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This is to certify that the

thesis entitled

INFLUENCE OF GLUCOSAMINE ON MATRIX
METALLOPROTEINASE EXPRESSION AND ACTIVITY
IN LPS-STIMULATED EQUINE CHONDROCYTES

presented by

Christopher R. Byron

has been accepted towards fulfillment
of the requirements for

MS degree in Wﬁences

 

 

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6/01 c:/CIRC/DateDue.p65-p, 15

INFLUENCE OF GLUCOSAMINE ON MATRIX METALLOPROTEINASE
EXPRESSION AND ACTIVITY IN LPS-STIMULATED EQUINE
CHONDROCYTES.

By

Christopher Robert Byron

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Large Animal Clinical Sciences

2002

ABSTRACT

INFLUENCE OF GLUCOSAMINE ON MATRIX METALLOPROTEINASE
EXPRESSION AND ACTIVITY IN LPS-STIMULATED EQUINE CHONDROCYTES

By

Christopher Robert Byron

The objective of this project was to characterize potential mechanisms of action of
glucosamine inhibition of MMP expression and activity in lipopolysaccharide (LPS)-
stimulated equine chondrocytes. The inﬂuence of glucosamine (50 mM, 25 mM, 6.25
mM, 3 mM, and 1.5 mM) on MMP activity in conditioned medium from LPS-stimulated
cartilage explants was determined by a colorimetric assay using azocoll as a substrate.
The inﬂuence of glucosamine on MMP synthesis in equine chondrocytes in pellet
cultures was determined using a commercial MMP-13 ELISA and azocoll digestion in
organomercurial-activated medium. The effects of glucosamine on MMP mRNA levels
in similarly treated equine chondrocytes was determined by Northern blotting using
MMP-1, -3, and -13 probes. Statistical analysis was carried out with two-way ANOVA,
and P was set at 0.05. Glucosamine had no effect on activated MMP activity.
Glucosamine inhibited MMP protein expression, as determined by azocoll digestion at 3
to 50 mM, and as determined by MMP-13 ELISA at 1.5 to 50 mM. Resting mRNA levels
for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed glucosamine
at concentrations of 50 mM and 25 mM. These results indicate that glucosamine is
capable of pre-translational, and possibly also translational, regulation of MMP
expression and provide a potential mechanism of action for previously reported

chondroprotective effects of this aminomonosaccharide.

ACKNOWLEDGMENTS

I would like to thank Tonia Peters for her indispensible technical help. I would
also like to thank Dr. Venta for his insight and suggestions. Finally, many thanks go to
the members of my committee, Dr. Orth, Dr. Lloyd, and Dr. Caron for their intellectual
input and service on the committee.

Special thanks go to my wife, Rachael, who stood with me through the rough

times and helped make the journey worthwhile.

iii

TABLE OF CONTENTS

LIST OF TABLES ........................................................................... vi
LIST OF FIGURES ......................................................................... vii
LIST OF ABBREVIATIONS .............................................................. ix
INTRODUCTION ............................................................................ 1
CHAPTER 1
LITERATURE REVIEW ..................................................................... 4
Synovial Joint Structure and Function ............................................. 4
Articular Cartilage .................................................................... 4
Extracellular Matrix of Cartilage ................................................... 5
Chondrocytes .......................................................................... 6
Synovium and Synovial Fluid ....................................................... 7
Joint Biomechanics ................................................................... 8
Matrix Metalloproteinases and Their Inhibitors ................................. 10
In Vitro Assessment of MMPs ..................................................... 15
Osteoarthritis ......................................................................... 17
Drugs in OA Treatment ............................................................. l9
Symptomatic Drugs .................................................................. 19
Chondroprotective Drugs ............................................................ 21
Glucosamine ........................................................................... 22
Glucosamine Clinical Trials ......................................................... 24
Glucosamine Side Effects ........................................................... 26
Glucosamine Mechanism of Action: Research To Date ......................... 27
In Vitro Chondrocyte Culture Systems ............................................ 29
Lipopolysaccharide in In Vitro Osteoarthritis Models ........................... 31
References ............................................................................. 32
CHAPTER 2

INFLUENCE OF GLUCOSAMINE ON MATRIX METALLOPROTEINASE
EXPRESSION AND ACTIVITY IN LPS-STIMULATED EQUINE

CHONDROCYTES ............................................................................ 43
Summary ................................................................................ 43
Introduction ............................................................................. 44
Materials and Methods ................................................................ 46
Results ................................................................................... 54
Discussion .............................................................................. 54
References .............................................................................. 58

iv

CHAPTER 3
CONCLUSION ................................................................................. 71

LIST OF TABLES

TABLE 1. Matrix metalloproteinases and their preferred substrates ................... 13

vi

LIST OF FIGURES

Figure 1. Mean (iSEM) MMP activity as determined by azodye release into the
supernatant at pH 7.4. Neg = o-phenanthroline-treated; Pos = APMA-treated; G50 =
glucose 50 mM; GLCN = glucosamine at the indicated mM concentrations. * Indicates
statistical signiﬁcance at p < 0.05 compared to positive control ......................... 62

Figure 2. Mean (iSEM) MMP activity as determined by azodye release into the
supernatant at pH 5.0. Neg = o-phenanthroline—treated; Pos = APMA-treated; G50 =
glucose 50 mM; GLCN = glucosamine at the indicated mM concentrations. * Indicates
statistical signiﬁcance at p < 0.05 compared to positive control ......................... 63

Figure 3. Mean (iSEM) MMP production of equine chondrocyte pellet cultures
stimulated with LPS (20 ug/ml). MMP production was determined by azodye release
using azocoll as a digestion substrate at pH 7.4. Neg = unstimulated; Pos = LPS-
stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. "' Indicates statistical signiﬁcance at p < 0.05 compared to positive
control ............................................................................................ 64

Figure 4. Mean (iSEM) MMP-13 concentration in media supernatant of equine
chondrocyte pellet cultures stimulated with LPS (20 ug/ml). MMP-13 concentration was
determined using a commercially available ELISA. Neg = unstimulated; Pos = LPS-
stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive
control ............................................................................................ 65

Figure 5. Mean (iSEM) relative MMP-1 mRN A expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 ug/ml). MMP-1 mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 288 rRNA bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive
control ............................................................................................. 66

Figure 6. Mean (iSEM) relative MMP-3 mRNA expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 ug/ml). MMP-3 mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 288 rRNA bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; 050 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive
control ............................................................................................. 67

vii

Figure 7. Mean (irSEM) relative MMP-13 mRNA expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 ug/ml). MMP-l3 mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 28S rRNA bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; GSO = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive
control ........................................................................................... 68

Figure 8. Representative example of a methylene-blue stained nylon transfer membrane.
The top and bottom bands represent 28s (approximately 4.7 kb) and 18 s rRNA
(approximately 1.9 kb), respectively. Nylon membranes were stained in methylene blue
(0.03% w/v methylene blue in 0.3 M sodium acetate, pH 5.2) for 45 seconds, destained in
water for 2 minutes, and dried between sheets of ﬁlter paper ............................. 69

Figure 9. Representative autoradiographs of northern blots with probes for MMP-1, -3,
and —13 after detection with the DIG system. Lane 1 = Negative control; 2 = Positive
control; 3 = Glucose 50 mM; 4 = GLCN 50 mM; 5 = GLCN 25 mM; 6 = GLCN 6.25

mM;7=GLCN3mM;8=GLCN1.5mM ................................................. 70

viii

LIST OF ABBREVIATIONS

Extracellular Matrix ............................................................................. ECM
Matrix Metalloproteinase ....................................................................... MMP
Non-Steroidal Antiinﬂammatory Drug ..................................................... NSAID
Lipopolysaccharide ................................................................................ LPS
Glucosamine .................................................................................... GLCN
Tissue Inhibitor of Metalloproteinase ........................................................ TIMP
Enzyme-Linked Immunosorbent Assay .................................................... ELISA
Aminophenylmercuric Acetate ............................................................... APMA
Phospholipase .................................................................................... PLA
Nitric Oxide ........................................................................................ NO
Polysulfated Glycosaminoglycan .......................................................... PSGAG
Glycosaminoglycan .............................................................................. GAG
Randomized Controlled Trial .................................................................. RCT
Tumor Necrosis Factor-or .................................................................. TNF-a
Interleukin .......................................................................................... IL
Toll-Like Receptor .............................................................................. TLR

ix

INTRODUCTION

Osteoarthritis (OA) causes substantial morbidity in the form of lameness and may
seriously curtail quality of life in addition to athletic serviceability of affected horses
(Rossdale et al. 1985, Todhunter and Lust 1990). It can be initiated by a number of
causes and ultimately affects all articular tissues. The halhnark of the disease is the
degeneration of articular cartilage, a highly specialized tissue consisting of relatively few
cells but having a rich extracellular matrix. Although there are a number of possible
causes of matrix degradation, experimental evidence suggests that proteolytic degradation
of cartilage by matrix metalloproteinases (MMPs) is a characteristic feature of OA
(McIlwraith 1996, Goldring 2000).

Matrix metalloproteinases (MMPs, matrixins) are a family of enzymes that
degrade the extracellular matrix of tissues. Several subgroups of MMPs based on
substrate preference and structural domains including collagenases, gelatinases, and
stromelysins are active in OA (Vu and Werb 2000, Nagase and Woessner 1999, Pozzi et
al. 2000, Rabbani et al. 2000). Since collagen damage is an important initial event in OA
(Roberts et al.1986, McIlwraith 1996, Goldring 2000), MMPs, particularly collagenases,
may serve as a pivotal component of OA initiation and progression. Stromelysin (MMP-
3) is also important in that it not only degrades non-collagenous ECM components,
including aggrecan, but also serves to activate latent collagenases (Goldring 2000).

Glucosamine therapy for the treatment of OA has been the subject of several
clinical studies in human patients. One recent review of 16 randomized control trials

utilizing glucosamine for the treatment of human OA reported that glucosamine is both

effective and safe for its treatment of OA (Towheed et a1. 2001). When glucosamine was
compared to an NSAID for treatment of OA, its effectiveness was found to be equivalent
in two trials and superior in two. While ibuprofen effects a more rapid decrease in patient
pain scores, oral glucosamine therapy yields an ultimately lower pain score after eight
weeks of treatment (Barclay et al. 1998). Glucosamine has also been shown to prevent
knee joint space loss in OA patients during the course of a three-year study (Reginster et
al. 2001).

The effects of glucosamine on in vitro cellular metabolism have received recent
attention. Proteoglycan production is stimulated by exogenous glucosamine in human
chondrocyte cultures (Bassleer et a1. 1998) and glucosamine has been shown to prevent
the repression of beta-1,3-g1ucuronosyltransferase I, a key biosynthetic enzyme in
glycosaminoglycan synthesis. Importantly, glucosamine prevents the up-regulation of
MMP-3 mRNA expression by interleukin-1 beta stimulated rat chondrocytes (Gouze et
al. 2001). While glucosamine has been shown to inhibit proteoglycan loss, inducible
nitric oxide synthase, and MMP activity in equine cartilage explants stimulated with LPS
or recombinant interleukin-1 (Fenton et al. 2000 a,b), the speciﬁc level at which these
effects occur remain unclear.

