Hervé Lemaréchal

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 (O2•-) 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). O2•- 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 O2•- 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 O2•- than those from controls. Nifedipine treatment considerably decreased O2•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2•- 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 O2•- 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.

Protein oxidative stress markers in peritoneal fluids of women with deep infiltrating endometriosis are increased

STUDY QUESTION Are protein oxidative stress markers [thiols, advanced oxidation protein products (AOPP), protein carbonyls and nitrates/nitrites] in perioperative peritoneal fluid higher in women with histologically proven endometriosis when compared with endometriosis-free controls? SUMMARY ANSWER Protein oxidative stress markers are significantly increased in peritoneal fluids from women with deep infiltrating endometriosis with intestinal involvement when compared with endometriosis-free controls. WHAT IS KNOWN ALREADY Endometriosis is a common gynaecologic condition characterized by an important inflammatory process. Various source of evidence support the role of oxidative stress in the development of endometriosis. STUDY DESIGN, SIZE, DURATION We conducted a prospective laboratory study in a tertiary-care university hospital between January 2011 and December 2012, and included 235 non-pregnant women, younger than 42 year old, undergoing surgery for a benign gynaecological condition. PARTICIPANTS/MATERIALS, SETTING, METHODS After complete surgical exploration of the abdomino-pelvic cavity, 150 women with histologically proven endometriosis and 85 endometriosis-free controls women were enrolled. Women with endometriosis were staged according to a surgical classification in three different phenotypes of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deeply infiltrating endometriosis (DIE). Perioperative peritoneal fluids samples were obtained from all study participants. Thiols, AOPP, protein carbonyls and nitrates/nitrites were assayed in all peritoneal samples. MAIN RESULTS AND THE ROLE OF CHANCE Concentrations of peritoneal AOPP were significantly higher in endometriosis patients than in the control group (median, 128.9 µmol/l; range, 0.3-1180.1 versus median, 77.8 µmol/l; range, 0.8-616.1; P < 0.001). In a similar manner concentrations of peritoneal nitrates/nitrites were higher in endometriosis patients than in the control group (median, 24.8 µmol/l; range, 1.6-681.6 versus median, 18.5 µmol/l; range, 1.6-184.5; P < 0.05). According to the surgical classification, peritoneal fluids protein AOPP and nitrates/nitrites were significantly increased only in DIE samples when compared with controls (P < 0.001 and P < 0.05; respectively), whereas the others forms of endometriosis (SUP and OMA) showed non-statistically significant increases. We found positive correlations between peritoneal fluids AOPP concentrations, nitrites/nitrates levels and the total number of intestinal DIE lesions (r = 0.464; P < 0.001 and r = 0.366; P = 0.007; respectively). LIMITATIONS, REASONS FOR CAUTION Inclusion of only surgical patients may constitute a possible selection bias. In fact, our control group involved women who underwent surgery for benign gynaecological conditions. This specificity of our control group may lead to biases stemming from the fact that some of these conditions, such as fibroids, ovarian cysts or tubal infertility, might be associated with altered peritoneal proteins oxidative stress markers. WIDER IMPLICATIONS OF THE FINDINGS We demonstrate the existence of a significantly increased protein oxidative stress status in peritoneal fluid from women with endometriosis especially in cases of DIE with intestinal involvement. This study opens the way to future more mechanistics studies to determine the exact role of protein oxidative stress in the pathogenesis of endometriosis. Even if an association does not establish proof of cause and effect, these intrinsic biochemical characteristics of endometriosis may lead to the evaluation of therapeutic approaches targeting oxidative imbalance. STUDY FUNDING/COMPETING INTERESTS No funding was used for this study. The authors have no conflict of interest.

trans-Resveratrol downregulates Txnip overexpression occurring during liver ischemia-reperfusion.

