| 1. |
- Bart, Genevieve, et al.
(författare)
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Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:18, s. 11479-11490
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Tidskriftsartikel (refereegranskat)abstract
- In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.
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| 2. |
- Inkinen, Ritva, et al.
(författare)
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Hyaluronan distribution in the human and canine intervertebral disc and cartilage endplate.
- 1999
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Ingår i: The Histochemical Journal. - 0018-2214 .- 1573-6865. ; 31:9, s. 579-587
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Tidskriftsartikel (refereegranskat)abstract
- A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis. Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.
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| 3. |
- Parkkinen, Jyrki, et al.
(författare)
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Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan.
- 1996
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Ingår i: Histochemistry and Cell Biology. - : Springer. - 0948-6143 .- 1432-119X. ; 105:3, s. 187-194
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Tidskriftsartikel (refereegranskat)abstract
- The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 microns-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47-51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8-15% of total proteoglycans. Analysis of the extract revealed that 24-50% of its hyaluronan was directly detectable with the probe, while 50-76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-micrograms-thick tissue sections.
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| 4. |
- Pasonen-Seppänen, Sanna, et al.
(författare)
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EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.
- 2003
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Ingår i: Journal of Investigative Dermatology. - : Nature Publishing Group. - 0022-202X .- 1523-1747. ; 120:6, s. 1038-1044
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.
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| 5. |
- Pienimaki, Juha-Pekka, et al.
(författare)
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Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
- 2001
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Ingår i: Journal of Biological Chemistry. - : American Society of Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 276:23, s. 20428-20435
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately 21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.
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| 6. |
- Qu, Chengjuan, 1967-, et al.
(författare)
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Extensive CD44-dependent hyaluronan coats on human bone marrow-derived mesenchymal stem cells produced by hyaluronan synthases HAS1, HAS2 and HAS3
- 2014
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Ingår i: International Journal of Biochemistry and Cell Biology. - : Elsevier. - 1357-2725 .- 1878-5875. ; 48:3, s. 45-54
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.
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| 7. |
- Rilla, Kirsi, et al.
(författare)
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Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.
- 2002
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Ingår i: Journal of Cell Science. - Cambridge : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 115, s. 3633-3643
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.
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| 8. |
- Rilla, Kirsi, et al.
(författare)
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Hyaluronan synthase-2 (HAS-2) regulates migration of epidermal keratinocytes
- 2002
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Ingår i: Hyaluronan, Vol 1: Chemical, Biochemical and Biological Aspects. - Great Britain : Woodhead Publishing Limited. - 1855735709 ; , s. 557-560
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Konferensbidrag (refereegranskat)abstract
- Hyaluronan (HA) is a linear polysaccharide abundant in the extracellular space between epidermal keratinocytes. It is synthesized at the inner face of the plasma membrane by hyaluronan synthases (Has). We probed the importance of hyaluronan in keratinocytes by establishing cell lines carrying exogenous hyaluronan synthase 2 (Has2) gene(s) in sense and antisense orientations in order to increase and decrease their hyaluronan synthesis, respectively.The cell lines with the sense Has2 cDNA showed increased HA synthesis, while most cell lines with Has2 antisense cDNA contained less HA. Has2 antisense cells differed from control cell lines; they spread at a slower rate and retained a rounded morphology for a longer time. Further, during the first 24 hours after plating, proliferation was delayed in the antisense cell lines. In an in vitro wounding assay the antisense cells showed a significantly decreased migration rate as compared to controls. Cell lines with the Has2 sense cDNA were similar to the control cell lines in spreading and proliferation rates. However, they migrated faster than control cell lines.
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| 9. |
- Tammi, Markku, et al.
(författare)
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EGF regulates HAS-2 expression, controls epidermal thickness and stimulates keratinocyte migration
- 2002
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Ingår i: Hyaluronan, Vol 1: Chemical, Biochemical and Biological Aspects. - Great Britain : Woodhead Publishing Limited. - 1855735709 ; , s. 561-570
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Konferensbidrag (refereegranskat)abstract
- High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.
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| 10. |
- Törrönen, Kari, et al.
(författare)
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Hyaluronan stimulates keratinocyte migration and activates the transcription factor AP-1 in keratinocytes through the JNK pathway
- 2002
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Ingår i: Hyaluronan, Vol 1: Chemical, Biochemical and Biological Aspects. - Great Britain : Woodhead Publishing Limited. - 1855735709 ; , s. 551-556
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Konferensbidrag (refereegranskat)abstract
- Hyaluronan (HA) has been considered a passive extracellular matrix (ECM) polysaccharide, but recent studies have shown its importance in controlling many cell functions including motility, proliferation and adhesion, which imply signaling from ECM to cytosol. Hyaluronan is a major ECM component in stratified epithelia such as skin epidermis. We found that hyaluronan added to the growth medium of newly plated human skin keratinocytes increased cell migration in an in vitro wound-healing assay. Hyaluronan also increased the transcription factor AP-1, as determined by gel shift assays. The kinase signals that apperently led to the increased AP-1 level were associated with the activation of c-Jun, mainly via the JNK pathway as early as 10 min after the addition of hyaluronan, and with the minimum concentration of 10 ng/ml. ERK1 was also slightly activated, while p38 MAPkinase was not affected.
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| 11. |
- Arokoski, Jari, et al.
(författare)
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Nivelrikon etiopatogeneesi [Etiopathogenesis of osteoarthritis].
