Elisa Pagnin

Rosiglitazone Reduces Glucose-Induced Oxidative Stress Mediated by NAD(P)H Oxidase via AMPK-Dependent Mechanism

Objective—Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. Methods and Results—Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPAR&ggr;, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. Conclusions—This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPAR&ggr;-independent mechanism.

Gender Differences in Endothelial Progenitor Cells and Cardiovascular Risk Profile The Role of Female Estrogens

Objective—Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state. Methods and Results—As measured by flow-cytometry in 210 healthy subjects, CD34+KDR+ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17&bgr;-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo. Conclusions—EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.

Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II.

Context RGS2 (regulators of G-protein signaling) is a negative regulator of Gαq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. Objective To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. Methods PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca2+ measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3′ untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). Results RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca2+ increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca2+ mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Conclusion Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

Co-stimulatory modulation in rheumatoid arthritis: The role of (CTLA4-Ig) abatacept

Associations between rheumatoid arthritis (RA) susceptibility and polymorphism in multiple immunoregulatory genes suggest a role of altered T cell function in the disease. The growing relevance of the oxidative stress in RA synovitis, which results in a number of T cell signalling abnormalities, is reinforced by the demonstration of a direct NO inducing activity through the shared epitope of the HLA class II molecules HLA-DRbeta1, with secondary lymphocytes oxidative damage. Direct T cell/macrophage contact-dependent activation, one of the driving mechanisms of synovitis, is mediated by co-stimulatory molecules as well as cell membrane cytokines and may also result in an impaired suppressive function of T regulatory cells (Treg) in RA joints. The fusion of CTLA4 extracellular binding domain to the Fcgamma1 allows to obtain a soluble CTLA4 receptor, the dimeric recombinant human fusion protein abatacept (CTLA4-Ig). The improved knowledge of the CTLA4-B7 co-stimulation regulatory mechanisms by signals delivered into DCs and Tregs provides multiple potential targets for the abatacept treatment. CTLA4-Ig shows the capacity, either ex vivo or in vivo, to interrupt at multiple steps the ongoing inflammatory and destructive process, and to concur in restoring the immunoregulatory balance in RA.

At the crossroads of longevity and metabolism: the metabolic syndrome and lifespan determinant pathways

The metabolic syndrome is becoming increasingly prevalent in the general population and carries significant incremental morbidity and mortality. It is associated with multi‐organ involvement and increased all‐cause mortality, resembling a precocious aging process. The mechanisms that account for this phenomenon are incompletely known, but it is becoming clear that longevity genes might be involved. Experiments with overactivation or disruption of key lifespan determinant pathways, such as silent information regulator (SIR)T1, p66Shc, and mammalian target of rapamycin (TOR), lead to development of features of the metabolic syndrome in mice. These genes integrate longevity pathways and metabolic signals in a complex interplay in which lifespan appears to be strictly dependent on substrate and energy bioavailability. Herein, we describe the roles and possible interconnections of selected lifespan determinant molecular networks in the development of the metabolic syndrome and its complications, describing initial available data in humans. Additional pathways are involved in linking nutrient availability and longevity, certainly including insulin and Insulin‐like Growth Factor‐1 (IGF‐1) signaling, as well as FOXO transcription factors. The model described in this viewpoint article is therefore likely to be an oversimplification. Nevertheless, it represents one starting platform for understanding cell biology of lifespan in relation to the metabolic syndrome.

Oxidative stress in kidney transplant patients with calcineurin inhibitor-induced hypertension: effect of ramipril.

In patients with cyclosporine-induced hypertension, upregulation of the nitric oxide system and oxidative stress were shown, which could induce hypertension, remodeling, and chronic rejection by increasing nitric oxide catabolism. However, it is still debated whether cyclosporine and tacrolimus exert a different action. The aim of the current study was to compare the effects of cyclosporine and tacrolimus on markers of oxidative stress and endothelial dysfunction in kidney transplant patients with posttransplant hypertension. Monocyte p22phox, a NADH/NADPH system subunit, transforming growth factor-&bgr; (TGF-&bgr;), heme oxygenase-1 (HO-1), and endothelial NOS gene expression were measured in 16 patients. Angiotensin II is a potent stimulator of oxidative stress and angiotensin-converting enzyme inhibition may blunt this effect. Therefore, the same parameters were measured before and after 2 months of treatment with ramipril (5 mg/d). At baseline, in cyclosporine-and tacrolimus-treated patients, p22phox and TGF-&bgr; mRNA were similarly increased in comparison with normotensive healthy controls (0.90 ± 0.05 d.u. and 0.83 ± 0.05 in cyclosporine, 0.89 ± 0.07 and 0.84 ± 0.05 in tacrolimus; 0.53 ± 0.07 and 0.75 ± 0.03 in controls, respectively; p < 0.001). Endothelial NOS mRNA was increased in cyclosporine-and tacrolimus-treated patients in comparison with controls (0.92 ± 0.09, 0.96 ± 0.04, and 0.37 ± 0.05 respectively; p < 0.001), whereas no difference was found between patients and controls in HO-1 mRNA. Ramipril reduced blood pressure (from 140 ± 11/91 ± 7 mm Hg to 129 ± 6/85 ± 5 mm Hg in cyclosporine and from 138 ± 7/92 ± 7 mm Hg to 127 ± 10/82 ± 6 mm Hg in tacrolimus group; p < 0.02 with no difference between groups). Ramipril also reduced p22phox (to 0.83 ± 0.05 in cyclosporine, p < 0.03 and to 0.81 ± 0.08 in tacrolimus; p < 0.01) and TGF-&bgr; mRNA (to 0.72 ± 01 in cyclosporine, p < 0.02, and to 0.73 ± 0.05 in tacrolimus; p < 0.01) with no difference between groups, but it did not change HO-1 and ecNOS mRNA. Cyclosporine and tacrolimus induce a comparable oxidative stress in kidney transplant patients with posttransplant hypertension. The association of ramipril normalizes blood pressure and reduces the oxidative stress induced by both drugs.

