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Biomedical subjects

L Robert

Publications and source records attributed to L Robert.

At least 163 records · Page 9Linked to original sources
PubMed1989

Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging.

Acute local gamma-irradiation of porcine skin induces, as in human skin, extensive mutilating sclerosis, characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of extracellular matrix macromolecules. Collagen synthesis, content, and types were studied by measuring the incorporation of the radiolabeled precursor [3H]-proline into confluent primary cultures and subcultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. A significant increase in collagen biosynthesis and deposition, with a preferential enhancement of type III collagen, was observed in primary cultures of fibrotic fibroblasts when compared to those of normal dermis. However, the percentage (36%) of neosynthesized collagen in relation to the total neosynthesized non-collagenous and collagenous proteins remained unchanged. In subcultures of normal cells, collagen synthesis and content remained constant until about the 10th passage and then progressively declined until death of the fibroblasts, at around the 15th passage. During the senescence phase, normal fibroblasts acquired the capacity to synthesize higher levels of non-collagenous proteins. On the contrary, collagen synthesis and content diminished in subcultures of fibrosis-induced fibroblasts from the second passage, and then progressively declined as a function of aging; particularly the ratio of type III to type I collagen returned to normal values from the second passage. Moreover, these fibroblasts did not become senescent, persisted in synthesizing low levels of collagen, and acquired later (around the 40th passage) a higher capacity to synthesize non-collagenous proteins. These results demonstrate that primary cultures of porcine fibroblasts obtained from normal or radiation-induced fibrotic dermis reproduce the in vivo situation. The results strongly suggest that: 1) the modification of collagen synthesis, content, and types observed in primary cultures of fibroblasts might be due to factors causing long-lasting changes in phenotypic expression and/in stimulation of the expansion of some fibroblast clones engaged in the accelerated synthesis of extracellular matrix macromolecules, such as collagen or non-collagenous proteins. 2) the rapid decrease in this active capacity to synthesize and accumulate collagen, observed in subcultures of fibrosis-induced fibroblasts, might be related to the loss of some activation factors or to the dedifferentiation of the cells. 3) the switch from collagen to non-collagenous protein synthesis during later subcultures of fibroblasts obtained from radiation-induced fibrosis, might be due to molecular modification at the transcriptional or DNA level.

Animals↗
PubMed1989

Elastonectin and the elastin receptor.

Two different types of interactions were described between cells and elastin: 1) the adhesion of cells to insoluble fibrous elastin and 2) the binding of soluble elastin derived peptides by cells. We could show that these are two different mechanisms underlying those two types of interactions. The adhesion of cells to elastin fibers is mediated by a cell-membrane complex with a 120 kD protein as the main adhesive compound which we proposed to designate elastonectin. Three other proteins (67, 60 and 45 kD) were coisolated with elastonectin. Adhesion of insoluble elastin to mesenchymal cells (fibroblasts, smooth muscle cells) is inducible with soluble elastin peptides. Highly metastatic Lewis lung carcinoma cells and human melanoma cells also exhibited the adhesion mechanism, but without a lag phase (constitutive adhesion). Binding curve (Scatchard plots) obtained with radiolabelled elastin peptides indicated the presence of high affinity elastin receptors on mesenchymal cells and human white blood cells (monocytes and PMNs) with Kd-s in the nanomolar range. The binding of elastin peptides triggers several cellular reactions such as chemotaxis, Ca++ influx (increase of Ca++i) and with mononuclear blood cells release of lytic enzymes and oxygen radicals. All these cells which exhibited elastin receptor function also exhibited adhesion although the two processes could be inhibited selectively. The receptor action is mediated by a G-protein-phospholipase-IP3 mechanism, involved in the increase of intracellular Ca++. It appears that this action also triggers the biosynthesis and membrane localization of elastonectin. The coupling of these 2 mechanisms between receptor and adhesion appears to be modified in transformed cells.

Cell Adhesion↗
PubMed1989

Determination of anti-elastin peptide antibodies in normal and arteriosclerotic human sera by ELISA.

