RESUMO
Circulating extracellular vesicles (EVs) regulate signaling pathways via receptor-ligand interactions and content delivery, after attachment or internalization by endothelial cells. However, they originate from diverse cell populations and are heterogeneous in composition. To determine the effects of specific surface molecules, the use of synthetic EV mimetics permits the study of specific EV receptor-ligand interactions. Here, we used endogenous EVs derived from the circulation of rats, as well as ligand-decorated synthetic microparticles (MPs) to examine the role of integrin αvß3 in platelet adhesion under flow in structurally intact cerebral arteries. At an intraluminal pressure of 50 mmHg and flow rate of 10 µl/min, platelets were delivered to the artery lumen and imaged with whole-field fluorescent microscopy. Under basal conditions very few platelets bound to the endothelium. However, adhesion events were markedly increased following the introduction of arginine-glycine-aspartate (RGD)-labelled synthetic MPs or endogenously-derived EVs from experimental stroke animals carrying excess RGD proteins, including vitronectin, CD40-ligand and thrombospondin-1. These data, which were generated in a dynamic and physiologically relevant system, demonstrate the importance of vesicle-carried RGD ligands in platelet adherence to the cerebrovascular endothelium and highlight the ability of synthetic EVs to isolate and identify key components of the molecular handshake between EVs and their targets.
Assuntos
Plaquetas/metabolismo , Artérias Cerebrais/metabolismo , Circulação Cerebrovascular , Vesículas Extracelulares/metabolismo , Integrina alfaVbeta3/metabolismo , Microvasos/metabolismo , Adesividade Plaquetária , Acidente Vascular Cerebral/metabolismo , Animais , Plaquetas/patologia , Artérias Cerebrais/patologia , Modelos Animais de Doenças , Vesículas Extracelulares/patologia , Camundongos , Microvasos/patologia , Acidente Vascular Cerebral/patologiaRESUMO
The nonselective ß-adrenergic receptor antagonist (ß-blocker) carvedilol has been shown to protect against myocardial injury, but the detailed underlying mechanisms are unclear. We recently reported that carvedilol stimulates the processing of microRNA (miR)-199a-3p and miR-214 in the heart via ß-arrestin1-biased ß1-adrenergic receptor (ß1AR) cardioprotective signaling. Here, we investigate whether these ß-arrestin1/ß1AR-responsive miRs mediate the beneficial effects of carvedilol against simulated ischemia/reperfusion (sI/R). Using cultured cardiomyocyte cell lines and primary cardiomyocytes, we demonstrate that carvedilol upregulates miR-199a-3p and miR-214 in both ventricular and atrial cardiomyocytes subjected to sI/R. Overexpression of the two miRs in cardiomyocytes mimics the effects of carvedilol to activate p-AKT survival signaling and the expression of a downstream pluripotency marker Sox2 in response to sI/R. Moreover, carvedilol-mediated p-AKT activation is abolished by knockdown of either miR-199a-3p or miR-214. Along with previous studies to directly link the cardioprotective actions of carvedilol to upregulation of p-AKT/stem cell markers, our findings suggest that the protective roles of carvedilol during ischemic injury are in part attributed to activation of these two protective miRs. Loss of function of miR-199a-3p and miR-214 also increases cardiomyocyte apoptosis after sI/R. Mechanistically, we demonstrate that miR-199a-3p and miR-214 repress the predictive or known apoptotic target genes ddit4 and ing4, respectively, in cardiomyocytes. These findings suggest pivotal roles for miR-199a-3p and miR-214 as regulators of cardiomyocyte survival and contributors to the functional benefits of carvedilol therapy.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Carbazóis/farmacologia , MicroRNAs/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Propanolaminas/farmacologia , Traumatismo por Reperfusão/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carvedilol , Linhagem Celular , Células Cultivadas , Simulação por Computador , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert "sponge-like" effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.