The purpose of the study presented here was to determine the nature of the
inhibition of MMP activity attributed to glucosamine HCl in an in vitro model of OA.
The speciﬁc aims were to determine if glucosamine HCl inﬂuences 1) the catalytic
activity of chondrocyte MMPs, 2) synthesis of MMPs at the protein level, and 3) resting

expression of MMP mRNA levels in LPS-stimulated equine chondrocytes.

The following chapters describe the in vitro research and results that address these
speciﬁc aims. Chapter 1 is a review of the current literature relating to the project which
provides the necessary background information. Chapter 2 contains a description of the
methods, the results, and a discussion of the project. Chapter 3 contains concluding
comments regarding the project, the results, and questions that may be addressed through

future research.

Chapter 1

LITERATURE REVIEW

Synovial Joint Structure and Function

The two major functions of synovial joints are the facilitation of joint movement
and the transfer of weight-bearing loads. Proximal limb joints which have freedom to
move in several planes and have the capability for rotational movement largely derive
their stability from large muscle masses and tendons surrounding them. Distal limb joints
rely more on ligamentous support, joint capsule integrity, and joint contour for stability
(Todhunter 1996). Certain joints such as the femorotibial and temporomandibular joints

incorporate menisci that lend stability (Mankin and Radin 1997).

Articular Cartilage

The subchondral bone plate beneath articular cartilage is composed of dense
cortical bone with haversian system orientation parallel to the joint surface. The hyaline
cartilage overlies this subchondral bone plate and is bound to the bone by collagen ﬁbers
which originate at right angles to the haversian system (Mankin and Radin 1997). These
collagen ﬁbrils impart tensile strength and provide a scaffold within which chondrocytes
and proteoglycans are embedded (Walsh et al. 1997).

Articular cartilage is organized into four distinct histological zones (Mankin and
Radin 1997). The surface tangential zone exhibits collagen ﬁbers organized parallel to
the articular cartilage surface and elongated chondrocytes whose long axis is also oriented

parallel to the articular surface. The deeper transitional zone exhibits rounder

chondrocytes with the appearance of random distribution. Radial zone chondrocytes are
arranged in short, irregular columns. Chondrocytes in the deepest calciﬁed zone are

surrounded by hydroxyapatite.

Extracellular Matrix of Cartilage

Articular cartilage is composed of 70% water and 30% matrix. The extracellular
matrix (ECM) is composed of 50% collagen, 35% proteoglycan, 10% glycoproteins, 3%
mineral, and 1% lipid. Chondrocytes account for l to 12% of the articular cartilage by
volume.

The major contributor to cartilage tensile strength is collagen type H which is
highly cross linked in mature hyaline cartilage. Type H collagen has a greater percentage
of lysine residue hydroxylation and greater glycosylation than type I collagen in equids
(Todhunter et al. 1994). Other minor collagen components include collagens type VI, IX,
X (physeal and hypertrophic cartilage), XI, XII, and XIV. These minor collagens have
important structural and functional properties as they serve to further organize the type H
collagen ﬁbrils and mediate ECM-ECM interactions as well as ECM-cell interactions.
Type VI collagen is a dimer that forms microﬁbrils which may link larger collagen ﬁbrils
and chondrocyte cell surface receptors. Type IX collagen is a triple helix that has a
chondroitin sulfate side chain and mediates interaction between major collagen ﬁbrils and
other ECM molecules. Type X collagen is an accessory collagen that is present
exclusively within the hypertrophic calciﬁed zone of articular cartilage. Type XI collagen
is interspersed between type H ﬁbrils. Type XH collagen is present in small amounts in

articular cartilage and other connective tissues. Type XIV collagen is structurally related

to type D( (Todhunter 1996, Mayne and Brewton 1993). Collagen types H, D(, X and XI
are found exclusively in cartilage (Walsh et al. 1997).

Proteoglycans are largely responsible for the elasticity of hyaline cartilage (Walsh
et al. 1997). They are composed of a linear protein core to which are attached the
glycosaminoglycans chondroitin 6—sulfate, chondroitin-4-sulfate, and keratan sulfate.
Proteoglycans include aggrecan, biglycan, decorin, ﬁbromodulin, and lumican. Aggrecan
is the major proteoglycan in articular cartilage and contributes the greater part of cartilage
resistance to compressive stresses through interaction of highly negatively charged
glycosaminoglycan side chains (Goldring 2000). As many as 50 GAG chains
(chondroitin 6-sulfate, chondroitin-4-sulfate, and keratan sulfate) may be attached to a
single core protein. The electronegativity of these side chains allows the proteoglycan
unit to maintain a rigid structure interspersed with water molecules, lending resiliency to
the articular cartilage (Mankin and Radin 1997).

Collagen turnover rates have been estimated at 120 years for canine cartilage and
350 years for human cartilage (Akizuki et al. 1987). Overall proteoglycan turnover,
however, is approximately 300 days in rabbit and canine cartilage and approximately
1800 days in human cartilage (Maroudas 1980). These data suggest that matrix tumover
is generally slow, and collagen turnover in particular is easily slower than the life

expectancy of the animal or person.

Chondrocytes
Chondrocytes are interspersed throughout articular cartilage in small groups or as

single cells. They are responsible for the synthesis and breakdown of articular cartilage

ECM components. They respond to mechanical and chemical stimuli in the articular
cartilage to regulate these diverse activities.

In osteoarthritis the metabolic responses of the chondrocyte to its environment are
altered by disruption of the chondrocyte-ECM interaction (Radin et al. 1991, Pool 1997).
Disruption of matrix interactions leads to early clonal growth, increased production of
cartilage matrix components, and increased production and release of catabolic cytokines
and matrix-degrading enzymes (Goldring 2000). Chondrocyte proliferation leads to local
chondron formation. Type H collagen and aggrecan production are increased in early CA
but decrease in late-stage OA. Interleukin-1 and IL-1 receptor type 1 are increased
serving as autocrine and paracrine catabolic signals. Important proteinases in OA
cartilage degradation produced by chondrocytes include matrix metalloproteinases,

aggrecanase, plasminogen activator, and cathepsins.

Synovium and Synovial Fluid

Lining the joint is a modiﬁed mesenchyme called synovium. The intima is
between one and four synoviocytes thick with no basement membrane and overlies a
layer of connective tissue. Synoviocytes have both secretory and phagocytic functions,
and have been shown to synthesize MMPs and hyaluronan, a major component of
synovial ﬂuid.

Synovial ﬂuid is an ultraﬁltrate of plasma containing only small proteins. The
ﬂuid contains fewer than 500 nucleated cells/pl, composed of 90% mononuclear cells
with 10% neutrophils. Hyaluronan is present in normal equine synovial ﬂuid at a

concentration of 0.5 mg/ml. Normal human synovial ﬂuid contains hyaluronan at a

concentration of 2 to 3 mg/ml. Fluid exchange between plasma and synovium is
governed by Starling forces and driven by motion-assisted lymphatic ﬂow and

intrasynovial ﬂuid pressures (Todhunter 1996).

Joint Biomechanics

Diarthrodial joints function with an extremely low coefﬁcient of ﬁiction (Palmer
and Bertone 1996). Whereas the coefﬁcient of friction of good artiﬁcial joints is between
0.1 and 0.5, healthy biologic diarthrodial joints have a much lower 0.002 coefﬁcient of
friction (Simon and Radin 1997). Contributing to the low coefﬁcient of ﬁiction is the
parallel orientation of collagen ﬁbers to the joint surface, the porosity of cartilage, the
presence of hyaluronan, and the presence of lubricating glycoproteins (lubricin) within
the synovial ﬂuid.

Hyaluronan lends thixotropic ﬂow characteristics to synovial ﬂuid (Mankin and
Radin 1997). This means that the more slowly synovial ﬂuid ﬂows the more viscous it
becomes. Due to the extremely low ﬁiction of normal cartilage-on-cartilage contact, the
periarticular soft tissues provide the majority of the frictional resistance to joint
movement. It is this peripheral friction which hyaluronan mitigates, serving as a
boundary lubricant rather than as a lubricant of the cartilage surface itself (Mankin and
Radin 1997).

The glycoprotein lubricin serves as the lubricant of cartilage surfaces against each
other during high load, low speed joint movement (Palmer and Bertone 1996). Lubricin
decreases the surface tension of joint surfaces, adhering to the cartilage and the synovial

surface, to effectively decrease shear stresses. Lubricin prevents the fusion or binding of

surfaces and may also play a role in aiding articular tracking during joint movement
(Palmer and Bertone 1996).

Aside from the boundary lubrication described above which functions in low
speed motion, there are two mechanisms currently thought to function in high speed
motion joint lubrication. Squeeze ﬁlm lubrication occurs when ﬂuid is forced out of the
high load areas of the porous cartilage, increasing pressure and assisting in load carrying.
Hydrodynamic lubrication occurs when joint surfaces move tangentially to one another
forming a wedge of ﬂuid ahead of the motion that acts to support and separate the
cartilage. Two relatively new hydrodynamic~based theories have addressed additional
properties of diarthrodial joint biomechanics. Elastohydrodynamic lubrication takes into
account cartilage deformation, which increases surface area of the loaded cartilage
surface, thereby increasing load-bearing capacity. Microelastohydrodynamic lubrication
theory proposes that natural irregularities in the cartilage surface are smoothed out during
weight bearing due to lubrication at the peaks and lack of weight bearing in trough area.
This increase in surface area would serve to increase the weight bearing area of cartilage
(Palmer and Bertone 1996).

Natural lubrication theory considers the biomechanics of the cartilage as well as
the viscoelastic principles listed above. In this theory, cartilage becomes less permeable
as load increases due to decreased space in the ECM. As this occurs, ﬂuid is forced out
of the cartilage ECM into the joint, thereby increasing lift and separation between the
cartilage surfaces (Palmer and Bertone 1996).

Collagen and proteoglycans within the cartilage ECM retain ﬂuid within the

matrix and offer resistance to interstitial ﬂuid ﬂow, which contributes to the cartilages

ability to withstand high compressive loads. Carboxyl and sulfate groups of cartilage
glycosaminoglycans have a ﬁxed negative charge. Water within the ECM neutralizes this
charge. During weight bearing water is forced out of the ECM, thereby increasing the
negative charge density as the GAGs come into closer contact. These electrical properties
contribute to the ability of cartilage to resist compressive loads (Simon and Radin 1997).

Additional support during compressive loading is offered by the subchondral bone
plate and trabecular bone, which expand to support and distribute loads during weight
bearing as well as absorbing concussive forces through micromotion (Simon and Radin
1997)

Alteration of the function of any component of the synovial joint can create
imbalances in the normal biomechanics of movement, resulting in cartilage breakdown.
Osteoarthritic processes can result in cartilage ECM compositional changes which may

alter the biomechanical attributes described above (McIlwraith 1996).

