Modulation of tight junction structure in blood-brain barrier endothelial cells Effects of tissue culture, second messengers and cocultured astrocytes

Abstract

Tight junctions between endothelial cells of brain capillaries are the most important structural elements of the blood-brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier characteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junctional strands, decreased 5 hours after culture but thereafter remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continuously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face association of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resistance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.

In Vitro Models of the Blood–Brain Barrier for the Study of Drug Delivery to the Brain

“…Especially important is the effect of astrocytes during adulthood on the interendothelial junctionswhich largely determines permeabilityand also on the modulation of transporter expression. Astrocytes play a crucial role in the development of the complexity of tight junctions 52 and upregulation of the efflux transporters ABCB1 53 and ABCG2. 54 Recent proteomic analysis revealed changes in 55 proteinsinvolved mainly in cell structure and motility and protein metabolism and modificationafter induction of bovine brain endothelial BBB functions by coculture with rat glial cells; 55 and a later study was mainly confirmatory.…”

Targeting Fibronectin to Overcome Remyelination Failure in Multiple Sclerosis: The Need for Brain- and Lesion-Targeted Drug Delivery

“…As the composition of the BM plays a pivotal role in providing endothelial cells with a supportive growth substrate, changes in the BM protein composition induce functional changes in endothelial cell phenotypes [ 118 ]. The importance of the supporting cell types for BBB integrity is highlighted by the lower endothelial barrier integrity and an altered cytoplasmic anchoring of tight junctions (TJs) in monocultures [ 119 ] compared with co-cultures [ 120 , 121 ]. Indeed, endothelial cells grown on an ECM produced by astrocytes and pericytes show enhanced barrier impermeability compared with cells grown on non-astrocyte/pericyte-derived ECM [ 122 ].…”

Role of the Endothelium in Neonatal Diseases

“… 44 Brain Endothelium: In the central nervous system, endothelial cells regulate plasma filtration and the movement of circulating cells through the blood-brain barrier, most likely via the assembly of tight junctions. 45 , 46 Cerebral microvasculature likely originates from the meningeal vessels, but the subsequent angiogenesis involves the whole brain. 47 …”