VEGF signalling enhances lesion burden in KRIT1 deficient mice

Abstract

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial-specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.

Keywords: angiogenesis; cerebral cavernous malformation; haemorrhagic stroke; vascular endothelial growth factor.

Figure 1

VEGFR2 Inhibition Reduces Lesion Number in Krit1^ieKO mice. A, Tyrosine 1175 phosphorylation (pY1175), total VEGFR2 and actin expression in microvascular brain endothelial cells isolated from wild‐type (Krit1^WT) or Krit1^ieKO mice treated with vehicle or SU5416. Blots are representative, n = 3. B, Average mouse weight (g) at 16 wk of age ± SEM, *P = .008 vs vehicle‐treated Krit1^ieKO mice by 2‐way ANOVA. C, Representative gross images of whole brains from wild‐type or Krit1^ieKO mice. Lesions are noticeable at the junction between the cerebrum and cerebellum. 4× magnification. D, Representative images of 2 mm coronal slices of brains from Krit1^WT or Krit1^ieKO mice. 4× magnification. E, Average number of lesions per mouse from vehicle or SU5416 treated Krit1^ieKO mice. Data shown are mean ± SEM, n = 7, P = .0013 by unpaired t test

Figure 2

Distribution of lesion size in vehicle and SU5416 treated Krit1^ieKO mice. A, Average maximum lesion diameter ± SEM. Vehicle, n = 593; SU5416, n = 391. B, Histogram distribution of percent total lesion number by lesion diameter (mm)

Figure 3

VEGFR2 activation increases in vivo microvessel permeability following loss of KRIT1. A, Cremaster microvessel permeability in wild type (Krit1^WT), heterozygous (Krit1 ^+/−) or Krit1^ieKO treated with vehicle or SU5416. Data shown are mean P_s ± SEM, n = 15 vessel sites, P < .001 by ANOVA, *P < .001 by post hoc testing vs vehicle treated Krit1^WT and **P < .001 vs vehicle‐treated Krit1^ieKO. B, Permeability of brain vasculature to fluorescein. Data shown are mean tissue fluorescence, normalized to serum fluorescein levels ± SEM, n = 4 mice/group, P < .01 by ANOVA, *P < .01 by post hoc testing vs vehicle treated Krit1^WT and **P < .001 vs vehicle‐treated Krit1^ieKO

Figure 4

VEGFR2 activation increases lesion haemorrhage following loss of KRIT1. A, Haematoxylin and eosin (H&E) and free iron staining of 5‐µm sections of coronal brain slices from Krit1^ieKO mice treated with vehicle or SU5416. Images are representative. Iron staining appears blue in image. B, Percentage of lesions that are positive for iron staining from vehicle and SU5416 treated Krit1^ieKO mice. Data shown are averaged from 2 distinct 5‐µm sections per mouse ± SEM, n = 11 per treatment type, *P = .017 by unpaired t test. C, Average area of iron staining per lesion. Box is standard deviation, with mean ± 95% CI (whiskers). Vehicle, n = 117; SU5416, n = 32. *P = .002 by unpaired t test. D, Average maximum lesion diameter of iron‐positive lesions. Vehicle, n = 117; SU5416, n = 32. *P = .03 vs vehicle treated by unpaired t test

Figure 5

Loss of CCM protein family members increases VEGFR2 tyrosine phosphorylation. A, Tyrosine phosphorylation of VEGFR2 in HPAEC transfected with negative control (NC), anti‐KRIT1, anti‐CCM2 and anti‐CCM3 siRNA. IB, immunoblot; IP, immunoprecipitation. Blots are representative, n = 3. B, Densitometric quantification of blots in (A). Data shown are P‐Tyr normalized to total VEGFR2, ± SEM, P = .0223 by ANOVA, *P < .05 by post hoc testing vs NC transfected cells. C, VEGF protein (pg/mL) in conditioned media harvested from cells expressing anti‐KRIT1, anti‐CCM2 and anti‐CCM3 siRNA P = .032 by ANOVA, *P < .05 by post hoc testing vs NC transfected cells