ATVB In Focus
Novel Mediators and Mechanisms in Angiogenesis and Vasculogenesis
Series Editor:
Stephanie Dimmeler
Previous Brief Reviews in this Series:
• Ferguson JE III, Kelley RW, Patterson C. Mechanisms of endothelial differentiation in embryonic vasculogenesis.
2005;26:2246 –2254.
• Werner N, Nickenig G. Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy?
2006;26:257–266.
Pericellular Proteases in Angiogenesis and Vasculogenesis
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Victor W.M. van Hinsbergh, Marten A. Engelse, Paul H.A. Quax
Abstract—Pericellular proteases play an important role in angiogenesis and vasculogenesis. They comprise (membranetype) matrix metalloproteinases [(MT-)MMPs], serine proteases, cysteine cathepsins, and membrane-bound aminopeptidases. Specific inhibitors regulate them. Major roles in initiating angiogenesis have been attributed to MT1-matrix
metalloproteinase (MMP), MMP-2, and MMP-9. Whereas MT-MMPs are membrane-bound by nature, MMP-2 and
MMP-9 can localize to the membrane by binding to ␣v␤3-integrin and CD44, respectively. Proteases switch on
neovascularization by activation, liberation, and modification of angiogenic growth factors and degradation of the
endothelial and interstitial matrix. They also modify the properties of angiogenic growth factors and cytokines.
Neovascularization requires cell migration, which depends on the assembly of protease–protein complexes at the
migrating cell front. MT1-MMP and urokinase (u-PA) form multiprotein complexes in the lamellipodia and focal
adhesions of migrating cells, facilitating proteolysis and sufficient support for endothelial cell migration and survival.
Excessive proteolysis causes loss of endothelial cell-matrix interaction and impairs angiogenesis. MMP-9 and cathepsin
L stimulate the recruitment and action of blood- or bone-marrow-derived accessory cells that enhance angiogenesis.
Proteases also generate fragments of extracellular matrix and hemostasis factors that have anti-angiogenic properties.
Understanding the complexity of protease activities in angiogenesis contributes to recognizing new targets for
stimulation or inhibition of neovascularization in disease. (Arterioscler Thromb Vasc Biol. 2006;26:716-728.)
Key Words: endothelium 䡲 neovascularization 䡲 matrix metalloproteinases 䡲 MT-MMPs 䡲 cathepsins
F
rom the sequences of the human and murine genomes the
presence of ⬇30 000 genes has been estimated. An even
10-fold larger number of actual proteins is anticipated on the
basis of splicing and posttranslational modifications. This
increase not only reflects glycosylated proteins and different
splice products but also proteolytically modified proteins and
formation of new blood vessels from existing ones by
sprouting enforced by intussusception of blood-borne cells,1,2
the involvement of proteases is well-established.3 Angiogenesis is enforced by vasculogenesis, a process that comprises
the recruitment of blood-borne bone marrow-derived cells
and the subsequent intussusception of these cells in the newly
forming vessels.2 Recruitment and invasion of cells during
vasculogenesis also involve proteolytic activities. New functions of split products of parent matrix molecules, receptors,
and growth factors are becoming recognized in conditions
See cover
peptides that acquire new and often unexpected biological
functions after proteolytic tailoring. In angiogenesis, the
Original received November 8, 2005; final version accepted January 25, 2006.
From the Laboratory for Physiology (V.W.M.H., M.A.E.), Institute for Cardiovascular Research, VU University Medical Center, Amsterdam; TNO
Quality of Life (P.H.A.Q.), Gaubius Laboratory, Dept. Biomedical Research, Leiden, The Netherlands.
Correspondence to Victor W.M. van Hinsbergh, PhD, Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center,
Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
716
DOI: 10.1161/01.ATV.0000209518.58252.17
van Hinsbergh et al
Pericellular Proteases in Angiogenesis
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Figure 1. Schematic representation of
secreted and membrane-bound MMPs and
related ADAMs and ADAMTSs involved in
angiogenesis. The proteins have various
domains with specific functions in common.
Key regulators of angiogenesis are indicated
by the yellow squares. MT-MMP-1, -2, -3,
and probably -5 enhance angiogenesis by
their pericellular action (see Figure 4). Similarly MMP-2 and MMP-9 stimulate angiogenesis and can act pericellular by binding
to membrane-anchored proteins. ProMMP-23 has a transmembrane domain, but
this is removed during activation of the protease, making the active enzyme a soluble
MMP. Part of the ADAM family members
has proteolytic activity, of which ADAM-10,15, and -17 are known to affect angiogenesis. ADAMTS-1 and the related ADAMTS-4
and -8 also can affect angiogenesis mainly
via their thrombospondin (TSP) domains.
The MMPs and MT-MMPs are depicted
according to Sato50 with some
modifications.
associated with angiogenesis.4,5 These split products have an
important effect on the delicate balance between factors that
enhance cell migration and growth and factors that keep the
endothelial cells in a quiescent and more differentiated state,
and can shift this balance toward the onset and progression of
angiogenesis.6
Proteases that are involved in angiogenesis can act intracellularly or extracellularly. Among the intracellular proteases several are involved in protein maturation and processing, such as furin. 7 Furthermore, proteasome-related
proteases degrade not only deformed proteins but also are
involved in controlling the survival of transcription factors,
such as hypoxia-induced factor-1␣ (HIF-1␣), that regulates
expression of angiogenesis factors.8 Lysosomal proteases
control degradation of internalized proteins and also digest
extracellular matrix proteins that are ingested in endosomal
bodies. Caspases, a group of cysteinyl aspartate-specific
proteases, are key regulators of apoptosis,9 a process pivotal
in endothelial death and vascular pruning.
Extracellular proteases are involved in the degradation of
matrix proteins. On the basis of devastating diseases such as
cancer, rheumatoid and bone diseases the focus has been
initially on destruction of matrix mass in these tissues and
during invasive growth. We also recognize a more delicate
pericellular degradation of matrix proteins and cellular receptors during the progression of cell migration and invasion.
Furthermore, well-coordinated pericellular proteolytic activities control the availability of active and modified angiogenic growth factors and cytokines.