Matrix Metalloproteinases and Their Inhibitors
MMP Family

Matrix metalloproteinases (MMPs, matrixins) are a family of enzymes that
degrade the extracellular matrix of tissues as required during embryonic development,
morphogenesis, reproduction, and tissue remodeling. Most MMPs are synthesized as
proenzyrnes and secreted as inactive pro-MMPs. MMPs are transcriptionally regulated
by growth factors, cytokines, and cellular transformation (N agase and Woessner 1999),
and their proteolytic activities are controlled by activators of pro-MMPs, endogenous

inhibitors, alpha-macroglobulins, and tissue inhibitors of matrix metalloproteinases

10

(TIMPS). These enzymes are dependent on Ca2+ and Zn2+ for activity. Several MMP
subgroups exist that are based on primary substrate preference and structural domains,
including collagenases which are most active against ﬁbrillar collagen, gelatinases which
are most active against denatured collagens, stromelysins that primarily degrade non-
collagen components of the extracellular matrix, and membrane-type MMPs (MT-MMPs)
that are transmembrane enzymes (V 11 and Werb 2000). In addition to primary
preferences, most MMPs have a wide range of available substrates (Table 1). In addition
to ECM turnover, MMPs also serve to modify the biological effects of ECM components
by changes in structure and unmasking of biologically active domains (N agase and
Woessner 1999). MMPs also serve a role in many pathological processes including
arthritis, cancer, cardiovascular disease, corneal ulceration (N agase and Woessner 1999,

Pozzi et al. 2000, Rabbani et al. 2000).

MMPs in OA

Several matrix metalloproteinases are involved in cartilage ECM degradation
including collagenases (MMP-1, -8, -13), stromelysins (MMP-3, -10, -11), gelatinases
(MMP-2, -9), and membrane-type (MMP-14) (Goldring 2000, review). Levels of TIMPS
are also elevated in synovial ﬂuid of arthritis patients however there may be a local
imbalance of MMP to TIIVIP at areas of continued degradation (Dean et al. 1989). Since
collagen damage may be an initial event in osteoarthritis disease progression (Roberts
1986, McIlwraith 1996), MMPs, particularly collagenases, may serve as a focal
component of OA initiation and progression. While proteoglycan degradation is also

observed early in OA progression, collagen damage may carry more serious and

11

permanent consequences due to its slow rate of turnover (Aigner and McKenna 2002).
Stromelysin (MMP-3) is also important in that it not only degrades non-collagenous
cartilage ECM components, including aggrecan, but also serves to activate latent
collagenases (Goldring 2000). Collagenases that degrade type H collagen, the primary
collagenous cartilage component, include MMP—l, -8, and ~13. Evidence of MMP-13
expression in cartilage and its effectiveness at degrading type H collagen suggests a major
role for this enzyme in OA progression (Knauper et al. 1996, Mitchell et al. 1996,
Billinghurst et al. 1997, Cawston et al. 1999, Dahlberg et al. 2000).

Matrix metalloproteinases-l and -13 have similar substrate speciﬁcities, with
MMP-l having a slightly broader range of substrates. MMP-1 and -13 both degrade
collagens type I, H, HI, VH, and X, as well as gelatins and aggrecan. Additionally, MMP-
1 degrades tenascin and link protein (Martel-Pelletier et al. 2001). Although similar
substrate preferences are apparent for these two collagenases, human MMP-13 has been
shown to have a higher catalytic efﬁciency for collagen type H than human MMP-1
(Jeffrey 1998). The roles of MMP-1 and MMP-13 in OA progression also appear to
differ, with MMP-1 associated with superﬁcial cartilage and MMP-13 associated with the
intermediate and deep cartilage zones. This suggests that MMP-1 may be associated with
acute tissue destruction during inﬂammatory phases, whereas MMP-13 may have a role
in remodeling (Martel-Pelletier et al. 2001). Activation of both collagenases occurs

extracellularly.

12

Table 1. Matrix metalloproteinases and their preferred substrates. Adapted from

Martel-Pelletier et al. 2001

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MMP Enzyme Name(s) Preferred Substrates
MMP-1 Collagenase-l, Collagen types I, H, HI, VH, X, gelatins, aggrecan,
interstitial collagenase tenascin, link protein

MMP-2 Gelatinase A, gelatinase Gelatins, collagens I, H, H], IV, V, VH, X, XI,

(MMP-5) 72 kDa type IV collagenase, ﬁbronectin, laminin,
aggrecan, elastin, tenascin, vitronectin

MMP-3 Stromelysin-1 Aggrecan, gelatin, ﬁbronectin, laminin, link

(MMP-4) protein, elastin, collagen types 1, HI, IV, V, VH1,

(MMP-6) D(, X, procollagenase-l (activation), vitronectin,
tenascin, decorin

MMP-7 Matrilysin Aggrecan, ﬁbronectin, vitronectin, tenascin,
laminin, gelatin, collagen type IV, elastin,
procollagenase-l (activation), link protein

MMP-8 Neutrophil collagenase Collagen types I, H, IH, VIH, X, aggrecan, link
protein

MMP-9 Gelatinase B, gelatinase Gelatins, collagen types I, IH, IV, V, VH, X, XI,

92 kDa XIV, aggrecan, elastin, vitronectin

MMP-10 Stromelysin-2 Aggrecan, ﬁbronectin, laminin, collagen types I,
111, VI, V, VIH, IX, gelatin, elastin, laminin

MMP-11 Stromelysin-3 Fibronectin, laminin, collagen type IV, aggrecan,
gelatin, alphaI-antitrypsin, serpin

MMP-12 Metalloelastase Elastin

MMP-13 Collagenase-3 Collagen types I, H, H1, VH, X, aggrecan, gelatins

MMP-14 MTl-MMP Pro-MMP-Z (activation), collagen types I, H, H],
dermatan sulfate, laminin, ﬁbronectin, gelatin,
vitronectin

MMP-15 MT 2-MMP

MMP-16 MT3-MMP Pro-MMP-2 (activation)

MMP-17 MT4-MMP

MMP-18 Collagenase-4

MMP-19 RASI-l

MMP-20 Enamelysin Enamel matrix

MMP-21 X-MMP

MMP-22 C-MMP

MMP-23 CA-MMP, MIFR-l

MMP-24 MTS-MMP

MMP-25 MT6-MMP

MMP-26 Endometase

MMP-27 Epilysin

MMP-28 GeneBank AAG41981.1

 

 

13

 

T IMPs

Tissue inhibitors of matrix metalloproteinases (TIMPS) are a family of proteins
which regulate the activity of MMPs by formation of a 1:1 MMP:TIMP complex.
Mammalian TIMPS are two-domain molecules and belong to one of four subgroups
(TIMP-1 through TIMP-4). The N-terminal domain consists of approximately 125 amino
acids and the C-terminal domain contains approximately 65 amino acids, with each
domain being stabilized by three disulﬁde bonds (Williamson et al. 1990). The N-
terrninal domain is capable of MMP inhibitory activity when separated from the C-
terminal domain (Murphy et al. 1991). TIMP inhibitory activity is mediated through a
conformational change in the MMP. This is brought about by TIMP binding to the MMP
active site, which causes disruption of amino acid residue ionic interactions (Brew et al.
2000, review).

The four known TIMPS are evolutionarily related. They are the products of gene
duplication events (Brew et al. 2000, review). TIMPS bind tightly to most MMPs
(Murphy and Willenbrock 1995), however there appears to be speciﬁcity in their
interactions with MMPs and with other proteins. For example, TIMP-2 plays a critical
role in the activation of proMMP-2 in the presence of MTl-MMP (Strongin et al. 1995),
while TIMP-1 does not have this ability (Brew et al. 2000, review). As another example,
TIMP-3, but not any other TIMP, binds tightly to ECM components (Pavloff et al. 1992,
Yang et al. 1992)

In addition to active MMP inhibition, TIMPS have been shown to aid in pro-MMP

activation (Strongin et al. 1995), matrix binding (Pavloff et al. 1992, Yang et al. 1992),

14

inhibition of angiogenesis (Murphy et al. 1993), induction of apoptosis (Baker et al.

1999), and promote cell growth (Bertaux et al. 1991).

In Vitro Assessment of MMPs

Options for detection of enzymes in a ﬂuid medium include various activity
assays that detect active enzyme indirectly, or Western blotting and enzyme linked
immunosorbent assay (ELISA) which detect proenzymes or active enzymes.

Measurement of MMP activity can be performed using zymography (Lein et al.
1997), utilizing substrate digestion within a gel matrix, or colorimetric substrate
degradation assays which rely on dye liberation upon substrate degradation (Chavira et al.
1984, Vachova and Moracova 1993).

Recently, commercially available ELISA-based detection systems have become
available (Lein et al. 1997) and have been validated for use in detecting and quantifying
MMPs from venous blood of healthy human adults. These systems detect both inactive
and active enzyme directly through antibody-based binding that is coupled to a colored
reporter substrate.

Detection of MMP mRNA may be performed using either Northern blotting or
polymerase chain reaction (PCR) methods. Collagenases, gelatinases, and stromelysins
have been detected using the northern blot and reverse transcriptase-PCR methods
(Maquoi et al. 2002, Han et al. 2002). Reverse transcriptase-PCR has a higher
sensitivity than northern blotting for the detection and quantitation of MMP mRNA (Fehr

et al. 2000).

15

Azocoll-based MMP detection

MMP activity may be detected using the colorimetric substrate azocoll, an
insoluble ground collagen which liberates a red azodye when degraded by proteolytic
action (Chavira et al. 1984). Azocoll digestion has been used to verify the presence of
MMPs in experimental laboratory samples and ﬂuids ﬁ'om clinical patients. Increased
azocoll digestion has been correlated with the presence of collagenases, gelatinases, and
stromelysins in puriﬁed MMP samples (Ko et al. 2000) and wound ﬂuids (Trengove et a1.
1999, Ashcroft et al. 1997). The technique has been applied to many ﬁelds of MMP
research, including dentistry (Broverrnan et al. 1998) and rheumatology (Okada et al.
1990). The azocoll assay has proven to be a rapid and reliable method of characterizing
the relative MMP content of experimental ﬂuid samples.

Total matrix metalloproteinase activity against azocoll may be measured at pH 7.0
since both stromelysin and gelatinases are active at neutral pH (Okada et al. 1990, Nagase
1998). Azocoll reactions run under acidic conditions favor the activity of stromelysin
(MMP-3) since this enzyme’s peak activity occurs at pH 5.5 (Nagase 1998). Inhibition of
proteolytic activity with the chelating agent 1,10-phenanthroline indicates activity is the

result of MMPs (Young and Grinnell 1994).

In vitro MMP Activation

In vitro -activity assays require activation of MMPs. This is often accomplished
through use of (4-aminophenyl)mercuric acetate (APMA), an‘ organomercurial compound
which causes a disruption of the pro-MMP, resulting in an unstable intermediate which

rapidly converts by autocatalysis to the stable active MMP form (N agase et al. 1990).

16

The activation of MMPs with APMA has been utilized in many studies involving
explant and cultured cells. Differentiation between latent and active enzyme may be
discerned through APMA activation of latent MMPs in both explant and cultured
chondrocytes (Beekrnan et al. 1998, van Lent et al. 2002, Yasuda et al. 2002).
Localization of matrix degradation in time may be correlated with the activation of
MMPs through use of APMA and subsequent MMP activity assay (Milner et al. 2001).
APMA has been used to discern the effects of PSGAG on proteoglycan degradation in
normal and chronic OA cartilage (Sevalla et al. 2000). In addition to studies utilizing
supernatant ﬁ'orn cultured and explant chondrocytes, APMA may also be used to assay
MMP activity in synovial ﬂuid samples of osteoarthritic and rheumatoid arthritic joints

(Yoshihara et al. 2000).

