow mesenchymal stem cell; CAM, chorioallantoic
membrane; CM, conditioned medium; Cyr61, cysteine-rich, angiogenic inducer 61; EC, endothelial cell; ECM, extracellular matrix; EGF, epidermal growth factor; EGM-2, endothelial
growth medium-2; FGF-2, fibroblast growth factor-2; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor 1; IL-6, interleukin-6; ISCT, International Society for Cell
Therapy; MCP-1, monocyte chemoattractant protein-1; MI, myocardial infarction; MMP, matrix metalloprotease; MSC, mesenchymal stromal/stem cell; PAD, peripheral artery disease;
PDGF, platelet-derived growth factor; PLGF, placental growth factor; SDF-1, stromal cell derived factor-1; TGF-α, transforming growth factor-α; tMCAO, transient middle cerebral artery
occlusion; TNF-α, tumor necrosis factor α; tPA, tissue plasminogen activator; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; vWF, von Willebrand Factor; WHO, World
Health Organization.
⁎ Corresponding author at: Department of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek, Belgium. Tel.: +32 11 26 92 77; fax: +32 11 26 85 99.
E-mail address: annelies.bronckaers@uhasselt.be (A. Bronckaers).

http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
0163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
2 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
1. Identification and characterization of mesenchymal stromal/
stem cells
Since their discovery in the 1960s, the fascinating nature of mes-
enchymal stromal/stem cells (MSCs) attracted many scientists and
this is reflected by the 26,968 PubMed citations on MSCs at the mo-
ment of this writing.
Almost 5 decades ago, the pioneer Friedenstein and his coworkers
identified within the bone marrow (BM) a minor subpopulation of
cells that had osteogenic potential in vivo (Friedenstein et al., 1966).
These cells could be distinguished from other BM cells (such as the
hematopoietic cells) by both their rapid adherence to plastic and
their fibroblast-like morphology. Since these cells showed to be
highly clonogenic as they could efficiently generate single-cell
derived colonies, they were designated as colony-forming unit fibro-
blasts (Friedenstein et al., 1970). The true physiological and biologi-
cal role for these cells appeared to be support of the hematopoiesis in
the bone marrow (Clark & Keating, 1995). In analogy to the hematopoi-
etic stem cell system, Caplan introduced the popular name ‘mesen-
chymal stem cell (MSC)’ referring to the multipotent differentiation
potential of these cells (Goshima et al., 1991). Later on, it was confirmed
that the progeny of such a single BM-derived cell was able to differenti-
ate into multiple mesenchymal tissues such as bone, cartilage, adipose
and fibrous tissues (Pittenger et al., 1999). Besides this mesodermal dif-
ferentiation potential, MSCs are also suggested to form myocardial cells
(Toma et al., 2002; Kawada et al., 2004), hepatocytes (K. D. Lee et al.,
2004), neuron-like and neuronal cells (Kopen et al., 1999; Wislet-
Gendebien et al., 2005) under the appropriate induction conditions. In
addition to the bone marrow, MSC populations can be isolated from a
wide variety of tissues such as (and not limited to) adipose tissue
(Zuk et al., 2002), dental pulp (Gronthos et al., 2000), amniotic fluid
(Nadri & Soleimani, 2007), umbilical cord blood (Erices et al., 2000)
and even breast milk (Patki et al., 2010). A great contribution to the
popularity of the MSCs can be attributed to their immune-suppressive
profile: Le Blanc et al. (2003) reported that MSCs do not express MHC
class II surface markers and no co-stimulatory molecules such as
CD40, CD80 and CD86, suggesting that MSCs can be “invisible” for the
host immune system and therefore could be clinically applied in allo-
genic settings.
In spite of their promising and remarkable features, it remains
disputable whether MSCs really fulfill all hallmark ‘stem cell’ charac-
teristics, such as self-renewal and differentiation in vivo, as adequate
test systems for these two properties are still lacking (Bianco et al.,
2008). Therefore, it is generally considered that the name ‘mesen-
chymal stromal cell’ (also abbreviated as MSC) is more appropriate
than the widely used historical description ‘mesenchymal stem cell’
(Bianco et al., 2008; Prockop & Oh, 2012).
Another bottleneck in MSC research in the years 1990–2000 was
the fact that due to differences and inconsistencies in isolation, ex-
pansion and identification methodology between various research
groups, it was difficult to compare the results of published MSC studies
(Caplan & Correa, 2011; Keating, 2012; Prockop & Oh, 2012). In order to
clearly define MSCs, the International Society for Cell Therapy (ISCT)
proposed the following minimal criteria for MSCs: MSCs have a
fibroblastoid phenotype, adhere to plastic and possess a trilineage
mesodermal differentiation capacity towards chondrocytes, adipo-
cytes and osteocytes in vitro. Additional requirements include the
expression of the cell surface molecules CD105 (endoglin), CD73
(ecto 5′ nucleotidase) and CD90 (Thy-1) and the absence of the he-
matopoietic markers CD34, CD45, CD14 (or CD11b), CD79α and the
MHC II class cellular receptor HLA-DR (Horwitz et al., 2005). While
it is assumed that expression of this broad set of markers defines
cells as MSCs, most of these markers are also found on fibroblastic
cells from any tissue (Bianco et al., 2008). Although various surface
molecules such as CD146 (Sacchetti et al., 2007), STRO-1 (Simmons
& Torok-Storb, 1991) and CD271 (Jones et al., 2002) have been
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
used to enrich BM-derived MSCs, the fact that to date there is no single
specific unambiguous determinant to designate MSCs in situ, remains a
particular struggle (Bianco et al., 2008; Caplan & Correa, 2011; Keating,
2012). On the other hand, it is possible that the ISCT marker profile def-
inition of MSCs is too simplistic and unsuitable for MSCs isolated from
other tissues than the BM, as for example MSCs derived from adipose
tissue in fact do express CD34 (Traktuev et al., 2008; Keating, 2012). An-
other drawback is that the expression of these markers may be deter-
mined or may even be induced by culture conditions rather than
being intrinsic characteristics of MSCs in situ. In mice, even strain differ-
ences influence the expression of surface epitopes on BM-MSCs (Peister
et al., 2004). As there is still no consensus on the surface marker expres-
sion profile of MSCs among leading investigators within the field, new
and more advanced molecular tools that help unravel the MSC prote-
ome and secretome could provide a solution to uniformly define MSCs
(Keating, 2012).
Traditionally, MSCs are considered to be of ‘pure’ mesodermal origin
as most skeletal bones are derived from the axial and lateral mesoderm
(Bianco et al., 2008; Caplan & Correa, 2011). However, in the embryonic
development of craniofacial bones and their marrow, the ectodermal
neural crest cells are also involved (Hall, 2008). Furthermore, there is
accumulating evidence that for almost all organs in the body, an MSC
population is present in the perivascular niche associated with blood
vessels in vivo. These perivascular cells, principally called pericytes,
are located at the abluminal side of blood vessels and communicate
with the endothelial cells. Based on immunoselection for CD146, NG2
and the pericyte marker PDGF-Rβ, Crisan et al. purified SC from various
tissues such as skeletal muscle, pancreas, adipose tissue and placenta. In
addition to their osteogenic, chondrogenic and adipogenic differentia-
tion potentials, all cells appeared to be myogenic in vitro and in vivo, re-
gardless of their origin of skeletal muscle or non-muscle tissues (Crisan
et al., 2008). Therefore, it is now speculated that all MSCs indeed are
pericytes and that their biological role is to fuel local tissue growth
and repair (Caplan & Correa, 2011).
As MSCs display a broad differentiation capacity in vitro, it was orig-
inally hypothesized that MSC transplantation would induce tissue re-
pair by replacing the damaged host tissue. Despite their long-lasting
therapeutic efficacy in a wide variety of in vivo models and clinical set-
tings (such as bone and cartilage repair, cardiovascular and neurological
diseases), the incidence of MSC engraftment remained to be surprising-
ly low. For example, children with osteogenesis imperfecta who re-
ceived MSC transplantation showed a significant improvement in
bone mineral density and growth velocity, while the population of
engrafted MSCs is less than 1% (Horwitz et al., 2002). Another striking
example of this low incorporation into the host was demonstrated by
Wu et al., who studied the rate of mouse BM-MSC engraftment in a mu-
rine wound healing model. By using sex-mismatched GFP + mBM-
MSCs, the number of cells engrafted into the wounded skin was found
to be 27% at 7 days post-injection and even decreased to only 2.5% at
28 days after administration (Y. Wu et al., 2007). This unexpected low
engraftment efficacy implied a major challenge for the field to explain
the beneficial effects of the MSCs (as they in most cases only temporar-
ily resided in the host) which was translated into a ‘back to the bench’
effect. There is now accumulating evidence that the general therapeutic
effects of MSC treatment are due to their ability to alter the host micro-
environment rather than their capacity to (trans)differentiate and in-
corporate into the host tissue. The basic mechanisms, other than
differentiation, by which MSCs are suggested to affect the host include:
(1) the modulation of the immune system (the ‘immunomodulatory
effect’),
(2) the secretion of factors that induce tissue repair (the trophic or
paracrine effect),
(3) recruitment of endogenous MSCs to the site of injury
(4) possibly transfer of mitochondria or vesicular components con-
taining mRNA, microRNA and proteins (Fig. 1) (Spees et al.,
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx 3

Paracrine/Trophic effects Immunomodulatory effects

MSC CD8+ T cell
Neutrophil

Treg cell NK cell B cell

Angiogenesis
CD4+ T cell

Plasma cell

Monocyte Dendritic cell

Proliferation and differentiation of host cells Differentiation

MSC

Neuron
Anti-apoptosis Cardiomyocyte
Chondrocyte Adipocyte
Osteocyte

Transfer of vesicular components

Recruitment of endogenous stem cells
MSC

Fig. 1. Proposed mechanisms of action of MSC transplantation. MSCs secrete a wide variety of proteins that affect host cells, a phenomenon that is called the trophic or paracrine effect. This
includes the induction of angiogenesis, protection of the host cells against apoptosis, stimulation of host cell proliferation and local stem cell or precursor cell differentiation and the re-
cruitment of endogenous stem cells to the site of engraftment. The immunomodulatory effects of MSC consist of inhibiting the proliferation and activity of neutrophils, NK cells, B cells,
CD4+ cells and CD8+ T cells, preventing the maturation of monocytes into dendritic cells, suppressing plasma cell immunoglobulin production but stimulating proliferation of regulatory
T cells. In some physiological settings, MSCs are able to differentiate themselves into multiple cell types and to transfer vesicles containing mRNA, proteins, microRNA and perhaps mito-
chondria to the host cells.

