Yasutaka Yutani

Local Application of Basic Fibroblast Growth Factor Minipellet Induces the Healing of Segmental Bony Defects in Rabbits

Abstract. Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 μg or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 μg of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice.

Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/Colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9

Previously, we showed that gene expression of the rheumatoid arthritis‐related antigen RA‐A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFα. Associated with this phenomenon, RA‐A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by downregulation of RA‐A47 expression with ra‐a47‐specific anti‐sense oligonucleotide. Here, we show that the enhanced surface expression of RA‐A47 on a chondrocytic cell line, HCS‐2/8 was also a direct result of RA‐A47 downregulation by ra‐a47 anti‐sense oligonucleotide, independent of the cytokine effects. Moreover, cell‐surface expression of CD9, a β1 integrin‐associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra‐a47 anti‐sense oligonucleotide‐treated cells. The CD9 was colocalized with RA‐A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin‐V binding to the cell surface and the level of a number of apoptosis‐related genes including caspase‐9 were increased after ra‐a47 anti‐sense oligonucleotide treatment, suggesting that enhanced surface expression of RA‐A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metallothionein‐III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti‐sense treatment, but not by TNFα. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFα. The surface‐exposed RA‐A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheumatoid arthritis.

CCN family member 2/connective tissue growth factor (CCN2/CTGF) has anti-aging effects that protect articular cartilage from age-related degenerative changes.

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.

Measurements of ζ potentials of particulate biomaterials in protein-rich hyaluronan solution with changes in pH and protein constituents

This study was undertaken to determine the zeta potentials of particulate biomaterials in three types of protein-rich hyaluronan solution with changes in pH; a microelectrophoretic method was used. For the purpose of determining the pH value of synovial fluid in various inflammatory conditions, we collected synovial fluid samples from joints with osteoarthritis (OA), rheumatoid arthritis (RA), and those undergoing revisions arthroplasties. The mean values of the pH in the synovial fluid from joints with OA, RA, and revision arthroplasty were shown to be 7.9, 7.5, and 8.1, respectively. The pH-zeta potential curves obtained differed, depending on the biomaterial and the medium. Addition of gamma-globulin to the medium reduced the absolute value of the zeta potentials of some of the biomaterials. The findings of this study suggest that the electrophoretic behaviors of the particulate biomaterials tested in this study are affected by the protein constituents of and pH changes in protein-rich synovial fluid. The values we obtained will be useful as reference standards and will also aid in the study of the surface phenomena of biomaterials.

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Abstract Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes.

We previously isolated a rheumatoid arthritis‐related antigen (RA‐A47) protein that had reactivity with RA sera from a human chondrosarcoma‐derived chondrocytic cell line, HCS‐2/8. Sequencing analysis of ra‐a47 cDNA revealed RA‐A47 as a product of the colligin‐2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra‐a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) β in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) α, interferon (IFN) β, and interleukin (IL)‐6 downregulated the expression of ra‐a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS‐2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)‐9 mRNAs were strongly stimulated by TNFα. We also found that cell‐surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA‐A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA‐A47 was detected in the cultured medium of TNFα‐treated HCS‐2/8 cells and of IL‐1‐treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA‐A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA‐A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFα. The altered localization of RA‐A47 to the surface or outside of cells may represent the mechanism for the recognition of RA‐A47 as an autoantigen during rheumatoid arthritis. J. Cell. Physiol. 186:268–281, 2001.

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

Abstract We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47 kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Downregulation of a Rheumatoid Arthritis-Related Antigen (RA-A47) by ra-a47 Antisense Oligonucleotides Induces Inflammatory Factors in Chondrocytes

Previously we have shown that the expression of RA‐A47 (rheumatoid arthritis‐related antigen) which is identical to HSP47, a collagen‐binding chaperon, is downregulated in chondrocytes by tumor necrosis factor α (TNFα). RA‐A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFα. To understand the significance of RA‐A47 downregulation in chondrocytes independent from other effects of TNFα, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS‐2/8 by ra‐a47 antisense S‐oligonucleotides significantly reduced the expression of ra‐a47 both at mRNA and protein level. Interestingly, this TNFα‐independent RA‐A47 downregulation was associated with a strong induction of matrix metalloproteinase (MMP)‐9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active‐type MMP‐9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS‐2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFα upregulation, since the ra‐a47 antisense oligonucleotide treatment did not enhance TNFα synthesis. These observations indicate that downregulation of RA‐A47 induces TNFα‐independent cartilage‐degrading pathways involving iNOS and MMP‐9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells. J. Cell. Physiol. 197: 94–102, 2003© 2003 Wiley‐Liss, Inc.

Effects of tumor necrosis factor α on proliferation and expression of differentiated phenotypes in rabbit costal chondrocytes in culture

SummaryTumor necrosis factor α (TNFα) decreased the synthesis of glycosaminoglycan (GAG) in rabbit costal chondrocytes in culture, but did not stimulate the release of GAG from cell layers. Like chondrocytes cultured in control medium, chondrocytes cultured in the presence of TNFα produced putative “cartilage-specific” proteoglycans identified by density gradient centrifugation under dissociative conditions. Although TNFα decreased the synthesis of the proteoglycans, it did not change their monomeric size, which is a marker of cartilage phenotypes. Moreover, TNFα did not affect the responsiveness to parathyroid hormone, insulin-like growth factor I, or transforming growth factor β, which is known to stimulate GAG synthesis in cultured chondrocytes. TNFα decreased the alkaline phosphatase activity in the chondrocytes dose dependently. On the other hand, it stimulated their DNA synthesis slightly, but significantly. The stimulatory effect of TNFα on DNA synthesis was potentiated by fibroblast growth factor, epidermal growth factor, and fetal bovine serum. These findings suggest that in the presence of hormones and growth factors, TNFα promotes the proliferation of chondrocytes while suppressing their further differentiation at the stage of synthesis of cartilage-specific proteoglycans.

Specific serum antibodies against membranous proteins of a human immortal chondrocytic cell line (HCS-2/8) in rheumatoid arthritis and their relationship to the natural history of this disease

An immortal human chondrocytic cell line (HCS-2/8) derived from a chondrosarcoma was used as a source of human antigens to find humoral antibodies to cell surface proteins of human chondrocytes in sera from patients with rheumatoid arthritis (RA). Membrane fractions prepared from the cell line were subjected to Western blot analysis using RA and normal sera as probes. RA sera recognized about a dozen bands, but three of these bands, with molecular weights of 105 kDa, 65 kDa, and 47 kDa, were found to be specific for the RA sera (P<0.05). These bands disappeared following V8 protease digestion, indicating that they were proteins. Among patients with 4 years or more of RA disease activity, reactivity against 105-kDa and 68-kDa proteins was relatively high in those whose joints showed a high degree of erosion. We suspect that levels of these two antibodies are suggestive of changes associated with the natural course of RA.