Walkyria O. Sampaio

Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways

Angiotensin-(1-7) [Ang-(1-7)] causes endothelial-dependent vasodilation mediated, in part, by NO release. However, the molecular mechanisms involved in endothelial NO synthase (eNOS) activation by Ang-(1-7) remain unknown. Using Chinese hamster ovary cells stably transfected with Mas cDNA (Chinese hamster ovary-Mas), we evaluated the underlying mechanisms related to receptor Mas–mediated posttranslational eNOS activation and NO release. We further examined the Ang-(1-7) profile of eNOS activation in human aortic endothelial cells, which constitutively express the Mas receptor. Chinese hamster ovary-Mas cells and human aortic endothelial cell were stimulated with Ang-(1-7; 10−7 mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10−6 mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10−6 mol/L). Changes in eNOS (at Ser1177/Thr495 residues) and Akt phosphorylation were evaluated by Western blotting. NO release was measured using both the fluorochrome 2,3-diaminonaphthalene and an NO analyzer. Ang-(1-7) significantly stimulated eNOS activation (reciprocal phosphorylation/dephosphorylation at Ser1177/Thr495) and induced a sustained Akt phosphorylation (P<0.05). Concomitantly, a significant increase in NO release was observed (2-fold increase in relation to control). These effects were blocked by A-779. Wortmannin suppressed eNOS activation in both Chinese hamster ovary-Mas and human aortic endothelial cells. Our findings demonstrate that Ang-(1-7), through Mas, stimulates eNOS activation and NO production via Akt-dependent pathways. These novel data highlight the importance of the Ang-(1-7)/Mas axis as a putative regulator of endothelial function.

Endothelial dysfunction and elevated blood pressure in MAS gene-deleted mice.

Mas codes for a G protein–coupled receptor that is implicated in angiotensin-(1-7) signaling. We studied the cardiovascular phenotype of Mas-deficient mice backcrossed onto the FVB/N genetic background using telemetry and found that they exhibit higher blood pressures compared with controls. These Mas−/− mice also had impaired endothelial function, decreased NO production, and lower endothelial NO synthase expression. Reduced nicotinamide-adenine dinucleotide phosphate oxidase catalytic subunit gp91phox protein content determined by Western blotting was higher in Mas−/− mice than in controls, whereas superoxide dismutase and catalase activities were reduced. The superoxide dismutase mimetic, Tempol, decreased blood pressure in Mas−/− mice but had a minimal effect in control mice. Our results show a major cardiovascular phenotype in Mas−/− mice. Mas-deletion results in increased blood pressure, endothelial dysfunction, and an imbalance between NO and reactive oxygen species. Our animals represent a promising model to study angiotensin-(1-7)–mediated cardiovascular effects and to evaluate Mas agonistic compounds as novel cardioprotective and antihypertensive agents based on their beneficial effects on endothelial function.

Angiotensin-(1-7) and its receptor as a potential targets for new cardiovascular drugs.

The identification of novel biochemical components of the renin–angiotensin system (RAS) has added a further layer of complexity to the classical concept of this cardiovascular regulatory system. It is now clear that there is a counter-regulatory arm within the RAS that is mainly formed by the angiotensin-converting enzyme 2–angiotensin (1-7)–receptor Mas axis. The functions of this axis are often opposite to those attributed to the major component of the RAS, angiotensin II. This review will highlight the current knowledge concerning the cardiovascular effects of angiotensin-(1-7) through a direct interaction with its receptor Mas or through an indirect interplay with the kallikrein–kinin system. In addition, there will be a discussion of its role in the beneficial effects of angiotensin-converting enzyme inhibitors and angio-tensin receptor type 1 (AT1) antagonists, and the potential of this peptide and its receptor as a novel targets for new cardiovascular drugs.