- 2001
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Ingår i: Duodecim. - : Duodecim. - 0012-7183 .- 2242-3281. ; 117:16, s. 1617-1626
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Tidskriftsartikel (refereegranskat)abstract
- Nivelrikon patofysiologia tunnetaan huonosti. Nykykäsityksen mukaan artroosissa ei olekyse nivelruston passiivisesta kulumisesta vaan biokemiallisesta tapahtumasarjasta, jossasoluväliaineen tuhoutuminen saa ylivallan rustoa korjaavista prosesseista. Nivelrikon alkuvaiheessarustosoluissa eli kondrosyyteissä aktivoituvat sekä ruston aineosien synteesitoimintaettä rustoa hajottavien entsyymien ilmentyminen ja niitä koodaavien geenientoiminta. Nivelrikko on koko nivelen sairaus, joka aiheuttaa muutoksia niin nivelrustossa,luussa kuin pehmytosissakin. Vallitsevan käsityksen mukaan nivelrikko käynnistyynivelruston pinnallisesta vyöhykkeestä. On myös esitetty, että nivelalueen altistuminenliialliselle kuormitukselle aiheuttaisi ensin rustonalaisen luun paksunemisen ja jäykkenemisen,mikä puolestaan altistaisi nivelruston suuremmille kuormittaville voimille. Riskitekijöistätärkeimpiä ovat ikääntyminen, liikapaino, niveleen kohdistuvat vammat ja ruumiillisentyön aiheuttama liikarasitus. Perinnöllisten tekijöiden osuus on myös merkittävä.Ruston kollageenien rakennevirheiden tiedetään altistavan nivelrikolle.
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| 12. |
- Haapala, Jussi, et al.
(författare)
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Coordinated regulation of hyaluronan and aggrecan content in the articular cartilage of immobilized and exercised dogs.
- 1996
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Ingår i: Journal of Rheumatology. - : Journal of Rheumatology. - 0315-162X .- 1499-2752. ; 23:9, s. 1586-1593
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the influence of joint loading and immobilization on articular cartilage hyaluronan concentration and histological distribution in the knee joints of young dogs subjected to 11 weeks' immobilization by splinting, and 15 weeks' running exercise at a rate of 40 km/day.METHODS: The amount of hyaluronan in articular cartilage was determined by a competitive binding assay using a biotinylated hyaluronan binding complex (HABC) of aggrecan and link protein. Histologic sections were stained for the localization of hyaluronan with the HABC probe. Extracted proteoglycans were characterized by sodium dodecyl sulfate agarose gel electrophoresis.RESULTS: Immobilization significantly reduced the concentration of hyaluronan in all sites studied (tibial and femoral condyles, patellar surface of femur). The proportion of hyaluronan to total uronic acid (mainly from aggrecan) remained unchanged because of a concurrent decrease in aggrecan. The ratio of hyaluronan and aggrecan remained constant also in runners. The staining pattern of free hyaluronan in the tissue sections and the electrophoretic mobility of the extracted proteoglycans were not affected by the different loading regimes.CONCLUSION: Reduced joint loading due to splint immobilization significantly decreases both hyaluronan and aggrecan in the articular cartilage. The remarkably parallel changes in aggrecan and hyaluronan content suggest that joint loading exerts a coordinated influence on their metabolism.
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| 13. |
- Haapala, Jussi, et al.
(författare)
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Remobilization does not fully restore immobilization induced articular cartilage atrophy.
- 1999
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Ingår i: Clinical Orthopaedics and Related Research. - : Lippincott Williams & Wilkins. - 0009-921X .- 1528-1132. ; :362, s. 218-229
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Tidskriftsartikel (refereegranskat)abstract
- The recovery of articular cartilage from immobilization induced atrophy was studied. The right hind limbs of 29-week-old beagle dogs were immobilized for 11 weeks and then remobilized for 50 weeks. Cartilage from the immobilized knee was compared with tissue from age matched control animals. After the immobilization period, uncalcified articular cartilage glycosaminoglycan concentration was reduced by 20% to 23%, the reduction being largest (44%) in the superficial zone. The collagen fibril network showed no significant changes, but the amount of collagen crosslinks was reduced (13.5%) during immobilization. After remobilization, glycosaminoglycan concentration was restored at most sites, except for in the upper parts of uncalcified cartilage in the medial femoral and tibial condyles (9% to 17% less glycosaminoglycans than in controls). The incorporation of 35SO4 was not changed, and remobilization also did not alter the birefringence of collagen fibrils. Remobilization restored the proportion of collagen crosslinks to the control level. The changes induced by joint unloading were reversible at most sites investigated, but full restoration of articular cartilage glycosaminoglycan concentration was not obtained in all sites, even after remobilization for 50 weeks. This suggests that lengthy immobilization of a joint can cause long lasting articular cartilage proteoglycan alterations at the same time as collagen organization remains largely unchanged. Because proteoglycans exert strong influence on the biomechanical properties of cartilage, lengthy immobilization may jeopardize the well being of articular cartilage.
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| 14. |
- Helminen, Heikki, et al.
(författare)
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Kuormituksen vaikutus nivelrustoon [The effects of loading on articular cartilage].
- 1992
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Ingår i: Duodecim. - : Duodecim. - 0012-7183 .- 2242-3281. ; 108:12, s. 1097-1107
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Forskningsöversikt (refereegranskat)abstract
- Nivelen kuormitus on tärkeimpiä nivelruston aineenvaihduntaan ja rakenteeseen vaikuttavia fysiologisia tekijöitä. Kohtuullinen rytminen kuormitus lisää nuoren ihmisen nivelruston proteoglykaanipitoisuutta. Tämän vaikutuksesta rusto jäykistyy ja kasvaa paksuutta. Hyvin voimakas kuormitus ei aiheuta tällaista positiivista vastetta. Toisaalta nivelkuormituksen puuttuminen pienentää ruston proteoglykaanipitoisuutta ja heikentää kimmo-ominaisuuksia. Nämä surkastumismuutokset ovat suurimmaksi osaksi–elleivät kokonaan–korjautuvia. Kohtuullisella nivelkuormituksella voidaan siis ylläpitää ja parantaa nivelruston ominaisuuksia. Pitkäaikaisen liikkumattomuuden jälkeen nivelrusto on heikompi kuin normaalisti ja voi vaurioitua niveltä voimakkaasti kuormitettaessa. Siksi nivelen kuormitusta pitää lisätä toipumisvaiheessa vähitellen.
