Experimental Induction of Physeal Injuries by Fracture, Drill, and Ablation Techniques: Analyses of Immunohistochemical Findings.

BACKGROUND: Although physeal fractures and physeal bars can result in significant clinical consequences to growth and development of the injured physis, little orthopaedic research has focused upon this topic. Our objective was to extend a previously developed rat model to examine the immunohistochemical features following surgical application of techniques disrupting the physis. METHODS: Physes were surgically disrupted using fracture (control), epiphyseal scrape (ES), or epiphyseal drill (ED). After 1, 3, 6, 10, or 21 days, animals were euthanized, sites processed for histology and immunohistochemical localization of vascular endothelial growth factor (VEGF), Factor VIII, Sox-9, PTHrP (parathyroid hormone-related protein) and PTHrP-R (parathyroid hormone-related protein receptor) in resting, proliferative, and hypertrophic physeal zones. Incidence of physeal bars, vertical septa and islands within the metaphysis was quantified. Semiquantitative analysis of immunohistochemistry was performed. RESULTS: Physeal bars, vertical septa, and displaced cartilage islands were present each of the surgical treatments. Fisher's exact test showed a statistically significant increase in the presence of physeal bars (P=0.002) and vertical septa (P=0.012) in the ED group at 10 and 21 days. Analysis of VEGF showed significant differences among the surgical treatments involving the resting zone, and the proliferative zone for days 1, 6, and 21 (P≤0.02) with greater mean scores present in the fracture (control) group, followed by the ED group; the lowest scores were present in the ES group. PTHrP-R immunolocalization showed significant differences among treatments in the hypertrophic zone at days 6 and 21 (P=0.022 and 0.044, respectively). CONCLUSIONS: On the basis of the type of surgical treatment, results show significant differences in the presence of VEGF (reflecting the vascular bed) in the resting and proliferating zones at days 1, 6, and 21. VEGF localization was less abundant in the ED group (which had more physeal bars), suggesting that lack of vascular ingrowth plays a role in physeal bar formation. CLINICAL RELEVANCE: Basic science data presented here provide insight into the importance of the various regions of the physis and its repair and continued growth after physeal fracture. We suggest that a better understanding of the cellular basis of physeal arrest following physeal fracture may have future relevance for the development of treatments to prevent or correct arrest.

Mucin 1, a signal transduction membrane-bound mucin, is present in human disc tissue and is downregulated in vitro by exposure to IL-1ß or TNF-α.

BACKGROUND: Back pain and disc degeneration have a growing socioeconomic healthcare impact. Mucin 1 (MUC1) is a transmembrane glycoprotein whose extracellular and intracellular domains participate in cellular signaling. Little is currently known about the presence or role of MUC1 in human disc degeneration. METHODS: In this IRB-approved research study, 29 human disc specimens were analyzed for MUC1 immunohistochemical localization and gene expression, and annulus fibrosus (annulus) cells were also isolated and cultured in 3D. Microarray analysis assessed expression levels of MUC1 in healthy and degenerated disc tissue and in cells exposed to proinflammatory cytokines (IL-1ß or TNF-α). RESULTS: MUC1 was shown to be present in annulus cells at the protein level using immunochemistry, and its expression was significantly upregulated in annulus tissue from more degenerated grade V discs compared to healthier grade I-II discs (p = 0.02). A significant positive correlation was present between the percentage of MUC1-positive cells and disc grade (p = 0.009). MUC1 expression in annulus cells cultured in 3D was also analyzed following exposure to IL-1ß or TNF-α; exposure produced significant MUC1 downregulation (p = 0.0006). CONCLUSIONS: Here we present the first data for the constitutive presence of MUC1 in the human disc, and its altered expression during disc degeneration. MUC1 may have an important role in disc aging and degeneration by acting as a regulator in the hypoxic environment, helping disc cells to survive under hypoxic conditions by stabilization and by activation of HIF-1α as previously recognized in pancreatic cancer cells.

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.