In the present survey, we focus on pericellular proteolytic
activities involved in angiogenesis and vasculogenesis. This
comprises pericellular proteolysis involved in cell migration
and invasion of endothelial cells and cells that support the
formation of new and stable microvessels; the activation and
modification of growth factors; and the generation of new
epitopes in tailored proteins and protein fragments with new
angiogenic and anti-angiogenic properties. After a short
survey of the major classes of proteases involved, we discuss
these proteases according to the biological effects that they
perform during neovascularization.
Types of Proteases Involved in Matrix
Degradation and Angiogenesis
Three major groups of endoproteases play an important role
in the processes that regulate angiogenesis, including the
remodeling of the extracellular matrix, cell migration, and
invasion, and the liberation and modification of growth
factors. They comprise the metalloproteinases, in particular
the matrix metalloproteinases (MMPs), the cathepsin cysteine
proteases and the serine proteases. The activities of these
proteases are controlled by specific activation mechanisms
and specific inhibitors, of which tissue inhibitors of metalloproteinases (TIMPs), cystatins, and inhibitors of serine proteases called serpins represent major classes. Many of these
proteases act on the cell surface either because they contain a
membrane spanning or binding domain or as the result of
their interaction with specific receptors on the cell surface. In
addition, inhibitor studies with fumagillin derivatives have
pointed to a role of aminopeptidases in angiogenesis.
Metalloproteinases: MMPs, ADAMs
and ADAMTSs
MMPs belong to a family of zinc-dependent endopeptidases
that digest specific extracellular matrix components, and also
are able to aid in the activation of proteins such as other
MMPs and growth factors.3,10,11 These enzymes are important
for cell migration and invasion, and regulate developmental
growth and many remodeling processes, such as angiogenesis, arterial remodeling and wound healing.12–14 The MMPs
have a specific structure bearing several homologous domains as depicted in Figure 1. They can be arranged in 2
groups, as soluble MMPs and membrane-type MMPs
(MT-MMPs).
The soluble MMPs are expressed as inactive pro-enzymes
that only become activated once they are present in the
extracellular environment. This family of MMPs harbors
collagenases (MMP-1, MMP-8, and MMP-13), gelatinases
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(MMP-2, and MMP-9), stromelysins (MMP-3, MMP-10 and
MMP-11), matrilysins (MMP-7 and MMP-25), and others
(MMP-12 and MMP-26).10,11 The MT-MMPs encompass 6
members (MT1-MMP to MT6-MMP) that are activated
intracellularly by furin-like enzymes, and anchored to the
plasma membrane with either a type-I transmembrane domain (MT1/2/3/5-MMP) or a glycosylphosphatidylinositol
(GPI) anchor (MT4/6-MMP). The MT-MMP family members were first identified as activators of soluble MMPs, and
subsequently also shown to be able to degrade extracellular
matrix components such as fibrillar collagen, laminin-1, and
laminin-5, aggrecan and fibronectin, and the plasma-derived
matrix proteins vitronectin, and fibrin.11,15 Furthermore, they
can proteolytically liberate and modify growth factors and
cytokines.4
Importantly, MT-MMPs at the cell surface pinpoint MMPdependent matrix degradation to the close vicinity of the cell.
MT1-MMP can also locally activate the proforms of MMP-2
and MMP-13, which focuses proteolytic activity on specific
sites on the cell surface that are involved in cell migration and
invasion.11,16 Furthermore, MT1-MMP processes cell adhesion molecules CD44 and pro-␣v-integrin and tissue transglutaminase.17–19 In this context it is of interest that CD44 and
␣v␤3-integrin have been recognized as binding moieties for
MMP-9 and MMP-2, respectively.20,21
Because of the unexpected effects of broadly acting metalloproteinase inhibitors, much interest was generated to
search for other types of metalloproteinases. These searches
recognized two additional large families of metalloproteinases, the ADAM (a disintegrin and metalloproteinase domain) family, which are membrane-spanning proteins, and
the ADAMTS (a disintegrin and metalloproteinase with
thrombospondin motifs) family, which are secreted into the
interstitial space and bind to extracellular matrix proteins.22,23
ADAMs have a transmembrane domain and can act as
sheddases on the surface of the cell. At least 34 ADAMs are
known, of which 19 have proteolytic activity.13,22 Three
members (ADAM-10, ADAM-15, ADAM-17) are known
that have properties directly important for the regulation of
angiogenesis, but other members may add, as they have not
yet been investigated sufficiently.
The ADAMTS proteins are a family of secreted metalloproteinases that contain one or several thrombospondin domains, by which many of them bind to extracellular matrix
proteins.23 Nineteen human members of the ADAMTS family
are known, of which ADAMTS-1, -4, -5, -8, and -15
represent a vertebrate subfamily that originated from gene
duplication. These latter proteases are involved in the degradation of the proteoglycans versican and aggrecan, and
several members of this family, ADAMTS-1 and
ADAMTS-8, have an inhibitory effect on angiogenesis.24,25
ADAMTS-4 was indirectly implicated in angiogenesis, as it
was differentially expressed in endothelial cells that formed
capillary-like tubular structures in a fibrin matrix.26
Inhibitors of Metalloproteinases
In general, inhibition of MMPs occurs in tissues through
specific inhibitors, designated as TIMPs, of which at least 4
members are known. MMPs can also be inhibited by ␣2-
macroglobulin and a membrane-anchored glycoprotein called
RECK (reversion-inducing cysteine-rich protein with Kazal
motifs).27,28 TIMPs bind activated MMPs to form equimolar
complexes. Although most soluble MMPs can be inhibited by
all TIMPs, MT-MMPs have a more restricted pattern of
TIMP inhibition. TIMP-2 and TIMP-3 efficiently inhibit
MT1-MMP, while TIMP-1 is rather ineffective in this respect.16 TIMPs also inhibit proteolytically active ADAMs and
ADAMTSs, but this inhibition is usually more selective, ie, a
higher selectivity for individual TIMPs is observed.13 TIMP-2
can also abrogate angiogenic factor-induced endothelial cell
proliferation in vitro and angiogenesis in vivo, independent of
MMP inhibition.29 TIMP-2 can enhance the expression of
RECK via Rap1 signaling, which causes an indirect, timedependent inhibition of endothelial cell migration.30 TIMP-3
is sequestered at the cell surface by association with the
glycosaminoglycan chains of proteoglycans, especially heparan sulfate, and may play a role in the regulation of ADAM17.31 TIMP-3 by itself can block vascular endothelial growth
factor (VEGF) binding to its receptor and reduce angiogenesis, independent of MMP inhibition.32
Cysteine Cathepsins
In humans, the cathepsin cysteine proteases or cysteine
cathepsins consist of a family of 11 members, which are
indicated as cathepsins B, C, F, H, L, K, O, S, V, W, and
X/Z.33 In the mouse, 19 cathepsins are known including
several additional placentally expressed cathepsins that have
no human homologue.34 The cysteine cathepsins are synthesized as inactive zymogens and are activated by proteolytic
removal of their N-terminal propeptide. Their activity is
regulated intracellularly by a specific cystatin subgroup
called stefins and extracellularly by cystatins and
kininogens.35
The majority of cysteine cathepsins are endopeptidases.33
Cathepsins are primarily known as lysosomal enzymes, but
new data indicate that they also play a role in antigen
processing by T cells, in cell invasion, extracellular matrix
and bone remodeling, in tumor growth, and in apoptosis.35,36
Cathepsins have their enzymatic activity optimum at acidic
pH. This facilitates their activities in lysosomes and endosomal vesicles, in which they are involved in the terminal
degradation of proteins. In addition, several cathepsins, particularly cathepsin B, K, and L, are also encountered outside
the cells or on the cell membrane,12,37,38 and contribute to
angiogenesis and recruitment of endothelial progenitor cells
into the angiogenic region. The close association of cathepsin
K with a proton pump in the membrane may cause a local
pericellular acidic environment that enhances cathepsin
activity.37
Serine Proteases
The serine proteases are endopeptidases that have a catalytic
site made up by three amino acids, serine, aspartic acid, and
histidine, the “catalytic triad.” They represent a vast class of
proteases with members with a broad variety of functions.