We adapted a highly sensitive and reproducible ELISA technique for the determination of anti-elastin peptide antibodies of IgG type AEAb-IgG) and IgM type AEAb-IgM) in human sera. The determination was performed in the sera of 265 normal and diseased persons. The pathologies studied included obliterative arteriosclerosis of the legs, ischemic heart disease, stroke, diabetes mellitus, type IIb and IV hyperlipoproteinemia and hypertension. No clearcut correlation could be found between AEAb and age. In contrast, in arteriosclerotic patients and especially in obliterative arteriosclerosis of the legs and ischemic heart disease, the concentration of AEAb-IgG was significantly increased. The AEAb-IgM showed no change in the studied diseases. Both types of AEAb were decreased in type IV hyperlipoproteinemia. Anti-elastin antibodies may be involved in the pathomechanisms of the above diseases and the determination of antibody concentrations may be of some help in obliterative arteriosclerotic diseases.

Adult↗
PubMed1989

[Effect of procyanidolic oligomers on cultured mesenchymal cells. I. Effect on attachment, proliferation and detachment of cells].

The effect of procyanidolic oligomers (OPC) was studied on mesenchymal cells in culture: human skin fibroblasts (FB) and porcine aorta smooth muscle cells (CML). In presence of OPC part of the freshly seeded FB did not attach. There was no significant effect on the attachment of CML. Proliferation of FB was also slowed down in a dose-dependent manner. Proliferation of CML-s was also decreased, but much less than for FB-s. The detachment of the cells was also studied by adding trypsin to previously attached cells. Detachment of FB-s was strongly inhibited in presence of OPC in a dose-dependent manner. Much less effect was seen on CML. It appears therefore that OPC may interact with some components of the FB cell membrane and modify the attachment, proliferation and detachment of these cells. The only cell kinetic parameter significantly influenced by OPC for CML was their rate of proliferation. This may be due to the different constitution of the CML cell surface as compared to the FB cell surface.

Adult↗
PubMed1988

Influence of heparin fragments on the biological activities of elastase(s) and alpha 1 proteinase inhibitor.

The in vitro and in vivo effects of heparin fragments (CY 216; CY 222) towards elastase(s) and elastase inhibitor (alpha 1 Pi) were studied. Heparin as well as its lower Mr fragments were shown to inhibit rat leucocyte elastase. The interaction between this enzyme and heparins appears to occur via electrostatic forces. Porcine pancreatic elastase is unaffected by heparin(s) but CY 216 and CY 222 could partly abolish the hydrolytic activity of hamster serum on Suc-Ala-Ala-Ala-N-PhNO2. N desulphated N acetylated CY 142 and CY 143 had no effect. CY 216 and CY 222 decreased in vitro the inhibitory potential of alpha 1 proteinase inhibitor (alpha 1 Pi) as well as the elastase inhibitory capacity of hamster serum. Maximum effect (30% decrease) was observed at ng concentrations of CY 216 and CY 222. Their N desulphated N acetylated counterparts (CY 142 and CY 143), but not heparin, exhibited similar effects. CY 216 and CY 222 were administered daily subcutaneously to hamsters and blood was collected 1, 2, 4, 7 and 24 hr after treatment for determining both serum elastase activity (E.A.) and serum elastase inhibitory capacity (E.I.C.). E.A. levels dropped by 30% 2 hr after CY 216 or CY 222 injection but returned to original values 4-7 hr later. This effect is independent of the duration of the treatment. Hamster serum E.I.C. was significantly increased (greater than 30%) after 3-4 weeks of treatment with CY 216 and CY 222. These findings point towards the potential use of these compounds in elastase-related diseases such as emphysema.

Acetylation↗
PubMed1988

Effect of cytokines and growth factors on the expression of elastase activity by human synoviocytes, dermal fibroblasts and rabbit articular chondrocytes.

