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A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension but remains elusive in humans. While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (superoxide, hydrogen peroxide, and peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly. In this review, we summarize recent advances in delineating the primary and secondary sources of reactive oxygen species (nicotinamide adenine dinucleotide phosphate oxidases, uncoupled endothelial nitric oxide synthase, endoplasmic reticulum, and mitochondria), the posttranslational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension. We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins, and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension. Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies.
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Hipertensão/etiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Humanos , Hipertensão/metabolismo , Inflamassomos/fisiologia , Rim/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredução , Transdução de Sinais/fisiologia , Superóxidos/metabolismo , Doenças Vasculares/metabolismoRESUMO
OBJECTIVE: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR-TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. CONCLUSIONS: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.
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Fator de Crescimento Epidérmico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Magnésio/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese , Músculo Liso Vascular/crescimento & desenvolvimento , Fosforilação , Cultura Primária de Células , Ratos Endogâmicos WKYRESUMO
NEW FINDINGS: What is the topic of this review? This review provides a comprehensive overview of Nox5 from basic biology to human disease and highlights unique features of this Nox isoform What advances does it highlight? Major advances in Nox5 biology relate to crystallization of the molecule and new insights into the pathophysiological role of Nox5. Recent discoveries have unravelled the crystal structure of Nox5, the first Nox isoform to be crystalized. This provides new opportunities to develop drugs or small molecules targeted to Nox5 in an isoform-specific manner, possibly for therapeutic use. Moreover genome wide association studies (GWAS) identified Nox5 as a new blood pressure-associated gene and studies in mice expressing human Nox5 in a cell-specific manner have provided new information about the (patho) physiological role of Nox5 in the cardiovascular system and kidneys. Nox5 seems to be important in the regulation of vascular contraction and kidney function. In cardiovascular disease and diabetic nephropathy, Nox5 activity is increased and this is associated with increased production of reactive oxygen species and oxidative stress implicated in tissue damage. ABSTRACT: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), comprise seven family members (Nox1-Nox5 and dual oxidase 1 and 2) and are major producers of reactive oxygen species in mammalian cells. Reactive oxygen species are crucially involved in cell signalling and function. All Noxs share structural homology comprising six transmembrane domains with two haem-binding regions and an NADPH-binding region on the intracellular C-terminus, whereas their regulatory systems, mechanisms of activation and tissue distribution differ. This explains the diverse function of Noxs. Of the Noxs, NOX5 is unique in that rodents lack the gene, it is regulated by Ca2+ , it does not require NADPH oxidase subunits for its activation, and it is not glycosylated. NOX5 localizes in the perinuclear and endoplasmic reticulum regions of cells and traffics to the cell membrane upon activation. It is tightly regulated through numerous post-translational modifications and is activated by vasoactive agents, growth factors and pro-inflammatory cytokines. The exact pathophysiological significance of NOX5 remains unclear, but it seems to be important in the physiological regulation of sperm motility, vascular contraction and lymphocyte differentiation, and NOX5 hyperactivation has been implicated in cardiovascular disease, kidney injury and cancer. The field of NOX5 biology is still in its infancy, but with new insights into its biochemistry and cellular regulation, discovery of the NOX5 crystal structure and genome-wide association studies implicating NOX5 in disease, the time is now ripe to advance NOX5 research. This review provides a comprehensive overview of our current understanding of NOX5, from basic biology to human disease, and highlights the unique characteristics of this enigmatic Nox isoform.
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NADPH Oxidase 5/genética , Animais , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas , NADPH Oxidase 5/biossíntese , NADPH Oxidase 5/metabolismo , Bombas de Próton , Espécies Reativas de OxigênioRESUMO
Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.
