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© Schattauer 2013 Thrombosis and Haemostasis 109.3/2013
1Theme Issue Article
The vascular biology of macrophage migration inhibitory factor (MIF)
Expression and effects in inflammation, atherogenesis and angiogenesis
Yaw Asare1; Martin Schmitt2,3; Jürgen Bernhagen1
1Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen, Germany; 2Institute of Molecular Cardiovascular Research (IMCAR), RWTH Aachen
University, Aachen, Germany; 3Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands
Summary
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine
with chemokine-like functions. MIF is a critical mediator of the host
immune and inflammatory response. Dysregulated MIF expression has
been demonstrated to contribute to various acute and chronic inflam-
matory conditions as well as cancer development. More recently, MIF
has been identified as an important pro-atherogenic factor. Its block-
ade could even aid plaque regression in advanced atherosclerosis.
Promotion of atherogenic leukocyte recruitment processes has been
recognised as a major underlying mechanism of MIF in vascular pa-
thology. However, MIF’s role in vascular biology is not limited to im-
mune cell recruitment as recent evidence also points to a role for this
mediator in neo-angiogenesis / vasculogenesis by endothelial cell acti-
vation and endothelial progenitor cell recruitment. On the basis of in-
troducing MIF’s chemokine-like functions, the current article focusses
on MIF’s role in vascular biology and pathology.
Keywords
Chemokine, monocyte/macrophage, cardiovascular disease, athero-
sclerosis, (neo-)angiogenesis/vasculogenesis
Correspondence to:
Univ.-Prof. Dr. rer. nat. Jürgen Bernhagen
Institute of Biochemistry and Molecular Cell Biology
RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany
Tel.: +49 241 80 88 840/31/41, Fax: +49 241 80 82 427
E-mail: jbernhagen@ukaachen.de
Received: November 18, 2012
Accepted after minor revision: December 3, 2012
Prepublished online: January 17, 2013
doi:10.1160/TH12-11-0831
Thromb Haemost 2013; 109: ■■■
Introduction:
chemokines and vascular biology
The processes governing vascular homeostasis, vascular repair
after acute injury and vascular remodelling during chronic disease
are controlled and driven by a plethora of factors. Among them
chemokines play a pivotal role at all levels of regulation.
Chemokines are small chemoattractant cytokines which have a
molecular weight of 8-12 kDa. Chemokines exert a broad variety
of functions in physiology and pathophysiology. In the context of
the current review article, we will focus on their role in leukocyte
chemotaxis, extravasation, as well as augmentation and/or attenu-
ation of angiogenesis (1-3). Based on the arrangement of four con-
served cysteine residues, chemokines are divided into four families
(CC, CXC, CX3C, and XC) (4). Numerous examples of key roles of
chemokines in vascular function, atherogenesis and vascular re-
pair exist. A joint role for several of these molecular players can
easily be rationalised, if one considers that the different steps of
leukocyte recruitment process, i.e. rolling, firm adhesion, and
transmigration are controlled by functionally specialised chemo-
kines, which act in a sequential and cooperative manner. However,
in the context of this review, we can only refer to some excellent
previous review articles covering the aspects of a balance between
chemokine redundancy and cooperativity in both the mainten-
ance of vascular homeostasis and vascular pathology (3, 5-8).
Chemokine structure and function may be modulated by di-,
tetrameric or perhaps even higher-order interactions. These inter-
actions are usually homomeric (for a comprehensive review please
see [9]), but chemokine function can also be modulated by hetero-
merisation, e.g. between CC and CXC chemokines. This may serve
to promote chemokine/receptor interactions. An example for the
latter is the heterodimerisation between CXCL4/PF4 and
CCL5/RANTES, which takes place in α-granules of human pla-
telets deposited at the glycosaminoglycan surface of endothelial
cells in vivo. In vitro and in vivo studies revealed a pathophysiologi-
cal function of such chemokine heterodimers (10, 11). In fact, von
Hundelshausen et al. demonstrated that heterodimerisation of
CCL5 and CXCL4 enhances CCL5-mediated monocyte recruit-
ment, while Koenen et al. additionally identified peptidic in-
hibitors specifically interrupting the CCL5/CXCL4 interface and
inhibiting vascular lesion formation in atherosclerotic Apoe–/–
mice (10, 11).
