α2β1 integrin–deficient tumor angiogenesis is PLGF dependent. (A) Tumor volume of LLC cells injected into α2-null mice (KO) and their wild-type (WT) littermates as a function of time. The data are presented as the mean plus or minus SEM with 5 animals of each genotype (P = .99, statistical analysis by ANOVA). (B) Immunohistochemical analysis of CD31⁺ vessels in LLC tumor sections. No difference in vascularity was observed (scale bar = 50 μm; 20×/0.75 NA objective). (C) Immunofluorescence analysis of VEGFR1 (red) expression on tumor endothelial cells in LLC tumors from wild-type and α2-null mice. Nuclei were stained with DAPI (blue; 20×/0.75 NA objective). (D) Light microscopic images of representative wild-type and α2-null aortic ring microvessels embedded in growth factor–reduced Matrigel supplemented with either PLGF or VEGF (10×/0.75 NA objective). (E) Quantitation of the number of vascular sprouts in the presence of PLGF. PLGF stimulated a significantly increased number of sprouts from the α2-null, but not wild-type aortic rings. Fifteen aortic rings of each genotype are included in the analysis (P = .002, statistical analysis by ANOVA). (F) Light microscopic images of neoangiogenesis in growth factor–reduced Matrigel supplemented with either PLGF, VEGF-A, or VEGF-C after 8 days (20×/0.75 NA objective). (G) Quantitation of the number of microvessels infiltrating into the Matrigel plug in response to either PLGF, VEGF-A, or VEGF-C (3 animals of each genotype and each experimental condition; *P < .001, statistical analysis by t test).