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Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Acetilcisteína/farmacologia , Animais , Células Cultivadas , Sequestradores de Radicais Livres/farmacologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/metabolismo , Oxirredução , Fosforilação , Proteína Fosfatase 1/efeitos dos fármacos , Pirazóis/farmacologia , Pirazolonas , Piridinas/farmacologia , Piridonas , Ratos , Ratos Endogâmicos WKY , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/genética , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/genética , Vasodilatadores/farmacologiaAssuntos
Microbioma Gastrointestinal , Hipertensão , Angiotensina II , Encéfalo , Humanos , MicrogliaRESUMO
OBJECTIVE: intra-articular co-injection of kaolin with carrageenan (CGN) in rodents is widely used as an experimental model of arthritis. However, the ability of kaolin to cause arthritis and related immune responses when administered alone is unclear. We evaluated the contribution of prostanoids and sensory C-fibres (and their neuropeptide substance P) to kaolin-induced inflammation in the rat knee. METHODS: Wistar rats, 8-10 weeks old, received an intra-articular injection of kaolin (1-10 µg/joint) or saline into the knee joint. Knee inflammation, proinflammatory cytokines, pain behaviour and secondary tactile allodynia were assessed over 5 h, when synovial leukocyte counts, histopathological changes and proinflammatory cytokine levels were evaluated. RESULTS: The intra-articular injection of kaolin caused a dose- and time-dependent knee swelling and impairment of motion that were associated with secondary tactile allodynia, elevated concentrations of IL-1ß, IL-6 and TNFα, leukocyte infiltration, and histopathological changes in the ipsilateral hindpaw. The neurokinin-1 (NK1) receptor antagonist SR140333 or neonatal treatment with capsaicin markedly reduced the inflammatory parameters, cytokines and allodynia but failed to significantly inhibit the impaired motion. The cyclo-oxygenase inhibitor indomethacin partially inhibited knee oedema and allodynia but did not affect the leukocyte influx, myeloperoxidase activity or impaired motion in the kaolin-injected rat. CONCLUSIONS: We show the first evidence that intra-articular injection of kaolin without CGN produced severe acute monoarthritis. This was highly dependent on substance P (released from C-fibres) and NK1 receptor activation, which stimulated local production of proinflammatory cytokines. This model may be of critical importance for mechanistic studies and screening new anti-inflammatory/analgesic drugs.
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Antidiarreicos/toxicidade , Artrite/induzido quimicamente , Caulim/toxicidade , Receptores da Neurocinina-1/metabolismo , Animais , Animais Recém-Nascidos , Artrite/complicações , Artrite/tratamento farmacológico , Capsaicina/toxicidade , Citocinas/metabolismo , Modelos Animais de Doenças , Edema/etiologia , Inibidores Enzimáticos/uso terapêutico , Hiperalgesia/etiologia , Indometacina/uso terapêutico , Articulação do Joelho/patologia , Masculino , Medição da Dor , Peroxidase/metabolismo , Piperidinas/uso terapêutico , Quinuclidinas/uso terapêutico , Ratos , Ratos Wistar , Líquido Sinovial/metabolismoRESUMO
Hypertension is a leading risk factor for stroke, heart disease and chronic kidney disease. Multiple interacting factors and organ systems increase blood pressure and cause target-organ damage. Among the many molecular elements involved in the development of hypertension are reactive oxygen species (ROS), which influence cellular processes in systems that contribute to blood pressure elevation (such as the cardiovascular, renal, immune and central nervous systems, or the renin-angiotensin-aldosterone system). Dysregulated ROS production (oxidative stress) is a hallmark of hypertension in humans and experimental models. Of the many ROS-generating enzymes, NADPH oxidases are the most important in the development of hypertension. At the cellular level, ROS influence signalling pathways that define cell fate and function. Oxidative stress promotes aberrant redox signalling and cell injury, causing endothelial dysfunction, vascular damage, cardiovascular remodelling, inflammation and renal injury, which are all important in both the causes and consequences of hypertension. ROS scavengers reduce blood pressure in almost all experimental models of hypertension; however, clinical trials of antioxidants have yielded mixed results. In this Review, we highlight the latest advances in the understanding of the role and the clinical implications of ROS in hypertension. We focus on cellular sources of ROS, molecular mechanisms of oxidative stress and alterations in redox signalling in organ systems, and their contributions to hypertension.
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Inflammatory responses in small vessels play an important role in the development of cardiovascular diseases, including hypertension, stroke, and small vessel disease. This involves various complex molecular processes including oxidative stress, inflammasome activation, immune-mediated responses, and protein misfolding, which together contribute to microvascular damage. In addition, epigenetic factors, including DNA methylation, histone modifications, and microRNAs influence vascular inflammation and injury. These phenomena may be acquired during the aging process or due to environmental factors. Activation of proinflammatory signaling pathways and molecular events induce low-grade and chronic inflammation with consequent cardiovascular damage. Identifying mechanism-specific targets might provide opportunities in the development of novel therapeutic approaches. Monoclonal antibodies targeting inflammatory cytokines and epigenetic drugs, show promise in reducing microvascular inflammation and associated cardiovascular diseases. In this article, we provide a comprehensive discussion of the complex mechanisms underlying microvascular inflammation and offer insights into innovative therapeutic strategies that may ameliorate vascular injury in cardiovascular disease.
