L. Robert

Identification and signal transduction mechanism of elastin peptide receptor in human leukocytes

The existence of a novel receptor on human polymorphonuclear leukocytes (PMNLs) and monocytes was demonstrated, named soluble elastin peptide receptor. Soluble elastin peptides, like K‐elastin, which are liberated from elastin fibres, can be found in the sera, and they possess several biological activities such as chemotaxis. Studying the effects of elastin peptides on leukocytes, it was found that: (i) the elastin peptide stimulates the oxidative burst, the intracellular free Ca2+ elevation through a specific receptor; and (ii) in the signal transduction mechanism of this elastin peptide receptor, the phosphatidylinositol breakdown is involved.

Molecular biology of elastin as related to aging and atherosclerosis

Abstract The authors consider in some detail the structure of elastin at the molecular level as related to its normal function and to the modifications accompanying aging and atherosclerosis. 1. (1) The peculiar physicochemical properties of elastin can be best explained by assuming the prevalence of hydrophobic interactions as the main stabilizing forces of its tertiary and quaternary structure. Detailed investigation of the acceleration of alkalin hydrolysis of elastin by a variety of organic solvents confirms this assumption (Kornfeld-Poullain and Robert, 1968). It is postulated that lipidic substances do share with organic solvents their affinity for elastin and many accelerate its “unfolding” and degradation through such “denaturing” effect. Elastin surface are measurements with 85 Kr and by flow calorimetry confirmed the direct accessibility of elastin in native stroma of aorta to small molecules (Robert et al. , 1970b; Stack and Robert, 1970). 2. (2) The study of the sugar components of elastin suggested the presence of structural glycoproteins (SGP) as “impurities” in all purified elastin samples investigated. These SGP-components are very probably identical with the “microfibrils” seen on electron-micrograms of elastin. 3. (3) In vitro incorporation of 14 C-lysine were carried out in aortas of rabbits immunized with purified elastin and SGP or fed on cholesterol rich diet. A strong labelling of the SGP-fraction was obtained and a significant labelling of the polymeric collagen and elastin fractions also. Incorporation was higher in strongly atheromatous aortas. It is concluded that at least a partial “derepression” of elastin synthesis may occur in such conditions. 4. (4) The antigenic properties of elastin can be ascribed also to the presence of both components, polymerized pro- or tropoelastin and SGP-s (microfibrils). The presence of anti-elastin antibodies in human sera was described (Stein et al. , 1965; Crouzet et al. , 1970). These antibodies appear at about 20 yr of age and decrease or disappear at 70–80 yr. The lower titres found in severely atheramatous sera was ascribed to the adsorbtion of these antibodies to degrading elastic fibers in the aorta and elsewhere. Arguments in favor of our immunological theory of atheromatosis are discussed (see Fig. 6) in the light of recent experiments. Such recent findings are the production of atheromatosis in rabbits immunized with elastin or aorta SGP-preparations and the isolation of a platelet protease capable of attacking elastin (Robert et al. , 1969).

Interaction between elastin and elastases and its role in the aging of the arterial wall, skin and other connective tissues. A review ☆

Elastic fibers are progressively lysed during maturation and aging and in an accelerated fashion in several aging diseases such as diabetes, arteriosclerosis, emphysema and several skin diseases. Several enzymes (elastase-type proteases) were isolated in recent years in our laboratory which appear to be involved in these processes. A cell membrane bound serine protease was isolated from arterial smooth muscle cells and was shown to increase with in vitro aging of the cells. A metallo-protease was isolated from skin fibroblasts and was shown to be capable of attacking the constituents of elastic fibers, mainly the microfibrillar glycoproteins and also the desmosine cross linked elastin in vivo. This partially purified fibroblast enzyme was shown to attack these elastic fibers when injected into the dermis. A new selective staining procedure was used to visualise and quantitate, by computerized image analysis, the skin elastic fibers in normal and pathological human or animal skin biopsies. This method, combined with the injection of elastase in rabbit skins, alone or together with inhibitors, enables the ex vivo/in vivo study of elastase action (and of its inhibition).

Extraction and fractionation of the media of the thoracic aorta:: Isolation and characterization of structural glycoproteins☆

The purpose of this work was to reach a better understanding of the structure and composition of the arterial wall in terms of macromolecular architecture. First we applied to the arterial wall an extraction-fractionation procedure that had already given useful results with other connective tissues. The method comprises four stages. 1. (1) Exhaustive extraction of the freely diffusible molecules with a 10% CaCl2 solution. 2. (2) The remaining polymeric fibrous network was “solubilized” by controlled hydrolysis in 2.7% TCA. 3. (3) This was followed by 8 M urea with 0.1 M mercaptoethanol. 4. (4) The final residue corresponds to elastin, and was brought into solution with 1 M KOH in 80 % ethanol (K elastin). The CaCl2 extract contained the freely diffusible and soluble proteins (plasma proteins), proteoglycans and salt-soluble collagen. The second extract contained polymeric collagen (as acid-processed gelatin) and the attached proteoglycans. The third extract contained the structural glycoprotein fraction (SGP) with some “bound lipids” and hydrophobic peptides, partly derived from elastin. A preliminary characterization of these fractions is reported, based on the quantitative determination of the sugar components, hydroxyproline and total protein, and on the chromatographic characterization of phosphorus containing compounds and amino acids. The crude urea extract (No. 3) was subfractioned, and the fractions characterized by the same methods. The purified structural glycoprotein fraction was separated into a water-soluble and a urea-soluble fraction. Some of the physicochemical (sedimentation velocity, cellogel electrophoresis) and chemical properties of these fractions were determined, as was the nature of the carbohydrate moiety and the amino acid composition. The water-soluble and urea-soluble fractions of the SGP gave similar results. The composition of the aorta-SGP fraction was in many respects similar to that of the SGP fraction isolated from other connective tissues. The small differences in the sugar and amino acid compositions can however explain the organ and species specificity of this fraction found by immunochemical techniques.

