The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis

The Role of Nitric Oxide in

Chondrocyte Models of Osteoarthritis

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

des Fachbereichs für Biologie an der Universität Konstanz

vorgelegt von

Anna Helena Mais

Tag der mündlichen Prüfung: 31.1.2006 1. Referent: Prof. Dr. Volker Ullrich

2. Referent: Priv. Doz. Dr. Christian Schudt

ACKNOWLEDGEMENTS

This work was performed in the Department of Biochemistry (RDR/B1), ALTANA Pharma AG, Konstanz.

I would like to express my deep and sincere gratitude to my supervisor Prof. Dr. Volker Ullrich for so much of his time, ideas and support.

I would like to thank Dr. Christian Schudt for his guidance, inspiring ideas and especially for always keeping his door open.

I would also like to thank the organizers of the graduate program “Biomedical Drug Research” Prof. Dr. Albrecht Wendel and Prof. Dr. Klaus Schäfer for giving me the opportunity to participate in this excellent program.

My special thanks go to Dr. Gereon Lauer for his support and motivation during the years. His scientific and technical advice was essential for the completion of this dissertation.

I would like to thank Dr. Thomas Klein especially for his enthusiasm. Thomas continually stimulated me with new ideas and encouraged me to develop independent thinking not only in the research area.

I would like to thank Marion Hirscher for her expert technical assistance and Nadine Kellner for help with chondrocyte culture.

I am grateful to colleagues from RDR/PX and RDR/IT especially to Silke Müller, Dr. Sascha Dammeier, Dr. Hubert Paul, Dr. Gordana Bothe, Lydia Willems,

Klaus Hägele and Gisela Schüßler for great support performing 2D-electrophoresis and protein identification as well Affymetrix analysis.

Colleagues and friends at ALTANA, especially Kathrin and Claudi, I thank for support at work but also friendship and help to make Germany my home.

Je dis merci beaucoup à Pierre pour son aide avec la langue anglaise.

My husband Georg for being my best friend, his patience and that he always believed in me. Special thanks to my family:

Mamie i Tacie, Babci i cioci Krysi dziękuję, za to że mnie zawsze kochali i wspierali, szczególnie za Nawojową mojego dzieciństwa.

Tą pracę dedykuję pamięci Stanisława Ciołkiewicza, który kochał mnie tak jak nikt inny, co dało mi siłę i wiarę w siebie na całe życie.

Konstanz, 8^th of December 2005

Some parts of this thesis have been already published:

Mais A, Klein T, Ullrich V, Schudt C, Lauer G: Prostanoid pattern and iNOS expression during chondrogenic differentiation of human mesenchymal stem cells.

JCB, in press (2006 Jan 26 Epub

Brenner SS, Klotz U, Alscher DM, Mais A, Lauer G, Schweer H, Seyberth HW, Fritz P, Bierbach U:

Osteoarthritis of the knee-clinical assessments and inflammatory markers.

Osteoarthritis Cartilage 12: 469-475 (2004)

Zusammenfassung

Osteoarthrose (OA) ist die häufigste musko-skeletale Erkrankung in der westlichen Welt. Die derzeit übliche Therapie der Erkrankung erlaubt zwar eine effektive Schmerzbehandlung aber es gibt keine pharmakologischen Ansätze, die die Progression des Knorpelabbaus verhindern, oder die Gelenkfunktion verbessern würden.

Die Entwicklung der OA ist durch eine fortschreitende Zerstörung der Knorpelmatrix und einen fehlgeschlagenen Reparaturmechanismus charakterisiert. Als Grund wird hierbei ein Ungleichgewicht von anaboler und kataboler Aktivität vermutet. Da der Chondrozyte die einzige Zelle des Gelenkknorpels ist, hat jede Einschränkung seiner Funktion bzw.

Vitalität eine Auswirkung auf das Knorpelgewebe.

Auch bei der Zerstörung des Knorpelgewebes spielen die Chondrozyten eine wichtige Rolle, da sie inflammatorische Mediatoren und MMPs sezenieren. Zusätzlich produzieren die aktivierten Zellen auch große Mengen von NO. Dieses wasser- und fettlösliche, gasförmige Molekül fungiert nun selber als Mediator einer Vielzahl zellulärer Prozessen.

Im Knorpel wird die Synthese von NO durch die iNOS vermittelt, die u.A. durch katabolische Zytokine induziert wird.

Dem Interleukin-1 (IL-1) kommt in der OA eine zentrale Rolle zu, da es sowohl die iNOS Expression erhöht, als auch andere degenerative Prozesse induziert. Dabei gehen viele Studien davon aus, dass ein großer Teil der negativen Effekte des IL-1 durch NO vermittelt ist. Aus diesem Grund könnte eine pharmakologische Inhibition der iNOS durchaus als Behandlungsoption bei der OA Therapie in Frage kommen.

Das Ziel der vorliegenden Arbeit war es den Einfluss von NO auf die Physiologie des Chondrozyten zu untersuchen, um mögliche positive und negative Effekte einer Inhibition der iNOS auf den humanen Gelenkknorpel zu identifizieren.

Um die Rolle von NO aufzuklären wurden verschiedene Zellkulturmodelle verwendet: in situ Kultur von Knorpelstücken, isolierte OA-Chondrozyten in Monolayer oder in 3- dimensionaler Alginatmatrix und nicht zuletzt, als Alternative zur Kultur von gesunden Chondrozyten, ein chondrogenes hMSC basiertes Differenzierungsmodell.

Dieses Differenzierungsmodell ist nicht in der Lage gewesen „normalen“ Gelenkknorpel zu generieren. Viel mehr zeigten unsere Analysen, dass ein Knorpel–ähnliches Gewebe entsteht, dass sich in den Expressionsanalysen vom hyalinen Knorpel signifikant unterscheidet. Trotzdem konnten wir zeigen, dass die differenzierten hMSCs in der Lage waren iNOS zu Expremieren und NO freizusetzen.

Zusätzlich wurde eine genaue Charakterisierung des Prostanoid-Spektrums während der Differenzierung durchgeführt. Da die Übereinstimmungen zwischen diesen Inflammationsmarkern in den hMSCs während der chondrogenen Differenzierung und Chondrozyten sehr gut war, vermuten wir, dass das Differenzierungsmodell in diesem Zusammenhang als tatsächliche Alternative zu primären Chondrozyten dienen kann.

Des weiteren wurde die Regulation der iNOS Expression in Chondrozyten untersucht.

Dabei konnten wir zeigen, dass sowohl Zellen aus OA-Patienten als auch aus gesundem Knorpel (Proben aus Schweine und Rindergelenken) mikromolare Mengen von NO nach der Stimulation mit IL-1 freisetzen. Zusätzliche Experimente zeigten, dass die Expression der iNOS in humanen Chondrozyten zentral von dem Transkriptionsfaktor NFκB reguliert wird. Auch die Beteiligung von intrazellulären cAMP an der iNOS-Regulation konnte gezeigt werden.

Eine Besonderheit der iNOS-Regulation in Chondrozyten stellt die Nichtbeeinflussung durch Glukokortikoide dar. Wir konnten diese Befunde eindeutig bestätigen und zusätzlich zeigen, dass diese „Dex-Resistenz“ nicht nur bei Chondrozyten von OA- Patienten zu finden ist, sondern auch für andere Spezies und gesunden humanen Knorpel gilt. Darüber hinaus fanden wir diesen Effekt auch in dem hMSC-Modell und weitere Analysen zeigten, dass die „Dex-Resistenz“ nicht mit der NFκB oder cAMP- Signaltransduktion assoziiert ist. Anhand dieser Daten kammen wir zu der Schlussfolgerung, dass Induktion durch IL-1, sowie die fehlende Inhibition der NO- Produktion nach Glukokortikoidgabe, neben den bisher verwendeten Knorpelmatrix- Proteinen, als Marker des Differenzierungsstatus von Chondrozyten verwendet werden kann.

