Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.     

Gene expression profile analysis of human intervertebral disc degeneration

In this study, we used microarray analysis to investigate the biogenesis and progression of intervertebral disc degeneration. The gene expression profiles of 37 disc tissue samples obtained from patients with herniated discs and degenerative disc disease collected by the National Cancer Institute Cooperative Tissue Network were analyzed. Differentially expressed genes between more and less degenerated discs were identified by significant analysis of microarray. A total of 555 genes were significantly overexpressed in more degenerated discs with a false discovery rate of < 3%. Functional annotation showed that these genes were significantly associated with membrane-bound vesicles, calcium ion binding and extracellular matrix. Protein-protein interaction analysis showed that these genes, including previously reported genes such as fibronectin, COL2A1 and β-catenin, may play key roles in disc degeneration. Unsupervised clustering indicated that the widely used morphology-based Thompson grading system was only marginally associated with the molecular classification of intervertebral disc degeneration. These findings indicate that detailed, systematic gene analysis may be a useful way of studying the biology of intervertebral disc degeneration.     

Connective tissue growth factor expression in human intervertebral disc: implications for angiogenesis in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is strongly associated with chronic low back pain, one of the most common causes of morbidity in the West. While normal healthy IVD is avascular, angiogenesis is a constant feature of IVD degeneration and has been shown to be associated with in-growth of nerves. Connective tissue growth factor (CTGF) plays a pivotal role in angiogenesis. To investigate the expression of CTGF in both normal and degenerated IVD, 21 IVDs were obtained from patients at surgery or postmortem examination and grouped according to the severity of histological degeneration. The immunohistochemical expression of CTGF was correlated with the degree of degeneration. CD31 immunohistochemistry was used to correlate IVD degeneration with vasculature. Our results showed that CTGF is expressed in non-degenerated and degenerated human IVDs and increased expression of CTGF is associated with degenerated discs, particularly within areas of neovascularization. We suggest that CTGF may play a role in angiogenesis in the human degenerated IVD.     

Loss of tenomodulin expression is a risk factor for age‐related intervertebral disc degeneration

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.     

PTEN promotes intervertebral disc degeneration by regulating nucleus pulposus cell behaviors

Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus‐mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX‐9 and matrix metalloproteinase‐3 (MMP‐3). Using small‐molecule AKT inhibitor GDC‐0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.     

Upregulated miR-154 promotes ECM degradation in intervertebral disc degeneration

Intervertebral disc degeneration (IDD), a common global health issue, is a major cause for low back pain (LBP). Given the complex etiology of IDD, micro RNA (miRNA) recently has been demonstrated to play essential roles in the progression of IDD. Therefore, this study aims to investigate functions of the miR-154, which is well-documented in a series of cell activities, IDD, and other relevant mechanisms. Lumbar nucleus pulposus (NP) samples were collected from patients with IDD and the control group. After solexa sequencing and bioinformatical analysis, the results showed that miR-154 was increased in NP cells of patients with IDD. Inhibition of miR-154 increased type II collagen and aggrecan and decreased mRNA expressions of collagenase-3 (MMP13) and aggrecanase-1 (ADAMTS4), whereas overexpression of miR-154 reversed such effects in NP cells. In addition, the luciferase reporter assay revealed that fibroblast growth factor 14 (FGF14) is a direct target of miR-154 and that the overexpression of FGF14 leads to similar effects as inhibition of miR-154 did. In conclusion, the results suggested that miR-154 participates in the development of IDD and its effects are mediated via targeting FGF14. Thus, miR-154 may be thought as a potential etiological factor for IDD and may provide insights into a therapeutic target to treat IDD.     

