The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis

Abstract

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.<br />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.<br />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.<br /><br />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.<br />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.<br />Interestingly in the present study we have demonstrated for the first time iNOS expression and NO production in hMSCs during chondrogenic differentiation.<br /><br />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.<br />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.<br />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.<br />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.<br />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-.<br />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.