Auch konnten wir zeigen, dass humane Chondrozyten nach IL-1 Stimulus COX-2 abhängig, große Mengen PGE2 produzieren. Diese COX-2 Expression ließ sich im Gegensatz zu der iNOS-Expression durch Dex hemmen.

Außerdem war kein deutlicher Effekt von NO auf die Prostanoid-Synthese in unseren Modellen zu beobachten. Das COX-2 Enzym ließ sich nicht durch NO oder seine Derivate inhibieren. Im Gegensatz dazu konnte mit Peroxynitrit ein höherer „Peroxid Tonus“ generiert werden, der zur Aktivierung der COX-Enzyme beitrug.

Als weiterer Aspekt wurde der Einfluss von NO auf die Apoptose von Chondrozyten untersucht, da frühere Studien hier einen Zusammenhang hergestellt hatten. Wir konnten jedoch zeigen, dass NO alleine keinen zytotoxischen Effekt auf die Zellen ausübt, dem gegenüber führten hohe Dosen von Peroxynitrit tatsächlich zu einer verstärkten Apoptose der Chondrozyten.

Oxidativer Stress scheint im Knorpelgewebe eine Rolle zu spielen. So konnten wir Nitrotyrosin im Knorpelproben nachweisen. Zusätzlich konnten wir eine Reihe von Proteinen identifizieren bei denen Tyrosin-Reste nitriert waren. Interessanterweise waren darunter einige Proteine die mit dem Glukose-Metabolismus assoziiert sind.

Leider war es uns jedoch nicht möglich die O2- -Entstehung in Chondrozyten direkt nachzuweisen.

Zusammenfassend kann also gesagt werden, dass sich Chondrozyten leicht durch IL-1 zur NO-Freisetzung stimulieren lassen. Außerdem scheinen diese Zellen ein hohes antioxidatives Potential zu besitzen und sind damit relativ resistent gegen die Folgen von oxidativem bzw. nitrosativem Stress. Da NO alleine offensichtlich keinen negativen Effekt auf die Vitalität der Chondrozyten hat, scheint es, als ob cytotoxische Effekte von NO nur in Kombinationen mit O2- entstehen können.

Wir vermuten dementsprechend, dass nicht nur das Gleichgewicht zwischen anabolen und katabolen Prozessen, sondern auch das antioxidative und oxidative Potential zentrale Bedeutung für die uneingeschränkte Funktion der Chondrozyten und damit für den Erhalt des Knorpelgewebes hat.

So konnten wir zwar zeigen, dass IL-1 eine Vielzahl von Genen induziert die mit der Pathophysiologie der OA assoziiert sind, aber entgegen früheren Annahmen, sind diese Veränderungen im Expressionsprofil nicht die Folge der NO-Freisetzung, sondern werden direkt durch IL-1 vermittelt. Aus diesen Gründen zeigt die vorliegende Studie, dass eine Inhibition der iNOS bei OA-Patienten, vermutlich keinen positiven Effekt auf die Physiologie und Funktion von Chondrozyten hat.

Summary and conclusions

Osteoarthritis (OA) is the most common form of musculo-skeletal disorders in the Western world. Current OA therapy relieves pain but there is no pharmacological treatment available that retards the disease progression and improves joint function.

The development of OA is characterized by excessive cartilage destruction and defective cartilage repair due to imbalance of the anabolic and catabolic activity of chondrocytes. Chondrocyte viability and function are crucial to articular cartilage as this is the only cell responsible for maintenance of this tissue. However, the chondrocyte plays also an active role in cartilage degradation in OA by releasing inflammatory mediators and matrix metalloproteases. Beside this, activated chondrocytes release high levels of nitric oxide (NO). NO is a gaseous water and lipid soluble molecule that serves as a mediator of a number of cellular processes. In cartilage elevated levels of NO are due to induction of inducible NO synthase (iNOS) in response to stimuli e.g.

catabolic cytokines. Interleukin 1 (IL-1) has been shown to play a pivotal role in cartilage damage and is also a very potent stimulator of NO production in articular chondrocytes.

It has been implicated that NO mediates many of the destructive effects of IL-1 in cartilage. Therefore pharmacological iNOS inhibition has been proposed as a treatment of OA.

The aim of the present study was to investigate the role of NO in the homeostasis of chondrocytes to evaluate potential beneficial or detrimental effects of iNOS inhibition in human articular cartilage.

To study the role of NO several cartilage-related cell culture models were used as OA cartilage in situ, isolated OA chondrocytes in monolayer and in 3-dimensional alginate matrix and as an alternative to a healthy cartilage a hMSCs chondrogenic differentiation model was established. However, differentiation of hMSCs led to the formation of cartilage-like tissue, which phenotypically differs, from hyaline cartilage in terms of gene expression. Interestingly in the present study we have demonstrated for the first time iNOS expression and NO production in hMSCs during chondrogenic differentiation. We

have also provided a detailed characterization of prostanoid production during chondrogenesis. Our investigations suggest that hMSCs undergoing chondrogenic differentiation could be used to investigate the regulation of the production of these inflammatory mediators in a cell system relevant to chondrocytes.

We first studied iNOS expression and its regulation in articular chondrocytes. We could show that OA and normal cartilage (bovine and porcine samples) and chondrocytes released micromolar amounts of NO in response to IL-1 stimulation indicating that NO production is characteristic not only for OA chondrocytes.

Further investigation on the regulation of iNOS expression revealed that NFκB is a key transcription factor regulating iNOS expression in human chondrocytes. Intracellular cAMP levels are also involved in iNOS regulation in chondrocytes.

It was reported that iNOS expression in human OA chondrocytes is glucocorticoid insensitive. We demonstrated that Dex-resistant iNOS expression is not restricted to human OA chondrocytes but is true for human healthy chondrocytes and chondrocytes from different species. Additionally, glucocorticoid-resistant iNOS expression was even true for hMSCs differentiating to chondrocytes. However, glucocorticoid-insensitivity is not related to NFκB and cAMP signaling pathways.

The chondrocyte differentiation status was classically categorized via gene expression of cartilage matrix proteins. We propose corticosteroid – insensitive NO production in response to IL-1β stimulation as additional marker of the chondrocyte differentiation status.

Human chondrocytes after stimulation with IL-1 release high levels of PGE2 due to expression of COX-2. In contrast to iNOS COX-2 expression in chondrocytes is glucocorticoid sensitive. In regard to our study under physiologic conditions the effect of NO on prostanoid synthesis in chondrocytes is not very pronounced. COX-2 was not inhibited by NO or its derivates. On the contrary peroxynitrite can provide a higher

“peroxide tone” and activate COX-enzymes in human chondrocytes.

In many previous studies NO production was correlated with the level of apoptosis in cartilage. Our results implicate that endogenous or exogenous NO is not cytotoxic to

chondrocytes, however peroxynitrite at high concentrations can lead to apoptotic cell death.

Oxidative and nitrosative stresses are present in human OA cartilage as we detected nitrotyrosine in cartilage samples. We identified a number of nitrated proteins;

interestingly several of them were related to glucose metabolism. However direct measurement of ·O2- generation in stimulated chondrocytes was impossible.

To conclude, we have shown that chondrocytes are easily stimulated by IL-1. These cells seem to have a high antioxidative potential and are therefore quite resistant against oxidative and nitrosative stress. NO by itself is not cytotoxic to chondrocytes and can exert detrimental effects only in combination with ·O2-.

We suggest that not only a balance between anabolic and catabolic activity of chondrocytes but also in antioxidative and oxidative potential of the cells are critical for proper function of chondrocytes and maintenance of hyaline cartilage.

