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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23802号 / 医博第4848号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 永井 洋士, 教授 羽賀 博典, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).

Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation.

Nous avons étudié l’expression et la régulation de la lipogenèse dans les aortes et CMLV et déterminé si elle est modifiée par l’insulino-résistance et le diabète. Les rats Zucker obèses (ZO), diabétiques et Psammomys obesus accumulent plus de lipides dans leurs aortes que leurs contrôles. Cependant l’expression des gènes de la lipogenèse et ceux impliqués dans la captation des acides gras, n’est pas augmentée. Un résultat similaire a été retrouvé dans des pièces d’endartériectomies chez l’homme. Le milieu adipogénique, le glucose ou l’insuline seule stimulent modérément la lipogenèse uniquement dans les CML de Zucker contrôles, aucun effet n’a été observé dans les CML de ZO. Nous avons montré que les effets du TO901317 sur la lipogenèse dans les CMLV sont dus uniquement à l’activation du récepteur nucléaire LXRα, PXR n’a aucun effet. En conclusion, la lipogenèse n’est pas augmentée dans la paroi artérielle durant l’insulino-résistance et le diabète. / We investigated the expression and regulation of lipogenesis in aortas and VSMC and determined if it is modified during metabolic abnormalities. Zucker obese (ZO), diabetic (ZDF) rats, and the high fat diet fed Psammomys obesus accumulated more triglycerides in their aortas than control rats. However the expression of lipogenic genes, or of genes involved in fatty acids uptake, was not increased. Lipogenesis was not increased in human carotid endarterectomy of diabetic compared to non-diabetic patients. The adipogenic medium (ADM), glucose or insulin stimulated moderately lipogenesis but only in VSMC from control rats. No effect was observed in VSMC from ZO. We showed that the lipogenic effects of TO901317observed in VSMC from Zucker control rats are due solely to the nuclear receptor LXRα, PXR agonist had no effect. Conclusion: Lipogenesis is not increased in arterial wall during insulin-resistance and diabetes.

Die Migration und Proliferation vaskulärer Zellen spielt eine entscheidende Rolle in der Pathogenese atherosklerotischer Gefäßwandveränderungen und trägt zudem in hohem Maße zu restenosebedingten Komplikationen interventioneller Therapien der Atherosklerose bei. Dies bedingt ein großes Interesse an pharmakologischen Interventionsmöglichkeiten zur Prävention / Therapie atherosklerotischer und restenotischer vaskulärer Läsionen. Dabei kommen neben lokal applizierbaren Substanzgruppen insbesondere Pharmaka in Betracht, die bereits Anwendung zur Behandlung metabolischer Risikofaktoren kardiovaskulärer Erkrankungen finden. Hierzu gehören insbesondere die oralen Antidiabetika vom Typ der Thiazolidindeone, die als Liganden für den "Peroxisome Proliferator-Activated Receptor gamma" (PPARg) agieren. PPARs sind eine Gruppe neuer Regulatoren der Genexpression, für die in den vergangenen Jahren eine Reihe vaskulärer Wirkungen nachgewiesen wurden. Wir konnten zeigen, daß die Proliferation und Migration von Gefäßmuskelzellen durch PPARg-Liganden gehemmt wird. Untersuchungen zu den beteiligten Signalübertragungsschritten ergaben, daß die pharmakologische Aktivierung von PPARg in Gefäßmuskelzellen insbesondere die durch die Mitogen-aktivierten Protein Kinasen ERK1/2 vermittelte Signaltransduktion beeinflußt. Diesbezüglich haben wir nachgewiesen, daß PPARg in Gefäßmuskelzellen die mitogene Signaltransduktion via ERK1/2 MAPK -> Elk-1 -> c-fos und die chemotaktische Signalübertragung via ERK1/2 MAPK -> Ets-1 -> Matrixmetalloproteinase-9 hemmt. Wir konnten ferner zeigen, daß PPARg-Liganden die Endothelzellmigration hemmen, die durch die Neovaskularisation atherosklerotischer Plaques und der damit verbundenen erhöhten Vulnerabilität einer Plaqueruptur eine Rolle in der Pathobiologie der Atherosklerose spielt. Diese migrationshemmende Wirkung der PPARg-Liganden basiert vermutlich auf einer Inhibition der für die Endothelzellmigration erforderlichen Signaltransduktion über den PI3 Kinase -> Akt -> eNOS