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Biomedical subjects

L Robert

Publications and source records attributed to L Robert.

At least 109 records · Page 6Linked to original sources
PubMed1995

Elastin derived peptides protect elastic fibres degradation by human neutrophil elastase: in vitro and in vivo studies using a mechanically induced rat gingival inflammatory model.

An elastin peptide (kE57) obtained from organoalkaline hydrolysis of calf ligamentum nuchae insoluble elastin, was isolated by gel permeation on Sephadex G150 and high performance liquid chromatography on a TSK G 3000 SW column. It possessed an average Mr = 57,000 and similar amino acids composition as its insoluble counterpart. kE57 behave as a competitive inhibitor of human neutrophil elastase (HNE) with Ki = 1.4 microM; it also inhibited porcine pancreatic elastase (PPE) but less efficiently Ki = 180 microM. Identification of elastic fibres in rat gingiva was ascertained by light and electron microscopic studies. Morphometric studies indicated that rat gingiva contained similar levels of elastic fibres (= 2%) as human skin; elastic fibres networks from both tissues also displayed high structural analogy. Gingival chronic inflammation was induced in rats by mechanical impaction associated with an hyperglucidic diet. After 5 weeks, the levels of rat gingiva elastic fibres, decreased from Vv = 1.94 +/- 0.1% to Vv = 1.02 +/- 0.06%. Local injections of kE57: 100 micrograms per day, 5 days a week for 5 weeks did restore the integrity of the gingiva elastic fibres network: Vv = 1.84 +/- 0.1. Without influencing leucocyte infiltration, it is proposed that elastin-derived peptides, acting as potent competitive inhibitor of neutrophil elastase involved in periodontitis, might be of therapeutic value.

Animals↗
PubMed1995

[Elastase-type endopeptidase of fibroblasts. Effect of metalloprotease inhibitors].

Human skin fibroblasts produce in culture an elastase-type metalloendopeptidase which can hydrolyze synthetic elastase-substrate as Suc ala3 pNA and degrade also elastic fibers when injected in the dermis or deposited on cryostat-skin sections [3-8]. Here we describe further characterization of this enzyme activity using metallo-enzyme inhibitors as well as specific inhibitors of known Zn-endopeptidases such as angiotensin converting enzyme and enkephalinase. Among the metal complexing agents tested only EDTA and o-phenanthrolin could inhibit the elastase-type activity of fibroblasts, other known metal complexing substances capable of reacting with Zn (2,2' dipyridyl, diethyl dithiocarbamate and other metal chelators) were ineffective as was also lisinopril, an ACE-inhibitor [13]. Phosphoramidon and retrothiorphan, specific enkephalinase inhibitors [12] did strongly inhibit the elastase type activity of human skin fibroblasts (IC50 10(-8) M). Ethanol at conc-s used to dissolve organic, water insoluble inhibitors (50-100 microliters/ml) strongly inhibited the enzyme. It appears that the metal prosthetic group of fibroblast elastase (presumably, Zn) is not directly accessible to several of the low M. Wt complexing agents. The efficiency of enkephalinase inhibitors suggest a possible relationship between this enzyme and fibroblast-elastase.

Adult↗
PubMed1995

[Role of glycosoaminoglycans in venous disease. Mode of action of some flavonoid drugs].

Varicose vein walls differ from normal venous walls by an important loss of their collagen content and an increase of their glycosaminoglycan content, essentially of hyaluronan. The decrease in fibrous protein content can be attributed to increased proteolytic (collagenolytic) activity as well as to free radicals. Glycosaminoglycan increase reflects a disregulation of the normal program of matrix biosynthesis by the cells of varicose vein wall, essentially smooth muscle cells. Some flavonoid drugs are capable of correcting these deviations by decreasing proteolytic attack on fibrous proteins and the accumulation of proteoglycans and hyaluronan. These effects, due to interactions between flavonoid drugs and the cells and fibrous proteins of the venous wall differ according to the nature of such drugs. A hypothesis is proposed to explain these differences in the intensity of action of flavonoid drugs with apparently closely related structures, based on the conformation of these drugs and their interaction with the triple helical structure of collagen fibers as well as with the cell membranes.

