The blood-brain barrier (BBB) is an important internal barrier. Herein, the electron microscope examination of duck BBB was performed during the brain development. Meanwhile, the genes/proteins of tight junctions (TJs) including zonula occludens-1, occludin, and claudin-5 in the duck brain were detected by Q-PCR and immunohistochemistry. The results showed the density of capillaries in the brain gradually increased during the embryonic period. The generation of the BBB and the specialization of its components occurred mainly in the embryonic stage. During this period, the endothelial cells (ECs) became thinner and pinocytic vesicles decreased; the TJs between EC membranes became longer and more electron-dense; the basement membrane surrounding ECs and pericytes gradually thickened; and the astrocyte foot processes appeared to wrap around the vessels. By the day of hatching (P1), the whole set of duck BBB structures was completely assembled and gradually improved in the subsequent growth process. Interestingly, compared with the cerebrum and cerebellum, the maturity level of the midbrain BBB was earlier seen during the embryonic stage. The expression of TJs increased during the embryonic period and remained stable by post-hatching. The study systematically investigated the histochemical and ultrastructural features of duck BBB during development and explored the corresponding relationship between structure and function.

Haemozoin is a by-product of haemoglobin digestion by intraerythrocytic malaria parasites, which induces immunologic responses on different tissues, including endothelial cells. In the present paper, the incubation of human microvascular endothelial cells with haemozoin significantly inhibited MTT reduction, a measure of cytotoxicity, without increasing the release of cytoplasmic lactate dehydrogenase. Moreover, haemozoin did not induce apoptosis or cell cycle arrest nor decreased the number of live cells, suggesting that cells viability itself was not affected and that the inhibition of MTT reduction was only apparent and probably due to accelerated MTT-formazan exocytosis. After 30 min of MTT addition, a significant increase in the % of cells exocytosing MTT formazan crystals was observed in haemozoin-treated cells compared with control cells. Such an effect was partially reversed by the addition of genistein, an inhibitor of MTT-formazan exocytosis. The rapid release of CXCL-8, a preformed chemokine contained in Weibel–Palade bodies, confirmed that haemozoin induces a perturbation of the intracellular endothelial trafficking, including the exocytosis of MTT-formazan containing vesicles. The haem moiety of haemozoin is responsible for the observed effect. Moreover, this work underlines that MTT assay should not be used to measure cytotoxicity induced by haemozoin and other methods should be preferred.

The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood–milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium.

The work was intended to explore the effect of the widely available cationic polymer polyethylenimine (PEI) on small diameter poly(ɛ-caprolactone) (PCL) blood vessel grafts. PEI was blended with PCL and electrospun into nanofibrous vascular scaffolds. The morphologies, wettabilities, mechanical properties, and biological activities of the PCL/PEI electrospun nanofibers were investigated. It was found that by increasing the content of PEI to 5% within the scaffolds, the fiber diameters decreased from 469.7 ± 212.1 to 282.5 ± 107.1 nm, the water contact angle was reduced from 126.6 ± 1.1° to 27.6 ± 3.9°, while the Young's modulus increased from 2.0 ± 0.2 to 4.1 ± 0.1 MPa, the suture retention strength increased from 4.2 ± 0.4 to 6.1 ± 0.7 N, and the burst pressure increased from 801.2 ± 14.1 to 926.2 ± 22.8 mmHg. The in vitro evaluations demonstrated that the nanofibers containing 2% PEI promoted the attachment and proliferation of human umbilical vein endothelial cells (HUVECs).

Amla (Emblica officinalis Gaertn.) has been used for many centuries in traditional Indian Ayurvedic formulations for the prevention and treatment of many inflammatory diseases. The present study evaluated the anti-inflammatory and anticoagulant properties of amla fruit extract. The amla fruit extract potentially and significantly reduced lipopolysaccharide (LPS)-induced tissue factor expression and von Willebrand factor release in human umbilical vein endothelial cells (HUVEC) in vitro at clinically relevant concentrations (1–100 μg/ml). In a leucocyte adhesion model of inflammation, it also significantly decreased LPS-induced adhesion of human monocytic cells (THP-1) to the HUVEC, as well as reduced the expression of endothelial-leucocyte adhesion molecule-1 (E-selectin) in the target cells. In addition, the in vivo anti-inflammatory effects were evaluated in a LPS-induced endotoxaemia rat model. Oral administration of the amla fruit extract (50 mg/kg body weight) significantly decreased the concentrations of pro-inflammatory cytokines, TNF-α and IL-6 in serum. These results suggest that amla fruit extract may be an effective anticoagulant and anti-inflammatory agent.

