Abstract
Joint injuries are highly associated with cell death and development of posttraumatic osteoarthritis (PTOA). The present study focused on necroptosis as a possible modality of chondrocyte death after cartilage trauma and its relevance in OA disease in general. For this purpose, apoptosis- and necroptosis-associated markers were determined in highly degenerated (ICRS ≥ 3) as well as macroscopically intact cartilage tissue (ICRS ≤ 1) by means of real-time PCR and immunohistochemistry (IHC). Moreover, influence of blunt trauma and/or stimulation with cycloheximide (CHX), TNF-a, and caspase-inhibitor zVAD were investigated in cartilage explants (ICRS ≤ 1). Further characterization of necroptosis was performed in isolated chondrocytes. We found that gene expression levels of RIPK3 (4.2-fold, P < 0.0001) and MLKL (2.7-fold, P < 0.0001) were elevated in highly degenerated cartilage tissue, which was confirmed by IHC staining. After ex vivo trauma and/or CHX/TNF stimulation, addition of zVAD further enhanced expression of necroptosis-related markers as well as release of PGE2 and nitric oxide, which was in line with increased cell death and subsequent release of intracellular HMGB1 and dsDNA in CHX/TNF stimulated chondrocytes. However, trauma and/or chemically induced cell death and subsequent release of pro-inflammatory mediators could be largely attenuated by RIPK1-inhibitor necrostatin 1 or antioxidant N-acetylcysteine. Overall, the study provided clear evidence of necroptotic cell death in OA disease. Moreover, a possible link between cartilage injury and necroptotic processes was found, depending on oxidative stress and cytokine release. These results contribute to further understanding of cell death in PTOA and development of novel therapeutic approaches.
Highlights
Cartilage trauma is known to result in induction of catabolic gene expression, inflammation, and progressive cell death
Relevance of necroptosis in human OA disease To estimate the relevance of necroptotic processes during the pathogenesis of OA disease, the gene expression of necroptosis- and apoptosis-associated markers was evaluated in highly degenerated tissue samples (ICRS grade ≥ 3)
After ex vivo cartilage trauma we found significant cellprotective effects of Necrostatin 1 (Nec-1), which were stronger than that of caspase-inhibitor zVAD; besides, the combination of both therapeutics revealed additional effects
Summary
Cartilage trauma is known to result in induction of catabolic gene expression, inflammation, and progressive cell death. These trauma-induced pathomechanisms drive the proceeding degradation of the extracellular matrix (ECM) and are considered as the main risk factors for the development of posttraumatic osteoarthritis (PTOA). The mechanical impact and the subsequent compression of the ECM leads to an immediate necrosis of the embedded cells. There are complex molecular pathways, primarily provoked by extrinsic stimuli, deciding upon survival or subsequent regulated cell death[4]. Typical death-associated triggers are pro-inflammatory tumor necrosis factor alpha (TNF-a), reactive oxygen/nitrogen species (ROS/RNS) or Official journal of the Cell Death Differentiation Association
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