Figure 5.
Endothelial TIE2 expression and activity are increased following loss of CCM function or gain of KLF4 function, acting upstream of PI3K in CCM pathogenesis. (A) Schematic representation of adult mice, aged 8–10 wk, undergoing craniotomy followed by focal AAV-Cre injection. Brains were harvested on POD 7, followed by histology studies. (B) Representative live-animal fluorescence microscopy showing tdTomato reporter signals following focal AAV-Cre injection through the cranial window. (C) Representative images showing immunostaining for TIE2 and pTIE2 in adult brains harvested on POD 7 from (a) Ai14 only, (b) Krit1fl/fl; Ai14, and (c) iPik3caH1047R; Ai14. Asterisks indicate autofluorescence emitted from lumenal red blood cells. (D) Quantification of MFI of pTIE2Y992, normalized to total TIE2 in the tdTomato-positive regions, as compared with the tdTomato-negative regions in the brain. A total of three to six mice in each group were measured and quantified. (E) Representative images showing immunostaining for CD31 and pS6, with DAPI counterstaining in neonatal brains harvested from of P11 littermates. White arrows indicate CD31 and pS6 double-positive (CD31+; pS6+) cells and yellow arrows indicate CD31-positive but pS6-negative (CD31+; pS6−) cells at the lesional and non-lesional neonatal cerebellum. (F) Quantification of pS6-positive (CD31+; TIE2+) and pS6-negative (CD31+; TIE2−) cerebellar endothelial cells. A total of three independent litters were measured and quantified. (G) Immunoblot detection of TIE2 and KLF4 in control and KRIT1-KO HUVECs. Cells were generated by lentiviral transduction of Cas9 with control (gCTL) or KRIT1-targeting (gKRIT1) gRNAs. (H) Quantification of immunoblotting for TIE2 and KLF4 relative to TUB protein (n = 4 independent experiments). (I) Immunoblot detection of TIE2 and KLF4 in isolated mouse brain endothelial cells carrying Cdh5-CreERT2; Krit1fl/fl alleles. GAPDH was used as a loading control. (J) Quantification of immunoblotting for TIE2 relative to GAPDH protein (n = 3 independent experiments). (K) Immunoblot detection of TIE2 and KLF4 in HUVECs transduced with doxycycline (Dox)-inducible control and V5-KLF4–encoding lentiviruses. Cells were treated with 200 ng/ml Dox for 48 h. (L) Quantification of immunoblotting for TIE2 relative to TUB protein (n = 3 independent experiments). (M) Immunoblot detection of TIE2, KLF4, and MEKK3 in CRISPR/Cas9-edited HUVECs (gCTL or gKRIT1) following 48 h siRNA-mediated knockdown of KLF2/4 (siKLF2/4), MAP3K3 (siMAP3K3), or scrambled control (siCTL). (N) Quantification of immunoblotting for TIE2 relative to TUB protein (n = 3 independent experiments). (O) Immunoprecipitation of TIE2 from gCTL and gKRIT1 HUVECs treated with recombinant human ANGPT1 and the TIE2 inhibitor BAY-826, followed by immunoblot analysis using the indicated antibodies. (P) Quantification of p-TYR, p-AKT, and p-S6 levels normalized to total TIE2, AKT, and S6, respectively (n = 3 independent experiments). (Q) Model depicting the regulation of TIE2 expression and function downstream of CCM-MEKK3–KLF2/4 signaling that links to PI3K-mTORC1 signaling to drive the formation of CCMs. Scale bar: 100 μm. Data shown are means ± SEM. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001 by two-tailed independent t test (H, J, and L) and one-way ANOVA, followed by Tukey HSD post hoc test (D, F, N, and P). No statistically significant (n.s.) differences are observed in P (left to right: P = 0.7395, 0.9998, 0.9946, 0.7696, and 0.7401). Source data are available for this figure: SourceData F5. Refer to the image caption for details. Panel A shows experimental timeline of A A V-C r e injection and brain histology. Panel B shows live fluorescence imaging of t d Tomato reporter activation. Panel C shows T I E 2 and p T I E 2 immunostaining in adult brains. Panel D shows a vertical bar graph quantifying p T I E 2 phosphorylation levels. Panel E shows C D 31 and p S 6 immunostaining in neonatal cerebellum. Panel F shows a vertical bar graph quantifying p S 6-positive endothelial cells. Panel G shows a western blot of T I E 2 and K L F 4 in K R I T 1-K O H U V E C s. Panel H shows a vertical bar graph quantifying T I E 2 and K L F 4 protein levels. Panel I shows a western blot of T I E 2 and K L F 4 in brain endothelial cells. Panel J shows a vertical bar graph quantifying T I E 2 protein expression levels. Panel K shows western blot of inducible K L F 4 and T I E 2 expression. Panel L shows a vertical bar graph quantifying T I E 2 expression after K L F 4 induction. Panel M shows western blot after K L F 2 slash 4 or M A P 3 K 3 knockdown. Panel N shows a vertical bar graph quantifying T I E 2 expression after knockdown. Panel O shows T I E 2 immunoprecipitation and downstream signaling analysis. Panel P shows vertical bar graphs quantifying T I E 2, A K T, S 6 phosphorylation. Panel Q shows a schematic model linking C C M signaling to T I E 2 pathway.