Antihypertensive Agents↗
PubMed1995

Interaction of tumour cells with elastin and the metastatic phenotype.

It is now well established that the interaction of tumour cells with elastin is important during invasion and metastasis. This is due to the fact that the elastin receptor complex is widely expressed by tumour cells and is overexpressed in highly metastatic variants. There is evidence that the elastin receptor complex is associated with a signal system involving G proteins, phospholipase C, the phosphoinositol cycle and protein kinase C. Therefore, activation of the elastin receptor system results in activation of protein kinase C-dependent cellular processes such as enzyme secretion and migration. Accordingly, soluble elastin can be used in vivo to interfere with tumour cell dissemination into elastin-rich tissues such as lung, skin or blood vessels. The importance of elastin-tumour cell interactions is emphasized by the observation that the 67 kDa receptor for laminin may well be identical to the 67 kDa elastin receptor of the elastin receptor complex. Interference with the function of this receptor system by the use of both laminin peptides and elastin ligands may provide the basis for a novel and more powerful antimetastatic intervention.

Amino Acid Sequence↗
PubMed1995

[Elastolysis, aging and atherogenesis].

In order to explore the potential role and importance of elastin fiber degradation in atherogenesis we determined in more than 1,400 individuals (males and females between 59 and 71 years of age), [the EVA epidemiological study] serum parameters related to elastin fiber degradation: serum elastase activity, circulating elastin peptides and serum elastase inhibitor titers. Significant correlations were found these between parameters and several other serum constituents considered as risk-factors of atherogenesis--essentially serum lipid-parameters and glycemia as well as several other biological factors. These correlations confirm the validity of the underlying hypothesis concerning the interest of the clinical determinations of these elastin-related parameters and the potential role of the permanent activation of the endothelial elastin-receptor in atherogenesis.

Aged↗
PubMed1995

[Vascular aging. Role of elastin receptor].

A distinction is made between atheromatous plaque formation and arteriosclerosis, only this second process is strictly age-dependent. Interactions between lipids and constituents of the vascular wall are however involved in both processes, although by different mechanisms. The progressive increase with age of elastase activity is a second important factor in the age-dependent progression of arteriosclerosis. The fragmentation of elastic fibers produces elastin peptides, present in the circulating blood in microgram/ml conc-s, increasing in several arteriopathies. The constant presence of elastin peptides in the circulation maintains activated the elastin receptor (16) on endothelial cells producing NO* with vasorelaxing activity (18). The simultaneous production of superoxyde leads to the formation of peroxynitrate, neutralized by reduced glutathion. This process, maintained over decades may well impair the antiradical defense mechanisms of the cells and deprive the endothelium from the vasorelaxing activity of NO*. We propose therefore that the maintained activation of the elastin receptor with free radical and lytic enzyme production might well represent the initiating process underlying atherogenesis.

Aging↗
PubMed1994

Interaction between cells and elastin fibers: an ultrastructural and immunocytochemical study.

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting "pseudopodia" that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types.

Adult↗
PubMed1994

Effect of hyaluronan on the elastase-type activity of human skin fibroblasts.

The influence of hyaluronan (HA) on the expression of human skin fibroblast elastase-type protease (HSFEp) (Homsy et al, 1988) was studied. At confluency of HSF cultures, hyaluronan increased the level of HSFEp in a time and dose-dependent fashion. Optimal effect was observed after 48 h of culture and at 2 mg/ml HA concentration; the stimulatory effect of HA could be suppressed by 1 microM cycloheximide. The enhancement of enzyme biosynthesis by HA was dependent on cell proliferation but quasi invariant with HSF passage number (from 7-21).

