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Expression of metalloproteinases and their inhibitors in degenerated and extruded intervertebral disks in chondrodystrophic dogs.

ABSTRACT: We analyzed the mRNA expression of matrix metalloproteinases (MMPs), metalloproteinases with thrombospondin motifs (ADAMTSs), and tissue inhibitors of metalloproteinases (TIMPs) in degenerated and herniated intervertebral disks (IVDs) in chondrodystrophic dogs. In degenerated IVDs, MMP3, 7, 13, and 14; ADAMTS4 and 5; and TIMP1-3 expression was significantly higher vs healthy controls (P<0.05). In herniated IVDs, MMP2, 3, 9, 13, and 14; ADAMTS4 and 5; and TIMP1 expression was significantly greater, and MMP7 expression was significantly lower vs degenerated IVDs (P<0.05). These results suggest that metalloproteinase may play a role in extracellular matrix degradation in IVD degeneration. Decreased MMP7 transcription may prevent proteoglycan degradation and reduces macrophage infiltration, which might affect the resorption process of herniated IVDs.

SUBMITTER: Ishino H

PROVIDER: S-EPMC8111347 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Expression of metalloproteinases and their inhibitors in degenerated and extruded intervertebral disks in chondrodystrophic dogs.

Ishino Hirokazu H Sakonju Iwao I

The Journal of veterinary medical science 20210205 4

We analyzed the mRNA expression of matrix metalloproteinases (MMPs), metalloproteinases with thrombospondin motifs (ADAMTSs), and tissue inhibitors of metalloproteinases (TIMPs) in degenerated and herniated intervertebral disks (IVDs) in chondrodystrophic dogs. In degenerated IVDs, MMP3, 7, 13, and 14; ADAMTS4 and 5; and TIMP1-3 expression was significantly higher vs healthy controls (P<0.05). In herniated IVDs, MMP2, 3, 9, 13, and 14; ADAMTS4 and 5; and TIMP1 expression was significantly greate ...[more]

PMID: 33551382

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Project description:IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Project description:ObjectiveTurnover of cartilage endplate extracellular matrix (ECM) may play an important role in disc degeneration and low back pain (LBP). However, the expression pattern of pro-inflammatory factors, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) in the cartilage endplates (CEP) of intervertebral discs (IVD) is not understood. We aimed to examine the transcriptional levels of MMP, TIMP, and interleukins (IL), and the correlations between them.MethodsThirty degenerated cartilage endplate samples from patients with LBP who underwent lumbar fusion surgery were included in the degenerated group. Ten patients without LBP history who underwent lumbar surgery because of vertebral burst fractures were included in the control group. The degenerative severity of the samples was evaluated by MRI, and hematoxylin-eosin and safranin O-fast green (SO-FG) staining. Real-time polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3, IL-1α, IL-1β, and IL-6. The correlations between the levels of these genes were tested using Spearman's rho test.ResultsHematoxylin-eosin and SO-FG staining confirmed a decrease in cell number and proteoglycans in the degenerated cartilage endplate. MRI showed significant signal changes in degenerated cartilage endplates. Patients in the degenerated group showed a higher rate of endplate Modic changes when compared with the control group. MMP-3, MMP-9, TIMP-3, IL-1α, and IL-1β were elevated with statistical significance, while MMP-1, MMP-13, TIMP-1, TIMP-2, and IL-6 were changed without statistical significance or remained unchanged. Expression of MMP-3 was positively correlated with IL-1α (Spearman coefficient, 0.486; P < 0.05); expression of TIMP-3 was positively correlated with MMP-9, IL-1α, and IL-1β (Spearman coefficient, 0.577, 0.407, and 0.571, respectively; P < 0.05).ConclusionMMP-3, MMP-9, TIMP-3, IL-1α, and IL-1β may play a role in the process of cartilage endplate degeneration. MMP-3 may be regulated by IL-1α, and TIMP-3 might be associated with MMP-9 and regulated by IL-1α and IL-1β.

Project description:BackgroundDegeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells.MethodsHuman disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay.ResultsExpression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered.ConclusionsPMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Dataset Information

Expression of metalloproteinases and their inhibitors in degenerated and extruded intervertebral disks in chondrodystrophic dogs.

Publications

Expression of metalloproteinases and their inhibitors in degenerated and extruded intervertebral disks in chondrodystrophic dogs.

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