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Inflamação , Animais , Humanos , Artérias/metabolismo , Doenças Cardiovasculares/metabolismo , Epigênese Genética , Inflamação/metabolismo , Inflamação/imunologia , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Vasculite/metabolismo , Vasculite/imunologiaRESUMO
Hypertension is the primary cause of cardiovascular diseases and is responsible for nearly 9 million deaths worldwide annually. Increasing evidence indicates that in addition to pathophysiologic processes, numerous environmental factors, such as geographic location, lifestyle choices, socioeconomic status, and cultural practices, influence the risk, progression, and severity of hypertension, even in the absence of genetic risk factors. In this review, we discuss the impact of some environmental determinants on hypertension. We focus on clinical data from large population studies and discuss some potential molecular and cellular mechanisms. We highlight how these environmental determinants are interconnected, as small changes in one factor might affect others, and further affect cardiovascular health. In addition, we discuss the crucial impact of socioeconomic factors and how these determinants influence diverse communities with economic disparities. Finally, we address opportunities and challenges for new research to address gaps in knowledge on understanding molecular mechanisms whereby environmental factors influence development of hypertension and associated cardiovascular disease.
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Doenças Cardiovasculares , Hipertensão , Humanos , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Hipertensão/epidemiologia , Hipertensão/etiologia , Estilo de Vida , Mediastino , Fatores de RiscoRESUMO
Under physiologic conditions, reactive oxygen species (ROS) function as signalling molecules that control cell function. However, in pathologic conditions, increased generation of ROS triggers oxidative stress, which plays a role in vascular changes associated with hypertension, including endothelial dysfunction, vascular reactivity, and arterial remodelling (termed the vasculopathy of hypertension). The major source of ROS in the vascular system is NADPH oxidase (NOX). Increased NOX activity drives vascular oxidative stress in hypertension. Molecular mechanisms underlying vascular damage in hypertension include activation of redox-sensitive signalling pathways, post-translational modification of proteins, and oxidative damage of DNA and cytoplasmic proteins. In addition, oxidative stress leads to accumulation of proteins in the endoplasmic reticulum (ER) (termed ER stress), with consequent activation of the unfolded protein response (UPR). ER stress is emerging as a potential player in hypertension as abnormal protein folding in the ER leads to oxidative stress and dysregulated activation of the UPR promotes inflammation and injury in vascular and cardiac cells. In addition, the ER engages in crosstalk with exogenous sources of ROS, such as mitochondria and NOX, which can amplify redox processes. Here we provide an update of the role of ROS and NOX in hypertension and discuss novel concepts on the interplay between oxidative stress and ER stress.
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Hipertensão , Estresse Oxidativo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Estresse do Retículo Endoplasmático/genética , OxirreduçãoRESUMO
The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels that function as cellular sensors for a wide range of physical, chemical, and environmental stimuli. According to sequence homology, TRP channels are categorized into 6 subfamilies: TRP canonical, TRP vanilloid, TRP melastatin, TRP ankyrin, TRP mucolipin, and TRP polycystin. They are widely expressed in different cell types and tissues and have essential roles in various physiological and pathological processes by regulating the concentration of ions (Ca2+, Mg2+, Na+, and K+) and influencing intracellular signalling pathways. Human data and experimental models indicate the importance of TRP channels in vascular homeostasis and hypertension. Furthermore, TRP channels have emerged as key players in oxidative stress and inflammation, important in the pathophysiology of cardiovascular diseases, including hypertension. In this review, we present an overview of the TRP channels with a focus on their role in hypertension. In particular, we highlight mechanisms activated by TRP channels in vascular smooth muscle and endothelial cells and discuss their contribution to processes underlying vascular dysfunction in hypertension.
