Matrix Metalloproteinases and Collagen Remodeling in Tissue Engineered Heart Valves

Matrix Metalloproteinases and
Collagen Remodeling
A Literature Review
Mieke M.H. van Marion
July 2006
BMTE 06.55
Committee:
prof. dr. ir. Frank P.T. Baaijens
dr. Anita Mol
dr. Maarten Merkx
Advisors:
ir. Mirjam P. Rubbens
Eindhoven University of Technology

Department of Biomedical Engineering
Division Soft Tissue Biomechanics and Engineering
Contents
1 Introduction
2 The Aortic Heart Valve

2.1 Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Matrix Metalloproteinases in Heart Valves . . . . . . . . . . . . . . . . . . . .
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3 Collagen
3.1 Structure . . . . . . . . . .
3.2 Biosynthesis . . . . . . . . .
3.3 Degradation . . . . . . . . .
3.4 Mechanical Characteristics .
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4 Matrix Metalloproteinases
4.1 Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 Role in Matrix Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5 Matrix Metalloproteinase Inhibition

5.1 Endogenous Inhibitors of MMPs . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Synthetic MMP Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6 Collagen Remodeling
6.1 Mechanically Induced Collagen Remodeling . . . . . . . . . . . . . . . . . . .
6.2 The Role of MMPs in Mechanically Induced Collagen Remodeling . . . . . .
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7 Quantification of Collagen Remodeling

7.1 Quantification and Visualization of Collagen . . . . .
7.1.1 Analysis of Collagen Content and Cross-links
7.1.2 Visualization of Collagen Formation . . . . .
7.2 MMP Analysis Methods . . . . . . . . . . . . . . . .
7.2.1 RNA Analysis . . . . . . . . . . . . . . . . .
7.2.2 ELISA . . . . . . . . . . . . . . . . . . . . . .
7.2.3 Western Blotting . . . . . . . . . . . . . . . .
7.2.4 Zymography . . . . . . . . . . . . . . . . . .
7.2.5 Peptide Substrates . . . . . . . . . . . . . . .
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8 Future Research
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3
Contents
Bibliography
51
Chapter 1
Introduction
The most commonly used method to treat heart valve diseases is replacement of the valve.
Currently available heart valve substitutes are mechanical prostheses, xenografts/ heterografts, and human aortic allografts/ homografts. Although these grafts have proved to be
functional, they have some important drawbacks. Trombogenicity, a limited life time, and
immunological reactions are the major limitations of these grafts. Tissue engineering uses patients own cells seeded on a biocompatible and biodegradable scaffold for mechanical integrity.
In time, the synthetic scaffold will degrade and the cells will build up their own functional
matrix. In this way, the tissue engineered heart valve has the potential to grow, repair, and
remodel. Therefore, tissue-engineered heart valve constructs will be able to offer improved
material and biological properties compared to conventional heart valve replacements.
Tissue engineered heart valve leaflets have already been implanted successfully in sheep at
the pulmonary position [44]. However, the pulmonary circulation is characterized by lower
pressure and stresses than the aortic circulation, resulting in an improved valve functioning
at this site. For application at the aortic position, more advanced biomechanical properties
are necessary to develop a fully functional heart valve substitute.
The mechanical properties of connective tissue are mainly determined by the collagen architecture (fibre density, structure, orientation, cross-linking etc.). Therefore, collagen remodeling
is thought to be an important factor in improving the mechanical properties of a tissue engineered heart valve. Specific collagenases (matrix metalloproteinases, (MMPs) and their
inhibitors (TIMPs) are essential in the regulation of collagen remodeling in vivo. By breaking
down collagen fibres at specific sites in the tissue, these enzymes regulate the degradation of
collagen and play an important role in the determination of its structure and orientation. It is
furthermore established that mechanical conditioning induces an increased collagen orientation and production [21, 43, 60]. In addition to this, mechanical conditioning also influences
the expression and activity of MMPs and TIMPs [4, 51, 65, 73]. A combination of mechanical
conditioning, and controlled MMP activity thus strongly influences the collagen architecture
and will enhance the mechanical properties of the tissue engineered heart valve (figure 1.1).
Chapter 1
Introduction
Figure 1.1: Relation between mechanical conditioning, MMPs, collagen architecture, and the mechanical properties of the tissue.
This literature review focusses on collagen remodeling and MMP/TIMP functions. It discusses the effects of mechanical conditioning on these aspects and the implications for the
mechanical properties of the tissue engineered heart valve.
Chapter 2
The Aortic Heart Valve

2.1
Anatomy
The aortic heart valve, or semiluminar valve, separates the left ventricle from the aorta. It
opens during systole, resulting in a blood flow into the aorta, and throughout the body. Closure occurs during diastole, preventing back flow from the aorta into the left ventricle. The
aortic heart valve is composed of three pocket-like leaflets, shaped like a half-moon, three
sinuses, and the aortic ring (figure 2.1). The leaflets and the sinusses together are called the
cusps. During opening and closure of the valve, the cusps have to withstand large cyclical
deformations (up to 50%), and high pressures [62]. To prevent from prolapsing under these
high pressures, the adjacent cusps have a contact area of approximately 40% of their surfaces.
Figure 2.1: Schematic representation of the aortic heart valve: (a) side view of the complete valve,
(b) after removal of one leaflet and the corresponding sinus, and (c) view from the aortic side (from
Hart et al. 2002 [17]).
In the aortic heart valve two general cell types are present: the endothelial cells, that form a
one layered cover between the valve tissue and the blood, and the interstitial cells. The interstitial cells have both fibroblast and smooth muscle cell features and are able to synthesize
and remodel the extracellular matrix (ECM). Furthermore, the valve is composed of three
distinct tissue layers with different ECM components (figure 2.2). The ventricularis, just below the inflow surface, comprises collagen and radially aligned elastin fibres. The spongiosa is
7
Chapter 2
located in the center, and is composed of randomly arranged collagen and glycosamoglycans
(GAGs). The GAGs are able to absorb the shear and cushion shock between the ventricularis
and the fibrosa during valvular motions. The third layer, the fibrosa, is situated below the
outflow surface, and is composed of densely packed collagen fibres that are arranged parallel
to the free edge of the cusp. Its function is to provide strength and stiffness during diastole.
The cusps are relatively thin and can be perfused by the blood that flows through the valve.
Therefore, the cusps contain almost no blood vessels.
Figure 2.2: Histological cross section of a porcine valve cusp showing the three major layers. Approximately the lower one-third represents the ventricularis, the middle third the spongiosa, and the top
third the fibrosa. El = elastin; outflow at the top (from Schoen et al. 1999 [62]).
Collagen and elastin are arranged perpendicularly to each other in the cusp layers, and cooperate during valve motion (figure 2.3) [62]. During opening, at minimal loads, the elastin
fibres extend and the collagen fibres unwrinkle and uncrimp. Near full closure, the load has
increased significantly and stresses rise. The load-bearing element now shifts from elastin to
collagen that is further extended by stretching of the fibres. In the systole, the elastin fibres
contract, and restore the configuration of the cusps.
Chapter 2
Figure 2.3: Structural and biomechanical features of the aortic valve. At the top: Schematic representation of cuspal conformation, and collagen and elastin architecture in systole and diastole. At the
bottom: Schematic representation of the biomechanical cooperation between collagen and elastin during
valve motion (from Schoen et al. 1999 [62]).
2.2
Matrix Metalloproteinases in Heart Valves
Matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs)are the principle enzymes that regulate collagen remodeling, and play
an important role in the determination of the collagen architecture of the tissue. Both MMPs
and TIMPs are differently expressed in the distinct zones of the aortic valve and are mainly
located within and around the interstitial cells. Dreger et al. [19] demonstrated that the
leaflets express MMP-1, -2 and TIMP-2 , the belly and freewall regions express MMP-1, and
that in the commissure zone MMP-2, and TIMP-1 are expressed. In this study, MMP-3, and
TIMP-3 could not be detected within the cusps, and surprisingly also not MMP-9, while in
another study by Koullias et al. [36], MMP-9 was mildly present in healthy aortic valvular
tissue. In both studies MMP presence was determined using immunohistochemistry. Koullias
et al also demonstrated the presence of MMP-1, and -2 and TIMP -1, and -2 in healthy aortic
heart valves. Diseased valves showed an enhanced presence of MMP-2 and/or -9. Moreover,
Rabkin-Aikawa et al. demonstrated significantly increased levels of MMP-1, -2, -9, and -13 in
myxomatous heart valves compared to healthy valves [57], and a significantly enhanced level
of MMP-1, and MMP-13 in fetal aortic heart valves compared to adult valves [2].
Chapter 2
A suitable cell type for use in tissue engineered heart valve constructs is the human saphenous
vein myofibroblast or smooth muscle cell (SMC). Galis et al. [24] investigated the capacity
of these cells to express MMPs and TIMPs in a monolayer and under serum free conditions.
Human saphenous vein SMCs showed a constitutively secreted level of MMP-2 and TIMP-1,
and -2. MMP-1, -3, and -9 were only present after cytokine treatment. They furthermore
revealed that TIMP-2 is mostly present in its complexed form with MMP-2 and that the same
SMC can produce both MMPs and TIMPs. In addition to this, Meng et al. [42] demonstrated
an increased level of active MMP-2, and pro MMP-9 by zymography and western blot in
injured human saphenous vein SMCs.
10
Chapter 3
Collagen
3.1
Structure
Collagens are the most abundant proteins of the extracellular matrix (ECM) and the major
structural component of connective tissues. They are characterized by three left handed helices, the chains, which together form a right handed triple helix. This triple helix can be
formed out of three identical chains (homotrimer, e.g. type I collagen) or out of two or
more different chains (heterotrimer, e.g. type V collagen). The primary structure of every
collagen molecule is a repeated unit of three amino acids. Every first amino acid of this
unit is a Glycine (Gly), the smallest amino acid. The other two are mostly proline and/ or
hydroxyproline, but it is also possible that a lycine is incorporated. An important feature
of hydroxyproline is its ability to form intermolecular H-bonds which are essential for the
stability of the triple helix. In the triple helix, the glycines are positioned at the center, while
the larger amino acids are placed at the outer sides, resulting in a dense packing around
the central axis of the molecule. At the N- and C-terminus, collagen contains a non-helical
structure, the telopeptides. These structures are able to form covalent cross-links to collagen
and other molecules, which is important for the mechanical integrity of the molecule.
Until now, 26 genetically different collagen types have been identified. The different types
are characterized by their complexity and diversity in structure, splice variants, presence of
additional non-helical domains, assembly and function. They can be classified in the following groups: fibril forming collagens, fibril associated collagens, network forming collagens,
anchoring fibrils, transmembrane collagens, and others. Type I collagen is the most abundant
and is present widespread in the body, except in cartilage, and synthesized in response to
injury. Table 3.1 lists the molecular composition and tissue distribution of the collagen types
[25].
11
Chapter 3
Collagen
Type Molecular composition

