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.

La sclérose systémique (ScS) est une maladie auto-immune dont l’un des principaux auto-anticorps, dirigé contre la protéine centromérique B (CENP-B), est fortement associé à l’hypertension artérielle pulmonaire, l’une des causes majeures de décès dû à la ScS. L’hypertension résulte de l’occlusion progressive des vaisseaux suite à une hyperactivation des cellules musculaires lisses (CML) de la paroi vasculaire. Cependant, les facteurs responsables de ce remodelage vasculaire restent inconnus. Plusieurs études récentes ont démontré que certains auto-antigènes possèdent des fonctions biologiques additionnelles lorsqu'ils se retrouvent dans le milieu extracellulaire. En effet, une fois libérés par nécrose ou apoptose, ces auto-antigènes adoptent une activité biologique qui s'apparente à celles des cytokines et peuvent ainsi participer aux processus normaux de réparation de blessure et/ou acquérir une activité pathogène qui contribue au développement de certaines maladies auto-immunes. Nos résultats suggèrent que la CENP-B peut être ajoutée à cette liste de molécules bifonctionnelles. À l'aide des techniques d'immunofluorescence, d'ELISA cellulaire et de cytométrie en flux, nous avons démontré que la CENP-B se liait spécifiquement à la surface des CML vasculaire de l’artère pulmonaire avec une plus grande affinité pour le phénotype contractile que synthétique. Cette liaison provoquait la migration des cellules ainsi que la sécrétion de cytokines pro-inflammatoires telles que l’interleukine 6 et 8. Les mécanismes par lesquels la protéine exerçait ces effets impliquaient la phosphorylation de FAK et Src ainsi que la voie des MAP kinases, avec ERK1/2 et p38. Des études de signalisation intracellulaire effectuées à l’aide de plusieurs inhibiteurs spécifiques ainsi que des études de désensibilisation nous ont permis d’identifier le récepteur de la CENP-B en plus d’identifier les mécanismes complets de sa signalisation membranaire. Nous avons démontré que la CENP-B se liait de manière spécifique aux CML vasculaire via le récepteur de chémokine 3 (CCR3) pour ensuite transactiver le récepteur EGF, selon un mécanisme métalloprotéase-dépendant qui implique le relargage du HB-EGF. Cette transactivation est un processus important dans l’activation de la voie des MAP kinases ainsi que dans la sécrétion d’IL-8 induite par la CENP-B. Finalement, nous avons démontré que les auto-anticorps anti-CENP-B pouvaient abolir cette cascade de signalisation, empêchant ainsi la CENP-B d’exercer son rôle de cytokine. L’identification de la CENP-B comme ligand du CCR3 ouvre donc plusieurs perspectives quant à l’étude du rôle pathogène des auto-anticorps anti-CENP-B dans la ScS. / CENP-B is a highly conserved, centromere associated protein and is a major autoantigen in systemic sclerosis (SSc). Anti-CENP-B autoantibodies are associated with prominent vascular manifestations such as pulmonary arterial hypertension (PAH) in the limited cutaneous subset of SSc. PAH occurs as a consequence of progressive obliteration of small arteries due to vascular smooth muscle cell dysfunction, migration and proliferation. However, the factors driving this obliteration are unknown. Earlier in vitro studies have demonstrated that some autoantigens have an additional role when they are released in the extracellular environment during the course of injurious insults resulting in cell death. Indeed, it was previously suggested that extracellular autoantigens participate in normal wound repair processes by acting like cytokines and/or chemokines and subsequently displaying pathogenic activities that contribute to the development of autoimmune diseases. Our present findings suggest that the nuclear autoantigen CENP-B can be added to this set of bifunctional molecules. The present study clearly indicates that exogenous CENP-B bound specifically to the surface of human pulmonary artery SMCs. Binding of CENP-B to SMC stimulated their migration during in vitro wound healing assays, as well as their secretion of interleukins 6 and 8. The mechanism by which CENP-B mediated these effects involved the focal adhesion kinase, Src, ERK1/2, and p38 MAPK pathways. Moreover, CENP-B released from apoptotic endothelial cells was found to bind to SMC, thus indicating a plausible in vivo source of extracellular CENP-B. Here, we also report several lines of evidence indicating that CENP-B, which has no obvious primary or secondary structural homology to chemokines, induced SMC activation by interacting with CCR3. Moreover, the present study clearly demonstrates the involvement of EGFR in CENP-B signaling leading to IL-8 secretion. Finally, anti-CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular SMCs. The present study sheds new light on the possible role of extracellular CENP-B and its potent biological effects on human pulmonary artery SMCs. The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti-CENP-B autoantibodies.

