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14 15 11 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Masumi Ai
Department of Life Sciences and Bioethics, Graduate School of Medicine, Tokyo Medical
and Dental University, and Nutrition Clinic, Kagawa Nutrition University, Tokyo, Japan
Loris Borghi
Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy
Marı́a Paula Ceballos
IFISE, CONICET, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad
Nacional de Rosario, Rosario, Argentina
Félix Fernández-Madrid
Division of Rheumatology, Department of Medicine, School of Medicine; Karmanos
Cancer Institute, School of Medicine, and Center for Molecular Medicine and Genetics,
School of Medicine, Wayne State University, Detroit, Michigan, USA
Gordon A.A. Ferns
Division of Medical Education, Brighton & Sussex Medical School, University of Brighton,
Brighton, United Kingdom
Masaki Q. Fujita
Department of Legal Medicine, Keio University School of Medicine, Tokyo, Japan
Naveen Garg
Department of Cardiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences
(SGPGIMS), Lucknow, India
Majid Ghayour-Mobarhan
Cardiovascular Research Centre, School of Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran
Sergio Ghersevich
Area of Clinical Biochemistry, Department of Clinical Biochemistry, Facultad de Ciencias
Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
Masafumi Koga
Department of Internal Medicine, Kawanishi City Hospital, Kawanishi, Japan
Surendra Kumar
Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences
Giuseppe Lippi
Laboratory of Clinical Chemistry and Hematology, Department of Pathology and Laboratory
Medicine, Academic Hospital of Parma, Parma, Italy
Marie-Claire Maroun
Division of Rheumatology, Department of Medicine, School of Medicine, Wayne State
University, Detroit, Michigan, USA
ix
x Contributors
Camilla Mattiuzzi
Service of Clinical Governance, General Hospital of Trento, Trento, Italy
Tiziana Meschi
Avshesh Mishra
Balraj Mittal
Takeaki Nagamine
Graduate School of Health Sciences, Gunma University, Maebashi, Japan
Katsuyuki Nakajima
Graduate School of Health Sciences, Gunma University, Maebashi; Department of Legal
Medicine, Keio University School of Medicine; Nutrition Clinic, Kagawa Nutrition
University, Tokyo, Japan, and Lipid Metabolism Laboratory, Jean Mayer USDA Human
Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, USA
Antonio Nouvenne
Ernst Schaefer
Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on
Aging at Tufts University, Boston, Massachusetts, USA
Anshika Srivastava
Akira Tanaka
Nutrition Clinic, Kagawa Nutrition University, Tokyo, Japan
Marzie Zilaee
Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran
PREFACE
The second volume of the Advances in Clinical Chemistry series for 2014 is
presented.
In Chapter 1, the role of matrix metalloproteinases in cardiovascular dis-
ease is presented. This family of enzymes degrades the extracellular matrix
and has numerous functions in cell adhesion and migration and may influ-
ence plaque stability. In Chapter 2, the cardiovascular disease theme is con-
tinued with an interesting review of heat-shock proteins as a potential risk
marker for atherosclerosis. Heat-shock proteins are highly conserved pro-
teins that are upregulated in response to environmental stress and appear
to be expressed in atherosclerotic plaques. In Chapter 3, the biochemistry
and functional role of apolipoprotein B-48 is explored. This unique
molecule is the only specific marker of intestinal chylomicron particles. In
Chapter 4, neutrophil gelatinase-associated lipocalin is reviewed with respect
to its potential role in cancer. While predominantly known for its role in acute
kidney injury, this protein is overexpressed in malignancy and is associated
with tumor size, stage, and invasiveness. In Chapter 5, the importance of auto-
antibodies in breast cancer is debated. While traditionally known for their
involvement in systemic rheumatologic conditions, these antibodies appear
to be promising markers in oncology and many target of specific group of
tumor-associated antigens. In Chapter 6, mammaglobin A, a protein of the
secretoglobulin family, is introduced with respect to its role in breast cancer.
This promising marker may provide a unique approach to identify and isolate
circulating tumor cells as well as potential therapeutic targets for treatment of
this disease. In Chapter 7, two markers of glycemic control are highlighted. In
this review, the role of glycated albumin and 1,5-anhydroglucitol in diabetes is
explored and compared to traditional markers such as hemoglobin A1c.
I thank each contributor of Volume 64 and extend my thanks to col-
leagues who contributed to the peer review process. I would also like to
thank Helene Kabes for her expert editorial support at Elsevier.
I hope the second volume for 2014 will be enjoyed. Your comments and
feedback are always appreciated.
I would like to dedicate Volume 64 to Nyle and Henry on the occasion
of their engagement.
GREGORY S. MAKOWSKI
xi
CHAPTER ONE
Matrix Metalloproteinases in
Coronary Artery Disease
Balraj Mittal*,1, Avshesh Mishra*, Anshika Srivastava*,
Surendra Kumar*, Naveen Garg†
*Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow,
India
†
Department of Cardiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow,
India
1
Corresponding author: e-mail address: balraj@sgpgi.ac.in; bml_pgi@yahoo.com
Contents
1. Introduction 2
2. Classification, Structure, and Function of MMP 3
3. Regulation of MMP 8
3.1 Inactive zymogens 8
3.2 Transcriptional regulation 9
3.3 Translational regulation 10
3.4 Tissue inhibitors of metalloproteinases 11
4. MMP in Cardiovascular System Development 12
4.1 MMP in vasculogenesis and angiogenesis 12
4.2 MMP in cardiac tube formation and looping 13
4.3 MMP in heart septation 15
4.4 MMP in embryonic and fetal cardiac remodeling 15
5. MMP in Coronary Artery Disease 16
5.1 MMP in atherosclerosis 17
5.2 MMP in plaque instability 21
5.3 MMP in acute coronary syndrome 25
5.4 MMP in vascular remodeling 26
5.5 MMP in restenosis 28
6. Genetics of Matrix Metalloproteinases 29
6.1 Studies on genetically modified animals in vascular remodeling
and atherosclerosis 29
6.2 Genetic predisposition to CAD in humans 35
7. Matrix Metalloproteinases and Therapeutic Aspects 48
8. Prognostic Value of ECM Biomarkers 50
9. Conclusion 50
Acknowledgments 51
References 51
Advances in Clinical Chemistry, Volume 64 # 2014 Elsevier Inc. 1

ISSN 0065-2423 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800263-6.00001-X
2 Balraj Mittal et al.
Abstract
Matrix metalloproteinases (MMP) are a family of zinc-containing endoproteinases that
degrade extracellular matrix (ECM) components. MMP have important roles in the
development, physiology and pathology of cardiovascular system. Metalloproteases
also play key roles in adverse cardiovascular remodeling, atherosclerotic plaque forma-
tion and plaque instability, vascular smooth muscle cell (SMC) migration and restenosis
that lead to coronary artery disease (CAD), and progressive heart failure. The study of
MMP in developing animal model cardiovascular systems has been helpful in deci-
phering numerous pathologic conditions in humans. Increased peripheral blood
MMP-2 and MMP-9 in acute coronary syndrome (ACS) may be useful as noninvasive
tests for detection of plaque vulnerability. MMP function can be modulated by certain
pharmacological drugs that can be exploited for treatment of ACS. CAD is a polygenic
disease and hundreds of genes contribute toward its predisposition. A large number
of sequence variations in MMP genes have been identified. Case-control association
studies have highlighted their potential association with CAD and its clinical manifes-
tations. Although results thus far are inconsistent, meta-analysis has demonstrated that
MMP-3 Glu45Lys and MMP-9 1562C/T gene polymorphisms were associated with
CAD risk.
1. INTRODUCTION
Matrix metalloproteases (MMP) are an ever-expanding family of
endopeptidases with common functional domains that degrade extracellular
matrix (ECM) components. MMP actions have been implicated in both
physiologic and pathologic tissue reshaping, including organ development,
wound healing, inflammation, and cancer [1]. MMP have important roles in
cardiovascular physiology and pathology. MMP and their inhibitors play
major roles in ECM degradation and in mediating cell–cell adhesion, cell
migration and invasion, cell proliferation and apoptosis, and growth factor
and cytokine signaling [2]. MMP have been implicated in the degradation of
the ECM of coronary plaque as well as remodeling of coronary arteries [3].
Functional polymorphisms of MMP have proved to be associated with the
concentration of circulating MMP, and over the past few decades, consid-
erable effort has been devoted to exploring the relationships between MMP
polymorphisms and coronary artery disease (CAD) risk among various
populations [4].
This review will focus on role of MMP in normal cardiovascular system
development and clinical manifestations of CAD as well as the relevance of
MMP genetic variants with CAD.
Matrix Metalloproteinases in Coronary Artery Disease 3
2. CLASSIFICATION, STRUCTURE, AND FUNCTION

OF MMP
MMP are classified as the matrixin subfamily of zinc metalloprotease
family M10 in the MEROPS database (http://www.merops.sanger.ac.uk/).
MMP family members have 40% of their primary structure in common.
Approximately 24 different MMP have been discovered and classified based
on their chromosome location and substrate specificity (Table 1.1). Number
designations MMP-1 to -28 are used for classification, but some have not
been identified [5,6]. Although MMP-4, -5, -6, and -22 designations are
missing, they appear identical to other members. MMP are extracellular pro-
teins, but recent studies have indicated that MMP-1 [7], -2 [8], and -11 [9]
are also found intracellularly and may act on intracellular proteins. A typical
MMP consists of a propeptide of 80 amino acids, a catalytic domain of
170 amino acids, a linker peptide of variable lengths (also called the hinge
region), and a hemopexin (Hpx) domain of 200 amino acids. Exceptions
are MMP-7 (matrilysin 1), MMP-26 (matrilysin 2), and MMP-23 that lack
the linker peptide and the Hpx domain. MMP-23 has a unique cysteine-rich
domain and an immunoglobulin-like domain following the catalytic
domain. Two gelatinases, A (MMP-2) and B (MMP-9), have three fibro-
nectin type II repeats in the catalytic domain. The zinc binding sequence
HEXXHXXGXXH in the catalytic domain and the “cysteine switch”
motif PRCGXPD in the propeptide region are common structural features.
The three histidine residues in the zinc binding domain and the cysteine res-
idue in the propeptide region coordinate the catalytic zinc ion. This
Cys-Zn2þ coordination keeps proMMP inactive by preventing a water
molecule essential for catalysis from binding zinc. The catalytic domain also
contains a conserved methionine, forming a “Met-turn” eight residues after
the zinc binding motif, which forms a base to support the structure around
the catalytic zinc [10].
Based on substrate preference, MMP are grouped into collagenases,
gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMP, and
others (Table 1.1).
Collagenases (MMP-1, -8, -13, and -18 in Xenopus) cleave interstitial
collagens I, II, and III into characteristic 3/4 and 1/4 fragments and can
digest other ECM molecules and soluble proteins. Recent studies indicate
that MMP-1 activates protease-activated receptor 1 by cleaving the same
thrombin-sensitive Arg-Ser bond promoting growth and invasion of breast
Table 1.1 Classification of matrix metalloproteinases (MMPs)