Fibril-forming collagens
I
[1(I)2 2(I)]
II
III
[1(II)]3
[1(III)]3
1(V),2(V),3(V)
Tissue distribution
bone, tendons, skin, ligaments, cornea, many interstitial
connective tissues (of e.g. heart valves)
hyaline cartilage
collagen I containing tissues, interstitial tissue of lungs, liver,
dermis, spleen, and vessels
forms heterofibrils with collagen I and III, present in bone
matrix, corneal stroma, and interstitial matrix of muscle,
lungs, liver, and placenta
codistributed with collagen II in articular cartilage
XI
1(VI),2(VI),3(VI)
Fibre Associated collagens
IX
1(IX),2(IX),3(IX)
codistributed with collagen II in cartilage and vitreous body
XII
[(XII)]3
resp. associated or colocalized with collagen I in skin, perichonand
[(XIV)]3
drium, periosteum, tendons, lung, liver, placenta, and vessel
XIV
walls
XIX
[1(XIX)]3
human rhabdomyosarcoma
XX
[1(XX)]3
corneal epithelium, embryonic skin, strenal cartilage, tendon
XXI
[1(XXI)]3
blood vessel wall
Microfibrillar Collagens
VI
1(VI),2(VI),3(VI)
all connective tissues, except bone
Hexogonal network-forming collagens
X
[3(X)]3
hypertrophic cartilage in fetal and juvenile growth plate,
ribs, and vertebrae
VIII
[1(VIII)]2 2(VIII)
endothelial cells, Descemets membrane (cornea)
Basement membrane collagens
IV
[1(IV)]2 2(IV)
basement membrane
Anchoring fibrils
VII
[1(VII)]3
skin, dermal-epidermal junctions, oral mucosa, cervix
Multiplexins
XV
[1(XV)]3
fibroblasts, smooth muscle cells, kidney, pancreas
XVI
[1(XVI)]3
hyaline cartilage and skin, associated with coll II fibres
XVIII [1(XVIII)]3
lungs, liver
Table 3.1: Collagen types, molecular composition and tissue distribution.
12
Chapter 3
3.2
Collagen
Biosynthesis
Collagen molecules are synthesized in the cells and released in the extracellular space after
which fibril formation takes place [25] (figure 3.1). In the cells, pre-procollagen is formed after
translation of the mRNA. The pre-procollagen is transported to the endoplasmatic reticulum,
where the signal pre-domain of the peptide is cleaved off. The proline and lysine of the procollagen are hydroxylated after which three chains assemble and form a triple helix. Then, the
procollagen helices fold and form intra- and inter chain disulphide bonds. After processing
and procollagen assembly, the triple helices are packed into secretory vesicles within the Golgi
apparatus and released into the extracellular space. Here, the C- and N-propeptides, important for triple helix formation respectively regulation of primary fibril diameters, are cleaved
off. In the extracellular space, the fibril forming collagens spontaneously aggregate into ordered fibrillar structures by a self-assembling process. The fibrils form a ordered cross-striated
structure, resulting in the typical banding pattern of collagen (figure 3.2). The generated fibrils have a blunt end, and a pointed paraboidal tip, from which growth proceeds [32]. When
growth proceeds, the blunt end can transform in a pointed tip for growth in the other direction. The fibrils can be stabilized by the formation of inter-fibrillar cross-links. Cross-links
are formed between the lysine and hydroxylysine of the telopeptides, which is initiated by the
enzyme lysyl oxidase, resulting in intermediate cross-links, or the enzyme lysyl hydroxylase
(PLOD), resulting in more stable bonds. Cross-linking of collagen is crucial for the stability
of its final fibrillar structure, and therefore plays an important role in the determination of
the mechanical properties of the tissue [5].
13
Chapter 3
Collagen
Figure 3.1: Schematic representation of the extracellular events in the synthesis of fibrillar collagens.
Procollagen is secreted from the cells and the prodomain is cleaved off by specific proteinases. The
generated collagen spontaneously self-assembles into cross-striated fibrils. These fibrils are stabilized
by the formation of covalent cross-links. (From Kadler et al. 1996 [32]).
Figure 3.2: Representation of a collagen fibril. (a) Transmission electron micrograph of a positively
stained bipolar fibril from a 18-day chick embryo metatarsal tendon, (b) enlargement of the banding
pattern, and (c) schematic representation of the arrangements of the helices in the transmission region
of the fibril (from Kadler et al. [32]).
14
Chapter 3
3.3
Collagen
Degradation
Extracellular matrix remodeling requires the degradation of its components. In general,

four types of proteolytic enzymes, capable of ECM degradation, exist: (1) matrix metalloproteinases (MMPs), (2) serine proteinases (e.g. plasmin), (3) cysteine proteinases (e.g.
cathepsin K), and (4) aspartic proteinases. The MMPs are considered to be essential for the
degradation of the main matrix component, collagen. Of the MMPs, the collagenases are
responsible for the first degradation step of collagen, in which the fibres are cleaved into the
characteristic 1/4 and 3/4 fragments. Gelatinases and cysteine proteases can further degrade
the collagen fragments. The necessity of the first cleavage step of collagen by collagenases,
is demonstrated by Krane et al. [40] by studying the effect of mutations at the collagen
cleavage site on tissue remodeling in mice. Mutant mice died, or developed skin and uterus
abnormalities due to collagen accumulation, suggesting that cleavage of collagen at the 14 / 34
site by collagenases is essential for its further degradation by other proteases.
For effective collagenolytic activity, the enzyme must be able to (1) bind to collagen, (2)
unwind the triple helix, and (3) cleave the individual strands of the helix. Lauer-Fields et al.
[37] developed a MMP collagen cleavage model. They suggest that collagen cleavage occurs at
the border of a tight triple helical region (high imino acid content) and a region of loose triple
helical conformation (low imino acid content) (figure 3.3). They furthermore established that
the ability of MMPs to cleave collagen depends on the cooperation of the catalytic domain,
the C-terminal hemopexin, and the hinge region of the enzyme. The hemopexin domain is
responsible for the binding of MMPs to the fibers and the orientation and destabilization of
the fibers. The catalytic domain unwinds the triple helix and cleaves the individual strands.
The precise role of the hinge region is still unknown, but removal of this region results in a
loss of collagenolytic activity of the MMP, which proves its necessity.
Figure 3.3: Model of the collagenase cleavage site in interstitial collagens. Cleaving occurs at the
border of a tight triple helix region, which is rich in imino acids, and a loose triple helix region, which
is deficient of imino acids. (From Lauer-Fields et al. 2002 [37]).
15
Chapter 3
3.4
Collagen
Mechanical Characteristics
Improving the mechanical properties of a tissue requires knowledge of the mechanical characteristics of its components. Collagen is the main structural component of the ECM and has
thus a major contribution to the mechanical properties of a tissue engineered construct. A
general method to analyze the mechanical properties of a material is to determine its stress/
strain curve. Therefore, the stress in the material is measured as function of the elongation.
The elongation of the material, expressed in percentages of its original length, is referred to
as the strain, and is a measure for tissue extensibility. The stress is the force per unit area
that the material experiences in response to strain and is a measure for tissue strength. The
slope of the linear part of a stress/ strain curve is referred to as the stiffness of the material
(also called modulus of elasticity or Youngs modulus).
The mechanical properties of collagen are dependant on its fibrillar structure [23]. A typical
stress/ strain curve of collagen consists of three zones: the toe, heel, and linear region (figure
3.4). In the toe region, small strains cause the removal of crimp in the collagen fibres. At
higher strains (3% and more), the kinks in the collagen molecules are straightened, leading to
an elongation of the fibrils and a reduction of the entropy. This part of the stress strain curve
is referred to as the heel region and has a typical upwards curvature. When all kinks in the
collagen molecule are straightened, the triple helices and cross-links will be stretched (linear
region). This causes a gliding of the neighboring molecules and eventually rupture. The
contribution of cross-links and the inter fibrillar substance to the the mechanical behavior of
collagen is still unknown. However, cross-links stabilize the collagen fibrils, which will result
in enhanced mechanical properties of the tissue.
Figure 3.4: Typical stress strain curve of rat tail tendon, representing the toe, heel and linear region.
(a) Removal of the macroscopic crimp of the fibrils in the toe region, visualized using polarized light.
(b) Further structural changes of collagen occur at the fibrillar level: straightening of the molecular
kinks in the gaps in the heel region, and gliding of the molecules in the linear region. (From Fratzl et
al. [23]).
16
Chapter 4
Matrix Metalloproteinases
Matrix metalloproteinases (MMPs), also called matrixins, are a group of zinc-dependant
proteolytic enzymes responsible for extracellular matrix (mainly collagen) remodeling and
degradation. In healthy tissue some MMPs are expressed at low levels, while others are only
marginally expressed or even absent. However, during tissue remodeling, diseases, or inflammation, MMP levels are often elevated. This chapter describes the structure and function of
MMPs and their role in matrix remodeling.
4.1
Structure and Function
In humans 23 different MMPs have been identified. They generally consist of a prodomain, a
catalytic domain, containing three histidines that bind zinc, a hinge region and a hemopexin
domain (figure 4.1). Based on their substrate specificity, MMPs can be divided into 6 groups.
The collagenases (MMP-1, -8, and -13) cleave the interstitial collagens I, II, and III by hydrolyzing the peptide bond after the Gly at 3/4 of the N-terminal end, resulting in the characteristic 1/4, and 3/4 fragments. The denatured collagen, or gelatin, is further degraded by
the gelatinases (MMP-2, and -9). Other groups are the stromelysins (MMP-3, -10, and -11),
the matrilysins (MMP-7, and -26), the membrane-type MMPs/ MT-MMPs (MMP-14, -15,
-16, -17, -24, and -25) and others (MMP-12, -19, -20, -21, -22, 23, -27, and -28)[48, 66, 67, 69].
Table 4.1 summarizes the MMPs and their substrates.
17
Chapter 4
Enzyme
MMP
Substrate [Collagen
Type][66] (additions
from [67, 69])
Other Substrates
Collagenases
Collagenase -1
MMP-1
gelatin
Collagenase -2
Collagenase -3
MMP-8
MMP-13
I, II, III, VII, X (VIII,

