Biomedical subjects

H Larjava

Publications and source records attributed to H Larjava.

At least 55 records · Page 3Linked to original sources

PubMed1993

Immunohistochemical localization of proteoglycans in human periodontium.

Proteoglycans (PGs) are extracellular and cell surface-associated macromolecules that regulate cell adhesion, cell growth, matrix formation, and bind growth factors. In this work we studied the distribution of core proteins of four PGs (decorin, biglycan, a large molecular weight PG, and CD44) in human gingiva and periodontal ligament by immunohistochemical staining of frozen tissue sections with specific antibodies. Decorin, a major PG of this tissue, was localized on collagen fiber bundles in the gingival and periodontal connective tissues. Staining for decorin was most intense at the subepithelial region. Biglycan was a minor PG component of the human periodontium, showing some accumulation in connective tissue under the oral epithelium. At the immunohistochemical level, biglycan appeared to form fine filament-like structures on extracellular matrix fibers. Localization of large molecular weight PG differed from that of decorin and biglycan. It was concentrated in deep connective tissue areas of the gingiva and in the periodontal ligament, and was only weakly present at the subepithelial region. CD44 was mainly concentrated in cell-cell contact areas of basal and spinous layers of oral epithelium. In the connective tissue of gingiva and periodontal ligament, CD44 was localized on fibroblast cell surfaces. Connective tissue area under the junctional epithelium contained relatively small amounts of PGs. The results indicate that different parts of human periodontium contain a typical variety of PGs, suggesting a specific function for each PG species in the location at which they accumulate.

Adolescent↗

PubMed1993

Expression of integrin family of cell adhesion receptors in rheumatoid synovium. Alpha 6 integrin subunit in normal and hyperplastic synovial lining cell layer.

Integrins are heterodimeric cell adhesion receptors. The beta 1 integrin subunit can be in a complex with multiple a subunits and form receptors for collagen, laminin, fibronectin, and vitronectin. We have characterized the distribution of eight integrin subunits in rheumatoid synovium, with special interest in the lining cell layer. The beta 1 integrin subunit was found in abundance in synovial stroma and in lining cells. The only alpha subunit seen constantly in lining cells was alpha 6. In complex with alpha beta subunit, alpha 6 forms a laminin receptor usually seen in epithelial or endothelial cells or in macrophages. The fact that laminin was found in the extracellular matrix around synovial cells suggests the importance of alpha 6 integrin in the adhesion of synovial lining cells. Furthermore, alpha 6 expression was noticeably weaker in strongly proliferative lining cell layers, indicating that the inflammatory process may regulate integrin expression. A potential connection between altered expression of cell adhesion receptors and the pathological behavior of rheumatoid lining cells is suggested.

Adolescent↗

PubMed1992

A biochemical analysis of human periodontal tissue proteoglycans.

Proteoglycans synthesized by periodontal (gingival, periodontal ligament, dental follicle) fibroblasts were analysed by SDS/polyacrylamide and agarose gel electrophoresis after being labelled with radioactive sulphate. Medium, cell membrane and extracellular matrix fractions were analysed separately. Samples were treated with chondroitinase AC, chondroitinase ABC, heparitinase or a combination of chondroitinase ABC and heparitinase before electrophoretic separation of proteoglycans. Antibodies to versican and decorin were used to identify these molecules by Western immunoblots. For steady-state metabolic radiolabelling of fibroblasts, medium and cell membrane fractions contained about equal proportions of radiolabelled proteoglycans (about 43%), whereas less radioactivity (about 14%) was found in proteoglycans of the matrix fraction. Periodontal fibroblasts produce six major proteoglycans: versican, a high-molecular-mass chondroitin sulphate proteoglycan (CSPG); decorin, a dermatan sulphate proteoglycan (DSPG); a membrane-associated heparan sulphate proteoglycan (HSPG); two medium- or matrix-associated HSPGs; and a 91 kDa membrane-associated CSPG. Variation in decorin molecular size was observed in mass cultures of fibroblasts. Similar polydispersity in molecular size of decorin was seen in several clones established from one mass culture.

Cell Fractionation↗

PubMed1992

Immunolocalization of beta 1 integrins in human gingival epithelium and cultured keratinocytes.

