Abstract
The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd − / −) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd − / − Chm1 − / −) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration.
Highlights
Intervertebral disc (IVD) degeneration is a common condition and is thought to be an initiating factor for back pain (Nguyen, Poiraudeau, & Rannou, 2015)
We focused our investigation on the outer annulus fibrous (OAF) zone in 6-month-old mice in Student's t test; n = 3 animals, and 200 fibrils were analyzed per genotype). (c) Plots of compressive stiffness data obtained by indentation AFM demonstrated that extracellular matrix (ECM) compressive stiffness was markedly lower in the OAF regions of Tnmd−/− intervertebral disc (IVD) but not noticeably different in the inner annulus fibrous (IAF) regions. (d) Significant downregulation of numerous IVD- and tendon/ligamentrelated genes was detected in Tnmd−/− OAF by quantitative real-time PCR (qRT–PCR) analysis
Following our observation of increased angiogenesis and macrophage infiltration in Tnmd−/− OAF, we investigated the human umbilical vein endothelial cells (HUVECs) migratory capacity toward WT or Tnmd−/− OAF cells by implementing transwell assays and found that loss of Tnmd in OAF supernatant promoted their migratory capacity compared to co-culture with WT OAF cells (Figure 6a–c)
Summary
Intervertebral disc (IVD) degeneration is a common condition and is thought to be an initiating factor for back pain (Nguyen, Poiraudeau, & Rannou, 2015). 2.3 | Tnmd deficiency results in abnormal diameters and biomechanical properties of IVD collagen fibrils, accompanied by reduced expression of multiple IVD- and tendon/ ligament-related genes in the OAF in WT mice (Figure 2b). We compared the mRNA levels of IVD- and tendon/ligament-related genes between tendon and OAF tissues from both genotypes, and showed that the absence of Tnmd in the tendon and the OAF causes opposite effects on the mRNA expression levels of Scx, Mkx, Col14a1, Col15a1, and Prg (Figure S1c) suggesting tissue-specific regulation Taken together, these findings demonstrate that Tnmd is a critical factor required to maintain the structural and biomechanical properties of the OAF collagen fibrils likely through the modulation of ECM gene expression. Following our observation of increased angiogenesis and macrophage infiltration in Tnmd−/− OAF, we investigated the human umbilical vein endothelial cells (HUVECs) migratory capacity toward WT or Tnmd−/− OAF cells by implementing transwell assays and found that loss of Tnmd in OAF supernatant promoted their migratory capacity compared to co-culture with WT OAF cells (Figure 6a–c)
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