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V \ UNIVERSITY
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dissertation entitled

GLUCOSAMINE AND CHONDROITIN SULFATE REGULATE
INTERLEUKlN-1 INDUCED MEDIATORS OF
OSTEOARTHRITIS IN ARTICULAR CARTILAGE EXPLANTS

presented by

POOl-SEE CHAN

has been accepted towards fulfillment
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GLUCOSAMINE AND CHONDROITIN SULFATE REGULATE INTERLEUKIN-l
INDUCED MEDIATORS OF OSTEOARTHRITIS 1N ARTICULAR CARTILAGE
EXPLANTS
By

Pooi-See Chan

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Animal Science

2005

ABSTRACT
GLUCOSAMIN E AND CHONDROITIN SULFATE REGULATE INTERLEUKIN-l
INDUCED MEDIATORS OF OSTEOARTHRITIS IN ARTICULAR CARTILAGE
EXPLANTS
By
Pooi-See Chan
Osteoarthritis (0A) is a significant problem for both humans and animals.

Nutraceuticals such as glucosamine and chondroitin sulfate (CS) are widely used to
alleviate symptoms of 0A. However, the mechanism(s) of action of these nutraceuticals
remains unclear. Bovine articular cartilage explants added with 50 ng/ml interleukin-1
(IL-1) to stimulate cartilage catabolism were used to determine the molecular events of
the nutraceuticals. Glucosamine (5 ug/ml) and CS (20 ug/ml), at concentrations
attainable in viva, were supplemented individually or in combination. Glucosamine and
CS regulated transiently nitric oxide (NO) synthesis and the expression of genes encoding
inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E synthase-l
(mPGEsl). There was concomitant repression of IL-1 induced expression of
cyclooxgenase—Z (COX-2) transcript with prostaglandin E2 (PGEz) production.
Glucosamine and CS reduced cytokine up-regulated expressions of matrix
metalloproteinase (MMP)-3, MMP-13, aggrecanase (Agg)-l and Agg-Z. The
nutraceutical combination was more effective in antagonizing some gene expression than
if glucosamine and CS were used individtrally. There were no treatment effects on gene
expression of tissue inhibitor of metalloproteinase (TIMP)-l, TIMP-Z, type II collagen
and aggrecan although the glucosamine and CS combination tended to elevate TIMP-3

transcript. In a separate study, bovine articular cartilage explants were induced with a

subsaturating dose of IL-1 (15 ng/ml) and treated with glucosamine (10 pg/ml) and CS
(20 ug/ml). The regulation of molecular events was also characterized at 8, 16 and 24
hours after culture. The combination abrogated IL-l induced gene expression of iNOS,
COX-2, mPGEsl and nuclear factor kappa beta p65 subunit at all time points. This was
accompanied by decreases in NO and PGE2 synthesis. Stimulated MMP-l3, Agg-l and
Agg-2 mRNA abundance were down-regulated while TIMP-3 was increased by the
nutraceutical combination at all time points. TIMP-3 protein abundance was elevated by
the combination. Proteoglycan release, an indicator of proteoglycan loss was suppressed
by glucosamine and CS combination. Thus, glucosamine and CS in combination repress
the synthesis and expression of genes encoding inflammatory mediators and matrix
catabolic enzymes while up-regulating TIMP-3. This provides a plausible mechanism for

their mild anti-inflammatory and chondroprotective properties.

This dissertation is dedicated to my dearest husband, Boon-Sam for his unconditional
love.

iv

ACKNOWLEDGEMENTS

I would like to thank Peggy Wolf, Angie Schlueter, John Wheeler, Tonya Skuse,
Dene Elliot, and Tonia Peters, past and present members from Dr. Orth’s laboratory for
all their help. Many thanks also go to Chris Colvin, Xiaoning Ren and Sue Sipkovsky
from the Center For Animal Functional Genomics laboratory for technical assistance. I
would also like to thank Ling-Chu Chang, Shih—Kai Chiang, Dr. Qinglei Li and Dr. Patty
Weber for their aid and advice concerning molecular techniques. Sincere appreciation
also goes to Jackie Christie and Barb Sweeney for help pertaining to paper work and
administrative issues.

My deepest appreciation goes to Dr. Mike Orth for giving me the opportunity to
learn from him, for his guidance and encouragement throughout my stay at MSU. I am
also indebted to Dr. John Caron for his continuous guidance, support and invaluable
inputs. My gratitude extends to the rest of my guidance committee members, Dr. Jeanne
Burton, Dr. Paul Coussens and Dr. George Smith for their brilliant ideas on experimental
designs, for their advice, for allowing me to use their facilities and for their constructive
criticisms. I would also like to thank Dr. Guilherme Rosa for all his help on statistical
analysis.

Special thanks goes out to all my brothers and sisters-in-Christ and Auntie Khoo
for their friendship, concern and prayers. Appreciation is expressed to my mom, Sau-
Lung for her continuous love and support. Love and gratitude also goes to my husband,
Boon-Sam for loving and supporting me, and for believing in me. Lastly, I would like to

thank the Lord for all the blessings that He has showered upon me.

TABLE OF CONTENTS

LIST OF TABLES ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, viii

LIST OF FIGURES ________________________________________________________________________________________________________________ 1X

LIST OF ABBREVIATIONS ________________________________________________________________________________________________ xi

INTRODUCTION ..................................................................................................................... 1
CHAPTER 1

LITERATURE REVIEW .......................................................................................................... 6

Articular Cartilage _________________________________________________________________________________________________________ 6

Osteoarthritis .................................................................................................................. 8

Mediators of OA ___________________________________________________________________________________________________________ 9

Cytokines ___________________________________________________________________________________________________________ 9

Inflammatory Mediators ________________________________________________________________________________ 14

Nitric Oxide _______________________________________________________________________________________ l4

Prostaglandin E2 _________________________________________________________________________________ 16

Matrix Enzymes _____________________________________________________________________________________________ 17

Matrix Metalloproteinases ________________________________________________________________ 17

Aggrecanases ______________________________________________________________________________________ 19

Tissue Inhibitor of Metalloproteinases _____________________________________________ 20

Glucosamine and Chondroitin Sulfate as Therapeutic Agents for CA ___________________ 21

Structure and Composition ________________________________________________________________ 21

Pharmacokinetics _______________________________________________________________________________ 23

Dosage and Administration _______________________________________________________________ 24

Safety Profile ______________________________________________________________________________________ 25

Clinical Trials _____________________________________________________________________________________ 26

Mechanism(s) of Action ____________________________________________________________________ 28

Rationale for experiments __________________________________________________________________________________________ 31

References ................................................................................................................... 36
CHAPTER 2

GLUCOSAMINE AND CHONDROITIN SULFATE REGULATE GENE
EXPRESSION AND SYNTHESIS OF NITRIC OXIDE AND PROSTAGLANDIN E2
IN ARTICULAR CARTILAGE EXPLANT S

Summary ______________________________________________________________________________________________________________________ 65
Introduction ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 66
Materials and Methods ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 68
Results __________________________________________________________________________________________________________________________ 72
Discussion ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 75
Acknowledgements _____________________________________________________________________________________________________ 80
References 88

...................................................................................................................

vi

CHAPTER 3

GLUCOSAMINE AND CHONDROITIN SULFATE REGULATE GENE
EXPRESSION OF PROTEOLYTIC ENZYMES AND THEIR INHIBITORS IN
INTERLEUKIN-l CHALLENGED BOVINE ARTICULAR CARTILAGE EXPLANTS

Abstract ________________________________________________________________________________________________________________________ 96
Introduction ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 97
Materials and Methods ________________________________________________________________________________________________ 98
Results ________________________________________________________________________________________________________________________ 101
Discussion ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 103
Footnotes ____________________________________________________________________________________________________________________ 108
References _________________________________________________________________________________________________________________ 1 15
CHAPTER 4

SHORT TERM GENE EXPRESSION CHANGES IN INTERLEUKIN-IB
STIMULATED CARTILAGE EXPLANT S WITH GLUCOSAMINE AND

CHONDROITIN SULFATE
Abstract ______________________________________________________________________________________________________________________ 122
Introduction ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 123
Materials and Methods _____________________________________________________________________________________________ 125
Results ________________________________________________________________________________________________________________________ 131
Discussion __________________________________________________________________________________________________________________ 13 3
References _________________________________________________________________________________________________________________ 149

CHAPTER 5

CONCLUSION l55

.....................................................................................................................

vii

LIST OF TABLES

 

Chapter I
Table 1. Summary of in vitro mechanistic studies with glucosamine and Chondroitin
sulfate using articular cartilage explants or articular chondrocytes 33
Chapter 2
Table 1. Description of components found in treatment media of explant cultures ............ 81

Table II. Forward and reverse primers (5'—>3') used for quantitative real-time polymerase
chain reaction 32

 

Table III. Relative gene expression of explants stimulated with rhIL-l B and
physiologically relevant concentrations of glucosamine and Chondroitin sulfate ,,,,,,,,,,,,,,, 83

Chapter 3
Table I. Description of components found in treatment media of explant cultures .......... 109

Table 11. Forward and reverse primer sequences (5'—->3') of genes of interest used for

 

quantitative real-time polymerase chain reaction __________________ 110
Chapter 4

Table I. Forward and reverse primer sequences (5'—-)3') of genes of interest used for
quantitative real-time polymerase chain reaction 139

 

Table II, Nitrite and PGE2 release from explants at 8, l6 and 24 hours post-stimulation
140

 

viii

LIST OF FIGURES

Chapter 2

Figure 1 (next page). Mean (:hSE) nitrite release into conditioned media 24 hours (Panel
A) and 48 hours (Panel B) afier treatments are added. Ctrl= fetal bovine serum control;
IL-1= 50 ng/ml rhIL-lB (human recombinant interleukin-1 beta); IL-l + GLN= 50 ng/ml
rhIL-IB and 5 ug/ml glucosamine; IL-l + CS= 50 ng/ml rhIL-IB and 20 ug/ml
chondroitin sulfate; IL—l + GLN + CS= 50 ng/ml rhlL-lB, 5 ug/ml glucosamine and 20
ug/ml Chondroitin sulfate. Different superscripts indicate significant differences at
P<0.05 84

..........................................................................................

 

 

Figure 2 (next page). Mean (iSE) prostaglandin E2 (PGE2) release into conditioned
media 24 hours (Panel A) and 48 hours (Panel B) after the addition of treatments. Ctrl=
fetal bovine serum control; IL-l= 50 ng/ml rhlL-IB (human recombinant interleukin-1
beta); IL-l + GLN= 50 ng/ml rhIL-IB and 5 ug/ml glucosamine; IL-l + CS= 50 ng/ml
rhIL-IB and 20 ug/ml Chondroitin sulfate; IL-l + GLN + CS= 50 ng/ml rhIL-lB, 5 ug/ml
glucosamine and 20 ug/ml Chondroitin sulfate. Different superscripts indicate significant
differences at P<0.05. * Tended to be different than the IL-1 treatment (P<O.10)

...........................................................................................................................

 

Chapter 3

Figure 1 (next page). Mean :1: SE expression of genes encoding proteolytic enzymes
relative to F BS control for cartilage explants stimulated for 24 hours (Panel A) and 48
hours (Panel B). FBS = fetal bovine serum. IL-1 = 50 ng/ml human recombinant
interleukin-1. IL-1 + GLN = 50 ng/ml human recombinant interleukin-1 with the addition
of 5 pg/ml glucosamine HCl. [L] + CS = 50 ng/ml human recombinant interleukin-1
with the addition of 20 ug/ml Chondroitin sulfate. IL-l + GLN + CS = 50 ng/ml human
recombinant interleukin-1 with the addition of 5 ug/ml glucosamine HCl and 20 ug/ml
Chondroitin sulfate. MMP-3 = matrix metalloproteinase-3. MMP-l3 = matrix
metalloproteinase-13. MMP-14 = matrix metalloproteinase-14. Agg-l = aggrecanase-1.
Agg-2 = aggrecanase-2. Different letters within a gene indicate significant (P<0.05)
differences between treatments ”1 l l

 

Figure 2 (next page). Mean :1: SE expression of genes encoding inhibitors of proteolytic
enzymes and cartilage macromolecules relative to PBS control for cartilage explants
stimulated for 24 hours (Panel A) and 48 hours (Panel B). F BS = fetal bovine serum. IL-1
= 50 ng/ml human recombinant interleukin-1. IL-1 + GLN = 50 ng/ml human
recombinant interleukin-1 with the addition of 5 pg/ml glucosamine HCl. IL-l + CS = 50
ng/ml human recombinant interleukin-l with the addition of 20 51ng Chondroitin
sulfate. IL-l + GLN + CS = 50 ng/ml human recombinant interleukin-1 with the addition
of 5 ug/ml glucosamine HCl and 20 ug/ml Chondroitin sulfate. TIMP-l = tissue inhibitor
of metalloproteinase-1. TIMP-3 = tissue inhibitor of metalloproteinase-3. Col 11 = type H
collagen. Different letters within a gene indicate that treatments tended (P<0.10) to be
different 113

 

ix

Chapter 4

Figure 1 (next page): Prostaglandin E2 (PGE2) release i SE in conditioned media with
increasing interleukin-1 (IL-1) concentration (A). Percentage inhibition of PGE2 release :1:
SE from 0 rig/ml glucosamine (GLN) or chondroitin sulfate (CS) in conditioned media
with 15 ng/ml IL-1 and increasing GLN or CS concentration (B). Different letters for

values within each line indicate significant (P<0.05) differences between concentrations
ofIL-l, GLN or CS 141

ooooooooooooooooooooooooooooooooooooooooooooooooooooooo

 

 

Figure 2 (next page): Mean relative gene expression t SE of inflammatory mediators at 8
hours (A), 16 hours (B) and 24 hours (C) post-stimulation. Different letters for values
within each gene indicate significant (P<0.05) differences between treatments. iNOS =
inducible nitric oxide synthase; COX-2 = cyclooxygenase-Z; mPGEsl = microsomal
prostaglandin E synthase-l; NFKB = nuclear factor kappa beta p65 subunit; FBS = fetal
bovine serum; IL-1 = 15 ng/ml human recombinant interleukin-1 beta (rhIL-lB); IL-l +
GLN + CS = 15 ng/ml rhlL-IB with the addition of 10 ug/ml glucosamine and 20 ug/ml
chondroitin sulfate 143

oooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

 

 

Figure 3 (next page): Mean relative gene expression i SE of enzymes at 8 hours (A), 16
hours (B) and 24 hours (C) post-stimulation. Different letters for values within each gene
indicate significant (P<0.05) differences between treatments. MMP-3 = matrix
metalloproteinase-3; MMP-13 = matrix metalloproteinase-13; Agg-l = aggrecanase-l;
Agg-2 = aggrecanase-2; ”PIMP-3 = tissue inhibitor of metalloproteinase-3; FBS = fetal
bovine serum; IL-1 = 15 ng/ml human recombinant interleukin-1 beta (rhIL-IB); IL-l +
GLN + CS = 15 ng/ml rhIL-IB with the addition of 10 ug/ml glucosamine and 20 ug/ml
chondroitin sulfate 145

oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

 

Figure 4 (next page): Proteoglycan release i SE in conditioned media at 24 and 48 hours
(A) post-stimulation. Different letters for values within each time point indicate
significant (P<0.01) differences between treatments. Representative western blots of
TIMP-3 and B-actin protein expression at 48 hours post-culture (B). Mean relative
abundance of TIMP-3 proteins 1 SE, as determined by densitometry (C). Different
letters indicate significant (P<0.05) differences between treatments. FBS = fetal bovine
serum; IL-1 = 15 ng/ml human recombinant interleukin-1 beta (rhIL-l B); GLN + CS = 10
ug/ml glucosamine and 20 pug/ml chondroitin sulfate; IL-l + GLN + CS = 15 ng/ml rhIL-
IB with the addition of 10 ug/ml glucosamine and 20 ug/ml chondroitin sulfate ____________ 147

Conclusion

Figure 1 (next page): Proposed mechanism(s) of action for glucosamine and CS as
indicated by results from the present in vitro experiments. X indicates suppression by the
glucosamine and CS combination while V indicates up-regulation by the glucosamine
and CS combination 161

LIST OF ABBREVIATIONS

Activating transcription factor ___________________________________________________________________________________________ ATF
Activator protein-1 _____________________________________________________________________________________________________________ AP-l

A disintegrin and metalloproteinase with thrombospondin motifs ,,,,,,,,,,,,,,,,,,,,,,,,, ADAMT S
Aggrecanase-l ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Agg-l

Aggrecanase—Z ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Agg-Z
CCAAT-box enhancer binding protein __________________________________________________________________________ c/EPB
Cellular myelocytomatosis oncogene _____________________________________________________________________________ c-Myc
Chondroitin sulfate ________________________________________________________________________________________________________________ CS
c-Jun N terminal kinase ______________________________________________________________________________________________________ INK
Cycle to threshold __________________________________________________________________________________________________________________ CT
Cyclic adenosine monophosphate response element binding protein __________________________ CREB
Cyclooxygenase-l ___________________________________________________________________________________________________________ COX- 1
Cyclooxygenase-Z ___________________________________________________________________________________________________________ COX-2
Dimethylmethylene blue ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, DMB

Dulbecco’s modified Eagle’s medium ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, DMEM
Endothelial nitric oxide synthase ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, eNOS
Extracellular matrix ___________________________________________________________________________________________________________ ECM
Extracellular signal related kinase ______________________________________________ ERK
Evolutionary conserved signaling intermediate in Toll pathways ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ECSIT
Fetal bovine serum ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, FBS
Glucosamine hydrochloride ______________________________________________________________________________________________ GLN

xi

Glucosamine sulfate GS

..............................................................................................................

Glucuronosyltransferase I ____________________________________________________________________________________________ GchT-l
Glutamine fi'uctose-6-phosphate amidotransferase _______________________________________________________ GFAT
Glyceraldehyde phosphate dehydrogenase _________________________________________________________________ GAPDH
Glycosaminoglycan _______________________ l _________________________________________________________________________________ ,._GAG
Human recombinant interleukin-1 beta ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, rhIL-l B
Inducible nitric oxide synthaserNOS
Inhibitor of kappa beta ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, IKB
Inhibitor of kappa beta kinase ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, IKK
Insulin-like growth factor 1 ____________________________________________________________________________________________ “IGF-l
Interleukin-l ________________________________________________________________________________________________________________________ IL-l
Interleukin-2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, IL-2
Interleukin-4 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, IL-4
Interleukin-6 ________________________________________________________________________________________________________________________ [14-6
Interleukin-8 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, IL-8
Interleukin-10 ____________________________________________________________________________________________________________________ IL- 1 0
Interleukin-1 receptor-activated kinase ___________________________________________________________________________ IRAK
Interleukin-1 receptor antagonist ___________________________________________________________________________________ IL-lRa
Interleukin-1 receptor associated protein __________________________________________________________________ IL-lRacP
Interleukin-1 receptor type I ___________________________________________________________________________________________ IL- lRI
Interleukin-1 receptor type II ________________________________________________________________________________________ IL- 1 R11
Intravenous ............................................................................................................................. IV
Immunoglobulin ...................................................................................................................... lg

xii

Matrix metalloproteinase ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, MMP

Microsomal prostaglandin E synthase 1 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, mPGEsl

Mitogen activated protein/extracellular signal related kinase kinase ____________________________ MEK

Mitogen activated protein/extracellular signal related kinase kinase kinase ______________ MEKK
Mitogen activated protein kinase ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, MAPK
Mitogen activated protein kinase kinase ,,,,,,,,,,,,,,,, I ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, MKK
Myeloid differentiation factor 88 __________________________________________________________________________________ MyD88
N-acetylglucosamine _____________________________________________________________________________________________________ GlcNac
Nitric oxide ____________________________________________________________________________________________________________________________ N0
Neuronal nitric oxide synthase ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, nNOS

Non-steroidal anti-inflammatory drugs _______________________________________________________________________ NSAIDs
Nuclear factor kappa beta ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, _. ,,,,,,,,,,,,,,,, NFKB
Nuclear factor kappa beta-inducing kinase ....................................................................... NIK
Osteoarthritis _________________________________________________________________________________________________________________________ 0A
Prostaglandin E ___________________________________________________________________________________________________________________ PGE
Prostaglandin E2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, . ..................................... PGE2
Prostaglandin G2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, PGGz
Prostaglandin H2 ________________________________________ _ ...................................................................... PGHz
Proteoglycan .......................................................................................................................... PG
Quantitative real-time polymerase chain reaction _________________________________________________ Q-RT-PCR
Rheumatoid arthritis ............................................................................................................. RA
Signal transducers and activators of transcription __________________________________________________________ STAT
Specificity protein,_m_,____m_,_______. __________________________________________________________________________________________ SP

xiii

Tissue inhibitor of metalloproteinase ______________________________________________________________________________ TIMP

Transforming growth factor _______________________________________________________________________________________________ TGF
Tumor necrosis factor alpha _____________________________________________________________________________________________ TNFa
Tumor necrosis factor receptor associated factor 6 _____________________________________________________ TRAF6
Type II collagen ________________________________________________________________________________________________________________ Col II

xiv

INTRODUCTION

Osteoarthritis (0A) is a debilitating disease that plagues not only humans but
animals as well. Companion animals, zoo animals, and livestock also have significant
problems with 0A. 0A is characterized by degeneration of the cartilage leading to loss of
the critical material necessary to absorb shock. The consequences of articular cartilage
destruction are pain, swelling, mild inflammation, joint stiffness, and distortion of
articulation leading to a loss of function and mobility.

