BBB endothelial cells

Most electron microscopic studies of BBB endothelial cells suggest the presence of relatively few observable endocytic vesicles in the cytoplasm of these cells compared with other endothelia. For example, the BBB contains only a fifth to a sixth of the endocytic profiles seen in muscle capillary endothelia [54], although they may increase to comparable levels with inflammation of the BBB [55]. However, when a comparison is made of the ability of capillary endothelia in a variety of different tissues to trancytose protein, there is a very poor correlation between the protein permeability of a microvessel and the number of observable endocytic profiles [54]. Brain capillary endothelia are very thin cells, the luminal and ablum-inal membranes only being separated by some 500 nm or less (5000 A), and caveoli are 50-80 nm in diameter and thus the events of transcytosis may be difficult to capture within the cell using conventional electron microscopical techniques. 

An increase in therapeutic efficacy and lower toxicity was reported with liposomes where the bradykinin analogue RMP-7 was chemically attached at the end of PEG molecules of PEGylated liposomes (approximate size 70nm) [384], RMP-7 exhibits high selectivity for the B2 receptor of the BBB endothelial cells, which shrunk and let the RMP-7-PEG liposomes to pass into the brain. Actually the mechanism used in that study was based on opening the tight junctions of the BBB. Liposome-incorporated nerve growth factor (NGF) concentration increased 10 times in comparison to free NGF, while they accumulated mainly in striatum, hippocampus, and cortex. 

The physico-chemical characteristics of dmgs (e.g., hydrophilicity, lipophili-city, hydrogen bonding potential) largely determine the passive transport of dmgs across the BBB. This comprises hydrophilic paracellular and lipophilic transcel-lular transport. Passive hydrophilic transport is mainly restricted by the tight junctions of the BBB endothelial cells. This paracellular permeability is, next to size (5), further dependent on the charge of the molecules and the possibility to form... 

Another method of Pgp regulation has been demonstrated by adrenomedullin (AM) (89). It is produced by endothelial cells in the brain and acts as a vasodilator in the cerebral circulation. It was shown that AM antisense decreased the transendo-thelial electrical resistance across endothelial monolayers. Treatment of these cells with AM activated Pgp, suggesting that AM acts as an autocrine mediator in the regulation of the properties of BBB endothelial cells. In addition, AM incubation decreased BBB permeability for sodium fluorescein (376 Da) but not for Evan s blue albumin (67 kDa). An interesting observation was that it also attenuated fluid-phase endocytosis. 

Fig. 8.1 Schematic drawing of the in vitro BBB model. Astrocytes are seeded on the bottom of the collagen-coated filter at a density of 45.000 cells per filter, allowed to adhere for 8 min, and cultured for 2 or 3 days. BBB endothelial cells (BCECs) are seeded at a density of 30.000 cells per filter. BCEC-astrocyte co-cultures are cultured to tight monolayers in growth medium for the first 2 or 3 days and on differentiation medium for the last 2 or 3 days. Transport or transendothelial electrical resistance (TEER), drug transport or receptor characterization experiments are performed after a total of 9 or 10 days after the brain capUlaries are seeded...

The blood-brain barrier (BBB) forms a physiological barrier between the central nervous system and the blood circulation. It consists of glial cells and a special species of endothelial cells, which form tight junctions between each other thereby inhibiting paracellular transport. In addition, the endothelial cells of the BBB express a variety of ABC-transporters to protect the brain tissue against toxic metabolites and xenobiotics. The BBB is permeable to water, glucose, sodium chloride and non-ionised lipid-soluble molecules but large molecules such as peptides as well as many polar substances do not readily permeate the battier. 

Fig. 15.3 D iagram showing a longitudinal cross-section of the blood-brain barrier, with the brain capillary endothelial cells sealed by the tight junctions and surrounded by pericytes and astrocyte foot processes. These cellular components of the BBB are separated by a basement membrane.

Lastly, pharmacogenomics could provide new tools for the design of more specific and active CNS pharmaceuticals. The efficacy of a broad spectrum of neuro-pharmaceutical drugs is often complicated by their inability to reach their site of action because of the BBB. One way to overcome this is to use carrier-mediated transport at the luminal and/or abluminal membranes of the endothelial cells of the BBB. This will provide a physiologically based drug delivery strategy for the brain by designing new chemical entities or fused proteins that can cross the BBB via these transporters. 

Although the absence of paracellular transport across the BBB impedes the entry of small hydrophilic compounds into the brain, low-molecular-weight lipophilic substances may pass through the endothelial cell membranes and cytosol by passive diffusion [7]. While this physical barrier cannot protect the brain against chemicals, the metabolic barrier formed by the enzymes from the endothelial cell cytosol may transform these chemicals. Compounds transported through the BBB by carrier-mediated systems may also be metabolized. Thus, l-DOPA is transported through the BBB and then decarboxylated to dopamine by the aromatic amino acid decarboxylase [7]. 

Like the glucose carrier, the carriers for large neutral amino acids, the so-called L-system - now designated LAT - are present at both sides of the endothelial cell membranes and transport at least 10 essential amino acids. The L-transporter at the BBB has a much higher transport capacity than those in other tissues. Its marked preference for phenylalanine analogs explains why the anticancer drugs melphalan and d,l-NAM-7 are transported by the L-system, as is the L-Dopa used to treat Parkinson s disease [42]. 

Fig. 15.5 D iagram showing some of the nutrient and drug transport processes associated with the brain capillary endothelial cells that form the BBB. Local transporters in the luminal or/and abluminal membranes are depicted as filled circles and ones whose location is more questionable or that are present at the BBB are depicted in open circles. GLUT1, LAT1, MCT1, oatp2 are present on both the luminal and abluminal membranes. This diagram shows that transport may be unidirectional or bidirectional.

The presence at the BBB of members of the multidrug resistance-associated protein (MRPs) family, whose members preferentially transport anionic compounds, is still controversial. The seven members of the MRP family belong, like P-gp, to the ATP-binding cassette (ABC) protein superfamily. Mrpl has been found at the BBB in isolated rat brain capillaries, primary cultures of brain capillary endothelial cells and in immortalized capillary endothelial cells, but not in human brain capillaries [59]. Another member, MRP2 has been found at the luminal membrane of the brain endothelial cells [60]. However, further studies are required to show that there are MRP transporters at the BBB (Figure 15.5). As for P-gp, a functional Mrpl was found in primary cultured rat astrocytes [56] and it has been shown to take part in the release of glutathione disulfide from brain astrocytes under oxidative stress [61]. 

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