Figure 3.
Endothelial deletion of Kdr does not prevent CCM formation in the adult or neonatal mouse model. (A) Schematic representation of adult Krit1fl/fl; iPik3caH1047R mice or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl mice, aged 8–10 wk, underwent craniotomy and received focal AAV-Cre injections. Live-animal microscopic images were captured through the cranial window from POD 7 to POD 21 to observe lesion formation and growth. (B) Representative images showing immunostaining for tdTomato, CD31, and VEGFR2, with DAPI counterstaining in brains harvested from Kdrfl/fl; Ai14 mice on POD 7. The boxed regions in the tdTomato-negative regions (a) and in the tdTomato-positive regions (b) are shown in a’–a’’’ and b’–b’’’, respectively, at higher magnification. Yellow arrows indicate CD31-positive but VEGFR2-negative endothelial cells in the tdTomato-positive regions (tdTomato+, CD31+, and VEGFR2−). Scale bar: 100 μm. (C) Quantification of VEGFR2-positive (CD31+; VEGFR2+) and VEGFR2-negative (CD31+; VEGFR2−) endothelial cells in both tdTomato-negative and -positive regions. (D) Serial of microscopic images through the cranial windows showing CCM growth in either Krit1fl/fl; iPik3caH1047R or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl mouse brains from POD 10 to POD 21. (E and F) Representative visual images (left panels) and microCT renders (right panels) (E), and (F) microCT quantification of CCM lesion volumes in Krit1fl/fl; iPik3caH1047R (n = 7) or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl (n = 7) mouse brains harvested on POD 21. The same microCT renders are also shown in Fig. S2 C to provide a comprehensive overview of genotype-dependent effects on lesion burden across the entire study cohort. Scale bar: 1 mm. (G) Schematic representation of neonatal induction of Krit1 deletion with or without Kdr deletion. 4-OHT was intragastrically injected on P1, and brains were harvested on P11. (H) Immunoblot detection of VEGFR2 in neonatal cerebellum harvested from P11 littermates. β-Actin was used as a loading control. (I) Quantification of immunoblotting for VEGFR2 protein level. A total of three paired littermates were measured and quantified. (J) Representative images showing immunostaining for CD31, VEGFR2, and pVEGFR2, with DAPI counterstaining in neonatal brains harvested from P11 littermates. White arrows indicate CD31 and VEGFR2 double-positive (CD31+; VEGFR2+) cells and yellow arrows indicate CD31-positive but VEGFR2-negative (CD31+; VEGFR2−) cells at the lesional and non-lesional neonatal cerebellum. Asterisks indicate autofluorescence emitted from lumenal red blood cells. Scale bar: 100 μm. (K) Quantification of VEGFR2-positive (CD31+; VEGFR2+) and VEGFR2-negative (CD31+; VEGFR2−) cerebellar endothelial cells. (L) Quantification of MFI of pVEGFR2Y951 in Krit1ECKO (Cdh5-CreERT2; Krit1fl/fl) neonatal cerebellum, normalized to total VEGFR2, compared with vasculature in the normal littermate control (Krit1fl/fl). (M–O) Representative visual images (top panels) and microCT renders (bottom panels), and microCT quantification of (N) CCM lesion volumes and (O) total brain volumes in Cdh5-CreERT2; Krit1fl/fl (n = 13), Cdh5-CreERT2; Krit1fl/fl; Kdrfl/+ (n = 18), and Cdh5-CreERT2; Krit1fl/fl; Kdrfl/fl (n = 8) mouse brains harvested on P11. The same brain specimens and corresponding microCT renders are also shown in Fig. S3 A to provide a global view of the whole brain. Representative images in this panel are presented with emphasis on cerebellar lesions. Scale bar: 1 mm. Data shown are means ± SEM. **P <0.01 and ****P <0.0001 by two-tailed Mann–Whitney U test (F and L), unpaired two-tailed t test (I), and one-way ANOVA, followed by Tukey HSD post hoc test (N and O). No statistically significant (n.s.) differences are observed in (F) P = 0.3036, (N) P = 0.1443, and (O) P = 0.1900. Source data are available for this figure: SourceData F3. Tukey HSD, Tukey’s honestly significant difference. Refer to the image caption for details. Panel A shows timeline of craniotomy, A A V injection, imaging, lesion development monitoring. Panel B shows immunofluorescence staining of t d Tomato, C D 31, V E G F R 2, DAPI brain sections. Panel C shows a vertical bar graph quantifying V E G F R 2 positive and negative endothelial populations. Panel D shows serial cranial window images tracking lesion growth over time. Panel E shows brain images and micro C T renders comparing C C M lesion presence. Panel F shows a vertical bar graph quantifying C C M lesion volumes between mouse genotypes. Panel G shows a schematic timeline of neonatal induction and tissue harvest experiment. Panel H shows an immunoblot detecting V E G F R 2 protein expression in neonatal cerebellum samples. Panel I shows a vertical bar graph quantification of V E G F R 2 protein immunoblot expression levels. Panel J shows immunofluorescence images of C D 31, V E G F R 2, p V E G F R 2 neonatal cerebellum vessels. Panel K shows a horizontal bar graph quantifying V E G F R 2 positive and negative endothelial cells. Panel L shows a vertical bar graph quantifying p V E G F R 2 T y r 951 phosphorylation levels in cerebellum. Panel M shows representative brain images and micro C T renders highlighting cerebellar lesions. Panel N shows bar graph quantifying C C M lesion volume across neonatal genotypes. Panel O shows a vertical bar graph quantifying total brain volume across neonatal genotypes.

