William Hornebeck

Evidence by in vivo and in vitro studies that binding of pycnogenols to elastin affects its rate of degradation by elastases

Procyanidol oligomers and (+) catechin bound to insoluble elastin markedly affect its rate of degradation by elastases. Insoluble elastin pretreated with procyanidol oligomers (PCO) was resistant to the hydrolysis induced by both porcine pancreatic and human leukocyte elastases. The quantitative adsorption of pancreatic elastase was similar on either untreated or PCO-treated elastin suggesting that the binding of this compound to elastin increases the non-productive catalytic sites of elastase molecules. (+) Catechin-insoluble elastin complexes were partially resistant to the degradation induced by human leukocyte elastase but were hydrolysed at the same rate as untreated samples by a constant amount of pancreatic elastase. In addition, the coacervation profile of kappa-elastin peptides as a function of temperature is greatly modified in presence of these flavonoids. We conclusively evidenced that PCOs bind to skin elastic fibres when injected intradermally into young rabbits. As a result, these elastic fibres were found more resistant to the hydrolytic action of porcine pancreatic elastase when injected to the same site. These in vivo studies further emphasized the potential effect of these compounds in preventing elastin degradation by elastase(s) as occurred in inflammatory processes.

FGF protection and inhibition of human neutrophil elastase by carboxymethyl benzylamide sulfonate dextran derivatives

Several derivatized dextrans (DxD) containing defined percentage of carboxymethyl, carboxymethyl benzylamide and carboxymethyl benzylamide sulfonate groups have been shown to stimulate tissue repair in various in vivo models including skin, bone, muscle and cornea. These selected DxD were also shown to mimic heparin or heparan sulfate by their ability to interact with, stabilise and protect the heparin-binding growth factor of the fibroblast growth factor family against trypsin digestion (Tardieu et al., J. Cell. Physiol. 1992; 150: 94). The wound healing action of these DxD was explained by postulating that the endogenously released heparin-binding growth factors could be protected within the wound. To further understand the action of these DxD on tissue repair, we have studied their effect on the human neutrophil elastase (HNE) activity, one of the proteases involved in wound repair. These DxD inhibited HNE in an hyperbolic non-competitive manner. Extent of HNE inhibition by DxD increased with their molecular weight and benzylamide sulfonate substitution levels. One DxD, RGT11, was the best inhibitor (Ki 40 pM) and efficiently inhibited FGF-2 proteolysis by HNE, restoring its growth-promoting activity towards human skin fibroblasts. The data contribute to a better understanding of the wound-healing property and anti-inflammatory activity of these polymers.

Fatty acid peptide derivatives as model compounds to protect elastin against degradation by elastases

Peptide sequences which fit the extended binding sites of porcine pancreatic elastase and human leukocyte elastase were covalently coupled to oleic acid. These compounds behave as competitive inhibitors towards both elastases. The coupling of fatty acid moiety to the peptide greatly decreases its inhibitor constant (Ki) vs human leukocyte elastase (Ki for Oleoyl(Ala)2ProValine: 3.0 (10(-6)M). It is less active on porcine pancreatic elastase (Ki for Oleoyl(Ala)2ProAlanine: 3.8 10(-4)M). The modifications of the carboxylic end group of the peptide to an aldehyde further greatly enhanced the inhibition capacity of the compound towards leukocyte elastase (Ki for Oleoyl(Ala)2ProAlaninal: 0.7 microM). Oleoyl peptide derivatives were seen to bind in a saturable fashion to purified insoluble elastin, and decreased the susceptibility of the macromolecule to hydrolysis by both pancreatic and leukocyte elastases. As stoichiometric quantities of elastase (vs inhibitor) could not desorb 3H-oleoyl(Ala)2Pro-Val bound to insoluble elastin, it is postulated that oleoyl peptide derivatives may act as bifunctional agents. This contention was further strengthened by the comparison of the adsorption curves of elastase to untreated insoluble elastin and elastin saturated with oleoyl peptide derivatives respectively. It was shown finally that Oleoyl(Ala)2Pro-Valine was also capable of inhibiting elastases in their adsorbed form to insoluble elastin.

Predictions of the secondary structure and antigenicity of human and bovine tropoelastins

Secondary structure and antigenicity predictive methods have been applied to the sequences of human and bovine tropoelastins in order to have some insight into the molecular structure of its insoluble counterpart, i.e., elastin. For both tropoelastins, all the predictions yielded 11 major regions, in which the pleated conformation was predominant, separated by 10 strong helical segments of various lengths located within alanyl rich regions of the chains. The overall conformations of human and bovine tropoelastins were estimated to contain 18 ± 5% α-helices, 63 ± 17% β-sheets, 13 ± 13% β-turns and 6 ± 6% random coil. For both tropoelastins, antigenicity predictions indicated the presence of seven synthetic decapeptides corresponding to continuous linear epitopes of the molecule. Some of the predicted epitopes are located in the same regions in both species while others are not. These predictions have allowed us to propose an α/β conformation for tropoelastin. Therefore this extracellular matrix macromolecule might be more structured (10 helical segments for about 18% of the overall structure) than previously suggested.

Inhibition of the human leukocyte endopeptidases elastase and cathepsin G and of porcine pancreatic elastase by N-oleoyl derivatives of heparin

N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.