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| 15. |
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| 16. |
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| 17. |
- Hyttinen, Mika, et al.
(författare)
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Age matters : collagen birefringence of superficial articular cartilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading.
- 2001
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Ingår i: Osteoarthritis and Cartilage. - : Saunders Elsevier. - 1063-4584 .- 1522-9653. ; 9:8, s. 694-701
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To compare responses of the collagen network and glycosaminoglycans (GAGs) of articular cartilage to physiological type of joint loading in young growing and adult mature guinea-pigs.DESIGN: 10- and 44-week-old guinea-pigs were accustomed to treadmill running for 3 weeks. Thereafter the animals ran 2500 m/day, 5 days a week, for 15 weeks. Articular cartilage specimens from knee joints were collected at 28 and 62 weeks. Osteoarthritis (OA) prevalence and severity was evaluated by aid of light microscopy. The degree of collagen fibril network organization and content was analyzed with quantitative polarized light microscopy. The local concentration of GAGs was determined from cartilage sections with digital densitometry after safranin-O staining.RESULTS: In the young guinea-pigs, running increased up to 24% the optical retardation of polarized light by collagen in the superficial articular cartilage of femur, indicating either a higher degree of fibril assembly and organization or increased amount of collagen, or both. In contrast, in the adult mature animals the optical retardation decreased almost 50% after joint loading (P< 0.01-0.001). Running did not increase cartilage fibrillation. Significant changes in GAG content of cartilage were not found either in the young or adult mature runners.CONCLUSIONS: Increased birefringence of the superficial articular cartilage after joint loading in young guinea-pigs can be interpreted to be a sign of improved and decreased birefringence in older animals a sign of worsened property of the collagen network. It can be suggested therefore that joint loading strengthened the collagen network in the young runners. It can be hypothesized further that with time the inferior property of the collagen network predisposes the older runners to earlier OA than in controls.
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| 18. |
- Inkinen, Ritva, et al.
(författare)
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Relative increase of biglycan and decorin and altered chondroitin sulfate epitopes in the degenerating human intervertebral disc.
- 1998
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Ingår i: Journal of Rheumatology. - 0315-162X .- 1499-2752. ; 25:3, s. 506-514
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Proteoglycans are major components of the extracellular matrix of the intervertebral disc. They are vital for the biomechanical properties of the tissue, and are subject to changes in disc degeneration. We aimed to further define these changes and their relationship to normal aging.METHODS: Normal discs (age 13-53 years, n = 6) were analyzed from 5 different sites across the sagittal anterior-posterior direction. Degenerated anterior annulus fibrosus was collected from 7 patients aged 39-46 years. Extracted proteoglycans were separated using agarose and polyacrylamide gel electrophoresis and detected with toluidine blue staining and Western blotting.RESULTS: The center of the disc showed the highest level of total proteoglycans, but lowest levels of decorin and biglycan. Western blots displayed reduced signal for both glycanated and nonglycanated biglycan and decorin after adolescence, while an increased signal of biglycan was observed in degenerated annuli. The 7D4(-) and 3B3(-) epitopes on native chondroitin sulfate chains were present in the large proteoglycans of intervertebral discs, but their signal intensity had no correlation to degeneration. Chondroitinase ABC digestion of the blots brought up 7D4(+) signal in the small proteoglycans of degenerated, but not in healthy tissue. Decrease or total loss of 2B6(+) epitope (indicating 4-sulfated stubs of chondroitin sulfate chains) were found in the large proteoglycans of all degenerated annuli.CONCLUSION: Human intervertebral disc degeneration involves the accumulation of decorin and biglycan relative to other uronic acid containing proteoglycans, the disappearance of 4-sulfated core region in aggrecan-like large proteoglycans, and the emergence of a core structure in the chains of small proteoglycans reacting with the 7D4 antibody; these findings indicate a fundamental alteration in matrix properties that may contribute to the pathogenesis of the disease.
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| 19. |
- Jortikka, Matti, et al.
(författare)
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A high sensitivity dot-blot assay for proteoglycans by cuprolinic blue precipitation.
- 1993
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Ingår i: Connective Tissue Research. - : Informa Healthcare. - 0300-8207 .- 1607-8438. ; 29:4, s. 263-272
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Tidskriftsartikel (refereegranskat)abstract
- A highly sensitive blot-assay was developed for glycosaminoglycans (GAGs) and proteoglycans (PGs) utilizing a precipitation reaction by a cationic dye Cuprolinic Blue. The precipitates were deposited into 1-2 mm2 spots on nitrocellulose membrane by using a 96-well filtration apparatus. The dried sheet was digitized by a flat bed scanner and the intensity of the dots was quantitated by an image analysis software. The working range for chondroitin sulfate was 10-300 ng. The response of various GAGs differed according to the number of anionic groups, both sulphate and carboxyl groups being able to bind the dye. The sensitivity of the assay was decreased by high concentrations of GuC, CsC and protein, but not by nonionic detergents, common buffers and 8 M urea. Contact exposure to autoradiography film enabled quantitation of 25-250 DPM, and 1-10 DPM, of 35SO4-radioactivity in precipitated PGs after overnight and 14 days' exposures, respectively.
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| 20. |
- Jortikka, Matti, et al.
(författare)
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Immobilisation causes longlasting matrix changes both in the immobilised and contralateral joint cartilage.