Procyanidin B3 alleviates intervertebral disc degeneration via interaction with the TLR4/MD-2 complex

As a chronic musculoskeletal degeneration disease, intervertebral disc degeneration (IVDD) has been identified as a crucial cause for low back pain. This condition has a prevalence of 80% among adults without effective preventative therapy. Procyanidin B3 (Pro-B3) is a procyanidin dimer, which is widely present in the human diet and has multiple functions, such as preventing inflammation. But the inhibiting effect of Pro-B3 in IVDD development is still no known. Thus, our study aimed to demonstrate the therapeutical effect of Pro-B3 in IVDD and explain the underlying mechanism. In vitro studies, human nucleus pulposus (NP) cells were isolated and exposed in lipopolysaccharide (LPS) to simulate IVDD development. Pro-B3 pre-treatment inhibited LPS-induced production of inflammation correlated factors such as tumour necrosis factor α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and Nitric oxide (NO). On the other hand, LPS-medicated extracellular matrix (ECM) breakdown was blocked in Pro-B3 treated NP cells. Additionally, Pro-B3 treatment blocked the activation of NF-κB/toll-like receptor 4 pathway in LPS-exposed NP cells. Mechanistically, Pro-B3 could occupy MD-2's hydrophobic pocket exhibiting high affinity for LPS to intervene LPS/TLR4/MD-2 complex formation. In vivo, Pro-B3 treatment prevented the loss of gelatin NP cells and structural damage of annulus fibrosus in rat IVDD model. In brief, Pro-B3 is considered to be a treatment agent for IVDD.     

Calcitonin inhibits intervertebral disc degeneration by regulating protein kinase C

Intervertebral disc degeneration (IVDD) is the most critical factor that causes low back pain. Molecular biotherapy is a fundamental strategy for IVDD treatment. Calcitonin can promote the proliferation of chondrocytes, stimulate the synthesis of matrix and prevent cartilage degeneration. However, its effect and the underlying mechanism for IVDD have not been fully revealed. Chondrogenic specific matrix components’ mRNA expression of nucleus pulposus cell (NPC) was determined by qPCR. Protein expression of NPC matrix components and protein kinase C was determined by Western blotting. A rat caudal intervertebral disc degeneration model was established and tested for calcitonin in vivo. IL‐1 induced NPC change via decreasing protein kinase C (PKC)‐ε phosphorylation, while increasing PKC‐δ phosphorylation. Calcitonin treatment could prevent or reverse IL‐1‐induced cellular change on PKC signalling associated with degeneration. The positive effect of calcitonin on IVDD in vivo was verified on a rat caudal model. In summary, this study, for the first time, elucidated the important role of calcitonin in the regulation of matrix components in the nucleus of the intervertebral disc. Calcitonin can delay degeneration of the intervertebral disc nucleus by activating the PKC‐ε pathway and inhibiting the PKC‐δ pathway.     

Angiopoietin‐like protein 8 expression and association with extracellular matrix metabolism and inflammation during intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is considered the primary culprit for low back pain. Although the underlying mechanisms remain unknown, hyperactive catabolism of the extracellular matrix (ECM) and inflammation are suggested to play critical roles in IDD progression. This study was designed to elucidate the role of angiopoietin‐like protein 8 (ANGPTL8) in the progression of IDD, especially the relationship of ANGPTL8 with ECM metabolism and inflammation. A positive association between ANGPTL8 expression and degenerative grades of IDD was detected in the analysis of human nucleus pulposus tissue samples. Silencing of ANGPTL8 attenuated the degradation of the anabolic protein type collagen II, and reduced the expression of the catabolic proteins MMP3 and MMP9, and the inflammatory cytokine IL‐6 through inhibition of NF‐κB signalling activation. In addition, the effect of ANGPTL8 was evaluated in a rat model of puncture‐induced IDD. Based on the imaging results and histological examination in animal study, knockdown of ANGPTL8 was demonstrated to ameliorate the IDD progression. These results demonstrate the detrimental role of ANGPTL8 expression in the pathogenesis of IDD and may provide a new therapeutic target for IDD treatment.     

Serum CXCL12/SDF-1 level is positively related with lumbar intervertebral disc degeneration and clinical severity