Endothelial deletion of Kdr does not prevent CCM formation in the adult or neonatal mouse model. (A) Schematic representation of adult Krit1fl/fl; iPik3caH1047R mice or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl mice, aged 8–10 wk, underwent craniotomy and received focal AAV-Cre injections. Live-animal microscopic images were captured through the cranial window from POD 7 to POD 21 to observe lesion formation and growth. (B) Representative images showing immunostaining for tdTomato, CD31, and VEGFR2, with DAPI counterstaining in brains harvested from Kdrfl/fl; Ai14 mice on POD 7. The boxed regions in the tdTomato-negative regions (a) and in the tdTomato-positive regions (b) are shown in a’–a’’’ and b’–b’’’, respectively, at higher magnification. Yellow arrows indicate CD31-positive but VEGFR2-negative endothelial cells in the tdTomato-positive regions (tdTomato+, CD31+, and VEGFR2−). Scale bar: 100 μm. (C) Quantification of VEGFR2-positive (CD31+; VEGFR2+) and VEGFR2-negative (CD31+; VEGFR2−) endothelial cells in both tdTomato-negative and -positive regions. (D) Serial of microscopic images through the cranial windows showing CCM growth in either Krit1fl/fl; iPik3caH1047R or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl mouse brains from POD 10 to POD 21. (E and F) Representative visual images (left panels) and microCT renders (right panels) (E), and (F) microCT quantification of CCM lesion volumes in Krit1fl/fl; iPik3caH1047R (n = 7) or Krit1fl/fl; iPik3caH1047R; Kdrfl/fl (n = 7) mouse brains harvested on POD 21. The same microCT renders are also shown in Fig. S2 C to provide a comprehensive overview of genotype-dependent effects on lesion burden across the entire study cohort. Scale bar: 1 mm. (G) Schematic representation of neonatal induction of Krit1 deletion with or without Kdr deletion. 4-OHT was intragastrically injected on P1, and brains were harvested on P11. (H) Immunoblot detection of VEGFR2 in neonatal cerebellum harvested from P11 littermates. β-Actin was used as a loading control. (I) Quantification of immunoblotting for VEGFR2 protein level. A total of three paired littermates were measured and quantified. (J) Representative images showing immunostaining for CD31, VEGFR2, and pVEGFR2, with DAPI counterstaining in neonatal brains harvested from P11 littermates. White arrows indicate CD31 and VEGFR2 double-positive (CD31+; VEGFR2+) cells and yellow arrows indicate CD31-positive but VEGFR2-negative (CD31+; VEGFR2−) cells at the lesional and non-lesional neonatal cerebellum. Asterisks indicate autofluorescence emitted from lumenal red blood cells. Scale bar: 100 μm. (K) Quantification of VEGFR2-positive (CD31+; VEGFR2+) and VEGFR2-negative (CD31+; VEGFR2−) cerebellar endothelial cells. (L) Quantification of MFI of pVEGFR2Y951 in Krit1ECKO (Cdh5-CreERT2; Krit1fl/fl) neonatal cerebellum, normalized to total VEGFR2, compared with vasculature in the normal littermate control (Krit1fl/fl). (M–O) Representative visual images (top panels) and microCT renders (bottom panels), and microCT quantification of (N) CCM lesion volumes and (O) total brain volumes in Cdh5-CreERT2; Krit1fl/fl (n = 13), Cdh5-CreERT2; Krit1fl/fl; Kdrfl/+ (n = 18), and Cdh5-CreERT2; Krit1fl/fl; Kdrfl/fl (n = 8) mouse brains harvested on P11. The same brain specimens and corresponding microCT renders are also shown in Fig. S3 A to provide a global view of the whole brain. Representative images in this panel are presented with emphasis on cerebellar lesions. Scale bar: 1 mm. Data shown are means ± SEM. **P <0.01 and ****P <0.0001 by two-tailed Mann–Whitney U test (F and L), unpaired two-tailed t test (I), and one-way ANOVA, followed by Tukey HSD post hoc test (N and O). No statistically significant (n.s.) differences are observed in (F) P = 0.3036, (N) P = 0.1443, and (O) P = 0.1900. Source data are available for this figure: SourceData F3. Tukey HSD, Tukey’s honestly significant difference.

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