Degeneration characteristic of OA occurs mainly via degradation of the
extracellular matrix (ECM) components, mainly type II collagen and aggrecan. The exact
etiology of 0A is not completely understood. However, a combination of factors is
proposed to contribute to the development of 0A. These factors include sudden and
repeated traumatic injury to the joint, obesity, age, genetic predisposition, and joint
deformity such as congenital joint diseases (1-5). Molecules implicated to participate in
the catabolic process of 0A are cytokines, inflammatory mediators, and matrix enzymes.

Interleukin-1 (IL-1) is the most widely known proinflammatory cytokine that acts
in an autocrine and paracrine fashion and is responsible for modifying chondrocyte
metabolism in OA (6). IL-1 induced matrix enzymes that potentially catabolize type H
collagen and aggrecan are the matrix metalloproteinases (MMPs) and aggrecanases (7,
.3). 0A is also characterized by an imbalance between degradative enzymes and the tissue
inhibitors of metalloproteinases (TIMPs) that are endogenous inhibitors, with the former
predominating (9). The mitogen activated protein kinase (MAPK) and the nuclear factor

kappa beta (NF KB) pathways facilitate the catabolic effects of IL-1 on cartilage

metabolism (10). In particular, the NFKB pathway is responsible for activating genes
encoding inflammatory mediators while both NFKB and MAPK pathways appear to
regulate MMP genes (11). Inflammatory mediators that participate in OA pathogenesis
include nitric oxide (NO) and prostaglandin E2 (PGE2) (12, 13).

Conventional therapies available for treating 0A such as non-steroidal anti-
inflammatory drugs (N SAIDs) are basically only effective for symptomatic relief.
Although these medications are often efficacious in attenuating 0A symptoms, they are
far from ideal as therapeutic agents because they are associated with serious adverse
events. Moreover, NSAIDs have been associated with acceleration of joint damage (14).
Natural substances such as glucosamine and chondroitin sulfate (CS) are safer
alternatives with preliminary evidence of being able to elicit chondroprotection while
manifesting symptomatic relief.

Of the available nutraceutical supplements as therapeutic agents for improving
joint health, glucosamine and CS are the most popular by far, based on revenue.
Although many clinical trials have reported on the benefits of glucosamine and CS in
relieving symptoms of OA (15-17), the exact mechanism of action remains unclear. In
vitro studies with glucosamine and CS found that both increase proteoglycan synthesis,
collagen synthesis and prevent loss of ECM components (18-20). Both glucosamine and
CS besides being chondroprotective, also have potential anti-inflammatory properties
(21-24). However, most in vitro mechanistic experiments with glucosamine and CS have
used concentrations of the nutraceuticals that are not biologically relevant. This was a
reason given by rheumatologists for their resistance in recommending these

nutraceuticals to patients (25). Many of the commercially available glucosamine and CS

are marketed as a combination but most studies have been conducted with the individual
compound. Several studies showed that the combination of glucosamine and CS have
elevated chondroprotective properties when given together (22, 23). Hence, combining
them in studies is important.

The present research is aimed at determining the effects of glucosamine and CS,
at concentrations achievable in vivo in the blood after oral and intravenous
administration, alone and in combination, on molecular events of OA mediators using an
IL-l induced bovine articular cartilage explant model. Chapter 1 represents a current
literature review while chapter 2 and chapter 3 depict the effects of glucosamine and CS,
supplemented individually and in combination on inflammatory mediators and on matrix
enzymes respectively. Chapter 4 features the effects of the nutraceutical combination on
both inflammatory mediators and matrix enzymes using a subsaturating dose of IL-1 on
cartilage explant culture. Lastly, chapter 5 concludes the findings of the previous

chapters.

References

1. Donohue JM, Buss D, Oegema TR Jr., Thompson RC Jr. The effects of indirect blunt
trauma on adult canine articular cartilage. J Bone Joint Surg Am 1983; 65:948-57.

2. Englund M, Roos EM, Lohmander LS. Impact of type of meniscal tear on radiographic
and symptomatic knee osteoarthritis: a sixteen-year followup of meniscectomy with
matched controls. Arthritis Rheum 2003; 48:2178-87.

3. Yoshimura N, Nishioka S, Kinoshita H, Hori N, Nishioka T, Ryujin M, et. al. Risk
factors for knee osteoarthritis in Japanese women: heavy weight, previous joint injuries,
and occupational activities. J Rheum 2004; 3 1 : 157-62.

4. Felson DT, Anderson JJ, Naimark A, Walker AM, Meenan RF. Obesity and knee
osteoarthritis. The Framingham Study. Ann Intern Med 1988; 109:18-24.

5. Knowlton RG, Katzenstein PL, Moskowitz RW, Weaver EJ, Malemud CJ, Pathria
MN, et. al. Genetic linkage of a polymorphism in the type II procollagen gene (COL2A1)
to primary osteoarthritis associated with mild chondrodysplasia. New Eng J Med 1990;
322:526-30.

6. Dinarello CA. Biology of interleukin 1. FASEB 1988; 2: 108-15.

7. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory
cytokine production by chondrocytes of human osteoarthritic cartilage: associations with
degenerative changes. Arthritis Rheum 2001; 44:585-94.

8. Tortorella MD, Malfait AM, Deccico C, Amer E. The role of ADAM-T84
(aggrecanase-l) and ADAM-T85 (aggrecanase-2) in a model of cartilage degradation.
Osteoarthritis Cart 2001; 9:539-52.

9. Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends In
Gen 1990; 62121-5.

10. Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of interleukin-l-stimulated
MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF -kappa B)
transcription factors down-regulates matrix metalloproteinase gene expression in articular
chondrocytes. Matrix Biol 2002; 21 :251-62.

11. Malemud CJ, Islam N, Haqqi TM. Pathophysiological mechanisms in osteoarthritis
lead to novel therapeutic strategies. Cells, tissues, organs 2003; 174:34-48.

12. Melchiorri C, Meliconi R, Frizziero L, Silvestri T, Pulsatelli L, Mazzetti I, et. al.
Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide
synthase by chondrocytes from patients with osteoarthritis. Arthritis Rheum 1998;
41:2165-74.

l3. Kirker-Head CA, Chandna VK, Agarwal RK, Morris EA, Tidwell A, O'Callaghan
MW, et. al. Concentrations of substance P and prostaglandin E2 in synovial fluid of
normal and abnormal joints of horses. Am J Vet Res 2000; 61:714-8.

14. Palmoski MJ, Brandt KD. Effects of some nonsteroidal antiinflammatory drugs on
proteoglycan metabolism and organization in canine articular cartilage. Arthritis Rheum
1980; 23:1010-20.

15. de los Reyes GC, Koda RT, Lien EJ. Glucosamine and chondroitin sulfates in the
treatment of osteoarthritis: a survey. Prog Drug Res 2000; 55:81-103.

16. McAlindon TE, LaValley MP, Gulin JP, F elson DT. Glucosamine and chondroitin for
treatment of osteoarthritis: a systematic quality assessment and meta-analysis. J Am Med
Assoc 2000; 283: 1469-75.

17. Leeb BF, Schweitzer H, Montag K, Smolen J S. A metaanalysis of chondroitin sulfate
in the treatment of osteoarthritis. J Rheum 2000; 27:205-11.

18. Bassleer C, Rovati L, F ranchimont P. Stimulation of proteoglycan production by
glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage
in vitro. Osteoarthritis Cart 1998; 62427-34.

l9. Bassleer CT, Combal JP, Bougaret S, Malaise M. Effects of chondroitin sulfate and
interleukin-1 beta on human articular chondrocytes cultivated in clusters. Osteoarthritis
Cart 1998; 6:196-204.

20. F enton J1, Chlebek-Brown KA, Caron JP, Orth MW. Effect of glucosamine on
interleukin-l-conditioned articular cartilage. Equine Vet J Suppl 2002; 34:219-23.

21. Largo R, Alvarez-Sofia MA, Diez-Ortego I, Calvo E, Sénchez-Pernaute O, Egido J,
et. a1. Glucosamine inhibits IL-lbeta-induced NFkappaB activation in human
osteoarthritic chondrocytes. Osteoarthritis Cart 2003; 11:290-8.

22. Lippiello L, Woodward J, Karpman R, Hammad TA. In vivo chondroprotection and
metabolic synergy of glucosamine and chondroitin sulfate. Clin Orthop 2000; 381:229-
40.

23. Orth MW, Peters TL, Hawkins JN. Inhibition of articular cartilage degradation by
glucosamine-BC] and chondroitin sulphate. Equine Vet J Suppl 2002; 34:224-9.

24. Nakamura H, Shibakawa A, Tanaka M, Kato T, Nishioka K. Effects of glucosamine
hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases
by chondrocytes and synoviocytes in osteoarthritis. Clin Exp Rheumatol 2004; 22:293-9.

25. Manson JJ, Rahman A. This house believes that we should advise our patients with
osteoarthritis of the knee to take glucosamine. Arthritis Rheum 2004; 43: 100-1.

CHAPTER 1

LITERATURE REVIEW

1. Articular Cartilage

Articular cartilage is a highly ordered, specialized connective tissue that is
aneural, alymphatic and avascular. It provides a smooth surface and frictionless weight
bearing support to protect joints by absorbing mechanical stresses and loads. Mature
articular cartilage is composed of chondrocytes that are sparsely distributed in an
extracellular matrix (ECM). Chondrocytes are the only cell type in cartilage and they
make up 10% or less of the total volume of articular cartilage. Chondrocytes derive their
nutrients from the synovial fluid and require the presence of growth factors for survival
(1). These cells are responsible for synthesizing, maintaining and degrading ECM
components. The ECM is constituted mainly by a network of crosslinked triple-helical
collagen fibrils, proteoglycans, and glycoproteins that interact together to give the tissue
its unique mechanical and physical properties. Proteolytic enzymes are responsible for
regulating the constant remodeling process of the ECM during grth and development.

Collagens are protein trimers with 3 alpha polypeptide chains intertwined into a
triple helix as their monomers. These monomers are about 300, 000 in molecular weight.
The peptide sequence within the triple helix is (Gly-X—Y)n with a high content of the
irnino acids, proline and hydroxyproline in the X and Y positions respectively. Collagens
are synthesized as large procollagen molecules that are eventually cut by proteinases
before they are secreted by the chondrocytes. These collagens are then assembled into

fibrils that are as long as 300 nm and are stabilized by covalent cross-linking. At least 19

different types of collagens are distinguished by different lengths, procollagen, and
glycosylation processes. The major type of collagen in articular cartilage is type II
collagen (90%) while collagen types VI, IX and XI are present in articular cartilage'to a
lesser extent (2). The presence of a rigid network of collagen provides cartilage with its
tensile strength by preventing articular cartilage from swelling to its maximum (3).

The structure of proteoglycans resembles that of a ‘bottle brush’ or a ‘comb’.
Proteoglycans are usually large molecules of up to 3.5 X 106 molecular weight (4).
Aggrecan, a supramolecule consisting of about 100 chondroitin sulfate chains (5) and 50
keratan sulfate chains (6) is the predominant proteoglycan in articular cartilage. A
multiaggregate forms when aggrecan interacts with hyaluronic acid via a stabilizing link
protein. Chondroitin sulfate (CS) and keratan sulfate are glycosaminoglycans (GAGs)
covalently attached to the core protein. GAGs are increasingly hydrated because they are
highly negatively charged (7) and this enables articular cartilage to withstand
compressive stresses (4). CS, the most common GAG in articular cartilage, is the
heteropolymer of N-acetylgalactosamine and glucuronic acid with a B-1,3 linkage. This
GAG can be sulfated at the C-4 or C-6 position of the amine sugar producing chondroitin
4-sulfate and chondroitin 6-sulfate respectively. Both C-4 and C-6 sulfated disaccharides
exist as a c0polymer in articular cartilage with chondroitin 6-sulfate predominating (8).
The GAGs found less abundantly are keratan sulfate and dermatan sulfate. Hyaluronic
acid is a GAG abundant in joint space responsible for viscosity of the synovial fluid. This
GAG is of widely varying molecular weight ranging from 0.5 to 1.6 X 106 (4) and unlike
the others, it is not sulfated. It consists of repeating disaccharide units, composed of N-

acetylglucosarnine linked B-l,3 to glucuronic acid.

2. Osteoarthritis

Osteoarthritis (0A), a degenerative joint disease, is the result of both mechanical
and biologic events that uncouple the normal balance between degradation and synthesis
by chondrocytes (9). This chronic musculoskeletal disorder has a significant social and
economic impact on both humans and animals. It is the major cause of lower extremity
disability and is prevalent among the elderly and former athletes involved in weight-
bearing sports (10-12). The National Institute of Arthritis and Musculoskeletal and Skin
Diseases estimates that more than 20 million people in the United States have the disease
to date and by 2030, about 70 million Americans will be at risk for CA. Chronic joint
disease cost the US economy $42.6 billion in medical bills and $82.2 billion. from lost
productivity annually (13).

Osteoarthritis commonly affects weight-bearing joints such as the knee, hip, neck
and the lower back and it is a crippling disease. Major symptoms include pain, swelling,
mild inflammation, joint stiffness and distortion of articulation leading to a loss of
function and mobility. Evidence for structural changes that occur in OA affected joints is
assessed by radiographic evaluation (14, 15). Earliest morphological changes include loss
of cartilage elasticity and cartilage flakes from the surface. As OA progresses, thinning,
fibrillation and splitting of the articular cartilage become inevitable resulting in joint
space narrowing. Osteophytes, subchondral sclerosis and synovitis also occur in the 0A
affected joint. At late stages of severe 0A, articular cartilage is eroded down to the
subchondral bone.

Osteoarthritis is the most common cause of lameness significantly impacting

livestock and companion animals particularly the equine industry resulting in poor

performance and early retirement (16, 17). Degenerative joint disease is the major reason
for euthanasia in military working dogs and as many as 20% of dogs greater than a year
old are affected with GA (18, 19). The feline (20), porcine (21) and ovine (22) species are
not spared from OA.

Degeneration of articular cartilage occurs mainly via degradation of type II
collagen and aggrecan (23). The exact etiology of 0A is not completely understood.
However, a combination of factors is proposed to contribute to the development of 0A.
These factors include sudden and repeated traumatic injury to the joint (24-26), obesity
(27), age (28), genetic predisposition (29), and joint deformity caused by congenital joint
diseases (30). In healthy articular cartilage, chondrocytes balance synthesis with
degradation to maintain consistent levels of proteoglycans and collagen in the matrix. In
0A however, stimuli of biological, mechanical or hormonal nature perturb this balance
resulting in cartilage loss (31-34). Molecules implicated to participate in the catabolic

process of 0A are cytokines, inflammatory mediators and matrix enzymes.

3. Mediators of 0A
3.1 Cytokines

Cytokines exert multiple biological and regulatory effects by providing
intercellular communication. Interleukin-1 (IL-1) is the most widely known
proinflammatory cytokine that acts in an autocrine and paracrine fashion and is
responsible for modifying chondrocyte metabolism in CA (35-3 7). It mounts an
inflammatory response similar to that in naturally occurring 0A. This cytokine is

predominantly produced by macrophages but is also secreted by synoviocytes and

chondrocytes (3 8-40). Patients with CA and rheumatoid arthritis (RA) have elevated
levels of IL-1 in the synovial fluid (41, 42). There are two distinct lL-l genes, one
encoding acidic IL-la and one encoding basic IL-IB, with the latter being more
prominently reported to enhance cartilage destruction (43-45). These isoforms share 26%
homology in their amino acid sequence and show a similar spectrum of biological effects
(35).

IL- 10 is primarily synthesized as a precursor and is activated by IL-lB-converting
enzyme that is located in the plasma membrane (46). This proteolytic enzyme, a serine
protease, is elevated in OA cartilage (47). Two receptors, IL-1 receptor type I (IL-1R1)
and II (IL-1RII) are present. IL-lRI has higher binding affinity for IL-lB. Both IL—lRI
and IL-lRII belong to the immunoglobulin (1g) superfamily. IL-lRI is an 80 kD
glycoprotein that has an extracellular segment containing 3 domains homologous to
immunoglobulins. This extracellular segment has several sites for glycosylation and there
is a transmembrane portion and a cytosolic region. IL-IRII also has 3 immunoglobulin-
like domains in the extracellular segment and a transmembrane segment. IL-lRII has a
truncated cytoplasmic portion and is not involved in signal transduction, unlike IL-lRI.
IL-lRII acts as a sink for IL-1 and is termed a decoy receptor. IL-l binds to the cell
membrane associated IL-lRI and the co-receptor for IL-1 signaling which is a receptor-
associated protein (IL-lRacP) that increases binding aflinity. Activation of the IL-1
receptor with IL-1 induces the recruitment of an adapter molecule, the myeloid
differentiation factor 88 (MyD88). Subsequently, MyD88 recruits the IL-1 receptor-
activated kinase (IRAK), a serine-threonine kinase and IRAK becomes

autophoshorylated. The Toll-interacting protein is involved in IRAK recruitment via

10

association with IL-lRacP. IRAK then interacts with another adapter molecule, TNF
receptor associated factor 6 (TRAF6) that eventually activates one of two pathways,
either the nuclear factor kappa beta (NF KB) or mitogen activated protein kinase (MAPK)
pathway (48). Thus, the binding of IL-1 to its receptor will result in the activation of a
number of phosphorylation dependent signaling pathways leading to the nuclear
translocation of transcription factors and activation of proteins participating in translation
of mRNA, modulating chondrocyte responses and gene expression.

The NFKB and MAPK pathways involve the transcription factors NFKB and
activator protein-l (AP-1) respectively. For the activation of the NFKB pathway, TRAF6
is linked to the NFKB-inducing kinase (N 1K). NIK in turns activates the inhibitor of
kappa beta (IKB) kinase (IKK) that then phosphorylates IKB. Phosphorylation of IKB
initiates the ubiquitin-proteasome mediated degradation of IKB liberating and activating
NFKB. NFKB is a ubiquitous protein residing in the cytoplasm in an inactive form when
bound to IKB. The most predominant form of NFKB is a heterodimer of the p50 and p65
subunits that has the transactivation domains needed for gene induction. Activated NFKB
then translocates to the nucleus and binds to NFKB enhancer elements in the promoter
region of a number of genes regulating transcription of these genes.

As for the MAPK pathway, TRAF6 associates with evolutionary conserved
signaling intermediate in Toll pathways (ECSIT). ECSIT then interacts with mitogen
activated protein/extracellular signal related kinase' kinase kinase (MEKK) that in turns
activate mitogen activated protein/extracellular signal related kinase kinase (MEK)1/2,
mitogen activated protein kinase kinase (MKK)4/7 and/or MKK3/6. MEK1/2 then

activates extracellular signal related kinase (ERK)1/2 while MKK4/7 activates c-Jun N

11

terminal kinase (JNK) and MKK3/6 responsible for p38 kinase activation. ERK1/2, JNK,
and p38 are all MAPK. The MAPK can then activate a number of transcription factors
including Elk-1, c-Fos, c-Jun, cellular myelocytomatosis oncogene (c-Myc), cyclic
adenosine monophosphate response element binding protein (CREB), activating
transcription factor (ATF)-2, specificity protein (SP)-1, signal transducers and activators
of transcription (STAT)-1, CCAAT-box enhancer binding protein (c/EPB). Following
activation, c-Jun moves to the nucleus to interact with c-Fos forming AP-l. The AP-l is a
transcription factor composed of two proteins of the F os and Jun families and the most
common is a heterodimer of c-Jun/c-Fos. AP-l then binds to the promoter of a number of
genes regulating their transcription.

The elucidation of specific transcription factors involved in IL-1 response in
articular cartilage could serve as therapeutic targets for CA. The catabolic effects of IL-1
on cartilage metabolism is facilitated by the MAPK and the NFKB pathways since p38
kinase, ERK and JNK, AP-l and NFKB transcription factors are all activated in the
presence of this cytokine in articular chondrocytes (49-53). In particular, NFKB pathway
is responsible for activating genes encoding inflammatory mediators while both NFKB
and MAPK pathways appears to regulate MMP genes (53). The calcium/calmodulin
dependent pathway in signal transduction is also suspected to be the mediator in up-
regulating MMPs in the presence of IL-1 (54, 55). IL-l induced down-regulation of type
II collagen is mediated by Sp-l and Sp-3 transcription factors (56).

A cascade of events is postulated to occur with IL-1 being produced fi'om the
synovial tissue and secreted into the synovial fluid (57). IL-1 in the synovial fluid induces

chondrocytes to produce proteolytic enzymes, inflammatory mediators and other

12

catabolic factors responsible for the pathogenesis of 0A (58). IL-l induced matrix
degrading enzymes that potentially catabolize type II collagen and proteoglycan are the
matrix metalloproteinases (MMPs) and aggrecanases (59—68).

Gene expression and synthesis of latent MMPs and MMPs were increased while
production of tissue inhibitor of metalloproteinases (TIMPs) decreased in response to IL-
] stimulation (61, 63, 69-72). IL-l increases proteoglycan release (73), proteoglycan loss
in vivo (74) and in vitro (75) and serine protease release (76). IL-l also inhibits synthesis
of proteoglycan (77-79), aggrecan (80), GAGs (81), and type II collagen (80, 82). IL-lB
treatment results in a marked inhibition of mRN A expression and activity of
glucuronosyltransferase l, the rate-limiting enzyme for GAG synthesis (83). In addition,
IL-1 has been implicated in regulation of glucose transporters by alteration of transporter
affinity resulting in glucose imbalance intervening with matrix synthesis (84, 85).
Degradation of the ECM in vivo and in vitro with IL-1 stimulation have been reported in
the human, bovine, porcine, equine and lapine species (86). Other factors up-regulated by
IL-1 include nitric oxide (NO) (77, 87) and prostaglandin E2 (PGE2) (35, 76, 88). The
administration of interleukin-1 receptor antagonist (IL-lRa) was reported to suppress IL-
1 mediated release of MMP and PGE2 attenuating matrix degradation (89).