Human synoviocytes, rabbit articular chondrocytes and human skin fibroblasts in culture were examined for their ability to express elastase activity. Latent enzyme activity degrading insoluble elastin was detected in the culture media of the three cell types and was completely abolished by metal chelating agents. Triton X-100 cell extracts were found to degrade a synthetic elastase substrate, N Succinyl-(Ala)3p-nitroanilide (SANA). The SANA-degrading activity of cell extracts could be attributed to a metalloprotease for fibroblasts and synoviocytes (100%) and to a metalloprotease associated with a cysteine protease for chondrocytes (70 and 30% respectively). This SANA-degrading activity was partly due to the combined action of an endo and an exopeptidase. Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) significantly enhanced the elastin degrading activity present in the culture media of both synoviocytes and chondrocytes. Interleukin-1 beta significantly increased the secretion of elastase by chondrocytes. By contrast, Transforming Growth Factor-beta (TGF-beta) reduced by 80 per cent the secretion of elastinolytic activity by chondrocytes but had not effect on other cell types.

Animals↗
PubMed1988

Inhibition of leucocyte elastase by heparin and its derivatives.

Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. Ki values ranged from 40 nM to 100 microM and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di- and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established.

Animals↗
PubMed1988

Modification of collagen and noncollagenous proteins in radiation-induced muscular fibrosis.

Six months after acute local gamma irradiation of the pig skin and adjacent muscle, the muscular tissue is replaced by a large mutilating and proliferative fibrosis deliminated by a perifibrotic inflammatory zone. The content and biosynthesis of collagen and noncollagenous proteins were studied in both fibrotic and perifibrotic zones after incubation of the biopsies with [14C]proline or [35S]methionine for 24 hr. Cells of perifibrotic and fibrotic regions synthesize about 10 times more proteins than those in the nonirradiated muscle. When compared to normal muscle tissue, our results indicate an important increase in collagen content and biosynthesis in fibrotic tissue. The increase in collagen biosynthesis in the irradiated tissue is more pronounced for type III collagen than for type I collagen. Biosynthesis of type III and type I collagens increases 20- and 10-fold, respectively, compared to the normal muscle. Type I to III collagen ratio in irradiated tissue decreases from 2.3 in normal tissue to 1.1 in fibrotic tissue. Histological examination of the biopsies as well as the protein pattern by polyacrylamide gel electrophoresis show striking differences in the perifibrotic and fibrotic areas as compared to the normal muscular tissue with a progressive disappearance of the myotubes replaced by a dense sclerotic tissue. The results indicate that the perifibrotic inflammatory area is engaged in a remodeling process and that the fibrotic tissue remains active in the neosynthesis of the extracellular matrix macromolecules with a high proportion of type III collagen. This high biosynthetic activity of the irradiated tissue may explain the pseudosarcomatous character of the radiation-induced lesions.

Animals↗
PubMed1988

Aging of the extracellular matrix and its pathology.

Recent concepts on the mechanisms of aging of extracellular matrix (EM) are reviewed as well as its involvement in age-associated diseases. Cell differentiation, histogenesis and organogenesis can be analyzed in terms of the program of the biosynthesis of EM macromolecules during development, maturation and aging. The most important biological role of EM is the integration of cells in tissues, of tissues in organs and of organs in the whole organism. EM can directly influence cell behavior through the contact between EM and the genome mediated by structural glycoproteins (fibronectin, laminin, elastonectin, etc.) interacting with other EM macromolecules (collagen, proteoglycans, elastin) and the cytoskeleton by trans-membrane receptors (integrins). Most age-associated diseases exhibit a deviation (qualitative or quantitative) from the normal program of EM biosynthesis. Three examples are analyzed in some detail: atherosclerosis, diabetes and malignant tumors. The degradation of elastic fibers catalyzed by cellular elastase-type enzymes is observed in atherosclerosis and also in emphysema and skin aging. Several of these enzymes were isolated and characterized from platelets, fibroblasts, smooth muscle cells and lipoproteins. The biosynthesis of some of them increases with age and facilitates cell migration. Plasma fibronectin increases with age exponentially. This increase is absent or strongly attenuated in diabetes and some cancers. Tissue fibronectin increases in diabetes, Werner syndrome and in the peritumoral desmoplastic reaction while most tumor cells can no more retain fibronectin on their membrane facilitating their movement in the organism. These examples demonstrate the importance of the study of cell matrix interactions for gerontology.