Beta 1 integrins are cell surface receptors for extracellular matrix binding. We have recently shown that these receptors may also play a role in cell-cell binding of human epidermal keratinocytes. In this study we used immunofluorescence and confocal laser scanning microscopy to localize beta 1 integrins in frozen sections of human gingiva and cultures of human gingival keratinocytes. The results show that beta 1 integrin polypeptides, localized by monoclonal and polyclonal antibodies, were detected mainly in the basal layer of the keratinized epithelium. There was also scattered staining in connective tissue fibroblasts, nerves, and blood vessel walls. In the basal layer, the integrins were found around the entire periphery of the basal keratinocytes. Furthermore, confocal laser scanning microscopy (CLSM) revealed that most of the staining was in fact localized in dot-like structures at the lateral cell membranes of neighboring basal cells. In cultured human gingival keratinocytes maintained in low calcium (0.15 mM) conditions beta 1 integrins were localized in several different structures: trails which were left behind when the cells moved in culture, dots underneath the cells, around the nucleus, and in cell-cell contacts. The trails were also found to contain fibronectin and type IV collagen but not laminin. Switching the keratinocytes to high calcium (1.2 mM) conditions induced the formation of cell-cell contacts which were strongly positive for beta 1 integrins. No fibronectin or type IV collagen was found in cell-cell contact sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells.

The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Characterization of fibroblast clones from periodontal granulation tissue in vitro.

Connective tissues are known to be composed of heterogeneous fibroblast subpopulations. The significance of this heterogeneity in different physiological and pathological conditions is poorly understood. Granulation tissue is formed in connective tissue during wound healing, chronic inflammation, and certain pathological conditions. In this study, heterogeneity of fibroblasts from granulation tissue was investigated by cell-cloning techniques. Granulation-tissue fibroblasts (GTFs) from both chronically inflamed periodontal lesions and healing wound granulation tissue behaved similarly. GTFs showed a more pronounced decrease in proliferative capacity with increasing cumulative population doubling levels (CPDLs) and 30-40% lower cloning efficiency compared with normal gingival fibroblasts (HGFs). Morphologically, cells in GTF cultures were mainly large, whereas HGFs were mainly small in size. Both cell-line types showed heterogeneity in cell morphology. Clones composed of large stellate-like cells predominated in GTF cultures, whereas clones composed of small spindle-shaped or epithelioid cells predominated in HGF cultures. In both cell-line type the proportion of clones composed of large cells increased without increasing CPDL. These findings show that the properties of the fibroblasts changed during their in vitro life spans. The finding that normal connective tissue and granulation tissues contain morphologically distinct fibroblast clones in different proportions suggests that local factors could stimulate local fibroblasts to differentiate into GTFs. Alternatively, local factors could select some fibroblast subpopulations to overgrow the others to form granulation tissue.

Actin Cytoskeleton↗

PubMed1991

A novel cell adhesion molecule, G-CAM, found on cultured rat glia.

Using a monoclonal antibody (mAb), designated AMP1, a novel cell adhesion molecule was identified on rat astrocytes and oligodendrocytes. When confluent monolayers of cultured rat astrocytes were labeled with AMP1, the antigen was discretely localized to the cell surface in regions of cell-cell contacts. The antibody did not label embryonic rat cortical neurons plated on monolayers of neonatal astrocytes, indicating that the antigen is neither present on cultured neurons nor does it reorganize on the glial surface under the neurons. On immunoblots of astrocytic or brain proteins, mAb AMP1 recognized a 106 kDa protein. In the present paper, data are presented demonstrating that the AMP1 antigen is a cell adhesion molecule and is distinct from all the known cell adhesion molecules present on astrocytes: N-CAM, N-cadherin, or members of the beta 1 integrin family. We have tentatively termed this molecule 'glial cell adhesion molecule' (G-CAM).

Animals↗

PubMed1991

Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes.

During the wound healing process lysis of basement membranes precedes keratinocyte migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming growth factor-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming growth factor-beta 1 was found to increase the expression of both 92- and 72-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal keratinocytes. The 92-kDa enzyme predominated in both unstimulated and stimulated cultures. The 92-kDa form was stimulated over 5-fold, and the other form by a factor of 2-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 92-kDa enzyme was similar in culture medium containing either 0.15 or 1.2 mM calcium chloride. Rat mucosal keratinocytes secreted only 92-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.2-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human keratinocytes were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 92-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human keratinocytes, offering a possible explanation of how keratinocytes detach from basement membranes prior to the migration over the wound bed.

Animals↗

PubMed1991

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture.

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.

Biglycan↗

PubMed1991

Expression of beta 1 integrins in normal human keratinocytes.

The majority of cell adhesive events to the extracellular matrix are mediated by cell surface receptors, beta 1 integrins. Keratinocytes express at least six different polypeptides of beta 1 integrin class, namely beta 1, alpha 2, alpha 3, alpha 5, and alpha 6 (alpha 6 is mainly associated with beta 4 polypeptide). These epithelial cells use alpha 2 beta 1 as a collagen receptor and alpha 3 beta 1 as a fibronectin receptor, while alpha 6 beta 4 is the major basement membrane receptor. Expression of alpha 5 beta 1 complex is low. Processing of beta 1 integrins is fast in keratinocytes; half-maximal maturation takes only 3 hours. In addition to their function in cell-matrix interactions, beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) have also a role in maintaining keratinocyte cell-cell interactions. It is possible that resting basal keratinocytes use beta 1 integrins as cell-cell adhesion receptors, and during activation, like in wound healing, these receptors relocalize to mediate events involving cell-matrix interactions.