The pathology of 0A is also mediated by tumor necrosis factor alpha (TNFa), an
essential cytokine stimulating synovium inflammation and matrix degradation via
activation of MMPs (37, 58, 71). TNFa, however, acts more slowly, is 10 fold less potent
than IL-1 and believed only to be involved in the onset of 0A unlike IL-l that is
intimately involved with cartilage degeneration (90-93). Furthermore, signaling receptors

for IL-1 and We are not expressed concurrently (94).

13

Interleukin-6 (IL-6) and interleukin-8 (IL-8) are other proinflammatory cytokines
elevated in OA cartilage. However, the exact role played by these cytokines in OA
pathogenesis remains unknown. Chondrocytes stimulated with IL-IB or TNFa produce
these cytokines excessively (95, 96). IL-6 is a 26 kDa glycosylated polypeptide that is
produced mainly by chondrocytes (97) and synovial fibroblasts (98) of inflamed joints. It
is present excessively in synovial fluid of OA patients (99). IL-6 is believed to reduce
cartilage destruction during CA by enhancing the presence of IL-lRa (100), TIMPs (72,
101, 102), reducing the production of collagenase (72) and TNFa (103) and ultimately
preventing matrix degradation (61). Unlike IL-1, IL-6 did not suppress proteoglycan
synthesis in humans, mice, and cows (104-106). IL-8 is a potent chemoattractant peptide
that induces neutrophils to release elastase resulting in proteoglycan release (107, 108).
Also produced by chondrocytes, IL-8 up-regulates inducible nitric oxide synthase (iNOS)
and enhances NO synthesis (109, 110). Other cytokines with anti-inflammatory activities
are interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (71). The synthesis of
IL-1 and TNFa were inhibited by IL-4 and IL-10. Their implications in OA remain to be
elucidated, though an imbalance in the destructive and protective cytokines may

contribute to the development of 0A (111, 112).

3.2 Inflammatory Mediators
3.2.1 Nitric Oxide

NO is an inorganic free radical produced from arginine catalyzed both by iN OS
and constitutive forms of nitric oxide synthase, the endothelial nitric oxide synthase

(eNOS) and neuronal nitric oxide synthase (nNOS). It is a highly reactive agent that

14

degrades very quickly into its stable end products, nitrate or nitrite (113, 114). NO is an
important cellular signaling molecule associated with the pathogenesis of 0A (115).
Articular chondrocytes increase production of NO in the presence of cytokines. In fact,
activated articular chondrocytes produce more NO than any other cells (87).

The expression and protein levels of iNOS are higher in OA and RA cartilage
relative to healthy articular cartilage and increases with severity of the joint disease (115).
NO is excessively produced by cartilage and synovial membrane in OA and RA patients
(116). Inflammation and cartilage destruction, increased synthesis of cytokines,
inhibition of IL-lRa, proteoglycan and collagen synthesis, activation of MMPs, and
inactivation of TIMPs are several pathological and molecular events induced by NO
(117-122). An inverse relationship between nitrite and proteoglycan synthesis has also
been reported (123). Other effects of NO on cartilage include inhibition of transforming
growth factor (TGF)-B; as well as a decline in matrix synthesis in the presence of insulin-
like grth factor-1 (IGF-l) (124, 125). Cell death by apoptosis is another mechanism of
cartilage damage mediated by NO (126).

Mice with genetically disrupted iNOS demonstrated a decline in proteoglycan
loss, diminished cartilage lesions and osteophyte formation by 50% (92). The
administration of iNOS inhibitor led to a decline in cartilage degradation,
cyclooxygenase-2 (COX-2) expression, levels of PGE2 and MMPs. Inhibitors of iNOS
also restored proteoglycan synthesis and decreased cartilage lesions (123, 127). Cytokine
induced iN OS and eventually NO production in chondrocytes requires the MAPK
cascade and NFKB pathway (128, 129). iNOS mRNA expression and NO production

were inhibited with a p38 inhibitor but not an ERKl/2 inhibitor (129, 130).

15

3.2.2 Prostaglandin E2

PGE2 plays a critical role in various biological firnctions and pathological
processes. Its synthesis is catalyzed by prostaglandin H synthases or cyclooxygenases.
There are two forms of cyclooxygenases, cyclooxygenase-1 (COX-1) being the
constitutive enzyme that regulates physiological functions while COX-2 is the inducible
form of the enzyme (131, 132). COX-2, their mRNAs and products but not COX-1 was
elevated in articular cartilage stimulated with cytokines (133). These enzymes share 60%
similarity in sequence (134). Both isozymes catalyze the reaction where the intermediate
prostaglandin G2 (PGG2) is formed fiom arachidonic acid. Peroxidase then reduces PGG2
to prostaglandin H2 (PGH2) that gets converted to PGE2 by a specific isomerase, the
prostaglandin E (PGE) synthase.

The functions of PGE2 include regulation of vasodilation, platelet aggregation,
blood pressure, smooth muscle contraction, pain perception and inflammation. PGE2 is
the most abundant prostanoid found in diseased joints (135) and is spontaneously
produced in OA cartilage specimens (136, 137). PGE2 mediates the inflammatory
response and is responsible for pain and swelling of the inflamed joint together with other
leukotrienes. Stimulation of chondrocytes with IL-1 resulted in a dose-dependent increase
in COX-2 mRNA expression and protein synthesis (138). COX-2 gene expression is
augmented 10 to 80 fold upon stimulation with cytokines or endotoxin (139, 140). The
induction of COX-2 is accompanied by rising concentrations of PGE2 (141). COX-2
selective inhibitors were effective in reducing PGE2 levels, preventing acute and chronic
inflammation and exhibiting analgesic effects (142-145). An increase in microsomal

prostaglandin E synthase 1 (mPGEsl), one of the inducible isoforms of the isomerase

l6

was observed with IL-1 treatment in synovial cells (146) and inarticular chondrocytes
(147, 148). The elevation of mPGEsl is in concert with COX-2 and PGE2 synthesis (146,
149, 150). PGE2 elicits its catabolic effect on articular cartilage by enhancing
proteoglycan degradation and release, the production of MMP-3 and inhibiting TIMP-l
synthesis (60, 73, 151, 152).

Signaling by PGE2 is mediated via the NFKB and MAPK pathway either in
concert or independently. Similar to NO, the use of a selective p38 but not ERK1/2
MAPK inhibitor reduced COX-2 transcript, COX-2 protein, and PGE2 synthesis in in
vitro models of OA (153-156). The p38 MAPK also stabilizes COX-2 transcript (157).
NFKB is involved in regulating COX-2 gene expression and PGE2 production (158).
Beta-catenin also plays a role in regulating COX-2 expression in chondrocytes and
synovial cells (159, 160). Other transcription factors such as members of c/EBP and
STAT families may participate in COX-2 modulation since the promoter of COX-2 gene

contains sites for these factors (161-163).

3.3 Matrix Enzymes
3.3.1 Matrix Metalloproteinases

MMPs are a family of neutral zinc dependent extracellular proteinases that are
responsible for ECM remodeling. They play a prominent role in cartilage pathology and
are significantly elevated in OA cartilage (164, 165). These enzymes are regulated at
several points. They are synthesized and secreted in a latent form, converted from the
proenzyme form to an active enzyme extracellularly, and form complexes with inhibitors,

the TIMPs (166). MMPs are regulated at the transcriptional level. MMPs are responsible

l7

for matrix degeneration in OA and their release are elevated with IL-1 stimulation (167,
168). MMP inhibitors are effective in abolishing proteoglycan release mediated by
MMPs (169). Osteoarthritis and IL-1 mediated catabolism of articular cartilage are also
attributed to an imbalance between MMPs and TIMPs where TIMPs are less abundant
than MMPs (31, 170).

MMPs can be generally classified as collagenases, gelatinases, stromelysins, and
membrane type MMPs (171). The collagenases cleave the native triple helix of types I, II
and III collagen and they include MMP-l , MMP-8 and MMP-13. The levels of these
collagenases are significantly raised in response to IL-1 (64, 165). MMP-13 has greater
importance in OA because it has the broadest substrate specificity, degrades aggrecan and
type II collagen preferentially, and its expression is significantly higher in OA cartilage
(64, 65, 67, 172, 173). MMP-l3 also cleaves type II collagen more rapidly than MMP-1
(174). Both AP-l and NFKB transcription factors are necessary for IL-1 stimulation of
MMP-1 and MMP-13 gene expression (50, 165, 175). Of the collagenases, MMP-8
mRNA expression was present only in minute amounts in lL-l stimulated and 0A
cartilage both in vivo and in vitro (176).

Two gelatinases, MMP-2 and MMP-9 that break down unwound collagen fibrils,
link protein, and proteoglycan have been detected in higher concentrations in OA
samples relative to normal cartilage (177-179). These gelatinases were also detected in
synovial fluid of horses affected with CA (180). Both zymogens can be stimulated with
IL-1 and have the ability to activate MMP-3 and MMP-13. MMP-2 is also constitutively

expressed (180). MMP-2 and MMP-9 form complexes with TIMP-2 and TIMP-l

respectively (181).

18

The stromelysins are MMP-3, MMP-10 and MMP-11, which have a more general
proteolytic activity against aggrecan, fibronectin, collagen, gelatin, laminin, and elastin.
MMP-3 plays a central role in remodeling of the ECM and fully activating the
procollagenases, specifically MMP-1, and MMP-9 (182, 183). 0A chondrocytes and
those responding to cytokines demonstrate significant up-regulation of MMP-3 coupled
with a decrease in aggrecan expression and proteoglycan concentration (64, 71, 184-186).

The membrane type MMPs with proteolytic and collagenolytic activities are
MMP-l4, Mlva-IS, MMP-16, and MMP-l7. These also participate in activating MMP-2,
MMP-9 and MMP-13 (179, 187-190). MMP-14 is predominant and plays a pivotal
function in remodeling connective tissues (191). The mRNA expression of MMP-14 is
elevated in OA cartilage and by cytokines (179, 192). The exact roles of the membrane

type MMPs in the pathophysiological digestion of the matrix remain to be studied.

3.3.2 Aggrecanases

Aggrecanase-l (Agg-l) and aggrecanase-2 (Agg-2) are other endopeptidases
reported to be involved in cartilage catabolism (193, 194). Agg-l and Agg-2 are enzymes
from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs)
protein family (195-198) that have been detected in the joint capsule, synovium, and
cartilage (199). Agg-l is also known as ADAMTS-4 while Agg-2 is synonymous with
ADAMTS-5. Aggrecanases cleave the interglobular domain of aggrecan between
residues Glu373 and Ala374, unlike MMPs that cleave between Asn341 and Phe342
(200, 201). Agg-l acts primarily at the Glu373-Ala374 site and secondarily at the MMP

site (202). Agg-l is induced by cytokines while Agg-2 is reported to be constitutively

l9

expressed (193, 203). Both proteins have been implicated as the primary enzymes
responsible for aggrecan release from CA and injured joints (204, 205). Cleavage of
aggrecan precedes collagen breakdown and is a critical event in matrix degeneration.
Aggrecanases mediate aggrecan turnover in bovine and porcine cartilage cultured with

IL-1 or TNFa (68, 193, 206).

3.3.3 Tissue Inhibitor of Metalloproteinases

TIMPs are major endogenous regulators of matrix degrading enzymes. Their
abundance is slightly greater than MMPs in normal cartilage but decreases below that of
MMPs in OA and cytokine-treated cartilage. TIMPs have the potential to reduce
proteoglycan degeneration and those identified to participate in cartilage metabolism are
TIMP-l, TIMP-2, and TIMP-3 (207). Collagen release was correlated with low levels of
TIMPs (208). TIMP-l forms a bimolecular complex with proMMP-9 and active forms of
MMP-1, MMP-3 and MMP-9 (209). Bovine chondrocytes responding to IL-1
demonstrate down-regulation of TIMP-1 expression (210). The administration of TIMP-1
and TIMP-3 inhibits IL-l induced loss of GAGs (211, 212). TIMP-2 has a 40% sequence
identity with TIMP-1 and binds covalently to both pro and active form of MMP-2 (209).
Its synthesis decreases in OA explants (213). TIMP-3 is localized in the ECM and
exhibits inhibitory activities against MMP-1, MMP-2, MMP-3, MMP-9, Agg-l and Ag-
2 (214-217). Activation of latent proMMP-2, 9, and 13 is inhibited by TIMP-2 and

TIMP-3 (190).

20

4. Glucosamine and Chondroitin Sulfate as Therapeutic Agents for CA
Conventional therapies available for treating 0A are basically only effective for
symptomatic relief. These are primarily non-steroidal anti-inflammatory drugs (N SAIDs)
such as ibuprofen and naproxen, and corticosteroids and COX-2 inhibitors, which are
analgesics and are anti-inflammatory. While these medications often are efficacious in
attenuating OA symptoms such as pain and reversing the loss of joint function, they are
far from ideal as therapeutic agents. NSAIDs can cause serious adverse effects including
gastrointestinal bleeding, peptic ulcer, and liver and kidney failure. They are responsible
for about 16, 000 deaths and 100, 000 hospitalizations a year (218). COX-2 inhibitors
have been linked with increasing risk of cardiovascular disease (219). Not only do
conventional medications fail to prevent the progression of OA, they have been
associated with acceleration of joint damage (220, 221). The availability of therapeutic
agents that can provide greater symptomatic relief and less adverse reactions will be
beneficial in managing OA. Natural substances such as glucosamine and CS are some
safer alternatives with preliminary evidence of being able to retard and repair cartilage
degradation while manifesting symptomatic relief. Glucosamine and CS have been
marketed as nutraceutical supplements for over 3 decades. They are termed nutraceuticals
because they have both nutrient and pharmaceutical properties. They are slow acting

compounds and hence, are also called symptomatic slow acting drugs.

4.1 Structure and Composition

Glucosamine is a naturally occurring amino monosaccharide formed by the

transfer of the amide nitrogen of glutamine to fructose on carbon 2. It is a precursor

21

molecule for components in cartilage matrix, synovial fluid, and other tissues (222, 223).
Glucosamine is the principal building block and intermediate substrate used in the
synthesis of N and O-linked GAGs and thus, can be rate-limiting for proteoglycan
production. Once glucosamine gets acetylated to N-acetylglucosamine, it becomes a
component of GAGs such as keratan sulfate and hyaluronic acid. Glucosamine can also
be converted to galactosamine via isomerization to become precursors of CS and
dermatan sulfate. Radioligand studies have shown that glucosamine is incorporated into
cartilage on the GAG chains via the hexosamine pathway (224, 225). Glucosamine has a
relatively low molecular weight (179.17 kDa). This nutraceutical is normally extracted
from exoskeletons of shellfish and is commercially marketed either as glucosamine
hydrochloride (GLN), glucosamine sulfate (GS) or N-acetylglucosamine (GlcNac).

CS is a complex GAG that is a major component of aggrecan (226). It is a
heteropolymer consisting of dissacharide units of sulfated N-acetylgalactosamine and
glucuronic acid. Sulfation of CS can occur either at carbon 4 or 6 of N-
acetylgalactosamine producing chondroitin-4-sulfate or chondroitin-6 sulfate
respectively. CS found in supplements is typically obtained from bovine tracheal
cartilage although it has also been obtained from many types of connective tissue from
animals such as the nasal cartilage, articular cartilage, bones, sclera, leukocytes, blood
platelets, skin, umbilical cord, and cardiac valves. The molecular weight of CS is
variable, ranging from 6 to 50 kDa, since they are marketed in varying chain length,

charge density and location of the sulfate groups.

22

4.2 Pharmacokinetics

Glucosamine is rapidly absorbed by the small instestine via glucose transporters
(227). At least 90% of orally administered glucosamine is absorbed in both human and
animals (222, 228). The tissue distribution of a single dose of glucosamine following oral
and intravenous (IV) administration is prompt and it has an affinity for cartilage as shown
in autoradiographic studies (224). Plasma concentration of orally administered
glucosamine peaked between 2 to 4 hours and at about 18 to 50 ug/ml (222, 229, 230).
Concentrations of glucosamine in the synovial fluid of horses given the nutraceutical via
the oral or IV route was 0.05 to 2.5 jig/ml (230).

The absorption of CS via the gastrointestinal tract varies depending on molecular
weight, chain length, location of the sulfate groups, charge density, and the source of CS
(231). CS is absorbed as disaccharide metabolites with low molecular weight and low
charge density CS being preferentially absorbed (232). Initially, there was much debate
regarding the absorption of CS following oral administration. This was due to detection
methods that lack sensitivity and specificity to differentiate between constituents of CS
disaccharides. The development of techniques to detect CS disaccharides has improved
recently. The absorption of CS taken by the oral route is at least 70% and it is
approximately 13% bioavailable (226, 233, 234). The absorption of CS obtained from
(shark cartilage was slower than that extracted from bovine origin (235, 236). Similar to
glucosamine, CS has tropism for articular cartilage (237) and accumulates in synovial
fluid (238). In humans, the peak plasma concentration of orally administered CS ranges

from about 5 to 11 ug/ml and peaked at about 8 hours (233, 235, 236).

23

The pharmacokinetics of GLN and low molecular weight CS (8 and 16.9 kDa)
administered concomitantly was reported in dogs and horses (239, 240). In beagle dogs,
plasma concentration of glucosamine reached a maximum at about 7 to 12 ug/ml with a
mean bioavailability of 12% after single and multiple dosing. The peak plasma
concentration and bioavailability of single dosing of CS were about 22 ug/ml and 5%
respectively. Bioavailability of CS increased significantly with multiple dosing in dogs,
implicating a carry over effect of CS. After oral dosing in horses, plasma concentration of
glucosamine peaked at 10.6 rig/ml and the bioavailability was 2.5%. As for CS following

oral and IV administration, peak plasma concentrations were between 36.5 to 210 ug/ml

and CS was about 22% bioavailable.

4.3 Dosage and Administration

Both glucosamine and CS are normally taken through oral administration in
humans as tablets or capsules. Effective treatment doses of oral glucosamine given to
human are 1500mg for GLN or 1884mg for GS, taken once daily or divided into two or
three equal doses (241-245). Typical oral dose for CS ranges fiom 800 to 1200mg, also
taken once daily or divided into two or three equal doses (241, 246-248). Some
glucosamine and CS are marketed in liquid form or as topical ointrnents. No
pharmacokinetic or clinical data exist for these forms of administration of glucosamine or
CS. Another concern regarding the liquid form is the lack of stability in the compound
since glucosamine needs to be stabilized with salts to maintain its potency. There is also

no proof as to how topical glucosamine or CS can penetrate the skin.

24

4.4 Safety Profile

Glucosamine and CS have a remarkable safety record. There are almost no cases
of adverse reactions or overdoses with these nutraceuticals. The good safety profile of
glucosamine is evident by pharmacokinetic studies and clinical trials investigated in
humans, dogs, and rats (222, 228, 249-252). Glucosamine has a smaller number of side
effects and is more tolerable when compared to other palliative analgesics (243, 253-
255). Glucosamine was also more favorable than NSAIDs in pharmacological therapeutic
index as its toxicity on the gastrointestinal tract is notably lower or practically absent,
even after prolonged administration. Studies in small animals and humans using very
high doses of glucosamine given through the IV route has raised concerns that
glucosamine may induce insulin resistance and thus, may be contraindicated in diabetic
patients (256, 257). To date, these claims have not been substantiated (258, 259). Studies
on patients with Type 2 diabetes mellitus have shown no alteration in blood glucose
markers with glucosamine supplementation (259). Another concern with glucosamine
pertains to allergies since this compound is extracted from shellfish (260, 261). Ig E that
initiates an allergic response does not come from the shell but flesh of shellfish (262) and
hence, this problem only occurs with low quality glucosamine products. A long-term
study reported no significant difference in any safety measures including allergies with
glucosamine (263).

Similar to glucosamine, CS also has an outstanding safety profile.
Pharmacokinetic studies and clinical trials demonstrated that CS is very well tolerated
and does not produce any toxic side effects in human, rats, and dogs (235, 237, 246, 248).

Concerns regarding possible anticoagulant activity with CS are unfounded as it does not

25

interact with antithrombin (264). Horses and small animals given glucosamine and CS in
combination at doses above the recommended daily dose showed no significant effect on
hemoglobin levels, clotting time or white blood cell count (232, 265). Oral CS

demonstrated an excellent toxicity profile when compared to various treatments for knee

OA such as NSAIDs, surgery and non-pharmacological therapy (255).

