Sirolimus

All experimental animal protocols were approved by the Yale University Institutional Animal Care and Use Committee. Rapamycin (sirolimus; EMD Millipore) and MEK inhibitor PD0325901 (Selleckchem) were diluted in ethanol and mixed with an equal volume (30 µl) of Cremophor EL (Sigma-Aldrich). These drug mixtures were dissolved in Dulbecco’s PBS at a final volume of 0.2 ml/mouse and administered via intraperitoneal injection. Female C.B-17 SCID/beige mice (Taconic) mice were injected with 3 mg/kg sirolimus, 25 mg/kg PD0325901, or 3 mg/kg sirolimus + 25 mg/kg PD0325901 every 24 h for 3 d. Control mice were injected with a mixture containing ethanol and Cremophor EL in PBS. After the final Rapamycin treatment, mice were injected with 30 µg/kg recombinant human TNF (R&D Systems) diluted in 0.2 ml PBS via the tail vein. After 16 h, mice were anesthetized and perfused with saline, and portions of the aorta, kidney, liver, and heart were harvested. Tissue samples were then either flash frozen in liquid nitrogen or frozen in optimal cutting temperature (OCT) compound (Sakura). Serial 5- or 30-µm transverse sections were cut for immunofluorescence or quantitative real-time RT-PCR analysis, respectively.