Flavanones are found specifically and abundantly in citrus fruits. Their beneficial effect on vascular function is well documented. However, little is known about their cellular and molecular mechanisms of action in vascular cells. The goal of the present study was to identify the impact of flavanone metabolites on endothelial cells and decipher the underlying molecular mechanisms of action. We investigated the impact of naringenin and hesperetin metabolites at 0·5, 2 and 10 μm on monocyte adhesion to TNF-α-activated human umbilical vein endothelial cells (HUVEC) and on gene expression. Except hesperetin-7-glucuronide and naringenin-7-glucuronide (N7G), when present at 2 μm, flavanone metabolites (hesperetin-3′-sulphate, hesperetin-3′-glucuronide and naringenin-4′-glucuronide (N4′G)) significantly attenuated monocyte adhesion to TNF-α-activated HUVEC. Exposure of both monocytes and HUVEC to N4′G and N7G at 2 μm resulted in a higher inhibitory effect on monocyte adhesion. Gene expression analysis, using TaqMan Low-Density Array, revealed that flavanone metabolites modulated the expression of genes involved in atherogenesis, such as those involved in inflammation, cell adhesion and cytoskeletal organisation. In conclusion, physiologically relevant concentrations of flavanone metabolites reduce monocyte adhesion to TNF-α-stimulated endothelial cells by affecting the expression of related genes. This provides a potential explanation for the vasculoprotective effects of flavanones.

We investigate the cytoprotective effects and the molecular mechanism of genistein in oxidative stress-induced injury using an endothelial cell line (EA.hy926). An oxidative stress model was established by incubating endothelial cells with H2O2. According to the present results, genistein pretreatment protected endothelial cells against H2O2-induced decreases in cell viability and increases in apoptosis. Genistein also prevented the inhibition of B-cell lymphoma 2 and the activation of caspase-3 induced by H2O2. Genistein increased superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels and attenuated the decrease in these antioxidants during oxidative stress. We also found that genistein induced the promoter activity of both nuclear factor erythroid 2-related factor 2 (Nrf2) and PPARγ. Additionally, genistein induced the nuclear translocation of Nrf2 and PPARγ. While genistein caused the up-regulation of both Nrf2 and PPARγ, it also activated and up-regulated the protein expression and transcription of a downstream protein, haem oxygenase-1 (HO-1). Moreover, the use of Nrf2 small interfering RNA transfection and HO-1- or PPARγ-specific antagonists (Znpp and GW9662, respectively) blocked the protective effects of genistein on endothelial cell viability during oxidative stress. Therefore, we conclude that oxidative stress-induced endothelial cell injury can be attenuated by treatment with genistein, which functions via the regulation of the Nrf2 and PPARγ signalling pathway. Additionally, the endogenous antioxidants SOD, CAT and GSH appear to play a role in the antioxidant activity of genistein. The present findings suggest that the beneficial effects of genistein involving the activation of cytoprotective antioxidant genes may represent a novel strategy in the prevention and treatment of cardiovascular endothelial damage.

Extracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors—platelet derived growth factor-BB and heparin binding-epidermal growth factor—are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formation in vitro and in vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.

In the present study, we examined the effect of repeated and long-term treatment with resveratrol on NO production in endothelial cells as a model of routine wine consumption. Repeated treatment with resveratrol for 5 d resulted in an increase in endothelial NO synthase (eNOS) protein content and NO production in human umbilical vein endothelial cell (HUVEC) in a concentration-dependent manner. A significant increase in functional eNOS protein content was observed with resveratrol, even at 50 nm. In contrast, eNOS phosphorylation was not stimulated and inducible NO synthase (iNOS) was not detected after resveratrol treatment. Both eNOS protein and mRNA expression were promoted by 50 nm-resveratrol in a time-dependent manner. Increased eNOS mRNA expression in response to resveratrol was not decreased by an oestrogen receptor (ER) antagonist ICI182780, a PPARα inhibitor MK886 or a sirtuin inhibitor Salermide. However, a combination of ICI182780 and MK886 significantly inhibited resveratrol-induced eNOS mRNA expression. Salermide had no effect even in the presence of ICI182780 or MK886. These results demonstrate that resveratrol within the physiological range increases eNOS mRNA and protein expression through ER and PPARα activation, thereby promoting NO production in endothelial cells. eNOS induction might result from the accumulative effect of nanomolar concentrations of resveratrol. The present study results can account in part for the observation that cardiovascular benefits of red wine are experienced with routine consumption, but not with acute consumption.

