CSF1 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.

<p>(<b>A</b>) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1α<i>^fl/fl</i>/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1α<i>^fl/fl</i>/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (<b>B</b>) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (<i>vehicle</i>), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (<i>LY294002</i>), the MEK inhibitor U0126 (10 µM) (<i>U0126</i>), or the NF-Kb inhibitor PDTC (100 µM) (<i>PDTC</i>) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (<i>Vehicle+CSF1</i>), LY294002+CSF1 (100 ng/ml) (<i>LY294002+CSF1</i>), U0126+CSF1 (100 ng/ml) (<i>U0126+CSF1</i>), or PDTC+CSF1 (100 ng/ml) (<i>PDTC+CSF1</i>) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (<b>C</b>) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1α<i>^fl/fl</i>/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (<i>LY294002+CSF1</i>) or not (<i>vehicle</i>) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1α<i>^fl/fl</i>/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.