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.

Novel Murine Models of Cerebral Cavernous Malformations

“…The research community has identified a number of different signaling pathways dysregulated following loss of the CCM genes: RhoA/ROCK [15][16][17][18], MEKK3-KLF2/4 [19][20][21], ICAP-1 and β1 integrin [22,23], DELTA-NOTCH [24], angiopoietin-2 [25], thrombomodulin and endothelial protein C receptor [26], reactive oxygen species (ROS) [27], autophagy [28], and endothelial-to-mesenchymal transition (EndMT) [29]. A nearly equal number of therapeutics have also been proposed or tested in CCM models: statins [15], fasudil [30], TGF-β inhibitors [29], sulindac [31], tempol [32], vitamin D 3 [32], angiopoietin-2 neutralizing antibody [25], fluvastatin and zoledronate [33], indirubin-3′-monoxime [34], thrombospondin1 replacement [35], propranolol [36,37], ponatinib [38], BA1049 [39], and VEGFR2 inhibitor [40]. Several of these compounds arose from unbiased, high-throughput in vitro and in vivo screens of libraries containing thousands of compounds [32][33][34].…”

“…Since their development, the inducible CCM alleles have required deletion during the first three days of life to induce CCMs. Postnatal developmental angiogenesis in the cerebellum and retina is a strong sensitizer of CCM formation [62] and the inhibition of angiogenesis, with multiple different therapies, reduces CCM formation [25,40]. We hypothesized that deleting Pdcd10 later in developmental angiogenesis may lead to a reduced CCM burden that is conducive to drug studies lasting several weeks.…”

Novel hemorrhage models of cerebral cavernous malformations

“…The CCMs that develop in these inducible models occur nearly exclusively in the cerebellum, suggesting a strong sensitizing role of angiogenesis. Inhibition of vascular endothelial growth factor (VEGF) signaling with SU5416, a VEGFR2 specific antibody, in an inducible CCM1 mouse model reduced CCM formation and hemorrhage [40]. By contrast, an exploratory biomarker study found plasma levels of VEGF to be lower in CCM patients who had a hemorrhage in the past 3 months when compared to CCM patients without hemorrhage [68].…”

“…Chronic CCM hemorrhage is a hallmark feature of the human disease for which few animal models exist for therapeutic testing [53,51,52,58] . Very few studies have been able to use an inducible CCM mouse model to study a chronic CCM hemorrhage, none of which have been with the more aggressive CCM3 mouse model [40]. One reason for the paucity of studies measuring chronic hemorrhage is the need to conduct experiments over several months for the chronic hemorrhage phenotype to develop.…”

“…The research community has identified a number of different signaling pathways dysregulated following loss of the CCM genes: RhoA/ROCK [15][16][17][18], MEKK3-KLF2/4 [19][20][21], ICAP-1 and b1 integrin [22,23], DELTA-NOTCH [24], angiopoietin-2 [25], thrombomodulin and endothelial protein C receptor [26], reactive oxygen species (ROS) [27], autophagy [28], and endothelial-to-mesenchymal transition (EndMT) [29]. A nearly equal number of therapeutics have also been proposed or tested in CCM models: statins [15], fasudil [30], TGF-b inhibitors [29], sulindac [31], tempol [32], vitamin D3 [32], angiopoietin-2 neutralizing antibody [25], fluvastatin and zoledronate [33], indirubin-3'-monoxime [34], thrombospondin1 replacement [35], propranolol [36,37], ponatinib [38], BA1049 [39], and VEGFR2 inhibitor [40] . Several of these compounds arose from unbiased, highthroughput in vitro and in vivo screens of libraries containing thousands of compounds [32][33][34].…”