High levels of BMP4 increase VEGF expression in leukemic cells and exacerbate angiogenesis in CNS of xenografted mice. (A) Representative images of CD31⁺ endothelial cells (green) and GFAP⁺ astrocytes (red) in sagittal brain cryosections of healthy, Nalm6, and Nalm6-BMP4 mice (Scale bars, 50 μm). (B) Quantitation of microvessel density using CD31 immunofluorescence. Three random cortical areas per section were imaged at magnification ×40. Fiji software was used for computerized quantification of immunostained vascular structures, and positive pixels were quantified and expressed as a percentage (±standard deviation) of CD31⁺ area per tissue total area (4 cryosections/3 mice per group; #P ≤ .05 and ***P ≤ .001; 2-tailed Student t test). (C) VEGFα production in Nalm6 and Naml6-BMP4 cell cultures (n = 4 independent experiments). Results are shown as increments relative to Nalm6 control cultures (*P ≤ .05; 2-tailed Mann-Whitney U test). (D) VEGFA mRNA expression in leukemic cells recovered from spleens of Nalm6 (n = 4) and Nalm6-BMP4 (n = 6) mice. Results are presented as increments relative to Nalm6 control cells (**P ≤ .01; 2-tailed Mann-Whitney U test). (E) Pearson’s correlation coefficient (r) and P value between VEGFA and BMP4 mRNA expression in primary BCP-ALL samples at diagnosis are shown (n = 57).