Previous cell culture-based studies have shown potential health beneficial effects on gene expression of dietary polyphenols, including those found in red wine and green tea. However, these studies have tended to use higher concentrations (2–100 μm) than those observed in blood (0·1–1 μm) after consuming polyphenol-rich foods or beverages. The present study investigated effects of physiological concentrations of different classes of dietary polyphenol on the expression of genes important in cardiovascular health (endothelial NO synthase (eNOS), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF)) by cultured vascular endothelial cells (human umbilical vein endothelial cells) in the absence or presence of H2O2. Resveratrol and quercetin (0·1–1 μm) increased eNOS and VEGF mRNA expression particularly in the absence of H2O2 (50 μm) and decreased H2O2-induced ET-1 mRNA expression (P < 0·001 for polyphenol × H2O2 interactions). Similarly, resveratrol and quercetin decreased endothelin secretion into the media, blocking the stimulatory effect of 50 μm-H2O2 (P < 0·001 for polyphenol × H2O2 interaction). Of the nine other polyphenols tested, only epigallocatechin gallate had similar effects on both the eNOS and ET-1 mRNA expression, but to a lesser extent than resveratrol at an equimolar concentration (0·1 μm). The observed effects on gene expression would be expected to result in vasodilation and thereby reduced blood pressure. Since only three of the eleven polyphenols tested had biological activity, it is unclear whether particular structures are important or whether the effects might relate to the relatively high antioxidant capacities of the three active polyphenols.

We have studied the effect of human serum, collected after red wine consumption (RWS), on TNF-α-dependent activation of transcription factors (NF-κB, activator protein-1 (AP-1) and cAMP response element-binding proteins) and on the expression of selected genes involved in cell adhesion or fibrinolysis processes in human primary endothelial cells (human umbilical vein endothelial cells (HUVEC)). Our data indicate that RWS containing RW metabolites, isolated after 40 min from an acute consume of wine (5 ml/kg body weight), induces nuclear translocation of NF-κB and AP-1 in the absence of any further stimulus. On the other hand, TNF-α treatment in the presence of RWS is associated with a delay in transcription factor activation and to a negative modulation on the expression of specific genes. Moreover, RWS stimulates c-jun binding to the tissue-type plasminogen activator cAMP responsive element consensus site modulating the expression of the specific gene downstream. These results confirm that RW metabolites affect the activity of different transcription factors playing an important preconditioning role in the modulation of the inflammatory pathway in endothelial cells. This is the first report on the effects of a complex food matrix, on the molecular mechanisms associated with inflammatory response in HUVEC cultured in condition that reproduces the physiological environment occurring in vivo.

The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2′7′-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

Factors which affect adhesion of cultured Trypanosoma congolense bloodstream forms to mammalian feeder cells have been examined. Using an in vitro binding assay, the initial events following interaction of trypanosomes with bovine aorta endothelial (BAE) cells were monitored by both light- and electron microscopy. Metabolic inhibitors and other biochemicals were incubated with either cells or parasites, to test whether any inhibited the process. Our findings suggest that adhesion of the parasites is an active process requiring metabolic energy from the trypanosomes, but not from endothelial cells. We also provide data suggesting that T. congolense bloodstream forms possess a lectin-like domain, localized at distinct sites on their flagellar surface, which interacts with specific carbohydrate receptors, most likely sialic acid residues, on the endothelial cell plasma membrane. We also suggest that the cytoskeletal protein actin is probably involved in this interaction.

Adhesion of parasitized red blood cells to vascular endothelium is thought to play an important role in the development of the ischaemic complications associated with severe falciparum malaria. Using a novel, flow-based assay, we have investigated the adhesion of parasitized red blood cells to formalin-fixed human umbilical vein endothelial cells (HUVEC), for isolates obtained from 32 Gambian subjects with mild or severe falciparum malaria. Red cells infected with wild strains of Plasmodium falciparum were able to adhere to HUVEC under physiologically relevant flow conditions, but the level of adhesion was highly variable, ranging from 1 to 688 adherent cells per mm2 of HUVEC. Within isolates, some adherent parasitized cells remained stationary, whilst other formed less stable interactions and rolled slowly over the cell surface. There was no significant difference in adhesion of parasitized cells between isolates obtained from mild or severe cases of malaria, although a subset of isolates did show very high levels of adhesion. The results suggest that there is not a simple relationship between the adhesion of parasitized cells to cultured endothelial cells (presumably via the receptor ICAM-1) and the clinical severity of the disease, although variation in microvascular adhesion in vivo may still be a determinant of ischaemic complications.

