Congenital hydrocephalus : new Mendelian mutations and evidence for oligogenic inheritance

Peer reviewe

Adrenomedullin: view on a novel vasodilatory peptide with natriuretic properties.

Although initially described in human pheochromocytoma, adrenomedullin has been isolated in several animal and human peripheral organs, including cardiovascular tissues. In experimental models, adrenomedullin exerts potent vasodilatory and natriuretic properties which could participate to maintain physiological cardiovascular and renal homeostasis. Whether adrenomedullin is powerful in humans remains to be proven. On the basis of increased plasma levels in hypertension and heart failure, adrenomedullin is suspected to contribute to the pathogenesis of these diseases. A reduced clearance is another possibility but has not yet been investigated in these pathological states. Finally, the ubiquitous distribution of adrenomedullin suggest various other biological activities that need to be established in future

Contribution of the proteomics platform to molecular imaging targets?

Our role in the “KeyMarker” project is to characterize specific biomarkers involved into different pathologies. These pathologies are linked to the concerned associates of the project. We proposed to use original proteomic methods to identify these biomarkers. The first part of our work is to characterize these predictive markers into the plasma of women treated for breast cancer chemotherapy. These potential biomarkers are present into the plasma as low abundant proteins. They are entirely masked by highly abundant proteins and significantly complicate the discovery process by limiting the detection of low abundance proteins. We proposed to use protein depletion to remove highly abundant proteins from plasma with Ig-based immunoaffinity columns. These columns capture the more abundant proteins from human biofluids such as plasma. We tested 2 different immunoaffinity colums. One is designed to remove twelve high-abundant proteins and the other can remove twenty high-abundant proteins. The depleted fraction is analyzed by two independent strategies. The first one is two-dimensional gel electrophoresis (2D gel) followed by mass spectrometry for protein identification (MALDI/MS). The second one is one-dimensional gel electrophoresis (SDS-PAGE gel) followed by liquid chromatography and mass spectrometry (1D-LC/ESI). Removal of high-abundant proteins enables improved resolution for one-dimensional and two-dimensional gel electrophoresis. These complementary strategies should provide us a maximum number of low abundant proteins. Type I and type II diabetes are pathologies characterized by abnormal apoptosis of beta-cells in pancreatic islets. Apoptosis is a physiological process triggering programmed cell dead. -cell apoptosis can be induced by the inflammatory mediator cytokine interleukin (IL)-1 β. This (IL)-1 β is used in vitro to promote -cell « decision » to undergo apoptosis. Cell lines or primary cell cultures of rat pancreatic beta-cells are cell models employ by Pr. Decio Eizirik. This group identified ~700 genes and EST that are modulated in extensive microarray experiments. Following these studies, Pr. Decio Eizirik wishes to use the experience from the Pr. Edwin De Pauw’s laboratory to characterize protein expression pattern of -cell undergoing apoptosis or not. To be able to use these specific -cell protein biomarkers for medical imaging (PET scan: Positron Emission Tomography), these makers should be accessible to antibodies used for imaging and could be potential therapeutic targets. Pr. Edwin De Pauw suggest to make a sub-proteome containing beta-cell transmembrane proteins treated or not with interleukin (IL)-1 β. Protein fractionation will be performed by labelling of their extracellular domains with biotin. Biotinylated proteins can react with streptavidin link to a streptavidin-sepharose column. Trypsin could be use to release labelled proteins. Biotinylated peptides will be identified after separation by 2D-LC and mass spectrometry (ESI). Type I and type II diabetes are pathologies characterized by abnormal apoptosis of b-cells in pancreatic islets. Apoptosis is a physiological process triggering programmed cell dead. b-cell apoptosis can be induced by the inflammatory mediator cytokine interleukin (IL)-1 β. (IL)-1 β is used in vitro to promote cell cultures of rat pancreatic b-cells « decision » to undergo apoptosis. Pr. D. Eizirik identified ~700 genes and EST that are modulated by (IL)-1 β in microarray experiments. Following these studies, Pr. D. Eizirik wishes to use the experience from the Pr. E. De Pauw’s laboratory to characterize protein expression pattern of b-cell undergoing apoptosis or not. To be able to use these specific b-cell protein biomarkers for medical imaging, they should be accessible to antibodies. Pr. E. De Pauw suggest to make a sub-proteome containing b-cell transmembrane proteins treated or not with interleukin (IL)-1 β. Protein fractionation will be performed by labelling of their extracellular domains with biotin. Biotinylated proteins can react with streptavidin link to a streptavidin-sepharose resin. Trypsin could be use to release labelled proteins. Biotinylated peptides will be identified after separation by 2D-LC and mass spectrometry (ESI). Outlook Our goal is to use molecular biomarker for medical imaging (PET scan: Positron Emission Tomography). Diagnostic imaging procedures are crucial for further treatment planning in many diseases. Pr. D. Eizirik wishes to characterize specific biomarkers induced during apoptosis process into the cells. These cells are undergoing apoptosis during type I and II diabetes. To use these markers for medical imaging, the marker should be a plasma cell membrane protein and should be accessible to the antibody. Pr. D. Eizirik will use the proteomic plateform at the Giga center to identify potential molecular imaging targets.‘Validation of tumoral biomarkers using proteomic methods