Nearly all members are synthesized as zymogens, which are
activated by proteolytic removal of an N-terminal part of the
molecule that covers and hides the active side. Although
van Hinsbergh et al
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many serine proteases may indirectly affect proteins involved
in angiogenesis, plasminogen activators (PAs) and plasmin
received specific attention. For several decades the serine
proteases urokinase-type PA (u-PA) and plasmin have been
recognized as proteases that are involved in cell migration
and invasion.39 u-PA can activate the zymogen plasminogen
into the broadly acting protease plasmin. This process is
facilitated by the u-PA receptor (uPAR).40 A second mammalian PA is tissue-type plasminogen activator (t-PA), which
activates plasminogen in the presence of fibrin. Its primary
function is fibrinolysis in the blood and body cavities,
whereas u-PA plays a dominant role in cell invasion. Both
u-PA and t-PA are inhibited by plasminogen activator inhibitors (PAIs), in particular PAI-1. Plasmin activity is highly
effectively inhibited by ␣2-antiplasmin and also controlled by
␣2-macroglobulin. Although dispensable for development of
the vascular tree, plasmin, u-PA and PAI-1 have been
reported to modulate angiogenesis in various pathological
conditions.41,42
Other serine proteases can also indirectly affect angiogenesis,
such as the protein convertases furin and PC6, which are major
regulators of the activation of MT-MMPs.7 This occurs intracellularly during transport of MT-MMPs to the membrane. Furthermore, several members of the coagulation cascade have an
effect on the early development of the vascular system that is
independent of their contribution to coagulation.43
Several groups of membrane-bound serine proteases were
recently recognized and are expanding rapidly.44 They comprise
proteins that are bound with a GPI-anchor and proteins that have
a transmembrane domain either in their C-terminal part or at
their N-terminal part. By their surface-bound nature they may
locally modify the biological activities of cytokines and vasoactive agents, as was shown for DPPIV/CD26 and the related
fibroblast activation peptide-␣.45– 47
Aminopeptidases
Aminopeptidases are exopeptidases that split off 1 or 2 amino
acids from the unblocked amino-terminus of proteins. Interest
in these proteases was stimulated by the finding that the target
for the antiangiogenic compound fumagillin and its derivative
TNP-470 was a methionine aminopeptidase-type 2 (MeAP2).48,49 Sato50 recently reviewed the potential role of this
and several other aminopeptidases in angiogenesis, particularly aminopeptidase N (CD13/APN) and puromycininsensitive leucyl-specific aminopeptidase (PILSAP), also
called adipocyte-derived leucine aminopeptidase. PILSAP is
expressed in endothelial cells and plays a stimulatory role in
postnatal angiogenesis.51 Sato50 suggested that the effect of
fumagillin might be via PILSAP instead via Me-AP2. The
availability of new inhibitors that discriminate between these
enzymes can elucidate this aspect.52
Aminopeptidase N (CD13/APN) was recognized as being
highly expressed on the endothelium of growing vessels53 after
earlier observations by Saiki et al54 that inhibitors of CD13/APN
impaired tumor growth. This recognition has prompted studies
on using CD13/APN as a target for inhibiting tumor vascularization53,55 and on the role of this receptor with protease activity
in angiogenesis.56,57
Pericellular Proteases in Angiogenesis
719
Switching on Angiogenesis: Matrix Proteolysis
When leukocytes, tumor cells, or endothelial cells invade into
a tissue they must pave their way and create space. It is
therefore quite understandable that the first studies on the
involvement of proteases in these processes regarded the
degradation of the extracellular matrix. With respect to
angiogenesis this encompasses in particular the degradation
of the endothelial basement membrane to enable endothelial
and accessory cells to migrate into the area of neovascularization, and proteolysis of matrix components to create space
for a vascular lumen. Although each member has its own
specific substrate specificity, MMPs in concert are able to
degrade a wide if not the whole spectrum of matrix proteins
and therefore they are considered to be the prime class of
proteases involved in degradation of the endothelial basement
membrane and interstitial matrix degradation. Quiescent endothelial cells produce little or no MMPs, whereas the
expressions of several MMPs are strongly upregulated in
activated endothelial cells in vitro,58,59 and in the endothelium
of vessels in wound healing, inflammation, and tumors.10,12,13
Various reviews have extensively surveyed the substrate
specificities of the various MMPs.3,10
A number of studies including gene deletions in mice have
pointed to the essential role of MMP-2, MMP-9, and MT1MMP in the onset of angiogenesis in tumors and in development
and bone formation.60 – 64 Although their involvement in the
onset of angiogenesis, the so-called angiogenic switch,6 may
suggest that these MMPs stimulate angiogenesis primarily by
matrix degradation, it should be noted that the activities of these
proteases are complex and may include other effects as well,
such as the activation of growth factors and cytokines, the
recruitment of endothelial progenitor cells, and the degradation
of inhibitors. This complex role is underlined by the observation
that after the onset of tumor angiogenesis MMP-9 also generates
angiogenesis inhibitors, such as tumstatin, by which angiogenesis becomes then retarded.65 A recent comparison of the ability
to enhance capillary-like tube formation in a collagen-rich
matrix indicated that MT1-MMP enables endothelial cells to
form invading tubular structures, whereas MMP-2, MMP-9,
their cognate cell-surface receptors ␤3-integrin and CD44, or
plasminogen did not.66
With respect to the involvement of other MMPs, it should be
noted that redundancies exist, which should be taken in account
when interpreting data of gene silencing toward the requirement
of these genes in angiogenesis. Furthermore, specific attention
should be given in translating data from animals to man. For
example, the major interstitial collagenase MMP-1 in humans is
absent in mice,67 in which its function is largely taken over by
MMP-13. Notwithstanding this, MT1-MMP, MMP-9 and
MMP-2 can be considered as important regulators of angiogenesis in a broad spectrum of pathological conditions.60 – 64
Other proteases, such as plasmin and cathepsin-B, can cooperate with MMPs. Once activated, plasmin can act by itself on
matrix proteins and is also able to activate various MMPs,
including MMP-1,-2,-3, and -9.68,69 In pathological conditions,
often an additional temporary matrix is formed, which consists
primarily of fibrin, but also contains fibronectin and vitronectin.
