Abnormal vascular smooth muscle cell (VSMC) proliferation is thought to play an important role in the pathogenesis of atherosclerosis. Adipocytes produce several paracrine bioactive substances that can affect VSMC growth and migration. Our study focused on the ability of epicardial adipocytes to produce bioactive substances together with studying of direct effect of these substances on the VSMC proliferation rate. The gene expression of human cytokines (IL-6, IL-8, IL-18, RANTES and MCP-1) and adipokines (leptin and adiponectin) was measured in primary cell lines of epicardial and visceral adipocytes, both in undifferentiated and mature statuses. Moreover, adipokine production (IL-6, IL-8, MCP-1, VEGF and adiponectin) in conditioned media obtained from above mentioned primary cell cultures of adipocytes was measured by a Luminex assay. The VSMC proliferation rate was measured after co-culturing with CM obtained from primary cell cultures of adipocytes. The epicardial preadipocytes showed an increased expression of IL-8 (3,25-fold, p<0,05) compared with visceral preadipocytes. The expression of the adiponectin in epicardial preadipocytes was markedly decreased in comparison of the expression in visceral preadipocytes (p< 0,0001). Moreover, the gene expression was dependent on the differentiation...

The effect of the new vasodilatory and antianginal compound, bepridil (CERM-1978), was examined on the electrical activity of the vascular smooth muscle of isolated dog coronary arteries. Tetraethylammonium (10 mm) was used to induce excitability in the muscle in the form of Ca2+-dependent overshooting action potentials, whose inward current is carried almost exclusively by Ca2+ ion through voltage-dependent slow channels. Bepridil (5 × 10-7-1 × 10-5 M) produced a dose-dependent depression of the rate of rise and amplitude of these Ca2+ spikes. Complete blockade of the action potentials occurred at 1 × 10-5 M bepridil. These effects of bepridil were antagonized by elevation of external Ca2+ concentration ([CA]o). The effects of bepridil were substantially reversed by washout after about 30 min. Bepridil (10-5 M) also produced a small but significant (p < 0.05) increase in resting membrane resistance (input resistance increased from a mean of 10.1 to 12.4 mΩ), accompanied by a small but significant (p < 0.05) depolarization of 6 m V (from a mean of -51 to -45 mV). These latter effects are consistent with a diminution of the resting K+ conductance (gK) by bepridil. It is concluded that the vasodilatory and antianginal properties of bepridil may be explained by the action of this drug in depressing and blocking the Ca2+ influx into the cells, presumably by acting directly on the voltage-dependent slow channels in the cell membrane, and thereby lowering [Ca]i and thus the degree of contraction. Bepridil has Ca2+-antagonistic (or Ca2+ entry blocking or slow channel blocking) properties much like verapamil, but it is somewhat less potent than verapamil in this action (i.e., complete blockade occurred at 10-5 M bepridil vs. 2 × 10 -6 M verapamil.).

Atherosclerotic disease is characterized by the formation of lipid-ladden plaques in artery walls. During later stages of disease, these plaques become calcified by mechanisms involving the trans-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells. Although vascular calcification was thought to be a passive mechanism, evidence shows that this process is heavily modulated by various cell signaling mechanisms, including CB2 endocannabinoid receptors. Previous studies have shown that known CB2 antagonists accelerate VSMCs trans-differentiation to an osteoblast-like phenotype, indicating that this receptor serves an anti-calcification signal. The goal of this investigation is to determine if a series of 11-[2-arylmethylene)hydrazono]-PBD derivatives with established CB2 binding affinity function as CB2 antagonists or agonists in a cell culture model of VSMC osteoblastic trans-differentiation. MOVAS cells were grown in standard media or osteogenic media (to induce trans-differentiation) supplemented with and without the various PBD derivatives. Following the treatment period, the extent of osteoblast-like activity was evaluated by alizarin red staining for calcium deposition. To quantify the staining present, the dye was extracted using cetylpyridinium chloride hydrate solution and then analyzed via UV-Vis spectroscopy at 570 nm. The ability of the derivatives to modulation of osteoblastic transdifferentiation of MOVAS cells was further evaluated by performing Western blot analysis for expression of Runx2, an essential transactivator of osteoblast differentiation. Results of this work determined that some of the PBD derivatives increased the calcification compared to the control, indicating that they likely act as CB2 receptor antagonists, while others decreased calcification compared to the control, indicating that they likely act as CB2 receptor agonists. Not only do these results characterize the interactions of these compounds with CB2 receptors, they demonstrate that these PBD derivatives have biological activity. These results also further implicate CB2 receptors as a regulator of VSMC cell calcification, which could lead to novel drug therapies for the treatment of atherosclerotic plaques.

