Didier Borderie

High N-terminal pro-brain natriuretic peptide levels and low diffusing capacity for carbon monoxide as independent predictors of the occurrence of precapillary pulmonary arterial hypertension in patients with systemic sclerosis.

OBJECTIVE To evaluate predictors of pulmonary arterial hypertension (PAH) in a prospective cohort of patients with systemic sclerosis (SSc). METHODS Routine clinical assessments as well as measurements of the diffusing capacity for carbon monoxide/alveolar volume (DLCO/VA) ratio and N-terminal pro-brain natriuretic peptide (NT-proBNP) level were performed in a prospective cohort of 101 SSc patients who did not have PAH or severe comorbidities. After a planned 36-month followup, we evaluated the predictive value of these parameters for the development of precapillary PAH, as demonstrated by cardiac catheterization, disease progression, and death. Criteria for cardiac catheterization were a systolic pulmonary artery pressure (PAP) of >40 mm Hg on echocardiography, a DLCO value of <50% without pulmonary fibrosis, and unexplained dyspnea. RESULTS Eight patients developed PAH, 29 had disease progression, and 10 died during a median followup of 29 months. Kaplan-Meier analysis identified the following baseline parameters as being predictors of PAH: DLCO/VA ratio <70% or <60% (P<0.01 for each comparison), elevated plasma NT-proBNP level (>97th percentile of normal; P = 0.005), echocardiographically estimated systolic PAP >40 mm Hg (P=0.08), and erythrocyte sedimentation rate >28 mm/hour (P=0.015). In multivariate analyses, an elevated baseline NT-proBNP level (hazard ratio [HR] 9.97 [95% confidence interval (95% CI) 1.69-62.42]) and a DLCO/VA ratio <60% (HR 36.66 [95% CI 3.45-387.6]) were predictors of the occurrence of PAH during followup. An increased NT-proBNP level together with a decreased DLCO/VA ratio of <70% was highly predictive of the occurrence of PAH during followup (HR 47.20 [95% CI 4.90-450.33]). CONCLUSION This prospective study identified a decreased DLCO/VA ratio and an increased NT-proBNP as predictors of PAH in SSc. Use of these markers should result in improved PAH risk stratification and allow earlier initiation of therapy.

Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis

Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N‐acetyl‐cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob/ob mice generated more O2°− than those of lean littermates (P < .01). Ex vivo, all three SOD mimics decreased O2°− generation (P < .001) and totally inhibited lipid peroxidation (P < .001) versus untreated ob/ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight (P < .02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob/ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans. (HEPATOLOGY 2004;39:1277–1285.)

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.

Disturbed angiogenesis in systemic sclerosis: high levels of soluble endoglin

OBJECTIVE SSc is a CTD characterized by early generalized microangiopathy with disturbed angiogenesis. Soluble endoglin (sENG), a serum anti-angiogenic protein, has recently been described as a major actor in pre-eclampsia, another severe vascular disease with abnormal angiogenesis. The aim of this study was to investigate, in a cross-sectional study, sENG levels together with other serum vascular markers. METHODS Serum levels of sENG were assessed by ELISA in consecutive SSc patients and controls matched for age and sex. We also measured by ELISA serum levels of VEGF and asymmetric dimethylarginine (ADMA), as respective markers of angiogenesis and endothelial dysfunction. RESULTS We included 235 unrelated subjects: 187 SSc patients and 48 controls. Higher concentrations of sENG (P = 0.002) and sVEGF (P < 0.0001) were found in SSc patients compared with controls whereas there was no difference for ADMA. In multivariate analysis, sENG levels were significantly increased in SSc patients with cutaneous ulcerations (P = 0.0003), positive for ACAs (P = 0.009) and with abnormal diffusing capacity for carbon monoxide divided by alveolar volume (P = 0.03). Soluble ENG levels negatively correlated with ADMA, but no relationship was found between sENG and sVEGF. CONCLUSION This study shows increased values of sENG in a large SSc cohort and a relevant association with a vascular phenotype. The predictive value of the biomarker sENG and its potential role on cellular endothelial disturbances remain to be determined.