Plasmin and its plasminogen activators, probably in concert with
MMPs, play a role in the recanalization of thrombi and in the
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neovascularization of fibrinous exudates, as occurs in healing
wounds, infarcted myocardium and tumor stroma.41,42,70 In
addition to plasmin, MT1-MMP can act as a fibrinolysin and
facilitate capillary outgrowth in a fibrinous environment.71,72 In
a fibrin matrix both plasmin and MT1-MMP have been indicated to be involved in endothelial tubule formation.41,71,73–75 In
endothelial cells in vitro both tubular outgrowth and MT1-MMP
activity were inhibited by TIMP-2 and-3 but not by TIMP-1.74,75
Interestingly, tube formation by endothelial cells of human
endometrium was equally inhibited by TIMP-1 and TIMP-3 and
may depend on MT3-MMP activity.76 Overexpression of MT1-,
MT2-, or MT3-MMP, but not MT4-MMP, each enhances the
fibrin-invasive activity of endothelial cells.72
Switching on Angiogenesis: Proteolytic
Activation and Modification of
Growth Factors
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Besides the proteolytic action of proteases on proteins involved
in cell–matrix interaction, proteases can control the onset and
progression of angiogenesis by the activation and modification
of the biological properties of angiogenic growth factors and
cytokines.3,4 The angiogenesis-inducing growth factor hepatocyte growth factor (HGF) is activated by HGF activating factor,
a serine protease related to plasminogen.77 Growth factors, such
as bFGF and VEGF, can become liberated from the extracellular
matrix after degradation of proteoglycans.78,79 In addition to
enhancing the expression of VEGF,80 – 82 MT-MMP can release
VEGF from the connective tissue growth factor/VEGF complex
by proteolytic cleavage of connective tissue growth factor.83
Growth factors that indirectly affect angiogenesis are also targets
of proteolytic activation. For example, plasminogen activators
drive the activation of latent transforming growth factor-␤ from
bone extracellular matrix and thus modulate angiogenesis in
bone.84,85
The proteolytic modification of growth factors not only
causes their activation but also can modify their properties. This
was elegantly shown for VEGF165, which on cleavage by either
MMP-3 or MMP-9 is reduced to a smaller molecule with
properties similar to VEGF121.86 Whereas VEGF165 induces a
regular vessel pattern during neovascularization in tumors,
VEGF121 and the shortened VEGF165 cleaved by MMP-3 or
MMP-9 cause an irregular pattern of neovascularization, probably because these molecules do not bind to heparin sulfates and
therefore do not provide spatial information that is buried in the
extracellular matrix.86 The properties of another important factor
in angiogenesis, stromal cell derived factor-1 (SDF-1) are also
modified by proteases, but in this case by carboxy- and aminoterminal truncations. The 2 isoforms of SDF-1, SDF-1a and
SDF-1b, are both are modified by the aminodipeptidase DPPIV/
CD26, by which their heparan sulfate affinities and interactions
with their receptor CXCR4 are reduced.87 SDF-1a is additionally
shortened by 1 amino acid by carboxypeptidase N,88 which
further reduces the affinity for heparan sulfates. MT1-MMP,
MMP-1, MMP-2, and MMP-13 can cleave and inactivate SDF-1
and its receptor CXCR4.89 VEGF and SDF-1 often participate
simultaneously in angiogenesis, particularly when bone marrow
recruited progenitor cells are involved.
Among the proteases that can alter the balance between
pro-and anti-angiogenic factors members of the ADAMs family
of proteins are receiving attention. ADAM-17 (tumor necrosis
factor [TNF]␣ converting enzyme [TACE]) proteolytically releases TNF␣ and HB-EGF from their membrane-bound precursors.90 These factors can indirectly modulate angiogenesis. Both
ADAM-17 and ADAM-10 may contribute to Notch signaling by
performing a specific cleavage essential for Notch receptor
activation on ligand binding, or by shedding the Notch ligand
Delta from the cell surface.22,91 This may affect angiogenesis as
Notch signaling is involved in endothelial differentiation and in
embryonic and tumor angiogenesis.92,93 ADAM-10 was also
shown to cleave and shed Eph receptors EphA2 and EphA3.94,95
Soluble EphA inhibits tumor angiogenesis in mice.96
Finally, the proteolytic modification not only regards growth
factors and cytokines, but also their receptors. Proteolytic modification of neuropeptide Y by CD26 generated a truncated3–36neuropeptideY, thereby reducing its affinity for Y1 receptors and
increasing that for Y2 receptors.46,97 Neuropeptide Y is liberated
in ischemic areas and enhanced angiogenesis via Y2/Y5 receptors. Y2⫺/⫺ mice have reduced angiogenesis in ischemic
tissue/muscle.