Les complications vasculaires telles que l’augmentation de la contractilité et la prolifération cellulaire sont les complications les plus communes observées dans le diabète et l’hyperglycémie chronique est un facteur important dans ces processus. La voie de signalisation de Gαq joue un rôle important dans la régulation du tonus vasculaire et l’altération de celle-ci peut contribuer aux complications vasculaires observées dans les cas de diabète et d’hyperglycémie. Il a été observé que les taux et l’activité des protéines kinase C (PKC) et du diacylglycérol (DAG) sont augmentés dans ces conditions. Cependant, aucune étude n’a démontré l’implication de Gαq/11 et des PLCβ, molécules de signalisation en amont de PKC/DAG. Plusieurs études révèlent que l’augmentation des taux et de l’activité des PKC et du DAG induite par l’hyperglycémie dans des cellules du muscle lisse vasculaire (CMLV) est attribuée à l’augmentation du stress oxydatif. De plus, les niveaux de certains peptides vasoactifs, tels que l’angiotensine II et l’endothéline-1, augmentés dans les conditions de diabète/d’hyperglycémie, peuvent contribuer à l’augmentation du stress oxydatif observée. Le travail présenté dans cette thèse avait pour but d’examiner les effets de l’hyperglycémie sur les niveaux d’expression protéique de Gαq/11 et de ses molécules associées, ainsi que d’étudier le mécanisme moléculaire par lequel l’hyperglycémie module la voie de signalisation de Gαq dans les CMLV. Dans la première étude, nous avons examiné si l’hyperglycémie pouvait moduler l’expression des protéines Gαq, Gα11, PLCβ1 et PLCβ2. Le prétraitement des CMLV A10 avec 26 mM de glucose durant 72 heures augmente l’expression des protéines Gαq, Gα11, PLCβ-1 et PLCβ-2 en comparaison avec les CMLV témoins. Le traitement avec des antagonistes aux récepteurs AT1 de l’Ang II, et ETA/ETB de l’ET-1, atténue la hausse de Gαq, de Gα11, de PLCβ1 et de PLCβ2 induite par l’hyperglycémie. De plus, la formation d’IP3 stimulée par l’ET-1 était plus élevée dans les CMLV exposée à 26 mM de glucose. Le traitement des CMLV A10 avec l’Ang II et l’ET-1 augmente également les niveaux d’expression des protéines Gα q/11 et PLCβ. Cette augmentation de l’expression est restaurée au niveau des CMLV témoins par les antagonistes des récepteurs AT1, ETA et ETB. Ces résultats suggèrent que l’augmentation de l’expression des protéines Gαq/11 et PLCβ dans les CMLV induite par l’hyperglycémie est attribuée à l’activation des récepteurs AT1, ETA et ETB. Dans la seconde étude, nous avons examiné l’implication du stress oxydatif dans l’augmentation des niveaux d’expression des protéines Gαq/11 et PLCβ et de leur signalisation induite par l’hyperglycémie. Nous avons également déterminé le mécanisme responsable de l’augmentation du stress oxydatif induite par l’hyperglycémie. L’augmentation de l’expression des protéines Gαq/11 et PLCβ des CMLV A10 exposées à 26 mM de glucose est revenue au niveau basal après un traitement avec l’antioxydant diphenyleneiodonium (DPI), et la catalase, un chélateur du peroxyde d’hydrogène, mais pas par le 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP) ni par l’acide urique, des chélateurs du peroxynitrite. De plus, l’augmentation de la formation d’IP3 stimulée par l’ET-1 dans les CMLV exposées à 26 mM de glucose est revenue au niveau basal après un traitement avec le DPI et la catalase. Ces résultats suggèrent que l’augmentation du stress oxydatif induite par l’hyperglycémie contribue à l’augmentation de l’expression des protéines Gαq/11 et les molécules associées à la voie de signalisation de Gq. De plus, l’augmentation de la production d’anion superoxyde (O2-), de l’activité de la NADPH oxydase et de l’expression des protéines p22(phox) et p47(phox) induite par l’hyperglycémie est revenue à un niveau basal après un traitement avec les antagonistes des récepteurs AT1, ETA et ETB. Ces résultats suggèrent que l’hyperglycémie