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First published online 23 December 2002
doi: 10.1242/jcs.00264

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Protection against hypoxia-induced increase in blood-brain barrier permeability: role of tight junction proteins and NFB

Department of Pharmacology, The University of Arizona College of Medicine, Tucson, AZ, USA

View larger version (23K): [in a new window]   Fig. 1. C6-conditioned media protects against hypoxia-induced permeability changes in BBMEC monolayers. BBMEC monolayers were incubated for three days in C6-CM or RA-CM and then subjected to 24 hours of normoxia or hypoxic stress (1% O2). Under normoxic conditions, neither C6-CM nor RA-CM has any effect on BBMEC monolayer permeability. However, after 24 hours of hypoxia, there is a significant increase in permeability in all three treatment groups. This increase in permeability after hypoxic stress is partially blocked by co-culture with C6-CM. ***P<0.001 versus comparable normoxic treatment group, ###P<0.001 versus hypoxic MEM/F12, ^P<0.05 versus hypoxic RA-CM, n=16.

View larger version (24K): [in a new window]   Fig. 2. C6-CM protective effect is time dependent. BBMEC monolayers were incubated with MEM/F12 or C6-CM for 1 or 3 days of exposure. Under normoxic conditions, neither 1 nor 3 days of exposure to C6-CM had any effect on monolayer permeability. However, after 24 hours of hypoxic stress, both 1 and 3 days of C6-CM exposure protected against hypoxia-induced monolayer breakdown. This effect was time dependent, with 1 day exposure being partly protective, and 3 day exposure resulting in almost complete protection against hypoxic stress. ***P<0.001 versus a comparable normoxic treatment group, ###P<0.001 versus hypoxic MEM/F12, ^P<0.01 versus hypoxic 1 day C6-CM, n=4-8.

View larger version (25K): [in a new window]   Fig. 3. C6-conditioned media increases NFB expression in BBMEC monolayers. BBMEC monolayers were incubated for three days in C6-CM or RA-CM and then subjected to 24 hours of normoxia or hypoxic stress (1% O2). NFB was immunoprecipitated and detected by western blotting. C6-CM significantly increases NFB expression under normoxic conditions; RA-CM also significantly increases NFB expression compared with MEM/F12. After 24 hours of hypoxia, NFB levels increase in both MEM/F12 and RA-CM-treated samples. There is no significant change in NFB expression in hypoxic C6-CM-treated samples compared to normoxic samples. #P<0.05 and ##P<0.01 versus normoxic MEM/F12, *P<0.05 and ***P<0.001 versus comparable normoxic treatment, n=3.

View larger version (28K): [in a new window]   Fig. 4. A potential model for C6-conditioned media protection against hypoxia-induced changes in paracellular permeability. (A) Cells grown in MEM/F12 and subjected to a 24-hour hypoxic stress undergo a breakdown of the TJ, with a resulting increase in paracellular permeability as measured by [14C]-sucrose flux. This breakdown of the TJ is probably caused by some dissociation of the component proteins, with resulting formation of actin stress fibers and removal of occludin and ZO-1 from their normal membrane-associated subcellular locations (Mark and Davis, 2002). We hypothesize that under C6-CM co-culture conditions (B), secreted factors in C6-CM trigger the activation of signal transduction mechanisms, linked to NFB or other as yet unidentified pathways. This treatment allows for an adaptive response in the BBMEC when they are exposed to 24 hours hypoxic stress, such that they respond by increasing their expression of claudin-1 and actin. These increases in claudin-1 and actin enable the BBMEC to build additional TJ, thereby preventing the increase in paracellular permeability seen under MEM/F12 conditions. However, C6-CM treatment may also protect via the maintenance of already existing TJ; the exact mechanisms remain to be elucidated.