The regenerative effects of caveolin-1 and TGF-β on nucleus pulposus cells
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Abstract Introduction: Intervertebral disc (IVD) degeneration is a common, painful disorder in dogs and humans that is also an important risk factor for spinal diseases. Current treatment options are not optimal, as they do not restore the health and function of the IVD. Hence, new regenerative treatment strategies are needed. Early IVD degeneration is characterized by the loss of notochordal cells and the subsequent replacement by nucleus pulposus cells (NPCs). Microarray analysis has shown a decrease in the expression of caveolin-1 within NPCs of canines suffering from early IVD degeneration. In addition, the nucleus pulposi of wild type mice were found to be rich in viable notochordal cells, whereas the nucleus pulposi of caveolin-1 knockout mice contained chondroid matrix with relatively few cells that were mainly apoptotic. Moreover, previous research has shown that caveolin-1 inhibits TGF-β signalling. Despite its anabolic effects, TGF-β signalling mainly results in ongoing matrix degradation in the degenerating in vivo IVD. These findings suggest that caveolin-1 may play an important role in maintaining a healthy notochordal cell-rich nucleus pulposus. We attempted to gain more insight into the regenerative effects of caveolin-1 on NPCs in order to assess the potential use of caveolin-1 as a therapeutic target in regenerative medicine. In this study, a caveolin-1 scaffolding domain (CSD) peptide was used to supplement NPCs in vitro. This peptide has been reported to mimic the function of caveolin-1 in vitro and in vivo. We hypothesized that CSD can be used to facilitate caveolin-1 signalling in vitro. Material and methods: Culture experiments were performed in which caveolin-1 scaffolding domain (CSD) (alone or in combination with TGF-β) was supplemented to pellets containing articular chondrocytes (ACs) and NPCs for 7 or 28 days. First, the optimal dose and culture conditions with respect to CSD treatment were determined in a toxicity pilot and a dose optimization pilot (chapter 2). The additional regenerative effects of CSD upon TGF-β1 treatment in NPCs from chondrodystrophic dogs (chapter 3), non-chondrodystrophic dogs (chapter 4) and human donors (chapter 4) were investigated. Cell proliferation and extracellular matrix production were specified as parameters for regeneration. The DNA content of the cell cultures was determined to measure cell proliferation. The extracellular matrix production was quantified by measuring the glycosaminoglycan (GAG) content and GAG release in the culture medium using a DMMB assay. Additionally, cell cultures were subjected to a Safranin O/Fast Green staining in order to visualize the GAG production. The expression levels of relevant genes (i.e. markers concerning cell proliferation, matrix production/remodelling, chondrogenesis, TGF-β and Wnt/β-catenin signalling and apoptosis) were determined using RT-qPCR. Results: Treatment with only CSD did not induce regenerative effects. On the contrary, supplemented in concentrations of 5 μM or higher, CSD inhibited DNA synthesis and extracellular matrix production in canine ACs. TGF-β1 treatment increased extracellular matrix production in canine and human NPCs. Also a proliferative effect on canine NPCs was observed. CSD exerted no additional regenerative effects upon TGF-β1 treatment of canine and human NPCs. Conclusion: CSD (alone or in combination with TGF-β1) did not elicit regenerative effects on canine or human NPCs. Hence, the scaffolding domain of caveolin-1 did not demonstrate to be a promising target for regenerative medicine in the current (anabolic) culture experimental set-ups. On the other hand, in 3 individual donor cases CSD exerted additional regenerative effects upon TGF-β1 treatment, indicating that CSD’s effects could be donor-dependent. Given that the CSD may also interfere with the intrinsic caveolin-1 function, we cannot exclude that the CSD may have functioned as an antagonist by interfering with the caveolin-1 dependent signalling. Furthermore, considering that caveolin-1 has anti-inflammatory properties, it cannot be excluded that under inflammatory and catabolic culture conditions, CSD treatment may exert beneficial effects. However, it can also not be excluded that CSD antagonized caveolin-1 function by interfering with caveolin-1 dependent signalling, and that caveolin-1 itself does have regenerative effects upon NPC cells.