A subfamily of the CXC chemokine family is known as the
ELR+ chemokines and is defined by a glutamic acid-leucine-argi-
nine (ELR)-motif near the CXC sequence. In contrast to non-ELR
chemokines (ELR– CXC chemokines), chemokines from the ELR+
subfamily are potent inducers of physiological and pathological
angiogenesis. They play important roles in diseases like cancer, fi-
broproliferative disorders and chronic inflammation like athero-
sclerosis (12-14). A representative example for an ELR+ chemo-
kine is CXCL8/interleukin (IL-8). Thus, besides being a potent
neutrophil chemoattractant, CXCL8 also plays an important role
in neovascularisation in human non-small cell lung cancer (15),
human gastrointestinal cancers (16) and human ovarian and pros-
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2Asare et al. Vascular biology of MIF
tate cancer (17), where the CXCL8-CXCR2 axis regulates tumour
angiogenesis accompanied by correlative reduction or enhance-
ment of tumour growth. Further evidence for CXCL8-mediated
angiogenesis in diseased tissue was found in human coronary ar-
tery plaques, where CXCL8 has been shown to be overexpressed
(18). This might point to CXCL8-mediated growth of intra-plaque
vessels and plaque destabilisation (19).
As an example for a prominent CC chemokine, the
CCL2/CCR2 ligand receptor axis is important in monocyte che-
moattraction and transendothelial migration into areas of vascular
inflammation. CCL2 expression has been shown in atherosclerotic
lesions, likely exacerbating lesion progression through extensive
monocyte attraction and also through triggering firm monocyte
adhesion to the inflamed endothelium (20, 21). A deletion or at-
tenuation of CCL2 expression in atherosclerotic mouse models re-
sulted in decreased lesion formation (22, 23).
In addition to the four canonical chemokine classes, a group of
molecules sharing functional similarities with chemokines has
emerged as a fifth subclass. This class has been referred to as ‘che-
mokine-like function’ (CLF) chemokines, non-canonical chemo-
kines, or micro-chemokines. By definition, the CLF family of che-
mokines encompasses certain inflammatory and immune proteins
that exhibit chemokine-like functions such as chemotactic proper-
ties or leukocyte arrest-promoting effects but which neither for-
mally carry the typical N-terminal cysteine motif of the classical
chemokines nor the chemokine fold. Most members of this func-
tional family have been found to act as non-canonical ligands for
classical chemokine receptors. CLF chemokines thus further ex-
pand the degree of redundancy and promiscuity in chemokine/
chemokine receptor interactions, with consequences for angiogen-
esis regulation, as discussed below.
Examples are a cleavage fragment of tyrosyl tRNA synthetase
(TyrRS) which has been shown to act as a non-canonical CXCR1
ligand via the presence of an ELR-like motif (24). The N-terminal
‘mini-TyrRS’ domain has proangiogenic properties and induces
neutrophil chemotaxis through interaction with CXCR1 but not
CXCR2 (24, 25). Interestingly, the ELR motif is only exposed and
available for interaction with CXCR1 in the cleaved fragment (26).
Similarly, autoantigenic aminoacyl-RS, released under apoptotic
conditions, have leukocyte recruitment properties by triggering
CC receptors. Both histidyl-RS and its N-terminal fragment are
chemoattractants for several CCR5-expressing immune cells. As-
paraginyl-RS interacts with CCR3 (27). For these aminoacyl-RS,
the presence of specific surface charge distributions has been sug-
gested to mediate chemokine receptor usage. Furthermore, the
human antimicrobial peptides β-defensin-1 and -2 were identified
as non-cognate ligands for CCR6, mediating chemotaxis (28).
