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Biomedical subjects

C Lafuma

Publications and source records attributed to C Lafuma.

At least 19 recordsLinked to original sources
PubMed1992

Accumulation of glycosaminoglycans in radiation-induced muscular fibrosis.

The content and biosynthesis of glycosaminoglycans (GAGs) were studied in the pig thigh muscle after acute local gamma-irradiation. Seven months following irradiation, the muscular tissue next to the irradiation cone was replaced by severe mutilating fibrosis delimited by an intermediary perifbrotic zone. Fibrosis, perifibrotic tissue and normal muscle, were sampled and incubated with [3H]glucosamine and [35S]sulphate, and GAGs were isolated following pronase digestion. Results showed a parallel increase of collagen and GAG content in perifibrotic and fibrotic tissues. Sulphated GAGs, heparan sulphate and dermatan sulphate were preferentially accumulated in fibrotic tissue, while the hyaluronic acid content increased only slightly. Synthesis of sulphated GAGs was more elevated in fibrotic tissue than in perifibrotic zone as compared with normal muscle. Seven months after irradiation well-developed fibrotic tissue continued to synthesize and to accumulate extracellular matrix macromolecules, indicating the invasive aspect of post-irradiation fibrosis.

Animals↗
PubMed1991

Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments.

Heparin and its derivatives inhibit human leucocyte proteinases i.e. elastase and cathepsin G, but do not inhibit porcine pancreatic elastase and Pseudomonas aeruginosa elastase. In vitro experiments, reported here, also indicate that elastin, one of the physiological substrates of human leucocyte elastase (HLE), could decrease by 30-fold the inhibitory potential of an hexadecasaccharide heparin fragment (dp 16) isolated from CY 222. Nevertheless, the inhibitory capacity of the heparin fragment still remains elevated with IC50 = 2.7 x 10(-7) M and still inhibits HLE in its free and adsorbed state to elastin. These overall data prompted us to evaluate the influence of CY 222 in HLE-induced emphysema. Emphysema was induced in mice eight weeks old, following a single instillation of 200 micrograms of HLE. CY 222 treated animals received 2.5 mg.kg-1 subcutaneously once daily, 6 days per week during 4 weeks prior to HLE instillation, and for eight weeks following HLE instillation. The heparin fragment treatment of the mice halved the mortality rate observed early following HLE instillation. After 8 weeks, surviving animals were examined for lung histological and morphometrical changes: mean linear intercept (MLI) and internal alveolar area (ISA). The CY 222 heparin fragments exerted a protective effect against HLE-induced emphysema by decreasing by 70% the MLI; these heparin fragments exerted no effect on emphysema induced by pancreatic elastase in hamsters or mice. Heparin derivatives represent a new class of physiological HLE low molecular weight inhibitors capable of preventing HLE-induced emphysema.

Animals↗
PubMed1991

[Matrix metalloproteinase (MMP)].

Matrix metalloproteinases (MMP) are a group of zinc endopeptidases responsible for the degradation of most extracellular matrix macromolecules as observed during many physiological and pathological processes; their role in tissue remodeling with aging is postulated. Members of this family share high level of structural analogy and are secreted by several cell types as zymogens. Many factors (cytokines, chemicals, growth factors, cell aging...) are able to interfere with MMP's expression at the transcriptional level. The activation of MMP's zymogens can be catalyzed by different agents: proteinases, Hg salts, disulfides, superoxide ions... Within the extracellular space, the activities of MMPs are controlled by a family of inhibitors: the tissue inhibitors of metalloproteinases or TIMPs.

Enzyme Precursors↗
PubMed1990

Fibronectin and collagen gene expression during in vitro ageing of pig skin fibroblasts.

The fibronectin, collagen type I, and collagen type III genes code for three major proteins of the cell matrix. The age-related alterations in their expression were measured during the in vitro lifespan of pig skin fibroblasts. We observed changes in the transcription rate of these specific genes during ageing. The levels of fibronectin and type III collagen mRNA rose markedly during the senescence phase. The level of collagen type I mRNA decreased during cell ageing, while that of beta-actin did not change. As regards proteins, we observed a sharp increase in the secreted noncollagenous proteins and in the total proteins of the cell layer during senescence. On the contrary, the secretion of the collagenous proteins decreased during senescence. Moreover, most of the newly synthesized molecules of collagen were immediately degraded in the cells, before their extracellular secretion. The terminal phenotype of pig senescent cells was therefore characterized by overexpression of fibronectin and type III collagen genes and reduced expression of the type I collagen gene. Surprisingly, for fibronectin and type III collagen, that terminal phenotype resembled the one normally found in the fibroblasts during the processes of tissue repair, cicatrization, and development.

