Autophagy in the Degenerating Human Intervertebral Disc: In Vivo Molecular and Morphological Evidence, and Induction of Autophagy in Cultured Annulus Cells Exposed to Proinflammatory Cytokines-Implications for Disc Degeneration.

STUDY DESIGN: Autophagy-related gene expression and ultrastructural features of autophagy were studied in human discs. OBJECTIVE: To obtain molecular/morphological data on autophagy in human disc degeneration and cultured human annulus cells exposed to proinflammatory cytokines. SUMMARY OF BACKGROUND DATA: Autophagy is an important process by which cytoplasm and organelles are degraded; this adaptive response to sublethal stresses (such as nutrient deprivation present in disc degeneration) supplies needed metabolites. Little is known about autophagic processes during disc degeneration. METHODS: Human disc specimens were obtained after institutional review board approval. Annulus mRNA was analyzed to determine autophagy-related gene expression levels. Immunolocalization and ultrastructural studies for p62, ATG3, ATG4B, ATG4C, ATG7, L3A, ULK-2, and beclin were conducted. In vitro experiments used IL-1ß- or TNF-α-treated human annulus cells to test for autophagy-related gene expression. RESULTS: More degenerated versus healthier discs showed significantly greater upregulation of well-recognized autophagy-related genes (P ≤ 0.028): beclin 1 (upregulated 1.6-fold); ATG8 (LC3) (upregulated 2.0-fold); ATG12 (upregulated 4.0-fold); presenilin 1 (upregulated 1.6-fold); cathepsin B (upregulated 4.5-fold). p62 was localized, and ultrastructure showed autophagic vacuolization and autophagosomes with complex, redundant whorls of membrane-derived material. In vitro, proinflammatory cytokines significantly upregulated autophagy-related genes (P ≤ 0.04): DRAM1 (6.24-fold); p62 (4.98-fold); PIM-2 oncogene, a positive regulator of autophagy (3-fold); WIPI49 (linked to starvation-induced autophagy) (upregulated 2.3-fold). CONCLUSION: Data provide initial molecular and morphological evidence for the presence of autophagy in the degenerating human annulus. In vivo gene analyses showed greater autophagy-related gene expression in more degenerated than healthier discs. In vitro data suggested a mechanism implicating a role of TNF-α and IL-1ß in disc autophagy. Findings suggest the importance of future work to investigate the relationship of autophagy to apoptosis, cell death, cell senescence, and mitochondrial dysfunction in the aging and degenerating disc. LEVEL OF EVIDENCE: N/A.

Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.

STUDY DESIGN: A study using cultured human annulus cells and human annular tissue. OBJECTIVE: To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo. SUMMARY OF BACKGROUND DATA: Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis. METHODS: High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis. RESULTS: Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P < 0.00001 by analysis of variance). A significant negative correlation for mitochondrial potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P < 0.0001). When exposed to the K ionophore valinomycin at sublethal levels to induce apoptosis, a significant reduction in mitochondrial potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1). CONCLUSION: Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration. LEVEL OF EVIDENCE: N/A.

High-mobility group box-1 gene, a potent proinflammatory mediators, is upregulated in more degenerated human discs in vivo and its receptor upregulated by TNF-α exposure in vitro.

Mechanisms which control and enhance proinflammatory cytokine expression during human disc degeneration are still poorly understood. The high-mobility group box-1 gene (HMGB1) produces a protein which can itself act as a cytokine, or can function as a potent proinflammatory mediator. Little is known about expression of HMGB1 in the human disc. Since proinflammatory cytokines increase significantly during human disc degeneration, in this work we hypothesized that HMGB1 may show upregulation with advancing stages of degeneration, and upregulation in cells exposed to TNF-α. Immunohistochemistry was performed to confirm the presence of HMGB1 in the human disc, and human annulus cells were cultured and challenged with 10(3)pM TNF-α for 14days in 3D culture. Cells with positive HMGB1 immunolocalization were abundant in the outer annulus. Molecular analysis of cultured cells showed an 8-fold significant increase in HMGB1 expression in more degenerated Thompson grade V discs compared to healthier grade I/II discs (p=0.033). Human disc tissue was assessed in molecular studies. Herniated specimens showed a 6.3-fold significantly greater expression level than that seen in control specimens (p=0.001). In culture experiments, expression of the receptor to HMGB1, toll-like receptor 2, showed a 24-fold upregulation in vitro in cells exposed to TNF-α vs. controls (p=0.0003).

Proinflammatory cytokines modulate the chemokine CCL2 (MCP-1) in human annulus cells in vitro: CCL2 expression and production.