Again, although the sequence similarity between the β-defensins
and CCL20 is limited, it appears that a cluster of cationic amino
acids and shared electrostatic charge patterns account for the over-
lap in chemotactic activities. Promiscuity and mimicry mechan-
isms not only can be found endogenously in the host, but there are
several examples of parasite or viral chemokine mimicry, en-
compassing both mimicry of classical host chemokine structures
and CLF-type mimicry mechanisms (29, 30). Most prominently,
HIV-1 capsid protein gp120 interacts with host CXCR4 (and
CCR5) to direct leukocyte infection (29, 31). The nuclear protein
high-mobility-group binding protein 1 (HMGB1) has been shown
to exert numerous extracellular inflammatory functions. HMGB1
signals through several receptors (32, 33) and most recently, het-
eromeric complex formation between HMGB1 and CXCL12 was
identified to mediate HMGB1 chemokine activities through
CXCR4 (34).
In this review, we will focus on macrophage migration in-
hibitory factor (MIF), one of the first cytokines to be discovered
(35) and another recent addition to the CLF family of chemokines
(30, 36). In the next chapters, we will briefly outline both the
physiology and the pathophysiologic roles of MIF in inflammatory
and vascular disease, including underlying mechanisms such as its
non-cognate interactions with the chemokine receptors CXCR2
and CXCR4. With regard to MIF’s mechanisms of action, we will
solely focus on aspects relating to vascular biology and patho-
physiology.
MIF: structure, mechanism of action, and role
in inflammatory disease
MIF is an evolutionarily-conserved protein that is abundantly ex-
pressed in humans and non-primate mammals. In addition to its
functions as cytokine/chemokine and angiogenic factor (see
below), it has been suggested that MIF also has anti-oxidative in-
tracellular effects. The MIF structure is unique among cytokines.
MIF consists of 114 amino acids and has a molecular weight of
12.5 kDa. The three-dimensional structure of human MIF shows
that MIF crystallises as a trimer of three identical subunits, but
studies at more physiological concentrations imply that the
monomer may have crucial functions in vivo as well. Despite its
wide tissue distribution, the secretion of MIF is tightly regulated,
with relevant triggers such as hypoxia/ischaemia or oxidised low-
density lipoprotein (oxLDL) of particular importance for this re-
view article. Moreover, MIF expression is strongly up-regulated in
several disease conditions most importantly in vascular pathology
and tumourigenesis (37-39).
CD74, the membrane-expressed form of invariant chain (Ii)
and an MHC class II chaperone, was identified as the first MIF
plasma membrane receptor (40). CD74 expression is typically re-
stricted to class II-positive cells, but under inflammatory condi-
tions as well as in several tumour cell types, CD74 can be up-regu-
lated, even in the absence of class II expression. MIF binds to
CD74 by high affinity interaction in the nanomolar range, but sig-
nalling additionally requires the recruitment of signalling-compet-
ent co-receptors such as CD44 or CXCR2 and CXCR4 (see below).
CD74 alone mediates MIF binding, but MIF-induced MAPK sig-
nalling requires the coexpression of CD44. MIF signalling through
CD74/CD44 also involves the serine phosphorylation of the cyto-
plasmic tails of CD74 and CD44 and the recruitment of a Src-type
tyrosine kinase (41). An architectural similarity between the MIF
monomer and the CXCL8 dimer instigated biochemical investi-
gations to probe potential interactions between MIF and CXCR2.
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Receptor binding studies then revealed that MIF engages in a non-
cognate, high affinity interaction with CXCR2 (42). CXCR2 had
been known to be promiscuous previously, as it also is the receptor
for six other ELR+ CXC chemokines including CXCL8. This as
well as numerous functional data then suggested that MIF belongs
to the family of CLF chemokines. The signal transduction path-
ways triggered by MIF/CXCR2 have not been studied systemati-
cally, but initial evidence shows that MIF binding to CXCR2 leads
to Gi coupling and can elicit calcium transients. A limited screen of
other chemokine receptors as well as an observed chemotactic ef-
fects of MIF on T cells, which do not express CXCR2 and only low
levels of CD74, unraveled yet another MIF/chemokine receptor in-
teraction, i.e. that with CXCR4. MIF/CXCR4 interaction is less af-
fine than that between MIF and CXCR2 but still in the nanomolar
range. Further research into the receptors of MIF revealed that
CD74 forms heteromeric complexes with either CXCR2 or CXCR4
(42, 43).