4.5 Clinical Trials

The first clinical trial with glucosamine as a therapeutic agent in humans was
conducted in Germany in 1969 followed by a number of short-term double-blind studies
in Europe, Asia, and the United States in the 19803 and 19905. The findings indicated
glucosamine was beneficial in modifying OA symptoms and possessed chondroprotective
properties while maintaining a good safety profile. Glucosamine can reduce joint
tenderness and swelling, decrease pain, and improve joint mobility, gait functions, and
quality of life. In double blind placebo controlled studies, patients treated with
glucosamine for at least 30 days experienced improvement in pain, tenderness and overall
joint function (251, 266). Alleviation of symptoms with glucosamine occurred faster than
patients on placebo (251). Trials comparing glucosamine to ibuprofen showed that the
nutraceutical was at least equal in potency to ibuprofen in reducing pain, but safer and
more tolerable (243, 254, 267). This amino sugar has also demonstrated improvement in
structural joint changes where joint space narrowing declined profoundly as assessed by
radiological methods (245, 268). The therapeutic effects of glucosamine are also
substantially conserved even well after therapy was discontinued (252, 267, 269). Recent

and more long term clinical trials, comparative studies plus meta-analyses substantiated

26

previous findings (245 , 270-274). In a meta-analysis, patients with hip or knee OA who
consumed glucosamine experienced about a 40% decline in pain and improvement in
mobility (275). Two hundred and twelve patients with knee OA given glucosamine for 3
years had a reduction in pain and no loss in joint space compared to placebo (245).

Although initial clinical trials with CS, similar to glucosamine, were relatively
short-term, CS was effective in alleviating 0A symptoms. Patients with knee, hip, and
finger OA taking low molecular weight CS used significantly less NSAIDs or other
analgesics and experienced pain relief (248, 276, 277). The residual effects of CS also
persisted longer than the NSAIDs therapy where patients experienced positive response
up to 3 months after discontinuation of the compound (276). Other beneficial effects of
CS include improvement in joint mobility, joint space narrowing and a reduction of
erosive 0A (247, 278, 279). CS was also advantageous in having an overall cost-
lowering effect (280, 281). There was a reduction in lameness and improvement in
function for horses treated with CS (282). Meta-analysis with CS supports its efficacy as
an OA therapeutic agent (283).

Glucosamine is increasingly marketed as a combination with CS and the
combination has enhanced efficacy. The combination of glucosamine and CS has been
proven efficacious in reducing pain, improving joint firnction, halting or reversing joint
degeneration in humans with mild to moderate OA of the knee (284, 285). In animals, the
combination is effective in improving temporomandibular joint disorders (241),
alleviating lameness in dogs (286, 287) and horses (288, 289) and managing pain in cats
(290). Dogs treated with the combination experienced a reduction in synovitis (287).

Severe cartilage lesions in an in vivo rabbit instability model of 0A were diminished with

27

glucosamine and CS supplementation (291). The improvement of lesions was more
apparent with the combination rather than the supplements given alone indicating
synergism of the agents (291). Besides possessing the ability to slow the progression of
OA, both agents are a natural product that poses almost no adverse effects and drug
interactions. The effectiveness of these modalities in OA therapy has been reviewed (274,

292).

4.6 Mechanism(s) of Action

Both glucosamine and CS are considered as structure-modifying agents with the
ability to retard the pathology of 0A. They have been claimed to possess
chondroprotective, anti-inflammatory, anti-arthritic, and anti-rheumatic properties
providing symptomatic relief, particularly in ameliorating joint pain. However, the exact
mechanism of action underlying the properties of the compounds and joint pain relief is
unknown. Outcomes from laboratories that only recently started exploring the
mechanistic aspect of these nutraceuticals by mostly employing concentrations between
0.1 to 10 mg/ml (Table 1), indicate that these agents are capable of supporting anabolic
events and arresting catabolic activities of chondrocytes. Both glucosamine and CS have
chondroprotective properties in viva and in vitra as evidenced by their ability to stimulate
proteoglycan synthesis (291, 293) and increase hyaluronic acid content and GAG
synthesis in synovial fluid (294, 295). An in viva study suggested that glucosamine and
CS may be synergistic in improving cartilage lesions while an in vitro experiment with
the nutraceuticals reported complementary effects in regulating catabolic mediators (291,

296). Both agents enhance the metabolic response of chondrocytes to stress in vitra

28

(297). The structure modifying effects of glucosamine and CS are also confirmed through
histological studies in rats and dogs as experimental models (294, 298). The combination
also decreases cartilage degradation by reducing the activity of MMP-9 and MMP-13
(296) and reverse the decrease in GAG synthesis (297).

The anabolic activity of glucosamine is supported by the fact that OA
chondrocytes treated with the hexosamine sugar demonstrated a dose-dependent increase
in proteoglycan synthesis, expression of aggrecan, GAG content, and elevated synovial
hyaluronic acid production (243, 295, 299-301). Glucosamine was a preferential
precursor for the galactosamine moieties of CS in cartilage explants (225). This was
recently contradicted by another study with human chondrocytes (302), possibly because
there was a lack of stress or stimulation on the cells. Glucosamine prevented the
inhibition of glucuronosyltransferase I gene expression and activity in IL-1 stimulated
chondrocytes (83). Over expression of glutamine fi'uctose-6-phosphate amidotransferase
(GFAT), a rate limiting enzyme in glucosamine biosynthesis, prevented lL-l provoked
decrease in proteoglycan synthesis (303). Aggrecan degradation was suppressed over a
long-term culture supplemented with glucosamine (304). Glucosamine was effective in
attenuating proteoglycan release slowing OA progression (305, 306). The anti-catabolic
activity of glucosamine treatment that halts cartilage breakdown is possibly mediated by
inhibition of proteolytic enzymes. Glucosamine inhibited aggrecanase activity, general
MMP, MMP-3, and collagenase activities in vitra (300, 305-308). Glucosamine was also
capable of pre-translational and translational regulation of MMPs by repressing mRNA
expression of MMP-1, MMP-3 and MMP-13, reducing MMP-3 protein by 18% to 65%

and decreasing MMP-13 activity (300, 309).

29

Pain amelioration with glucosamine may be attributed to a reduction in
inflammatory mediators possibly via regulation of lL-l signaling pathways such as
NFKB and MAPK. Glucosamine increased the expression of IL-lRII, a decoy receptor
that is unable to generate the IL-1 signaling pathway in chondrocytes (310). Explant
cultures treated with glucosamine demonstrated a decline in NO release into the media
(83, 305, 306, 311-314). Cytokine stimulated NO release was also depressed in
synoviocyte and chondrocyte co-culture transduced with GFAT (303). The iNOS
transcript and protein in cartilage stimulated with IL-1 was suppressed by glucosamine
(311, 315). Glucosamine also inhibited NFKB activity and translocation, COX-2 mRNA,
and protein expression in a dose-dependent manner coupled with an increase in the
inhibitor of NFKB in IL-1 induced articular cartilage (310, 316). Parallel with the
inhibition of COX-2, PGE2 production and release was also inhibited with glucosamine
(83, 306, 316, 317) and with over expression of GP AT that catalyzes glucosamine
synthesis (303). The anti-inflammatory properties of glucosamine may also be mediated
by inhibitory actions on neutrophils and p38 MAPK phosphorylation, although this study
was conducted with a relatively high glucosamine concentration (6 mg/ml) (318).

CS can regulate cartilage metabolism in vitra and in viva; it increases RNA
synthesis that correlates with increases in synthesis of proteoglycan and collagen (319-
321). CS also reversed IL-l inhibition of proteoglycan synthesis (321, 322). The
chondroprotective property of this GAG also includes cartilage repair by increasing
hyaluronic acid production (246, 293, 323). Hyaluronic acid is essential for maintaining
synovial fluid viscosity (246, 295). Oral administration of CS reduced proteoglycan loss

from articular cartilage in humans (277) and rats (324). In addition, CS inhibits cartilage

30

damage by suppressing aggrecanase, elastase, lysosomal enzymes and collagenolytic
activity (246, 323, 325, 326). CS possesses anti-inflammatory properties by mechanisms
involving the reduction of PGE2, reactive oxygen species and free radical release (246,

321, 327, 328).

5. Rationale for experiments

Of the available nutraceutical supplements as therapeutic agents for improving
joint health, glucosamine and CS are the most popular by far based on revenue. Although
quite a number of clinical trials have reported on the benefits of glucosamine and CS in
providing symptomatic relief, the exact mechanism of action remains unclear. Most in
vitra mechanistic studies that revealed potential chondroprotective and anti-inflammatory
properties of glucosamine and CS have used concentrations of the nutraceuticals which
are not biologically relevant or far from what is attainable in viva. Many of the
commercially available glucosamine and CS products are marketed as a combination, but
most studies have been conducted with the individual compounds. Several short-term
studies showed that the combination provides pain relief, inhibits cartilage degradation,
and have increased chondroprotective properties when given together (285, 289, 291,
296,298)

Thus, the present experiments are designed to determine the effects of
glucosamine and CS, at concentrations achievable in viva in the blood after oral and IV
administration, alone and in combination, on molecular events of OA mediators using an
IL-l induced bovine articular cartilage explant model. The mRNA abundance of genes

encoding ECM catabolic mediators including MMP-2, MMP-3, MMP-9, MMP-13,

31

MMP-l4, Agg-l and Agg-l will be assessed. Gene expression of ECM anabolic
mediators measured will include TIMP-l, TIMP-2, TlMP-3, aggrecan and type II
collagen. The protein abundance of TIMP-3 will be assessed as well. Besides matrix
enzymes, genes encoding inflammatory mediators such as NFKB, COX-2, mPGEsl,
iNOS and eNOS will be other parameters measured. Biochemical variables assessed will

include NO and PGE2 synthesis, and proteoglycan release.

32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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299. Bassleer C, Rovati L, F ranchimont P. Stimulation of proteoglycan production by
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301. McCarty MF. Enhanced synovial production of hyaluronic acid may explain rapid
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302. Mroz PJ, Silbert J E. Use of 3H-glucosamine and 3SS-sulfate with cultured human
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49:577-8 1 .

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325. Baici A, Bradamante P. Interaction between human leukocyte elastase and
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327. Campo GM, Avenoso A, Campo S, Ferlazzo A, Altavilla D, Micali C, et al.
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328. Campo GM, Avenoso A, Campo S, Ferlazzo AM, Altavilla D, Calatroni A. Efficacy

of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats.
Arthritis Res Ther 2003; 52R122-31.

63

CHAPTER 2

Chan PS, Caron JP, Rosa GJM, Orth MW. Glucosamine and chondroitin sulfate
regulate gene expression and synthesis of nitric oxide and prostaglandin E2 in
articular cartilage explants. Osteoarthritis Cart 2005; 132387-94.

64

Summary

Objective: Glucosamine (GLN) and chondroitin sulfate (CS) are widely used to alleviate
symptoms of osteoarthritis (0A). However, the mechanism(s) of action of these
nutraceuticals remains unresolved. In the present study, we determined the effect of
physiologically relevant concentrations of GLN and CS on gene expression and synthesis
of nitric oxide (NO) and prostaglandin E2 (PGE2) in cytokine-stimulated articular
cartilage explants.

Methods: Using bovine articular cartilage explants in culture stimulated with IL-1, the
effects of physiologically relevant concentrations of GLN and CS on gene expression of
inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS),
cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGEsl) were
assessed with quantitative real-time polymerase chain reaction (Q-RT-PCR). The
production of NO and PGE2 was also quantified.

Results: Chondroitin sulfate and the GLN and CS combination at concentrations
attainable in the blood down-regulated IL-l induced mRNA expression of iNOS at 24
and 48 hours post-culture. Up-regulated iNOS expression at 24 hours by IL-1 was also
suppressed by GLN. Glucosamine and CS transiently repressed the cytokine-stirnulated
mPGEsl transcript. Synthesis of NO was reduced with CS alone and the combination
after 24 hours of culture. Repression of COX-2 transcripts by GLN and CS was
accompanied by concomitant reduction in PGE2 release.

Conclusion: Our results indicate that physiologically relevant concentrations of GLN and
CS can regulate gene expression and synthesis of NO and PGE2, providing a plausible

explanation for their purported anti-inflammatory properties.

65

Keywords: osteoarthritis, interleukin-l, nitric oxide, prostaglandin, glucosamine,
chondroitin sulfate

Running title: Glucosamine and chondroitin sulfate regulate inflammatory mediators

Introduction

Osteoarthritis is characterized by the degeneration of matrix components of
articular cartilage accompanied with an excess production of proinflammatory cytokines.1
Interleukin-1 beta is widely accepted as one of the proinflammatory cytokines that plays
a pivotal role in the pathophysiology of 0A.2 It induces a cascade of catabolic events in
chondrocytes including the upregulation in genes of matrix metalloproteinases (MMPs),
iNOS, COX-2 and mPGEsl and the release of NO and PGE2.“5 Interleukin-1 also retards
anabolic activities of the chondrocytes leading to declines in proteoglycan synthesis and
collagen synthesis.7’ 8

Nitric oxide is produced from L-arginine catalyzed by a family of NO synthases.9'
H The constitutive isoforms are eNOS and neuronal nitric oxide synthase (nNOS). An
elevated level of iNOS is responsible for the secretion of NO, an inorganic free radical, in
large amount from CA cartilage. Nitric oxide promotes inflammation and articular
cartilage degeneration by increasing cytokine production, suppressing matrix synthesis,
elevating MMPs and inactivating tissue inhibitor of metalloproteinases (TIMPs).'2'l4

Prostaglandin E2 mediates inflammatory and pain responses, and is the most
abundant prostanoid found in the diseased joint.15 Cyclooxgenases and prostaglandin E

synthases are rate-limiting enzymes responsible for generating PGE2. The inducible form

of this enzyme is COX-2, which catalyzes the conversion of arachidonic acid to

66

prostaglandin H2 (PGH2) and mPGEsl, which isomerizes PGH2 to PGE2. The synthesis
of PGE2 correlates with up-regulation of COX-2 and mPGEsl .6’ '6 Prostaglandin E2
elicits its catabolic effect on articular cartilage by enhancing MMP production and
proteoglycan degradation and by inhibiting synthesis of TIMPs.'7‘ ‘8

Various compounds have been used in palliative treatment of OA. Two
compounds that are increasing in favor as a treatment option for joint diseases both in
humans and animals are glucosamine and chondroitin sulfate. These compounds are
termed nutraceuticals because they have both nutrient and pharmaceutical properties.
They have been used over the past 30 years for the treatment of OA and shown in several
animal and human studies to be beneficial in reducing articular cartilage degeneration,
pain, and inflammation. In animals, the combination reduces lameness in dogs and horses
and is effective in managing pain in cats. ”'21 Glucosamine and CS used alone or in
combination can decrease joint space narrowing, alleviate pain, and improve joint
mobility in humans affected with knee OA.”'25

Elucidating the mechanism(s) of action of GLN and CS in vivo is an active area of
research. Pain amelioration with GLN and CS may be attributed to a reduction in
inflammatory mediators. In vitro, cytokine-stressed cartilage explants treated with GLN
demonstrated a decline in iNOS transcript and NO release into the media.”30 Parallel
with the inhibition of COX-2, PGE2 production and release was also inhibited with
GLN.26' 28’ 31 Chondroitin sulfate possesses anti-inflammatory properties.32

To date, the majority of in vitro research has been performed with concentrations

of GLN and CS that exceed those likely to be obtained by oral administration.26'28’ 3" 33‘”

This was one of the reasons given by rheumatologists in their objection to recommending

67

GLN to patients.36 The concentrations of GLN in serum after oral and intravenous
administration range from 1 to 20 ug/ml while CS concentrations are in the 5 to 200
ug/ml range depending on the route of administration, species and the molecular weight
of CS.3742 Thus, the aim of the present study was to determine the effect of
concentrations of GLN (5 rig/ml) and CS (20 ug/ml) that have been attained in blood on
mRNA expression of iNOS, eNOS, COX-2 and mPGEsl and the production of NO and
PGE2 in articular cartilage explants stimulated with IL-1, a cytokine associated with

cartilage catabolism.

Materials and methods
EXPLANT CULTURES

Articular cartilage was isolated from the carpal joints of Holstein steers (18-24
months old) obtained from a local abattoir within 3 hours of slaughter. Cartilage discs (6
mm) were biopsied from the articular surface and did not include cartilage with gross
characteristics of OA and calcified cartilage. Two explant discs (approximately 60 mg
total wet weight) were randomly picked and cultured in each well of a 24-well Falcon
culture plate (Fisher Scientific, Pittsburgh, PA, U.S.A.) containing 1 ml of 1:1 modified
version of Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (Ham) (Gibco,
Grand Island, NY, U.S.A.), as previously described.27 The media was supplemented with
20 amino acids (Sigma, St. Louis, MO, U.S.A.), 50 rig/ml ascorbic acid, and 100 units/ml
penicillin/streptomycin (Gibco). The concentrations of amino acids added were 50% of
those previously reported.43 Cartilage explants were housed in a humidified incubator at

37°C with 7% CO2.

68

Explants were maintained in media without serum for 2 equilibration days prior to
the addition of treatments. After equilibration, all treatments received 10% fetal bovine
serum (F BS, Gibco) and 50 ng/ml of human recombinant interleukin-1 beta (rhIL-l B, R
& D Systems, Minneapolis, MN, U.S.A.) added to induce cartilage catabolism. The
concentrations of glucosamine HCl (5 rig/ml; F CHG49®, Nutramax Laboratories,
Edgewood, MD, U.S.A.) and low molecular weight (16.9 kDa) CS (20 rig/ml; TRH122®,
Nutramax Laboratories) chosen were well within the range of levels measured in the
blood”‘ 41. There were 5 treatments per experiment (Table I), an F BS control (Ctrl), 50
ng/ml rhIL-lB (IL-1), 50 ng/ml rhIL-l B with the addition of 5 jig/ml GLN (IL-1 + GLN),
50 ng/ml rhIL-l B with the addition of 20 ug/ml CS (IL-1 + CS) and 50 ng/ml rhIL-l B
with the addition of both GLN and CS (IL-l + GLN + CS). Each treatment consisted of
24 discs (12 wells) collected from a pool of 3 steers for an experiment. Cartilage discs
were collected 6, 24 and 48 hours post-stimulation, frozen in liquid nitrogen and stored at
-80°C until RNA isolation. Conditioned media were collected at 24 and 48 hours and
stored at 4°C for nitrite and PGE2 analysis. Experiment was repeated a total of 3 times for
each time point, each time using tissue from a pool of 3 different animals.

NITRIC OXIDE ASSAY

Nitrite is a stable end product of NO metabolism and was measured in
conditioned media using the Griess reagent and sodium nitrite as standard.44 Briefly, 150
pl medium was incubated with 150 pl of 1.0% sulfanilamide, 0.1% N-l-
napthylethylenediamide hydrochloride and 25% phosphoric acid at room temperature for
5 minutes. Due to some precipitation of reagents with CS, 96-well plates were spun at

3250 rpm for 3 minutes at 4°C. The remaining supernatant was transferred to a new plate.

69

Absorbance was measured at 540 nm using a Spectromax 300 plate reader (Molecular
Devices, Sunnyvale, CA, U.S.A.).
PROSTAGLANDIN E2 ASSAY

Prostaglandin E2 release into conditioned media was quantified using a
commercially available competitive enzyme linked immunosorbent assay kit (R & D
Systems). Indomethacin (10 ug/ml) was added to conditioned media samples collected at
24 and 48 hours post-stimulation to inhibit any prostaglandin synthase present, and stored
at -20°C until analysis. Samples were run according to the manufacturer’s instructions.
Absorbance was determined at 405 nm with a wavelength correction set at 590 nm.
TOTAL RNA ISOLATION

The extraction of RNA from explants was performed following a modified
protocol.45 Briefly, cartilage was homogenized in TRIzol® reagent (Invitrogen, Carlsbad,
CA, U.S.A.), chloroform was added to extract total RNA followed by vigorous agitation
and a 2 minute incubation. The aqueous phase containing RNA was collected after
centrifugation and the RNA was precipitated with ethanol. Total RNA was then purified
further with RNeasy mini columns (Qiagen, Valencia, CA, U.S.A.) and quantified by UV
spetrophotometry (Beckman Coulter, Fullerton, CA, U.S.A.). Total chondrocyte RNA
was resolved on 1.2% agarose gel to validate spectrophotometric determinations and
RNA integrity.
cDNA SYNTHESIS

For each sample, 2 pg of RNA was treated with DNase I (Invitrogen) to degrade
contaminating single and double standed DNA. Treated RNA was converted to single

stranded cDNA using Superscript II reverse transcriptase (Invitrogen) as recommended

70

by the manufacturer. Single stranded cDNA was quantified with UV spectrophotometry
(Beckman Coulter) and diluted to 50 ng/ul.

QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (Q-RT-PCR)
Primers for glyceraldehyde phosphate dehydrogenase (GAPDH, used as a
housekeeping gene) and molecules from Table II were designed using the Primer Express

software version 2.0 (Applied Biosystems, Foster City, CA, U.S.A.). Optimal
concentrations of each set of primers were determined with a Primer Matrix (lowest
standard deviation with no change in cycle to threshold (Ct)). Quantitative real-time PCR
was performed with 50 ng cDNA templates in 96-well plates using the ABI PRISM 7000
sequence detection system (Applied Biosystems). The analysis of each sample was
performed in duplicate. The cDNA templates were combined with optimal concentrations
of primers and SYBR Green PCR dye mix (Applied Biosystems) in a total volume of 50
ul and the amplification conducted as recommended by manufacturer.

