Sébastien Faure

Reactive Oxygen Species and Cyclooxygenase 2-Derived Thromboxane A2 Reduce Angiotensin II Type 2 Receptor Vasorelaxation in Diabetic Rat Resistance Arteries

Angiotensin II has a key role in the control of resistance artery tone and local blood flow. Angiotensin II possesses 2 main receptors. Although angiotensin II type 1 receptor is well known and is involved in the vasoconstrictor and growth properties of angiotensin II, the role of the angiotensin II type 2 receptor (AT2R) remains much less understood. Although AT2R stimulation induces vasodilatation in normotensive rats, it induces vasoconstriction in pathological conditions involving oxidative stress and cyclooxygenase 2 expression. Thus, we studied the influence of cyclooxygenase 2 on AT2R-dependent tone in diabetes mellitus. Mesenteric resistance arteries were isolated from Zucker diabetic fatty (ZDF) and lean Zucker rats and studied using in vitro using wire myography. In ZDF rats, AT2R-induced dilation was lower than in lean rats (11% versus 21% dilation). Dilation in ZDF rats returned to the control (lean rats) level after acute superoxide reduction (Tempol and apocynin), cyclooxygenase 2 inhibition (NS398), or thromboxane A2 synthesis inhibition (furegrelate). Cyclooxygenase 2 expression and superoxide production were significantly increased in ZDF rat arteries compared with arteries of lean rats. After chronic treatment with Tempol, AT2R-dependent dilation was equivalent in ZDF and lean rats. Chronic treatment of ZDF rats with NS398 also restored AT2R-dependent dilation to the control (lean rats) level. Plasma thromboxane B2 (thromboxane A2 metabolite), initially high in ZDF rats, was decreased by chronic Tempol and by chronic NS398 to the level found in lean Zucker rats. Thus, in type 2 diabetic rats, superoxide and thromboxane A2 reduced AT2R-induced dilation. These findings are important to take into consideration when choosing vasoactive drugs for diabetic patients.

Plasmatic concentration of organochlorine lindane acts as metabolic disruptors in HepG2 liver cell line by inducing mitochondrial disorder

Lindane (LD) is a persistent environmental pollutant that has been the subject of several toxicological studies. However, concentrations used in most of the reported studies were relatively higher than those found in the blood of the contaminated area residents and effects of low concentrations remain poorly investigated. Moreover, effects on cell metabolism and mitochondrial function of exposure to LD have received little attention. This study was designed to explore the effects of low concentrations of LD on cellular metabolism and mitochondrial function, using the hepatocarcinoma cell line HepG2. Cells were exposed to LD for 24, 48 and 72 h and different parameters linked with mitochondrial regulation and energy metabolism were analyzed. Despite having any impact on cellular viability, exposure to LD at plasmatic concentrations led to an increase of maximal respiratory capacity, complex I activity, intracellular ATP and NO release but decreased uncoupled respiration to ATP synthesis and medium lactate levels. In addition, LD exposure resulted in the upregulation of mitochondrial biogenesis genes. We suggest that, at plasmatic concentrations, LD acts as a metabolic disruptor through impaired mitochondrial function and regulation with an impact on cellular energetic metabolism. In addition, we propose that a cellular assay based on the analysis of mitochondria function, such as described here for LD, may be applicable for larger studies on the effects of low concentrations of xenobiotics, because of the exquisite sensitivity of this organelle.

Increased Microparticle Production and Impaired Microvascular Endothelial Function in Aldosterone-Salt- Treated Rats: Protective Effects of Polyphenols

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h−1), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg−1.day−1), or spironolactone (30 mg.kg−1.day−1) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.

Cell Wall Modifications during Conidial Maturation of the Human Pathogenic Fungus Pseudallescheria boydii

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.

Red wine polyphenol compounds favor neovascularisation through estrogen receptor α-independent mechanism in mice.

