M-CSF induces mononuclear phagocyte recruitment <i>in vivo</i>.

<p>A) Matrigel™ was resuspended with PBS <i>(PBS)</i> or rmM-CSF (100 ng/ml) <i>(M-CSF)</i> at 4°C overnight and then injected subcutaneously into mice. Matrigel™ was left <i>in situ</i> for 10 days and then harvested. The plugs were stained for mononuclear phagocytes using an anti-mouse F4/80 antibody. Brown staining represents F4/80(+) cells within the plug. Pictures shown were taken using a dissecting microscope (1.5× objective lens) <i>(left)</i> to display overall mononuclear phagocyte influx between conditions as well as an inverted microscope (20× objective lens) <i>(right)</i> to show detailed staining of these cells. B) The percent of F4/80(+) cells (brown stain) per plug was evaluated from 15 digital images captured randomly in a blinded manner. Adobe Photoshop histogram pixel analysis of the brown stain was used for quantification. This data represents three plugs per group.</p

M-CSF induced VEGF production occurs through a HIF-independent mechanism.

<p>A) The 1.5 kb wild type VEGF promoter <i>(WT1.5 kb)</i> and the same promoter sequence containing a non-functional mutation of the hypoxia regulatory element (HRE) <i>(ΔHRE)</i> were inserted into the pGL3-basic vector to create constructs that produce luciferase upon VEGF promoter activation. Nucleotides changed from the original wild type sequence are designated in bold. Human monocytes were transfected with either the control empty construct (pGL3-Basic), or pGL3 containing 1 µg of each of the described VEGF promoter constructs above. B) Transfected monocytes containing <i>(WT1.5 kb)</i> or <i>(ΔHRE)</i> were allowed to adhere for 1 hour in RPMI/5% FBS followed by the addition of fresh media containing rhM-CSF (100 ng/ml) for 16 hours. The adherent cells were lysed and assayed for luciferase production using a luminometer. This data represents the mean+/−SEM of six individual blood donors. All data is represented as the fold change in luciferase over the pGL3 control construct.</p

M-CSF-induced VEGF production from human monocytes is blocked by ERK inhibition and mithramycin.

<p>A) Monocytes were pre-incubated with DMSO (vehicle control), 1 µM or 10 µM the PI3 kinase inhibitor (LY294002) for 30 minutes. The monocytes were either left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i>, with rhM-CSF (100 ng/ml) plus DMSO <i>(M-CSF+DMSO)</i>, or rhM-CSF (100 ng/ml) plus 1 or 10 µM LY294002 <i>(1)</i> and <i>(10)</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from four independent donors. B) Monocytes were pre-incubated with DMSO (vehicle control) or 10 µM of the inhibitor of ERK activity (U0126) for 30 minutes. The monocytes were either left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) plus DMSO <i>(M-CSF+DMSO)</i> or with rhM-CSF (100 ng/ml) plus U0126 <i>(M-CSF+U0126 (10 µM))</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from six independent donors. C) Freshly-isolated human monocytes were plated overnight in 5% FBS and M-CSF (20 ng/ml). The next day the cells were starved for 2 hours in minimal media. For the last 30 minutes, DMSO (vehicle control) or 10 µM U0126 was added to the appropriate samples. The cells were left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF+DMSO)</i> or <i>(M-CSF+U0126)</i> for 10 minutes. Cell lysates were probed for phospho-ERK MAP Kinase (Thr202) and total ERK MAP Kinase by western blot analysis. This data is representative of two independent monocyte donors. D) Monocytes were pre-incubated with methanol (vehicle control) or 8, 40, or 200 nM of the Sp1 transcription factor binding inhibitor, mithramycin, for 45 minutes. The cells were either left non-stimulated <i>(NS)</i> or stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i>, <i>(M-CSF+MeOH)</i>, <i>(M-CSF plus 8 nM, 40 nM, or 200 nM mithramycin)</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from ten independent blood donors.</p

M-CSF induces VEGF and regulates angiogenesis <i>in vivo</i>.

<p>A) Matrigel™ was resuspended with PBS <i>(PBS)</i>, rmVEGF (10 ng/ml) <i>(VEGF)</i> or rmM-CSF (100 ng/ml) <i>(M-CSF)</i>. The slides were stained with either an anti-mouse VEGF antibody or its isotype IgG. Brown staining represents VEGF within the plug. Pictures were taken using a 20× objective lens. B) The amount of brown stain (VEGF) within the plugs was determined by taking 15 digital images randomly using a 20× objective lens in a blinded manner. Quantification of VEGF within each plug was determined by Adobe Photoshop histogram pixel analysis of the brown stain. C) Matrigel™ was resuspended with PBS <i>(PBS)</i>, rmVEGF (10 ng/ml) <i>(VEGF)</i>, rmM-CSF (100 ng/ml) <i>(M-CSF)</i>, or rmM-CSF (100 ng/ml)+anti-VEGF neutralizing antibody (5 µg/ml) <i>(M-CSF+anti-VEGF neutralizing Ab)</i>. The plugs were stained for endothelial cells using an anti-mouse von Willebrand factor (vWf) antibody. Brown staining represents vWf(+) cells within the plugs. Pictures were taken using a 20× objective lens. D) Quantification of blood vessels within each plug was determined by counting the total number of vWf(+)-stained endothelial cells that formed a complete circle (red arrows) throughout each entire plug using a 20× objective lens in a blinded manner. This data represents three plugs per group.</p

M-CSF induces Sp1 nuclear localization in an ERK-dependent manner.

