Atherosclerosis is the chief cause of acute coronary syndromes and may progress for many years before any noticeable clinical syndromes occur. Hyperlipidemia is the common clinical problem for people adopting a western style of living and it can initiate a series of vascular events that result in atherosclerosis. The pathological processes include the accumulation of modified lipid, mainly oxidized low-density lipoprotein (oxLDL), endothelial cell dysfunction and activation, increase in expression of adhesion molecules, activation and recruitment of inflammatory cells and induction of proliferation and migration of vascular smooth muscle cells. / Endothelial-monocyte adhesion is crucial process for the recruitment of monocyte into intima. DG (7:3), Danshen and SAB were found to inhibit TNF-alpha-induced endothelial-monocyte adhesion. They also showed inhibition on TNF-alpha induced production of chemokines, MCP-1 which promotes the transmigration of monocyte. However, it did not inhibit the production of cytokine, IL-6 which stimulates the expression of adhesion molecules such as VCAM-1 and ICAM-1. / For the in vivo study, DG (7:3) exhibited no anti-hyperlipidemic or hypolipidemic effect against diet-induced hyperlipidemia, nor did it lower cholesterol level in hamsters. Also, it did not inhibit HMG-CoA reductase activity or increase the total fecal sterols excretion. However, DG (7:3) exhibited hypocholesterolemic effect on diet-induced hyperlipidemia in the rabbit model, wherein it could lower plasma total cholesterol and liver cholesterol level. Moreover, it could significantly decrease the atheroma formation. / In the present study, the anti-oxidative effects of herbal extract/compound were measured by three in vitro assays, namely the inhibition of 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced red blood cells hemolysis, AAPH-induced cardiomyocyte (H9c2) cells death and Cu2+-induced low-density lipoprotein oxidation. The results showed that the aqueous extract of the compound formula Danshen (D) and Gegen (G) (7:3), abbreviated as DG (7:3), and an aqueous extract of Danshen as well as salvianolic acid B (SAB) exhibited anti-oxidant effect, but Gegen did not produce such effect. It was found that SAB showed a stronger anti-oxidant effect than that of ascorbic acid. / Proliferation and migration of vascular smooth muscle cells (vSMCs) are important pathological processes involved in the development of atherosclerosis. DG (7:3), Danshen and SAB were found to inhibit PDGF-induced vSMCs proliferation through G1/S cell cycle arrest. Cyclin D, a main component that governs the transition of G1 phase to S phase, was found to be down-regulated by DG (7:3), Danshen and SAB, as assessed by measurements of both protein and mRNA levels. Moreover, DG (7:3), Danshen and SAB showed anti-migratory effect against platelet-derived growth factor-induced vSMCs migration. / To summarize, DG (7:3) was found to have potential to produce anti-atherosclerotic effect by inhibiting the LDL oxidation, proliferation and migration of vascular SMC, thereby preventing the formation of atheroma plaque. / Koon Chi Man. / "March 2006." / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6324. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 246-264). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Atherosclerosis is a multifactorial, chronic inflammatory condition characterised by endothelial dysfunction, hyperlipidaemia and the accumulation of fatty deposits within the tunica intima of medium-to-large sized muscular arteries. This disease can prove fatal with patients suffering lethal myocardial infarction or stroke. Recently, two studies investigating the role of G-protein-coupled receptor 55 (GPR55) in atherosclerosis reported conflicting results; one reported a pro-atherogenic role for GPR55 and the other, an anti-atherogenic role for this receptor. Interestingly, another study demonstrated that the activation of GPR55 by lysophosphatidylinositol (LPI) in cultured rat neonatal ventricular cardiomyocytes provokes distinct cellular functions that are dependent on the location of GPR55, leading to suggestions that GPR55 may regulate cardiomyocyte function at two cellular sites and be a potential therapeutic target for cardiac disorders. While it has been demonstrated that GPR55 is important in the maintenance of cardiac function of healthy mice, what is currently unknown is if GPR55 has a role in the cardiovascular remodelling and cardiac function of atherosclerosis prone mice. To address this, the present studies were conducted to investigate 1) the role of GPR55 in atherogenesis, 2) if GPR55 has a role in the cardiac function of mice suffering from atherosclerosis, 3) the signalling pathway by which LPI activates cardiomyocytes, 4) the impact of GPR55 activation on the outcome of myocardial ischaemia/reperfusion (I/R) injury and, 5) the signalling mechanisms by which GPR55 elicits any observed effects on the myocardium in response to such injury. Using C57BL/6 (wildtype; WT), apolipoprotein E knockout (ApoE-/-; mouse model of atherosclerosis), GPR55 knockout (GPR55-/-) and novel ApoE-/-/GPR55-/- mice, this study has established that in the presence of high fat feeding (to accelerate atherosclerosis), GPR55 has a complex role whereby it both regulates risk factors associated with atherosclerosis (i.e. body weight and fat mass) yet promotes the development of fatty streaks within the vasculature, via a