Aging↗
PubMed1988

Effect of elastin peptides and N-formyl-methionyl-leucyl phenylalanine on cytosolic free calcium in polymorphonuclear leukocytes of healthy middle-aged and elderly subjects.

The effect of elastin peptides (kappa-elastin, KE) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) on cytosolic free calcium in polymorphonuclear leukocytes (PMNLs) of healthy middle-aged (35-45 years) and elderly (greater than 60 years) patients with normal and high serum cholesterol level was investigated. The cytosolic free calcium [( Ca2+]i) elevation after stimulation with these compounds was decreased in PMNLs of the aged groups compared to the healthy middle-aged group. The guanine nucleotide binding regulatory Gi protein inhibitor, pertussis toxin (PT) prevented the enhancing effect of KE and FMLP on PMNL free calcium of healthy middle-aged subjects, but could not completely abolish the [Ca2+]i elevation in PMNLs of aged subjects.

Adult↗
PubMed1988

Characterization of human skin fibroblasts elastase activity.

We present evidence that enzyme activity hydrolyzing Succinoyl trialanine paranitroanilide (Suc(Ala)3NA) expressed by Human Skin Fibroblasts (HSF) in culture could be attributed to the concerted action of an endopeptidase and an aminopeptidase(s). Both endopeptidase and aminopeptidase activities were strongly inhibited by metal chelating agents and Copper and Zinc ions but were insensitive to Tissue Inhibitor of Metallo Proteases (TIMP). These protease activities coeluted on ion exchange chromatography (DEAE Tris acryl M) and were further separated by high-performance liquid chromatography HPLC (TSK 3000 SW). The endopeptidase activity, designated as HSF E1, was eluted at the position corresponding to an Mr equal to 94,000. It has only a limited elastinolytic potential as evaluated on 3H insoluble elastin, but it extensively degrades human skin elastic fibers as directly assessed on human skin tissue sections and further quantitated by automated image analysis. The level of HSF E1 increases with the number of fibroblast passages.

Adult↗
PubMed1988

Study of skin ageing as a function of social and professional conditions: modification of the rheological parameters measured with a noninvasive method--indentometry.

Skin ageing was studied with a noninvasive method: indentometry. We measured two rheological parameters: resistance to pressure, indentation, under the pressure of 10 g/cm2, and elastic rebound, elasticity, after the removal of the pressure. We studied three different populations: cloistered nuns, white collar and blue collar workers. We found in all populations a steady decrease in elasticity as a function of age; this effect was always steeper in females. The working women lost their elasticity more rapidly than the nuns, and the male blue collar workers lost their elasticity more rapidly than the male white collar workers. The development of indentation as a function of age is somewhat different. The white collar males showed a steady loss of resistance to pressure with age much more rapidly than their blue collar counterparts. The females showed either a biphasic change (the nuns), no change at all (the white collar workers) or a loss of resistance to pressure (the blue collar workers). These results show that professional as well as social factors may influence skin ageing.

Adult↗
PubMed1988

Elastase activity of bronchoalveolar cells in advanced pulmonary sarcoidosis.

Elastase activities were investigated in bronchoalveolar lavage (BAL) cells from healthy controls (C, n = 8) and patients with untreated mediastinopulmonary sarcoidosis at different stages: I (n = 5), II (n = 7), IIIA (n = 3), IIIB (n = 4). Elastase activities (EA and ELA) were measured against a synthetic substrate, succinyl-trialanine-paranitroanilide (SLAPN) and radiolabelled ligamentum nuchae 3H insoluble elastin (specific radioactivity 3.7 MBq.mg-1). A significant increase of ELA of BAL cells was observed in patients (S III), in particular those exhibiting fibrotic roentgenographic patterns and a higher number of polymorphonuclear cells. On the contrary, EA of BAL cells showed no variation between different stages of sarcoidosis. ELA was never detected in the macrophage culture medium and appeared to be mainly associated with non-adherent macrophages, whereas EA appeared to be preferentially associated with adherent macrophages. The inhibitory profiles of ELA and EA were investigated using general proteinase inhibitors and also compounds interacting specifically with leucocyte elastase. ELA was mainly due to a serine protease, which has a sensitivity to inhibitors similar to leucocyte elastase and is increased in advanced stages of sarcoidosis. EA appears to be only slightly modified, if at all, in sarcoidosis and is probably related to the action of both a serine protease and a metalloenzyme. Thus, only ELA yields diagnostic information concerning stage III of sarcoidosis associated with a fibrotic or bullous roentgenographic pattern.