XI)
I, II, III (VII, VIII, X)
I, II, III (IV, VI, X,
XIV)
Gelatinases
Gelatinase A
MMP-2
I IV, V, VII, X (II, III,

X)
IV, V, XIV (XI)
gelatin
gelatin
aggrecan, gelatin
gelatin
Gelatinase B
Stromelysins
Stromelysin-1
MMP-9
Stromelysin-2
Stromelysin-3
Matrilysins
Matrilysin-1
Matrilysin-2
MMP-10
MMP-11
III, IV, IX, X (II, VII,

XI)
IV (III, V)
IV
MMP-7
MMP-26
IV (I)
IV
fibronectin, laminin, gelatin

fibrinogen,
fibronectin,
gelatin
gelatin, fibronectin, laminin
MMP-3
Membrane Type MMPs

MT1-MMP
MMP-14
gelatin
laminin, fibonectin, elastin

fibronectin, laminin, aggrecan
MT2-MMP
MMP-15
MT3-MMP
MT4-MMP
MT5-MMP
MT6-MMP
Others
Macrophage metalloelastase
-
MMP-16
MMP-17
MMP-24
MMP-25
I, II, III ( and proMMP2)

general (and proMMP2)
- (III)
IV
MMP-12
- (I, IV)
elastin, fibronectin
MMP-19
IV (I)
Enamelysin
XMMP
CMMP
Epilysin
MMP-20
MMP-21
MMP-23
MMP-27
MMP-28
unknown
unkown
aggrecan, elastin, fibrillin,

gelatin
aggrecan
aggrecan
gelatin, casein, fibronectin
unknown
unkwown
Table 4.1: The MMP family.
18

fibrinogen, fibrin
gelatin
Chapter 4
Figure 4.1: Domain structure of MMPs. S, signalpeptide; Pro, prodomain; Cat, catalytic domain;
Zn, active zinc; Hpx, hemopexin domain; Fn, fibronectin domain; V, vitronectin insert; I, type I
transmembrane domain; II, type II transmembrane domain; G, GPI anchor, Cp, cytoplasmic domain;
Ca, cysteine array region; and Ig, IgG-like domain. (From Visse et al. 2003 [72]).
4.2
Regulation
MMP regulation takes place at different levels. Firstly, specific growth factors (e.g. transforming growth factor 1), cytokines (e.g. TNF and IL-1), and matrikines are important
regulators of MMP gene expression [38], and control the transcription of mRNA encoding for
the MMPs. In addition, EMMPRIN (an extracellular MMP inducer of the immunoglobulin
family) and mechanical stretch are also known to regulate MMP expression (see chapter 6).
Secondly, specific regulation mechanisms exists that control the translation of mRNA to the
actual protein. Lastly, MMP regulation can take place on protein levels, where the activation
the enzyme is controlled.
The majority of the MMP proteins are secreted as inactive proMMPs, also called latent
MMPs or zymogens, and activated in the extracellular space induced by serine proteases (e.g.
plasmin) or already activated MMPs [38, 67, 69, 72]. Latent MMPs become activated when
the sulphide bond between the cysteine in the prodomain and the zinc in the catalytic domain
is disrupted (the cysteine switch). After disruption, the sulphide group is replaced by a water
molecule, resulting in the intermediate active form (figure 4.2) [67]. The active form of MMPs
is formed when the prodomain is removed by autolytic cleavage or by other proteases.
The activation of MMP-2 is regulated differently, and takes place on the cell surface, mediated
by MT-MMPs and TIMPs (figure 4.3). A suggested mechanism is that TIMP-2 binds with its
C-terminus to the hemopexin domain of proMMP-2 [38, 72]. The N-termini of both molecules
are thus still free, so that both the MMP and the inhibitor maintain their properties. After
formation, the proMMP-2/TIMP-2 complex binds to the active site of MT1-MMP and the
bounded proMMP-2 can be activated by another MT-MMP. The crystal structure of TIMP2/proMMP-2 suggests that the complex does not work as a rate enhancing step for protease
inhibition, but that the TIMP has a bridging function to connect proMMP-2 to other MMPs
to induce activation [46]. In addition, it is established that at low concentrations, TIMP-2
serves as a receptor for proMMP-2, while at high concentrations TIMP-2 neutralizes MT19
Chapter 4
MMP and prevents MMP-2 activation [38].

Regulation of active MMPs takes place by specific inhibitors. These mechanisms will be discussed in more detail in chapter 5.
Figure 4.2: The stepwise activation of latent MMP into active MMP. The latent form becomes activated upon cleavage of the sulfide bond, and the uptake of a water molecule, resulting in the intermediate active form. The MMP is fully activated after removal of the prodomain, initiated by proteases
or autolytic cleavage. (From Snoek et al. 2005 [67]).
20
Chapter 4
Figure 4.3: Schematic representation of the activation of MMP-2 by TIMP-2, and MT1-MMP (from
Sternlicht et al. 2001 [69]).
21
Chapter 4
4.3
Role in Matrix Remodeling
It is well established that MMPs are essential for matrix remodeling and breakdown. For
example, collagen type IV degradation in the basement membrane of ex vivo cultured human
saphenous vein segments was shown to be mediated mostly by MMPs instead of cysteine
proteases [1]. However, it is still debatable which MMPs play the major roles in matrix these
processes.
It is generally assumed that MMP-1, -8, -13 , -14, and cathepsin K are the proteases responsible for the first degradation step of collagen, and that MMP-2, and -9 further digest
the degraded collagen fibres. In contrast to this, Creemers et al. [14] demonstrated that
MMP-2 is primarily responsible for collagen digestion in the soft connective tissue of the
rabbit periosteum. They also assessed that the amount of active MMP-2 increased with the
level of collagen digestion. Kerkvliet et al. [33] confirmed these findings and showed that
MMP-2 plays an important role in the digestion of collagen types I, II, IV, and V, the main
collagen types in periosteal tissue. This suggests that next to MMP-1, MMP-2 is important
for collagen degradation. Kerkvliet et al. furthermore assessed that the levels of MMP-1, -8,
-9 and -13, and cysteine proteases were not related to the level of collagen digestion in their
experimental set-up. The feature that MMP-2 has also collagenolytic activity is supported by
a study by Aimes et al. [3], in which it is demonstrated that MMP-2, in absence of TIMPs,
is capable of collagen I cleavage.
Rabkin-Aikawa et al. [58] investigated the role of MMPs in collagen remodeling. They demonstrated an increased MMP-13 expression in clinical early pulmonary-to-aortic valve (PAVs)
transplants compared to normal pulmonary and aortic valves, while in late PAVs MMP-13
levels decreased to normal levels. This initial increase in MMP-13 is probably related to the
cell activation caused by surgical trauma and subsequent collagen remodeling. It is suggested
that a change in mechanical environment causes an activation of the interstitial cells, resulting
in an upregulation of matrix related enzymes. When the extracellular matrix is fully remodeled and adapted to its new environment, the cells will return to their quiescent state. This
concept is confirmed by two other studies of Rabkin-Aikawa, which showed an significantly
increased level of MMP-1, -2, -9, and -13 in myxomatous valves compared to healthy controls
[57] (figure 4.4), and strongly elevated levels of MMP-1, and MMP-13 in fetal heart valves
compared to very low levels in adult valves [2] (figure 4.5). Moreover, during maturation
of the heart valves, collagen content, fiber alignment, fiber maturation, and elastin content
increased, indicating an important role for MMPs in collagen remodeling. Next to Rabkin et
al., Sung et al. [70] stated that MMP-9 is responsible for approximately 39 % of the collagen
remodeling in in vitro cultured mouse aortic smooth muscle cells. In addition to this, Defawe
et al. [18] demonstrated a dual effect of MMP-9 on rat aortic smooth muscle cell mediated
collagen gel contraction. At low MMP-9 concentrations collagen contraction was increased,
indicating a stimulating effect on collagen remodeling, while at high concentrations, collagen
contraction was inhibited. Altogether, these results suggest that MMP-1, -2, -9 and -13 are
important regulators of collagen remodeling.
Lastly, the effect of collagen on MMP levels is studied by Kerkvliet et al. [34]. They showed
that collagen amount and type modulate MMP levels in cultured rabbit periosteum fibroblasts. While MMP-2 increased in collagen coated cultures, the level of MMP-1, and -3 decreased. In addition, collagen digestion was highest in type I, and lowest in type V coated
cultures. MMP-2 and MMP-1, -3 thus showed an opposite behavior in the collagen coated
22
Chapter 4
cell cultures. It is therefore suggested that in situations where cells are in direct contact with
relatively low amounts of collagen (normal situation), MMP-1 levels are low, and collagen
digestion is mediated by MMP-2. Conversely, when the amount of degradable collagen increases (e.g. in inflammation), the levels of both MMP-2, and MMP-1, and -3 are likely to
increase.
Figure 4.4: Expression of proteolytic enzymes in the interstitial cells of healthy and myxomatous
aortic heart valves as analyzed by immunohistochemistry. Diseased valves show significantly increased
levels of MMP-1, -2, -9, and -13. (From Rabkin et al. 2001 [57]).
Figure 4.5: Protein expression in the interstitial cells of fetal, adolescent, and adult aortic heart valves
as analyzed by immunohistochemistry. Adult valves show a significantly lower expression of -SMA
(marker for the phenotype of myofibroblasts) and SMemb (marker for cell activation), and MMP-1,
and -13. (From Aikawa et al. 2006 [2]).
23
Chapter 4
24
Chapter 5
Matrix Metalloproteinase Inhibition

