Abstract
This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.
Highlights
Osteoarthritis (OA) poses an enormous burden on the individual patient that adds up to a profound socioeconomic impact on health care sectors worldwide [1]
Significant differences between groups are indicated with asterisks (*p < 0.05, n = 3, Friedman test). d, e OA chondrocytes from seven patients were exposed to the galectin mixture Gal-1/-3/-8 (5/1/5 μg/ml) over time and NF-κB activation was measured by In-Cell Western (2 technical replicates). d A scan of cells from one representative patient is shown. e Signal intensities of phosphorylated p65 were normalized to total p65 and shown as mean values and standard deviations (n = 7)
Further work in OA chondrocytes should aim at the identification of the binding partner(s) of the galectins that trigger the first steps of the post-binding signaling cascade toward NF-κB activation
Summary
Osteoarthritis (OA) poses an enormous burden on the individual patient that adds up to a profound socioeconomic impact on health care sectors worldwide [1]. This justifies vigorous efforts to systematically study the molecular basis of disease onset and progression with the aim to develop innovative therapeutic strategies that interfere with the complex OA pathogenesis [2, 3], currently culminating in multiomics approaches to identify relevant molecular signatures [4]. Two-dimensional (2D) cell cultures have been considered as the starting point on the way to mimic an in situ-like microenvironment in vitro. Since the status of 3D culture for joint degeneration has recently been assessed to be “still in infancy” [7], intensive research is urgently required to foster its maturation
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