lipid independent mechanism. In terms of cardiac function, GPR55 exerted a protective role by maintaining the systolic function of high fat fed ApoE-/- mice, yet negatively affected the contractile reserve of these mice. With regard to infarct size, the present study established that LPI-induced activation of GPR55 (pre-global ischaemia) exacerbates myocardial tissue injury via a Rho-associated protein kinase (ROCK) dependent mechanism. Finally, this study established that LPI signals through the same signalling pathway as it did in the isolated heart, in both mouse and human-induced pluripotent stem cell-derived cardiomyocytes thus suggesting a translational role for GPR55 in the human heart. In conclusion, despite further research being required, the data presented within this thesis provides evidence that GPR55 may have the potential to be targeted for therapeutic gains in atherosclerosis and myocardial I/R injury.

O presente trabalho buscou investigar a formação da placa de aterosclerose através de caracterização da autofluorescência do tecido e do plasma na presença de marcadores fluorescentes. Para realizar o estudo, coelhos foram divididos em dois grupos: um grupo controle onde os animais foram submetidos a uma dieta normal e um grupo experimental onde os animais foram submetidos a uma dieta hipercolesterolêmica. Foram realizadas duas experimentações animais: na primeira os animais foram sendo eutanasiados ao longo do experimento e suas artérias foram coletadas. Na segunda os animais foram acompanhados por no máximo 80 dias. Durante o experimento apenas o sangue foi coletado e os animais foram eutanasiados no final do experimento. Dois marcadores fluorescentes foram utilizados no trabalho: o complexo európio-clorotetraciclina (EuCTc) e o corante tioflavina T (ThT). Analisouse inicialmente a fluorescência dos marcadores na presença do plasma dos coelhos tanto para o grupo controle, quanto para o grupo experimental em função dos tempos de dieta. Para o complexo EuCTc observou-se duas bandas de emissão, com excitação em 400 nm, uma característica da clorotetraciclina, em 515 nm e uma em 617 nm característica do íon európio. A análise da banda do íon európio indicou um incremento da banda de emissão do complexo na presença do plasma do grupo experimental em relação ao grupo controle. Para o corante ThT também foi observado um aumento na banda de emissão em 480 nm, com excitação em 413 nm, para o grupo experimental em comparação com o grupo controle. A potencialidade de utilização do complexo EuCTc e EuCTcMg (EuCTc na presença do íon magnésio) para marcação da placa de aterosclerose nas artérias, foi estudada através da análise de microscopia de fluorescência. Observou-se que a emissão do complexo melhora muito a visualização da placa quando comparada com a autofluorescência. Observou-se, através de microscopia de tempo de vida de fluorescência, que há uma transferência de energia entre os fluoróforos presentes na placa e os complexos EuCTc e EuCTcMg. Essa transferência de energia ocasionou em uma diminuição drástica no tempo de vida de fluorescência dos fluoróforos nessa região. Por fim, estudou-se a geração de segundo harmônico do colágeno na placa de aterosclerose, sendo obtidas diferenças na quantidade e organização do colágeno para os diferentes grupos experimentais. / This study aimed to investigate the formation of atherosclerotic plaque by the characterization of the autofluorescence of the tissue and plasma in the presence of fluorescent markers. For this study, rabbits were divided into two groups: a control group and an experimental group submitted to a hypercholesterolemic diet. The animal experimentation was performed twice, the first animals were being euthanized during the experiment and their arteries were collected. In the second experiment, the animals were followed for a maximum of 80 days and only during the experiment the blood was collected. The animals were euthanized at the end of experimentation. Two fluorescent markers were used in this study: europiumchlortetracycline complex (EuCTc) and the dye Thioflavin T (ThT). Firstly, it was analyzed the markers fluorescence in the presence of rabbits plasma for both, the control and the experimental groups with different diet times. For EuCTc complex, it was observed two bands of emission with excitation at 400 nm, first, a characteristic of chlortetracycline at 515 nm and at 617 nm characteristic of the europium ion. Analyzing only the band of europium ion, it was observed a greater increase of the complex in the presence of plasma in the experimental group. For ThT dye the emission band at 480 nm with excitation at 413 nm for the experimental group, in comparison with the control group. I was analysed the possibility to use EuCTc and EuCTcMg complex (EuCTc the Mg ion present) for marking the atherosclerotic plaque in arteries by fluorescence microscopy analysis. The results showed that the emission of the complex increase dramatically compared to the autofluorescence. Also, there was evidence of energy transfer between the fluorophores present of the plaque and EuCTc and EuCTcMg complex by fluorescence lifetime microscopy. This energy transfer generated a drastic decrease in the fluorescence lifetime of fluorophores. Finally, the generation of second harmonic of collagen in the atherosclerotic plaque was studied and it was obtained differences in the amount of collagen and organization in the different experimental groups.

