Nifedipine decreases sVCAM-1 concentrations and oxidative stress in systemic sclerosis but does not affect the concentrations of vascular endothelial growth factor or its soluble receptor 1

Microvascular injury, oxidative stress, and impaired angiogenesis are prominent features of systemic sclerosis (SSc). We compared serum markers of these phenomena at baseline and after treatment with nifedipine in SSc patients. Forty successive SSc patients were compared with 20 matched healthy subjects. All SSc patients stopped taking calcium-channel blockers 72 hours before measurements. Twenty SSc patients were also examined after 14 days of treatment with nifedipine (60 mg/day). Quantitative ELISA was used to measure the serum concentrations of vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), carbonyl residues, and advanced oxidation protein products (AOPP). The median concentrations of VEGF, sVEGFR-1, sVCAM-1, carbonyl residues, and AOPP were significantly higher in SSc patients than in healthy subjects at baseline. A correlation was found between VEGF concentration and carbonyl residue concentration (r = 0.43; P = 0.007). Nifedipine treatment led to a significant decrease in concentrations of sVCAM-1, carbonyl residues, and AOPP but did not affect concentrations of VEGF and sVEGFR-1. Nifedipine treatment ameliorated endothelium injury in patients with SSc, as shown by the concentrations of adhesion molecules and oxidative damage markers. The fact that VEGF and sVEGFR-1 concentrations were not changed whereas oxidative stress was ameliorated by nifedipine is consistent with the hypothesis that VEGF signalling is impaired in SSc. However, more experimental evidence is needed to determine whether the VEGF pathway is intrinsically defective in SSc

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O(2)(•-)) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O(2)(•- )production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O(2)(•- )production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O(2)(•- )than those from controls. Nifedipine treatment considerably decreased O(2)(•- )production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O(2)(•- )production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O(2)(•- )overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress

Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes

SummaryObjectiveHypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions.MethodsHuman cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-β (IL-1β), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2− production, NOX subunit expression and nitrosylation were also assessed.ResultsiNOS expression and nitrite (but not peroxynitrite) production were significantly increased under H/R conditions when compared with to normoxia (P < 0.05). H/R conditions decreased O2− production from ∼0.20 to ∼0.12 nmol min−1 mg proteins−1 (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2− production was restored at normoxic levels (0.21 nmol min−1 mg of proteins−1).ConclusionsOur results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia

Production d'espèces radicalaires oxygénées dans la polyarthrite rhumatoïde (étude sur culture de synoviocytes humains)

La production de l anion superoxyde (O2·-) est étudiée sur des synoviocytes en culture provenant de patients atteints de polyarthrite rhumatoïde (PR). Notre travail montre une production basale d O2·-. L IL-1b et le TNF-a stimulent la génération d O2·-. Deux isoformes de NADPH oxydase (NOX2 et NOX4) sont impliquées. Les liens entre production d O2·- et phospholipase A2 (PLA2) sont explorés : l utilisation d inhibiteurs spécifiques montre l implication de 2 types de PLA2, l un cytosolique, l autre sécrétoire. L acide arachidonique, produit de ces enzymes, stimule la production d O2·-. Il agit essentiellement sur NOX2 en modulant le fonctionnement d un canal à protons. L importance du processus d hypoxie-reperfusion dans la PR nous conduit enfin à utiliser un modèle d hypoxie/réoxygénation (H/R). L H/R entraîne une diminution de la production d O2·- ; l activité NO synthase inductible est stimulée, ce qui conduit à une inactivation des sous-unités NOX2 et NOX4 par nitrosylation.We studied superoxide anion (O2·-) production by cultured synoviocytes obtained from patients with rheumatoid arthritis (RA). We identified a basal O2·- production. IL-1b and TNF-a stimulated this production. Two NADPH oxidase isoformes (NOX2, NOX4) are implicated. We evaluated the link between O2·- production and phospholipase A2 (PLA2). Using specific inhibitors, we showed the implication of both cytosolic and secretory PLA2. Arachidonic acid (AA), produced by these enzymes, induces O2·- production. AA potentiates NOX2 through the modulation of proton channel opening. As hypoxia/reoxygenation (H/R) is an important feature in rheumatoid arthritis, we lastly used a model of H/R. H/R leads to an inhibition of O2·- production that is associated to an increased inducible NO synthase expression and the inactivation of NOX2 and NOX4 by nitrosylationPARIS-BIUP (751062107) / SudocSudocFranceF

Implication du stress oxydant et propriètés anti-oxydantes des inhibiteurs calciques dihydropyridiniques au cours de la sclérodermie systémique

