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Unravelling the roles of tenomodulin at the nexus of early tendon healing and intervertebral disc homeostasis
Unravelling the roles of tenomodulin at the nexus of early tendon healing and intervertebral disc homeostasis
Tenomodulin (Tnmd) is a tendon/ligament-specific marker that also serves as an anti-angiogenic protein. It is a novel type II transmembrane glycoprotein class member, and exhibits a cysteine-rich C-terminal domain that can readily be cleaved. Chondromodulin I (Chm1) is the only other characterized protein with homology to Tnmd. Tnmd expression is mainly observed in hypovascular tissues, including ligaments, tendons, and eyes. There is increasing evidence that Tnmd may have diverse roles in various tissues and pathological conditions. Therefore, it was of great importance to further examine Tnmd-knockout (Tnmd-/-) model by subjecting it to disease-like conditions, namely tendon rupture, as well as to investigate its potential role in the intervertebral disc (IVD) being also avascular tissue. First, we carried out an Achilles tendon injury in 6-month-old Tnmd-/- mice and investigated the early tendon repair at day 8, at which time it is possible to observe clear evidence of the development of scar tissue, infiltration by inflammatory and vascular cells, increased cell migration, proliferation and extracellular matrix (ECM) deposition. When comparing wild type (WT) and Tnmd-/- tendons, we observed clear reductions in the expression of tendon-associated transcription factors and ECM genes, impaired cellular proliferation, altered scar organization, and enhanced apoptosis and blood vessel/adipocyte accumulation in Tnmd-/- tendons. We further found that the scars in Tnmd-/- tendons exhibited increased biglycan (Bgn), cartilage oligomeric matrix protein (COMP), and fibronectin (Fn) deposition within the ECM; an altered macrophage profile, with predominantly M1 macrophages; and reduced CD146-positive cell numbers, which may be tendon stem/progenitor cells (TSPCs). In vitro, Tnmd-/- TSPCs were found to be markedly less proliferative and migratory than were WT TSPCs. The supernatant protein levels of Bgn and Fn were also markedly elevated in media collected from Tnmd-/- TSPCs, and these cells were found to more rapidly undergo adipogenic differentiation and to exhibit higher mRNA-level expression of peroxisome proliferator-activated receptor gamma (Pparγ) and lipoprotein lipase (Lpl). We therefore concluded that Tnmd is capable of inhibiting the accumulation of adipocytes and the formation of a fibrovascular scar in the context of early tendon healing. Second, to investigate whether loss of Tnmd expression may lead to IVD degeneration, we performed immunolocalization and western blotting analyses of WT IVD tissues revealing that Tnmd was age-dependent and was detectable primarily in the outer annulus fibrosus (AF), with its expression being reduced at 6 months of age (the time at which mice begin to exhibit early signs of IVD degeneration). We also confirmed that TNMD was predominantly expressed within the ECM of the outer AF of human lumbar discs. IVD phenotypic analyses demonstrated more rapid progression of age-related IVD degeneration in Tnmd-/- IVD. This included reductions in the diameters of collagen fibrils, decreases in compressive stiffness, reductions in the expression of genes associated with tendons/ligaments and with IVD, increases in macrophage infiltration and angiogenic activity in the outer AF, and a greater number of hypertrophic-like chondrocytes within the nucleus pulposus (NP). Furthermore, Tnmd and Chm1 double knockout mice displayed an ectopic bone formation in the IVD. In vitro studies demonstrated reduced proliferation and migration but increased apoptosis when comparing Tnmd-/- versus WT outer AF-derived cells. Furthermore, these cells showed p65 and matrix metalloproteinases (MMPs) upregulation and promoted the migration of human umbilical vein endothelial cells. Hence, we concluded that Tnmd can inhibit angiogenesis in the context of homeostatic IVD maintenance while protecting against IVD degeneration that occurs as a result of aging. In summary, we reported that reduced Tnmd expression results in inferior tendon healing and increasing the risk of age-related IVD degeneration. These novel findings provided new information pertaining to the important role of Tnmd in tendon and IVD tissues, which can facilitate the development of novel therapeutic interventions that can prevent or treat tendon and IVD diseases.
