Monique David-Dufilho

Mitochondrial Arginase II Modulates Nitric-Oxide Synthesis through Nonfreely Exchangeable l-Arginine Pools in Human Endothelial Cells

Reduced synthesis of nitric oxide (NO) contributes to the endothelial dysfunction and may be related to limited availability of l-arginine, the common substrate of constitutive nitric-oxide synthase (NOS) and cytosolic arginase I and mitochondrial arginase II. To determine whether arginases modulate the endothelial NO synthesis, we investigated the effects of the competitive arginase inhibitor Nω-hydroxy-nor-l-arginine (Nor-NOHA) on the activity of NOS, arginases, and l-arginine transporter and on NO release at surface of human umbilical vein endothelial cells (HUVECs). In unstimulated cells, Nor-NOHA dose-dependently reduced the arginase activity with maximal inhibition at 20 μM. When HUVECs were stimulated by thrombin without extracellular l-arginine, Nor-NOHA dose-dependently increased the NOS activity and the NO release with maximal effects at 20 μM. Extracellular l-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 μM l-arginine, NOS activity and NO release were similar in untreated and Nor-NOHA-treated cells. However, despite activation of l-arginine uptake, the inhibition of arginase activity by Nor-NOHA was still significant. The depletion of freely exchangeable l-arginine pools with extracellular l-lysine did not prevent Nor-NOHA from increasing the NO release. This indicates the presence of pools, which are accessible to NOS and arginase, but not exchangeable. Interestingly, the mitochondrial arginase II was constitutively expressed, whereas the cytosolic arginase I was barely detectable in HUVECs. These data suggest that endothelial NO synthesis depends on the activity of arginase II in mitochondria and l-arginine carriers in cell membrane.

Protease-Activated Receptor-1 Antagonist F 16618 Reduces Arterial Restenosis by Down-Regulation of Tumor Necrosis Factor α and Matrix Metalloproteinase 7 Expression, Migration, and Proliferation of Vascular Smooth Muscle Cells

Wound healing after angioplasty or stenting is associated with increased production of thrombin and the activation of protease-activated receptor 1 (PAR1). The aim of the present study was to examine the effects of a new selective PAR1 antagonist, 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)-penta-1,3-dienyl]-benzonitrile (F 16618), in restenosis and vascular smooth muscle cell (SMC) proliferation and migration using both in vivo and in vitro approaches. Daily oral administration of F 16618 inhibited the restenosis induced by balloon angioplasty on rat carotid artery in a dose-dependent manner. Furthermore, single intravenous administration of F 16618 during the angioplasty procedure was sufficient to protect the carotid artery against restenosis. In vitro, F 16618 inhibited the growth of human aortic SMCs in a concentration-dependent manner with maximal effects at 10 μM. At that concentration, F 16618 also prevented thrombin-mediated SMC migration. In vivo, oral and intravenous F 16618 treatments reduced by 30 and 50% the expression of the inflammatory cytokine tumor necrosis factor α (TNFα) 24 h after angioplasty. However, only acute intravenous administration prevented the induction of matrix metalloproteinase 7 expression. In contrast, F 16618 treatments had no effect on early SMC de-differentiation and transcription of monocyte chemoattractant protein-1 and interleukin-6 and late re-endothelialization of injured arteries. Furthermore, F 16618 compensated for the carotid endothelium loss by inhibiting PAR1-mediated contraction. Altogether, these data demonstrate that PAR1 antagonists such as F 16618 are a highly effective treatment of restenosis after vascular injury, by inhibition of TNFα, matrix metalloproteinase 7, and SMC migration and proliferation in addition to an antithrombotic effect.

Superoxide release from interleukin-1β-stimulated human vascular cells: in situ electrochemical measurement

Release of superoxide anion by cultured vascular cells was investigated with the use of selective microelectrodes. Local concentration of superoxide anion (O2*-) was followed by differential pulse amperometry on a carbon microfiber at 0.1 V/SCE. The oxidation current allows O2*- detection in the 10(-8) M concentration range without interference of the other major oxygen species. Interleukin-1beta-stimulated O2*- release that progressively increased to reach local concentrations at the cell membrane level of 76 +/- 11 nm 40-60 min after stimulation in human cord vein endothelial cells, and 131 +/- 18 nm 1-2 h after stimulation in internal mammary artery smooth muscle cells. In the two types of cells, the O2*- oxidation signal was suppressed in the presence of superoxide dismutase. Spontaneous O2*-release from unstimulated cells was undetectable. These results demonstrate that selective microelectrodes allow direct and real-time monitoring of local O2*- released from vascular endothelial as well as from smooth muscle cells submitted to an inflammatory stimulus.