During inflammation (44), endothelial cells do not only get ac-
tivated but also adjust their phenotypes to the inflammatory re-
sponse (45). Activation of endothelial cells (ECs), however, con-
tributes to both acute and chronic inflammatory diseases such as
sepsis, inflammatory bowel disease, rheumatoid arthritis, inflam-
matory lung disease, and atherosclerosis (46). There is ample evi-
dence now that MIF is a key mediator of all of these disease condi-
tions (37, 38, 47-49). Thus, MIF has been proven to play a pivotal
role in the pathogenesis of both acute and chronic inflammatory
diseases. Two prominent examples are sepsis and rheumatoid ar-
thritis.
First evidence implicating MIF in systemic infection and sepsis
dates back two decades ago when pituitary-derived MIF was found
to contribute to serum levels of MIF in the post-acute phase of en-
dotoxaemia. Employing a mouse model of endotoxic shock, MIF
was found to promote lethal endotoxaemia in mice (50). Indeed,
several and diverse models of septic shock have demonstrated the
crucial role of MIF in mediating sepsis (51-53).
Rheumatoid arthritis, a systemic chronic inflammatory dis-
order of the joints, is characterised by key pathological events in-
cluding diapedesis of leukocytes and the active participation of cy-
tokines such as tumour necrosis factor (TNF). MIF has been ex-
tensively described to play a role in rheumatoid arthritis by e.g. in-
ducing the secretion of CCL2 and to promote TNF production to
amplify leukocyte recruitment at yet another level (38, 54). Of
note, polymorphisms in the MIF gene functionally enhancing the
transcriptional activity of MIF have been linked to increased dis-
ease severity of rheumatoid arthritis and other inflammatory con-
ditions (38, 55).
MIF in the vasculature
Vascular endothelial cells exhibit a profound heterogeneity and
organ specificity in terms of their phenotype and protein ex-
pression patterns. Depending on the vessel or tissue they inhabit,
ECs are either strictly continuous with tight junctions, e.g. to
maintain the blood-brain barrier or discontinuous in the case of
the liver to allow for maximal fluid exchange. On the other hand,
ECs lining the glomerulus are strongly fenestrated to allow for op-
timal filtration results. In large arteries, an additional requirement
to resist high pressure and shear flow is found. ECs express various
molecules that have been described to be pivotal in the pathogen-
esis of numerous vascular diseases such as atherosclerosis and an-
giogenesis. Among these molecules are various adhesion mol-
ecules, such as vascular cell adhesion molecules (VCAMs), inter-
cellular adhesion molecules (ICAMs), selectins, and junctional ad-
hesion molecules (JAMs).
The expression levels of MIF in human ECs of both microvas-
cular and large artery origin have been shown to be upregulated
upon treatment with oxLDL (56) or thrombin (57) in a time- and
dose-dependent manner, suggesting a role for MIF in the vascula-
ture. MIF released upon oxLDL stimulation contributes to athero-
genic leukocyte recruitment (56, 58) (
▶
Figure 1). Moreover, ex-
posing human ECs to hypoxia led to a release of substantial
amounts of MIF that was found to participate in the recruitment
and migration of endothelial progenitor cells (59). The expression
of MIF in the vasculature extends beyond the endothelial layer as
vascular smooth muscle cells (VSMCs) have also been shown to
express low levels of MIF (56); moreover, MIF expression was also
observed to be upregulated by oxLDL in VSMCs (60). Importantly,
VSMCs do not only express MIF but also migrate towards exogen-
ous MIF after 6 hours (h) of incubation (61) (
▶
Figure 1).
MIF signalling in the vasculature has also been pursued. Subse-
quent to showing MIF-induced expression of ICAM-1 in ECs (62),
Cheng et al. reported the expression of VCAM-1, E-selectin,
ICAM-1 and CCL2 to be MIF-dependent. Indeed, TNF-induced
leukocyte rolling and adhesion to MIF-depleted human umbilical
vein endothelial cells (HUVECs) were impaired, an observation
that was attributed to a reduction of p38 MAPK activation, result-
ing in reduced expression of chemokines and adhesion molecules
(63). In addition, MIF was found to upregulate mononuclear cell
adhesion molecules such as VCAM-1 and ICAM-1 in a Src-, nu-
clear factor-κB (NF-κB), and phosphatidylinositol-3-kinase
(PI3K)-dependent manner (64). The above findings are in line
with a previous report where oxLDL- and MIF-induced monocyte
arrest on HAoEC was abrogated by anti-MIF neutralising antibody
(58). Moreover, MIF blockade leads to reduced VSMC prolifer-
ation and neointimal thickening (65), while at later stages during
the atherogenic process MIF contributes to plaque instability (58).
