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Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells.

ABSTRACT: Loss of intervertebral disc (IVD) matrix and ultimately disc height as a result of 'degeneration' has been implicated as a major cause of low back pain (LBP). The use of anabolic growth factors as therapies to regenerate IVD matrix, hence restoring disc height and thus reversing degenerative disc disease, has been suggested. Cartilage-derived morphogenetic protein (CDMP) is a growth factor which stimulates proteoglycan production in chondrocyte-like cells and thus could be a useful growth factor for LBP therapies. However, little is known about the expression of CDMP or its receptor in human IVD, nor its effects on human disc cells.Using immunohistochemistry we investigated the localisation of CDMP in non-degenerate and degenerate human IVDs. Additionally, we investigated the effect of CDMP on aggrecan and type II collagen gene expression and proteoglycan synthesis in nucleus pulposus (NP) cells derived from degenerate IVDs.We demonstrated that CDMP 1 and 2 were expressed in the non-degenerate and degenerate IVD, particularly in cells of the NP. A small decrease in the number of CDMP 1 and 2 immunopositive cells was seen with degeneration. Treatment of human NP cells, (derived from degenerate IVD), with CDMP showed an increase in aggrecan and type II collagen gene expression and increased production of proteoglycan (GAGs).The data suggests that CDMP may be a useful growth factor to stimulate proteoglycan production in the human degenerate IVD and hence the repair of the extracellular matrix.

SUBMITTER: Le Maitre CL

PROVIDER: S-EPMC2787300 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells.

Le Maitre Christine L CL Freemont Anthony J AJ Hoyland Judith A JA

Arthritis research & therapy 20090915 5

<h4>Introduction</h4>Loss of intervertebral disc (IVD) matrix and ultimately disc height as a result of 'degeneration' has been implicated as a major cause of low back pain (LBP). The use of anabolic growth factors as therapies to regenerate IVD matrix, hence restoring disc height and thus reversing degenerative disc disease, has been suggested. Cartilage-derived morphogenetic protein (CDMP) is a growth factor which stimulates proteoglycan production in chondrocyte-like cells and thus could be a ...[more]

PMID: 19754961

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Project description:Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Project description:Intervertebral disc (IVD) degeneration is a pathological process, which may lead to lower back pain. The present study aimed to investigate the pathogenesis of IVD degeneration. GSE42611 was downloaded from Gene Expression Omnibus, including 4 nucleus pulposus samples isolated from degenerated IVDs and 4 nucleus pulposus samples separated from normal IVDs. The differentially expressed genes (DEGs) between the degenerated and normal samples were screened using the limma package in R. Functional and pathway enrichment analyses were conducted separately for the upregulated and downregulated genes, using Database for Annotation, Visualization and Integrated Discovery software. In addition, protein‑protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes database and Cytoscape software. Finally, module analyses were conducted for the PPI networks using the MCODE plug‑in in Cytoscape. A total of 558 DEGs were identified in the degenerated nucleus pulposus cells: 253 upregulated and 305 downregulated. Pathway enrichment analysis revealed that downregulated thrombospondin 1 (THBS1) was enriched in extracellular matrix‑receptor interaction. Interleukin (IL)‑6 in the PPI network for the upregulated genes and vascular endothelial growth factor A (VEGFA) in the PPI network for the downregulated genes had higher degrees. Additionally, four modules (µM1, µM2, µM3 and µM4) were identified from the PPI network for the upregulated genes. Four modules (dM1, dM2, dM3 and dM4) were identified from the PPI network for the downregulated genes. In the dM2 module, collagen genes and integrin subunit α4 (ITGA4) may interact with each other. Additionally, functional enrichment indicated that collagen genes were enriched in extracellular matrix organization. In conclusion, IL‑6, VEGFA, THBS1, ITGA4 and collagen genes may contribute to the progression of IVD degeneration. These results suggested that the manipulation of these genes and their products may have potential as a novel therapeutic strategy for the treatment of patients with IVD.

Project description:During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.

Dataset Information

Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells.

Publications

Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells.

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