2006; Meirelles Lda et al., 2009; Hocking & Gibran, 2010; Ben- and where MSCs can (functionally) improve blood vessel formation
Ami et al., 2011; Caplan & Correa, 2011; Bieback et al., 2012; will be essential for unmasking new contributions by these cells in
Prockop & Oh, 2012). a whole array of diseases.

The local immunosuppressive actions of MSCs have fuelled hope

of allogeneic MSC treatment in settings where the use of autologous
cells is limited or even impossible. The trophic and immunomodula-
tory activities of the MSCs are considered to account for the majority
of the beneficial effects of MSC and inspired Caplan to launch the
concept of MSCs as a ‘drugstore’, secreting specialized bioactive mol-
ecules which prevent unfavorable immune reactions and support the
injured tissue to repair (Caplan & Correa, 2011). This spectrum of
bioactive molecules is generally defined as the MSC secretome and
includes growth factors, chemokines and cytokines (Ranganath
et al., 2012).
Since MSC research is now predominantly focused on the thera-
peutic advantages of the secretome, more than on the potential dif-
ferentiation of engrafted MSCs, we will summarize the paracrine
effects of MSCs particularly in diseases that are caused by limited
or insufficient angiogenesis. First, the process of angiogenesis and
the pro-angiogenic role of MSCs in vitro and in vivo are described.
In addition, the transdifferentiation potential of MSCs towards endo-
thelial cells is overviewed in detail. A greater understanding of how
2. Current concepts of angiogenesis
Within the human body, a complex labyrinth of blood vessels
provides all cells and tissues with vital nutrients and oxygen needed
for survival and proliferation. This network includes large, stable ar-
teries and veins as well as small, dynamic microvascular structures
such as capillaries, arterioles and venules.
In the embryo, new blood vessels arise through vasculogenesis, mean-
ing the de novo formation of a basic vasculature out of mesoderm-derived
endothelial precursors, termed angioblasts (Friedenstein et al., 1970;
Herbert & Stainier, 2011). Subsequently, this primordial vascular bed is
enlarged by a process defined as angiogenesis: the branching of new vas-
cular sprouts out of pre-existing ‘parental’ blood vessels (Friedenstein
et al., 1970; Folkman, 1971; Ribatti & Crivellato, 2012). Other mechanisms
of vascular development include splitting of pre-existing blood vessels
(intussusception) or stimulation of vessel expansion by circulating pre-
cursor cells. However, the majority of the blood vessel network is consid-
ered to be built through angiogenic processes (Friedenstein et al., 1970).

Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
4 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

A B C

D E

F G

Figure legend:
Endothelial cell (EC) Basement membrane

Pericyte Matrix metalloproteinase

Chemokine
Tip cell
Chemokine receptor
Stalk cell Growth factor

Extracellular matrix (ECM) Growth factor receptor

Fig. 2. Key steps of angiogenesis. A) Small blood vessels consist of hollow tubes aligned with endothelial cells (EC) surrounded by pericytes, a basement membrane (BM) and extracellular
matrix (ECM). Angiogenesis is a multistep process involving the following steps: (B) pericyte detachment and breakdown of the extracellular matrix and basement membrane by, for ex-
ample matrix metalloproteinases, (C) EC activation by angiogenic factors such as growth factors and cytokines, (D–E) formation of a tip cell which guides the migration and a stack cell
which proliferates and forms a primitive tube (F), and (G) finally pericyte attachment, deposition of BM and EC followed by maturation into fully functional blood vessels.

Normal physiological angiogenesis is a fundamental event during et al., 2011; Carmeliet & Jain, 2011b). This type of blood vessel forma-
embryonic development, wound and fracture healing and for the tion is tightly regulated in a spatial and temporal manner by the ex-
growth and function of the female reproductive organs (Bhadada tensive interplay of a wide variety of molecules such as matrix

Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
metalloproteases, growth factors, chemokines, adhesion molecules,
enzymes and angiogenic inhibitors (Carmeliet, 2005; Ferrara &
Kerbel, 2005; Carmeliet & Jain, 2011a).
Fig. 2 visualizes the key steps of angiogenesis. Small blood capil-
laries consist of hollow tubes composed of endothelial cells (EC) sup-
ported by pericytes and surrounded by a basement membrane and
extracellular matrix (ECM) (Eble & Niland, 2009) (Fig. 2A). In the
adult, blood vessels and EC remain in a quiescent state and are rarely
activated to form new branches. The basement membrane and
pericytes surrounding the EC prevent the EC from leaving their posi-
tions. Only in response to specific stimuli such as hypoxia and inflam-
mation, the angiogenic process is initiated (Carmeliet & Jain, 2011b).
First, pericytes detach and both the ECM and basement membrane are
degraded by proteases such as the matrix metalloproteases (MMPs)
(Fig. 2B). This leads to destabilization of the blood vessels and an in-
crease in vascular permeability. In addition, the proteolytic breakdown
of ECM releases sequestered proangiogenic growth factors and
chemokines, such as vascular endothelial growth factor (VEGF), fibro-
blast growth factor-2 (FGF-2) and stromal cell derived factor-1 (SDF-
1) (Arroyo & Iruela-Arispe, 2010). Once liberated, these proteins are
able to bind to the receptors of the EC, thereby activating the EC and in-
ducing the start of branch formation (Fig. 2C). This initiation of
sprouting requires the specification of EC into ‘tip’ and ‘stalk’ cells
with different morphologies and functional properties, a process
which is tightly regulated by VEGF/Notch signaling (Phng & Gerhardt,
2009; Potente et al., 2011; Ribatti & Crivellato, 2012) (Fig. 2D). EC tip
cells have numerous filopodia and primarily migrate and coordinate
the direction of the branch formation, while stalk cells follow the tip
cells and proliferate during sprout elongation. These stalk cells also
form the lumen of the primitive blood vessel, either by the fusion of in-
tracellular pinocytic vesicles or by rearrangement of their shape and
junctions (Potente et al., 2011; Ribatti & Crivellato, 2012) (Fig. 2E).
Subsequently, the stable sprout begins to transform into a mature
blood vessel and fuses with an existing capillary (Fig. 2F–G). New
ECM is deposited around the new tube, recruited pericytes cover
the EC and finally, a stable blood vessel with a functional blood
flow is established (Fig. 2G). Without pericyte attachment, blood
vessels are considered to be unstable and can regress rapidly
(Potente et al., 2011).
The equilibrium between angiogenic stimulators and inhibitors
in the microenvironment, the so-called angiogenic balance, tightly
controls the rate of blood vessel formation (Bergers & Benjamin,
2003). Numerous angiogenic stimulators exist, including FGF-2,
SDF-1, placental growth factor (PLGF) and epidermal growth factor
(EGF). VEGF, one of the most studied angiogenic proteins, is consid-
ered to be the most potent mediator of neovascularization in health
and disease (Carmeliet & Jain, 2011a; Potente et al., 2011). However,
recent studies reveal the existence of alternative splicing variants of
VEGF (the VEGF-xxxb isoforms) which may have anti-angiogenic and
anti-tumoral properties (Woolard et al., 2009; Harris et al., 2012).
Thrombospondin, endostatin, angiostatin and angiopoietin-2 are other
examples of endogenous angiogenic inhibitors (Carmeliet & Jain,
2011a; Potente et al., 2011). Disturbance of angiogenic balance
can lead to a growing list of life-threatening pathological disorders.
Extensive angiogenesis, for instance, contributes to rheumatoid
arthritis, psoriasis, blinding eye diseases and cancer growth and
metastases. On the other hand, inadequate stimulation of the an-
giogenic process is one of the main causes of insufficient recovery
following ischemic insults as seen in atherosclerosis, stroke, myo-
cardial infarction and chronic wounds (Bhadada et al., 2011;
Carmeliet & Jain, 2011a). In addition, in the field of tissue engineer-
ing, the main reason of tissue transplantation failure is the lack of
proper angiogenesis, causing necrosis of the engrafted cells
(Laschke et al., 2006). It is in these clinical settings, where angio-
genesis is limited, that MSC transplantation might be promising
as an efficient therapy.
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
5
3. Role of MSCs in angiogenesis
3.1. The MSC secretome as a powerful inducer of angiogenesis
3.1.1. Angiogenic factors in the MSC secretome
Table 1 summarizes the angiogenic mediators and their functions
detected in MSCs of different tissue sources. Using ELISA, antibody
arrays and immunohistochemistry a broad repertoire of angiogenic
factors have been detected in the secretome of BM-MSCs including
VEGF, FGF-2, angiopoietin-1 (Ang-1), monocyte chemoattractant
protein-1 (MCP-1), interleukin-6 (IL-6) and placental growth factor
(PLGF) (Kinnaird et al., 2004a; Hung et al., 2007; Wu et al., 2007; L.
Chen et al., 2008; Boomsma & Geenen, 2012). Recently, Estrada et al.
identified the presence of the protein Cyr61 (cysteine-rich, angiogenic
inducer 61) in the mBM-MSCs with advanced mass spectrometry.
Addition of an antibody against Cyr61 inhibited BM-MSC-induced
EC tube formation in vitro and blood vessel formation in vivo in the
mouse matrigel assay, indicating that Cyr61 plays a key role in BM-
MSC-mediated angiogenesis (Estrada et al., 2009). Concerning
angiogenic receptors, it has been shown that BM-MSCs lack VEGF-
receptors, but they do express their co-receptor neuropilin-1,
which co-localizes with platelet-derived growth factor (PDGF)-re-
ceptor. This cross-talk is important in MSC proliferation, migration
and network formation (Ball et al., 2010). Placental MSCs produce
IL-6 (S. H. Liu et al., 2010), while adipose-derived MSCs (AD-MSCs)
secrete high amounts of VEGF, hepatocyte growth factor (HGF) and
transforming growth factor β (TGF-β) (Rehman et al., 2004). Elevat-
ed levels of VEGF and MCP-1 have also been found in human dental
pulp stem cells (hDPSCs) (Kandel & Pittenger, 1999; Aranha et al.,
2010).
The secretion of angiogenic factors by MSCs can be upregulated
by a range of chemokines and hypoxic conditions. De Luca et al. dem-
onstrated that transforming growth factor α (TGF-α) significantly
induces the secretion of various growth factors such as VEGF, HGF,
IL-6, IL-8, PDGF-BB and Ang-2 in BM-MSCs. Both the MEK/MAPK
and PI3K/AKT pathways were involved in the ability of TGF-α to in-
crease the levels of these factors in the BM-MSC secretome. Conse-
quently, conditioned medium (CM) from TGF-α treated MSCs
induced more blood vessel growth compared to CM from untreated
BM-MSCs in the in vivo chorioallantoic membrane (CAM) assay
(De Luca et al., 2011). In addition, Crisostomo et al. (2008) demon-
strated that hypoxia, tumor necrosis factor α (TNF-α) and LPS stim-
ulated the production of VEGF, FGF-2, HGF and insulin-like growth
factor 1 (IGF-1) via induction of the transcription factor NF-κB. In
AD-MSCs, LPS increased the secretion of several chemokines includ-
ing IL-6 and TNF-α, whereas FGF-2 and epidermal growth factor
(EGF) upregulated HGF production (Kilroy et al., 2007). BM-MSCs
preconditioned with interferon-β (IFN-β) produced higher levels of
MCP-1 and interferon gamma-induced protein 10 than untreated MSC
(Croitoru-Lamoury et al., 2007). Under hypoxic conditions, the CM of
BM-MSCs contained higher amounts of for instance VEGF, IGF-1, SDF-
1 and erythropoietin compared to CM of dermal fibroblasts (L. Chen
et al., 2008). It has also been shown that hypoxia elevates secretion of
VEGF but not of FGF-2 in hDPSC cultures due to upregulation of the
transcription factor HIF-1α (Aranha et al., 2010). Accordingly, also in
AD-MSCs, hypoxia selectively increased VEGF but not FGF-2, HGF and
TGF-β mRNA and protein levels (Rehman et al., 2004). Interestingly,
serum-deprivation during 30 days elevated the levels of VEGF, HGF,
IGF-1 and Ang in the secretome of human BM-MSCs (Bianco et al.,
2008). CM of these serum-deprived cells generated longer neovascular
sprouts in the ex vivo aortic ring assay compared to CM of control BM-
MSCs (Bianco et al., 2008).
3.1.2. MSCs alter the behavior of EC in vitro
As angiogenesis is a multi-step process involving EC survival, prolif-
eration, migration, tube formation and maturation, several in vitro
6 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Table 1
Angiogenic factors secreted by cultured MSCs.

