The effect of caveolin-1 on TGFB1 treatment of degenerated canine nucleus pulposus cells.

Abstract

Introduction. Intervertebral disc (IVD) degeneration is a common disorder that affects a significant proportion of the human population. Spontaneously occurring IVD degeneration is also a common problem in dogs, and therefore, the dog is considered an unique animal model for IVD degeneration research. The current purpose of a treatment is to alleviate pain. Because the current treatments do not solve the problem, there is increasing interest in regenerative therapies, like treatment with TGF-β. In this study, we have investigated whether caveolin-1, acknowledged for its physiological importance in tissue repair and fibrosis, has an additive effect on TGF-β treatment of degenerated canine nucleus pulposus cells (chondrocyte-like nucleus pulposus cells, CLCs). Materials and methods. Degenerated nucleus pulposus cells (CLCs) were used from twelve canine donors; the donors were all chondrodystrophic dogs (Beagles), aged 19 – 32 months, eight females and four males. The CLC pellets were cultured for 28 days in standard chondrogenic medium without (negative control) or with 2 or 10 ng/mL TGF-β1 (positive controls), with and without caveolin-1 supplementation. At day 0, 1, 7 and 28, cell proliferation was determined by measuring the pellets’ DNA content. Furthermore, the glycosaminoglycan (GAG) content was determined using a DMMB assay. At day 7 and day 28, gene expression was determined using RT-qPCR. Histology was performed at day 28. Results. TGF-β has a positive, dose-dependent effect on cell proliferation and matrix production of the CLC pellets. In general, no additive effect of caveolin-1 is observed on TGF-β treatment of the degenerated CLC pellets. But in one donor, a statistical significant additive effect of caveolin-1 on the CLC pellets GAG content is observed on day 28. Discussion. Although no additive effect of caveolin-1 is observed in the mean values of the pellets, it is interesting that if the donors are observed independently, donor A seems to have an additive effect of caveolin-1 on TGF-β 10 ng/mL treatment on day 28 of cell culture suggesting that the effect of caveolin-1 is donor-dependent. When the different donors are compared to each other, no differences in age or breed are present, so it is not certain why this effect only in donor A is seen. Although there is an effect of caveolin-1 on Axin2 expression observed, the exact role of caveolin-1 in the Wnt/β-catenin signaling is still not certain. There are a few hypotheses, but further research is necessary to find out the exact role of caveolin-1 in this pathway. Conclusion. No additive effect of caveolin-1 is found on TGF-β treatment of degenerated CLC pellets, except for donor A on GAG content. Further investigations are necessary to understand the different pathways caveolin-1 regulates an which role caveolin-1 plays in intervertebral disc degeneration.