The PCR conditions were 2 min at 50°C and 10 min at 95°C followed by 40
cycles of extension at 95°C for 15 sec and 1 min at 60°C, and data collected during the
last 30 sec. Threshold lines were adjusted to intersect amplification lines in the linear
portion of the amplification curves. The software automatically recorded the Ct. The
GAPDH gene was used as an endogenous control and run together with the samples for
each amplification reaction to allow for normalization of different samples for RNA
loading, cDNA synthesis, and amplification efficiencies and for comparison of samples
run at different times. The FBS control was used as a calibrator (i.e. the fold change for

control is 1.0). Replicated data was normalized with GAPDH and the fold change in gene

71

expression relative to FBS control treatment was calculated using the delta delta Ct
method.46
STATISTICAL ANALYSIS

Data for NO and PGE2 release into conditioned media were analyzed using a
linear mixed effects model, including the fixed effect of treatment and the random effects
of pool, the interaction between treatment and pool, and repeated measures within each
treatment and pool combination. Treatment effects were compared within each time point
using the multiple comparisons approach of Tukey. The computations were performed
using the MIXED procedure of SAS.47 Relative gene expression data determined using
Q-RT-PCR was analyzed considering the nonparametric ANOVA approach of
Friedman.48 Differences were declared statistically significant when P<0.05, unless
otherwise noted. Spearman’s rank correlations (r) between gene expression data and

biochemical data were computed using the CORR procedure of SAS.47

Results
THE EFFECT OF GLN AND CS ON GENE EXPRESSION

Bovine articular cartilage explants cultured with 50 ng/ml rhIL-lB for 6, 24 and
48 hours resulted in significant (P<0.01) up-regulation of iNOS expression relative to
control (Table H1). The large standard error for both IL-1 and IL-1 plus CS treatments at
6 hours was attributed to variation between pools of animal tissue and not technical error
within a replication. Glucosanrine (5 ug/ml) down-regulated iNOS mRNA expression
from 11.1 fold to 4.5 fold in explants cultured for 24 hours (Table III). Chondroitin

sulfate (20 ug/ml) added alone or in combination with GLN for 24 hours was as effective

72

as GLN in suppressing iNOS transcripts (Table III) but only the combination reduced
iNOS expression to control levels. After 48 hours, cytokine stimulated explants treated
with CS or GLN and CS repressed iNOS mRNA expression to control levels (Table III).
The iNOS transcript of explants treated with GLN alone at the 48 hour time point was not
different from IL-l stimulated explants (Table IH).

Cartilage explants stimulated with 50 ng/ml rhIL-l B demonstrated significant
elevation in COX-2 mRNA expression relative to control by 30 fold for 6 hours post-
stimulation (Table 111). Like iNOS mRNA expression at 6 hours, variability of COX-2
level of transcript at this time point was due to differences between replications. At the 6
hour time point, GLN or CS used individually and used in combination resulted in a
decline of COX-2 transcripts to control levels (Table III). The COX-2 gene for explants
treated with 50 ng/ml rhIL-l B was significantly up-regulated by 8 fold and 13 fold for 24
and 48 hours post-stimulation respectively (Table 111). At both time points, GLN
suppressed the activation of the COX-2 gene to control levels (Table III). Physiologically
relevant concentration of CS at 20 rig/ml did not repress COX-2 transcript increased by
IL-1 until 48 hours (Table III). The GLN and CS combination decreased COX-2
expression to 3.4 and 1.0 fold change for 24 and 48 hours respectively (Table HI), which
is not different from control.

The transcript for mPGEsl, the terminal enzyme involved in PGE2 synthesis was
elevated by about 9 fold (P=0.0008) relative to control, in explants cultured with 50
ng/ml rhIL-l B over a 6 hour period (Table HI). At this time point, GLN alone and the

combination nutraceuticals repressed mPGEsl mRNA expression to control levels (Table

73

 

111). No effect on mPGEsl expression was found for any of the treatments at 24 and 48
horns (Table III).
THE EFFECT OF GLN AND CS ON NO AND PGE2 SYNTHESIS

Nitric oxide released into conditioned media at 24 hours after the addition of
treatment was increased with 50 ng/ml rhIL-lB to 21.39 uM fiom 5.98 uM for FBS
control (Figure 1, Panel A). The addition of CS or GLN plus CS suppressed NO release
to 16.93 and 17.27 uM respectively (Figure 1, Panel A). At 48 hours, the elevation in NO
release with 50 ng/ml rhIL—l B was not reduced by either nutraceutical (Figure 1, Panel
B).

Explants stimulated with 50 ng/ml rhIL- 1 B released significantly more PGE2 into
media when compared to control. After 24 hours stimulation, PGE2 release was elevated
to 1986 pg/ml compared with basal PGE2 release at 169 pg/ml (Figure 2, Panel A). The
addition of GLN and CS, or in combination reduced PGE2 release induced by cytokine to
control levels (Figure 2, Panel A). The basal PGE2 release into media 48 hours post-
treatment was 171 pg/ml (Figure 2, Panel B). This level was elevated to 1122 pg/ml when
50 ng/ml rhIL-lB was added (Figure 2, Panel B). Glucosamine decreased PGE2 release
due to IL-1 by 68% (Figure 2, Panel B). Likewise, the combination resulted in a 76%
decline in PGE2 release induced by IL-1 (Figure 2, Panel B). Chondroitin sulfate addition

to the explants in vitro tended (P<0.10) to decrease PGE2 release (Figure 2, Panel B).

74

THE EFFECT OF GLN AND CS ON ENDOTHELIAL NO SYNTHASE GENE
EXPRESSION

Since the biochemical data on NO release conflicted with the Q-RT-PCR data on
iNOS gene expression profile, we decided to investigate the expression of the eNOS
gene, another isoform of the NO synthase enzyme. Only a transient pattern of eNOS
regulation by CS was witnessed. The IL-l mediated increase in eNOS transcript of 3.3
fold at 24 hours was significantly (P<0.05) reduced to 0.3 fold with 20 rig/ml CS. No

significant differences were seen with the other treatments (data not shown).

Discussion

To our knowledge, the present study is the first to describe the effect of GLN and
CS attainable in blood on gene expression and synthesis of inflammatory mediators using
a cartilage explant culture. The concentration chosen for GLN was 5 rig/ml while that for
CS was 20 ug/ml. These concentrations were at the low end of the range found in blood
after oral or intravenous administration.” 4" 42 Previous trials fiom our laboratory used
concentrations which are higher than the range found in the plasma of animals
administered GLN and CS.49 Another novelty of this research lies in the nutraceutical
combination investigated. Few in vitro studies have been conducted with the GLN and
CS combination although they are frequently marketed as a combination. One study
reported that they are synergistic,50 while another suggested they may be
complementary.49

Nitric oxide is implicated in the pathogenesis of arthritis. Normal cartilage

explants produce little NO.51 On the contrary, chondrocytes and the synovial membrane

75

in OA and rheumatoid arthritis (RA) patients produce NO abundantly and so does
cytokine-induced cartilage.5 "53 In fact, activated articular chondrocytes produce more
NO than any other cells, including synoviocytes, hepatocytes and macrophages.3
Inducible forms of NO synthase are found in cartilage and produce large amounts of NO
in response to IL-1.13 Both iNOS mRNA and protein concentrations are higher in OA
cartilage relative to healthy articular cartilage and increase with severity of the
degenerative joint disease.“ The ability of IL-1 to enhance iNOS transcript was also
evident in the present study (Table III).3 Nitric oxide suppresses proteoglycan synthesis
by inhibiting the process of sulfation on the glycosaminoglycan chains.”’ 55 Inhibition of
prolyl hydroxylase coupled with the suppression of type II collagen synthesis was
associated with the release of large amounts of NO.56 This inorganic free radical also
mediates the degradation of matrix components via activation of MMPs.” Besides
cartilage destruction, NO has also been implicated as a mediator of inflammatory
responses.‘2 Thus, the inhibition of NO synthesis may be effective as a therapeutic route
for DA.

Glucosamine and CS have been used successfirlly for treating chronic
inflammatory arthritides. One of the mechanisms of action of these compounds may be
the suppression of NO production. Previous work fi'om our laboratory using explants
from horses indicates decreased NO production with 250 ug/ml CS and with the
combination of 500 ug/ml GLN and 250 jig/ml cs.49 Inhibition ofNO with GLN
occurred at concentrations greater than 1mg/ml in equine explants and human
chondrocytes.27’ 30’ 49 Suppression of NO synthesis with GLN was associated with iNOS

mRNA and protein repression.29’ 30 In the current trial, CS alone and in combination with

76

I!”

GLN at physiological concentrations were effective in reducing NO synthesis induced by
IL-1 at 24 hours (Figure 1, Panel A). At 48 hours of culture, nitrite has accumulated to
the same level for all treatments except for control (Figure 1, Panel B). These levels of
NO in conditioned media may be explained by transcriptional regulation of genes
pertaining to the enzymes responsible for synthesizing NO. Although the levels of NO
were significantly reduced at 24 hours, these were marginal declines. Since NO and PGE2
can modulate the activity of the other”, even transient regulation of NO may affect the
anti-inflammatory response to some extent.

The inducible form of the NO synthases is iNOS and there are two constitutive
isoforms, eNOS and nNOS. The constitutive form of eNOS was initially thought to be
confined to endothelial cells while nN OS was localized to the central and autonomic
nervous systems.9 However, eNOS and nN OS have increasingly been identified in a lot
of different cell types including chondrocytes.58 Endothelial NO synthase may also be
inducible since human eNOS gene contains activator protein-l (AP-l) site in its promoter
region.59 The mitogen activated protein kinase (MAPK) pathway is activated by
cytokines leading to AP-l phosphorylation and up-regulation of a number of genes
associated with cartilage catabolism.60 Human OA chondrocytes express elevated levels
of nNOS.61 To date, only the genes for iNOS and eNOS and not nNOS in the bovine
species have been cloned and sequenced. Since eNOS mRNA levels were not markedly
affected at 48 hours, the lack of treatment effect on NO production could be in part due to
the activity of eNOS and potentially nN OS.

Prostaglandin E2, another catabolic mediator in the pathogenesis of 0A is formed

from a series of enzymatic reactions termed the arachidonic acid cascade. It mediates

77

synoviocyte proliferation and is responsible for pain and inflammatory responses.
Prostaglandin E2 enhances the degradative processes of proteoglycans.” ‘8 It is found in
diseased joints and is significantly elevated in synovial fluid of OA patients.” 18 Large
amounts of PGE2 are synthesized by chondrocytes that are exposed to IL-1 in vitro.18
Bovine explant cultures induced with IL-1 demonstrated significant increase in PGE2
from basal levels (Figure 2, Panel A & B). Elevated levels of PGE2 depleted matrix
components from intact articular cartilage cultured in vitro.62

The induction of COX-2 is largely responsible for elevated concentrations of
PGE2.31’63 Cyclooxygenase-2 expression is elevated in cartilage specimens of OA and
RA patients.“ 65 Findings from the present study demonstrated the significant up-
regulation of the COX-2 gene relative to control in response to IL-1 for all time points
(Table III) in accord with previous reports.3 1’ 66 Levels of PGE2 in our explant cultures
were positively correlated with COX-2 mRNA (r = 0.81, P<0.0001) in agreement with
Geng et. (11.66 Inhibiting PGE2 release is an increasingly common strategy for providing
relief to patients affected with arthritis as evidenced by the development of COX-2
inhibitors. '

In clinical studies, GLN and CS have been consistently beneficial in improving
the pain scores and inflammatory responses of patients affected with CA.”25 In vitro
results from previous work in our laboratory and others demonstrated declines in the
inflammatory mediator, PGE2, with GLN and CS supplementation.26’ 31’49’67 In the
present study, GLN, CS alone or in cambination reduced IL-l induced PGE2 levels. The
ability of GLN and CS to reduce PGE2 synthesis in the present study can be explained by

their effect on COX-2 gene expression. Since there is a lag time of about 12 to 18 hours

78

 

from the time the gene is expressed to the time it is translated into protein, the COX-2
mRNA data at 6 hours does correspond to the PGE2 levels at 24 hours while mRNA data
at 24 hours has a similar profile with PGE2 levels at 48 hours. The ability of GLN to
suppress COX-2 mRNA expression by about 2 fold at 24 hours post-stimulation parallels
the work by Largo et. al. in human chondrocytes.31 However, the concentration of GLN
used in their study was 1 mg/ml which is 200 times the concentration used in our study.
The present study substantiates the claim that repression of COX-2 transcripts and
eventually PGE2 is at least one of the mechanisms GLN alone or together with CS exert
their analgesic effects.

The effect of these chondroprotective agents on terminal PGE enzymes involved
in the formation of PGE2 has not been reported. In fact, we only know of one abstract that
has investigated this enzyme in chondrocytes.68 There are reports of concomitant
regulation of COX-2, mPGEsl and PGE2.'6’ 68 Transcripts of COX-2 and mPGEsl are
correlated in the present study (r = 0.43, P<0.05). However, differences in the timing for
COX-2 and mPGEsl induction may be due to dissimilar regulatory sequences in these
two genes.6 Explants treated either with GLN alone or in combination with CS repressed
IL-l induced mPGEsl expression at 6 hours post-culture (Table IH). Further studies on
the contribution of this brief regulation on mPGEsl in chondrocytes by the nutraceuticals
are warranted.

Determining the mechanism of action behind GLN and CS is essential to
elucidating the beneficial effects elicited by these compounds in combating arthritic
symptoms. Understanding how these agents may regulate NO and PGE2 is an active area

of research. Besides being pro-inflammatory, NO and PGE2 have the ability to increase

79

proteoglycan loss and decrease proteoglycan synthesis.”’ '8 Our results suggest a
transient modulation of mPGEsl and NO synthase gene expression, and NO synthesis by
the nutraceuticals. Physiologically relevant concentrations of GLN and CS repress COX-
2 expression that eventually translates into accompanying declines in PGE2 synthesis.
Thus, the findings in the present study may explain at least in part how IL-l induced
proteoglycan breakdown and decreased proteoglycan synthesis is prevented in vitroég’ 70.
Results fiom the present study indicate that GLN and CS may regulate the

expression and synthesis of NO and PGE2, providing a plausible explanation for the

symptomatic relief attributed to the administration of these compounds.

Acknowledgements
The authors are grateful to Angela E. Schlueter for technical assistance and Bellingar’s
Packing in Ashley, M1 for the provision of Holstein steers. This work was supported by

the Michigan Agricultural Experiment Station and Nutramax Laboratories, Inc.

80

 

Table I

Description of components found in treatment media of explant cultures

 

 

Treatment FBS (%) rhIL-l B (IL-1) Glucosamine Chondroitin
(GLbD sulfate (CS)

Ctrl 10 - - -

IL-1 10 50 ng/ml - -

IL-l + GLN 10 50 ng/ml 5 jig/ml -

IL-1 + CS 10 50 ng/ml - 20 rig/ml

IL-l + GLN + CS 10 50 11ng 5 Hg/ml 20 ug/ml

 

81

 

A2: ooov EUEUUGéiUOOtOOO Oz: cog UH<UH©UUOOHOOHGU<HO mice: 22 HmmOmE

2a 80
22a 80 0E0e0e0c<<<<0§00000e00 0E<00E~0~<0e0~<<<0<00 3.38.2. 380

A2: 80
<0<H0§<0<0p<00<0<0c<0<0§00<0 22a 80 <E0000E0~00e09000 22222 mozo

 

22.. ca 0E0<000H0<<0H<0c0<§00c 22.. oa<<00e0<<00e<00000 3202 002:
22a
22.. ca 0.205000550300000 oa<0c<t0<000<00e00e<00 32089.. 20.20
008::
A:o::b:00:00v BEE— 0E0>0m 3030:5058 208:: Heston ”8200.000: 0:00
0:50.50

 

:03000: 50:0 00808239083200: 03:82:56 :8 00m: amalhv 2082.2.» 0020.6: 0:0 0850a
= 030,—.

82

Table III

Relative gene expression of explants stimulated with rhIL-l B and physiologically
relevant concentrations of glucosamine and chondroitin sulfate

 

Mean fold change (:1: SE) in gene expression relative to control 6 hours post-stimulation

 

 

 

 

 

 

 

Treatment
Gene IL-1 IL-1 + GLN IL-l + cs IL-l + GLN + cs
iNOS 87.7 i 73.6 a 4.1 :l: 1.0 a 26.7 a: 24.9 a 4.1 3: 2.8 ‘
cox-2 30.0 :1: 13.3 b 1.5 :1: 0.9 a 2.7 :l: 2.5 a 3.3 a: 3.1 ‘
mPGEsl 8.7 i 2.8 b 4.5 i 1.0 a 5.4 a: 1.5 b 4.9 i 1.5 a
Mean fold change (0 SE) in gene expression relative to control 24 hours post-stimulation
iNOS 11.1131“ 4.53:1.9a 4.400.6‘ 3.03:0.8a
cox-2 7.7 :h 0.6 b 3.2 a; 2.0 a 7.1 :t 0.6 b 3.4 i 0.6 “
mPGEsl 0.7 :t 0.1 a 0.4 at 0.1 a 1.0 :t 0.3 a 0.6 3. 0.2 a
Mean fold change (d: SE) in gene expression relative to control 48 hours post-stimulation
iNOS 9.4027b 3.101.4" 2301.38 1.20103
cox-2 12.6 :t 4.0 b 3.4 :t 0.7 a 2.8 :l: 1.0 a 1.0 :l: 0.8 "
mPGEsl 4.8 i 2.0 a 1.6 :h 0.8 a 1.5 :l: 0.8 ° 0.8 i 0.3 '

 

Different superscripts for values within a row (i.e. one gene) denote significant
differences (P<0.05) between treatments.

83

Figure 1. Mean (iSE) nitrite release into conditioned media 24 hours (Panel A) and 48
hours (Panel B) afier treatments are added. Ctrl= fetal bovine serum control; IL-1= 50
ng/ml rhIL-l [3 (human recombinant interleukin-1 beta); IL-l + GLN= 50 ng/ml rhIL-l [3
and 5 ug/ml glucosamine; IL-l + CS= 50 ng/ml rhIL—lB and 20 ug/ml chondroitin
sulfate; IL-l + GLN + CS= 50 ng/ml rhIL-l B, 5 ug/ml glucosamine and 20 pg/ml

chondroitin sulfate. Different superscripts indicate significant differences at P<0.05.

84

25
l Panel A c

20-

10q

Nitrite (uM)

 

 

 

 

IL-l+GLN m1+cs m1+cm+cs
Treatment

   

18 -
Panel B

15-

12-

Nitrite (uM)

34

 

   

o4 .
cm

 

1 I

IL-l+GLN IL-l+CS lIrl+GLN+CS
Treatment

 

85

Figure 2. Mean (iSE) prostaglandin E2 (PGE2) release into conditioned media 24 hours
(Panel A) and 48 hours (Panel B) afier the addition of treatments. Ctrl= fetal bovine
serum control; IL-1= 50 ng/ml rhIL-l B (human recombinant interleukin-1 beta); IL-l +
GLN= 50 ng/ml rhIL-l B and 5 pg/ml glucosamine; IL-l + CS= 50 ng/ml rhIL-l B and 20
ug/ml chondroitin sulfate; IL-l + GLN + CS= 50 ng/ml rhIL-l B, 5 itng glucosamine

and 20 ug/ml chondroitin sulfate. Different superscripts indicate significant differences at

P<0.05. * Tended to be different than the IL-1 treatment (P<0.10).

86

PGE2 (pg/ml)

PGE2 (pg/ml)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

87

3500 1
Panel A
3000 4 '3
l
2500 4
2000 -
1500 0
1000 1 a
a
500 4 a I a
T l
1 I
0 U j I
Ctrl 11,1 IL—l + GLN 1L-1 + cs IL—l + GLN + cs
Treatment
1800 -
Panel B
b
1500 a
1200 a
900 4
bit
600 1 a i
1 l a
a
300 4 l T
J 1
l
0 1 a .
Ctrl IL-1 IL-1 + GLN IL-l + cs [L] + GLN + cs
Treatment

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94

CHAPTER 3

Chan PS, Caron JP, Orth MW. Glucosamine and chondroitin sulfate regulate gene
expression of proteolytic enzymes and their inhibitors in interleukin-l challenged
bovine articular cartilage explants. Am J Vet Res 2005; in press.

95

Abstract

Objective-To determine the effects of glucosamine (GLN) and chondroitin sulfate (CS),
at concentrations attainable in vivo, on expression of genes encoding proteolytic
enzymes, enzyme inhibitors and macromolecules of articular cartilage in interleukin-1
(IL-1) challenged bovine cartilage explants.

Sample Population-Bovine articular cartilage explants.

Procedures-Explants were cultured in a commercial medium for 48 hours. Cartilage was
then exposed to media containing 10% fetal bovine serum (F BS), 50 ng/ml IL-1, and 5
ug/ml GLN, 20 ug/ml CS alone or in combination for 24 and 48 hours. Cartilage was
frozen and RNA was extracted from the frozen cartilage. Gene expression of matrix
metalloproteinase (MMP)-2, 3, 9, 13 and 14; aggrecanase (Agg)-l and 2; tissue inhibitor
of metalloproteinase (TIMP)-1, 2 and 3; type II collagen (Col II) and aggrecan relative to
F BS control were assessed with quantitative real-time polymerase chain reaction.
Results-Up-regulated MMP-3, MMP-13 and Agg-l transcripts at 24 hours were
repressed by the GLN and CS combination by at least about 6 fold. Glucosamine was
effective in suppressing IL-l induced mRNA expression of MMP-1 3, Agg-l and Agg-2
while CS was effective in decreasing IL-l induced MMP-13 transcript at 24 hours. At 48
hours post-stimulation, GLN and CS added alone and in combination significantly
abrogated Agg—l and Agg-2 gene induction. The combination also decreased IL-l
stimulated MMP-13 transcript. Treatments had no effect on Col 1] or aggrecan
expression.

