Abdominal aortic aneurysm (AAA) is a disease of the aorta characterized by pathological remodeling and progressive weakening of the vessel resulting in the increased risk of rupture and sudden death. In a mouse model of the disease induced by chronic Angiotensin II (AngII) infusion, progression of AAAs is associated with reduced differentiation of smooth muscle cells (SMCs) at the site of lesion development. In the mouse model, the effectiveness of cyclooxygenase-2 (COX-2) inhibition for attenuating AAA progression is associated with maintenance of a differentiated SMC phenotype. However, the safety of COX-2 inhibitors is currently in question due to the increased risk of adverse cardiovascular events. Thus, it is crucial to identify mediators downstream of COX-2 that may provide new targets for treatment of this disease. Recent studies in humans and mouse models have suggested that the microsomal prostaglandin E synthase (mPGES-1) enzyme, which acts downstream of COX-2, may also be involved in the pathogenesis of the disease. We hypothesized that increased prostaglandin E2 (PGE2) synthesis resulting from the induction of both COX-2 and mPGES-1 may result in reduced differentiation of SMCs, and that disruption of this pathway would preserve the differentiated phenotype. To test this hypothesis, human aortic smooth muscle cells (hASMCs) were utilized to examine the effects of a variety of agents involved in AAA development and the COX-2 pathway. My findings suggest that one of the effects of exposing hASMCs to AngII involves a specific induction of mPGES-1 expression. Furthermore, although different COX-2-derived products may have opposing effects, mPGES-1-derived PGE2 may be the primary prostanoid synthesized by SMCs which functions to attenuate differentiation. Therefore, mPGES-1 inhibition may provide inhibition of PGE2 that is more specific than COX-2 inhibitor treatment and may serve as a therapeutic target for attenuating AAA progression by maintaining a differentiated SMC phenotype.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

The aim of the present thesis was to characterize the role of the different families of phosphodiesterases (PDEs), the enzymes degrading 3'-5'-cyclic adenosine monophosphate (cAMP), in controlling the cAMP signalling in two distinct smooth muscle cells (SMCs), the rat aorta SMC (RASMCs) and the rat bladder SMC (RBSMCs).In cultured RASMCs, we firstly characterized the pattern of cAMP-PDE expression and activity. We then showed, by using a FRET-based cAMP sensor to explore cAMP signals in living cells, that PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, whereas PDE3 and PDE4 act synergistically at the submembrane compartment. The mechanisms of this subcellular compartmentation need to be characterized. In neonatal RBSMCs, we showed that both PDE3 and PDE4 are involved in regulating the phasic contractions albeit through distinct mechanisms. PDE4 inhibition inhibits the carbachol-enhanced contractions through a protein kinase A-dependent pathway involving an increase in Ca2+ sparks frequency which activates BK channels to ultimately decrease Ca2+ transients, whereas PDE3 inhibition acts through a protein kinase G-dependent pathway through a still unknown mechanism.In conclusion, our work shows that in the SMC, the different cAMP-PDE families exhibit a specificity in their function and/or mechanism of action, thus participating to a subcellular signaling compartmentation.

国立情報学研究所で電子化したコンテンツを使用している。

Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response. Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions). Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene 5/viperin (Cig5), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection. In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene 5/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves [174]-186). Also available on the Internet.

Made available in DSpace on 2015-05-14T12:59:52Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 7372344 bytes, checksum: 1300f16a278dc5441d8295112a100645 (MD5) Previous issue date: 2013-07-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Many Annonaceae species are odoriferous due to the presence of essential oils and these have attracted great pharmacological interest because they have action on smooth muscle. Thus, we aimed to investigate a possible spasmolytic activity of essential oils of leaves from Xylopia langsdorfiana A. St.