Adult↗
PubMed1988

Comparative biochemical and morphometric studies on corneal wound healing.

Comparative biochemical and morphological studies were carried out on wounded corneas in order to correlate biochemical findings with morphometric observations during the healing process. Experimental production of corneal wounds, biochemical determinations and quantitative morphometric studies are described in an attempt to correlate corneal matrix macromolecules biosynthesis during the healing process with the morphological modifications of the tissue. The central part and the peripheral part of the corneas were examined separately and compared to the central and peripheral parts of the controlateral corneas. The DNA content of the central portion of the wounded corneas progressively increased and reached after 60 days the same level as the corresponding portion of the controlateral corneas. The DNA contents of the peripheral portions of wounded and controlateral corneas are persistently higher than the DNA contents in the central portion. In peripheral portions of wounded corneas the DNA content is higher than in controlateral corneas and continues to increase steadily during the 60 days of experimentation. Cell density, as determined by morphometry, increased also during that period. The differences between the evolution of the DNA contents and cell density curves may be attributed to variations in cell sizes. The collagen content, estimated from hydroxyproline determinations remained lower in the wounded corneas as compared to the controlateral corneas, even 60 days after operation. This was true for the central as well as for the peripheral portions, suggesting a collagenolytic process as a result of wounding. This is confirmed by morphometric evaluation of fiber density.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

[Proteases of the elastase type].

Elastin in the most resistant fibrous protein of the organisms. Its degradation is catalysed by proteases designated as elastases. Elastic fibers appeared during phylogenesis at the level of the first Vertebrates and rendered possible the emergence of efficient circulatory and respiratory systems which were necessary for the development of the higher Vertebrates. Several pathological conditions, mostly age-dependent, are accompanied by the degradation of elastic fibers or their alteration due to increasing association with lipids and calcium salts. Several proteases (endopeptidases) of cellular origin were described over the last years, especially those of PMN leukocytes, platelets, monocytes-macrophages, smooth muscle cells and fibroblasts. Although less active on fibrous elastin than pancreatic elastase, these enzymes may well play an important role in the development of age-dependent pathologies such as athero-arteriosclerosis and emphysema. The involvement of cellular elastases in these pathologies is discussed in some detail. The age-dependent increase, both in vivo and in vitro of the elastase activity of fibroblasts and smooth muscle cells appears to play an important role in the modifications of cell behaviour observed in the above pathologies.

Aging↗
PubMed1987

Inhibition by elastase inhibitors of the formyl Met Leu Phe-induced chemotaxis of rat polymorphonuclear leukocytes.

Rat leukocyte elastase has been purified successively by AH-Sepharose Kappa-elastin affinity chromatography and by ion exchange chromatography on a carboxymethyl Sephadex resin. It has great similarities with human leukocyte elastase in its molecular weight, substrate specificity and inhibitory profile. The effect of rat leukocyte elastase inhibitors in influencing the chemotactic response of rat PMN to fMetLeuPhe has been compared to that of other proteinase inhibitors. The results indicated that oleoyl (Ala)2ProValCH2Cl, a specific inhibitor of human and rat leukocyte elastases and Eglin C, which also inhibits human and rat cathepsin G, are among the powerful inhibitors of rat PMN chemotaxis induced by the formyl oligopeptide. This suggests that these neutral proteinases, in addition to their known participation in connective tissue catabolism, could play a role in PMN locomotion and chemotaxis.

Animals↗