Chromosomal
location
Enzyme MMP (human) ECM substrate
Collagenases
Insterstitial MMP-1 11q22–q23 Collagens (I, II, III, VIII, and X),
collagenase gelatine, aggrecan, Lselectin, IL-1b
(Collagenase 1) proteoglycanes, entactin, ovostatin,
monocyte chemoatractant protein-
3, protease-activated receptor 1
Neutrophil MMP-8 11q21–q22 Collagens (I, II, III, V, VII, VIII, and
collagenase X), gelatine, aggrecan, fibronectin
(Collagenase 2)
Collagenase 3 MMP-13 11q22.3 Collagens (I, II, III, IV, IX, X, and
XIV), gelatine, plasminogen,
aggrecan, perlecan, fibronectin,
osteonectin, monocyte
chemoatractant protein-3
Collagenase 4 MMP-18 Not found in Collagens (I, II, III, VIIIa, and X),
(Xenopus) humans gelatin, aggrecan
Gelatinases
Gelatinase A MMP-2 16q13 Collagens (I, IV, V, VII, X, XI, and
XIV), gelatine, elastin, fibronectin,
aggrecan, osteonectin, laminin-1,
laminin 5g2 chain,
chondroitinsulphate proteoglycan,
IL-1b degradation, monocyte
chemoatractant protein-3, decorin,
big endothelin, adrenomedullin,
stromal cell-derived factor 1a (SDF-1)
Gelatinase B MMP-9 20q11.2– Collagens (IV, V, VII, X, and XIV),
q13.1 gelatine, entactin, aggrecan, elastin,
fibronectin, osteonectin,
plasminogen, galactin-3, IL-1b
degradation, IL-2Ra, precursor of
TGFb
Stromelysins
Stromelysin 1 MMP-3 11q23 Collagens (III, IV, V, and IX),
gelatine, aggrecan, perlecan,
Table 1.1 Classification of matrix metalloproteinases (MMPs)—cont'd

Chromosomal
location
decorin, laminin, elastin, casein,
osteonectin, ovostatin, antactin,
plasminogen, MBP, IL-1 b,
E-cadherin, perlecan, monocyte
chemoatractant protein-3
Stromelysin 2 MMP-10 11q22.3–q23 Collagens (III–V), gelatine, casein,
aggrecan, elastin
Matrilysins
Matrilysin 1 MMP-7 11q21–q22 Collagens (IV and X), gelatine,
aggrecan, decorin, fibronectin,
laminin, entactin, elastin, casein,
transferring, plasminogen
Matrilysin 2 MMP-26 11p15 Gelatin Ia, fibrinogen, fibronectin,
vitronectin
Stromelysin 3 MMP-11 22q11.2 IGFBP-1
Membrane-type MMP
(A) Transmembrane type
MT1-MMP MMP-14 14q11–q12 Type I collagen, CD44, laminin 5g2
chain, monocyte chemoatractant
protein-3, cell surface tissue
transglutaminase, MUC1, a
transmembrane mucin
MT2-MMP MMP-15 15q13–q21 Fibronectin, entactin, laminin,
perlekan, cell surface tissue
transglutaminase
MT3-MMP MMP-16 8q21 Collagen III, gelatine, casein,
fibronectin cell surface tissue
transglutaminase
MT5-MMP MMP-24 20q11.2 Unknown
(B) GPI-anchored
MT4-MMP MMP-17 12q24.3 Unknown
MT6-MMP MMP-25 16p13.3 Progelatinase A, fibrin, fibronectin,
collagen IV, gelatin
Continued
Table 1.1 Classification of matrix metalloproteinases (MMPs)—cont'd

Chromosomal
location
Others
Macrophage MMP-12 11q22.2– Collagen IV, gelatine, elastin, casein,
elastase q22.3 fibronectin, vitronectin, laminin,
entactin, MBP, fibrinogen, fibrin,
plasminogen
– MMP-19 12q14 Gelatin, aggrecan, fibronectin,
IGFBP-3, laminin 5g2 chain
Enamelysin MMP-20 11q22.3 Amelogrenein, aggrecan
– MMP-21 1p36.3 Unknown
CA-MMP MMP-23 1p36.3 Unknown
– MMP-27 11q24 Unknown
Epilysin MMP-28 17q21.1 Casein
carcinoma cells [11]. Two other matrixins, MMP-2 and MMP-14 (MT1-
MMP), have collagenolytic activity, but they are classified into other sub-
groups because of their domain composition.
Gelatinases (MMP-2 and -9) readily digest gelatin with the help of their
three fibronectin type II repeats that bind to gelatin/collagen. They also
digest a number of ECM molecules including type IV, V, and XI collagens,
laminin, aggrecan core protein, etc. MMP-2, but not MMP-9, digests col-
lagens I, II, and III in a manner similar to collagenases [12,13]. The col-
lagenolytic activity of MMP-2 is much weaker than MMP-1 in solution.
Interestingly, proMMP-2 is recruited to the cell surface and activated by
the membrane-bound MT-MMP. As such, it may express reasonable col-
lagenolytic activity on or near the cell surface.
Stromelysins (MMP-3, -10, and -11) have a domain arrangement similar
to collagenases but do not cleave interstitial collagens. MMP-3 and -10 are
similar in structure and substrate specificity, but MMP-11 (stromelysin 3) is
distantly related. The MMP-11 gene is located on chromosome 22, whereas
MMP-3 and -10 genes are located on chromosome 11, along with MMP-1,
-7, -8, -12, -20, -26, and -27. MMP-3 and -10 digest a number of ECM
molecules and participate in proMMP activation. MMP-11, on the other
hand, has very weak activity toward ECM molecules [14], but cleaves
serpins more readily [15]. MMP-11 has a furin recognition motif RX[R/K]
R at the C-terminal end of the propeptide, and therefore, it is activated
intracellularly [16]. An intracellular 40-kDa MMP-11 isoform (h-
stromelysin 3) is found in cultured cells and placenta [9]. This transcript
results from alternative splicing and promoter usage thus lacking the signal
peptide and prodomain. The function of this isoenzyme is not known.
Matrilysins (MMP-7 and -26) lack a hemopexin domain. MMP-7 is
synthesized by epithelial cells and is secreted apically. Besides ECM compo-
nents, it processes cell surface molecules such as pro-a-defensin, Fas-ligand,
protumor necrosis factor a, and E-cadherin. MMP-26 is expressed in normal
cells such as those of the endometrium and in some carcinomas. It digests
several ECM molecules, and unlike most other MMP, it is largely stored
intracellularly [17].
MT-MMP in mammals includes four type I transmembrane proteins
(MMP-14, -15, -16, and -24) and two glycosylphosphatidylinositol-
anchored proteins (MMP-17 and -25). They all possess a furin recognition
sequence RX[R/K]R at the propeptide C-terminus. They are activated
intracellularly, and active enzymes are likely to be expressed on the cell sur-
face. All MT-MMP, except MT4-MMP (MMP-17) [18], can activate
proMMP-2. MT1-MMP (MMP-14) has collagenolytic activity on collagens
I, II, and III [19]. MT1-MMP null mice exhibit skeletal abnormalities during
postnatal development attributed to the lack of collagenolytic activity [20].
Seven MMP are not grouped in the above categories although MMP-12,
-20, and -27 have similar structures and chromosomal location as
stromelysins. Metalloelastase (MMP-12) is not only expressed primarily in
macrophages but also found in hypertrophic chondrocytes [21] and osteo-
clasts [22]. It digests elastin and a number of ECM molecules. MMP-19
digests many ECM molecules including the components of basement mem-
branes [23]. Enamelysin (MMP-20) is expressed in newly formed tooth
enamel and digests amelogenin [24]. MMP-21 was originally found in
Xenopus [25] and more recently in mice and humans [26]. It is expressed
in various fetal and adult tissues and in basal and squamous cell carcino-
mas [27]. It digests gelatin, but information on its action on ECM compo-
nents is not known. MMP-23 is a unique member as it has unique a
C-terminal cysteine-rich immunoglobulin-like region instead of a
hemopexin domain [28]. The propeptide lacks a cysteine switch. It is pro-
posed to be a type II membrane protein having a transmembrane domain at
the N-terminal of the propeptide, but the enzyme is released from the cell as
the membrane-anchored propeptide cleaved by proprotein convertase [29].
MMP-27 was first found in chicken embryo fibroblasts [30]. MMP-27

digests gelatin and casein and causes autolysis of the enzyme, but little infor-
mation is available on the activity of mammalian enzyme.
Epilysin (MMP-28) is expressed in many tissues such as lung, placenta,
heart, gastrointestinal tract, and testis. The enzyme expressed in basal
keratinocytes in skin is considered to function in wound repair [31].
MMP-21, -23, and -28 have a furin recognition sequence that precedes
the catalytic domain and are therefore likely activated intracellularly and
secreted as active enzymes.
A disintegrin and metalloproteinases (ADAM) family of transmembrane
proteins also belong to the zinc protease superfamily. Members have a mod-
ular design, characterized by the presence of metalloprotease and integrin
receptor-binding activities, and a cytoplasmic domain that in many family
members specifies binding sites for various signal transducing proteins.
The ADAM family has been implicated in the control of membrane fusion,
cytokine and growth factor shedding, and cell migration, as well as processes
such as muscle development, fertilization, and cell fate determination.
Pathology such as inflammation and cancer also involve ADAM family
members [32].
3. REGULATION OF MMP
MMP activity is regulated at multiple levels: synthesis of inactive
zymogens and gene transcription, posttranslational activation of zymogens,
and interactions of secreted MMP with tissue inhibitors of
metalloproteinases (TIMP).
3.1. Inactive zymogens

Most MMP are synthesized and secreted as inactive proenzymes. Enzymatic
activation requires removal of their prodomain which can occur via degra-
dation by other proteases such as plasmin and cell-associated MT-MMP.
Enzyme activation is mediated essentially through cleavage of the
thiol-Zn2þ interaction site by three potential mechanisms including direct
prodomain cleavage and removal by another proteinase, thiol reduction by
nonphysiologic agents or reactive oxygen species (ROS), and allosteric inhi-
bition of the zymogen [33,34]. The molecular mechanisms underlying
direct proMMP activation or allosteric control are mostly unknown and
may involve proteases such as kallikrein, trypsin, other MMP in vivo or
chemical agents like alkylating agents or heavy metals, free radicals, and
detergents in vitro. Autocatalytic MMP activation has also been reported,

mediated by allosteric interaction with its substrate. Activation mechanisms
may occur via compartmentalization processes that specifically increase their
local concentration [33].
Alternatively, fully activated MMP can arise through prodomain autol-
ysis secondary to conformational changes to reveal the catalytic site. Such
conformational changes also allow substrate lysis by proMMP and are the
basis for zymographic detection of both latent and activated MMP following
exposure to sodium dodecyl sulfate. Once activated, MMP participate in a
broad spectrum of physiologic and pathologic processes. Important non-
matrix substrates include molecules whose biological activity is regulated
by MMP processing such as TNF-a [35], growth factors and their recep-
tors [36], plasminogen and its activators [37,38], and endothelin [39]. The
major cellular constituents of normal blood vessels, human endothelial cells
(EC) and SMCs, produce constitutively in vitro MMP-2, TIMP-1, and
TIMP-2 [40,41]. Immunohistochemical studies suggest that nondiseased
human arteries and experimental animal arteries uniformly express
MMP-2 and the inhibitors TIMP-1 and -2 across the wall. Interestingly,
no in situ activity was detected [42,43], a finding that suggests tight control
of MMP activity in the presence of zymogen abundance.
3.2. Transcriptional regulation

Transcriptional activity is an important event in the ultimate synthesis and
release of soluble MMP as well as MT-MMP. Following biologic and/or
mechanical stimuli, a cascade of intracellular events culminate in the forma-
tion of a number of transcription factors. These transcription factors can bind
to the promoter region of MMP genes and induce transcription. Two major
cis-acting elements are found in a majority of the MMP promoters: activator
protein-1 (AP-1) and polyoma enhancer A binding protein-3 (PEA-3),
which interact with the Fos-Jun family and Ets family of transcription fac-
tors, respectively. MMP-2 is interesting in that it lacks both the AP-1 and
PEA-3 elements and also lacks a TATA box [44]. Due to the fact that
MMP-2 lacks these canonical transcription factor binding sites, it was orig-
inally assumed that MMP-2 was constitutively expressed. Although MMP-2
is constitutively expressed in tissues at substantial levels, still there is evidence
that external stimuli can influence additional increase in MMP-2 production
[45,46]. While AP-1 and PEA-3 are found in most MMP types, there are
other elements found in individual MMP promoter regions. For example,
MMP-1, -7, -13, and MT1-MMP all have one or more TGF inhibitory ele-
ments which bind the family of SMAD transcription factors [47–50]. Other
transcription factors such as SP1 likely bind to the GC box contained within
the promoter regions of MMP-2, -9, and MT1-MMP [49]. In addition,
MMP-9 has a nuclear factor-kB (NF-kB) binding site [51]. MMP-1 and
-13 both have been described to have an NF-kB-like binding site which
responds to the NF-kB transcription factor [52]. Chromatin immunopre-
cipitation showed interaction of p50 and p65 units of NF-kB transcription
factor with the MMP-3 promoter in macrophages [53]. Thus, the transcrip-
tion factor binding sites, the number of these binding sites, and type of
upstream stimuli that are operative under certain cardiac disease states will
ultimately determine the transcriptional activity of select MMP types.
3.3. Translational regulation