Systemic neutralization of IL-17A significantly reduces breast cancer associated metastasis in arthritic mice by reducing CXCL12/SDF-1 expression in the metastatic niches.

BACKGROUND: IL-17A is a pro-inflammatory cytokine that is normally associated with autoimmune arthritis and other pro-inflammatory conditions. Recently, IL-17A has emerged as a critical factor in enhancing breast cancer (BC)-associated metastases. We generated immune competent arthritic mouse models that develop spontaneous BC-associated bone and lung metastasis. Using these models, we have previously shown that neutralization of IL-17A resulted in significant reduction in metastasis. However, the underlying mechanism/s remains unknown. METHODS: We have utilized two previously published mouse models for this study: 1) the pro-arthritic mouse model (designated SKG) injected with metastatic BC cell line (4T1) in the mammary fat pad, and 2) the PyV MT mice that develop spontaneous mammary gland tumors injected with type II collagen to induce autoimmune arthritis. Mice were treated with anti-IL-17A neutralizing antibody and monitored for metastasis and assessed for pro-inflammatory cytokines and chemokines associated with BC-associated metastasis. RESULTS: We first corroborate our previous finding that in vivo neutralization of IL-17A significantly reduced metastasis to the bones and lungs in both models. Next, we report that treatment with anti-IL17A antibody significantly reduced the expression of a key chemokine, CXCL12 (also known as stromal derived factor-1 (SDF - 1)) in the bones and lungs of treated mice. CXCL12 is a ligand for CXCR4 (expressed on BC cells) and their interaction is known to be critical for metastasis. Interestingly, levels of CXCR4 in the tumor remained unchanged with treatment. Consequently, protein lysates derived from the bones and lungs of treated mice were significantly less chemotactic for the BC cells than lysates from untreated mice; and addition of exogenous SDF-1 to the lysates from treated mice completely restored BC cell migration. In addition, cytokines such as IL-6 and M-CSF were significantly reduced in the lung and bone lysates following treatment. The data presented suggests that systemic neutralization of IL-17A can block the CXCR4/SDF-1 signaling pathway by reducing the expression of SDF-1 in the metastatic niches and significantly reducing metastasis in both mouse models. CONCLUSION: In our model, neutralization of IL-17A regulates SDF-1 expression in the metastatic niches either directly or indirectly via reducing levels of IL-6 and M-CSF.

Matrix metalloproteinase-12 immunolocalization in the degenerating human intervertebral disc and sand rat spine: Biologic implications.

Matrix metalloproteinase-12 (MMP-12; macrophage metalloelastase) degrades a number of extracellular matrix components which are present in the intervertebral disc, including type IV collagen, fibronectin, laminin, chondroitin sulfates, elastin and fibrinogen. MMP-12 has recently discovered relationships with cytokines and chemokines which also relate to disc cell biology. To date, no study has assessed immunolocalization of MMP-12 in degenerating human intervertebral disc tissue. Immunocytochemical localization was performed on 18 human disc specimens and on lumbar spines of the sand rat, a small animal model with well-recognized age-related disc degeneration. In the human disc, intracellular localization was present in both the annulus and nucleus portions of the disc. The sand rat degenerating disc also showed MMP-12 disc localization, with additional presence in chondrocytes of the vertebral endplate of older animals. This is the initial characterization of the presence of MMP-12 in the human and sand rat disc, and in chondrocytes of the vertebral endplate in older sand rats with degenerating discs. Findings are important because they document the presence of an additional MMP-12 in disc tissue, thus expanding our understanding of disc extracellular matrix remodeling, and because they provide novel information on the presence of MMP-12 in the cartilage endplate as it undergoes sclerosis during disc degeneration in the aging sand rat.

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.

A novel catechol-O-methyltransferase variant associated with human disc degeneration.