- 1997
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 56:4, s. 255-261
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The capacity of articular cartilage matrix to recover during 50 weeks of remobilisation after an atrophy caused by 11 weeks of immobilisation of the knee (stifle) joint in 90 degrees flexion starting at the age of 29 weeks, was studied in young beagle dogs.METHODS: Proteoglycan concentration (uronic acid) and synthesis ([35S]sulphate incorporation) were determined in six and three knee joint surface locations, respectively. Proteoglycans extracted from the cartilages were characterised by chemical determinations, gel filtration, and western blotting for chondroitin sulphate epitope 3B3.RESULTS: The proteoglycan concentrations that were reduced in all sample sites immediately after the immobilisation, remained 14-28% lower than controls after 50 weeks of remobilisation in the patella, the summit of medial femoral condyle, and the superior femoropatellar surface. In the contralateral joint, there was a 49% increase of proteoglycans in the inferior femoropatellar surface after remobilisation, while a 34% decrease was simultaneously noticed on the summit of the medial femoral condyle. Total proteoglycan synthesis was not significantly changed after immobilisation or 50 weeks' remobilisation in the treated or contralateral joint, compared with age matched controls. The chondroitin 6- to 4- sulphate ratio was reduced by immobilisation both in the radioactively labelled and the total tissue proteoglycans. In the remobilised joint, this ratio was restored in femur, while in tibia it remained at a level lower than controls. Neither immobilisation nor remobilisation induced epitopes recognised by the monoclonal antibody 3B3 on native (undigested) proteoglycans.CONCLUSION: These results show that the depletion of proteoglycans observed after 11 weeks of immobilisation was not completely restored in certain surface sites after 50 weeks of remobilisation. The significant changes that developed in the contralateral joint during the remobilisation period give further support to the idea that a permanent alteration of matrix metabolism results even from a temporary modification of loading pattern in immature joints.
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| 21. |
- Jortikka, Matti, et al.
(författare)
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The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure.
- 2000
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 374:2, s. 172-180
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Tidskriftsartikel (refereegranskat)abstract
- Chondrocytes of the articular cartilage sense mechanical factors associated with joint loading, such as hydrostatic pressure, and maintain the homeostasis of the extracellular matrix by regulating the metabolism of proteoglycans (PGs) and collagens. Intermittent hydrostatic pressure stimulates, while continuous high hydrostatic pressure inhibits, the biosynthesis of PGs. High continuous hydrostatic pressure also changes the structure of cytoskeleton and Golgi complex in cultured chondrocytes. Using microtubule (MT)-affecting drugs nocodazole and taxol as tools we examined whether MTs are involved in the regulation of PG synthesis in pressurized primary chondrocyte monolayer cultures. Disruption of the microtubular array by nocodazole inhibited [(35)S]sulfate incorporation by 39-48%, while MT stabilization by taxol caused maximally a 17% inhibition. Continuous hydrostatic pressure further decreased the synthesis by 34-42% in nocodazole-treated cultures. This suggests that high pressure exerts its inhibitory effect through mechanisms independent of MTs. On the other hand, nocodazole and taxol both prevented the stimulation of PG synthesis by cyclic 0. 5 Hz, 5 MPa hydrostatic pressure. The drugs did not affect the structural and functional properties of the PGs, and none of the treatments significantly affected cell viability, as indicated by the high level of PG synthesis 24-48 h after the release of drugs and/or high hydrostatic pressure. Our data on two-dimensional chondrocyte cultures indicate that inhibition of PG synthesis by continuous high hydrostatic pressure does not interfere with the MT-dependent vesicle traffic, while the stimulation of synthesis by cyclic pressure does not occur if the dynamic nature of MTs is disturbed by nocodazole. Similar phenomena may operate in cartilage matrix embedded chondrocytes.
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| 22. |
- Karppinen, Jaro, et al.
(författare)
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Effects of tiaprofenic acid and indomethacin on proteoglycans in the degenerating porcine intervertebral disc.
- 1995
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Ingår i: Spine. - : Wolters Kluwer. - 0362-2436 .- 1528-1159. ; 20:10, s. 1170-1177
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Tidskriftsartikel (refereegranskat)abstract
- STUDY DESIGN: Eighteen pigs were stabbed with a scalpel in the anterior part of the anulus fibrosus of a lumbar disc. After surgery, the pigs received either tiaprofenic acid or indomethacin daily, and a third group did not receive any medication.OBJECTIVES: Nonsteroidal anti-inflammatory agents are widely used in the treatment of low back patients, but their long-term effects on the matrix molecules in the degenerate disc are unknown.SUMMARY OF BACKGROUND DATA: Several in vitro and in vivo studies on articular cartilage have suggested that tiaprofenic acid may not have adverse effects on matrix metabolism, whereas indomethacin probably does.METHODS: Uronic acid, DNA, and water contents were determined from five different locations in each injured disc. Transport and incorporation of sulfate were examined by in vivo radioactive tracer analysis, and proteoglycan structures were analyzed by gel electrophoresis.RESULTS: Morphologically, there were no differences between the treatments. Tiaprofenic acid maintained a higher uronic acid content in the nucleus pulposus and outer anulus compared with that of the nonmedicated animals. Tiaprofenic acid decreased the incorporation of sulfate in the injured area and the water content at most sites. Indomethacin had no adverse effects compared with the nonmedicated group, and it increased water content in the posterior anulus fibrosus.CONCLUSIONS: Long-term administration of tiaprofenic acid and indomethacin did not have harmful effects on matrix metabolism after disc injury. On the contrary, tiaprofenic acid may slightly protect proteoglycans in the degenerating disc.
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| 23. |
- Király, Kari, et al.
(författare)
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Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation.
- 1996
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Ingår i: The Histochemical Journal. - : Chapman & Hill. - 0018-2214 .- 1573-6865. ; 28:2, s. 99-107
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Tidskriftsartikel (refereegranskat)abstract
- The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.
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| 24. |
- Kiviranta, Ilkka, et al.