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This work identifies the Fragile X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMC). FXR1 was identified as an HuR interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The-HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased, but not normal arteries. SiRNA knock down of FXR1 increases abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. RNA-EMSA and RIP demonstrate that FXR1 directly interacts with an ARE and a previously uncharacterized element in the 3’UTR of TNFa. FXR1 expression is increased in VSMC challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts. Additionally, we observed significantly increased poly-A-Binding protein (PABP) expression localizing to discrete punctate structures in both vascular smooth muscle (VSMC) and endothelial cells (EC) of the aortic arch of Ldlr-/- mice, as compared to WT controls. EIF2α phosphorylation, requisite for SG formation, was also induced by clotrimazole and oxLDL in these cells. Interestingly, VSMCs pre-treated with anti-inflammatory cytokine IL-19 followed by clotrimazole significantly reduced the formation of SGs and eIF2a phosphorylation, suggesting a relationship between inflammation and SG formation in vascular cells. Reduction of SG component G3BP1 by siRNA knockdown significantly reduced stress granule formation and inflammatory gene abundance in hVSMC. Microtubule inhibitors reduced SG formation in hVSMC. These results support the hypothesis that SG formation in atherosclerosis is driven by inflammation, SG may mediate the cellular response to inflammation, and that anti-inflammatory treatment may lessen atherosclerosis progression and plaque formation by reduction of SGs. / Biomedical Sciences

La sclérose systémique (ScS) est une maladie auto-immune dévastatrice d'étiologie inconnue. Le dysfonctionnement immunitaire, la fibrose et la vasculopathie sont les trois principales caractéristiques de cette maladie. Une récente étude a révélé un nouveau lien entre l'auto-immunité et la fibrose, par la présence d'auto-anticorps stimulant le récepteur du facteur de croissance dérivé des plaquettes (PDGFR) des fibroblastes. Ces auto-anticorps sont capables de stimuler les espèces réactives de l'oxygène et d’activer la kinase régulée par un signal extracellulaire (ERK1/2). L’hypothèse que nous formulons est que les cellules musculaires lisses vasculaires (VSMCs) exprimant conjointement les PDGFR, répondront elles aussi aux autoanticorps anti-PDGF-R. Le travail présenté ici vise à valider la présence d'auto-anticorps PDGFR dans les sérums de patients ScS, et à caractériser ensuite la réponse de VSMCs exposées à de l'immunoglobuline G (IgG) de ces sérums, en mesurant l’activation des cascades de signalisation spécifiques, ainsi que l'induction des gènes impliqués dans la réponse fibrotique. Nos résultats démontrent la présence d'une fraction IgG stimulant une réponse phénotypique dans les cultures de VSMCs. Notamment, d’importantes régulations positive et négative des gènes pro-fibrotiques tgfb1 et tgfb2 respectivement, ont été observées dans les VSMCs exposées à des fractions de ScS-IgG. Les fractions de IgG positives pour l'activation de ERK étaient présentes dans la plupart, mais pas dans tous les échantillons de SSc (68%, 19/28), et moins présentes dans les contrôles 27% (11/3). Bien que, les fractions de SSc-IgG ont pu considérablement immunoprécipiter le PDGFR, l'utilisation d'un inhibiteur spécifique des récepteurs au PDGF (AG1296), n'a pas inhibé l'activation de ERK médiée par les fractions de SSc-IgG. Globalement, nos résultats indiquent la présence d'autoanticorps stimulants avec activité pro-fibrotique dans les sérums des patients ScS. Des travaux sont en cours pour identifier l'entité moléculaire responsable de la réponse d’IgG observée dans les cultures de VSMCs. / Systemic Sclerosis (SSc) is a devastating autoimmune disease of unknown etiology. Immune dysfunction, fibrosis and vasculopathy are the three major features of the disease; however, the interactions between these components are poorly understood. A novel link between autoimmunity and fibrosis has been proposed by the presence of stimulatory autoantibodies to the platelet-derived growth factor receptor (PDGFR) on fibroblasts. These