Pathway. Die Inhibition dieses Signalwegs könnte die Folge der von uns beobachteten PPARg-Ligand-induzierten Expression der Phosphatase PTEN sein, die den PI3K -> Akt Signalweg negativ reguliert und die Aktivierung und Phosphorylierung von Akt inhibiert. Somit haben PPARg-aktivierende Liganden eine wichtige Funktion in der Behandlung der metabolischen Hauptrisikofaktoren kardiovaskulärer Erkrankungen, spielen aber gleichzeitig eine vermutlich ebenso wichtige Rolle in der Protektion atherosklerotischer und restenosebedingter Gefäßwandveränderungen durch direkte vaskuläre Effekte. / Migration and proliferation of vascular cells not only play an important role in the pathogenesis of atherosclerotic lesion formation, but also contribute to restenosis after therapeutic angioplasty. Therefore, pharmacological strategies for the prevention and/or treatment of atherosclerotic and restenotic vascular lesions are of great clinical interest. This involves substances that can be locally administered via stents, as well as agents that are already in clinical use for the treatment of metabolic risk factors. Among the latter, antidiabetic thiazolidinediones which function as ligands for the "peroxisome proliferator-activated receptor gamma" (PPARg), have been identified as promising drugs to target vascular lesion formation. PPARs constitute a group of novel regulators of gene expression, that exert several vascular effects. We report that vascular smooth muscle cell proliferation and migration is inhibited by PPARg-ligands. Investigating the signalling steps that are involved, we find that pharmacological activation of PPARg interferes with signal transduction through the mitogen-activated protein kinases ERK1/2 in vascular smooth muscle cells. We demonstrate that PPARg inhibits mitogenic signal transduction via ERK1/2 MAPK -> Elk-1 -> c-fos and also blocks chemotactic signalling through the ERK1/2 MAPK -> Ets-1 -> matrix metalloproteinase-9 pathway in vascular smooth muscle cells. We also showed that PPARg-ligands inhibit endothelial cell migration, which participates in the neovascularization of atherosclerotic plaques, thereby contributing to plaque destabilization and increased risk of plaque hemorrhage. This antimigratory action of PPARg-ligands results from an inhibition of signal transduction via PI3 Kinase -> Akt -> eNOS, a pathway that is crucial for endothelial cell migration. Since we observed a PPARg-ligand-induced upregulation of PTEN, a phosphatase that negatively regulates the PI3K -> Akt signalling pathway, this might constitute the mechanism by which PPARg-ligands inhibit endothelial cell migration. In conclusion, PPARg-activating ligands may provide a dual benefit in cardiovascular disease by ameliorating metabolic risk factors, as well as protecting the vasculature from atherosclerotic and restenotic alterations through direct vascular effects.

As metaloproteinases, abundantes em venenos de serpentes da família Viperidae, apresentam homologia estrutural e funcional com as metaloproteinases de mamíferos (MMPs), cujos níveis estão elevados em doenças de natureza inflamatória, como a aterosclerose. A metaloproteinase BaP1, do veneno da serpente Bothrops asper, apresenta potente atividade inflamatória e constitui ferramenta científica importante para o estudo das ações das MMPs. Durante a aterosclerose, as células de músculo liso vascular (CMLVs) mudam do fenótipo contrátil para sintético, migram para a camada subendotelial do vaso, liberam mediadores inflamatórios e expressam MMPs. No entanto, o papel destas enzimas na resposta inflamatória das CMLVs e a potencial relação deste efeito com a migração das mesmas, não foi esclarecida. Neste estudo, investigou-se os efeitos da BaP1 em CMLVs, quanto à 1) indução da migração; 2) liberação de diferentes classes de mediadores inflamatórios e expressão de moléculas de adesão; 3) indução da mudança fenotípica das CMLVs; 4) expressão e participação de enzimas de síntese de prostaglandinas e de receptores de PGE2 na liberação deste eicosanoide; 5) participação de eicosanoides e da IL-1&#946 na migração e mudança de fenótipo das CMLVs. Os resultados obtidos, a partir dos ensaios de transwell e wound