Thus, it is not surprising that there is extensive literature on the
functional role of vascular MIF in the pathogenesis of vascular dis-
ease like atherosclerosis or neointimal growth after vascular injury.
MIF in atherosclerosis
Inflammatory processes are key contributors to the pathogenesis
of atherosclerosis (66). The proinflammatory chemokine-like cy-
tokine MIF has been broadly implicated in atherogenesis (39)
(
▶
Figure 1). Expression of MIF in developing atherosclerotic
plaques in humans was minimal in ECs and smooth muscle cells
(SMCs) in non-lesion-associated areas but was upregulated in
ECs, SMCs, macrophages and T cells upon atheroprogression sug-
gesting a role for MIF in plaque instability (56, 67). Interestingly,
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4Asare et al. Vascular biology of MIF
an interaction between MIF and CSN5/JAB1, a negative regulator
of NF-κB which is a key transcription factor involved in the in-
flammatory and immune processes associated with atherosclerosis
(68, 69), was revealed in human atherosclerotic lesions (56). As a
negative regulator of CSN5, MIF might synergise with JAB1 in
regulating NF-κB-driven inflammatory and immune signalling in
atherosclerosis (70).
Atherosclerosis may be preceeded by vascular injury and MIF
has been shown to regulate the biological response to the injured
tissue. In a carotid artery injury model of atherosclerosis-suscep-
tible mice, MIF was shown to potentiate neointimal thickening by
promoting the accumulation of inflammatory cells in the neointi-
ma and the proliferation of medial and intimal cells (65). Indeed,
neutralising MIF resulted in a reduction of neointimal macro-
phage content and an increase in SMCs and collagen type I content
in the neointimal lesions in a model of vascular injury-induced ac-
celerated lesion formation. This reduction in neointimal macro-
phage content was attributed to impaired monocyte recruitment as
exogenous MIF increased the number of monocytes adhering to
HAoECs, an effect that was dampened by neutralising MIF mAbs
(58). The notion that MIF is critical in the development of athero-
sclerosis was corroborated by Mif gene-inactivation in Ldlr–/–
mice. These mice showed impaired atherogenic diet-induced
lesion initiation and progression when compared with corre-
sponding wild-type mice. The ability of MIF to induce prolifer-
ation of SMCs which has previously been observed mainly by
using mAb was confirmed by this genetic study (71). In a sponta-
neous atherogenic Apoe–/– mouse model, MIF was found to be
elevated at the aortic wall and blocking aortic MIF with anti-MIF
neutralising antibody led to a reduction in intimal macrophage
content and inflammatory mediators (72). Notably, treatment with
a blocking antibody that targets MIF even resulted in athero-
sclerotic plaque regression and a more stable plaque phenotype in
diet-induced atherosclerotic Apoe–/– mice. This observation was
reasserted by the finding that MIF mediates atherogenic monocyte
and T cell recruitment in vivo by engaging its receptors CXCR2
and CXCR4, respectively (
▶
Figure 1). Dual action of MIF
through CXCR2 and CXCR4 also may explain why anti-MIF
Figure 1: Expression and functional role of vascular MIF in angiogen-
esis and atherosclerosis. Left (angiogenesis): After tissue injury, MIF along
with other chemokines such as CXCL12, CXCL1, and VEGF is released to acti-
vate and recruit EPCs and also monocytes to the site of injury where they are
embedded into tube structures. Importantly, MIF drives tube formation and
the formation of new vessels that follows. EPCs carry angiogenic factors
(‘cargo’) themselves. Right (atherosclerosis): MIF expressed by ECs and mac-
rophages in the atherosclerotic plaque is upregulated upon stimulation with
inflammatory and atherogenic mediators such as oxLDL and thrombin. MIF
induces the expression of chemokines (CCL2) and adhesion molecules
(VCAM-1, ICAM-1) which regulates the recruitment and adhesion of mono-
cytes to the surface of the endothelium. Alternatively, MIF may employ its
chemokine receptors CXCR2 and CXCR4 (expressed on recruited monocytes
and T cells, respectively; receptors not shown) in exerting its chemokine-like
functions. After transmigration into the subendothelial space, monocytes
subsequently differentiate into macrophages. MIF and other pro-atherogenic
factors (latter not shown) stimulate these macrophages to secrete TNF-
α
,
IL-1
β
, iNOS and NO, enhancing the inflammatory milieu in the lesion. Addi-
tionally, MIF potentiates foam cell formation by accelerating macrophage
oxLDL uptake, thereby enhancing lesion formation. VSMCs express and also
migrate towards MIF and this may contribute to plaque stability, although
long term exposure to MIF has also been shown to inhibit PDGF-BB-induced
VSMCs migration and MMP upregulation which points in the direction of
plaque destabilisation by MIF (for references see text).