autoantibodies were capable of stimulating reactive oxygen species and subsequent activation of ERK1/2. If the anti-PDGFR autoantibodies are present in the systemic circulation of SSc patients, they will most certainly encounter vascular smooth muscle cells (VSMCs). The latter are known to express the PDGFR and response to PDGF, which is a known phenotypic modulator of VSMCs. The work presented here seeks to readdress the presence of stimulatory anti-PDGFR autoantibodies in serum derived from SSc-patients and to characterize the effects of SSc-IgG on VSMCs by measuring the activation of specific signaling cascades and the induction of genes involved in fibrotic responses. Our results demonstrate the presence of an IgG fraction stimulating a phenotypic response in cultured VSMCs. Notably, a significant up-regulation of the pro-fibrotic gene tgfb1 and a significant down-regulation of the anti-fibrotic gene tgfb2 were observed in VSMC exposed to SSc-IgG fractions. Positive IgG fractions for ERK activation were present in most, but not all, SSc samples (68%, 19/28), and they were less present in controls (27%) (3/11). Although, the SSc-IgG fractions were able to significantly immunoprecipitate the PDGFR, the use of a selective PDGFR inhibitor, AG1296, did not inhibit the activation of ERK mediated by SSc-IgG fractions. Altogether, our findings suggest the presence of stimulatory autoantibodies with profibrotic activity in serum derived form SSc patients. Work is in progress to identify the molecular entity responsible for the IgG response observed in cultured VSMCs.

L’hypertension artérielle est le facteur de risque le plus important dans les maladies cardiovasculaires (MCV) et les accidents vasculaires cérébraux (AVC). L’hypertension artérielle essentielle est une maladie complexe, multifactorielle et polygénique. Même si on a identifié de nombreux facteurs de risque de l’hypertension artérielle, on ne comprend pas encore clairement les mécanismes qui la régissent. Les kinases hépatocytes produisant l’érythropoïétine (Eph) constituent la plus grande famille des récepteurs tyrosine kinase qui se lient à des ligands de surface cellulaire appelés éphrines sur les cellules avoisinantes. On sait que les interactions de Eph et des éphrines sont essentielles aussi bien dans les processus de développement que dans le fonctionnement des organes et des tissus adultes. Cependant on n’a pas encore étudié la relation entre Eph/éphrines et l’hypertension artérielle. Nous avons créé des modèles de souris knockout (K.O.) Ephb6-/-, Efnb1-/- et Efnb3-/- pour cette étude. Dans le modèle EphB6-/-, nous avons observé que les souris K.O. Ephb6 castrées, mais pas les femelles, ainsi que les souris mâles non castrées présentaient une tension artérielle élevée (TA) par rapport à leurs homologues de type sauvage (TS). Ceci suggère que Ephb6 doit agir de concert avec l’hormone sexuelle mâle pour réguler la TA. Les petites artères des mâles castrés Ephb6-/- présentaient une augmentation de la contractilité, une activation de RhoA et une phosphorylation constitutive de la chaîne légère de la myosine (CLM) lorsque comparées à celles de leurs homologues TS. Ces deux derniers résultats indiquent que la phosphorylation de CLM et de RhoA passe par la voie de signalisation de Ephb6 dans les cellules du muscle lisse de la paroi vasculaire (CMLV). Nous avons démontré que la réticulation de Efnbs mais non celle de Ephb6 aboutit à une réduction de la contractilité des CMLV. Ceci montre que l’effet de Ephb6 passe par la signalisation inversée à travers Efnb. Dans le modèle Efnb1-/- conditionnel spécifique au muscle lisse, nous n’avons observé aucune différence entre Efnb1-/- et les souris de TS concernant la mesure de la TA dans des conditions normales. Cependant, la TA des souris K.O. Efnb1 lors d’un stress d’immobilisation est supérieure à celle des souris de TS. Dans les petites artères des souris K.O. Efnb1, le rétrécissement et la phosphorylation de CLM étaient élevés. In vitro, la contractilité et l’activation RhoA de la CMLV des souris TS étaient augmentées quand leur Efnb1 était réticulé. Ces résultats corroborent ceux des souris KO Ephb6 et prouvent que l’effet de Ephb6 dans le contrôle de la TA se produit au moins par l’intermédiaire d’un de ses ligands