Ladislas Robert

Effect of Elastin Peptides on Vascular Tone

Elastin peptides are present in human blood. As elastin receptors exist on several cell types, especially endothelial cells, this investigation was carried out to study the effect of elastin peptides on vascular tone. For this purpose, rat aortic rings were mounted in an organ bath for isometric tension measurements. Elastin peptides (kappa-elastin) were added in the concentration range of 0.1 ng/ml to 1 microgram/ml, concentrations similar to those found in the circulating blood. In rat aortic rings, precontracted or not with noradrenaline (10(-6) M), elastin peptides induced an endothelium-dependent vasodilation. The pretreatment of aortic rings with N-omega-nitro-L-arginine methyl ester (10(-5) M), an inhibitor of nitric oxide (NO) production, or with indomethacin (10(-5) M), an inhibitor of cyclooxygenase, prevented elastin peptide-induced vasodilation. These findings suggest that elastin peptides act through the synthesis of prostanoids, leading to the production of NO. Moreover, this relaxant effect of elastin peptides was decreased or inhibited when aortic rings were treated with lactose (10(-5) to 10(-2) M) or laminin (10(-6) to 10(-4) mg/ml) whereas lactose or laminin was unable to inhibit acetylcholine-induced vasodilation. These findings suggest that the inhibitory effects of lactose and laminin are specific for elastin peptide receptors and are in agreement with previous studies on these receptors. As there is evidence of the degradation of elastin in several vascular diseases, the concept that elastin peptides may contribute to the control of vascular tone is discussed.

Action of Tropoelastin and Synthetic Elastin Sequences on Vascular Tone and on Free Ca2+ Level in Human Vascular Endothelial Cells

The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to microg/mL range. Similar concentrations of an elastin peptide preparation (kappa-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10(-13) to 10(-7) mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca2+]i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca2+]i, whereas PVGV and VGVA produced these effects only at low concentration (10(-11) mol/L). A likely candidate for mediating the elastin peptide-related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.

Elastin-elastase-atherosclerosis revisited

This review proposes reinvestigation of a topic studied in the authors laboratory over the last decades concerning the age-dependent modifications of the vascular extracellular matrix (ECM) as related to atherogenesis and its recognized risk-factors: blood lipids, lipoproteins. Most salient previous results are confronted with recent publications in this field. Age-dependent modifications of the vascular wall discussed in this review include upregulation of elastolytic enzymes, demonstrated for the first time in the vascular wall in this laboratory, matrix biosynthesis and receptor function. The progressive deposition of lipids in elastic tissues as well as the addition of lipoproteins or lipids to cell and organ cultures were shown to modify matrix biosynthesis and upregulate elastase expression. Lipid-elastin interactions exhibit a great deal of specificity as shown by the nature and amount of lipids accumulating in elastin in vivo and in vitro. Recent epidemiological studies (the EVA study) enables the confrontation of blood lipid parameters with matrix related components (serum elastase and inhibitors, elastin peptides, fibronectin) in the same blood samples. The elastin laminin receptor present on vascular cells was shown to trigger NO dependent vasodilation, and downregulation of cholesterol synthesis. Both of these functions decrease or disappear with age except the upregulation of elastase release which is preserved and increased. Recent experiments extended these findings to T-lymphocytes present also in the atherosclerotic plaque. Finally several recent publications are analyzed which give more precision on the cellular mechanisms underlying the above-described modifications.

Free radical depolymerization of hyaluronan by maillard reaction products: Role in liquefaction of aging vitreous

The degradation of hyaluronan was followed by viscosimetry and by HPLC in order to study the possible role of Maillard products (lysine-glucose) on the alteration of the vitreous gel in aging and diabetes. Lysine-glucose generated Maillard products produced a decrease of viscosity and of the number average molecular weight (Mn) of hyaluronan during a 1 h incubation at 37 degrees C. This effect was comparable to that produced by 1 U/ml of testicular hyaluronidase but was weaker than the effect of a Fenton-type reagent (Udenfriends reagent). The polydispersity of hyaluronan incubated with Maillard products appeared higher than with hyaluronidase suggesting a more random reaction. Antioxydant enzymes (SOD, catalase), the iron chelators (desferrioxamine, transferrin) and the free radical scavengers (uric acid, carnosine) inhibited the degradation by Maillard products confirming its free radical nature and the intervention of trace metals. Maillard products have been detected in diabetic vitreous and may play a role in its accelerated modifications (liquefaction) in diabetes as compared to normal aging.