Although we could show that IL-1 is a very potent inducer of OA-related genes, the observed gene expression changes are not mediated by NO, but are directly due to the action of IL-1. Therefore iNOS inhibition in OA would not be beneficial in regard of chondrocyte homeostasis.

1. Introduction... 1

1.1. Osteoarthritis ……… 1

1.2. Articular cartilage ………. 4

1.2.1. Chondrocytes ... 4

1.2.2. Collagen ……… 6

1.2.3. Aggrecan ……….. 7

1.3. Human mesenchymal stem cells differentiation model ……… 8

1.4. Pathophysiology of OA ... 10

1.4.1. Changes in cartilage by proinflammatory cytokines ……….. 10

1.4.1.1. Pivotal role of IL-1β in the pathophysiology of OA …………..…... 10

1.4.2. Apoptosis in the pathophysiology of OA ……….. 11

1.4.3. Prostanoids in the development of OA ……… 12

1.4.3.1. Biosynthesis of prostanoids ………. 12

1.4.3.2. The role of prostanoids in cartilage ………. 14

1.4.3.3. COX inhibition in OA ……….. 15

1.4.4. Nitrosative and oxidative stress ……… 15

1.4.4.1. Nitric oxide and nitric oxide synthases ……….…. 15

1.4.4.1.1. iNOS (NOS-2) ……….. 17

1.4.4.1.2. NOS inhibitors ...…. 19

1.4.4.2. Peroxynitrite ...…. 21

1.4.4.3. Formation of nitrotyrosine ……….…. 23

1.4.5 Interplay of prostaglandins and nitric oxide ………. 24

1.4.6 Nitric oxide and the development of OA ……….. 25

2. Aims of the study ………... 26

3. Materials and Methods ………... 28

3.1. Chemicals ………. 28

3.2. Cartilage processing and cell culture ……….. ………. . 28

3.2.1. Cartilage processing………. 28

3.2.2. Cartilage explants in culture ...…. 28

3.2.3. Monolayer culture ………. 29

3.2.4. Alginate culture ………... 29

3.3. human Mesenchymal Stem Cells from bone marrow ………... 29

3.3.1 Chondrogenic differentiation ………. ... 30

3.3.2. Osteogenic differentiation ……… 30

3.3.2.1. Assessment of osteogenic phenotype ……… 30

3.3.2.1.1. von Kossa staining ... 30

3.3.2.1.2. Alkaline phosphatase staining ... 31

3.3.3. Adipogenic differentiation ……… 31

3.3.3.1. Assessment of adipogenic phenotype ……….. 31

3.3.3.1.1. Oil red “O” staining ……… 31

3.4. Cell treatment with growth factors, cytokines and inhibitors ………. 32

3.4.1. Stimulation of chondrocytes ……… 33

3.4.2. Stimulation of hMSCs ……….. 33

3.5. Biochemical methods ………. 33

3.5.1. Determination of cellular viability ……… 33

3.5.2. Toxicity: release of lactate dehydrogenase (LDH) ……….. 33

3.5.3. Griess assay ……….. 33

3.5.4. PGE2 ELISA ………... 34

3.5.5. GC/MS/MS ………. 34

3.5.6. Cytochrome c assay ………. 34

3.5.7. Electron Spin Resonance (ESR) ……… 34

3.5.8. SDS-Page ... 36

3.5.9. Western blotting and protein detection ………. .. 36

3.5.10. Stripping of Western blots ……… 37

3.5.11. Two-dimensional gel electrophoresis ……… 37

3.5.11.1. Cell extract ... 37

3.5.11.2. Two-dimensional gel electrophoresis ... 38

3.5.11.3. Western blot analysis ... 39

3.5.11.4. Protein identification (MALDI-TOF)... 39

3.6. Staining of cartilage and chondrocytes ……….... 40

3.6.1. Preparation of cartilage sections ………. 40

3.6.2. . Histochemical and fluorescence staining of tissue samples ……. 41

3.6.3. Cytospin preparations ……… 42

3.6.4. Evaluation of antibodies ……… 42

3.6.4.1. iNOS TK2553 ………. 42

3.6.4.2. β-actin ………. 42

3.6.4.3. Nitrotyrosine ………. 43

3.6.4.4. PCS ... 43

3.6.5. TUNEL staining ... 44

3.6.6. Haematoxylin staining ……… 44

3.7. Molecular biology methods ………. 45

3.7.1. RNA Extraction and TaqMan PCR ……….. 45

3.7.2. Affymetrix and bioinformatics analysis ………. 47

3.7.2.1. Microarray ……….. 47

3.7.2.2. Microarray data analysis ………. 47

4. Results ……… 49

4.1. Characterization of hMSC differentiation model ………. 49

4.1.2. Collagen subtypes, aggrecan and SOX-9 expression during the course of chondrogenic differentiation ………. 53

4.1.3. Regulation of marker gene expression in pellets was similar to chondrocytes in alginate beads ……… 56

4.1.4. Affymetrix gene chip characterization of chondrogenic differentiation 58

4.1.5. Comparison of gene expression of healthy cartilage and chondrogenic pellets ……… 60

4.2. iNOS expression and NO production in chondrocytes ……….. 64

4.2.2. Detection of iNOS in human articular cartilage ………. 64

4.2.3. Production of NO in response to inflammatory stimuli: IL-1α, IL-1ß, TNFα, and LPS ………... 66

4.2.3.1. The effect of IL-1ß on the iNOS induction in cartilage explants ……… 68

4.2.3.2. Cellular localization of iNOS in primary chondrocytes ……… 69

4.2.4. Time course of NO production, iNOS mRNA and protein expression in human chondrocytes after IL-1ß stimulation ………… 70

4.2.5. The effect of NOS inhibitors on NO synthesis in human chondrocytes 72 4.2.6. The effect of cycloheximide and Byk 17790 on iNOS protein expression ………... 73

4.2.7. Nitric oxide production and iNOS gene expression during

the course of chondrogenic differentiation ……….. 75

4.3. Regulation of iNOS in chondrocytes ……… 77

4.3.1. The effect of Dexamethasone ……… 77

4.3.1.1. iNOS and COX-2 are differentially regulated in human chondrocytes …….. 77

4.3.1.2. Dexamethasone does not inhibit IL-1β induced NO formation in OA chondrocytes or in hMSCs undergoing chondrogenic differentiation . 78

4.3.1.3. Dexamethasone effect on NO production is species-independent ……….. 80

4.3.1.4. Dexamethasone effect on NO production is independent on stimuli …….. 81

4.3.2. Dexamethasone and the regulation mechanism of iNOS expression 82

4.3.2.1. NFκB ……….. 82

4.3.2.2. cAMP ………. 85

4.3.3. cAMP but not NFκB regulates PGE2 production in chondrocytes ….. 88

4.4. Effects of NO on the chondrocyte gene expression ………. 90

4.4.1. Effects of NO on chondrogenic differentiation ……… 90

4.4.2. Effects of NO on OA chondrocytes ………. 97

4.5. Effects of IL-1 on the chondrocyte gene expression ……… 103

4.5.1. Affymetrix gene chip characterization of IL-1β regulated genes in the hMSCs differentiation model ……… 103

4.5.2. Affymetrix gene chip characterization of IL-1β regulated genes in human chondrocytes, effect of iNOS inhibition ……….. 104