Conclusions and Clinical Relevance-Our results indicate that GLN and CS, at

concentrations that fall well within the range measured in synovial fluid and blood after

96

 

oral administration, may regulate expression of matrix degrading enzymes and their

inhibitors at the transcriptional level, providing a plausible mechanism for their purported

chondroprotective properties.

Introduction

Progressive and permanent articular cartilage degeneration is the hallmark
characteristic of osteoarthritis (OA). Biological and mechanical factors uncouple the
normal balance between articular cartilage degradation and synthesis. Cartilage
destruction primarily results from an imbalance between synthesis and degradation of the
extracellular matrix, particularly aggrecan and type II collagen (Col II). The expression
and activity of proteolytic enzymes such as matrix metalloproteinases (MMPs) and
aggrecanases exceeds that of endogenous inhibitors like tissue inhibitor of
metalloproteinases (TIMPs).l Excess production of matrix degrading enzymes is in large
part induced by the release of interleukin-1 (IL-l).2

Interleukin-1 is widely accepted as one of the proinflammatory cytokines that
plays a critical role in OA pathogenesis.3 It up-regulates proteolytic enzymes and retards
anabolic activities of the chondrocyte leading to declines in synthesis of collagen and
proteoglycan}5 Degradation of the extracellular matrix in vivo and in vitro with IL-1
stimulation is reported in the human, bovine, porcine, equine and lapine species.6

The nutraceuticals, glucosamine (GLN) and chondroitin sulfate (CS) have been
used over the past 30 years for treatment of OA and have reduced articular cartilage

degeneration in animal models and human clinical trials. In humans, both compounds

have demonstrated therapeutic effects on symptoms of OA, alleviating pain and

97

 

improving rnobility.7’9 In animals, the combination reduces lameness in dogs and horses
and is effective in managing pain in cats.”'2 Despite several studies with positive results,
use of these nutraceuticals is still not widely accepted due to small sample sizes and
short-term nature of the trials.

Many of the commercially available nutraceutical products contain both GLN and
CS. Glucosamine and CS were synergistic in protecting articular damage in vivo13 and
another study suggested they were complementary. '4 Thus, the chondroprotective
properties of each molecule may increase when taken together and combining them for
explant studies is important. Most in vitro studies have used concentrations of the
nutraceuticals that exceed those generally found in synovial fluid. '5 The concentrations of
GLN in synovial fluid and blood after oral and intravenous administration range from
0.05 to 20 [lg/1111.15.19 Depending on the route of administration, species, and the source
and molecular weight of CS, the concentration of CS in serum ranges from 5 to 200
rig/mlm'20 Thus, in the present study, we determined the effect of GLN (5 rig/ml) and CS
(20 ug/ml) individually and in combination, at concentrations attainable in viva, on gene
expression of IL-1 stimulated bovine articular cartilage explants. The genes of interest
included were matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, MMP-l3, MMP-14,
aggrecanase (Agg)-1, Agg—2, tissue inhibitor of metalloproteinase (TIMP)-l, TIMP-2,

TIMP-3, Col 11 and aggrecan.
Materials and Methods

Explant cultures-Articular cartilage was isolated from the carpal joints of

Holstein steers (18-24 months old) obtained fiom a local abattoir within 3 hours of

98

slaughter. Cartilage discs (6 mm in diameter) were biopsied from the articular surface and
did not include the calcified layer of the tissue and cartilage with gross characteristics of
OA. Two explant discs (approximately 60 mg total wet weight) were selected at random
and cultured in each well of a 24-well Falcon culture plate11 containing 1 ml of 1:1
modified version of Dulbecco’s modified Eagle’s medium (DMEM): nutrient mixture F -
12 (Ham)b, as previously described.” The media was supplemented with amino acids, 50
ug/ml ascorbic acid and 100 units/ml penicillin-streptomycin ”’21. Cartilage explants were
maintained in a humidified incubator at 37°C with 7% C02.

Explants were maintained in media without serum for 48 hours before the
addition of treatments. Media in the wells were exchanged daily. After equilibration, all
treatments received 10% fetal bovine serum (F BS) b. Human recombinant interleukin-1
beta (rhIL-l B)° was added at a concentration of 50 ng/ml to induce cartilage catabolism.
To examine the effects of GLN and CS, glucosamine HCld and low molecular weight
chondroitin sulfate6 were added to the wells at the same time as FBS and rhIL-l B. The
concentrations of GLN and CS chosen were 5 rig/ml and 20 rig/ml respectively. These
concentrations were well within the range of levels attainable in synovial fluid and blood
after oral and intravenous administration.” '20 There were 5 treatments per experiment
(Table I), an FBS control (Ctrl), 50 ng/ml rhIL-l B (IL-1), 50 ng/ml rhIL-lB with the
addition of 5 ug/ml GLN (IL-l + GLN), 50 ng/ml rhIL-lB with the addition of 20 rig/ml
CS (IL-1 + CS) and 50 ng/ml rhIL-lB with the addition of both GLN and CS (IL-l +
GLN + CS). Each treatment consisted of 12 wells (24 discs) collected from a pool of 3
steers for an experiment. Cartilage explants were collected 24 and 48 hours after

stimulation, frozen in liquid nitrogen and stored at -80°C until RNA isolation Experiment

99

was repeated a total of 3 times for each time point, each time using cartilage from a pool
of 3 different animals.

Total RNA isolation-Total RNA was extracted from cartilage explants following
a modified protocol.22 Briefly, cartilage was homogenized in TRIzol® reagentf and
chloroform was added to extract total RNA followed by vigorous agitation and a 2-
minute incubation. The aqueous phase containing RNA was collected after centrifugation
(4°C, 12,000 X g, 15 minutes) and RNA precipitated with an equal volume of 70%
ethanol. Total RNA was then purified further with RN easy mini columns8 and quantified
by UV spectrophotometryh. Total chondrocyte RNA was resolved on 1.2% agarose gel to
validate spectrophotometric determination and RNA integrity.

cDNA synthesis-F or each sample, 2 1.1g of total RNA was treated with DNase If to
degrade contaminating single and double stranded DNA. Treated RNA was converted to
single stranded cDNA using Superscript II reverse transcriptasef as recommended by the
manufacturer. Single stranded cDNA was quantified by UV spectrophotometry", and
diluted with RNase-free water to 50 ng/ul.

Quantitative real-time polymerase chain reaction (Q-RT-PCR)-Primers for
glyceraldehyde phosphate dehydrogenase (GAPDH, used as a housekeeping gene) and
molecules from Table II were designed using the Primer Express software version 2.0‘.
Nucleotide sequences used for primer design were obtained from public databases
(Genbank’). Optimal concentrations of each set of primers were determined with a primer
matrix [lowest standard deviation with no change in cycle to threshold (CT)]. Quantitative
real-time PCR was performed with 50 ng cDNA templates in 96-well platesi using the

ABI PRISM 7000 sequence detection systemi. The cDNA templates were combined with

100

 

optimal concentrations of primers and SYBR Green PCR dye mixi in a total volume of 50
111 and the amplification conducted as recommended by the manufacturer.

The PCR conditions were 2 min at 50°C and 10min at 95°C followed by 40 cycles
of extension at 95°C for 15 sec and l min at 60°C, and data collected during the last 30
sec. Threshold lines were adjusted to intersect amplification lines in the linear portion of
the amplification curves. The software automatically recorded the CT. The analysis of
each sample was performed in duplicate, and a standard deviation of S 0.5 between
replicates was set as a criterion for inclusion of data. The GAPDH gene was used as an
endogenous control and run together with the samples for each amplification reaction to
allow for normalization of different samples for RNA loading, cDNA synthesis, and
amplification efficiencies and for comparison of samples run at different times. The F BS
control treatment was used as a calibrator (i.e. the fold change for control is 1.0).
Replicated data was normalized with GAPDH and the fold change in gene expression
relative to FBS control was calculated using the 2(-AACT) method.23

Statistical analysis-Relative gene expression data determined using Q-RT-PCR
was analyzed with SAS", using the nonparametric ANOVA approach of Friedman. The
p-values of the gene-specific analyses were corrected for a False Discovery Rate of 5%
using the approach discussed by Benjarnini and Hochberg.24 Differences were declared

statistically significant when P<0.05, unless otherwise noted.
Results

The effect of glucosamine and chondroitin sulfate on gene expression of

proteolytic enzymes-Explants cultured with rhIL-l B for 24 hours resulted in significant

101

up—regulation of MMP-3, MMP-13, Agg—l and Agg-2 relative to control (Figure 1, Panel
A). Glucosamine repressed mRNA expression of MMP—l3, Agg-l and Agg—2 at 24 hours
after induction (Figure 1, Panel A). Chondroitin sulfate suppressed a 7.7 times resting
level induction of MMP-13 by IL-1 to control levels (Figure 1, Panel A). The GLN and
CS combination was effective in down-regulating IL-l induced gene expression of
MMP-3, MMP-13 and Agg-l (Figure 1, Panel A). There were no treatment effects for
MMP-2 and MMP-9 (data not shown), and MMP-l4 (Figure 1, Panel A & Panel B) at 24
and 48 hours post-culture.

The induction of MMP-13, Agg-l and Agg—2 mRNA expression by IL-1 at 24
hours were sustained up to 48 hours post-stimulation (Figure 1, Panel B). Glucosamine
and CS added alone or in combination for 48 hours significantly reduced IL-l stimulated
Agg-l and Agg-2 mRNA transcripts (Figure 1, Panel B). At 48 hours, the GLN and CS
combination decreased IL-l induced MMP-13 from 4.9 fold to 1.0 fold (Figure 1, Panel
B). No treatment effects were found for MMP-3 (Figure 1, Panel B) at the 48-hour time
point.

The effect of glucosamine and chondroitin sulfate on gene expression of
inhibitors of proteolytic enzymes-At 24 hours post-stimulation, the GLN and CS
combination tended (P<0.10) to up-regulate TIMP-3 transcript relative to CS (Figure 2,
Panel A). Treatment effect was not found for TIMP-1 (Figure 2, Panel A & Panel B) and
TIMP-2 (data not shown) at both 24 and 48-hour time points and TIMP-3 at 48 hours

(Figure 2, Panel B).

102

 

 

The effect of glucosamine and chondroitin sulfate on gene expression of
cartilage macromolecules-No treatment effect was found for expression of genes

encoding both Col 11 and aggrecan at all time points.

Discussion

Glucosamine and CS have been used for over 3 decades to treat OA. Clinical
trials showed that GLN and CS used individually or in combination are effective in
improving the symptoms of knee and hip OA.7'9’25’26 Their benefits also extend to animals
suffering from OA, which is a common and natural occurrence in dogs, horses and
catsm'12 Despite the fact they have been reported to be beneficial, their exact
mechanism(s) of action remains to be resolved. Previous experiments have provided
some clues about the chondroprotective properties of the nutraceuticals. However, one
major limitation to these studies is that concentrations of the nutraceuticals employed
have been quite high, ranging from 0.1 to 10 mg/ml.”'2 1’27'3 6 These concentrations are
most probably not attainable in viva. This is one reason given by rheumatologists for not
advocating the use of these nutraceuticals.” The current study represents an improved
approach to study the effects of GLN and CS in that concentrations of the nutraceuticals
were used at levels that have been reported in blood after oral and intravenous
administration. Now that we have determined the beneficial effects at these
concentrations, further experiments will be done looking at a range of concentrations
within the reported values. We also studied the effect of GLN and CS as a combination
since they are increasingly marketed as one entity and the combination has a putative

synergistic effect in decreasing the severity of cartilage lesions.l3

103

 

The predominate groups of enzymes responsible for cartilage breakdown are
MMPs and aggrecanases. The MMPs are a group of zinc containing proteinases that play
a central role in the degeneration of the extracellular matrix. During matrix breakdown,
the normal balance between MMPs and TIMPs is disrupted towards an increase in
activity of MMPs and a decrease in TIMPs.l Chondrocytes and synoviocytes in OA
specimens produce MMPs particularly MMP-3 and MMP-13.38 Cytokines stimulate the
expression of MMP-3 and MMP-133942 in agreement with the current study. MMP-3 has
a broad range of substrate specificity and deemed as one of the major enzymes
responsible for matrix proteolysis.43 MMP-13 plays a critical role in OA by breaking
down type II collagen preferentially.“ MMP-14 belongs to the membrane-type MMPs. It
hydrolyzes collagens and is capable of activating latent MMP-2.43 Thus, suppressing the
expression of these enzymes will likely be beneficial to OA patients.

The ability of GLN and CS to protect cartilage from being degraded
pathologically” may be explained at least in part by the regulation of MMP synthesis.
Specifically, the GLN and CS combination inhibited the cytokine-induced expression of a
number of the MMPs examined. Glucosamine and CS in combination exerted a transient
influence on the MMP-3 transcript in that a significant reduction in cytokine-induced
expression was observed at 24 but not at 48 hours. Previous reports have demonstrated
suppression of MMP-3 gene expression and enzyme activity by GLN.27’29’3 "34 However,
the concentration of GLN used was at least 100 ug/ml, far exceeding the concentration
employed in the present study. One study that used glucosamine sulfate at low
concentrations (~200 ng/ml) on human OA chondrocytes did show a decline in MMP-3

activity and production but not on mRN A expression.“ We observed that GLN and CS

104

used alone were effective in down-regulating MMP-13 for at least 24 hours and the
repression was prolonged by the combination. These findings supplements previous
studies that reported a decrease in MMP-13 protein and activity with the nutraceutical
combination[4 and with GLN alone at higher concentrations than we employed.27

To date, the effects of GLN and CS on MMP-l4 mRNA expression has not been
investigated. Neither the nutraceutical combination nor IL-l had a significant influence
on its expression. The absence of an effect of IL-1 on MMP-14 and MMP-2 is supported
by their reported constitutive expression in adult human cartilage.“46 Lack of an effect
with GLN and CS on the expression and activity of MMP-2 paralleled previous
studiesmiz"36 Expression patterns of MMP-2 also coincided with MMP-l4 since MMP-
14 activates it. The expression of MMP-9 is strongly inducible by inflammatory
cytokines.“ The activity of MMP-9 was, however significantly reduced by 1 mg/ml GLN
in equine cartilage explants.l4 The reason for an absence of IL-1 effect on MMP-9
mRN A expression in the present study is not known.

Aggrecanases plays a pivotal role in normal and pathological turnover of
aggrecan in cartilage.48 Together with MMPs, Agg-l and Agg-2 hydrolyzes the major
proteoglycan of articular cartilage and are induced by IL— 1 .28’42’49'50 The suppression of
IL-1 stimulated aggrecanase activity with GLN has been reported by Sandy et. al.28 and
[lie et. al.51 The concentrations of GLN used in the present study is about 80 and 200
times lower than that employed in studies by Sandy et. al.28 and Ilic et. al.5 1 respectively.
Chondroitin sulfate and the nutraceutical combination at pharmacological concentrations
successfully repressed both aggrecanases by 48 hours post-stimulation. Chondroitin

sulfate inhibited aggrecanase activity in culture.30 Simultaneous suppression of MMPs

105

and aggrecanases with GLN and CS supplementation may represent an effective way to
protect matrix components from catabolic processes.

Synthetic inhibitors of MMPs, TIMPs and agents that elevated TIMPs
demonstrated effectiveness towards reversing or halting catabolic activities associated
with OA.52 Addition of TIMP-1 and TIMP-2 reduced MMP-3 concentrations in canine
chondrocytes culture.53 TIMP-3 retarded IL-l stimulated proteoglycan release that is
mediated by aggrecanases4 Thus, upregulation of TIMPs may be one of many events to
counter catabolic activity in OA pathogenesis. TIMP-3 was up—regulated 2.4 fold by the
nutraceutical combination relative to IL-1 at 6 hours of culture (data not shown). In a
different study conducted in our laboratory employing 10 pg/ml GLN in combination
with 20 ug/ml CS found that TIMP-3 was significantly elevated relative to IL-1 at 8
hours post-stimulation (unpublished observation). Studies are ongoing in our laboratory
to verify if TIMP-3 transcript up—regulation is accompanied by an elevation in TIMP-3
protein when explants are supplemented with the nutraceutical combination.

Glucosamine and CS at these pharmacological concentrations did not affect the
expression of gene encoding aggrecan and type II collagen. Glucosamine sulfate at
concentrations of 10 ug/ml to 100 ug/ml had no effect on collagen production in
vitra.55'56 Horses affected with OA established no association of mRNA expression of
Ms with aggrecan and type II collagen gene expressions7 In normal human
chondrocytes, GLN had no effect on proteoglycan synthesis.58 Our findings on aggrecan
contrast the study by Dodge and co-worker45 that reported a dose dependent increase in
aggrecan mRNA with GLN (from about 200 11ng to 30 ug/ml) and this may be

attributed to the difference in species (bovine versus human), type of tissue (normal

106

 

 

 

versus OA), the source of GLN (GLN hydrochloride versus GLN sulfate), type of culture
used (explants versus monolayers), different concentrations of IL-1 (50 11ng versus 5
ng/ml), time length of culture (a maximum of 48 hours versus 72 hours) and the absence
versus the presence of preincubation with GLN. Lack of adequate stress may also prevent
changes seen in macromolecules with GLN and CS supplementation.”59 The synthesis
of these major macromolecules of cartilage is catalyzed by a number of enzymes. These
nutraceuticals may exert their effects on mediators of aggrecan and collagen synthesis or
affect macromolecule synthesis at a translational or post-translational level. Glucosamine
and CS increased sulfate incorporation in stressed cartilage59 and GLN prevented IL-l
mediated inhibition of galactose-betal, 3-glucuronosyltransferase I, an enzyme that
catalyzes the addition of the initial glucuronic acid residue in glycosaminoglycan
synthesis.29 Both GLN and cs also increase hyaluronic acid synthesis.“-61
With the recent controversy surrounding COX-2 inhibitors in humans and the
deleterious effects of nonsteroidal anti-inflammatory drugs on matrix homeostasis, the
use of alternative therapies for OA needs to be explored further. Since both GLN and CS
exhibited good therapeutic efficacy, remarkable tolerability and safety coupled with
effective symptomatic relief for humans and animals7'12’25’26’62, their continued use looks
promising. The present study shows that GLN and CS at concentrations that are
biologically relevant prevented some of the changes in gene expression associated with
IL-1. The inhibition of proteoglycan degradation with GLN and C855'59 may be in part
mediated by the repression of genes associated with some major cartilage-degrading

enzymes and possibly up-regulation of metalloproteinase inhibitors. The combination

was more effective in antagonizing some gene expression than if GLN and CS were used

107

 

individually. Further experiments should be conducted to determine if these changes in

gene expression can be replicated using an animal model for OA.

Footnotes

aFisher Scientific, Pittsburgh, PA.

I’Gibco, Grand Island, NY.

c R & D Systems, Minneapolis, MN.

dFCHG49®, Nutramax Laboratories, Edgewood, MD.
”TRH122®, Nutramax Laboratories, Edgewood, MD.
fInvitrogen, Carlsbad, CA.

gQiagen, Valencia, CA.

hBeckrnan Coulter, Fullerton, CA.

iPerkin-Elmer Applied Biosystems, Foster City, CA.
jGenbank Database, National Centre Biotechnology Information.

"SAs Institute Inc, Cary, NC.

108

Table I: Description of components found in treatment media of explant cultures

 

 

Treatment FBS (%) rhIL—lB (IL-1) Glucosamine Chondroitin
(GLN) sulfate (CS)

FBS control 10 - - -

IL-1 10 50 rig/ml - -

IL-l + GLN 10 50 ng/ml 5 rig/ml -

IL-l + CS 10 50 ng/ml - 20 rig/ml

IL—l + GLN + CS 10 50 ng/ml 5 “g/ml 20 pg/ml

 

F BS= fetal bovine serum; rhIL-1B= human recombinant interleukin-1 beta

109

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

05505550550000 55505005050550 3.380.... 5.20
0055005055550005 5550005505055 285 58000.
55505000050500 50050505000550 848x 0 60
50505055005000 55550500550000 53.2122 5-55
0505550550005005 55550050555000 53.2122 055
5050550000000 5050505055550 53.2122 :55
05505005505500 55000505000055 ESE... 0-0m?
55055500500000 55055505055000050 25052 70.0....
55500505000000 05050505005050 02.41.? 2.02:
50505050055555. 0555555005005<wwwm50 332122 2.52:
05050000055500 505055505550500 3542122 0.52:
00555550050005 05505000050500005 082:... 5.022
5050500000050005 550000550000505 2542122 ~50:

1038:: 5530000.
50.55 0E0>0¢ 50.5.:— Eazeh 0:03:0U 0:00

 

 

 

56000.. 32.0

00208.nen oat-.00.. 02:35:50 .55 con: 500585 .«e non—om we 5.0.41.3 000:0:000 50.5.5 09.0.5.— ES 203.—eh "~— 030,—.

 

110

Figure 1. Mean i SE expression of genes encoding proteolytic enzymes relative to F BS
control for cartilage explants stimulated for 24 hours (Panel A) and 48 hours (Panel B).
FBS = fetal bovine serum. IL-1 = 50 11ng human recombinant interleukin-1. IL-1 +
GLN = 50 ng/ml human recombinant interleukin-1 with the addition of 5 1.1ng
glucosamine HCl. IL-l + CS = 50 ng/ml human recombinant interleukin-l with the
addition of 20 ug/ml chondroitin sulfate. IL-l + GLN + CS = 50 ng/ml human
recombinant interleukin-l with the addition of 5 pg/ml glucosamine HC1 and 20 pg/ml
chondroitin sulfate. MMP-3 = matrix metalloproteinase-3. MMP-13 = matrix
metalloproteinase-13. MMP-l4 = matrix metalloproteinase-l4. Agg-l = aggrecanase-l.
Agg-Z = aggrecanase-2. Different letters within a gene indicate significant (P<0.05)

differences between treatments.