Studies on the interaction of cholesterol with soluble and insoluble elastins

Abstract The exchange of 3 H-cholesterol in an aqueous solution of taurodeoxycholate with insoluble elastin and kappa-elastin peptides has been quantitatively studied. At higher cholesterol concentrations, and in similar experimental conditions, 0.3 μg and 0.2 μg of cholesterol could be adsorbed, respectively, on to 1 mg insoluble elastin and 1 mg kappa-elastin coacervate. Therefore, the above amounts of cholesterol fixed per mg elastin represent minimal estimates. The replacement of Na + ions by Ca 2+ ions increased the cholesterol binding both of fibrous and of soluble elastins. The binding curves tend towards a limiting value in the presence of Na + , but no saturation effect was observed in the presence of Ca 2+ . The replacement of sodium ions by calcium ions in the taurodeoxycholate solutions also lowered the coacervation temperature of the kappa-elastin peptides. Fibrous elastin retained more cholesterol at 65°C than at 37°C, suggesting the importance of hydrophobic interactions in cholesterol elastin association. These results suggest that conformational changes could be induced in both soluble and insoluble forms of elastin by calcium ions increasing their affinity for cholesterol. This enhancement of cholesterol fixation by elastin in the presence of Ca 2+ ions may well have a physiopathological importance. So does also the fact that elastin-peptides exhibit a higher affinity in the presence of Ca 2+ for cholesterol than insoluble elastin, suggesting the possibility of increased cholsterol (and calcium) fixation as elastin is degraded in situ by elastases.

Neutrophil elastase as a target in lung cancer.

Human neutrophil elastase (HNE), a main actor in the development of chronic obstructive pulmonary diseases, has been recently involved in non-small cell lung cancer progression. It can act at several levels (i) intracellularly, cleaving for instance the adaptor molecule insulin receptor substrate-1 (IRS-1) (ii) at the cell surface, hydrolyzing receptors as CD40 (iii) in the extracellular space, generating elastin fragments i.e. morphoelastokines which potently stimulate cancer cell invasiveness and angiogenesis. Since decades, researchers identified natural compounds and/or synthesized agents which antagonize HNE activity that will be described in this review article. Some of these compounds might be of value as therapeutic agents in lung cancer. However, it is now widely accepted that lung tumor invasion and metastasis involve proteolytic cascades. Accordingly, we will here mainly focus our attention to natural substances able to display a dual inhibitory capacity (i.e. lipids and derivatives, phenolics) towards HNE and matrix metalloproteinases (MMPs), particularly MMP-2. To that purpose, we recently synthesize substances named LipoGalardin (Moroy G. et al., Biochem. Pharmacol., 2011, 81(5), 626-635) exhibiting such inhibitory bifunctionality. At last, we will propose an original synthetic scheme for designing a potent biheaded HNE/MMP-2 inhibitor.

Presence of gelatinase A and etalloelastase 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‐1β, 1L4, IL6), interferon γ (IFN γ) and tumor growth factor β (TGFβ) were studied for their ability to modify the levels of those plasma membrane associated proteinases. Among these mediators, only IL‐1β was found to enhance the amounts of HSF membrane‐bound HSFE1 and Gelatinase A.

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture

The properties and subcellular localization of the elastase-like activities of smooth muscle cells cultured from pig aortas have been investigated. Homogenates of the cells hydrolysed N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, a synthetic substrate for elastases, with a distinct pH optimum of 8.2 and hydrolysed insoluble elastin with a distinct pH optimum of 8.5. Both enzyme activities were directly proportional to the concentration of homogenate in the assay mixture. The activities toward both substrates were inhibited by phenylmethylsulphonyl fluoride and were therefore probably due to a serine peptidase(s). The activities were also inhibited by EDTA and, in a dose-related manner, by alpha 1-antiprotease. Pepstatin, which inhibits cathepsin D, and leupeptin, which inhibits cathepsin B, did not significantly inhibit the elastase-like activities in these cells. The cells were homogenized and a post-nuclear supernatant subjected to sucrose density gradient centrifugation. The distribution of elastase-like activity toward both substrates was similar to that of the plasma membrane marker 5-nucleotidase, and distinct from those of marker enzymes for the other organelles. Cells were also homogenized with digitonin, which selectively increases the equilibrium density of the plasma membrane. The equilibrium densities of both 5-nucleotidase and of the elastase-like activities were increased considerably, confirming the plasma membrane localization of the elastase-like activities. The subcellular localization of the elastase-like activities of arterial smooth muscle cells is therefore consistent with a role for them in the degradation of elastin in the normal arterial wall and in atherosclerotic lesions.

Ultrastructural and biochemical modifications of rabbit arteries induced by immunization with soluble elastin peptides

Immunization of rabbits with soluble elastin peptides (kappa 1-elastin) in complete Freunds adjuvant resulted in morphological and biochemical modifications in aorta and in lung arterioles. The elastic fibers of both tissues appeared fragmented at the light microscopical and ultrastructural levels. The presence of IgG at the site of lysed elastic fibers could be evidenced by immunological techniques. In agreement with these findings, a significantly increased elastase-type protease activity could be demonstrated in aorta extracts from the immunized animals as compared to those obtained from control animals. The biosynthetic activities of aorta explants of rabbits immunized with kappa 1-elastin maintained in organ culture conditions were considerably reduced, as shown by the decrease of incorporation of [14C]lysine and [14C]glucosamine in aorta macromolecules. These results show that anti-elastin antibodies may well be involved in the pathological modifications of the arterial wall and especially in the triggering of the degradation of elastic lamellae.