Development and characterization of a human articular cartilage-derived chondrocyte cell line that retains chondrocyte phenotype.

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage-derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3-dimensional matrix within 48 h post-infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3-dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL-1 or BMP-7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra-cellular matrix molecules. In addition, one clone (AL-4-17) showed similar responses as primary chondrocytes when treated with IL-1 or BMP-7. In summary, this report provides a novel procedure to develop human articular cartilage-derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology.

Synthesis and biological evaluation of ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides: a class of potent aggrecanase-1 inhibitors.

The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.

Immortalized mouse articular cartilage cell lines retain chondrocyte phenotype and respond to both anabolic factor BMP-2 and pro-inflammatory factor IL-1.

Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM-Sv/HP and MM-Sv/HP-2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q-PCR) were performed on cells in monolayer culture and cells embedded in a three-dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP-2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro-inflammatory IL-1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP-13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP-2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL-1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase-cleaved aggrecan fragments. In summary, MM-Sv/HP and MM-Sv/HP-2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology.

Involvement of protein kinase Czeta in interleukin-1beta induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-kappaB signaling in primary human osteoarthritic chondrocytes.

OBJECTIVE: Protein kinase Czeta (PKCzeta), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKCzeta in interleukin-1beta (IL-1beta)-induced NF-kappaB signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. METHODS: Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKCzeta. Western blot analysis was used to evaluate phosphorylation of PKCzeta and NF-kappaB. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. RESULTS: Phosphorylation of PKCzeta and NF-kappaB was induced by IL-1beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCzeta suppressed IL-1beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCzeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKCzeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. CONCLUSION: Our results show that PKCzeta is involved in the regulation of IL-1beta-induced NF-kappaB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCzeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.

Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis.

OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Immortalized cell lines from mouse xiphisternum preserve chondrocyte phenotype.

Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA.

Protein kinase Czeta is up-regulated in osteoarthritic cartilage and is required for activation of NF-kappaB by tumor necrosis factor and interleukin-1 in articular chondrocytes.

Protein kinase Czeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCzeta is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCzeta may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCzeta activity in NF-kappaB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-alpha caused an increase in NF-kappaB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCzeta-sparing inhibitor calphostin C. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Synthesis and biological evaluation of biphenylsulfonamide carboxylate aggrecanase-1 inhibitors.

Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of proteoglycan degradation at 10 microg/mL and has an oral bioavailability in rat of 35%.

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression.

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN-gamma and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-gamma. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN-gamma and IL-10, and down-regulation of TNF-alpha production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.

Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis.

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice.

OBJECTIVE: To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). METHODS: We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. RESULTS: ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. CONCLUSION: Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

Characterization and functionality of cell surface molecules on human mesenchymal stem cells.

We have characterized adhesion molecules on the surface of multipotential human mesenchymal stem cells (hMSCs) and identified molecules whose ligands are present on mature hematopoietic cells. Flow cytometric analysis of hMSCs identified the expression of integrins: alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta4, in addition to ICAM-1, ICAM-2, VCAM-1, CD72, and LFA-3. Exposure of hMSCs to IL-1alpha, TNFalpha or IFNgamma up-modulated ICAM-1 surface expression, whereas only IFNgamma increased both HLA-class I and -class II molecules on the cell surface. Whole cell-binding assays between the hMSCs and hematopoietic cell lines showed that T lymphocytic lines bound hMSCs with higher affinity than lines of either B lymphocytes or those of myeloid lineage. Experiments using autologous T lymphocytes isolated from peripheral blood mononuclear cells showed that hMSCs exhibited increased affinity for activated T-lymphocytes compared to resting T cells by quantitative whole cell binding and rosetting assays. Flow cytometric analysis of rosetted cells demonstrated that both CD4+ and CD8+ cells bound to hMSCs. To determine the functional significance of these findings, we tested the ability of hMSCs to present antigen to T lymphocytes. hMSCs pulsed with tetanus toxoid stimulated proliferation and cytokine production (IL-4, IL-10, and IFNgamma) in a tetanus-toxoid-specific T cell line. Maximal cytokine production correlated with maximal antigen-dependent proliferation. These data demonstrate physiological outcome as a consequence of interactions between hMSCs and human hematopoietic lineage cells, suggesting a role for hMSCs in vivo to influence both hematopoietic and immune function(s).

Role of apoptosis inhibition in various chondrocyte culture systems.

Apoptosis may limit the utility of cell-based therapies for articular cartilage disorders. We tested the hypothesis that chondrocyte apoptosis can be reduced by optimizing the conditions employed to expand chondrocyte numbers in culture. Chondrocyte apoptosis was examined in monolayer and suspension culture, in the presence of fetal bovine serum (FBS) or autologous serum, and for culture periods of 2 or 4 days. The effect of these variables was assessed by measuring cell viability, Annexin V labeling and mitochondrial membrane potential. After 2 days of culture, the greatest increase in viable cell number (3.7-fold) occurred in monolayer cultures with autologous serum. After 4 days of culture, the greatest increase in cell number (9.0-fold) occurred in monolayer cultures supplemented with FBS. By Annexin V staining, the proportion of cells undergoing apoptosis after 2 days was not affected by the type of serum used or by culture in monolayer versus suspension. After 4 days of culture, the proportion of apoptotic cells was significantly reduced (35% to 13%, p<0.02) in suspension cultures with autologous serum. Apoptosis assessed by loss of mitochondrial membrane potential was decreased in the presence of autologous serum. These data suggest that suspension culture with autologous serum is useful in simultaneously maintaining cell proliferation and minimizing apoptosis in cultured human articular chondrocytes.