4.6. Effects of NO on the eicosanoid production ……….. 109

4.6.1. Eicosanoid production in human chondrocytes and hMSCs ………… 109

4.6.2.COX-2 expression during chondrogenic differentiation ………. 112

4.6.3.COX-2 mRNA and protein expression in human chondrocytes ……… 113

4.6.4.Time course of eicosanoid production ………. 114

4.6.5.Influence of NO-donors and iNOS inhibitors on PGE2 synthesis ……. 115

4.6.6.Experiments with exogenous Arachidonic Acid (AA)……….. 119

4.6.6.1. AA concentration dependency of prostanoid formation ………. 119

4.6.6.2. Effect of NO-donors on AA stimulated prostanoid formation ……… 120

4.6.6.3. Effect of NO-donors on AA stimulated Isoprostane formation ………. 122

4.6.7. Prostacyclin synthase in human cartilage ……….. 123

4.6.7.1. Prostacyclin has no effect on the expression of extracellular matrix

proteins ……… 124

4.6.7.2. Influence of NO-donors and iNOS inhibitors on PGI2 synthesis …………. 125

4.6.7.3. Effect of NO-donors on AA stimulated PGI2 formation ……….. 126

4.7. The effect of NO on apoptosis (TUNEL) ……….. 128

4.8. Protein nitrotyrosine in chondrocytes ... 134

4.8.1. Detection of nitrotyrosine in OA cartilage ……….. 134

4.8.2. Nitrotyrosine immunostaining correlates with iNOS in human chondrocytes (staining of IL-1β stimulated cells) ……….. ….. 135

4.8.3. Detection of protein-nitrotyrosine in one- and two-dimensional gel electrophoresis ……….. 136

4.8.3.1. 2D-gel analyses revealed no COX-2 nitration ……….. 140

4.9. Superoxide ……… 142

4.9.1. Measurement of ESR signal in chondrocytes supernatant ………… 142

4.9.2. Measurement of ESR signal in cell suspension ……….. 143

4.9.3.Measurement of ESR signal generated by SIN-1 ……… 145

4.9.4.Measurement of superoxide production by human macrophages … 146

4.9.5. Cytochrome c assay (human chondrocytes) ……… 148

5. Discussion ……… 151

5.1. In vitro studies on cartilage metabolism ………. 151

5.2. NO production and iNOS expression in chondrocytes ……… 157

5.3. iNOS regulation in chondrocytes ………. 159

5.4. COX-2 and prostaglandin production in human chondrocytes ……….. 162

5.4.1. Non-enzymatic isoprostane formation in excess of AA ……… 166

5.4.2. Redox-regulation of prostanoid synthesis ……….. 167

5.5. Apoptosis in the development of OA and the role of NO in this process ….. 169

5.6. NO, O2- andONOO^- ……… 171

5.7. Protein tyrosine nitration ……… 176

5.8. IL-1 versus NO mediated effects ………. 180

5.9. Clinical implications ……… 183

6. References……… 186

7. Supplement ……….. 212

1. INTRODUCTION

1.1. Osteoarthritis

Osteoarthritis (OA) is one of the most common forms of musculo-skeletal diseases and the most common form of arthritis that affects millions of patients. According to data in the “Ärzte Zeitung” every second person in Germany aged above 60 suffers from OA. In Europe, a joint is replaced due to OA every 1,5 min, in the USA the situation is even worse (Wieland et al., 2005). Therefore Kofi Annan, the Secretary-General of the United Nations signed the declaration to launch the Bone and Joint Decade 2000-2010 for the treatment and prevention of musculo-skeletal disorders.

Clinically OA is characterized by joint paint, stiffness and movement limitation, crepitus, swelling, and finally instability and deformity of the affected joint.

The prevalence of OA in all joints is strikingly correlated with age. It is uncommon in adults aged under 40 and becomes extremely prevalent in those aged above 60 (over 70% of people who are 65 or older have OA). Additionally OA is more prevalent in women than in men.

OA affects both the small and large joints, either singly or in combination. However, there are joints commonly affected by OA as: knee, hip, interphalangeal joints of the hand, first MTP – metatarsophalangeal joint, cervical and lumbosacral spine. Notably, the ankle, wrist, elbow and shoulder are usually spared. The etiology of OA is unknown.

At present the concept that OA represents not a single disease entity but a group of overlapping distinct diseases, which may have different etiologies but with similar biologic, morphologic and clinical outcomes is one of the most accepted.

OA is a slowly developing degeneration of articular cartilage, but disease processes finally affect the entire joint including the subchondral bone, ligaments, capsule, synovial membrane, and periarticular muscles (Figure 1).

normal OA

Figure 1. Comparison of normal with OA joint.

To see changes in cartilage and bone with secondary

inflammatory changes, particularly in the synovium of OA joint.

Cartilage fibrillation and destruction is observed.

Bone may be most altered in subchondral sites: less stiff and less dense. In OA the

development of osteophytes is seen very often (this involves the formation of a cap of new peripheral articular cartilage as well as new bone formation as part of an endochondral process.

From: Wieland at el.

“Osteoarthritis – an untreatable disease? Nature Reviews 2005

Classically, the diagnosis of OA has relied on the characteristic radiographic changes described by Kellgren and Lawrence (Kellgren and Lawrence, 1957), which include as cardinal feature joint space narrowing, changes in subchondral bone and the formation of osteophytes.

The most potent systemic risk factors for developing OA are increasing age, obesity and female gender. The primary factors leading to joint susceptibility to OA are listed in the Table 1.

Table 1. Risk factors for development of OA:

- constitutional susceptibility:

• heredity

• gender / hormonal status

• race

• obesity - aging

- mechanical factors:

• trauma

• joint shape

• usage: occupational, recreational.

In contrast to rheumatoid arthritis (RA) osteoarthritis is considered a non-inflammatory arthritis, however episodic inflammation of the synovium (synovitis) is also observed in OA. Short comparison between RA and OA is given in the Table 2.

Table 2. Short comparison between OA and RA:

OA RA

general characteristic

strictly local cartilage damage

slowly developing

systemic disease -eyes, lung, heart,

kidneys, blood vessels fast developing prevalence increases with age

(>60 years)

younger people (prevalence much lower

than OA) origin bio-mechanical autoimmune disease

primary changes in cartilage synovium

inflammation secondary inflammation chronic inflammation

driven by chondrocytes T-cells, macrophages

cytokines IL-1, IL-6, (TNF-α) TNF-α, IL-1, IL-6

drugs with disease modifying efficacy

not available on the market

1.2. Articular cartilage

Articular cartilage is a type of hyaline cartilage (gr. hyalos = glass). Articular cartilage is a highly specialized tissue, 1-5 mm thick that covers the surface of synovial joints allowing smooth movement between adjacent bones.

The structure of cartilage matrix determines biomechanical properties of this tissue:

tensile strength and the ability to resist compression.

Cartilage matrix comprises:

- water (75%)

- proteoglycans (5%): mainly aggrecan - collagens (20%): type 2, 6, 9, 11.

1.2.1. Chondrocytes

Cartilage is synthesized and maintained by chondrocytes, the only type of cell found in the cartilage. In the adult human, these cells may occupy as little as 2% of the total volume of articular cartilage. Chondrocytes are located within lacunae (chondrons), which usually contain few chondrocytes and pericellular matrix bound within a discrete collagenous capsule (Poole, 1997) (Figure 2a). Under the light microscope chondrocytes appear round or oval in the deeper part of the cartilage, and lens-shaped near the surface (Figure 2b).

a) b)

tidemark

surface

Figure 2. Haematoxylin staining of human articular cartilage. a) lacunae (chondrons) surrounded by extracellular matrix; intensive staining of pericellular matrix due to higher content of proteoglycans

b) cartilage cross-section, description in text (Photos: Anna Mais).

Nutrition and oxygen are supplied to chondrocytes by the synovial fluid as cartilage is avascular, aneural and alymphatic tissue. Synovial fluid (stands for “egg white” in Greek) is produced by the synovial membrane and is similar in composition to blood plasma, except for the addition of hyaluronic acid, which is responsible for its viscosity.