Basal Cell Carcinoma↗

PubMed1991

Extracellular matrix molecules and their receptors: an overview with special emphasis on periodontal tissues.

Knowledge of extracellular matrix molecules and their cell receptors has increased exponentially during the last 2 decades. It is now known that the structure and function of each tissue is based on specific combinations of matrix molecules. The major constituents of the extracellular matrix are collagens, proteoglycans, and adhesive glycoproteins. The rapid development of biochemical, molecular biological, and immunological research has revealed a lot of interesting details pertaining to these molecules. Several new collagen types have been discovered. In addition to being responsible for the strength and form of tissues, each collagen type has specific sequences providing them with special features such as flexibility and the ability to interact with other matrix molecules and cells. Proteoglycans are another large group of matrix molecules with a variety of functions. Proteoglycans play an important role in tissue resilience and filtering. Some proteoglycans have a capacity to specifically bind other matrix molecules and growth factors, while others act as matrix receptors on the cell surface. An important part of regulation of the cell behavior is played by adhesive glycoproteins belonging to the fibronectin and laminin families. Several isoforms of fibronectin and laminin that result from alternative RNA splicing serve specific functions such as controlling the attachment, migration, and synthetic activity of cells. A major group of cell receptors for cell-matrix and cell-cell interactions is termed integrins. The integrins are cell surface proteins composed of two polypeptides whose structure dictates the specificity of each receptor. The cytoplasmic domain of the integrins interacts with cytoskeletal elements within the cell, and thereby relays the information from the extracellular space into the protein synthesis machinery. The expression of the integrins is controlled by the extracellular matrix and growth factors, most notably TGF beta. During periodontal diseases several aspects of the cell-matrix interactions may be disturbed. Therefore, an understanding of the special features of the extracellular matrix and their receptors in periodontal tissues is a prerequisite for developing new approaches to the prevention and treatment of periodontal diseases.

Extracellular Matrix Proteins↗

PubMed1991

Analyses of periodontal glycosaminoglycans and proteoglycans. Regulation by microbial, chemical and inflammatory factors.

During the last two decades one of the main lines of research in the Department of Periodontology in Turku has related to connective tissue glycosaminoglycans and proteoglycans. A general outline of current knowledge about proteoglycans is presented, with a review of most published and unpublished results on periodontal glycosaminoglycans and proteoglycans obtained in our Department.

Bacteria↗

PubMed1990

Differences in the biosynthesis and localization of the fibronectin receptor in normal and transformed cultured human cells.

We examined the biosynthesis and localization of the fibronectin receptor integrin from normal and transformed cultured human cells. Normal cells required a minimum of 20 h for the biosynthesis of completely mature fibronectin-receptor beta-subunit, while transformed cells required only 6-8 h. There was a correspondingly major decrease in the amount of the intracellular beta-chain precursor in the transformants. Immunostaining of normal fibroblastic cells with monoclonal antibodies indicated that both alpha- and beta-polypeptides of the fibronectin receptor are localized in cell surface streaks and focal contact areas. In contrast, both subunits lacked this clustering and had a more diffuse distribution on the surfaces of transformed cells, even though quantitative immunofluorescence experiments indicated that similar or larger amounts of each subunit were present on a per cell basis. Both immunostaining and biochemical analyses also indicated the presence of a relatively large intracellular pool of beta-polypeptides in normal fibroblasts that is not present in transformed cells. There was no major transformation-dependent change in total quantities of mature fibronectin receptor subunit expressed and inserted into the plasma membrane, when normalized to total protein synthesis. Our results indicate that malignant transformation of cultured human cells results in altered localization and processing of the fibronectin receptor. Such changes involving pathways of crucial cell surface molecules may contribute to alterations in their interactions with extracellular macromolecules, including during the process of cellular invasion.

Cell Transformation, Neoplastic↗

PubMed1990

Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe.

Biochemical data suggest that gingival epithelium contains hyaluronate, but there is little histochemical information about its localization. Hyaluronate was here visualized in gingival and buccal mucosa using a specific probe derived from the hyaluronate binding region of cartilage proteoglycan. Hyaluronate was found both in the gingival and buccal epithelium, but its localization was correlated with the type of keratinization. In the keratinized epithelium of gingiva, whether ortho- or parakeratotic, the intercellular spaces from basal to upper spinous layers displayed strong staining, most intense in the middle spinous cell layer. The uppermost vital cell layers as well as the cornified cell layer remained unstained. In the non-keratinized epithelium of buccal mucosa and the local non-keratinized areas of gingiva, only the basal cells and the lowermost spinous cell layers stained for hyaluronate, whereas the majority of the upper epithelium was negative. Electron microscopic examination of the basal and spinous cell layers displayed hyaluronate, both associated with the cell surface and free in the intercellular space. The subepithelial connective tissue showed positive but diffuse staining in all specimens.