(författare)
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Effects of mechanical loading and immobilization on the articular cartilage
- 1997
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Ingår i: Bailliere's Clinical Orthopaedics. - 1074-8814. ; 2:1, s. 109-122
-
Forskningsöversikt (refereegranskat)abstract
- Articular cartilage provides nearly frictionless surfaces for joint movemants and reduces contact pressures, protecting the underlying suchondral bone from excess stress. The unique properties of articular cartilage are based on the interaction of the main components of the extracellular matrix: proteoglycans (PGs), collagen and interstitial fluid. Animal experiments and in vitro studies demonstrate that one of the most important regulators of the extracellular matrix metabolism is mechanical loading acting on the joints. Unloading and immobilization leads to PG depletion and softening of articular cartilage, increasing the risk of permanent cartilage degeneration. Moderate running exercise and increased weight bearing increases cartilage thickness, PG concentration and improves biomechanical properties of articular cartilage. With further increase in training intensity this positive influence of exercise disappears and cartilage shows changes analogous to immobilization of the joint, i.e. PG depletion and softening of the tissue. In humans most epidemiological studies have failed to prove the connection between running training and cartilage degeneration, but there is evidence that sports activities exposing joints to impact loading might increase the risk of osteoarthrosis.
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| 25. |
- Kääpä, Eeva, et al.
(författare)
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Proteoglycan chemistry in experimentally injured porcine intervertebral disk.
- 1994
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Ingår i: Journal of spinal disorders. - : Lippincott Williams & Wilkins. - 0895-0385 .- 1531-2305. ; 7:4, s. 296-306
-
Tidskriftsartikel (refereegranskat)abstract
- An animal model of disk degeneration was used to study the concentration levels and types of proteoglycans in the different parts of the intervertebral disk. An annular incision was made with a scalpel blade into the anterior part of the porcine lumbar intervertebral disks via a retroperitoneal approach. Three months after injury the morphology of the injured disk had changed considerably. Disk height was diminished, and in the injured segment osteophytes had formed at the ventral edges of the vertebral body. The nucleus was small, fibrous, and yellowish. The annular lesion had healed by formation of granulation tissue, but the lamellar structure was partially destroyed. The concentration of inorganic [35S]sulfate had decreased across the whole disk, reflecting a decrease in the rate of solute transport. The concentration of incorporated [35S]sulfate had also decreased in the injured disks. The DNA concentration in the anterior annulus and in the nucleus had increased, whereas both the concentration of uronic acid and the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate in the nucleus had decreased. Agarose gel electrophoresis combined with chondroitinase B digestion suggested the presence of dermatan sulfate proteoglycans in the injured annulus fibrosus. The morphology and chemical composition of the disks adjacent to the injured one were normal, and only a slight increase in the concentration of incorporated [35S]sulfate was observed in the disks above the injured one.
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| 26. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.
- 1993
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Ingår i: Annals of the Rheumatic Diseases. - : British Medical Journal. - 0003-4967 .- 1468-2060. ; 52:5, s. 369-377
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles.METHODS: Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations.RESULTS: In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals.CONCLUSION: The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.
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| 27. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Autoradiographic quantitation of radiolabeled proteoglycans.
- 1991
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 22:4, s. 301-310
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Tidskriftsartikel (refereegranskat)abstract
- Radiolabeled proteoglycans or glycosaminoglycans were precipitated with the cationic dye safranin O onto a sheet of nitrocellulose filter using a dot-blot apparatus. An autoradiography film was exposed against the nitrocellulose sheet. The developed film and the nitrocellulose sheet were separately digitized in a flat-bed-gray-scale scanner connected to a microcomputer. An image analysis program of the microcomputer was used to quantify the density of the radioactivity dots produced in the film, and the intensity of the dye spots on nitrocellulose. With this procedure, a single sample containing the minimum of about 20 ng uronic acid and 5 dpm of incorporated 35SO4 was quantified for both total glycosaminoglycan content and radioactivity. Unincorporated 35SO4 and low molecular mass radioactivity (e.g. products of glycosaminoglycan degrading enzymes) did not interfere since they were quantitatively washed through the membrane before the assay.
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| 28. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O
- 1988
-
Ingår i: Analytical Biochemistry. - : Academic Press. - 0003-2697 .- 1096-0309. ; 168:2, s. 352-357
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 m urea, 1% Triton X-100, 30% sucrose, 0.02% NaN3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 m guanidinium chloride and 3 m CsCl reduced the sensitivity of the assay to 30–50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.
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| 29. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Effects of long-term running exercise on canine femoral head articular cartilage.
- 1993
-
Ingår i: Agents and actions. Supplements. - : Birkhäuser Verlag. - 0379-0363. ; 39, s. 95-99
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Tidskriftsartikel (refereegranskat)abstract
- After long-term running program (40 km/day) anterior and posterior tissue samples from canine femoral head were labeled ex vivo in the presence of 35S-SO4. Sulfate incorporation rates did not differ between runner and control groups. The statistically significant changes in runners included a decreased uronic acid concentration (p < or = 0.02) and proportion of extractable proteoglycans (p < or = 0.05) as well as increased concentration of tissue uronic acid after 4 M GuCl extraction (p < or = 0.05) in the posterior area. These results support an idea of strengthened cartilage tissue after this kind of motion and load.
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| 30. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.
- 1994
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 304, s. 723-730
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.