Red wine polyphenol compounds (RWPC) exert paradoxical effects depending on the dose on post-ischemic neovascularisation. Low dose RWPC (0.2 mg/kg/day) is pro-angiogenic, whereas high dose (20 mg/kg/day) is anti-angiogenic. We recently reported that the endothelial effect of RWPC is mediated through the activation of a redox-sensitive pathway, mitochondrial biogenesis and the activation of α isoform of the estrogen receptor (ERα). Here, we investigated the implication of ERα on angiogenic properties of RWPC. Using ovariectomized mice lacking ERα treated with high dose of RWPC after hindlimb ischemia, we examined blood flow reperfusion, vascular density, nitric oxide (NO) production, expression and activation of proteins involved in angiogenic process and muscle energy sensing network. As expected, high dose of RWPC treatment reduced both blood flow and vascular density in muscles of mice expressing ERα. These effects were associated with reduced NO production resulting from diminished activity of eNOS. In the absence of RWPC, ERα deficient mice showed a reduced neo-vascularisation associated with a decreased NO production. Surprisingly in mice lacking ERα, high dose of RWPC increased blood flow and capillary density in conjunction with increased NO pathway and production as well as VEGF expression. Of particular interest is the activation of Sirt-1, AMPKα and PGC-1α/β axis in ischemic hindlimb from both strains. Altogether, the results highlight a pro-angiogenic property of RWPC via an ERα-independent mechanism that is associated with an up-regulation of energy sensing network. This study brings a corner stone of a novel pathway for RWPC to correct cardiovascular diseases associated with failed neovascularisation.

Paradoxical effects of the topoisomerase inhibitor, ethoxidine, in the cellular processes leading to angiogenesis on EaHy.926 endothelial cells

1 Which importance for P 450 Cytochromes in drug interactions? A study from the French PharmacoVigilance Database AC Danton, A Sommet, G Durrieu, H Bagheri and JL Montastruc Service de Pharmacologie, Faculté de Médecine, Toulouse, France Objective: Cytochromes (CYPs) are a superfamily of isoenzymes involved in drug metabolism. The main isoenzymes are CYP 1A2, 2C9, 2D6 and 3A4. However, their relative importance in clinically significant interactions remains unknown. The aim of the present study was to investigate in the French PharmacoVigilance Database (FPVD) the number and characteristics of drug-drug interactions possibly explained by involvement of CYPs. Methods: Spontaneous notifications of Adverse Drug Reactions (ADRs) recorded by Toulouse Midi-Pyrénées PharmacoVigilance Centre between 1st January and 31st August 2008 were extracted from the FPVD. For each observation, we recorded the main characteristics of patients (age, gender), involved drug(s) (name, pharmacological class and involved CYP) and induced ADRs (type, ‘seriousness’, evolution). Results: Between 1st January and 31st August 2008, 1205 ADRs were registered by Toulouse Midi-Pyrénées PharmacoVigilance Centre into the FPVD. They involved 683 women (56.6%). In 12 cases, patients were less than 1 year old and in 14 cases, age was not informed. For other observations, mean age was 55.8 (median value: 58.0) years. Among these ADRs, 410 (34%) were ‘serious’ (including 24 fatal outcomes), 356 involved only one drug and 730 did not involve any drug interaction. Finally, 119 reports (i.e. 9.9% of registered ADRs) involved one (or more) drug interaction related to CYPs. Among these 119 notifications, the most frequently involved CYP was 3A4/5 (n = 81), followed by 2C19 (n = 20), 2C9 (n = 16), 2D6 (n = 16) and 2E1 (n = 1). In 13 notifications, several CYPs were involved. Drugs more frequently involved in this kind of drug interaction were amiodarone (n = 21) followed by proton pump inhibitors (n = 11), meprobamate (n = 9), antiretrovirals (n = 9) and rifampicine (n = 6). There was no major difference in ‘seriousness’ or evolution of ADRs between CYP-related interactions and others. In 22 cases, ADRs were possibly due to CYP-related drug interaction (18.5% of CYP-related drug interactions). Conclusion: CYP-related drug interactions are found in around 10% of ADRs registered in the FPVD. The most frequently involved CYPs are 3A4/5 (68%) followed by 2C19 (17%). This CYP-involving interaction leads to clinically significant consequences (ADR) in almost one case out of five. These data underline the importance of CYPs not only in drug interactions but also in mechanisms of ADRs.