-Hil. & Tul. (XL-OE), Xylopia frutescens Aubl. (XF-OE) e Rollinia leptopetala R. E. Fries (RL-OE) species on rat aorta, rat uterus and guinea pig trachea and ileum, and so elucidate the action mechanism of the essential oil that to present the better spasmolytic effect in one of the tested organs. Isometric and isotonic contractions and cytosolic Ca2+ were measured. In preliminary pharmacological screening all essential oils presented a higher spasmolytic potency and efficacy on guinea pig ileum, when compared with the other organs, being RL-OE the most promising natural product. So we decided to characterize the action mechanism of RL-OE on this organ. RL-OE inhibited cumulative concentration-response curves to histamine, and these were shifted to the right, in a non-parallel manner, with Emax reduction, discarding thus a competitive type antagonism and relaxed the guinea pig ileum contracted by KCl (40 mM), CCh (10-6 M) or histamine (10-6 M). How the common pathway of signaling of these contractile agents are the voltage-gated Ca2+ channels (CaV), we hypothesized that RL-OE would be preventing the Ca2+ influx through CaV. it was observed that RL-OE antagonized the CaCl2- induced contractions in a depolarizing medium namely without Ca2+, in addition to relax guinea pig ileum pre-contracted by S-(-)-Bay K 8644, a CaV-L selective agonist, demonstrating that the CaV subtype involved is CaV-L. How the relaxant potency of RL-OE was higher when the organ was pre-contracted by KCl this is suggestive that other mechanism would be involved on RL-OE spasmolytic effect. Since the CaV can be modulated by K+ channels, we decided to investigate these channels. The RL-OE spasmolytic action seems to involve the positive activation/modulation of valtege-gated (KV) and large conductance calcium-activated (BKCa) K+ channels, since there was a right-shift of RL-OE relaxation curve in the presence of the following blockers: 4-aminopyridine (4-AP), selective to KV; tetraethylammonium (TEA+) 1 mM and IbTX, selectives to BKCa. The confirmation of KV and BKCa participation was carried out by a simultaneous blockade (TEA+ and 4-AP) and verified since the relaxant potency was similar when in the presence of non-selective blocker (CsCl). In cellular experiments, the viability of longitudinal layer myocytes from guinea pig ileum was not altered in RL-OE (81 μg/mL) presence and the fluorescence intensity in these intestinal myocytes stimulated by histamine was reduced due to cytosolic Ca2+ concentration ([Ca2+]c). Thus the mechanism of action spasmolytic of RL-OE on guinea pig ileum involves blocking the Ca2+ influx, by CaV, in addition to positive activation/modulation of KV and BKCa, that would lead to reduced of [Ca2+]c and consequent relaxation of this organ. / Muitas espécies de Annonaceae são odoríferas, devido à presença de óleos essenciais e estes têm despertado um grande interesse farmacológico por apresentarem atividade sobre a musculatura lisa. Assim, decidiu-se investigar a possível atividade espasmolítica dos óleos essenciais das folhas das espécies Xylopia langsdorfiana A. St.-Hil. & Tul. (XL-OE), Xylopia frutescens Aubl. (XF-OE) e Rollinia leptopetala R. E. Fries (RL-OE) em aorta de rato, útero rata, traqueia e íleo de cobaia, além de elucidar o mecanismo de ação do óleo essencial que apresentasse uma melhor atividade espasmolítica em um dos órgãos testados. Foram realizadas medidas de contrações isotônicas e isométricas e do cálcio citosólico. Na triagem farmacológica preliminar todos os óleos essenciais apresentam maior eficácia e potência espasmolítica em íleo de cobaia, quando comparados aos outros órgãos, sendo o RL-OE o produto natural mais promissor. Assim, passou-se a caracterizar o mecanismo de ação do RL-OE nesse órgão. O RL-OE inibiu as curvas cumulativas à histamina, desviando-as para direita, de maneira não paralela e com redução do Emax, descartando um antagonismo do tipo competitivo, além de relaxar o íleo pré-contraído por KCl (40 mM), carbacol (10-6 M) ou histamina (10-6 M). Como o passo comum na via de sinalização destes agentes contráteis são os canais de Ca2+ dependentes de voltagem (CaV), hipotetizou-se que o RL-OE estaria impedindo o influxo de Ca2+ através destes canais. Foi observado que o RL-OE antagonizou as contrações induzidas por CaCl2 em meio despolarizante nominalmente sem Ca2+, além de relaxar o íleo pré-contraído por S-(-)-Bay K 8644, um agonista dos CaV-L, demonstrando que o subtipo de CaV envolvido é o CaV-L. Como a potência relaxante do RL-OE foi maior quando o órgão foi pré-contraído com KCl do que pelo S-(-)-Bay K8644 é sugestivo de que outros mecanismos estão envolvidos no efeito espasmolítico do RL-OE. Sabendo que os CaV podem ser modulados pelos canais de K+, decidiu-se investigar a participação desses canais. A ação espasmolítica do RL-OE parece envolver ativação/modulação positiva dos canais de K+ dependentes de voltagem (KV) e dos ativados por Ca2+ de grande condutância (BKCa), uma vez que houve desvio da curva de relaxamento do RL-OE para direita na presença dos bloqueadores: 4-aminopiridina (4-AP), seletivo para os KV; tetraetilamônio (TEA+) 1 mM e iberiotoxina, seletivos para os BKCa. A confirmação da participação dos KV e BKCa foi feita