Osteoarthritis

Osteoarthritis (OA) is a progressive joint disease that affects approximately 12%
of the population of the United States. The incidence increases with advancing age
(Hawker 1997). The disease is characterized by gradual loss of articular cartilage and, in
human patients, has traditionally been considered a noninﬂammatory process, although
most animal models as well as numerous human patients exhibit signs of local
inﬂammation and synovitis. Inﬂammatory cytokine concentrations are consistently
elevated in OA (Goldring 2000). The disease is often deﬁned by the presence of clinical
signs (pain, decreased range of motion) together with radiographic signs (osteophytosis,

joint space narrowing) (F elson 1997). The view of OA as a disease of articular cartilage

17

has given way to a more global understanding which takes into account the effects of the
total joint environment (Sokoloff 1987, McIlwraith 1996).

Chondrocytes, ﬁbroblast-like synovial cells, and macrophage-like synovial cells
serve as sources of cytokines that induce a cascade of cartilage digestion as chondrocytes
secrete matrix-degrading proteases (Goldring 2000). The inciting cause is generally
considered to be mechanical in nature, focusing on the extracellular matrix and the effects
of chondrocyte metabolism on this matrix, although pre-existing subchondral bone or
synovial pathology may also predispose the joint to OA development (McIlwraith 1996,
Burton-Wurster et al. 1999, Goldring 2000).

Articular cartilage degradation is characterized by a decrease in proteoglycan
content, changes in proteoglycan structure, increased cartilage water content, and changes
in collagen structure. Differences in individual joint predisposition to development of
OA has been linked to the molecular structure of matrix components (Cole et al. 2002).
Structurally, the cartilage becomes weaker in both compression due to proteoglycan
changes and in tension due to collagen changes (Kempson et al. 1971, Kempson et al.
1973, Wirth et al. 1980). Damage to the collagen component of ECM may be the earliest
event in OA degradation. Proteoglycan content of cartilage in early OA may be
unchanged which indicates that the collagen damage could be the initial event in disease
progression (Roberts 1986, McIlwraith 1996). However, early loss of matrix aggrecan is
also prominent event in OA progression (Mankin et al. 1971). While proteoglycan
degradation is also observed early in OA progression, collagen damage may carry more
serious and permanent consequences due to its slow rate of turnover (Aigner and

McKenna 2002).

18

Drugs in OA Treatment

The goals of OA treatment are to prevent the onset and progression of cartilage
degradation and to alleviate the effects of disease symptoms, particularly pain associated
with joint use (Lozada and Altman 1997). Drugs that primarily alleviate the pain
associated with CA, such as NSAIDS, are useful in that they increase mobility but may
not prevent progression of the disease itself. These drugs may speed progression of CA
by allowing increased use of damaged joints, thereby increasing cartilage destruction
(Blot et al. 2000). In addition, NSAID toxicities are commonly reported, although these
drugs are popular and usually safe if used in an appropriate manner. Similarly,
corticosteroids are used intra-articularly for their antiinﬂammatory and analgesic
activities. These drugs also have side effects associated with their use.

In contrast to drugs whose primary functions are antiinﬂammatory and analgesic
activities, chondroprotective drugs serve to slow, prevent, or reverse OA progression.
These drugs may also have some analgesic and antiinﬂammatory actions associated with

their use, secondary to their primary chondroprotective role.

Symptomatic Drugs
Corticosteroids

Corticosteroids modulate inﬂammatory cascades at several levels. Their effects
are mediated through cytoplasmic glucocorticoid receptors. Once bound, the receptor-
glucocorticoid complex localizes to the nucleus where it regulates gene transcription

through binding to glucocorticoid response elements in the promoter region of many

19

genes (Barnes and Addock 1993) or by direct interactions with transcription factors
(Y ang—Yen et al. 1990). Glucocorticoids may also modify RNA translation, protein
synthesis, and protein secretion through mechanisms other than DNA interactions
(Chrousos et al. 1993, Schleimer 1993).

Corticosteroids interrupt the arachidonic acid inﬂammatory cascade through
induction of lipocortins, which inhibit the actions of PLAz, preventing the conversion of
cell membrane phospholipid into arachidonic acid (May and Lees 1996). Corticosteroids
inhibit both the cyclooxygenase and lipoxygenase inﬂammatory pathways, and have other
effects independent of arachidonic acid metabolism, such as decreased vascular
permeability (Camuccio et al. 1987), inhibition of nitric oxide synthesis (Radomski et al.
1990), inhibition of collagenases, and inhibition of chemotactic factors (Schleimer 1993).
Corticosteroids are typically used intra-articularly in the treatment of osteoarthritis, but
can have several side effects such as retarded chondrocyte metabolism, inhibition of

normal bone metabolism, and potentiation of infection (Trotter 1996a).

Nonsteroidal Antiinﬂammatory Drugs

Nonsteroidal antiinﬂammatory drugs inhibit cyclooxygenase, the enzyme
responsible for conversion of arachidonic acid to prostaglandins (May and Lees 1996).
Two isoforrns of cyclooxygenase exist, the constitutively expressed COX-1 and the
inducible COX-2 (Clements and Harold 1997). The inhibition of constitutive COX-1 is
responsible for many NSAID-linked toxicities, including gastrointestinal ulceration, renal
papillary necrosis, and vascular thrombosis (May and Lees 1996). In addition to

cyclooxygenase regulation, certain NSAHDS, including ketoprofen, inhibit the

20

lipoxygenase pathway (Dawson et al. 1982). NSAIDS may alleviate OA symptoms
through pathways other than cyclooxygenase and lipoxygenase inhibition, including

inhibition of neutrophil activation (Abramson et al. 1985).

Chondroprotective Drugs
Hyaluronan

Hyaluronan is a nonsulfated glycosaminoglycan composed of repeating
disaccharide units of D-glucuronic acid and N-acetyl-D-glucosarnine. It is an integral
component of synovial ﬂuid and articular cartilage in normal joints (Todhunter 1996),
and exogenous hyaluronan may be administered intravenously or intra-articularly
(Howard and McIlwraith 1996, review). Exogenous hyaluronan has been hypothesized to
act in many ways in the abatement of OA clinical signs including acting as a joint
lubricant (Swann et al. 1974), an antiinﬂammatory agent (Howard and McIlwraith 1996,
review), an enzyme inhibitor (through steric hindrance) (Ogston and Sherman 1961,

Forrester and Wilkinson 1981), and a free radical scavenger (Sato et al. 1988).

Polysulfated Glycosaminoglycans

Polysulfated glycosaminoglycans are a normal component of articular cartilage
ECM. Exogenous PSGAGs may be administered intrarnuscularly and intra-articularly.
The proposed mechanisms of action of exogenous PSGAGs in OA have included
degradative enzyme inhibition, antiinﬂammatory action, and enhanced synthesis of

hyaluronic acid, glycosaminoglycans, and collagen (Trotter 1996b).

21

Pentosan Polysulfate

Pentosan polysulfate is a plant-derived polysulfated polysaccharide that has been
administered intrarnuscularly, subcutaneously, orally, intravenously, and intra-articularly.
Proposed mechanisms of action in OA include improved incorporation of proteoglycans
into the cartilage ECM, stimulation of hyaluronan synthesis by synovium, and inhibition

of proteoglycan loss from the ECM, by degradative enzymes (Little and Ghosh 1996).

Glucosamine

The amino sugar glucosamine (2-amino-2-deoxy-alpha—D-glucose), together with
galactosamine, is one of the major hexosamine sugars present in animal cells (Kelly
1998). The only difference between glucosamine and glucose is the replacement of the
glucose carbon-2 OH group with an NH; group. It is synthesized by the enzyme
glucosamine synthetase, which transfers an amide group ﬁom glutamine to fructose-6-
phosphate, to yield glucosamine-6-phosphate. This formation of glucosamine-6-
phosphate is the rate-limiting step in amino sugar biosynthesis. Glucosamine is then
acetylated to form N-acetylglucosamine, followed by conversion to either N-
acetylgalactosarnine or N-acetylmannosamine. This pathway provides the amino sugars
necessary for the synthesis of macromolecules, including glycolipids, glycoproteins,
glycosaminoglycans, hyaluronate, and proteoglycans (Kelly 1998). Glucosamine is the
hexosamine component of the glycosaminoglycan keratan sulfate and the proteoglycan
backbone hyaluronic acid (Barclay et a1. 1998).

The pharmacokinetics of glucosamine have been described in dogs and in humans

(Setnikar et al. 1986, Setnikar et a1. 1993). After oral administration glucosamine is

22

completely ionized in the stomach and is readily absorbed. Approximately 87% of the
administered dose is absorbed ﬁ'om the canine gastrointestinal tract. Glucosamine in
plasma exists as free glucosamine and is not bound to plasma proteins. It is therefore
unlikely that glucosamine will interact with other drugs pharmacokinetically. A single
2000 mg oral dose of glucosamine in a beagle dog achieves short-term plasma
concentrations between 15 and 20 pg/ml (Liang et al. 1999), and intravenous
administration can achieve much higher transient peaks of at least 150 rig/ml (Du et al.
2001). The free glucosamine is rapidly and selectively concentrated within organs,
particularly liver (incorporation into plasma globulins) and kidney (urinary excretion), as
well as the ECM of tissues such as cartilage.

Glucosamine is distributed to all connective tissues after intravenous or oral
administration, including the matrix of muscle, bone, and skin. Cartilage uptake of
glucosamine is greater than that of other musculoskeletal tissues, with approximately 2.5
times more glucosamine concentrated in cartilage compared to muscle at both 2 hours and
144 hours after intravenous administration in dogs (Setnikar et al. 1986). This
margination of glucosamine reached its peak between 30 and 60 minutes after
intravenous glucosamine administration and was maintained until at least 144 hours.

Clearance of glucosamine is primarily through urinary excretion by the kidneys

and secondarily as C02 in expired air and in feces.

23

Glucosamine Clinical Trials

Many clinical trials have utilized glucosamine as a treatment for CA since the
early 19808 (da Camara and Dowless 1998). Most trials support glucosamine therapy
with few minor side effects but many of the studies are plagued by several problems
rendering the usefulness of the results unclear. These trials have consistently enrolled
small numbers of patients, utilized short treatment duration, failed to report inclusion
criteria or severity of disease indices, had short wash—out periods for other OA
medications before the onset of the studies, included variable periods of bed rest, lacked
baseline studies for reported chondroprotective effects, had variable or unreported doses
and routes of administration, and utilized non-blinded scoring (da Camara and Dowless
1998). Furthermore, the studies have largely relied upon industry support for funding and
may be subject to conﬂict of interest problems (McAlindon et al. 2000). One recent
review of 16 randomized controlled trials (RCTs) utilizing glucosamine for the treatment
of osteoarthritis, reported that glucosamine is both effective and safe for the treatment of
osteoarthritis (Towheed et al. 2001). In 13 RCTs that compared glucosamine to a
placebo, glucosamine was found to be superior to placebo in 12. In the 4 RCTs in which
glucosamine was compared to a non-steroidal anti-inﬂammatory drug (N SAID)
glucosamine was found to be superior to NSAID in 2 and equivalent in 2. This review
indicated that while glucosamine appears to be safe and effective for the treatment of OA,
further research is needed to conﬁrm effectiveness and toxicity. Furthermore, the
effectiveness of different preparations by different manufacturers is unknown. There is,
in fact, a wide variability in the dose and form of glucosamine within marketed

glucosamine products (da Camara and Dowless 1998) and no study to date has compared

24

the effectiveness of these different preparations in a clinical trial. Another review of 17
controlled trials involving glucosamine reported poor study quality scores (McAlindon et
al. 2000). Combined results showed a moderate beneﬁt for glucosamine and a large
beneﬁt for chondroitin. However, the utility of these beneﬁt magnitudes remains unclear
due to study design inconsistencies as well as dependence on industry support for
ﬁrnding.