Assuntos
Doença/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Progressão da Doença , Regulação da Expressão Gênica , HumanosRESUMO
Cardiovascular disease is the leading cause of death in the United States, accounting for nearly one in every seven deaths. Over the last decade, various targeted therapeutics have been introduced, but there has been no corresponding improvement in patient survival. Since the mortality rate of cardiovascular disease has not been significantly decreased, efforts have been made to understand the link between heart disease and novel therapeutic targets such as non-coding RNAs. Among multiple non-coding RNAs, long non-coding RNA (lncRNA) has emerged as a novel therapeutic in cardiovascular medicine. LncRNAs are endogenous RNAs that contain over 200 nucleotides and regulate gene expression. Recent studies suggest critical roles of lncRNAs in modulating the initiation and progression of cardiovascular diseases. For example, aberrant lncRNA expression has been associated with the pathogenesis of ischemic heart failure. In this article, we present a synopsis of recent discoveries that link the roles and molecular interactions of lncRNAs to cardiovascular diseases. Moreover, we describe the prevalence of circulating lncRNAs and assess their potential utilities as biomarkers for diagnosis and prognosis of heart disease.
Assuntos
Doenças Cardiovasculares/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Biomarcadores , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Progressão da Doença , Humanos , PrognósticoRESUMO
Chronic treatment with the ß-blocker carvedilol has been shown to reduce established maladaptive left ventricle (LV) hypertrophy and to improve LV function in experimental heart failure. However, the detailed mechanisms by which carvedilol improves LV failure are incompletely understood. We previously showed that carvedilol is a ß-arrestin-biased ß1-adrenergic receptor ligand, which activates cellular pathways in the heart independent of G protein-mediated second messenger signaling. More recently, we have demonstrated by microRNA (miR) microarray analysis that carvedilol upregulates a subset of mature and pre-mature miRs, but not their primary miR transcripts in mouse hearts. Here, we next sought to identify the effects of carvedilol on LV gene expression on a genome-wide basis. Adult mice were treated with carvedilol or vehicle for 1 wk. RNA was isolated from LV tissue and hybridized for microarray analysis. Gene expression profiling analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes into pathways involved in tight junction, malaria, viral myocarditis, glycosaminoglycan biosynthesis, and arrhythmogenic right ventricular cardiomyopathy. Genes encoding proteins in the tight junction, malaria, and viral myocarditis pathways were upregulated in the LV by carvedilol, while genes encoding proteins in the glycosaminoglycan biosynthesis and arrhythmogenic right ventricular cardiomyopathy pathways were downregulated by carvedilol. These gene expression changes may reflect the molecular mechanisms that underlie the functional benefits of carvedilol therapy.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Carbazóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Propanolaminas/farmacologia , Animais , Cardiomiopatias/genética , Carvedilol , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/genética , Malária/genética , Camundongos Endogâmicos C57BL , Miocardite/genética , Proteínas/genética , Proteínas/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
AIMS: Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the ß-arrestin-biased ß-blocker, carvedilol that ß-arrestin1-biased ß1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown. METHODS AND RESULTS: Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes. CONCLUSION: These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.
Assuntos
MicroRNAs/genética , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular , Animais , Animais Recém-Nascidos , Apoptose , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Interferência de RNA , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
It is believed that obesity has detrimental effects on the coronary circulation. These include immediate changes in coronary arterial vasomotor responsiveness and the development of occlusive large coronary artery disease. Despite its critical role in regulating myocardial perfusion, the altered behavior of coronary resistance arteries, which gives rise to coronary microvascular disease (CMD) is poorly understood in obesity. A chronic, low-grade vascular inflammation has been long considered as one of the main underlying pathology behind CMD. The expanded adipose tissue and the infiltrating macrophages are the major sources of pro-inflammatory mediators that have been implicated in causing inadequate myocardial perfusion and, in a long term, development of heart failure in obese patients. Much less is known the mechanisms regulating the release of these cytokines into the circulation that enable them to exert their remote effects in the coronary microcirculation. This mini review aims to examine recent studies describing alterations in the vasomotor function of coronary resistance arteries and the role of adipose tissue-derived pro-inflammatory cytokines and adipokines in contributing to CMD in obesity. We provide examples of regulatory mechanisms by which adipokines are released from adipose tissue to exert their remote inflammatory effects on coronary microvessels. We identify some of the important challenges and opportunities going forward.