Txnip (thioredoxin-interacting protein) is a protein with multifunctional roles in cellular responses and stress-related diseases. Txnip is involved in intracellular redox regulation and has been recently described as a possible link between redox state and metabolism. trans-Resveratrol (T-res) is a natural phytoalexin with antiproliferative, antiapoptotic and antioxidative effects. However, to date there have been no reports of the implication of Txnip in a model of liver acute stress such as ischemia-reperfusion (I/R) and no work has looked for a T-res effect on Txnip. Here we studied the effects of a post-ischemic treatment of T-res on the liver thioredoxin (Trx)/Txnip system and investigated whether the T-res effects were dependent on *NO production. In this work, liver I/R induced hepatic Txnip expression and T-res inhibited I/R Txnip expression. This decrease in Txnip expression by T-res was associated with an increase in liver Trx redox activity and a decrease in hepatic I/R-induced Trx-1 expression with no effect on Trx-2, on plasma Trx redox activity or on liver and plasma Trx reductase activity, independently of *NO production. In conclusion, these results show that in our model, not only did T-res protect Trx redox activity by diminishing the Txnip protein expression; it also reduced secretion of Trx1. This is the first report of a major implication of the Trx1/Txnip system in hepatic I/R injuries. It also affirms the importance of the antioxidant effect of T-res on the Trx1/Txnip system.

Reactive oxygen species-mediated killing of activated fibroblasts by arsenic trioxide ameliorates fibrosis in a murine model of systemic sclerosis.

OBJECTIVE In patients with systemic sclerosis (SSc), activated fibroblasts produce reactive oxygen species (ROS) that stimulate their proliferation and collagen synthesis. By analogy with tumor cells that undergo apoptosis upon cytotoxic treatment that increases ROS levels beyond a lethal threshold, we tested whether activated fibroblasts could be selectively killed by the cytotoxic molecule arsenic trioxide (As(2) O(3) ) in a murine model of SSc. METHODS SSc was induced in BALB/c mice by daily intradermal injections of HOCl. Mice were simultaneously treated with daily intraperitoneal injections of As(2) O(3) . RESULTS As(2) O(3) limited dermal thickness and inhibited collagen deposition, as assessed by histologic examination and measurement of mouse skin and lung collagen contents. As(2) O(3) abrogated vascular damage, as shown by serum vascular cell adhesion molecule 1 level, and inhibited the production of autoantibodies, interleukin-4 (IL-4), and IL-13 by activated T cells. These beneficial effects were mediated through ROS generation that selectively killed activated fibroblasts containing low levels of glutathione. CONCLUSION Our findings indicate that treatment with As(2) O(3) dramatically improves skin and lung fibrosis in a mouse model of SSc, providing a rationale for the evaluation of As(2) O(3) treatment in patients with SSc.

Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes

The thioredoxin/thioredoxin reductase system is strongly induced in patients with rheumatoid arthritis (RA). We have investigated the impact on TR activity of doses of superoxide anion generated by the hypoxanthine (HX)/xanthine oxidase (XO) system and by hydrogen peroxide, H2O2, for various times and compared the findings with synoviocytes obtained from osteoarthritis (OA) patients. At baseline, TR activity in RA cells was significantly higher than in OA cells (2.31 ± 0.65 versus 0.74 ± 0.43 mUnit/mg protein, p < 0.01). HX/XO and H2O2 in RA cells decreased TR activity, which was found to be unchanged in OA cells. H2O2 and superoxide anion caused a time-dependent accumulation of oxidized TR and induced the formation of carbonyl groups in TR protein in RA cells rather than OA cells, and oxidized the selenocysteine of the active site. The oxidation in TR protein was irreversible in RA cells but not in OA cells. In conclusion, we report that the oxidative aggression generates modifications in the redox status of the active site of the TR and induces an alteration of the Trx/TR system, concomitant with those of the other antioxidant systems that could explain the causes of oxidative stress related to RA disease.

Increased Serum Oxidative Stress Markers in Women with Uterine Leiomyoma

Background Uterine leiomyomas (fibroids) are the most common gynaecological benign tumors in premenopausal women. Evidences support the role of oxidative stress in the development of uterine leiomyoma. We have analysed oxidative stress markers (thiols, advanced oxidized protein products (AOPP), protein carbonyls and nitrates/nitrites) in preoperative sera from women with histologically proven uterine leiomyoma. Methodology/Principal Findings We conducted a laboratory study in a tertiary-care university hospital. Fifty-nine women with histologically proven uterine leiomyoma and ninety-two leiomyoma-free control women have been enrolled in this study. Complete surgical exploration of the abdominopelvic cavity was performed in each patient. Preoperative serum samples were obtained from all study participants to assay serum thiols, AOPP, protein carbonyls and nitrates/nitrites. Concentrations of serum protein carbonyl groups and AOPP were higher in leiomyoma patients than in the control group (p=0.005 and p<0.001, respectively). By contrast, serum thiol levels were lower in leiomyoma patients (p<0.001). We found positive correlations between serum AOPP concentrations and total fibroids weight (r=0.339; p=0.028), serum AOPP and serum protein carbonyls with duration of infertility (r=0.762; p=0.006 and r=0.683; p=0.021, respectively). Conclusions/Significance This study, for the first time, reveals a significant increase of protein oxidative stress status and reduced antioxidant capacity in sera from women with uterine leiomyoma.

