Abstract
Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1 −/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1 −/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1 −/− BOECs which was observed in CCM1 +/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.
Highlights
Cerebral cavernous malformations (CCMs) are convolutes of dilated capillaries in the central nervous system
To model the hereditary and sporadic type of CCM disease in vitro (Figure 1), we used blood outgrowth endothelial cells (BOECs) isolated from peripheral blood of a CCM proband with a heterozygous CCM1 loss-of-function germline variant (CCM1+/−) and wild-type CI-huVECs (CCM1+/+)
NGS amplicon sequencing verified compound heterozygosity for the preexisting CCM1 germline variant and a second CRISPR/Cas9induced mutation that led to inactivation of the CCM1 wild-type allele (Figure 2B, Supplementary Table S1)
Summary
Cerebral cavernous malformations (CCMs) are convolutes of dilated capillaries in the central nervous system. A recent prospective population-based study revealed that antithrombotic therapy – anticoagulant as well as antiplatelet – is associated with a lower risk of intracranial hemorrhage and focal neurological deficits (Zuurbier et al, 2019). These counterintuitive data are consistent with a neuropathological study that anticipated a dysfunction of cavernous ECs which would result in local organizing thrombi as the primary step causing repeated secondary microhemorrhages and disease progression (Abe et al, 2005). Secretion of large amounts of VWF from WPBs can be triggered by shear stress or stimulation with Ca2+ raising agents like histamine, thrombin or vascular endothelial growth factor (VEGF) (Loesberg et al, 1983; Erent et al, 2007)
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