Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Impact du stress hyperoxique en période néonatale sur la structure vasculaire : implication des phénomènes de sénescence et rôle possible dans la programmation développementale de l'hypertension artérielle

Réalisé en cotutelle avec l'Université de Lorraine (France)

Role of Nuclear Angiotensin-II Receptor Mediated Signalling in Cardiovascular Remodelling

Le remodelage cardiaque est le processus par lequel la structure ou la fonction cardiaque change en réponse à un déséquilibre pathophysiologique tel qu'une maladie cardiaque, un contexte d'arythmie prolongée ou une modification de l'équilibre hormonal. Le système rénine-angiotensine (SRA) est un système hormonal largement étudié et il est impliqué dans de nombreuses activités associées au remodelage cardiovasculaire. L’existence d'un système circulatoire couplé à un système de tissus locaux est une représentation classique, cependant de nouvelles données suggèrent un SRA indépendant et fonctionnellement actif à l'échelle cellulaire. La compréhension de l'activité intracellulaire du SRA pourrait mener à de nouvelles pistes thérapeutiques qui pourraient prévenir un remodelage cardiovasculaire défavorable. L'objectif de cette thèse était d'élucider le rôle du SRA intracellulaire dans les cellules cardiaques. Récemment, les récepteurs couplés aux protéines G (RCPG), les protéines G et leurs effecteurs ont été détectés sur des membranes intracellulaires, y compris sur la membrane nucléaire, et les concepts de RCPG intracellulaires fonctionnels sont en voie d'être acceptés comme une réalité. Nous avons dès lors fait l'hypothèse que la signalisation du SRA délimitant le noyau était impliquée dans le contrôle de l'expression des gènes cardiaques. Nous avons démontré la présence de récepteurs d'angiotensine de type-1 (AT1R) et de type-2 (AT2R) nucléaires dans les cardiomyocytes ventriculaires adultes et dans une fraction nucléaire purifiée de tissu cardiaque. Des quantités d'Ang II ont été détectées dans du lysat de cardiomyocytes et des microinjections d'Ang-II-FITC ont donné lieu à des liaisons préférentielles aux sites nucléaires. L'analyse transcriptionnelle prouve que la synthèse d'ARN de novo dans des noyaux isolés stimulés à l'Ang-II, et l'expression des ARNm de NF-κB étaient beaucoup plus importants lorsque les noyaux étaient exposés à de l'Ang II par rapport aux cardiomyocytes intacts. La stimulation des AT1R nucléaires a engendré une mobilisation de Ca2+ via les récepteurs de l'inositol trisphosphate (IP3R), et le blocage des IP3R a diminué la réponse transcriptionnelle. Les méthodes disponibles actuellement pour l'étude de la signalisation intracrine sont limitées aux méthodes indirectes. L'un des objectifs de cette thèse était de synthétiser et caractériser des analogues d'Ang-II cellule-perméants afin d’étudier spécifiquement dans les cellules intactes l'activité intracellulaire du SRA. Nous avons synthétisé et caractérisé pharmacologiquement des analogues photosensibles Ang-II encapsulée en incorporant un groupement 4,5-diméthoxy-2-nitrobenzyl (DMNB) photoclivable sur les sites actifs identifiés du peptide. Chacun des trois analogues d'Ang II encapsulée synthétisés et purifiés: [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB et [Tyr(DMNB)4]Ang-II-ODMNB a montré une réduction par un facteur deux ou trois de l'affinité de liaison envers AT1R et AT2R dans les dosages par liaison compétitive et une activité réduite dans la contraction de l'aorte thoracique. La photostimulation de [Tyr(DMNB)4]Ang-II dans des cellules HEK a augmenté la phosphorylation d'ERK1/2 (via AT1R) et la production de cGMP (via AT2R) alors que dans les cardiomyocytes isolés elle générait une augmentation de Ca2+ nucléoplasmique et initiait la synthèse d'ARNr 18S et d'ARNm du NF-κB. Les fibroblastes sont les principaux générateurs de remodelage cardiaque structurel, et les fibroblastes auriculaires sont plus réactifs aux stimuli profibrotiques que les fibroblastes ventriculaires. Nous avons émis l'hypothèse que l’Ang-II intracellulaire et l'activation des AT1R et AT2R nucléaires associés contrôlaient les profils d'expression des gènes des fibroblastes via des systèmes de signalisation distincts et de ce fait jouaient un rôle majeur dans le développement de la fibrose cardiaque. Nous avons remarqué que les fibroblastes auriculaires expriment l’AT1R et l’AT2R nucléaire et l'Ang-II au niveau intracellulaire. L’expression d'AT1R nucléaire a été régulés positivement dans les cas d’insuffisance cardiaque (IC), tandis que l'AT2R nucléaire a été glycosylé post-traductionnellement. La machinerie protéique des protéines G, y compris Gαq/11, Gαi/3, et Gβ, a été observée dans des noyaux isolés de fibroblastes. AT1R et AT2R régulent l'initiation de la transcription du fibroblaste via les voies de transduction de signal d'IP3R et du NO. La photostimulation de [Tyr(DMNB)4]Ang-II dans une culture de fibroblastes auriculaire déclenche la libération de Ca2+ nucléoplasmique, la prolifération, et la synthèse et sécrétion de collagène qui ne sont pas inhibées par les bloqueurs d'AT1R et/ou AT2R extracellulaires.