This study aimed to examine whether stromal cell-derived factor-1 (SDF-1) or C-X-C chemokine ligand 12 (CXCL12) participates in the development of lumbar disc degeneration, as implicated earlier by the level of CXCL12 correlating with this disease. It enrolled 145 patients with symptomatic lumbar intervertebral disc degeneration (IDD) and 130 asymptomatic healthy controls with no indication of IDD. Radiological assessment of the IDD patients was targeted at the lumbar vertebra region, based on Pfirrmann grade. Degeneration of the multifidus and psoas major muscles was evaluated using Goutallier classification. Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI) scores were obtained for assessing the severity of manifestation. The levels of serum CXCL12, IL-6 and TNF-α were determined by ROC curve analysis, resulting in their prognostic value for Pfirrmann grading. Higher levels of serum CXCL12 were found in patients with IDD than in asymptomatic individuals, and were positively related to the Pfirrmann grade as well as multifidus muscle degeneration. Furthermore, serum CXCL12 concentration showed a significant correlation with the VAS and ODI scores. In addition, elevated serum CXCL12 levels were related to serum levels of TNF-α and IL-6. The ROC curve analysis implicated that CXCL12 could function as a biomarker of the early-mediate phase of IDD development. In summary, the serum CXCL12/SDF-1 level is positively related with lumbar IDD and its clinical severity.     

Down‐regulation of insulin‐like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.     

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.     

Melatonin ameliorates intervertebral disc degeneration via the potential mechanisms of mitophagy induction and apoptosis inhibition

Intervertebral disc degeneration (IDD) is a complicated disease in patients. The pathogenesis of IDD encompasses cellular oxidative stress, mitochondrion dysfunction and apoptosis. Melatonin eliminates oxygen free radicals, regulates mitochondrial homoeostasis and function, stimulates mitophagy and protects against cellular apoptosis. Therefore, we hypothesize that melatonin has beneficial effect on IDD by mitophagy stimulation and inhibition of apoptosis. The effects of melatonin on IDD were investigated in vitro and in vivo. For the former, melatonin diminished cellular apoptosis caused by tert‐butyl hydroperoxide in nucleus pulposus (NP) cells. Mitophagy, as well as its upstream regulator Parkin, was activated by melatonin in both a dose and time‐dependent manner. Mitophagy inhibition by cyclosporine A (CsA) partially eliminated the protective effects of melatonin against NP cell apoptosis, suggesting that mitophagy is involved in the protective effect of melatonin on IDD. In addition, melatonin was demonstrated to preserve the extracellular matrix (ECM) content of Collagen II, Aggrecan and Sox‐9, while inhibiting the expression of matrix degeneration enzymes, including MMP‐13 and ADAMTS‐5. In vivo, our results demonstrated that melatonin treatment ameliorated IDD in a puncture‐induced rat model. To conclude, our results suggested that melatonin protected NP cells against apoptosis via mitophagy induction and ameliorated disc degeneration, providing the potential therapy for IDD.     

椎间盘退变动物模型的研究进展

椎间盘退变是腰痛发生的主要原因,严重影响了人们的生活和工作。尽管具体发病机制尚不明确,但近年来其相关动物模型的研究有了很大的进步。造模方法包括结构损伤、应力改变及基因敲除等,本文综述并讨论了这些方法的优缺点和应用方向,以期为后续的研究奠定理论基础。     

MicroRNA-181a exerts anti-inflammatory effects via inhibition of the ERK pathway in mice with intervertebral disc degeneration

Enzymatic decomposition of extracellular matrix and possibly local inflammation may cause intervertebral disc degeneration (IDD). MicroRNAs have been reported to correlate with the development of IDD. In this experiment, we aim at finding out the role of miR-181a in the inflammation of IDD and the underlying mechanism. The targeting relationship between miR-181a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was verified. Following the establishment of IDD mouse models, disc height index (DHI) and the change of DHI (%DHI) were measured. The functional role of miR-181a in IDD was determined using ectopic expression and depletion and reporter assay experiments. Expression of miR-181a, TRAIL, extracellular signal-regulated kinase (ERK) pathway-related genes and inflammatory factors was evaluated. Also, the expression of collagen I and collagen II was observed. miR-181a directly targeted TRAIL. IDD mice exhibited significant degeneration of the intervertebral disc. miR-181a was downregulated while TRAIL was upregulated in mice with IDD. miR-181a upregulation and the ERK pathway inhibition could reduce expression of TRAIL, ERK pathway-related genes, inflammatory factors, and collagen I, but promote collagen II expression. Our results reveal that upregulation of miR-181a protects against inflammatory response by inactivating the ERK pathway via suppression of TRAIL in IDD mice. These results point to miR-181a as a potential therapeutic target for the clinical management of IDD.