Among the unsolved mysteries of modern biology is the nature of a lining of blood vessels called the ‘endothelial surface layer’ or ESL. In venous micro-vessels, it is half a micron in thickness. The ESL is 10 times thicker than the endothelial glycocalyx (eGC) at its base, has been presumed to be comprised mainly of water, yet is rigid enough to exclude red blood cells. How is this possible? Developments in physical chemistry suggest that the venous ESL is actually comprised of nanobubbles of CO2, generated from tissue metabolism, in a foam nucleated in the eGC. For arteries, the ESL is dominated by nanobubbles of O2 and N2 from inspired air. The bubbles of the foam are separated and stabilized by thin layers of serum electrolyte and proteins, and a palisade of charged polymer strands of the eGC. The ESL seems to be a respiratory organ contiguous with the flowing blood, an extension of, and a ‘lung’ in miniature. This interpretation may have far-reaching consequences for physiology.

Protocols designed for the adipogenic differentiation of human and mouse cells are commonly used for inducing the adipogenesis of bovine stromal vascular cells. However, likely due to metabolic differences between ruminant and non-ruminant animals, these methods result in only few cells undergoing complete adipogenesis with minimal lipid droplet accumulation. Here, we discuss the development of an adipogenic differentiation protocol for bovine primary cells through a three-dimensional spheroid culture. Stromal vascular cells derived from bovine intramuscular fat were isolated and stored in liquid nitrogen before culturing. Cells were cultured in hanging drops for 3 days to allow for the formation of spherical structures. The spheroids were then transferred to cell culture plates with endothelial basal medium-2 for 3 days and in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with a standard adipogenic cocktail for 3 additional days, which were then allowed to fully differentiate for 3 days in DMEM supplemented with insulin. Compared with conventional two-dimensional culture, cells in a three-dimensional spheroid culture system had higher adipogenic gene expression and consequently contained more adipocytes with larger lipid droplets. In addition, endothelial induction of spheroids prior to adipogenic differentiation is essential for efficient induction of adipogenesis of bovine stromal vascular cells, mimicking in vivo adipose development. In summary, the newly developed three-dimensional spheroid culture method is an efficient way to induce adipogenic differentiation and study adipose development of cells derived from ruminant animals, which also can be used for studying the role of angiogenesis in adipose development.

Obesity and type 2 diabetes lead to dramatically increased risks of atherosclerosis and CHD. Multiple mechanisms converge to promote atherosclerosis by increasing endothelial oxidative stress and up-regulating expression of pro-inflammatory molecules. Microvesicles (MV) are small ( < 1 μm) circulating particles that transport proteins and genetic material, through which they are able to mediate cell–cell communication and influence gene expression. Since MV are increased in plasma of obese, insulin-resistant and diabetic individuals, who often exhibit chronic vascular inflammation, and long-term feeding of a high-fat diet (HFD) to rats is a well-described model of obesity and insulin resistance, we hypothesised that this may be a useful model to study the impact of MV on endothelial inflammation. The number and cellular origin of MV from HFD-fed obese rats were characterised by flow cytometry. Total MV were significantly increased after feeding HFD compared to feeding chow (P< 0·001), with significantly elevated numbers of MV derived from leucocyte, endothelial and platelet compartments (P< 0·01 for each cell type). MV were isolated from plasma and their ability to induce reactive oxygen species (ROS) formation and vascular cell adhesion molecule (VCAM)-1 expression was measured in primary rat cardiac endothelial cells in vitro. MV from HFD-fed rats induced significant ROS (P< 0·001) and VCAM-1 expression (P= 0·0275), indicative of a pro-inflammatory MV phenotype in this model of obesity. These findings confirm that this is a useful model to further study the mechanisms by which diet can influence MV release and subsequent effects on cardio-metabolic health.