Adolescent↗

PubMed1990

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.

Actins↗

PubMed1990

In vivo analyses of integrin beta 1 subunit function in fibronectin matrix assembly.

Early development of the urodele amphibian Pleurodeles waltl is accompanied by a process of progressive fibronectin (FN) fibrillogenesis. FN begins to assemble into fibrils on the inner surface of the blastocoele roof at the early blastula stage and progressively forms a complex extracellular matrix. We have analyzed the mechanisms of FN-fibril formation under normal and experimental conditions in vivo with the following probes: iodinated FN, fluorescein-labeled FN, synthetic peptides containing the Arg-Gly-Asp (RGD) cell surface recognition sequence of FN, and polyclonal antibodies against both beta 1 subunit of the amphibian FN receptor and the cytoplasmic domain of beta 1 subunit. We report that in living embryos, exogenous labeled mammalian FN injected into the amphibian blastocoele undergoes FN-fibril formation in spatiotemporal patterns similar to those of endogenous FN. This indicates regulation of fibrillogenesis by the cell surface rather than by changes in the type of FN. Fibrillogenesis is inhibited in a dose-dependent manner both by the GRGDS peptide and monospecific antibodies to amphibian integrin beta 1 subunit. Furthermore, when injected intracellularly into uncleaved embryos or into selected blastomeres, antibodies to the cytoplasmic domain of integrin beta 1 subunit produce a reversible inhibition of FN-fibril formation that follows early cell lineages and cause delays in development. Together, these data indicate that in vivo, the integrin beta 1 subunit and the RGD recognition signal are essential for the proper assembly of FN fibrils in early amphibian development.

Amino Acid Sequence↗

PubMed1990

Differential localization of type I and type III procollagen messenger ribonucleic acids in inflamed periodontal and periapical connective tissues by in situ hybridization.

Inflammatory lesions of periodontal and periapical connective tissue were studied by in situ hybridization to detect cells responsible for type I and type III collagen production. Formalin-fixed and paraffin-embedded tissue specimens from patients with oral lesions of various stages of inflammation were hybridized with cDNA probes specific for human pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, and with bacteriophage lambda DNA as a control probe. This technique permitted us to localize fibroblasts active in type I collagen synthesis in the vicinity of inflammatory infiltrates in all the samples studied. Cells containing high levels of type III collagen mRNA were seen in early abscess formation and they were particularly abundant in pyogenic granuloma and irritation fibroma. Type I collagen mRNA was prominent in gingival fibrosis. In the infrabony lesions with active inflammatory infiltrations the production of collagen was confined mostly to the periphery of the lesions. These findings give indirect evidence that cytokines liberated during the early stages of the inflammatory process stimulate expression of the type III collagen gene by fibroblasts. In chronic lesions a gradual switch from type III to type I collagen gene expression occurs. The change in collagen types appears to underlie the observed isolation of the inflammation by a collagenous capsule. In all the samples studied fibroblasts exhibited marked variation in their levels of procollagen mRNAs, supporting previous views about their heterogeneity in connective tissues. The approach presented here offers new possibilities to study cellular interactions and metabolic activities in inflammatory lesions.

Adolescent↗

PubMed1989

Gingival crevicular fluid fibronectin degradation in periodontal health and disease.

The molecular forms of fibronectin (FN) in gingival crevicular fluid of five subjects with at least two sites exhibiting clinical signs of inflammation and pockets of at least 4 mm (test group) and five subjects with clinically healthy periodontium (control group) were investigated. Samples were collected with standard filter paper strips. In the test group samples from both diseased and healthy sites were collected. After collection the test group received one episode of periodontal treatment (scaling and root planning). The sampling and clinical recording were repeated for the diseased sites after about 2 wk. The crevicular fluid FN was analyzed using sodium dodecyl sulphate gel electrophoresis followed by western blotting with polyclonal antibodies against FN. Both intact FN and FN fragments were found in all samples. A larger proportion of FN was in degraded form in the diseased sites than in the healthy or the treated sites. FN was also degraded into smaller peptide fragments in the diseased than in the treated sites. These results suggest that crevicular fluid FN is partially degraded both in periodontal health and disease and that the degree of degradation of FN increases with periodontal inflammation and decreases with periodontal treatment.

Adult↗