healing, mostraram que a BaP1(50nM) induziu a migração das CMLVs, após 48 h, mas não a proliferação celular, observada pelo ensaio de ciclo celular. Além disso, a metaloproteinase induziu aumento da liberação de PGE2 (1-48h), LTB4 (1-3h), IL-1&#946 (12-24h), MCP-1 (24-48h) e fractalcina (24-48h), mas não de PGI2 e nem TXA2, analisados por ensaios de EIA e multiplex. Ainda, a BaP1 aumentou a expressão proteica de COX-2 (1° h) e de PGESm-1 (4° h), analisada por Western blotting, e expressão gênica das sFLA2-IIA (30 min) e cFLA2-IVA (30 min), verificada por PCR em tempo real, sem alterar os níveis de COX-1, dos receptores EP1, EP2, EP3 e EP4, de ICAM-1 e VCAM-1 e da iFLA2. A intervenção farmacológica com os inibidores de COX-2 ou de FLA2 intracelulares reduziu a liberação de PGE2 induzida pela BaP1. Além disso, o pré-tratamento das células com o inibidor da FLAP e com os antagonistas do receptor de IL-1&#946 ou do receptor EP3 reduziu a migração celular induzida pela BaP1. Esta metaloproteinase também induziu a mudança de fenótipo contrátil para o sintético, das CMLVs, verificada pela diminuição da expressão de &#945-actina pelo ensaio de citometria de fluxo. A inibição da COX-2 e da FLAP não alterou este efeito. Este conjunto de dados demonstra a capacidade da BaP1 estimular diretamente as CMLVs para migração, liberação de mediadores inflamatórios e a expressão de COX-2, PGESm-1, sFLA2-IIA e cFLA2-IVA. A produção de PGE2 induzida pela BaP1 depende das FLA2s intracelulares, com ativação das vias da COX-1 e -2. A migração das CMLVs, induzida pela BaP1, depende da PGE2 via ativação do receptor EP3, do LTB4 e da IL-1&#946. Ainda, esta metaloproteinase estimula a mudança fenotípica das CMLVs para o estágio sintético, em que as CMLVs migram e proliferam. Os dados deste estudo, ao demonstrarem que as metaloproteinases contribuem para o desenvolvimento de eventos inflamatórios, em CMLVs, apontam um papel adicional desta classe de enzimas em doenças de natureza inflamatória, como a aterosclerose. / Metalloproteinases are abundant enzymes in Viperidae family snake venoms and exhibit structural and functional homology with mammalian matrix metalloproteinases (MMPs). The levels of these enzymes are incresead in inflammatory diseases, such as atherosclerosis. The BaP1 metalloproteinase from Bothrops asper snake venom presents potent inflammatory activity and constitutes important scientific tool for the study of the actions of MMPs. During atherosclerosis, vascular smooth muscle cells (VSMCs) switch their phenotype from a contractile to a synthetic state, migrate into the subendothelial vessel layer, release inflammatory mediators and express high levels of MMPs. However, the role of these enzymes in the inflammatory response of VSMCs and the potential relationship of this effect with cell migration have not been clarified. In this study, we investigated the effects of BaP1 on CMLVs with focus on: 1) induction of cell migration; 2) release of different classes of inflammatory mediators and protein expression of adhesion molecules; 3) induction of VSMCs phenotype switching; 4) expression and participation of prostaglandin synthesis enzymes and PGE2 receptors in the release of this eicosanoid; 5) participation of eicosanoids and IL-1&#946 in migration and phenotype switching of VSMCs. Results obtained from the transwell and wound healing assays showed that BaP1 (50nM) induced VSMCs migration after 48 h, but not cell proliferation, observed by the cell cycle assay. In addition, this metalloproteinase caused release of PGE2 (1-48h), LTB4 (1-3h), IL-1 (12-24h), MCP-1 (24-48h) and fractalkine (24-48h), but not PGI2 and TXA2, analyzed by EIA and multiplex assays. Furthermore, BaP1 increased protein expression of COX-2 (1 h) and PGESm-1 (4 h), analyzed by western blotting and gene expression of sFLA2-IIA (30 min) and cFLA2-IVA (30 min), evaluated by real-time PCR, without altering COX-1, EP1, EP2, EP3 and EP4, ICAM-1 and VCAM-1 and iFLA2 levels. Pharmacological intervention with COX-2 or intracellular FLA2 inhibitors reduced PGE2 release induced by BaP1. In addition, pretreatment of cells with either a FLAP inhibitor, or IL-1&#946 receptor, or EP3 receptor antagonist