<p>A) Human monocytes were left non-stimulated <i>(NS)</i> or stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i> for 30 minutes. Nuclear lysates were isolated and normalized for total protein. Sp1 that translocated into the nucleus in response to M-CSF was analyzed using a biotinylated Sp1 DNA sequence bound to a streptavidin-coated plate, a polyclonal rabbit anti-Sp1 primary antibody, a HRP-conjugated goat anti-rabbit IgG secondary antibody, and TMB substrate. The absorbance was read at 450 nm to reflect Sp1 within the nucleus. These data represent the mean±SEM from four independent blood donors. B) Human monocytes were starved for 6 hours, inhibited for 30 minutes with U0126 (10 µM) or DMSO (vehicle control) and left non-stimulated <i>(Non-stimulated)</i> or treated with rhM-CSF for 6 hours <i>(M-CSF+DMSO)</i> and <i>(M-CSF+U0126)</i>. The cells were fixed, permeablized, and stained with a normal IgG control antibody <i>(top row)</i> or a primary antibody targeting Sp1 followed by subsequent staining with Alexa 594-conjugated secondary antibody, targeting Sp1 <i>(red)</i>, and with DAPI stain designating the nucleus <i>(blue)</i>. Images were captured using the Zeiss LSM 510 confocal microscope. These pictures are representative of four individual monocyte donors. C) Quantification of Sp1 localization to the nucleus of monocytes (horseshoe-shaped nuclei) using Image J software. This data represents mean+/−SEM of cells from four individual trials.</p

M-CSF regulates VEGF mRNA transcription through ERK and Sp1.

<p>A) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours in minimal media. The cells were stimulated with 100 ng/ml rhM-CSF for 6 hours followed by isolation of RNA. cDNA was synthesized from total cellular RNA, standardized, and subjected to SYBR Green real-time PCR using primers specific for VEGF-A. The data represents the mean+/−SEM of six individual donors. B) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hrs followed by pre-treatment with either DMSO (vehicle control) or U0126 (10 µM) in minimal media for 30 minutes. The cells were then stimulated with 100 ng/ml rhM-CSF <i>(M-CSF)</i>, <i>(M-CSF+DMSO)</i>, <i>(M-CSF+U0126 10 µM)</i> and analyzed as stated in A. The data represents the mean+/−SEM of three individual donors. C) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours followed by pre-treatment with either methanol (vehicle control) or mithramycin (200 nM) in minimal media. The cells were then stimulated with 100 ng/ml rhM-CSF <i>(M-CSF)</i>, <i>(M-CSF+MeOH)</i>, <i>(M-CSF+Mith (200 nM))</i> and analyzed as in A. The data represents the mean+/−SEM of four individual donors.</p

ERK and Sp1 are necessary for M-CSF induced functional activation of the VEGF promoter.

<p>A) The wild type VEGF promoter (region −88 to +54 relative to the transcription start site (0)) <i>(−88/+54 WT)</i> and the same promoter sequence containing mutations of Sp1 (<i>ΔSp1</i>), AP-2 (<i>ΔAP-2</i>), and a Sp1/AP-2 double mutant (<i>ΔSp1/ΔAP-2</i>) were inserted into the pGL3-basic vector to drive luciferase production upon VEGF promoter activation. Nucleotides changed in the mutated sequences are designated in bold. B) Transfected monocytes containing the pGL3 control vector or <i>(−88/+54 WT)</i> were allowed to adhere for 1 hour in RPMI/5% FBS followed by the addition of 10 µM U0126 <i>(−88/+54 WT+U0126)</i> for 30 minutes. The cells were washed and fresh media containing rhM-CSF (100 ng/ml) was added for 16 hours, when the cells were lysed and assayed for luciferase production using a luminometer. This data represents the mean+/−SD of six individual blood donors. C) Transfected monocytes containing <i>(−88/+54 WT)</i>, <i>(ΔSp1)</i>, <i>(ΔAP-2)</i>, or <i>(ΔSp1/ΔAP-2)</i> were allowed to adhere for 1 hour in RPMI/5% FBS followed by the addition of fresh media containing rhM-CSF (100 ng/ml). After 16 hours, adherent cells were lysed and assayed for luciferase production using a luminometer. This data represents the mean+/−SD of three individual blood donors.</p

CSF1 up-regulates Tie2 receptor on CD14+ human monocytes.

<p>(<b>A</b>) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (<i>Freshly isolated</i>) or after treated without (-<i>CSF1</i>) or with rhCSF1 (100 ng/ml) (+<i>CSF1</i>) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (<b>B</b>) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (<b>C</b>) CD14+ monocytes were left untreated (<i>Utx</i>) or treated with rhANG2 (100 ng/ml) (<i>ANG2</i>), rhCSF1 (100 ng/ml) (<i>CSF1</i>), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (<i>CSF1R NAb+CSF1</i>) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (<b>D</b>) CD14+ monocytes were left untreated (<i>Untreated</i>), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (<i>CSF1R NAb+CSF1</i>), or with rhCSF1 (100 ng/ml) alone (<i>CSF1</i>) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.</p

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.</p

CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.

<p>(<b>A</b>) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (<i>Media</i>), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (<b>B</b>) CD14+ monocytes were treated with rhANG2 (10 ng/ml) <i>(ANG2</i>) or rhCSF1 (10 ng/ml) (<i>CSF1</i>) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.</p