Ablation of angiotensin (1-7) receptor Mas in C57Bl/6 mice causes endothelial dysfunction

The Mas gene codes for an angiotensin (1-7) receptor. There is accumulating evidence that Mas is involved in vascular homeostasis. We have recently backcrossed Mas-knockout mice to two different genetic backgrounds, C57Bl/6 and FVB/N. FVB/NMas-deficient mice exhibited elevation in blood pressure (BP) and impaired endothelial function. In the present study, we aimed to address the question whether this phenotype is strain-specific. Therefore, we evaluated endothelial function in C57Bl/6Mas-deficient mice. Similar to FVB/NMas-knockout animals, Mas-deficiency in C57Bl/6 mice leads to endothelial dysfunction evaluated by the acute BP effect of acetylcholine administration. Measurements of nitric oxide (NO) and reactive oxygen species (ROS) and the systems involved in their metabolism revealed an imbalance between these vasoactive factors in C57Bl/6Mas-knockout mice, which may explain the impairment of endothelial function in these animals. However, endothelial dysfunction was less prominent in Mas-deficient mice on a C57Bl/6 background compared to FVB/N. Moreover, C57Bl/6Mas-deficient mice remained normotensive while FVB/N-based animals exhibited elevated BP. The impairment of endothelium-dependent vasodilatory response to acetylcholine in two different mouse strains with Mas deficiency indicates a key role of Mas in endothelial function by its effects on the generation and metabolism of NO and ROS.

Angioprotectin: an angiotensin II-like peptide causing vasodilatory effects

The family of angiotensin peptides has been steadily growing in recent years. Most are fragments of angiotensin II (Ang II) with different affinities to the known angiotensin receptors. Here, we describe a novel endogenous Ang II‐like octapeptide in plasma from healthy humans and patients with end‐stage renal failure, which acts as a stronger agonist at Mas receptors than Ang 1–7. Chromatographic purification and structural analysis by matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight (MALDI‐TOF/TOF) revealed an Ang II‐like octapeptide, angioprotectin, with the sequence Pro‐Glu‐Val‐Tyr‐Ile‐His‐Pro‐Phe, which differs from Ang II in Pro1 and Glu2 instead of Asp1 and Arg2. Pro‐Glu‐Val‐Tyr‐Ile‐His‐Pro‐Phe in angioprotectin is most likely generated enzymatically from Ang II. Angioprotectin antagonized the contractile actions of Ang II on rat aortic rings. The physiological antagonism of vasoconstrictor actions of Ang II by angioprotectin is mediated by the Mas receptor. Angioprotectin has a stronger affinity to the Mas receptor than Ang‐1–7. Plasma concentrations were ~15% of plasma Ang II concentrations in healthy volunteers and up to 50% in patients with renal failure. A commercially available Ang II antibody did not discriminate between angioprotectin and Ang II; thus, angioprotectin can contribute to Ang II concentrations measured by antibody‐based assays. This novel peptide is likely to be a relevant component of the human renin‐angiotensin‐system.—Jankowski, V., Tölle, M., Santos, R. A. S., Günthner, T., Krause, E., Beyermann, M., Welker, P., Bader, M., Pinheiro, S. V. B., Sampaio, W. O., Lautner, R., Kretschmer, A., van der Giet, M., Zidek, W., Jankowski, J. Angioprotectin: an angiotensin II‐like peptide causing vasodilatory effects. FASEB J. 25, 2987–2995 (2011). www.fasebj.org