Although the most important step regarding translational/posttranslational
modification is proteolytic activation of proMMP, oxidative stress can also
yield an active enzyme [54–57]. For example, myeloperoxidase, an enzyme
associated with oxidative stress, can cause transform the catalytic domain,
yielding an active enzyme [54,58]. Human myeloperoxidase is encoded
by the MPO gene and is abundantly expressed in neutrophil
granulocytes [59]. With the use of an isolated heart preparation, it was dem-
onstrated that peroxynitrite could induce the release and activation of
MMP-2 within the coronary circulation [60]. Thus, the effects of oxidative
stress on myocardial MMP are twofold. First, the formation of ROS causes a
de novo increase in MMP transcription. Second, oxidative stress causes a
posttranslational modification and activation of existing MMP reservoirs.
Since the MT-MMP, that is, MT1-MMP, are proteolytically active once
inserted into the cell membrane, then posttranslational events are critical
for yielding a fully functional enzyme. MT1-MMP contains extracellular
catalytic, transmembrane and intracellular domains, all of which are critical
for full protease function. Modifications of these domains will alter
MT1-MMP trafficking or activity [61–64]. It has been demonstrated that
trans-Golgi processing and intracellular activational steps such as those by
the intracellular protease furin play a critical role in processing the mature
full-length MT1-MMP [61,65]. MT1-MMP is well positioned to sense
and modify the extracellular environment by processing matrix compo-
nents, transmembrane proteins, and soluble factors [66].
3.4. Tissue inhibitors of metalloproteinases

In addition to autodigestion, a critical control point for MMP activity is
through the inhibition of the activated enzyme by the action of a group of
specific MMP inhibitors termed TIMP. There are four known TIMP species
of which TIMP-1 and -2 have been the most studied. TIMP are low-
molecular-weight proteins (190 amino acids) that can noncovalently complex
active MMP (1:1 molar ratio) with high efficiency [67–70]. TIMP bind the
catalytic domain of active MMP and thereby prevent substrate access [67,71].
Although the relative binding affinity of TIMP to different active MMP is
subject to assay conditions, it appears that TIMP-1 binds less MT-MMP less
efficiently [67]. TIMP-3 is unique among the known four TIMP because it
binds ECM proteins directly, thereby providing a means to stabilize MMP–
TIMP complexes in the interstitial space [72,73]. TIMP-3 binding to matrix
proteins would also require close proximity of the active MMP site for effec-
tive inhibitory binding effect. TIMP-3 has also been demonstrated to inhibit
ADAM, notably ADAM-17, or most commonly termed TNF convertase
(TACE) [74,75]. TIMP-4 is the most recently identified member of the
TIMP family in which a high degree of expression occurs within the cardio-
vascular system most notably the myocardium [76–78].
As with MMP, there are a number of transcription factor binding ele-
ments within the promoter region of TIMP that include AP-1, PEA-3,
Sp1, and Ets [52,79]. At first, this would seem to imply that an extracellular
stimulus would cause a concordant increase in both MMP and TIMP tran-
scriptional activity, and under normal circumstances, this may be the case.
However, there are distinct differences within the promoter sequences of
MMP and TIMP and individual TIMP that provide for unique regulation
and expression. For example, TIMP-2 contains a cluster of Sp1 and AP-2
motifs as well as a TATA-like element [80]. Furthermore, exposure to
phorbol esters, which in time would increase protein kinase C activity,
increases TIMP-1 transcriptional activity but does not change TIMP-2
expression from basal level [80,81]. TIMP-3 appears to be devoid of a
TATA sequence but contains multiple Sp1 sites [82,83]. Murine TIMP-4
has a GATA binding site and a unique initiator-like element [76]. While
the regulation of TIMP expression is highly complex, the different combi-
nations of cis-binding elements as well as the interaction of trans-activating
factors located on the TIMP promoter can provide a regional and temporal
regulatory mechanism for their expression. Although a predominant
characteristic of TIMP is inhibition of active enzymes, this appears
oversimplified. In early biochemistry studies in which the relative affinity for

TIMP binding to active MMP was under investigation, it was noted that
TIMP may actually bind to proMMP [84–86]. This TIMP binding domain
was distal from the MMP catalytic domain. There is now conclusive evi-
dence that TIMP bind to specific domains on inactive proMMP [87].
One of the most studied is TIMP-2 binding to proMMP-2 [87,88]. Under
these circumstances, a proMMP-2:TIMP-2 complex is formed which can
then bind a MT1-MMP dimer complex. This interaction causes the forma-
tion of a tetrameric structure that ultimately leads to proteolytic processing
and activation of bound MMP-2 [87]. However, excess TIMP, that is,
TIMP-2, can bind to MT1-MMP and thereby interrupt activation. It also
appears that TIMP-4 can bind proMMP-2, but whether this can yield an
activation complex with MT1-MMP remains unknown [89].
4. MMP IN CARDIOVASCULAR SYSTEM DEVELOPMENT

MMP have important roles in cardiovascular physiology and pathol-
ogy. MMP and their inhibitors play major roles in ECM degradation and in
mediating cell–cell adhesion, cell migration and invasion, cell proliferation
and apoptosis, tissue remodeling, and growth factor and cytokine signaling
[2]. Cardiovascular morphogenesis for normal vascular and heart develop-
ment requires dynamic changes in all these processes and involves MMP
gene expression and activation of proMMP. The role of MMP in
vasculogenesis and angiogenesis, cardiac tube formation and looping, heart
septation, and cardiac remodeling has been studied extensively during
embryonic development in animal models.
4.1. MMP in vasculogenesis and angiogenesis

Vasculogenesis is the process of de novo blood vessel formation via cell deter-
mination of angioblasts (EC precursors). It is distinct from angiogenesis which
involves the formation of new blood vessels from preexisting blood vessels via
sprouting or remodeling of existing vessels. Vasculogenesis and angiogenesis
require EC (or their precursors) to form three-dimensional aggregates and net-
works by directed cell migration, invasion, and ECM remodeling [90].
Fibroblast growth factors (FGF), VEGF, and their receptors have impor-
tant roles in the formation and maintenance of blood vessels during embryo-
genesis [91–94]. MMP, particularly MMP-9, release bioactive basic FGF
and VEGF sequestered within the ECM [95,96]. Active FGF and VEGF
stimulate the expression of several MMP necessary for angiogenesis
[97–99]. MMP-9-deficient mice are fertile but exhibit abnormal or delayed

angiogenesis. It is likely that other MMP family members compensate for
MMP-9 loss [100]. Vasculogenesis and angiogenesis require cell migration,
proliferation, and formation of branching endothelial chains and concomi-
tant lumen formation [101]. MMP play important roles in regulating these
events [2]. Migration of EC during angiogenesis also requires breakdown of
collagen components in the ECM [102].
MMP-mediated degradation of plasminogen generates angiostatin and
endostatins, potent inhibitors of angiogenesis [103]. Their formation pro-
vides an important braking mechanism for MMP-driven angiogenesis.
EC migration in adult blood vessels requires loosening of inter-EC contacts
and weakening of peri-EC support. In embryos lacking the angiogenic fac-
tor, angiopoietin-1, EC fail to associate with the underlying ECM and do
not recruit periendothelial support cells resulting in the formation of leaky
vessels [104]. Angiopoietin-1 increases plasmin generation and MMP-2
secretion in adult porcine pulmonary arterial EC while suppressing
TIMP-2 secretion [105]. Hence, the angiopoietin-1-dependent recruitment
of peri-EC and degree of EC adhesion and blood vessel permeability may
depend on particular levels of MMP and plasmin activity.
Integration of circulating EC into blood vessel walls may involve the
ephrin/EphB family which are transmembrane ligands for a family of EphB
receptor-tyrosine kinases [106,107]. The binding of ephrins to its receptors
stimulates transduction signals in the EphB-expressing cells but can also trans-
duce a reverse signal into the ephrin/ligand-expressing cell. Such interactions
and signaling events have been shown to play major roles in the development
of the vascular system including blood vessel remodeling and artery or vein
differentiation [108–110]. EphB4 receptors stimulate microvascular EC pro-
liferation and migration [111]. Stimulation of the EphB4 receptor by ephrin-
B2 increases activated forms of MMP-2 and -9 in vitro [111]. In ephrinB2- or
EphB4-deficient mouse embryos, there is a complete arrest of angiogenesis
[112]. Ephrin-B2 reverse signaling is required for proper development and
remodeling of the embryonic cardiac valves [112–114]. These observations
suggest that ephrins and EphB receptors have important roles in regulating
MMP activity during blood vessel development.
4.2. MMP in cardiac tube formation and looping

MMP-2 is the earliest MMP expressed during heart development. MMP-2
expression first appears within the lateral plate mesoderm and becomes
increasingly restricted to the splanchnic mesoderm adjacent either side of

the developing cranial foregut in the avian embryo [115]. Within this
splanchnic mesoderm, angioblasts organize into two primitive
endocardial-lined tubes that eventual fuse forming a single inner endocardial
tube surrounded by differentiating cardiomyocytes. The single heart tube is
suspended from the foregut into the primitive thoracic cavity by dorsal
mesocardium which eventually ruptures to allow looping of the primitive
heart tube. During the process, MMP-2 is expressed in the endocardium,
early differentiating cardiomyocytes, and dorsal mesocardium but is soon
lost within the myocardium [115,116]. Blocking MMP-2 activity prevents
midline fusion of the primitive heart tubes leading to cardiac bifida [116].
Both MMP-2 neutralizing antibodies and the broad-spectrum MMP inhib-
itor Ilomastat produce severe heart tube defects in chick embryonic stages 5
(primitive streak stage) to 12 (16-somites). Exposure to the MMP inhibitor
at stage 5 produces various degrees of cardia bifida. At the seven-somite
stage, MMP-2/Ilomastat inhibition causes a shift in normal left–right pat-
terning of cell proliferation within the dorsal mesocardium and mesoderm
of the anterior heart field that correlates with a change in looping direction.
MMP inhibition at the 10- to 12-somite stage results in an arrest of heart
tube bending by inhibiting the breakdown of the dorsal mesocardial
ECM. Experiments involving exposure of embryonic chick embryos with
MMP inhibitors also suggest that MMP activity regulates the coordination
of early heart organogenesis by affecting ventral closure of the heart and gut
tubes, asymmetric cell proliferation in the dorsal mesocardium to drive
looping direction and ECM degradation within the dorsal mesocardium all-
owing looping to proceed toward completion [116]. TIMP-2 is also
expressed within the endocardium of the single heart tube and in the dorsal
mesocardium just prior to the rupture of the dorsal mesocardium and the
onset of cardiac looping [116,117]. Cardiac looping converts the single,
straight tubular heart into a S-shaped tube and repositions the primitive
heart chambers into their adult anatomical positions before cardiac septation
is complete. Looping is thought to be driven by elongation and remodeling
of the heart tube at the cranial end [117]. As the heart tube continues to
lengthen during the looping process, TIMP-3 expression appears within
the endocardium and in the myocardium of the outflow region
[117–119]. The expression of TIMP-3 within this myocardium is precisely
at the place where lengthening and remodeling of cardiac tube takes place.
In addition, MMP-2-mediated growth also appears to be involved in
orchestrating the direction of cardiac looping [116].
4.3. MMP in heart septation