Bone Mineral Density Evolution After Successful Parathyroidectomy in Patients With Normocalcemic Primary Hyperparathyroidism

CONTEXT It is unclear whether bone mineral density (BMD) improves in patients with normocalcemic primary hyperparathyroidism (PHPT) after parathyroidectomy (PTX). OBJECTIVE The objective of the study was to evaluate and compare the impact of PTX on BMD change at 1 year in normocalcemic vs hypercalcemic PHPT. DESIGN This was a longitudinal cohort study. SETTING The study took place at a referral center. PATIENTS We included 60 PHPT patients (mean age 64.0 ± 10.1 years), successfully treated by PTX by the same surgeon. Two groups were individualized according to baseline serum total (albumin corrected) calcium: 39 patients with normal baseline serum total calcium (normocalcemic group) and 21 patients with hypercalcemia at baseline (hypercalcemic group). MAIN OUTCOME MEASURE BMD changes 1 year after PTX were measured. RESULTS In the normocalcemic group, BMD increased significantly by +2.3 ± 5.0% at the spine (P = .016) and +1.9 ± 5.7% at the hip (P = .048). In the hypercalcemic group, BMD increased significantly by +4.0 ± 3.8% at the spine (P = .0003) and +3.2 ± 4.2% at the hip (P = .003). There was no difference in these BMD gains between both groups (P > .1). The presence of multiple adenomas or hyperplasia was more frequent in the normocalcemic group than in the hypercalcemic group (P = .04). CONCLUSION Our results indicate for the first time that successful PTX in normocalcemic PHPT patients with osteoporosis is followed with mild but significant BMD improvement at the spine and hip at 1 year, comparable with that observed in hypercalcemic PHPT, suggesting that PTX may be beneficial in normocalcemic PHPT.

High DNA Oxidative Damage in Systemic Sclerosis

Objective. Several lines of evidence suggest that the generation of reactive oxygen species (ROS) is of major importance in the pathogenesis of SSc. Protein and lipid damage have previously been demonstrated, but scarce data are available on oxidative damage to DNA. In patients with SSc, we evaluated levels of 8-hydroxy-2’-deoxyguanosine (8-oxodG), the main validated biomarker of endogenous oxidative damage to DNA, compared to levels of F2-isoprostane, a product of free radical-mediated peroxidation of arachidonic acid. Methods. Urinary levels of 8-oxodG and 8-isoprostaglandin-F2α (8-iso-PGF2α) were determined by competitive ELISA method in consecutive SSc patients and controls matched for age and sex. Results. We included 80 unrelated SSc patients (72 women, mean age 56 ± 11 yrs) and 39 controls (33 women, mean age 64 ± 8 yrs). Urinary levels of 8-oxodG/creat and 8-iso-PGF2α/creat in SSc patients were found to be higher than in controls (6.5 ng/mg vs 3.7 ng/mg, p = 0.0001; and 11.4 ng/mg vs 4.2 ng/mg, p < 0.0001). In multivariate analysis, 8-oxodG levels were associated with the presence of pulmonary fibrosis on computerized tomography scan, decreased forced vital capacity, and decreased DLCO/alveolar volume. In patients with the diffuse cutaneous subset, a modified Rodnan skin score > 14 was independently associated with 8-oxodG levels. In SSc, 8-oxodG and 8-iso-PGF2α values were correlated (r = 0.32; p = 0.005). Conclusion. Our study confirmed marked oxidative stress in SSc. We also found increased values of 8-oxodG in SSc patients and a relevant association with a fibrotic phenotype. The predictive value of this marker and its potential influence on fibrotic disturbances remain to be determined.

Piceatannol is more effective than resveratrol in restoring endothelial cell dimethylarginine dimethylaminohydrolase expression and activity after high-glucose oxidative stress

Abstract Glucose-induced oxidative stress is involved in endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) and arginase are regulators of the endothelial NO synthase (eNOS). This study aimed to compare the effect of two polyphenolic antioxidants, resveratrol and piceatannol, on DDAH and arginase pathways in bovine aortic endothelial cells under 25 mM glucose for 24 h. DDAH activity and expression were decreased in these cells as compared to control cells, whereas arginase activity was unchanged. DDAH inhibition led to intracellular accumulation of asymmetric dimethylarginine (ADMA), a natural inhibitor of eNOS. Under these conditions, cell pre-treatment with resveratrol (0.1–10 μM) restored basal DDAH activity and ADMA level with a dose-dependent effect. Piceatannol acted as resveratrol on DDAH pathway but at 10-fold lower concentrations. Resveratrol and piceatannol restored DDAH activity even in the presence of splitomicin, a specific inhibitor of Sirtuin 1. These results suggest potential therapeutic intervention targeting resveratrol or piceatannol administration to improve endothelial dysfunction.

