Transcriptional profiling and pathway analysis of monosodium iodoacetate-induced experimental osteoarthritis in rats: relevance to human disease.

OBJECTIVE: The objective of this study was to characterize the rat monosodium iodoacetate (MIA)-induced model for osteoarthritis (OA) and determine the translatability of this model to human disease. This was accomplished through pathway, network and system level comparisons of transcriptional profiles generated from animal and human disease cartilage. METHODS: An OA phenotype was induced in rat femorotibial joints following a single injection of 200mug MIA per knee joint for a period of 2 or 4 weeks. Lesion formation in the rat joints was confirmed by histology. Gene expression changes were measured using the Agilent rat whole genome microarrays. Cartilage was harvested from human knees and gene expression changes were measured using the Agilent human arrays. RESULTS: One thousand nine hundred and forty-three oligos were differentially expressed in the MIA model, of these, approximately two-thirds were up-regulated. In contrast, of the 2130 differentially expressed oligos in human disease tissue, approximately two-thirds were down-regulated. This dramatic difference was observed throughout each level of the comparison. The total overlap of genes modulated in the same direction between rat and human was less than 4%. Matrix degradation and inflammatory genes were differentially regulated to a much greater extent in MIA than human disease tissue. CONCLUSION: This study demonstrated, through multiple levels of analysis, that little transcriptional similarity exists between rat MIA and human OA derived cartilage. As disease modulatory activities for potential therapeutic agents often do not translate from animal models to human disease, this and like studies may provide a basis for understanding the discrepancies.

Fibronectin and its fragments increase with degeneration in the human intervertebral disc.

STUDY DESIGN: This laboratory-based experiment correlates fibronectin content of intervertebral disc with a morphologic grade of degeneration. OBJECTIVES: To correlate the fibronectin content of the anulus fibrosus and nucleus pulposus with a gross morphologic grade of disc degeneration, and to determine the molecular size of the extractable fibronectin. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration increases with age and can lead to low back pain. Fibronectin helps to organize the extracellular matrix and provides environmental cues by interaction with cell surface integrins. In other tissues, its synthesis is elevated in response to injury. Fibronectin fragments can stimulate cells to produce metalloproteases and cytokines and inhibit matrix synthesis. METHODS: In this study, 17 anuli fibrosis and 18 nuclei pulposus from 11 spines were graded by Thompson's gross morphologic scale. Fibronectin was sequentially extracted with 4 mol/L guanidine hydrochloride and trypsin, and then quantitated by enzyme-linked immunoassay. The size of extractable fibronectin was determined by Western blot analyses. RESULTS: The fibronectin content of the disc increased with grade and was significantly elevated between Grades 3 and 4. The percentage of extractable fibronectin varied widely, but it was more extractable from the nucleus. In both the nucleus and anulus, 30% to 40% of the extractable fibronectin existed as fragments. Many of the fragments contained functional heparin or collagen-binding sites. CONCLUSIONS: Fibronectin is elevated in degenerated discs and frequently present as fragments. Elevated levels of fibronectin suggest that disc cells are responding to the altered environment. Fibronectin fragments resulting from normal or enhanced proteolytic activity could be a mechanism that induces the cell to degrade the matrix further.

Internalization of the hyaluronan receptor CD44 by chondrocytes.

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.

CD44 and beta1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells.

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells lining the peritoneal cavity. The aim of this study was to identify the adhesion molecules involved in the interaction of ovarian carcinoma cells with mesothelial cells. The human ovarian carcinoma cell lines SKOV3 and NIH:OVCAR5 as well as LP9 cells, a human peritoneal mesothelial cell line, were analyzed by flow cytometry for the expression of CD44 and the beta1 integrin subunit. An in vitro adhesion assay was developed whereby LP9 cells were grown as confluent monolayers, and radiolabeled ovarian carcinoma cells were monitored for their ability to adhere to the mesothelial monolayer in the presence of potential inhibitors. Each cell line was evaluated for the presence of a pericellular matrix by a particle exclusion assay. A monoclonal antibody (MAb) against the beta1 integrin subunit significantly reduced the adhesion of SKOV3 cells to LP9 cells, whereas NIH:OVCAR5 adhesion to LP9 cells was significantly inhibited by a CD44 MAb. The LP9 cells produced both hyaluronic acid (a ligand for CD44) as well as several extracellular matrix molecules (ligands for the beta1 integrin heterodimers). These results suggest that both CD44 and the beta1 integrin heterodimers may play a role in mediating the adhesion of ovarian carcinoma cells to mesothelial cells.

Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis.

The disappearance of notochordal cells is correlated with early degenerative changes in the intervertebral disc. With increased disc degeneration there is a marked decrease in proteoglycan synthesis, resulting in loss of mechanical function. One possible mechanism for the decrease in proteoglycan synthesis is the loss of notochordal cells from the tissue. In this study, nucleus pulposus cells cocultured with notochordal cells exhibit an increase in proteoglycan synthesis. Interestingly, purified notochordal cells synthesize little proteoglycan as observed by [35S]sulfate incorporation into proteoglycans. The observed increase in proteoglycan synthesis does not appear to be dependent on cell-cell contact; rather it is the result of soluble factor(s) produced by notochordal cells. Finally, no difference in chondroitin sulfate chain size in notochordal-stimulated nucleus pulposus cells was observed which is consistent with an up-regulation in aggrecan core protein synthesis. These results are consistent with canine breeds where notochordal cells persist into adult age and disc degeneration is not observed. This suggests notochordal cells play a vital role in maintaining disc integrity.

CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis.

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.