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 inactivate zebrafish ccm2 resulting in novel lethal multi-cavernous lesions in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic intussusceptive angiogenesis; however, 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 resulting aberrant flow signaling. Surviving adult fish manifested histologically-typical hemorrhagic CCM. Formation of mammalian CCM requires flow-regulated transcription factors, KLF2 and KLF4; 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 the first zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.

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

“…adrb1 -/embryo also displayed a marked decrease of blood flow in CVP (Supplementary Video 2, and Supplementary Figure 1B), A similar reduction of heart rate and contractility by 2,3-BDM, a cardiac myosin ATPase inhibitor, prevented CVP dilation (Figure .4F). We previously found that arresting blood flow prevents aberrant intussusceptive angiogenesis and the resulted CVP cavernomas in ccm2 CRISPR embryos 3 . Taken together, these data suggest that reduced blood flow secondary to reduced cardiac function accounts for the protective effect of loss of β1AR signaling on CVP lesions and on CCM.…”

“…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…”

“…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.…”

“…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.…”