Angiogenic factor Full-name Function MSC source REF

Angiogenin Angiogenin Cell migration, invasion, proliferation and tube formation hBM-MSC Hung et al., 2007
Ang-1 Angiopoietin-1 Vessel stabilization, EC survival, recruitment of pericytes mBM-MSC Wu et al., 2007
Ang-2 Angiopoietin-2 EC migration and sprouting mBM-MSC Wu et al., 2007
Cyr61 Cysteine-rich 61 Cell adhesion and EC migration mBM-MSC Estrada et al., 2009
FGF-2 Fibroblast growth factor-2 EC proliferation, migration, remodeling of the extracellular matrix mBM-MSC Kinnaird et al., 2004a,
HGF Hepatocyte growth factor EC and SMC proliferation, migration rBM-MSC Tögel et al., 2007
hAD-MSC Rehman et al., 2004
IGF-1 Insulin-like-growth factor-1 EC proliferation, survival, induction VEGF and plasminogen activators rBM-MSC Tögel et al., 2007
IL6 Interleukin-6 EC proliferation and migration hBM-MSC Hung et al., 2007
hPl-MSC Liu et al., 2010
IL8 Interleukin-8 EC proliferation, survival, migration and tube formation hBM-MSC Majumdar et al., 1998;
De Luca et al., 2011
MCP-1 Monocyte chemoattractant protein-1 EC migration mBM-MSC Kinnaird et al., 2004a;
Boomsma & Geenen, 2012
hBM-MSC Hung et al., 2007
MIG Monokine induced by interferon-gamma Chemoattractant of T-cells, inhibition of angiogenesis mBM-MSC Boomsma & Geenen, 2012
MIP-1α Macrophage inflammatory protein-1alpha Proinflammation, chemoattractants of immune cells mBM-MSC Boomsma & Geenen, 2012
MIP-1β Macrophage inflammatory protein-1beta Proinflammation, chemoattractants of immune cells mBM-MSC Boomsma & Geenen, 2012
NAP-2 (CXCL7) Neutrophil activating protein 2 Neutrophil recruitment, EC migration, release of VEGF and MMP hBM-MSC Hung et al., 2007
PLGF Placental growth factor (PLGF) Induction of vessel formation mBM-MSC Kinnaird et al., 2004a,
TGF-beta Tumor growth factor-beta Tube formation, vessel stabilization, ECM synthesis hAD-MSC Rehman et al., 2004
TIMP-1 Tissue inhibitor of metalloproteinase-1 Inhibitor of metalloproteinases hBM-MSC Hung et al., 2007
TIMP-2 Tissue inhibitor of metalloproteinase-2 Inhibitor of metalloproteinases hBM-MSC Hung et al., 2007
VEGF Vascular endothelial growth factor Increase vessel permeability, ECM degradation, EC proliferation, mBM-MSC Kinnaird et al., 2004a,
migration, tube formation and survival Wu et al., 2007
hBM-MSC Hung et al., 2007
hDPSC Bronckaers et al., 2013,
Tran-Hung et al., 2008
hAD-MSC Rehman et al., 2004

models exist that mimic each of these essential events. There is detailed
and elegant literature that describes the effect of MSCs on each of these
phases in vitro.
AD-MSCs have been shown to induce EC proliferation (Rehman
et al., 2004), while MSCs derived from pulp tissue had no effect on
EC cell growth (Kandel & Pittenger, 1999; Bronckaers et al., 2013).
For BM-MSCs, contradictory results have been reported: Two studies
indicated that the CM of BM-MSCs had no effect (Gruber et al., 2005;
Potapova et al., 2007), whereas others demonstrated that BM-MSCs
do increase EC proliferation (Kinnaird et al., 2004b; Potapova et al.,
2007; Duffy et al., 2009). These conflicting results might be ex-
plained by variations in isolation methods, species (mouse versus
human) and EC sources used.
In addition, EC migration in vitro was induced by various MSC
populations, including Wharton Jelly-derived umbilical vein MSCs
(Choi et al., 2013), amniotic MSCs (Kim et al., 2013), AD-MSCs
(Takahashi et al., 2010), human dental pulp stem cells (DPSCs)
(Bronckaers et al., 2013) and BM-MSCs (Gruber et al., 2005;
Potapova et al., 2007; Burlacu et al., 2013). Furthermore, the ability
of EC to degrade the surrounding ECM in vitro (in the so-called inva-
sion assay) is stimulated by MSC. AD-MSCs as well as BM-MSCs have
been shown to induce EC invasion, albeit through upregulation of
distinct proteases (Kinnaird et al., 2004b; Kachgal & Putnam,
2011). Potapova et al. (2007) demonstrated that CM of BM-MSCs
cultured in special three-dimensional aggregates had a more pro-
nounced effect on EC proliferation, migration and invasion than CM
of monolayer BM-MSCs, probably due to a higher concentration of
VEGF, FGF-2, angiogenin and IL-11.
BM-MSC and CM of BM-MSC induce EC tube formation on colla-
gen and matrigel in vitro (Wu et al., 2007; Duffy et al., 2009;
Estrada et al., 2009; Boomsma & Geenen, 2012). In addition, it was
demonstrated that human BM-MSCs in direct co-culture with EC on
matrigel increased the persistence of pre-existing blood vessels, sug-
gesting that BM-MSCs not only promote tube formation, but also
play an active role in stabilization and maturation of newly formed
vessels. This effect was considered to be caused by direct cell contact
between the BM-MSCs and EC (Duffy et al., 2009). Another study
described that BM-MSCs co-aligned with tubular EC structures in a
pericyte-like manner and some BM-MSC populations enhanced the
complexity and extent of these tubes in a 3D matrigel co-culture sys-
tem (Sorrell et al., 2009). Accordingly, Au et al. (2008) have demon-
strated that hBM-MSCs efficiently stabilized nascent blood vessels
in vivo by functioning as perivascular precursor cells in an in vivo tis-
sue engineered blood vessel construct. These findings are in concor-
dance with the current hypothesis that MSCs are indeed pericytes
(Caplan & Correa, 2011). On the other hand, Otsu et al. (2009)
demonstrated that addition of high numbers of BM-MSCs caused an
inhibition of tube formation in a matrigel assay due to the production
of cytotoxic reactive oxygen species. The observed anti-angiogenic
effect was considered to be caused by the large amount of administered
MSCs, as the EC cytotoxicity was significantly reduced when the num-
ber of added BM-MSCs also decreased. These results strongly indicate
that administration of high MSC concentrations should be avoided in
MSC-based treatments as it can potentially induce apoptosis of newly
formed blood vessels (Otsu et al., 2009).
MSCs also have the capacity to protect EC from apoptosis in vitro. The
results published by Hung et al. (2007) demonstrated that the secretome
of BM-MSCs inhibited hypoxia-induced apoptosis of EC, probably
through activation of the PI3K/Akt and not the ERK pathway. In coculture
with BM-MSCs, apoptosis of UV-irradiated fibroblasts and also of lung
epithelial cells incubated under hypoxic and low pH conditions was de-
creased through the secretion of the peptide hormone stanniocalcin-1
(Block et al., 2009). Placental MSCs significantly reduced oxidative
stress-related apoptosis (induced by the chemical tert-butyl hydroperox-
ide) via the STAT3 signal transduction cascade (S. H. Liu et al., 2010). CM
of hypoxic AD-MSCs abrogated EC cell death induced by a cocktail of cy-
cloheximide and TNF-α (Rehman et al., 2004).
In conclusion, MSCs are involved in all stages of angiogenesis, not
only in the early steps such as proliferation or migration of EC but also
in the later phases which involve blood vessel maturation.
3.1.3. MSCs have angiogenic properties in vivo
In this section, we will first discuss the in vivo angiogenic capacities in
animal models that allow one to exclusively investigate neoangiogenesis

Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
in the absence of any pathology, such as the ‘chicken chorioallantoic
membrane (CAM) assay’ and the ‘mouse matrigel plug assay’. Next, we
discuss the benefits of MSC-treatment in animal models and human clin-
ical trials that display a wide variety of disorders caused by limited angio-
genesis. A summary of all published scientific data is beyond the scope of
this review, and hence, only key observations that exemplify the pro-
angiogenic impact of MSC administration will be highlighted. In addition,
contradictions in the literature will also be discussed critically.
3.1.3.1. CAM assay. The chicken chorioallantoic membrane (CAM) is an
extra-embryonic, highly vascularized tissue that serves as a transient
gas exchange surface for the embryo (comparable to the lungs in
adult vertebrates). The main cellular components of the CAM are
EC and mural cells such as pericytes. Traditionally, this model has
been successfully used to investigate angiogenic factors in developmen-
tal biology and to select anti-angiogenic compounds for anticancer
treatments. As the early chicken embryo lacks a complete immune sys-
tem, mammalian cells or tissues can be placed onto the CAM without re-
jection allowing a simple assessment of the angiogenic potential of the
transplant (Laschke et al., 2006). BM-MSCs, CM of BM-MSCs, hDPSCs
and placental MSCs have been shown to stimulate blood vessel growth
in this small and accessible in vivo model (Kandel & Pittenger, 1999;
Gruber et al., 2005; M. Y. Lee et al., 2009). Serum-deprived BM-MSCs
proved to cause significantly more chicken blood vessel growth than
normal BM-MSCs (Bianco et al., 2008). In addition, CAM angiogenesis
induced by EC-differentiated placental MSCs was inhibited by addition
of antibodies against integrins α5 and β1 (M. Y. Lee et al., 2009).
3.1.3.2. Mouse matrigel plug assay. The matrigel plug angiogenesis assay
is an in vivo technique to detect newly formed blood vessels in a gel
plug which is transplanted into immunocompromised mice. Matrigel
is a reconstituted basement membrane preparation that is extracted
from the Engelbreth–Holm–Swarm mouse sarcoma, a tumor rich in
ECM proteins such as laminin, collagen IV and entactin. Usually, a mix-
ture of stem cells and matrigel is subcutaneously injected into mice and
directly after injection, this matrigel solidifies and forms a plug. Several
weeks post injection, these plugs are removed and the number of blood
vessels is assessed.
mBM-MSCs dose-dependently induced angiogenesis in the
mouse matrigel assay and the blood vessel sprouting was more pro-
nounced than in matrigels incubated with recombinant FGF-2 or
VEGF (Al-Khaldi et al., 2003b; Estrada et al., 2009). A study by the
research group of Verfaillie indicated that BM-MSCs are indeed able to
attract host blood vessels to subcutaneous matrigel transplants (mea-
sured 3 weeks post injection), but these tubes are leaky as evidenced
by the presence of red blood cell pools in the matrigel (Roobrouck et al.,
2011). Recently, subcutaneous DPSC-matrigel transplants in mice were
shown to induce more vessel formation than BM-MSC plugs. This
DPSC-induced neovascularization was inhibited by addition of soluble
VEGF-receptor 1, which neutralizes VEGF-A (Janebodin et al., 2013).
3.1.3.3. Wound healing. Wound healing is a very complex multifactorial
process involving the balanced interaction of inflammation, re-
epithelialization, angiogenesis and deposition and remodeling of
the ECM. Poor wound healing is one of the most serious complications
in patients who suffer from diabetes. As the incidence of diabetes
mellitus is reaching pandemic proportions worldwide and current
treatments only result in partial and temporal healing, there is a grow-
ing need for an effective therapy (Hocking & Gibran, 2010).
Numerous researchers already described the beneficial effects of
BM-MSCs in wound healing (reviewed in Hocking & Gibran, 2010).
For example, BM-MSC treatment of wounds in diabetic mice resulted
in accelerated wound closure, increased re-epithelialization and
wound vascularity probably through the release of angiopoietin-1
and VEGF (Y. Wu et al., 2007). In this case, the rate of MSC engraft-
ment 28 days after injection was demonstrated to be extremely
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
7
low (less than 1%). In addition, no integration of the transplanted
GFP + BM-MSCs into the host vasculature was observed (Y. Wu et al.,
2007). Some investigators state that besides induction of angiogenesis,
MSCs contribute to wound repair by differentiation into keratinocytes
(Sasaki et al., 2008), although evidence of this transdifferentiation is
not provided in all studies (Hocking & Gibran, 2010). Moreover, the ap-
plication of concentrated CM of BM-MSCs or CM of hypoxia-pretreated
AD-MSCs to murine excisional wounds significantly enhanced wound
healing (L. Chen et al., 2008; E. Y. Lee et al., 2009). Addition of neutral-
izing antibodies against VEGF and FGF-2 to the wounds treated with
the AD-MSC CM reduced the wound repair, indicating that upregulation
of angiogenesis caused the observed amelioration (E. Y. Lee et al., 2009).
In contrast, another study addresses the beneficial effect of the BM-MSC
CM to the induced recruitment of macrophages and endothelial progen-
itor cells to the site of injury (L. Chen et al., 2008). Interestingly, a small
pilot study in patients with acute or severe chronic wounds showed
that topical administration of autologous BM-MSCs with a fibrin spray
significantly reduced wound size (Falanga et al., 2007).
3.1.4. MSCs as a therapy for diseases caused by limited angiogenesis
3.1.4.1. Peripheral artery disease (ischemia). Ischemia is a restriction of
blood supply to certain organs or tissues, caused by pathological
changes in the vascular system, such as vasoconstriction or thrombo-
sis (obstruction of a blood vessel). A common pathology associated
with ischemia is peripheral artery disease (PAD), referring to ob-
struction of the large limb arteries caused by atherosclerosis and in-
flammation. PAD can cause a wide variety of symptoms including
weakness and cramping in the muscles, chronic wounds and ulcers,
changes in color and temperature of the tissue, muscle atrophy and
gangrene. Since effective pharmacological treatments do not exist
yet, amputation of the affected limb is needed to solve the unbear-
able symptoms in a third of the cases. Owing to their angiogenic po-
tential, MSCs can be considered as an effective treatment option
since restoration of the blood flow might eventually restore the func-
tionality of the affected extremity. PAD is mimicked in animal
models by blocking the blood flow by (temporary) ligation or resec-
tion of important hind limb arteries, such as the femoral artery,
resulting in ischemia of this extremity (Iwase et al., 2005).
In a murine model of hindlimb ischemia induced by distal femoral
ligation, local intramuscular injection of GFP + BM-MSCs improved
limb perfusion, increased blood vessel density, reduced the inci-
dence of auto-amputation, attenuated muscle atrophy and signifi-
cantly improved limb function. These actions occurred without
observable MSC incorporation into the blood vessels, but were probably
caused by the paracrine actions of the MSCs as VEGF and FGF-2 levels
were increased in the MSC-treated muscles and VEGF colocalized with
the administered GFP + MSC (Kinnaird et al., 2004b). In a rat model
of hindlimb ischemia, treatment with BM-MSCs was found to be supe-
rior compared to administration of BM mononuclear cells. In contrast
to the aforementioned mouse study of Kinnaird et al., transplanted
MSCs were considered to differentiate into EC as von Willebrand Factor
(vWF)-positive transplanted cells were found in the tissue 3 weeks
after transplantation (Iwase et al., 2005). Also, Al-Khaldi et al. (2003a)
found that administered BM-MSCs expressed EC markers (such as
CD34 and the EC marker factor VIII) 4 weeks after transplantation in a
rat model of ischemia. Recently, a phase II clinical trial demonstrated
that intra-arterial infusion of autologous bone marrow significantly in-
creased both peripheral blood perfusion and the percentage of
amputation-free patients with critical limb ischemia one year after the
treatment (Schiavetta et al., 2012). Moreover, intravenous injection of
hAD-MSCs improved blood perfusion in a mouse hind limb ischemia
model (Rehman et al., 2004; Moon et al., 2006). Implanted DiI-labeled
hAD-MSCs also seemed to differentiate into EC as vWF/DiI double pos-
itive cells were detected within the muscle tissue. Nevertheless, the ac-
tual incorporation rate of these stem cells into the host vascular
8 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
