Mécanismes moléculaires de l’hypertrophie vasculaire dans le modèle animal d’hypertension essentielle (SHR)

La voie de signalisation des phosphoinositides joue un rôle clé dans la régulation du tonus vasculaire. Plusieurs études rapportent une production endogène de l’angiotensin II (Ang II) et de l’endothéline-1 (ET-1) par les cellules musculaires lisses vasculaires (CMLVs) de rats spontanément hypertendus (spontaneously hypertensive rats : SHR). De plus, l’Ang II exogène induit son effet prohypertrophique sur les CMLVs selon un mécanisme dépendant de la protéine Gqα et de la PKCẟ. Cependant, le rôle de l’axe Gqα/PLCβ/PKCẟ dans l’hypertrophie des CMLVs provenant d’un modèle animal de l’hypertension artérielle n’est pas encore étudié. L’objectif principal de cette thèse est d’examiner le rôle de l’axe Gqα/PLCβ1 dans les mécanismes moléculaires de l’hypertrophie des CMLVs provenant d’un modèle animal d’hypertension artérielle essentielle (spontaneously hypertensive rats : SHR). Nos premiers résultats indiquent que contrairement aux CMLVs de SHR âgés de 12 semaines (absence d’hypertrophie cardiaque), les CMLVs de SHR âgés de 16 semaines (présence d’hypertrophie cardiaque) présentent une surexpression protéique endogène de Gqα et de PLCβ1 par rapport aux CMLVs de rats WKY appariés pour l’âge. L’inhibition du taux d’expression protéique de Gqα et de PLCβ1 par des siRNAs spécifiques diminue significativement le taux de synthèse protéique élevé dans les CMLVs de SHR. De plus, la surexpression endogène des Gqα et PLCβ1, l’hyperphosphorylation de la molécule ERK1/2 et le taux de synthèse protéique élevé dans les CMLVs de SHR de 16 semaines ont été atténués significativement par des antagonistes des récepteurs AT1 (losartan) et ETA (BQ123), mais pas par l’antagoniste du récepteur ETB (BQ788). L’inhibition pharmacologique des MAPKs par PD98059 diminue significativement la surexpression endogène de Gqα/PLCβ1 et le taux de synthèse protéique élevé dans les CMLVs de SHR. D’un côté, l’inhibition du stress oxydatif (par DPI, inhibiteur de la NAD(P)H oxidase, et NAC , molécule anti-oxydante), de la molécule c-Src (PP2) et des récepteurs de facteurs de croissance (AG1024 (inhibiteur de l’IGF1-R), AG1478 (inhibiteur de l’EGFR) et AG1295 (inhibiteur du PDGFR)) a permis d’atténuer significativement la surexpression endogène élevée de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. D’un autre côté, DPI, NAC et PP2 atténuent significativement l’hyperphosphorylation de la molécule c-Src, des RTKs (récepteurs à activité tyrosine kinase) et de la molécule ERK1/2. Dans une autre étude, nous avons aussi démontré que la PKCẟ montre une hyperphosphorylation en Tyr311 dans les CMLVs de SHR comparées aux CMLVs de WKY. La rottlerin, utilisée comme inhibiteur spécifique de la PKCẟ, inhibe significativement cette hyperphosphorylation en Tyr311 dépendamment de la concentration. L’inhibition de l’activité de la PKCẟ par la rottlerin a été aussi associée à une atténuation significative de la surexpression protéique endogène de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. De plus, l’inhibition pharmacologique de l’activité de la PKCẟ, en amont du stress oxydatif, a permis d’inhiber significativement l’activité de la NADPH, le taux de production élevée de l’ion superoxyde ainsi que l’hyperphosphorylation de la molécule ERK1/2, de la molécule c-Src et des RTKs. À notre surprise, nous avons aussi remarqué une surexpression protéique de l’EGFR et de l’IGF-1R dans les CMLVs de SHR à l’âge de 16 semaines. L’inhibition pharmacologique de l’activité de la PKCẟ, de la molécule c-Src et du stress oxydatif a permis d’inhiber significativement la surexpression protéique endogène de ces RTKs. De plus, l’inhibition de l’expression protéique de l’EGFR et de la molécule c-Src par des siRNA spécifiques atténue significativement le taux d’expression protéique élevé de Gqα et de PLCβ1 ainsi que le taux de synthèse protéique élevé dans les CMLVs de SHR. Des siRNAs spécifiques à la PKCẟ ont permis d’atténuer significativement le taux de synthèse protéique élevé dans les CMLVs de SHR et confirment le rôle important de la PKCẟ dans les mécanismes moléculaires de l’hypertrophie des CMLVs selon une voie dépendante du stress oxydatif. En conclusion, ces résultats suggèrent un rôle important de l’activation endogène de l’axe Gqα-PLCβ-PKCẟ dans le processus d’hypertrophie vasculaire selon un mécanisme impliquant une activation endogène des récepteurs AT1/ETa, de la molécule c-Src, du stress oxidatif, des RTKs et des MAPKs.