Aterosclerose é uma doença inflamatória e proliferativa que tem início quando fatores de risco alteram o endotélio vascular. Células inflamatórias e vasculares liberam citocinas e fatores de crescimento que estimulam a proliferação e migração das células de músculo liso e a síntese de componentes da matriz extracelular. A nanoemulsão lipídica LDE se concentra em regiões onde a proliferação celular e a inflamação são maiores, tornando-se um veículo para fármacos. O etoposide, fármaco antiproliferativo, ainda não foi explorado no tratamento da aterosclerose. O paclitaxel recobre stents utilizados em angioplastia para evitar a reestenose. A associação do LDE-etoposide ao LDE-paclitaxel aumentaria o efeito antiproliferativo dos fármacos isolados, por agirem em diferentes fases do ciclo celular. Este estudo tem por objetivos avaliar a eficácia do tratamento com LDE-etoposide e com a combinação LDE-etoposide/LDE-paclitaxel da aterosclerose induzida por dieta rica em colesterol em coelhos; comparar a eficácia dos tratamentos; no grupo de tratamento mais eficaz e no grupo Controle, avalia a expressão protéica de receptores de lipoproteínas, citocinas, MMP9 e marcadores de proliferação celular. Para tanto, 27 coelhos receberam dieta rica em colesterol por 8 semanas. Depois de 4 semanas, foram divididos em 3 grupos: grupo Controle, que recebeu 6 mL de solução salina intravenosa; LDE-etoposide, que recebeu dose de 6mg/kg; e grupo Combinação, que recebeu LDE-etoposide/LDE-paclitaxel nas doses de 6mg/kg e 4mg/kg, respectivamente. Os tratamentos foram administrados uma vez por semana durante 4 semanas. Foram avaliados perfil lipídico, hematológico, ponderal e o consumo de ração. Após a eutanásia, as lesões ateroscleróticas macroscópicas foram medidas. Depois, o arco aórtico foi analisado por morfometria e por imunohistoquímica. Foi observado que não houve diferença no perfil ponderal e no consumo de ração entre os grupos de estudo. No perfil lipídico, ao final do estudo, as concentrações de colesterol total e de triglicérides aumentaram em todos os grupos. O perfil hematológico mostrou redução do número de hemácias nos grupos de tratamento ao final do estudo. Em comparação com o grupo Controle, os animais tratados com LDE-etoposide apresentaram lesões ateroscleróticas 11 vezes menores e o grupo Combinação, apresentou lesões 3 vezes menores. Na análise morfométrica, a área total e a espessura da aorta foram menores no grupo LDE-etoposide quando comparado com os outros dois grupos. O grupo tratado com LDE-etoposide apresentou presença de macrófagos e de células de músculo liso menor que os grupos Controle e Combinação. Como o tratamento com LDE-etoposide apresentou uma maior eficácia, a avaliação dos outros fatores foi feita apenas neste grupo e no grupo Controle para comparação. O grupo LDE-etoposide apresentou menor expressão protéica dos receptores de lipoproteínas, das citocinas inflamatórias, da MMP9 e dos marcadores de proliferação celular topoisomerase II e tubulina. As avaliações mostraram que o tratamento com LDE-etoposide foi mais eficaz no tratamento das lesões ateroscleróticas induzidas em coelhos do que a combinação entre LDE-etoposide/LDE-paclitaxel / Atherosclerosis is an inflammatory and proliferative disease that is triggered by risk factors damaging vascular endothelium. Inflammatory and vascular cells release cytokines and growth factors, promoting the proliferation and migration of smooth muscle cells and extracellular matrix elements synthesis. LDE, an artificial nanoemulsion, concentrates in areas of greater proliferation and inflammation rates and can be used as a vehicle to direct drugs to those cells. Etoposide, an antiproliferative drug, have not been studied in atherosclerosis treatment. Paclitaxel is used in drug-eluting stents to avoid restenosis. The association of LDE-etoposide and LDE-paclitaxel would enhance the antiproliferativo effect of the isolated drugs, due to act in different cell cycle phase. The aim of this study was to evaluate the effectiveness of the treatment with LDE-etoposide and with LDE-etoposide/LDE-paclitaxel of atherosclerosis induced by a cholesterol-rich diet in rabbits; compare the effectiveness of these treatments; in the most effective treatment group and in Control group, evaluate the protein expression of lipoprotein receptors, cytokines, MMP9 and cell proliferation markers. To do so, 3 groups of 9 rabbits