structures was less than 1%, indicating that the paracrine effects of AD-
MSCs principally account for the beneficial effect on hindlimb ischemia
(Moon et al., 2006). In addition, intramuscular injection of a side popu-
lation of porcine DPSCs (CD31−/CD146− DPSCs) increased blood flow
and capillary density in a murine hindlimb ischemia model. These
transplanted cells were present in close proximity to the vessel and in
situ hybridization revealed that these cells expressed several angiogenic
proteins such as VEGF and MMP-3 (Iohara et al., 2008).
3.1.4.2. Myocardial infarction. According to the World Health Organiza-
tion (WHO), ischemic heart disease is the leading cause of mortality in
western societies. Myocardial infarction (MI) leads to heart dilatation
or cardiac failure often resulting in significant disability or death. The ra-
tionale behind MSC-treatment for MI is to repair the damaged heart tis-
sue by cardiomyocyte differentiation and by supplying growth factors
that induce angiogenesis or stimulate resident cardiac stem cell migra-
tion and commitment to cardiomyocytes (Ranganath et al., 2012). In an-
imal models, MI is caused by a ligation of one of the coronary arteries,
such as the left artery descending and left circumflex artery.
Abundant evidence demonstrates the therapeutic benefits of
bone marrow (Dai et al., 2005), adipose (Miyahara et al., 2006),
pulp (Gandia et al., 2008) and even amniotic-derived MSCs (Kim
et al., 2013) in various mouse or rat models of MI. Transplantation
of these cells significantly improved ventricular function, which is
associated with the induction of myogenesis and angiogenesis. AD-
MSCs transplanted as a monolayer, improved cardiac function after
MI through the secretion of HGF and VEGF (Miyahara et al., 2006),
while intramyocardial injection of amniotic MSCs did increase the
levels not only of VEGF but also of Ang-1 in the affected heart tissue
(Kim et al., 2013). In a hamster model of cardiomyopathy, injection
of porcine BM-MSCs into the hamstring skeletal muscles improved
cardiac repair. In this model, MSCs secreted multiple IL-6-like cyto-
kines, which induced activation of the JAK/STAT pathway and in-
creased the levels of VEGF and HGF in the cardiac tissue. The
reparative impact of MSCs on heart failure was reversed by addition
of the JAK/STAT pathway inhibitor WP1066 (Shabbir et al., 2010).
Furthermore, a recent meta-analysis of large animal models (such
as pigs, dogs and sheep) of MI indicated that the transplantation of
BM-MSCs leads to a significant improvement of left ventricle ejection
fraction. In addition, a trend towards greater advantage in the case of
administration of MSCs when compared with BM-mononuclear cells
was reported (van der Spoel et al., 2011). In all available documenta-
tions, the ability of the engrafted MSCs to transdifferentiate/fuse into
cardiomyocytes was found to be extremely scarce (Dai et al., 2005;
Gandia et al., 2008; van der Spoel et al., 2011). In contrast to the re-
ports of Otsu et al., who claimed that high numbers of administrated
MSC can cause toxicity, the meta-analysis of MI models of large ani-
mals indicated that the best results were obtained with the highest
amount of injected cells (Otsu et al., 2009; van der Spoel et al.,
2011). This contradiction might be explained by the differences in
animal model or by alternative ways of administration. Indeed, as in
MI, cells are mostly injected intramyocardially or in the intracoronary
artery (and immediately diluted into the blood flow or a larger tissue
area), whereas in the study of Otsu et al., MSCs were locally applied.
Currently, the safety, dose and impact associated with MSC-
treatment for MI in humans are intensively studied and already a few
clinical trials have been published (Tongers et al., 2011; Ranganath
et al., 2012). For example, a double-blinded, randomized clinical trial
by Hare et al. (2009) indicated that intravenous allogenic BM-MSC
treatment is safe and that cell-treated patients had improved outcomes
with regard to cardiac arrhythmias, pulmonary function and left
ventricular function compared to the placebo group. In addition, S. L.
Chen et al. (2004) showed that intracoronary application of BM-MSCs
also resulted in improved left ventricular function and exercise
capability.
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
3.1.4.3. Cerebral ischemia/stroke. Stroke, also referred to as encephalic
vascular accident, is a focal neurologic deficit caused by a disturbance
in the blood supply of the brain. The most recurrent type of stroke is is-
chemic stroke, caused by a blockage of the blood circulation by a throm-
bosis or an arterial embolism leading to a decrease in oxygen and
nutrient delivery to the brain. Depending on the affected brain area, is-
chemic stroke is associated with temporary or permanent loss of brain
tissue and function, resulting in a wide variety of devastating symptoms
comprising motor, sensory, balance and cognitive dysfunction. Follow-
ing MI, stroke is the most common cause of death worldwide and the
most common cause of adult disability, with 250,000 people in Europe
becoming disabled after their first stroke each year.
Currently, tissue plasminogen activator (tPA) is the only available
intervention to reduce the infarct size (Tissue plasminogen activator
for acute ischemic stroke. The National Institute of Neurological
Disorders and Stroke rt-PA Stroke Study Group, 1995). However, op-
timal results are only observed if tPA is given within 90 min follow-
ing the primary insult (Hacke et al., 2004), thereby strongly
reducing its therapeutic capacities. This narrow therapeutic window
in combination with many contraindications makes tPA therapy only
available to about 5% of stroke victims (Brown, 2005). Therefore,
there is still a high demand for novel treatment options that aim at
long-term recovery of the affected brain area. MSC therapy might
offer an alternative approach to induce functional recovery by
means of improving revascularization, affecting neural plasticity or
inducing cell replacement near the stroke area. Over the years,
many animal stroke models have been developed in order to simu-
late what happens during stroke in humans. Among them, the tran-
sient middle cerebral artery occlusion (tMCAO) model is by far the
most frequently used. The main advantage of the tMCAO model lies
in its physiological relevance being the occlusion of one of the main
cerebral arteries. Other models include a photochemical, autologous
thromboembolic and the more recently developed endothelin-1
model (Kudo et al., 1982; De Ryck et al., 1989; Windle et al., 2006).
Numerous reports demonstrated that delivery of BM-MSCs by a
wide variety of routes (intravenous, intra-arterial and intracerebral)
reduced infarct size and improved the functional outcome in ische-
mic stroke (Banerjee et al., 2012; Ma et al., 2013). Although the ini-
tial idea was that MSCs would enhance recovery after stroke by
transdifferentiation into neurons and glial cells, it is now suggested
that MSCs have a beneficial impact primarily through the inhibition
of apoptosis, neuroprotection and enhancement of endogenous
neurogenesis and angiogenesis (Zhang et al., 2009; Banerjee et al.,
2012). Indeed, multiple studies describe the production of angiogenic
factors by MSCs indicating a key role for these factors in the improved
recovery seen in transplanted stroke animals (Kurozumi et al., 2005).
Chen et al. (2003) demonstrated in a tMCAO rat model of ischemic
stroke, that intravenous injection of human BM-MSCs enhanced angio-
genesis in the ischemic brain, probably by increasing the endogenous
VEGF and VEGFR2 expression. Another research group reported a sig-
nificant upregulation of Ang-1 and Ang-2 mRNAs in BM-MSC treated
rats using the same animal model (Ma et al., 2013). Moreover, MSCs iso-
lated from the peripheral blood reduced lesion volume, improved re-
gional cerebral blood flow and stimulated functional improvement
after transplantation (Ukai et al., 2007). A recent study compared the
beneficial effect of intravenous administration of BM-MSCs and AD-
MSCs in a rat stroke model. AD-MSCs were found to be as effective as
BM-MSCs in promoting functional recovery, reducing cell death as
well as increasing cellular proliferation, neurogenesis, and the expres-
sion of angiogenesis markers (such as VEGF) at 14 days post-
infarction (Gutierrez-Fernandez et al., 2013). Even the intraperitoneal
injection of a cell-free extract derived from MSCs significantly de-
creased the ischemic volume and improved motor function (D. Jeon
et al., 2013).
This extensive preclinical work on MSC treatment in stroke, encour-
aged the study of these promising cells in phase I/II clinical trials