MMPs are responsible for the degradation and remodeling of collagen. A disturbed balance of
MMPs and TIMPs will therefore affect the collagen content and, consequently, the mechanical
properties of the tissue. Inhibition of MMPs can be regulated at three different levels: (1)
inhibition of protein expression and synthesis, (2) inhibition of the activation mechanism,
and (3) inhibition of the active enzyme (figure 5.1). This chapter focusses on the last level
and discusses the endogenous regulation of MMPs and the possibilities to use synthetic/
non-specific inhibitors to control MMP activity.
Figure 5.1: MMP inhibition can be regulated at three levels: (1) inhibition of MMP expression and
protein synthesis, (2) inhibition of MMP activation, and (3) inhibition of MMP activity (from Sluijter
et al. 2005 [66]).
25
Chapter 4
5.1
Matrix Metalloproteinase Inhibitors
Endogenous Inhibitors of MMPs
Endogenous inhibition of MMPs is performed by their natural inhibitors: the tissue inhibitors
of metalloproteinases (TIMPs), of which four different types (TIMP-1, -2, -3, and -4) have
been identified [72]. The N-terminal domain of TIMPs folds as a separate unit, and is responsible for its inhibitory properties. The overall shape of TIMPs is wedge-like and fits in the
active site of an MMP, similar to the substrate. The formed complex is tight and irreversible.
TIMPs are capable of inhibiting all MMPs, with the exception of TIMP-1, which is unable
to inhibit MT-MMPs. They have a very short half-life in vivo, which makes them unsuitable
for pharmacologic applications [38]. Their use in in vitro experiments may be possible by
constant administration or overexpression. For example, Aguilera et al. [1] demonstrated by
immunocytochemistry that adenovirus-mediated overexpression of TIMPs in cultured human
saphenous vein segments resulted in an increased amount of smooth muscle cells surrounded
by collagen type IV (92% in TIMP-1, and 97% in TIMP-3 treated samples compared to 83%
in controls after 14 days of culture). Moreover, TIMP overexpression led to a decreased MMP
activity (15% 3% and 12% 2% for TIMP-1 respectively TIMP-3, and 49% 6% for controls after 14 days of culture, as demonstrated by in situ zymography). Admission of TIMPs
thus results in a decreased MMP activity and an elevated collagen density in vitro.
TIMPs are produced by connective tissues to control local extracellular MMP activity. In
serum and synovial fluid, the 2 -macroglobulins (2 M) are responsible for the inhibition of
MMPs [10, 72, 69]. These high molecular weight general endopeptidase inhibitors inhibit
most proteinases by trapping them in the macroglobulin. MMPs cleave the the bait region of
2 M, initiating a conformational change in the macroglobulin, resulting in entrapment of the
enzyme. When entrapped in 2 M, MMPs have still proteolytic activity but cannot interact
anymore with their natural substrates (collagen) [69]. Small/ low molecular weight substrates
can still be cleaved by MMPs entrapped in 2 M. Eventually, the 2 M/MMP complex is endocytosed and cleared. Collagenases bind preferentially to 2 M, probably because its binding
sites resembles more the collagen binding site than the binding site of TIMPs. Therefore, it
is suggested that TIMPs only regulate MMP activity locally, because there 2 M is excluded
for its size [10]. 2 M is important for a controlled MMP activity in acute situations (e.g.
inflammation) and responsible for the clearance of MMPs.
Tchetverikov et al. [71] demonstrated that a surplus of MMPs over TIMPs results in an
increased level of 2 M/MMP complexes in the circulation using low molecular weight fluorogenic substrates that can still be cleaved by MMPs entrapped in 2 M. They investigated
MMP-13 activity in presence and absence of 2 M and in addition of TIMP (0.1 M) or BB-94
(0.1 M), a synthetic MMP inhibitor, in buffer (containing 10 nM MMP-13) or serum (supplemented with 10 nM MMP-13). It was demonstrated that in buffer TIMP-1 fully inhibits
MMPs in absence of 2 M, but no inhibition was achieved in presence of 2 M. In contrast,
BB-94 in buffer effectively inhibited MMP-13 in presence and absence of 2 M. This indicates
that BB-94 can still inhibit MMPs entrapped in 2 M. In serum, TIMP had no effect on the
activity of MMP-13, while BB-94 did induce a reduced activity of MMP-13. Thus, when
serum is present, all active MMPs that are not complexed to TIMPs will be entrapped in
2 M.
26
Chapter 4
5.2
Synthetic MMP Inhibitors
Next to endogenous inhibition, MMP activation can also be controlled using non-selective
synthetic inhibitors. Similar to endogenous inhibitors, these synthetic inhibitors can regulate MMP inhibition at different levels. For example, Batimastat (BB-94), and its oral form
Marimastat, entrap the active MMP form and inhibit at protein levels, while statins and
doxycyclines inhibit MMPs at gene regulation levels. Most synthetic MMP inhibitors are
non-selective, and thus inhibit all MMPs present in the tissue. An exception are the doxycyclines that are able to selectively inhibit certain MMPs.
Synthetic MMP inhibitors are of particular intrest in clinical situations, to prevent extreme matrix turnovers in arterial remodeling (e.g. (re)stenosis, plaque vulnerability, and
aneurysms), tumor growth, wound healing, arthritis etc. In these situations, MMP inhibitors
prevent the migration of smooth muscle cells and subsequent matrix formation, by decreasing
the rate of collagen degradation. For example, Batimastat and Marimastat have an inhibitory
effect on the formation of a restenosis after balloon dilation [66] and dose dependently inhibit
the rate of tumor marker secreted in the serum [59]. Doxycycline reduces the concentration
of MMP-1 in carotid plaques in rats, and statins are known to inhibit MMP-1, -2, -3, and -9
secretion from rabbit and human smooth muscle cells and macrophages [66]. Most synthetic
MMP inhibitors were discontinued during clinical development due to side effects at concentrations required for efficient inhibition. Until now, the only successfully launched MMP
inhibitor is Periostat, a doxycycline [52]. Despite their limited function in vivo, the use of
synthetic inhibitors in in vitro studies showed some promising results with respect to MMP
inhibition and diminished collagen breakdown. Tables 4.2, 4.3, and 4.4 show the results of in
vitro tissue culture studies in which synthetic MMP inhibitors are used.
Next to their important role in matrix degradation, MMPs also mediate other biological
processes, such as cell death, cell proliferation, and cell differentiation. Broad-spectrum inhibition of MMPs can therefore result in numerous side effects, because next to collagen
breakdown also other physiological processes will be affected, or even blocked. Inhibition of
specific MMPs might therefore be more successful [13]. Moreover, the synthetic compounds
theirselves can have an unwanted effects on other physiological processes. For example, next
to their inhibitory effects on MMPs, statins are known to have a large effect on smooth muscle cell proliferation and function by inhibiting certain cellular mechanisms [12]. Porter et
al. [55] demonstrated a decreased SMC proliferation of 32 to 92 % in human saphenous vein
segments treated with simvastatin (0.1 - 5 M). Therefore, statins are considered less suitable
for use in tissue engineered heart valve constructs. In the same study, Marimastat showed
to have no inhibiting effects on SMC proliferation, and may thus be more suitable for this
application. Another synthetic MMP inhibitor that next to MMP inhibition also affects other
cellular mechanisms is doxycyline that inhibits MMPs by binding to the active Zn site and a
non-active Ca site, causing conformational changes and a loss of enzyme activity. Porter et al.
[56] demonstrated a significantly reduced MMP-9 level (zymography) and intima thickness
in response to doxycycline treatment. However, next to MMP inhibition, doxycycline also
influences the enzyme nitric oxide synthase (NOS), which can significantly affect intima hyperplasia. It is thus unsure whether the observed loss in reduced intima thickness was caused
by the reduced MMP-9 activity, by a change in NOS activity, or by both.
27

Chapter 4
Kerkvliet et al.
1999 [33]
Porter et al. 1998

[54]
Study
Creemers et al.
1998 [14]
Human saphenous vein

segments cultured in
medium
containing
serum and antibiotics
for 14 days.
Culture
Rabbit periosteal explants
cultured
in
medium with BSA and
antibiotics for 72 h.
Porter et al. 1999

[56]
Rabbit periosteal cells

coated on mouse tail
collagen type I, cultured
in medium with BSA
and antibiotics for 72 h.
medium with serum and
antibiotics for 14 days.
Inhibitor
2.5 nM - 25 M
SC44463
1-500 nM CT1166
0.1, 1, 10 M
Marimastat
0.1 M CT1166,
0.2 M CT1399,
0.2
M
and
CT1746 (respectively).
10 g/mL Doxycycline
Analysis methods
Hydroxyproline in conditioned medium.
Gelatin
zymography (MMP activity)
of tissue extracts,
and
immunohistochemistry (neointima
thickness.)
Hydroxyproline
released in medium.
Zymography (MMP-2,
-9) and western blot
(MMP-2,
TIMP-1,
-2) of tissue extracts,
and
immunohistochemistry
(intimal
thickness, immunolocalisation of MMP-2,
-9).
Results
Dose dependant inhibition of hydroxyproline release up to 80 % at 25 M.
Dose dependant inhibition of hydroxyproline release up to 90 % at 50 nM.