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy

Objective: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. Methods: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. Results: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (P

Emerging roles of urotensin-II in cardiovascular disease

Urotensin-II (UII) is a highly potent endogenous peptide within the cardiovascular system. Through stimulation of Galphaq-coupled UT receptors, UII mediates contraction of vascular smooth muscle and endothelial-dependent vasorelaxation, and positive inotropy in human right atrium and ventricle. A pathogenic role of the UT receptor system is emerging in cardiovascular disease states, with evidence for upregulation of the UT receptor system in patients with congestive heart failure (CHF), pulmonary hypertension, cirrhosis and portal hypertension, and chronic renal failure. In vitro and in vivo studies show that under pathophysiological conditions, UII might contribute to cardiomyocyte hypertrophy, extracellular matrix production, enhanced vasoconstriction, vascular smooth muscle cell hyperplasia, and endothelial cell hyper-permeability. Single nucleotide polymorphisms of the UII gene may also impart a genetic predisposition of patients to diabetes. Therefore, the UT receptor system is a potential therapeutic target in the treatment of cardiac, pulmonary, and renal diseases. UT receptor antagonists are currently being developed to prevent and/or reverse the effects of over-activated UT receptors by the endogenous ligand. This review describes UII peptide and converting enzymes, and UT receptors in the cardiovascular system, focusing on pathophysiological roles of UII in the heart and blood vessels. (C) 2004 Elsevier Inc. All rights reserved,

Dog peritoneal and pleural cavities as bioreactors to grow autologous vascular grafts

Objective: The purpose of this study was to grow artificial blood vessels for autologous transplantation as arterial interposition grafts in a large animal model (dog). Method and results: Tubing up to 250 mm long, either bare or wrapped in biodegradable polyglycolic acid (Dexon) or nonbiodegradable polypropylene (Prolene) mesh, was inserted in the peritoneal or pleural cavity of dogs, using minimally invasive techniques, and tethered at one end to the wall with a loose suture. After 3 weeks the tubes and their tissue capsules were harvested, and the inert tubing was discarded. The wall of living tissue was uniformly 1-1.5 mm thick throughout its length, and consisted of multiple layers of myofibroblasts and matrix overlaid with a single layer of mesothelium. The myofibroblasts stained for a-smooth muscle actin, vimentin, and desmin. The bursting strength of tissue tubes with no biodegradable mesh scaffolds was in excess of 2500 mm Hg, and the suture holding strength was 11.5 N, both similar to that in dog carotid and femoral arteries. Eleven tissue tubes were transplanted as interposition grafts into the femoral artery of the same dog in which they were grown, and were harvested after 3 to 6.5 months. Eight remained patent during this time. At harvest, their lumens were lined with endothelium-like cells, and wall cells stained for alpha-actin, smooth muscle myosin, desmin and smoothelin; there was also a thick adventitia containing vasa vasorum. Conclusion: Peritoneal and pleural cavities of large animals can function as bioreactors to grow myofibroblast tubes for use as autologous vascular grafts.

Structural basis for control of secondary vessels in the long-finned eel, Anguilla reinhardtii

Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel Anguilla reinhardtii were examined by light and transmission electron microscopy. Interarterial anastomoses were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to anastomose with a secondary vessel running in parallel with the primary counterpart. In contrast to findings from other species, secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, with a single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these appeared more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it is anticipated that flow through secondary vessels to some extent is affected by the vascular tone of the primary vessel. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. No immunoreactivity was observed on primary or secondary arteries against neuropeptide Y or calcitonin gene-related peptide.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT)

1 The aim was to test the hypothesis that nitric oxide ( NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2 The NO donors, MAHMA/NO ( a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen- 1-ium-1,2-diolate), SIN-1 ( a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 ( an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt ( source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [H-3]- 5-HT in COS-7 cells expressing human SERT. 3 Superoxide dismutase (150 U ml(-1)) plus catalase ( 1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 ( which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[ 1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one; 3 muM). 5 L-Cysteine ( 1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6 It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7 Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-1, inhibin beta A, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

High glucose transactivates the EGF receptor and up-regulates serum glucocorticoid kinase in the proximal tubule

