The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis

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2005
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Mais, Anna
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Titel in einer weiteren Sprache
The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis
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Zusammenfassung

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.
Chondrocyte viability and function are crucial to articular cartilage as it is the only cell responsible for maintenance of this tissue. It has been implicated that nitric oxide (NO) released due to induction of inducible nitric oxide synthase (iNOS) by activated chondrocytes mediates a number of destructive processes in cartilage.
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, namely OA cartilage in situ, isolated OA chondrocytes in monolayer and in 3-dimensional alginate matrix and as an alternative to healthy cartilage a hMSCs chondrogenic differentiation model was established.
We could show that OA and normal cartilage (bovine and porcine samples) as well as isolated chondrocytes released micromolar amounts of NO in response to IL-1 stimulation indicating that NO production is characteristic not only for OA chondrocytes.
Interestingly in the present study we have demonstrated for the first time iNOS expression and NO production in hMSCs during chondrogenic differentiation.

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 characteristic as well 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.
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.
Additionally we assessed the effect of NO on prostanoid production, gene expression and apoptosis of chondrocytes. Our results indicate that NO by itself is not cytotoxic to chondrocytes and can exert detrimental effects only in combination with O2-.
Beside this we could show that IL-1 is a very potent inducer of OA-related genes, however the observed gene expression changes are not mediated by NO, but are directly due to the action of IL-1. Therefore concluded, that iNOS inhibition in OA would not be beneficial in regard of chondrocyte homeostasis.

Zusammenfassung in einer weiteren Sprache

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.
Da der Chondrozyt die einzige Zelle des Gelenkknorpels ist, hat jede Einschränkung seiner Funktion bzw. Vitalität eine Auswirkung auf das Knorpelgewebe.
Viele Studien gehen davon aus, dass in Chondrozyten durch das von der iNOS freigesetzte NO ein großer Teil der negativen Effekte auf den Knorpel vermittelt wird. Das Ziel der vorliegenden Arbeit war es den Einfluss von NO auf die Physiologie des Chondrozyten zu untersuchen, um positive bzw. 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-Kultur oder in 3-dimensionaler Alginatmatrix und nicht zuletzt ein chondrogenes hMSC basiertes Differenzierungsmodell.
In den oben genannten Modellen konnten wir zeigen, dass OA- und gesunde Chondrozyten, als auch die differenzierten hMSCs in der Lage waren iNOS zu Expremieren und NO freizusetzen.
Des weiteren wurde die Regulation der iNOS Expression in Chondrozyten untersucht.
Hier konnten wir zeigen, dass die Expression der iNOS in humanen Chondrozyten zentral von dem Transkriptionsfaktor NFκB reguliert wird. Auch die Beteiligung von intrazellulärem cAMP an der iNOS-Regulation konnte gezeigt werden.
Eine Besonderheit der iNOS-Regulation in Chondrozyten stellt die Nichtbeeinflussung durch Glukokortikoide dar. Diese Befunde konnten wir 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 unabhängig von der NFκB oder cAMP-Signaltransduktion ist. Anhand dieser Daten kamen wir zu der Schlussfolgerung, dass die 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.
Oxidativer und nitrosativer Stress sind im OA-Knorpelgewebe nachweisbar. So 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. Zusätzlich untersuchten wir den Effekt von NO auf die Expression von knorpelrelevanten Genen, die Prostanoid-Produktion sowie die Apoptose-Induktion.
Hier zeigte sich das IL-1 eine Vielzahl von Genen induziert, die mit der Pathophysiologie der OA assoziiert sind. Entgegen früherer Annahmen sind diese Veränderungen im Expressionsprofil, aber nicht die Folge der NO-Freisetzung, sondern werden direkt durch IL-1 vermittelt. Zusätzlich wiesen unsere Ergebnisse daraufhin, dass cytotoxische Effekte von NO nur in Kombinationen mit O2- entstehen können.

Zusammenfassend kann also gesagt werden, dass sich Chondrozyten leicht durch IL-1 zur NO-Freisetzung stimulieren lassen, aber die vorliegende Studie lässt auch darauf schließen, dass eine Inhibition der iNOS bei OA-Patienten, vermutlich keinen positiven Effekt auf die Physiologie und Funktion von Chondrozyten hat.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Induzierbare NO-Synthase, Interleukin 1 (IL-1), Chondrozyt, cartilage, chondrocyte, hMSCs, nitric oxide (NO), inducible NO synthase (iNOS), interleukin 1 (IL-1)
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ISO 690MAIS, Anna, 2005. The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Mais2005Nitri-8300,
  year={2005},
  title={The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis},
  author={Mais, Anna},
  address={Konstanz},
  school={Universität Konstanz}
}
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January 31, 2006
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