Article

Annulus cells from more degenerated human discs show modified gene expression in 3D culture compared with expression in cells from healthier discs

Department of Orthopaedic Surgery, Carolinas Medical Center, PO Box 32861, Charlotte, NC 28232, USA.
The spine journal: official journal of the North American Spine Society (Impact Factor: 2.43). 08/2010; 10(8):721-7. DOI: 10.1016/j.spinee.2010.05.014
Source: PubMed

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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    • "Collagen VI, expressed in AF and NP cells, significantly increases through degeneration, which might be considered as a repair mechanism in response to tissue damage (Eskola, 2012). COL6A2 encodes a subunit of collagen VI and is down-regulated in disc degeneration (Gruber et al., 2010), as well as COL16A1 (α1 subunit of collagen XVI), COL6A1 (α1 subunit of collagen VI), COL29A1 (α1 subunit of collagen XXIX), and α1 subunit of collagen SVI. On the contrary, COL4A2, COL3A1, and COL8A2 are up-regulated (Gruber et al., 2011). "
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    ABSTRACT: Intervertebral disc degeneration (IVDD) is one of the common causes of low back pain. Similar to many other multifactorial diseases, it is affected by environmental and genetic factors. Although not completely understood, genetic factors include a wide spectrum of variations, such as single nucleotide polymorphisms, which could play a significant role in the etiology of this disease. Besides, the interactions with environmental factors could make the role of genetic factors more complicated. Genetic variations in disc components could participate in developing degenerative disc disease through altering the normal homeostasis of discs. Gene polymorphisms in disc proteins (collagens I, II, III, IX, and XI), proteoglycans (aggrecan), cytokines (interleukins I, VI, and X), enzymes (matrix metalloproteinases II, III, and IX), and vitamin D receptor seem to play considerable roles in the pathology of this disease. There are also many other investigated genes that could somehow take part in the process. However, it seems that more studies are needed to clarify the exact role of genetics in IVDD.
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    • "Disc tissues from human, bovine, or canine have been used to reveal the differential gene expression profiles between degenerative and nondegenerative intervertebral discs [4] [10]. Cells from discs with higher grades (Thompson Grades IV and V) and lower grades of disc degeneration (Thompson Grades I, II, and III) were grown in three-dimensional cultures and have been compared by complementary DNA (cDNA) microarray analysis [11]. Laser capture microdissection has also been applied to harvest cells from human annulus fibrosus, and gene expression profiles were derived using microarray analysis [12]. "
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    ABSTRACT: BACKGROUND CONTEXT: Disc degeneration is a multifactorial disease that may cause clinical symptoms such as chronic back pain or radiculopathy in the extremities. Periostin, an extracellular matrix protein involved in the process of fibrosis, expressed in tissues subjected to mechanical stress such as intervertebral disc. However, the expression of periostin during disc degeneration has not yet been studied. PURPOSE: The aim of this study is to elucidate the difference in gene expression profiles between degenerative and nondegenerative intervertebral discs for a better understanding of disc degeneration. STUDY DESIGN: Degenerative and nondegenerative nucleus pulposus cells were isolated from elderly patients with degenerative disc disease and younger patients with adolescent idiopathic scoliosis, respectively. METHODS: Affymetrix GeneChip Human arrays were used to derive gene expression profiles for disc degeneration, and gene expressions of periostin and other degeneration-related markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analysis. Immunohistochemical analysis of periostin and Gomori trichrome stain was performed to show the relationship of periostin, fibrosis, and disc degeneration. The mechanical stress experiment was designed to demonstrate the relationship of periostin, stress, and disc degeneration. RESULTS: Fourteen genes were identified to express at significantly different levels between degenerative and nondegenerative groups. An increase of periostin gene expression was observed in human degenerative nucleus pulposus cells for the messenger RNA and protein levels. Histological examination demonstrated an increased positive staining of periostin in degenerative discs from human tissues and rat needle-punctured tails and more fibrosis with architectural disorder and fragmentation in human degenerative disc as compared with nondegenerative discs. The expression of periostin was significantly induced by stress in human degenerative nucleus pulposus cells but not in nondegenerative cells. CONCLUSIONS: This study demonstrates for the first time an upregulation of periostin in addition to the expression levels of Type I collagen and matrix metalloproteinase-2 in human disc degeneration. It suggests that periostin may be a candidate gene that shows promise as a new prognostic marker and a therapeutic target that is worth further study to expand our knowledge of its role in disc degeneration.
    Full-text · Article · Feb 2013 · The spine journal: official journal of the North American Spine Society
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    • "Genes significantly altered during degeneration included cell senescence, cell division, hypoxia-related and other genes important for cell survival. Similarly, in AF cells cultured in 3D carriers, differences in gene expression patterns between healthier and more degenerative discs were observed for a variety of molecules involved in AF metabolism (Gruber et al., 2010). Important genes included those related to ECM synthesis or degradation, cell proliferation, apoptosis, growth and differentiation, or inflammation. "
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