Les radicaux libres pourraient être un des médiateurs clés des désordres successifs vasculaires, immunitaires et fibrotiques qui sont associés à la sclérodermie systémique. Notre travail met en avant le rôle des monocytes circulant dans la production excessive d'anion superoxyde qui pourrait expliquer les augmentations concomitantes de différents marqueurs du dommage oxydatif constatées dans cette affection. Nous avons pu montrer qu'un traitement par inhibiteurs calciques dihydropyridiniques diminue cette activation monocytaire et les concentrations des marqueurs circulant du stress oxydant. Cette classe médicamenteuse agit sur la production d'anion superoxyde par l'inhibition des phosphorylations protéiques normalement induites par le PMA et en diminuant l'activité des protéines kinasesPARIS5-BU-Necker : Fermée (751152101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Le système thiorédoxine / thiorédoxine réductase dans la polyarthrite rhumatoïde (étude sur synoviocytes humains en culture)

Le rôle du système thiorédoxine/thiorédoxine réductase dans le maintien de l homéostasie redox cellulaire est étudié dans la polyarthrite rhumatoïde. Dans une première partie, la valeur de ces paramètres dans la circulation générale est établie en les reliant à l activité de la maladie évaluée par le DAS (disease activity score) et aux marqueurs classiques de l oxydation protéique. Puis, sur synoviocytes humains en culture, nous étudions l action des espèces radicalaires (anion superoxyde, peroxyde d hydrogène et peroxynitrite) et celle de cytokines pro-inflammatoires (tumor necrosis factor-a, interleukine-1b) en déterminant l activité de ce système et sa régulation. Enfin, nous mettons en évidence la génération d une forme tronquée de la thiorédoxine, nommée thiorédoxine80, molécule possédant des propriétés prolifératives. Au final, il apparaît que ce système joue un rôle important dans cette pathologie et puisse être au centre d une nouvelle approche thérapeutiqueThe role of the thioredoxin/thioredoxin reductase system implied in the homeostasis redox maintenance, is studied in rheumatoid arthritis disease. In a first part, we establish the value of these parameters in the general circulation by correlating them to the DAS (disease activity score), and to the protein oxidation markers. In a second part, on human synoviocytes in culture, we investigated the action of reactive species (e.g. superoxide anion, hydrogen peroxide, and peroxynitrite) and of pro-inflammatory cytokines (tumor necrosis factor-a, interleukine-1 b) by determining the activity of this system and its regulation. Lastly, we highlight the generation of a truncated form of the thioredoxin, named thioredoxin80 that exhibits proliferative properties. Thus, it appears that this system plays an important role in this disease, and can be a target in a therapeutic new approachPARIS-BIUP (751062107) / SudocSudocFranceF

Increased plasma soluble CD40 ligand concentrations in systemic sclerosis and association with pulmonary arterial hypertension and digital ulcers

Objective: To compare sCD40L concentrations in patients with systemic sclerosis and healthy controls. Methods: Quantitative sandwich ELISA was used to measure plasma sCD40L in systemic sclerosis (n = 50) and matched healthy controls (n = 20). Patients with systemic sclerosis had limited cutaneous disease (29), digital ulcers (14), pulmonary arterial hypertension (PAH) (10), pulmonary fibrosis on CT (23), positive anti-Scl70 (14), and anti-centromere antibodies (10). Calcium channel blockers were discontinued 72 hours before measurements. Results: Median (range) sCD40L concentration (pg/ml) was higher in systemic sclerosis than in controls (495 (10 to 7720) v 79 (50 to 118); p = 0.003), in limited cutaneous disease v diffuse disease (620 (20 to 7720) v 250 (10 to 2690); p = 0.005), in patients with digital ulcers v those without (1430 (36 to 7720) v 370 (10 to 2320); p = 0.002), and in those with PAH v those without (995 (15 to 3850) v 400 (10 to 7720); p = 0.048). sCD40L correlated with pulmonary arterial pressure estimated by Doppler echocardiography (r = 0.41; p = 0.005). Conclusions: The soluble form of CD40L is increased in plasma in systemic sclerosis and may be associated with vascular complications of the disease

Effects of nifedipine (NIF) on phorbol myristate acetate (PMA)-induced protein phosphorylation in monocytes as detected with the fluorescent probe ProQ-Diamond

<p><b>Copyright information:</b></p><p>Taken from "Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis"</p><p>Arthritis Research & Therapy 2004;7(1):R93-R100.</p><p>Published online 16 Nov 2004</p><p>PMCID:PMC1064885.</p><p>Copyright © 2004 Allanore et al.; licensee BioMed Central Ltd.</p> Representative gels of phosphorylated protein from homogenates of monocytes from healthy donors, treated with the indicated concentrations of nifedipine (a, b) or calphostin (CAL) (b) for 30 min and then stimulated with 100 nM PMA for 10 min. Right panels show a representative densitometric analysis of 10 protein bands (molecular masses from 15 to 60 kDa) that were strongly phosphorylated in the presence of PMA. Effects of nifedipine on the cytosolic fraction of control monocytes treated with the indicated concentrations of nifedipine for 30 min before stimulation of protein kinase C with a mixture of calcium and diacylglycerol. Results are the net phosphorylation induced by PMA, expressed relative to the PMA lane (taken as 100%)