Excessive Proteolysis: No Grip, No Invasion
The proteolytic activities involved in matrix destruction and
remodeling require spatial and temporal control. Excessive
proteolysis can cause unwanted damage to the tissue and might
dissolve the matrix needed for anchoring the migrating cells.
This was elegantly shown in mice deficient for PAI-1.98,99
Because PAI-1 inhibits plasminogen activators and hence plasmin activation, one would expect that PAI-1 deficiency would
increase angiogenesis and tumor growth. However, when PAI1– deficient mice were challenged with xenografted cancer cells
on a collagenous matrix, angiogenesis and vascular stabilization
were severely impaired, thereby hampering tumor growth.
PAI-1 protects the surrounding extracellular matrix from excessive degradation by plasmin, thus maintaining a foothold for the
endothelial cells that migrate and form capillary structures to
nourish the tumor.99 –101 Improper proteolytic processing also
underlies the disrupted vascular development and premature
deaths of murine embryos deficient of the inhibitor RECK. This
pathology is likely caused by uncontrolled MMP activity,
because a partial rescue was obtained in mice that were deficient
for both RECK and MMP-2.27
Angiogenesis on the Move: Assembling
Pericellular Proteolysis
Migration and invasive growth of cells involved in angiogenesis
requires a delicately balanced interplay between detachment and
new formation of cell adhesions to enable the cell to crawl
forward through the extracellular matrix.40 To this end, the cell
generates limited proteolytic activity at individual focal adhesions often via the formation of multiprotein complexes. In
recent years it has become clear that multiprotein complexes are
built up in lipid rafts on the cell surface, and that membranebound proteases often take part therein. For example in invading
leukocytes, a complex containing u-PA, uPAR and integrins has
been recognized that participates in cell adhesion and inva-
van Hinsbergh et al
Figure 2. The u-PA receptor acts as a coordinating protein in
the assembly of a multiprotein complex involved in cell adhesion
and cell signaling, and supports u-PA activation and removal of
u-PA:PAI-1 complexes.
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sion.102 uPAR acts here as an organizing center being able to
form noncovalent complexes with integrins, LRP-like proteins
and u-PA or urokinase plasminogen activator (uPA):PAI-1
(Figure 2). Such complexes also occur on endothelial cells.
The uPAR–Integrin Complex
The uPAR is a GPI-anchored protein present on the surface of
endothelial cells, leukocytes, and many other cell types.40 When
the inactive pro-form of u-PA is secreted, it binds to uPAR and
can subsequently be activated. A number of proteases have been
reported to activate u-PA on the cell surface, including cathepsin
B and kallikrein. Cathepsin B is found on various cells including
endothelial cells. Cathepsin B is primarily a lysosomal enzyme,
but it can bind to annexin II heteromer and p11 protein on the
cell surface and activate uPAR-bound u-PA.103 The uPARbound u-PA can activate cell-bound plasminogen efficiently,
and subsequently formed plasmin can degrade extracellular
matrix proteins and, at least in vitro, activate several MMPs. In
addition u-PA can also directly act on several matrix proteins,
such as fibronectin.104 Active u-PA acts only for a short period
of time, because its inhibitor PAI-1 rapidly inhibits its activity.
The uPA:PAI-1/uPAR complex is subsequently internalized,
after which uPAR is separated from u-PA:PAI-1 and released
unoccupied to the plasma membrane, whereas the u-PA:PAI-1
complex
is degraded in the lysosomes. The internalization of the uPA:PAI-1/uPAR complex is inhibited by the Receptor associated
protein (Rap), which indicates that LRP or an LRP-like protein
is involved.40,105
Similar as in leukocytes,106 an increase in functional uPAR/
uPA has been detected at the cell surface of the leading edge of
migrating endothelial cells.105,107 By repetitive activation of the
u-PA/plasmin system at adhesion sites, migrating endothelial
cells and leukocytes become able to tunnel through fibrinous
matrices. The focally localized uPA/uPAR is bound to integrins,
including ␤1-integrins and ␣v␤3-integrin.102,108 This interaction
can be on the cell surface, but also between uPAR and integrins
on another cell, suggesting that the uPAR-integrin interaction
facilitates cell–matrix and even cell– cell interactions. In endothelial cells and smooth muscle cells interactions of uPAR with
Pericellular Proteases in Angiogenesis
721
␣5␤1 and ␣v␤3-integrin have been described.108,109 When
uPAR is occupied by active uPA, interaction of a ␤1-integrin
with this complex results in a conformational change in the
integrin, as demonstrated for ␣5␤1-integrins,110 and causes
intracellular activation of extracellular signal regulated kinase
(ERK) and Jak/Stat pathway.40,111 This activation probably
explains how the u-PA-uPAR complex, which is bound to the
cell by its GPI-anchor and has no transmembrane moiety, can
signal into the cell. Signaling may also involve other proteins,
namely the G-protein receptor FPRL1 and epidermal growth
factor receptor.40
Although many data on cell-bound u-PA and plasmin have
been obtained in in vitro studies, the in vivo evidence for the
involvement of this system is less generally evident. Mice
deficient in plasminogen develop normal blood vessels, but are
disturbed in VEGF-induced and bFGF-induced angiogenesis in
the cornea; data on u-PA– deficient animals are unequivocal.112,113 Neovascularization after myocardial infarction depends equally on u-PA/plasmin activities as on MMPs.42 However, one cannot discriminate on the basis of the data available
whether the u-PA/plasmin contribution acts largely via endothelial cells on angiogenesis or that the invasion of leukocytes and
endothelial progenitor cells, which may supply additional
growth factors, also contributes to this effect.
Pericellular Activities of MMPs and MT-MMPs
It is likely that also other membrane-associated metalloproteinases, such as MT-MMPs, can participate in similar multiprotein
complexes. Analogous to the interaction of integrins with uPA/
uPAR, the localization of MMP-2 on the cell membrane can be
associated with ␣V␤3-integrin, which aids in focusing proteolytic
activity.60,114 MT1-MMP colocalizes with ␤1-integrins in cellcell contacts, whereas it was encountered with ␣v␤3-integrins in
migrating endothelial cells.115 Similarly, MMP-9 interacts with
the cell adhesion molecule CD44,20 which on its turn is processed by MT1-MMP.116 Furthermore, MMP-2 binds, in a
complex reaction with TIMP-2, to MT1-MMP on the cell
surface.11 This interaction facilitates the activation of MMP-2 by
a second adjacent MT1-MMP molecule.
