ABSTRACT
O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)
The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)
Subject(s)
Humans , Chemokines/metabolism , Cytokines/metabolism , Fibroblasts/physiology , Interleukin-1beta/physiology , Renin-Angiotensin System/physiology , Analysis of Variance , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II/analysis , Angiotensin II/physiology , Angiotensin I/analysis , Angiotensin I/physiology , Blotting, Western , Cells, Cultured , Chemokines/analysis , Cytokines/analysis , Gingiva/cytology , Losartan/pharmacology , Peptide Fragments/analysis , Peptide Fragments/physiology , Peptidyl-Dipeptidase A/analysis , Peptidyl-Dipeptidase A/physiology , Periodontal Ligament/cytology , Polymerase Chain Reaction , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/physiology , Receptor, Angiotensin, Type 1/analysis , Receptor, Angiotensin, Type 1/physiologyABSTRACT
Antecedentes: Recientemente hemos propuesto en un modelo experimental de infarto al miocardio una significativa interregulación entre los niveles de la enzima convertidora de angiotensina I (ECA) y su homóloga (ECA-2), junto con que angiotensina (Ang)-(1-9) más que Ang-(1-7) actuaría como uncontrarregulador de Ang II. Sin embargo tal relación no se ha investigado en el remodelado aórtico hipertensivo. Objetivo: Determinar la expresión de ECA y ECA-2, los niveles de Angs I, II, (1-7) y (1-9) y los parámetros de remodelado de la pared aórtica de ratas hipertensas. Métodos: Ratas normotensas Lewis (n=18) fueron randomizadas a hipertensión (HTA) por sobrecarga de presión (modelo Goldblatt, GB, 2 riñones-1 pinzado, n=9). Ratas pseudo-operadas se usaron como controles (S, n=9). A las 6 semanas post cirugía, se determinó la masa cardíaca relativa (MCR) y la presión arterial sistólica (PAS). En la aorta torácica se determinó el grosor de la túnica media (GTM), área de la TM (ATM), niveles de mRNA de ECA y ECA-2, factor de crecimiento transformante tipo beta (TGF-beta), inhibidor del activador de plasminógeno (PAI-1) y de la proteína quimioatractante de monocitos (MCP-1) por RT-PCR. La actividad y niveles proteicos de ECA y ECA-2 por fluorimetría y Western blot y los niveles de Angs I, II, (1-7) y (1-9) por HPLC y radioinmunoensayo. Resultados: La MCR y la PAS aumentaron significativamente (p<0,05) en el grupo GB respecto a su control S. Las ratas hipertensas mostraron un aumento significativo (p<0.05) del GTM (18 por ciento), ATM (31 por ciento), niveles de mRNA de ECA (164 por ciento), TGF-beta (105 por ciento), PAI-1(51 por ciento), MCP-1 (53 por ciento) junto con mayor actividad (89 por ciento), niveles proteicos de ECA (130 por ciento) y Ang II (48 por ciento). Esos efectos se asociaron a una significativa disminución del mRNA, los niveles proteicos y actividad...
Background: In experimental models of myocardial infarction we have recently proposed a significantinter-regulation between levels of Angiotensin I converting enzyme (ACE) and its homologous, ACE-2; in addition, we have proposed that Angiotensin 1-9 (Ang-(1-9)) rather than Ang-(1-7) counter regulates Ang II. These relations have not been investigated in hypertensive aortic wall remodeling. Aim: To measure de expression of ACE and ACE-2, the aortic wall levels of Ang I, Ang II, Ang-(1-7) and Ang-(1-9), along with parameters of aortic wall remodeling in hypertensive rats. Methods: 18 Lewis rats were randomized to Goldblatt (2 kidneys, 1 clamped) induced hypertension (n=9) or sham operation (controls, n=9). Six weeks after surgery, relative cardiac mass (RCM), systolic blood pressure (SBP), medial layer aortic wall thickness (MLT) and ML area (MLA) were measured. The aortic wall levels of ACE and ACE-2, tissue growth factor beta (TGF- beta), plasminogen activator inhibitor (PAI-1) and monocyte chemoattractant protein (MCP-1) were determined by RT-PCR. Activity and protein levels of ACE and ACE-2 were measured by fluorometry and Western Blot and ANG I, Ang II, Ang-(1-7) and Ang-(1-9) levels were determined using HPLC and radioimmunoassay. Results: RCM and SBP increased significantly in hypertensive as opossed to sham operated rats...
