Artikel
Novel regulating factor of collagen I fibrillogenesis in tendons: the role of tenomodulin
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Veröffentlicht: | 5. Oktober 2015 |
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Gliederung
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Objectives: Collagen I, the most abundant tendon protein, forms fibers via a complex fibrillogenesis process modulated by several enzymes and proteoglycans. The collagen fiber quality and quantity define the strength and tissue function of tendons, and when affected they predispose progression of tendon diseases. Up to date, the exact mechanism of collagen fibrillogenesis is still not fully explained. We hypothesized that tenomodulin (Tnmd), a well-known gene marker for tendons, exerts a regulatory effect in this process based on electron microscopy findings, which demonstrated thicker collagen fibers in the tendons from Tnmd-knockout (KO) mice (Docheva et al, 2005). Hence, to understand the molecular pathway behind the observed fiber phenotype we performed analyses of collagen cross-linking as well as atomic force microscopy (AFM) in Achilles tendons from wild type (WT) and Tnmd KO mice.
Methods: Achilles tendons from 12 months old mice were obtained and analyzed by AFM for fiber size quantification and stiffness measurements on single fiber level. Tendon-derived RNAs/cDNAs were implicated in RT-PCR analyses of 8 different gene markers for collagen cross-linking: the enzymes lysyl oxidase, lysyl hydroxylase and transglutaminase 1, and the proteoglycans biglycan, decorin, asporin, fibromodulin and lumican. Last, immunohistochemical stainings were performed to validate the RT-PCR data. 3-5 animals per genotype were investigated and statistical analysis was performed using unpaired t-tests.
Results and Conclusion: AFM investigation on tendon sections demonstrated in WT a Gaussian distribution of collagen fiber diameters spanning from 100-260 nm with peak of 160-180 nm. In contrast, the KO showed a shift of distribution towards fiber size with a min. of 170 nm and reaching a max. of 380 nm. Moreover, calculation of Young's modulus revealed a significant stiffening of the collagen fibers in the KO. Next, RT-PCR analyses showed an altered expression of 5 different cross-linkers. Specifically, lysyl oxidase was strongly upregulated in Tnmd KO, followed by lysyl hydroxylase, asporin, fibromodulin and lumican. Immunohistochemical stainings confirmed the RT-PCR results, with the exception that asporin and lumican were weakly detected on protein level in both genotypes.
In the absence of Tnmd, Achilles tendons display pathologic alterations in collagen fibrillogenesis, consisting of significantly larger as well as stiffer fibers. Furthermore, clear upregulation in the expression of lysyl oxidase, lysyl hydroxylase, asporin, fibromodulin and lumican was identified in Tnmd KO, explaining the molecular mode of action. However, at present due to the unknown direct binding partners of Tnmd, we cannot clarify how Tnmd affects the above cross-linkers. In sum, the loss of Tnmd leads to distorted collagen fibrillogenesis and enhanced cross-linking, a phenotype resembling a status of tendon tissue aging found in humans, thus suggesting Tnmd KO mice as a suitable model to study aged-related diseases in tendons.