augmente les niveaux endogènes de l’Ang II et de l’ET-1, ce qui augmente le stress oxydatif par la formation d’O2- et de H2O2 et peut contribuer à l’augmentation des niveaux de Gq/11α et de leurs molécules de signalisation. Puisqu’il a été observé que l’hyperglycémie transactive les récepteurs aux facteurs de croissance tels que le récepteur au facteur de croissance épidermique (EGF-R) et le récepteur au facteur de croissance dérivé des plaquettes (PDGF-R), nous avons entrepris d’examiner, dans la troisième étude, l’implication d’EGF-R et de PDGF-R dans l’augmentation des niveaux de Gαq/11, de PLCβ et de leur signalisation induite par l’hyperglycémie. L’augmentation des niveaux d’expression des protéines Gαq, Gα11, PLCβ-1 et PLCβ-2 induite par l’hyperglycémie est revenue au niveau basal après un traitement avec les inhibiteurs d’EGF-R (AG1478) et de PDGF-R (AG1295) et par l’inhibiteur de c-Src, PP2. L’augmentation de la phosphorylation d’EGF-R et de PDGF-R induite par l’hyperglycémie a été abolie par AG1478, AG1295 et PP2. De plus, l’augmentation des niveaux de Gαq/11, et de PLCβ induite par l’hyperglycémie est atténuée par l’inhibiteur des MAPK, le PD98059, et par l’inhibiteur d’AKT, le wortmannin. L’augmentation de la phosphorylation d’ERK et d’AKT était également atténuée par AG1478 et AG1295. Ces résultats suggèrent que la transactivation des récepteurs aux facteurs de croissance induite par c-Src peut contribuer à l’augmentation des niveaux de Gα q/11/PLC et de leur signalisation par la voie des MAPK/PI3K. En conclusion, les études présentées dans cette thèse indiquent que l’hyperglycémie augmente les niveaux de Gαq/11 et de PLCβ. Nous avons émis des évidences qui démontrent que l’augmentation endogène de l’Ang II et de l’ET-1 par l’hyperglycémie peut contribuer à l’augmentation de la production d’O2- et de H2O2 résultant ainsi en une augmentation du stress oxydatif qui pourrait être responsable de l’augmentation de Gαq/11/PLC et de leur signalisation dans les conditions d’hyperglycémie. Finalement, nous avons démontré que la transactivation des récepteurs aux facteurs de croissance induite par l’hyperglycémie peut être responsable de l’augmentation de Gαq/11/PLC et les molécules associées à la voie de signalisation de Gq dans les cas de diabète et d’hyperglycémie. / Vascular complications including increased contractility and cell proliferation are most common complications in diabetes, and chronic hyperglycemia seem to be an important contributing factor in this process. Gqα signaling pathway plays an important role in the regulation of vascular tone and aberration of these mechanisms may contribute to vascular complications in hyperglycemia/diabetes. The levels and activity of protein kinase C (PKC) and diacylglycerol (DAG) were shown to be up-regulated in diabeteshyperglycemia. In addition, studies on the expression of upstream signaling molecules of phosphatidyl inositol (PI) turnover were lacking. The enhanced activity/levels of protein PKC and DAG induced by high glucose in VSMC have been shown to be attributed to the increased oxidative stress. Furthermore, the levels of various vasoactive peptides including Ang II and ET-1 which are augmented in diabetes and under hyperglycemic conditions, may also contribute to the enhanced oxidative stress in diabetes/hyperglycemia. The work presented in this thesis was therefore undertaken to examine if hyperglycemia/diabetes could also modulate the expression of Gqα and phospholipase Cb (PLCβ) proteins and associated PI turnover signaling in A10 VSMC exposed to high glucose and to explore the molecular mechanisms by which high glucose modulates Gqα/PLC signaling. The first study was undertaken to investigate if hyperglycemia can modulate the expression of Gqα, G11α, PLCβ-1 and PLCβ-2 and associated signaling. Pre-treatment of A10 VSMC with high glucose (26 mM) for 3 days augmented the levels of