Sphingosine-1-phosphate receptor type 1 (S1PR1), one of the five S1PRs that signals in response to bioactive lysosphingolipid S1P, regulates several fundamental processes in distinct cell types and is implicated in atherosclerosis. Using the cre-lox recombination system, previous studies identified that knocking out S1PR1 in myeloid and endothelial cells promotes plaque development in atherogenic mouse models. In the process of generating S1pr1lox/lox; ApoEKO/KO control mice, we unexpectedly noticed that S1pr1lox/lox mutation alone, in the absence of cre recombinase, reduces high-fat (HF) diet-induced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice compared to S1pr1WT/WT; ApoEKO/KO mice. Although S1pr1lox/lox allele partially suppressed S1pr1 levels in macrophages and vascular smooth muscle cells (VSMC), the presence of this mutation in a non-BM derived cell type was responsible for this reduced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice. We speculated that it could be VSMCs due to their abundance in the vascular wall and their role in foam cell formation. In this thesis, we directly tested the effects of inactivating S1PR1 in smooth muscle cells (Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice) on atherosclerosis. Our results demonstrated that deleting S1PR1 in smooth muscle cells drastically reduces atherosclerosis in apoE-deficient mice. The aortic SMCs isolated from these mice also exhibited reduced cell proliferation and lipid droplet formation in response to S1PR1 agonist SEW2871 compared to S1PR1-WT VSMCs. Furthermore, we also tested the effects of directly inhibiting S1PR1 with S1PR1 selective antagonist Ex26 at a dosage of 0.1 mg/kg/hr in S1pr1WT/WT; ApoEKO/KO mice and Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice. The prolonged exposure to Ex26 substantially reduced atherosclerotic plaque development in apoE KO mice on an HFD compared to DMSO-treated apoE KO mice. However, this protection was completely lost in mice that lack the S1pr1 gene in VSMCs. Overall, our results suggest that knocking out S1PR1 in VSMCs results in atheroprotection that surpasses the effects of inactivating S1PR1 in macrophages and endothelial cells which are known to promote atherosclerosis. / Dissertation / Doctor of Philosophy (PhD)

Orientador: Eduardo Rochete Ropelle / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T08:41:08Z (GMT). No. of bitstreams: 1 Silva_VagnerRamonRodrigues_M.pdf: 4649552 bytes, checksum: 7f2da3d7826eb50aa18465d79242f6d6 (MD5) Previous issue date: 2013 / Resumo: A ingestão alimentar e o gasto energético são minuciosamente regulados por neurônios específicos localizados no hipotálamo. Durante as duas últimas décadas, a localização dos receptores da leptina em núcleos hipotalâmicos, bem como a descrição da via de transmissão intracelular disparado por este hormônio em neurônios hipotalâmicos, foi determinante para o entendimento do controle da ingestão alimentar e do gasto energético. Cada vez mais os distúrbios alimentares associados a doenças como obesidade são relacionados à disfunções na transmissão do sinal da leptina no hipotálamo. O processo inflamatório subclínica frequentemente observado em modelos experimentais de obesidade estão diretamente associados à distintos mecanismos de resistência à leptina no hipotálamo e resultam em aumento da ingestão alimentar e ganho de peso corporal. Por outro lado, estudos demonstram que o exercício físico é capaz de aumentar a sensibilidade da leptina no hipotálamo de animas obesos através de citocinas anti-imflamatórias, contudo, esses mecanismos permanecem apenas parcialmente conhecidos. Recentemente, a proteína S1PR1 (sphingosine-1-phosphate receptor-1) foi descrita como uma molécula com alta capacidade de exercer potentes efeitos sinérgicos sobre a via de sinalização da leptina, sustentando a ativação da via Jak2/STAT3 em algumas linhagens celulares. Assim, o presente estudo tem por objetivo investigar o os efeitos do exercício físico sobre a atividade da SIPR1 e a sensibilidade à leptina em hipotálamo de roedores obesos. Acreditamos que a realização do presente estudo contribuirá para caracterizar a participação da S1PR1 na sinalização da leptina no hipotálamo, bem como determinar os efeitos do exercício físico sobre a atividade da S1PR1 neuronal / Abstract: The food intake and energy expenditure are closely regulated by specific neurons in the hypothalamus. During the last two decades, the location of the leptin receptor in hypothalamic nuclei as well as the description of the route of transmission Intracellular triggered by this hormone in hypothalamic neurons, were crucial to the understanding of the control of food intake and energy expenditure.Increasingly, eating disorders, diseases associated with obesity are related to signal transmission malfunction of leptin in the hypothalamus. The subclinical inflammatory process frequently observed in experimental models of obesity are directly associated with distinct mechanisms of leptin resistance in the hypothalamus and result in increased food intake and body weight gain. Furthermore, studies have shown that physical exercise can increase the sensitivity of leptin in the hypothalamus of obese animals, through of antiinflammatory cytokines, however, these mechanisms remain only partially understood. Recently, the protein S1PR1 (sphingosine-1-phosphate receptor-1) was described as a molecule with high ability to exert potent synergistic effects on the signaling pathway of leptin, supporting the activation of Jak2/STAT3 in some cell lines. Thus, this project aims to investigate the effects of exercise on the activity of SIPR1 and leptin sensitivity in hypothalamus of obese rodents. We believe that the completion of this project will contribute to characterize the involvement of S1PR1 in leptin signaling in the hypothalamus, and to determine the effects of exercise on the activity of neuronal S1PR1 / Mestrado / Metabolismo e Biologia Molecular / Mestre em Ciências da Nutrição e do Esporte e Metabolismo