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.     

Tenascin in the human intervertebral disc: alterations with aging and disc degeneration

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.     

Damage accumulation location under cyclic loading in the lumbar disc shifts from inner annulus lamellae to peripheral annulus with increasing disc degeneration

It is difficult to study the breakdown of lumbar disc tissue over several years of exposure to bending and lifting by experimental methods. In our earlier published study we have shown how a finite element model of a healthy lumbar motion segment was used to predict the damage accumulation location and number of cyclic to failure under different loading conditions. The aim of the current study was to extend the continuum damage mechanics formulation to the degenerated discs and investigate the initiation and progression of mechanical damage. Healthy disc model was modified to represent degenerative discs (Thompson grade III and IV) by incorporating both geometrical and biochemical changes due to degeneration. Analyses predicted decrease in the number of cycles to failure with increasing severity of disc degeneration. The study showed that the damage initiated at the posterior inner annulus adjacent to the endplates and propagated outwards towards its periphery in healthy and grade III degenerated discs. The damage accumulated preferentially in the posterior region of the annulus. However in grade IV degenerated disc damage initiated at the posterior outer periphery of the annulus and propagated circumferentially. The finite element model predictions were consistent with the infrequent occurrence of rim lesions at early age but a much higher incidence in severely degenerated discs.     

Morphologic complexity of the pericellular matrix in the annulus of the human intervertebral disc

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.     

Morphologic complexity of the pericellular matrix in the annulus of the human intervertebral disc

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.     

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.     

Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus

ABSTRACT: BACKGROUND: In spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following institutional review board approval. RESULTS: Analyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus. CONCLUSIONS: Findings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.     

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.     

Cellular immunohistochemical localization of the matricellular protein myocilin in the intervertebral disc

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc.     

Cellular immunohistochemical localization of the matricellular protein myocilin in the intervertebral disc

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc.     

Expression of serglycin in human disc is increased in degenerated discs and up-regulated in vitro by exposure to IL-1ß or TNF-α

We investigated whether the multifunctional intercellular proteoglycan, serglycin, is expressed in human intervertebral disc cells and assessed its localization. We also investigated expression levels of serglycin in human annulus fibrosus (annulus) cells exposed to IL-1ß and TNF-α, which are two proinflammatory cytokines that are expressed during disc degeneration. Immunolocalization of serglycin was common in many cells of the human annulus, but less common in the nucleus pulposus (nucleus). Both intracellular and cell membrane localization were observed. Annulus cells from Thompson grades III, IV and V degenerated discs exhibited a 4.69 fold up-regulation in serglycin expression vs. cells from healthier grades I and II discs. In monolayer annulus cell culture, cells from more degenerated discs exhibited a 9.4 fold up-regulation of serglycin expression compared to cells from healthier discs. Exposure of cultured cells to IL-1ß or TNF-α caused significant up-regulation of serglycin expression. We found that serglycin expression increased with increasing disc degeneration both in vivo and in vitro, and also increased with exposure in vitro to IL-1ß and TNF-α.     

Periostin is expressed by cells of the human and sand rat intervertebral discs

Abstract

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.     

IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P<0.001 for all comparisons). This is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in human disc cells. IL-1beta treatment appeared to "sensitize" annulus cells to mechanical load. This increased responsiveness to mechanical load in the face of inflammatory cytokines may imply that the sensitivity of annulus cells to shear increases during inflammation and may affect initiation and progression of disc degeneration.     

Disruption of the thrombospondin-2 gene alters the lamellar morphology but does not permit vascularization of the adult mouse lumbar disc

Introduction

The biological basis for the avascular state of the intervertebral disc is not well understood. Previous work has suggested that the presence of thrombospondin-1 (TSP-1), a matricellular protein, in the outer annulus reflects a role for this protein in conferring an avascular status to the disc. In the present study we have examined thrombospondin-2 (TSP-2), a matricellular protein with recognized anti-angiogenic activity in vivo and in vitro.

Methods

We examined both the location and expression of TSP-2 in the human disc, and its location in the disc and bordering soft tissues of 5-month-old normal wild-type (WT) mice and of mice with a targeted disruption of the TSP-2 gene. Immunohistochemistry and quantitative histology were utilized in this study.

Results

TSP-2 was found to be present in some, but not all, annulus cells of the human annulus and the mouse annulus. Although there was no difference in the number of disc cells in the annulus of TSP-2-null mice compared with that of WT animals, polarized light microscopy revealed a more irregular lamellar collagen structure in null mouse discs compared with WT mouse discs. Additionally, vascular beds at the margins of discs of TSP-2-null mice were substantially more irregular than those of WT animals. Counts of platelet endothelial cell adhesion molecule-1-positive blood vessels in the tissue margin bordering the ventral annulus showed a significantly larger vascular bed in the tissue bordering the disc of TSP-2-null mice compared with that of WT mice (P = 0.0002). There was, however, no vascular ingrowth into discs of the TSP-2-null mice.

Conclusion

These data confirm a role for TSP-2 in the morphology of the disc and suggest the presence of other inhibitors of angiogenesis in the disc. We have shown that although an increase in vasculature was present in the TSP-2-null tissue in the margin of the disc, vascular ingrowth into the body of the disc did not occur. Our results point to the need for future research to understand the transition from the well-vascularized status of the fetal and young discs to the avascular state of the adult human disc or the small mammalian disc.     

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.     

Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.     

Morphologic features of spontaneous annular tears and disc degeneration in the aging sand rat (Psammomys obesus obesus)

The sand rat, a member of the gerbil family, is a valuable small animal model in which intervertebral disc degeneration occurs spontaneously as the animal ages. Radiographic features of cervical and lumbar degeneration resemble those in human spines. We conducted a retrospective analysis of spines of 140 animals 3?41 months old focusing specifically on the presence of annular tears that are not visible by radiography and have not been described previously in the sand rat disc. During degeneration of the nucleus pulposus, notochordal cell death occurs and granular material, which stains with Alcian blue for proteoglycans, accumulates. Lamellar architecture also deteriorates and annular tears occur that are morphologically similar to the concentric, radiating and transdiscal annular tears in human discs. These tears contain granular material that provides a “marker” that can be used to distinguish the annular tears from artefactual separations during sectioning. We observed lamellar degeneration and separation in the annulus fibrosus at 4 months with associated tears that contained granular material in the nucleus. Tears that contained granular material and displacement of the degenerating nucleus were common in cervical and lumbar discs of animals older than 9 months; some specimens showed tears at 4 and 5 months. With advanced degeneration, granular globules were displaced dorsally adjacent to and into the spinal cord area and also ventrally into regions where osteophytes formed. We present morphologic data that expand the utility of this rodent model of spontaneous age-related disc degeneration and provide novel information on annular tears and disc degeneration.