The septation of the atria and ventricles and division of the cardiac outflow
tract into the aorta, and pulmonary artery requires the migration, proliferation,
and differentiation of two distinct mesenchymal populations, endocardial-
derived cushion cells and invading neural crest (NC) cells [120]. The
MMP have been implicated in regulation of epithelial-to-mesenchymal tran-
sitions (EMT) responsible for forming both cell populations. MMP-2 is
expressed by endocardial cells prior to and during EMT of the endocardium
in atrioventricular as well as outflow tract regions of the developing heart
[115,121]. MT3-MMP is expressed just prior to endocardial EMT, suggesting
that proMMP-2 activation is a prerequisite for EMT [121,122]. Activation of
proMMP-2 requires TIMP-2 or -3 via formation of a ternary complex with
either MT1-MMP or MT3-MMP. Both TIMP-2 and -3 are expressed in the
endocardium of the atrioventricular and outflow tract regions prior to and
during EMT of the endocardium [116]. Collagen type IV integrity is lost
within the endocardial basement membrane just prior to endocardial EMT.
In the heart, blocking MMP activity decreases active MMP-2, prevents loss
of basement membrane integrity, and inhibits endocardial EMT [122,123].
Endocardial cells undergoing EMT and migrating cushion tissue cells activate
proMMP-2 necessary for migration. This activity may be directed to the lead-
ing edges of their invadopodia.
Many congenital heart and great vessel defects stem from aberrant NC
morphogenesis [124]. Blocking MMP activity inhibits NC cell migration
both in vivo and in vitro [115,125,126]. Patch mice, which exhibit
NC-related craniofacial and cardiac defects [127,128], have deficiencies in
MMP-2 and MT1-MMP expression. NC-derived mesenchyme from these
embryos has decreased migratory capacity in vitro [129].
4.4. MMP in embryonic and fetal cardiac remodeling

MMP and their inhibitors have been shown to be temporally and spatially
expressed during various stages of embryonic and fetal heart remodeling.
The developing valve leaflets surrounding the atrioventricular orifices
express MMP-2 [115,121]. During the formation and remodeling of the
muscular septa, cells of septum primum adjacent to ostium primum express
MMP-2, TIMP-2, and TIMP-3. TIMP-3 is also expressed within remo-
deling myocardium [115,117]. With the onset of cushion cell formation,
the atrioventricular and conal myocardium begin expressing TIMP-3 in a
pattern, suggesting that TIMP-3 participates in separating the atrial
myocardium from the ventricular myocardium and in realigning the atrio-

ventricular canals with the developing ventricles [117]. Overlapping expres-
sion of TIMP-2 and MMP-2 within the epicardium and developing
coronary vasculature also points out importance of MMP activity in the
development of this tissue layer as well [115,117].
MMP are also involved in remodeling events for transfiguring the prim-
itive ventricular myocardial wall into a compact layer and inner trabecular
layer. In mouse embryo, MMP-2, -3, -9, and -13 are expressed in prenatal
hearts, particularly over the trabeculae and epicardial tissue, as early as
embryonic day 12, and continue to be expressed there during the remainder
of fetal period [130].
5. MMP IN CORONARY ARTERY DISEASE

CAD is the most common type of heart disease and cause of heart
attacks. The disease is caused by atheromatous plaque building up along
the inner walls of the arteries of the heart which narrows the arteries and
restricts blood flow to the heart (http://www.nhlbi.nih.gov/health/
health-topics/topics/cad/). After decades of progression, some of these ath-
eromatous plaques may rupture and start limiting blood flow to the heart
muscle. As CAD progresses, there may be near-complete obstruction of
the lumen of the coronary artery. Individuals with this degree of CAD typ-
ically have suffered from one or more myocardial infarctions (MI) and may
have signs and symptoms of chronic coronary ischemia, including symptoms
of angina at rest (https://www.metrohealth.org/body.cfm?id¼1484). Previ-
ously considered a cholesterol storage disease, but atherogenesis is a complex
interaction of risk factors including cells of the artery wall and the blood and
molecular messages that they exchange. It is now believed that inflammation
plays a major role in all stages of atherogenesis [131] including local, myo-
cardial, and systemic complications of atherosclerosis. As a major conse-
quence of the inflammatory ferment underway in the early atheroma,
SMCs migrate from the tunica media into the intima. These cells proliferate
and elaborate a rich and complex ECM. In concert with EC and monocytes,
they secrete MMP in response to various oxidative, hemodynamic, inflam-
matory, and autoimmune signals. MMP, in concert with their endogenous
tissue inhibitors, modulate numerous functions of vascular cells, including
activation, proliferation, migration, and cell death, as well as new vessel for-
mation, geometric remodeling, healing, or destruction of ECM of arteries
and the myocardium [132].
Aberrant MMP catalytic activity has been linked to atherosclerotic

plaque formation and plaque instability, vascular SMC migration and reste-
nosis, development of aortic aneurysm, and progressive heart failure in
both animal models and humans [2]. Because any lasting change in blood
vessel structure entails remodeling of its matrix scaffold, MMP involvement
has recently been proposed with respect to vascular pathology in CAD.
In vitro studies with cultured cells and histologic observations of normal
and diseased human and experimental blood vessels indicate that both vas-
cular and inflammatory cells produce MMP, although the spectra of MMP
secreted basally or in response to stimuli are distinctive. On the other hand,
focally increased MMP expression and activity have been observed in dis-
eased human arteries [43,133–135], and in association with arterial mor-
phologic changes in experimental models of atherosclerosis and
restenosis [136–138]. These findings indicate that MMP enable blood ves-
sel reshaping including those associated with pathology. Further evidence
has resulted from in vitro studies of cultured vascular and inflammatory cells
that tested stimuli characteristic for the diseased vessel environment.
Increased risk for fatal coronary heart disease events has been reported
in subjects with increased plasma MMP-9 [139]. This association was inde-
pendent of conventional cardiovascular risk factors. Increased TIMP-1 has
been observed in patients with coronary heart disease. The Framingham
study group has reported that increased TIMP-1 predict cardiovascular
disease/death [140].
Several MMP system components are expressed in atherosclerotic tissue.
These are associated with almost all steps of plaque formation, erosion and
rupture, with the latter leading to the clinical manifestations of CAD.
In this section, we will discuss clinical characteristics of CAD and their
association with MMP.
5.1. MMP in atherosclerosis

Atherosclerosis is a specific form of arteriosclerosis in which an artery wall
thickens as a result of the accumulation of fatty materials such as cholesterol
and triglyceride. Initial lesions in atherosclerosis consist of fatty streaks that
develop into fibroproliferative lesions. However, mature lesions mainly
comprise foam cells, SMC, a necrotic core, and a fibrous cap containing
ECM components. Clinical complications often involve rupture of unstable
plaques, In addition, thinning of the vessel wall due to elastin and collagen
degradation and media necrosis may result in aneurysm formation and
bleeding. Further proteolysis may contribute to neovascularization and rup-

ture of plaques or to ulceration and rupture of aneurysm.
It is widely accepted that atherosclerosis is initiated by chemical and/or
mechanical injury of the endothelium. This is followed by transendothelial
infiltration of circulating monocytes into the intima where they become
activated and elaborate a variety of cytokines and growth factors [141]. In
response to these stimuli, VSMC migrate from the media to the intima
and undergo proliferation. Matrix degradation is a prerequisite for both
the recruitment of monocytes and migration of VSMC. The cells have to
transverse the extracellular barriers including the basement membrane (con-
sisting of collagen type IV and laminin) underlying the endothelium and sur-
rounding each smooth muscle, as well as a dense mesh of interstitial collagen.
In mice, electrical injury to femoral arteries to stimulate intimal thickening
caused enhanced expression of MMP-2 and -9 [142]. In TIMP-1-deficient
mice, intimal thickening was significantly higher versus wild-type controls
[143]. Together, these observations support a role for MMP involvement in
intimal thickening, particularly in migration of VSMC. Previous studies
demonstrated that lipid-laden macrophages from human atherosclerotic
plaques elaborate MMP-1 and -3 [138]. Macrophage cultures exposed to
fibrous caps of human atherosclerotic plaques induced MMP-dependent
collagen breakdown [144].
MMP are not produced in normal arteries, except for a small amount of
MMP-2 and -4 [145]. MMP, however, have highly variable expression in
atherosclerotic disease reflecting their detrimental effects on the vasculature.
Henney et al. detected MMP-3 transcripts in coronary atherosclerotic
lesions which were colocalized with large clusters of lipid-laden
macrophages in the shoulder areas of the plaque [146]. Galis et al. reported
that atherosclerotic plaque and lesion-free arteries had different patterns of
MMP expression. MMP-2 together with TIMP-1 and -2 was expressed by
VSMC in all layers of nonatherosclerotic arteries, whereas MMP-1, -3, and
-9 were localized to macrophages, VSMC and the endothelium in the
fibrous cap and shoulders of the lesions [43,147]. Other researchers have
also detected the expression of MMP-1 and -2 in the shoulder areas of
plaque [133,134,148–150].
MMP favor monocyte infiltration of the vascular wall. An increase in the
expression of certain MMP, that is, MMP-12, leads to macrophage infiltra-
tion, rupture of the internal elastic lamina, and the acceleration of athero-
sclerosis [151]. The induction and activation of the MMP, particularly
MMP-14 (MT1-MMP), favors the invasion of plaques by VSMC and
fibroblasts. The migration and proliferation of these cell types is important in

the development of intimal hyperplasia [152,153]. The activity of MMP-2
and -9, among others, is indispensable for the neovascularization of athero-
sclerotic plaques. This process, induced by proangiogenic and inflammatory
stimuli, appears necessary for growth and is associated with vulnerability of
advanced lesions [154,155].
The expression and activity of MMP-9 in an atherosclerotic plaque,
which is mainly associated with macrophages, may be the consequence of
increased NADPH-dependent superoxide anion because it coincided with
NADPH oxidase expression and free radical production [156]. However,
gelatinases and collagenases are not the only MMP associated with athero-
sclerosis. Increased MMP-10 (stromelysin 2) has also been observed in
advanced carotid plaques [157]. MMP-10 has also been associated with aor-
tic aneurysms characterized by destructive remodeling of the vascular ECM
and wall rupture [158]. Finally, increased TIMP-1 has been reported in
calcified areas of human atherosclerotic plaques further indicating that
inhibition of MMP activity may be related to greater plaque instability
[159]. Clinical manifestation of atherosclerosis, that is, unstable angina
and MI, is the result of unstable lesions in symptomatic patients rather than
obstruction of blood flow through the narrowed lumens of the arteries [4].
MMP-9 is related in greater degree with this kind of lesion in symptomatic
patients versus stable lesions in asymptomatic patients independent of plaque
size [160]. Atheromatous plaque and its formation within the arteries is a
complex and evolving process involving remodeling of the endothelium
as well as of the surrounding tissues, such as ECM. Since the first observation
of MMP-9 within the atherosclerotic plaques [161], several studies have
documented the existence of MMP-9 within atherosclerotic lesions
[138,162,163]. Luttun et al. [164] demonstrated that MMP-9 is basic to
plaque growth because it promotes the basement membrane breakdown,
thereby allowing SMC migration and proliferation within the plaque. At
the same time, MMP-9 is responsible for the initiation of a vicious cycle
in which it facilitates the accumulation of monocytes and macrophages
[165]. These, in turn, convert to foam cells thus contributing to the lipid
core of the plaque. In addition, these cells secrete chemoattractant and
proinflammatory molecules, that is, TNF-a, which can stimulate MMP-9
activation [40,41,166]. These cells are the main source of MMP-9 within
atherosclerotic plaques and are detrimental to emerging lesions [167].
MMP-9 allows for plaque growth and stabilization via its direct effect on
VEGF which promotes plaque neovascularization in this inflammatory
environment [168,169]. In late disease, MMP-9 [170] secreted by macro-

