Endothelial cell monolayer barrier

MV Shah, KL Audus, RT Borchardt. The application of bovine brain micro vessel endothelial-cell monolayers grown onto polycarbonate membranes in vitro to estimate the potential permeability of solutes through the blood-brain barrier. Pharm Res 6 624-627, 1989. 

Audus KL, Borchardt RT. Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier. Ann N Y Acad Sci 1987 507 9-18. 

Two compounds had to be excluded from the data set. They were outliers (1.3 log units deviation) as also found in the analysis by Abraham et al. [26], It must also be remembered when considering the derived equations that log BB is a complex parameter that encompasses brain partitioning and permeability and may also depend on other processes such as metabolism, active transport, and so forth. Thus, the standard deviations of these determinations may fall within the range of the mean values. The authors could demonstrate that the derived equation could also estimate log BB outside of the training data set. Therefore, the value of the above correlation with the solvation free energy lies in its power to rank compounds for their ability to cross the blood-brain barrier before synthesis. Interestingly, it was also shown by Lombardo et al. [71] that the calculated AG°W correlated well with the determined permeability coefficient, PC, using endothelial cell monolayers from bovine brain mi-... 

Mackic JB, Stins M, McComb JG, Calero M, Ghiso J, et al. 1998. Human blood-brain barrier receptors for Alzheimer s amyloid-beta 1-40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J. Clin. Invest. 1024 734 13... 

Plateel, M., Dehouck, M.P., Torpier, G., Cecchelh, R., and Teissier, E., Hypoxia increases the susceptibility to oxidant stress and the permeabihty of the blood-brain barrier endothelial cell monolayer, /. Neurochem., 65(5), 2138,1995. 

The presence of only two dietary carotenoids in the retina, lutein and zeaxanthin, out of about 14 normally present in the plasma indicates their highly specific uptake and retention (Bernstein et al., 2001 Bone and Landrum, 1992 Bone et al., 1988,1997,1993 Davies and Morland, 2004 Khachik et al., 1997, 2002). The retina-blood barrier is formed by the tight zonulae occludentes of the endothelial cells in the inner retina and of the RPE, a monolayer of cells which separates the outer retina from its choroidal blood supply (Strauss, 2005). 

Freshly isolated or subcultured brain microvascular endothelial cells offer a notable in vitro tool to study drug transport across the blood-brain barrier. Cells can be grown to monolayers on culture plates or permeable membrane supports. The cells retain the major characteristics of brain endothelial cells in vivo, such as the morphology, specific biochemical markers of the blood-brain barrier, and the intercellular tight junctional network. Examples of these markers are y-glutamyl transpeptidase, alkaline phosphatase, von-Willebrandt factor-related antigen, and ZO-1 tight junctional protein. The methods of... 

Figure 2.5. Setup for in vitro measurement of blood-brain barrier permeability with a co-culture of bovine brain microvascular endothelial cells (BBMEC) and an astro ioma cell line, C6. The BBMEC are grown on top of a filter insert. The C6 cells are either grown on the opposite side of the filter or on the bottom of the wells. Transport across the BBMEC monolayer is measured by adding the test substance to the upper chamber and sampling from the lower chamber. The tightness of the monolayer is also characterized by the transendothelial electrical resistance (TEER). Courtesy of T. Abbruscato.

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

It is not clear why LA and none of the saturated fatty acids that were studied disrupted endothelial barrier function. The injurious effects of LA on cultured endothelial cells may be mediated, in part, by the induction of peroxisomes and, thus, by excessive hydrogen peroxide formation. In addition, enrichment of endothelial lipids with selective fatty acids can modify specific cellular lipid pools and alter the morphology of cultured cell monolayers. Such fatty acid-mediated compositional changes may be sufficient to alter membrane properties, e.g., fluidity and activities of membrane-bound enzymes. One may speculate from these and other data that high dietary intakes of certain unsaturated fatty acids, such as LA, might not be entirely safe. 

Terasaki T, Takakuwa S, Saheki A, Moritaui S, Shimura T, Tabata S, Tsuji A (1992) Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier Studies with monolayers of primary cultured bovine brain capillary endothelial cells. PharmRes 9 529—534. 

Atherosclerosis is accepted as a common mechanism underlying all CVDs [2], Atherosclerosis is a disease of large and medium-sized arteries. It affects all three coats of the arterial wall in its more advanced stages. The arterial wall consists of three layers intima, media, and adventitia. The most inner luminal part of the intima is a monolayer of endothelial cells lining the whole wall. The intact endothelial layer is a selective barrier for plasma lipids and also has antitrombotic properties [3,7]. The pathogenesis of atherosclerosis can be divided into three main stages. 

The SLC transporters mediate either drug uptake or efflux, whereas ABC transporters mediate only unidirectional efflux. Asymmetrical transport across a monolayer of polarized ceUs, such as the epithelial and endothelial cells of brain capillaries, is called vectorial transport (Figure 2-5). Vectorial transport is important in the efficient transfer of solutes across epithelial or endothelial barriers it plays a major role in hepatobiliary and urinary excretion of drugs from the blood to the lumen and in the intestinal absorption of drugs and nutrients. In addition, efflux of drugs from the brain via brain endothelial cells and brain choroid plexus epithelial cells involves vectorial transport. 

Another barrier of interest for drug delivery studies is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells of brain capillaries. The primary characteristics of the BBB are its high resistance to chemical diffusion and transport due to the presence of complex tight junctions that inhibit paracellular transport and its low endocytic activity. Several in vitro models of the BBB have been developed, and several authors have reviewed the models and their possible uses as permeability and toxicity screens (Reinhardt and Gloor, 1997 Gumbkton and Audus, 2001 Lundquist and Renftel, 2002). The most common in vitro BBB model consists of a monolayer of primary isolated brain capillary endothelial cells, primary isolated endothelial cells from elsewhere in the body, or an endothelial cell line cultured on a membrane insert. The endothelial cells are often cocultured with astrocytes or astroglial cells. In cocultures, the barrier properties of the BBB model increase. 

Current technologies provide for availability of cell culture models from a selection of the significant tissue barriers as either primary cell culture systems or continuous cell line cultures. A number of endothelial and epithelial cell systems can now be grown onto permeable supports as monolayer systems to facilitate transcellular transport. These cell culture models provide powerful tools for the pharmaceutical chemist to characterize fundamental cellular transport mechanisms at the biochemical and molecular levels and effective screening systems to facilitate appropriate drug design and development. Moreover, they all have applications in... 

Occludin phosphorylation may provide a molecular mechanism to control barrier properties. Studies from our group have demonstrated that both VEGF and shear stress induce permeability across endothelial monolayers associated with a rapid phosphorylation of occludin (67,68). The occludin phosphorylation was attenuated by a non-hydrolyzable cAMP analog that also inhibits shear-induced permeability (68). This phosphorylation of occludin appears to be serine or threonine directed since immunoprecipitation of occludin and phosphotyrosine blotting did not reveal any evidence of occludin tyrosine phosphorylation in this cell system (unpublished observation). However, in epithelial cells, evidence of occludin tyrosine phosphorylation exists (69). In addition, others have identified occludin phosphorylation in response to histamine (70) and use of brain extracts has helped identify casein kinase II as an occludin kinase (71). Collectively, this work demonstrates a close association of occludin phosphorylation with permeability. Future studies identifying specific occludin phosphorylation sites, followed by mutational analysis, should reveal the functional significance of occludin phosphorylation. 

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