Introduction: Cyanotic congenital heart disease is associated with functional limitation and vascular events. The nature and extent of endothelial dysfunction in cyanotic adults is poorly understood. We sought to characterise endothelial function in this setting. Methods: A total of fourteen adults with cyanotic congenital heart disease (40±3 years) together with age- and sex-matched healthy controls underwent assessment of nitric oxide-dependent vascular responses, including flow-mediated dilatation of the brachial artery and dynamic vessel analysis of the retina in response to flickering light. Plasma levels of the endothelium-derived vasoconstrictor endothelin-1 and the nitric oxide antagonist, asymmetric dimethylarginine, were measured. Circulating endothelial progenitor cells were assessed by flow cytometry. Results: Flow-mediated dilatation was significantly lower in cyanosed adults than controls (4.0±0.8 versus 7.2±1.0%, p=0.019, n=11 per group). Retinal arterial and venous dilatory responses were also impaired (2.9±0.8 versus 5.0±0.6%, p=0.05 and 3.4±0.3 versus 5.2±0.7%, p=0.04, n=13). Serum levels of endothelin-1 and asymmetric dimethylarginine were higher in cyanosed adults (3.0±0.6 versus 1.1±0.1 pg/ml, p=0.004 and 0.68±0.05 versus 0.52±0.02 μmol/L, p=0.03, n=11). Endothelial progenitor cells (CD34+CD45dimCD133+KDR+) were reduced in those with chronic cyanosis (17±4 versus 40±6 per million white blood cells, p=0.005, n=11). Conclusions: Endothelial function is impaired in the systemic arteries and retinal vessels in adults with cyanotic congenital heart disease, suggesting a widespread endotheliopathy. Diminished numbers of endothelial progenitor cells might potentially contribute to these observations.

The consumption of cocoa and dark chocolate is associated with a lower risk of CVD, and improvements in endothelial function may mediate this relationship. Less is known about the effects of cocoa/chocolate on the augmentation index (AI), a measure of vascular stiffness and vascular tone in the peripheral arterioles. We enrolled thirty middle-aged, overweight adults in a randomised, placebo-controlled, 4-week, cross-over study. During the active treatment (cocoa) period, the participants consumed 37 g/d of dark chocolate and a sugar-free cocoa beverage (total cocoa = 22 g/d, total flavanols (TF) = 814 mg/d). Colour-matched controls included a low-flavanol chocolate bar and a cocoa-free beverage with no added sugar (TF = 3 mg/d). Treatments were matched for total fat, saturated fat, carbohydrates and protein. The cocoa treatment significantly increased the basal diameter and peak diameter of the brachial artery by 6 % (+2 mm) and basal blood flow volume by 22 %. Substantial decreases in the AI, a measure of arterial stiffness, were observed in only women. Flow-mediated dilation and the reactive hyperaemia index remained unchanged. The consumption of cocoa had no effect on fasting blood measures, while the control treatment increased fasting insulin concentration and insulin resistance (P= 0·01). Fasting blood pressure (BP) remained unchanged, although the acute consumption of cocoa increased resting BP by 4 mmHg. In summary, the high-flavanol cocoa and dark chocolate treatment was associated with enhanced vasodilation in both conduit and resistance arteries and was accompanied by significant reductions in arterial stiffness in women.

Epidemiological studies have reported a greater reduction in cardiovascular risk and metabolic disorders associated with diets rich in polyphenols. The antioxidant effects of polyphenols are attributed to the regulation of redox enzymes by reducing reactive oxygen species production from mitochondria, NADPH oxidases and uncoupled endothelial NO synthase in addition to also up-regulating multiple antioxidant enzymes. Although data supporting the effects of polyphenols in reducing oxidative stress are promising, several studies have suggested additional mechanisms in the health benefits of polyphenols. Polyphenols from red wine increase endothelial NO production leading to endothelium-dependent relaxation in conditions such as hypertension, stroke or the metabolic syndrome. Numerous molecules contained in fruits and vegetables can activate sirtuins to increase lifespan and silence metabolic and physiological disturbances associated with endothelial NO dysfunction. Although intracellular pathways involved in the endothelial effects of polyphenols are partially described, the molecular targets of these polyphenols are not completely elucidated. We review the novel aspects of polyphenols on several targets that could trigger the health benefits of polyphenols in conditions such as metabolic and cardiovascular disturbances.