K012 Pharmacological blockade or genetic deletion of AT2R abolishes flow-induced structural remodeling in rat and mice resistance arteries in vivo

The renin—angiotensin system (RAS) plays a major role in regulating the cardiovascular system. Angiotensin II (AngII), the main effector of the RAS, is involved in both endothelial and smooth muscle cells functions, acting by two receptors, type 1 (AT1R) and type 2 (AT2R). If the role of the AT1R has been widely investigated in vivo and in vitro, the role of the AT2R remains controversial. It is admitted that AT2R induces vasodilation and possesses anti-proliferative properties. Nevertheless, recent studies have shown that the AT2R is vasoconstrictor in hypertension and has proliferative effects in tumors. AT2R has a role in vascular reactivity to flow (shear stress) and in cell growth and migration. Moreover, the AT2R decreases MMP-2 and increases elastin. Thus we hypothesized that changes in vascular wall mechanical strain, extracellular matrix deposition, and MMPs activity could be modulated differentially by the AngII receptors. We investigated the role of the AT2R on the capacity of resistance arteries to remodel in response to chronic changes in local blood flow. We investigated flow-mediated remodeling in resistance arteries in PD123319 (AT2R blocker)-treated rats and in AT2R-knockout mice. Second order mesenteric arteries were ligated in vivo, generating low flow (LF) and high flow (HF) arteries, compared to normal flow (NF) vessels. In these vessels a biochemical (protein expression and mRNA level) and a structural study (lumen diameter, intima media thickness and media cross-sectional area) will be performed in order to determine the mechanism involved in the remodeling. After 2 weeks, outward hypertrophic remodeling occurred in HF arteries (increased diameter and medial cross-sectional area) in NaCl treated-rats or wild type mice. Nevertheless, high blood flow-induced remodeling in mesenteric resistance arteries failed to occur in PD123319-treated-rats and in AT2R-KO mice. Inward eutrophic remodeling, which occurred in LF arteries, was not affected to the absence of the AT2R or its blockade. Thus, the diameter enlargement due to a chronic rise in blood flow in resistance arteries involves the AT2R, suggesting a vasodilator and trophic effect of the receptor in arteriolar remodeling in vivo.

D004 Pharmacological blockade of angiotensin II type 2 receptor inhibits tumor growth decreasing cell proliferation and tumor vascularization

Angiotensin II type 2 receptor (AT2R) stimulation is thought to induce vasodilation and apoptosis. Nevertheless, its role remains controversial. In hypertension and aging, AT2R might promote vasoconstriction and cell growth. In cancer cells, AT2R is highly expressed but its role remains poorly understood. Thus, we investigated the role of AT2R in tumor growth, hypothesizing that AT2R affects cell proliferation and/or tumor vascularization. Tumor induction by 3-methylcholanthrene was significantly delayed in AT2R knockout mice, suggesting a role for AT2R in tumor initiation. Tumor growth after subcutaneous injection of LL/2 cancer cells in C57/BL6 mice treated with PD123,319, an AT2R antagonist, was decreased when PD123,319 was given at an early stage, suggesting that AT2R is also involved in tumor promotion. In vitro proliferation of LL/2 cells was reduced by PD123,319 with a significant decrease in Ki67 expression and ERK1/2 phosphorylation. In addition, tumor vascular density, measured by CD31 labeling and angiogenesis using the aortic ring assay on Matrigel®, were reduced by PD123,319 and in AT2R-null mice. Therefore, we uncovered a novel mechanism by which AT2R promotes tumor development favoring both malignant cancer cell proliferation and tumor angiogenesis. Thus, blocking AT2R could be a new approach in tumor therapy.