One recent clinical trial has addressed some of the fundamental ﬂaws of earlier
trials by using a larger number of patients over a longer trial period (Reginster et al.
2001). This trial utilized 212 patients, dividing them equally and randomly into placebo-
treated and glucosamine-treated groups. Investigators were blinded to treatment
assignments. Patients were allowed access to NSAID medication as needed for rescue
analgesia with a washout period of at least 5 half-lives before symptom assessment.
Glucosamine was administered as a once daily oral dose of 1500 mg for 3 years.
Symptoms were scored on the Western Ontario and McMaster Universities (WOMAC)
index and mean joint-space of the medial femorotibial joint was assessed by digital image
analysis. Patients were examined upon enrolment, at 1 year, and at 3 years. Patients in
the glucosamine treatment group showed a signiﬁcant improvement in WOMAC index
scores compared to a slight worsening of index scores in placebo-treated patients. Mean
joint-space width of the medial femorotibial joint in glucosamine-treated patients showed
no signiﬁcant decrease while placebo-treated patients exhibited progressive joint—space
narrowing. The authors of this study concluded from the results that glucosamine could

be a disease-modifying agent in osteoarthritis.

25

Overall, clinical trial data support the clinical use of glucosamine for treatment of
OA. In general, analgesic effects take much longer than those of NSAIDS, however the
maximal effect may be greater. Additionally, where NSAIDS do not appear to prevent
the progression of disease indices such as joint space narrowing, glucosamine has a
beneﬁcial effect. These ﬁndings support glucosamine as a chondroprotective substance

that provides symptomatic relief with long-term use.

Glucosamine Side Effects

There is no currently established LDso for glucosamine since no mortality has
been observed in laboratory animals, even at the extremely high doses of 5000 mg/kg
after oral administration (Kelly 1998). This is well below the dose of 500 mg orally three
times daily that has been used in most human clinical studies (Barclay et al. 1998).
Glucosamine has been administered to arthritic human patients with a variety of diseases
such as cardiovascular disease, liver disease, diabetes, pulmonary disease, and depression
with no adverse effects on the course of the disease or interference with the
pharmacologic treatments of these diseases (Kelly 1998). In comparison to NSAID
therapy for osteoarthritis, glucosamine has been reported to have a lower incidence of
side effects (Wright 2001). Adverse side effects when they do occur are generally related
to the gastrointestinal system (mild abdominal pain, nausea, diarrhea, constipation)
(Kelly 1998, Reginster et al. 2001). Individual reports of other side effects such as
immediate hypersensitivity reactions, photosensitization, moderate systolic hypertension,
proteinuria, and elevated creatine phosphokinase levels in blood have also been reported

(Matheu et al. 1999, Danao-Camara 2000).

26

Glucosamine Mechanisms of Action: Research to Date

Proteoglycan production is stimulated by exogenous glucosamine in human
chondrocyte cultures maintained in a 3-dimensional culture system (Bassleer et al. 1998).
The chondrocytes were collected from osteoarthritic femoral head cartilage and
maintained in DMEM media for up to 16 days in a gyratory shaker under standard culture
conditions. This effect was noted for glucosamine concentrations of 10 rig/ml and 100
jig/ml, but was not demonstrated at 1 jig/ml. Additionally, the stimulatory effect of 100
rig/m1 glucosamine was still present 4 days after removal of glucosamine from the culture
medium.

Glucosamine has also been shown to inhibit nitric oxide production at 2.5 and
25.0 mg/ml in equine antebrachiocarpal and middle carpal explant cultures (F enton et al.
2000 a,b). Cultures were treated with either LPS (10 rig/ml) or rhIL-l B (50 ng/ml). Nitric
oxide production was inhibited by glucosamine at 0.1, 0.5, 1, and 2 mM in cultured rat
macrophages stimulated with 1 jig/ml LPS (Meininger et al. 2000). Glucosamine (4.5
g/l) has also been shown to inhibit nitric oxide production in HA [3 (25 and 250 units/ml)
stimulated rat chondrocytes grown in monolayer culture (Gouze et al.2001). Nitric oxide
is an important mediator of OA progression. Nitric oxide, produced by equine
chondrocytes in response to lipopolysaccharide and interleukin-1 (F rean et al. 1997,
Fenton et al. 2000a), decreases proteoglycan synthesis, and may also up-regulate
activation of MMPs (Taskiran et al. 1994, Murrell et al. 1995). Osteoarthritic cartilage
shows enhanced production of nitric oxide (Del Carlo and Loeser 2002). Despite these

ﬁndings, the role of nitric oxide in the pathogenesis of osteoarthritis is not ﬁrlly

27

characterized. NO has been shown to acutely prevent bovine chondrocyte catabolism
when exposed to 20 U/ml th.—l (Stefanovic-Racic et al. 1996), and chondrocyte cell
death in human articular chondrocytes grown in alginate bead and monolayer cultures has
been shown to require both reactive oxygen species and NO (Del Carlo et al. 2002).

Beta-1,3-glucuronosyltransferase I is a key biosynthetic enzyme in
glycosaminoglycan synthesis. Glucosamine at 2 and 4.5 mg/ml prevented the repression
of beta—l,3-glucuronosyltransferase 1 expression in rat articular chondrocyte monolayer
cultures stimulated with IL-1 [3 (Gouze et al. 2001). Glucosamine at 1 mg/ml was not
effective in preventing repression of this enzyme. Glucosamine at 2 and 4.5 mg/ml also
increased proteoglycan synthesis in rat articular chondrocytes cultured in alginate bead
suspension and stimulated with IL-1 [3. The same study showed that 4.5 mg/ml
glucosamine prevented the up—regulation of MMP-3 mRNA expression by interleukin-10
stimulated rat chondrocytes grown in monolayer culture.

Glucosamine (0.25, 2.5, and 25.0 mg/ml) inhibited cartilage proteoglycan loss and
gelatinase/collagenase activities in equine cartilage explants stimulated with LPS (10
ug/ml) or rhIL-lB (F enton et al. 2000 a,b). These effects were shown for both
glucosamine HCl and for glucosamine-3-sulfate, but reportedly not for N-acetyl-
glucosamine (Fenton et al. 2000b).

These activities support the role of glucosamine as a chondroprotective agent and
support further investigation into its actions. Induction of chondrocyte proteoglycan
production, prevention of cartilage proteoglycan loss, inhibition of NO production,

inhibition of gelatinase/collagenase activity, inhibition of MMP production, and

28

protection of GAG-synthesis enzymes are all mechanisms which may be responsible for

the observed chondroprotective properties of exogenous glucosamine.

In Vitro Chondrocyte Culture Systems

In vitro, chondrocytes demand high seeding density and minimal cell-substratum
interactions in order to maintain their chondrocytic phenotype. Cell culture systems
designed to meet these goals have included cartilage explants, monolayer cultures, cell
suspension cultures, agarose gel cultures, and pellet cultures (Solursh 1991, Stewart et al.
2000). Cartilage explants maintain the local ECM by transferring an entire island of
cartilage to culture without further processing. Suspension cultures rely on constant
agitation to maintain isolated chondrocytes in a three-dimensional organization. Agarose
gel and alginate bead cultures use a substrate to maintain isolated chondrocytes in a three-
dimensional organization.

Monolayer cultures maintain isolated chondrocytes in a single conﬂuent layer.
However, cells eventually dedifferentiate from the chondrocytic to a ﬁbroblastic cell
phenotype as they lose collagen type H and aggrecan expression, and begin to express
collagen type I (Stewart et al. 2000).

Monolayer cultures have been used repeatedly in the study of chondrocyte
metabolism. Recent experiments in cartilage tissue engineering have also relied on in
Vitro monolayer culture propagation of chondrocytes prior to implantation in vivo.
Recently, rabbit autologous chondrocytes have been cultured in monolayer cultivation
systems under standard cell culture conditions (37 C, 5% C02) and used for chondral

defect resurfacing (Bacenkova et al. 2001). Monolayer cultures have also been used in

29

 

the experimental infection of rabbit, sheep, cattle, and human chondrocytes with cell
markers for the purposes of cell tracking after knee joint chondral defect repair
(Hirschmann et al. 2002). F irst-passage porcine chondrocytes grown in monolayer have
been shown to produce cartilage in vivo after transplantation (Passaretti et al. 2001).

Despite their utility, monolayer cultures of chondrocytes can be phenotypically
unstable. Cells grown in monolayer culture over long periods of time begin to express
collagen type I with decreasing collagen type H and aggrecan expression (Velikonja et al.
2001). Recently, cartilage oligomeric matrix protein and collagen type D( down-
regulation have been identiﬁed as earlier phenotypic markers of chondrocyte
dedifferentiation (Zaucke et al. 2001). Differences in collagen type H expression have
been attributed to a decrease in transcription factor Spl binding activity in
dedifferentiated chondrocytes (Dharmavaram et al. 1997).

Pellet cultures are formed by gentle centrifugation of isolated chondrocytes within
culture tubes. Pellet cultures allow chondrocytes to form an extracellular matrix (Xu et
al. 1996) and are easily adaptable to laboratory protocols requiring a high number of
replicates with closely controlled cell numbers in each replicate.

Pellet cultures may be used to maintain cartilage-like appearance and normal
metabolism of chondrocytes (Solursh 1991). Extracellular matrix gene expression
proﬁles of pelleted horse chondrocytes have been shown to be similar to tissue levels.
This includes similar expression of collagen type H, collagen type I, and aggrecan

(Stewart et al. 2000).

30

Lipopolysaccharide in In Vitro Osteoarthritis Models

Several models have been used to recreate aspects of osteoarthritis in in vitro
cartilage systems. Two popular methods of inducing an OA-like state is the use of either
bacterial lipopolysaccharide (LPS) or interleukin-1 of animal or human origin. Bacterial
lipopolysaccharide and recombinant human interleukin-1 initiate similar metabolic
responses in cultured equine chondrocytes (F enton et al. 2000 a,b). LPS is efﬁcient in
the induction of cartilage damage and is at least 10 times more potent in horses than most
other mammals (MacDonald and Benton 1996). In addition to mimicking septic arthritis
and reactive arthritis conditions, LPS is a useful and economical model for the study of
osteoarthritis.

LPS mediates IL-1 expression by chondrocytes (MacDonald and Benton 1996),
which in turn stimulates expression of MMPs associated with osteoarthritis (Fenton et al.
2000 a,b). Mammalian cells respond to LPS stimulation by releasing several
inﬂammatory cytokines including TNF-or, H.-6, and HA [3 (U levitch et al. 1995),
initiating conditions similar to those encountered in OA cartilage such as enhanced MMP
activity, proteoglycan release, and nitric oxide production (F enton et al. 2000b).
Mammalian cells respond to pathogen-associated molecules through a family of Toll-like
receptors (Taro et al. 2001, Axtelle and Pribble 2001). E. coli LPS, which was used in
this study, signals through the Toll-like receptor 4 (TLR4) complex (Pulendran et al.
2001). This receptor includes an intracellular region belonging to the IL-lR family and
therefore uses the same signaling pathways as IL-1 (Bowie and O’Neill 2000). In light of
these studies LPS is an appropriate stimulus for the induction of chondrocyte responses

similar to those observed in OA.

