The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

No abstract

The endothelial cells (ECs) lining every blood vessel wall are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. To ensure proper EC mechano-sensing and transduction, there are a variety of mechano-sensors and transducers that have been identified on the EC surface, intra- and trans-EC membrane and within the EC cytoskeleton. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix-like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins, and is close to other prominent EC mechano-sensors and transducers. The ESG thickness was found to be in the order of 0.1–1 μm by different visualization techniques and in different types of vessels. Detailed analysis on the electron microscopy (EM) images of the microvascular ESG revealed a quasi-periodic substructure with the ESG fiber diameter of 10–12 and 20 nm spacing between adjacent fibers. Atomic force microscopy and optical tweezers were applied to investigate the mechanical properties of the ESG on the cultured EC monolayers and in solutions. Enzymatic degradation of specific ESG glycosaminoglycan components was used to directly elucidate the role of the ESG in EC mechano-sensing and transduction by measuring the shear-induced productions of nitric oxide and prostacyclin, two characteristic responses of the ECs to the flow. The unique location, composition, and structure of the ESG determine its role in EC mechano-sensing and transduction.

RationaleIt is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.ObjectiveTo evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.Methods and ResultsUsing confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.ConclusionAll GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.