were fed a cholesterol-rich diet during 8 weeks then the animals were separated in 3 groups: Control, intravenously injected with 6mL of saline solution; LDE-etoposide, injected with a dose 6mg/kg; and Combination, injected with LDE-etoposide/LDE-paclitaxel in a dose of 6mg/kg and 4mg/kg, respectively. Treatments were administered once a week during 4 weeks. Lipids, blood cell count, weight and food intake were evaluated. The animals were sacrificed and the macroscopic atherosclerotic lesions were measured. Later, the aortic arch was analyzed by microscopic morphometry and by immunohistochemistry. It was seen that there was no difference in food intake and weight between study groups. Total cholesterol and triglycerides concentration increased in all groups. Blood cell count showed reduction of red blood cell in treatment groups at the end of the study. Animals treated with LDE-etoposide showed 11-fold less lesions and Combination group showed 3-fold less lesions than Control group. By morphometric analysis, total area and thickness of aorta were smaller in LDE-etoposide group than the other groups. LDE-etoposide group showed less macrophages and smooth muscle cells than Control and Combination groups. As treatment with LDE-etoposide showed a better efficacy, evaluation of other factor were performed only in LDE-etoposide and Control group, to perform a comparison. LDE-etoposide showed less protein expression of lipoprotein receptors, cytokines, MMP9, and of cell proliferation markers topoisomerase II and tubulina. The analysis showed that LDE-etoposide treatment was more effective in treating atherosclerotic-induced lesions in rabbits than the combination between LDE-etoposide/LDE-paclitaxel

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-17T18:43:17Z No. of bitstreams: 1 SuramaSousa.pdf: 1969308 bytes, checksum: c138cabc7138f8f84f270091f116db73 (MD5) / Made available in DSpace on 2017-05-17T18:43:17Z (GMT). No. of bitstreams: 1 SuramaSousa.pdf: 1969308 bytes, checksum: c138cabc7138f8f84f270091f116db73 (MD5) Previous issue date: 2014-11-21 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Introduction: Cardiovascular disease is the leading cause of morbidity and mortality worldwide and in Brazil. Women from the climateric have increased cardiovascular risk and atherosclerosis. Evaluation methods of noninvasive atherosclerosis are important to detect early changes. Objectives: compare the methods intima-media thickness of the carotid, ankle brachial index, scores of risk stratification and Framingham Global Risk Score with coronary angiography for detection of atherosclerosis in the climacteric. Methods: A cross-sectional study with 51 climacteric women undergoing coronary angiography, the Hemodynamic Service of the University Hospital of Federal University of Maranhão, covering the period from January to December 2013. It was performed coronary angiography for medical evaluation and was considered normal, examination with obstructive lesion <29% of stenosis. We collected sociodemographic and health information, laboratory tests including ultrasensitive C-reactive protein, carotid ultrasound and ankle-brachial index were performed. We applied the Framingham risk score, score of overall risk, and we evaluated the presence of metabolic syndrome. The sample was divided into two groups by the presence or absence of coronary artery disease. We analyzed the data with the Fisher exact test or chi-square and Mann-Whitney or Test-t, with p <0.05%. Results: Thirty-five percent of participants had confirmed coronary artery disease. There was an association between coronary artery disease and ankle brachial index, p value = 0.004, there was no association between carotid intimal media thickness, C-reactive protein and metabolic syndrome with coronary artery disease. In the assessment by Framingham risk score were predominant findings in low-risk (94.12%) and average risk (5.88%), the overall risk score that included the aggravating risk factors ranked (72.55%) in high risk (21.57%) at average risk, and (5.88%) at low risk. Conclusion: In this population there was association between the change in ankle brachial index and the presence of coronary artery disease. / Introdução: As doenças cardiovasculares são a principal causa de morbimortalidade no mundo e no Brasil. As mulheres, a partir do climatério, apresentam aumento do risco cardiovascular e