Figure 4
Figure 4. VEGFR1 but not VEGFR2 expression is up-regulated on α2-null endothelial cells in vitro and in vivo. (A) The levels of VEGFR1 and VEGFR2 expression by primary pulmonary microvascular endothelial cells were evaluated by immunoblot analysis. β-actin was used as loading control. Representative images are shown. Quantitative data are presented as the mean plus or minus SEM (4 immunoblots per genotype pair from 4 endothelial cell preparations; P < .001, statistical analysis by t test). (B) Immunofluorescence analysis of VEGFR1 (red) or VEGFR2 (green) expression on tumor endothelial cells in B16F10 melanoma. Nuclei were stained with DAPI (blue; 20×/0.75 NA objective). VEGFR1 intensity was quantitated and the data presented as mean plus or minus SEM (5 tumors per genotype repeated in 2 separate experiments; *P < .001). (C) Immunofluorescence analysis colocalized CD31 (red) and VEGFR1 (green) on endothelial cells in the tumors of the α2-null mouse (20×/0.75 NA objective). (D) Inhibition of VEGFR1 reduced endothelial cell proliferation in vitro. Proliferation of primary pulmonary microvascular endothelial cells from wild-type and α2-null animals was inhibited by anti-VEGFR1–neutralizing antibodies (10 μg/mL), as designated. Absolute counts per minute (cpm) incorporated after 72 hours is shown. Bars and errors indicate the mean and SEM (3 experiments were performed in quadruplicate; *P < .01 for both wild-type and α2-null endothelial cells). (E) Rescue of α2-null endothelial cells in vitro. Primary α2-null endothelial cells were transfected with lentiviral vectors expressing either the full-length human α2 integrin subunit cDNA or GFP-control vector. Endothelial cell expression of the human α2 integrin subunit was analyzed by flow cytometric analysis using PE-conjugated antihuman α2 integrin subunit antibody. Histograms show the expression of the α2β1 integrin by cells transfected with the α2 integrin subunit cDNA, but not by control transfectants. (F) Re-expression of human α2β1 integrin resulted in decreased expression of VEGFR1 to levels similar to wild-type endothelial cells. Expression of VEGFR1 by α2-null primary pulmonary endothelial cells transfected with the α2 integrin cDNA was compared with α2-null or wild-type primary pulmonary endothelial cells transfected with GFP by immunoblot analysis.

VEGFR1 but not VEGFR2 expression is up-regulated on α2-null endothelial cells in vitro and in vivo. (A) The levels of VEGFR1 and VEGFR2 expression by primary pulmonary microvascular endothelial cells were evaluated by immunoblot analysis. β-actin was used as loading control. Representative images are shown. Quantitative data are presented as the mean plus or minus SEM (4 immunoblots per genotype pair from 4 endothelial cell preparations; P < .001, statistical analysis by t test). (B) Immunofluorescence analysis of VEGFR1 (red) or VEGFR2 (green) expression on tumor endothelial cells in B16F10 melanoma. Nuclei were stained with DAPI (blue; 20×/0.75 NA objective). VEGFR1 intensity was quantitated and the data presented as mean plus or minus SEM (5 tumors per genotype repeated in 2 separate experiments; *P < .001). (C) Immunofluorescence analysis colocalized CD31 (red) and VEGFR1 (green) on endothelial cells in the tumors of the α2-null mouse (20×/0.75 NA objective). (D) Inhibition of VEGFR1 reduced endothelial cell proliferation in vitro. Proliferation of primary pulmonary microvascular endothelial cells from wild-type and α2-null animals was inhibited by anti-VEGFR1–neutralizing antibodies (10 μg/mL), as designated. Absolute counts per minute (cpm) incorporated after 72 hours is shown. Bars and errors indicate the mean and SEM (3 experiments were performed in quadruplicate; *P < .01 for both wild-type and α2-null endothelial cells). (E) Rescue of α2-null endothelial cells in vitro. Primary α2-null endothelial cells were transfected with lentiviral vectors expressing either the full-length human α2 integrin subunit cDNA or GFP-control vector. Endothelial cell expression of the human α2 integrin subunit was analyzed by flow cytometric analysis using PE-conjugated antihuman α2 integrin subunit antibody. Histograms show the expression of the α2β1 integrin by cells transfected with the α2 integrin subunit cDNA, but not by control transfectants. (F) Re-expression of human α2β1 integrin resulted in decreased expression of VEGFR1 to levels similar to wild-type endothelial cells. Expression of VEGFR1 by α2-null primary pulmonary endothelial cells transfected with the α2 integrin cDNA was compared with α2-null or wild-type primary pulmonary endothelial cells transfected with GFP by immunoblot analysis.

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