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5Asare et al. Vascular biology of MIF
blockade was more efficacious in the clinically highly relevant re-
gression model than treatment with anti-CXCL1 or anti-CXCL12
(42). Indeed, a MIF N-loop-derived peptide that disrupts the inter-
action between MIF and CXCR2 blocked MIF-induced leukocyte
adhesion in carotid arteries in vivo (73).
Taken together, data from several groups using either neutralis-
ing antibody or genetic deletion underpins the pathogenic role of
MIF in promoting atherogenic changes in the arterial vessel wall.
Importantly, the proatherogenic role of MIF was confirmed in epi-
demiologic studies where single nucleotide polymorphisms
(SNPs) in the human MIF gene were identified as a risk factor for
coronary heart disease, showing an association of a haplotype con-
taining the rs755622C allele, which has been reported before to in-
crease the susceptibility for various other proinflammatory condi-
tions (74), and showing that the GG genotype of the MIF SNP
rs1007888 was associated with myocardial infarction (MI) in
Czech female patients (75). Moreover, Makino et al. showed that
high plasma levels of MIF are associated with adverse long-term
outcome in patients with stable coronary artery disease and im-
paired glucose tolerance or type 2 diabetes mellitus (76). Similarly,
Müller et al. found that MIF expression is enhanced in acute cor-
onary syndromes (ACS), that it is associated with various markers
of the inflammatory response, that it correlates with the extent of
cardiac necrosis marker release after percutaneous intervention
and that it is increased in ACS patients with respective lesions (77).
Role of MIF in (neo-)angiogenesis /
vasculogenesis
MIF’s role in atherogenesis has been extensively studied (see
above); however, its roles in the processes controlling physiologic
and pathophysiologic angiogenesis are less well understood.
Angiogenesis is the growth of blood vessels from a pre-existing
vasculature. It occurs throughout life and is an important compo-
nent of different physiological and pathophysiological conditions
such as wound healing and pregnancy (78). Regulation of angio-
genesis is achieved by balancing angiogenic and angiostatic
triggers. If not properly controlled, angiogenesis can promote to
tumour growth, rheumatic arthritis, and retinopathies (78). The
therapeutic value of angiogenesis has become of great interest. In-
hibiting or decreasing angiogenesis possesses therapeutic potential
in treating cancer and rheumatic arthritis, while stimulation of an-
giogenesis can be helpful in ischaemic heart disease, peripheral ar-
terial disease, and wound healing responses by increasing reperfu-
sion of the tissue. Moreover, vasculogenesis, a process formerly
considered to be restricted to the de novo formation of vascular
structures from mesenchymal angioblasts in early embryonic vas-
cular development (79, 80), has now also been discussed to occur
postnatally, where it is triggered by endothelial progenitor cells
(EPCs1) (81-84), opening-up promising novel therapeutic avenues
as well (85, 86).
