Cardiovascular disease is the leading cause of non-communicable disease in the whole world killing 17 million people in 2012. Among the many vascular diseases is vascular calcification (VC) which is the mineral build up in the walls of blood vessels. Medial calcification is the plaque buildup in the medial layer of the blood vesicle that is characterized by arterial stiffness and high blood pressure. Current calcification research involves two dimensional (2D) lab methods such as flat petri dishes to investigate the mechanism that causes and inhibits vascular calcification. Research has shown that the use of three-dimensional (3D) models can be beneficial in mimicking the in vitro environment of the human body for lab practices. We aim to create a 3D benchtop model for vascular calcification research from decellularized carotid porcine arteries to understand the more accurate mechanisms that lead to the plaque buildup in the medial layer of the artery wall.

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Contexte : l'aldostérone (Aldo) est impliquée dans la rigidité artérielle et l'insuffisance cardiaque (IC), mais les mécanismes sous-jacents restent méconnus. La galectine-3 (Gal-3), une lectine se liant aux bêta-galactoside, joue un rôle important dans la fibrose et l'IC. Dans cette étude, nous avons recherché si la Gal-3 était impliquée dans la fibrose vasculaire induite par l'Aldo. Méthodes et résultats : Des cellules musculaires lisses vasculaires de rat (CMLVs) ont été stimulées avec de l'Aldo en combinaison avec des antagonistes du récepteur minéralocorticoïde (MR) ou des inhibiteurs de la Gal-3. L'Aldo régule l'expression de la Gal-3 via le MR dans les CMLVs. De plus, la surexpression de la Gal-3 augmente spécifiquement la synthèse de collagène de type I. Les inhibiteurs de la Gal-3 ou sa sous-expression (siRNA) bloquent la synthèse de collagène de type I induite par l'Aldo. Des rats ont été traités avec de l'Aldo + sel combiné avec du spironolactone ou de la pectine de citron modifiée (MCP) pendant 3 semaines. Les rats hypertensifs traités à l'Aldo ont présenté une hypertrophie vasculaire, une fibrose et une augmentation de l'expression aortique de Gal-3. Les traitements avec le spironolactone ou le MCP préviennent tous ces effets. Des souris sauvages (WT) et mutées pour la Gal-3 (KO) ont été traitées avec de l'Aldo pendant 6 heures. Le bolus d'Aldo augmente l'expression de la Gal-3 et du collagène de type I dans l'aorte des souris WT alors qu'aucun changement ne se produit dans les souris KO pour la Gal-3. Conclusions : Nos donnés indiquent que la Gal-3 est indispensable à la réponse fibrotique de l'Aldo dans les CMLVs in vitro et in vivo, suggérant un rôle clef pour la Gal-3 dans la fibrose vasculaire / Background. Aldosterone (Aldo) is involved in arterial stiffness and heart failure (HF), but the mechanisms have remained unclear. Galectin-3 (Gal-3), a beta-galactoside-binding lectin, plays an important role in fibrosis and HF. We here investigated whether Gal-3 is involved in Aldo-induced vascular fibrosis. Methods and Results. Rat vascular smooth muscle cells (VSMCs) were stimulated with Aldo combined with mineralocorticoid receptor (MR) antagonists and Gal-3 inhibitors. Aldo upregulated Gal-3 expression via MR in VSMCs. Moreover, Gal-3 over-expression specifically enhanced collagen type I synthesis. Gal-3 inhibitors or Gal-3 silencing (siRNA) blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin (MCP) for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, fibrosis and increased aortic Gal-3 expression. Spironolactone or MCP treatment reversed all the above effects. Wild type (WT) and Gal-3 knock-out (KO) mice were treated with Aldo for 6 hours. Aldo bolus increased aortic Gal-3 and collagen type I expression in WT mice whereas no changes occurred in Gal-3 KO mice. Conclusions. Our data indicate that Gal-3 is required for the fibrotic response to Aldo in VSMCs in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis

Il est admis que le Transforming Growth Factor-bêta (TGF-ß) est athéroprotecteur. Son apport bénéfique dans l'athérosclérose semble être contrarié par l'Interleukine-1-bêta (IL-1ß) qui est l'archétype des cytokines pro-inflammatoires. Notre but est, d'une part, de comprendre la régulation transcriptionnelle du TGF-ß dans les cellules musculaires lisses vasculaires (CMLv) humaines, dans l'athérome carotidien humain à partir des données du transcriptome obtenues sur 32 patients à la fois dans les plaques athéromateuses (ATH) et dans le tissu avoisinant macroscopiquement sain (TMS) et, d'autre part, d'étudier l'antagonisme des effets du TGF-ß et de l'IL-1ß sur des CMLv en culture provenant du TMS. Nous avons abordé nos problématiques par deux approches : (1) une analyse bioinformatique de données transcriptomiques issues de biopuces. (2) une étude in vitro sur des CMLv de la carotide humaine. in vitro. Grace à l'analyse in silico nous avons montré que l'implication de KLF6 dans la transcription du TGFB1 était assez spécifique des cellules musculaires lises vasculaires (CMLv). Nos résultats permettent également de proposer de nouveaux FT potentiels, spécifiques des CMLv (SLC2A4RG, GABP et SALL2), qui pourraient favoriser le rôle athéroprotecteur de TGFB1 dans les CMLv de la carotide. Enfin, il semble qu'il existe un équilibre entre les FT activateurs ou inhibiteurs de l'expression de TGFB1 qui permet d'en moduler finement la transcription. Notre approche in vitro, à montre que l'IL-1ß induisait un phénotype inflammatoire associé à une activité élastolytique consécutive, pour l'essentiel, à l'augmentation de l'expression de la cathépsine S (CTSS). La neutralisation du TGF-ß endogène dé-réprime l'expression de la CTSS et exerce un effet additif à celui de l'IL-1ß sur l'expression de l'enzyme. En conclusion, l'expression du TGFB1 dans les CMLv étant soumise à un contrôle transcriptionnel spécifique du type cellulaire, il est envisageable de développer des approches pharmacologiques qui permettent de maintenir l'expression du TGFB1 dans la paroi artérielle au niveau requis pour qu'il y exerce ses effets athéroprotecteurs / It is accepted that the TGF-β is atheroprotective. Its beneficial contribution atherosclerosis seems to be opposed by IL -1β that is the archetype of the pro-inflammatory cytokines. Our goal is, first, to understand the transcriptional regulation of TGF-β in human vSMCs in carotid atherosclerosis from the transcriptomic data obtained from 32 patients in both atherosclerotic plaques (ATM) and in the adjacent macroscopically intact tissue (MIT) and, secondly, to study the antagonism of the effects of TGF-β and IL-1β on vSMCs cultured from MIT samples. We followed two approaches: (1) a bioinformatic analysis of transcriptomic data from the microarrays; (2) an in vitro study of the human vSMCs. In silico, we have shown that the involvement of KLF6 in the transcription of TGFB1 was specific to the vSMCs. Our results also identify potential new transcription factors (SLC2A4RG, GABP and SALL2) that are vSMC-specific and could promote the atheroprotective role of TGFB1 in carotid vSMCs. The balance between the FT activating or inhibiting the expression of TGFB1 allows the fine tuning of its transcription. Our in vitro approach showed that IL-1β induces in the vSMCs an inflammatory phenotype associated with an elastolytic activity resulting mainly from the increase in the expression of cathepsin S (CTSS). Neutralization of endogenous TGF-β in the vSMCs de-represses the expression of the CTSS and exerts an additive effect to that of IL-1β on the expression of the enzyme. In conclusion, the expression of TGFB1 in the vSMCs is submitted to a cell-specific transcriptional control. It is possible to develop pharmacological approaches that maintain the expression the expression of TGFB1 in the arterial wall at the level required allowing TGF-β1 to exert its atheroprotective effects

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.