Gqα, G11α, PLCβ-1 and β-2 proteins as compared to control cells which were restored to control levels by endothelin-1 (ET-1) ETA and ETB and angiotensin II (Ang II) AT1 receptor antagonists. In addition, ET-1-stimulated IP3 formation was also significantly higher in VSMC exposed to high glucose. Furthermore, treatment of A10 VSMC with Ang II and ET-1 also increased significantly the levels of Gq/11α and PLCβ proteins which were restored towards control levels by ETA/ETB and AT1 receptor antagonists. These results suggest that high glucose augmented the expression of Gq/11α, PLCβ and -mediated signaling in VSMC which may be attributed to activation of AT1, ETA and ETB receptors. The second study was undertaken to investigate the implication of oxidative stress in high glucose-induced enhanced expression of Gq/11α and PLCβ1/2 proteins and associated signaling in A10 VSMC and to explore the mechanism responsible for high glucose induced enhanced oxidative stress. We showed that the increased levels of Gqα, G11α, PLCβ-1 and PLCβ-2 proteins in A10 VSMCs exposed to high glucose were restored to control levels by the antioxidant diphenyleneiodonium (DPI), and catalase, a scavenger of hydrogen peroxide, but not by 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP) and uric acid, scavengers of peroxynitrite. In addition, endothelin-1 (ET-1)-stimulated production of IP3 that was enhanced by high glucose was also restored towards control levels by DPI and catalase. These results suggest that high glucose-induced enhanced oxidative stress that contributes to the enhanced expression of Gq/11α and PLCβ protein and signaling. Furthermore, the enhanced production of superoxide anion (O2-), NADPH oxidase activity and enhanced expression of p22(phox) and p47(phox) proteins induced by high glucose was restored to control levels by losartan, BQ123 and BQ788, the antagonists of angiotensin AT1 and endothelin-1 ETA/ETB receptors respectively. These results suggest that high glucose-induced enhanced levels of endogenous Ang II and ET-1, by increasing oxidative stress may contribute to the increased levels of Gq/11α and-mediated signaling in A10 VSMC. Since high glucose has been shown to increase growth factor receptor activation, we investigated, in the third study, the role of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) transactivation in high glucose-induced enhanced expression of Gq/11α and PLCβ. The increased levels of Gqα, G11α, PLCβ-1 and PLCβ-2 proteins induced by high glucose were restored to control levels by AG1478, an inhibitor of EGF-R, and AG1295, an inhibitor of PDGF-R as well as by PP2, an inhibitor of c-Src. High glucose-induced increased phosphorylation of EGF-R and PDGF-R which were abolished by AG1478, AG1295 and PP2. High glucose-induced enhanced levels of Gq, G11α and PLCβ were also attenuated by PD98059, an inhibitor of mitogen-activated protein kinase (MAPK), and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). In addition, high glucose-induced enhanced phosphorylation of ERK1/2 and AKT was also attenuated by AG1478 and AG1295. These results suggest that c-Src-induced transactivation of growth factor receptor contributes to the high glucose-induced enhanced expression of Gq/11α/PLC and-mediated cell signaling through MAPK/PI3K pathway. In conclusion, the studies presented in this thesis indicate that hyperglycemia increased the levels of Gq/11α and PLCβ1/2 proteins and mediated signaling. We provided evidence that high glucose-induced increased levels of Ang II and ET-1 may contribute to the enhanced production of O2- and H2O2 and results in enhanced oxidative stress which may be responsible for the high glucose-induced enhanced expression of Gq/11α and PLCβ. Finally, we demonstrated that high glucose-induced transactivation of growth factor receptors may also be responsible for the high glucose-induced enhanced expression of Gq/11α and PLCβ1/2.

