Made available in DSpace on 2017-07-10T14:17:11Z (GMT). No. of bitstreams: 1 FINALIZADA _ABRIL2015.pdf: 3170133 bytes, checksum: 1d4668fdaca4f714ff23eb80e3ac0b68 (MD5) Previous issue date: 2015-06-30 / The prevalence of obesity has increased in recent decades in many countries, and harmful effects to the body can begin in childhood and persist into adulthood. Obesity is associated with disturbances of reproductive function in women and in female rodents, such as early onset of puberty, change in menstrual / estrous cycle and infertility, with impaired ovulation or anovulation (lack of ovulation). Angiotensin II (Ang II) appears to exert effects on reproduction and obesity, contributing to the development of the deleterious effects of obesity and affecting pre-ovulatory surge of luteinizing hormone (LH), estradiol and progesterone, thus reducing ovulation in adult rats. The objective of this study was to evaluate losartan effect, an antagonist of Ang II AT1 receptor, administrated in adulthood, in follicular development of adult Wistar rats with obesity induced by cafeteria diet. After weaning at 21 days of life, female Wistar rats were divided into 02 groups: control (CTL) that received standard chow diet; Cafeteria (CAF) received the cafeteria diet. From 70 days of life began losartan administration by gavage. The CTL group received water in gavage (CTL) and CAF group was separated into 02 groups, CAF (which received water in gavage) and CAF + LOS (who received losartan in gavage), in total 03 groups were performed. 05 animals were used per group. Euthanasia was performed on the first proestrous after 30 days of administration of losartan or water. The retroperitoneal, perigonadal and subcutaneous fat were removed and weighed. Morphological analysis of ovaries was performed, proceeding to count the number of primary follicles, secondary, antral and mature follicles per ovary. Was also collected blood sample for determination of FSH, LH, prolactin and progesterone. Body weight and the weight of the 03 fats were measured, and the number of antral follicles were higher in group CAF in relation to the group CTL (p <0.001). However, FSH and LH levels were lower in the CAF animals compared to the animals of the group CTL (p <0.001). The administration of losartan normalized the body weight and accumulation of retroperitoneal and subcutaneous fat as well as the number of antral follicles. Thus, we suggest that the use of the antagonist of Ang II AT1 receptor, losartan, in adulthood, can improve follicular development in females with cafeteria diet-induced obesity and, can be, in the future, an coadjuvant drug in the treatment of infertility associated to obesity. / A prevalência da obesidade aumentou nas últimas décadas em vários países, tendo efeitos prejudiciais ao organismo que podem iniciar na infância e persistir até a vida adulta. A obesidade está associada a distúrbios da função reprodutiva, em mulheres e em fêmeas de roedores, como início precoce da puberdade, alteração do ciclo menstrual/estral e infertilidade, com alterações da ovulação (disovulias) ou anovulação (ausência da ovulação). A Angiotensina II (Ang II) parece exercer efeitos sobre a reprodução e a obesidade, contribuindo para o desenvolvimento dos efeitos deletérios da obesidade e afetando o pico pré-ovulatório do hormônio luteinizante (LH), progesterona e estradiol, reduzindo, consequentemente, a ovulação em ratas adultas. O objetivo deste estudo foi avaliar o efeito do losartan, um antagonista do receptor AT1 da Ang II, sobre o desenvolvimento folicular de ratas Wistar adultas, com obesidade induzida pela dieta de cafeteria. Após o desmame, aos 21 dias de vida, ratas Wistar foram separadas em 02 grupos: controle (CTL) que recebeu ração padrão e Cafeteria (CAF) que recebeu a dieta de cafeteria. A partir dos 70 dias de vida iniciou-se a administração de losartan por gavagem. O grupo CTL recebeu água na gavagem (CTL) e o grupo CAF foi separado em 02 grupos, CAF (que recebeu água na gavagem) e CAF+LOS (que recebeu losartan na gavagem), totalizando 03 grupos. Foram utilizados 05 animais por grupo. A eutanásia foi realizada no primeiro proestro após 30 dias da administração de losartan ou de água. As gorduras retroperitoneal, perigonadal e subcutânea foram retiradas e pesadas. Foi realizada a análise morfológica dos ovários, procedendo-se a contagem do número dos folículos primários, secundários, antrais e maduros por ovário. Também foi coletado o sangue total para dosagens de FSH, LH, Prolactina e Progesterona. O peso corporal, bem como, o peso das 03 gorduras foram avaliados, e, o número de folículos antrais foi estatisticamente maior no grupo CAF em relação ao CTL (p<0,001). Todavia, as concentrações de FSH e LH foram menores nos animais CAF em relação ao CTL (p<0,001). A administração do losartan normalizou o peso corporal e o acúmulo das gorduras retroperitoneal e subcutânea, bem como, o número de folículos antrais. Dessa forma, sugerimos que o uso do antagonista do receptor AT1 da Ang II, o losartan, na vida adulta, possa contribuir para melhorar o desenvolvimento folicular em fêmeas com obesidade induzida pela dieta de cafeteria e, futuramente, ser uma droga coadjuvante no tratamento da infertilidade associada à obesidade.

