Jean Verdetti

Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats

Glutathione peroxidase (GSh-Px), superoxide dismutase (SOD), catalase (CAT) activities and malon-dialdehyde (MDA) content were determined in heart, liver, kidney and brain of rats. Two different age groups (4 months; 24 months) were considered. GSH-Px and SOD activities decrease significantly for the aged liver and kidney. During aging, the activity of catalase increase in cardiac muscle and, in contrast, decrease in other organs. Lipids peroxidation, expressed in term of MDA formation, decrease in all the organs of the aged rats. The results indicate that: 1) the liver and kidney antioxidative defense decrease with age; 2) the enzymatic activities evolve in a different manner for different enzymes and organs. Furthermore, the results suggest that there is not any correlation between the SOD, CAT, and GSH-Px activities and the peroxidative status of the organs; thus, the age-related increase in the MDA content proposed as a criterion of aging process should be considered with caution.

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.

Effect of age on the vasodilatory action of elastin peptides

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin-laminin receptor and, at least in part, by EDRF (NO). Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4-6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10(-6)-10(-3) mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10(-3) mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin-laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin-laminin receptor changes with age. It could be hypothesized that in young animals (0-2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals. (4-12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin-laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.

Reduction of Ischemia-Induced Myocardial Necrosis in the Rat with Permanent Coronary Artery Occlusion under the Effect of Diltiazem*

Abstract The purpose of the study was to assess the ability of the calcium channel blocker diltiazem to limit infarct size in rat. Myocardial infarct size was measured by planimetry of histologic sections of serial slices of ventricular mass 48 h after left coronary artery ligation. Each section was stained for succinic dehydrogenase activity. Diltiazem was used as an intravenous infusion (30 or 100 μg.kg −1 .min −1 ) given for 20 min before and 60 min after ligation. Diltiazem-treated hearts showed a significant reduction in the volume of necrotic myocardium. However, the topography of the protection was complex. In diltiazem-treated hearts, the extent of apical necrosis was generally greater than in untreated hearts, whereas the larger degree of protection was constantly observed at the basal level. Mean arterial blood pressure and heart rate were significantly lower in diltiazem-treated rats. On the other hand, 48 h after coronary artery ligation diltiazem-treated rats maintained significantly higher cardiac contents of ATP and creatine phosphate than did untreated rats. Such a beneficial effect of diltiazem is probably related to its ability to limit calcium overloading which usually develops during ischemia and/or to its hemodynamic action which reduces cardiac work.

Highly Protective Effects Of Chronic Oral Administration Of Nicorandil On The Heart Of Ageing Rats

1. We have tested the effects of 2 month oral treatment with the KATP opener, nitric oxide (NO) donor and anti‐oxidant molecule nicorandil (0.1 mg/kg per day) on major physiological parameters and heart function of 4‐, 12‐ and 24‐month‐old rats.

Age-dependent differences in energetic status, electrical and mechanical performance of rat myocardium

Transmembrane action potential (AP), isotonic contraction and biochemical measurements were performed in 12-day, 1-, 3-, 14- and 24-month-old rat hearts. The major findings of this study are: (1) the AP and contraction duration decrease between 12 days and 1 month of age (growth period) and increase between 1 month and 24 months of age; (2) as compared with 1 month and 14 months, respectively, isotonic contraction peak shortening is lower at 12 days and 24 months of age; (3) the phosphorylation potential is higher during the postnatal period and decreases in an age-correlated manner; (4) the inorganic phosphate and glycogen contents are higher in the senescent heart. We conclude that, during the postnatal period the particular AP and the lower mechanical performances could be the result of immature sarcolemma and sarcoplasmic reticulum properties rather than modifications in the oxidative phosphorylation mechanism and by contrast, in senescent heart, that AP and contraction modifications could result from metabolic modifications.

Microfibrils and fibrillin-1 induce integrin-mediated signaling, proliferation and migration in human endothelial cells

Microfibrils are macromolecular complexes associated with elastin to form elastic fibers that endow extensible tissues, such as arteries, lungs, and skin, with elasticity property. Fibrillin-1, the main component of microfibrils, is a 350-kDa glycoprotein for which genetic haploinsufficiency in humans can lead to Marfan syndrome, a severe polyfeatured pathology including aortic aneurysms and dissections. Microfibrils and fibrillin-1 fragments mediate adhesion of several cell types, including endothelial cells, while fibrillin-1 additionally triggers lung and mesangial cell migration. However, fibrillin-1-induced intracellular signaling is unknown. We have studied the signaling events induced in human umbilical venous endothelial cells (HUVECs) by aortic microfibrils as well as recombinant fibrillin-1 Arg-Gly-Asp (RGD)-containing fragments PF9 and PF14. Aortic microfibrils and PF14, not PF9, substantially and dose dependently increased HUVEC cytoplasmic and nuclear calcium levels measured using the fluorescent dye Fluo-3. This effect of PF14 was confirmed in bovine aortic endothelial cells. PF14 action in HUVECs was mediated by αvβ3 and α5β1 integrins, phospholipase-C, inosital 1,4,5-trisphosphate, and mobilization of intracellular calcium stores, whereas membrane calcium channels were not or only slightly implicated, as shown in patch-clamp experiments. Finally, PF14 enhanced endothelial cell proliferation and migration. Hence, fibrillin-1 sequences may physiologically activate endothelial cells. Genetic fibrillin-1 deficiency could alter normal endothelial signaling and, since endothelium dysfunction is an important contributor to Marfan syndrome, participate in the arterial anomalies associated with this developmental disease.

Early effects of acute γ-radiation on vascular arterial tone

1 To determine the acute effects of irradiation on the functionality of vessel, rat aortic rings were mounted in an organ bath for isometric tension measurements and irradiated (60Co, 1 Gy min−1, 15 min). 2 Irradiation, which is without effect on non‐contracted or endothelium‐denuded vessels, led to an immediate and reversible increase in vascular tone on (−)‐phenylephrine (1 μM)‐precontracted aortic rings. The tension reached a plateau about 5 min after the beginning of irradiation. 3 The maximal radiation‐induced contraction occurred on aortic rings relaxed by acetylcholine (ACh) (1 μM). In this condition, the addition of catalase (1000 u ml−1), which reduces hydrogen peroxide, and DMSO (0.1% v/v), which scavenges hydroxyl radical, had no influence on tension level while superoxide dismutase (SOD) (100 u ml−1), a superoxide anion scavenger, reduced the observed contraction. A similar result was obtained in the presence of indomethacin (10 μM), a cyclo‐oxygenase blocker. 4 Pretreatment of rings with the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME) (10–100 μM) inhibited the radiation‐induced contraction. 5 This effect was dose rate‐dependent and even occurred for a very low dose rate (0.06 Gy min−1). 6 The present results indicate that γ‐radiation induces an instantaneous vascular tone increase that is endothelium and dose rate‐dependent. This effect is (i) maximal when nitric oxide (NO) is produced, (ii) greatly reduced by SOD and (iii) inhibited by L‐NAME, suggesting a major involvement of complexes between NO and superoxide anion.

Identification of Membrane Calcium Channels Essential for Cytoplasmic and Nuclear Calcium Elevations Induced by Vascular Endothelial Growth Factor in Human Endothelial Cells

Abstract Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the fmt stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.