Characterisation of anti-inflammatory and pro-inflammatory influences on blood-brain barrier phenotype using an in vitro human brain microvascular endothelial model

Introduction: Within the central nervous system, the cerebral endothelial cells have highly specialised structural and functional properties. Compared to the endothelium of the periphery, brain endothelial cells are phenotypically unique in that they have enhanced inter-endothelial junction complexes that provide a highly restrictive yet controlled paracellular barrier for the brain from the constituents of the circulation, effectively embodying a blood-brain barrier. Disruption of the proteins that form these junctional complexes (i.e. occludin, claudin-5, VE-cadherin and ZO-1) has therefore been implicated in several CNS disease states. Endothelial functions can be modulated by both local and systemic environmental factors. The ability of the endothelium to sense its humoral and biomechanical environment and modify its functional phenotype accordingly plays a pivotal role in the maintenance of vascular homeostasis or the development of vascular pathology. However, much remains unknown about how anti- and pro- inflammatory stimuli modulate blood-brain barrier phenotype at the molecular level, thus framing the context of this thesis. In the current study, in view of the opposing physiological and pathophysiological actions demonstrated thus far in similar yet distinctly different in vivo and in vitro endothelial models, we propose to investigate how anti-inflammatory laminar shear stress and pro-inflammatory cytokines can differentially modulate BBB phenotype, with functional consequences for endothelial homeostasis, particularly with regards to the coordination and maintenance of the BBB interendothelial junction complex. Results: Exposure of cultured human brain microvascular endothelial cells (HBMvECs) to physiological levels of laminar shear stress (8 dynes cm-2) resulted in a reduction in monolayer permeability. The transcription and translation of occludin, claudin-5, VE-cadherin and ZO-1 were all upregulated, with an enhanced localisation of said proteins at the cell-cell junctions. Moreover, our studies demonstrated laminar shear stress caused a substantial reduction in pTyr and pThr levels on each protein, with functional consequences for barrier integrity as determined using dephostatin and genistein. In addition, laminar shear stress promoted a number of anti-inflammatory mechanisms, which, in the presence of inflammatory cytokines, could partially ameliorate the injurious effects of the latter. Sophisticated co-IP techniques, coupled with mass spectrometry analysis, identified several isoforms of the 14-3-3 family of proteins as intracellular binding partners to the interendothelial junction complex. This family of proteins was implicated in contributing to the protective, barrier- stabilising effect of laminar shear, whilst inhibition of their activity exacerbated cytokine injury. In parallel studies, exposure of cultured HBMvECs to pathophysiological levels of inflammatory cytokines, TNF-alpha and IL-6, resulted in an increase in monolayer permeability, an effect directly attributable to the reduction in the transcription and translation of the aforementioned tight and adherens junction proteins. Moreover, each cytokine caused a substantial increase in pTyr/Thr levels on each protein. Noteworthy, all cytokine effects were dose- and time-dependent. This increase in injury can be possibly attributed to a correlative reduction in anti-inflammatory mechanisms coupled with a correlative increase in the production and release of inflammatory mediators such as ROS and other cytokines such as IL-6 into the local environment. Conclusions: Physiological levels of laminar shear stress and pathophysiological levels of inflammatory cytokines, TNF-alpha and IL-6, modulate the expression and post-translational properties of BBB tight and adherens junction proteins in an opposing manner. Inflammatory cytokines mediate their effects in part through the induced release of injury potentiating agents such as ROS and IL-6. A novel role for 14-3-3 isoforms in the modulation of interendothelial junction assembly is also implicated in these studies

Pulmonary endothelial permeability and tissue fluid balance depend on the viscosity of the perfusion solution

Fluid filtration in the pulmonary microcirculation depends on the hydrostatic and oncotic pressure gradients across the endothelium and the selective permeability of the endothelial barrier. Maintaining normal fluid balance depends both on specific properties of the endothelium and of the perfusing blood. Although some of the essential properties of blood needed to prevent excessive fluid leak have been identified and characterized, our understanding of these remains incomplete. The role of perfusate viscosity in maintaining normal fluid exchange has not previously been examined. We prepared a high-viscosity perfusion solution (HVS) with a relative viscosity of 2.5, i.e., within the range displayed by blood flowing in vessels of different diameters in vivo (1.5–4.0). Perfusion of isolated murine lungs with HVS significantly reduced the rate of edema formation compared with perfusion with a standard solution (SS), which had a lower viscosity similar to plasma (relative viscosity 1.5). HVS did not alter capillary filtration pressure. Increased endothelial shear stress produced by increasing flow rates of SS, to mimic the increased shear stress produced by HVS, did not reduce edema formation. HVS significantly reduced extravasation of Evans bluelabeled albumin compared with SS, indicating that it attenuated endothelial leak. These findings demonstrate for the first time that the viscosity of the solution perfusing the pulmonary microcirculation is an important physiological property contributing to the maintenance of normal fluid exchange. This has significant implications for our understanding of fluid homeostasis in the healthy lung, edema formation in disease, and reconditioning of donor organs for transplantation

TRAIL inhibits oxidative stress in human aortic endothelial cells exposed to pro-inflammatory stimuli

