Figure 4.
Genetic deletion of TIE2 prevents CCM formation in both adult and neonatal mouse brains. (A) Schematic representation of adult Krit1fl/fl; iPik3caH1047R mice or Krit1fl/fl; iPik3caH1047R; Tekfl/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 TIE2, with DAPI counterstaining in brains harvested from Tekfl/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 TIE2-negative endothelial cells in the tdTomato-positive regions (tdTomato+, CD31+, and TIE2−). Scale bar: 100 μm. (C) Quantification of TIE2-positive (CD31+; TIE2+) and TIE2-negative (CD31+; TIE2−) 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; Tekfl/fl mouse brains from POD 10 to POD 21. (E and F) Representative visual images (left panels) and microCT renders (right panels), and (F) microCT quantification of CCM lesion volumes in Krit1fl/fl; iPik3caH1047R (n = 12) or Krit1fl/fl; iPik3caH1047R; Tekfl/fl (n = 20) mouse brains harvested on POD 21. The same microCT renders are also shown in Fig. S4 A 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 Tek deletion. 4-OHT was intragastrically injected on P1, and brains were harvested on P11. (H) Immunoblot detection of TIE2 in neonatal cerebellum harvested from P11 littermates. β-Actin was used as a loading control. (I) Quantification of immunoblotting for TIE2 protein level. A total of three paired littermates were measured and quantified. (J) Representative images showing immunostaining for CD31, TIE2, and pTIE2, with DAPI counterstaining in neonatal brains harvested from P11 littermates. White arrows indicate CD31 and TIE2 double-positive (CD31+; TIE2+) cells and yellow arrows indicate CD31-positive but TIE2-negative (CD31+; TIE2−) 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 TIE2-positive (CD31+; TIE2+) and TIE2-negative (CD31+; TIE2−) cerebellar endothelial cells. (L) Quantification of MFI of pTIE2Y992 in Krit1ECKO (Cdh5-CreERT2; Krit1fl/fl) neonatal cerebellum, normalized to total TIE2, 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 = 7), Cdh5-CreERT2; Krit1fl/fl; Tekfl/+ (n = 9), and Cdh5-CreERT2; Krit1fl/fl; Tekfl/fl (n = 7) mouse brains harvested on P11. Scale bar: 1 mm. Data shown are means ± SEM. *P <0.05 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 (N) P = 0.9997 and (O) P = 0.8649. Source data are available for this figure: SourceData F4. Tukey HSD, Tukey’s honestly significant difference. Refer to the image caption for details. The image illustrates the experimental setup and results of studying the impact of T I E 2 deletion on C C M formation in mouse brains. Panel A shows a schematic of adult mice undergoing craniotomy and A A V-C r e injections, with live-animal imaging from P O D 7 to P O D 21. Panel B displays immunostaining images for t d Tomato, C D 31, T I E 2, and DAPI in T E K f l slash f l; A i 14 mice brains on P O D 7, highlighting C D 31-positive but T I E 2-negative endothelial cells. Panel C quantifies T I E 2-positive and T I E 2-negative endothelial cells in t d Tomato-negative and -positive regions. Panel D shows serial microscopic images of C C M growth in mouse brains from P O D 10 to P O D 21. Panels E and F present visual images and micro C T renders of C C M lesion volumes in different mouse brains on P O D 21, with quantification shown in Panel F. Panel G outlines the neonatal induction of K r i t 1 deletion with or without T e k deletion, with brains harvested on P 11. Panel H shows immunoblot detection of T I E 2 in neonatal cerebellum, with quantification in Panel I. Panel J displays immunostaining images for C D 31, T I E 2, p T I E 2, and DAPI in neonatal brains, indicating C D 31 and T I E 2 double-positive cells and C D 31-positive but T I E 2-negative cells. Panel K quantifies T I E 2-positive and T I E 2-negative cerebellar endothelial cells. Panel L quantifies the mean fluorescence intensity of p T I E 2 Y 992 in K r i t 1 E C K O neonatal cerebellum. Panels M to O show visual images, microC T renders, and quantification of C C M lesion volumes and total brain volumes in different mouse brains on P 11.

Genetic deletion of TIE2 prevents CCM formation in both adult and neonatal mouse brains. (A) Schematic representation of adult Krit1fl/fl; iPik3caH1047R mice or Krit1fl/fl; iPik3caH1047R; Tekfl/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 TIE2, with DAPI counterstaining in brains harvested from Tekfl/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 TIE2-negative endothelial cells in the tdTomato-positive regions (tdTomato+, CD31+, and TIE2−). Scale bar: 100 μm. (C) Quantification of TIE2-positive (CD31+; TIE2+) and TIE2-negative (CD31+; TIE2−) 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; Tekfl/fl mouse brains from POD 10 to POD 21. (E and F) Representative visual images (left panels) and microCT renders (right panels), and (F) microCT quantification of CCM lesion volumes in Krit1fl/fl; iPik3caH1047R (n = 12) or Krit1fl/fl; iPik3caH1047R; Tekfl/fl (n = 20) mouse brains harvested on POD 21. The same microCT renders are also shown in Fig. S4 A 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 Tek deletion. 4-OHT was intragastrically injected on P1, and brains were harvested on P11. (H) Immunoblot detection of TIE2 in neonatal cerebellum harvested from P11 littermates. β-Actin was used as a loading control. (I) Quantification of immunoblotting for TIE2 protein level. A total of three paired littermates were measured and quantified. (J) Representative images showing immunostaining for CD31, TIE2, and pTIE2, with DAPI counterstaining in neonatal brains harvested from P11 littermates. White arrows indicate CD31 and TIE2 double-positive (CD31+; TIE2+) cells and yellow arrows indicate CD31-positive but TIE2-negative (CD31+; TIE2−) 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 TIE2-positive (CD31+; TIE2+) and TIE2-negative (CD31+; TIE2−) cerebellar endothelial cells. (L) Quantification of MFI of pTIE2Y992 in Krit1ECKO (Cdh5-CreERT2; Krit1fl/fl) neonatal cerebellum, normalized to total TIE2, 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 = 7), Cdh5-CreERT2; Krit1fl/fl; Tekfl/+ (n = 9), and Cdh5-CreERT2; Krit1fl/fl; Tekfl/fl (n = 7) mouse brains harvested on P11. Scale bar: 1 mm. Data shown are means ± SEM. *P <0.05 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 (N) P = 0.9997 and (O) P = 0.8649. Source data are available for this figure: SourceData F4. Tukey HSD, Tukey’s honestly significant difference.

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