tenomodulin, tendon healing, tendon stem/progenitor cell, intervertebral disc degeneration, angiogenesis, knockout mice
Lin, Dasheng
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Lin, Dasheng (2020): Unravelling the roles of tenomodulin at the nexus of early tendon healing and intervertebral disc homeostasis. Dissertation, LMU München: Faculty of Medicine
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Abstract

Tenomodulin (Tnmd) is a tendon/ligament-specific marker that also serves as an anti-angiogenic protein. It is a novel type II transmembrane glycoprotein class member, and exhibits a cysteine-rich C-terminal domain that can readily be cleaved. Chondromodulin I (Chm1) is the only other characterized protein with homology to Tnmd. Tnmd expression is mainly observed in hypovascular tissues, including ligaments, tendons, and eyes. There is increasing evidence that Tnmd may have diverse roles in various tissues and pathological conditions. Therefore, it was of great importance to further examine Tnmd-knockout (Tnmd-/-) model by subjecting it to disease-like conditions, namely tendon rupture, as well as to investigate its potential role in the intervertebral disc (IVD) being also avascular tissue. First, we carried out an Achilles tendon injury in 6-month-old Tnmd-/- mice and investigated the early tendon repair at day 8, at which time it is possible to observe clear evidence of the development of scar tissue, infiltration by inflammatory and vascular cells, increased cell migration, proliferation and extracellular matrix (ECM) deposition. When comparing wild type (WT) and Tnmd-/- tendons, we observed clear reductions in the expression of tendon-associated transcription factors and ECM genes, impaired cellular proliferation, altered scar organization, and enhanced apoptosis and blood vessel/adipocyte accumulation in Tnmd-/- tendons. We further found that the scars in Tnmd-/- tendons exhibited increased biglycan (Bgn), cartilage oligomeric matrix protein (COMP), and fibronectin (Fn) deposition within the ECM; an altered macrophage profile, with predominantly M1 macrophages; and reduced CD146-positive cell numbers, which may be tendon stem/progenitor cells (TSPCs). In vitro, Tnmd-/- TSPCs were found to be markedly less proliferative and migratory than were WT TSPCs. The supernatant protein levels of Bgn and Fn were also markedly elevated in media collected from Tnmd-/- TSPCs, and these cells were found to more rapidly undergo adipogenic differentiation and to exhibit higher mRNA-level expression of peroxisome proliferator-activated receptor gamma (Pparγ) and lipoprotein lipase (Lpl). We therefore concluded that Tnmd is capable of inhibiting the accumulation of adipocytes and the formation of a fibrovascular scar in the context of early tendon healing. Second, to investigate whether loss of Tnmd expression may lead to IVD degeneration, we performed immunolocalization and western blotting analyses of WT IVD tissues revealing that Tnmd was age-dependent and was detectable primarily in the outer annulus fibrosus (AF), with its expression being reduced at 6 months of age (the time at which mice begin to exhibit early signs of IVD degeneration). We also confirmed that TNMD was predominantly expressed within the ECM of the outer AF of human lumbar discs. IVD phenotypic analyses demonstrated more rapid progression of age-related IVD degeneration in Tnmd-/- IVD. This included reductions in the diameters of collagen fibrils, decreases in compressive stiffness, reductions in the expression of genes associated with tendons/ligaments and with IVD, increases in macrophage infiltration and angiogenic activity in the outer AF, and a greater number of hypertrophic-like chondrocytes within the nucleus pulposus (NP). Furthermore, Tnmd and Chm1 double knockout mice displayed an ectopic bone formation in the IVD. In vitro studies demonstrated reduced proliferation and migration but increased apoptosis when comparing Tnmd-/- versus WT outer AF-derived cells. Furthermore, these cells showed p65 and matrix metalloproteinases (MMPs) upregulation and promoted the migration of human umbilical vein endothelial cells. Hence, we concluded that Tnmd can inhibit angiogenesis in the context of homeostatic IVD maintenance while protecting against IVD degeneration that occurs as a result of aging. In summary, we reported that reduced Tnmd expression results in inferior tendon healing and increasing the risk of age-related IVD degeneration. These novel findings provided new information pertaining to the important role of Tnmd in tendon and IVD tissues, which can facilitate the development of novel therapeutic interventions that can prevent or treat tendon and IVD diseases.