Significantly reduced neointima thickness
(median values) in the 105 M (-18.5 m),
and 106 M (-15.5 m) treatment group,
but not in the 107 M group, compared
to controls. MMP-2 levels were significantly reduced in all treatment groups,
and MMP-9 levels in the 105 M, and
106 M treatment group, compared to controls.
Diminished collagen breakdown upto 80%.
Less intimal thickening (5.5 m vs 21 m

in controls), and a significantly reduced
MMP-9 level (0.607 vs. 1.198 in controls). No significant differences could be
observed in pro-, and active MMP-2 levels
and TIMP-1, and -2 levels.
Table 5.1: The effect of synthetic MMP inhibitors on MMP activity, and ECM remodeling.
28
al.
Chapman et al.
2003 [11]
Porter et al. 2002

[55]
Study
Peterson et
2000 [53]
29
10 and 100 M
PD166793, and
40 nM BB-94
0.1, 1, 5 M Simvastatin
Inhibitor
1 and 10
Marimastat
Zymography of conditioned medium and

cell lysates (MMP-2,
-9), immunoblotting
of cell lysates (MT1MMP,
MMP-13),
ELISA (MMP and
TIMP), and MMP9 activity assay of
conditioned medium.
Gelatin zymography of
tissue extracts (MMP
activity),
and immunohistochemistry
(intimal thickness).
Analysis methods
Immunohistochemistry,
zymography and western blots of tissue
extracts.
Results
Significantly reduced neointima formation
(5.5 m in controls, 0.75 m in 10 M, and
1.7 m in 1 M treated samples). Significantly reduced latent and active MMP-2,
and -9 levels in treated samples compared
to controls, more profound in the active
MMP forms and the 10 M treated samples.
Significantly reduced intima formation
(median values were 19.3 m for controls,
8.0 m for 0.5 M (-59%), 6.2 m for 1
M (-68%), and 4.9 m for 5 M (-75%)
treated samples) and MMP-9 levels (median ratio with respect to the levels observed in HT-1080 cells was 1.37 for controls, 1.08 for 0.5 M, 0.67 for 1.0 M, and
0.68 for 5.0 M treated samples.)
Treatment with 10 M PD166793 had no
significant effect on MMP-2, and -9 levels. Exposure to 100 M PD166793 resulted in increased MMP-9 levels (zymography) and activity (activity assay), but
had no effect on TIMP-1 and -2, expression, MMP-2 levels, or MT1-MMP and
MMP-13 levels. In response to BB-94 a
significant increase in MMP-9 levels was
observed, which is in contrast with the
pharmacological effects of this drug.
Human left ventricular myocardial fibroblasts in medium with

for 24-36 h.

Culture
Cultured human internal mammary artery in
Chapter 4

Chapter 4
Study
Daniels et
2003 [15]
al.
Kerkvliet et al.
2003 [35]
Luan et al. 2003

[41]

SMCs (pretreated with
Il-1 and PDGF) in
serum free medium
with antibiotics for 48
h.
Culture
Human tenons capsule
fibroblasts seeded in a
free collagen (type I)
lattice model, cultured
in medium containing
for 7 days.
Rabbit periosteal explants,
cultured in
medium with BSA and
antibiotics for 72 h.
Corpataux et al.
2005 [12]

explants in medium
with serum and antibiotics for 14 days.
50 nM Cerivastatin
CT1166, CT1399,
and CT1746 at
0.1-10 M
Inhibitor
Ilomastat, BB-94,
and BMS-275291,
0.1 nM - 100 M
Zymography and western blot of conditioned

media.
Zymography
and
western blot (MMP-2,
-9) on conditioned
medium, and rt-PCR
(MT1-MMP) on tissue
extracts.
Analysis methods
ELISA, zymography of
conditioned medium,
and collagen lattice
contraction.
Reduced expression of MMP-1 (94%

5%), MMP-2 (57% 2.3%), MMP-3 (78%
24%), and MMP-9 (74% 9.8%).
All statins significantly reduced neointimal thickening (17 m in controls, 6-10
m in treatment groups) and pro-MMP2, and -9, and active MMP-2, and 9 levels
(based on median values).
Concentrations
up
to
0.2
M
CT1166/CT1399 or 0.5 M CT1746
resulted in increased MMP-2 levels,
higher concentrations caused a decreased
MMP-2 activity, which was completely
abolished at 10 M. MMP-9 levels
remained constant, the expression of
MT1-MMP only significantly decreased
at high inhibitor concentration (10 M).
Reduced expression of MMP-1 (52%
19%), MMP-2 (57% 2.3%), MMP-3
(71% 18%), and MMP-9 (73% 17%).
Results
Dose dependant inhibition of latent MMP2 and MMP1/MMP-3 (56kDa) levels and
collagen lattice contraction. The effects
were most pronounced in the Ilomastat
treated samples.
5 M Lovastatin
Gelatin zymography of
tissue extracts (MMP
activity),
and immunohistochemistry
(intimal thickness).
Simvastatin,
Lovastatin, Atorvastatin,
and
Pravastatin at 1
M, Fluvastatin
and Cerivastatin
at 0.1 M.
30
Chapter 6
Collagen Remodeling
6.1
Mechanically Induced Collagen Remodeling
Mechanical conditioning can improve both the morphology (more organized collagen fibre
structure) and mechanical properties of the tissue [63]. It is established that strained tissue engineered constructs show more matrix and tissue formation, and more organization
[43]. In addition, the strained tissue shows more pronounced and viable cells, and matrix
formation in the direction of the applied strain. Mol et al. [43] demonstrated that cyclic
strains of 9% or more, applied for 14 days, resulted in pronounced matrix formation and an
increased stiffness and ultimate tensile strength of tissue engineered constructs composed of a
PGA/P4HB scaffold seeded with human saphenous vein myofibroblasts. An increased matrix
or collagen formation thus seems to result in enhanced mechanical properties. This suggestion is confirmed by a study of Engelmayr et al. [21], who indicated a positive correlation
between collagen content and material stiffness in PGA/PLLA constructs seeded with ovine
vascular smooth muscle cells. Although the relation between collagen content and mechanical
properties of the tissue seems to be logical, there is still no strong evidence for this feature
and more research in this field is necessary. Table 6.1 lists some studies in which mechanical conditioning is applied to improve collagen architecture and mechanical properties of the
tissue.
31
Collagen Remodeling
Chapter 6
Culture
Adult rat SMCs
seeded in rat tail
type I collagen gel
(Seliktar et al. 2000
[63]).
Human saphenous
myofibroblasts
seeded on a PGAP4HB scaffold (Mol
et al. 2003 [43]).
Ovine
vascular
SMCs seeded on a
PGA/PLLA scaffold
(Engelmayr et al.
2005 [21]).
Load and Analysis

10% cyclic strain at 1
Hz for 4 and 8 days, in
medium containing serum.
Analysis by collagen contraction (rate of collagen
remodeling), histology, and
mechanical testing (strainto-failure).
Increased hydroxyproline content from

116% 74% in constructs strained
upto 7% (not significant) to 171%
23% in constructs strained upto 10%,
compared to controls. (Total DNA
content, and glycosaminoglycan content increased in a similar manner).
Collagen architecture
Conditioned constructs were significantly shorter that controls (-28% at
4 days, -68% at 8 days). Volume of
4 day conditioned constructs decreased
by 35% and had thinner walls compared to controls (not significant for
8 days conditioned constructs). Organized fibre orientation in conditioned
constructs, random in controls.
Collagen concentration increased 63%

in the flex group compared to static
(DNA concentration did not differ significantly between static and flex). No
changes in sample thickness were observed, which indicates a more dense
collagen fibre packing in the flex group.
2%, 7%, 9%, and 10%