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
(Banerjee et al., 2012). A small study with 5 patients that intravenously
received autologous BM-MSCs, showed that the MSC-treated group had
the tendency to have a better functional outcome, although this differ-
ence was not statistically significant (Bang et al., 2005). The same
authors published the results of a long-term follow-up study of intrave-
nous autologous BM-MSC transplantation in patients with ischemic
stroke. These experiments revealed that the BM-MSC treatment im-
proved functional outcome and survival, even 5 years after MSC admin-
istration (J. S. Lee et al., 2010). A recent non-blinded trial (in 12 patients)
with autoserum-expanded autologous BM-MSCs, indicated that intra-
venous administration of the cells 36–133 days after stroke was not
only safe and feasible, but also could reduce infarct size by 20% at one
week post-infusion (Honmou et al., 2011). Although these clinical trials
had a small sample size and the study of Honmou et al. was non-blinded
and did not exclude placebo effects, all investigations demonstrated the
potential benefit of MSC-treatment in stroke and justified additional
blinded, placebo-controlled studies.
3.1.4.4. Ambiguous role of MSC in tumor formation. As MSCs are able to in-
duce blood vessel formation in vivo and since tumor growth and metas-
tases are angiogenesis-dependent, the contribution of MSC to cancer
development has been the subject of intense investigations. Although
several studies suggest that MSCs have a tumor-suppressive function
(Khakoo et al., 2006; Otsu et al., 2009), other reports indicate that
MSCs promote tumor growth in various cancer models such as adeno-
carcinoma (E. S. Jeon et al., 2010), melanoma (Djouad et al., 2003) and
breast cancer (Galie et al., 2008) (reviewed in Klopp et al., 2011 and
Cuiffo & Karnoub, 2012). These contradictory results might be explained
by variations in MSC source, isolation method of MSCs, tumor cell type,
contamination of the applied MSCs with tumor cells, and differences in
timing, dose and mode (systemic injection versus local application into
the tumor) of MSC administration (Klopp et al., 2011). Although the
molecular mechanisms behind the tumor-promoting capacities of
MSCs are still not fully understood, several papers indicate that MSCs in-
deed stimulate tumor growth by enhancing angiogenic processes (Jeon
et al., 2010; Liu et al., 2011; Suzuki et al., 2011; Cuiffo & Karnoub, 2012).
For example, Y. Liu et al. (2011) demonstrated that VEGF or HIF-1α
siRNA effectively reduced MSC-favored colon tumor growth in vivo.
Other proposed mechanisms of promoting tumor progression include
malignant transformation of MSCs, protection against apoptosis, vascu-
lar support and immunomodulation. The tumor-suppressing capacities
of MSCs, on the other hand, are attributed to induction of apoptosis and
inflammation, inhibition of angiogenesis and alteration of the cell cycle
progression (Klopp et al., 2011).
Another intriguing capacity of MSCs is that they are able to home to
developing tumors with great affinity. They do selectively home not
only to solid tumors, but also to metastases far removed from the prima-
ry site of the tumor (Nakamizo et al., 2005; Dwyer et al., 2010). Accord-
ingly, they have been explored as delivery vehicles of anticancer (gene)
therapy (see Section 4). Before MSCs find their way to the clinic as a
therapeutic tool in the fight against cancer, the exact conditions under
which MSCs are able to promote tumor growth have yet to be fully elu-
cidated. Nevertheless, a recent systemic meta-analysis of clinical trials
with MSCs showed that, so far, no de novo malignancies in patients
have been reported in over 1000 patients treated with MSCs for a vari-
ety of disorders (Lalu et al., 2012).
3.2. MSC transdifferentiation towards endothelial cells (EC)
3.2.1. EC differentiation in vitro
As previously mentioned, the differentiation of MSCs towards EC,
both in vitro and after transplantation, remains a controversial as-
pect. Supported by the idea of the tremendous plasticity of MSCs,
as they were proposed to have the ability to transdifferentiate into
other cell types than mesodermal lineage cells, numerous efforts
have been made to differentiate MSC to EC in vitro (summarized in
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
9
Table 2). MSC commitment to EC is mostly demonstrated by the up-
regulation of typical EC surface molecules such as CD31,CD34, VEGF-
receptor1 (VEGFR1), VEGFR2 and vWF. As change in surface marker
profile is insufficient to evidence differentiation, functional tests are
also performed, including in vitro tube formation on matrigel and up-
take of acetylated-low density lipoproteins (although macrophages
and pericytes also internalize these molecules) (Voyta et al., 1984).
Based on these commonly used in vitro assays, it might be inaccurate
to designate these differentiated MSCs as fully mature and functional
EC. It is therefore more correct to define these cells as EC-like cells.
Although the in vitro differentiation approaches listed in Table 2
demonstrate indications for EC-like characteristics such as EC mark-
er expression, matrigel tube formation and Ac-LDL uptake, there is
almost no literature available which shows morphological and ultra-
structural evidence of EC transdifferentiation. For example, the pres-
ence of typical EC characteristics such as Weibel–Palade bodies and
tight junctions, which could be demonstrated by transmission elec-
tron microscopy, have never been reported for EC-directed MSCs
(except in Jazayeri et al., 2008).
Skewing MSCs to EC differentiation has been commonly achieved
by using VEGF. In one of the earliest attempts of EC differentiation, it
was demonstrated that confluent hBM-MSCs grown in medium with
2% FCS and 50 ng/ml VEGF for 7 days, displayed a substantial increase in
EC surface markers such as VEGFR1, VEGFR2, VE-Cadherin, VCAM-1 and
vWF. Moreover, when incubated on matrigel in vitro, treated BM-MSCs
formed characteristic capillary-like structures. These differentiation ex-
periments were only performed with cells at low passage number
(below 5 passages) (Oswald et al., 2004). Furthermore, hBM-MSCs in-
cubated in medium with 5% FCS, IGF and VEGF were immunopositive
for CD31, vWF, Tie2, VCAM1 and VE-cadherin. Electron microscopic
analysis showed the presence of typical EC morphological features
including Weibel–Palade bodies, tight junctions and caveolae
(Jazayeri et al., 2008). In addition, multipotent stem cells isolated
from the placenta (hPMSCs) showed an increased expression of EC
markers CD31, CD34, VEGFR1 and VEGFR2 after incubation with
50 ng/ml VEGF (M. Y. Lee et al., 2009). Recently, Bento et al. de-
scribed that MSCs derived from exfoliated teeth (the so-called milk
teeth), differentiated into VEGFR2/CD31 positive EC-like cells. Inhibi-
tion of ERK signaling with siRNA against MEK1 or with the chemical
compound U0126 completely abrogated this differentiation, unveiling
that the VEGF/MEK1/ERK signaling transduction cascade is key in EC
commitment of this type of MSC (Bento et al., 2013). Furthermore,
hBM-MSCs cultured for 3 weeks in ‘endothelial growth medium-2’
(EGM-2, which contains VEGF, EGF, FGF-2, IGF-1, hydrocortisone, hepa-
rin, ascorbic acid and 2% FCS), displayed significantly elevated expres-
sion of CD31, CD144, VEGFR2, CD105 and CD34 and were also able to
form tubes on matrigel in vitro (J. W. Liu et al., 2007). In contrast,
Roobrouck et al. (2011) demonstrated that incubation of hBM-MSC in
basal medium containing VEGF significantly increased mRNA expres-
sion of CD34, VEGFR1, and VEGFR2 but not of Tie-2 and vWF and the
mRNA level of CD31 was even decreased. These hBM-MSC did not
form a tubular network when plated onto matrigel in vitro, thereby
failing the functional test of full EC differentiation (Roobrouck
et al., 2011).
Several other recent reports question the role of VEGF in EC com-
mitment of MSCs. For example, Fan et al. (2011) demonstrated that
hBM-MSCs cultured in the presence of 20, 40 or 80 ng/ml of VEGF
had no increase in CD31, vWF or VEGFR2. Furthermore, Galas and
Liu suggested that VEGF treatment does not accelerate EC differentia-
tion of MSCs: confluent BM-MSCs incubated with EGM-2 had increased
PECAM and vWF mRNA levels, VE-cadherin, VEGFR2 and vWF protein
expression and Ac-LDL uptake compared to untreated (and proliferat-
ing) BM-MSCs. Addition of VEGF (at 50 or 100 ng/ml) to this EGM-2 me-
dium did not further accelerate EC marker expression. These data
suggest that either a component of EGM-2 or the confluent MSC cell–
cell contacts induced the EC-specification of BM-MSC (Galas & Liu,
 10
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate

Table 2
Media used to induce EC transdifferentiation of MSCs in vitro.

Reference MSC type Incubation Differentiation medium Resulting marker expression Functional evidence of EC
time transformation

Al-Kaldi et al., 2003b mBM-MSC 14 days 10% FCS, 50 ng/ml VEGF CD31 Matrigel tube formation
Alviano et al., 2007 hAM-MSC 7 days 2% FCS and 50 ng/ml VEGF VEGFR1,VEGFR2, vWF, CD34, ICAM-1 Matrigel tube formation

A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Bai et al., 2010 rBM-MSC 7 days VEGF alone CD31, Factor VII, VEGFR2, tPA ND
Bai et al., 2010 rBM-MSC 8 days Shear stress (24 h) + VEGF (7 days) CD31, Factor VII, VEGFR2, tPA ND
Bento et al., 2013 hSHED 28 days EGM-2 + 50 ng/ml VEGF VEGFR2, CD31 Elongated morphology
Chen et al., 2009 BM-MSC 12 days 5% FCS + 100 ng/ml VEGF + 50 ng/ml EGF + 1 μg/ml VEGFR2, vWF, VE cadherin Matrigel tube formation and LDL uptake
hydrocortisone
Chen et al., 2009 WJ-MSC 12 days 5% FCS + 100 ng/ml VEGF + 50 ng/ml EGF + 1 μg/ml VEGFR2, vWF, VE cadherin Matrigel tube formation and LDL uptake
hydrocortisone
Chung et al., 2009 hBM-MSC 21 days EGF + VEGF + hydrocortisone VEGF, vWF, VE-cadherin Matrigel tube formation
Fischer et al., 2009 hAD-MSC 18 days ECGS (14 days) + shear stress (4 days) CD31 LDL uptake
Gang et al., 2006 UM-BMSC 21 days EGF + VEGF + hydrocortisone VEGFR1, VEGFR2, VE-Cadherin, vWF, VCAM-1, Tie-1 and Matrigel tube formation and LDL uptake
Tie-2
Iohara et al., 2008 DPSC 10 days On matrigel, EGM-2 CEACAM-1, occludin, CD146 (mRNA) Matrigel tube formation
Iohara et al., 2008 DPSC 3 days EBM-2 + 2% serum + 10 ng/ml VEGF + 10 ng/ml FGF-2 vWF, VE-cadherin, CD31 Matrigel tube formation and LDL uptake
Jazayeri et al., 2008 BM-MSC 5 days 5% FCS, 50 ng/ml VEGF and 20 ng/ml IGF-1 CD31, vWF, VEGFR2, VEGFR1, Tie2, VCAM1 and VE-cadherin Matrigel tube formation
Karbanova et al., 2011 hDPSC 10 days 10% FCS + 1% ITS + 20 ng/ml VEGF 10% of cells, upregulation of CD31, CD34, vWF ND
Konno et al., 2010 mAD-MSC 12 days EGM-2 BulletKit (without the FGF-2) + ITS + 10 ng/ml FGF-2 CD34, Tie-2, VEGFR2, VE-cadherin Matrigel tube formation and LDL uptake
M.Y. Lee et al., 2009 Placental MSC 21 days EGM-2 + 2% FCS + 50 ng/ml VEGF CD31, CD34, VEGFR-1 and VEGFR2, vWF and VE-cadherin ND
Liu et al., 2007 hBM-MSC 21 days EGM-2 + 2% FCS CD31, CD144, VEGFR2, CD105 and CD34 Matrigel tube formation and LDL uptake
Marchionni et al., 2009 hDPSC 7 days 2% FCS and 50 ng/ml VEGF vWF,ICAM-1,CD34 Matrigel tube formation
Moon et al., 2006 hAD-MSC 2 days On matrigel, EGM-2 + 50 ng/ml VEGF + 10 ng/ml FGF-2 vWF ND
Oswald et al., 2004 hBM-MSC 7 days 2% FCS + 50 ng/ml VEGF VEGFR1, VEGFR2, VE-Cadherin, VCAM-1 and vWF Matrigel tube formation
Pankajakshan et al., 2013 Porcine BM-MSC 10 days EGM-2 + 50 ng/ml VEGF vWF, VE-cadherin, CD31 Matrigel tube formation and LDL uptake
Portalska et al., 2013 hBM-MSC 11 days EGM-2 + shear stress (10 days) + 1 day on matrigel VEGFR1, vWF, CD31 LDL uptake
Roobrouck et al., 2011 hBM-MSC 14 days Basal medium + 100 ng/ml VEGF CD34, VEGFR1, and VEGFR2 but not of Tie-2 and vWF Failure matrigel tube formation
Takahashi et al., 2010 AD-MSC 7 days EGM-2 + 5% FCS VEGFR1 and Ang-1 not VEGFR2, VE-cadherin nor CD31 ND
Whyte et al., 2011 hBM-MSC 14 or 28 days Basal medium VE-cadherin, PECAM-1, vWF, and VEGFR1 Matrigel tube formation and LDL uptake
Wu et al., 2005 rBM-MSC 5 days Co-culture with rabbit EC VEGFR1, no vWF ND
Xu et al., 2008 rBM-MSC 7 days 2% FCS + 0.5 μM simvastatin CD31, vWF, VE-cadherin, VEGFR1, VEGFR2 Matrigel tube formation
Zhang et al., 2008 hBM-MSC 21 days 5% FCS + 50 ng/ml VEGF vWF, VEGFR1,VEGFR2, Tie-1, Tie-2 Matrigel tube formation and LDL uptake
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
2014). This is in agreement with a study of Whyte et al., who could
prove that culture density of BM-MSC regulates EC commitment.
Human BM-MSCs incubated at high densities (10,000 cells/cm2) for
14 or 28 days obtained an EC-like phenotype expressing VE-cadherin,
PECAM-1, vWF, and VEGFR1, displaying Ac-LDL-uptake and forming
tubes in the matrigel assay. These high density plated hBM-MSCs had
an increased expression of VEGF. This autocrine VEGF secretion
plays a role in the EC differentiation of hBM-MSCs as inhibition of
VEGF with neutralizing antibodies resulted in a decrease of VEGFR1
or VE-cadherin. Nevertheless, incubation of low density plated BM-
MSC with VEGF did not induce EC marker expression. Taken togeth-
er, these data indicate that VEGF alone is not sufficient to initiate EC
differentiation, but this growth factor enhances EC commitment in
high cell density cultures (Whyte et al., 2011).
These contradictory findings on the role of VEGF in EC differentiation
of MSCs might be caused by the heterogeneity of MSC, differences in
MSC isolation, tissue sources and species, differences in assays used to
demonstrate EC characteristics and artifacts associated with culturing
techniques. It might also be worth to mention that not in all cases and
reports MSCs have VEGF-receptors (Whyte et al., 2011). In addition
to VEGFR1 and VEGFR2, VEGF is able to bind various co-receptors in-
cluding neuropilins and heparan sulfate proteoglycans (Ruiz de
Almodovar et al., 2009). It is possible that, depending on the receptor
profile, MSCs respond differently to soluble VEGF in the medium.
Xu et al. demonstrated that the Notch-signaling pathway plays a key
role in MSC commitment to EC. Rat BM-MSCs incubated for 7 days in 2%
FCS and 0.5 μM simvastatin (a commonly used cholesterol-lowering
agent), had increased surface expression of CD31, vWF, VE-cadherin,
VEGFR1 and VEGFR2 and showed tube formation on the matrigel
assay. Targeted inhibition of Notch signaling with Notch 1 siRNA
suppressed this EC differentiation (Xu et al., 2009). Moreover, the
aforementioned study of Whyte et al. also showed the fundamental
role of Notch signaling in EC differentiation. High density plated
hBM-MSCs were immunopositive for Notch transcription factor HES-1
and the Notch receptors 1, 2 and 3 during EC specification. Knockdown
of the Notch receptors with specific siRNA or addition of the chemical
Notch inhibitor DAPT abrogated the EC commitment of these hBM-
MSCs. In addition, incubation of hBM-MSCs seeded at low density
with EDTA (which activates Notch) for 24 h resulted in an upregulation
of vWF and VEGFR1 (Whyte et al., 2011). In addition, it was shown that
the Notch-pathway in cross-talk with VEGF signaling, is also involved in
the arterial-venous fate of EC-differentiated BM-MSCs. Treatment of
confluent BM-MSC with 50 ng/ml VEGF for 14 days increased expres-
sion of the venous marker EphB4, while at the concentration of
100 ng/ml, venous ephrin B4 expression decreased while the arterial
markers ephrin B2, Dll4 and Notch4 were strongly upregulated. This
VEGF-dependent arteriogenesis was blocked by inhibition of the
Notch pathway, resulting in a shift from arterial to venous EC fate
(G. Zhang et al., 2008). A recent study by Chung et al. showed that
during endothelial differentiation, the expression pattern of 4 HOX
genes changed significantly: incubation of hBM-MSCs with EGF and
VEGF for 21 days resulted in a dramatic increase of HOXA7 and
HOXB3 while HOXA3 and HOXB13 decreased. Although the exact
role of each HOX gene in EC differentiation remains to be elucidated,
these data indicate that these HOX genes might be involved in EC
commitment of MSCs (Chung et al., 2009).
Furthermore, Lozito et al. described that hBM-MSCs expressed EC
marker CD31 when directly co-cultured with EC. As this effect was not
seen in the indirect co-culture systems but could be mimicked by grow-
ing the BM-MSC on decellularized EC matrices, this indicated that not
the soluble factors but EC-matrix acts as a critical regulator of BM-MSC
commitment to EC (Lozito et al., 2009).
Classically, the control of stem cell fate has been mainly assigned
to molecular mediators such as growth factors. New data indicate
that in addition to soluble factors, physical cues can also provide a
successful method to induce MSC EC differentiation. Such tools
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
11
include mechanical stimuli (Maul et al., 2011), shear stress (Fischer
et al., 2009; Bai et al., 2010) and seeding the cells on elastic nanofiber
hydrogels (Wingate et al., 2012) or on 3-dimensional matrices
(Valarmathi et al., 2009; Zhang et al., 2010). For example, placenta-
derived MSC subjected to shear stress for 24 h showed a significant
increase in vWF and PECAM-1 expression, LDL-uptake and matrigel
tube formation (Wu et al., 2008). Despite the characterization of sev-
eral key actors for EC commitment such as the Notch signal cascade,
additional cues for functional and homogenous EC differentiation
must be identified to fully understand the underlying molecular
mechanisms. Successful homogenous EC differentiation of MSCs
could be of great use for the development of functional blood vessels
in the field of tissue engineering.
3.2.2. EC differentiation in vivo
As described above, the rate of engraftment of MSCs remains very
low in most settings. Therefore, it is assumed that the angiogenic effect
of MSCs is predominantly caused by their paracrine actions rather than
their EC transdifferentiation potential. For example, Al-Khaldi et al.
showed that by using LacZ transgenic BM-MSCs in the murine matrigel
plug assay over 99% of the generated blood vessels originated from
host-derived EC and that transdifferentiation of BM-MSCs towards EC
in that setting was very rare. Only 10% of injected BM-MSCs were
found in the close proximity of- or in blood vessels (Al-Khaldi et al.,
2003b). Another striking example is a study with GFP + BM-MSC in
the treatment of wounds in diabetic mice. The rate of MSC engraftment
28 days post-injection was extremely low (less than 1%) and no partic-
ipation of the transplanted GFP + BM-MSCs into the host vasculature
was observed (Wu et al., 2007). Although the low engraftment of
MSCs in most settings and the lack of incorporation into host blood
vessels is abundantly described, it may not be ruled out that in par-
ticular settings or time frames, the EC differentiation of MSC exists.
For example, a recent study by Yue, where rBM-MSCs were injected
in a rat vein grafting model, showed that the injected DAPI labeled
BM-MSCs differentiated into EC 2 weeks after transplantation as
they were capable of adhering to the injured vascular wall and
showed immunopositivity for CD31 and endothelial nitric oxide syn-
thase (Yue et al., 2008).
Although the evidence of full EC differentiation of MSCs in vivo is
debatable, preconditioning MSCs in the so-called EC-differentiation
medium could be useful as this pretreatment might transform the
MSCs towards a more ‘angiogenic’ cell type. For example, placental
MSCs incubated with 50 ng/ml VEGF displayed higher angiogenic activ-
ity than normal placental MSCs in the CAM assay (M. Y. Lee et al., 2009).