MT1-MMP is directed toward the lamellipodia at the front of
migrating cells (Figure 3), suggesting an interaction between
MT1-MMP and the actin cytoskeleton. The hemopexin domain,
which is located extracellularly, rather than the cytoplasmic
domain is responsible for the association of MT1-MMP with the
cortical actin net within the cell. The CD44 isoform CD44H,
also the dominant form in endothelial cells,117 interacts both with
the hemopexin domain of MT1-MMP and actin.116 This interaction facilitates the di-/oligomerization of MT1-MMP molecules that is required for activation of MMP-2. Once activated,
both MMP-2 and MT1-MMP can degrade various proteins of
the extracellular matrix. Furthermore, MT1-MMP enhances
VEGF gene expression in various cell types, a process that
requires its proteolytical activity as well as its cytoplasmic
domain, and involves src tyrosine kinase.80 – 82 MT1-MMP can
also cause intracellular activation of ERK, which requires the
cytoplasmic part of MT1-MMP.118 Multifunctional gC1qR protein and a recently cloned MT1-MMP cytoplasmic tail binding
protein-1 (MTCDP-1) have been recognized as intracellular
proteins that bind to the MT1-MMP cytoplasmic tail.119,120 In
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Figure 3. After synthesis, MT1-MMP is activated by furin or furinlike proteases and reaches the plasma membrane subsequently. It
is associated with actin fibers in the lamellipodia of migrating cells
via CD44H. The blue line represents the cell membrane. MT1-MMP
has a number of proteolytic functions including MMP-2 activation,
matrix degradation, and modification of growth factors, cytokines,
transglutaminase and CD44, after which it is internalized. Finally,
recycling MT1-MMP to the plasma membrane or degrading it in
the lysosomes regulates its activity.
addition, MT1-MMP can process several membrane proteins.
This includes the maturation of ␣v-integrins and the shedding of
tissue transglutaminase, syndecan-1, and CD44.17,18,116,121 CD44
cleavage appeared to be needed for migration to proceed,
because a mutant CD44H that could not be cleaved by MT1MMP prevented cell migration.116 Subsequently, MT1-MMP is
internalized via caveolae and clathrin-coated vesicles.19,122,123
The internalized MT1-MMP can either be degraded in lysosomes or be recycled to the plasma membrane. Internalization
appears to be required for cell migration.124 Possibly the treadmilling of a complex of MT1-MMP and adjacent (receptor)
proteins is required for the locomotion of the cell. Although most
of these data were obtained from tumor cells and fibroblasts, it
is likely that a similar mechanism also acts in endothelial cells.
The important role of MT1-MMP in endothelial cell migration
and angiogenesis was already mentioned. Galvez et al123 demonstrated that internalization of MT1-MMP via caveolae was
involved in MT1-MMP–mediated migration of endothelial cells
in a collagen substrate/matrix.
The mechanisms of MT1-MMP and u-PA/uPAR have much
in common. Prager et al125 showed that the uPAR is redistributed
to focal adhesions at the leading edge of endothelial cells in
response to VEGF, and that subsequent cell migration depended
on u-PA activation, PAI-1 interaction and internalization of the
uPAR. VEGF-dependent activation of the uPAR-bound u-PA
activation depended on a change in integrin affinity and MMP-2
activity bound to MT1-MMP on these cells. Other investigators
reported on MT-MMPs and u-PA/plasmin as additionally acting
mechanisms,74,75 each of which predominated depending on the
matrix conditions. Furthermore, MT1-MMP has been implied in
the activation of MMP-2 and MMP-13 and in the processing of
cellular receptors and membrane-bound proteins, as already
mentioned above.
Pericellular Activity of Cathepsin B
In tumor cells the secreted procathepsin B binds to the cell
surface via the light chain of the annexin II heterotetramer and
the therewith-associated protein p11, which facilitates its conversion into active cathepsin B.12 This complex associates with
lipid raft structures, similar as the uPAR complex. Cathepsin B
can be activated from its zymogen by various proteases including t-PA, u-PA, cathepsins G and D, and elastase. Cathepsin B
has also been indicated to activate uPAR-bound pro-u-PA and
thus to enhance angiogenesis.126 Rao and colleagues have shown
in a series of elegant experiments that simultaneous inhibition of
cathepsin B and uPAR by siRNAs markedly reduced the growth
of gliomas and their vasculature in mice in vivo, and that the
simultaneous inhibition of the expression of both proteins was
more effective than inhibition of one of them.127 Similarly, they
showed that the simultaneous inhibition of cathepsin B and
MMP-9 also inhibited glioma growth in mice.128
Kostoulas et al129 proposed a potent additional mechanism by
which cathepsin B may enhance angiogenesis, namely degradation of TIMP-1 and TIMP-2. This degradation leads to increased
activities of MMPs. Furthermore, cathepsin B is involved in the
activation of growth factors.
Other Cysteine Cathepsins and Angiogenesis
Other cathepsins may also be active pericellularly either
after binding to membrane-bound chaperones130 or by
association with a proton-pump, which causes a local
acidification.37 The cysteine cathepsins B, K, L, and H are
highly expressed in various tumor cells. A number of
studies show the presence of cathepsins in the vasculature
of human tumors (incl. glioma and prostate) associated
with an increase in angiogenesis and tumor growth.131,132
In addition to the already mentioned studies on cathepsin
B, the expression and role of other cathepsins in tumor
angiogenesis was further investigated in RIP1-Tag2
mice.133 These mice develop multiple pancreatic islet
tumors as a consequence of expressing the SV40 T antigen
(Tag) oncogene in insulin-producing ␤-cells. The cathepsins B, H, L, and X/Z were expressed in the tumor cells,
infiltrating leukocytes, and the endothelial cells of the
tumor. Inhibition of cathepsin activity by a broad-spectrum
cysteine cathepsin inhibitor reduced tumor vascularization
and vascular branching during pancreatic islet tumorigenesis. Remarkably the core of the tumors was more affected
than the outer part, possibly related to co-option of existing
vessels in the outer region of the tumor. Endothelial
progenitor cells express various cysteine cathepsins, of
which cathepsin L was shown to contribute to angiogenesis
in mice.38 Mice deficient of cathepsin S show an impaired
development of microvessels during wound repair.134 A
role of cathepsin S in angiogenesis was underscored by the
in vitro finding that endothelial tube formation and invasion in Matrigel and collagen 1 matrix was facilitated by
cathepsin S.134 These data strengthen the idea that cathepsins indeed play a role in angiogenesis in tumors and
tissue repair.