N-terminal pro-brain natriuretic peptide in systemic sclerosis: a new cornerstone of cardiovascular assessment?

Background: Cardiac involvement, a common and often fatal complication of systemic sclerosis (SSc), is currently detected by standard echocardiography enhanced by tissue Doppler echocardiography (TDE). Objective: The performance of the biomarker of cardiovascular disease, N-terminal pro-brain natriuretic peptide (NT-proBNP), in the detection of cardiac involvement by SSc was examined. Methods: A total of 69 consecutive patients with SSc (mean (SD) age 56 (13) years, 56 women) were prospectively studied with standard echocardiography and TDE measurements of longitudinal mitral and tricuspid annular velocities. Plasma NT-proBNP was measured in all patients. Results: Overall, 18 patients had manifestations of cardiac involvement, of whom 7 had depressed left ventricular and 8 depressed right ventricular myocardial contractility, and 8 had elevated systolic pulmonary arterial pressure. Patients with reduced contractility had increased mean (SD) NT-proBNP (704 (878) pg/ml versus 118 (112) pg/ml in patients with normal myocardial contractility, p<0.001). Similarly, NT-proBNP was higher in patients with (607 (758) pg/ml) than in patients without (96 (78) pg/ml) manifestations of overall cardiac involvement (p<0.001). Receiver operating characteristic analysis showed NT-proBNP reliably detected depressed myocardial contractility and overall cardiac involvement (area under the curve 0.905 (95% CI 0.814 to 0.996) and 0.935 (95% CI 0.871 to 0.996), respectively). Considering patients with SSc with normal echocardiography and TDE as controls, and using a 125 pg/ml cut-off concentration, sensitivity and specificity were 92% and 71% in the detection of depressed myocardial contractility, and 94% and 78% for overall cardiac involvement. Conclusions: NT-proBNP reliably detected the presence of cardiac involvement and appears to be a very useful marker to risk stratify patients presenting with SSc.

Increased Plasma S-Nitrosothiol Concentrations Predict Cardiovascular Outcomes among Patients with End-Stage Renal Disease: A Prospective Study

The plasma concentrations of S-nitrosothiols, which are circulating nitric oxide metabolites with potential biologic activity, are increased among patients undergoing chronic hemodialysis (HD). However, the ability of S-nitrosothiols to release nitric oxide at physiologically relevant sites may be reduced among HD patients, because of impaired availability and/or activity of factors involved in S-nitrosothiol breakdown. The resultant lack of S-nitrosothiol bioavailability could contribute to the high cardiovascular risk for such patients. A possible relationship between plasma S-nitrosothiol levels and cardiac outcomes, as well as all-cause mortality rates, was investigated in a cohort of 250 chronic HD patients and who were undergoing regular dialysis three times per week were monitored for 1 yr. During that follow-up period, major cardiac events and all-cause deaths were prospectively recorded. At baseline, high plasma S-nitrosothiol levels (>2 micro M, corresponding to the top quartile of all measured values) were independently associated with pulse pressure in an adjusted multivariate analysis (odds ratio, 1.03; 95% confidence interval, 1.01 to 1.05; P = 0.007). During the follow-up period, 36 patients died (16 as a result of cardiac causes) and 33 patients experienced major adverse cardiac events. In an adjusted Cox proportional-hazards model, high plasma S-nitrosothiol concentrations (i.e., the top quartile versus the three other quartiles) were an independent predictor of cardiac events (hazard ratio, 3.30; 95% confidence interval, 1.61 to 6.76; P = 0.001) but not of all-cause death. Therefore, among chronic HD patients, markedly elevated plasma S-nitrosothiol levels are associated with pulse pressure and predict cardiovascular outcomes. These findings support the hypothesis that impaired S-nitrosothiol bioavailability in uremia is an important factor for the excessive cardiovascular risk among HD patients.

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.