Chemokines are important secondary inflammatory mediators released in response to stimuli which act as second-order cytokines with specialized functions in inflammation. The role of many of these specialized mediators is as yet poorly understood in the human intervertebral disc. Here we investigated CCL2 (chemokine (C-C motif) ligand 2, also known as monocyte chemotactic protein-1 (MCP-1)) in a study of its immunolocalization in disc tissue, and then hypothesized that exposure of cultured human annulus cells to proinflammatory cytokines might alter CCL2 gene expression and CCL2 production. CLL2 was localized to many disc cells in both herniated and non-herniated tissue specimens. Molecular analyses showed that cells exposed to IL-1ß showed a 5.5 fold upregulation in CCL2 gene expression vs. controls, p=0.017. Cells exposed to TNF-α showed a 7.7 fold upregulation vs. controls, p=0.005. Cultured cells (grades II-V) showed increased MCP-1 production in IL1-ß-treated cells vs. controls (p=0.016), with no significant difference in production in TNF-α-treated cells. Local production of CCL2 in vivo and vitro suggests that annulus cells may be primary effector cells (as well as target cells), with the ability to mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

Cortistatin is endogenous to the human intervertebral disc and exerts in vitro mitogenic effects on annulus cells and a downregulatory effect on TNF-α expression.

BACKGROUND CONTEXT: Cortistatin (CST) is a recently discovered cyclic neuropeptide with biologic anti-inflammatory properties relevant to disc degeneration. PURPOSE: To test whether CST is present in the disc tissue, whether its expression is influenced by tumor necrosis factor-α (TNF-α), and whether it influences cell proliferation. STUDY DESIGN: Institutional review board-approved study using immunohistochemistry on human disc tissue, in vitro annulus cultures to determine the effect of CST on cell proliferation, and the effect of TNF-α on CST gene expression. PATIENT SAMPLE: Discs from 12 subjects used for immunohistochemistry, four annulus specimens used for cell culture with proinflammatory cytokines, and 11 used for cell proliferation analyses. OUTCOME MEASURES: Immunohistochemical localization of CST, gene expression of CST, and cell proliferation analyses. METHODS: Immunohistochemistry localized CST in disc tissue. Microarray analysis measured CST gene expression. Human annulus cells were exposed to CST for proliferation tests or cultured for the effect of TNF-α on CST expression. Standard statistical analyses were performed. RESULTS: Immunohistochemistry identified CST in outer annulus, inner annulus, and nucleus tissue. Annulus cells exposed to TNF-α revealed significantly lower CST expression (p=.013). Exposure to CST significantly increased proliferation. Quantitative real-time polymerase chain reaction also confirmed expression of CST in vitro. CONCLUSIONS: Data provide the first evidence that CST is present in the human disc. Addition of CST significantly increased cell proliferation. Cortistatin expression was significantly downregulated by TNF-α exposure in vitro. Findings suggest possible in vivo reduction of the anti-inflammatory actions of CST because of elevated proinflammatory cytokines during degenerating disc.

Growth and differentiation factor-5 (GDF-5) in the human intervertebral annulus cells and its modulation by IL-1ß and TNF-α in vitro.

Growth and differentiation factor-5 (GDF-5) is a member of the TGF-ß superfamily which regulates cell division and differentiation. GDF-5 attracted high interest because of its role in skeletal development, especially in cartilaginous sites. Little is known, however, about the role of GFD-5 in disc cell biology. The present work demonstrated the immunohistologic presence of GDF-5 in human outer and inner annulus tissue. Microarray analysis of annulus cells showed significant upregulation of GDF-5 expression in herniated vs. non-herniated lumbar discs (2.14-fold change, p=0.021). In vitro three-dimensional culture studies challenged human annulus cells with IL-1ß and TNF-α, two proinflammatory cytokines known to be elevated in the human degenerating disc. Exposure resulted in significant downregulation of GDF-5 during both TNF-α exposure (5.83-fold change, p=0.044) and IL-1ß exposure (3.38-fold change, p=0.015). In vitro findings suggest that the degenerating disc milieu, with high proinflammatory cytokine levels, may limit expression of GDF-5, resulting in limited regenerative capacity of the intact disc.

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-ß and TNF-α in 3D culture.

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C-C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p=0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-ß and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p<0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p<0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

Mitochondrial gene expression in the human annulus: in vivo data from annulus cells and selectively harvested senescent annulus cells.