31

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42

Chapter 2

INFLUENCE OF GLUCOSAMINE ON MATRIX METALLOPROTEINASE
EXPRESSION AND ACTIVITY IN LPS-STIMULATED EQUINE CHONDROCYTES.

Summary
Objective: To characterize potential mechanisms of action of glucosamine inhibition of

MMP expression and activity in LPS-stimulated equine chondrocytes.

Design: The inﬂuence of glucosamine and suitable controls on MMP activity in
conditioned medium from LPS-stimulated cartilage explants was determined by a
colorimetric assay using azocoll as a substrate. Treatments consisted of negative and
positive controls, glucose (50 mM), and glucosamine (50 mM, 25 mM, 6.25 mM, 3 mM,
and 1.5 mM). The inﬂuence of glucosamine on MMP synthesis was determined using
equine chondrocytes in pellet culture incubated with LPS (20 ug/ml). MMP 13 was
quantiﬁed in spent medium using a commercial ELISA, and non-speciﬁc MMP activity
was determined using azocoll digestion in organomercurial-activated medium. The
effects of glucosamine on MMP mRNA levels in similarly treated equine chondrocytes
was determined by Northern blotting using MMP-1, -3, and -13 probes. Statistical

analysis was carried out with two-way ANOVA, and P was set at 0.05.

Results: Glucosamine had no effect on activated MMP activity. Glucosamine inhibited
MMP protein expression, as determined by azocoll digestion at 3 to 50 mM, and as

determined by MMP-13 ELISA at 1.5 to 50 mM. Resting mRNA levels for MMP-1, -3,

43

and -13 mRNA were signiﬁcantly lower in cultures exposed to glucosamine at

concentrations of 50 mM and 25 mM.

Conclusion: These results indicate that glucosamine is capable of pre-translational, and
possibly also translational, regulation of MMP expression and provide a potential
mechanism of action for previously reported chondroprotective effects of this

aminomonosaccharide.

Introduction

Osteoarthritis (OA) is a well-known and expensive problem that causes
substantial morbidity in the form of lameness and may seriously curtail quality of life in
addition to athletic serviceability of affected horses (Rossdale et al. 1985, Todhunter and
Lust 1990). It can be initiated by a number of causes and ultimately affects all articular
tissues. The hallmark of the disease is the degeneration of articular cartilage, a highly
specialized tissue consisting of relatively few cells but having a rich extracellular matrix.
Although there are a number of possible causes of matrix degradation in OA, abundant
experimental evidence suggests that proteolytic degradation of cartilage by matrix
metalloproteinases (MMPs) is a predominant event (McIlwraith 1996, Goldring 2000).

Matrix metalloproteinases (MMPs, matrixins) are a family of enzymes that
degrade the extracellular matrix of tissues. There are several subgroups of MMPs based
on substrate preference and structural domains including collagenases, gelatinases, and
stromelysins, which are active in OA (Vu and Werb 2000, Nagase and Woessner 1999,

Pozzi et al. 2000, Rabbani et al. 2000). Since collagen damage is an important initial

event in OA (McIlwraith 1996, Goldring 2000, Roberts et al. 1986), MMPs, particularly
collagenases, may serve as a pivotal component of OA initiation and progression.
Another MMP, stromelysin (MMP-3) is also important in that it not only degrades non-
collagenous extracellular matrix components, including the principal aggregating
proteoglycan of cartilage (aggrecan), but also serves to activate latent collagenases
(Goldring 2000).

Oral administration of glucosamine as a symptomatic treatment of OA has been
the subject of several clinical studies in human patients. One recent review of 16
randomized controlled clinical trials utilizing glucosamine suggested that glucosamine is
both an effective and safe treatment for the disease (Towheed et al. 2001). When
glucosamine was compared to an NSAID (ibuprofen), it was equally effective in two
trials and superior in two other trials. While ibuprofen effects a more rapid decrease in
patient pain scores, oral glucosamine therapy yields an ultimately lower pain score after
eight weeks of treatment (Barclay et al. 1998). A chondroprotective effect has been
supported by data indicating that glucosamine prevents knee joint space loss in OA
patients over the course of a three-year study (Reginster et al. 2001).

Certain effects of glucosamine on in vitro cellular metabolism have received
recent attention. Proteoglycan production is stimulated by exogenous glucosamine in
human chondrocyte cultures (Bassleer et al. 1998) and glucosamine prevents the
repression of beta-1,3-g1ucuronosyltransferase I, a key biosynthetic enzyme in
glycosaminoglycan synthesis. Irnportantly, glucosamine prevents the up-regulation of
MMP-3 mRN A expression by interleukin-1 beta stimulated rat chondrocytes (Gouze et

al. 2001). While glucosamine inhibits proteoglycan loss, inducible nitric oxide synthase,

45

and MMP activity in equine cartilage explants stimulated with LPS or human and equine
recombinant interleukin-1 (F enton et al. 2000a,b), the speciﬁc level(s) at which these
effects occur require further characterization.

The purpose of the study presented here was to determine the nature of the
inhibition of MMP activity attributed to glucosamine HCl in an in vitro model of OA.
The speciﬁc aims were to test the hypothesis that glucosamine HCl inﬂuences 1) the
catalytic activity of chondrocyte MMPs, 2) synthesis of MMPs at the protein level, and 3)
resting MMP-l, MMP-3, and MMP-13 mRNA levels in LPS-stimulated equine

chondrocytes.

Materials and methods
Tissue Sources

Grossly normal metacarpophalangeal articular cartilage was obtained from horses
between 2 and 8 years of age that had died or were euthanized for reasons other than joint
disease. Cartilage was dissected from the subchondral bone and incubated in penicillin
(500 U/ml) and streptomycin (500 mg/ml) (25 C, 1 hour). Chondrocytes were isolated by
sequential digestion with pronase and collagenase as previously described (Caron et al.
1996). After digestion, the cells were separated by centrifugation (300 X g, 10 minutes),

washed, and re-suspended in 10 ml DMEM, with 10% F BS, penicillin, and streptomycin.

Pellet Cultures
Cell concentration was determined using a hemocytometer and aliquots of 1 X 106

cells were transferred to 15 ml polypropylene centrifuge tubes in 2 ml DMEM with 10%

46

FBS, penicillin, and streptomycin. Following centrifugation (300 X g, 5 min), pellets
were incubated under standard cell culture conditions (37 C, 95% RH, 5% C02).
Medium was exchanged every 3 to 4 days. Pellet cultures were deprived of serum for 3
to 5 days prior to the start of an experiment. Medium from treated pellet cultures was

collected to determine activity and concentration of MMPs as described below.

Monolayer Cultures

After centrifugation and washing, high-density monolayer cultures were
established in DMEM with 10% FBS, penicillin, and streptomycin under standard cell
culture conditions. Experiments were performed using ﬁrst-passage, high-density
cultures which were deprived of serum for 3 to 5 days prior to the start of an experiment.
RNA from treated monolayer chondrocytes was collected to determine relative amount of

MMP RNA present as described below.

LPS-Conditioned Medium Preparation

To prepare an MMP-rich medium for subsequent MMP activity assays,
metacarpophalangeal joint cartilage was placed in 50 m1 polypropylene centrifuge tubes
in 30 ml DMEM with 10% F BS, penicillin, and streptomycin. LPS was added at a
concentration of 160 ug/ml and cartilage was incubated under standard cell culture
conditions for 24 hours. LPS-conditioned medium was stored at -80 C until analysis for

MMP activity by colorimetric methods as described in mere detail below.

47

Eﬁfect 0f Glucosamine 0n MMP Protein Synthesis

Pellet cultures were placed in 600 pl fresh DMEM with penicillin and
streptomycin. Treatment media contained glucose at a concentration of 50 mM or
glucosamine HCl at a concentration of 50 mM, 25 mM, 6.25 mM, 3 mM, or 1.5 mM.
LPS was added at a ﬁnal concentration of 20 ug/ml to all pellet cultures except negative
controls. Positive controls contained LPS with no treatment. Pellet cultures were
incubated under standard cell culture conditions for 24 hours with loose caps. Media was
analyzed for MMP activity by azocoll digestion as well as for MMP-13 by ELISA as

described below.

Quantiﬁcation OfPre-Formed MMP Activity And MMP Synthesis

Azocoll (Calbiochem, San Diego, CA) (200 mg) was placed in 10 ml reaction
buffer (Tris, pH 7.4 or MES, pH 5.0), vortexed brieﬂy, incubated at 25 C for 2 to 3 hours,
ﬁltered, and re-suspended (Chavira et al. 1984). The azocoll suspension was ﬁltered
through number 1 ﬁlter paper and re-suspended in 10 ml reaction buffer with vigorous
agitation. Test media (200ul) was placed into centrifuge tubes along with 25 pl APMA
(12 mM solution) and 100 pl azocoll suspension. 25 ul 1,10-phenanthroline (120 mM
solution) was added to negative control tubes as an inhibitor of MMP activity. Reaction
buffer (Tris, pH 7.4 or MES, pH 5.0) was added to a ﬁnal volume of 600 al. Treatment
tube reaction buffer contained glucose at a concentration of 50 mM or glucosamine at a
concentration of 50 mM, 25 mM, 6.25 mM, 3 mM, or 1.5 mM. Centrifuge tubes were
capped tightly and incubated at 37 C for 72 hours. Reaction tubes were centrifuged

(12,000 X g, 3 minutes) and absorbance read at 520 nm. Reactions were carried out at

48

pH 7.4 in order to detect total MMP activity (Okada et al. 1990, Nagase 1998) and at pH
5.0 in order to favor stromelysin activity (Nagase 1998).

Preparation of treatment-containing buffers consisted of weighing the appropriate
amount of glucose or glucosamine for 50 ml of pre-made buffer (Tris or MES). The
sugars were then dissolved in buffer at room temperature by vigorous agitation. Each
treatment-containing buffer was then adjusted to proper pH (pH = 7.4 for Tris; pH = 5.0
for MES) individually and stored at 4 C.

To quantify MMP synthesis, test media (200pl) was placed into centrifuge tubes
along with 25 pl APMA (12 mM solution) and 100 pl azocoll suspension. Reaction
buffer (Tris, pH 7 .4) was added to a ﬁnal volume of 600 pl. Centriﬁrge tubes were
capped tightly and incubated at 37 C for 72 hours. Reaction tubes were centrifuged
(12,000 X g, 3 minutes) and absorbance read at 520 nm. Reactions were canied out at

pH 7 .4 in order to detect total MMP activity (Okada et al. 1990, Nagase 1998).

MMP-13 Elisa

Stored media from pellet cultures was assayed for proMMP-13 and MMP-13
using a commercially available kit (Amersham Biosciences, Inc., Piscataway, NJ) using
manufacturer’s directions. Brieﬂy, 100 p1 prepared standard and test media (diluted 1:4)
were incubated in assay wells coated with anti-MMP-13 antibody (2 hours, 25 C). Wells
were washed with supplied phosphate buffer and incubated for 1 hour at 25 C with 100 p1
anti-MMP-13 peroxidase conjugate. Wells were washed and incubated with 100 pl

3,3’5,5’-tetramethylbenzidine/hydrogen peroxide in 20% (w/v) dimethylformamide for

49

exactly 30 minutes at 25 C. The reaction was stopped by the addition of 1.0 M sulfuric

acid and optical density measured at 450 nm.