Cyclic strain at 1 Hz for 14
days in medium containing
serum. Biochemical assays
for hydroxyproline to determine collagen content,
tensile failure test to determine mechanical properties.
Unidirectional cyclic flexure (central displacement
of 6.35 mm) in medium
with serum. Collagen assay, DNA analysis, and
effective stiffness measurements were performed.
Mechanical Properties
Increased yield stress in conditioned constructs: 7.7 kPa at 4 days (+84%), 26 kPa
at 8 days (+200%) compared to 4 kPa resp.
9 kPa for controls. Higher ultimate stress
in conditioned constructs: 17 kPa at 4 days
(+57%), and 58 kPa at 8 days (+240%), compared to 10 kPa resp. 16 kPa for controls.
Elevated Youngs modulus (E) in 8 day conditioned constructs (142 kPa, 108% higher)
compared to controls (68 kPa). 57% higher
yield strain in 4 day , and 35% in 8 day conditioned constructs.
Ultimate tensile strength increased in
strained constructs: 120 42% at 7% strain
(not significant), 141 39% at 9% strain,
255 55% at 10% strain, compared to
controls. Youngs modulus (E) increased
93 45% (not significant) in 7% strained
constructs to 200 75%, and 308 151% in
9, resp. 10% strained constructs compared
to controls.
Non significant increase in Youngs modulus
(E) in flexed samples (978 228 kPa) compared to static (748 130 kPa).
Table 6.1: The effect of mechanical conditioning on collagen architecture and mechanical properties of the construct/ tissue.
32
Chapter 6
6.2
Collagen Remodeling
The Role of MMPs in Mechanically Induced Collagen Remodeling
Mechanical stretch and shear influence the MMP expression, production, and activity causing
an imbalance between MMPs and TIMPs [48]. This results in a change in the remodeling
of the tissue that affects its mechanical properties. In addition, it is known that mechanical
stress may lead to the release of reactive oxygen species, which may activate MMPs [4].
Rabkin-Aikawa et al. [58] showed an increase in MMPs in response to an altered mechanical
environment. When the mechanical equilibrium was restored, the level of MMPs decreased
to normal levels. This indicates an important role for MMPs in the development of tissues.
In addition to this, Ruberti et al. [60] showed that loaded fibrils in presence of a general
collagenase preferentially degrade in a manner that is related inversely to the applied strain,
suggesting that the load bearing collagen fibres are stabilized against enzymatic degradation.
This suggests that the orientation of collagen fibres, induced by mechanical loading, is mediated by MMPs. Furthermore, Seliktar et al. [65] demonstrated that there is a relation
between mechanical load, amount of MMPs, and the stiffness of the tissue, which indicates
that only a controlled balance between mechanical load and MMPs will lead to improved
mechanical properties of the tissue.
Altogether, these studies indicate that MMPs play an important role in the mechanically
induced collagen remodeling of tissues. Tables 6.2, 6.3, and 6.4 summarize the effect of mechanical conditioning on MMPs, collagen architecture, and mechanical properties in different
studies.
The effect of mechanical strain on the expression, amount, activity, and type of MMP is
thoroughly studied over the last years. Firstly, Seliktar et al. [65] investigated MMP activation (by zymography), collagen expression, and mechanical properties in cyclically strained
(10% at 1 Hz, for 4-8 days) collagen constructs seeded with human aortic smooth muscle
cells. They demonstrated that strain increased the MMP-2 level (strongest after 8 days).
Furthermore, strain enhanced the mechanical properties of the constructs after 4 days, but
diminished properties after 8 days. In addition, constructs showed the highest collagen and
elastin expression (mRNA) after 4 days of strain. This suggests that MMP-2 has a 2-fold
function. On one hand, it facilitates remodeling, associated with cyclic strain, which results in
enhanced mechanical properties of the construct. On the other hand, when present in large
amounts, MMP-2 accumulation causes a loss of the mechanical integrity of the construct.
Inhibition of MMP-2 with high ribose medium (which limits the expression of MMP-2) resulted in decreased mechanical properties at 4 days, but elevated mechanical properties after
8 days (figure 6.1). This indicates that inhibition of MMPs can result in enhanced mechanical
properties. An optimum between MMP inhibitors and applied strain should be determined.
Secondly, Asanuma et al. [4] investigated the effect of static and cyclic strain on MMP
production in human saphenous vein smooth muscle cell monolayers on a collagen coated
membrane. They demonstrated increased MMP-2 expression (ELISA), pro-and active MMP2 levels (zymography) in cells, and pro-MMP-2 levels in medium in response to stationary
stretch (5%). Conversely, cyclic stretch (10% at 1 Hz) resulted in decreased pro-MMP-2 levels
in the medium, which indicates that in a normal vascular environment, the amount of proMMP-2 is restricted, probably also resulting in a decreased active MMP-2 level. They also
showed that pro-MMP-9 levels were very low, and were only elevated in conditioned medium
33
Chapter 6
Collagen Remodeling
of stationary stretched constructs after 3 days, suggesting that mainly MMP-2 is affected by
mechanical conditioning of human saphenous vein smooth muscle cells.
In contrast to Asanuma et al., OCallaghan et al. [50] showed increased MMP-2 levels, as
demonstrated by gelatin zymography, in cyclically stretched (10-16% at 1 Hz) human aortic or
venous smooth muscle cells. They furthermore demonstrated that both static and stretched
cells produced MMP-2, and -9, but not MMP-1, and -3. The release of MMP-2 was dominant,
and showed a time-dependant increase in response to stretch.
Lastly, Solan et al. [68] showed an increased collagen content in cyclically stretched (1.44
0.46% at 1.5 and 2.6 Hz) swine carotid or femoral smooth muscle cells on a PGA mesh.
MMP-1 levels did not significantly increase in response to cyclic stretch, but TIMP-1 levels
showed an elevated expression, as demonstrated by ELISA. This suggests that the enhanced
collagen production results from an altered MMP-1/TIMP-1 balance.
Conversely, Yang et al. [73] demonstrated a decrease in MMP-1, and TIMP-1 expression and
levels in response to small cyclic strains (1-4% at 1 Hz) in human saphenous vein smooth
muscle cell monolayers as demonstrated by western- and northern blot, and zymography. Expression, and levels of MMP-2, -9, and TIMP-2 did not change.
Overall, it can be concluded that the role of MMPs in mechanically induced collagen remodeling is debatable. Which MMPs and TIMPs are up-, or down regulated remains still unclear
and seems to differ among species, and cell type. Furthermore, the amount and type of load
(static vs. dynamic) has also a large effect in the enzymatic degradation of collagen. Low
strains (around 4 %, corresponding to the level of strain where the collagen fibres are completely uncrimped, but not stretched yet) result in an increased stiffness of the tissue, which
is probably caused by altered kinetics of the collagenase-collagen interaction [27, 47]. In contrast to this, high strains result in an increased collagenase activity causing deterioration of
the material stiffness [20, 27]. This effect is most pronounced in dynamically loaded tissue
[20]. Altogether, these results suggest that there is a strong relation between the mechanical
load, MMP activity, collagen architecture, and the stiffness of the tissue. Investigating this
relation may contribute to the development of an optimal conditioning regime and improved
mechanical properties of a tissue engineered construct.
Until now, MMP activity is determined only for a few MMPs per study, mostly due to a limited availability of suitable analysis methods. Therefore, it is unclear which MMPs, or other
proteases, play the major role in strain stimulated collagen remodeling in (tissue engineered)
tissues. Future research should focuss on this aspect and elucidate the essential proteases
in this process. With this knowledge, protease activity can be controlled specifically, which,
together with an optimal conditioning regime, may result in enhanced mechanical properties
of the engineered tissue.
34
Chapter 6
Collagen Remodeling
Figure 6.1: Mechanical properties of human aortic SMC (HASM) seeded constructs in response to
cyclic stretch. Top, ultimate stress; bottom, modulus; asterisks indicate a significant difference between
constructs cultured in high ribose medium (HRM) and untreated constructs. In absence of any treatment, HASM constructs have increased mechanical properties after 4 days of stretch, but decreased
properties after 8 days. HRM treated constructs (to reduce the expression of MMP-2) resulted in significantly higher ultimate stress and modulus after 8 days of stretch stimulation. (From Seliktar et al.
[65]).
35
Collagen Remodeling
Chapter 6
Human
saphenous vein SMCs
monolayers on a
fibronectin coated
membrane, pretreated
with
PDGF (Yang et
al. 1998 [73]).
Culture
1% and 4% cyclic stretch at
1 Hz, for 24 h in serumfree
medium. MMP and TIMP
analysis with zymography
and western blot of conditioned medium and northern blot of cells.
Load and Analysis
Suppression of MMP-1 expression (-37% 15% at 1%, and 50% 15% at 4% stretch compared to controls), and TIMP-1
expression (-27% 7% at 1%,
and 48% 8% at 4% stretch
compared to controls) Expression and levels of MMP-2, -9, and
TIMP-2 were unaffected.
Time dependent increase of
MMP-2 (latent) levels in response to stretch. No differences
in MMP-9 levels.
MMP
MMP-2 total enzyme levels increased 5.7 fold compared to

static; 1.6 fold increased MMP2 activity ratio (active/latent)
compared to static.
Human
aortic and venous
SMCs monolayers
(OCallaghan et
al. 2000 [50]).
Human
aortic
SMCs seeded in a
rat tail collagen
type I gel (Seliktar et al. 2001
[64])
10-16% stretch at 1 Hz for

96 h in low serum medium
(1%). MMP analysis by
gelatin zymography, collagen production by 3 Hproline labeling (of conditioned medium)
10% cyclic stretch for 4
days at 1 Hz, in medium
containing serum. MMP
analysis by western blot
and gelatin zymography of
homogenized constructs,
collagen contraction by
measuring
gel
dimension at the end of each
experiment.
Collagen
tecture
archi-
Significantly
increased
collagen
production
in
response to stretch.
59% more contraction in stretched

constructs after 4
days compared to
static.
Yield stress 6.2 kPa (290%

higher), ultimate stress
22 kPa (390% higher),
Youngs modulus 4.9 kPa
(192% higher) after 4 days
with respect to static.
Table 6.2: The effect of mechanical conditioning on MMPs, collagen architecture, and mechanical properties of the construct/ tissue.
36
37
Significantly enhanced collagen

synthesis in 2
mN
preloaded
samples.
Significantly
increased intima
thickness (1.77
0.11 mm in high
shear, 1.26
0.10 mm in low
shear compared
to 0.41 0.02 in
controls).
Increased
Youngs modulus (E) in 2 mN
preloaded samples; decreased
initial failure
load
in
10
mN preloaded
samples.
Mechanical
Properties
Human dermal fibroblasts seeded

in a collagen type
I gel (Berry et al.
2003 [8]).
10% cyclic stretch at 1 Hz

for 24 h to 2 mN, or 10 mN
preloaded samples, in medium
containing serum. Analysis
by [3 H] proline incorporation
to measure collagen synthesis,
gelatin and casein zymography
(of constructs) for MMP activity, and a tensile failure test
to determine the mechanical
properties.
Increased total MMP-2 activity in high

shear stress samples.
Significantly increased MMP-1, -2, and TIMP-1, and 2 protein levels in tissue extracts of high
shear stress samples compared to controls.
In medium TIMP-2 was increased in high
shear stress samples, TIMP-1 in high, and
low shear stress samples (highest level in
low shear) compared to controls.
Significant higher MMP-2 expression (50%,
80%, and 110% at 1, 2, and 3 days resp.),
cell associated pro and active MMP-2 at
days 2 and 3, medium pro MMP-2 at day
3, medium active MMP-2 after 3 days,
and medium pro MMP-9 levels after 2
and -3 days in stationary constructs compared to controls. Significantly lower level
of medium pro-MMP-2 in cyclic stretched
constructs after 2, and 3 days. No changes
in TIMP-1, or -2 levels.
Increased latent MMP-1 level in 2 mN
preloaded samples, increased latent MMP1, -2, -9, and active MMP-1, -2, and -3
levels and decreased latent MMP-3 levels
in 10 mN preloaded samples compared to
controls.
Low shear flow (2-4 dyn/cm2 )

or high shear flow (9-12
dyn/cm2 ) for 7 days in
medium containing serum.
Analysis
by
zymography
(MMP-2, -9 activity), ELISA
(MMP-2, -9, and TIMP-1,
-2 expression), and histology
(intima thickness).
5% stationary stretch, and
10% cyclic stretch at 1 Hz for
3 days in serum free medium.
RT-PCR (total SMC RNA),
western blotting and gelatin
zymography (of cell lysates
and conditioned medium were
performed to assess MMP levels and activity.
Human
saphenous vein segments (Patterson
et al. 2001 [51].
Human
saphenous vein SMCs
seeded on a silicon membrane
coated
with
collagen type I
(Asanuma et al.
2003 [4])
MMP
Load and Analysis
Culture
Chapter 6
Collagen Remodeling
Collagen Remodeling
Chapter 6
Human
aortic
SMCs seeded on
rat collagen type
I gel (Seliktar et
al. 2003 [65])
Culture
Load and Analysis
Swine carotid or
femoral
SMCs
seeded on a cylindrical PGA mesh
(Solan et al. 2003
[68]).
10% cyclic stretch for