Mortalidad por trauma cardiaco penetrante en un hospital de Bogotá, Colombia : análisis de factores asociados

Objetivó: Caracterizar los pacientes con heridas cardiacas penetrantes grado II a VI, describir las características del trauma, tratamiento quirúrgico, evolución clínica e identificar los factores asociados a un desenlace. Metodología: Se diseñó un estudio de asociación en 308 pacientes que ingresaron a cirugía con diagnóstico de herida penetrante de corazón entre enero de 1999 y octubre de 2009. Se excluyeron 68 casos. La serie analizada incluyó 240 pacientes con heridas cardiacas. Se analizaron variables demográficas, clínicas, quirúrgicas y de evolución, tabulados en EXCEL® y analizados en SPSS 20®. Resultados: El promedio de edad fue 27.8 años, principalmente hombres (96%), lesiones por arma cortopunzante 93% y un 7% por proyectil arma de fuego. El estado hemodinámico al ingreso (según Ivatury) fue normal 44%; Shock profundo 34%; Agónicos 18% y 3% fatales. El 67% (n=161) presentaron taponamiento cardiaco. Los grados de lesión cardiaca según la clasificación OIS-AAST fueron: grado II 33%, grado III 13%, grado IV 29%, grado V 22% y grado VI 3%. La ventana pericárdica fue el método diagnóstico confirmatorio de lesión en 63% y las incisiones de abordaje quirúrgico fueron la esternotomía 63% y la toracotomía anterolateral 35%. La mortalidad fue 15% (n=36). Las diferencias en mortalidad entre el estado hemodinámico al inicio de cirugía, mecanismo de lesión y grado de herida, demostraron ser estadísticamente significativas (valor de p<0.001). Conclusiones: El estado hemodinámico y las heridas por arma de fuego son factores asociados a mortalidad. La ventana pericárdica subxifoidea favorece la preferencia y buenos resultados de la esternotomía como vía de abordaje quirúrgico.

Avaliação do apêndice atrial esquerdo pela ecocardiografia transtorácica com imagem harmônica após evento neurológico agudo

Introdução: Embora a imagem com segunda harmônica esteja largamente disponível na maioria dos aparelhos de ultra-sonografia, sua acurácia para avaliar a morfologia e a função do apêndice atrial esquerdo (AAE) permanece precariamente caracterizada. Objetivos: Explorar o desempenho diagnóstico da ecocardiografia transtorácica com segunda harmônica (ETTsh) na avaliação do AAE após eventos neurológicos agudos. Métodos: Realizamos um estudo transversal em pacientes com eventos neurológicos isquêmicos agudos, encaminhados para realização de ETTsh e Ecocardiografia Transesofágica (ETE). As análises da área longitudinal máxima e do pico da velocidade de esvaziamento de fluxo do AAE foram realizadas por observadores cegos. Resultados: Foram avaliados 51 pacientes (49% femininas, 62 ± 12 anos) com eventos neurológicos isquêmicos agudos. Contraste ecocardiográfico espontâneo foi observado em 11 (22%) pacientes no AE, em 7(14%) no AAE e em 3 (6%) na aorta torácica descendente. Trombo no AAE foi identificado em apenas 2 (4%) pacientes. O mapeamento e a análise do AAE foi factível na maioria dos casos (98%), tanto para o estudo com Doppler quanto para avaliação da área do AAE. Observamos uma associação positiva e significativa entre o ETTsh e o ETE, tanto para a avaliação das velocidades máximas de esvaziamento do AAE (r=0,63; p<0,001) quanto para a área longitudinal máxima do AAE (r=0,73; p<0,001). Ademais, todos os pacientes com trombos no AAE ou contraste espontâneo (n=7) tiveram velocidade de esvaziamento inferior a 50 cm/s no mapeamento transtorácico (valor preditivo negativo de 100%). Na análise multivariada ajustada para diversos potenciais preditores transtorácicos de risco, a velocidade máxima de esvaziamento do AAE permaneceu independentemente associada com trombos no AAE ou contraste espontâneo. Conclusão: ETTsh pode fornecer informações relevantes a respeito da morfologia e dinâmica do AAE. Em particular, pacientes com velocidades altas de esvaziamento do AAE podem não necessitar de avaliação adicional com ETE.

