Heli Salminen

Expression of Sox9 and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoarthritis

OBJECTIVE To determine the capacity of chondrocytes in aging and degenerating articular cartilage to produce major components of the extracellular matrix and maintain the normal structure of articular cartilage in a transgenic mouse model of osteoarthritis. METHODS Transcription factor Sox9 was used as an indicator of the activation and maintenance of the articular chondrocyte phenotype. Knee joints of Del1 mice carrying 6 copies of the pro alpha1(II) collagen transgene with a short deletion mutation were analyzed at the age of 10 days and at 2, 3, 4, 6, 9, and 15 months by Northern hybridization, RNase protection assay, quantitative reverse transcription-polymerase chain reaction, and immunohistochemistry. Nontransgenic littermates were used as controls. RESULTS We demonstrated the presence of Sox9 in articular chondrocytes during development, growth, and aging, with the highest messenger RNA levels during the period of rapid growth. With the appearance of degenerative lesions in articular cartilage, 2 repair processes were observed. Local proliferation and activation of chondrocytes rich in Sox9, surrounded by type IIA procollagen and proteoglycans, was seen in articular cartilage. In contrast, metabolically inactive chondrocytes were observed at the margins of the defects. They were devoid of Sox9 and were surrounded by a proteoglycan-poor matrix. Sometimes, the lesions were filled with repair tissue that contained type III collagen but little proteoglycan or type II collagen. CONCLUSION The results indicate that chondrocytes in mature articular cartilage are capable of inducing the production of Sox9 and type IIA procollagen, which is typical of early chondrogenesis. Degenerative defects in the knee joints of transgenic Del1 mice are associated with local activation of chondrocytes, which probably contributes to the repair process. In other areas, the repair process produces a noncartilaginous matrix, which is insufficient to maintain the integrity of articular cartilage and which allows degeneration to proceed.

Decorin is produced by capillary endothelial cells in inflammation-associated angiogenesis.

Decorin is a small extracellular chondroitin/dermatan sulfate proteoglycan that has previously been shown to be involved in the angiogenesis-like behavior of endothelial cells (ECs) in vitro. There is also evidence that decorin plays a role in angiogenesis in vivo. In this study we sought to further explore the involvement of decorin in angiogenesis in vivo, especially in that associated with inflammation. We found by CD31 immunostaining of ECs that in giant cell arteritis there are capillary blood vessels not only in the adventitia as in uninvolved temporal artery wall, but also in the media and the external zone of the thickened intima. Localization of decorin by antiserum LF-30 in adjacent sections showed that in normal temporal artery wall decorin resides mainly in the media and the adventitia, whereas in inflamed temporal artery wall decorin is distributed throughout the vessel wall including the intima. Furthermore, the most intense reaction for decorin was evident in ECs of capillary neovessels within the media and the thickened intima of inflamed temporal artery wall. Decorin was also found in capillary ECs in certain pathological and physiological conditions in which the pivotal role of angiogenesis is more generally accepted. Pyogenic granulomas, granulation tissue of healing dermal wounds, and ovaries at different phases of follicle and corpus luteum formation all contained widely distributed CD31-positive capillaries. Decorin, on the other hand, was found in capillary ECs in pyogenic granulomas and granulation tissue, but not in those in the ovaries. The assessment of the degree of inflammation in the specimens with the presence of CD68-positive macrophages showed that the pyogenic granuloma, granulation tissue, and giant cell arteritis specimens were rich in macrophages around the decorin-positive capillaries. In contrast, the ovarian specimens were populated with fewer macrophages and even they were not located in close vicinity of capillaries negative for decorin. Our results confirm that decorin is involved in angiogenesis in vivo and, particularly, in conditions in which the inflammatory component is dominant.

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.

Type X collagen, a natural component of mouse articular cartilage: Association with growth, aging, and osteoarthritis

OBJECTIVE To perform a systematic study on the production and deposition of type X collagen in developing, aging, and osteoarthritic (OA) mouse articular cartilage. METHODS Immunohistochemistry was employed to define the distribution of type X collagen and Northern analyses to determine the messenger RNA levels as an indicator of the synthetic activity of the protein. RESULTS Type X collagen was observed in the epiphyseal and articular cartilage of mouse knee joints throughout development and growth. Type X collagen deposition in the transitional zone of articular cartilage became evident toward cessation of growth, at the age of 2-3 months. The most intense staining for type X collagen was limited to the tidemark, the border between uncalcified and calcified cartilage. Northern analysis confirmed that the type X collagen gene is also transcribed by articular cartilage chondrocytes. Intense immunostaining was observed in the areas of OA lesions, specifically, at sites of osteophyte formation and surface fibrillation. Type X collagen deposition was also seen in degenerating menisci. CONCLUSION This study demonstrates that type X collagen is a natural component of mouse articular cartilage throughout development, growth, and aging. This finding and the deposition of type X collagen at sites of OA lesions suggest that type X collagen may have a role in providing structural support for articular cartilage.