através do bloqueio simultâneo (TEA+ e 4-AP) e constatada, uma vez que, a potência relaxante foi similiar quando o bloqueio era realizado por um bloqueador não seletivo dos canais de K+ (CsCl). Nos experimentos celulares, a viabilidade dos miócitos da camada longitudinal do íleo de cobaia não foi alterada na presença do RL-OE (81 μg/mL) e a intensidade de fluorescência nos miócitos intestinais estimulados por histamina foi reduzida em consequência da redução da concentração citosólica de Ca2+ ([Ca2+]c). Assim, o mecanismo de ação espasmolítico do RL-OE em íleo de cobaia envolve bloqueio do influxo de Ca2+, via CaV, além da ativação/modulação positiva dos KV e BKCa, o que levaria a redução da [Ca2+]c e, consequente, relaxamento desse órgão.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-09T11:18:39Z No. of bitstreams: 2 Dissertação - Marco Túlio Lacerda Ribeiro - 2018.pdf: 1380967 bytes, checksum: 194ed48e2af9c60ef94be3f60db75ef7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-09T11:27:43Z (GMT) No. of bitstreams: 2 Dissertação - Marco Túlio Lacerda Ribeiro - 2018.pdf: 1380967 bytes, checksum: 194ed48e2af9c60ef94be3f60db75ef7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-07-09T11:27:43Z (GMT). No. of bitstreams: 2 Dissertação - Marco Túlio Lacerda Ribeiro - 2018.pdf: 1380967 bytes, checksum: 194ed48e2af9c60ef94be3f60db75ef7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-06-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Background: Some studies relate the use of paracetamol with the exacerbation of asthma symptoms and other respiratory problems. Other studies are inconclusive or deny this correlation. This makes the association between paracetamol and airway hypersensitivity very controversial and still under debate. Objectives: To investigate the effect of chronic treatment with paracetamol on rats on the contraction and relaxation of isolated airways. Methods: Wistar rats were divided into 2 groups (n = 5-8). One group was treated for 2 weeks with paracetamol (400 mg / kg, v.o.) in drinking water and another control group (pure water). The animals were euthanized after two weeks, blood was collected for biochemical analyzes (ALT, AST, TBARs reaction) and tracheal rings were isolated and prepared in an organ bath to measure the isometric tension against contractile stimuli (concentration- effect for carbachol and KCl, concentration- 2+ effect curve for Ca stimulated with carbachol or KCl) and relaxants (isoprenaline, sodium nitroprusside and verapamil). Results: Liver enzymes ALT, AST and lipid peroxidation (TBARs) were significantly increased after paracetamol treatment. Acupuncture of the airway tracheal smooth muscle response does not present any alteration, either to the contractile or relaxing stimulus, for example: cholinergic agonist, membrane 2+ 2+ 2+ depolarization, Ca influx by the sarcolemma, internal release of Ca from the sarcoplasmic reticulum, Ca , β-receptor agonist or NO donor-induced relaxation. Conclusion: Chronic treatment with paracetamol does not induce hyper or hyporeativity of the airways isolated from rats. / Introdução: Alguns estudos relacionam o uso de paracetamol com a exacerbação dos sintomas da asma e outros problemas respiratórios. Outros estudos não são conclusivos ou negam essa correlação. Isso torna a associação entre o paracetamol e a hipersensibilidade das vias aéreas muito controversa e ainda em debate. Objetivos: Investigar o efeito do tratamento crônico com paracetamol em ratos sobre a contração e relaxamento de vias aéreas isoladas. Métodos: Ratos Wistar foram divididos em 2 grupos (n=5-8). Um grupo foi tratado por 2 semanas com paracetamol (400 mg/Kg, v.o.) na água de beber e outro, o grupo controle, foi tratado apenas com água potável. Os animais foram eutanasiados após duas semanas, o sangue foi coletado para análises bioquímicas (ALT, AST, reação de TBARs) e os aneis de traqueias foram isolados e preparados em banho de órgãos para medir a tensão isométrica mediante estímulos contráteis 2+ (curva concentração-efeito para carbacol e KCl; curva concentração-efeito para Ca estimulados com carbacol ou KCl) e relaxantes (isoprenalina, nitroprussiato de sódio e verapamil). Resultados: As enzimas hepáticas ALT, AST e a peroxidação lipídica (TBARs) foram significativamente aumentadas após o tratamento com paracetamol. A capacidade de resposta do músculo liso traqueal das vias aéreas não apresentam qualquer alteração, seja ao estímulo contrátil ou relaxante, como exemplo: agonista 2+ 2+ colinérgico, despolarização da membrana, influxo de Ca pelo sarcolema, liberação interna de Ca do 2+ retículo sarcoplásmico, bloqueio do canal Ca , agonista de receptores β ou relaxamento induzido por doador de NO. Conclusão: O tratamento crônico com paracetamol não induz a hiper ou hiporreatividade das vias aéreas isoladas de ratos.