reduced cell migration induced by BaP1. This metalloproteinase also induced conversion of contractile VSMCs to an synthetic phenotype, as evidenced by decrease of -actin expression, analyzed by flow cytometry assay. Inhibition of COX-2 and FLAP did not alter this effect. Altogether, these data demonstrate the ability of BaP1 to directly stimulate VSMCs for migration, release of inflammatory mediators and expression of COX-2, PGESm-1, sFLA2-IIA and cFLA2-IVA. PGE2 production induced by BaP1 depends on the intracellular FLA2s, with activation of COX-1 and -2 pathways. VSMCs migration induced by BaP1 depends on PGE2 via EP3 receptor engagement, LTB4 and IL-1&#946. Furthermore, this metalloproteinase stimulates VSMCs phenotypic switching to a synthetic phenotype, in which these cells migrate and proliferate. These data demonstrate that metalloproteinases can contribute to the development of inflammatory events in VSMCs, evidencing an additional role of this class of enzymes in inflammatory diseases, such as atherosclerosis.

Une des propriétés majeures de la thrombine est le caractère pléiotropique de ses effets physiologiques et pathologiques, à la fois dans le compartiment sanguin et tissulaire de la paroi. Notre hypothèse est que les changements phénotypiques des cellules musculaires lisses vasculaires (CMLVs) participent aux modifications des propriétés pro- et anticoagulantes de la paroi. Les objectifs ont été d’étudier : (i) le rôle prothrombotique des CMLVs dans l’hypertension chez le rat SHR et le syndrome métabolique (Smet) chez le rat Zucker, (ii) les mécanismes de régulation de la génération de thrombine par l’intégrine αvβ3 des CMLVs (récepteur de la pro- thrombine), et de développer des glyco-peptides fluorés pour l’imagerie permettant d’évaluer l’activité de cette intégrine dans la paroi, et (iii) d’évaluer l’effet de variants génétiques du locus 9p21 de susceptibilité aux maladies coronariennes sur le phénotype de coagulation. Résultats : Les CMLVs sont responsables du phénotype prothrombotique de la paroi artérielle associée à l’hypertension chez le rat SHR. Les acides gras libres et l’inflammation vasculaire augmentent la génération de thrombine dans les 2 compartiments ce qui se traduit par une fibrinolyse diminuée et une activité métallo-protéinase augmentée chez Le rat Zucker. L’invalidation de l’intégrine αvβ3 des CMLVs diminue la génération de thrombine dans les 2 compartiments et ralentit la survenue de thrombose carotidienne en réponse à une stimulation l’angiotensine. Le traçage de l’intégrine αvβ3 par des glyco-peptides comprenant une séquence RGD a été validé au niveau plaquettaire et des CMLVs. La souris invalidée pour le locus 9p21 exprime un phénotype pro-thrombotique qui est retrouvé chez l’homme pour certains variants (rs10120688 et rs1333040) dans ce locus. En conclusion, la CML est un support cellulaire clé de réactions procoagulants et pourrait être impliqué via les intégrines et/ou ses récepteurs pour la thrombine dans un couplage thrombine tissulaire – rigidité cellulaire dans les pathologies vasculaires / One of the major properties of thrombin is the pleiotropic character of its physiological and pathological effects, both in the blood compartment and the tissue of the arterial wall. We hypothesized that the phenotypic changes of vascular smooth muscle cells (VSMCs) are involved in modifications of pro- and anti-coagulant properties of the arterial wall. The objectives were to examine: (i) the prothrombotic role of VSMCs in hypertension of SHR rats and in the metabolic syndrome (Smet) of Zucker rats, (ii) regulatory mechanisms of thrombin generation by integrin αvβ3 of VSMCs (a pro-thrombin receptor), and to develop fluorinated glyco- peptides for imaging, to assess the activity of this integrin in the wall, and (iii) evaluate the effect of genetic variants of the 9p21 locus that give a susceptibility to coronary heart disease on the coagulation phenotype. Results: The VSMCs are responsible for the prothrombotic phenotype of the arterial wall associated with hypertension in SHR rats. Free fatty acids and vascular inflammation increase thrombin generation in the two compartments resulting in decreased fibrinolysis