Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.

Angiopoietin like-2: a pro-inflammatory and pro-oxidative protein that contributes to endothelial dysfunction

Le vieillissement vasculaire est caractérisé par une dysfonction de l’endothélium. De nombreux facteurs de risque cardiovasculaire tels que l’obésité et l’hypertension prédisposent l’endothélium à un stress oxydant élevé aboutissant à une dysfonction endothéliale, celle-ci étant communément accompagnée d’une diminution de la biodisponibilité du monoxyde d’azote. Bien que la fonction endothéliale soit un déterminant majeur de la prédiction du risque cardiovasculaire des patients, son évaluation individuelle reste très limitée. En conséquence, il existe un intérêt scientifique grandissant pour la recherche de meilleurs biomarqueurs. L’Angiopoiétine like-2 (angptl2), une protéine identifiée récemment, joue un rôle pro-inflammatoire et pro-oxydant dans plusieurs désordres causés par une inflammation chronique allant de l’obésité à l’athérosclérose. L’inflammation et un stress oxydant accru ont été établis comme des mécanismes sous-jacents à l’apparition d’une dysfonction endothéliale, c’est pourquoi ce travail met l’accent sur le rôle de l’angptl2 dans la dysfonction endothéliale. Plus précisément, ce travail vise à: 1) déterminer les effets aigus de l’angptl2 sur la fonction endothéliale, 2) caractériser la fonction endothéliale et la contribution des différents facteurs relaxants dérivés de l'endothélium (EDRF) dans plusieurs lits vasculaires, et ce, dans un modèle de souris réprimant l’expression de l’angptl2 (knock-down, KD), et 3) examiner si l'absence d'expression angptl2 protège contre la dysfonction endothéliale induite par un régime riche en graisses (HFD) ou par perfusion d'angiotensine II (angII) chez la souris. Dans la première étude, l’incubation aigue avec de l’angptl2 recombinante induit une dysfonction endothéliale dans les artères fémorales isolées de souris de type sauvage (WT), probablement en raison d’une production accrue d'espèces réactives oxygénées. Les artères fémorales de souris angptl2 KD présentent une meilleure fonction endothéliale en comparaison aux souris WT, vraisemblablement par une plus grande contribution de la prostacycline dans la vasodilatation. Après 3 mois d’une diète HFD, les principaux EDRF respectifs des artères fémorales et mésentériques sont conservés uniquement dans les souris angptl2 KD. Cette préservation est associée à un meilleur profil métabolique, une moindre accumulation de triglycérides dans le foie et des adipocytes de plus petite taille. De plus, l’expression de gènes inflammatoires dans ces tissus adipeux n’est augmentée que chez les souris WT. Dans la seconde étude, l’absence d’angptl2 résulte en une production accrue de monoxyde d’azote dans les artères cérébrales isolées par rapport à celles des souris WT. La perfusion chronique d’angII provoque, seulement chez les souris WT, une dysfonction endothéliale cérébrale probablement par le biais d’une augmentation de la production d’espèces réactives oxygénées, probablement dérivé des NADPH oxydase 1 et 2, ainsi que l'augmentation des facteurs constricteurs dérivés de l’endothélium issus de la cyclo-oxygénase. En revanche, l’apocynine réduit la dilatation cérébrale chez les souris KD traitées à l’angII, ce qui suggère le recrutement potentiel d’une voie de signalisation compensatoire impliquant les NADPH oxydases et qui aurait un effet vaso-dilatateur. Ces études suggèrent fortement que l’angptl2 peut avoir un impact direct sur la fonction endothéliale par ses propriétés pro-inflammatoire et pro-oxydante. Dans une optique d’application à la pratique clinique, les niveaux sanguins d’angptl2 pourraient être un bon indicateur de la fonction endothéliale.

Protección miocárdica con custodiol y trastornos de la osmolalidad

En análisis retrospectivo evaluamos 91 pacientes llevados a cirugía cardiaca entre 2013 y 2014 en la Fundación Cardioinfantil, en quienes se administro Custodiol, analizando los niveles de sodio y osmolalidad plasmática efectiva antes, durante y después del procedimiento quirúrgico. Nosotros evaluamos la relación entre administración de Custodiol y cambios en el sodio y osmolalidad plasmática del paciente llevado a cirugía cardiaca.

Perinatal lead exposure affects nitric oxide and cyclooxygenase pathways in aorta of weaned rats

Perinatal Pb exposure may modulate arterial tone through nitric oxide (NO) and cyclooxygenase products. To investigate this, Wistar dams received 1000 ppm of Pb or sodium acetate (control) in drinking water during pregnancy and lactation. Curves were constructed in phenylephrine-precontracted intact and/or denuded rings of thoracic aortas of weaned (23-day-old) male pups from their responses to N-omega-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and ACh in the absence or presence of indomethacin (10(-5)M, cyclooxygenase inhibitor) or L-NAME (3 x 10(-7)M and 3 x 10(-4)M). Blood lead concentration and systolic blood pressure (SBP) were higher in intoxicated than control pups (blood lead mu g/dl: control < 3.0, Pb 58.7 +/- 6.5*; SBP mmHg: control 111.4 +/- 2.3, Pb 135.5 +/- 2.4*). In L-NAME-treated rings maximal responses increased in Pb-exposed rats, and were higher in intact than in denuded aortas (contraction [% of phenylephrine] intact: control 184.3 +/- 23.7, Pb 289.1 +/- 18.3*; denuded: control 125.1 +/- 4.5, Pb 154.8 +/- 13.3*). ACh-induced relaxation in intact aortas from Pb-exposed rats presented rightward shift in L-NAME presence (EC50 x 10(-7)M: control 1.32 [0.33-5.18], Pb 4.88 [3.56-6.69]*) but moved left under indomethacin (EC50 x 10(-7)M: control 8.95 [3.47-23.07], Pb 0.97 [0.38-2.43]*). *p < 0.05 significant relative to the respective control; N = 7-9. Endothelium removal abolished ACh-induced relaxation. Perinatal Pb exposure caused hypertension associated with alterations in the production and/or release of basal and stimulated endothelium-derived relaxing factors-NO and constricting cyclooxygenase products. These findings may help explain the contribution of NO and cyclooxygenase products to the etiology and/or maintenance of Pb-induced hypertension and could ultimately lead to therapeutic advantages in plumbism.