Aging↗
PubMed1990

Early modifications of outgrowth and metallo-endopeptidase activity in fibroblasts from rat cutaneous explants after local gamma-irradiation: dermis cellularity and collagen fibre alterations.

The initial response to local gamma-irradiation of skin was investigated in fibroblasts from cutaneous explants after doses of 4, 8, 12, 16 or 20 Gy. On the day of irradiation, fibroblast outgrowth was inhibited in a dose-dependent manner, but by day 7 post-irradiation, cell restoration occurred especially in explants exposed to 4 or 8 Gy. The dose-dependent inhibition of fibroblast outgrowth correlated with the decrease in cellular metallo-endopeptidase (MEP) activity against succinyl trialanine paranitroanilide. However, the secretion of this MEP activity was 10-fold higher in the culture medium after the lowest irradiation dose (4 Gy). Its inhibition profile was not modified after local irradiation, whatever the dose. In vivo, the cell density of mastocytes, pericytes and endothelial cells decreased after irradiation. Moreover, damaged collagen was observed in the superficial dermis after local irradiation. These results strongly suggest that this MEP may be involved in the alterations occurring in dermal connective tissue components after skin irradiation. The rapid decrease with the dose in fibroblast outgrowth and MEP activity also suggests that these two parameters may provide useful tools for dosimetric assay of the heterogeneity and extent of irradiated areas.

Aminopeptidases↗
PubMed1990

Susceptibility of baboon aorta elastin to proteolysis.

Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants V and [S50] of aortic elastin hydrolysis by HLE (0.15 microM) were 0.00286 mg x ml-1 x min-1 and 0.158 mg x ml-1, respectively. One mg of this elastin could be saturated with 5.6 micrograms of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine alpha-chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.

Animals↗
PubMed1989

Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis.

Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor [3H]proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.

Animals↗
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

Fibronectin and glycosaminoglycan synthesis by fibrotic pig fibroblasts in primary culture.

Synthesis of fibronectin and glycosamingoglycans (GAGs) was studied in fibroblasts from pigs with post-irradiation subcutaneous fibrosis. Fibrosis was developed in the femoral muscle by local gamma irradiation with a dose of 60 Gy. Normal fibroblasts were obtained from the healthy skin of the same animal. To measure GAG and fibronectin synthesis fibrotic and normal fibroblasts were labeled with 3H-glucosamine, 35S-sulfate and 35S-methionine. Fibrotic fibroblasts synthesized 2.5 times as much fibronectin as normal skin fibroblasts but total protein synthesis did not change. Parallel enhanced secretion of hyaluronic acid and dermatan sulfate into the cell culture medium were also observed. GAGs from the pericellular layer of trypsin-digested fibrotic fibroblasts exhibited increased 3H incorporation, but reduced 35S-sulfate incorporation. The largest reduction in the latter was observed for heparan sulfate. These results indicate that the fibroblasts from the well developed fibrotic tissue maintain enhanced synthesis of matrix macromolecules in primary cultures. Structural and/or metabolic changes in secreted GAGs, combined with the stimulation of tissue repair by growth factors may be responsible for the excessive deposition of collagen in post-irradiation fibrosis.

Animals↗
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

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

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

[Elastases and pulmonary pathologies].

Various elastases classes normally reside in alveolar structure and are liable to degrade the elastin as well as the other macromolecular components of pulmonary extracellular matrix (collagen, proteoglycans, fibronectin...), during lung injury. The most are the polymorphonuclear or monocyte serine elastase and the macrophage metallo and cysteine elastases. Metalloelastase may also arise from pathogenic bacteria as Pseudomonas aeruginosa. In another part proteases elastase-type from fibroblasts, endothelial cells or alveolar macrophages might to be involved into the remodelling of lung connective tissue or pulmonary cells differentiation and activation. The regulation of elastolytic activities, is supported both by activators (as plasminogen activator...) and inhibitors (alpha 1 Pi, 2M, BrI, TIMP, bacterial inhibitors...). These inhibitors are mostly generated in situ from macrophages, monocytes or polymorphonuclear cells so allowing to control fast local elastolytic activity. Since alveolar macrophage can internalize leucocyte elastase, synthetize metalloelastases, and secrete their inhibitors and activators, it plays a complex role in the lung defense and during various pulmonary pathogenesis. In conclusion, the lung response to bacterial or viral infections, the intensity of alveolitis, the nature and the gravity of emphysematous or fibrotic lung lesions, as well as the tumour growth or metastatic pulmonary invasion may depend upon the lung elastolytic activities.

Animals↗
PubMed1987

Prevention by calcitonin of the pathological modifications of the rabbit arterial wall induced by immunization with elastin peptides: effect on vascular smooth muscle permeability to ions.