Le stress oxydatif est impliqué dans l’expression du récepteur B1 des kinines (RB1) dans différents modèles de diabète et d'hypertension. Puisque l'angiotensine II (Ang II) et l'endothéline-1 (ET-1) sont des peptides prooxydants impliqués dans les maladies cardiovasculaires, leur contribution dans l'augmentation de l'expression du RB1 a été étudiée dans des cellules musculaires lisses vasculaires (CMLV). Le QRT-PCR et l’immunobuvardage de type Western ont été utilisés pour mesurer l’expression du RB1 dans des CMLV dérivées de la lignée A10 et de l’aorte de rats Sprague-Dawley. Cette étude montre que l’Ang II augmente l’expression du RB1 (ARNm et protéine) en fonction de la concentration et du temps (maximum 1 μM entre 3-6 h). Cette augmentation implique le récepteur AT1, la PI3K et le NF-κB, mais non le récepteur AT2 et ERK1/2. Aussi, le récepteur ETA de l’ET-1 est impliqué dans la réponse à l’Ang II à 6-8 h et non à 1-4 h. Par contre, l’ET-1 augmente l’expression du RB1 (maximum 2-4 h) via la stimulation des récepteurs ETA et ETB. L’augmentation du RB1 causée par l’Ang II et l’ET-1 est bloquée par les antioxydants (N-acétyl-cystéine et diphénylèneiodonium). Ces résultats suggèrent que l’Ang II induit le RB1 dans les CMLV par le récepteur AT1 dans la première phase, et par la libération d’ET-1 (majoritairement par ETA) dans la phase tardive, via le stress oxydatif et l’activation de la PI3K et du NF-κB. Ces résultats précisent le mécanisme impliqué dans la surexpression du RB1 ayant des effets néfastes dans le diabète et l'hypertension. / Oxidative stress is involved in the overexpression of kinin B1 receptor (B1R) in various models of diabetes and hypertension. Since angiotensin II (Ang II) and endothelin-1 (ET-1) are pro-oxidative peptides involved in cardiovascular diseases, their contribution in increasing the expression of B1R was examined in vascular smooth muscle cells (VSMC). QRT-PCR and Western blot analysis were used to measure the expression of B1R in VSMC derived from A10 cell line and the aorta of Sprague-Dawley rats. This study shows that Ang II increases the expression of B1R (mRNA and protein) in a concentration- and time-dependent manner (maximum 1 μM between 3-6 h). This increase involves AT1 receptor, PI3K and NF-κB, but not AT2 receptor and ERK1/2. Moreover, ETA receptor is involved in the effect of Ang II at 6-8 h but not at 1-4 h. However, ET-1 increases expression of B1R (maximum 2-4 h) via stimulation of ETA and ETB receptors. Ang II and ET-1-induced increase expression of B1R is blocked by antioxidants (N-acetyl-L-cysteine and diphenyleneiodonium). These results suggest that Ang II induces B1R in VSMC by AT1 receptor in the first phase and by releasing ET-1 (predominantly by ETA receptor) in the late phase, via oxidative stress and activation of PI3K and NF-κB. These results clarify the mechanism involved in the overexpression of B1R which has adverse effects in diabetes and hypertension.

Nous avons précédemment montré que les cellules musculaires lisses vasculaires(CMLV) des rats spontanément hypertendus (SHR) présentent une expression augmentée des protéines G inhibitrices (Gi) et une prolifération cellulaire accrue par rapport aux CMLV des rats Wystar-Kyoto (WKY). Le niveau d'AMPc s’est également avéré plus faible dans les CMLV de SHR. La présente étude a donc été entreprise afin d'examiner la contribution de la diminution du niveau intracellulaire d'AMPc à l’augmentation de l'expression des protéines Gi et à la prolifération accrue des CMLV de SHR et de continuer à explorer les mécanismes moléculaires sous-jacents responsables de cette réponse. Les CMLV de SHR ont montré par rapport aux CMLV des WKY une expression accrue de Giα-2 et Giα-3 qui a été diminué d'une manière dépendante de concentration par le dbcAMP, un analogue d'AMPc perméable à la membrane cellulaire. En outre, les fonctions augmentées des protéines Gi comme démontrées par l'amplification de l’inhibition de l'adénylate cyclase par les hormones inhibitrices et l'activité forskoline (FSK)-stimulée de l’adénylate cyclase par une faible concentration de GTPγS dans les CMLV de SHR ont également été restaurées aux niveaux de WKY par le dbcAMP. La prolifération accrue des CMLV de SHR a également été atténuée par le dbcAMP et la forskoline, un activateur de l'adénylate cyclase. De plus, dbcAMP a restauré la production augmentée d'anion superoxyde (O2-), l'activité de la NAD(P)H oxydase et l’expression accrue des protéines Nox 4 et p47phox observée dans les CMLV de SHR jusqu’au niveau contrôle. Par ailleurs, la phosphorylation accrue des PDGF-R, EGF-R, c-Src et ERK1/2 énoncée par les CMLV de SHR a également été diminuée par le dbcAMP d'une manière dépendante de concentration. Ces résultats suggèrent que le niveau réduit d'AMPc intracellulaire montré par les CMLV de SHR contribue à