Membrane and cytoplasmic proteome biology of anterior mice hippocampus

The prevalence of neurodegenerative diseases increases steadily and proteomics on disease-related brain areas become more and more complex, whereas suitable samples are scarce. Due to this difficulty, animal models are indispensable in understanding human biology and disease, and the most commonly used model is the mouse (Mus musculus). Mice share many genetic and physiological characteristics with humans, breed rapidly, and can be genetically modified leading to the functional characterisation of many proteins. As for human, the mouse hippocampus is one of the most important areas of the brain and has therefore been an object of intensive investigation for many years. Mouse hippocampus has served as models for degenerating brain diseases associated with Alzheimer's disease, Parkinson's disease, ischemia, epilepsy, synaptic plasticity and underlying mechanisms of learning and memory. Using 2 anterior parts of normal mouse hippocampi, we intend to characterize membrane and cytoplasmic proteome using a modified procedure previously established (WISNIEWSKI et al., 2008). This method permit the preparation and analysis of 2 individual fractions enriched in membrane and cytoplasmic proteins. The method for separation of membrane and cytoplasm fractions comprises a stepwise depletion of non-integral membrane proteins from entire tissue homogenate by high-salt, carbonate, and urea washes. The cytoplasmic proteins obtained from high-salt depletion are considered as the soluble fraction. The membrane proteins obtained from the stepwise depletion are considered as the insoluble fraction. Enzymatic digestion of both membrane and cytoplasmic fractions are carried out without use of detergents by double digestion with endoproteinase Lys-C and trypsin. Digested peptide fractions are loaded on StageTips for rapid desalting and are separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC). Separated peptides are analyzed by electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). The entire procedure allows rapid processing and preparation of samples from minute amounts as 30-40 mg frozen tissue leading to about 70 g of membrane proteins and 500 g of cytoplasmic proteins. This can be extremely helpful for proteomic profiling of small pieces of tissue and clinical material. Analyses of membrane fraction identified about 45% total membrane proteins. This fraction includes glutamate receptor 1 and 2, proteins involved into the trafficking of synaptic vesicles, lipid-anchor proteins, G proteins involved in various transmembrane signalling systems, NMDAR signalling complex proteins, voltage channel proteins… Analyses of the cytoplasmic fraction identify various proteins belonging to different compartments and/or pathways. A large amount of these proteins are specifically expressed into the hippocampus and/or into the nervous system.‘Validated Predictive Dynamic Model of Complex Intracellular Pathways Related to Cell Death and Survival’ AND ‘Validation of tumoral biomarkers using proteomic methods

Endothelin-1 receptor antagonism does not influence myocardial function in hypertensive dogs.