Papel das espécies reativas de oxigênio na hiporreatividade de aortas à fenilefrina observada no final da prenhez de Ratas Espontaneamente Hipertensas (SHR)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Endothelium dependent expression and underlying mechanisms of des-Arg(9)-bradykinin-induced B1R-mediated vasoconstriction in rat portal vein

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B-1 receptor (B1R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B1R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B1R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B1R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B1R increased phospholipase A(2)/COX-2-derived thromboxane A(2) (TXA(2)) levels, which in turn mediated portal vein contraction through binding to TXA(2) receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B1R expression and identify a critical role for the endothelial B1R in the modulation of portal vein vascular tone. Our study suggests a potential role for B1R antagonists as therapeutic tools for diseases where portal hypertension may be involved. (C) 2012 Elsevier Inc. All rights reserved.

Theoretical investigations of intercellular communication in the microcirculation: Conducted responses, myoendothelial projections and endothelium derived hyperpolarizing factor

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. ^ Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca 2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. ^ Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP 3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca 2+ transient generated a hyperpolarizing feedback of ∼ 2–3mV. ^ Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC V m, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.^

The Gastrointestinal Peptide Obestatin Induces Vascular Relaxation Via Specific Activation of Endothelium-Dependent Nitric Oxide Signalling.

Background and purpose: Obestatin is a recently-discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation. Experimental approach: Cumulative relaxation responses to obestatin peptides were assessed in isolated rat aorta and mesenteric artery (n=8) in the presence/absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). Key results: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, L-NAME (NO synthase inhibitor), high extracellular K(+) , GDP-ß-S (G protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked G protein-coupled receptor, PI3K/Akt, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and Akt phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarising factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. Conclusions and Implications: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterised by endothelial dysfunction and cardiovascular complications.

Endothelium-derived agents in pericyte function/dysfunction

The major components of blood vessels are the vascular endothelium and its supporting smooth muscle. Significant strides have been made in the understanding of the cellular and molecular biology of these two cell types and in particular their interactions have been the subject of much interest and debate over the past two decades. The vascular endothelium is now known to profoundly influence the synthetic and motor functions of the underlying smooth muscle and participate in the pathogenesis of all the major vascular disorders. Similarly, the vascular smooth muscle has important effects on the overlying endothelium, and any disruption in the cellular physiology of either cell type can result in dysfunction with important effects on blood flow and vascular permeability The majority of this accumulated knowledge relates to the vascular cells of the macrocirculation. Pericytes are the supporting cells of the microvasculature and a body of evidence is now available to show that similar regulatory mechanisms and vessel-wall cross-talk exists between these cells and the microvascular endothelium. Nowhere are these interactions more important than in the retinal microcirculation where autoregulation is vital for the maintenance of smooth and uninterrrupted blood flow. This review focuses on the interactions between retinal microvascular endothelial cells and their associated pericytes and examines the role of the endothelial cell and the pericyte in the pathogenesis of disease.

Endothelium-derived intermedin/adrenomedullin-2 protects human ventricular cardiomyocytes from ischaemia-reoxygenation injury predominantly via the AM1 receptor

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM1 receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1 nmol L−1, applied either throughout ischaemia (3 h) and re-oxygenation (1 h) or during re-oxygenation (1 h) alone, attenuated HCM injury (P < 0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P < 0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P < 0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM1 receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Myocardial angiogenesis induction with bone protein derived growth factors (animal experiment).

Myocardial angiogenesis induction with vascular growth factors constitutes a potential strategy for patients whose coronary artery disease is refractory to conventional treatment. The importance of angiogenesis in bone formation has led to the development of growth factors derived from bovine bone protein. Twelve pigs (mean weight, 73 +/- 3 kg) were chosen for the study. In the first group (n = 6, growth factor group) five 100 micrograms boluses of growth factors derived from bovine bone protein, diluted in Povidone 5%, were injected in the lateral wall of the left ventricle. In the second group (n = 6, control group), the same operation was performed but only the diluting agent was injected. All the animals were sacrificed after 28 days and the vascular density of the left lateral wall (expressed as the number of vascular structures per mm2) as well as the area of blood vessel profiles per myocardial area analysed were determined histologically with a computerised system. The growth factor group had a capillary density which was significantly higher than that of the control group: 12.6 +/- 0.9/mm2 vs 4.8 +/- 0.5/mm2 (p < 0.01). The same holds true for the arteriolar density: 1 +/- 0.2/mm2 vs 0.3 +/- 0.1/mm2 (p < 0.01). The surface ratios of blood vessel profiles per myocardial area were 4900 +/- 800 micron 2/mm2 and 1550 +/- 400 micron 2/mm2 (p < 0.01) respectively. In this experimental model, bovine bone protein derived growth factors induce a significant neovascularisation in healthy myocardium, and appear therefore as promising candidates for therapeutic angiogenesis.