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion.

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production.

Métaux de transition et production de monoxyde d’azote par les cellules endothéliales humaines

Abstract The bioavailability of endothelial nitric oxide (NO) is regulated by transition metals but their mechanisms of action on NO synthesis and degradation are not clearly understood. Using differential pulse amperometry and NO microelectrodes, local NO concentration was measured at the surface of cultured human umbilical vein endothelial cells (HUVECs) stimulated by histamine or thrombin in the presence of transition metal chelators. The agonist-activated NO release required both extracellular Ca 2+ and transition metals. In the presence of 1 mM external Ca 2+ , a low concentration of EGTA (5 μM) inhibited by 40 % the NO release from stimulated HUVECs. In the presence of extracellular L-arginine, the inhibitory effect of EGTA was even more marked and, in its absence, it was suppressed by adding exogenous superoxide dismutase. The decrease in NO release induced by the copper chelators, cuprizone and DETC, suggests that extracellular traces of Cu 2+ could regulate NO availability.

Thiosulfinates modulate platelet activation by reaction with surface free sulfhydryls and internal thiol-containing proteins

Thiosulfinates are characteristic flavors of Allium vegetables, with a highly reactive S–S=O group, that we previously showed to inhibit platelet aggregation through calpain-dependent mechanisms. With the aim to clarify the mode of action of these redox phytochemicals, we studied their effect on extracellular free sulfhydryls in relation to their effect on platelet responses (Ca2+ signals, release reaction, and aIIbβ3 integrin activation state). At the platelet surface, thiosulfinate dose-dependently increased the basal level of free sulfhydryls, independently of protein disulfide isomerase activity. This generation of new free sulfhydryls was associated with: (i) a three fold increase in labeling of resting platelets with an anti ligand-induced binding site antibody and (ii) marked inhibition of subsequent aIIbβ3 activation by agonists. Thiosulfinates increased the basal intracellular Ca2+ level of platelets. In activated platelets, they markedly inhibited the Ca2+ mobilization independently of the external Ca2+, the calpain-induced SNAP-23 cleavage and the granule release. In platelet free systems, thiosulfinates inhibited the activity of purified calpain and the free sulfhydryl of glutathione without any reducing properties on disulfides. The results demonstrate for the first time that thiosulfinates rapidly interact with sulfhydryls both at the platelet surface and inside the cell on intracellular cysteine-proteins, especially calpain. Inhibition of free cysteine and glutathione in whole blood may also contribute to their anti-aggregant properties. Such sulfur compounds are of interest for the development of a new class of antithrombotic agents.

0386 : Direct thrombin inhibitors prevent atrial myocardial hypertrophy and atrial fibrillation susceptibility associated with heart failure in the rat

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia, which is characterized by a high risk of stroke due to increased thrombogenesis and thrombin formation in poorly contractile atria. In addition to its role in the coagulation cascade, thrombin has pleiotropic effects on cardiovascular system through the activation of protease activated receptor 1 (PAR 1). Here we examined whether thrombin contributes to the atrial myocardial remodeling that occurs during heart failure (HF) and whether direct thrombin inhibitors can prevent this remodeling process and AF progression. The study was conducted in a well established rat model of atrial dilation and susceptibility to AF associated with ischemic HF secondary to myocardial infarction (MI). The endogenous thrombin potential increased in plasma after MI, and remained elevated in HF rats. Treating MI rats for one or two months with direct thrombin inhibitors reduced the area and diameter of left atria. After two months, there was also a reduction of the duration of burst pacing induced AF and a downregulation of the expression of hypertrophic markers such as brain natriuretic peptide and β myosin heavy chain. The transcription factor NFATc3, known to be activated in cardiac hypertrophy, and the plasminogen activator inhibitor 1, which has both thrombotic and fibrogenic activities were also downregulated. Treating rats with a PAR 1 antagonist reproduced the effect of thrombin inhibitors. To study the role of thrombin on atrial myocardium in absence of other cardiac stimuli, atrial explant cultures were performed. In cultured atria, thrombin upregulated hypertrophic markers and plasminogen activator inhibitor 1 through PAR 1 and the Rho/Rho kinase pathway. These results indicate that thrombin is a potent hypertrophic factor for atrial myocardium; they point to chronic inhibition of thrombin/PAR1 pathways as a potential therapeutic option to prevent atrial remodeling and AF substrate formation. The author hereby declares no conflict of interest