Up-regulation of cartilage oligomeric matrix protein at the onset of articular cartilage degeneration in a transgenic mouse model of osteoarthritis

OBJECTIVE To investigate the suitability of cartilage oligomeric matrix protein (COMP) as a marker for articular cartilage degeneration in a transgenic mouse model of osteoarthritis (OA). METHODS Northern blot analysis of total RNA extracted from the knee joints of transgenic Del1 mice, which harbor a short deletion in a type II collagen transgene, and of their nontransgenic littermates was used to monitor changes in COMP messenger RNA (mRNA) levels during cartilage degeneration. Immunohistochemistry was used to determine the distribution of COMP in articular cartilage, and serum levels of COMP were measured by immunoassay. RESULTS Transient up-regulation of COMP mRNA was seen in articular cartilage of transgenic Del1 mice at the onset of OA lesions at the age of 3 months. Compared with nontransgenic controls, COMP immunostaining of articular cartilage in 3-9-month-old transgenic mice was increased, especially at the border of uncalcified and calcified cartilage. There was also a change from predominantly interterritorial to pericellular/territorial deposition of COMP. This difference persisted until the age of 15 months, when the nontransgenic controls also demonstrated articular cartilage degeneration and increased COMP immunostaining. Increased serum levels of COMP were seen in Del1 mice at the age of 4 months, correlating temporally with the onset of cartilage degeneration. CONCLUSION These findings suggest that upregulation of COMP mRNA and redistribution of the protein are characteristic of the early stages of articular cartilage degeneration in the transgenic mouse model in which OA results from a dominant-negative mutation in the type II collagen gene. The data provide additional support for the notion that COMP is a useful marker for altered cartilage metabolism in developing OA.

Chicken B-Cell-Activating Factor: Regulator of B-Cell Survival in the Bursa of Fabricius

Previous studies on mammals have demonstrated that a tumour necrosis factor family member, B‐cell‐activating factor (BAFF) (BlyS, TALL‐1), is mainly produced by myeloid and dendritic cells and that BAFF promotes B‐cell differentiation and survival in a paracrine fashion. We have recently shown that BAFF is upregulated at the bursal stage of the avian B‐cell development. We now show that the avian bursal B cells and B‐cell lines, RP‐9, RP‐13 and DT40, express chicken BAFF (cBAFF). In situ hybridization confirms strong cBAFF expression within the bursal follicles. Like mammals, cBAFF is expressed in the avian myeloblast and myelomonocytic cell lines but not in the peripheral blood αβ and γδ T cells. The binding of recombinant human BAFF (hBAFF) to the bursal B‐cells indicates a conserved receptor–ligand binding. Furthermore, the recombinant hBAFF has a positive effect on bursal cell proliferation and transiently inhibits cell death in vitro. In conclusion, cBAFF is highly conserved structurally, but as a novel observation we suggest cBAFF to function in an autocrine fashion to promote the growth and maturation of follicular B cells in bursa of Fabricius.

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton.

Abstract. Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.

Expression of Sox9 and type IIA procollagen during ocular development and aging in transgenic Del1 mice with a mutation in the type II collagen gene.

Purpose To study the expression and distribution of transcription factor Sox9 and type IIA procollagen in the developing and aging eyes of normal and transgenic Del1 mice carrying proa1(II) collagen transgenes with a short deletion mutation, which cause ocular abnormalities in this mouse line. Methods The eyes of Del1 mice were studied on embryonic days E14.5, E16.5 and E18.5, and at the ages of 4 and nine months, using their nontransgenic littermates as controls. Sox9 and proa1(IIA) collagen were detected by RNase protection assay and immunohistochemistry. Results RNase protection assay revealed Sox9 transcripts in the eyes of Del1 and control mice during development and aging. The mRNA for type IIA procollagen had a similar temporal expression pattern. On embryonic days E14.5, E16.5 and E18.5, Sox9 was located by immunohistochemistry in the nuclei and type IIA procollagen in the extracellular space of the developing retina. During growth and aging, the ocular expression of Sox9 mRNA and the immunohistochemical reaction for Sox9 antibody diminished, concomitant with the reduction in type II procollagen mRNA. However, at the age of nine months, levels of Sox9 and type IIA procollagen mRNAs were higher in the degenerating eyes of Del1 and control mice. Conclusions The similarities in the temporo-spatial distribution of Sox9 and type IIA procollagen suggest that this transcription factor is involved in the activation of type II collagen expression in the eye, as has been demonstrated in prechondrogenic mesenchyme and immature cartilage. The increased production of Sox9 and type IIA procollagen in the aging retina and vitreous is analogous to degenerating articular cartilage where attempted tissue repair has also been observed.

Ultrastructural characterization of developmental and degenerative vitreo-retinal changes in the eyes of transgenic mice with a deletion mutation in type II collagen gene.

Purpose. Molecular genetic analyses have clearly associated vitreoretinal degeneration with mutations in the type II collagen gene, but lack of experimental models has prevented systematic analyses of the occurrence of phenotypic changes and of the pathogenetic mechanisms involved. The present study is a detailed morphological and ultrastructural analysis of the vitreoretinal consequences of a small deletion mutation in the type II collagen gene. Methods. The eyes of Del1 mice carrying six copies of proa1(II) collagen transgene with a small deletion mutation were analyzed during embryonic development, postnatal growth and aging using their nontransgenic littermates as controls. Tissue samples were processed for light and electron microscopy for morphological and ultrastructural analyses. Transcription of proa1(II) collagen gene was localized by in situ hybridization, and type II collagen was detected by immunohistochemistry. Results. In this mouse model most components of the eye are ultrastructurally unaltered. However, the transgenes caused a dose-dependent dominant negative effect seen as a reduced number of type II collagen fibrils in the vitreous. In concert with this, dose-dependent accumulation of amorphous material was observed in the dilated rough endoplasmic reticulum of cells responsible for the production of type II collagen molecules. In mice homozygous for the transgene locus, the vitreoretinal degenerative lesions appeared already during late embryonic development. In mice heterozygous for the locus, such changes were milder and appeared only during postnatal growth and progressed gradually upon aging. Conclusions. The observed ultrastructural changes suggest that defective structure and function of collagen fibrils in Del1 mice result from a partial block in the post-translational processing and secretion of the mutated procollagen chains, and partly from secretion of mutated procollagen molecules which interfere with normal fibrillogenesis.