So far, a protective influence of phytosterols on the human organism and atherogenesis has been suggested. Most studies have concentrated on the cytotoxic efficacy of phytosterols on cancer cells. However, there are only a few reports showing their influence on normal cells. The aim of the present study was to determine whether dietary plant sterols and their thermal processing products could influence the viability of normal, abdominal endothelial cells that play a crucial role in atherogenesis. Thus, we studied the effect of rapeseed oil-extract components, β-sitosterol, cholesterol and their epoxy-derivatives, 5α,6α-epoxy-β-sitosterol and 5α,6α-epoxycholesterol, on the proliferation and viability of human abdominal aorta endothelial cells HAAE-2 in vitro. We showed strong cytotoxic properties of β-sitosterol in HAAE-2 cells (half maximal inhibitory concentration (IC50) = 1·99 (sem 0·56) μm) and, interestingly, a weaker cytotoxic effect of 5α,6α-epoxy-β-sitosterol (IC50>200 μm). Moreover, we observed a significantly stronger cytotoxic activity of β-sitosterol than cholesterol (IC50 = 8·99 (sem 2·74) μm). We also revealed that β-sitosterol as well as cholesterol caused apoptosis, inducing caspase-3 activity in the cells (60 % increase compared with control cells) that corresponded to the DNA fragmentation analysis in a terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) study. Although absorption of plant sterols is low compared with cholesterol, they can still influence other physiological functions. Since they effectively reduce serum LDL-cholesterol and atherosclerotic risk but also decrease the viability of cancer cells as well as normal cells in a time- and dose-dependent manner in vitro, their influence on other metabolic processes remains to be elucidated.

Diabetes mellitus is an important risk factor for CVD. A previous study showed that high glucose induced the apoptosis of human umbilical vein endothelial cells (HUVEC) via the sequential activation of reactive oxygen species, Jun N-terminal kinase (JNK) and caspase-3. The apoptosis cascade could be blocked by ascorbic acid at the micromolar concentration (100 μm). In addition to ascorbic acid, quercetin, the most abundant dietary flavonol, has been recently actively studied in vascular protection effects due to its antioxidant effect at low micromolar concentrations (10–50 μm). Quercetin sulfate/glucuronide, the metabolite of quercetin in blood, however, has been rarely evaluated. In the present study, we investigated the effect of quercetin sulfate/glucuronide on the prevention of high glucose-induced apoptosis of HUVEC. HUVEC were treated with media containing high glucose (33 mm) in the presence or absence of ascorbic acid (100 μm) or quercetin sulfate/glucuronide (100 nm, 300 nm and 1 μm). For the detection of apoptosis, a cell death detection ELISA assay was used. The level of intracellular H2O2 was measured by flow cytometry. JNK and caspase-3 were evaluated by a kinase activity assay and Western blot analysis. The results showed that high glucose-induced apoptosis was inhibited by quercetin sulfate/glucuronide in a dose-dependent manner. The effect of quercetin sulfate/glucuronide on H2O2 quenching, inhibition of JNK and caspase-3 activity at the nanomolar concentration (300 nm) was similar to that of ascorbic acid at the micromolar concentration (100 μm). The findings of the present study may shed light on the pharmacological application of quercetin in CVD.

We investigated the effects of the flavonols kaempferol and quercetin on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), endothelial cell selectin (E-selectin), inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), and on the activation of the signalling molecules NF-κB and activator protein-1 (AP-1), induced by a cytokine mixture in cultured human umbilical vein endothelial cells. Inhibition of reactive oxygen and nitrogen species generation did not differ among both flavonols at 1 μmol/l but was significantly stronger for kaempferol at 5–50 μmol/l. Supplementation with increasing concentrations of kaempferol substantially attenuated the increase induced by the cytokine mixture in VCAM-1 (10–50 μmol/l), ICAM-1 (50 μmol/l) and E-selectin (5–50 μmol/l) expression. A significantly inhibitory effect of quercetin on VCAM-1 (10–50 μmol/l), ICAM-1 (50 μmol/l) and E-selectin (50 μmol/l) expression was also observed. Expression of adhesion molecules was always more strongly inhibited in kaempferol-treated than in quercetin-treated cells. The inhibitory effect on iNOS and COX-2 protein level was stronger for quercetin at 5–50 μmol/l. The effect of kaempferol on NF-κB and AP-1 binding activity was weaker at high concentrations (50 μmol/l) as compared with quercetin. The present study indicates that differences exist in the modulation of pro-inflammatory genes and in the blockade of NF-κB and AP-1 by kaempferol and quercetin. The minor structural differences between both flavonols determine differences in their anti-inflammatory properties and in their efficiency in inhibiting signalling molecules.

Cardiopulmonary bypass makes a mess of homeostasis. Extracorporeal perfusion produces pulseless flow without physiologic controls, showers microemboli throughout the body, and brings blood diluted with additives to an enzymatic boil. Much of the morbidity associated with open heart surgery is due to contact of blood with the synthetic surfaces of the extracorporeal perfusion circuit. Bypass temporarily impairs function of every organ, alters concentrations of over 25 vasoactive substances within the plasma, and causes massive retention of fluid. In short, cardiopulmonary bypass makes patients sick.