Session Title: Osteoarthritis & Joint Biology – Basic Science Poster
Session Type: Poster Session (Tuesday)
Session Time: 9:00AM-11:00AM
Background/Purpose: Osteoarthritis (OA) is mainly characterized by the progressive damage of articular cartilage, where chondrocytes acquire a hypertrophic-like phenotype. Indian Hedgehog levels are increased in OA cartilage. An induction of Hedgehog (Hh) signaling pathway induces OA-like cartilage lesions, whereas its pharmacological downregulation prevents articular destruction in animal models. Mutations in EVC or EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld skeletal dysplasia. Hence, Evc deletion would hamper the expression of Hh target genes, Runx2, ColX and MMP13 among others, preventing the expression of hypertrophic mediators and protecting cartilage against OA progression. Our aim was to study whether Hh pathway inhibition restrains chondrocyte hypertrophy and the effect of Evc deletion on joint damage in an Evc tamoxifen induced conditional knock out (cKO) (EvccKO-/-) model of OA in mice.
Methods: C57BL6 wild-type (WT) and Evc-/- primary murine chondrocytes were isolated from cartilage explants from E18.5 embryos and cultured in micromass. Hypertrophy was induced with insulin for 14 days (n≥6). Cyclopamine (5-10 µM) was used for Hh pharmacological inhibition. Gene expression of hypertrophic markers and Hh genes was measured by qRT-PCR, and proteoglycan content visualized by Alcian Blue staining. To study the effect of Evc deletion in vivo, OA was induced by surgical knee destabilization in WT (n=6) and EvccKO-/- (n=6) adult mice and let evolve for 8 weeks. Healthy WT (n=8) and EvccKO-/- (n=3) mice were used as controls. Cartilage damage and chondrocyte size were evaluated in Safranin-O and haematoxylin/eosin stained joint sections. MMP levels were assessed by western blot and hypertrophic markers gene expression by qRT-PCR.
Results: Cyclopamine prevented the expression of ColX, alkaline phosphatase (ALP), Adamts5 and Runx2 (p< 0.05) and reduced proteoglycan content (p=0.004) evoked by insulin in cultured WT chondrocytes. Evc-/- chondrocytes showed a partial inhibition of insulin-induced hypertrophy, with a decrease in ALP and Runx2 gene expression (p=0.03) and in proteoglycan content (p=0.02). However, Evc deletion did not modify articular cartilage damage in OA mice, since similar OARSI scores were observed in OA WT and OA EvccKO-/- mice (p=0.64). MMP13, MMP1 and MMP3 protein levels were increased in OA WT knees (p< 0.01 vs Healthy WT), and reduced levels of MMP3 (p=0.09) and MMP13 (p=0.18) were found in OA EvccKO-/- mice when compared to OA WT. However, gene expression of Hh genes was induced in OA WT knees, and completely inhibited in OA EvccKO-/-. Furthermore, ColX (p=0.002), ALP (p=0.13) and Runx2 (p=0.21) gene expressions were decreased in OA EvccKO-/- mice in comparison with OA WT. Likewise, chondrocyte size was increased in the OA WT calcified cartilage (p=0.098 vs. Healthy WT), while it was diminished in OA EvccKO-/- (p=0.11 vs OA WT).
Conclusion: Although the inactivation of Hh signaling pathway prevented chondrocyte hypertrophy, it was not able to prevent cartilage damage progression in OA EvccKO-/-. Therefore, our results suggest that chondrocyte hypertrophy is not a pathogenic event in the progression of articular cartilage damage in OA.
To cite this abstract in AMA style:Lamuedra A, Gratal P, Ruiz-Perez V, Palencia-Campos A, Portal-Nuñez S, Herrero-Beaumont G, Largo R. What Is the Pathogenic Meaning of Chondrocyte Hypertrophy in Osteoarthritis? Effect of Evc Deletion Through Hedgehog Signaling [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/what-is-the-pathogenic-meaning-of-chondrocyte-hypertrophy-in-osteoarthritis-effect-of-evc-deletion-through-hedgehog-signaling/. Accessed April 12, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/what-is-the-pathogenic-meaning-of-chondrocyte-hypertrophy-in-osteoarthritis-effect-of-evc-deletion-through-hedgehog-signaling/