BACKGROUND: Disc degeneration and its associated low back pain are a major health care concern causing disability with a prominent role in this country's medical, social and economic structure. Low back pain is devastating and influences the quality of life for millions. Low back pain lifetime prevalence approximates 80% with an estimated direct cost burden of $86 billion per year. Back pain patients incur higher costs, greater health care utilization, and greater work loss than patients without back pain. METHODS: Research was performed following approval of our Institutional Review Board. DNA was isolated, processed and amplified using routine techniques. Amplified DNA was hybridized to Affymetrix Genome-Wide Human SNP Arrays. Quality control and genotyping analysis were performed using Affymetrix Genotyping Console. The Birdseed v2 algorithm was used for genotyping analysis. 2589 SNPs were selected a priori to enter statistical analysis using lotistic regression in SAS. RESULTS: Our objective was to search for novel single nucleotide polymorphisms (SNPs) associated with disc degeneration. Four SNPs were found to have a significant relationship to disc degeneration; three are novel. Rs165656, a new SNP found to be associated with disc degeneration, was in catechol-O-methyltransferase (COMT), a gene with well-recognized pain involvement, especially in female subjects (p=0.01). Analysis confirmed the previously association between COMT SNP rs4633 and disc degeneration. We also report two novel disc degeneration-related SNPs (rs2095019 and rs470859) located in intergenic regions upstream to thrombospondin 2. CONCLUSIONS: Findings contribute to the challenging field of disc degeneration and pain, and are important in light of the high clinical relevance of low back pain and the need for improved understanding of its fundamental basis.

Spontaneous age-related cervical disc degeneration in the sand rat.

BACKGROUND: Disc space narrowing, osteophytes, and disc degeneration are common and increase with aging. Few animal models are appropriate for the study of spontaneous age-related cervical disc degeneration. QUESTIONS/PURPOSES: We used the sand rat, a member of the gerbil family with well-recognized age-related lumbar disc degeneration, to determine whether spontaneous cervical disc degeneration differed from lumbar degeneration when evaluated by (1) radiologic and (2) histologic measures. Animals 2 to 25 months of age were used in these analyses. METHODS: Cervical and lumbar discs of 99 sand rats were analyzed with radiology, and cervical discs of 67 sand rats were studied with histology. Lateral digital radiographs of cervical and lumbar spines were scored for presence or absence of wedging, disc space narrowing, osteophytes, end plate calcification, and irregular disc margins at C2-C3 through C6-C7 and T12-L1 through L7-S1. Percentages for presence were calculated and statistically analyzed for younger (range, 2-11.9 months old) versus older (range, 12.0-25 months old) animals. RESULTS: Cervical discs in younger animals exhibited a greater proportion of irregular margins compared with lumbar sites (94% versus 83%; p = 0.02; 95% CI for difference, 2.7, 19.0%). In older animals, cervical discs showed a greater proportion of osteophytes than did lumbar discs (7% versus 0%; p < 0.0001). The incidence of disc space narrowing was greater in cervical versus lumbar sites (99% versus 90%; p = 0.0008). Cervical spine sites which contained osteophytes morphologically showed irregular disc margins and revealed an extrusion of herniated disc material in the osteophytes. CONCLUSIONS: Radiologic and morphologic studies confirmed age-related disc degeneration in the cervical spine of the sand rat. CLINICAL RELEVANCE: Clinical cervical aging studies have shown that 14% of asymptomatic subjects younger than 40 years have abnormal MRI scans with an increase to 50% by 50 years old. We studied an economic rodent model for cervical age-related spontaneous disc.

Arthritis augments breast cancer metastasis: role of mast cells and SCF/c-Kit signaling.