aterosclerose. Os métodos de avaliação de aterosclerose não invasivos são importantes para detectar alterações precocemente. Objetivos: comparar os métodos espessura médio-intimal de carótidas, índice tornozelo braquial, escores de estratificação de risco de Framingham e Escore de risco Global com a cinecoronariografia na detecção de aterosclerose no climatério. Métodos: Estudo transversal analítico, com 51 mulheres no climatério submetidas à cinecoronariografia, no Serviço de hemodinâmica do Hospital Universitário da Universidade Federal do Maranhão, compreendendo o período de janeiro a dezembro de 2013. Realizou-se a cinecoronariografia por indicação médica e foi considerado normal o exame com lesão obstrutiva <29% de estenose. Coletou-se informações sociodemográficas e de saúde, realizou-se exames laboratoriais incluindo proteína C reativa ultrassensível, ultrassonografia de carótidas e índice tornozelo-braquial. Aplicou-se o escore de risco de Framingham, escore de risco global, e avaliou-se a presença de síndrome metabólica. Dividiu–se a amostra em dois grupos pela presença ou não de doença coronariana. Analisou-se os dados com o teste exato de Fisher ou qui quadrado e Mann-Whitney ou Test –t, com valor de p<0,05%. Resultados: Trinta e cinco por cento das participantes tiveram doença arterial coronariana confirmada. Houve associação entre doença arterial coronariana e o índice tornozelo braquial, p=0,004, não houve associação entre espessura média intimal de carótida, proteína C reativa e síndrome metabólica com doença arterial coronariana. Na avaliação pelo escore de risco de Framingham houve predomínio de conclusões em baixo risco (94,12%) e médio risco (5,88%), o escore risco global que incluiu os fatores agravantes de risco classificou (72,55 %) em alto risco, (21,57%) em médio risco, e (5,88%) em baixo risco. Conclusão: Na população estudada houve associação entre a alteração do índice tornozelo braquial e a presença de doença arterial coronariana.

Atualmente, as doenças cardiovasculares são apontadas como as principais causas de mortes no Brasil e no mundo. Essas doenças são causadas pelo comprometimento das artérias, principalmente as artérias coronárias. Essas artérias possuem a importante função de transportar nutrientes ao próprio coração, possibilitando que o mesmo exerça sua tarefa de suprir todo o resto do corpo com elementos essenciais à sobrevivência do indivíduo. Este trabalho é sobre um estudo do comportamento dessas artérias quando ocorre a difusão de um fluido através de suas paredes. Primeiramente, estudou-se a composição das artérias coronárias, suas funções e patologias, para extrair elementos para compor um modelo fisicamente realístico e analiticamente tratável. Devido à sua composição histológica em três túnicas, a artéria coronária foi modelada como um cilindro elástico composto de cilindros ocos e concêntricos. Investigou-se então a solução do problema da difusão de um fluido em duas e três camadas, sendo esta última configuração geométrica mais próxima da realidade. Por fim, estudou-se a difusão de um fármaco contido em um stent farmacológico, cuja função é desobstruir uma artéria aterosclerosada e evitar sua reestenose. / Currently, cardiovascular diseases are known to be the primary cause of death in Brasil and worldwide. These diseases are caused by the malfunction of the arteries, especially the coronary arteries. These arteries have the important role of transporting nutrients to the heart itself, allowing it to exert its main task of providing the rest of the body with essential elements to the survival of an individual. This work is about a study of the diffusion of a fluid through the walls of a coronary artery. First, the composition of the coronary arteries, their functions, and pathologies were studied to extract elements that could be used to construct a model that is both physically realistic and analytically amenable to analysis. Due to its histologic composition in three layers, a coronary artery was modeled as an elastic cylinder composed of hollow and concentric cylinders. The solution of the diffusion problem of a fluid in two and three layers was studied, being this last geometrical configuration the closest to reality. Finally, the diffusion of a drug contained in a pharmacological stent was investigated. The main task of this type of stent is to clear an atherosclerotic artery and to avoid its restenosis.