Endothelial TIE2 expression and activity are increased following loss of CCM function or gain of KLF4 function, acting upstream of PI3K in CCM pathogenesis. (A) Schematic representation of adult mice, aged 8–10 wk, undergoing craniotomy followed by focal AAV-Cre injection. Brains were harvested on POD 7, followed by histology studies. (B) Representative live-animal fluorescence microscopy showing tdTomato reporter signals following focal AAV-Cre injection through the cranial window. (C) Representative images showing immunostaining for TIE2 and pTIE2 in adult brains harvested on POD 7 from (a) Ai14 only, (b) Krit1fl/fl; Ai14, and (c) iPik3caH1047R; Ai14. Asterisks indicate autofluorescence emitted from lumenal red blood cells. (D) Quantification of MFI of pTIE2Y992, normalized to total TIE2 in the tdTomato-positive regions, as compared with the tdTomato-negative regions in the brain. A total of three to six mice in each group were measured and quantified. (E) Representative images showing immunostaining for CD31 and pS6, with DAPI counterstaining in neonatal brains harvested from of P11 littermates. White arrows indicate CD31 and pS6 double-positive (CD31+; pS6+) cells and yellow arrows indicate CD31-positive but pS6-negative (CD31+; pS6−) cells at the lesional and non-lesional neonatal cerebellum. (F) Quantification of pS6-positive (CD31+; TIE2+) and pS6-negative (CD31+; TIE2−) cerebellar endothelial cells. A total of three independent litters were measured and quantified. (G) Immunoblot detection of TIE2 and KLF4 in control and KRIT1-KO HUVECs. Cells were generated by lentiviral transduction of Cas9 with control (gCTL) or KRIT1-targeting (gKRIT1) gRNAs. (H) Quantification of immunoblotting for TIE2 and KLF4 relative to TUB protein (n = 4 independent experiments). (I) Immunoblot detection of TIE2 and KLF4 in isolated mouse brain endothelial cells carrying Cdh5-CreERT2; Krit1fl/fl alleles. GAPDH was used as a loading control. (J) Quantification of immunoblotting for TIE2 relative to GAPDH protein (n = 3 independent experiments). (K) Immunoblot detection of TIE2 and KLF4 in HUVECs transduced with doxycycline (Dox)-inducible control and V5-KLF4–encoding lentiviruses. Cells were treated with 200 ng/ml Dox for 48 h. (L) Quantification of immunoblotting for TIE2 relative to TUB protein (n = 3 independent experiments). (M) Immunoblot detection of TIE2, KLF4, and MEKK3 in CRISPR/Cas9-edited HUVECs (gCTL or gKRIT1) following 48 h siRNA-mediated knockdown of KLF2/4 (siKLF2/4), MAP3K3 (siMAP3K3), or scrambled control (siCTL). (N) Quantification of immunoblotting for TIE2 relative to TUB protein (n = 3 independent experiments). (O) Immunoprecipitation of TIE2 from gCTL and gKRIT1 HUVECs treated with recombinant human ANGPT1 and the TIE2 inhibitor BAY-826, followed by immunoblot analysis using the indicated antibodies. (P) Quantification of p-TYR, p-AKT, and p-S6 levels normalized to total TIE2, AKT, and S6, respectively (n = 3 independent experiments). (Q) Model depicting the regulation of TIE2 expression and function downstream of CCM-MEKK3–KLF2/4 signaling that links to PI3K-mTORC1 signaling to drive the formation of CCMs. Scale bar: 100 μm. Data shown are means ± SEM. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001 by two-tailed independent t test (H, J, and L) and one-way ANOVA, followed by Tukey HSD post hoc test (D, F, N, and P). No statistically significant (n.s.) differences are observed in P (left to right: P = 0.7395, 0.9998, 0.9946, 0.7696, and 0.7401). Source data are available for this figure: SourceData F5.

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