Efnb1 dans les CMLV. Dans le modèle Efnb3-/-, on a observé une augmentation de la TA et du rétrécissement des vaisseaux chez les femelles Efnb3-/-, mais non chez les mâles; l’échographie a aussi révélé une résistance accrue au débit sanguin des souris K.O. femelles. Cependant la mutation de Efnb3 ne modifie pas la phosphorylation de la CLM ou l’activation de RhoA in vivo. Dans l’expérience in vitro, les CMLV des souris femelles Efnb3-/- ont présenté une augmentation de la contractilité mais pas celle des souris mâles Efnb3-/-. La réticulation des CMLV chez les mâles ou les femelles de TS avec solide anti-Efnb3 Ab peut réduire leur contractilité. Notre étude est la première à évaluer le rôle de Eph/éphrines dans la régulation de la TA. Elle montre que les signalisations Eph/éphrines sont impliquées dans le contrôle de la TA. La signalisation inverse est principalement responsable du phénotype élevé de la TA. Bien que les Efnb1, Efnb3 appartiennent à la même famille, leur fonction et leur efficacité dans la régulation de la TA pourraient être différentes. La découverte de Eph/Efnb nous permet d’explorer plus avant les mécanismes qui gouvernent la TA. / Hypertension is the most important risk factor for the cardiovascular diseases (CVD) and strokes. The essential hypertension is a complex, multifactorial and polygenic disease. Although many hypertension risk factors have been identified, the comprehensive understanding of mechanisms remains elusive. Erythropoietin-producing hepatocyte kinases (Ephs) are the largest family of receptor tyrosine kinases, which bind to cell surface ligands called ephrins on neighboring cells. Eph and ephrin interactions are known to be essential in developmental processes, as well as in functions of adult organs and tissues. However the relationship between Ephs/ephrins and hypertension has not been studied. Ephb6-/-, Efnb1-/- and Efnb3-/-knockout mice models were established for this study. In the EphB6-/- model, we observed that the castrated Ephb6 KO mice but not female or uncastrated male mice presented heightened blood pressure (BP) compared to the wild type (WT) counterparts. This suggests that Ephb6 needs to act in concert with sex hormone to regulate blood pressure. Small arteries from castrated Ephb6-/- males showed increased contractility, RhoA activation and constitutive myosin light chain (MLC) phosphorylation compared to their WT counterparts. The latter two findings indicate that RhoA and MLC phosphorylation are in the signaling pathway of Ephb6 in vascular smooth muscle cell (VSMC). We demonstrated that, crosslinking of Efnbs but not Ephb6 resulted in reduced VSMC contractility. This indicates that the effect of Ephb6 is via reverse signaling through Efnbs. In smooth muscle-specific conditional Efnb1-/- model, no difference was observed between Efnb1-/- and WT mice in BP measurement under a normal condition. However, the BP of Efnb1 KO mice during immobilization stress were higher than that of WT mice. In the small arteries from Efnb1 KO mice, the constriction and MLC phosphorylation were elevated. In vitro, the contractility and RhoA activation of WT VSMC were augmented when their Efnb1 was crosslinked. These results corroborate the findings from Ephb6 KO mice, and prove that the effect of Ephb6 in BP control is at least via one of its ligand Efnb1 in VSMC. In the Efnb3-/- model the heightened BP and increased vessel constriction were observed in Efnb3-/-females but not males; the echography also revealed the increased blood flow resistance of female KO mice. However the mutation of Efnb3 doesn’t alter the MLC phosphorylation or RhoA activation in vivo. In in vitro experiment, VSMCs from Efnb3-/- female mice showed increased contractility but did not Efnb3-/- male mice. Crosslinking of VSMCs from WT males or females with solid anti-Efnb3 Ab can reduce their contractility. Our study is the first to assess the role of Eph/ephrins in BP regulation. Eph/ephrins signalings are involved in the regulation of BP. The reverse signaling is mainly responsible for the elevated BP phenotype. Although the Efnb1, Efnb3 belongs to the same family, their function and effectiveness in the regulation of BP might be different. The discovery of Eph/Efnbs allows us to further explore the mechanism in BP.