Angiogenesis Enforced: Recruitment of Bone
Marrow-Derived Cells
Leukocytes and endothelial progenitor cells can contribute to the
initiation and guidance of new blood vessels.135,136 u-PA/uPAR
and MT1-MMP play a role in monocyte recruitment during
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inflammation,106,137 as MMP-9 does also. Monocytes produce
various pro-angiogenic factors.135 Furthermore, a special population of CD34⫹ cells which can acquire endothelial-like properties, such as the expression of VE-cadherin and VEGF
receptor-2 (kdr) are thought to markedly influence the progression of angiogenesis.136,138 Their absence or dysfunction is
associated with impaired vascularization in cardiac and diabetes
patients.139,140 MMP-9 plays a role in the recruitment of these
so-called endothelial progenitor cells (EPCs). Furthermore, these
cells produce high amounts of cathepsins including the
angiogenesis-stimulating cathepsin L. For a discussion of the
types of circulating endothelial/progenitor cells the reader is
referred to Ingram et al.141
MMP-9 plays a role in the mobilization of hemopoietic and
endothelial progenitor cells from the bone marrow. After suppression of the vascular niche, the transfer of hemopoietic and
endothelial precursor cells to the proliferation compartment in
the bone marrow requires cell activation by soluble c-kit ligand,
which is set free by MMP-9.142 Deficiency of MMP-9 in mice
retards recruitment of EPCs and angiogenesis. Similarly,
MMP-9 plays a crucial role in the recruitment of bone marrowderived leukocytes to the vasculature of neuroblastomas.143 This
role appears critical for the formation of a mature vasculature
and coverage of endothelial capillaries by pericytes. Heissig et
al144 recently proposed an amplifying mechanism, in which: (1)
circulating VEGF induced MMP-9 expression in the bone
marrow; (2) MMP-9 released c-kit ligand; (3) c-kit activation
recruits hemopoietic, endothelial and mast cell progenitor cells;
and (4) mast cells accumulating in the angiogenic area produce
large amounts of VEGF. This cycle was elevated by low-dose
irradiation.144
Not only MMP-9 is involved in EPC-enhanced angiogenesis.
Urbich et al38 found that cathepsin L is required for EPCinduced neovascularization. They selected cathepsin L as a
potential regulator of EPC-enhanced neovascularization after
studying the expression profiles of various proteases and protease inhibitors in EPCs, endothelial cells and CD14⫹ monocytes. These EPCs are able to stimulate neovascularization and
blood flow in the ischemic murine hindleg after their injection in
affected hindleg. A high expression of a number of cysteine
cathepsins occurred specifically in EPCs, including cathepsins
D, H, L, K, and X/Z, as became obvious from mRNA array
analysis. The antigen and activity of cathepsin L was considerably higher in EPCs than in monocytes and endothelial cells.
This activity may be, at least in part, extra- or pericellular.
Mature cathepsin L can remain active extracellularly at neutral
pH by the chaperone action of a p41 splice variant of the MHC
class II-associated invariant chain,130 which indeed is strongly
expressed in EPCs.38 Such activity may facilitate EPC invasion,
remodeling of matrix collagens and gelatin, and neovascularization. Deficiency of cathepsin L in mice caused a significant
impairment of blood flow restoration in ischemic limbs, indicative for an impaired neovascularization. Furthermore, neovascularization was reduced in mice treated with bone marrow
derived cells deficient of cathepsin L as compared with wild–
type cells. Cathepsin L was also required for the recruitment and
infiltration of EPCs into glioma xenografts and their vasculature
in mice.38 The target via which cathepsin L stimulates angiogenesis is not yet resolved.
Pericellular Proteases in Angiogenesis
723
Proteolytic Degradation Products as Endogenous Inhibitors
of Angiogenesis
Proteolytic fragments of the extracellular matrix
Thrombospondin-1
Collagen type XVIII fragment:
Endostatin
Collagen type IV fragments:
Arresten
Canstatin
Tumstatin
Metastatin
Collagen VIII fragment: vastatin
Collagen XV fragment: restin
Laminin fragments
Nidogen fragment
Fibronectin fragment: anastellin
Histidine-rich glycoprotein (HGRP) fragment
Perlecan fragment: endorepellin
Proteolytic fragments of hemostasis factors
Kringles
Plasminogen fragments: Angiostatin, kringle 5
HMW-kininogen fragment: Kininostatin (kringle 5 of HMWK)
Prothrombin fragments: Kringle 2 fragments of kringles 1 and 2
Antithrombin III fragment
Fibrinogen fragments
Fibrinogen degradation peptide E
Alphastatin: N-terminal 24 aminoacids of the ␣-chain of fibrinogen
Urokinase fragment (Amino terminal fragment, Kringle)
For review see references 4, 150, 155.
Switching Off Angiogenesis: Matrikines as
Endogenous Inhibitors Generated
by Proteases
Invasive growth and angiogenesis are accompanied by proteolytic degradation of matrix proteins. Interestingly, among the
proteolytic degradation products derived from extracellular matrix proteins and hemostasis factors a number of fragments have
potent angiogenesis inhibiting properties (Table). A thrombospondin fragment was initially recognized in 1990.145,146 In a
search for primary tumor factors that suppressed the growth of
metastases, Folkman et al isolated angiostatin and endostatin
from the urine and tumor tissue of mice.147,148 When these
compounds were administrated to tumor-bearing mice, these
investigators observed growth arrest, regression, and dormancy
of the tumor grafts.148,149
Presently, a considerable number of extracellular matrix
fragments are known that inhibit angiogenesis (Table)4,150.