BACKGROUND: As endothelin-1 exerts positive inotropic effects, the present study evaluated whether the hypotensive effects of the endothelin-1 receptor antagonist bosentan were partially related to a decrease in myocardial performance. METHODS: In group I, eight anaesthetized open-chest dogs with perinephritic hypertension received four cumulative doses of bosentan (B1-B4). In group II, eight animals received the same doses of bosentan after autonomic blockade. Indices of heart function were derived from the pressure-length loops obtained during vena cava occlusion. RESULTS: In group I, bosentan decreased left ventricular systolic pressure (LVSP) and mean aortic pressure (MAP) dose dependently, reaching 21% and 23% respectively at B4 (LVSP from 190 +/- 8 to 150 +/- 5 mmHg, P < 0.001; MAP from 167 +/- 7 to 128 +/- 5 mmHg, P < 0.001). These effects were only related to peripheral vasodilatation, without depression of myocardial contractility, as systemic vascular resistance dropped (from 670 +/- 83 to 446 +/- 53 mmHg mL-1 min-1 x 10(4); P < 0.05), and the end-systolic pressure-length relationship (ESPLR) remained unchanged (4.0 +/- 0.4 vs. 4.3 +/- 0.7 mmHg mm-1 kg-1). Concomitantly with pressure decline, heart rate tended to increase in this group (from 150 +/- 4 to 156 +/- 6 beats min-1). When autonomic system was blocked (group II), administration of bosentan induced similar hypotensive effects as in group I (26% and 28% reduction in LVSP and MAP respectively, P < 0.001) whereas ESPLR did not change (3.0 +/- 0.9 vs. 3.1 +/- 0.5mmHg-1 mm kg-1 ). Under these sympathetically blocked conditions, heart rate significantly fell after bosentan infusion (from 120 +/- 4 to 110 +/- 6 beats min-1, P < 0.001). CONCLUSIONS: Without influencing heart function, bosentan is an efficient and safe therapy that opens up new therapeutic perspectives in human essential hypertension

Carvedilol and lacidipine prevent cardiac hypertrophy and endothelin-1 gene overexpression after aortic banding.

Carvedilol and lacidipine have been shown to exert cardioprotective effects in rat models of chronic hypertension. We investigated their effects in an acute model of pressure overload produced by suprarenal aortic constriction, in which enhanced myocardial production of endothelin-1 could play a crucial role. In the absence of drug treatment, after 1 week, aortic banding provoked an increase in carotid pressure associated with left ventricular hypertrophy (29%; P<0.01). These changes were accompanied by increased myocardial expression of preproendothelin-1 (2.5 times; P<0.05) and skeletal alpha-actin (3.6 times; P<0.05), but the expression of cardiac alpha-actin was not modified. Oral administration of carvedilol at a dose of 30 mg. kg(-1). d(-1) to rats with aortic banding normalized carotid pressure and left ventricular weight as well as preproendothelin-1 and skeletal alpha-actin gene expression. Carvedilol at a lower dose (7.5 mg x kg(-1) x d(-1)) and lacidipine 1 mg x kg(-1) x d(-1) had only moderate and nonsignificant effects on carotid pressure but largely prevented left ventricular hypertrophy (P<0.01) and preproendothelin-1 overexpression (P<0.05). Labetalol (60 mg x kg(-1) x d(-1)) tended to exert similar effects but insignificantly. These results show that the antihypertrophic properties of carvedilol and lacidipine are partly independent of their antihypertensive effects and may be related to their ability to blunt myocardial preproendothelin-1 overexpression. Moreover, carvedilol at a dose of 7.5 mg x kg(-1) x d(-1) did not prevent myocardial overexpression of skeletal alpha-actin, which suggests that, in this model, reexpression of a fetal gene can be activated by pressure overload independently of cardiac hypertrophy

Cardiovascular effects of beta 3-adrenoceptor stimulation in perinephritic hypertension.

