endothelin-1 and Intervertebral-Disc-Degeneration

Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown.. The purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway.. The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated.. To establish a model for CEP degeneration, three lumbar discs (L3-L4, L4-L5, and L5-L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/β-catenin signaling pathway was also investigated by Western blotting.. After 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect.. Endothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.

Topics: Aggrecans; Animals; beta Catenin; Collagen; Endothelin Receptor Antagonists; Endothelin-1; Intervertebral Disc; Intervertebral Disc Degeneration; Peptides, Cyclic; Rabbits; Wnt Signaling Pathway

Inflammatory cytokines are involved in intervertebral disc (IVD) degeneration. Endothelin-1 (ET-1), a 21-amino-acid cytokine implicated with cartilage degradation, is secreted by vascular endothelial cells and also by many other cell types. The expression of ET-1 in human IVD cartilage endplate (CEP) and its role in disc degeneration have not been explored.. The expression of ET-1 in degenerated CEP was analyzed by immunohistochemical staining and Western blotting; ET-1 was demonstrated in cartilaginous endplate cells (CECs) by immunofluorescent staining. The ET-1 mRNA expression and protein production by CECs stimulated by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, were determined by real-time PCR analysis and Western blotting, respectively. The matrix metalloprotease-1 (MMP-1), MMP-13 and tissue inhibitor of metalloproteases-1 (TIMP-1) levels in the supernatant of cultured CECs treated with ET-1 were determined using enzyme-linked immunosorbent assays. Nitric oxide (NO) release and nitric oxide synthase (NOS) activity were measured using a spectrophotometric assay. The apoptosis of CECs by ET-1 was measured by an Annexin V-FITC detection assay. The production of ET-1 in degenerated cartilage endplate was significantly higher than normal CEP. The results showed that ET-1 was expressed by CECs and modulated by TNF-α in a dose-dependent manner. ET-1 increased production of MMP-1 and MMP-13, decreased TIMP-1 production, and induced NO and NOS release by cultured CECs. The direct stimulation of CECs by ET-1 did not promote cell apoptosis.. The study results suggest that ET-1 played a pivotal role in human CEP degeneration, and may be a new target for development of therapies for this condition.

Topics: Adult; Aged; Apoptosis; Cartilage; Chondrocytes; Endothelin-1; Female; Humans; Intervertebral Disc Degeneration; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Protein Biosynthesis; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tumor Necrosis Factor-alpha; Young Adult