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Abstract The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.

Abstract Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non‐invasive pharmacological therapies. Here, we present the results of several unbiased small‐molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology‐based target prediction tool to integrate the results with the whole‐transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small‐molecule‐based therapies. We found indirubin‐3‐monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss‐of‐CCM phenotypes in human endothelial cells. Our multi‐organism‐based approach reveals new components of the CCM regulatory network and foreshadows novel small‐molecule‐based therapeutic applications for suppressing this devastating disease in patients.

Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.

Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7(+) cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.

DNA-protein crosslinks pose a significant challenge to genome stability and cell viability. Efficient repair of DPCs is crucial for preserving genomic integrity and preventing the accumulation of DNA damage. Despite recent advances in our understanding of DPC repair, many aspects of this process, especially at the organismal level, remain elusive. In this study, we used zebrafish as a model organism to investigate the role of TDP2 (Tyrosyl-DNA phosphodiesterase 2) in DPC repair. We characterized the two tdp2 orthologs in zebrafish using phylogenetic, syntenic and expression analysis and investigated the phenotypic consequences of tdp2 silencing in zebrafish embryos. We then quantified the effects of tdp2a and tdp2b silencing on cellular DPC levels and DSB accumulation in zebrafish embryos. Our findings revealed that tdp2b is the main ortholog during embryonic development, while both orthologs are ubiquitously present in adult tissues. Notably, the tdp2b ortholog is phylogenetically closer to human TDP2. Silencing of tdp2b, but not tdp2a, resulted in the loss of Tdp2 activity in zebrafish embryos, accompanied by the accumulation of DPCs and DSBs. Our findings contribute to a more comprehensive understanding of DPC repair at the organismal level and underscore the significance of TDP2 in maintaining genome stability.

DNA–protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.