Oxygen plays a crucial role in controlling angiogenesis. Hy-
poxic conditions stimulate vessel growth by activation of ECs (i.e.
proliferation and migration). The cellular response to hypoxia is
mediated via up-regulation of hypoxia-inducible transcription fac-
tors (HIFs), most prominently HIF-1α. HIFs upregulate the tran-
scription of numerous genes, thereby affecting endothelial cell
growth, SMC recruitment, and leukocyte attraction. HIF-1α is the
best characterised inducer of the expression of vascular endothelial
growth factor (VEGF), the major endothelial growth factor in an-
giogenesis and a key trigger of angiogenesis. Hypoxia-triggered
VEGF production and the subsequent increase in oxygen supply
following newly formed vessel growth reciprocally regulate each
other to restore homeostasis following limited blood supply
(86-88).
MIF was first implicated in angiogenesis some 14 years ago,
when Chesney et al. found that MIF blockade by a neutralising
antibody reduces tumour vascularisation and tumour growth in a
murine model of B-cell lymphoma (89). Although several distinct
mechanisms have been suggested to underlie MIF’s potent pro-tu-
mourigenic capacity, a role for MIF in tumour angiogenesis was
confirmed in a model of colon adenocarcinoma formation, in
which blockade of MIF led to reduced microvessel formation (90).
Moreover, MIF expression is seen in non–small-cell lung cancer, a
tumour entity in which MIF now has been firmly established as a
crucial player (91-95). MIF expression occurs in association with
angiogenic CXC chemokines and increased vessel density (70). As
discussed above, MIF is a non-cognate ligand for CXCR2, the cog-
nate receptor for angiogenic CXCL8, and MIF also promiscuously
engages CXCR4, the cognate receptor for CXCL12. Although
CXCL12 is an ELR– CXC chemokine, both CXCR2 and CXCR4
have been found involved in numerous pro-angiogenic effects in
various models of postnatal angiogenesis, including post-is-
chaemic adaption (71–73), underscoring the notion that the che-
mokine receptors of MIF could be critical in mediating MIF-
driven pro-angiogenic responses, although direct evidence from
knockout mouse models is yet missing for this assumption (
▶
Fig-
ure 1).
As for angiogenic factors such as VEGF, MIF expression was
also identified to be regulated by HIF-1α activity, as demonstrated
in lung tissue, ECs, hepatic stellate cells, and VSMCs (59, 96-98). A
number of studies have since confirmed MIF’s pro-angiogenic
properties and explored the underlying molecular and cellular
mechanisms. MIF mediates EC migration and tube formation in
matrigel assays and induces angiogenesis in matrigel plugs and the
cornea angiogenesis assay (
▶
Figure 1). These effects rely on mi-
togen-activated protein kinase (MAPK) and PI3K signalling, ac-
tivities known to be triggered by MIF (42, 99-101). Accordingly,
1 Endothelial progenitor cells (EPCs) were initially considered to represent a single
entity of progenitor cells capable of supporting post-natal de novo blood vessel
formation and have been assigned a crucial role in neo-angiogenesis. However,
since their discovery in 1997 their phenotype has been refined and become more
complex. In fact, EPCs exhibit different characteristics: (i) the so-called early out-
growth EPCs (EOCs) are derived from circulating CD34-positive mononuclear
cells and additionally express CD45 and CD14; they exert enhanced adhesion
proprieties but fail to proliferate in vitro; (ii) the so-called late outgrowth EPCs
(LOCs), lack hematopoietic markers but have the ability to proliferate. Both sub-
types respond to angiogenic stimuli, express CD31 and secrete angiogenic factors
such as VEGF and angiogenic cytokines/chemokines by themselves (79-82).
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MIF could be detected in the tumour-associated neovasculature
and neointima following vascular injury in pro-atherogenic mouse
models (58, 89, 99).
Tissue repair after MI heavily relies on neoangiogenesis of the
infarcted area. Chemokines and their receptors play important
roles during these ‘repair’ processes. Both exacerbating and pro-
healing responses occur. For example, the genetic absence of CC
chemokine receptor Ccr1 in a corresponding mouse model re-
duces functional impairment and structural remodelling after MI
due to an abrogated early inflammatory recruitment of neutro-
phils and improved tissue healing including vessel regeneration
(102). Interestingly, the CXCL12/MIF receptor CXCR4 was re-
cently found to have a dual role in neo-angiogenesis after MI.