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Hipertensão , Canais de Potencial de Receptor Transitório , Humanos , Células Endoteliais/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Músculo Liso Vascular/metabolismo , Íons/metabolismoRESUMO
COVID-19, caused by SARS-CoV-2, is a respiratory disease associated with inflammation and endotheliitis. Mechanisms underling inflammatory processes are unclear, but angiotensin converting enzyme 2 (ACE2), the receptor which binds the spike protein of SARS-CoV-2 may be important. Here we investigated whether spike protein binding to ACE2 induces inflammation in endothelial cells and determined the role of ACE2 in this process. Human endothelial cells were exposed to SARS-CoV-2 spike protein, S1 subunit (rS1p) and pro-inflammatory signaling and inflammatory mediators assessed. ACE2 was modulated pharmacologically and by siRNA. Endothelial cells were also exposed to SARS-CoV-2. rSP1 increased production of IL-6, MCP-1, ICAM-1 and PAI-1, and induced NFkB activation via ACE2 in endothelial cells. rS1p increased microparticle formation, a functional marker of endothelial injury. ACE2 interacting proteins involved in inflammation and RNA biology were identified in rS1p-treated cells. Neither ACE2 expression nor ACE2 enzymatic function were affected by rSP1. Endothelial cells exposed to SARS-CoV-2 virus did not exhibit viral replication. We demonstrate that rSP1 induces endothelial inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication. We define a novel role for ACE2 in COVID-19- associated endotheliitis.
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COVID-19 , Células Endoteliais , Humanos , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2 , SARS-CoV-2 , Inflamação , Replicação Viral , RNA de Cadeia DuplaRESUMO
Microcirculation is pervasive and orchestrates a profound regulatory cross-talk with the surrounding tissue and organs. Similarly, it is one of the earliest biological systems targeted by environmental stressors and consequently involved in the development and progression of ageing and age-related disease. Microvascular dysfunction, if not targeted, leads to a steady derangement of the phenotype, which cumulates comorbidities and eventually results in a nonrescuable, very high-cardiovascular risk. Along the broad spectrum of pathologies, both shared and distinct molecular pathways and pathophysiological alteration are involved in the disruption of microvascular homeostasis, all pointing to microvascular inflammation as the putative primary culprit. This position paper explores the presence and the detrimental contribution of microvascular inflammation across the whole spectrum of chronic age-related diseases, which characterise the 21st-century healthcare landscape. The manuscript aims to strongly affirm the centrality of microvascular inflammation by recapitulating the current evidence and providing a clear synoptic view of the whole cardiometabolic derangement. Indeed, there is an urgent need for further mechanistic exploration to identify clear, very early or disease-specific molecular targets to provide an effective therapeutic strategy against the otherwise unstoppable rising prevalence of age-related diseases.
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Artérias , Inflamação , Humanos , Doença Crônica , MicrocirculaçãoRESUMO
AIMS: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSION: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.
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Hipertensão , Músculo Liso Vascular , Actinas/metabolismo , Angiotensina II/metabolismo , Animais , Células Cultivadas , Humanos , Meliteno/metabolismo , Meliteno/farmacologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , NADPH Oxidase 5/farmacologia , Oxirredução , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hyperaldosteronism causes cardiovascular disease as well as hypomagnesemia. Mechanisms are ill-defined but dysregulation of TRPM7, a Mg2+-permeable channel/α-kinase, may be important. We examined the role of TRPM7 in aldosterone-dependent cardiovascular and renal injury by studying aldosterone-salt treated TRPM7-deficient (TRPM7+/Δkinase) mice. Plasma/tissue [Mg2+] and TRPM7 phosphorylation were reduced in vehicle-treated TRPM7+/Δkinase mice, effects recapitulated in aldosterone-salt-treated wild-type mice. Aldosterone-salt treatment exaggerated vascular dysfunction and amplified cardiovascular and renal fibrosis, with associated increased blood pressure in TRPM7+/Δkinase mice. Tissue expression of Mg2+-regulated phosphatases (PPM1A, PTEN) was downregulated and phosphorylation of Smad3, ERK1/2, and Stat1 was upregulated in aldosterone-salt TRPM7-deficient mice. Aldosterone-induced phosphorylation of pro-fibrotic signaling was increased in TRPM7+/Δkinase fibroblasts, effects ameliorated by Mg2+ supplementation. TRPM7 deficiency amplifies aldosterone-salt-induced cardiovascular remodeling and damage. We identify TRPM7 downregulation and associated hypomagnesemia as putative molecular mechanisms underlying deleterious cardiovascular and renal effects of hyperaldosteronism.