Origem das artérias celíaca e mesentérica cranial em bubalinos (Bubalus bubalis, L. 1758)

Com o intuito de conhecer melhor o sistema arterial nesta espécie, descrevemos a origem das artérias celíaca e mesentérica cranial dos bubalinos. Utilizamos 30 fetos e 1 animal adulto. Os fetos variavam de 4 a 8 meses de idade e tiveram seus vasos injetados com solução de látex - Neoprene, tiveram seus vasos dissecados, e um animal adulto, que teve seus vasos dissecados no local de abate. Observamos que as artérias celíaca e mesentérica cranial originavam-se da porção torácica da aorta em todos os casos, estando isoladas em 90,33% deles e em tronco comum (tronco celíaco mesentérico) em 9,67% dos casos.

Descrição anátomo-topográfica do coração da paca (Agouti paca)

Background: The domestic animals heart is a conical hollow viscera, surrounded by pericardium, laterally compressed, accompanying the thorax shape. Atriums constituted the heart basis and their auricles partially bound the initial portion of the aorta and pulmonary trunk. In mammals, heart is kept suspended in the thoracic cavity and the pericardic sac is fixed dorsally by great veins and arteries roots, and ventrally fixed to the sternum, although its fixation to the diaphragm varies among species. This paper aimed to describe morphological aspects of the heart of the paca, the second biggest Brazilian rodent.Materials, Methods & Results: There were used 12 hearts of adult pacas for this study, obtained from the UNESP, Jaboticabal, SP, which died due fights or anesthesia during bandages or radiograph exams. The thoracic aorta was filled with colored latex and the animal was set in a 10% formaldehyde solution for at least 72 hours. The thoracic cavity was dissected and hearts individualized and measured with a paquimeter, lateromedially, craniocaudally and dorsoventrally. The paca heart is placed between the first and fifth intercostal space (ICS), in a craniocaudal oblique position; its basis is craniodorsally positioned, on the middle third between the first and second ICS and its apex is located near the sternodiaphragmatic joint, on the fifth ICS, tilted to the left antimere. The heart is surrounded by pericardium, which from ventrocaudally is originated the sternopericardic ligament, that continues as phrenopericardic ligament. At the heart basis, the rising of the pulmonary trunk was observed and the conus arteriosus formed a typical projection. The aorta also rised from the heart basis and its arch, which was caudally curved, crossed dorsally the pulmonary trunk; the right cranial and caudal cava veins drained to the right atrium. There is a left cranial cava vein, which surrounded the left atrium and joined the right caudal cava vein on the right atrium. The azygos vein joins the right cranial cava vein and four pulmonary veins drained to the left atrium. At palpation, a hard structure on the rising of the aorta was observed, similarly to a cartilaginous tissue, which would be part of the cardiac skeleton. The left and right coronary arteries were observed in all hearts.Discussion: The paca heart is anatomica and topographically similar to those of domestic mammals, differing from them for being placed one intercostal space more cranial and due to the presence of two cranial cava veins, the left and the right ones, besides the presence of the caudal cava vein. This vascular description is similar to that of small rodents, as rats and mice. In paca heart, the sinus venous, the terminal crest, the oval fossa, the atrioventricular valvae, the papillary muscles and tendinous cords, besides smooth atriums and auricles covered by pectinate muscles, were observed. The sternopericardic ligament, which is dorsally elongated as phrenopericardic ligament, is similar to the one present in humans, pigs, castors, and different from the one observed in carnivorous, that presents the phrenopericardic ligament and from the one of horses and ruminants, which present the sternopericardic ligament.