Aging of the vascular wall: serum concentration of elastin peptides and elastase inhibitors in relation to cardiovascular risk factors. The EVA study

The relations of biological markers of extracellular matrix (plasma elastin peptides and elastase inhibitors) to the clinical history of cardiovascular diseases and risk factors for atherosclerosis were examined in a large population study (the EVA Study) on vascular and cognitive aging performed in 1389 men and women aged 59-71 years. A moderate decrease in elastin peptides was observed in women with a self-reported history of coronary heart disease (P < 0.091) and stroke (P < 0.03) as well as with diabetes (P < 0.043). Similar but non-significant trends were found in men. Furthermore, elastin peptides were significantly and positively correlated to HDL-cholesterol and apolipoprotein A1 in both sexes. On the other hand, elastase inhibitor titers were significantly higher in women than in men. A moderate increase was also found in men (P < 0.097) and women (P < 0.068) with a history of coronary heart disease that reached significance level after pooling both sexes (P < 0.014). Furthermore, elastase inhibitor titers were significantly and positively related to fibrinogen and C reactive protein in either sex. No consistent associations were observed between both biological markers of extracellular matrix and age, blood pressure, body mass index and tobacco or alcohol consumption. These results suggest that a decrease in elastin peptides and an increase in elastase inhibitors might be associated with risk factors of atherogenesis as well as with atherosclerosis-related diseases.

Biological effects of hyaluronan in connective tissues, eye, skin, venous wall. Role in aging

Hyaluronan, as most macromolecules of the extracellular matrix, are produced by the differentiated mesenchymal cells. These cells produce also enzymes degrading hyaluronan. This results in the presence of several hyaluronan pools of different molecular weights, all capable of interacting with surrounding cells, mediated by hyaluronan binding proteins and receptors. These interactions modulate cell phenotype and produce a variety of effects conditioning the specific functions of tissues. We shall discuss here several examples studied in our laboratory, concerning skin, cornea and the venous wall. Some of these actions might even be harmful, and could play an important role in aging of connective tissues with loss of function. Some of these age-dependent modifications mediated by hyaluronan will be reviewed and commented, especially the upregulation of matrix degrading enzymes as MMP-2 and MMP-9. We shall also mention some of our experiments for finding molecules capable of counteracting the harmful effects mediated by hyaluronan.

EFFECT OF HYALURONAN ON MMP EXPRESSION AND ACTIVATION

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form.

Age-related alterations in the signal transduction pathways of the elastin-laminin receptor

With aging we assist to alterations in the vascular structure and function. One important factor in these vascular wall changes is the degradation of the elastin fibre major protein: elastin. Elastin peptides derived from the degradation are present in human sera. Elastin peptides induce on fibroblasts, phagocytic cells, lymphocytes, smooth muscle cells and endothelial cells, a variety of biological effects mediated by the elastin-laminin receptor which has been demonstrated to be present on the membrane of these cells. The transduction pathway of the ELR receptor involves the activation of phospholipase C (PLC) by a pertussis toxin sensitive G-protein. PLC induces the production of inositol trisphosphate (IP3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of PKC leading to the phosphorylation of members of the MAPK family, such as p42/p44 MAPK. A progressive age dependent uncoupling of the elastin-laminin receptor occurs impairing its transduction pathway and which results in alteration of the calcium signaling and loss in calcium homeostasis of the cells. These alterations in the signal transduction of the elastin-laminin receptor result in modified activities of parenchymal and phagocytic cells with aging, such as free radical production and elastase release. Thus, these age-related alterations in the elastin-laminin receptor signal transduction may be involved in the atherogenesis.

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.

Serum elastase activity is elevated in migraine

Migraine has been associated with diseases considered to be related to extracellular matrix disorders—in particular, cervical artery dissection. In this population‐based study, we found a highly significant association between migraine and the activity of serum elastase, a metalloendopeptidase degrading specific elastin‐type amino acid sequences. Such enzymes are involved in matrix degradation. This association was seen in both sexes and was stronger for migraine with aura. These findings could help in the understanding of why patients with migraine are at higher risk of stroke. Further study is needed to establish whether extracellular matrix abnormalities play a broader role in the pathophysiology of migraine. Ann Neurol 2000;47:648–651