In this study, we formed GW572016 an in vitro model of the BBB using human cerebral microvascular endothelial cell cultures to study the effects of soluble TWEAK on the properties and integrity of the BBB. We showed that sol uble TWEAK induces an inflammatory profile on HCMEC, especially by promoting secretion of cytokines, by modulating production and activation of MMP 9, and expression of cell adhesion molecules. We also demonstrated that these effects of TWEAK are asso ciated with increased permeability of the HCMEC monolayer in the in vitro BBB model. Methods Cells and culture Inhibitors,Modulators,Libraries reagents The human brain endothelial cell line hCMECD3 is described in.

hCMECD3 cells were seeded on TranswellW filters coated with type I collagen, at a density of 350,000 cellscm2 in commercially available complete medium EGMW 2, supplemented with vascular endothelial growth factor, insulin like growth factor 1, epidermal growth factor, basic fibroblast growth factor, hydrocortisone, ascorbate, penicillin streptomycin, and 2. 5% FCS, Inhibitors,Modulators,Libraries in an incu bator at 37 C with 5% CO2. For differentiation and ex pression of junction related proteins, the hCMECD3 cells were grown at confluence in a growth factor depleted medium. Primary HCMECs were grown on 0. 2% gelatin coated tissue culture plates in M199 medium supplemen ted with 20% Inhibitors,Modulators,Libraries fetal bovine serum, 5% heat inactivated human serum, 1% penicillin streptomycin, and 12 ngml endothelial cell growth factor. Human umbilical vein endothelial cells and a human acute monocytic leukemia cell line were obtained from ATCC and were cultivated, respectively, in EBM 2 medium supplemented with EBM 2 bullet kit and RPMI 1640 supple mented with 10% FCS and 1% penicillin streptomycin.

For stimulation assays, cells were incubated Inhibitors,Modulators,Libraries for 3 h, 12 h, or 24 h with recombinant human TWEAK, Fc TWEAK, its isotype control P1. 17, or recombinant human TNF. In some experiments, cells were incubated with recombinant human MMP 9 from Calbiochem. All reagents were endotoxin free. Flow cytometry After trypsination, differentiated unstimulated or TWEAK stimulated hCMECD3 cells were pre incubated on ice for 20 min with a solution containing PBS, 1% FCS, 0. 02% sodium azide, and 25% purified human serum Immuno globulin G to inhibit binding to Fc recep tors. After washes with a solution containing PBS, 1% FCS, and 0.

02% sodium azide, cells were incubated Inhibitors,Modulators,Libraries on ice for 20 min with fluorescein conjugated anti human ICAM 1 antibody, fluorescein conjugated Temsirolimus anti human E selectin antibody, or anti human Fn14 phycoerythrin conjugated antibody. After three more washes, cells were centrifuged and resuspended in PBS with 2% paraformaldehyde. Fluorescence activated cell sorting analysis was performed on a FACSCanto II using BD FACSDiva software. RNA extraction and RT PCR analysis Total RNA was prepared from cultures of hCMECD3, HUVECs, and THP 1 using the RNeasy Lipid Tissue Mini kit.