Transendothelial transport

R. Soda and M. Tavassoli. Transendothelial transport (transcytosis) of iron-transferrin complex in bone and marrow. J. Ultrastruct. Res. 88 18-25 (1984). 

S. Yokota. Immunocytochemical evidence for transendothelial transport of albumin and fibrinogen in the rat heart and diaphragm. Biomed Res. 4 577-586 (1983). 

Before initiating this work, we started from a hypothesis derived from the work of Palade and the Simionescus [17,29-33] and described by us in a review article in 1984 [21]. The hypothesis suggested that the process of receptor-mediated transcytosis (Fig. 1) was likely responsible for the movement of specific biological macromolecules across the endothelial barrier. A significant controversy exists with respect to the relative importance of the transcellular and paracellular pathways [1,14,15]. The experiments described in Tables 5 and 6 indicate that both pathways are plausible but more importantly that the process of transendothelial transport may be receptor dependent. 

Numerous modifications of in vitro culture systems have been developed for the estimation of BBB transfer [52]. Culture systems in use are either primary cultures of brain microvessel endothelial cells (BMEC) or immortalized endothelial cell hues. BMEC may be grown in co-culture with astrocytes or in astrocyte-conditioned medium. Astrocyte-derived factors increase the tightness of the barrier as measured by transendothelial electrical resistance (TEER) and by the permeability of hydrophUic markers such as sucrose. They also up-regulate the expression of BBB-enriched enzymes such as y-glutamyl transpeptidase (y-GTP) and alkaline phosphatase. A setup of the in vitro technique in a transwell system for transport studies is depicted in Figure 2.5. 

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.

Transendothelial IgG transport may be mediated by binding of endogenous IgG or exogenously administered monoclonal antibodies to FcyRIIa,b on endothelium (e.g., FcyRIIa on skin microvessels [27]) FcyRIIb on placenta villous (fetal) endothelium [28,29] or FcRn on placental syncytiotrophoblasts and fetal endothelium [28], Transcellular transport is further described below under FcRn-mediated transport, I cyRIIa-mediated transport, and specialized skin microvessel transport mechanisms. 

Drags are tested at 10 p,M concentration and in two directions (apical to basolateral (a-b) and basolateral to apical (b-a)). Monolayer efflux studies are conducted at 37 °C in a humidified incubator with shaking (90 rpm) for 60 min. Transendothelial electrical resistance is measured with an Endohm Meter (World Precision Instruments, New Haven, CT). Reference drags for paracellular transport (14C-mannitol), tran-scellular transport (3H-propranolol), and Pgp efflux (amprenavir) should be included in each experiment. Concentrations of 14C-mannitol and 3H-propranolol are measured by liquid scintiallation counting. Amprenavir is analyzed by cassette LC/MS/MS analysis along with the test drags. 

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 main component of the blood-brain barrier is the brain endothelium, which exhibits a physical, an efflux and a metabolic barrier for the transport of drugs into the CNS. The physical barrier, an efflux, is a result of the tight junctions between adjacent endothelial cells, which are around 50-100 times tighter than in the peripheral endothelium, so that penetration across the endothelium is effectively confined to transcellular mechanisms [26, 27]. These junctions significantly restrict even the movement of small ions such as Na" " and Cl , so that the transendothelial electrical resistance (TEER), which is typically 2-20 2 cm in peripheral capillaries, can be over 1000 1 cm in brain endothelium [28]. 

Fig. 3. Transvascular exchange. Transport pathways in normal capillary endothelium. (1) endothelial cell (2) lateral membrane diffusion (3) interendothelial junctions—(a) narrow, (b) wide (4) endothelial fenestrae—(a) closed, (b) open (5) vesicular transport—(a) transcytosis, (b) transendothelial channels. Note that water and lipophilic solutes share pathways (1), (3), and (4). Lipophilic solutes may use pathway (2) as well. Hydrophilic solutes and macromolecules use pathways (3) and (4). Macromolecules may also follow pathway (5). Note that in tumors these pathways have a leakier structure. [From Jain (1987a), with permission.]...

FIGURE 2-5 Transepithelial or transendothelialflux. Transepithelial or transendothelial flux of drugs requires distinct transporters at the two surfaces of the epithelial or endothelial barriers. These are depicted diagrammatically for transport across the small intestine (absorption), the kidney and liver (elimination), and the brain capillaries that comprise the blood-brain barrier. 

To characterize the transport properties of in vitro BBB models, the solute permeability P of the in vitro BBB was determined by measuring the flux of the selected tracer. The most commonly used cell culture substrate consists of a porous membrane support submerged in the culture medium (Transwell apparatus). The Transwell system is characterized by a horizontal side-by-side or vertical diffusion system. During the experiment, the flux of tracers into the abluminal compartment of the Transwell system is recorded as a function of the time and the solute permeability P is calculated from the slope of the flux. The tracers used in the transport experiments are labeled by a fluorescent dye or isotope whose intensity can be measured quantitatively. Another index, transendothelial electrical resistance (TEER), or the ionic conductance of the monolayer, is also a measurement of the tightness of the in vitro BBB models. 

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