La « semicarbazide-sensitive amine oxidase » (SSAO) catalyse la déamination oxydative des amines primaires en aldéhyde, peroxyde d’hydrogène et ammoniac. Elle participe à la différenciation cellulaires, l’inflammation et la transmigration leucocytaire à travers l’endothélium lymphatique. Nos objectifs ont été d’étudier le rôle de la SSAO (i) dans la différenciation chondrocytaire hypertrophique, en relation avec le développement de l’arthrose en utilisant des chondrocytes de rat en culture primaire et des genoux arthrosiques de patients (ii) dans le développement de l’athérosclérose en invalidant des souris ApoE-/- qui développent naturellement l’athérosclérose pour le gène de la SSAO. Au niveau articulaire, la SSAO a été détectée dans le cartilage de rat et humain. In vitro, la SSAO (activité et expression) augmentent au cours de la différenciation terminale de chondrocytes de rat. Son inhibition par le LJP1586 entraîne un retard de différenciation chondrocytaire. La SSAO augmente également dans les zones arthrosiques du cartilage humain parallèlement à l’augmentation de l’hypertrophie. La SSAO jouerait donc un rôle dans la différenciation terminale des chondrocytes (hypertrophie) possiblement via le transport de glucose et dans le développement de la maladie. Au niveau vasculaire, les souris femelles ApoE-/-SSAO-/- de 25 semaines présentent une augmentation de la surface des plaques d’athérome par rapport aux ApoE-/-. Ceci est associée à une diminution de l’expression d’α-actine dans le média sous les plaques et de smMHC dans l’aorte abdominale (AA) sans modification ni de l’infiltration des lymphocytes T; ni des monocytes/ macrophages dans la paroi artérielle, ni du profil cytokinique pro-/anti-inflammatoire dans la rate. A 15 semaines, les souris femelles ApoE-/-SSAO-/-, sm-MHC a diminué dans les AA de ces souris par rapport aux ApoE-/- ainsi qu’une réorientation du trafic des cellules immunitaires vers la paroi aortique sans modification significative de la surface des plaques a été détecté. La SSAO jouerait donc un rôle précoce dans le développement de l’athérosclérose via une modification du trafic des cellules immunitaires et du phénotype des CML dans la paroi / The semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidative deamination of primary amines into aldehydes, hydrogen peroxide and ammonia. The SSAO was implicated in cellular differentiation, inflammation and transmigration of leukocyte through the lymphatic. The objectives of this work were to study the role of SSAO (i) in chondrocyte differentiation and in the development of osteoarthritis using rat chondrocyte primary cell culture and osteroarthritic samples from patients. (ii) in the development of atherosclerosis using ApoE-/- mice, which develop naturally atherosclerosis, invalidated for the SSAO gene. Concerning the articulation, the SSAO (expression and activity) was detected in the rat and human cartilage. In vitro, SSAO increases during chondrocyte terminal differentiation (hypertrophy) and the inhibition of its activity by LJP1586, decreases the level of differentiation. In human arthritic cartilage, SSAO was higher that in healthy cartilage, in association with an increase in hypertrophic markers. The SSAO plays a role in the terminal differentiation of chondrocytes and might be involved in the development of osteoarthritis. At the vascular level, 25 week-old female ApoE-/-SSAO-/- mice presented a 50% increase in plaque surface associated with an 80% decrease in α-actin expression in the media of aortic sinus and a decrease in sm-MCH in abdominal aortas (AA) compared to ApoE-/- mice. These results were not due neither to a modification of monocytes/ macrophages, Tcell infiltration in the plaque nor in a pro- or anti-inflammatory cytokine change in spleen. In 15 week-old ApoE-/-SSAO-/- mice, even if no modification of plaque surface was found, a decrease in sm-MHC was noticed in the AA from ApoE-/-SSAO-/- compare to ApoE-/- mice. More over, the immune cell trafficking was increased in the aortic wall of ApoE-/-SSAO-/- compared to ApoE-/- mice. Thus, SSAO is involved in the early development of atherosclerosis in changing the immune cell trafficking and the VSMC phenotype