Aterosclerose é uma doença inflamatória e proliferativa que tem início quando fatores de risco alteram o endotélio vascular. Células inflamatórias e vasculares liberam citocinas e fatores de crescimento que estimulam a proliferação e migração das células de músculo liso e a síntese de componentes da matriz extracelular. A nanoemulsão lipídica LDE se concentra em regiões onde a proliferação celular e a inflamação são maiores, tornando-se um veículo para fármacos. O etoposide, fármaco antiproliferativo, ainda não foi explorado no tratamento da aterosclerose. O paclitaxel recobre stents utilizados em angioplastia para evitar a reestenose. A associação do LDE-etoposide ao LDE-paclitaxel aumentaria o efeito antiproliferativo dos fármacos isolados, por agirem em diferentes fases do ciclo celular. Este estudo tem por objetivos avaliar a eficácia do tratamento com LDE-etoposide e com a combinação LDE-etoposide/LDE-paclitaxel da aterosclerose induzida por dieta rica em colesterol em coelhos; comparar a eficácia dos tratamentos; no grupo de tratamento mais eficaz e no grupo Controle, avalia a expressão protéica de receptores de lipoproteínas, citocinas, MMP9 e marcadores de proliferação celular. Para tanto, 27 coelhos receberam dieta rica em colesterol por 8 semanas. Depois de 4 semanas, foram divididos em 3 grupos: grupo Controle, que recebeu 6 mL de solução salina intravenosa; LDE-etoposide, que recebeu dose de 6mg/kg; e grupo Combinação, que recebeu LDE-etoposide/LDE-paclitaxel nas doses de 6mg/kg e 4mg/kg, respectivamente. Os tratamentos foram administrados uma vez por semana durante 4 semanas. Foram avaliados perfil lipídico, hematológico, ponderal e o consumo de ração. Após a eutanásia, as lesões ateroscleróticas macroscópicas foram medidas. Depois, o arco aórtico foi analisado por morfometria e por imunohistoquímica. Foi observado que não houve diferença no perfil ponderal e no consumo de ração entre os grupos de estudo. No perfil lipídico, ao final do estudo, as concentrações de colesterol total e de triglicérides aumentaram em todos os grupos. O perfil hematológico mostrou redução do número de hemácias nos grupos de tratamento ao final do estudo. Em comparação com o grupo Controle, os animais tratados com LDE-etoposide apresentaram lesões ateroscleróticas 11 vezes menores e o grupo Combinação, apresentou lesões 3 vezes menores. Na análise morfométrica, a área total e a espessura da aorta foram menores no grupo LDE-etoposide quando comparado com os outros dois grupos. O grupo tratado com LDE-etoposide apresentou presença de macrófagos e de células de músculo liso menor que os grupos Controle e Combinação. Como o tratamento com LDE-etoposide apresentou uma maior eficácia, a avaliação dos outros fatores foi feita apenas neste grupo e no grupo Controle para comparação. O grupo LDE-etoposide apresentou menor expressão protéica dos receptores de lipoproteínas, das citocinas inflamatórias, da MMP9 e dos marcadores de proliferação celular topoisomerase II e tubulina. As avaliações mostraram que o tratamento com LDE-etoposide foi mais eficaz no tratamento das lesões ateroscleróticas induzidas em coelhos do que a combinação entre LDE-etoposide/LDE-paclitaxel / Atherosclerosis is an inflammatory and proliferative disease that is triggered by risk factors damaging vascular endothelium. Inflammatory and vascular cells release cytokines and growth factors, promoting the proliferation and migration of smooth muscle cells and extracellular matrix elements synthesis. LDE, an artificial nanoemulsion, concentrates in areas of greater proliferation and inflammation rates and can be used as a vehicle to direct drugs to those cells. Etoposide, an antiproliferative drug, have not been studied in atherosclerosis treatment. Paclitaxel is used in drug-eluting stents to avoid restenosis. The association of LDE-etoposide and LDE-paclitaxel would enhance the antiproliferativo effect of the isolated drugs, due to act in different cell cycle phase. The aim of this study was to evaluate the effectiveness of the treatment with LDE-etoposide and with LDE-etoposide/LDE-paclitaxel of atherosclerosis induced by a cholesterol-rich diet in rabbits; compare the effectiveness of these treatments; in the most effective treatment group and in Control group, evaluate the protein expression of lipoprotein receptors, cytokines, MMP9 and cell proliferation markers. To do so, 3 groups of 9 rabbits were fed a cholesterol-rich diet during 8 weeks then the animals were separated in 3 groups: Control, intravenously injected with 6mL of saline solution; LDE-etoposide, injected with a dose 6mg/kg; and Combination, injected with LDE-etoposide/LDE-paclitaxel in a dose of 6mg/kg and 4mg/kg, respectively. Treatments were administered once a week during 4 weeks. Lipids, blood cell count, weight and food intake were evaluated. The animals were sacrificed and the macroscopic atherosclerotic lesions were measured. Later, the aortic arch was analyzed by microscopic morphometry and by immunohistochemistry. It was seen that there was no difference in food intake and weight between study groups. Total cholesterol and triglycerides concentration increased in all groups. Blood cell count showed reduction of red blood cell in treatment groups at the end of the study. Animals treated with LDE-etoposide showed 11-fold less lesions and Combination group showed 3-fold less lesions than Control group. By morphometric analysis, total area and thickness of aorta were smaller in LDE-etoposide group than the other groups. LDE-etoposide group showed less macrophages and smooth muscle cells than Control and Combination groups. As treatment with LDE-etoposide showed a better efficacy, evaluation of other factor were performed only in LDE-etoposide and Control group, to perform a comparison. LDE-etoposide showed less protein expression of lipoprotein receptors, cytokines, MMP9, and of cell proliferation markers topoisomerase II and tubulina. The analysis showed that LDE-etoposide treatment was more effective in treating atherosclerotic-induced lesions in rabbits than the combination between LDE-etoposide/LDE-paclitaxel

Vascular calcification is strongly correlated with the clinical manifestations of atherosclerosis, heart attacks and strokes. The calcification process resembles bone formation and involves the osteogenic trans-differentiation of smooth muscles cells within the arterial wall. Cannabinoid receptors are known to modulate bone formation and are present in atherosclerotic vessels, suggesting they may also play a role in modulating calcification. Therefore, we evaluated the effects of cannabinoids on the expression of osteogenic proteins by vascular smooth muscle cells undergoing calcification.

Rupture of atherosclerotic plaque can cause acute life-threatening events such as myocardial infarction and ischemic stroke; therefore, there is much interest in developing therapies aimed at increasing plaque stability. More stable lesions are characterized as having high collagen content and containing a large number of vascular smooth muscle cells (SMCs) of contractile/differentiated phenotype. In our previous studies using an apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis, we found that insulin-like growth factor-1 (IGF-1)-infusion not only reduced total plaque burden, but also increased collagen expression and the number of alpha-smooth muscle actin (αSMA)-positive cells in plaque. In this study, we identify cellular mechanisms responsible for these observations. We found that in human aortic smooth muscle cells (HASMCs) grown in culture, IGF-1 post-transcriptionally upregulated expression of the procollagen type I alpha-1 subunit (pro-α1(I)) as well as contractile proteins, αSMA and smooth muscle 22-alpha (SM22α), via a PI3K-dependent but Erk1/2- and mTOR-independent signaling mechanism. Furthermore, experiments using an inhibitor of collagen synthesis or a blocking antibody against the alpha2beta1-integrin (α2β1) suggested that interaction with collagen type I promotes HASMC contractile phenotype. To elucidate mechanisms underlying IGF-1 upregulation of collagen synthesis we investigated the effect of IGF-1 on the mRNA-binding protein, la ribonucleoprotein domain family member 6 (LARP6), which had been shown to bind a conserved stem-loop secondary motif in the 5’UTR of COL1a1 and COL1a2 mRNA. IGF-1 rapidly increased LARP6 expression in HASMCs leading to increased COL1a1 and COL1a2 mRNA bound LARP6 and increased synthesis of collagen type I. Mutation of the 5’stem-loop of Col1a1 mRNA (that inhibited binding by LARP6) or overexpression of a 5’stem-loop RNA molecular decoy (that sequesters LARP6) both prevented the ability of IGF-1 to increase pro-α1(I) synthesis as well as mature α1(I) expression in cultured medium. Furthermore, IGF-1-infusion in Apoe-/- mice increased LARP6 and pro-α1(I) expression in aortic lysates, and SMC-specific IGF-1-overexpression in transgenic mice robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, this work identifies LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, and uncovers key mechanisms whereby IGF-1 promotes atherosclerotic plaque stability. / acase@tulane.edu