Cell Division↗
PubMed1994

Presence of gelatinase A and metalloelastase type protease at the plasma membrane of human skin fibroblasts. Influence of cytokines and growth factors on cell-associated metalloendopeptidase levels.

Gelatinase A and elastase type proteinase (Homsy, et al., 1988) present at plasma membranes of human skin fibroblasts (HSF) were separated by anion exchange chromatography on a DEAE Tris acryl M column. Elastase type proteinase (HSFE1) was able to convert 72 kDa progelatinase A to a lower 66 kDa M.W. active enzyme. Several cytokines (IL-1 beta, IL4, IL6), interferon gamma (IFN gamma) and tumor growth factor beta (TGF-beta) were studied for their ability to modify the levels of those plasma membrane associated proteinases. Among these mediators, only IL-1 beta was found to enhance the amounts of HSF membrane-bound HSFE1 and Gelatinase A.

Cell Membrane↗
PubMed1994

Heparin and its derivatives modulate serine proteinases (SERPS) serine proteinase inhibitors (SERPINS) balance. Physiopathological relevance.

Heparin and heparan sulfate, exhibiting wide biological interactions, are constituted of block structures. A defined pentasaccharide motif was found responsible for the enhancement of the rate of inactivation of factor Xa by antithrombin III. Heparin also interacts with other serine proteinase inhibitors as protease nexin I, and thus possibly modulates extracellular matrix proteolysis by serine proteinases in the pericellular environment. Human neutrophil elastase (HNE) activity is inhibited by heparin with Ki = 75 pM. This strong interaction is electrostatic, involving HNE/arginine residues disposed in a "cluster shoe" arrangement on the surface of the molecule and mainly OSO3- groups of heparin. HNE-heparin interactions also interfere with HNE associations with its natural inhibitors: it decreases the rate of association of HNE with alpha 1 proteinase inhibitor (alpha 1 P(i)) by 3 orders of magnitude, while increasing kass between HNE and mucus bronchial inhibitor (MBI) by > 10 fold. In vivo experiments demonstrated that heparin fragments lacking anticoagulant activity were able to nearly completely abolish emphysematous lesions induced in mice by a single intratracheal administration of 200 micrograms HNE. Long chain unsaturated fatty acids peptide conjugates were described as competitive HNE inhibitors (Hornebeck W. et al. 1985). We synthesized N-oleoyl heparin derivative (3 oleoyl groups/one molecule of heparin); such a lipophilic glycosaminoglycan (LipoGAG), although acting as an elastin protecting agent, possessed lower HNE inhibitory capacity as compared with heparin. In contrast, however, it was able to inhibit other serine proteinases such as urokinase, plasmin, porcine pancreatic apha-chymotrypsin and elastase. Such Lipo GAG's can be therefore useful to control matrix metalloproteinases (MMPs) during tissue remodeling or tumor invasion.

Animals↗
PubMed1994

Retarded fibronexus formation and cell attachment on type V collagen.

We previously found that type V collagen repressed the attachment and spread of aortic smooth muscle cells. The present study was carried out to investigate the effects of type V collagen on the formation of fibronectin and F-actin filaments of human dermal fibroblasts in relation to cell attachment and spread, using an immunofluorescent technique and morphometry. The number and area of the cells attached to type V collagen at 1 and 3 hours after seeding were significantly lower than those of cells on other substrates, including collagen types I, III and IV, and bovine serum albumin. However, there was no significant difference in the attachment and spread among the cells on these substrates after 24 hours. Cultured fibroblasts exhibited two patterns of fibronectin; one was a clear, linear fibronectin localized mainly in the cellular margins, and the other was a granular or flocculent fibronectin found in the perinuclear areas. The former was stained in non-permeabilized cells, but not in trypsin-treated cells (cell surface fibronectin). In contrast, the latter was not detected in nonpermeabilized cells, but was found in trypsin-treated cells (perinuclear fibronectin). Most of the cells cultured on type V collagen did not form either linear cell surface fibronectin or F-actin filaments at 3 hours. In contrast, many cells on collagen types I, III, and IV developed both cell surface fibronectin and F-actin filaments, the distributions of which were partially coincident. Colocalization of linear cell surface fibronectin and F-actin filaments was found in cells on all of the substrates after 24 hours. Perinuclear fibronectin showed similar patterns, and was not colocalized with F-actin filaments on different substrates at 3 and 24 hours of culture. Solid-phase substrates induced a better cellular attachment at 3 hours than serum adhesive factors. The administration of monensin, which inhibits the secretion of protein products, decreased the intensity of the fluorescence of cell surface fibronectin in fibroblasts, which was observed in a clear line. These results suggest that the retardation of the initial attachment and spread of fibroblasts on type V collagen is related to an inhibition in the formation of the fibronexus, a close transmembranous association of individual fibronectin fibers and F-actin filaments.