and an increased metallo-proteinase activity in the Zucker rats. The invalidation of integrin αvβ3 of VSMCs reduced thrombin generation in the two compartments and slowed angiotensin-induced carotid thrombosis. Tracing of the integrin αvβ3 by glyco-peptides including RGD was validated at the platelet level and VSMCs. Mice invalidated for the 9p21 locus express a prothrombotic phenotype that is found in humans for certain variants (rs10120688 and rs1333040) in this locus. In conclusion, the VSMC is a cell supported key to procoagulant reactions and may be involved via integrins and/or its receptors for thrombin in the ”tissular thrombin - cell rigidity” coupling in vascular pathologies

To this day heart failure with preserved ejection fraction (HFpEF) remains a poorly understood malady. Half of all heart failure (HF) cases are HFpEF, and the prevalence of HF is on the rise. Unlike HF with reduced ejection fraction, HFpEF has no treatment options and is often times difficult to diagnose because victims of HFpEF often have pre-existing conditions. Vascular endothelial growth factor (VEGF) has been implicated in maintaining myocardial health and is thought to play a role in HFpEF. We sought to test the hypothesis that VEGF-A plays a role in HFpEF in a hypertensive murine model of HFpEF. Using Western blot analysis we found that there was an up regulation of VEGF-A in the homogenized left ventricle (LV) of our HFpEF mice. Unexpectedly, there was a down regulation of VEGF-A in the homogenized tissue from the aorta in those mice. To study the circulating levels of VEGF in our HFpEF mice we used an ELISA. We found that our HFpEF mice had similar levels of circulating VEGF as our control. This suggests that VEGF has paracrine/autocrine role in our HFpEF model rather than endocrine, like our human data suggested. To identify the cells responsible for the expression profile we saw in the homogenized tissue data we looked at the response of adult rat ventricular myocytes (ARVM) and vascular smooth muscle cells (VSMC) to aldosterone stimulation at short (1hr) and long (24hr) time points at both physiological (50nm) and pathological (1μm) concentrations. To do this analysis we recruited the help of Western blot, ELISA and RT-PCR techniques to construct a consistent VEGF expression profile. The Western blot ARVM data showed statistically significant (P<0.05) increase in VEGF-A to pathological doses of aldosterone, especially at the longer time point. When we tested the VSMC using Western blot analysis, we found that the trend of our n=1 sample suggested a strong response to the physiological dose of aldosterone in the short term. Using the more sensitive ELISA technique to measure the VEGF content of our VCMS we increasing our sample size to n=4 and found no statistically significant (p=NS) response to aldosterone stimulation from the VSMC. However, looking at the trends in the data it is clear that VSMC increases VEGF in response to long-term physiological doses of aldosterone. This is contrary to what we found using Western blot analysis, so we queried the VEGF mRNA from the VSMC to settle the score. Unfortunately, this too proved fruitless. The RT-PCR data was not significant and the trend was that of the ARVM expression profile. We initially turned to VSMC because we hypothesized that they could contribute to the paracrine/autocrine activity similar to what we saw in the LV from the ARVM. It is unclear if VSMC play a role in HFpEF progression, but their lack of consistent response to aldosterone could potential explain the down regulation of VEGF-A we observed in the aorta of our HFpEF mice. We initially sough to test the hypothesis that VEGF-A plays a role in our HFpEF mouse model, what we found was that ARVM contribute to localized VEGF-A increased production in the LV while in the aorta there is a down regulation of VEGF-A in our HFpEF model, we are unable to make any conclusion about VSMC response to aldosterone because of insufficient sample size. Thus in conclusion, it appears that VEGF-A does play a role in our HFpEF model specifically in a paracrine/autocrine manner in the LV where the ARVM contributes to the increased production of the cytokine.