Assuntos
Adipocinas/imunologia , Tecido Adiposo/imunologia , Doença das Coronárias/etiologia , Citocinas/imunologia , Microvasos/imunologia , Obesidade/complicações , Adipocinas/sangue , Circulação Coronária/imunologia , Circulação Coronária/fisiologia , Doença das Coronárias/imunologia , Doença das Coronárias/patologia , Citocinas/sangue , Humanos , Microcirculação/imunologia , Microcirculação/fisiologia , Microvasos/patologia , Obesidade/imunologia , Obesidade/patologiaRESUMO
Conducted vasodilation is essential to coordinate vascular resistance along distances to ensure adequate tissue perfusion. We hypothesized that conducted vasodilation of coronary resistance arteries declines with age. Coronary arterioles were dissected from right atrial appendage of patients (n = 27) undergoing cardiac surgery. Arterioles (~100 µm) were cannulated and pressurized (80 mmHg), and developed spontaneous myogenic tone. Conducted vasodilation was initiated by locally administering the endothelium-dependent agonist bradykinin (BK; 100 µM) ejected from a glass micropipette (~3 µm tip opening, positioned in close proximity to the vessel wall). Diameter changes were measured at local and upstream sites (500 and 1,000 µm from the stimulus) with videomicroscopy. Local administration of BK elicited vasodilation, the magnitude of which increased with the duration of stimulus (69 ± 6, 81 ± 6, 90 ± 2%, after 1, 3, and 5 × 100 ms, respectively). BK-induced dilation remained substantial at upstream sites (500 µm: 53 ± 7%; 1,000 µm: 46 ± 9%). The gap junction uncoupler carbenoxolone or 18-α-glycyrrhetinic acid did not affect local responses, but diminished conducted vasodilation. Inhibitors of small/intermediate conductance calcium-activated potassium channels (SKCa/IKCa), apamin and TRAM34, reduced dilations both at local and remote sites. We found that conducted dilation, but not the local response, was significantly reduced in older (≥64 yr) patients. The nitric oxide (NO) synthesis inhibitor N(ω)-nitro-l-arginine methyl ester did not affect local responses, but markedly reduced conducted dilation in younger (<64 yr) individuals. Collectively, we show that human coronary arterioles exhibit SKCa/IKCa-mediated hyperpolarization spread through gap junctions, which contributes to conducted vasodilation initiated by focal application of BK. We demonstrate that conducted dilation declines with age, likely due to reduced NO availability, which plays a permissive role in propagating longitudinal vasomotor signaling.
Assuntos
Envelhecimento/fisiologia , Arteríolas/fisiopatologia , Vasos Coronários/fisiopatologia , Canais de Potássio Cálcio-Ativados/fisiologia , Vasodilatação/fisiologia , Idoso , Idoso de 80 Anos ou mais , Arteríolas/efeitos dos fármacos , Bradicinina/farmacologia , Vasos Coronários/efeitos dos fármacos , Feminino , Humanos , Masculino , Microscopia de Vídeo , Pessoa de Meia-Idade , Miografia , Óxido Nítrico/metabolismo , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
The antioxidant and reactive-oxygen-species-scavenging activity of stobadine has been demonstrated in previous studies. Recently, chemical modification of this leading structure led to the synthesis of other pyridoindole derivatives with significantly increased intrinsic antioxidant efficacy. Further structural modifications of stobadine provided the opportunity to increase bioavailability and attenuate unwanted side effects, such as α-adrenolytic activity. The aim of the work was to evaluate the direct effect of a novel pyridoindole, SMe1EC2, on the vascular wall ex vivo. The vasomotor effect of SMe1EC2 (1×10(-8)-1×10(-4) mol/l) was measured on isolated and pressurized rat cerebral and coronary arterioles using video-microscopy. The effect of SMe1EC2 (1×10(-6) and 1×10(-5) mol/l) on high potassium-, phenylephrine- or serotonin-induced contraction or acetylcholine-induced relaxation was also determined in aortic rings. We found that SMe1EC2 (1×10(-8)-1×10(-4) mol/l) elicited significant dilatations in both cerebral and coronary arterioles (max dilatation: 25±8% and 18±5% respectively). Yet, SMe1EC2 (1×10(-6) and 1×10(-5) mol/l) did not influence the tone of aortic rings nor did it affect high potassium-, phenylephrine- or serotonin -induced contractions and acetylcholine-induced relaxation. Thus SMe1EC2 was able to dilate resistance arteries but did not affect aortic contractility. It is likely that SMe1EC2 does not possess α1-adrenolytic and anti-serotoninergic activity in the vascular wall.