BACKGROUND: A new beta 3-adrenoceptor (beta3-AR) has been shown to mediate peripheral vasodilation. This study was conducted to evaluate effects of the beta3-AR agonist, SR58611 in normal and hypertensive dogs. MATERIALS AND METHODS: In protocol 1, SR58611 was infused in normal dogs after placebo, after beta1/beta2 blockade with nadolol, after beta1/beta2/beta3 blockade with bupranolol and after combined autonomic blockade (CAB). In protocol 2, perinephritic hypertension was produced in dogs, which received SR58611 at 3 and 6 weeks of hypertension. Effects of SR58611 were evaluated at 7 weeks of hypertension after CAB. RESULTS: In normal dogs, SR58611 produced a dose-dependent decrease in mean aortic pressure (AOP) (from 116 +/- 19 to 100 +/- 19 mmHg, - 14%; P < 0.05) that was accompanied by baroreflex activation (heart rate increased by 70%; P < 0.01). This hypotensive effect resulting from peripheral vasodilation persisted after nadolol or CAB while baroreflex activation was blunted or abolished. A biphasic response of cardiac output, characterized by a rise and a decline (P < 0.05) reflected a reduction in after- and pre-load. After CAB, SR58611 did not modify cardiac contractility. SR58611 stimulated lipolysis as reflected by a 4-fold increase in blood free fatty acids (FFA) (P < 0.0005). Under CAB, the rise of FFA was reduced (P < 0.01). In hypertensive dogs, SR58611 produced a dose-dependent decrease in mean AOP (from 168 +/- 32 to 125 +/- 35 mmHg; - 26%, P < 0.0001), that was greater than in normal dogs (P < 0.05). Reflex-mediated tachycardia also occurred but at higher blood pressure values. Blood FFA rose similarly (P < 0.0001). Under CAB, heart rate remained unchanged but SR58611 still induced a decrease (P < 0.0001) in mean AOP concomitantly with a rise of (dP/dt)/DP40 (P < 0.005), an effect not observed in normal dogs. CONCLUSIONS: Beta3-AR stimulation exerts hypotensive effects, increases cardiac contractility and stimulates lipolysis in hypertensive dogs

MCF-7/BOS cells membrane proteome profiling for the analysis of plasmic proteins in pancreatic beta cells

Type I and type II diabetes are diseases characterized by the activation of the apoptotic processes in pancreatic islet of beta-cells. To highlight specific proteins of beta cells in medical imaging (positron emission tomography or PET), the development of antibodies directed against membrane markers of the beta cells is undertaken. These membrane markers have to be accessible to antibodies and should be potential therapeutic targets. In order to isolate membrane proteins of beta cells, preliminary tests were conducted on MCF-7 / BOS cell line, immortal and easier to cultivate. The goal of this work is to obtain a pure membrane fraction to facilitate the analysis of the sample. To isolate transmembrane proteins, we compared two methods. The first one used a differential centrifugation to isolate membrane proteins. The second method labelled accessible extracellular domains at the surface of MCF-7 cells with biotin prior to differential centrifugation. Our results shown that the labelling technique can provide 50% of membrane proteins. This second method will be used to isolate membrane proteins of pancreatic beta cells.‘Validation of tumoral biomarkers using proteomic methods

Protéomique différentielle de la fraction membranaire des cellules beta des ilots de Langerhans du pancréas

Le diabète de type I et de type II sont des pathologies caractérisées par le déclenchement des processus apoptotiques des cellules β des ilots de Langerhans du pancréas. L’apoptose des cellules β, induite par l’interleukin (IL)-1 β, déclenche la mort cellulaire génétiquement programmée. Le groupe du Pr D. Eizirik utilise comme modèle les cellules β du pancréas du rat pour identifier environ 700 gènes dont l’expression est modulée après exposition à l’interleukine (IL)-1 β (1 et 2). Pour mettre en évidence les protéines spécifiques des cellules en imagerie médicale (tomographie par émission de positron ou PET), le développement d’anticorps dirigés contre des marqueurs membranaires des cellules est envisagé. Ces marqueurs membranaires doivent être des protéines transmembranaires pour être accessibles aux anticorps et pour être des cibles thérapeutiques potentielles. Ces marqueurs spécifiques du domaine extracellulaire des protéines des cellules devraient voir leur expression modulée dans les cellules qui entrent en apoptose. Pour isoler les protéines transmembranaires, un marquage à la biotine des domaines extracellulaires accessibles à la surface des cellules est réalisé. Les protéines biotinylées sont isolées sur une résine streptavidine puis précipitées et dosées pour être déposées sur un gel SDS-PAGE. Ces protéines sont éluées du gel après digestion par la trypsine. Les peptides obtenus sont séparés par chromatographie HPLC (2D-LC) et détectés par spectrométrie de masse du type trappe à ions. L’identification des protéines est réalisée au moyen d’un moteur de recherche en base de données. Afin d’isoler un ou des biomarqueurs potentiels, les sous protéomes membranaires des cellules traitées ou non avec l’interleukin (IL)-1 β sont comparés par analyse protéomique différentielle. (1) M.Cnop et al. Diabetes. 2005 (54):S97-107 (2) DL. Eizirik et al. Ann N Y Acad Sci. 2003 (1005)55-74‘Validation of tumoral biomarkers using proteomic methods