Also Whyte et al. (2011) demonstrated that high density plated BM-
MSCs (which differentiate into EC-like cells) were found to induce sig-
nificantly more blood vessels in this in vivo assay. In addition, AD-
MSCs incubated with EGM-2 for 7 days, secreted higher amounts of
angiopoietin-1, promoted HUVEC migration and significantly sup-
pressed neointimal formation in a wire injury model of the rat femoral
artery compared to control AD-MSCs (Takahashi et al., 2010).
4. MSC as vehicles for targeted
(anti-)angiogenic drug and gene delivery
Although MSCs naturally possess an enormous therapeutic poten-
tial, gene therapy is applied to modify MSCs to further improve their ef-
ficacy and even extend the spectrum of diseases for which MSCs could
provide a successful cure. MSCs can be readily transduced with all the
clinically available viral vector systems including those based upon ret-
rovirus, lentivirus, adenovirus and adeno-associated virus. Viral trans-
duction of MSCs is relatively easy and results in efficient production of
cytoplasmic, membrane-bound and secreted protein products. As viral
vectors are able to integrate into the host genome, this method could re-
sult in a long-term gene expression in vivo (Porada & Almeida-Porada,
2010). Numerous animal studies have reported the success of MSCs as
12 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
a gene delivery vehicle in a wide variety of pathologies in vivo. How-
ever, many clinical trials in which viral vectors were used, have been
terminated since application of these vectors had induced adverse ef-
fects including toxicities, immune- and oncogenicity (Marshall, 1999;
Hacein-Bey-Abina et al., 2003; Santos et al., 2011). Therefore, an in-
creasing number of non-viral vectors are currently being developed
such as calcium phosphate, liposomes, niosomes and nanoparticles. As
the gene expression by such a non-viral system is transient and the
transfection efficiency is relatively low, current studies on these gene
delivery methods are still limited to in vitro settings (Hu et al., 2010).
4.1. MSCs designed as carriers for pro-angiogenic factors
MSCs genetically engineered to secrete cytokines and other angio-
genic factors have been used in a wide variety of animal models. For ex-
ample, using a lentiviral vector, Xu and coworkers designed BM-MSCs
overexpressing Ang-1, an angiogenic protein inducing endothelial sur-
vival and vascular stabilization. In an in vivo mouse model, the LPS-
induced lung injury was significantly alleviated in the group treated
with BM-MSCs carrying Ang1, compared with groups treated with
BM-MSCs or Ang1 alone (Xu et al., 2008). In a MCAO-induced rat stroke
model, animals receiving FGF-2-modified BM-MSCs demonstrated sig-
nificant functional recovery compared with rats treated with normal
BM-MSCs (Ikeda et al., 2005). In addition, both HGF- and VEGF-
overexpressing mBM-MSC treatments resulted in smaller scar sizes, in-
creased angiogenesis and better preserved left ventricular function
when compared with MSCs transfected with empty vector in a mouse
model of MI (Deuse et al., 2009).
4.2. MSCs engineered for anti-cancer treatments
As MSCs are able to migrate to the tumor side (cf. Section 3.1.4.4),
they represent a very powerful and unique vehicle to selectively de-
liver anti-cancer gene products. The delivery of interleukins via
MSCs has been explored in order to improve the anti-cancer surveil-
lance by activating immune cells such as cytotoxic lymphocytes and
natural killer cells (Shah, 2012). For example, interleukin-12 secret-
ing MSCs reduced tumor growth in renal cell and cervical tumor mice
models and prevented metastasis in mice bearing pre-established
metastases of melanoma, hepatoma and breast tumors (X. Chen
et al., 2008; Shah, 2012). Also treatment of MSCs containing anti-
proliferating and proapoptotic genes such as IFN-α, IFN-β and
TRAIL displayed a significant antitumor activity in several mouse
models. In addition, gene therapy is applied to specific target the
tumor-associated angiogenesis. In a recent study, it was shown that
single administration of stem cells overexpressing thrombospondin
markedly reduced tumor vessel-density and inhibited tumor-
progression in mice bearing human gliomas (van Eekelen et al.,
2010). Systemic administration of MSCs designed to express NK4,
an antagonist of HGF, efficiently decreased metastases by blocking an-
giogenesis and lymphangiogenesis in mice bearing lung metastases
(Kanehira et al., 2007). Another strategy in the fight against cancer is
the so-called prodrug therapy, which involves delivery of ‘suicide
genes’ encoding enzymes that are able to convert nontoxic prodrugs
into active antitumoral antimetabolites. Examples of such genes that
are currently investigated in clinical trials are cytosine deaminase and
herpes simplex virus thymidine kinase which respectively activate 5-
fluorocytosine and Ganciclovir (Menon et al., 2008; Shah, 2012). Com-
bined therapy of cytosine deaminase expressing AD-MSCs and the
prodrug 5-fluorocytosine resulted in a significant production of the che-
motherapeutic 5-fluorouracil and inhibited tumor formation in mouse
models of melanoma (Kucerova et al., 2008). In addition, BM-MSCs
overexpressing herpes simplex virus thymidine kinase injected into
the tumor or the vicinity of the tumor were shown to successfully re-
duce tumor volume in rats bearing glioma and treated with ganciclovir
(Miletic et al., 2007).
Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013
5. Therapeutic expectations and challenges
Although MSCs originally captured the attention as a therapeutic
agent for tissue repair due to their presumed in vivo plasticity, there is
now emerging evidence suggesting that immunomodulatory and para-
crine actions predominantly contribute to their broad therapeutic effi-
cacy. The last few years, intensive research has led to the identification
of a multitude of bioactive angiogenic molecules such as VEGF, FGF-2
and MCP-1 in the conditioned medium of MSCs. Additionally, MSCs
and the broad repertoire of angiogenic mediators they secrete, have
been shown to induce angiogenesis and thereby enhancing tissue repair
and functional outcome in a wide variety of pathologies associated with
insufficient angiogenesis.
Some unresolved issues, however, still impede these promising cells
to become a realistic treatment option for the future. The absence of a
unique marker of MSCs as well as the heterogeneity/inconsistency of
MSCs isolation and expansion techniques are still major hurdles,
which makes it a huge struggle to compare preclinical and clinical re-
sults from different research groups. Furthermore, as in most settings
the rate of engraftment of MSCs is extremely low, the question arises
concerning the fate of the administered MSCs. There is the possibility
that they home to other tissues or undergo necrosis or apoptosis. Ad-
vances in cell labeling and imaging techniques are essential to clarify
this issue. In addition, recent preclinical and clinical studies have now
begun to demonstrate that MSC treatment is safe and feasible, although
the long-term biosafety is still an essential concern and warrants further
long-term follow-up of the patients. Another key obstacle before MSC-
based treatment can enter the clinic as a standard therapy is the current
inefficient protocol of MSC expansion. Since the existing culture tech-
nology (serial passaging in plastic flasks) is still time-consuming and
costly, there is an urgent need to develop large scale-up production of
MSCs in serum-free conditions (for example in bioreactors).
Despite the increasing number of studies indicating the beneficial
effects of MSC treatment, optimization of these therapies remains a
huge challenge. Transduction of angiogenic factors in MSCs or pre-
conditioning of MSCs by hypoxia or certain chemical agents has
been shown to boost the angiogenic effects of MSCs, which might
be a valuable strategy to maximize their clinical potential (although
there might be some biosafety concerns, which also warrant in-
depth examination). Also, besides adapting the treatment protocol
to multiple administrations, the identification and use of a certain su-
perior subpopulation of MSCs could ameliorate existing MSC therapies.
Since the evidence arises that the paracrine mechanisms mostly ac-
count for the beneficial effects of MSCs, the use of the MSC secretome
instead of cellular therapy requires further exploration in clinical set-
tings. Since the application of autologous (or allogeneic) MSCs requires
the time-consuming expansion to sufficient numbers, the use of an allo-
geneic MSC secretome could overcome this problem. It could be pre-
pared in large amounts in advance and is then directly available as an
‘off-the-shelf’ product. This may be especially helpful in clinical settings
where immediate treatment is desired, for example after MI or acute
stroke. This secretome-approach could lead to the development of spe-
cialized stem cell products, designed to contain specific trophic factors
to suit the specific subtype of disorder. Nevertheless, clear understand-
ing of the in vivo MSC secretome and its potential effect on the microen-
vironment remain crucial before further (pre)clinical studies can be
performed.
In conclusion, over the last decade, tremendous advances have been
made in the understanding of the angiogenic role of MSCs. This is evi-
denced by the successful application and neovascularization in a wide
variety of in vivo settings of MI, stroke, ischemia and wound healing,
resulting in an increasing optimism of both basic scientists and clini-
cians. Though the road remains long and hard before these puzzling
cells can become a realistic everyday therapeutic option, routine clinical
application of MSCs for a wide variety of pathologies associated with
limited angiogenesis is an exciting prospect.
 A. Bronckaers et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
Conflicts of interest
The authors declare that there are no conflicts of interest
Acknowledgments
The preparation of the manuscript was supported by the Re-
search Foundation—Flanders (‘Fonds Wetenschappelijk onderzoek
Vlaanderen—FWO’, grant Nr. 1.5.060.13N). Petra Hilkens benefits
from a PhD scholarship of the FWO and Annelies Bronckaers is a
postdoctoral fellow of the FWO. The FWO was not involved in
study design collection, analysis and interpretation of the data, in
writing the report and in the decision to submit the paper for pub-
lication. We would also like to thank Nathalie Geurts for critical re-
vision of the manuscript.
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Please cite this article as: Bronckaers, A., et al., Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate
angiogenesis, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.013