INTRODUCTION: Breast cancer remains the second leading cause of cancer-related deaths for women in the United States. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We recently reported that mice with autoimmune arthritis have significantly increased incidence of bone and lung metastasis and decreased survival associated with breast cancer. In this study, we evaluated the mechanism underlying the increased metastasis. METHODS: We used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and another that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mast cell levels and metastasis were monitored. RESULTS: First, we confirmed that breast tumor-bearing arthritic mice have a significantly higher incidence of bone and lung metastasis than do their nonarthritic counterparts. Next, we showed increased recruitment of mast cells within the primary tumor of arthritic mice, which facilitates metastasis. Next, we report that arthritic mice without any tumors have higher numbers of mast cells in the bones and lungs, which may be the underlying cause for the enhanced lung and bone metastases observed in the arthritic mice. Next, we showed that once the tumor cells populate the metastatic niches (bones and lungs), they further increase the mast cell population within the niche and assist in enhancing metastasis. This may primarily be due to the interaction of c-Kit receptor present on mast cells and stem cell factor (SCF, the ligand for ckit) expressed on tumor cells. Finally, we showed that targeting the SCF/cKit interaction with an anti-ckit antibody reduces the differentiation of mast cells and consequently reduces metastasis. CONCLUSION: This is the first report to show that mast cells may play a critical role in remodeling not only the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis through SCF/cKit interaction in breast cancer with arthritis.

Human annulus cells regulate PAPP-A and IGFBP-4 expression, and thereby insulin-like growth factor bioavailability, in response to proinflammatory cytokine exposure in vitro.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-ß or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p < 0.001). PAPP-A production following exposure to IL-1ß was significantly greater in cells derived from more degenerated versus healthier discs (p = 0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1ß- or TNF-α-exposed cells (p = 0.01-0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1ß- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.

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

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.

Fukutin-related protein is essential for mouse muscle, brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies.

Mutations in fukutin-related protein (FKRP) cause a common subset of muscular dystrophies characterized by aberrant glycosylation of alpha-dystroglycan (α-DG), collectively known as dystroglycanopathies. The clinical variations associated with FKRP mutations range from mild limb-girdle muscular dystrophy type 2I with predominantly muscle phenotypes to severe Walker-Warburg syndrome and muscle-eye-brain disease with striking structural brain and eye defects. In the present study, we have generated animal models and demonstrated that ablation of FKRP functions is embryonic lethal and that the homozygous-null embryos die before reaching E12.5. The homozygous knock-in mouse carrying the missense P448L mutation almost completely lacks functional glycosylation of α-DG in muscles and brain, validating the essential role of FKRP in the functional glycosylation of α-DG. However, the knock-in mouse survives and develops a wide range of structural abnormalities in the central nervous system, characteristics of neuronal migration defects. The brain and eye defects are highly reminiscent of the phenotypes seen in severe dystroglycanopathy patients. In addition, skeletal muscles develop progressive muscular dystrophy. Our results confirm that post-translational modifications of α-DG are essential for normal development of the brain and eyes. In addition, both the mutation itself and the levels of FKRP expression are equally critical for the survival of the animals. The exceptionally wide clinical spectrums recapitulated in the P448L mice also suggest the involvement of other factors in the disease progression. The mutant mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-related muscular dystrophies.

Matrix metalloproteinase-26, a novel MMP, is constitutively expressed in the human intervertebral disc in vivo and in vitro.

Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I-IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration.

Collagen induced arthritis increases secondary metastasis in MMTV-PyV MT mouse model of mammary cancer.

BACKGROUND: Several studies have demonstrated that sites of chronic inflammation are often associated with the establishment and growth of various malignancies. A common inflammatory condition in humans is autoimmune arthritis (AA). Although AA and cancer are different diseases, many of the underlying processes that contribute to the disorders of the joints and connective tissue that characterize AA also affect cancer progression and metastasis. Systemically, AA can lead to cellular infiltration and inflammation of the lungs. Several studies have reported statistically significant risk ratios between AA and breast cancer. Despite this knowledge being available, there has been minimal research linking breast cancer, arthritis, and metastasis associated with breast cancer. Notably both diseases are extremely prevalent in older post-menopausal women. METHODS: To establish the novel link between arthritis induced inflammation and secondary metastasis associated with breast cancer, PyV MT mice that spontaneously develop mammary gland carcinoma were injected with Type II collagen (CII) to induce arthritis at 9 and 18 weeks of age for pre-metastatic and metastatic condition. The sites of secondary metastasis and the associated inflammatory microenvironment were evaluated. RESULTS: A significant increase in breast cancer-associated secondary metastasis to the lungs and bones was observed in the arthritic versus the non-arthritic PyV MT mice along with an increase in primary tumor burden. We report significant increases in the levels of interstitial cellular infiltrates and pro-inflammatory cytokines such as interleukin-17 (IL-17), interleukin-6 (IL-6), Pro- Matrix metallopeptidase 9 (Pro-MMP9), insulin like growth factor-II (GF-II) and macrophage colony stimulating factor (M-CSF) in the arthritic lung and bone milieu as well as in the circulation. These pro-inflammatory cytokines along with the inflammatory microenvironment may be the underlying factors facilitating tumor progression and metastasis in arthritic PyV MT mice. This was further substantiated by treatment with celecoxib, an anti-inflammatory drug + αIL-17 antibody that significantly reduced the secondary metastasis to lung and bone. CONCLUSIONS: The data generated not only reveal the underlying mechanism of high susceptibility to bone and lung metastasis in an arthritic condition but our combination therapies may lead to treatment modalities that will be capable of reducing tumor burden, and preventing relapse and metastasis in arthritic patients with breast cancer.

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.

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease.

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62(P392L) mutation contributes to the development of this disease. To determine the effects of p62(P392L) expression on osteoclasts in vitro and in vivo, we introduced either the p62(P392L) or WT p62 gene into normal osteoclast precursors and targeted p62(P392L) expression to the osteoclast lineage in transgenic mice. p62(P392L)-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-kappaB ligand (RANKL) and TNF-alpha and showed increased NF-kappaB signaling but did not demonstrate increased 1,25-(OH)(2)D(3) responsivity, TAF(II)-17 expression, or nuclear number per osteoclast. Mice expressing p62(P392L) developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62(P392L) expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Senescent vs. non-senescent cells in the human annulus in vivo: cell harvest with laser capture microdissection and gene expression studies with microarray analysis.

BACKGROUND: Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells. METHODS: We employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens. RESULTS: Microarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified. CONCLUSIONS: Our LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.

Analysis of meniscal degeneration and meniscal gene expression.

BACKGROUND: Menisci play a vital role in load transmission, shock absorption and joint stability. There is increasing evidence suggesting that OA menisci may not merely be bystanders in the disease process of OA. This study sought: 1) to determine the prevalence of meniscal degeneration in OA patients, and 2) to examine gene expression in OA meniscal cells compared to normal meniscal cells. METHODS: Studies were approved by our human subjects Institutional Review Board. Menisci and articular cartilage were collected during joint replacement surgery for OA patients and lower limb amputation surgery for osteosarcoma patients (normal control specimens), and graded. Meniscal cells were prepared from these meniscal tissues and expanded in monolayer culture. Differential gene expression in OA meniscal cells and normal meniscal cells was examined using Affymetrix microarray and real time RT-PCR. RESULTS: The grades of meniscal degeneration correlated with the grades of articular cartilage degeneration (r = 0.672; P < 0.0001). Many of the genes classified in the biological processes of immune response, inflammatory response, biomineral formation and cell proliferation, including major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1), integrin, beta 2 (ITGB2), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis, progressive homolog (ANKH) and fibroblast growth factor 7 (FGF7), were expressed at significantly higher levels in OA meniscal cells compared to normal meniscal cells. Importantly, many of the genes that have been shown to be differentially expressed in other OA cell types/tissues, including ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) and prostaglandin E synthase (PTGES), were found to be expressed at significantly higher levels in OA meniscal cells. This consistency suggests that many of the genes detected in our study are disease-specific. CONCLUSION: Our findings suggest that OA is a whole joint disease. Meniscal cells may play an active role in the development of OA. Investigation of the gene expression profiles of OA meniscal cells may reveal new therapeutic targets for OA therapy and also may uncover novel disease markers for early diagnosis of OA.