Loss of Pdcd10 increased expression of brain endothelial TM. (A) Pdcd10^ECKO hindbrains show prominent hemorrhagic lesions. Scale bar, 2 mm. (B) Hematoxylin and eosin staining of cerebellar sections from Pdcd10^ECKO mice and Pdcd10^fl/fl littermate controls. Arrows indicate bleeding into the brain parenchyma, and arrowheads indicate normal cerebral vasculature. Asterisks denote the vascular lumen of CCM lesions. Higher-magnification images are shown (right panels). Scale bars, 100 μm (n = 4 mice in each group). (C) Immunofluorescence staining of blood cells using TER119 (red) and labeling of the microvasculature, using isolectin B4 (white), of cerebellar sections from Pdcd10^ECKO or Pdcd10^fl/fl mice at P9; DAPI staining (blue) was used to reveal nuclei (left and middle panels). Arrows indicate bleeding into the brain parenchyma. Scale bar, 100 μm. Higher-magnification images are shown (right panels; scale bar, 50 μm). (n = 3 mice in each group). (D) Immunofluorescence staining of TM (green) or isolectin B4 (IB4; white) of cerebellar sections from Pdcd10^ECKO and Pdcd10^fl/fl mice. Asterisks denote the vascular lumen of CCM lesions. Scale bar, 50 μm (n = 4 mice in each group). (E) Quantification of TM in cerebellar tissue in Pdcd10^ECKO and control Pdcd10^fl/fl littermates, as assessed by western blot analysis (SEM, n = 4 or 7 mice in each group). (F) Analysis of Thbd mRNA levels by RT-qPCR in cerebellar, heart, intestine, and lung tissue from Pdcd10^ECKO mice compared with control Pdcd10^fl/fl littermates (SEM, n = 6 or 8 mice in each group). (G) Analysis of Klf2 and Klf4 mRNA levels by RT-qPCR in cerebellar tissue in Pdcd10^ECKO mice compared with control Pdcd10^fl/fl littermates (SEM, n = 7 mice in each group). (H) Whole-mount P9 retinas show hemorrhage in Pdcd10^ECKO mice but not in littermate control Pdcd10^fl/fl mice. (I) Maximum-intensity projection of whole-mount P9 retinal vasculature at the angiogenic growth front stained for TM (green) and for red blood cells using TER119 antibodies (red); retinal vasculature was labelled with isolectin B4 (IB4; turquoise) (n = 4). (J) Measurement of hemoglobin content of extravasated erythrocytes from retinal vasculature at P10 in Pdcd10^ECKO mice and control Pdcd10^fl/fl littermates. **P < .01, ***P < .001, Student t test and 1-way ANOVA, followed by the Tukey post hoc test.