8 days in medium containing serum. Western
blot and gelatin zymography of construct
homogenates
were
performed to determine MMP presence
and activity.
Collagen expression was
determined by PCR.
1.44 0.46% Cyclic
stretch at 90, or 165
bpm in medium with
serum. Analysis with
ELISA of conditioned
medium (amount of active MMP-1, and total
TIMP-1), and hydroxyproline content of vein
segments (collagen content).
MMP
MMP-2 activity ratio (active/latent) 61% higher after 4 days,
and 313% higher after 8 days
compared to static.
Non significant increased amount

of active MMP-1 in 90 and 165
bpm pulsed constructs (194.90
38.66 ng/mL resp. 149.48
91.34 ng/mL) compared to
static (119.30 122.61 ng/mL)
and a significantly increased
amount of TIMP-1 in 90 and 165
bpm pulsed constructs (143.83
37.67 ng/mL (not significant)
resp. 195.94 28.84 ng/mL)
with respect to controls (125.46
55.88 ng/mL).
The amount of type
I collagen mRNA
indicated a 3-fold
increase after 4 days
and remained elevated after 8 days.
Static
constructs
showed a sustained
increase.
Increased
collagen
content in 90 bpm
(30.48 3.29 % (not
significant)) and 165
bpm (35.37 8.28
%) pulsed constructs
compared to static
(24.90 8.28 %).
Ultimate stress 22 kPa

(390% higher) after 4 days,
and 4.9 kPa (48% higher)
after 8 days. Modulus 48
kPa (192% higher) after 4
days, and 14 kPa (31%
higher) after 8 days, compared to static.
38
Chapter 7
Quantification of Collagen
Remodeling
The mechanical stability and tensile strength of collagen are highly dependant of its architecture. Mechanical conditioning can alter this architecture in such a way that the mechanical
properties of the tissue will improve. In addition, mechanical conditioning can alter the expression and activity of MMPs, which also affects the collagen architecture. To get insight in
the effects of mechanical conditioning on the collagen architecture of the tissue, techniques are
needed to analyse collagen remodeling, comprising collagen content, amount of cross-links,
fibre structure and orientation, and MMP expression and activity. This chapter discusses
these techniques.
7.1
7.1.1
Quantification and Visualization of Collagen

Analysis of Collagen Content and Cross-links
Collagen Content
The amount of collagen, or collagen peptides in tissue extracts can be determined by measuring the hydroxyproline content [28]. Free hydroxyprolines arise from the alkaline hydrolysis
of the collagen proteins. Oxidation of the hydroxyprolines at low pH results in chromophore
formation and color development, which is stable for 8 h. Absorbance of the color (at 550 nm)
can be measured and quantified. Advantage of this technique is that there is a linear relationship between the hydroxyproline content and absorbance, also in presence of other proteins.
Furthermore, the method is quick, sensitive, efficient, and the color yield is not affected by
column solvents, except high concentrations of urea. A disadvantage of the technique is that
it might not be specific enough to analyse collagen formation, because the hydroxyproline
content in free collagen peptides and collagen fibres is the same [45]. Fibre formation can be
studied by fluorescence assays (section 7.1.2).
Cross-links
The mechanical stability and tensile strength of collagen are highly dependant on the amount
of cross-links present in the collagen network. To measure the collagen cross-link concentration, high performance liquid chromatography (HPLC) can be used. This technique is based
39
Chapter 7
Quantification of Collagen Remodeling
on the intrinsic fluorescent properties of the cross-links and the acid hydrolysis of all peptide
bound cross-links. Collagen extracts are first hydrolyzed, after which they are run trough a
column, containing an ion-pairing substrate. Compounds have distinct binding properties to
the column, and will be separated based on their retarded elution. The elution fractions can
be analyzed by a fluorometer at specific excitation and emission wavelengths.
In collagen three different types of cross-links can be formed:
immature cross-links:
dehydrohydroxylysinohydroxynorleucine (DHHLHNL)
dehydrohydroxylysinonorleucine (DHHLNL)
dehydrolysinonorleucine (DHLNL)
mature cross-links:
pyridinoline (hydroxylysylpyridinoline, HP)
deoxypyridinoline (lysylpyridinoline, LP)
histidinohydroxylysinonorleucine (HHLNL)
senescent crosslinks:
pentosidine
Pyridinoline and deoxypyridinoline are the major cross-links in mature collagen. Pyridinoline
is dominant in connective tissues, while deoxypyridinoline is dominant in mineralized tissue.
When the tissue develops, the immature cross-links will disappear, while the mature ones will
become more pronounced, resulting in an increased strength. Since mechanical conditioning
and a controlled MMP activity strongly affect the collagen content and mechanical properties
of the tissue, it would be interesting to study also their effect on the maturation of collagen
by determining the amount and types of cross-links present in the tissue.
The presence of pyridinoline and deoxypyridinoline in urine, tissue extracts [22], and serum or
synovial fluid [30], can also be measured using HPLC. Measurements in serum or synovial fluid
require advanced ion-paired substrates and narrow-bore columns. Bank et al. [6] described
a sensitive method to quantify simultaneously HP, LP and pentosidine in a wide variety of
connective tissues by single-run reversed-phase HPLC. Furthemore, Saito et al. [61] developed an advanced HPLC method to analyse the non-autofluorescent cross-links DHHLHNL,
DHHLNL, DHLNL, and HHLNL. By post column O-phthalaldehyde derivation, fluorescence
of these cross-links can be detected using an excitation of 350 nm and an emission of 450
nm. Pyridinoline and deoxypyridinoline can be detected with an excitation of 295 nm and an
emission of 395 nm. Wavelengths for the detection of Pentosidine are resp. 335 nm and 385
nm for excitation and emission. The method by Saito et al. can be useful to study changes
in the maturation of collagen.
40
Chapter 7
7.1.2
Visualization of Collagen Formation
Fluorescent Staining
Collagen fibre formation can be studied using picro sirius red staining (figure 7.1). Sirius red
stains collagen by reacting via its sulphonic acid groups, with basic groups present in the
collagen molecule. The elongated dye molecules are attached to the collagen fibres in such a
way that their long axis are parallel, which results in an enhanced birefringency [31]. Using
polarized microscopy, very small fibres can be visualized by this technique. The difference between thin and thick fibres can be observed in a shift in polarization colors from green-yellow
to yellow-orange [16]. Disadvantages of this technique are that it is not specific to collagen
types, and that quantification is not possible [29].
Figure 7.1: Picro sirius red staining of human saphenous vein myofibroblasts seeded on a PGA/P4Hb
scaffold after six weeks of culturing. Birefringent fibres are present (from Mol et al. 2004 [45])
Real-time Visualization
Orientation of collagen fibres can be studied by the collagen specific probe CNA35-OG488,
developed by Krahn et al. [49]. This probe has significant higher specificity and sensitivity
than conventional probes (figure 7.2). It is composed of the highly specific collagen binding domain present in bacterial proteins (CNA-35), and a fluorescent label, Oregon Green
(OG488), for visualization. The binding between CNA-OG488 and collagen is reversible,
which allows monitoring collagen production in living tissues over a prolonged period of time.
Other advantages are that much smaller newly formed fibrils can be visualized than currently
used probes, and that the probe is smaller than conventional antibodies, which is beneficial
for tissue penetration.
41
Chapter 7
Figure 7.2: Fluorescent staining of living human vena saphena myofibroblasts with cell tracker Orange to visualize cells (red), and (a) CNA35-OG488, (b) GST-I-OG488, and (c) DTAF to visualize
collagen (green); bar = 50 m (from Krahn et al. 2006 [49]). Staining with CNA35-OGG488 results
in an significantly increased collagen detection compared to conventional staining methods.
42
Chapter 7
7.2
7.2.1
MMP Analysis Methods