phages in the shoulder region cleaves the fibrous cap that stabilizes the ath-
eroma. This leads to thrombus formation and clinical sequelae, that is,
unstable angina or MI. MMP-9 is related to severity of the disease. Its
increase in three-vessel CAD relative to one- or two-vessel disease implies
an association with atherosclerotic severity. As such, measurement of serum
MMP-9 could be useful to detect and assess disease severity in CAD patients
with stable angina [171]. Increased MMP-9 has also been observed in cor-
onary plaques of patients with unstable angina versus those with stable angina
[160,172], a finding related to postinfarct ventricular remodeling [173,174].
Increased MMP-9 has also been reported in human brain tissue following
ischemic and hemorrhagic ictus. Induction of MMP-9 has been reported
in the cerebral vasculature following fibrinolysis. This indicates that this
enzyme may contribute to ischemic cerebral lesions and perihematomal
edema as well as to cerebral hemorrhages and neurovascular lesions follow-
ing fibrinolysis [175,176]. These local alterations can be detected at the sys-
temic level. Patients with angiographic proof of coronary heart disease have
fibrinolysis/proteolysis imbalance in peripheral blood [177]. After acute MI
(AMI), urinary MMP-9 was increased while TIMP-1 was reduced [177].
Thus, MMP-9 may be considered as an attractive tool for the early detection
of myocardial ischemia [139,178–182].
Imbalanced MMP/TIMP ratio contributes to atherosclerotic plaque
destabilization and rupture. Using antioxidants, specific inhibitors, and
siRNA, Gargiulo et al. [183] demonstrated that the oxysterol mixture
induced MMP-9 expression via overproduction of ROS, probably by
NADPH-oxidase and mitochondria; upregulation of mitogen-activated
protein kinase signaling pathways via protein kinase C; and upregulation
of activator protein-1- and nuclear factor-kB-DNA binding. These results
suggest, for the first time, that oxysterols accumulating in advanced athero-
sclerotic lesions significantly contribute to plaque vulnerability by promot-
ing MMP-9/TIMP-1/-2 imbalance in phagocytic cells [183]. The role of
MMP in the formation of atherosclerotic plaques and atherothrombosis is
shown (Fig. 1.1).
5.1.1 MMP and atherosclerotic risk factors

The majority of the classic risk factors for atherosclerosis have been related to
changes in the concentration of various ECM markers [184,185]. The
mechanisms behind this, however, are unclear. Risk factors include age,
gender, dyslipidemia, hypertension, diabetes, obesity, metabolic syndrome,
Figure 1.1 The role of matrix metalloproteases (MMP) in the synthesis and breakdown
of the ECM in atherothrombosis: MMP contribute to the plaque formation, mediated by
vascular smooth muscle cells and macrophage-derived foam cells. In addition, they con-
tribute in the extra cellular matrix and fibrous cap degradation as well as the digestion of
external elastic lamina.
tobacco, alcohol, and inflammation. They can influence the production or

activities of MMP and TIMP and modulate the vascular structure and sta-
bility of atherosclerotic plaques. MMP involved in these risk factors are
shown (Table 1.2).
5.2. MMP in plaque instability

Atherosclerotic plaques are terminated through structural disruption of the
arterial wall, further triggering thrombosis, causing occlusion and the major-
ity of acute vascular events [204]. Plaque disruption takes one of two forms,
frank rupture and superficial erosion [205,206]. Rupture is associated with
fracture of the fibrous cap with exposure of the prothrombotic core [207].
Peripheral blood MMP-2 and -9 are increased in ACSs and as such may have
potential use to detect plaque vulnerability [208]. A vulnerable plaque, a
type of atheromatous plaque, is a collection of white blood cells (primarily
macrophages) and lipids (including cholesterol) in an arterial wall that is
Table 1.2 Association of MMP with atherosclerotic risk factors

Risk factors MMP/TIMP associated References
Age Advanced age has been related to elevated [184]
concentrations of TIMP-1
Gender Male sex has been related to elevated concentrations of [184]
TIMP-1
Hormone therapy has been reported to reduce MMP-9 [186]
concentrations in postmenopausal women
Dyslipidemia MMP-9 has been associated with high levels of low- [185]
density lipoprotein cholesterol (LDL-C)
MMP-3, MMP-9, and TIMP1 have been associated [187]
with carotid atherosclerotic lesions
MMP-9 has been associated in patients with familial [188]
hypercholesterolemia and coronary heart disease
TIMP-1 has been associated with the total cholesterol/ [184]
high-density lipoprotein cholesterol (HDL-C) ratio
Vascular expression of MMP-1 increases in a porcine [189]
hypercholesterolemia model
In in vitro experiments, oxidized LDL has been shown [190]
to increase the production of MMP-1, 3, and 9, while
that of TIMP-1 is reduced. In contrast, HDL reduces
the production of several MMP types
In monocytes/macrophages, it has been reported that [191]
oxidized LDL acts synergistically with proinflammatory
factors to induce the expression of MMP-1 and
MMP-9, while HDL prevents the induction of
MMP-1 via these same agents
Tobacco Smoking shows increased concentrations of MMP-9 [192]
and TIMP-1, the extent of which is partly related to the
duration of tobacco exposure
Alcohol An inverse association has been reported between [184]
moderate alcohol consumption and circulating
TIMP-1 concentrations
No association has been reported between the abusive [193]
intake of alcohol and circulating TIMP-1 and MMP-2
concentrations
Table 1.2 Association of MMP with atherosclerotic risk factors—cont'd

Risk factors MMP/TIMP associated References
Diabetes Increased MMP-9 and TIMP-1 have been associated [194]
with diabetes patients
Increased glucose levels induce the expression of [195]
MMP-1 and 2 by ECs, and of MMP-9 by macrophages,
but have no effect on TIMP-1
In a clinical study involving patients with diabetes, the [196]
control of blood sugar and atherosclerotic risk factors
reduced TIMP-1 without modifying MMP-9 or
TIMP-2
Obesity The concentration of TIMP-1, but not of MMP-9, has [184]
been related to body mass index
A study involving obese women, a reduction in [197]
MMP-1 was noticed 1 year after performing stomach
reduction surgery
Metabolic Increased MMP-9 and TIMP-1 have been associated [198]
syndrome with metabolic syndrome
Hypertension An increase in circulating not only MMP-9 but also [199]
TIMP-1 has been reported in patients with high blood
pressure and general thickening of the artery walls
High blood pressure is associated with an increase in [200]
collagen synthesis and a reduction in its breakdown
Inflammation MMP-2 and -9 have been associated with increased [201]
inflammation in atherosclerotic plaques
C-reactive protein, an inflammatory biomarker of [157,202]
atherosclerotic risk, induces the expression of MMP-1
by macrophages, and of MMP-10 by ECs, without
affecting TIMP-1 concentrations
Higher concentrations of circulating MMP-9 in [203]
patients with CHD have been directly associated with
the concentrations of inflammation markers such as
plasma C-reactive protein, interleukin 6, and
fibrinogen
particularly unstable and prone to sudden major events, that is, heart attack
or stroke. Human coronary atherectomy specimens revealed uniform and
active synthesis of MMP-9 by macrophages and SMC in lesions from
patients with unstable versus stable angina, suggesting a role for this specific
MMP in ACS [135]. Resident macrophage-derived foam cells, characteris-
tic of unstable plaques, have been identified as a major source of MMP.
The mechanism responsible for focally increased expression and activa-
tion of macrophage-derived MMP, which may enable arterial remodeling
and precipitate plaque destabilization, is likely related to oxidative stress
[209]. The capacity of human monocyte-derived macrophages to induce
collagen breakdown in the fibrous caps of atheromas via release of MMP
was demonstrated ex vivo [144]. Using a cleavage-specific antibody,
degraded collagen was found to colocalize with MMP-1 and -13 positive
macrophages in atheromatous human carotid arteries in situ [210]. Alterna-
tive or complementary systems for the activation of latent MMP in athero-
sclerotic plaques have been suggested. Thrombin has been shown to
proteolytically activate purified proMMP-2 in vitro and thus could provide
cell-independent MMP activation at sites of vascular injury [211]. In com-
plicated atherosclerotic plaques, thrombin could promote plaque instability
in episodes of intraplaque hemorrhage or superimposed plaque thrombosis
by increasing the local matrix-degrading activity of MMP. The mutually
activating MMP/thrombin system may serve as an important positive-
feedback loop in ACS [212]. As acute plaque disruption leads to local throm-
bin production at the site of vascular injury, this may facilitate proteolytic
activation of MMP-2, shown to be able to mediate platelet aggregation
and further generate thrombin and additional MMP-2 activation [213].
Pericellular activation of proMMP-2 can be achieved by MT-MMP
expressed by vascular EC and SMC in response to cytokines and oxidized
lipoproteins [214]. The plasminogen cascade represents another
proteolytic-activating mechanism for MMP. Its contribution to the devel-
opment of experimental neointimal lesions after injury and to aortic medial
destruction was demonstrated in urokinase-type plasminogen activator
(u-PA) and plasminogen activator inhibitor-1 null mice [215] and apolipo-
protein E (ApoE) null mice, [216] respectively.
MMP-3, -8, and -9 have been found in atheromatic lesions [210,217]. In
lesions prone to rupture, MMP-8, -11, -14, and -16 are abundantly present,
while MMP-1, -2, -8, -13, and -14 can be observed in stable lesions [218].
MMP-1 and -13 are also colocalized with cleaved collagen in plaques [210].
EC produce MMP-1 in response to high glucose [195], while MMP-1, -3,
-8, -9 can also be upregulated in stimulation of tumor necrosis factor alpha

(TNF-a) and interleukin 1a (IL-1a) [41,219]. CD40 ligation denotes the
production of MMP-1, -3, -8, -9, and -11 in these cells [220,221]. In
VSMC, MMP-1, -3, and -9 are upregulated due to the response by IL-1,
IL-4, and TNF-a [222,223]. Cell contact with T-lymphocytes and CD40
ligand induced MMP-1, -3, -8, and -9 in VSMC [221,224]. Transforming
growth factor beta (TGFb) and platelet-derived growth factor (PDGF) also
triggered TIMP-1 and -3 production [166,225]. These findings suggested
that stretch, injury, and inflammation shifted the balance to proteolysis
and advanced plaque formation and rupture. In macrophages, cytokines
such as IL-1b, TNF-a, macrophage colony stimulating factor, and PDGF
upregulated MMP-14 and -16 [226]. Secreted MMP-12 enhanced mono-
cyte tissue migration [227]. MMP-1 secretion was also found to take place in
these cells by the contact with EC [228]. Although T-lymphocytes have
been implicated in MMP-1, -2, -3, and -9 secretion, the evidence was
not clear [43]. These findings imply that MMP secretion was essential during
the inflammatory process of atherosclerosis and may be key to plaque for-
mation and stabilization as well as plaque erosion and rupture. As such, these
molecules may be worthy of further study to assess disease and develop ther-
apeutic strategies.
5.3. MMP in acute coronary syndrome