Chondrocytes are mesenchymal cells specialized to produce cartilage-specific matrix, with collagen type 2 and aggrecan, that are responsible for the tensile strength and resistance to mechanical stresses. However, the phenotype of chondrocytes shows multiple modulations. Phenotypic changes are occurring in differentiating chondroprogenitor cells of the fetal growth plate cartilage in vivo. The cells can differentiate from progenitor cells characterized by the expression of the alternative splice variant of type 2 collagen, through mature chondrocytes expressing the typical cartilage proteins: collagen type 2, type 9 and type 11 as well aggrecan to hypertrophic chondrocytes, which are characterized by synthesis of type 10 collagen (Aigner et al., 1993; von der Mark et al., 1992). These cells are found in the lowest zone of the cartilage of the fetal growth plate (Sandell and Aigner, 2001). Chondrocyte hypertrophy has been shown to be an initial event leading to cartilage mineralization (Kirsch et al., 1992) and endochondral ossification (Topping et al., 1994). Premature chondrocyte differentiation to hypertrophic chondrocytes was indicated by von der Mark (von der Mark et al., 1992) in OA cartilage.

In vitro, after isolation from cartilage and expansion in monolayer culture chondrocytes shift toward fibroblast-like morphology and lose their characteristic spherical shape. In this culture conditions not only cell shape is altered, but also production of collagens.

Instead of cartilage-specific collagen 2, collagen type 1 and type 3 are synthesized. This process is known as dedifferentiation of chondrocytes. To preserve the chondrocyte specific phenotype 3-dimensional cell culture models were developed, as suspension of these cells in alginate beads.

1.2.2. Collagen network is the major structural component of articular cartilage.

Collagen fibers are bio-mechanically very stable and show an extremely long half-life estimated to be about 117 years (Verzijl et al., 2000). Type 2 collagen (80-90% of articular cartilage collagens) is the most important component of these fibers and a molecule specific for cartilage. However, other collagens such as collagen type 6, found mostly in the territorial matrix closer to the cell, collagen type 11 and collagen type 9 are also present in the ECM but to a much lower extent. These last two collagens may have a role in determining the thickness and assembly of the collagen 2 fibers. Some other molecules, which are bound to collagen fibrils, are fibromodulin, decorin, biglycans and other leucine-rich repeat proteins.

The fiber orientation is different in various parts of articular cartilage (see Figure 3).

In the superficial (tangential) zone collagen fibers run parallel to the surface, and this layer has the greatest ability to resist shear stress.

In the middle zone (transitional) collagen fibers run in variable directions and this structure is responsible for transition between the shearing forces of the surface layer to compression forces in the deeper layer. The middle zone is the richest of proteoglycans.

In the deep (radial) zone of cartilage collagen fibers are perpendicular to the surface and anchored in the tidemark. The structure of this zone is important for the distribution of loads and compression resistance.

The calcified zone is located between cartilage layer and subchondral bone. This zone contains the Tidemark Layer (basophilic line which straddles the boundary between calcified and uncalcified cartilage). Collagen 10 is charactreristic for calcified cartilage.

Figure 3. Cross sections cut through the thickness of articular cartilage, showing the four zones of the cartilage: superficial, intermediate, radiate, and calcified. The foreground shows the

organization of collagen fibers. From: Joseph M. Mansour Biomechanics of Cartilage

1.2.3. Aggrecan is the major non-collagenous component of articular cartilage.

Aggrecan is a very large molecule consisting of a central protein core (about 2000 amino acids), to which numerous glycosaminoglycan chains of chondroitin sulfate are attached. Many molecules of aggrecan monomers are attached to a long, hyaluronic acid chain forming large aggregates. The glycosaminoglycan chains of aggrecan are negatively charged. Therefore they can bind many water molecules and generate an osmotic swelling pressure making articular cartilage resistant to compression. During loading, when cartilage is compressed, water is squeezed out from ECM. However, when the compressive force is removed, aggrecan again binds water molecules, cartilage swells generating a force equal to the compressive force of the next loading.

A net loss of proteoglycan content is one of the hallmarks of OA cartilage degradation (Mankin et al., 1971; Mankin et al., 1981).

All matrix molecules of the articular cartilage are synthesized by chondrocytes and this process is controlled by hormones, cytokines and mechanical stimuli. There are several

endogenous anabolic factors released by chondrocytes that stimulate cartilage generation and remodelling. Among them are transforming growth factor β (TGFβ), bone morphogenic proteins (BMPs) and insulin growth factor 1 (IGF1). These factors are also very important during chondrogenesis. In normal adult cartilage matrix remodelling is a very slow process.

1.3. Human mesenchymal stem cells differentiation model

Human mesenchymal stem cells (hMSCs) are resident in the bone marrow throughout adult life and have the capacity to differentiate along a number of connective tissue lineages, including bone, cartilage and adipose tissue (Jaiswal et al., 1997; Johnstone et al., 1998; Mackay et al., 1998; Pittenger et al., 1999; Murphy et al., 2002).

The availability of human chondrocytes is limited and they are difficult to expand in vitro.

This is obviously a limiting factor for investigations on the regulation of the above- mentioned mediators. Bone marrow human mesenchymal stem cells (hMSCs) can be differentiated into chondrocytes and are able to synthesize cartilage matrix (You et al., 1998; Neumann et al., 2002). What makes hMSCs such an attractive alternative to primary chondrocytes is that they can be relatively easily expanded and harvested (Solchaga et al., 2004). hMSCs differentiated into chondrocytes could thus provide a solution for drug development projects and tissue replacement therapies targeting OA.

Indeed, in recent years there has been increasing interest in hMSCs as an alternative to primary chondrocytes for drug discovery projects and tissue transplant therapies (Redman et al., 2005). However, it is known that currently used protocols are not able to induce homogenous differentiation leading to uniform hyaline cartilage, but rather to chondrocytic phenotype specific for OA cartilage or as recently reported intervertebral disc-like cells (Winter et al., 2003; Steck et al., 2005).

Still, most studies were focussed on the expression of ECM-markers such as collagen subtypes. Beside this there was only limited interest in the inflammatory mediator production by hMSCs during and following chondrogenesis, although production of high levels of NO and PGE2 after stimulation of IL-1β is specific for chondrocytes.

Recently subpopulations of mesenchymal progenitor cells were identified in normal and OA cartilage (Alsalameh et al., 2004; Fickert et al., 2004).

These cells are potential sources of cartilage tissue repair, but also lead to the development of osteophytes often observed in OA (Gelse et al., 2003).

hMSCs

chondroblast chondrocyte

osteoblast osteocyte

preadipocyte adipocyte

Figure 4. The diagram illustrates the sequence of events involved in the formation of cartilage, bone and adipose tissue from adult hMSCs. hMSCs proliferate (i.e., undergo multiple divisions) to generate increased numbers of MSCs. Many of these expanded hMSCs then undergo a commitment step and enter a particular lineage pathway, leading ultimately to the formation of differentiated, tissue-specific cells (adapted from Osiris Therapeutics, Inc.).

hMSCs undergo chondrogenic differentiation when cultured in serum-free conditions and stimulated with TGFβ.

Several transcription factors have been shown to be involved in the regulation of chondrogenesis. Sox-9 (SRY-related high mobility group-Box gene 9) is a key regulator of chondrogenic differentiation. This transcription factor regulates expression of cartilage specific collagen 2a1 (de Crombrugghe et al., 2001; de Crombrugghe et al., 2000; Lefebvre et al., 2001; Lefebvre et al., 1997) . Mice with mutated Sox-9 show numerous cartilage-derived skeletal disorders (Wright et al., 1995).

Another transcription factor Runx2 (Cbfa 1) was shown to play a central role in skeletal development, primarily in osteoblast differentiation and bone formation. However recently it has been demonstrated that Runx2 promotes also early chondrogenic differentiation (Stricker et al., 2002).