BACKGROUND CONTEXT: Mitochondrial dysfunction is recognized during cell senescence and apoptosis, two important components of human disc aging/degeneration. We hypothesize that mitochondrial dysfunction is present in the degenerating and senescent annulus cells. The objective of the present study was to analyze gene expression profiles related to mitochondrial function in vivo. PURPOSE: This study had two objectives in the analysis of gene expression patterns related to mitochondria in the human annulus: First, to assess human annulus cells in a genome-wide microarray analysis approach to evaluate mitochondrial gene expression in annulus tissue from degenerated compared with healthier discs. Second, to use laser capture microdissection (LCM) to selectively isolate senescent versus nonsenescent annulus cells to evaluate their mitochondrial gene expression patterns. STUDY DESIGN: Following approval by our Human Subjects Institutional Review Board, annulus cells from 20 human lumbar discs were analyzed for gene groups related to mitochondrial function; a subset was also analyzed, which focused on senescent versus nonsenescent annulus cells in a study of annulus cells from 10 lumbar discs. PATIENT SAMPLE: Human annulus tissue was used in molecular studies following institutional review board approval. OUTCOME MEASURES: Gene expression levels identified with microarray analyses were statistically evaluated using GeneSifter Web-based software (VizX Labs, Seattle, WA, USA). METHODS: Human annulus specimens were assessed for gene expression related to mitochondrial function. Approaches used whole annulus tissue and senescent or nonsenescent annulus cells selectively harvested using LCM. Microarray data were analyzed using gene ontology searches and GeneSifter Web-based software. RESULTS: Analysis of annulus cells compared mitochondrial gene expression patterns in annulus cells from more degenerated discs with patterns in annulus cells derived from healthier discs. Important findings included significant upregulation of p53 and several proapoptotic genes (including apoptosis-inducing factor, mitochondrion-associated 1, BCL2-like 11 [an apoptosis facilitator]; caspase 7 apoptosis-related cysteine peptidase; proteasome 26S subunit nonadenosine triphosphatase 10, programmed cell death 6, and reticulon 3). Methionine sulfoxide reductase (Msr), a repair enzyme that reduces methionine sulfoxide residues in proteins damaged by oxidation, was also significantly upregulated (2.02-fold increase). The gene "membrane-associated ring finger (C3HC4) 5" was significantly upregulated and relevant because it is believed to play a role in preventing cell senescence acting to regulate mitochondrial quality control. Nitric oxide synthase 3 (endothelial nitric oxide synthase [eNOS]) showed a 5.9-fold downregulation in more degenerated versus healthier annulus cells. In LCM-harvested senescent cells, Msr was significantly downregulated in senescent versus nonsenescent cells, a finding previously recognized in other types of senescent cells. CONCLUSIONS: Novel data showed that significant gene expression patterns are present in the human annulus related to mitochondrial dysfunction; changes were identified in important genes involving apoptosis, eNOS and Msr expressions, and solute carrier genes. Because current research efforts are focusing on bioactive compounds for mitochondria, we suggest that future biologic cell-based therapies for annulus degeneration should also consider mitochondrial-focused therapies.

Variations in aggrecan localization and gene expression patterns characterize increasing stages of human intervertebral disk degeneration.

During disk degeneration, annulus dehydration and matrix fraying culminate in the formation of tears through which nucleus and annulus disk material may rupture, causing radicular pain. Annular tears are present in more than half of the patients in early adulthood and are almost always present in the elderly. Aggrecan, which provides the disk with a shock absorber function under loading, is a key disk extracellular matrix (ECM) component. The objective of the present study was to assess the immunolocalization of aggrecan in the annulus, and to assess molecular gene expression patterns in the annulus ECM utilizing microarray analysis. Immunohistochemistry was performed on 45 specimens using an anti-human aggrecan antibody. Affymetrix microarray gene expression studies used the extracellular matrix ontology approach to evaluate an additional 6 grade I-II, 9 grade III, and 4 grade IV disks. Grade III/IV disks were compared to healthier grade I/II disks. Healthy and less degenerated disks showed a general uniform aggrecan immunolocalization; more degenerated disks contained regions with little or no identifiable aggrecan localization. In degenerated disks, molecular studies showed a significant downregulation of aggrecan, ADAMTS-like 3, and ADAMTS10. Collagen types III and VIII, fibronectin, decorin, connective tissue growth factor, TIMP-3, latent TGF-ß binding protein 2 and TGF-ß1 were significantly upregulated with fold changes ranging from 2.4 to 9.8. Findings here help us better understand changes in the immunohistochemical distribution of a key proteoglycan during disk aging. Such information may have application as we work towards biologic therapies to improve the aging/degenerating disk matrix.

IGF-1 rescues human intervertebral annulus cells from in vitro stress-induced premature senescence.

The aging human intervertebral disc contains a sizeable population of senescent cells. Since senescent cells cannot divide, senescence reduces the disc's ability to generate new cells to replace existing ones lost to necrosis or apoptosis. The objectives of the present work were: (1) to develop a reliable in vitro model for stress-induced premature senescence in human annulus cells, and (2) to investigate the potential for insulin-like growth factor-1 (IGF-1) to prevent or ameliorate senescence in vitro. The developed experimental model employs a 2 h exposure to 50 microM hydrogen peroxide; immunocytochemical localization of senescence associated-beta-galactosidase at pH 6.0 was used as the marker for senescent cells, and the percentage of senescent cells quantified after 3 days of culture. Nine sets of annulus cells were obtained from eight human surgical disc specimens; cells were tested with 0, 50, 100 or 500 ng/ml IGF-1. Although 50 or 100 ng/ml IGF-1 did not significantly alter the percentage of senescent cells, a significant reduction was present following exposure to 500 ng/ml IGF-1 (control, 56.3% +/- 8.5 (9); mean +/- SEM, (n) vs. treated, 39.6% +/- 6.6 (9), p = 0.0009). These novel findings point to the value of continued research towards development of future biologic therapies designed to reduce cell senescence in degenerating human discs.