ACS refers to a group of symptoms attributed to coronary artery obstruction.
The syndrome usually occurs as a result of ST elevation MI, non-ST eleva-
tion MI, or unstable angina. Acute coronary ischemia is usually initiated by
atherosclerotic plaque rupture, leading to intracoronary thrombosis and
occlusion. Collagen content contributes critically to the plaque stability.
Cheung et al. reported that the rat myocardium subjected to ischemia–
reperfusion injury released MMP-2 [229]. Brown et al. observed that
MMP-9 was commonly expressed in coronary atherectomy specimens from
patients with recent plaque rupture [135]. Recently, Kai et al. reported that
circulating MMP-2 and -9 were increased in AMI and unstable angina [208].
Inokubo et al. reported that plasma MMP-9 was significantly increased in the
coronary circulation in patients with AMI and unstable angina versus control
subjects, suggesting a process of active plaque rupture in ACS [230]. Hirohata
et al. observed increased plasma MMP-1 [231], and Hojo et al. observed
increased plasma MMP-2 [232] in AMI. Increased MMP expression may
modulate vascular and ventricular remodeling in ACS. Inokubo et al.
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'Hallo, Kelly!' said Antony, 'from the little a landsman like me knows of
the sea, yonder ship seems in a nasty place.'
'Ay, sir, that she be; and though the tide isn't out so far as it will be, the
Partans' Rock, sir, is all awash. I mind,' he added, 'when the Fair Maid o'
Wales went on shore on that same ugly rock three year and more agone.'
'Went to pieces, did she?'
'Went to pieces? Ay, that she did. A cat couldn't have lived out there that
night.'
'Any one lost?'
'Nobody saved, sir—not a living soul.'
'Is there a lifeboat anywhere near?'
'No nearer nor B—— and it's there I'm going as fast's I can; but it's ten
good miles yet.'
'Won't detain you,' said Antony. 'But could nothing be done to warn the
station?'
Kelly was moving on.
'Ay,' he shouted up the wind, 'if the fools on the bark show a rocket or
two; but they won't do that till they strike, and then'—— Kelly was supposed
to have added the words 'too late,' but they were not distinctly heard.
Antony took Lotty's hand, so that together they might run down the
distance that intervened betwixt this high outlook-cliff and the camp. The
child was trembling all over.
'You're not afraid, are you, dear?'
'No—that is, not much, Mr Blake,' she replied; 'but, oh, wouldn't it be
dreadful if the ship went to pieces on the Partans' Rock, and poor dear dead
sailors were washed up through the firth to our camp?'
'But I don't think it will be so bad as that, Lotty. Those coastguards
generally make the worst of things.'
'Did not Kelly say something about rockets?'
'Yes, dear.'
'And we have lots in our camp.'
'But, child, the bark may have some; anyhow, our sending up rockets
would, I feel certain, only confuse the coastguard. They would know even in
the dark that these were sent up from the shore, and imagine we were only
just having some fireworks.'
'That is true; but, oh dear, it is terrible!' Then suddenly she added, 'I think
we should light a fire just here; that would warn them off.'
'Yes, Miss Lotty.'
The child let go Antony's hand now, and he knew she meant business.
There was dead wood, with withered furze, lying about here, and all hands
commenced at once to collect it. Even Wallace brought piece after piece and
wisely laid it down beside the rest. And in five minutes' time a red, roaring
fire was gleaming through the darkness far across the sea.
Those on that devoted bark saw the fire even if they could not perceive
the treacherous rock; and, knowing too well what it meant, tried to luff and
get farther out to northwards. From the lights which now appeared upon her,
Lotty could tell that she had put about and was trying to work into the wind's
eye.
They left the cliff, and left Chops there to feed the flames, and by making
all haste were not long before they reached the camp. It would now be about
nine o'clock, or hardly; and so heavy were the clouds that nothing was to be
seen, never a star above, and only the shifting lights in the camp. Little to be
heard either except the wash and swell of the breakers and the occasional
wild scream of a sea-bird. That was an ugly coast at night for vessels to get
near when the shore was a lee one, and more especially in the dark o' the
neap, with a falling tide.
As they were still gazing, with hands above their eyes, out into the pitchy
gloom of that wild sea, suddenly there was a flash as of red lightning, then a
gun roared out, awakening a hundred echoes among the rocks.
'Oh, Mr Blake, Mr Blake, the bark has struck!'
'I fear she has, Lotty. But nothing can be done. Why, I wonder, do they
not fire a rocket?'
'Because, dear Mr Blake, perhaps they haven't one.'
'Well, calm yourself, Lotty. We can only pray for them, and before long
the coastguard may come with the lifeboat crew.'
'Ay, but they will never know. Kelly will take two long hours to reach B
—— and by that time, oh dear, oh! there may not be a soul left on board the
bark, nor a plank for a drowning man to cling to.'
Biffins Lee himself had come up.
'A sad business out yonder in the dark o' the neap, I fear, Mr Blake.'
There was no sign of sorrow in the man's voice. In fact, he seemed but
looking upon the whole affair from a spectacular point of view.
Another flash and another reverberating roar.
'One more gun!' said Biffins coolly. 'She can't hold together long if she be
on the Partan,' he added, 'or even inside it, which is more likely. I'm going to
tell Mary to heat water and to set men to watch the surf for dark things—
bodies—coming in. We may save life, Mr Blake, and what a fine
advertisement for the Queerest Show on Earth!'
Probably Frank Antony had never troubled himself to hate the man
before, but he did now; and, though the young man's arm was held down by
his side, he clinched his fist and gnashed his teeth. Then came the
questioning thought to his mind, just as it had often come before: 'Can this
brute really be the father of the sweet child who is standing by my side?'
And as Lee went out of sight, up out of the dark o' the neap seemed to
arise the answer: 'He cannot be her father, be the mystery what it may!'
Antony turned now to speak to Lotty. He missed her. She was here not a
minute ago, but now she was gone.
'Lotty, Lotty!' he called, and even Wallace raised his voice, barking a
strange, querulous kind of bark, and that was Antony's only answer.
'She is off to light the lamps in the "Gipsy Queen,"' he said to himself. So
he lit a cigar, and went smoking towards the caravan. But it was all in
darkness.
He met Mary.
'Have you seen Lotty?' he said.
'A few moments ago. Yes, Mr Blake. She was going towards the big
marquee.'
Ee-yowf barked Wallace again. It was the strangest sort of bark ever the
young man had heard him utter—speakingly strange, in fact. Then he started
off in the direction of the swinging, swaying petroleum lights of the camp.
He found the honest dog in the marquee looking for his mistress, and it
was evident enough he was on trail. He went dashing out now with his head
low towards the turf; and, as far as Antony could see him, he appeared to be
making tracks for the river. And Antony followed as quickly as he could, a
strange, wild thought having suddenly taken possession of his very soul. He
felt it was foolish, but he could not help it.
He found Wallace standing by the little boathouse looking helplessly out
towards the sea. Antony quickly opened the back-folding doors and threw
the gleam of his flashlight inside. It was empty! The Jenny Wren was gone!
He had not a single doubt now in his mind as to what must have
happened.
'Oh, how daring! how mad!' he said to himself half-aloud.
The little gipsy lass had evidently gone right to the big marquee to find
rockets, and with these in her possession she had no doubt set out in the little
boat towards the stranded bark, over that wild and stormy sea, in the dark o'
the neap.
CHAPTER XXI.
THE WRECK OF THE 'CUMBERLAND.'
T HAT next half-hour seemed to be the longest for Antony that ever he had
passed in his lifetime. That is what he said to his sister Aggie when he
wrote and told her all about the events of this fearful night. He first went
back towards his caravan, closely followed by Wallace, who appeared to
watch his every movement, certain in his canine mind that Antony would do
something to find his mistress. For a master stands in the place of a god to
his faithful dog, and the latter believes him omnipotent. Hardly knowing
what he did, he lit the great lamps, and through their dark-crimson shades
their light streamed like blood across the sand and on the surf.
For long minutes he sat beside Wallace, the dog giving vent occasionally
to that long, sad sigh that shows every one who knows and understands such
an animal that he is in grief—the dog sighing, the man chafing at his own
helplessness.
Presently he got up, and descended the steps. He met Mary bustling
around, and told her his fears.
All she answered was, 'Then God help our bonny bairn this dread and
awful night!'
No comfort in that quarter.
'I fear the worst, Mary,' he said.
'Let us hope for the best, then,' said Mary.
Then he set himself to walking rapidly up and down the beach as if to
work away his awful anxiety. And thus a good half-hour was passed. 'Had
she succeeded in reaching the doomed ship,' he told himself, 'there would
have been some sign ere now. Hallo!'
Another flash, another report, and almost immediately after the meteor-
track of a splendid rocket rising high in air, turned a little towards the west,
then bursting with a dull sound into brightest flame.
'Thank God!' cried Antony, 'the daring child has got safely on board.'
Something cold and damp touched his hand, and he found Wallace by his
side; and the gentle touch gave him both comfort and hope. But just at that
moment Antony made a strange discovery, which he put into words, though
these were not spoken aloud, but to his heart, as it were.
'I love that child,' he told himself with the frankness of youth, 'more than
is good for my peace of mind, more than is good for my future happiness or
probably hers; for if she is spared this sad night to get safely on shore, soon
now the parting must come, and I go away on my road in life and Lotty on
hers. Heaven help the child and me!'
Just as he was communing with himself, the dark o' the neap was riven
once more by a rocket's gleam that swayed and rose and burst as before.
It will be better to go back a little and follow the gipsy lass's movements
as she went sweeping down the river that night and headed straight for that
boiling sea. Surely she had more than the strength and skill of any two man-
o'-war's men, and more than the daring of a Grace Darling to venture forth
on such a forlorn hope.
But she had reasoned thus with herself: 'Even if I fail to reach the ship, I
can, I think, sweep my light skiff quickly round and go rushing back to the
river on the scud of the furious sea, and if I miss that the surf will rush me
right up upon the beach, and I will not lose either my life nor Antony's Jenny
Wren.'
Both wind and waves tossed the little boat about, but bravely did Lotty
keep her head to the mountain seas. So high and wild were some of the
breakers that they lifted the bows high in air and almost sank the skiff stern
first. The first quarter of an hour's pull was the worst. After this, though the
waves were higher, they did not break so much. Ah! but Lotty soon saw that
they were sweeping high and horrible right over the stranded vessel, and for
quite a long time she hovered near, hesitating how and when to approach.
At last she said to herself, 'It must be now or never.'
So high on a wavetop was she as the boat went dashing on from
windward that the brave girl could see right down on to the wave-washed
deck. Perhaps it was well for Lotty that the bark was so firmly, so steadfastly
fixed on the rocks; bad for the ship, it is true, for from this position never
could she move except in staves and broken timbers.
The sailors had seen the coming skiff, and three of them, at imminent
peril, rushed to the side and seized it in time; and next moment Lotty, safe
for the time being, was on the slippery upper deck of the bark, and even the
Jenny Wren was hauled on board. Right aft in the skiff was a little locker,
and it was in this Lotty had stowed the rockets.
On the beach, about an hour after this, shapeless black things were driven
up by the spume and the rush of the waves, and these were quickly seized by
the hands of the ready fishermen who had been attracted to the spot. Antony
himself was there, in fear and trembling lest one of the bodies washed in
might be—oh, terrible!—Lotty's own.
. . . . . . .
Bob Stevens was a hard, rosy-faced man, bold and blue-eyed, strong in
muscle, without one superfluous ounce of fat. Those eyes of his had peered
into the darkness overcanopying many a stormy sea, those hard brown hands
were at home with either tiller or oar, and more than at home with tack or
sheet; a fisherman by trade, a sailor bold as ever trod a slippery deck, and
master of the B—— lifeboat. Bob had gone quietly into the bar-parlour of
the 'Lovat Arms' on that evening, with three of his pals, all life-boatmen, and
they were smoking and enjoying modest glass and yarn when a man in
oilskins rushed hurriedly in.
'Bob, you'll be wanted,' he said. 'There's a ship on the Partan Rocks.'
'God help her if she's there to-night!' said one of Bob's crew.
'Up with your drams, lads, and we'll get the Maiden out at once.'
In an almost incredibly short space of time, and just as the second red
rocket cleft the darkness of the sky, the Maiden was launched and standing
out to sea. So quickly had they gone that the men's wives knew nothing of
their going until they had made good their offing and were swallowed up in
the dark o' the neap. The Maiden could sail as close to the wind as any boat
on the coast; but it needed all her seaworthiness and all Bob's skill to-night
to battle with these fierce and seething seas.
Never in this world will all the brave deeds done by our British lifeboats'
crews be recorded. Perhaps—quien sabe?—their stories may be told on the
shores of the Heavenly Canaan, where it is to be hoped we shall all meet.
But now, through the darkness, the ocean lit up by the white of the
curling waves, the Maiden toils on, up the watery hills, down with a rush
into the vales between, hit, buffeted, overwhelmed, and shivering, but still
striving on and on and on.
Will she be in time?
Perhaps hardly not, for the last rocket has been fired, and out of the
goodly crew of seventeen men and a boy that left London but two weeks ago
only nine are now alive. The rest have been swept away into the black,
yawning seas, and washed shorewards to death on the turn of the tide.
Lotty is down below, for here is the captain's wife and baby, whom the
little gipsy lass is doing her best to comfort. And she is thus engaged when
her quick ear is sensible of knocking and scraping along the leeside
bulwarks, and presently she cries aloud with joy, her eyes sparkling, her face
sweetly flushed in the light of the lamp in gimbals.
'Saved, dear lady, saved!' she says to the skipper's wife. For she has heard
strange voices on the deck, manly voices shouting strange orders high above
the wail of wind and dash of raging sea.
She and her companions are soon lifted by some of the rescuers and
carried as if babies in the strong arms of the rough but kindly men; and in a
few minutes all are on board the lifeboat, the last man to throw himself in
being the brave skipper himself.
Bob Stevens presently feels a tug at his arm, and a young girl's voice says
in his ear, 'Do not try to beach her; the sea is high and the bottom is rock. Up
the Burn o' Bogie with her. I'm going forward,' continued the voice; and Bob
said, many a time after this, it sounded to him like the voice of a seraph
—'I'm going forward with my flashlight, and will guide you safe up the
burn.'
It is needless to say that the voice was Lotty's, and next minute she was as
far forward as one could get in a boat like this, with the light in her hand.
She could see the fearful, roaring white of the seas that dashed on shore; but
there were hills with their black heads yonder too, and it was by these she
kept the course, till, with boiling waves high and threatening on both sides of
her, the lifeboat glided into the still, deep waters of the Burn o' Bogie.
It was Antony himself who lifted Lotty from the bows and landed her
safe on shore; and in all the vast crowd, that had gathered from every part of
the country, hardly was there a dry eye or a heart that did not throb with joy
in thinking of the brave deed done this night by the little gipsy lass.
. . . . . . .
'And to think, my dear,' said Mrs Oak the skipper's wife of the lost bark
Cumberland, as she was leaving for the south two or three weeks after this,
'that I may never have a chance of doing you a favour for all your brave
kindness to us! Oh surely,' she added as she pressed Lotty's hand, and would
not let it go, 'the King himself will hear of your gallantry and pluck, and
thank you. Good-bye, Lotty, oh good-bye, and God be with you aye!—Kiss
the child, James,' she said to her husband; 'kiss the darling that saved our
lives.'
Big, brown-bearded James did as he was told. And Lotty was made to
promise that if ever she came to Shepherd's Bush she would pay Capstan
Cottage a visit, where she, this burly skipper's wife, lived when her husband
was far at sea.
But as she made this promise, little did Lotty think how soon she would
meet this kindly woman once again.
. . . . . . .
They say it is an ill wind that blows nobody good. The storm that caused
the sad and awful wreck of the good bark Cumberland had been by no
means an ill-wind to the showman Biffins Lee. No one knew better than he
did how to take advantage of a windfall. Immediately after the disaster his
people had all to work extra time. No extra pay—that was quite another
matter. But the Queerest Show had to be got into full swing, for hundreds of
people from all directions flocked towards it, not only to see the scene of the
wreck and to pick up souvenirs thereof on the beach, but to see Lotty the
heroine, or to listen to the story of her daring and courage from the lips of
Biffins Lee; for the girl herself was far too modest and shy to say one word
concerning it. In fact, she thought very little about the matter, and could not
have been made to believe that she had given evidence of any extra courage
in doing what she had done. She knew from the first that in her Jenny Wren
there would be no great difficulty in taking out a few rockets to the stranded
ship, and she did so. Of course, it had been a little awkward getting
alongside, and all that; but then—well, she was successful, and that perhaps
as much by chance, she thought, as her own good management.
She shrank instinctively, therefore, from being made a hero of, and
lionised; and when she saw newspapers effulgent with flowery language
descriptive of her deed of daring, and calling her the second Grace Darling,
and all that, she could have torn the pages out and burned them. Indeed, that
is precisely what she would have done, only the papers belonged to the show
and not to her.
But Biffins Lee had extracts made from these articles, printed in big,
attractive capitals, and posted up near to the show, and great posters here and
there at roadsides, with big black hands and pointing fingers on them:
THIS WAY TO THE QUEEREST SHOW ON EARTH.
————
GRACE DARLING,
WHO SAVED THE CREW OF THE 'CUMBERLAND.'
EVERY VARIETY OF AMUSEMENT,