Immunization of rabbits with elastin peptides prepared from purified bovine ligamentum nuchae elastin produces calcified arteriosclerotic lesions and fragmentation of elastic lamellae. Simultaneous administration of porcine calcitonin largely prevents the development of lesions. Experiments were carried out to clarify the mechanisms involved in the development of lesions as well as those involved in the preventive effect of calcitonin. Control experiments were carried out using bovine serum albumin (BSA) as antigen. Circulating antibodies and soluble immune complexes increased steadily in the sera of animals immunized with elastin peptides or BSA. The cellular immune reaction was weak as assessed by [3H]thymidine incorporation into lymphocytes in the presence of antigen or phytohemagglutinin. Arterial lesions appeared only in the animals immunized with elastin peptides, not in those immunized with BSA. Ion flux measurements were also carried out on strips of aorta obtained from immunized and control animals. Immunization with elastin peptides significantly increased the ouabain-insensitive 22Na+ efflux, the 86Rb efflux (indicator of K+ efflux), and the 45Ca2+ influx. Simultaneous calcitonin administration prevented the increase in Ca2+ influx but did enhance passive permeability to Na+ and K+ as well as the sodium pump. When calcitonin was administered without immunization, it decreased arterial smooth muscle permeability to Na+ and K+ and also decreased the basal Ca2+ influx. It is concluded that the pathological modifications of the arterial wall triggered by immunization with elastin peptides is at least partly mediated by the effect of antielastin antibodies and immune complexes on the ion permeability of arterial smooth muscle. Prevention of the increased Ca2+ influx by calcitonin is probably a key effect in the prevention of the development of lesions. The fact that calcitonin alone can modify the ion permeability of arterial smooth muscle suggests that this hormone may play a role in the regulation of vascular homeostasis.

Animals↗
PubMed1987

Effect of low-level NO2 chronic exposure on elastase-induced emphysema.

The effect of chronic exposure to 2 ppm nitrogen dioxide (NO2) for 8 hr a day, 5 days a week, for 8 weeks was assessed in normal and emphysematous hamsters by measuring (1) lung morphometry (mean linear intercept [Lm] and internal surface area [ISA]), (2) lung mechanics (lung volume, compliance and coefficient of static deflation, pressure-volume curve fitted to an exponential equation), and (3) serum elastolytic activity and protease inhibitor capacity. Emphysema was induced by a single intratracheal injection of 6 IU porcine pancreatic elastase. Four groups of animals were used; Control, NO2-exposed, elastase-treated, and NO2-exposed postelastase. Our results show that NO2 exposure alone induced mild emphysematous lesions whose degree of severity estimated by morphometry increase in Lm and decrease in ISA. P less than 0.01) was of the same order as that of the lesions induced by 6 IU elastase. Exposure to 2 ppm NO2 enhanced elastase-induced emphysema (further increased Lm and further reduced ISA. P less than 0.01). By contrast, study of lung mechanics revealed no difference between the control and NO2-exposed groups or between the elastase-treated animals exposed to NO2 and those not so exposed. This apparent discrepancy between results of morphometry and lung mechanics may be due to the lower sensitivity of lung mechanics parameters and their consequent inability to reflect changes in the emphysematous lesions induced by elastase injection or 2 ppm NO2 inhalation. In vivo, serum elastolytic activity and protease inhibitor capacity were not modified in any group, indicating that either serum does not reflect the degree of protease inhibitor capacity in the alveolar spaces or chronic inhalation of low concentrations of NO2 is not sufficient to cause elastase/antielastase imbalance. Lastly, our results suggest that chronic exposure to 2 ppm NO2 may cause individuals with inherited or acquired emphysematous lesions to develop more severe emphysema.

Animals↗
PubMed1987

Parallel increase of plasma fibronectin and perchlorosoluble serum glycoproteins in radiation-induced lung damage.

A study of the behavior of plasma fibronectin during a radiation-induced inflammatory lung reaction was carried out. Rats were exposed to a single homogenous thoracic irradiation of 15 Gy from a 60Co gamma source. Perchlorosoluble glycoproteins were determined (as sialic acid) as well as plasma fibronectin at 2, 8, 27, 60 and 120 days after irradiation. A parallel increase of the perchlorosoluble glycoproteins and plasma fibronectin was found. Morphological and histological studies of the irradiated lungs showed a predominating endothelial cell injury in the lung microcapillaries. The parallel increase of plasma fibronectin and perchlorosoluble serum glycoproteins suggests that plasma fibronectin can be considered as an acute phase reactant. Its early increase may be the result of the inflammatory reaction and endothelial injury induced by the radiation.

Animals↗