Redox unbalance: NADPH oxidase as therapeutic target in blood pressure control

Several studies refer to reactive oxygen and nitrogen species (RONS) as important agents in the pathogenesis of a number of heart diseases, including high blood pressure, arteriosclerosis and heart failure. Such species are highly bioactive molecules and a short life due chiefly to reduction of molecular oxygen. The enzyme complex of NADPH oxidase is the main source of these reactive species in vascular system. Under physiological conditions, formation and elimination of these substances seem balanced in vascular wall. During redox Unbalance, nonetheless, there is increase in NADPH oxidase activity and predominance of pro-oxidizing agents, surpassing the anti-oxidant capacity of the organism self-defense. Besides this, such enzyme hyperactivity reduces the bioavailability of nitric oxide, capital for vasodilation and maintenance of normal vascular function. In spite of NADPH oxidase being directly connected to the endothelial dysfunction, it was firstly described as for its expression in phagocytes, where its activity determines efficiency of organism defense mechanisms against pathogens. Slight differences between structural units of NADPH oxidases, depending on the type of cell which expresses it, may create therapeutic implications, allowing to selectively inhibiting redox unbalance triggered by NADPH oxidase, without compromising, however, its participation in physiological cellular signaling which make sure protection against micro-organisms.Varios estudos destacam as especies reativas de oxigenio e nitrogenio (ERONs) como importantes contribuintes na patogenese de numerosas doencas cardiovasculares, incluindo hipertensao, aterosclerose e falencia cardiaca. Tais especies sao moleculas altamente bioativas e com vida curta derivadas, principalmente, da reducao do oxigenio molecular. O complexo enzimatico da NADPH oxidase e a maior fonte dessas especies reativas na vasculatura. Sob condicoes fisiologicas, a formacao e eliminacao destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanco redox, entretanto, ha um aumento na atividade da NADPH oxidase e predominio de agentes pro-oxidantes, superando a capacidade de defesa orgânica antioxidante. Alem disso, tal hiperatividade enzimatica reduz a biodisponibilidade do oxido nitrico, crucial para a vasodilatacao e a manutencao da funcao vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente a disfuncao endotelial, foi primeiramente descrita por sua expressao em fagocitos, onde sua atividade determina a eficacia dos mecanismos de defesa orgânica contra patogenos. As sutis diferencas existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicacoes terapeuticas, permitindo a inibicao seletiva do desequilibrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participacao nas vias fisiologicas de sinalizacao celular que garantem a protecao contra microorganismos.

Desbalanço redox: NADPH oxidase como um alvo terapêutico no manejo cardiovascular

Several studies refer to reactive oxygen and nitrogen species (RONS) as important agents in the pathogenesis of a number of heart diseases, including high blood pressure, arteriosclerosis and heart failure. Such species are highly bioactive molecules and a short life due chiefly to reduction of molecular oxygen. The enzyme complex of NADPH oxidase is the main source of these reactive species in vascular system. Under physiological conditions, formation and elimination of these substances seem balanced in vascular wall. During redox Unbalance, nonetheless, there is increase in NADPH oxidase activity and predominance of pro-oxidizing agents, surpassing the anti-oxidant capacity of the organism self-defense. Besides this, such enzyme hyperactivity reduces the bioavailability of nitric oxide, capital for vasodilation and maintenance of normal vascular function. In spite of NADPH oxidase being directly connected to the endothelial dysfunction, it was firstly described as for its expression in phagocytes, where its activity determines efficiency of organism defense mechanisms against pathogens. Slight differences between structural units of NADPH oxidases, depending on the type of cell which expresses it, may create therapeutic implications, allowing to selectively inhibiting redox unbalance triggered by NADPH oxidase, without compromising, however, its participation in physiological cellular signaling which make sure protection against micro-organisms.Varios estudos destacam as especies reativas de oxigenio e nitrogenio (ERONs) como importantes contribuintes na patogenese de numerosas doencas cardiovasculares, incluindo hipertensao, aterosclerose e falencia cardiaca. Tais especies sao moleculas altamente bioativas e com vida curta derivadas, principalmente, da reducao do oxigenio molecular. O complexo enzimatico da NADPH oxidase e a maior fonte dessas especies reativas na vasculatura. Sob condicoes fisiologicas, a formacao e eliminacao destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanco redox, entretanto, ha um aumento na atividade da NADPH oxidase e predominio de agentes pro-oxidantes, superando a capacidade de defesa orgânica antioxidante. Alem disso, tal hiperatividade enzimatica reduz a biodisponibilidade do oxido nitrico, crucial para a vasodilatacao e a manutencao da funcao vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente a disfuncao endotelial, foi primeiramente descrita por sua expressao em fagocitos, onde sua atividade determina a eficacia dos mecanismos de defesa orgânica contra patogenos. As sutis diferencas existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicacoes terapeuticas, permitindo a inibicao seletiva do desequilibrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participacao nas vias fisiologicas de sinalizacao celular que garantem a protecao contra microorganismos.