Actins↗
PubMed1994

[Role of the elastin-laminin receptor in the vasoregulation].

The elastin-laminin receptor was shown to be present on several benign and malignant cell types and to mediate several important cell reactions such as chemotactic movements of fibroblasts and monocytes, release of lytic enzymes and oxygen free radicals from leucocytes, increased adhesion of mesenchymal cells to elastin fibers as well as modifications of ion fluxes-increase of calcium and sodium influxes and decrease of ouabain-dependent potassium influx. We now demonstrated that the addition of elastin peptides to rat aorta rings precontracted with noradrenaline produced an endothelium-dependent vasorelaxation. The inhibition of this effect by laminin and lactose is in favor of the mediation of this action of elastin peptides by the 67 kDa subunit of the elastin-laminin receptor which possesses a lectin site. As elastin peptides are present in the circulating blood and their concentration was shown to increase in some pathological conditions, this phenomenon may well have physiopathological significance.

Animals↗
PubMed1993

[Biology of aging].

The biology of aging, a modest branch of medical sciences at the beginning of this century experienced a considerable development as a result of the importance and urgency of problems it has to solve. In this short review only a schematic presentation of this science can be tempted. Clinical epidemiological studies revealed the differential decline of quantifiable physiological functions with quite different kinetics. The fastest decline was noticed for the elastic tissues such as the lung and the blood vessels, both rich in extracellular matrix (ECM), that is connective tissues. Such tissues contain cells able to proliferate (mitotic cells) and extracellular matrix. Aging studies of such tissues should thus comprise the analysis of cellular aging and the aging of the ECM as well as the age-dependent modifications of cell-matrix interactions. The "Hayflick limit" is the limited number of population doubling for such cells in culture is obeyed by the cells of these tissues. It appears however that this limit is not reached during the actual life time of our species. The mechanisms underlying this limit at the level of the genome are now actively studied and start to be understood. The age-dependent modifications of the relative rates of biosynthesis of ECM-macromolecules appears to be regulated independently of cell proliferation. Post-synthetic matrix undergoes also age-dependent modifications such as crosslinking attributed to the Maillard reaction, proteolytic degradation and free radical damage. The result is a progressively modified ECM in its structure and function.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Elastin peptide concentration in human serum: variation with antibodies and elastin peptides used for the enzyme-linked immunosorbent assay.

Discrepancies exist between the reported values for the mean elastin peptide (EP) concentration in human sera. In order to understand these discrepancies, several EP preparations were obtained in vitro and monoclonal and polyclonal antibodies were produced against them. These different EP preparations and antibodies were used in an enzyme-linked immunosorbent assay (ELISA) to study cross-reactivity between EP preparations and to quantitate EP concentration in human sera. The method of purification of elastin, the method of hydrolysis of elastin and the molecular weight of EP influence their reactivity with antibodies and the results of EP measurements in human sera. However, there is a good correlation between EP measurements carried out in several human sera with the different EP preparations and different antibodies. Although absolute values of the EP concentrations varied with the EP preparation and antibodies used for the ELISA, the variations of this EP concentration measured from one human serum to another are significant.

