Figure 7
Figure 7. Stage-specific regulation of stromal myeloid cell recruitment and retention. (A) Representative CD31/CD11b coimmunofluorescence demonstrating recruitment and stromal accumulation of CD11b myeloid cells during the TetON-HIF-1 DOX day 0 to day 14 neovascular development stage, with HIF-1–dependent retention of these cells from day 14 to day 28. Myeloid cell recruitment is markedly inhibited by VEGFR1 (MF1) or VEGFR2 (DC101). Notably, neovascular development proceeds despite marked inhibition of myeloid cell recruitment in MF1-treated TetON-HIF-1 mice. AMD3100 partially inhibits myeloid cell recruitment (supplemental Figure 8B). DOX day 14 to day 28 myeloid cell retention is independent of either VEGFR1 or VEGFR2 but dependent on signaling from both receptors. Myeloid cell retention is incompletely inhibited by AMD3100 (supplemental Figure 8). The maintenance stage neovasculature persists despite profound stromal myeloid depletion in the day 14 to day 28 DC101/MF1 inhibitor cocktail-treated TetON-HIF-1 mice. Stromal myeloid cells rapidly disappear after 3 (14/3) and 14 (14/14) days of DOX withdrawal. (B) Plasma VEGF (left), PlGF (middle), and SDF-1α (right) expression levels in TetON-HIF-1 mice and DOX NTG controls determined by ELISA. TetON-HIF-1 data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; **P < .01; ***P < .001. (C) Model of epithelial neovascularization regulated by HIF-1. Continuous HIF-1 activation produces multistage intrinsically regulated neovascular development, maintenance, and transgene-dependent regression. Mechanisms of myeloid cell recruitment and retention were stage-specific. Resistance to VEGFR2 immuno-blockade was also stage-specific. The cyan bar represents peak proliferation achieved at day 3; dark orange arrow, neovascular growth and maintenance independent of myeloid cells; purple arrow, 14 days of continuous DOX induction; purple bar, 14 days of DOX withdrawal; yellow bar, peak apoptosis and L-PAM–determined capillary luminal constriction 3 days after DOX withdrawal. TetON-HIF-1 data at each DOX day were compared with NTG data, using unpaired Student t test: *P < .05; **P < .01; ***P < .001. Bar represents 200 μm.

Stage-specific regulation of stromal myeloid cell recruitment and retention. (A) Representative CD31/CD11b coimmunofluorescence demonstrating recruitment and stromal accumulation of CD11b myeloid cells during the TetON-HIF-1 DOX day 0 to day 14 neovascular development stage, with HIF-1–dependent retention of these cells from day 14 to day 28. Myeloid cell recruitment is markedly inhibited by VEGFR1 (MF1) or VEGFR2 (DC101). Notably, neovascular development proceeds despite marked inhibition of myeloid cell recruitment in MF1-treated TetON-HIF-1 mice. AMD3100 partially inhibits myeloid cell recruitment (supplemental Figure 8B). DOX day 14 to day 28 myeloid cell retention is independent of either VEGFR1 or VEGFR2 but dependent on signaling from both receptors. Myeloid cell retention is incompletely inhibited by AMD3100 (supplemental Figure 8). The maintenance stage neovasculature persists despite profound stromal myeloid depletion in the day 14 to day 28 DC101/MF1 inhibitor cocktail-treated TetON-HIF-1 mice. Stromal myeloid cells rapidly disappear after 3 (14/3) and 14 (14/14) days of DOX withdrawal. (B) Plasma VEGF (left), PlGF (middle), and SDF-1α (right) expression levels in TetON-HIF-1 mice and DOX NTG controls determined by ELISA. TetON-HIF-1 data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; **P < .01; ***P < .001. (C) Model of epithelial neovascularization regulated by HIF-1. Continuous HIF-1 activation produces multistage intrinsically regulated neovascular development, maintenance, and transgene-dependent regression. Mechanisms of myeloid cell recruitment and retention were stage-specific. Resistance to VEGFR2 immuno-blockade was also stage-specific. The cyan bar represents peak proliferation achieved at day 3; dark orange arrow, neovascular growth and maintenance independent of myeloid cells; purple arrow, 14 days of continuous DOX induction; purple bar, 14 days of DOX withdrawal; yellow bar, peak apoptosis and L-PAM–determined capillary luminal constriction 3 days after DOX withdrawal. TetON-HIF-1 data at each DOX day were compared with NTG data, using unpaired Student t test: *P < .05; **P < .01; ***P < .001. Bar represents 200 μm.

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