l'expression accrue des protéines Gi et à l’hyperprolifération cellulaire à travers l’augmentation du stress oxydatif, la transactivation des EGF-R, PDGF-R et la voie de signalisation des MAP kinases. / We have previously shown that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of inhibitory G proteins (Gi) and enhanced cell proliferation as compared to VSMC from Wystar-Kyoto rats (WKY). The levels of cAMP were shown to be decreased in VSMC from SHR. The present study was therefore undertaken to examine the contribution of the decreased intracellular level of cAMP in the enhanced expression of Gi proteins and increased proliferation of VSMC from SHR and to further explore the underlying molecular mechanisms responsible for this response. VSMC from SHR showed an enhanced expression of Giα-2 and Giα-3 as compared to VSMC from WKY which was decreased in a dose-dependent manner by dbcAMP, a cell-permeable cAMP analog. In addition, the enhanced functions of Gi proteins as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin (FSK)-stimulated adenylyl cyclase activity by low concentration of GTPγS in VSMC from SHR were also restored to the WKY levels by dbcAMP. The enhanced proliferation of VSMC exhibited by SHR was also attenuated by dbcAMP and forskolin, an activator of adenylyl cyclase. In addition, dbcAMP also restored the increased production of superoxide anion (O2-), NAD(P)H oxidase activity and enhanced expression of Nox 4 and p47phox proteins observed in VSMC from SHR to control levels. Furthermore, the increased phosphorylation of PDGF-R, EGF-R, c-Src and ERK1/2 exhibited by VSMC from SHR were also decreased by dbcAMP in a dose-dependent manner. These results suggest that decreased levels of intracellular cAMP exhibited by VSMC from SHR contributes to the enhanced expression of Gi proteins and hyperproliferation through increasing oxidative stress and transactivation of EGF-R, PDGF-R and MAP kinase signaling pathway.

Este estudo se propôs a avaliar o potencial inflamatório das partículas eliminadas na exaustão do diesel (PED) e 1,2-naftoquinona (1,2-NQ) sobre outros compartimentos, como o músculo liso vascular (aorta torácica; RTA) e do corpo cavernoso isolados de ratos (RCC) e os mecanismos envolvidos via ensaios funcionais e bioquímicos. A injeção i.tr. das PED e 1,2-NQ em ratos Wistar causou inflamação e hiporreatividade das vias aéreas associados ao aumento significativo do relaxamento evocado pela ACh na RTA. No RCC desses mesmos animais, tanto o GTN quanto o estímulo elétrico (EFS) causou maior relaxamento. O conteúdo basal de TBARs na RTA e pulmão foi reduzido, embora outros testes indicadores de estresse oxidativo e / ou atividade antioxidante não mostraram diferenças entre os grupos. As taxas de expressão gênica / protéica da nNOS no RCC de ratos não diferiram do grupo controle, mas a expressão da eNOS e iNOS foi reduzida na RTA e RCC. Não foram quantificadas concentrações séricas do TNF<font face=\"Symbol\">&#945 ou IL-1<font face=\"Symbol\">b, sugerindo que os efeitos sistêmicos ocorrem independentemente destas citocinas. Conclui-se que o tratamento agudo de ratos com a mistura de poluentes induziu inflamação das vias aéreas (e hiporreatividade), capaz de afetar outros compartimentos, como a musculatura lisa vascular (RTA) e do RCC. / We tested the hypothesis that local inflammation in the airways evoked by intra-tracheal instillation of the environmental chemical 1,2-naphthoquinone (1,2-NQ) and diesel exhaust particles (DEP) is capable of targeting other systemic compartments, such as vessels (rat thoracic aorta; RTA) and corpus cavernosum (RCC), and possible involved mechanisms. After 3h, this treatment induced airways hyporresponsiveness to ACh and local inflammation. This effect was associated with decreased numbers of leukocyte in the blood and spleen and increased number of leukocytes in the bone marrow. Pollutant treatment also markedly increased ACh-induced relaxation in RTA and by both GTN- and electrical stimulation-induced relaxation in RCC. Exposure to pollutants did not affect FE-induced contraction in RTA. Neither serum levels of cytokines (TNF<font face=\"Symbol\">&#945 e IL-1<font face=\"Symbol\">b) nor basal concentration of total nitrate were different amongst groups. No evidence of increased catalase activity in RTA, RCC and lung was found. The treatment reduced the eNOS e iNOS gene expression in RTA e RCC, without significantly affecting the nNOS gene expression in RCC. Our results are consistent with the hypothesis that DEP-induced airways hiporresponsiveness and inflammation can account to produce systemic changes, such as structural and functional changes in the RTA and RCC by means of substances derived from endothelium or due to the ability of these pollutants to act to stimulate the production of scavenger of free radical.