Neste estudo, avaliou-se o efeito da exposição intermitente à Ang II, levando em consideração uma dose que tenha uma ação não pressora, sobre a liberação de citocinas inflamatórias como a interleucina-6 (IL-6) e fator de crescimento transformante beta (TGF&beta;), bem como PAI-1 e plasminogênio/plasmina. O estudo foi realizado em camundongos machos C57Bl/6, submetidos ao tratamento com Ang II (30ng/kg), com losartan (30mg/kg) ou uma combinação destes, nos tempos de: 30 minutos, 1, 3 e 10 dias. As avaliações mostraram que a Ang II não altera pressão arterial, sugerindo que os aumentos observados de IL-6 e TGF&beta; sejam decorrentes de ação direta da Ang II. A Ang II promove aumento tanto agudo de TGF&beta;, possivelmente associado à ação proteolítica da plasmina e alterações vasculares transitórias compatíveis com aumento de permeabilidade, como crônico de TGF&beta;, possivelmente associado ao aumento da expressão gênica, levando ao aumento da deposição de colágeno vascular. / This study, evaluated the effect of the intermittent exposure to Angiotensin II (Ang II), taking in account a non-pressor dose on the release of inflammatory cytokines such as interleukin-6 (IL-6), transforming growth factor beta (TGF&beta;) as well PAI-1 and plasminogen /plasmin. The study was conducted on male mice C57BL/6 subjected to the treatment with angiotensin II (30 ng/kg), losartan (30 mg/kg) or a combination thereof, at times: 30 minutes, 1, 3 and 10 days. The evaluations showed that Ang II did not change blood pressure, suggesting that the increases of IL-6 and TGF&beta; may be by due to direct action of Ang II. Ang II promotes both acute increase of TGF&beta;, possibly associated with the proteolytic action of plasmin and transient vascular changes consistent with increased permeability, such as chronic increase of TGF&beta;, possibly associated with increased gene expression, leading to increased vascular collagen deposition.

Ainda existe controvérsia se a terapia hormonal estrogênica confere benefício ou dano cardiovascular. O dano potencial causado por doses padrão de estrogênio incluindo doença cardíaca coronariana e trombose venosa pode ser atenuada por uso de doses mais baixas. O objetivo deste estudo foi avaliar o efeito do tratamento com conjugado estrogênio equino, no leito mesentérico venular (LVM) de SHR ovariectomizadas (OVX), com dose padrão (SD) vs dose mínima (LD). Angiotensina II (AngII) foi perfundida de forma concentração-dependente. Hiperreatividade à Ang II, aumento na geração de EROS, redução da atividade da SOD e catalase e da biodisponibilidade do NO foram encontrados em OVX e SD vs controle e LD. Por outro lado, a resposta reduzida à AngII no LVM de ratas LD foi relacionado ao aumento da atividade da eNOS, redução na geração de EROS e aumento da expressão dos receptores AT2,ER<font face=\"Symbol\">&#945; e GPR30. Sugerimos que a dose mínima tem efeito protetor sobre o LVM das OVX e que a dose SD pode aumentar o risco para a doença venular induzindo alterações na reatividade venular. / Controversy still exists whether estrogen hormonal therapy confers cardiovascular benefit or harm. The potential harm caused by standard dosages of estrogen including coronary heart disease and venous thrombosis may be mitigated by use of lower doses. The aim of this study was evaluated the effect of conjugated equine estrogen (CEE) treatment, in isolated mesenteric venular bed (MVB) of ovariectomized SHR (OVX), at standard therapeutic (SD) vs low dose (LD). Angiotensin II (Ang II) was perfused in a concentration-dependent manner. Hyperreactivity to Ang II,augmented ROS generation, reduced SOD, catalase activity and NO availability were found in OVX and SD vs. control and LD. However, the reduced MVB response to Ang II in LD rats was related to increased endothelial NO synthase activity, reduced ROS generation and increased Ang II AT2, ER<font face=\"Symbol\">&#945; and GPR30 receptor expression. We suggest that CEE at a low dose has a protective effect in OVX mesenteric venular bed. The standard dose might increase the risk for venular disease by inducing alterations in venular reactivity.

<p>Important information on the bioactive conformation of biologically active peptides may be obtained by studies of rigid peptides or well-defined secondary structure mimetics incorporated into pseudopeptides. The structural requirements for the interaction of angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) with its AT₁ and AT₂ receptors were the subject of this study.</p><p>The main objectives of this work were to synthesize secondary structure mimetics and incorporate these into Ang II. Ang II has been suggested to adopt a turn conformation around Tyr⁴ when interacting with its AT₁ receptor. Therefore, two <i>γ</i>- and one <i>β</i>-turn mimetic scaffolds based on the benzodiazepine structure were synthesized and decorated with side chains. The scaffolds replaced the turn region around Tyr⁴. Most of the pseudopeptides obtained after incorporation into Ang II exhibited high AT₂/AT₁ selectivity and nanomolar affinity to the AT₂ receptor. One pseudopeptide encompassing a <i>β</i>-turn mimetic also displayed AT₁ receptor affinity.</p><p>We hypothesized that the position of the guanidino group of the arginine residue and the N-terminal end, in relation to the tyrosine side chain, was critical for AT₂ receptor affinity. Conformational evaluation of the pseudopeptides revealed that in all the compounds with AT₂ receptor affinity the arginine side chain and the N-terminal end could reach common regions, not accessible to the inactive compound. It is proposed that Ang II has a more extended bioactive conformation when binding to the AT₂ receptor than when binding to the AT₁ receptor.</p><p>Furthermore, in a Gly scan of Ang II only replacement of the arginine residue reduced the affinity for the AT₂ receptor considerably. Some N-terminal modified Ang II analogues were also synthesized and it was concluded that truncated Ang II analogues interact with the AT₂ receptor differently than Ang II.</p><p>Three of the synthesized pseudopeptides were evaluated in AT₂ receptor functional assays and were found to act as agonists.</p>

<p>Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT₂ receptor.</p><p>The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT₂ receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan.</p><p>To further examine ligand binding, a 3D model of the AT₂ receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT₂ receptor.</p><p>By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT₂ receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT₁ and AT₂ receptor affinity as well as selectivity were derived. </p>

Important information on the bioactive conformation of biologically active peptides may be obtained by studies of rigid peptides or well-defined secondary structure mimetics incorporated into pseudopeptides. The structural requirements for the interaction of angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) with its AT1 and AT2 receptors were the subject of this study. The main objectives of this work were to synthesize secondary structure mimetics and incorporate these into Ang II. Ang II has been suggested to adopt a turn conformation around Tyr4 when interacting with its AT1 receptor. Therefore, two γ- and one β-turn mimetic scaffolds based on the benzodiazepine structure were synthesized and decorated with side chains. The scaffolds replaced the turn region around Tyr4. Most of the pseudopeptides obtained after incorporation into Ang II exhibited high AT2/AT1 selectivity and nanomolar affinity to the AT2 receptor. One pseudopeptide encompassing a β-turn mimetic also displayed AT1 receptor affinity. We hypothesized that the position of the guanidino group of the arginine residue and the N-terminal end, in relation to the tyrosine side chain, was critical for AT2 receptor affinity. Conformational evaluation of the pseudopeptides revealed that in all the compounds with AT2 receptor affinity the arginine side chain and the N-terminal end could reach common regions, not accessible to the inactive compound. It is proposed that Ang II has a more extended bioactive conformation when binding to the AT2 receptor than when binding to the AT1 receptor. Furthermore, in a Gly scan of Ang II only replacement of the arginine residue reduced the affinity for the AT2 receptor considerably. Some N-terminal modified Ang II analogues were also synthesized and it was concluded that truncated Ang II analogues interact with the AT2 receptor differently than Ang II. Three of the synthesized pseudopeptides were evaluated in AT2 receptor functional assays and were found to act as agonists.

Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT2 receptor. The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT2 receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan. To further examine ligand binding, a 3D model of the AT2 receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT2 receptor. By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT2 receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT1 and AT2 receptor affinity as well as selectivity were derived.

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

PKCe plays a very important role in mediating the protection against myocardial ischemia and reperfusion injury induced by ischemic preconditioning (IPC) and pharmacologic preconditioning (PPC). The redistribution of PKCe was assessed by subcellular fractionation and western blotting in the Langendorff-perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine A1 and/or A3 agonists, bradykinin, angiotensin II, and d1-opioid agonists resulted in PKCe redistribution from the cytosol to the mitochondria. This effect of IPC on PKCe redistribution was visible up to at least 30min of reperfusion, while that of PPC was lost by 10min of drug washout, indicative of the transient nature of PKCe redistribution. PKCe redistribution to mitochondria by IPC was also visualized using immunogold electron microscopy. Thus, IPC and PPC caused PKCe redistribution from the cytosol to the mitochondria, which was longer-lasting in IPC than in PPC.