Extraction OfMonolayer RNA

Total RNA was extracted, using a commercial extraction preparation (TriReagent,
Molecular Research Center, Cincinnati, OH) and following manufacturer’s instructions.
Brieﬂy, 1 ml of this agent was added to each monolayer well and incubated (25 C, 5
minutes). The solution was transferred to centrifuge tubes and 200 p1 chloroform was
added. The tubes were agitated vigorously and incubated at 25 C for 10 minutes. After
centriﬁrgation (4 C, 12,000 X g, 15 minutes) the aqueous phase was removed and the
RNA precipitated with isopropanol. After centrifugation (4 C, 12,000 X g, 15 minutes)
the pellets were washed with 75% ethanol. Pellets were resuspended in sterile water

treated with 0.1% diethylpyrocarbonate.

Northern Blot Hybridization

Probe preparation was as previously described (Tung et al. 2002). Brieﬂy,
ampliﬁcation of cDNA was accomplished, using RT-PCR with 2 pg of total RNA from a
chondrocyte culture that had been stimulated (6 h) with 1 pg/ml of LPS using standard
protocols. Speciﬁc sets of equine oligonucleotide primers for MMP-13 genes were
synthesized based on published human cDNA sequences and were used at a ﬁnal
concentration of 5 pM/pl. The cDNA fragments were ligated directly to PCR 2.1 TOPO
vector (TOPO TA Cloning Kit; Invitrogen) and the sequencing performed manually. The

vector was subcloned into competent E. coli (TOPO TA Cloning Kit; Invitrogen). Clones

50

of MMP-1 and MMP-3 were prepared in pBluescript vector. The transformed bacteria
were propagated in NZY broth at 37 C and 260 rpm in an orbital shaker overnight. The
plasmid was isolated using standard protocols. Brieﬂy, cell lysis in NET buffer/lysis
solution was performed, followed by phenol/chloroform extraction and ethanol
precipitation. Precipitate was re-suspended in 1 ml sterile water with 1 pl RNAse A and
stored at -80 C.

Restriction endonuclease digest was carried out to free the MMP segment of
interest from the plasmid. MMP-1 and MMP-3 plasmid digestion was carried out as
follows: 400 p1 puriﬁed plasmid, 160 p1 Eco R1 (20 U/pl), 240 pl Xho I (400 U/pl), 400
pl React-2 buffer, and 40 pl RNAse (DNAse free) were brought to a ﬁnal volume of 4 ml
with sterile water in a 15 ml polypropylene centrifuge tube and incubated overnight at 37
C. MMP-13 plasmid digestion was carried out as follows: 400 pl puriﬁed plasmid, 200
pl Eco R1 (20 U/ pl), 200 pl Hind IH (10 U/pl), 400 p1 React-2 buffer, and 40 pl RNAse
(DNAse free) were brought to a ﬁnal volume of 4 ml with sterile water in a 15 ml
polypropylene centriﬁge tube and incubated overnight at 37 C. MMP fragments were then
puriﬁed by ammonium acetate/ethanol precipitation and re-suspended in 1 m1 sterile
water. The MMP fragments were isolated by 1.2 % agarose gel puriﬁcation, retaining the
band of interest, followed by extraction using a commercial gel puriﬁcation kit (Qiaquick
Kit; Qiagen). MMP-l bands were approximately 1.7 kb, MMP-3 bands were
approximately 1.6 kb, and MMP-13 bands were approximately 1.3 kb. Conditions of
fragment extration from agarose included solubilization in 3 volumes of gel buffer with
sodium acetate and isopropanol at 50 C, followed by spin column extraction with 3 spin

column tubes per gram of gel. MMP fragment DNA was eluted from the columns using

51

50 p1 Qiagen EB buffer per tube and stored at — 80 C. Concentration was determined by
running 8 pl puriﬁed fragment on a 1.2% agarose gel and estimating the amount based on
band density.

MMP DNA probes were labeled by random primer labeling incorporating
digoxigenin-l l-UTP using a commercially available kit (DIG High Prime DNA Labeling
and Detection Kit H; Roche Molecular Biochemicals, Indianapolis, Indiana). For labeling
reactions, DNA was added at a concentration of 62.5 ng/ pl to DIG High Prime in a 4:1
ratio. DIG-labeled probe concentration was estimated using DIG detection compared to a
known pre-labeled DNA control. DIG-labeled probe was added to DIG Easy Hyb
granules to obtain to appropriate concentration for northern blot hybridization.

Northern blot hybridization was conducted using a commercially available kit by
following manufacturer’s instructions (DIG, Roche Molecular Biochemicals,
Indianapolis, IN). Brieﬂy, total chondrocyte RNA was resolved on 1.2% agarose-
formaldehyde gels, using 3 pg of RNA. After overnight transfer to nylon membranes at
25 C, RNA was cross-linked to the membranes by exposure to UV light. After
prehybridization for 1 hour at 50 C the membranes were hybridized overnight at 50 C
with labeled probe in DIG Easy Hyb (MMP-1 = 75 ng/ml, MMP-3 = 200 rig/ml, MMP-13
= 120 ng/ml). Serial post-hybridization washes were canied out at 25 C and 68 C, with
decreasing concentrations of sodium-citrate-based buffer, followed by washing in maleate
buffer at 25 C. Detection was accomplished using a chemiluminescent method with
digoxigenin-l l-uridyl triphosphate (DIG High Prime DNA Labeling and Detection
Starter Kit H, Roche Molecular Biochemicals, Indianapolis, IN) and CSPD (3,4-

methoxyspiro { 1,2-dioxetane-3 ,2 ’-(5 ’-chloro)tricyclo {3 .3. 1 . 13’7]decan-4-y1} phenyl

52

 

phosphate) (Roche, Indianapolis, IN) as a substrate for alkaline phosphatase conjugated to
digoxigenin antibody Fab fragments. Chemiluminescence was detected by exposure to
scientiﬁc imaging ﬁlm (Eastman Kodak, Rochester, NY) for 10 minutes at 25 C.
Developed ﬁlm was scanned (Epson scanner, Adobe Photoshop) and relative MMP band
intensities were analyzed using available software (Scion Image Beta 4.02, Scion Corp,
Frederick, MD). Nylon membranes were stained in methylene blue (0.03% w/v
methylene blue in 0.3 M sodium acetate, pH 5.2) for 45 seconds, de-stained in water for 2
minutes, and dried between sheets of ﬁlter paper. The 28S ribosomal band densities were
then analyzed. Chemiluminescent bands were standardized against methylene-blue
stained 28S ribosomal RNA bands on the nylon transfer membranes. Speciﬁc mRNA
expression was calculated as the ratio of the intensity of the MMP bands to the intensity

of the 28S rRNA bands.

Statistical Analysis

Sample sizes were determined using calculations based on desired resolution of
differences between treatments and expected variability in data based on previous studies.
Comparison of means for the activity of MMP in conditioned medium, medium
concentrations of MMP-13, and relative expression of MMPs were performed using a
two-way AN OVA (blocked by horse) followed by a Student-Newman-Keuls multiple

comparisons procedure. A P value < 0.05 was considered signiﬁcant.

53

 

 

Results
Eﬂect 0f Glucosamine 0n MMP Activity And MMP-13 Production

Substrate digestion by pre-formed, organomercurial-activated MMP was not
signiﬁcantly affected by glucosamine (Fig. 1 and 2).

In pellet cultures, LPS (20 pg/ml) signiﬁcantly stimulated the synthesis of MMP
as determined by azocoll digestion. This effect was signiﬁcantly reduced by glucosamine
at all concentrations tested except the lowest (1.5 mM) (Fig. 3). This effect was
paralleled by a signiﬁcant reduction in MMP-13 levels at all glucosamine concentrations

tested (Fig. 4).

Effect OfGlucosamine 0n Relative MMP-1, MMP-3 And MMP-13 mRNA Expression
LPS (20 pg/ml) signiﬁcantly stimulated expression of MMP-1, MMP-3, and
MMP-13. This effect was signiﬁcantly reduced by glucosamine at 50 mM and 25 mM

for all three MMPs (Fig. 5, Fig. 6, and Fig. 7).

Discussion

These data support the ﬁndings of prior studies suggesting chondroprotective
properties of glucosamine (Fenton et al. 2000a,b, Sandy et al. 1998) and offer further
information on the mechanism of action of glucosamine with respect to inhibition of
MMP synthesis.

Glucose was used as a sugar moiety control for the glucosamine HCl molecule.
The only difference between these two molecules is the addition of an amine group in the

case of the glucosamine (F enton et al. 20003). Previous studies of a similar nature have

54

been conducted using glucose as a control (Fenton et al. 2000a,b). No effects were
observed for MMP synthesis, NO levels, or proteoglycan release, indicating that the non-
aminated monosaccharide is devoid of chondroprotective effects.

The glucosamine dose range used in this study correlates well with doses used in
prior in vitro studies (Gouze et al. 2001, F enton et al. 2000a,b, Sandy et al. 1998,
Goldberg et al. 2000, Shikhman et al. 2001). Moreover, one in vivo study utilizing
intravenous glucosamine infusion in rats measured circulating glucosamine
concentrations within this dose range (V irkam and Yki-Jarvinen 1999). Although
glucosamine is absorbed readily after oral administration and is selectively concentrated
in cartilage (Setnikar et al. 1986), the dose range used in this study may not approximate
in vivo levels achievable with exogenous oral glucosamine supplementation. A single
2000 mg oral dose of glucosamine in a beagle dog achieves short-term plasma
concentrations between 15 and 20 pg/ml (Liang et al. 1999). Although ultimate cartilage
concentrations of glucosamine may be higher than those in plasma due to selective
concentration, they may not achieve the levels required to demonstrate in vitro effects.
Additionally, this study utilizes a model in which glucosamine treatment and stimulus
(LPS treatment) occur simultaneously. Treatment with glucosamine prior to irreversible
ECM structural damage may require lower doses than those needed to mitigate severe
concurrent chondrocyte stimulation. Therefore, glucosamine concentrations required in
vivo to achieve chondroprotective effects may be much lower than those required in this
in vitro osteoarthritis model.

Results of our experiments using azocoll digestion with APMA-activated,

conditioned medium, indicate that glucosamine HCl has negligible effects on the activity

55

of pre-formed MMP. Matrix metalloproteinase activity assays were carried out at pH 5.0
and at pH 7.4 to favor the activity of stromelysin and total MMP activity, respectively
(Okada et al. 1990, Nagase 1998).

The observation that glucosamine did not inhibit MMP activity at either pH tested
indicates that the activity of the well-characterized MMPs are not signiﬁcantly inhibited
by glucosamine.

Results of MMP synthesis experiments (pellet culture medium azocoll digestion
and MMP-13 ELISA) indicate that glucosamine modulates expression of MMPs at or
before protein translation. This effect was signiﬁcant over the dose range of 3 to 50 mM
as determined by azocoll digestion assay and was mirrored by signiﬁcant reductions in
MMP-13 production at all ﬁve doses of glucosamine tested. ELISA kits were tested for
detection of MMP-1 and MMP-3 as well, however the human antibodies did not cross-
react with the equine forms of these MMPs and the kits could therefore not be validated
for equine use. These results are supportive of one another, and the difference in
signiﬁcance at the 1.5 mM level may be explained by differences between the detection
methods used or due to real differences in total MMP production compared to MMP-l 3
production. The data does suggest a real difference between MMP-13 levels and levels of
other MMPs, as glucosamine treatment at the 1.5 mM level yielded a much larger
reduction in MMP-13 content compared to positive controls (70%) than did results of the
azocoll digestion assay (8%) (Figs. 3 and 4).