RESUMO
Coronary artery disease (CAD) remains the leading cause of death in the Western societies. Diabetes mellitus (DM) is one of the highly prevalent diseases, which remarkably accelerates the development of CAD. Experimental evidence indicates that decreased bioavailability of coronary endothelial nitric oxide (NO) contributes to the development of CAD in DM. There are recent studies showing that a selective impairment of NO synthesis occurs in coronary arteries of DM patients, which is mainly due to the limited availability of endothelial NO synthase (eNOS) precursor, l-arginine. Importantly, these studies demonstrated that DM, independent of the presence of CAD, leads to selective up-regulation of arginase-1. Arginase-1 seems to play an important role in limiting l-arginine availability in the close proximity of eNOS in vessels of DM patients. This brief review examines recent clinical studies demonstrating the pathological role of vascular arginase-1 in human diabetes. Whether arginase-1, which is crucial in the synthesis of various fundamental polyamines in the body, will represent a potent therapeutic target for prevention of DM-associated CAD is still debated.
RESUMO
The etiology of diabetic complications is strongly associated with increased oxidative stress. The aim of the present study was to evaluate the effect of the potent antioxidant stobadine (STB) on global ischemia-reperfusion cardiac injury in the rat model of diabetes mellitus (DM). Diabetes was induced by multiple low doses of streptozotocin. The effect of STB was compared with that of a high dose of α-lipoic acid (ALA). All experiments were performed on isolated Langendorff-perfused hearts 10 weeks after streptozotocin administration. Diabetic hearts showed to be more resistant to ischemia-reperfusion than the control hearts, as shown by the reduced number of reperfusion dysrhythmias. The effect of the therapy with ALA (100 mg/kg i.p., 5 times a week during 8 weeks) was comparable to that of STB (25 mg/kg i.p., 5 times a week during 8 weeks) resulting in lowering the heart rate and coronary flow as well as the number of serious reperfusion dysrhythmias. Though the protective effect of STB on the reperfusion-induced dysrhythmias was comparable with that of ALA, both substances failed to enhance functional recovery of the diabetic rat heart.
Assuntos
Antioxidantes/administração & dosagem , Carbolinas/administração & dosagem , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/fisiopatologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ácido Tióctico/administração & dosagem , Animais , Masculino , Traumatismo por Reperfusão Miocárdica/etiologia , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
Generation of reactive oxygen species is a major, well-known cause of heart injury induced by ischemia-reperfusion. This injury is manifested through myocardial stunning, reperfusion and lethal reperfusion injury of cardiocytes. The pyridoindole stobadine has been shown to exhibit significant antioxidant, free-radical scavenging and hypoxic-tissue-protecting properties. The present study examined the effects of stobadine and two novel derivatives, SMe1 and SMe1EC2, which exhibit improved pharmacodynamic and toxicity profiles, on the functional properties and reperfusion dysrhythmias of the isolated rat heart in ischemia-reperfusion conditions. All experiments were performed on isolated Langendorff-perfused hearts isolated from 3-month-old male Wistar rats. After 15 min of stabilization, the hearts were subjected to a 30-minute period of global no-flow ischemia, followed by a 30-minute reperfusion period. Stobadine, SMe1 and SMe1EC2 were applied at a concentration of 1 x 10(-5) 10 min before the onset of ischemia, and during reperfusion through the perfusion medium. As compared to the untreated group, addition of SMe1EC2 during reperfusion significantly increased left ventricular developed pressure, decreased pathologically elevated left ventricular end-diastolic pressure and enhanced recovery of the stunned myocardium after ischemia. Both SMe1 and stobadine failed to influence these parameters; however, all derivatives tested inhibited serious life-threatening reperfusion dysrhythmias such as ventricular tachycardia and ventricular fibrillation. Our findings suggest that SMe1EC2 promotes an improved recovery of the left ventricular function following ischemia compared to either stobadine or SMe1. However, both SMe1EC2 and SMe1 manifested a significant anti-dysrhythmic effect comparable with that of stobadine and partially reduced myocardial ischemia-reperfusion-induced injury.
Assuntos
Carbolinas/farmacologia , Indóis/farmacologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Piridinas/farmacologia , Animais , Antioxidantes/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Técnicas In Vitro , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio Atordoado/tratamento farmacológico , Miocárdio Atordoado/etiologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/etiologiaRESUMO
One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo.