Date: Monday, November 9, 2015
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
In osteoarthritis, cartilage degradation is partly due to chondrocytes gaining a hypertrophy-like phenotype. TGFβ1 is considered to be a protective factor for articular cartilage by blocking this hypertrophy and by inducing production of matrix molecules like proteoglycans (e.g. aggrecan). However, these functions of TGFβ1 have been identified using very young cartilage whereas TGFβ1-signaling is known to change upon maturation (ageing). Therefore, we investigated in healthy mature cartilage the effect of TGFβ1 on chondrocyte phenotype, viability and total amount of glycosaminoglycans (GAGs, as measure for proteoglycans) in long term ex vivo culture.
Methods: Articular cartilage explants were isolated from the metacarpophalangeal joint of healthy 5 yr old adult cows. Subsequently, explants were cultured in DMEM/F12 alone or supplemented with either 10% Fetal Calf Serum (FCS), rhTGFβ1 or rhIGF1 for the duration of 2 weeks. To investigate via which TGFβ1 receptor, ALK1 or ALK5, the observed TGFβ1 effects run, the ALK5-kinase inhibitor SB-505124 (5 μM) was used. Sulfated-GAGs were measured using dimethylmethylene blue (DMB). Cellular viability was measured with the use of XTT. To correct for the amount of cells, total DNA content of explants was measured using PicoGreen. Furthermore, gene expression of chondrocyte hypertrophy markers was measured using qPCR.
Results: Two weeks of ex vivo explant culture resulted in GAG loss; total GAG content was reduced from approximately 4% of wet weight in freshly isolated explants to 1% of wet weight in cultured explants. Addition of 10% FCS fully prevented this GAG loss. In contrast, TGFβ1 (10 ng/ml) was unable to inhibit GAG loss. Replacement of 10% FCS with 20 ng/ml of IGF1 also prevented GAG loss, but surprisingly, when IGF1 was combined with TGFβ1, TGFβ1 inhibited the positive effect of IGF1 on total GAG content. Compared to freshly isolated samples, expression of the chondrocyte-hypertrophy markers Col10a1 and Mmp13 was profoundly upregulated in explants cultured for 2 weeks. The addition of 10% FCS to the medium did not inhibit the upregulation of these genes, and even induced expression of Alkaline phosphatase (Alpl), another marker of chondrocyte hypertrophy. In contrast, addition of TGFβ1 (1 ng/ml or 10 ng/ml) fully blocked induction of Col10a1 and Mmp13 expression, and lowered expression of Alpl 4-fold. Additionally, TGFβ1 induced Col2a1 expression 16-fold. TGFβ1 also enhanced chondrocyte viability, because after 2 weeks, mitochondrial activity was 50% higher in TGFβ1 treated samples compared to 10% FCS treated samples. All these effects of TGFβ1 were blocked by 5 μM of the ALK5-kinase inhibitor SB-505124.
Based on our results, we conclude that in adult cartilage TGFβ1 is a potent inhibitor of chondrocyte hypertrophy and that this inhibition runs via ALK5. Additionally, TGFβ1 profoundly induced Col2a1 production, and maintained chondrocyte viability in long term culture. In contrast, TGFβ1 was not able to counteract GAG depletion over time and even inhibited the beneficial effect of IGF1 on total GAG-content in cartilage, demonstrating that TGFβ1 does not positively regulate GAG content in adult cartilage.
To cite this abstract in AMA style:van Caam A, Madej W, van Beuningen H, Blaney Davidson E, van der Kraan P. TGFß1 Blocks Chondrocyte Hypertrophy and Increases Cell Viability in Cultured Cartilage Explants but Does Not Protect Against Proteoglycan Loss [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/tgfs1-blocks-chondrocyte-hypertrophy-and-increases-cell-viability-in-cultured-cartilage-explants-but-does-not-protect-against-proteoglycan-loss/. Accessed September 17, 2019.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tgfs1-blocks-chondrocyte-hypertrophy-and-increases-cell-viability-in-cultured-cartilage-explants-but-does-not-protect-against-proteoglycan-loss/