No description available.

O remodelamento vascular é um determinante fundamental do lúmen em doenças vasculares, porém os mecanismos envolvidos não estão completamente elucidados. Nós investigamos o papel da chaperona redox residente do retículo endoplasmático Dissulfeto Isomerase Proteica (PDI) e sua fração localizada na superfície celular (peri/epicelular=pecPDI) no calibre e arquitetura vascular durante reparação à lesão. Em artérias ilíacas de coelho submetidas à lesão in vivo, houve importante aumento do mRNA e expressão proteica (~25x aumento 14 dias pós-lesão vs. controle) da PDI. O silenciamento da PDI por siRNA (cultura de órgãos) acentuou o estresse do retículo e apoptose, diferentemente da inibição da pecPDI com anticorpo neutralizante (PDI Ab). Bloqueio in vivo da pecPDI por aplicação de gel perivascular contendo PDI Ab no 12° dia após lesão, com análise após 48 h, promoveu ca.25% redução no calibre vascular analisado por arteriografia e diminuição similar na área total do vaso detectada por tomografia de coerência óptica. Neste processo, não ocorreu alteração no tamanho da neoíntima, indicando assim, que PDI Ab acentuou remodelamento constrictivo. Neutralização da pecPDI promoveu importantes alterações na arquitetura da matriz de colágeno e citoesqueleto, resultando em fibras com orientação invertida e desorganizadas. Diminuição na produção de espécies reativas de oxigênio e óxidos de nitrogênio também ocorreu. Análise de propriedades viscoelásticas nas artérias indicou redução na ductilidade vascular, evidenciada pela menor distância para ruptura. As alterações subcelulares no citoesqueleto observadas in vivo após PDI Ab foram recapituladas em um modelo de estiramento cíclico em células musculares lisas vasculares, com importante redução na formação das fibras de estresse. Em modelo de migração randômica de células musculares lisas, a exposição a PDI Ab reduziu a resiliência de regulação da polaridade. Embora a neutralização da pecPDI não tenha afetado a atividade global de RhoA, ela promoveu alterações no padrão de marcação em resposta ao estiramento, na redistribuição de RhoA na superfície celular e na associação com regiões contendo caveolina. Além disso, em aterosclerose nativa em humanos, a expressão da PDI correlacionou-se inversamente com remodelamento constrictivo. Dessa forma, PDI é fortemente expressa após a lesão e sua fração peri/epicelular remodela a arquitetura da matriz e citoesqueleto, promovendo um efeito anti-remodelamento constrictivo / Whole-vessel remodeling is a critical lumen caliber determinant in vascular disease, but underlying mechanisms are poorly understood. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI(peri/epicellular=pecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and pecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis, contrarily to pecPDI neutralization with PDI-antibody(PDI Ab). PecPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. PecPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and disorganization. Decreased ROS and nitrogen oxide production also occurred. Viscoelastic artery properties assessment showed decreased ductility, evidenced by decreased distance to rupture. Subcellular cytoskeletal disruption by PDI Ab was recapitulated in vascular smooth muscle cell stretch model, with marked decrease in stress fiber buildup. Also, PDI Ab incubation promoted decreased regulation resilience of vascular smooth muscle migration properties. While pecPDI neutralization did not affect global RhoA activity, there was altered RhoA redistribution to the cell surface and association with caveolin-containing clusters, which mislocalized after stretch. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect

Durante o processo de reparação tecidual, o fator de transformação de crescimento <font face=\"symbol\">b1 (TGF-<font face=\"symbol\">b1) apresenta um importante papel na regulação da expressão da <font face=\"symbol\">a-actina de músculo liso (<font face=\"symbol\">a-AML) e portanto, na diferenciação miofibroblástica. Como os fibroblastos pulpares apresentam características peculiares, com a expressão de proteínas específicas que os diferem de fibroblastos de outros tecidos conjuntivos, o presente estudo avaliou in vitro se o TGF-<font face=\"symbol\">b1 aumenta a expressão de <font face=\"symbol\">a-AML em fibroblastos pulpares humanos comparando-os com fibroblastos de gengiva. Para tal, diferentes doses de TGF-<font face=\"symbol\">b1 (5 à 10 ng/ml) foram adicionadas às culturas de células, sendo a expressão da <font face=\"symbol\">a-AML analisada por imunofluorescência e western-blotting. Ambos os tipos celulares imunoexpressaram <font face=\"symbol\">a-AML mesmo sem o tratamento com o TGF-<font face=\"symbol\">b1, estando aumentada consideravelmente, quando o TGF-<font face=\"symbol\">b1 foi adicionado às culturas. Os resultados do presente estudo demonstraram que o TGF-<font face=\"symbol\">b1 induz a expressão de <font face=\"symbol\">a-AML, sugerindo a indução do fenótipo miofibroblástico em fibroblastos pulpares. / Transforming growth factor-beta 1 (TGF-<font face=\"symbol\">b1) has been related to induce the expression of <font face=\"symbol\">a-smooth muscle actin (<font face=\"symbol\">a-SMA) in fibroblasts during repair. Since pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-<font face=\"symbol\">b1 enhances the expression of <font face=\"symbol\">a-SMA in human pulpal fibroblasts. TGF-<font face=\"symbol\">b1 was added in doses between 5-10 ng/ml to cultures of both dental pulp and gingiva human fibroblasts. The expression of <font face=\"symbol\">a-SMA was analyzed by immunofluorescence and western-blotting. Both cell types were immunoreactive for <font face=\"symbol\">a-SMA even without TGF-<font face=\"symbol\">b1. When TGF-<font face=\"symbol\">b1 was added to cell cultures, the expression of <font face=\"symbol\">a-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the western blot analysis. The present findings showed that TGF-<font face=\"symbol\">b1 up-regulated the expression of <font face=\"symbol\">a-SMA thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.

Wong Cheuk Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 101-124). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vi / List of Abbreviations --- p.vii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Methods --- p.4 / Chapter 2.1 --- Measurement of cAMP and cGMP in VSMCs --- p.4 / Chapter 2.1.1 --- Cell culture --- p.4 / Chapter 2.1.2 --- Enzyme-immunoassay colorimetric measurement for cAMP and cGMP --- p.5 / Chapter 2.1.3 --- Statistical analysis --- p.6 / Chapter 2.2 --- Measurement of apoptosis in VSMCs and bone marrow-derived stem cells --- p.6 / Chapter 2.2.1 --- Cell culture --- p.6 / Chapter 2.2.2 --- Hoechst33258 --- p.7 / Chapter 2.2.3 --- Cell Death ELISA plus --- p.7 / Chapter 2.2.4 --- Protein extraction and Western blot analysis of PKG expression --- p.8 / Chapter 2.2.5 --- Statistical analysis --- p.9 / Chapter 2.3 --- Measurement of cell proliferation in VSMCs and bone marrow-derived stem cells --- p.9 / Chapter 2.3.1 --- Cell culture --- p.9 / Chapter 2.3.2 --- Cell count --- p.10 / Chapter 2.3.3 --- MTT assay --- p.11 / Chapter 2.3.4 --- BrdU-(5`Bromo-2-deoxyuridine) ELISA colorimetric assay --- p.11 / Chapter 2.3.5 --- Statistical analysis --- p.12 / Chapter Chapter 3. --- Effects of hyperglycemia on CGRP-induced cAMP response in VSMCs / Chapter 3.1 --- Introduction --- p.13 / Chapter 3.2 --- Results --- p.18 / Chapter 3.3 --- Discussion --- p.22 / Chapter Chapter 4. --- Role of cGMP and protein kinase G in regulation of apoptosis in VSMCs / Chapter 4.1 --- Introduction --- p.26 / Chapter 4.2 --- Results --- p.30 / Chapter 4.3 --- Discussion --- p.44 / Chapter Chapter 5. --- Role of protein kinase G in regulation of proliferation in VSMCs / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Results --- p.58 / Chapter 5.3 --- Discussion --- p.67 / Chapter Chapter 6. --- Effects of aging and eNOS- and iNOS-gene deletion (using eNOS- and iNOS-knockout mice) on apoptosis of VSMCs / Chapter 6.1 --- Introduction --- p.73 / Chapter 6.2 --- Results --- p.76 / Chapter 6.3 --- Discussion --- p.79 / Chapter Chapter 7. --- Role of protein kinase G in regulation of apoptosis and proliferation of bone marrow stromal stem cells / Chapter 7.1 --- Introduction --- p.81 / Chapter 7.2 --- Results --- p.84 / Chapter 7.3 --- Discussion --- p.92 / Chapter Chapter 8. --- Overall discussion --- p.95 / Chapter Chapter 9. --- References --- p.101