CXCR4 was found to play a crucial role in endogenous remodell-
ing processes after MI, contributing to inflammatory/progenitor
cell recruitment and neovascularisation, whereas its deficiency li-
mits infarct size and causes adaptation to hypoxic stress (103).
MIF has now been amply implicated as a protective factor in
MI-ischaemia/reperfusion (I/R) injury. Although the protective
mechanisms involved have been suggested to span from AMP ki-
nase activation to promotion of anti-oxidative pathways
(104-107), it is likely that MIF-driven angiogenic/vasculogenic
processes are equally important. Indeed, MIF secreted from ECs
by hypoxic stimulation has been identified to promote EPC che-
motaxis in a CXCR4- (59) and CXCR2- (D. Simons and J. Bern-
hagen, unpublished observations) dependent manner (
▶
Figure
1). This finding was intriguing, because previously CXCL12 was
considered the main protagonist in driving EPC recruitment fol-
lowing hypoxic/ischaemic triggers (108, 109). However, these
seemingly divergent findings may be readily reconciled if one con-
siders the kinetics of chemokine production. Hypoxia-stimulated
HUVECs and human aortic endothelial cells (HAoECs) did not
secrete detectable CXCL12 levels within an early time window of 2
h, when MIF was predominantly secreted, peaking at 60 minutes
(59). This nicely coincides with the findings by Ceradini et al.
reporting that CXCL12 production from hypoxically challenged
HUVECs and ischaemic tissue in vivo occurred in a time interval
of 6-24 h and correlated subsequent EPC trafficking in vivo (109).
Interestingly, a second MIF secretion peak was observed 8 h after
the hypoxic trigger (59), indicating that within this intermediate
time window MIF and CXCL12 may jointly act to drive EPC re-
cruitment and neovascularisation (
▶
Figure 1).
EPC recruitment and neovascularisation also represent impor-
tant mechanisms driving the healing process of acute or chronic
skin wounds. Accordingly, MIF was defined as a potential inducer
of EPC mobilisation after flap operations. In flap patients, the
number of circulating EPCs and serum levels of MIF but not
CXCL12 serum levels was increased, especially in the patient
group of free microvascular flaps. Also serum MIF and EPC levels
correlated and MIF blockade, and to a lesser extent CXCL12 in-
hibition, partially reverted the chemotactic effect of patient serum
on isolated human EPCs (110). The study also indicated that MIF-
mediated EPC mobilisation is dependent on the degree of ischae-
mia (110).
As mentioned above, the neovascularisation potential of EPCs
is in part due to the fact that these cells are carriers of numerous
potent angiogenic/vasculogenic factors (‘angiogenic cargo’).
Prominent cargo factor are VEGF and thymosin-β4, but interest-
ingly also MIF and other chemokines (111, 112). The differential
usage of these factors was recently further refined by in vivo im-
plantation experiments, indicating that the MIF/chemokine recep-
tor axis has an important role in differentiation towards an en-
dothelial and SMC phenotype (113). As murine embryonic EPCs
were previously found to induce blood vessel growth and cardio-
protection under conditions of severe acute and chronic ischaemia
in a mouse I/R model and a rat hind-limb ischaemia model, the
above study confirms the therapeutic neovascularisation potential
of EPCs in combination with selected sets of angiogenic chemo-
kines/factors, including MIF (112, 113).
Acknowledgements
We thank Elisa Liehn, Heidi Noels, David Simons, Nancy Tuch-
scheerer and other doctoral researchers of the EuCAR program as
well as numerous (inter)national collaborators and friends for
helpful discussions over the past years. Our studies on the role of
MIF and other chemokines in neovascularisation and athero-
sclerosis were supported by an IZKF Aachen grant of the Faculty
of Medicine, RWTH Aachen University to J. B. and by Deutsche
Forschungsgemeinschaft (DFG) grants BE1977/4-2/FOR 809 to J.
B. and DFG-GRK1508/1 to J. B., Y. A., and M. S.
Conflicts of interest
None declared.
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