The potential to reduce cardiovascular morbidity through dietary modification remains an area of intense clinical and scientific interest. Any putatively beneficial intervention should be tested within a randomised controlled trial which records appropriate endpoints, ideally incident CVD and death. However, the large sample sizes required for these endpoints and associated high costs mean that the majority of dietary intervention research is conducted over short periods among either healthy volunteers or those at only slightly increased risk, with investigators using a diverse range of surrogate measures to estimate arterial health in these studies. The present review identifies commonly employed techniques, discusses the relative merits of each and highlights emerging approaches.

Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLAB's efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.

Due to their ubiquitous distribution, flavonoids from different classes are commonly present together in foods. However, little is known about the interactions between them. The flavonol quercetin and the flavan-3-ol (+)-catechin are among the most abundant flavonoids in the diet. In the present study, we have analysed the interactions between these two flavonoids on vascular function using two pure compounds and mixtures of these flavonoids in 1:0·1, 1:1 or 1:10 proportions. Quercetin induced a more potent concentration-dependent relaxant effect than catechin in the isolated rat aorta, and the isobolographic analysis of the mixtures showed no synergistic or antagonistic effects between them, i.e. their effects were additive. Quercetin was more potent in mesenteric than in pulmonary arteries. Catechin had weak effects in these vessels and did not modify the effects of quercetin. Endothelial dysfunction induced by increased oxidative stress by the superoxide dismutase inhibitor diethyldithiocarbamate was prevented by quercetin, whereas catechin showed a weak effect and the 1:1 mixture an intermediate effect compared with the pure compounds. Quercetin but not catechin showed a pro-oxidant and NO-scavenging effect, which was not prevented by catechin. In conclusion, catechin was less potent than quercetin as a vasodilator, pro-oxidant or to prevent endothelial dysfunction, and there were no synergistic interactions between quercetin and catechin.

Impaired endothelial vasodilatation may contribute to the exaggerated blood pressure (BP) responses to exercise in individuals who are overweight/obese. The present study investigated whether consumption of cocoa flavanols, which improve endothelium-dependent flow-mediated dilatation (FMD), can modify BP responsiveness to exercise. Twenty-one volunteers (eight females and thirteen males, 54·9 (se 2·2) years, BMI 31·6 (se 0·8) kg/m2, systolic BP 134 (se 2) mmHg, diastolic BP (DBP) 87 (se 2) mmHg) were randomised to consume single servings of either a high-flavanol (HF, 701 mg) or a low-flavanol (LF, 22 mg) cocoa beverage in a double-blind, cross-over design with 3–7-d washout between treatments. Two hours after cocoa consumption, FMD was measured, followed by continuous beat-to-beat assessment (Finapres™) of BP before and during 10 min of cycling at 75 % of age-predicted maximum heart rate. Averaged data from two assessments on each type of beverage were compared by analysis of covariance using pre-exercise BP as the covariate. Pre-exercise BP was similar after taking LF and HF (153 (se 3)/88 (se 3) v. 153 (se 4)/87 (se 2) mmHg, respectively, P>0·05). However, the BP response to exercise (area under BP curve) was attenuated by HF compared with LF. BP increases were 68 % lower for DBP (P = 0·03) and 14 % lower for mean BP (P = 0·05). FMD measurements were higher after taking HF than after taking LF (6·1 (se 0·6) % v. 3·4 (se 0·5) %, P < 0·001). By facilitating vasodilation and attenuating exercise-induced increases in BP, cocoa flavanols may decrease cardiovascular risk and enhance the cardiovascular benefits of moderate intensity exercise in at-risk individuals.