SERIOUS AND COMICAL.
BEARS, APES, LIVING SKELETON, AND

THE DREADED DOOROOCOOLIE.
ONLY LIVING MERMAN IN THE WORLD.
Well, Lotty could not help this; but when told that she must appear on the
stage in front of a specially prepared scenic screen—rocks, sea, and wrecked
ship, with the little Jenny Wren bounding over the waves, and she in it; and
that, moreover, she must dress as 'Grace Darling Redivivus' and describe her
adventure, then for the first time in her life she became a beautiful but tearful
little rebel.
'Father, father,' she cried, 'I cannot, will not do this!'
'Will not, eh? Well,' was the reply, 'and when I tell you that you must and
shall, what is your answer to be?'
'That I sha'n't.'
The scene that followed is one which no author would care to dwell upon.
But, losing all control over himself, the fat and burly showman advanced
with his fist clenched and eyes aflame, and no doubt might have done poor
innocent Lotty serious injury. But just at that moment came a fresh actress
on the scene, in the person of Crona herself. Dressed as usual in the clean
starched mob-cap or mutch, her tartan plaid and garments fluttering around
her in the breeze blowing off the sea, a long staff in her hand, and Joe the
raven on her shoulder, she appeared as suddenly as if she had sprung from
the earth, and interposed her presence 'twixt the showman and child.
'Back!' she almost screamed, 'back, Biffins Lee. Lay but a finger on that
child, and there is an end to your show and to your career as well.'
'How dare you, woman! Who are you that interferes with the legal right
of a parent to reprimand a disobedient child?'
'Who am I, Biffins Lee?' she repeated. Then she made one step towards
him and hissed something in his ear.
'Father, father,' she cried, 'I cannot, will not do this.'
LASS. Page 224.
Lee started as if shot, and stood there before the witch, pale, perspiring,
and trembling.
But Crona only laughed.
'Come with your fairy godmother, darling.' She placed an arm around
Lotty, and together they left the stage.
CHAPTER XXII.
THE AMBITIONS OF CHOPS JUNIOR.
'TO—run—away—Miss
say—run—away?'
Lotty?' Chops was gasping. 'Miss Lotty, did ye
'Oh yes, Chops, I fear I had to say—run away. But you won't tell
anybody ever, will you?'
'Never, never, never,' said Chops with curious solemnity.
'Because, you know, Chops, I've always told you everything, and I dare
not go away without telling you this. Mary is good, and so is Skeleton; but
they would try to argue me out of going, and if he knew he would kill me I
think.'
'Miss Lotty, who is 'e?'
'Mr Biffins Lee,' said Lotty quietly, sadly, with her eyes turned towards
the stars, but seeing them not.
'Yer father, Miss Lotty?'
'The man who says he is my father,' she said slowly and deliberately.
Then, 'Oh Chops, there is a mystery, a strange, strange mystery that I must
not even tell you yet. But Crona knows it all.'
'Mustn't—tell—me yet? Ye said "yet," didn't ye, Miss Lotty?'
'I said "yet."'
'But then some day I'll know, won't I? So I'll live in 'opes o' that some
day. An' some day,' this queer boy went on, 'some day summat helse be
agoin' to 'appen. Ye know the song, Miss Lotty, "'E never told 'is love"?
Well, that's me. An' I'm goin' to be honest an' straight with ye as ever was.
An' till the day w'en by savin' an' savin' I makes a bit o' money, an' is old
enough to lead ye to the halter, I'm goin' to be a helder brother to ye. So 'elp
me, Billy-o.'
Who Billy-o was it would be difficult to say; but Chops's adjuration
certainly sounded a strong one. But evidently the lad liked Lotty very much,
and it would be wrong to laugh at love even in the crude.
Chops was silent for a time.
Wallace was lying down perilously close to the edge of the cliff—so
close, indeed, that Lotty feared to call him lest he should miss his foothold
and tumble over to destruction. But it was only the dog's way, and he was
perfectly safe.
But Lotty's newly constituted elder brother broke the silence at last.
'Miss Lotty,' he said, 'Chops, yer friend, will neither give sleep to 'is
heyes nor slumber to 'is heyelids until 'e 'as prayed hover an' thought hall
about the scheme for runnin' away. If so be,' he added, 'that it seems best for
ye, Chops will tell ye to-morrer mornin' has ever was. No more at present
from yours truly till death do us part—Chops junior.'
Wallace had drawn away from the cliff, much to Lotty's relief, and come
forward as if to listen to the conversation.
'Oh, Chops, by the way,' said the girl, 'you said Chops junior. I've been
often going to ask you had you ever, ever a father, Chops?'
'Wot then, Miss Lotty? Think I 'ad two mothers hinstead? Or that baby
Chops floated ashore on a 'urdle? My father, Miss Lotty, is a jobbin'
gardener, as does hodd jobs in 'Ighgate 'Eath, as good a man, t'old pa'son
says, has hever drew the breath o' life on a Monday mornin'.'
'And your mother, Chops?'
'Does laundryin'. 'As a sweet little cottage—three rooms an' a hattic, a
porch afore the door, nice little garden, an' a nice wash'ouse behind. Oh! my
people be swells in a way, Miss Lotty.'
'Any brothers or sisters, Chops?'
'Just one bit o' a sister o' ten, Miss Lotty, an' she be a girl like. There is
no boys to be brothers. As sharp's they makes 'em Mariar is. W'en not at
school she runs herrands, goes for poor father's 'arf pint o' cold fourpenny,
an' takes the washin' 'ome o' Saturday nights to the haristocracy.'
'Chops, why didn't you ever tell me all this before?'
‘’Cause, Miss Lotty, t' old pa'son says to me, "Chops," 'e says, "it's allers
manners to wait till you're haxed."'
'Highgate Heath, Chops—is that a nice part?'
'W'ich it's puffikly lovely, an' wot they calls a charmin' locality, an'
freekwented mostly by the hupper-ten.'
After a pause, Lotty put her hand on her companion's shoulder.
'Chops!'
'That's me, Miss Lotty.'
'Did your mother ever take a lodger?'
'No, little sister—but—but'——
Then at that moment Chops began to dance and to caper so wildly there
in the starlight that Wallace was forced to back astern to bark at him.
'I 'ave it, Miss Lotty. Lor' love ye, I've got it!' he cried. 'Wonder it didn't
strike me at onct.' Then he lowered his voice to almost a whisper. 'If so be
as ye does run away, Lotty, an' does get as far as Lunnon town, my dear, it's
straight to 'Ighgate 'Eath ye goes, to the 'ouse o' my parents, an' lodge there
as 'appy as a May Queen till ye gets summat to do.'
'Oh,' said Lotty, 'this is so very kind of you, Chops. I hope I'll be able to
thank you some day, Chops—some day.'
'W'en I leads ye to the halt'——
Lotty put her little hand on his mouth. 'You shall always be my friend,
won't you?'
'Oh lor', wot a question to hax!'
'But if I go to London—if I get to London—I'll be sure to call on your
parents at Highgate Heath. Then, Chops'—— she said somewhat anxiously.
'Me again?' said Chops.
'Does my father—does Mr Lee—know your parents, or know where they
live? Did he meet you there first?'
'Miss Lotty, Biffins Lee, the man as was yer father, but ain't now no
more, doesn't know nuffin', an' I wouldn't go for to trust 'im not the
valuedom of a tin whistle. Biffins Lee picked me up at a penny gaff, w'ere I
were a hactin' the horphan chee-ild.'
'Well,' said Lotty, thoughtfully but cheerfully, 'I think if I went to your
mother's cottage till I got teaching or something, I would be very happy.'
This somewhat adipose lad had a very good-humoured face and a kindly
eye; but he could have struck no one as being a devotee at the histrionic
shrine. Yet, nevertheless, his one ambition was to become some day a
tragedian, and strut the boards, perhaps even as Othello himself. At this
very moment he must strike an attitude, and with arm uplifted towards the
eastern stars give voice as follows:
'The lot in life o' this poor son o' toil 'as not been a joyful one 'itherto.
Torn from the harms o' 'is weepin' parents at a hearly age, 'e was attached to
the great conglomeration o' Biffins Lee. Though fed like a queen-bee in
order that 'e might take the part of Roly Poly in the Christmas pant., or
'Umpty Dumpty on the Wall, with every bite 'e got to heat 'e received a
buffet, an' on the cold, cold ground on w'ich 'e slept tears used to chase each
other hover 'is face as large as limpet-shells.
'But who would pity the sorrows o' the poor fat boy? No heye was
bedewed with tears to see 'im rolled across the stage as the livin' football,
carried in a net as a string o' honions, trussed an' carried in a tray with
carrots an' turmots on a baker's 'ead to represent "ye first-prize Christmas
goose." An' no one wept w'en 'e was placed on the table to be carved by
Prince de Gourmand—no, not w'en 'e swore 'e was old an' tough, an' then
threw 'im at the 'ead o' the pantaloon. I've been a roast suckin'-pig with
brown gravy, I've been a turkey with sassingers, I've been a pigeon-pie with
my 'ead an' my toes a-stickin' through the crust, an' I've figured as cold side-
dish at the board o' the King o' the Cannibul-high-lows. An' against these
indignities my proud soul 'as burned within me, an' I kept silence honly
because somefin allers told me my day would one day come. An' I see it
now a-comin'. I see a glorious vista openin' up before my mental heyes o'
triumph hafter triumph as Hengland's greatest tragedian.
'An', Miss Lotty,' he said, coming down to the terrestrial, 'if ye goes to
Lunnon, who knows but what Chops junior will follow ye, an' in the
fullness o' time strut the stage as a star o' the fust magnitude?'
'Don't you think, Chops, it is time we went to camp to dinner now?'
Next day—because it was after the 'fast,' and everybody in the parish
had been to church—was a great one in the camp of the Queerest Show on
Earth. Bruin danced his very drollest, the Skeleton performed the most
wonderful tricks. The dreaded dooroocoolie, it was said, had got loose;
