We recently showed that unstimulated microglia can degrade fibronectin. In molecular weight calculator the absence of microglia, the substrate fluorescence was uniform. Re gardless of treatment, microglial Inhibitors,Modulators,Libraries cells degraded fibronec tin, leaving cell sized patches of reduced fluorescence. The invasion capacity of microglia was then analyzed using an assay in which migration to the underside of each filter requires degradation of Matrigel. Inhibitors,Modulators,Libraries IL4 treated microglia invaded 1. 7 fold more than control cells whereas, LPS treated cells invaded 66% less. Adding ATP to the lower well increased the invasiveness of unstimulated cells by 2. 6 fold, and IL4 treated cells by 3. 2 fold. IL4 treated microglia had a 2. 2 fold greater invasion capacity than unstimulated cells. LPS treated cells were not analyzed because they migrated and invaded very poorly.

IL4 treated microglia use a wide range of degradative enzymes for invasion Degradation of ECM can involve any or all of three broad classes of degradative enzymes MMPs, cathep sins, and heparanase. To analyze their contributions to microglia transmigration Inhibitors,Modulators,Libraries and invasion, we first used three class specific but broad spectrum inhibi tors GM6001, E 64, OGT2115. Then, based on the results, we tested selective inhibi tors of Cat S or Cat K 2 propanone. For each inhibitor, we used a single concentration. Because the invasion assay was for 24 hr, during which the inhibitor efficacy might decrease, for the broad spectrum inhibitors, we chose a high concentration in an attempt to inhibit all the subtypes within the relevant enzyme class.

Inhibitors,Modulators,Libraries Then, for the selective Cat S and Cat K inhibitors we used a concentration that Inhibitors,Modulators,Libraries was 10 to 20 times the IC50, which is expected to inhibit 90% of the enzyme activity. Importantly, none of the inhibitors was toxic at con centrations and times used. For control, unstimulated microglia, none of the enzyme inhibitors affected trans migration through open holes in the filter, which also demonstrates lack of non specific effects or toxicity. Only IL4 treated microglia were compared with controls because LPS treated cells migrated very poorly. IL4 treatment greatly in creased transmigration, and this was reduced back to the control level by the Cat S inhibitor. The apparent trend toward reduced transmigration by three other inhibitors did not reach statistical significance with the sample size used.

None of the inhibitors affected cell viability at the concentrations and times tested. Interestingly, invasion through the same ECM sub strate required different enzymes in un treated and IL4 treated microglia. In unstimulated microglia, invasion was inhibited selleck compound only by the broad spectrum cysteine cathepsin inhibitor, E 64, which decreased invasion to approximately 50% below the control level. Invasion was not altered by the selective Cat S and K inhibitors, suggesting that E 64 acts through a different enzyme. IL4 treatment increased invasion about 2 fold, and all the enzyme inhibitors then reduced it to the baseline level.