Several of them act as competitive inhibitors of specific integrinmatrix interactions,3,150 whereas others affect endothelial metabolism. Endostatin, the C-terminal part of collagen type XVIII, is
generated by cathepsin-L and elastase. It inhibits VEGF-induced
endothelial migration and induces apoptosis.151,152 An important
aspect of the biological activity of endostatin is its ability to bind
via heparin sulfates to ␣5␤1-integrin.152,153 This complex affects
cellular signaling through caveolin and Src and disturbs the
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proper organization of the F-actin cytoskeleton.153 The collagen
type IV fragment tumstatin inhibits focal adhesion kinase (FAK)
through interaction with ␣v␤3-integrin interaction. It inhibits
activation of various signaling kinases (PI3-kinase, Akt,
mTOR), and causes endothelium-specific inhibition of protein
synthesis.154 It is likely that additional extracellular matrix
fragments will be recognized that act as angiogenesis
modulators.
Proteolytic fragments of factors involved in the hemostatic
system represent an additional group of anti-angiogenic regulatory molecules (Table).155 Anti-angiogenic activities in vitro as
well in vivo have also been identified in the proteolytically
generated kringle domains of plasminogen,147,156 prothrombin,157 HMW-kininogen,158 and u-PA.159 Although angiostatin
(kringles 1 to 4, generated from plasminogen by various proteases, including MMP-3, MMP-7, MMP-9 and u-PA) was the
first degradation product recognized to have anti-angiogenic
properties, the kringle-5 domain of human plasminogen has been
shown to possess powerful anti-angiogenic activity of the
plasminogen fragments as well.156,160 These plasminogenderived kringles target to several proteins on the endothelial
membrane including ATP synthase, angiomotin, ␣v␤3-integrin,
and the HGF receptor c-met and induce endothelial cell death.160
The N-terminal 24 amino acid peptide of the ␣-chain of human
fibrinogen has been recognized to harbor the anti-angiogenic
activity that was originally observed to be present in fibrinogen
degradation product E.161
Stabilization of New Vessels: Proteases and
the Recruitment of Pericytes
The present paradigm of angiogenesis considers the smoothmuscle-like pericyte as key regulator of the stabilization of
newly formed vessels.162 MMP-9 was present in pericytes
present in the stroma of tumors of breast cancer patients.163 The
recruitment of pericytes was disturbed in animals that were made
deficient of MMP-9.164 This markedly affects the stability of
vessels and the degree of vascularization of neuroblastomas.164
Aminopeptidase A was also present in activated pericytes in
various pathological conditions associated with angiogenesis,
whereas its expression was low in pericytes of quiescent vessels.165 The mechanism how this protease contributes to angiogenesis has not yet been investigated. Pericyte-derived TIMP-2
and TIMP-3 can inhibit MT1-MMP dependent MMP-2 activation on the endothelial cells, and thus may contribute to the
stabilization of newly formed microvessels.166,167
Without pericyte covering, the immature vessels remain
dependent on the continuous exposure to angiogenic growth
factors.162 Once their supply ceases, the endothelial cells do no
longer survive.168,169 Caspases are involved in endothelial apoptosis, while other proteases take part in the degradation and
removal of the cell remains.
Increasing Proximal Blood Supply
When angiogenesis is effective many new microvascular structures are formed. However, to acquire an adequate perfusion of
these newly formed vessels, the proximal vascular tree has to be
remodeled. Enlargement of small arteries and arterioles will be
necessary to provide a suitable amount of blood per unit of time.
This requires remodeling of the smooth muscle cell layer of
these vessels, a process that also involves various proteases that
were described above to be involved in matrix remodeling and
migration. The reader is referred to the work of Schaper et
al170,171 for further details.
Perspective
From this article, it is clear that proteases can influence neovascularization in many different ways, both in development and
particularly in pathological conditions. Because they are major
regulators of tissue destruction and cell migration, it was initially
expected that inhibition of matrix-degrading proteinases would
be beneficial to counteract growth of tumors and their vasculature. Although promising results were obtained in animal studies, clinical trials failed to show an improving effect and often
resulted in unacceptable side effects.172 This has stimulated
further research, from which new families of proteases, such as
MT-MMPs, ADAM, and ADAMTS, were discovered. More
importantly, a new view has become apparent on how proteases
modulate growth factors and cytokines and generate new biologically active fragments from matrix and circulating proteins.
Protease inhibitors also appear to have additional unexpected
effects by themselves, of which much has still to be learned. The
new insights in the modulation of cytokines by proteases
contribute to the understanding of inadequate cytokine-mediated
response in disease, as well as in improving the use of growth
factors and cytokines in treating disease. In addition, the new
biological entities that inhibit or stimulate angiogenesis can
provide new leads for developing agents that inhibit or stimulate
angiogenesis, depending on the disease to be combatted.
The complexity of the effects of proteases in disease is
enhanced by parallel activities of proteases from similar or even
different families. Better understanding of these mechanisms
will be helpful in establishing strategies to deal with these
redundancies and in avoiding side effects. The use of agents that
target to specific cells and receptors on the cell surface173 and
combined use of either specific protease inhibitors or virally and
nonvirally delivered siRNAs127 are options that may contribute
to reduce the burden of specific malignancies.
Although mainly studied from the angle of tumor growth and
treatment, the role of proteases in angiogenesis extends to many
other aspects of human life, starting with implantation of the
embryo and placentation and growth of the fetus. Proper wound
healing and tissue repair after inflammation depend on proteases.
Furthermore, the lack of blood perfusion or the excessive growth
of vessels is pivotal in many pathological conditions.174,175 It is
likely that the substrates and products of proteases will be the
preferred targets for treating these diseases, but successful
applications often depend on knowledge how these proteins and
peptides are activated, altered, and generated by proteases.
Acknowledgments
This work was supported by grants of ZON-MW (902-90-017),
STW/DTPE (VGT.6747), the Netherlands Heart Foundation
(M93.001), and the European Vascular Genomics Network (LSHMCT-2003-503254). The authors thank Dr Roeland Hanemaaijer for
critical reading the manuscript and advice.
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Pericellular Proteases in Angiogenesis and Vasculogenesis
Victor W.M. van Hinsbergh, Marten A. Engelse and Paul H.A. Quax
Arterioscler Thromb Vasc Biol. 2006;26:716-728; originally published online February 9, 2006;
doi: 10.1161/01.ATV.0000209518.58252.17
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