Differential modulation of transendothelial electrical resistance by TRPV4 agonists is mediated by apoptosis and/or necrosis.
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ABSTRACT:
SUBMITTER: Pairet N
PROVIDER: S-EPMC6804647 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The blood-brain barrier (BBB) represents the tightest endothelial barrier within the cardiovascular system characterized by very low ionic permeability. Our aim was to describe the setups, electrodes, and instruments to measure electrical resistance across brain microvessels and culture models of the BBB, as well as critically assess the influence of often neglected physical and technical parameters such as temperature, viscosity, current density generated by different electrode types, surface size, circumference, and porosity of the culture insert membrane. We demonstrate that these physical and technical parameters greatly influence the measurement of transendothelial electrical resistance/resistivity (TEER) across BBB culture models resulting in severalfold differences in TEER values of the same biological model, especially in the low-TEER range. We show that elevated culture medium viscosity significantly increases, while higher membrane porosity decreases TEER values. TEER data measured by chopstick electrodes can be threefold higher than values measured by chamber electrodes due to different electrode size and geometry, resulting in current distribution inhomogeneity. An additional shunt resistance at the circumference of culture inserts results in lower TEER values. A detailed description of setups and technical parameters is crucial for the correct interpretation and comparison of TEER values of BBB models.
| S-EPMC8231150 | biostudies-literature
Project description:Good in vitro blood-brain barrier (BBB) models that mimic the in vivo BBB phenotype are essential for studies on BBB functionality and for initial screening in drug discovery programmes, as many potential therapeutic drug candidates have poor BBB permeation. Difficulties associated with the availability of human brain tissue, coupled with the time and cost associated with using animals for this kind of research have led to the development of non-human cell culture models. However, most BBB models display a low transendothelial electrical resistance (TEER), which is a measure of the tightness of the BBB. To address these issues we have established and optimised a robust, simple to use in vitro BBB model using porcine brain endothelial cells (PBECs). The PBEC model gives high TEER without the need for co-culture with astrocytes (up to 1300 O cm(2) with a mean TEER of ~800 O cm(2)) with well organised tight junctions as shown by immunostaining for occludin and claudin-5. Functional assays confirmed the presence of high levels of alkaline phosphatase (ALP), and presence of the efflux transporter, P-glycoprotein (P-gp, ABCB1). Presence of the breast cancer resistance protein (BCRP, ABCG2) was confirmed by TaqMan real-time RT-PCR assay. Real-time RT-PCR assays for BCRP, occludin and claudin-5 demonstrated no significant differences between batches of PBECs, and also between primary and passage 1 PBECs. A permeability screen of 10 compounds demonstrated the usefulness of the model as a tool for drug permeability studies. Qualitative and quantitative results from this study confirm that this in vitro porcine BBB model is reliable and robust; it is also simpler to generate than most other BBB models. This article is part of a Special Issue entitled Electrical Synapses.
| S-EPMC3694297 | biostudies-literature
Project description:Three-dimensional (3D) cell cultures and organs-on-a-chip have been developed to construct microenvironments that resemble the environment within the human body and to provide a platform that enables clear observation and accurate assessments of cell behavior. However, direct observation of transendothelial electrical resistance (TEER) has been challenging. To improve the efficiency in monitoring the cell development in organs-on-a-chip, in this study, we designed and integrated commercially available TEER measurement electrodes into an in vitro blood-brain barrier (BBB)-on-chip system to quantify TEER variation. Moreover, a flowing culture medium was added to the monolayered cells to simulate the promotion of continuous shear stress on cerebrovascular cells. Compared with static 3D cell culture, the proposed BBB-on-chip integrated with electrodes could measure TEER in a real-time manner over a long period. It also allowed cell growth angle measurement, providing instant reports of cell growth information online. Overall, the results demonstrated that the developed system can aid in the quantification of the continuous cell-pattern variations for future studies in drug testing.