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Hiperaldosteronismo , Canais de Cátion TRPM , Aldosterona/farmacologia , Animais , Fibrose , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Rim/metabolismo , Magnésio/metabolismo , Camundongos , Proteína Fosfatase 2C/metabolismo , Cloreto de Sódio , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Inflammatory arthritis, such as rheumatoid arthritis (RA), stands out as one of the main sources of pain and impairment to the quality of life. The use of hemopressin (PVNFKFLSH; Hp), an inverse agonist of type 1 cannabinoid receptor, has proven to be effective in producing analgesia in pain models, but its effect on neuro-inflammatory aspects of RA is limited. In this study, antigen-induced arthritis (AIA) was evoked by the intraarticular (i.art.) injection of methylated bovine serum albumin (mBSA) in male Sprague Dawley rats. Phosphate buffered saline (PBS)-injected ipsilateral knee joints or AIA contralateral were used as control. Nociceptive and inflammatory parameters such as knee joint oedema and leukocyte influx and histopathological changes were carried out in addition to the local measurement of interleukins (IL) IL-6, IL-1ß, tumor necrosis factor-α and the immunoreactivity of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord (lumbar L3-5 segments) of AIA rats. For 4 days, AIA rats were treated daily with a single administration of saline, Hp injected (10 or 20 µg/day, i.art.), Hp given orally (20 µg/Kg, p.o.) or indomethacin (Indo; 5 mg/Kg, i.p.). In comparison to the PBS control group, the induction of AIA produced a significant and progressive mono-arthritis condition. The degree of AIA severity progressively compromised the normal walking pattern and impaired mobility over the next four days in relation to PBS-injected rats or contralateral knee joints. In AIA rats, the reduction of the distance between footprints and disturbances of gait evidenced signs of nociception. This response worsened at day 4, and a loss of footprint from the ipsilateral hind paw was evident. Daily treatment of the animals with Hp either i.art. (10 and 20 µg/knee) or p.o. (20 µg/Kg) as well as Indo (5 mg/Kg, i.p.) ameliorated the impaired mobility in a time-dependent manner (P < 0.05). In parallel, the AIA-injected ipsilateral knee joints reach a peak of swelling 24 h after AIA induction, which persisted over the next four days in relation to PBS-injected rats or contralateral knee joints. There was a significant but not dose-dependent inhibitory effect produced by all dosages and routes of Hp treatments on AIA-induced knee joint swelling (P < 0.05). In addition, the increased synovial levels of MPO activity, total leukocytes number and IL-6, but not IL-1ß, were significantly reduced by the lower i.art. dose of Hp. In conclusion, these results successfully demonstrate that Hp may represent a novel therapeutic strategy to treat RA, an effect which is unrelated to the proinflammatory actions of the neuropeptides CGRP and SP.
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Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Hemoglobinas/farmacologia , Dor Nociceptiva/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Citocinas/metabolismo , Edema/tratamento farmacológico , Marcha/efeitos dos fármacos , Hemoglobinas/administração & dosagem , Inflamação/tratamento farmacológico , Injeções Intra-Articulares , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Leucócitos/efeitos dos fármacos , Masculino , Fragmentos de Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Substância P/metabolismoRESUMO
Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Whether cholesterol-rich microdomains (lipid rafts/caveolae) are involved in these processes is unclear. Here we examined the sub-cellular compartmentalization of Nox isoforms in lipid rafts/caveolae and assessed the role of these microdomains in VSMC ROS production and pro-contractile and growth signaling. Intact small arteries and primary VSMCs from humans were studied. Vessels from Cav-1-/- mice were used to test proof of concept. Human VSMCs express Nox1, Nox4, Nox5 and Cav-1. Cell fractionation studies showed that Nox1 and Nox5 but not Nox4, localize in cholesterol-rich fractions in VSMCs. Angiotensin II (Ang II) stimulation induced trafficking into and out of lipid rafts/caveolae for Nox1 and Nox5 respectively. Co-immunoprecipitation studies showed interactions between Cav-1/Nox1 but not Cav-1/Nox5. Lipid raft/caveolae disruptors (methyl-ß-cyclodextrin (MCD) and Nystatin) and Ang II stimulation variably increased O2- generation and phosphorylation of MLC20, Ezrin-Radixin-Moesin (ERM) and p53 but not ERK1/2, effects recapitulated in Cav-1 silenced (siRNA) VSMCs. Nox inhibition prevented Ang II-induced phosphorylation of signaling molecules, specifically, ERK1/2 phosphorylation was attenuated by mellitin (Nox5 inhibitor) and Nox5 siRNA, while p53 phosphorylation was inhibited by NoxA1ds (Nox1 inhibitor). Ang II increased oxidation of DJ1, dual anti-oxidant and signaling molecule, through lipid raft/caveolae-dependent processes. Vessels from Cav-1-/- mice exhibited increased O2- generation and phosphorylation of ERM. We identify an important role for lipid rafts/caveolae that act as signaling platforms for Nox1 and Nox5 but not Nox4, in human VSMCs. Disruption of these microdomains promotes oxidative stress and Nox isoform-specific redox signalling important in vascular dysfunction associated with cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/metabolismo , Caveolina 1/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Músculo Liso Vascular/metabolismo , NADPH Oxidase 1/metabolismo , NADPH Oxidase 5/metabolismo , Animais , Doenças Cardiovasculares/patologia , Caveolina 1/genética , Humanos , Camundongos , Camundongos Knockout , Músculo Liso Vascular/patologia , Oxirredução , Estresse Oxidativo , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
