Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

ABSTRACT: Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies have considerable drawbacks and fail to replace the normal disc in facilitating spinal movements and absorbing load. Therefore, the focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about the cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis system. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 17 dysregulated molecular markers and various dysregulated cellular functions, including cell proliferation and inflammatory response, in the human degenerative annulus fibrosus. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon signalling pathway. In conclusion, this study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation which may affect cellular function in human degenerative disc. 48 samples of intervertebral disc tissue - annulus fibrosus and nucleus pulposus - displaying varying degrees (grades) of degeneration

ORGANISM(S): Homo sapiens

SUBMITTER: Peadar O'Gaora

PROVIDER: E-GEOD-70362 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus.

Kazezian Zepur Z Gawri Rahul R Haglund Lisbet L Ouellet Jean J Mwale Fackson F Tarrant Finbarr F O'Gaora Peadar P Pandit Abhay A Alini Mauro M Grad Sibylle S

Scientific reports 20151022

Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies fail to replace the normal disc in facilitating spinal movements and absorbing load. The focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about cascades of disc degeneration and allow for the design of specific therapies. We hypothesi ...[more]

PMID: 26489762

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Project description:Autologous stem cell therapy has potential for biologic treatment of disc degeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal stem cells (AD-MSC) are readily available. Our objectives were: 1) To develop/validate methods to harvest AD-MSC and direct them to a disc-like phenotype by three-dimensional (3D) culture and TGF-Ã3 exposure; 2) To perform gene expression profiling for human AD-MSC and annulus cells in 3D culture; 3) To test whether disc cell-AD-MSC co-culture could augment proteoglycan production. Stem cell plasticity offers potential for future biologic therapies for disc degeneration. Data indicated that human AD-MSC can successfully be manipulated in 3D culture to express gene products important in the disc ECM (types I and II collagen, chondroitin sulfate, keratin sulfate, decorin), and that co-culture of annulus cells with AD-MSC enhances proteoglycan production. Studies defined gene expression patterns of AD-MSC and human annulus cells in 3D culture, important as we explore the potential of MSC in biologic therapies for disc degeneration. Experiment Overall Design: AD-MSC were extracted from human adipose tissue, and characterized as stem cells using accepted criteria (direction into osteoblasts or chondrocytes; and cell surface marker criteria). Three AD-MSC cultures were grown in 3D with or without TGF-Ã3 for 2-3 weeks. Disc Tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Seven disc cultures were also grown in 3-D for 2 weeks. RNA was harvested according to instructions with the Trizol isolation method, checked for quality using the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA USA), reverse-transcribed to double-stranded cDNA, subjected to two rounds of transcription, and hybridized to the DNA microarray in the Affymetrix Fluidics Station 400. Affymetrix human U133 X3P arrays were used. Using Genesifter, gene expression in AD-MSC with and witout TGF-B3 was compared to annulus cells. Gene expression of stem cells with TGF-Ã3 was also compared to stem cells grown in the absence of TGF-Ã3.

Project description:Asporin, also known as periodontal ligament-associated protein 1 (PLAP1), is a member of the family of small leucine-rich proteoglycan (SLRP) family. It is present within the cartilage extracellular matrix (ECM), and is reported have a genetic association with osteoarthritis. Its D14 allele has recently been found to be associated with lumbar disc degeneration in Asian subjects. There have been no studies, however, of this gene’s normal immunohistochemical localization within the human intervertebral disc, nor of expression levels in Caucasian individuals with disc degeneration. Studies were approved by our human subjects Institutional Review Board. Methods included immunohistochemical localization of asporin in the disc of humans and the sand rat (a small rodent with spontaneous age-related disc degeneration), and Affymetrix microarray analysis of asporin gene expression in vivo and in vitro. mmunohistochemical studies of human discs revealed that some, but not all, cells of the outer annulus expressed asporin. Fewer cells in the inner annulus contained asporin, and it was rarely present in cells in the nucleus pulposus. Similar patterns were found for the presence of asporin in lumbar discs of sand rats. Substantial relative gene expression levels were seen for asporin in both disc tissue and in annulus cells grown in three-dimensional culture. More degenerate human discs (Thompson grade 4) showed higher expression levels of asporin than did less degenerate (grade 1, 2 and 3) discs, p = 0.004. In the discs of Caucasian subjects studied here, and in the sand rat, greater immunolocalization levels were found in the outer compared to inner annulus. Localization was rare in the nucleus. Gene expression studies showed greatest expression of asporin in the more degenerate human discs in vivo. Disc Tissue samples were obtained via the National Cancer Institute Cooperative Tissue Network (CHTN) as well as surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Tissue was fixed and paraffin embedded. Standard laser capture microdissection (LCM) techniques were used to collect the cells from which total RNA was isolated and analyzed via microarray. Based on the Thompson scouring system, unhealthy discs (grade 4) were compared to healthy discs (grades 2,3).

Dataset Information

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

Publications

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus.

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