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| 31. |
- Lammi, Mikko, 1961-
(författare)
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Influences of in vivo and in vitro loading on the proteoglycan synthesis of articular cartilage chondrocytes
- 1993
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this study, the biosynthesis of proteoglycans (PGs) was examined in articular cartilage of canine hip joint after long-distance running experiment and in bovine chondrocyte cultures during in vitro loading with hydrostatic pressure. In addition, new assays were developed for more sensitive quantitation of glycosaminoglycans (GAGs) and PGs.Anterior (weight-bearing) and posterior (less weight-bearing) areas of the femoral head from young beagles were labeled after long-term, longdistance running exercise. Total sulpahte incorporation rates were determined and distribution of of the incorporated sulphate in the tissue was localized by quantitative autoradiography. Concentration and extractability of the PGs were determined, and PG structures were studied by gel filtration, agarose gel electrophoresis, and chemical determinations. In the less weight-bearing area, the amount of extractable PGs was decreased, simultaneously with an increased concentration of residual GAGs in the tissue after 4M GuCl extraction. In the weight-bearing area, no marked alterations were noticed. The congruency of the femoral head seems to protect the cartilage from untoward alterations that occur in the femoral head condyles subjected to the same running program.The effect of hydrostatic pressure on PG metabolism of chondrocyte cultures was examined during 20 hours' exposure of chondrocytes to 5 and 30 MPa pressures. The continuous 30 MPa pressure reduced total PG synthesis by 37 % as measured by [35S]sulphate incorporation, in contrast to the 5 MPa which had no effect. Continuous 30 MPa hydrostatic pressure also reduced the steady-state mRNA level of aggrecan. The cyclic pressures showed a frequency dependent stimulation (0.5 Hz, + 11 %) or inhibition (0.017 Hz, -17 %). Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75 % SDS-agarose gel electrophoresis and also eluted earlier on Sephacryl S-1000 gel filtration, indicative of larger molecular size. The results demonstrate that high hydostatic pressure can influence the synthesis of PGs in chondrocytes both at the transcriptionl and translational/posttranslational levels.
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| 32. |
- Lin, Chun-Yu, 1976-
(författare)
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The role of hyaluronan and its CD44 receptor in inflammation and cancer
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hyaluronan, an important extra-cellular matrix molecule, was thought to be interstitial connecting glue decades ago. However, recent evidence has revealed that hyaluronan and its binding proteins also play crucial roles in various pathophysiological conditions in humans, including inflammation and infection.Study I focused on dengue virus infection and found that elevated serum hyaluronan levels during early infection phase was an independent predictor for occurrence of warning signs, and thus severe dengue. High circulating levels of the viral non-structural protein 1 (NS1) correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity. Study II reports a negative correlation between the expression of genes encoding hyaluronan synthase HAS2, its natural antisense transcript HAS2-AS, the chromatin modulating factor HMGA2 and transforming growth factor-β (TGFβ), and survival of patients with invasive breast cancer. TGFβ induction of Hmga2, Has2as and Has2 in mouse mammary epithelial cells, and synthesis of hyaluronan were accompanied with activation of Akt and Erk1/2 MAP-kinase signaling and were required for breast cancer cell motility. Importantly, the hyaluronan receptor Cd44, but not Hmmr, was required for TGFβ-mediated epithelial-mesenchymal transition phenotype. Has2as was found to contribute to the maintenance of stem cell factors and breast cancer stemness. Study III explored the physical interaction between the inhibitor of the apoptosis-stimulating protein of p53 (iASPP) and the hyaluronan receptor CD44. The CD44 standard isoform (CD44s), but not the variant isoform, bound to iASPP via the ankyrin-binding domain in CD44s. iASPP was required for hyaluronan-induced CD44-dependent migration and adhesion of fibroblasts. CD44 altered the sub-cellular localization of the iASPP-p53 complex; thus, ablation of CD44 promoted translocation of iASPP from the nucleus to the cytoplasm, resulting in increased formation of a cytoplasmic iASPP-p53 complex in fibroblasts. Overexpression of iASPP decreased the level of intracellular reactive oxygen species, while overexpression of CD44 increased. Knock-down of CD44s, in the presence of p53, led to increased cell growth and cell density of fibroblasts by suppression of p27 and p53.In summary, we investigated the interaction of hyaluronan and its transmembranous receptor, CD44, as well as the modulation of hyaluronan synthesis, in several different pathophysiological conditions.
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| 33. |
- Parkkinen, Jyrki, et al.
(författare)
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A mechanical apparatus with microprcessor controlled stress profile for cyclic compression of cultured articular cartilage explants
- 1989
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Ingår i: Journal of Biomechanics. - : Elsevier. - 0021-9290 .- 1873-2380. ; 22:11-12, s. 1285-1291
-
Tidskriftsartikel (refereegranskat)abstract
- An apparatus was designed for mechanical compression of cultured articular cartilage explants with a cylindrical plain-ended loading head (diameter 2–5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa–12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.
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| 34. |
- Parkkinen, Jyrki, et al.
(författare)
-
Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes.
- 1993
-
Ingår i: Annals of the Rheumatic Diseases. - : British Medical Journal. - 0003-4967 .- 1468-2060. ; 52:3, s. 192-198
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Articular cartilage proteoglycan content is controlled by joint loading. This study aimed to elucidate the role of hydrostatic pressure in this regulation.METHODS: Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure, applied continuously or cyclically at 0.125 or 0.05 Hz. The Golgi apparatus was visualised either by a fluorochrome coupled wheat germ agglutinin or by transmission electron microscopy. Proteoglycan synthesis was studied by the incorporation of sulphur-35 labelled sulphate.RESULTS: After 30 MPa continuous hydrostatic pressure, the Golgi apparatus was observed in a compact form with a concomitant decrease in proteoglycan synthesis. The normal stacked appearance of the Golgi apparatus was no more visible in the electron microscopy preparation of the pressurised chondrocytes. This effect was reversible and was also noticed after 15 MPa continuous load, though to a minor extent. Cyclic pressures (5-30 MPa) caused no apparent change in the Golgi apparatus. The shape of some cells changed to a more retracted form after 30 MPa continuous pressure. Nocodazole, which causes disassembly of the microtubules, blocked the compacting influence of pressurisation on the Golgi apparatus, and reduced proteoglycan synthesis to about half of the control level.CONCLUSIONS: The packing of the Golgi apparatus is dependent on microtubules and may contribute to the inhibition of proteoglycan synthesis observed in articular cartilage subjected to high hydrostatic pressure.