111

Panel A

D FBS control I IL-1 I lL-1 + GLN [D lL-1 + CS B lL-1 + GLN + CS

161

Mean relative gene expression

 

 

Panel B

1“

N

Mean relative gene expression

 

OJ

 

112

Figure 2. Mean i SE expression of genes encoding inhibitors of proteolytic enzymes and
cartilage macromolecules relative to F BS control for cartilage explants stimulated for 24
hours (Panel A) and 48 hours (Panel B). FBS = fetal bovine serum. IL-1 = 50 ng/ml
human recombinant interleukin-1. IL-1 + GLN = 50 ng/ml human recombinant
interleukin-l with the addition of 5 ug/ml glucosamine HCI. IL-l + CS = 50 ng/ml
human recombinant interleukin—1 with the addition of 20 pg/ml chondroitin sulfate. IL-l
+ GLN + CS = 50 11ng human recombinant interleukin-1 with the addition of 5 ug/ml
glucosamine HCl and 20 pg/ml chondroitin sulfate. TIMP-1 = tissue inhibitor of
metalloproteinase-1. TIMP-3 = tissue inhibitor of metalloproteinase-3. Col II = type II
collagen. Different letters within a gene indicate that treatments tended (P<0.10) to be

different.

113

Panel A

El FBS control I lL-1 I IL-1 + GLN ll] lL-1 + CS a lL-1 + GLN + CS

ab

Mean relative gene expression

 

 

Panel B

&

u

21

 

Mean relative gene expression

 

IMP-1 TIMP-3 Col ll Aggrocan

114

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CHAPTER 4

Chan PS, Caron JP, Orth MW. Short term gene expression changes in interleukin-
IB stimulated cartilage explants with glucosamine and chondroitin sulfate. Arthritis
Rheum 2005; submitted.

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ABSTRACT

Objective. To determine the short-terrn effects of glucosamine (GLN) and chondroitin
sulfate (CS) on expression of genes encoding inflammatory mediators and matrix
enzymes in interleukin-1 (IL-1) induced bovine cartilage explants.

Methods. Dose response experiments were conducted for IL-1, GLN and CS to select
concentrations of each that will help improve the sensitivity of detecting treatment effects
on cartilage explants. Based on the dose response experiments, treatments included F BS
control, 15 ng/ml IL-1, and 15 ng/ml IL-l with the addition of 10 ug/ml GLN and 20
rig/m1 CS. Media were measured for nitric oxide (NO) and prostaglandin E2 (PGE2) while
explants were frozen for RNA extraction at 8, l6 and 24 hours. Gene expression relative
to FBS control for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2),
microsomal prostaglandin E synthase-1 (mPGEs 1 ), nuclear factor kappa beta p65 subunit
(NF KB), matrix metalloproteinase (MMP)-3, l3, aggrecanase (Agg)—1, 2 and tissue
inhibitor of metalloproteinase-3 (TIMP-3) were assessed with quantitative real-time
polymerase chain reaction (Q-RT-PCR). In a separate study using incubation of explants
with the aforementioned treatments for 48 hours, proteoglycan release was measured with
dimethylmethylene blue assay and TIMP-3 protein was evaluated with Western blots.
Results. The GLN and CS combination abrogated IL-l induced gene expression of iNOS,
COX-2, mPGEsl, NFKB at all time points. NO, PGE2 and proteoglycan release were
reduced with the combination. Stimulated MMP-13, Agg-l and Agg-2 mRNA abundance
were repressed whereas TIMP-3 was up-regulated by the combination at all time points.

TIMP-3 protein abundance was increased by the combination relative to IL-1 at 48 hours.

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Conclusion. Glucosamine and CS in combination suppress synthesis and expression of
genes encoding inflammatory mediators and proteolytic enzymes while up regulating
TIMP-3. This provides a plausible mechanism for GLN and CS purported mild anti-

inflammatory and chondroprotective properties.

INTRODUCTION

Glucosamine (GLN) and chondroitin sulfate (CS) are the predominant
nutraceutical supplements marketed for improving joint health. Glucosamine is an amino
monosaccharide and a major building block of proteoglycans. Clinical trials show that
GLN administered orally was effective in decreasing pain and improving joint mobility in
patients with osteoarthritis (OA) (1). Chondroitin sulfate, a complex glycosaminoglycan,
is a major component of aggrecan. Beneficial effects of CS include improved joint
mobility, reduced rate of joint space narrowing and a reduction of erosive OA (2, 3).
Glucosamine is commonly combined with CS in many of the commercially available
nutraceutical products. The combination has been proven efficacious in reducing pain,
improving joint function, and halting or reversing joint degeneration in humans with mild
to moderate OA of the knee (4). Severe cartilage lesions in an in vivo rabbit instability
model of CA were diminished with GLN and CS supplementation (5). Very few in vitro
studies aimed at determining the mode of action of these nutraceuticals have employed
the combination although clinical studies have reported enhanced efficacy with co-
administration of GLN and CS.

Despite several studies reporting favorable results, the exact mechanism of action

of these nutraceuticals still remains to be resolved. The applicability of many in vitro

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mechanistic studies has been questioned because concentrations of the nutraceuticals
used greatly exceed those generally found in blood and synovial fluid (6, 7). The
concentrations of GLN in blood and synovial fluid after oral and intravenous (IV)
administration range from about 0.05 to 20 ug/ml (7-10). Depending on the route and
frequency of administration, species, and the source and molecular weight of CS, the
concentration of CS in serum ranges from 5 to 200 uglml (6, 8, 11, 12).

In past studies, cartilage explants or cell cultures stimulated with IL-1
demonstrated suppression of nitric oxide (N O) and prostaglandin E2 (PGE2) release to the
media with supplementation of GLN and CS in the concentration range of 0.1 to 10
mg/ml (13-22). Two other studies employed concentrations of GLN and CS that are
attainable in vivo and have shown that these nutraceuticals were able to prevent a decline
in proteoglycan synthesis typically induced by catabolic agents (23, 24). Recently, we
provided preliminary data demonstrating that biologically relevant concentrations of
GLN (5 rig/ml) and CS (20 ug/ml) to regulate both gene expression and protein synthesis
of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in addition to a
reduction in the microsomal prostaglandin E synthase-1 (mPGEsl) gene in 24 hour
incubations (25). These concentrations of GLN and CS were also able to repress major
cartilage proteolytic enzymes implicated in OA pathogenesis at the pre-translational level
(26).

Most in vitro studies have used IL-l as a catabolic agent in the range of 1 to 10
11ng (19, 24, 27-29). The objective of the current study is to further characterize the
effects of biologically relevant concentrations of these nutraceuticals using a

subsaturating IL—l induction of bovine cartilage explants. Specifically, we investigated

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the effect of GLN and CS in combination on IL-1 induced proteoglycan degradation,
gene expression and protein synthesis on an array of inflammatory mediators and
cartilage matrix degrading enzymes and one of their inhibitors in short-terrn explant

cultures.

MATERIALS AND METHODS

Explant cultures. Articular cartilage was isolated from the carpal joints of
Holstein steers (18-24 months old) obtained fiom a local abattoir within 3 hours of
slaughter. Cartilage discs (6 mm in diameter) were biopsied from the articular surface and
did not include the calcified layer of the tissue nor cartilage with gross characteristics of
OA. Two explant discs (approximately 60 mg total wet weight) were selected at random
and cultured in each well of a 24-well Falcon culture plate (Fisher Scientific, Pittsburgh,
PA) containing 1 ml of 1:1 modified version of Dulbecco’s modified Eagle’s medium
(DMEM): nutrient mixture F -12 (Ham) (Gibco, Grand Island, NY), as previously
described (14). The media was supplemented with 50 ug/ml ascorbic acid, 100 units/ml
penicillin-streptomycin (Gibco) and 20 amino acids (Sigma, St. Louis, MO). The
concentrations of amino acids added were 50% of those previously reported (30).
Cartilage explants were maintained in a humidified incubator at 37°C with 7% CO2.

Dose response experiment with IL-1. Explants were maintained in media
without serum for 48 hours before the addition of treatments. Media in the wells were
exchanged daily. Afier equilibration, all wells received 10% fetal bovine serum (F BS,
Gibco) and varying concentrations (0, 5, 10, 20 and 50 ng/ml) of human recombinant

interleukin-1 beta (rhIL-l B, R & D Systems, Minneapolis, MN) for 24 hours to determine

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the subsaturating concentration of IL-1 that would result in stimulation of nitric oxide
(NO) and PGE2 release. There were 8 wells per IL—l concentration. Conditioned media
collected at 24 hours were stored at 4°C for NO and PGE2 analysis. Experiment was
repeated a total of 3 times, each time using tissue from a different animal.

Dose response experiments with GLN and CS. Similar to the methods above,
afier a 48 hour equilibration with serum free media, all explants were treated with 10%
FBS, 15 ng/ml IL-l (approximated subsaturating concentration of lL-l), and varying
concentrations of glucosamine HCl (FCHG49®, Nutramax Laboratories, Edgewood,
MD) for 24 hours. The concentrations of GLN chosen were 0, 1, 5, 10 and 20 rig/ml.
These concentrations were within the range of levels attainable in blood after oral and IV
administration (6-10). Conditioned media collected at 24 hours were stored at 4°C for
NO and PGE2 analysis. There were 8 wells for each GLN concentration. The experiment
was repeated a total of 3 times, each time using tissue from a different steer. These
procedures for GLN were repeated for CS. Concentrations of low molecular weight (16.9
kDa) CS (TRH122®, Nutramax Laboratories) chosen were 0, 5, 20, 50 and 100 ug/ml.

GLN and CS in combination. Explants were maintained in media without serum
for 48 hours prior to addition of treatments. Afier equilibration, all treatments received '
10% FBS and 15 ng/ml IL-1 for 8, 16 and 24 hours to induce inflammatory mediators
and cartilage catabolism. To examine the effects of GLN and CS, they were added to the
wells at the same time as F BS and IL-1. The concentrations of GLN and CS chosen were
10 ug/ml and 20 ug/ml respectively (discussed in results). There were 3 treatments per
experiment, an FBS control, 15 ng/ml IL-1 (IL-1) and 15 ng/ml IL-l with the addition of

10 ug/ml GLN and 20 ug/ml CS (IL-1 + GLN + CS). Each treatment consisted of 12

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wells (24 discs) per time point. Cartilage explants were collected at 8, 16 and 24 hours
after stimulation, frozen in liquid nitrogen, and stored at -80°C until RNA isolation.
Conditioned media were collected at all time points and stored at 4°C for NO (all time
points) and PGE2 (only the 24-hour samples) analysis. The experiment was repeated a
total of 3 times, each time using cartilage from a different animal.

Proteoglycan and TIMP-3 study. Explants were maintained in media without
serum for 48 hours prior to addition of treatments. After equilibration, all treatments
received 10% FBS. To examine the effects of GLN and CS on proteoglycan release (an
indicator of metalloproteinase and aggrecanase activity) and TIMP-3 protein, they were
added to the wells at the same time as F BS and IL-1 . There were 4 treatments per
experiment, an F BS control, 15 ng/ml lL-l (IL-1), 10 ug/ml GLN and 20 ug/ml CS (GLN
+ CS) and 15 ng/ml IL-l with the addition of 10 ug/ml GLN and 20 ug/ml CS (IL-1 +
GLN + CS). Each treatment consisted of 6 wells (12 discs). Media was collected and
replaced daily. Cartilage explants were collected 48 hours after stimulation, frozen in
liquid nitrogen and stored at -80°C until protein extraction. Conditioned media were
collected at 24 and 48 hours post-stimulation and stored at 4°C for dimethyhnethylene
blue (DMB) analysis. The experiment was repeated a total of 3 times, each time using
cartilage from a different animal.

Nitric oxide assay. Nitrite was measured in conditioned media using the Griess
reagent and sodium nitrite as standard (32). Briefly, 150 pl medium was incubated with
150 pl of 1.0% sulfanilamide, 0.1% N-l-napthylethylenediamide hydrochloride and 25%
phosphoric acid at room temperature for 5 nrinutes. Due to some precipitation of reagents

with CS, 96-well plates were spun at 1000 X g for 3 rrrinutes at 4°C. The remaining

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supernatant was transferred to a new plate. Absorbance was measured at 540 nm using a
Spectromax 300 plate reader (Molecular Devices, Sunnyvale, CA).

Prostaglandin E2 assay. Prostaglandin E2 release into conditioned media was
quantified using a commercially available competitive enzyme linked immunosorbent
assay kit according to manufacturer’s instructions (R & D Systems). Conditioned media
samples were stabilized with indomethacin (10 ug/ml) and stored at -20°C until analysis.

Dimethylmethylene blue assay. Proteoglycan release into conditioned media
was measured using a DMB assay as previously described (33). Proteoglycan
concentration was determined by measuring sulfated glycosaminoglycan (GAG) content
compared to a chondroitin sulfate standard. Absorbance was measured at 530 nm with a
wavelength correction set at 590nm using a Spectromax 300 plate reader (Molecular
Devices). For samples containing media with 20 ug/ml CS (GLN + CS and IL-1 + GLN
+ CS), GAG content was subtracted with DMB readings from media measured only with
20 ug/ml CS.

Total RNA isolation. Total RNA was extracted from cartilage explants following
a modified protocol (34). Briefly, cartilage was homogenized in TRIzol® reagent
(Invitrogen, Carlsbad, CA) and chloroform was added to extract total RNA followed by
vigorous agitation and a 2-minute incubation. The aqueous phase containing RNA was
collected after centrifugation (4°C, 12,000 X g, 15 minutes) and RNA precipitated with
an equal volume of 70% ethanol. Total RNA was then purified further with RNeasy mini
columns (Qiagen, Valencia, CA) and quantified by UV spectrophotometry (Beckman
Coulter, Fullerton, CA). Total chondrocyte RNA was resolved on 1.2% agarose gel to

validate spectrophotometric determination and RNA integrity.

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cDNA synthesis. For each sample, 2 ug of total RNA was treated with DNase I
(Invitrogen) to degrade contaminating single and double stranded DNA. Treated RNA
was converted to single stranded cDNA using Superscript II reverse transcriptase
(Invitrogen) as recommended by the manufacturer. Single stranded cDNA was quantified
by UV spectrophotometry (Beckman Coulter), and diluted with RNase-free water to 10
ng/ul.

Quantitative real-time polymerase chain reaction (Q-RT-PCR).
Glyceraldehyde phosphate dehydrogenase (GAPDH) was validated as an appropriate
housekeeping gene. Primers for GAPDH and molecules from Table I were designed
using the Primer Express software version 2.0 (Perkin-Ehner Applied Biosystems, Foster
City, CA). These molecules were chosen from other studies that have demonstrated
significant induction with higher concentrations of IL-1 (25, 26). Nucleotide sequences
used for primer design were obtained from public databases (Genbank). Optimal
concentrations of each set of primers were determined with a primer matrix [lowest
standard deviation with no change in cycle to threshold (C7)]. Quantitative real-time PCR
was performed with 50 ng cDNA templates in 96-well plates (Perkin-Elmer Applied
Biosystems) using the ABI PRISM 7000 sequence detection system (Perkin-Elmer
Applied Biosystems) as previously described (25). The FBS control treatment was used
as a calibrator (i.e. the fold change for control is 1.0). Replicated data was normalized
with GAPDH and the fold change in gene expression relative to FBS control was
calculated using the 2(-AACT) method (3 5).

Protein extraction. Protein was extracted from cartilage explants using a

modified protocol (36). Explants were rinsed with sterile phosphate buffer solutions

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(PBS) and homogenized. Pulverized explants were placed in microcentrifuge tubes with
stir bars and 10 ul extraction buffer (50 mmol/l Tris HCl, 10 mmol/l CaCl2, 2 mol/l
guanidine HCl and 0.05% Brij-35, pH 7.5) per mg tissue. The mixture was stirred
overnight at 4°C and then centrifuged at 18, 000 g for 30 minutes at 4°C. The supernatant
was dialyzed for 24 hours against assay buffer (50 mmon Tris HCl, 10 mmol/l CaCl2,
0.2 mol/l NaCl and 0.05% Brij-35, pH 7.5) using Spectrapor 3 dialysis tubing with a 3.5-
kd cutoff (Spectrum Medical Industries, Los Angeles, CA). Dialysis was continued for 24.
hours with distilled water.

Western blots. The amount of protein in the explant extract was quantified using
the Pierce Micro BCA Protein Assay (Pierce, Rockford, IL) with bovine serum albumin
as the standard. Protein extracts (80 rig/lane) were loaded on 12.5% sodium dodecyl
sulfate (SDS)-polyacrylamide gels following denaturation by boiling for 5 minutes in
SDS loading buffer and electrophoresed. Following electrophoresis, proteins were
transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules,
CA). Protein transfer and size determinations were validated using prestained protein
markers. Membranes were blocked with 5% nonfat dry milk in TTBS (lOmmol/l Tris-
HCl, pH 8, 0.05% Tween-20, 150mmol/l NaCl) for 1 hour at room temperature and
subsequently probed with antibodies directed against TIMP-3 (Chenricon International,
Temecula, CA). Si gnals were detected using horseradish peroxidase-conjugated
secondary antibodies and an enhanced chemiluminescence detection kit (ECL,
Amersham Biosciences, Piscataway, NJ). Blots were developed (Futura Model B film

processor), stripped (62.5 mmol/l Tris-HCl, pH 6.8, 100 mmol/l B-mercaptoethanol, and

2% SDS; 50°C for 30 minutes), and reprobed with anti-B-actin antibody (Abcam Inc.,

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Cambridge, MA). Films were scanned after development and the density of each band
quantified using computer-aided densitometry. Amounts of TIMP-3 proteins were
normalized relative to amounts of B-actin detected in each sample.

Statistical analysis. Data for NO, PGE2, inhibition of NO and PGE2 (expressed as
percentage inhibition relative to negative controls), and proteoglycan release into
conditioned media were analyzed using a linear mixed effects model, including the fixed
effect of concentration! treatment and the random effect of steer. Concentration/
treatment effects were compared within each time point using the multiple comparisons
approach of Tukey. The computations were performed using the MD(ED procedure of
SAS (3 7). Relative gene expression data determined using Q-RT-PCR and densitometry
measurements from Western blots were analyzed using the nonparametric ANOVA
approach of Friedman using SAS (3 7). Differences were declared statistically significant
when P<0.05, unless otherwise noted. Spearman’s rank correlations (r) between Q-RT-
PCR data and data for NO and PGE2 release were computed using the CORR procedure

of SAS (37).

RESULTS

The effect of increasing IL-l concentration on NO and PGE2 release. There
were dose dependent increases in NO and PGE2 release with increasing IL-l
concentration. Nitrite release increased from 12.84 M to 65.95 M with increasing IL-l

concentrations from 0 ng/ml to 50 ng/ml respectively (data not shown). The release of

PGE2 increased from its basal level of 65.56 pg/ml in the absence of IL-1 to 1501.03

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pg/ml with 50 ng/ml IL-l (Figure 1A). Based on these data, a subsaturating concentration
of IL-1 (15 ng/ml) was selected for subsequent experiments.

The effect of increasing GLN and CS concentrations on NO and PGE2
release. Increasing GLN concentration abrogated IL-l stimulated release of PGE2. The
release of PGE2 was inhibited by about 36 %, 47 %, 49 % and 50 % with 1, 5, 10 and 20
ug/ml GLN respectively (F igurelB). Increasing CS concentration inhibited PGE2 release
hour 18 %, 20 %, 30 % and 34 % with 5, 20, 50 and 100 ug/ml CS respectively (Figure
1B). There was no significant effect of GLN or CS at any concentration on NO (data not
shown). Numerically, maximal reduction of NO occurred with 10 ug/ml GLN (16%) and
20 ug/ml CS (11%). Based on these results, we selected 10 ug/ml of GLN and 20 rig/ml
of CS for the subsequent experiments.

The GLN and CS combination suppressed inflammatory mediators.
Interleukin-l stimulated increases in iNOS, COX—2, mPGEsl and NFKB transcripts at all
time points were down-regulated by the GLN and CS combination (Figures 2A, 2B and
2C). Nitrite release was not different between treatments at 8 hour post-stimulation. Both
NO and PGE2 release were significantly induced by IL-1 at 16 (N 0 only) and 24 hours
post-culture. The elevation in these inflammatory mediators was effectively reduced by
the GLN and CS combination (T able II).

The GLN and CS combination repressed gene expression of matrix enzymes
and reduced proteoglycan release. There was significant up-regulation of MMP-3
mRN A abundance by about 4 fold and 42 fold at 8 and 24 hours after IL-1 stimulation
respectively. Glucosamine and CS suppressed IL-l induced expression of the MMP-3

gene by 35 fold at 24 hours. Cytokine induced mRNA expression of MMP-1 3, Agg-l and

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Agg-2 were repressed at all time points by the combination (Figures 3A, 3B and 3C).
Induction of proteoglycan release with IL-1 was significantly (P<0.01) reduced by the
GLN and CS combination at both 24 and 48 hours post-stimulation (Figure 4A).

