Annulus cells from more degenerated human discs show modified gene expression in 3D culture compared with expression in cells from healthier discs.
ABSTRACT Understanding gene expression patterns of disc cells in culture is important as we develop biologic therapies for disc degeneration. The objective of the present study was to determine if cells from more degenerated discs expressed different genes, or differed in their expression patterns, compared with patterns of cells from healthier discs.
To determine if annulus cells from more degenerated discs expressed different gene expression patterns compared with patterns of cells from healthier discs using genome-wide analysis.
Cells from human annulus tissue were grown in three-dimensional (3D) culture and their gene expression patterns analyzed with Affymetrix microarray analysis. Gene expression patterns of cells from more degenerated discs (Thompson Grades IV and V) were compared with patterns from cells from healthier discs (Thompson Grades I, II, and III).
After approval by our human subjects institutional review board, annulus cells were obtained from lumbar discs of seven subjects with Thompson Grades I, II, or III and from five subjects with discs of Thompson Grades IV and V. Cells were grown in 3D culture for 2 weeks; 3D cultures were used because this microenvironment more closely mimics the in vivo condition. mRNA was harvested, processed for Affymetrix genome-wide gene analysis, and data analyzed with p values adjusted so as to compensate for false discovery rates.
GeneSifter analyses showed that cells from more degenerated discs had 320 genes significantly upregulated, and 104 genes significantly downregulated compared with cells from healthier discs. Important genes included those related to: 1) the extracellular matrix (ECM) (keratin-associated protein 1-1, hyaluronan synthase 2, and nexin were upregulated; biglycan, collagen type VI alpha 2, thrombospondin 3, laminen alpha 1, fibronectin type III domain-containing protein 1, elastin microfibril interfacer 2, fibulin 2, and nidogen 1 and 2 were downregulated); 2) ECM proteolysis (ADAMTS6 was upregulated); 3) cell proliferation (never in mitosis gene 1-related kinase 3, cell division cycle 2-like 5 [cholinesterase-related cell division controller], RAB42 [member of RAS oncogene family], and cyclin-dependent kinase 6 were upregulated; RAS-like GTP-binding 1 was downregulated); 4) apoptosis (BCL2-like 11 and p53-inducible nuclear protein 1 were upregulated; caspase recruitment domain family, member 10, caspase-1 dominant-negative inhibitor pseudo-ICE, and caspase 9 and FADD-like apoptosis regulator were downregulated); and 5) growth factors, inflammatory mediators, and other genes (fibroblast growth factor 1, pregnancy-associated plasma protein-A, interleukin 1 alpha, and interleukin 7 were upregulated; TGF-beta-induced transcript 1, interleukin 26 and interleukin 1 receptor-like 1, tumor necrosis factor, alpha-induced protein 2, and chemokine (C-X3-C motif) ligand 1 were downregulated).
Data presented here show that annulus cells from more degenerated discs show modified gene expression in 3D culture. Important gene variations involved expression of interleukins, cytokines, ECM components, and apoptosis regulators. Results presented here have potential application in future cell-based biologic therapies for disc degeneration.
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ABSTRACT: Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-α, interleukin-1β, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo.Journal of Cellular and Molecular Medicine 03/2014; · 3.70 Impact Factor
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ABSTRACT: Background. MYB is predicted to be a favorable prognostic predictor in a breast cancer population. We proposed to find the inferred mechanism(s) relevant to the prognostic features of MYB via a supervised network analysis. Methods. Both coefficient of intrinsic dependence (CID) and Galton Pierson's correlation coefficient (GPCC) were combined and designated as CIDUGPCC. It is for the univariate network analysis. Multivariate CID is for the multivariate network analysis. Other analyses using bioinformatic tools and statistical methods are included. Results. ARNT2 is predicted to be the essential gene partner of MYB. We classified four prognostic relevant gene subpools in three breast cancer cohorts as feature types I-IV. Only the probes in feature type II are the potential prognostic feature of MYB. Moreover, we further validated 41 prognosis relevant probes to be the favorable prognostic signature. Surprisingly, two additional family members of MYB are elevated to promote poor prognosis when both levels of MYB and ARNT2 decline. Both MYBL1 and MYBL2 may partially decrease the tumor suppressive activities that are predicted to be up-regulated by MYB and ARNT2. Conclusions. The major prognostic feature of MYB is predicted to be determined by the MYB subnetwork (41 probes) that is relevant across subtypes.Computational and Mathematical Methods in Medicine 01/2014; 2014:813067. · 0.79 Impact Factor
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ABSTRACT: Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5% (non-injured) or 30% (injured) of disc height. 30% strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteolglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased MMP and aggrecanase activity. Injured-IVD conditioned media contained significantly elevated IL-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG, and ELISA analysis showed significantly increased NGF levels compared to non-injured media. Injured-disc media caused significant neurite srpouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.European cells & materials 09/2014; 28:98-111. · 4.89 Impact Factor