there was the most fearsome roaring, and the struggle in surrounding it once
more with its clanking chains was more dreadful than the battle of St
George and the ten-clawed Dragon. But the performances of Lotty Lee were
encored till the poor child was well-nigh tired to death. Then the whole
strength of the band discoursed the sweetest selection while the limelights
were turned on the merman's tank.
The astonished rustics gaped with dread and with curiosity when Biffins
Lee, stepping forward, announced to the ladies and gentlemen that to-night
they would see something that they would not only marvel at, but remember
as long as their toes held together on the face of the earth. The merman, it
was said, expected his daughter early next week, the real live and beautiful
mermaiden who had been seen afloat in Partan Bay; and so her father to-
night had determined to have a great spring cleaning of his cave. The public
were requested to inspect for themselves; and, sure enough, there was the
merman in all his hideous ugliness asleep in the corner of the tank. Then
Biffins waved the audience back, and almost immediately afterwards they
would have gladly gone still farther back if they had had a chance, for the
awful old merman appeared suddenly on the surface of the tank, and began
to eat pieces of fried fish that were handed to him.
'He used to eat this raw,' the showman explained, 'but he is far more
civilised now.'
The trumpets brayed, the drums beat louder, the crimson light played
across the tank, and with an eldritch shriek the merman disappeared. But
presently, to the surprise of everybody, up to the surface of the water
bobbed a cane-bottomed chair.
Now, there is not by any means a confusing amount of romance about a
cane-bottomed chair. No one would go so far as to say that. And this chair
was a very ordinary one, the seat of it even calling out aloud for repair.
Nevertheless, it had been sent up by the merman in a business-like way, and
the people with one accord lifted up their voices and cheered that old
bedroom chair; and when a small deal table came to the surface next,
followed by a somewhat dilapidated washstand, they cheered all the more,
for the British public is certainly a strange animal; but when a three-legged
stool next appeared the British public mingled laughter with its cheers.
Meanwhile busy hands of supernumeraries seized article after article as it
came to the top of the tank, and commenced scrubbing it with hot water and
soap.
'Ladies and gentlemen,' said Biffins, with his most artistic bow, 'I feel
constrained to apologise to you for the somewhat meagre, not to say
shabby, appearance of the furniture sent up to undergo spring cleaning. I
must admit that it is scarcely the sort of articles one would naturally expect
in a merman's cave. We might have looked forward to a better display than
this: furniture inlaid with gold and precious stones—the onyx, the jasper,
the opal, and coral white and crimson—carpets of green sea-moss, and
curtains of the velvety sea-weeds, with candelabra that would have dazzled
the eyes, and articles of bigotry and virtue. But our merman came here in a
hurry, and left his caves and his marble halls all behind him. I have not
myself been down to the bottom of this mighty tank, and have not,
therefore, had a peep inside the merman's cave; but, judging from these
specimens, I should fancy the poor merman has had to "furnish throughout
on the hire system."'
At this moment the merman himself came to the surface, puffing and
blowing as if choking with dust. He was wearing an old flannel shirt with
the sleeves rolled up, and had a dust-pan in one hand and sweeping-brush in
the other, while round his head was pinned a dirty old rag of a towel. He
signed to Biffins Lee for a drink, and a tumbler of some brown liquor like
rum was handed to him. This he tossed off, threw the glass at Biffins's head,
and dived below to work again.
By-and-by he came up once more, and, receiving article after article,
critically examined it for specks of dirt, then dived with it and appeared
again to grasp another, and so on until all the furniture was down below.
After this the creature came to the surface, tore off the old woollen garment
and the disreputable-looking towel, rolled them together, and threw them
disdainfully at the showman. Then it yawned in a tired way, stretched itself
as if much fatigued, and dived below.
'Now,' said Biffins Lee, 'the great spring cleaning is all over, and next
week I hope to have the extreme felicity of introducing to your notice the
merman's only daughter, the beautiful mermaid herself.'
But Biffins Lee was too confident. He was at the height of his glory at
present with his Queerest Show on Earth, and he little knew what was in
store for him.
CHAPTER XXIII.
'WELL, CHOPS, TO RUN AWAY.'
F RANK ANTONY BLAKE had gone! He had been summoned away

suddenly—it is ever thus that sorrows come—to visit the sickbed of his
mother. And this was the first dark cloud that had arisen on the horizon
of Lotty's young life. It was sweet for her to know next evening by telegram
that her hero's dear mother was better, and that a letter would follow. But—
ah!—Antony had gone, and somehow everything was so changed now all
about and around her.
It was springtime. Spring, indeed, was in its first fresh glory. The sea she
loved, the sea out yonder stretching away and away to the illimitable north,
may have lost none of its beauty—the blue of its waves when the sun had
climbed the mountains, its opal and silver-gray on that cloud-streaked noon,
its emerald streaks where sky's blue mixed with the yellow of half-hidden
sandbanks, the pearl where the billows broke lazily over the brown-black of
weed-capped rocks, its ineffable glory of sunset or moonlight clear
gleaming. No, the sea must be the same, and yet it awakened less response,
less sympathy, in the heart of the little gipsy lass.
Towards the forest, where she went wandering away alone with Wallace,
because she wanted to think, things seemed strangely altered somehow. Was
the moss that carpeted the beech-woods less soft and bright, or the bark of
the birch-trees less snowy? Were the clouds of needled foliage on the
brown-stemmed pines more black and solemn, and had the tasselled larches
with buds of crimson lost already their spring-green tints? And where was
the glory of the golden furze? Where the music of the rose-linnet? Ah!
surely the fluting melody of the blackbird and the wild, ringing song of
mavis, ay, and the bold lilt of the chaffinch, were less loud and stirring. Yes,
and the cur-r-r and croodle of wood-pigeon in the planting's green shade,
that used to thrill the heart, sounded farther away now and grown more sad
and mournful.
The hero was gone. And, girl-like—well, childlike then—this wee gipsy
maiden sat down upon a stone and burst into tears, much to the concern of
ever-faithful Wallace, who did his best to kiss those tears away. But sorrow

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Advances in Clinical Chemistry 64 1st

Edition Gregory S. Makowski (Eds.)

Advances in Clinical Chemistry, Volume 73 Gregory S.

Advances in Clinical Chemistry 1st Edition Gregory

Advances in Clinical Chemistry 62 1st Edition Gregory

Advances in Clinical Chemistry 63 1st Edition Gregory

Advances in Clinical Chemistry 74 1st Edition Gregory

Advances in Clinical Chemistry 75 1st Edition Gregory

Advances in Clinical Chemistry 76 1st Edition Gregory

Advances in Clinical Chemistry 77 1st Edition Gregory

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First edition 2014

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Advances in Clinical Chemistry, Volume 64 # 2014 Elsevier Inc. 1

2. CLASSIFICATION, STRUCTURE, AND FUNCTION

Table 1.1 Classification of matrix metalloproteinases (MMPs)

Table 1.1 Classification of matrix metalloproteinases (MMPs)—cont'd

Table 1.1 Classification of matrix metalloproteinases (MMPs)—cont'd

MMP-27 was first found in chicken embryo fibroblasts [30]. MMP-27

3.1. Inactive zymogens

detergents in vitro. Autocatalytic MMP activation has also been reported,

3.2. Transcriptional regulation

3.3. Translational regulation

3.4. Tissue inhibitors of metalloproteinases

oversimplified. In early biochemistry studies in which the relative affinity for

4. MMP IN CARDIOVASCULAR SYSTEM DEVELOPMENT

4.1. MMP in vasculogenesis and angiogenesis

[97–99]. MMP-9-deficient mice are fertile but exhibit abnormal or delayed

4.2. MMP in cardiac tube formation and looping

increasingly restricted to the splanchnic mesoderm adjacent either side of

4.3. MMP in heart septation

4.4. MMP in embryonic and fetal cardiac remodeling

myocardium from the ventricular myocardium and in realigning the atrio-

5. MMP IN CORONARY ARTERY DISEASE

Aberrant MMP catalytic activity has been linked to atherosclerotic

5.1. MMP in atherosclerosis

bleeding. Further proteolysis may contribute to neovascularization and rup-

fibroblasts. The migration and proliferation of these cell types is important in

environment [168,169]. In late disease, MMP-9 [170] secreted by macro-

5.1.1 MMP and atherosclerotic risk factors

tobacco, alcohol, and inflammation. They can influence the production or

5.2. MMP in plaque instability

Table 1.2 Association of MMP with atherosclerotic risk factors

Table 1.2 Association of MMP with atherosclerotic risk factors—cont'd

-8, -9 can also be upregulated in stimulation of tumor necrosis factor alpha

5.3. MMP in acute coronary syndrome

THE WRECK OF THE 'CUMBERLAND.'

EVERY VARIETY OF AMUSEMENT,

BEARS, APES, LIVING SKELETON, AND

THE AMBITIONS OF CHOPS JUNIOR.

'WELL, CHOPS, TO RUN AWAY.'

F RANK ANTONY BLAKE had gone! He had been summoned away

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