Antibodies, Monoclonal↗
PubMed1993

Inhibition of human leucocyte elastase by fatty acyl-benzisothiazolinone, 1,1-dioxide conjugates (fatty acyl-saccharins).

Derivatives of benzisothiazolinone 1,1-dioxide (saccharin) N-acetylated with aliphatic and aromatic substituted aliphatic acyl groups were prepared. The inhibitory activity of the compounds was assayed against human leucocyte elastase (EC 3.4.21.37) and several other proteases. The IC50 values for inhibition of the human leucocyte elastase decreased with increasing length of the acyl residue, and reached a minimum value at C16 (2 microM). This phenomenon and the decrease of the inhibition by surfactants or by saturation of the enzyme with palmitic acid, indicates that in addition to acylation, hydrophobic interactions are also involved in the inhibition of this proteinase by compounds substituted with acyl groups containing at least 12 carbon atoms. The inhibitory activity of N-palmitoyl-benzisothiazolinone 1,1-dioxide (palmitoyl-saccharin) is about 14 times higher toward human leucocyte elastase than for thrombin (EC 3.4.21.5), and several hundred times, compared to porcine pancreatic elastase (EC 3.4.21.36) and to plasmin (EC 3.4.21.7). Fatty acylated saccharin derivatives were seen to bind in a saturable fashion to insoluble elastin, and decreased the susceptibility of this protein to hydrolysis by human leucocyte elastase.

Amino Acid Sequence↗
PubMed1993

Dog genetic polymorphism revealed by synthetic tandem repeats.

We are studying the genetic polymorphism associated with Variable Number of Tandem Repeat (VNTR) loci in 13 breeds of dogs, namely: Alaskan Malamute, Barzoi, Beagle, Belgian Shepherd, Fox Terrier, Griffon, Labrador, Irish Setter, Spaniel, Dachshund, Irish Terrier, Shar Pei and Poodle. Our approach is based upon synthetic tandem repeats (STRs). Using a panel of these arbitrary unit polymers to detect minisatellites, we are attempting to develop paternity testing systems on pure bred dog pedigrees. We are evaluating the potential importance of STRs as a tool for the isolation of minisatellites in dogs, as well as for the characterization of dog genetic markers.

Animals↗
PubMed1993

Tropoelastin gene expression in the developing vascular system of the chicken: an in situ hybridization study.

Temporal and spatial patterns in the accumulation of Tropoelastin (TE) mRNA during development of the chick embryo were established by in situ hybridization. Radiolabeled oligonucleotide probes of high specific activity were hybridized to serial sections of the cardiovascular system from embryonic day 3.5 (ED 3.5) to ED 19. Tropoelastin mRNA was observed as early as ED 3.5 in the dorsal part of the arterial trunk. During septation varying levels of TE mRNA were seen in the pulmonary trunk, the aorta and the aorticopulmonary septum. Thereafter TE mRNA levels increased up to ED 12, and the appearance of message was distributed distally in the walls of developing arteries. From ED 4.5 on, we found a decreasing proximo-distal gradient of the hybridization signal along the trunks and later along the main arteries (longitudinal gradient), and a radial gradient through the arterial vessel wall with the highest levels of TE mRNA in the outer layers of the media. Both gradients persisted in all major arterial vessels except in the proximal systemic and pulmonary trunks, where the original radial gradient was inverted or locally bimodal during the second half of development. The valvular region of aortic and pulmonary trunks showed particularly striking patterns of TE mRNA distribution, notably a prominent label on the endothelial cell layer on aortic and pulmonary valves. Outside the cardiovascular system, TE mRNA was mainly present in prochondral or perichondral cells in trachea and growing skeleton, and in the gap of growing joints. In kidney or nephric primordia, TE mRNA was only detectable in the wall of renal arteries. A hybridization signal was observed on mesenchyme of pulmonary septae at ED 16. Our results suggest a complex regulation of elastin gene expression during development, particularly within the proximal regions of the large arterial vessels.

Animals↗