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Project description:BackgroundAdiponectin's effects on systemic physiology and cell-specific responses are well-defined, but little is known about how this insulin-sensitizing and anti-inflammatory adipokine reaches its target cells. All molecules face active and passive transport limitations, but adiponectin is particularly noteworthy due to the diverse size range and high molecular weights of its oligomers. Additionally, its metabolic target organs possess a range of endothelial permeability.MethodsFull-length recombinant murine adiponectin was produced and oligomer fractions isolated by gel filtration. Adiponectin complex sizes were measured by dynamic light scattering to determine Stokes radii. Transendothelial transport of purified oligomers was quantitatively assessed under a number of different conditions in vitro using murine endothelial cells and in vivo using several mouse models of altered endothelial function.ResultsAdiponectin oligomers exhibit large transport radii that limit transendothelial transport. Oligomerization is a significant determinant of flux across endothelial monolayers in vitro; low molecular weight adiponectin is preferentially transported. In vivo sampled sera from the heart, liver, and tail vein demonstrated significantly different complex distribution of lower molecular weight oligomers. Pharmacological interventions, such as PPARγ agonist treatment, differentially affect adiponectin plasma clearance and tissue uptake. Exercise induces enhanced adiponectin uptake to oxidative skeletal muscles, wherein adiponectin potently lowers ceramide levels. In total, endothelial barriers control adiponectin transport in a cell- and tissue-specific manner.ConclusionsAdiponectin oligomer efficacy in a given tissue may therefore be endothelial transport mediated. Targeting endothelial dysfunction in the metabolic syndrome through exercise and pharmaceuticals may afford an effective approach to increasing adiponectin's beneficial effects.
| S-EPMC3932731 | biostudies-literature
Project description:In this paper, we describe the use of 3D printed devices for both static and flow studies that contain electrospun collagen scaffolds and can accommodate transepithelial/transendothelial electrical resistance (TEER) measurements. Electrospinning was used to create the collagen scaffold, followed by an optimized 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) cross-linking procedure to produce stable collagen fibers that are similar in size to fibers in vivo. LC/MS was used to study the leaching of solvent and NHS from the scaffold, with several rinsing steps being shown to eliminate the leaching and promote the culture of Madin-Darby Canine Kidney (MDCK) epithelial cells on the scaffold. Both static and flow 2-part devices were successfully fabricated by 3D printing using either VeroClear or MED610 material (PolyJet printing) and assembling the scaffold between laser cut Teflon gaskets. The devices were designed to easily accommodate commonly used STX2 chopstick electrodes for TEER measurements. A detailed comparison was made between the use of collagen scaffolds vs other electrospun materials for cell culture. The collagen extracellular matrix model displayed a high barrier functionality for up to 7 days. In addition, a different 3D printed device with a collagen scaffold is described to incorporate continuous flow and replenishment of media under the cell layer in a manner that also enables periodic recording of TEER measurements. Overall, this work shows that the combination of biological ECM materials such as collagen into microfluidic devices that incorporate flow have great potential to form more realistic cell culture models in areas such as blood brain barrier research.
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Project description:A robust, nanoelectromechanical switch is proposed based upon an asymmetric pendant moiety anchored to an organic backbone between two C60 fullerenes, which in turn are connected to gold electrodes. Ab initio density functional calculations are used to demonstrate that an electric field induces rotation of the pendant group, leading to a nonlinear current-voltage relation. The nonlinearity is strong enough to lead to negative differential resistance at modest source-drain voltages.
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Project description:Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell-bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-alpha is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-alpha receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-alpha. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.
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Project description:Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.
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Project description:It is known that infection with different pathogens, including helminths, can alter the progression of malignant or other diseases. We studied the effect of chronic Trichinella spiralis infection or muscle larvae excretory-secretory (ES L1) antigens on the malignant tumour growth in the mouse melanoma model system in vivo and in vitro. Our results confirmed that chronic infection with T. spiralis possesses the capacity to slow down the progression of tumour growth, resulting in an impressive reduction in tumour size. We found that the phenomenon could, at least partially, be related to a lower level of tumour necrosis compared to necrosis present in control animals with progressive malignancy course. An increased apoptotic potential among the low percentage of cells within the total tumour cell number in vivo was also observed. ES L1 antigen, as a parasitic product that is released during the chronic phase of infection, reduced the survival and slightly, but significantly increased the apoptosis level of melanoma cells in vitro. Our results imply that powerful Trichinella anti-malignance capacity does not rely only on necrosis and apoptosis but other mechanisms through which infection or parasite products manipulate the tumor establishment and expansion should be considered.
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