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a long-lived, redox-active product of protein oxidation, is capable of functioning as both a pro- and anti-oxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Furthermore, as free DOPA is currently the major treatment available for Parkinson?s disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. The hypothesis presented in this thesis is that under physiological conditions, when little redox active transition metal is available, PB-DOPA can function as a redox signalling molecule, triggering an enhancement of cellular antioxidant defences, with a potentially specific role in the regulation of defences targeted against protein oxidation. Physiological levels of PB-DOPA are very low, however the level on individual proteins can change to a proportionally large degree during oxidative stress, an appropriate property for a signalling molecule. In addition, remarkably elevated levels occur in some pathologies, including atherosclerosis. As an initial and commonly formed product of protein oxidation, PB-DOPA is well placed for a signalling role, promoting a significant up-regulation of antioxidant defences in the early stages of oxidative stress, before extensive damage has occurred. As an initiator of antioxidant defences, PB-DOPA would be potentially useful as a therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage. The main objective of this thesis was, therefore, to examine the effect of PB-DOPA on the cellular antioxidant defence system using monocytic and macrophage-like cells, key cells involved in the formation of atherosclerotic plaques. The incorporation of free DOPA into protein during protein synthesis, a process previously shown to occur both in vitro and in vivo, was used to generate PB-DOPA. Neither free nor PB-DOPA were found to be toxic to monocytic or macrophage-like cells in culture, but rather were both capable of protecting these cells from oxidative stress. Free DOPA was shown to be capable of directly scavenging radicals, a process that was thought to be in part responsible for the protection induced during oxidative stress. The presence of free and PB-DOPA up-regulated the activity of catalase and NAD(P)H:quinone oxidoreductase, two enzymatic antioxidants, however the activity of superoxide dismutase and the concentration of oxidised and reduced glutathione were not affected. Whilst it was thought that PB-DOPA would have a specific effect on the activity of antioxidant defences targeted against protein oxidation, proteolysis and bulk chaperone activity were not affected by a combination of free and PB-DOPA. Oxidatively-induced protein aggregation, however, was inhibited by the presence of free and PB-DOPA, suggesting that a more specific chaperone regulation may be taking place. The regulation of gene and protein expression was thought to be one possible mechanism by which PB-DOPA could function as a signalling molecule. To test this hypothesis, the effect of free and PB-DOPA on transcription factor activation and protein expression were investigated. Free and PB-DOPA did not induce the expression or activation of Nrf2, AP-1 or NFJB, three transcription factors thought to be involved in the expressional regulation of genes involved in the antioxidant defence system. However, the expression of a number of proteins, including antioxidants, chaperones and proteins involved in cell cycle progression, were regulated in monocytic and macrophage-like cells following the administration of free DOPA under conditions that resulted in either a high or low level of PB-DOPA generation. The regulated proteins differed between the two conditions, suggesting that the level of PB-DOPA may be a key factor in determining the specific defences targeted. The results presented in this thesis support the hypothesis that PB-DOPA can function as a signalling molecule, triggering an enhancement of cellular antioxidant defences, with a specific role in the regulation of the chaperone system, a key defence targeted against protein oxidation. This thesis may provide the basis for the potential use of free or PB-DOPA as a therapeutic for diseases known to involve oxidative stress or oxidative damage, however more research will be required to determine if the effects demonstrated in this thesis are also capable of occurring in vivo.