A expressão dos receptores AT1 e AT2 da Angiotensina II (Ang II) em astrocitomas humanos está associada a um pior prognóstico. Para investigar os mecanismos moleculares da ação da Ang II em gliomas, foi analisado por DNA microarray o transcriptoma da linhagem celular C6 tratada com Ang II e antagonistas de AT1 e AT2 nos intervalos de 3 e 6 horas. Os genes diferencialmente expressos obtidos foram submetidos a análises de enriquecimento funcional, diagramas de Venn e interatomas. As alterações observadas no transcriptoma a partir do tratamento com Ang II revelaram genes envolvidos em funções biológicas e vias de sinalização pró-tumorais tais como ciclo celular, migração, ErbB, MAPK e mTOR. Além disso, a identificação das proteínas centrais nos interatomas avaliados forneceu evidências para estudos futuros com foco nesses alvos. Embora a transdução de sinal de AT2 seja distinta da observada em AT1, ambas apresentaram categorias funcionais em comum que podem estar relacionadas ao pior prognóstico observado em pacientes cujos gliomas expressam esses receptores. / The expression of AT1 and AT2 receptors of Angiotensin II (Ang II) in human astrocytomas was associated with a worse prognosis. To investigate the molecular mechanisms of Ang II actions in gliomas, the transcriptomic profile of C6 cells treated with Ang II and antagonists of AT1 and AT2 was evaluated. The differentially expressed genes obtained were submitted to functional enrichment, Venn diagram and transcriptome network analyzes. Ang II treatment caused gene expression changes involved in pro-tumor functions and signaling pathways such as cell cycle, migration, ErbB, mTOR and MAPK. Furthermore, the identification of central proteins in the interactomes provided evidences for future studies focused on these targets. Taken together, these results provided insights into how the transcriptome of glioma cells is affected by Ang II. Moreover, although the signal transduction of AT2 is distinct from the observed for AT1, they present functional categories in common that may be underlying the worst prognosis observed in gliomas expressing Ang II receptors.

(en) Charles University in Prague Faculty of Pharmacy Hradec Králové Department of Pharmacology  Toxicology Student: Růžena Kubíčková Supervisors: Prof. Manuela Morato PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Differential reactivity of the longitudinal and circular muscle of the rat distal colon Physiological regulation of the function of the large intestine is neurohumoral. The neural part of this regulation implies the vegetative nervous system, which is crucial for the control of the gastrointestinal tract (GIT) motility. Concerning the humoral part of the regulation, recent studies showed that angiotensin II (Ang II) causes contraction of the colonic smooth muscle and, thus, can also influence the motility of the colon. However, there are no known studies that have described this process in detail. The aims of this work were (1) to compare the reactivity of the longitudinal and circular muscles of the rat distal colon to potassium chloride (KCl), acetylcholine (ACh) and Ang II, (2) to compare the observed results between male and female rats, and (3) to characterize the receptors mediating the response to Ang II. For purpose of the project, adult, 10-12 weeks old Wistar Han rats of both genders were used. Strips of the the rat distal colon were mounted in organ baths along their...

L’hypertension artérielle chronique représente le premier facteur de risque de l’AVC ischémique, mais elle en est aussi le principal facteur aggravant. Les mécanismes à l’origine du risque ischémique lié à l’hypertension ne sont pas encore entièrement compris. Plusieurs études ont montré l’existence d’une forte composante inflammatoire délétère impliquée dans la physiopathologie de l’hypertension. Au niveau cérébral, la présence d’une intense réactivité microgliale hypothalamique, participant à l’aggravation de la pathologie a été observé. Dans le travail présenté ici, nous nous sommes intéressés à l’impact de l’hypertension artérielle sur l’état inflammatoire du cortex cérébral, une région particulièrement touchée lors d’un AVC ischémique chez le patient. Nous avons tiré parti de la souche transgénique de souris CX3CR1GFP/+ pour l’imagerie de la dynamique microgliale, principal acteur de l’immunité cérébrale. Par une analyse in vivo réalisée en microscopie biphotonique, nous avons montré que l’hypertension induite par infusion chronique d’angiotensine-II altère la morphologie de la microglie, mais surtout sa capacité de surveillance du parenchyme cérébral et sa capacité cicatricielle. Nous avons aussi montré que ce type d’hypertension endommage la structure et la fonctionnalité des vaisseaux corticaux. L’ensemble de ces résultats pourrait expliquer, au moins en partie, la sensibilisation du cerveau aux lésions ischémiques par l’hypertension artérielle, avant même la survenue de l’AVC. / Chronic high blood pressure is ischemic stroke’s leading risk factor, but it is also its main aggravating factor. The mechanisms underlying hypertension-induced ischemic brain lesion exacerbation are not yet fully understood. Several studies highlighted the existence of a strong inflammatory component in the pathophysiology of hypertension. In the brain, the presence of intense hypothalamic microglial reactivity, contributing to the pathology worsening has been shown. In this work, we focused on the impact of high blood pressure on the inflammatory state of the cerebral cortex, a region particularly affected by ischemic stroke in the patient. We took advantage of the CX3CR1GFP/+ mice transgenic strain for imaging microglia dynamics. By using in vivo two-photon microscopy, we have shown that hypertension induced by chronic infusion of angiotensin-II alters the microglia morphology, especially its parenchymal surveilling activity and its cicatricial capacity. We have also shown that this type of hypertension disrupts the structure and the functioning of cortical vessels. All of these results can explain, at least in part, the brain sensitization to ischemic lesions under arterial hypertension, before the onset of stroke.