The redox enzyme p66Shc contributes to diabetes and ischemia-induced delay in cutaneous wound healing

OBJECTIVE The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing. RESEARCH DESIGN AND METHODS Skin wounds were created in wild-type (WT) and p66Shc−/− control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and β-catenin. Response to hind limb ischemia was also evaluated. RESULTS Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of β-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc−/− than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and β-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc−/− dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia. CONCLUSIONS p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

Silencing regulator of G protein signaling-2 (RGS-2) increases angiotensin II signaling: insights into hypertension from findings in Bartter's/Gitelman's syndromes.

Objective Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartters syndrome/Gitelmans syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling. The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ ( ) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses. Methods Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced using Fura-2 AM. Results RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 ± 0.02 vs. 0.19 ± 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 ± 13.2 vs. 130.33 ± 13.64 mmol/l, P = 0.011 and 0.64 ± 0.08 vs. 0.91 ± 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 ± 10.8 (peak-basal) vs. 40.5 ± 14.1 nmol/l, P = 0.017 and 0.84 ± 0.06 vs. 0.64 ± 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 ± 4.3 and 0.91 ± 0.03 nmol/l). Integrating the response over time showed increased area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013). Conclusion This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.

Absence of vascular remodelling in a high angiotensin-II state (Bartter's and Gitelman's syndromes): implications for angiotensin II signalling pathways

BACKGROUND Angiotensin II (Ang II) is a powerful proinflammatory cytokine and growth factor that activates NF-kappaB, as well as NAD(P)H oxidase, and thus is a key factor for the induction and progression of cardiovascular diseases. Our previous studies have shown high Ang II and high blood pressure-driven proatherogenic remodelling in an animal model. To further explore Ang II in proatherogenic vascular remodelling independent of blood pressure, we used Bartters/Gitelmans syndrome (BS/GS) patients given their elevated plasma Ang II, yet normo/hypotension, because extensive mechanistic studies in these patients suggest they are a good model to explore Ang II-mediated signalling. METHODS The study evaluated BS/GS patients for nitric oxide-dependent (FMD) and -independent vasodilation and intima-media thickness (IMT) of the carotid arteries compared with healthy subjects and essential hypertensive patients. RESULTS The results showed the absence of IMT growth in BS/GS patients as cumulative mean-IMT and mean maximum-IMT levels in BS/GS did not differ from normotensives: 0.58 +/- 0.09 mm versus 0.60 +/- 0.09 and 0.67 +/- 0.09 versus 0.70 +/- 0.13 respectively, P = ns, but were significantly lower compared with hypertensive patients: 0.69 +/- 0.13, P < 0.046 and 0.85 +/- 0.19, P < 0.018, respectively. FMD was increased in BS/GS versus hypertensives or normotensive controls (10.8 +/- 2.7% versus 6.5 +/- 2.3 and 8.7 +/- 1.9, P < 0.002 respectively) while endothelium-independent dilation did not differ (10.2 +/- 3.6% versus 7.2 +/- 1.9 and 8.2 +/- 3.3, P = ns) between groups. CONCLUSIONS Our study in BS/GS provides to our knowledge the first clinical data that point to a direct proatherogenic role for Ang II. However, because the data are derived from findings in BS/GS and therefore are indirect, further studies in this and other models using more direct approaches should be pursued to demonstrate a direct proatherogenic effect of Ang II as well as further studies on Ang II type 2 receptor (AT2R) signalling that the spectrum of findings of this and other studies indicate as involved in the lack of vascular remodelling.

OXIDATIVE STRESS AND TGFBETA IN KIDNEY-TRANSPLANTED PATIENTS WITH CYCLOSPORIN-INDUCED HYPERTENSION. EFFECT OF CARVEDILOL AND NIFEDIPINE

Cyclosporin is a powerful stimulator of oxidative stress signaling, leading to TGFbeta production, NO degradation, endothelial dysfunction, hypertension and post-transplant nephropathy. Carvedilol, alpha1-beta-blocker with strong antioxidant activity, may interfere with this chain of events. Therefore, we measured monocyte ecNOS, TGFbeta and heme oxygenase-1 (HO-1) mRNA level and plasma nitrite/nitrate, 3-nitrotyrosine, an estimate of peroxynitrite, and total plasma antioxidant power in kidney-transplanted patients with post-transplant hypertension, before and after treatment with carvedilol, 25 - 50 mg o.d. orally for 4 months (n = 15). The dihydropyridine calcium channel blocker nifedipine (n = 10) was used as comparator antihypertensive drug. Blood pressure fell to a similar extent with both drugs. Carvedilol increased plasma antioxidant power and HO-1 mRNA and reduced 3-nitrotyrosine and TGFbeta mRNA levels, while the same was not observed with nifedipine. Monocyte ec NOS mRNA levels and plasma nitrite/nitrate were higher in the patients than in a normotensive healthy control group and were unaffected by either treatment. In conclusion, carvedilol reduces the oxidative stress and corrects the altered cellular signaling mediated by oxidative stress in CsA-induced post-transplant hypertension. Therefore, it may prevent long-term complications, such as endothelial dysfunction, fibrogenesis and post-transplant nephropathy by decreasing NO degradation and production of TGFbeta, a key fibrogenic cytokine, and by activating HO-1 production.