Ticks are blood-feeding arthropods widely distributed in the world and vectors of several diseases. As haematophagy demands evasion strategies and repeatedly infested hosts develop protective immune responses, we investigated the mechanisms of the Rhipicephalus (Boophilus) microplus saliva anti-haemostatic activity and the possible relationship between the acquired natural anti-tick host resistance and anti-haemostatic action. For this purpose, we studied the effects of R. microplus saliva on different pathways of haemostasis and tested whether repeated infested bovine sera (RIBS) are able to abolish salivary anti-haemostatic activities. R. microplus saliva (i) displays inhibitory activity upon collagen-induced platelet aggregation; (ii) inhibits the induction of endothelial pro-coagulant state; and (iii) reduces thrombogenesis in vivo. RIBS were shown to be able to partially block the delay of coagulation and the anti-thrombotic effect of saliva, and to totally abolish the modulation of endothelium activation. Conversely, RIBS has no effect on the inhibition of platelet aggregation. These results show, for the first time, the neutralization ability of sera from acquired resistance hosts against tick anti-haemostatics. Moreover, this is the first report of a haematophagous parasite able to modulate endothelial cell pro-coagulant state, and addresses the presence of anti-platelet and anti-thrombotic activity in R. microplus saliva.

Conjugated linoleic acids (CLA) are biologically highly active lipid compounds that inhibit the development of atherosclerotic plaques in experimental animals. The underlying mechanisms of action, however, are only poorly understood. Since cell-culture experiments are appropriate to provide a detailed view into the mechanisms of action of a compound, the present review summarises results from in vitro studies dealing with the effects of CLA isomers and CLA mixtures on functional properties of cells of the vascular wall, such as endothelial cells, smooth muscle cells and monocyte-derived macrophages, which are amongst the major cells contributing to atherosclerotic lesion development. Based on these studies, it can be concluded that CLA exert several beneficial actions in cells of the vascular wall through the activation of nuclear PPAR. These actions of CLA, which may, at least partially, explain the inhibition of atherogenesis by dietary CLA, include modulation of vasoactive mediator release from endothelial cells, inhibition of inflammatory and fibrotic processes in activated smooth muscle cells, abrogation of inflammatory responses in activated macrophages, and reduction of cholesterol accumulation in macrophage-derived foam cells.

Pomace olive oil (POM), an olive oil subproduct traditionally used in Spain, is a good source of minor components from the unsaponifiable fraction such as triterpenoids, mainly in the form of oleanolic acid, which induces vascular protection and vasodilatation. Our aim was to evaluate the effects of long-term intake of diets enriched in POM with high concentration in oleanolic acid on endothelial dysfunction associated to hypertension in small mesenteric arteries (SMA) from spontaneously hypertensive rats (SHR). During 12 weeks, rats (six rats per group) were fed either a control 2 % maize oil diet (BD), or high-fat diets containing 15 % refined olive oil (OL), pomace olive oil (POM), or pomace olive oil supplemented in oleanolic acid (POMO; up to 800 parts per million). Endothelial and vascular functions were assessed by relaxing or contracting responses to acetylcholine (ACh) or phenylephrine, respectively. The involvement of endothelium-derived relaxing factors in these responses was evaluated. In contrast to BD, SHR fed high-fat diets showed a biphasic response to ACh related to changes in eicosanoid metabolism. POM enhanced the endothelial function in SMA from SHR by increasing the endothelium-derived hyperpolarising factor (EDHF)-type component, whereas administration of POMO resulted in a similar contribution of NO/EDHF in the endothelial response to ACh. The present study shows that despite the lack of changes in blood pressure, consumption of POM improves endothelial function in SMA from SHR by improving the agonist-mediated EDHF/NO response. Thus, triterpenoids confer a protective role to POM against endothelial dysfunction in hypertension.