The ACE2/Angiotensin-(1–7)/MAS Axis of the Renin-Angiotensin System: Focus on Angiotensin-(1–7)

The renin-angiotensin system (RAS) is a key player in the control of the cardiovascular system and hydroelectrolyte balance, with an influence on organs and functions throughout the body. The classical view of this system saw it as a sequence of many enzymatic steps that culminate in the production of a single biologically active metabolite, the octapeptide angiotensin (ANG) II, by the angiotensin converting enzyme (ACE). The past two decades have revealed new functions for some of the intermediate products, beyond their roles as substrates along the classical route. They may be processed in alternative ways by enzymes such as the ACE homolog ACE2. One effect is to establish a second axis through ACE2/ANG-(1–7)/MAS, whose end point is the metabolite ANG-(1–7). ACE2 and other enzymes can form ANG-(1–7) directly or indirectly from either the decapeptide ANG I or from ANG II. In many cases, this second axis appears to counteract or modulate the effects of the classical axis. ANG-(1–7) itself acts on the receptor MAS to influence a range of mechanisms in the heart, kidney, brain, and other tissues. This review highlights the current knowledge about the roles of ANG-(1–7) in physiology and disease, with particular emphasis on the brain.

Ibuprofen arginate retains eNOS substrate activity and reverses endothelial dysfunction: implications for the COX-2/ADMA axis

Nonsteroidal antiinflammatory drugs, including ibuprofen, are among the most commonly used medications and produce their antiinflammatory effects by blocking cyclooxygenase (COX)‐2. Their use is associated with increased risk of heart attacks caused by blocking COX‐2 in the vasculature and/or kidney, with our recent work implicating the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), a cardiotoxic hormone whose effects can be prevented by L‐arginine. The ibuprofen salt ibuprofen arginate (Spididol) was created to increase solubility but we suggest that it could also augment the NO pathway through codelivery of arginine. Here we investigated the idea that ibuprofen arginate can act to simultaneously inhibit COX‐2 and preserve the NO pathway. Ibuprofen arginate functioned similarly to ibuprofen sodium for inhibition of mouse/human COX‐2, but only ibuprofen arginate served as a substrate for NOS. Ibuprofen arginate but not ibuprofen sodiumalso reversed the inhibitory effects of ADMA and NG‐nitro‐L‐argininemethyl ester on inducible NOS (macrophages) and endothelial NOS in vitro (aorta) and in vivo (blood pressure). These observations show that ibuprofen arginate provides, in one preparation, a COX‐2 inhibitor and NOS substrate that could act to negate the harmful cardiovascular consequences mediated by blocking renal COX‐2 and increased ADMA. While remarkably simple, our findings are potentially game‐changing in the nonsteroidal antiinflammatory drug arena.—Kirkby, N. S., Tesfai, A., Ahmetaj‐Shala, B., Gashaw, H. H., Sampaio, W., Etelvino, G., Leão, N.M., Santos, R.A., Mitchell, J.A. Ibuprofenarginate retains eNOS substrate activity and reverses endothelial dysfunction: implications for the COX‐2/ADMA axis. FASEB J. 30, 4172–4179 (2016). www.fasebj.org

Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPARβ/δ-Dependent Vasodilator PathwayNovelty and Significance

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2–dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPAR&bgr;/&dgr; (peroxisome proliferator-activated receptor-&bgr;/&dgr;). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPAR&bgr;/&dgr;-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPAR&bgr;/&dgr; axis as a therapeutic target in renal disease.