Subject(s)
Animals , Rats , Angiotensin I/physiology , Angiotensin II/physiology , Aorta, Thoracic/enzymology , Hypertension , Peptidyl-Dipeptidase A/physiology , Renin-Angiotensin SystemABSTRACT
Angiotensin-(1-7) (Ang-(1-7)) is now considered to be a biologically active member of the renin-angiotensin system. The functions of Ang-(1-7) are often opposite to those attributed to the main effector component of the renin-angiotensin system, Ang II. Chronic administration of angiotensin-converting enzyme inhibitors (ACEI) increases 10- to 25-fold the plasma levels of this peptide, suggesting that part of the beneficial effects of ACEI could be mediated by Ang-(1-7). Ang-(1-7) can be formed from Ang II or directly from Ang I. Other enzymatic pathways for Ang-(1-7) generation have been recently described involving the novel ACE homologue ACE2. This enzyme can form Ang-(1-7) from Ang II or less efficiently by the hydrolysis of Ang I to Ang-(1-9) with subsequent Ang-(1-7) formation. The biological relevance of Ang-(1-7) has been recently reinforced by the identification of its receptor, the G-protein-coupled receptor Mas. Heart and blood vessels are important targets for the formation and actions of Ang-(1-7). In this review we will discuss recent findings concerning the biological role of Ang-(1-7) in the heart and blood vessels, taking into account aspects related to its formation and effects on these tissues. In addition, we will discuss the potential of Ang-(1-7) and its receptor as a target for the development of new cardiovascular drugs.
Subject(s)
Animals , Humans , Angiotensin I/physiology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Peptide Fragments/physiology , Angiotensin I/antagonists & inhibitors , Angiotensin I/biosynthesis , Blood Pressure/drug effects , Cardiovascular Physiological Phenomena/drug effects , Coronary Vessels/drug effects , Endothelial Cells , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/biosynthesis , Renin-Angiotensin System/physiologyABSTRACT
For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption
Subject(s)
Animals , Angiotensin II/physiology , Angiotensin I/physiology , Extracellular Space/enzymology , Kidney Tubules, Proximal/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/urine , Extracellular Space/physiology , Receptors, Angiotensin/physiologyABSTRACT
The heptapeptide angiotensin-(1-7) is considered to be a biologically active endproduct of the renin-angiotensin system. This angiotensin, which is devoid of the most known actions of angioatensin II such as induction of drinking behavior and vasoconstriction, has several selective effects in the brain and periphery. In the present article we briefly review recent evidence for a physiological role of angiotensin-(1-7) in the control of hydroelectrolyte balance.
Subject(s)
Rats , Animals , Male , Angiotensin III/physiology , Angiotensin II/physiology , Angiotensin I/physiology , Angiotensins/physiology , Drinking/physiology , Kidney Glomerulus/physiology , Kidney Tubules, Distal/physiology , Kidney Tubules, Proximal/physiology , Receptors, Angiotensin/physiology , Renin-Angiotensin System/physiology , Vasopressins/physiology , Kidney/physiology , Rats, WistarABSTRACT
Hace aproximadamente un siglo (en 1898), se realizaron los primeros estudios que llevaron al descubrimiento de la renina y, posteriormente, a todo el complejo sistema de la renina-Angiotensina-aldosterona. En los últimos veinte años, se ha aprendido a manipular farmacológicamente la enzima que en ese sistema transforma la Angiotensina I (decapéptido) en Angiotensina II (octapéptido); tal enzima "convertidora" es una dipeptidilcar-boxipeptidasa obicua y no específica, cuya amplia distribución y su capacidad de endo y exopeptidasa sobre péptidos como la bradikinina, Angiotensina I, encefalina, neurotensina, substancia P, hormona liberadora de la hormona luteinizante, etc., hace que su inhibición pueda afectar múltiples mecanismos de control en todo el organismo. Entre mediados de los años 70's y durante la década de los 80's, diversos inhibidores orales de la ECA han tenido gran aplicación en enfermedades como la hipertensión arterial y la insuficiencia cardíaca, donde se han demostrado alteraciones neurohumorales y tisulares del sistema renina-Angiotensina-aldosterona, así como de otros sistemas que pueden ser afectados al inhibir la ECA. En los proximos años, la investigación de los IECA deberá responder dudas básicas como las que se refieren a la especificidad de algunos de estos inhibidores sobre la ECA en algunos tejidos; así mismo, deberá dilucidar aspectos farmacológicos (ejemplo: cronofarmacología) o terapéuticos (ejemplo: efecto sobre los mecanismos básicos de la ateroesclerosis, isquemia miocárdica, hiperactividad vascular del paciente hipertenso).