Abortive intussusceptive angiogenesis causes multi-cavernous vascular malformations

Abstract

Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a over-expression and resultant aberrant flow signaling. Surviving adult fish manifested histologically-typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2

“…Finally, accumulating evidence implicates this transcription factor in vessel size regulation. Mammalian and piscine studies have found that mutations in Cerebral Cavernous Malformations (CCM) complex components and their partners induce pathogenic blood vessel dilations with elevated klf2 endothelial expression, and suppressing klf2 levels ameliorates these vascular lesions[34, 36, 37, 94-96]. Additionally, klf2a - and klf2a - ; klf2b -/+ fish mutants show axial vessels with progressively smaller outer diameters[35], consistent with the possibility that these animals have reduced vascular calibers.…”

Genetic Inactivation of the β1 adrenergic receptor prevents Cerebral Cavernous Malformations in zebrafish

“…Inactivation of CCM genes leads to increased endothelial KLF2 expression 17, 18 , a transcription factor important in cavernoma formation 3, 19 . Nevertheless, silencing of adrb1 did not prevent the expected increased endothelial klf2a 17 expression in ccm2 morphant Tg(klf2a:H2b-EGFP) fish in which the nuclear EGFP expression is driven by the klf2a promoter (Figure 4A and B).…”

“…Adult ccm2 CRISPR zebrafish display highly penetrant CCMs throughout the central nervous system 3 . Brains from adult ccm2 CRISPR fish on adrb1 -/- (12 brains) or wild type (13 brains) background were treated with CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) 15 , and these transparent brains were then scanned with light-sheet microscopy and lesions were enumerated and volumes were estimated with NIH ImageJ.…”

“…Familial CCMs are due to heterozygous loss of function mutations in the KRIT1, CCM2 , or PDCD10 genes with a second mutation inactivating the normal allele in random brain endothelial cells 2 . We previously developed a zebrafish CCM model using CRISPR-Cas9 to inactivate ccm2 in a manner that replicates the mosaic genetic background of the human disease 3 . This zebrafish model exhibits two phenotypic phases: lethal embryonic caudal venous plexus (CVP) cavernomas at 2 days post-fertilization (dpf) in ∼30% of embryos and histologically- typical CNS CCMs in ∼100% of surviving 8 week old fish 3 .…”

“…We previously developed a zebrafish CCM model using CRISPR-Cas9 to inactivate ccm2 in a manner that replicates the mosaic genetic background of the human disease 3 . This zebrafish model exhibits two phenotypic phases: lethal embryonic caudal venous plexus (CVP) cavernomas at 2 days post-fertilization (dpf) in ∼30% of embryos and histologically- typical CNS CCMs in ∼100% of surviving 8 week old fish 3 . Both phases of this model, like their murine counterpart, depend on Krüppel-like factor 2 (KLF2), confirming shared transcriptional pathways.…”