ISOLATION, PURIFICATION AND PROPERTIES OF AORTIC ELASTASE

An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16].

Lésions artérielles produites chez le lapin par immunisation avec l'élastine et les glycoprotéines de structure de l'aorte Etudes biochimiques et morphologiques

Abstract Arterial lesions produced in rabbits by immunisation with elastin and structural glycoproteins of aorta Biochemical and morphological studies The production of arterial lesions is described by immunisation with purified extracts of human and porcine aorta, as well as the morphological study of the lesions produced. The antigens used are: 1. (a) CTC-extract of human aorta, containing water soluble proteins and proteoglycans of the aorta, 2. (b) 8 M urea-extract of human and porcine aorta, containing the structural glycoprotein (SGP) component and part of the “bound lipids” of the aorta, 3. (c) purified elastin from human and porcine aorta. The chemical (carbohydrate and amino acid composition) and immunochemical properties of these antigens are described. Cross-reactions between these antigens and their antisera indicate the presence of a water soluble form of SGP and of elastin in the CTC extract and the presence of a small amount of SGP in purified elastin. Serum cholesterol rose significantly in the sera of all rabbits (control and treated), during the 1st month, the highest elevation has been observed in sera of rabbits immunised with the urea-extract. β-Lipoprotein rose continuously in the sera of all rabbits. These effects may be attributed to the diet and to aging of the rabbits. The antibody titer was highest in the sera of rabbits immunised with the CTC-extract of aorta, somewhat lower with the urea-extract and lowest with elastin as sensitizing antigen. All antigens produced specific delayed type of hypersensitivity reactions. About 25% of all sensitized rabbits developed macroscopic arterial lesions, covering the whole ascending aorta, the arch and the thoracic aorta, with strongly fibrous, calcifying lesions. More than 70% of the elastin-treated rabbits and 50% of the SGP-treated rabbits showed lesions, macroscopically visible, or detected by optical or electron microscopy. The CTC-extract was much less efficient in inducing such lesions and the control rabbits did not show any of these lesions. All the lesions were characterised by early and massive destruction of the elastic tissue, which became fragmented and calcified. Intercellular fibrosis has been noted in the media and intima, together with cellular necrosis. The pathogenic efficiency of the antigens appeared to be inversely related to the circulating antibody titer they elicited. No signs of a cellular immuno-reaction could be demonstrated in the lesions. These results confirm the pathogenic efficiency of the antigens derived from the insoluble polymeric stroma of the arterial wall in inducing arterial lesions on prolonged immunisation. This result is in agreement with the theory in which we tried to explain the immunological mechanism of atherosclerosis.

On the multiplicity of cellular elastases and their inefficient control by natural inhibitors.

Several elastic tissue diseases are mainly characterized by the conspicuous fragmentation and loss of orientation of the elastic fibers and it is now generally accepted that the degradation of elastin plays a primordial role in the development of human diseases as emphysema and arteriosclerosis1,2. This generalized age-dependent lysis of elastin, which is accelerated under pathological circumstances, has been attributed to the proteolytic action of endopeptidases designated as elastases3. Such an example is the specific degradation of elastin in lung tissue which is considered as a necessary requirement for the experimental induction of emphysema. The severity of the induced lesion is directly related to the elastolytic efficiency of the intratracheally infused protease4.

Isolation and Partial Characterization of an Elastase-Like Protease from Rat Aorta Smooth Muscle Cells. Possible Role in the Regulation of Elastin biosynthesis

An elastase-like protease was isolated from rat aorta smooth muscle cells and partially characterized. It appears to behave as an intracellular enzyme and may be involved in the regulation of elastin biosynthesis.

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.

Composition of glycopeptides obtained by proteolytic digestion of the media of porcine aorta.

Abstract Glycopeptides were isolated from the pronase-collagenase digested polymeric fibrous stroma of porcine aortic media. The glycopeptides were separated by gel filtration and high voltage electrophoresis and their sugar and aminoacid composition was determined. Two major types of glycopeptides could be isolated: one (type I) similar to the heteropolysaccharides isolated from structural glycoproteins of other connective tissues containing galactose, glucose, mannose, glucosamine, galactosamine, fucose and sialic acid. The other (type II) of low molecular weight corresponding to the hydroxylysylgalactoside and hydroxylysylgalactosido-glucoside, was previously isolated from several purified collagen preparations. Type I glycopeptides turned out to be very heterogenous and the seven fractions separated by high voltage electrophoresis all had different carbohydrate compositions. One fraction was devoid of sialic acid and fucose. The sialic acid/fucose ratio increased regularly with the anodic mobility of these fractions. The following chemical parameters were estimated from these results and are considered to be characteristic of the morphological pattern of aortic media: hexose content of the polymeric stroma of aorta, 6.6%; ratio of hydroxylysylglycopeptide/ heteropolysaccharides (ratio of glycopeptides of type II/type I), ∼ 1; ratio of hydroxylysyl mono- and disaccharide ( Hyl-Gal Hyl-GaIGIc ), ∼1. These chemical parameters were previously shown to be closely related to the morphological parameters (as fiber diameter) of the fibrous stroma of different connective tissues.