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1.
J Pediatr Orthop ; 39(9): 479-486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31503237

RESUMO

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.


Assuntos
Lâmina de Crescimento/metabolismo , Fraturas Salter-Harris/metabolismo , Técnicas de Ablação , Animais , Epífises/lesões , Epífises/metabolismo , Fator VIII/metabolismo , Lâmina de Crescimento/cirurgia , Imuno-Histoquímica , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Ratos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fraturas Salter-Harris/cirurgia , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Stem Cells Dev ; 27(3): 147-165, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29241405

RESUMO

The aim of this study was to examine the complexity of the stem cell populations in the intervertebral disc (IVD) and understand their role in disc degeneration, with a view of determining whether the resident stem cells could be developed for therapeutic purposes to combat IVD degeneration. Stem cells have been isolated from disc and paradiscal tissues, including the notochord, annulus fibrosus (AF), nucleus pulposus (NP), cartilaginous endplate (CEP), ligamentum flavum, and vertebral body. Resident AF and NP cells are relatively sparsely distributed occurring as single or occasional doublet cells surrounded by an extensive extracellular matrix (ECM). Small clusters of 4-12 cells also occur close to annular lesions in experimental ovine and canine disc degeneration, these are indicative of an attempted repair response by resident stem cells. The rat IVD also has notochordal and peripheral cell populations in the outer AF, which express CS sulfation motifs (7-D-4, 4-C-3, 3-B-3[-]) characteristic of activated stem cells, the murine IVD also has a cell population in the outer AF adjacent to the vertebral growth plate with characteristics of a progenitor cell population. These have also been observed in rabbit, minipig, ovine, and human IVDs. Chondroid cell nests in the ovine NP may represent a progenitor/stem cell reserve. Such human chondroid cells express CS sulfation motifs (7-D-4, 4-C-3, 3-B-3[-]), cytokeratin-8 and 19, and CD cell surface markers typical of stem cells, including OCT3/4, CD105, CD90, STRO-1, NOTCH1, and JAGGED1. Similar stem cell populations are present in grade IV degenerate human IVDs. A greater understanding of the biology of this chondroid cell population may identify them as a therapeutic resource. A resident therapeutic cell type adapted to the demanding IVD environment may be advantageous in repair strategies.


Assuntos
Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/fisiologia , Regeneração/fisiologia , Células-Tronco/metabolismo , Animais , Cães , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Camundongos , Ratos , Ovinos , Células-Tronco/patologia
3.
BMC Musculoskelet Disord ; 18(1): 182, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28482827

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo/fisiologia , Interleucina-1beta/farmacologia , Disco Intervertebral/metabolismo , Mucina-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Disco Intervertebral/química , Disco Intervertebral/efeitos dos fármacos , Pessoa de Meia-Idade , Mucina-1/análise , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Adulto Jovem
4.
Spine (Phila Pa 1976) ; 42(20): 1529-1537, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28306638

RESUMO

STUDY DESIGN: Institutional review board-approved research using human annulus cells cocultured with F11 nerve cells. OBJECTIVE: To perform functional, kinetic assays of neurite dynamics and media neurotrophin measurements to test whether proinflammatory cytokines influence annulus cells' signaling cues for neurite growth/repulsion. SUMMARY OF BACKGROUND DATA: Nerves grow in response to signaling molecules called neurotrophins, which disc cells produce (e.g., brain-derived neurotrophic factor [BDNF], glial cell line-derived neurotrophic factor [GDNF], and neurotrophin 3 [NT3]) and which influence neuron survival, differentiation, and migration. How proinflammatory cytokines influence disc signaling cues for neurite growth/repulsion is poorly understood. METHODS: Studies used our previous model of 4-day human annulus cell-F11 nerve cell coculture to assess effects of added proinflammatory cytokines interleukin 1 beta (IL-1ß; 10 pmol/L) or tumor necrosis factor alpha (TNF-α) (10 pmol/L). Annulus cells were cultured from 6 Thompson grade I, 9 grade II, 8 grade III, 11 grade IV, and 7 grade V discs. Neurite lengths were measured following control conditions or with added IL-1ß or TNF-α, and conditioned media assayed with RayBiotech Growth Factor Arrays. Standard statistical methods used analysis of variance and Spearman correlation coefficient testing associations of neurite length with neurotrophin levels. RESULTS: IL-1-ß or TNF-α significantly increased neurite lengths (P < 0.001) and BDNF, NT3, and GDNF media levels (P ≤ 0.01) versus controls. Significant positive correlations were present between media neurotrophin levels for BDNF, NT3, and GDNF and neurite lengths under control conditions, following addition of IL-1ß, and following addition of TNF-α. Novel data showed production of the neurotrophin amphiregulin. CONCLUSION: In vitro data supported the hypothesis that nerve-disc cell interactions may be influenced by the heightened proinflammatory milieu present in degenerating discs, leading to increased nerve migration. Data may have direct clinical relevance/implications for nerve ingrowth and pain in the outer annulus (where disc cell numbers are high), and in regions where nerves penetrate into the disc via annular tears. LEVEL OF EVIDENCE: N/A.


Assuntos
Anel Fibroso/metabolismo , Interleucina-1beta/farmacologia , Neuritos/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Adolescente , Adulto , Idoso , Anel Fibroso/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Sinais (Psicologia) , Citocinas/metabolismo , Citocinas/farmacologia , Feminino , Humanos , Lactente , Recém-Nascido , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/metabolismo , Neuritos/efeitos dos fármacos , Neurotrofina 3 , Estudos Prospectivos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
5.
J Orthop Res ; 34(8): 1351-60, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27249627

RESUMO

Back pain and intervertebral disc degeneration have growing socioeconomic/health care impacts. Increasing research efforts address use of stem and progenitor cell-based replacement therapies to repopulate and regenerate the disc. Data presented here on the innate human annulus progenitor cells: (i) assessed osteogenic, chondrogenic and adipogenic potentials of cultured human annulus cells; and (ii) defined progenitor-cell related gene expression patterns. Verification of the presence of progenitor cells within primary human disc tissue also used immunohistochemical identification of cell surface markers and microarray analyses. Differentiation analysis in cell cultures demonstrated a viable progenitor cell pool within Thompson grades III-IV discs. Osteogenesis was present in 8 out of 11 cultures (73%), chondrogenesis in 8 of 11 (73%), and adipogenesis in 6 of 6 (100%). Immunolocalization was positive for CD29, CD44, CD105, and CD14 (mean values 80.2%, 81.5%, 85.1%, and 88.6%, respectively); localization of CD45 and CD34 was negative in disc tissue. Compared to controls, surgical discs showed significantly downregulated genes with recognized progenitor cell functions: TCF7L2 (2.7 fold), BMI1 (3.8 fold), FGF receptor 2 (2 fold), PAFAH1B1 (2.3 fold), and GSTP1 (9 fold). Compared to healthier grade I/II discs, grade III/IV discs showed significantly upregulated XRCC5 (3.6 fold), TCF7L2 (6 fold), GSTP1 (3.7 fold), and BMI1 (3 fold). Additional significant cell marker analyses showed expression of platelet-derived growth factor receptor alpha, CD90, CD73, and STRO-1. Statement of Clinical Significance: Findings provide the first identification of progenitor cells in annulus specimens from older, more degenerate discs (in contrast to earlier studies of healthier discs or nondegenerative specimens from teenagers). Findings also increase knowledge on progenitor cells present in the disc and suggest their value in potential future utilization for regeneration and disc cell therapy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1351-1360, 2016.


Assuntos
Células-Tronco Adultas/fisiologia , Anel Fibroso/citologia , Adipogenia , Adulto , Idoso , Condrogênese , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese
6.
J Orthop Res ; 34(8): 1456-65, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27155444

RESUMO

The relationship between neurotrophins produced by human annulus cells, such as neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF) which function in neurite survival and outgrowth, and nerve ingrowth into the disc remains poorly understood. In this work, we tested F11 neurite growth during exposure to control media, media with added nerve growth factor (NGF), conditioned media (CM) harvested from previous human annulus culture, or co-culture with annulus cells. Co-culture of F11 cells with annulus cells significantly increased media levels of amphiregulin, BDNF, glial-derived neurotrophic factor, and vascular endothelial growth factor compared to levels from in culture of F11 cells alone (p ≤ 0.04). Cell-based assays of neurite growth revealed that BDNF levels present in CM bore a significant (p = 0.01) positive relationship to neurite length and accounted for 38.5% of the change in neurite length. NT4 levels produced during co-culture with annulus cells bore a significant (p = 0.04) positive relationship to neurite length and accounted for 40.9% of the change in length. Statement of clinical significance: In vitro findings point to a potential role of annulus cells related to nerve ingrowth in vivo, and may have relevance in the outer annulus (where cell numbers are high) or in regions where nerves penetrate into annular tears or fissures. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1456-1465, 2016.


Assuntos
Anel Fibroso/inervação , Dor Lombar/etiologia , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Adolescente , Adulto , Idoso , Anel Fibroso/metabolismo , Técnicas de Cocultura , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto Jovem
7.
Spine (Phila Pa 1976) ; 40(11): 773-82, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26091153

RESUMO

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.


Assuntos
Autofagia/genética , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/ultraestrutura , Vértebras Lombares , RNA Mensageiro/análise , Sacro , Adulto , Idoso , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/genética , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Proteínas de Transporte/genética , Catepsina B/genética , Células Cultivadas , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Interleucina-1beta/farmacologia , Disco Intervertebral/química , Degeneração do Disco Intervertebral/metabolismo , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Presenilina-1/genética , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Fator de Necrose Tumoral alfa/farmacologia , Enzimas Ativadoras de Ubiquitina/análise , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/análise , Enzimas de Conjugação de Ubiquitina/genética , Regulação para Cima/efeitos dos fármacos , Adulto Jovem
8.
Spine (Phila Pa 1976) ; 40(12): 876-82, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25909354

RESUMO

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.


Assuntos
Apoptose , Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/patologia , Potencial da Membrana Mitocondrial , Mitocôndrias/patologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Forma do Núcleo Celular , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Potássio/metabolismo , Valinomicina/toxicidade
9.
Exp Mol Pathol ; 98(3): 427-30, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25746662

RESUMO

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).


Assuntos
Proteína HMGB1/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Receptor 2 Toll-Like/metabolismo , Regulação para Cima , Adulto , Idoso , Células Cultivadas , Feminino , Proteína HMGB1/genética , Humanos , Lactente , Recém-Nascido , Disco Intervertebral/efeitos dos fármacos , Degeneração do Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/farmacologia
10.
JBJS Case Connect ; 5(4): e96, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-29252802

RESUMO

CASE: A thirty-nine-year-old man with alkaptonuria presented with low back pain. Imaging demonstrated lumbar scoliosis, extensive irregularities of end-plate vertebral margins, and Thompson Grade-V disc degeneration. Six months later, the patient returned with a herniated L2-L3 disc. Minimally invasive disc surgery was performed, and harvested disc tissue showed marked extracellular matrix changes and ochronotic pigment deposits. CONCLUSION: Scattered previous literature is available regarding disc changes in alkaptonuria. Although rare, alkaptonuria appears to severely impact the disc as reflected by cellular and matrix inclusions that contribute to disc-cell pathology.

11.
Exp Mol Pathol ; 98(1): 102-5, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25527176

RESUMO

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.


Assuntos
Quimiocina CCL2/metabolismo , Interleucina-1beta/farmacologia , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Lactente , Recém-Nascido , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/imunologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/imunologia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Adulto Jovem
12.
Int J Med Sci ; 11(7): 748-53, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24904231

RESUMO

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.


Assuntos
Catecol O-Metiltransferase/genética , Degeneração do Disco Intervertebral/genética , Dor Lombar/genética , Dor/genética , Adulto , Feminino , Estudos de Associação Genética , Humanos , Degeneração do Disco Intervertebral/complicações , Degeneração do Disco Intervertebral/patologia , Dor Lombar/complicações , Dor Lombar/patologia , Masculino , Pessoa de Meia-Idade , Dor/complicações , Dor/patologia , Polimorfismo de Nucleotídeo Único , Caracteres Sexuais
13.
Arthritis ; 2014: 812678, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24963403

RESUMO

Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

14.
Spine J ; 14(12): 2995-3001, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24912122

RESUMO

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.


Assuntos
Anti-Inflamatórios/farmacologia , Disco Intervertebral/efeitos dos fármacos , Neuropeptídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Feminino , Humanos , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Fator de Necrose Tumoral alfa/genética
15.
Exp Mol Pathol ; 97(1): 1-5, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24768589

RESUMO

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.


Assuntos
Degeneração do Disco Intervertebral/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Adulto , Fatores Etários , Idoso , Animais , Pré-Escolar , Modelos Animais de Doenças , Feminino , Gerbillinae , Humanos , Lactente , Recém-Nascido , Vértebras Lombares/metabolismo , Masculino , Metaloproteinase 12 da Matriz/análise , Pessoa de Meia-Idade
16.
Exp Mol Pathol ; 96(2): 225-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24582800

RESUMO

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.


Assuntos
Fator 5 de Diferenciação de Crescimento/biossíntese , Interleucina-1beta/metabolismo , Deslocamento do Disco Intervertebral/genética , Fator de Necrose Tumoral alfa/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 5 de Diferenciação de Crescimento/metabolismo , Humanos , Interleucina-1beta/farmacologia , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/patologia , Análise em Microsséries , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
17.
BMC Cancer ; 14: 225, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24674692

RESUMO

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.


Assuntos
Artrite/complicações , Neoplasias Ósseas/patologia , Quimiocina CXCL12/metabolismo , Interleucina-17/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Experimentais/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Artrite/induzido quimicamente , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-6/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Fator Estimulador de Colônias de Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CXCR4/metabolismo
18.
Clin Orthop Relat Res ; 472(6): 1936-42, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24515407

RESUMO

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.


Assuntos
Envelhecimento , Vértebras Cervicais , Degeneração do Disco Intervertebral/etiologia , Vértebras Lombares , Fatores Etários , Animais , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/patologia , Modelos Animais de Doenças , Feminino , Gerbillinae , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Masculino , Radiografia
19.
Exp Mol Pathol ; 96(2): 133-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24468005

RESUMO

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.


Assuntos
Quimiocina CCL5/biossíntese , Interleucina-1beta/biossíntese , Disco Intervertebral/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Técnicas de Cultura de Células , Linhagem Celular , Quimiocina CCL5/genética , Humanos , Disco Intervertebral/citologia , Análise em Microsséries , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Connect Tissue Res ; 54(6): 432-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24060054

RESUMO

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.


Assuntos
Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Disco Intervertebral/citologia , Proteína Plasmática A Associada à Gravidez/metabolismo , Somatomedinas/metabolismo , Disponibilidade Biológica , Demografia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Interleucina-1beta/farmacologia , Proteína Plasmática A Associada à Gravidez/genética , Fator de Necrose Tumoral alfa/farmacologia
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