Heart failure is a syndrome in which the heart is unable to supply the entire body with oxygen. It is manifested in shortness of breath and exercise intolerance. One class of drugs that have proven effective in managing the progression of heart failure is β-blockers. These drugs bind to β-adrenergic receptors with high affinity, thus preventing the binding of endogenous catecholamines such as epinephrine and norepinephrine to the receptors by outcompeting them. The most common explanation of how β-blockers help manage the progression of heart failure is that by slowing the heart rate, it reduces the strain put on the heart. There may however be other ways that β-blockers help decrease morbidity and mortality of heart failure. Alternative reasons to how β-blockers aid the treatment of heart failure have been proposed based on the literature. It was found that the compensatory mechanisms intended to alleviate failure may be the main reasons that actually worsen it. Prolonged stimulation by epinephrine and norepinephrine damage the myocardium through oxidative damage, signaling for apoptosis and cardiac remodeling, as well as causing an increase in blood volume through the RAS-system. By blocking these maladaptive responses, β-blockers such as Carvedilol, Metoprolol and Nebivolol, together with other drugs such as ACE-inhibitors, and lifestyle changes help manage the progression of heart failure as well as increase the quality of life for the patients suffering from it

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Angiotensin-(1-7) (Ang-(1-7)) is reported to be equipotent with angiotensin II (AII) in producing some central biological effects but the receptors responsible for these actions have not been defined. Three classes of receptor have been proposed: AT1, AT2, and a putative Ang-(1-7) selective receptor. This study specifically evaluates Ang-(1-7) competition at AII binding sites (AT1 and AT2) in the rat brain. 125I Sar1 Ile8 AII (269-312 pM) was used to conduct receptor autoradiographic binding assays in brain sections. Competition with Ile5 AII and Val5 AII was similar at nuclei in which either AT1 or AT2 receptor subtypes predominate (Ki = 11-18 nM). Ang-(1-7) competed 150-fold less effectively than native AII at AT1 predominant brain nuclei (Ki = 2.4 μM). At brain regions where AT2 receptors predominate, Ang-(1-7) showed a very low affinity (Ki = 104 μM) for the majority of the 125I Sar1 Ile8 AII binding sites (AT2). A small proportion of 125I Sar1 Ile3 AII binding sites showed an affinity of 2.0 μM, presumably AT1 receptors present in those brain regions. For biological responses where Ang-(1-7) is reported to be equipotent with AII, it is unlikely that these actions are mediated by the widely distributed AT1 or AT2 receptor subtypes which recognize 125I Sar1 Ile8 AII.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AIIα subtype) or PD123177 (designated AIIβ subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AIIα and AIIβ subtypes.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-KB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Thrombospondin-1 (TSP-1) is synthesized, secreted, and incorporated into the extracellular matrix by a variety of cells, including the endothelial cells. Addition of angiotensin II (AII) significantly induced TSP-1 mRNA in rat heart-derived endothelial cells. TSP-1 mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 5 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, could abolish the induction of TSP-1 mRNA by AII. Phorbol 12-myristate 13-acetate (TPA) was found to enhance TSP-1 mRNA level whereas a protein kinase C inhibitor, H7, was shown to block the induction. Immunoblot analysis revealed that TSP-1 was detectable in the medium 4 h after AII stimulation. Our results suggest that the upregulation of TSP-1 by All represents an important mechanism leading to perivascular fibrosis in the heart.

Angiotensin II (All) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of All. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that All stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of All-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after All stimulation. Since endothelial cells line the blood vessels and sense the rise in All associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.