To ascertain whether other inflammatory mediators induce MMP 9 launch from pericytes, we treated cells with interferon g, interleukin 1b, IL 6 topical Hedgehog inhibitor and LPS for 24 h. None of these inflammatory mediators induced MMP 9 release from pericytes. Pericytes would be the major source of MMP 9 produced from cells constituting the BBB in response to TNF a We identified the TNF a stimulated MMP 9 launch from three cellular components of the BBB after-treatment with 100 ng/mL TNF a for 24 h. TNF a notably enhanced the release of MMP 9 from pericytes and astrocytes in to the supernatant. Pericytes showed noticeable MMP 9 launch, although astrocytes and RBECs produced lower levels of MMP 9. That TNF an activated MMP 9 launch from pericytes was 3. 3 and 2. 5 fold higher than from RBECs and astrocytes, respectively. TNF a release of MMP 9 in the three cell types increased with time, as shown in Figure 2B. This increased reaction appeared within 12 h in each culture. As TNF a can bind to 2 structurally different membrane receptors on target cells, TNFR1 and TNFR2, we examined their expression levels in astrocytes, RBECs and pericytes. There were no significant differences in the Protein biosynthesis expression levels of TNFR1 among RBECs, astrocytes and pericytes. The expression amount of TNFR2 in pericytes was about 2. 2 fold higher-than in astrocytes and RBECs. TNF an induces MMP 9 release from pericytes via the p38 MAPK pathways, JNK, and p42/ p44 MAPK We investigated whether MAPKs get excited about TNFa induced MMP 9 release from pericytes. ALK inhibitor When pericytes were pre-treated with a MEK1/2 inhibitor, a JNK inhibitor and a p38 MAPK inhibitor for 15 min prior to a 24 h exposure to TNF a, TNF an induced MMP 9 release was blocked by each inhibitor in a concentration dependent manner. U0126, SP600125 and SB203580 inhibited TNF an activated MMP 9 release by about 80, 75 and 35%, respectively. TNF an increased the phosphorylation levels of p42/ p44 JNK, MAPK and p38 MAPK in pericytes by 110-story and 102, 75 of vehicle, respectively. TNF a causes MMP 9 release from pericytes via the phosphoinositide 3 kinase /Akt cascade Pretreatment with the PI3K inhibitor, LY294002, considerably inhibited TNF an induced MMP 9 release by about 30 and 800-919, respectively. To test whether TNF a stimulates phosphorylation of Akt, a direct downstream target of PI3K, western blot analysis of pericytes was performed utilizing an anti phospho Akt antibody. Phospho Akt amounts were increased in TNF a treated pericytes, compared with vehicletreated pericytes. Up regulation of MMP 9 is necessary for the induction of pericyte migration To judge the functional activity of the MMP 9 expression induced by TNF a, we examined the cellular migration of pericytes employing a scratch wound healing assay in vitro. Representative photographs demonstrate that TNF a stimulated pericytes to migrate over the wound edge into the region 72 h after scratching. The extent of TNF a stimulated pericyte migration somewhat increased to 1896-1910 of vehicle.