The etiology of hypertension involves complex interactions among genetic, environmental, and pathophysiologic factors that influence many regulatory systems. Hypertension is characteristically associated with vascular dysfunction, cardiovascular remodelling, renal dysfunction, and stimulation of the sympathetic nervous system. Emerging evidence indicates that the immune system is also important and that activated immune cells migrate and accumulate in tissues promoting inflammation, fibrosis, and target-organ damage. Common to these processes is oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the oxidants that leads to a disruption of oxidation-reduction (redox) signalling and control and molecular damage. Physiologically, reactive oxygen species (ROS) act as signalling molecules and influence cell function through highly regulated redox-sensitive signal transduction. In hypertension, oxidative stress promotes posttranslational modification (oxidation and phosphorylation) of proteins and aberrant signalling with consequent cell and tissue damage. Many enzymatic systems generate ROS, but NADPH oxidases (Nox) are the major sources in cells of the heart, vessels, kidneys, and immune system. Expression and activity of Nox are increased in hypertension and are the major systems responsible for oxidative stress in cardiovascular disease. Here we provide a unifying concept where oxidative stress is a common mediator underlying pathophysiologic processes in hypertension. We focus on some novel concepts whereby ROS influence vascular function, aldosterone/mineralocorticoid actions, and immunoinflammation, all important processes contributing to the development of hypertension.
Assuntos
Hipertensão/fisiopatologia , Estresse Oxidativo/fisiologia , Aldosterona/metabolismo , Angiotensina II/metabolismo , Endotelina-1/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Fatores Sexuais , Transdução de Sinais/fisiologia , Remodelação Vascular/fisiologia , Rigidez Vascular/fisiologia , Vasoconstrição/fisiologiaRESUMO
AIMS: Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis. METHODS AND RESULTS: TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFß) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFß in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment. CONCLUSIONS: We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.
Assuntos
Cardiomegalia/metabolismo , Cardiomiopatias/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Miocárdio/metabolismo , Canais de Cátion TRPM/metabolismo , Remodelação Ventricular , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Migração e Rolagem de Leucócitos , Macrófagos/metabolismo , Macrófagos/patologia , Magnésio/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Transdução de Sinais , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Migração Transendotelial e TransepitelialRESUMO
SIGNIFICANCE: NADPH oxidases (Noxs), of which there are seven isoforms (Nox1-5, Duox1/Duox2), are professional oxidases functioning as reactive oxygen species (ROS)-generating enzymes. ROS are signaling molecules important in physiological processes. Increased ROS production and altered redox signaling in the vascular system have been implicated in the pathophysiology of cardiovascular diseases, including hypertension, and have been attributed, in part, to increased Nox activity. Recent Advances: Nox1, Nox2, Nox4, and Nox5 are expressed and functionally active in human vascular cells. While Nox1, Nox2, and Nox4 have been well characterized in models of cardiovascular disease, little is known about Nox5. This may relate to the lack of experimental models because rodents lack NOX5. However, recent studies have advanced the field by (i) elucidating mechanisms of Nox5 regulation, (ii) identifying Nox5 variants, (iii) characterizing Nox5 expression, and (iv) discovering the Nox5 crystal structure. Moreover, studies in human Nox5-expressing mice have highlighted a putative role for Nox5 in cardiovascular disease. CRITICAL ISSUES: Although growing evidence indicates a role for Nox-derived ROS in cardiovascular (patho)physiology, the exact function of each isoform remains unclear. This is especially true for Nox5. FUTURE DIRECTIONS: Future directions should focus on clinically relevant studies to discover the functional significance of Noxs, and Nox5 in particular, in human health and disease. Two important recent studies will impact future directions. First, Nox5 is the first Nox to be crystallized. Second, a genome-wide association study identified Nox5 as a novel blood pressure-associated gene. These discoveries, together with advancements in Nox5 biology and biochemistry, will facilitate discovery of drugs that selectively target Noxs to interfere in uncontrolled ROS generation.