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| 35. |
|
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| 36. |
- Parkkinen, Jyrki, et al.
(författare)
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Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants.
- 1993
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 300:1, s. 458-465
-
Tidskriftsartikel (refereegranskat)abstract
- Primary chondrocyte cell cultures and explants of bovine articular cartilage were subjected to cyclic hydrostatic pressure in a novel computer-controlled pressure chamber designed for this purpose. The cultures were labeled with 5 microCi/ml 35SO4 and simultaneously pressurized with 5 MPa load for 1.5 or 20 h with pressure cycles of 0.0167, 0.05, 0.25, and 0.5 Hz. The chondrocyte cell cultures were also subjected to 0.0082 and 0.0034 Hz cycles. Sulfate incorporation was significantly inhibited in cell cultures subjected to the 0.5, 0.25, or 0.05 Hz cyclic loads for 1.5 h, but stimulated in explant cultures with a 0.5 Hz cyclic 1.5-h load. Chondrocyte cultures subjected to longer (20 h) loading showed a stimulation of sulfate incorporation with 0.5 and 0.25 Hz cycles, but an inhibition with 0.0167 Hz. The results indicate that cyclic hydrostatic pressures of presumably physiological magnitude have significant influences on proteoglycan synthesis in articular cartilage chondrocytes. Comparison of the cell and explant cultures under identical pressure conditions suggested that chondrocyte interactions with extracellular matrix are involved in this regulation by cyclic hydrostatic pressure. The responses of the chondrocytes to pressurization also varied according to the total length of the treatment, a finding compatible with the idea of multiple metabolic steps in chondrocytes, both pre- and post-translational, controlled by the ambient hydrostatic pressure.
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| 37. |
- Parkkinen, Jyrki, et al.
(författare)
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Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.
- 1995
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Ingår i: Journal of Orthopaedic Research. - : John Wiley & Sons. - 0736-0266 .- 1554-527X. ; 13:4, s. 495-502
-
Tidskriftsartikel (refereegranskat)abstract
- The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.
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| 38. |
- Parkkinen, Jyrki, et al.
(författare)
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Local stimulation of proteoglycan synthesis in articular cartilage explants by dynamic compression in vitro.
- 1992
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Ingår i: Journal of Orthopaedic Research. - : John Wiley & Sons. - 0736-0266 .- 1554-527X. ; 10:5, s. 610-620
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Tidskriftsartikel (refereegranskat)abstract
- Cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa compressions repeated at intervals of 2-60 s for 1.5 h and simultaneously labeled with 35SO4. The compression was delivered with a 4-mm-diameter nonporous loading head on an 8-mm-diameter cartilage explant. This method created directly compressed (central) and uncompressed (border) areas within the tissue. Analysis of the whole explant under a 0.5 MPa load showed significantly increased 35SO4 incorporation by compression repeated at 2- and 4-s but not at 20- and 60-s intervals. When the incorporation was studied separately in the border and central areas, a statistically significant stimulation was noticed in the central area with a 4-s cycle, while the border area was stimulated with a 2-s cycle. Autoradiography of the central area showed that the stimulation with 0.5 MPa and a 4-s cycle occurred through the whole depth of the cartilage, while raising the pressure to 1 MPa or the frequency to 2 s reduced the stimulation, particularly in the superficial cartilage. In the border area the stimulation with 0.5 MPa and a 2-s cycle was noted in the superficial zone only. The stimulation of proteoglycan synthesis is thus limited to certain loading frequencies and pressures and occurs in specific areas under and around the loaded site. Its rapid appearance suggests enhanced glycosylation or sulfation of core proteins or enhanced speed of posttranslational processing.
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| 39. |
- Parkkinen, Jyrki, et al.
(författare)
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Polyamine-dependent alterations in the structure of microfilaments, Golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells.
- 1997
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 66:2, s. 165-174
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Tidskriftsartikel (refereegranskat)abstract
- The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a beta-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [3H]glucosamine and [35S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation.
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| 40. |
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| 41. |
- Parkkinen, Jyrki, et al.
(författare)
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Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer.
- 1990
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Ingår i: Histochemistry. - : Springer. - 0301-5564. ; 93:3, s. 241-245
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Tidskriftsartikel (refereegranskat)abstract
- A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.
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| 42. |
- Puustjärvi, Kaija, et al.
(författare)
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Flat bed scanner in the quantitative assay of 35SO4-incorporation by X-ray film autoradiography of intervertebral disc sections.
- 1993
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Ingår i: Histochemistry. - : Springer. - 0301-5564. ; 99:1, s. 67-73
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Tidskriftsartikel (refereegranskat)abstract
- A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of 35SO4 and prepared as histological slides. For comparison, sulphate incorporation rates in the C5-C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of 35SO4-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2 +/- 0.9, 5.2 +/- 0.8, 4.5 +/- 0.6 and 4.1 +/- 0.8 pmol/mm3 per h (+/- SE, n = 10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.
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| 43. |
- Puustjärvi, Kaija, et al.
(författare)
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Proteoglycan synthesis in canine intervertebral discs after long-distance running training.