Biomechanical and histologic analysis in aortic endoprosthesis using fibrin glue

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Association between atherosclerotic aortic plaques and left ventricular hypertrophy in patients with cerebrovascular events

Background and Purpose - the purpose of this research was to evaluate whether an association exists between the presence of atherosclerotic plaque in the thoracic aorta and left ventricular hypertrophy (LVH) in patients with a cerebrovascular event.Methods - We included 116 consecutive patients ( 79 men; mean age, 62 +/- 12.4 years) with previous history of stroke or transient ischemic attack in a cross-sectional study. Transthoracic echocardiogram was performed to diagnose LVH and transesophageal echocardiogram for the detection of atheromas of the thoracic aorta. Continuous variables were analyzed by Student t or Mann-Whitney tests and categorized variables by Goodman test. From the significant association of LVH and age with atheromatous disease of the aorta, an adjustment to the multivariate logistic model was made using high blood pressure history or age as covariates. All of the statistical tests were carried out at a level of 5% significance.Results - Almost half of the patients (43.1%) presented atherosclerotic lesions in the aorta. LVH was present in 90.0% of patients with plaque and in only 30.3% of patients without plaque. Using high blood pressure as a covariate, the risk of patients with LVH presenting atherosclerotic plaque in the aorta was 18.23-fold greater than the risk for patients without LVH (95% CI, 5.68 to 58.54; P < 0.0001). Adding age into the model, the risk increased to 26.36 ( 95% CI, 7.14 to 97.30; P < 0.0001).Conclusions - LVH detected by conventional echocardiogram is associated with high risk of atherosclerotic plaque in the aorta and would be used as a criterion for indication of transesophageal echocardiography in patients with previous stroke or transient ischemic attack LVH.

Some segmental features on the structure of the aortic wall of the dog

Structural features of segmental parts of the aorta of the dog were studied by light microscopy (LM) and scanning electron microscopy (SEM). The variability in the wall architecture composition and vascular thickness of the ascending (T2-3 level), thoracic (T7-8 level) and abdominal (L6-7 infrarenal level) segments of the aorta was analysed. Morphological features such as presence of intimal folds, pattern of the medial myoconnective components with segmental variations in the number of elastic lamellae, whose relative number was higher in the thoracic aorta (ascending and descending parts), compared with the abdominal aorta, and a network of connective (stromal) elements formed by elastic and collagen lamellae and fibres in the adventitia were observed. The results were discussed on a histophysiological basis, because small but significant segmental differences had been characterized in the aortic wall structure of the dog.

Vascular adaptive responses to physical exercise and to stress are affected differently by nandrolone administration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações vasculares em ratos obesos por dieta rica em gordura: papel da Via L-arginina/NO Endotelial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The rate of force generation by the myocardium is not influenced by afterload

The influence of afterload on the rate of force generation by the myocardium was investigated using two types of preparations: the in situ dog heart (dP/dt) and isolated papillary muscle of rats (dT/dt). Thirteen anesthetized, mechanically ventilated and thoracotomized dogs were submitted to pharmacological autonomic blockade (3.0 mg/kg oxprenolol plus 0.5 mg/kg atropine). A reservoir connected to the left atrium permitted the control of left ventricular end-diastolic pressure (LVEDP). A mechanical constriction of the descending thoracic aorta allowed to increase the systolic pressure in two steps of 20 mmHg (conditions H1 and H2) above control values (condition C). After arterial pressure elevations (systolic pressure C: 119 ± 8.1; H1: 142 ± 7.9; H2 166 ± 7.7 mmHg; P<0.01), there were no significant differences in heart rate (C: 125 ± 13.9; H1: 125 ± 13.5; H2: 123 ± 14.1 bpm; P>0.05) or LVEDP (C: 6.2 ± 2.48; H1: 6.3 ± 2.43; H2: 6.1 ± 2.51 mmHg; P>0.05). The values of dP/dt did not change after each elevation of arterial pressure (C: 3,068 ± 1,057; H1: 3,112 ± 996; H2: 3,086 ± 980 mmHg/s; P>0.05). In isolated rat papillary muscle, an afterload corresponding to 50% and 75% of the maximal developed tension did not alter the values of the maximum rate of tension development (100%: 78 ± 13; 75%: 80 ± 13; 50%: 79 ± 11 g mm-2 s-1, P>0.05). The results show that the rise in afterload per se does not cause changes in dP/dt or dT/dt