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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

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

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

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

Abstract 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