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Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies.

Smolders LA, Meij BP, Onis D, Riemers FM, Bergknut N, Wubbolts R, Grinwis GC, Houweling M, Groot Koerkamp MJ, van Leenen D, Holstege FC, Hazewinkel HA, Creemers LB, Penning LC, Tryfonidou MA - Arthritis Res. Ther. (2013)

Bottom Line: With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs.NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not.Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

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The intervertebral disc phenotype of Caveolin-1 knock-out mice. Typical examples of the intervertebral discs from 3-month-old wild-type (A and C) and Caveolin-1 knock-out (B and D) mice, stained with H&E (A and B) and alcian blue/pricrosirius red (C and D). The pictures on the right are magnifications of the corresponding pictures on the left. The wild-type nucleus pulposus (NP) consisted of centrally located, viable notochordal cells (arrowhead) and a relatively limited amount of chondroid-like intercellular matrix (*), which stains blue in the sections stained with alcian blue/picrosirius red. The NP of the Caveolin-1 KO mice contained apoptotic (arrowheads) and rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar aspect (arrows), and a relatively large amount of chondroid-like intercellular matrix (*).
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Figure 5: The intervertebral disc phenotype of Caveolin-1 knock-out mice. Typical examples of the intervertebral discs from 3-month-old wild-type (A and C) and Caveolin-1 knock-out (B and D) mice, stained with H&E (A and B) and alcian blue/pricrosirius red (C and D). The pictures on the right are magnifications of the corresponding pictures on the left. The wild-type nucleus pulposus (NP) consisted of centrally located, viable notochordal cells (arrowhead) and a relatively limited amount of chondroid-like intercellular matrix (*), which stains blue in the sections stained with alcian blue/picrosirius red. The NP of the Caveolin-1 KO mice contained apoptotic (arrowheads) and rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar aspect (arrows), and a relatively large amount of chondroid-like intercellular matrix (*).

Mentions: The IVD of 3-month-old Caveolin-1 KO mice was significantly different from that of wild-type mice of the same age (Figure 5). The NP of the wild-type IVD consisted of a centrally located area of large cells with highly vacuolated cytoplasm and hyperchromatic nuclei, consistent with the morphological characteristics of viable NCs. A limited amount of chondroid-like intercellular matrix was visible within the area of NCs and there was a large rim of this matrix in the zone between the NCs and the endplate. In contrast, the NP of the Caveolin-1 KO mice contained rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar appearance. Over 75% of these cells did not contain recognizable nuclei and were characterized by cytoplasmic eosinophilia suggestive of necrosis or apoptosis. The NP of Caveolin-1 KO mice contained a large amount of chondroid-like intercellular matrix between the cells within the NP.


Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies.

Smolders LA, Meij BP, Onis D, Riemers FM, Bergknut N, Wubbolts R, Grinwis GC, Houweling M, Groot Koerkamp MJ, van Leenen D, Holstege FC, Hazewinkel HA, Creemers LB, Penning LC, Tryfonidou MA - Arthritis Res. Ther. (2013)

The intervertebral disc phenotype of Caveolin-1 knock-out mice. Typical examples of the intervertebral discs from 3-month-old wild-type (A and C) and Caveolin-1 knock-out (B and D) mice, stained with H&E (A and B) and alcian blue/pricrosirius red (C and D). The pictures on the right are magnifications of the corresponding pictures on the left. The wild-type nucleus pulposus (NP) consisted of centrally located, viable notochordal cells (arrowhead) and a relatively limited amount of chondroid-like intercellular matrix (*), which stains blue in the sections stained with alcian blue/picrosirius red. The NP of the Caveolin-1 KO mice contained apoptotic (arrowheads) and rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar aspect (arrows), and a relatively large amount of chondroid-like intercellular matrix (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672710&req=5

Figure 5: The intervertebral disc phenotype of Caveolin-1 knock-out mice. Typical examples of the intervertebral discs from 3-month-old wild-type (A and C) and Caveolin-1 knock-out (B and D) mice, stained with H&E (A and B) and alcian blue/pricrosirius red (C and D). The pictures on the right are magnifications of the corresponding pictures on the left. The wild-type nucleus pulposus (NP) consisted of centrally located, viable notochordal cells (arrowhead) and a relatively limited amount of chondroid-like intercellular matrix (*), which stains blue in the sections stained with alcian blue/picrosirius red. The NP of the Caveolin-1 KO mice contained apoptotic (arrowheads) and rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar aspect (arrows), and a relatively large amount of chondroid-like intercellular matrix (*).
Mentions: The IVD of 3-month-old Caveolin-1 KO mice was significantly different from that of wild-type mice of the same age (Figure 5). The NP of the wild-type IVD consisted of a centrally located area of large cells with highly vacuolated cytoplasm and hyperchromatic nuclei, consistent with the morphological characteristics of viable NCs. A limited amount of chondroid-like intercellular matrix was visible within the area of NCs and there was a large rim of this matrix in the zone between the NCs and the endplate. In contrast, the NP of the Caveolin-1 KO mice contained rounded cells, with a smaller amount of cytoplasm lacking the typical vacuolar appearance. Over 75% of these cells did not contain recognizable nuclei and were characterized by cytoplasmic eosinophilia suggestive of necrosis or apoptosis. The NP of Caveolin-1 KO mice contained a large amount of chondroid-like intercellular matrix between the cells within the NP.

Bottom Line: With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs.NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not.Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

Show MeSH
Related in: MedlinePlus