Interestingly, it has been shown that the chondrogenic and adipogenic activity of OA patient-derived hMSCs is reduced in comparison to hMSCs donors without any symptoms of OA (Murphy et al., 2002). The same authors reported the loss of proliferative capacity of cells from OA patients, which was not age- or site-dependent, but associated with the disease.

1.4. Pathophysiology of OA

1.4.1. Changes in cartilage by proinflammatory cytokines

In pathological conditions such as OA, cartilage turnover may accelerate, leading to early regenerative changes, such as synthesis of extracellular matrix which are accompanied by chondrocyte proliferation (hyperplasia) as well as chondrocyte enlargement (hypertrophy). However this early attempt to repair the damaged matrix is followed by degenerative changes, such as insufficient synthesis of ECM, chondrocyte cell death and cartilage matrix erosion.

Chondrocytes, but to a lesser extent also synovial cells, can release several catabolic cytokines among others interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), which drive the breakdown of articular cartilage. IL-1β is the major pro-inflammatory cytokine found in synovial fluid (Thomas et al., 2002).

1.4.1.1. Pivotal role of IL-1β in the pathophysiology of OA

Evidence has accumulated that IL-1 is by far a more destructive mediator for cartilage than TNFα (van Lent et al., 1995; van de Loo et al., 1995; Probert et al., 1995).

IL-1β is synthesized by cells as a precursor, which is intracellulary converted by interleukin-1 converting enzyme (ICE) to produce the active form. IL-1 can stimulate its

own production through autocrine and paracrine mechanisms. IL-1 has been shown to activate matrix metalloproteinases (MMPs), which degradate cartilage matrix. MMP1, MMP8 and MMP13 cleave collagen; MMP3 cleaves proteoglycans (Shingu et al., 1995;

Mengshol et al., 2000; Fernandes et al., 2002).

It has been demonstrated that IL-1β induces the dedifferentiation process of chondrocytes by decreasing the expression of type 2 and 9 collagens and increasing type 1 and 3 collagen expression (Goldring et al., 1988).

IL-1β stimulates the expression of all enzymes of PGE2 biosynthesis pathway:

phospholipase A2, cyclooxygenase 2 (COX-2) and prostaglandin E synthase 1 (PGES) resulting in increased PGE2 production (Massaad et al., 2000; Thomas et al., 2000;

Masuko-Hongo et al., 2004).

IL-1β is a very potent inducer of the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production.

The pathophysiological importance of IL-1β in OA has been confirmed by use of an ICE inhibitor pralnacasan (Vertex/Sanofi-Aventis) in two murine models of OA, and also by gene transfer of IL-1β receptor antagonist, where both treatments reduced joint damage (Rudolphi et al., 2003; Zhang et al., 2004).

1.4.2. Apoptosis in the pathophysiology of OA

Several authors have suggested that apoptosis of chondrocytes is characteristic for OA, and is a reason for cartilage degeneration (Blanco et al., 1998; Hashimoto et al., 1998a;

Kim et al., 2000; Goggs et al., 2003; Kirsch et al., 2000; Heraud et al., 2000). There are also opinions, contrary to previous suggestions, that apoptotic cell death is not a widespread phenomenon in aging or OA cartilage (Aigner et al., 2001).

Chondrocyte apoptosis in vivo seem to differ from “classical apoptosis”. Unlike classical apoptosis chondrocyte apoptosis involves an initial increase in endoplasmic reticulum (ER) and Golgi apparatus, reflecting an increase in protein synthesis. Increase in ER membranes provides compartments within which cytoplasm and organells are digested what leads to the complete self-destruction of the chondrocyte and remaining empty lacunae. This mechanism may be of importance as there are no phagocytic cells within

the cartilage. It has been proposed to give chondrocyte apoptosis even a special name

“chondroptosis” (Roach et al., 2004).

Previous studies have linked NO and chondrocyte apoptosis, especially the data from animal models of OA suggested that NO could be a signal for apoptosis.

Anterior cruciate ligament transaction in dogs resulted in an increase in NO levels in the affected joint and increase in apoptotic cells (Hashimoto et al., 1998b). Treatment with selective iNOS inhibitor (L-NIL) resulted in the reduced level of chondrocyte apoptosis and reduction of OA progression in this model (Pelletier et al., 2000). The data revealed an association between NO and apoptosis in vivo, however it is still unclear if chondrocytes underwent apoptosis in direct response to NO. Interestingly, in the rabbit model of OA hyaluronan injections decreased the level of apoptosis but not NO (Takahashi et al., 2000).

Whether apoptosis or cell disintegration is primary or secondary to the destruction of cartilage matrix is difficult to answer. There is also the possibility that chondrocyte death and matrix loss form a cycle, with the progression of one having effects on the other.

1.4.3. Prostanoids in the development of OA 1.4.3.1 Biosynthesis of prostanoids

Prostanoids are a class of saturated fatty acid deriverates containing prostaglandins and thromboxanes. The prostanoid class is a subclass of eicosanoids (gr. Eicos – twenty) containing 20 carbon atoms in the molecular structure.

The first step in prostanoid biosynthesis is the liberation of arachidonic acid from the membrane phospholipids by phospholipase A (PLA2). Arachidonic acid (AA) serves as precursor of all prostanoids. AA undergoes the cyclooxygenase reaction in which two molecules of oxygen are added to form a bicyclic endoperoxide with a hydroperoxy group in position 15 (PGG2). This hydroperoxide is then reduced by a functionally coupled peroxidase reaction to form the 15-hydroxy-9,11-endoperoxide (PGH2). This conversion of AA is catalyzed by cyclooxygenase, named also prostaglandin endoperoxide H2 synthase (COX or PGHS, respectively). COX exists in at least two

isoforms: COX-1, expressed constitutively and COX-2, which expression is induced in various cell types after exposure to bacterial endotoxins or proinflammatory cytokines.

PGH2 is subsequently converted to a variety of prostanoids that include: prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2α (PGF2α) , prostacyclin (PGI2) and thromboxane (TX), as ilustrated in Figure 5.

Figure 5. The biosynthesis of prostanoids from arachidonic acid by cyclooxygenase pathway.

From: Narumiya et al., “Prostanoid receptors: Structures, Properties, and Functions”, Physiological Reviews, 1999, Vol. 79, 1193-1226

The proportions of the various enzymes of the prostanoid pathway and therefore produced prostaglandins differ according to the cell type.

In 1990 Morrow reported prostaglandin-like molecules in humans that were result of peroxidation of AA by a free radical mechanism independent of COX activity (Morrow et

al., 1990). As these compounds are isomeric to COX-derived PGF2α, they were termed F2-isoprostanes (isoprostanes). Interest in these molecules arises from the fact that they provide an index of free-radical induced lipid peroxidation. The level of isoprostanes can indicate several disorders, for example higher levels of 8-epi-PGF2α have been detected in the plasma of smokers.

However COX-dependent formation of isoprostanes has been also postulated (Klein et al., 1997)

1.4.3.2 The role of prostanoids in cartilage

Gene expression, cartilage matrix synthesis and proliferation have been shown to be regulated by prostaglandins in cartilage (Geng et al., 1995).

In articular cartilage prostaglandins have been discussed as mediators of inflammation and tissue destruction. However recently prostglandins have been shown to play a role in a cartilage formation by stimulating the differentiation of prechondroblasts to chondrocytes (Jakob et al., 2004). Schwartz et al. have proposed that the effect of PGE2 depends on its concentration, low levels promote differentiation, whereas high doses promote an anabolic response (Schwartz et al., 1998).

In regard to this PGE2 can exert both catabolic or anabolic effects in chondrocytes depending on the microenvironment and which of the four receptor subtypes are present. Indeed, PGE2 has also been shown to be involved in the development of OA.