The GLN and CS combination up-regulated TIMP-3. Gene expression of
TIMP-3 was elevated with the GLN and CS combination relative to IL-1 at all time
points and relative to FBS control at 8 and 24 hours post-stimulation (Figures 3A, 3B and
3C). TIMP-3 protein abundance was increased by the combination relative to IL-1 at 48

hours after culture (Figure 4B and 4C).

DISCUSSION
We performed the present study to expand on previous studies (25, 26) involving

the effects of GLN and CS on the expression and synthesis of putative mediators of OA.
Specifically, in this series of experiments, GLN and CS were employed at concentrations
achievable in blood after oral and IV administration. Moreover, a subsaturating dose of
the cytokine was used in this study to ensure the possibility of a two-tailed response to
the arthritogenic stimulus and gene expression was examined at additional time points
following stimulation and treatment. Pooling of animals in this experiment was avoided
to prevent masking of outliers that may contribute to large variation in the results. Our
results indicate that clinically relevant concentrations of this nutraceutical combination
retain cartilage-sparing effects in this model.

As expected IL-l significantly induced expression of the iN OS gene at all time
points in the current study, an event that has been associated with cartilage degradation

and suppression of ECM synthesis (3 8-40). Hence, limiting NO synthesis may be critical

133

to retarding OA progression and finds support in the documented beneficial effects of
iNOS inhibitors in a canine OA model (36). In the present experiment, GLN and CS
suppressed iNOS mRNA abundance and NO release. Individually, neither significantly
decreased NO production, suggesting the two had a synergistic effect (5). At
concentrations 10 fold or more than those employed in the present study, GLN and CS,
alone or in combination are capable of repressing iNOS mRNA expression and reducing
cytokine-induced release of NO (14, 15, 18-22, 27). The current study complements
previous reports supporting co-administration of GLN and CS (5, 22).

Prostaglandin E2 is the most abundant prostanoid found in diseased joints (41).
Formed from the arachidonic acid cascade, PGE2 mediates synoviocyte proliferation,
inflammatory and pain responses. Rate limiting enzymes responsible for making PGE2
include the cyclooxygenases and prostaglandin E synthases. The inducible forms of these
enzymes are COX-2 and mPGEsl respectively. The synthesis of PGE2 correlates well
with the elevation of COX-2 (r = 0.93, P<0.0002) and mPGEsl (r = 0.95, P<0.0001)
transcripts in agreement with other studies (25, 42, 43). The concomitant regulation of
COX-2, mPGEsl and PGE2 in the current study parallels other reports (25, 43). At
biologically relevant concentrations, GLN and CS in combination effectively decreased
IL-l induced gene expression of COX-2 and mPGEsl at all time points, and eventually
PGE2 synthesis. A difference observed for 24 hours post-culture in the current study and
in Chan et. al. (25) is that marginal reduction in mPGEsl transcript with GLN and CS is
now significantly detected. These findings of modulation of iNOS, COX-2 and mPGEsl
activities are in keeping with reductions in articular inflammation in OA patients and may

help explain the analgesic effects of GLN and CS (4, 44).

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The expression and activity of catabolic enzymes such as MMPs and
aggrecanases exceeds those of endogenous inhibitors like TIMPs in OA (45). The ability
of GLN and CS in combination to inhibit cartilage erosion (5) and prevent proteoglycan
release in the current study (Figure 4) may be partially attributed to regulation of these
enzymes. Concentrations of MMPs are elevated in OA cartilage specimens and they are
localized at the site of OA lesions (46, 47). The nutraceutical combination demonstrated
effectiveness in mitigating IL-l elevation of MMP-3 only at 24 hours post culture,
consistent with a previous report where there was no treatment effect at the 6 hour time
point (26). Past experiments with GLN, at higher concentrations, showed suppression of
MMP-3 gene expression and enzyme activity (15, 17, 19, 48). Suppression of the MMP-
13 transcript with the combination at 24 hours also agrees with Chan et. al. (26).
However, at a higher lL—l concentration, the ability of GLN and CS to reduce expression
of the MMP-13 gene at 6 hours was not detected. In this study, induction of MMP-13
occurred at all time points and was suppressed at all time points by the combination. The
findings on MMP-13 are also in agreement with other studies that reported a decrease in
MMP-13 protein and activity in equine cartilage with the nutraceutical combination (22)
and with GLN alone (17). Concentrations employed in the current study were at least 30
times lower for GLN and 6 times lower for CS than those reported in previous studies.

The aggrecanases have been implicated as the primary proteins responsible for
initiating aggrecan release from OA and injured joints (49). The present study
demonstrated that GLN and CS in combination repressed IL-l up-regulation of Agg-l
and Agg—2 at all time points. Our current findings roughly parallel those of Chan et. al.

(26). The effect of GLN and CS in combination on aggrecanases has not been extensively

135

studied. Glucosamine supplemented alone suppressed IL-l stimulated aggrecanase
activity at concentrations that were at least 40 times higher than the current study (28,
50). Chondroitin sulfate used individually in culture inhibited aggrecanase activity (51).

The TIMPs have the potential to reduce proteoglycan destruction. Localized in the
extracellular matrix, TIMP-3 exhibits potent inhibitory activities against MMPs and
aggrecanases (52). Interleukin-l stimulated glycosaminoglycan release via aggrecanase
was reversed by TIMP-3 (53). Our current study detected elevation in the TIMP-3
transcript with the combination relative to IL-1 throughout the 24 hour culture period.
Simultaneous suppression of MMPs and aggrecanases coupled with up-regulation of
TIMP-3 with GLN and CS supplementation may represent an effective way to protect
matrix components fiom being degraded, as evidenced by the decline in proteoglycan
release.

Interleukin-l is upstream of the activation of a number of phosphorylation
dependent signaling pathways leading to the nuclear translocation of transcription factors
and activation of proteins participating in translation of mRNA. NFKB stimulates
expression of iNOS and COX-2, and their end products, which contribute to the
inflammatory process in arthritis (54). There was simultaneous expression of NFKB with
iNOS (r = 0.48, P<0.01) and COX-2 (r = 0.55, P<0.003) in the present study. The down-
regulation of certain MMPs with GLN and CS is perhaps also a downstream event of the
repression of IL-1 signaling molecules. NFKB is one of two early response genes needed
for MMP transcription (5 5). The activation of MMP-3 and MMP-13 relies on NFKB (56,
57). The mRNA expression of NFKB in the present study is highly correlated with

MMP-13 (r = 0.76, P<0.0001) and IL-1 induction transcripts of both NFKB and MMP-13

136

increased significantly (P<0.05) with time of stimulation. Glucosamine, at 1 mg/ml,
prevented IL-l induced IKB degradation, NFKB activation and nuclear translocation of
p50 and p65 NFKB subunits and PGE2 release in human chondrocytes (13). In rat
chondrocytes, GLN at concentration 450 times higher than the current study decreased
NFKB activation (16). Our study shows the ability of GLN and CS in combination to
repress IL-l stimulated mRNA abundance of the p65 NFKB subunit. This is essential
since NFKB is able to regulate its own gene expression whereby IL-l induced increases
in NFKB translocation to the nucleus stimulates continuous synthesis of NFKB to replace
those that were translocated (5 8). Thus, the effect of GLN and CS on genes of iNOS,
COX-2, and the MMPs could be explained at least, in part, via the inhibition of NF KB.
Further studies are needed to substantiate the effect of these nutraceuticals on signaling
events in the chondrocytes.

Considering the adverse effects elicited by NSAIDs and COX-2 inhibitors,
availability of other compounds that can help those suffering from joint pain is essential.
The benefits of GLN and CS for symptomatic relief of OA have been clearly documented
(1-4). These nutraceuticals are safer alternatives judging from the paucity of reports of
adverse events (59). In vitro studies are revealing the mechanisms that underlie the in
vivo effects of GLN and CS. From the present study, suppression by GLN and CS in
combination of NO and PGE2 production may partially contribute to the anti-
inflammatory effects experienced by CA patients consuming these compounds. The
purported cartilage protective feature of GLN and CS (5) and the ability of these
nutraceuticals to prevent IL-l induced proteoglycan degradation, and decrease

proteoglycan synthesis (23, 24) is attributed, at least in part, to suppression of catabolic

137

matrix enzymes and up-regulation of TIMP-3, an important enzyme inhibitor. GLN and
CS may regulate signaling pathways upstream of the production of inflammatory
mediators and matrix enzymes, which translates into beneficial effects thereafter.
Glucosamine and CS may also impact positively the interactions that occur between all
these molecules that are associated with CA pathogenesis. Further experiments are being

planned to confirm these findings in vivo.

138

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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139

Table II: Nitrite and PGE2 release from explants at 8, 16 and 24 hours post-stimulation

 

 

 

 

 

 

 

Treatment

Variable FBS control IL—1 IL-1 + GLN + CS
Nitrite release (uM t SE) 1.80 i 0.37‘1 7.67 i- 1.251‘ 5.82 5: 0.44‘ll
at 8 hours post-stimulation
Nitrite release (uM i SE) 5.56 i 0.99a 28.17 i 386" 15.61 i 1.873
at 16 hours post-
stimulation
Nitrite release (uM 3: SE) 8.04 i 0.94“ 52.60 i 555° 22.54 i 2.315
at 24 hours post-
stimulation
PGE2 release (pg/ml t SE) 70.03 i 9.113 1159.16 i 248.09c 675.29 1 106.84”
at 24 hours post-
stimulation

 

 

 

 

F BS = fetal bovine serum; IL-1 = 15 11ng human recombinant interleukin-1 beta (rhIL-
IB); IL-l + GLN + CS = 15 ng/ml rhIL-IB with the addition of 10 pig/ml glucosamine

and 20 ug/ml chondroitin sulfate; PGE2 = prostaglandin E2
Different superscripts for values within a row (i.e. one variable) denote significant
(P<0.05) differences between treatments.

140

 

 

 

Figure l: Prostaglandin E2 (PGE2) release 3: SE in conditioned media with increasing
interleukin—1 (IL-1) concentration (A). Percentage inhibition of PGE2 release i SE from 0
ug/ml glucosamine (GLN) or chondroitin sulfate (CS) in conditioned media with 15

ng/ml IL-1 and increasing GLN or CS concentration (B). Different letters for values

within each line indicate significant (P<0.05) differences between concentrations of IL-1,

GLN or CS.

141

PGEZ (pg/ml)

 

 

 

 

Inhibition of PGE2 (%)

 

 

 

 

 

 

 

 

 

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142

Figure 2: Mean relative gene expression i SE of inflammatory mediators at 8 hours (A),
16 hours (B) and 24 hours (C) post-stimulation. Different letters for values within each
gene indicate significant (P<0.05) differences between treatments. iNOS = inducible
nitric oxide synthase; COX-2 = cyclooxygenase-2; mPGEsl = microsomal prostaglandin
E synthase-1;NFKB = nuclear factor kappa beta p65 subunit; F BS = fetal bovine serum;
IL-1 = 15 ng/ml human recombinant interleukin-1 beta (rhlL-IB); IL-l + GLN + CS = 15
ng/ml rhIL-lfi with the addition of 10 ug/ml glucosamine and 20 ug/ml chondroitin

sulfate.

143

 

 

 

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144

Figure 3: Mean relative gene expression 1: SE of enzymes at 8 hours (A), 16 hours (B)
and 24 hours (C) post-stimulation. Different letters for values within each gene indicate
significant (P<0.05) differences between treatments. MMP-3 = matrix metalloproteinase-
3; MMP-13 = matrix metalloproteinase-13; Agg—l = aggrecanase-1; Agg-2 =
aggrecanase-2; TIMP-3 = tissue inhibitor of metalloproteinase-3; F BS = fetal bovine
serum; IL-1 = 15 ng/ml human recombinant interleukin-1 beta (rhIL-l [3); IL—1 + GLN +
CS = 15 ng/ml rhIL-l B with the addition of 10 ug/ml glucosamine and 20 ug/ml

chondroitin sulfate.

145

Mean relative expression

 

Mean relative expression

 

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Gene
UFBS control I lL-1 a IL-1 + GLN + CS

146

Figure 4: Proteoglycan release i SE in conditioned media at 24 and 48 hours (A) post-
stimulation. Different letters for values within each time point indicate significant
(P<0.01) differences between treatments. Representative western blots of TIMP-3 and B-
actin protein expression at 48 hours post-culture (B). Mean relative abundance of TIMP-3
proteins 1: SE, as determined by densitometry (C). Different letters indicate significant
(P<0.05) differences between treatments. (1) FBS = fetal bovine serum; (2) IL-1 = 15
ng/ml human recombinant interleukin-1 beta (thL-l B); (3) GLN + CS = 10 ug/ml
glucosamine and 20 ug/ml chondroitin sulfate; (4) IL-1 + GLN + CS = 15 ng/ml rhIL-l B

with the addition of 10 ug/ml glucosamine and 20 rig/ml chondroitin sulfate.

147

 

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148

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154

CONCLUSION

Previous research findings with glucosamine and CS in our laboratory and others
provided some clues about the mechanism(s) of action for these nutraceuticals although
the concentrations used were far from relevant. Most of these studies were conducted
with glucosamine and very few with CS and the majority mainly assessed biochemical
parameters including NO, PGE2, and proteoglycan synthesis. Only a handful have looked
at enzymatic activity. Data from these studies point to the possibility of cartilage-sparing
properties and suppression of molecules that participate in the inflammation process.

The present research was targeted at expanding on previous results using
concentrations of glucosamine and CS that are achievable in vivo in the blood of humans,
dogs, and horses after oral administration of these nutraceuticals. Specifically, the mRN A
abundance of an array of proteolytic enzymes and inflammatory mediators were
determined since iNOS, COX-2, and several MMPs in particular are regulated at the
transcriptional level. Moreover, the effects of these nutraceuticals on chondrocyte gene
expression have rarely been studied.

Current research with biologically relevant concentrations of glucosamine (5
rig/ml) and CS (20 ug/ml) demonstrated their ability of pre-translational regulation of
genes encoding inflammatory mediators and cartilage matrix catabolic enzymes. Using
an lL-l concentration (50 ng/ml) reported in previous trials in our laboratory to stimulate
cartilage catabolism, explants supplemented with glucosamine and CS showed transient
suppression of molecules implicated in the progression of OA such as NO and genes of

iNOS, mPGEsl and MMPs. Glucosamine and CS down-regulation of COX-2 was

155

accompanied by reductions in PGE2 production. The ability of these nutraceuticals to
suppress NO and PGE2 is critical since both inflammatory mediators facilitate cartilage
degradation. Both NO and PGE2 are important regulatory molecules of MMPs. NO and
PGE2 also interact with one another. Studies have indicated that an NO-dependent
pathway mediates IL-l stimulation and inhibition of PGE2 and the opposite may also be
true. More studies are needed to determine how these two molecules affect each other in
the process of inflammation and cartilage destruction. The mRNA abundance of enzymes
(iNOS, COX-2 and mPGEsl) that catalyze the production of these inflammatory
mediators serves as another point of regulation affected by glucosamine and CS.
Researchers in the field of cartilage have just started studying mPGEsl since it was only
recently detected in chondrocytes. The ability of glucosamine and CS to affect mPGEsl
is pertinent since it is the terminal enzyme involved in PGE2 synthesis.

The positive effect of glucosamine and CS on MMPs and aggrecanases at the
gene level is important information. MMPs are the primary enzymes responsible for
ECM remodeling and found in elevated concentrations in the sites of OA lesions while
aggrecanases participate in pathological turnover of aggrecan. Moreover, the majority of
studies with biologically irrelevant concentrations of glucosamine and CS have only
assessed general activities of these enzymes. Lack of treatment effect on genes of matrix
components; type II collagen and aggrecan keeps up with current literature on the
possibility that glucosamine and CS may exert their effects on matrix constituents at a
translational and post-translational level. F urthennore, the synthesis of these
macromolecules is catalyzed by a number of enzymes that have yet to be investigated.

The nutraceutical combination antagonized some gene expression induced by IL-

156

1 better than when they were used individually. This finding is in support of past studies
that reported enhanced symptomatic efficacy in glucosamine and CS when taken
together. We are confident that these responses are highly specific to the nutraceuticals
added in culture since previous research in our laboratory with glucosamine has proven
that its effects on the release of inflammatory mediators is specific and not due to an
artifact of high sugar moieties in the culture media.

Given that most in vitro studies that used IL-l as a stimulant on cartilage explants
employ it at a much lower concentration than what was used in chapters 2 and 3,
detection in modulation of genes especially those with subtle changes was probably
limited by this factor. Another limitation was attributed to pooling of animals for tissue
sampling that caused much variability in the results. Preliminary studies also
demonstrated that chondrocytes become refiactory to IL-1 after 48 hours. Thus, a
subsaturating dose of IL-1 (15 ng/ml) with cartilage explants from individual animals was
used for a separate study with the duration of regulation characterized within 24 hours.
The data from this research provided further evidence for the proposed mechanism(s) of
action for glucosamine (10 rig/ml) and CS (20 uglml). Figure 1 depicts a summary of this
evidence.

Glucosamine and CS in combination effectively repressed genes of selected
molecules associated with inflammation and cartilage degradation. The NFKB p65
subunit that is downstream of IL-1 signaling and an important factor driving transcription
of most of the selected genes in this study was negatively regulated by glucosamine and
CS. This is an interesting finding as NF KB regulates its own expression. Decreasing its

mRN A abundance will probably interrupt transcription of some of the critical mediators

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of OA, in particular iNOS, COX-2, MMP-3 and MMP-13. Future studies focusing on the
effects of the nutraceuticals on nuclear translocation and activation of this signaling
pathway are warranted. Some research suggests that glucosamine may regulate proteins
such as the NF KB via the glycosylation process. Glycosylation may offset the ability of
the transcription factors to be phosphorylated, preventing their translocation to the
nucleus to affect transcription of other genes containing enhancer elements in their
promoter regions. This postulation remains to be proven in chondrocytes.

Other studies reported that glucosamine and CS are beneficial for CA patients by
providing building blocks for cartilage matrix synthesis. Our research suggests that these
nutraceuticals do more than just serve as precursors for matrix synthesis. The present
research demonstrated the ability of these nutraceuticals to regulate mediators of OA at
the pre-translational level, decreasing mRNA abundance of iNOS, COX-2, mPGEsl ,

NF KB, MMP-3, MMP-13, Agg-l and Agg-2. Repression of most of these genes may be
mediated by inhibition of NF KB. Another novel finding of this research pertains to up-
regulation of mRNA and protein abundance of TIMP-3. Since TIMP-3 has potent affinity
for many of the matrix proteolytic enzymes, the ability of glucosamine and CS to elevate
it is important. Moreover, these effects on the putative mediators of OA are coupled with
a reduction in proteoglycan loss and, production of NO and PGE2. Hence, these research
findings may explain, in part, the possible mechanisms for mild anti-inflammatory and
chondroprotective features of glucosamine and CS.

Further studies with the nutraceuticals should determine in depth how the selected
OA mediators are regulated. The drawback to the present study is the fact that only

abundance of genes was measured although a few biochemical variables were included,

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and they did match up well with the gene data. Future studies should investigate the
effects of glucosamine and CS on rates of transcription and translation, and the stability
of the transcript and protein. Parameters such as enzymatic activities, binding and
substrate assays would be useful to study post-transcriptional regulation. Studies are also
needed to determine how and if CS accesses the chondrocytes.

The anabolic aspects affected by glucosamine and CS should be considered as
well by exploring molecules such as the grth factors; TGFB, lGF-l , and osteogenic
protein-l , recently implicated to mediate proteoglycan synthesis. Other variables
associated with pain such as bradykinin, and neurotransmitters including serotonin and
dopamine should also be investigated. Bradykinin is capable of inducing proteoglycan
degradation. A comparison in responses of normal and healthy cartilage with CA
cartilage to glucosamine and CS will also be interesting. The concentrations of
glucosamine and CS chosen should be closer to those in the synovial fluid as more
pharmacokinetic and pharmacodynamic studies become available reporting on the
concentrations of the nutraceuticals measured in the synovial fluid after oral and
intravenous administration. Since cartilage does not exist in isolation, these in vitro
findings would be more convincing when confirmed in viva using for example, a rabbit
instability model or some other model of OA.

Overall, results from the present research complement those found in clinical
trials. Studies in vivo with animals and clinical trials with humans have reported on the
ability of glucosamine and CS to attenuate cartilage loss. Current findings with relevant
concentrations of the nutraceuticals demonstrated an association between reduced mRNA

abundance of catabolic enzymes and up—regulation of an important enzyme inhibitor with

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the inhibition of proteoglycan loss. Reductions in pain assessments also coincide with
suppression of inflammatory mediators measured in the present experiments. This finding
is useful information for physicians and veterinarians in their consideration whether to
recommend or not recommend these nutraceuticals to their patients. No longer can these
compounds be dismissed because their concentrations investigated in vitro are irrelevant.
Furthermore, millions of people suffer from OA and with the recent controversy
surrounding COX-2 inhibitors, the availability of safer alternatives such as glucosamine
and CS may be critical to a patient’s health and well-being. However, these nutraceuticals
are no “magic bullet”. Clinical trials have shown that they appear only to be beneficial to
those with mild to moderate OA. Until more studies are perform, we cannot say
definitively that glucosamine and CS is the answer to treating OA. Nevertheless, the
present research does provide additional information pointing to possible mechanism(s)

of action of glucosamine and CS that could explain the results seen from clinical trials.

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Figure 1: Proposed mechanism(s) of action for glucosamine and CS as indicated by
results from the present in vitro experiments. X indicates suppression by the glucosamine
and CS combination while / indicates up-regulation by the glucosamine and CS

combination.

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