Background. Serum glucocorticoid regulated kinase (SGK-1) is induced in the kidney in diabetes mellitus. However, its role in the proximal tubule is unclear. This study determined the expression and functional role of SGK-1 in PTCs in high glucose conditions. As the epidermal growth factor (EGF) receptor is activated by both EGF and other factors implicated in diabetic nephropathy, the relationship of SGK-1 with EGFR activity was assessed. Methods. mRNA and protein expression of SGK-1 and mRNA expression of the sodium hydrogen exchanger NHE3 were measured in human PTCs exposed to 5 mmol/L (control) and 25 mmol/L (high) glucose. The effects of SGK-1 on cell growth, apoptosis, and progression through the cell cycle and NHE3 mRNA were examined following overexpression of SGK-1 in PTCs. The role of EGFR activation in observed changes was assessed by phospho-EGFR expression, and response to the EGFR blocker PKI166. SGK-1 expression was then assessed in vivo in a model of streptozotocin-induced diabetes mellitus type 2. Results. A total of 25 mmol/L glucose and EGF (10 ng/mL) increased SGK-1 mRNA (P < 0.005 and P < 0.002, respectively) and protein (both P < 0.02) expression. High glucose and overexpression of SGK-1 increased NHE3 mRNA (P < 0.05) and EGFR phosphorylation (P < 0.01), which were reversed by PKI166. SGK-1 overexpression increased PTC growth (P < 0.0001), progression through the cell cycle (P < 0.001), and increased NHE3 mRNA (P < 0.01), which were all reversed with PKI166. Overexpression of SGK-1 also protected against apoptosis induced in the PTCs (P < 0.0001). Up-regulation of tubular SGK-1 mRNA in diabetes mellitus was confirmed in vivo. Oral treatment with PKI166 attenuated this increase by 51%. No EGF protein was detectable in PTCs, suggestive of phosphorylation of the EGFR by high glucose and downstream induction of SGK-1. Conclusion. The effects of high glucose on PTC proliferation, reduced apoptosis and increased NHE3 mRNA levels are mediated by EGFR-dependent up-regulation of SGK-1.

Ligand-gated channels

Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This paper will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which plays a critical role in the fast chemical transmission of electrical signals between nerve cells and between nerve and muscle cells. The superfamily will mainly be exemplified by the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels.

Correlation of non-uniform protein expression with variation in transmitter release probability

The strength of synaptic transmission is highly variable between different synapses. The present study examined some factors that may contribute to this variation in the strength of neurotransmission in sympathetic varicosities of the mouse vas deferens. Transmitter release was measured using a focal macropatch electrode placed over pairs of visualised varicosities. By regulating the calcium concentration of the solutions inside the recording electrode and in the bath independently of each other, transmitter release was restricted to one or two surface varicosities at each recording site. Using this technique, transmitter release probability was shown to be highly variable, even between adjacent varicosities on single axon branches. Very little variation was observed in the calcium influx following single impulse nerve stimulation between adjacent Oregon Green BAPTA-1 loaded varicosities. However, the staining intensities of three vesicular proteins, SV2, synaptophysin, and synaptotagmin 1, showed considerable variation between adjacent varicosities on single axon branches. This variation in staining intensity may be partly explained by variation in the density of synaptic vesicles. However, double staining experiments using two vesicular antigens showed some varicosities staining for one vesicular antigen, but not for the second, suggesting that the expression of these release machinery proteins is regulated locally within the varicosities. The results of the present study strengthen suggestions that synaptic strength is at least in part, regulated by variation in the expression of vesicular proteins. (C) 2004 Wiley-Liss, Inc.

EphA4 regulates central nervous system vascular formation

Molecules involved in axon guidance have recently also been shown to play a role in blood vessel guidance. To examine whether axon guidance molecules, such as the EphA4 receptor tyrosine kinase, might also play a role in development of the central nervous system (CNS) vasculature and repair following CNS injury, we examined wild-type and EphA4 null mutant (-/-) mice. EphA4-/- mice exhibited an abnormal CNS vascular structure in both the cerebral cortex and the spinal cord, with disorganized branching and a 30% smaller diameter. During development, EphA4 was expressed on endothelial cells. This pattern of expression was not maintained in the adult. After spinal cord injury in wild-type mice, expression of EphA4 was markedly up-regulated on activated astrocytes, many of which were tightly associated with blood vessels. In EphA4-/- spinal cord following injury, astrocytes were not as tightly associated with blood vessels as the wild-type astrocytes. In uninjured EphA4-/- mice, the blood-brain barrier (BBB) appeared normal, but it showed prolonged leakage following spinal cord injury. These results support a role for EphA4 in CNS vascular formation and guidance during development and an additional role in BBB repair.