CBA/T6 mice inoculated with Plasmodium berghei ANKA strain (PbA) exhibited cerebral symptoms and died from cerebral malaria 6–8 days p.i. whereas DBA/2J mice developed (around days 6–9) a non-fatal cerebral malaria, with milder cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop cerebral malaria. These mouse/parasite strain combinations were used, in conjunction with the retinal whole-mount technique, to elucidate factors critical in the pathology of murine cerebral malaria. CBA/T6 mice infected with PbA (PbA-CBA mice) demonstrated mild changes in vascular permeability as early as days 2–3, prior to the appearance on day 5 of cerebral symptoms, whereas mice with non-cerebral malaria did not show any vascular permeability changes until the very late stage of the disease (days 14–22). In the PbA infections, progressive deterioration of endothelial barrier properties, demonstrated by Evans' Blue leakage both generally and from specific focal areas, as well as a developing monocytosis and adherence of mononuclear cells to the endothelium of the retinal vessels continued until death (in CBA/T6 mice) or resolution (in DBA/2J mice). Adherent monocytes, particularly in PbA-CBA mice, were associated with reduced Hoechst staining of individual endothelial cells and a banking up proximally of both parasitized and non-parasitized blood cells in the small blood vessels, often with accompanying focal leakage of Evans' Blue from the retinal vessels. The occurrence and severity of these early changes in the microcirculation correlated with the subsequent development of cerebral symptoms. Monocyte margination appeared to be the most significant factor associated with the development of cerebral symptoms.

CBA/T6 and Balb/c mice inoculated with Plasmodium berghei ANKA strain (PbA) died from cerebral malaria 6–8 days post-inoculation. DBA/2J mice similarly inoculated developed a non-fatal cerebral malaria, with mild temporary cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop a cerebral malaria but died between days 15 and 22 from other malaria-related complications. These mouse strain/parasite strain combinations allow for detailed examination of factors critical in the pathology of murine cerebral malaria. Monastral Blue, a colloid dye, when injected intravascularly between days 0 and 2 into PbA-inoculated CBA (PbA-CBA) or Balb/c (PbA-Balb/c) mice prevented death from cerebral malaria. There was no evidence of increased vascular permeability at this stage. When Monastral Blue was injected between days 5 and 8, there was increased vascular permeability in the kidney, liver, lung, spleen and brain of PbA-CBA and PbA-Balb/c mice. Injection of Monastral Blue into these animals at this time also precipitated cerebral symptoms and death, but not in Pb-infected mice. Endothelial and mononuclear cells phagocytosed, and were coated with, the Monastral Blue particles when the dye was injected between days 5 and 8 into PbA-CBA and PbA-Balb/c mice. Control, uninfected mice did not demonstrate either of these features. Pb-infected mice only demonstrated coated mononuclear cells. Mononuclear cell attachment to the endothelium, increased vascular permeability and increased association of Monastral Blue particles with monocytes and endothelial cells were correlated with cerebral symptoms and death. Monastral Blue is thus a useful agent for studying the roles of mononuclear cells and endothelium in murine cerebral malaria.

The purpose of this study was to determine whether n-3 PUFA result in an effect on endothelial function that is in addition to that of acute exercise. For 4 weeks, male Sprague–Dawley rats were subjected to a diet based on n-3 PUFA or a standard diet. In each diet group, ten rats were submitted to an acute treadmill exercise while the remaining ten acted as sedentary controls. The running speed was progressively increased until the animals were exhausted. Endothelial function was then assessed by measuring isometric tension in rings of the thoracic aorta. In vessels precontracted with 0·1 μm-phenylephrine, responses to acetylcholine (ACh) were significantly improved following acute exercise in all diet groups. When PUFA supplementation was compared to the standard diet no significant difference was found in response to ACh, either at rest or after an acute exercise. Pretreatment of rings with Nω-nitro-l-arginine methyl esther (50 μm) inhibited the ACh-mediated vasorelaxation in all groups. Response to 10 μm-nifedipine, an L-type Ca2+ channel antagonist, was similarly enhanced after acute exercise in both standard and PUFA diets. Furthermore, response to 0·01 μm-nifedipine was significantly higher after acute exercise only in the PUFA diet. In conclusion, in our ‘healthy’ rat model with ‘normal’ baseline endothelial function, acute exercise improves response to ACh while PUFA supplementation alone or in combination with acute exercise has no effect on endothelium-dependent vasorelaxation. However, PUFA may potentiate the acute exercise effect on smooth muscle cell relaxation via L-type Ca2+ channel modifications.