RNA Analysis
Screening tissue extracts for the presence or absence of a gene coding for an enzyme or protein of interest, can be performed by determining the mRNA expression by Northern blotting
or PCR (polymerase chain reaction). For Northern blotting, the isolated RNA is separated
by size on an agarose gel, transferred to a membrane, and detected using a labeled probe.
Quantification is performed by determining the band intensities. Although northern analysis
is the standard method for the detection and quantification of RNA levels, its sensitivity is
lower than that of PCR.
For PCR, the mRNA encoding for the protein of interest, is isolated and amplified using
specific primers (figure 7.3). The amplified mRNAs can be visualized by agarose gel electrophoresis and quantified by determining the band intensities. Real time PCR (rt-PCR),
that next to primers also uses fluorescent probes, is used to monitor changes in mRNA formation, and to determine differences in mRNA expression between samples. An advantages
of rt-PCR for MMP analysis is that samples can be screened for the presence of all MMPs.
Furthermore, rt-PCR is more sensitive, has a higher resolution, and can be quantified more
accurate that traditional PCR. A disadvantage of both PCR and Northern Blot, is that only
the presence of the mRNA encoding for MMPs can be detected, which is not always directly
related to the amount and activation state of the enzyme.
Figure 7.3: Schematic representation of the PCR reaction. (1) Denaturation of double stranded DNA
at high temperatures (90 - 96 o C). (2) Binding of the forward and reverse primers (specific for the
gen of interest) to the singles stranded DNA molecules (template) at 45 - 60 o C. (3) Formation of the
complement strand using a DNA polymerase (at 72 o C). (4) Denaturation of the newly formed double
stranded DNA etc.
43
Chapter 7
7.2.2
ELISA
The presence of MMPs in tissue extracts or conditioned medium can be detected using
enzyme-linked immunosorbent assays (ELISA) (figure 7.4). Samples are incubated in wells,
precoated with antibodies. A secondary antibody is used to detect the protein of interest.
Specific antibodies for MMPs and TIMP are commercially available. Disadvantage of the
ELISA method is that it is not possible to measure MMP activity, only presence. Furthermore, commercially available MMP measurements kits are mostly only partial specific for
some MMPs forms, and cross react with other MMPs.
Figure 7.4: Schematic representation of the ELISA method.
7.2.3
Western Blotting
Western blotting detects the total amount of MMPs present in tissue extracts or conditioned
medium using antibody labeling. Proteins are separated on a polyacrylamide gel by electrophoresis under denaturing conditions (SDS-PAGE). After gel electrophoresis, proteins are
transferred on a nitrocellulose membraan and incubated with a primary antibody which is
MMP specific. A secondary antibody can be used for detection. Bands, corresponding to
the molecular weight of the MMP forms, become visible after chemiluminescence treatment,
and exposure to a film. Table 7.1 lists the molecular weight of frequently measured MMPs,
and TIMPs [39]. Quantification of the bands can be performed by determining the bandintensities.
44
Chapter 7
A disadvantage of western analysis is that measurements in samples containing serum or

plasma is difficult, because almost all free active MMPs will be complexed with 2 M (for
explanation, see section 5.1). The MMP/2 M complex is too large for separation by electrophoresis, and SDS cannot release the MMP from the macroglobulin. When no serum is
present, the free active MMPs will be complexed with TIMPs after some time. SDS causes
(partly) the separation of the MMP-TIMP complex, which makes it impossible to determine
which part of the active MMP band represents the MMPs that really have been active. Another disadvantage of western blotting is that only the presence of MMPs is determined,
which cannot be related to the activity of the enzyme.
MMP/TIMP
MMP-1
MMP-2
MMP-3
MMP-8
MMP-9
MMP-13
MT1-MMP
TIMP-1
TIMP-2
TIMP-3
TIMP-4
latent (kDa)
55
72
57
75
92
60
66
-
active (kDa)
45
66
46
58
86
48
56
28
21
24
22
Table 7.1: Molecular weight of MMPs and TIMPs
7.2.4
Zymography
Zymography identifies MMPs by the degradation of their preferential substrate and by their
molecular weight [67]. First, proteins are separated by electrophoresis under denaturing
(SDS), non-reducing conditions. The electrophoresis takes place in a polyacrylamide gel
containing a MMP specific substrate co-polymerized with the gel. SDS causes MMP denaturation, which results in inactivation of the enzyme. After running, the gel is washed to remove
the SDS. Enzymes will (partially) recover and become active again. Latent MMPs will be
auto-activated without cleavage. The pro-domain thus stays attached to the enzyme and
therefore active and latent MMP can still be distinguished on their molecular weight. During
incubation, the MMPs will digest the substrate in the gel. After staining with Coomassive
Blue, colorless bands become visible where MMPs degraded the substrate. These bands can
be ascribed to the corresponding MMPs based on their molecular weight (table 7.1). The
substrates can be adapted to the preference of the MMPs. Gelatin is used for MMP-2, and
-9, casein for MMP-1, -3, -7, and -13.
45
Chapter 7
A disadvantage of casein zymography is that MMP-1, -3, -13 have almost the same molecular
weight, which makes it difficult to distinguish between these MMPs. Furthermore the disadvantages of zymography are similar to those of western blotting: it is not possible to detect
the original activity of MMPs from the tissue or cells, because SDS causes the separation
of the MMP-TIMP complex, and measurements in presence of serum or plasma are difficult
since almost all free MMPs will be complexed with 2 M.
TIMPs can be detected using reverse zymography. In this technique, also MMP-2, which can
be inhibited by all TIMPs, is added to the gel. After electrophoresis, MMP-2 will only digest
the gel were no TIMPs are present. Staining with Coomassive Blue will result in a colorless
gel, with blue bands at the molecular weight of the TIMPs.
7.2.5
Peptide Substrates
MMP activity can also be measured using fluorogenic peptide substrates [9]. These substrates contain a short amino sequence that can be cleaved by MMPs. The substrate contains
a donor group, and a quenched fluorescent acceptor group. When MMPs cleave the substrate,
quenching is lost, which results in a decreased fluorescence (figure 7.5). Advantage of this
technique is that it measures the real MMP activity. However, the substrates are mostly not
MMP specific, which makes it impossible to discriminate between MMP type.
Hanemaaijer et al. [26] developed an immunocapture activity assay, based on the above described method, that is MMP specific. The method is based on the immobilization of MMPs
from biological fluids using a specific antibody, and a substrate pro-enzyme that upon activation by MMPs can be assayed using a chromogenic substrate (figure 7.6). The sensitivity
of the method is comparable to that of gelatin zymography.
A local excess of active MMPs over TIMPs in the tissue will be cleared by secretion into the
circulation, followed by neutralization by 2 macroglobulin. Free MMPs are therefore unlikely
to be present in serum or plasma, because they will be captured in the macroglobulin. However, the MMP/2 M complex has still the capacity to convert small substrates. Beekman et
al. [7] developed small fluorogenic substrates with catalytic efficiencies for MMP-1, -2, -3, -8,
and -9 (not specific) in presence of 2 M. This method enables to measure MMP activity in
presence of serum or plasma. However, not only the amount of active MMPs will be determined, also the local acces of active MMPs.
46
Chapter 7
Figure 7.5: Mechanism of fluorogenic peptide substrates. D, donor group; A, quenched fluorescent
acceptor group.
Figure 7.6: Schematic representation of the setup of the MMP-9 immunocapture activity assay. MMP9 is captured by an antibody, after which pro-urokinase (proUKCOL) is converted in urokinase (UKCOL). MMP activity can be measured using a urokinase specific chromogenic peptide substrate. (A)
Already activated MMP-9 converts proUKCOL directly and active MMP is measured. (B) Upon activation of latent MMP-9 with APMA, total MMP-9 (already activated MMP-9 plus MMP-9 activated
by APMA) can be measured. (From Hanemaaijer et al. 1998 [26].)
47
Chapter 7
48
Chapter 8
Future Research
This review points out that mechanical conditioning results in more ordered and pronounced
collagen fibres and improved mechanical properties of engineered tissues. In addition, it shows
that MMPs play an important role in the collagen remodeling of the tissue, and that the expression and/ or activation of these enzymes is influenced by mechanical stress and strain.
However, the exact mechanisms of MMPs and/ or other proteases in these processes remain
still unclear.
Future research will focus on these aspects and investigate the role of MMPs, TIMPs and
other proteases in collagen remodeling of mechanically conditioned tissue engineered constructs. The up- and/or down regulation of the gene expression of these enzymes will be
determined in response to static and dynamic stretch, to elucidate which protease is primarily responsible for collagen remodeling under these circumstances. The collagen content and/
or amount cross-links of the tissue engineered constructs can then be increased by specific inhibition or stimulation of this protease. Furthermore, a relation between protease inhibition/
stimulation, mechanical conditioning, collagen remodeling and mechanical properties of the
constructs can be established, resulting in an optimal conditioning regime. The outcomes of
these studies will contribute to the development of a fully mechanical and biological integer
tissue engineered heart valve construct.
49
50
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Matrix Metalloproteinases and Collagen Remodeling in Tissue Engineered Heart Valves

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Matrix Metalloproteinases and Collagen Remodeling in Tissue Engineered Heart Valves

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Matrix Metalloproteinases and

Eindhoven University of Technology

2 The Aortic Heart Valve

5 Matrix Metalloproteinase Inhibition

7 Quantification of Collagen Remodeling

The Aortic Heart Valve

The Aortic Heart Valve

The Aortic Heart Valve

Matrix Metalloproteinases in Heart Valves

The Aortic Heart Valve

Type Molecular composition

Extracellular matrix remodeling requires the degradation of its components. In general,

Structure and Function

I, II, III, VII, X (VIII,

I IV, V, VII, X (II, III,

III, IV, IX, X (II, VII,

fibronectin, laminin, gelatin

Membrane Type MMPs

laminin, fibonectin, elastin

I, II, III ( and proMMP2)

aggrecan, elastin, fibrillin,

Table 4.1: The MMP family.

gelatin, fibronectin, laminin

MMP and prevents MMP-2 activation [38].

Role in Matrix Remodeling

Matrix Metalloproteinase Inhibition

Matrix Metalloproteinase Inhibitors

Endogenous Inhibitors of MMPs

Matrix Metalloproteinase Inhibitors

Synthetic MMP Inhibitors

Matrix Metalloproteinase Inhibitors

Porter et al. 1998

Human saphenous vein

Porter et al. 1999

Rabbit periosteal cells

Dose dependant inhibition of hydroxyproline release up to 90 % at 50 nM.

Less intimal thickening (5.5 m vs 21 m

Porter et al. 2002

Zymography of conditioned medium and

Human left ventricular myocardial fibroblasts in medium with

Human saphenous vein

Matrix Metalloproteinase Inhibitors

Luan et al. 2003

Human saphenous vein

Human saphenous vein

Zymography and western blot of conditioned

Reduced expression of MMP-1 (94%

Mechanically Induced Collagen Remodeling

Load and Analysis

Increased hydroxyproline content from

Collagen concentration increased 63%

2%, 7%, 9%, and 10%

The Role of MMPs in Mechanically Induced Collagen Remodeling

Load and Analysis

MMP-2 total enzyme levels increased 5.7 fold compared to

10-16% stretch at 1 Hz for

59% more contraction in stretched

Yield stress 6.2 kPa (290%

Significantly enhanced collagen

Human dermal fibroblasts seeded

10% cyclic stretch at 1 Hz

Increased total MMP-2 activity in high