Relative mRNA expression of all three MMPs was signiﬁcantly inhibited by
glucosamine at 50 mM and 25 mM. This result indicates that glucosamine inhibits MMP

production or enhances MMP degradation at a pre-translational level. Similar to protein

56

expression data, MMP-13 mRNA levels appeared to be lower than those of the other
MMPs assayed, however this difference was not signiﬁcant.

Recent studies have supported the role of glucosamine in altering mRNA
expression of several key enzymes involved in OA progression. Glucuronosyltransferase
I, an enzyme involved in glycosaminoglycan biosynthesis is repressed by H.415 in rat
chondrocyte cultures. This phenomenon is prevented by the addition of 21 mM
glucosamine to the culture medium (Gouze et al. 2001). Additionally, the same study
reported that glucosamine prevented H.-1 B-induced upregulation of MMP-3 mRN A in rat
chondrocytes. Similarly, inclusion of 2 mmol/l glucosamine in culture medium for 4 days
has been shown to enhance DNA binding of the Spl transcription factor to the PAI-l
promoter site in rat glomerular mesangial cells (Goldberg et al. 2000). Moreover, it has
recently been shown that glucosamine is capable of inhibiting HA effects through
inhibition of the NF KB pathway (Gouze et al. 2002). These studies support our ﬁnding
that glucosamine HCl is capable of altering gene expression. Speciﬁcally, in this study
glucosamine mitigates the LPS-induced induction of relative MMP mRNA expression in
cultured equine chondrocytes.

The precise level of glucosamine action on decreased MMP mRNA production
may be due to inhibition of signal transduction pathways, regulation of transcription
factors, promoter efﬁciency, transcription itself, or post-transcriptional processing
resulting in premature MMP mRNA degradation. Several studies have indicated
regulation of MMP expression through the transcription factor activator protein-l (F ahmi
et al. 2002), Nmp4 gene splice variants (Torrungruang et al. 2002), and Sp family

transcription factors (Liang et al. 2002). Additionally, Ras-responsive elements have

57

been demonstrated in promoter regions (F utamura et al. 2001), suggesting MMP
expression may be regulated through a number of signaling pathways involving
regulation of gene expression. It remains to be determined how glucosamine may

inﬂuence these regulatory pathways.

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61

Activity (Arbitrary Until)

Figure 1

 

0.7

 

 

 

 

 

Figure 1. Mean (iSEM) MMP activity as determined by azodye release into the
supernatant at pH 7.4. Neg = o-phenanthroline-treated; Pos = APMA-treated; G50 =
glucose 50 mM; GLCN = glucosamine at the indicated mM concentrations. * Indicates

statistical signiﬁcance at p < 0.05 compared to positive control.

62

Flam! 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Acﬂvlty (Aroma Unh)
.0
Ur

 

Neg P08 650 GLCNSO GLM GLWJS (31.043 GINLS

Figure 2. Mean (iSEM) MMP activity as determined by azodye release into the
supernatant at pH 5.0. Neg = o-phenanthroline-treated; Pos = APMA-treated; G50 =
glucose 50 mM; GLCN = glucosamine at the indicated mM concentrations. * Indicates

statistical signiﬁcance at p < 0.05 compared to positive control.

63

 

Flour. 3

 

 

 

 

IMP Activity (Arum Units)

 

 

 

Neg P00 650 GLCNSO GLCN25 GLCN8.25 GLCN: GLCN1 .5

Figure 3. Mean (iSEM) MMP production of equine chondrocyte pellet cultures
stimulated with LPS (20 pg/ml). MMP production was determined by azodye release
using azocoll as a digestion substrate at pH 7.4. Neg = unstimulated; Pos = LPS-
stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive

control.

64

Figure 4

 

 

10 _._v —« I Lm

 

 

 

 

 

 

 

 

 

nap-1: Concentration (nutmll
a:
l
l

 

* * *

 

 

     

P03 650 GLCNSO GLCNZS GLCMZS GLCN3 GLCN1 .5

Figure 4. Mean (iSEM) MMP-13 concentration in media supernatant of equine
chondrocyte pellet cultures stimulated with LPS (20 pg/ml). MMP-13 concentration was
determined using a commercially available ELISA. Neg = unstimulated; Pos = LPS-
stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive

control.

65

Flour. 5

 

 

 

.5
0|

 

 

IMP-1 Expression (Arbitrary Units)

 

 

 

 

Neg P08 650 GLCN50 GLCN25 GLCMJS GLCN3 GLCN1.5

Figure 5. Mean (iSEM) relative MMP-1 mRNA expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 pg/ml). MMP-l mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 288 rRN A bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive

control.

66

Figure 6

 

2.5

 

 

 

HIP-3 Elm (MM UI'IIB)

 

 

 

 

Neg P0. 650 GLCN50 GLCN25 GLCNB.25 GLCN3 GLCN1.5

Figure 6. Mean (:tSEM) relative MMP-3 mRNA expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 pg/ml). MMP-3 mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 28S rRNA bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive

control.

67

Figure 7

 

 

 

IMP-13 Expression Minty Units)

 

 

 

Neg Fee 650 GLCN50 GLCN25 GLCMIS GLCN3 GLCN1.5

Figure 7. Mean (iSEM) relative MMP-13 mRNA expression of equine chondrocyte
monolayer cultures stimulated with LPS (20 pg/ml). MMP-l3 mRNA concentration is
expressed as the ratio of the intensity of northern hybridization bands to the intensity of
the 28S rRNA bands on methylene-blue stained nylon membranes. Neg = unstimulated;
Pos = LPS-stimulated; G50 = glucose 50 mM; GLCN = glucosamine at the indicated mM
concentrations. * Indicates statistical signiﬁcance at p < 0.05 compared to positive

control.

68

 

Figure 8. Representative example of a methylene-blue stained nylon transfer membrane.

The top and bottom bands represent 28s (approximately 4.7 kb) and 18 s rRNA
(approximately 1.9 kb), respectively. Nylon membranes were stained in methylene blue
(0.03% w/v methylene blue in 0.3 M sodium acetate, pH 5.2) for 45 seconds, destained in

water for 2 minutes, and dried between sheets of ﬁlter paper.

69

 

1 2 3 4 5 6 7 sJ

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. 7 . 9"" - - v MMP-3
hr '. *" ”'. “' - MMP-l3

 

 

Figure 9. Representative autoradiographs of northern blots with probes for MMP-l , -3,
and —l 3 after detection with the DIG system. Lane 1 = Negative control; 2 = Positive
control; 3 = Glucose 50 mM; 4 = GLCN 50 mM; 5 = GLCN 25 mM; 6 = GLCN 6.25

mM; 7 = GLCN 3 mM; 8 =GLCN1.5 mM.

70

Chapter 3

CONCLUSION

This study supports the ﬁndings of prior studies suggesting chondroprotective
properties of glucosamine and offers fruther information on the mechanism of action of
glucosamine with respect to inhibition of MMP synthesis. Osteoarthritis (OA) is a well-
known and expensive problem that causes substantial morbidity in the form of lameness
and may seriously curtail quality of life in addition to athletic serviceability of affected
horses. The halhnark of the disease is the degeneration of articular cartilage. Although
there are a number of possible causes of matrix degradation in OA, abundant
experimental evidence suggests that proteolytic degradation of cartilage by matrix
metalloproteinases (MMPs) is a predominant event. This study has shown the following:

1. Glucosamine does not signiﬁcantly affect organomercurial-activated MMP
activity.

2. Glucosamine signiﬁcantly reduces the expression of MMPs ﬁ'om LPS-
stimulated equine chondrocytes as determined by a substrate digestion assay,
and this effect is paralleled by a signiﬁcant reduction in MMP-l3 levels.

3. Glucosamine signiﬁcantly reduces the upregulation of mRNA in LPS-
stimulated equine chondrocytes.

Taken together, these results indicate that glucosamine is capable of pre-translational, and
possibly also translational, regulation of MMP expression and provide a potential

mechanism of action for previously reported chondroprotective effects of glucosamine.

71

Results of our experiments using azocoll digestion with APMA-activated,
conditioned medium, indicate that glucosamine HCl has negligible effects on the activity
of pre-forrned MMP. Matrix metalloproteinase activity assays were carried out at pH 5.0
and at pH 7.4 to favor the activity of stromelysin and total MMP activity, respectively.
The observation that glucosamine did not inhibit MMP activity at either pH tested
indicates that the activity of the well-characterized MMPs are not signiﬁcantly inhibited
by glucosamine.

Results of MMP synthesis experiments (pellet culture medium azocoll digestion
and MMP-l3 ELISA) indicate that glucosamine modulates expression of MMPs at or
before protein translation. This effect was signiﬁcant over the dose range of 3 to 50 mM
as determined by azocoll digestion assay. Additionally, MMP-13 production was
signiﬁcantly inhibited at all ﬁve doses of glucosamine tested. Due to the inability to
validate MMP-1 and MMP-3 kits for detection of equine MMPs we were unable to
speciﬁcally demonstrate inhibition of expression of these metalloproteinases as well.
These results are supportive of one another, and the difference in signiﬁcance at the 1.5
mM level may be due to real differences in total MMP production compared to MMP-13
production. Altemately, this difference may be explained by differences between the
detection methods used.

Relative mRNA expression of all three MNIPs was signiﬁcantly inhibited by
glucosamine at 50 mM and 25 mM. This result indicates that glucosamine inhibits MMP
production or enhances MMP degradation at a pre-translational level.

Numerous studies have indicated a potential role for glucosamine in altering

several cell signaling pathways. While it remains to be seen which pathways are affected

72

and in what ways, we feel conﬁdent that glucosamine does regulate cellular metabolism
through these interrelated pathways. Based on other recent research, glucosamine may
modulate MMP expression through pathways involving the transcription factor activator
protein-l, Nmp4 gene splice variants, Sp family transcription factors, Ras-responsive
elements, and the NF-kappa B pathway. MMP expression may be regulated by
glucosamine through a number of these pathways as well as others yet to be
demonstrated.

This project has demonstrated a deﬁnite role for glucosamine in inhibiting MMPs
in an osteoarthritis chondrocyte model. Considering the excellent bioavailability of orally
administered glucosamine, its tropism for cartilage, and nearly nonexistent toxicity, a
strong case can be made for the inclusion of glucosamine in an OA treatment regimen.
While glucosamine alone may not prove to be the proverbial “silver bullet” in medical
management of OA, it may potentiate other treatments and decrease the necessity of using
treatments with high side effect rates. Glucosamine may also play a role in delaying the
onset and slowing the progression of OA.

Future work will be required to validate the usefulness of glucosamine further,
and to demonstrate the precise mechanisms of action. The knowledge that glucosamine
downregulates resting mRNA levels for MMP-1, -3, and -13 mRNA can help guide future
investigations into these mechanisms, which may include inhibition of signal transduction
pathways, regulation of transcription factors, promoter efﬁciency, transcription itself, or
post-transcriptional processing resulting in premature MMP mRNA degradation.

Additionally, this study design may be used to investigate the effects of other potential

73

chondroprotective agents, and to investigate their effects on the expression of other

factors involved in OA.

74

  
 

   

 
 
   

     

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