PGE2 modulates proteoglycan and collagen synthesis (Abramson, 1999; Goldring et al., 1996), stimulates matrix metalloproteinase-2 expression (Choi et al., 2004) and enhances matrix metalloproteinase-3 production (Amin et al., 2000). In addition, PGE2

inhibits chondrocyte proliferation (Blanco and Lotz, 1995b) and induces chondrocyte apoptosis (Notoya et al., 2000; Amin et al., 1997). Strikingly, PGE2 was also the only parameter, which we recently found to correlate with the WOMAC-index scores of patients with knee OA (Brenner et al., 2004).

The overproduction and role of PGE2 in OA cartilage have been reported several times.

However, COX-2 overexpression in OA not only leads to the production of PGE2 but to a variety of prostanoid endproducts that have not been fully characterized in human cartilage.

1.4.3.3 COX inhibition in OA

COX inhibitors represent the most widely used class of drugs. Although aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of inflammation, pain and fewer for more than 100 years the mechanism of their action, namely COX inhibition, was demonstrated for the first time in 1971 by Sir John Vane.

NSAIDs inhibit both COX isoforms. COX-2 inhibition accounts for the therapeutic benefits and inhibition of COX-1 for the side effects of NSAIDs. The most common side effect of NSAIDs is irritation damage of gastrointestinal mucosa leading to the development of gastric erosions and ulcerations. This effect is due to COX-1 inhibition.

As COX-2 is primarily responsible for increased PG production in inflamed tissues specific COX-2 inhibitors have been developed to minimize the risk of side effects mainly in gastro-intestinal tract.

Both classes of COX inhibitors are very often prescribed for the treatment of OA.

Specific COX-2 inhibitors: celecoxib (Celebrex, Pfizer) and rofecoxib (Vioxx, Merck) have been shown to be more effective than placebo and similarly effective as standard doses of nonselective NSAIDs (Hochberg, 2005). Both classes of COX inhibitors provide effective relief from pain as a major subjective symptom of OA (Martel-Pelletier et al., 2003). However, clinical trial investigating rofecoxib revealed that patients at higher cardiovascular risk exhibit a significantly higher incidence of myocardial infraction receiving COX-2 specific inhibitor compared with NSAIDs (Bombardier et al., 2000).

Interestingly, recently celecoxib was reported to have chondroprotective effects by enhancing proteoglycan content and therefore matrix integrity in OA cartilage (Mastbergen et al., 2005).

1.4.4. Nitrosative and oxidative stress

1.4.4.1. Nitric oxide and nitric oxide synthases

Nitric oxide is a gaseous short lived regulatory molecule exerting a broad range of functions in many physiological and pathophysiological cell and tissue responses (Moncada et al., 1991). Under aqueous, aerobic conditions NO spontaneously oxidizes to its stable products nitrite and nitrate.

The first described physiological effect of NO was activation of soluble guanylyl cyclase (Ignarro et al., 1999). The rise in cGMP (3`,5`-cyclic guanosine monophosphate) level accounts for many of cellular responses to NO.

Relatively low reactivity combined with a high lipophilicity allow NO to diffuse away from the point of origin, and therefore to carry out its function as a messenger molecule beyond cell borders (Wink and Mitchell, 1998).

NO has the potential to interact directly or indirectly with metals, thiols and oxides, and therefore affects proteins, nucleic acids, lipids and sugars (Davis et al., 2001). Effects of NO depend on its concentration and the redox state of the cell.

NO is produced by nitric oxide synthases (NOS). These group of enzymes catalyse the production of NO and L-citrulline from L-arginine, NADPH and O2.

Figure 6. Nitric oxide biosynthesis.

NO synthesis from L-arginine is a reaction which involves two separate mono-oxygenation steps. N^ω-hydroxy- L-argninie is an intermediate formed by a reaction requiring O2 and NADPH in a presence of BH4. The second step results in the oxidation of N^ω-hydroxy- L-argninie to form citrulline and NO.

From: Knowles, (1994) Nitric oxide synthases in mammals. Biochem J 298: 249-258

There are three NOS isoforms identified: endothelial NOS (eNOS, NOS III), neuronal NOS (nNOS, NOSI) and inducible NOS (iNOS, NOSII). NOSes are enzymes active only

as dimers. They contain tightly-bound cofactors (6R)-5,6,7,8-tetra-hydrobiopterin (BH4), flavine-adenine dinucleotide (FAD), flavine mononucleotide (FMN) and iron protoporphyrin IX (haem)(Knowles and Moncada, 1994; Marletta et al., 1998; Alderton et al., 2001). The activity of eNOS and nNOS is calcium-dependent, as Ca²⁺ stabilizes the calmodulin binding to its binding site in these two NOS isoforms, thereby initiating NO synthesis (Bredt and Snyder, 1990; Abu-Soud and Stuehr, 1993). However in iNOS calmodulin is tightly bound and therefore its activity is calcium-independent (Vallance and Leiper, 2002; Ruan et al., 1996; Vallance and Leiper, 2002).

eNOS and nNOS are regarded as constitutively expressed, however it has been shown that expression of these enzymes is to some extent also regulated at the transcriptional level by local environmental conditions (Xu et al., 1995; Liu et al., 1996; Kleinert et al., 2000; Forstermann et al., 1998). After activation eNOS and nNOS produce nanomolar concentrations of NO, and are active for relatively short periods of time. Both isoforms are principally considered to participate in the regulation of physiological processes in the cardiovascular and nervous system, were they were first found (Marletta et al., 1998). However, their expression has been found later in other tissues and cell types.

1.4.4.1.1. iNOS (NOS-2)

iNOS was first described in activated macrophages (Hibbs et al., 1988). This isoform is not usually expressed in healthy quiescent cells, but is rapidly transcriptionally induced in multiple cell types in response to stimulation with bacterial endotoxins or proinflammatory cytokines (Vallance and Leiper, 2002). Once induced, iNOS produces high amounts of NO (about 100x higher concentrations as eNOS and nNOS) for a prolonged period of time (Nathan, 1992). These high levels of NO are important for a host defence against infectious organisms (Nathan, 1997; Stuehr et al., 1991; Schmidt and Walter, 1994; Nathan, 1997). NO produced by iNOS regulates also the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells (Coleman, 2001).

Expression of iNOS has been found in many cell types like in endothelium, epithelium and also in chondrocytes and synoviocytes after induction.

iNOS expression is regulated at the transcriptional level and at the level of iNOS mRNA stability (Kleinert et al., 2000). Activation of transcription factor NF-kappa B (NFκB) seems to be an essential step for iNOS induction in most cells (Förstermann and Kleinert, 1995). Glucocorticoids have been shown to interfere with iNOS expression in many cell types (Di Rosa et al., 1990). Inhibition of iNOS expression by glucocorticoids has been shown to result from inhibition of NFκB activation (Kleinert et al., 1996;

Mukaida et al., 1994). cAMP activated transcription factors like CREB, C/EBP and ATF2 seem also to be involved in the regulation of iNOS expression (Bhat et al., 2002;

Kleinert et al., 2003). However, regulation of iNOS expression is very complicated and can also involve other transcription factors like AP1 or STAT1.