Effects of pro- and anti-inflammatory cytokines and nitric oxide donors on hyaluronic acid synthesis by synovial cells from patients with rheumatoid arthritis.

The aim of the present study was to investigate the effects of (i) the pro-inflammatory cytokines IL (interleukin)-1beta, TNF-alpha (tumour necrosis factor-alpha), IFN-gamma (interferon-gamma) and anti-inflammatory cytokines IL-4 and IL-13, and (ii) NO (nitric oxide) donors on HA (hyaluronic acid) production by synovial cells from patients with rheumatoid arthritis. Synovial cells obtained from five patients with rheumatoid arthritis were incubated for 24 h without or with IL-1beta, TNF-alpha, IFN-gamma, or with this mixture for 24 h plus IL-4 or IL-13 for the last 6 h. The same cells were also incubated for 3-24 h without or with SNP (sodium nitroprusside) or SNAP (S-nitroso-N-acetyl-DL-penicillamine). HA secretion was determined by an immunoenzymic assay based on HA-specific binding by proteoglycan isolated from bovine cartilage. IL-1beta, TNF-alpha and IFN-gamma alone or in combination stimulated HA synthesis, whereas IL-4 and IL-13 dose-dependently inhibited HA production induced by Th1 cytokines. HA production was significantly increased by the presence of 1 mM SNP after 6 and 12 h (maximal effect). HA production was significantly increased by the presence of 0.01 and 0.1 mM SNAP after 12 h of incubation, and cells treated with 1 mM SNAP showed a maximal HA production after 24 h of incubation. In conclusion, the present study provides data concerning the regulatory role of pro- and anti-inflammatory cytokines and NO donors on HA metabolism in rheumatoid synovial cells and may help in understanding the pathophysiology of rheumatoid arthritis.

Pantethine Prevents Murine Systemic Sclerosis Through the Inhibition of Microparticle Shedding

Endothelial cell (EC) damage in systemic sclerosis (SSc) is reflected by the shedding of microparticles (MPs). The aim of this study was to show that inhibiting MP release using pantethine or by inactivating ATP‐binding cassette transporter A1 (ABCA1) ameliorates murine SSc.

Expression and extracellular release of Trx80, the truncated form of thioredoxin, by TNF-alpha- and IL-1beta-stimulated human synoviocytes from patients with rheumatoid arthritis.

Thioredoxin (Trx) plays several important roles, through changes to sulfhydryl reactions and protein interactions, in controlling cellular signalling processes in RA (rheumatoid arthritis). Trx80, the 10 kDa C-terminal truncated form of Trx, is a potent mitogenic cytokine and is involved in the Th1 response. In the present study, we have investigated the ability of synoviocytes from five RA patients to induce Trx80 after ex vivo stimulation by the pro-inflammatory cytokines IL-1beta (interleukin-1beta) and TNF-alpha (tumour necrosis factor-alpha) or by H(2)O(2). Synoviocytes from five OA (osteoarthritis) patients were used as controls. Immunoprecipitation assays using two different antibodies showed that RA, but not OA, cells expressed intact Trx80 protein in culture even when not stimulated. Treatment with pro-inflammatory cytokines alone or in combination enhanced this basal production and induced the extracellular release of Trx80 by all of the RA cells tested. Under our experimental conditions, the rate of Trx80 release from RA cells was approx. 30% of the total Trx produced. In contrast, Trx80 was not detected in response to H(2)O(2) in RA or OA synoviocyte lysates and their respective culture supernatants, indicating that the oxidative process induced by H(2)O(2) in synoviocytes was unable to modify Trx80 release. Moreover, Trx80 induced synoviocyte proliferation as evaluated by [(3)H]thymidine incorporation. These results highlight the effect of the inflammatory process on the release of both Trx and Trx80 from RA synoviocytes, and suggest that the cytokine-induced increase in Trx80 cell release may constitute a link between inflammation and the immune system in RA.