- 1993
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Ingår i: Journal of Orthopaedic Research. - : John Wiley & Sons. - 0736-0266 .- 1554-527X. ; 11:5, s. 738-746
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Tidskriftsartikel (refereegranskat)abstract
- The alterations and distribution of proteoglycan (PG) synthesis in the intervertebral discs of young dogs exercised with long-distance running (40 km/day) were studied with a method based on image analysis of tissue sections. Ten dogs were run on a treadmill daily for 55 weeks, and 10 dogs from the same litters served as controls. The daily running distance gradually was increased to 40 km and was maintained at that level for the final 15 weeks. Midsagittal disc segments C7-T1, T8-9, and L1-2 were labeled with 35SO4, and histological sections of the segments were apposed against autoradiographic film to determine the synthesis of PGs. Next, the same sections were stained with safranin O to estimate possible alterations in PG concentration. The radiographs and stained sections were digitized with a flatbed scanner and measured by image analysis. The lumbar discs of runners displayed a significantly lower rate of 35SO4 incorporation, while a tendency toward enhanced incorporation was seen in the cervical and thoracic discs. Safranin O staining showed a pattern just opposite to 35SO4 incorporation: decreased staining in the cervical and thoracic discs and increased staining in the lumbar discs of the runners. The results demonstrate qualitatively different influences of long-term running training on PG metabolism at the cervical, thoracic, and lumbar levels in young dogs.
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| 44. |
- Puustjärvi, Kaija, et al.
(författare)
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Proteoglycans in the intervertebral disc of young dogs following strenuous running exercise.
- 1994
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Ingår i: Connective Tissue Research. - : Informa Healthcare. - 0300-8207 .- 1607-8438. ; 30:3, s. 225-240
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Tidskriftsartikel (refereegranskat)abstract
- The proteoglycans (PGs) of intervertebral disc were studied in ten beagles which ran on a treadmill for one year (up to 40 km/day) and in ten non-running control dogs. Nucleus pulposus and annulus fibrosus from cervical (C5) and thoracic (T6 and T12) discs were labeled in vitro with 35SO4. The extractability, concentration and synthesis of PGs, and the electrophoretic subpopulations, aggregation and glycosaminoglycan chain lengths of newly-synthesized and total PGs were measured. Sulfate incorporation was significantly elevated by running in the C5 disc and reduced in the annulus of T6 discs. In the annulus of the T6 discs the concentration of total PGs was significantly lower although that of dermatan sulfate PGs was actually higher than in the controls. The results show that enhanced loading of the spine exerts significant alterations in the intervertebral disc PGs in a spine-level specific manner. In the most strained area of the spine (upper thoracic), the alterations in the runners suggest compromised biomechanical properties of the disc.
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| 45. |
- Sallisalmi, Marko, et al.
(författare)
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Plasma hyaluronan and hemorheology in patients with septic shock : a clinical and experimental study
- 2014
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Ingår i: Clinical hemorheology and microcirculation. - 1386-0291 .- 1875-8622. ; 56:2, s. 133-144
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUNDTotal plasma hyaluronan concentration is increased in septic shock. High-molecular-weight hyaluronan has a high intrinsic viscosity. Excessive release of high-molecular-weight hyaluronan in sepsis may induce hyperviscosity.METHODSPlasma viscosity and the molecular size of plasma hyaluronan were determined in 20 patients with septic shock and in 20 healthy controls. Ex vivo, the effects of 0.4% and 0.047% high-molecular-weight hyaluronan 1560 kDa, 0.9% saline, and 6% hydroxy-ethyl-starch 130 kDa were compared to plasma and whole blood viscosity and red blood cell aggregation at a systemic hematocrit of 0.4, and at a microcirculatory hematocrit of 0.2.RESULTSPlasma viscosity and total plasma protein content were low in septic shock patients on days one and four of treatment. Hyaluronan concentration was 10-fold higher in sepsis on day 1. Molecular weight of hyaluronan was relatively low, mostly 50-500 kDa, and did not change significantly in sepsis. Ex vivo, 0.4% high-molecular-weight hyaluronan 1560 kDa increased blood viscosity but did not promote red blood cell aggregation. Dilutions of 6% hydroxyl-ethyl-starch 130 kDa and 0.047% high-molecular-weight hyaluronan 1560 kDa had comparable effects on blood viscosity and red blood cell aggregation.CONCLUSIONSPlasma viscosity of the septic patients remained low for four days despite markedly elevated concentration of relatively small-molecular-weight hyaluronan.
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| 46. |
- Säämänen, Anna-Marja, et al.
(författare)
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Effect of running exercise on proteoglycans and collagen content in the intervertebral disc of young dogs.
- 1993
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Ingår i: International Journal of Sports Medicine. - : Georg Thieme Verlag KG. - 0172-4622 .- 1439-3964. ; 14:1, s. 48-51
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Tidskriftsartikel (refereegranskat)abstract
- Collagen and proteoglycans in the intervertebral disc (LI-II) of young beagle dogs (age 55 weeks) were analyzed following a 15 weeks' daily 20 km running training on a treadmill with 15 degree uphill inclination. In nucleus pulposus no statistically significant alterations were found in the content of proteoglycans or collagen. In annulus fibrosus the total tissue wet weight and total amount of collagen (hydroxyproline) increased by 34-36% in the runners as compared to age-matched, untrained controls. Since the total amount of proteoglycans did not increase, the annulus fibrosus became relatively depleted of proteoglycans, as indicated by the 27% reduction in uronic acid concentration, expressed either per wet weight or hydroxyproline. The average molecular size of the remaining nonaggregating proteoglycans was larger, and there was also a trend towards increased proportion of proteoglycans aggregating with hyaluronan. Most of the chondroitin sulfate side chains were 6-sulfated (65-66%). Running did not alter the sulfation or length of the chondroitin sulfate chains. The decreased proteoglycan/collagen ratio in annulus fibrosus may result in altered mechanical properties of the tissue and reflects its adaptation to enhanced motion and stress.
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| 47. |
- Visser, Nanette, et al.
(författare)
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Increase of decorin content in articular cartilage following running.
- 1998
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Ingår i: Connective Tissue Research. - : Informa Healthcare. - 0300-8207 .- 1607-8438. ; 37:3-4, s. 295-302
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Tidskriftsartikel (refereegranskat)abstract
- The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.
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