Studies suggest that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has vasoprotective potential, as low levels of TRAIL cause accelerated vascular calcification, whereas exogenous TRAIL administration exhibits anti-atherosclerotic activity. The mechanism of TRAIL-mediated vasoprotection remains unclear. We studied the effects of TRAIL (100 ng/ml) on human aortic endothelial cells (HAECs) exposed to pro-atherogenic conditions; (a) oscillatory shear stress (±10 dynes/cm2) using the ibidi µ-slide fluidic system; (b) pro-inflammatory injury, that is, tumor necrosis factor alpha (TNF-α, 100 ng/ml) and hyperglycemia (30 mM d-glucose). End-points examined included inflammatory gene expression and reactive oxygen species (ROS) formation. TRAIL shifted the net gene expression toward an antioxidant phenotype in HAECs exposed to oscillatory shear stress. TRAIL significantly reduced ROS formation in HAECs exposed to both TNF-α and hyperglycemia. Therefore, TRAIL appears to confer atheroprotective effects on the endothelium, at least in part, by reducing oxidative stress

Surface-to-Volume Ratio Affects the Toxicity of Nanoinks in Daphnids

The Organization for Economic Co-operation and Development (OECD) has set widely used guidelines that are used as a standardized approach for assessing toxicity in a number of species. Given that various studies use different experimental setups, it is difficult to compare findings across them as a result of the lack of a universally used setup in nano-ecotoxicology. For freshwater species, Daphnia magna, a commonly used filter feeding crustacean, can generate significant molecular information in response to pollutant exposure. One factor that has an effect in toxicity induced from nanomaterials in daphnids is the surface-to-volume ratio of the exposure vessels; however, there is limited information available about its impact on the observed effect of exposure. In this study, daphnids were exposed to silver nanoparticle ink in falcon tubes and Petri dishes for 24 h. Toxicity curves revealed differences in the observed mortality of daphnids, with animals exposed in Petri dishes displaying significantly higher mortality. Differences in the activities of a number of key enzymes involved in the catabolism of macromolecules and phosphate were also observed across the exposure setups, indicating possible differences in the toxicity mechanism of silver nano-ink. Understanding the impact of factors relevant to experimental setups in ecotoxicology can increase the reproducibility of testing, and also reduce experimental costs, time, generated waste, and daphnids used in research

A Role for Syntaxin 3 in the secretion of IL-6 from Dendritic Cells following activation of Toll-like Receptors

The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNARE) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of eighteen SNARE proteins in DCs in response to activation with a panel of three toll-receptor (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease

The emerging role of SNARE proteins in dendritic cell function

Dendritic cells (DCs) provide an essential link between innate and adaptive immunity. At the site of infection, antigens recognised by DCs via pattern-recognition receptors such as Toll like receptors (TLR), initiate a specific immune response. Depending on the nature of the antigen, DCs secrete distinct cytokines with which they orchestrate homeostasis and pathogen clearance. Dysregulation of this process can lead to unnecessary inflammation which can result in a plethora of inflammatory diseases. Therefore, the secretion of cytokines from DCs is tightly regulated and this regulation is facilitated by highly conserved trafficking protein families. These proteins control the transport of vesicles from the golgi complex to the cell surface and between organelles. In this review we will discuss the role of soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs) in DCs both as facilitators of secretion and as useful tools to determine the pathways of secretion through their definite locations within the cells and inherent specificity in opposing binding partners on vesicles and target membranes. The role of SNAREs in DC function may present an opportunity to explore these proteins as novel targets in inflammatory disease

Bioactive Ingredients from Dairy-Based Lactic Acid Bacterial Fermentations for Functional Food Production and Their Health Effects

Lactic acid bacteria are traditionally applied in a variety of fermented food products, and they have the ability to produce a wide range of bioactive ingredients during fermentation, including vitamins, bacteriocins, bioactive peptides, and bioactive compounds. The bioactivity and health benefits associated with these ingredients have garnered interest in applications in the functional dairy market and have relevance both as components produced in situ and as functional additives. This review provides a brief description of the regulations regarding the functional food market in the European Union, as well as an overview of some of the functional dairy products currently available in the Irish and European markets. A better understanding of the production of these ingredients excreted by lactic acid bacteria can further drive the development and innovation of the continuously growing functional food market

Antioxidant strategies.

<p>Summary of antioxidant strategies employed to attenuate the endothelial permeabilizing effects of proinflammatory cytokines. Key: ROS, reactive oxygen species; O₂⁻, superoxide; H₂O₂, hydrogen peroxide; SOD, superoxide dismutase; CAT, catalase; NAC, <i>N</i>-acetylcysteine; APO, apocynin; NSC23766, Rac-1 inhibitor; gp91⁻, gp91 siRNA; p47⁻, p47 siRNA.</p

Exopolysaccharides of Lactic Acid Bacteria: Production, Purification and Health Benefits towards Functional Food

Lactic acid bacteria (LAB) are capable of synthesising metabolites known as exopolysaccharides (EPS) during fermentation. Traditionally, EPS plays an important role in fermented dairy products through their gelling and thickening properties, but they can also be beneficial to human health. This bioactivity has gained attention in applications for functional foods, which leads them to have prebiotic, immunomodulatory, antioxidant, anti-tumour, cholesterol-lowering and anti-obesity activity. Understanding the parameters and conditions is crucial to optimising the EPS yields from LAB for applications in the food industry. This review provides an overview of the functional food market together with the biosynthesis of EPS. Factors influencing the production of EPS as well as methods for isolation, characterisation and quantification are reviewed. Finally, the health benefits associated with EPS are discussed