Table 3. Short characteristics of nitric oxide synthases.

iNOS eNOS nNOS

expression inducible constitutive constitutive

stimulated by LPS, proinflammatory

cytokines - -

calcium dependent - + +

present in

chondrocytes, macrophages, lymphocytes, epithelial

cells, smooth muscle cells etc.

endothelial cells neurons

The production of high NO levels in inflammation is considered to be responsible for many detrimental effects leading to tissue destruction. The overproduction of iNOS is implicated in a number of pathologies like septic shock (Titheradge, 1999), ulcerative colitis, Crohn`s disease (Cross and Wilson, 2003), asthma (Barnes and Liew, 1995;

Ricciardolo et al., 2004; Moncada, 1999), rheumatoid arthritis (Henrotin et al., 2003), but iNOS may be also involved in the pathogenesis of other disorders associated with a low grade chronic inflammation state, such as atherosclerosis (Cromheeke et al., 1999;

Behr-Roussel et al., 2000), diabetes (Shimabukuro et al., 1997; Shimabukuro et al., 1998; Zhou et al., 2000) or osteoarthritis (Amin and Abramson, 1998; Studer et al., 1999).

1.4.4.1.2. NOS inhibitors

As L-arginine is a substrate for NOS several NOS inhibitors are analogues of arginine competing for the active site of the enzyme. L-NMMA (N^G-monomethyl-L-arginine) and L-NAME (N^G-nitro-L-arginine methyl ester) represent the group of NOS unspecific substrate analogues and their inhibitory action on NOS can be antagonized by high concentrations of L-arginine. L-NAME exhibits slightly higher potency towards constitutive enzymes.

AMT (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine) is a very potent (about 1000-fold more potent than arginine analogues presented here) but a non-selective NOS inhibitor (Nakane et al., 1995; Boer et al., 2000).

In recent years a dogma was established that the constitutive forms of nitric oxide synthases, eNOS and nNOS, are critical to normal physiology and inhibition of these enzymes causes damage, whereas induction of the iNOS is harmful and specific inhibition of this enzyme would be beneficial. Therefore, specific iNOS inhibitors have been developed.

The first highly selective iNOS inhibitors were the bis-isothioureas reported by Garvey et al. (Garvey et al., 1994), which were ca. 200-fold more selective for iNOS than for eNOS, but only 5-fold in comparison to nNOS (Alderton et al., 2001).

Improvement of iNOS selectivity was achieved with 1400W an amidine-derived iNOS inhibitor. 1400W is not only highly selective as an iNOS inhibitor versus both eNOS and nNOS but also penetrates cells and tissues (Garvey et al., 1997). 1400W does exhibit an acute toxicity at high doses, which is likely to prevent its safe therapeutic use in humans, but it can be used as a pharmacological tool in a variety of animal models (Alderton et al., 2001).

BYK191023 is an imidazopyridine derivative developed by ALTANA Pharma.

Imidazpyridine compounds represent a novel class of NO-synthase inhibitors with high selectivity for the inducible isoform (Strub et al., 2005). BYK191023 is 200-fold more selective for human iNOS versus nNOS and 1000-fold in comparison to eNOS.

BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations (Strub et al., 2005).

Table 4. The selectivity of NOS inhibitors.

Selectivity (fold) Inhibitor Structure

iNOS/eNOS iNOS/nNOS nNOS/eNOS

L-NMMA 0,3 0,8 0,3

L-NAME 0,05 0,05 1

AMT 3 0,8 3

1400W 200 20 10

BYK191023 >1000 >200 -

Selectivity was determined on the basis of IC50 values obtained for purified NOS isoforms in the same experimental conditions (except values for BYK191023). Source: Boer et al. The Inhibitory Potency and Selectivity of Arginine Substrate Site Nitric-Oxide Synthase Inhibitors Is Solely Determined by Their Affinity toward the Different Isoenzymes 2000, Molecular Pharmacology; 58:1026-1034. Data for BYK191023 from Strub et al. The Novel Imidazopyridine BYK191023 is a Highly Selective Inhibitor of the Inducible Nitric Oxide Synthase 2005 Molecular Pharmacology;

There are also NOS inhibitors with distinct mechanisms of action than competing with arginine for the active site of these enzymes. For example BH4 analogs, which act at BH4 binding site, like 4-amino-H4-biopterin (Werner et al., 2003) or inhibitors of NOS dimerization like imidazole derivatives (Ohtsuka et al., 2002).

Clinical trials with NOS inhibitors were started in septic shock patients, however they were not successful till now and had to be stopped due to increased mortality. This was probably due to use of unspecific NOS inhibitors (L-NMMA) (Petros et al., 1991; Petros

et al., 1994). Still highly selective iNOS inhibitors have not yet been tested in published clinical trials.

1.4.4.2. Peroxynitrite

●NO reacts very rapidly with superoxide (^●O2-) to form peroxynitrite (ONOO^-).

The rate constant of this reaction is near the diffusion-controlled limit

(k= 6,7x10⁹ M^-1● s^-1) (Huie and Padmaja, 1993). Dismutation of ^●O2- by superoxide dismutases (SOD) was observed with k= 10⁷ M^-1● s^-1, which is 2-3 orders of magnitude slower than the reaction with NO, so formation of peroxynitrite is favored.

Superoxide anion can be generated in cells either enzymatically (NADPH oxidases) or by processes that produce reactive oxygen species (ROS) such as the electron transport chain in mitochondria. Cellular sources of superoxide are given in the

Figure 7.

●

O

●

NO

ONOO

•

•

•

•

•

H2O2

SOD

•OH RNS

Figure 7. Cellular sources and fates of superoxide (^●O2-). Superoxide reacts with NO much faster than its detoxification reaction with superoxide dismutase (SOD) takes place.

The product of superoxide and nitric oxide is peroxynitrate, which can undergo further reactions.

Although neither ^●NO nor ^●O2- is a strong oxidant, ONOO^- is a potent and versatile oxidant that can attack a wide range of biological targets (Pryor and Squadrito, 1995).

Some of biologically important reactions of peroxynitrite are listed below.

Biological reactions of peroxynitrite:

- nitration of tyrosine residues of proteins (Haddad et al., 1994; Reiter et al., 2000)

- triggering of lipid peroxidation (Radi et al., 1991b)

- inhibition of mitochondrial electron transport (Radi et al., 1994) - oxidation of thiol compounds (Radi et al., 1991a)

- DNA-strand breakage (Szabo, 2003; Szabo et al., 1996; Szabo, 1996;

Zingarelli et al., 1996)

- activation of MMP’s (Migita et al., 2005)

- inactivation of TIMP (tissue inhibitor for MMP), and α1-proteinase inhibitor (Brown et al., 2004)

- modulation of cyclooxygenase activity (Landino et al., 1996; Deeb et al., 2002; Schildknecht et al., 2005; Mollace et al., 2005)

- apoptosis (Lin et al., 1995; Salgo et al., 1995; Virag et al., 2003)

1.4.4.3. Formation of nitrotyrosine

Peroxynitrite is a potent protein-nitrating agent. Especially aromatic tyrosine residues of proteins are susceptible to reaction with ONOO^-, leading to the formation of 3- nitrotyrosine (Figure 8).

O

H NH₂

OH O

O

H NH₂

OH O O N O

N O O

O-

tyrosine 3-nitrotyrosine

Figure 8. Formation of 3-nitrotyrosine.

Protein nitrotyrosine formation alters the structure and function of proteins. It may prevent tyrosine phosphorylation leading to alterations in signal transduction, which has been considered as pathological event, but on the other hand it might be also a part of cellular signaling mechanisms.

Increased levels of 3-nitrotyrosine have been reported in many diseases, such as cancer, Parkinson`s disease, Alzheimers disease, COPD, RA and OA. However, the group of Stuehr postulated that protein nitration is observed under normal conditions in all tissues (Aulak et al., 2004). The role protein nitration plays in cell physiology is unclear.

Nitrotyrosine formation can be detected by immunostaining and has been used as a

“footprint” of peroxynitrite. However a second mechanism of tyrosine nitration via heme peroxidase dependent reactions using nitrite as substrate to generate the nitrating agent nitrogen dioxide have been proposed. Thus nitrotyrosine staining can be used as an indicator for nitrosative stress in the tissue rather than a specific marker of peroxynitrite.