Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells

Rath, Matthias; Schwefel, Konrad; Malinverno, Matteo; Skowronek, Dariush; Leopoldi, Alexandra; Pilz, Robin A.; Biedenweg, Doreen; Bekeschus, Sander; Penninger, Josef; Dejana, Elisabetta; Felbor, Ute

doi:10.1007/s00018-022-04355-6

Cited by 7 publications

(4 citation statements)

References 52 publications

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“…This also reinforces the important role of exogenous triggers for CCM formation. Even though additional factors occurring in vivo, e.g., cell-cell interactions with pericytes and astrocytes [ 30 , 44 ] or ECs with preserved CCM expression [ 25 ], are most likely also necessary to trigger the comprehensive pathophysiological changes after CCM protein loss, our study strengthens that a specific microenvironment is one important factor in the multi-step pathomechanism in CCM disease.…”

Section: Discussionmentioning

confidence: 54%

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Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature

Pilz

Skowronek

Mellinger

et al. 2023

IJMS

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Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1−/− iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1−/− precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.

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Section: Discussionmentioning

confidence: 54%

“…However, we did not observe major differences. Given that we have only examined a limited number of targets and that certain processes in CCM formation such as inflammatory mechanisms may only be activated under in vivo or co-culture conditions [ 25 , 26 ], further studies are needed to elucidate the proteomic effect of CCM1 knockout.…”

Section: Resultsmentioning

confidence: 99%

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature

Pilz

Skowronek

Mellinger

et al. 2023

IJMS

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“…Moreover, we demonstrate that CRISPR/Cas9 editing in iPSCs represents a powerful approach for variant interpretation and can provide a promising platform for basic research or therapeutic CCM studies. Using iPSC-derived human brain microvascular endothelial-like cells and mosaic vascular organoids, we were recently able to show abnormal proliferation of CCM3 mutant ECs in co-culture with wild-type ECs ( Rath et al, 2022 ). In the future, novel patient-specific, co-culture or three-dimensional iPSC-based cell culture models could give further insight into CCM pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion

Pilz

Skowronek

Hamed

et al. 2022

Front. Mol. Biosci.

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Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis.

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“…Expanding upon the cancer-like mechanisms identified for CCM formation 20,21 , we asked if cooperative migration strategies such as those used by fibroblasts to regulate tumor cell invasion 22,23 might also contribute to recruitment and reprogramming of WT ECs by CCM mutants. Since this multifactorial question requires an approach to systematically explore the mechanics of both cell-matrix and cell-cell interactions, we leveraged our three-dimensional (3D) biomimetic model of angiogenesis 24 integrated with biophysical computations using 3D traction force microscopy and molecular investigations using single-cell RNA sequencing.…”

Section: Introductionmentioning

confidence: 99%

Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth

Shapeti,

Barrasa-Fano,

Fattah

et al. 2023

Preprint

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Force-driven cellular interactions are known to play a critical role in cancer cell invasion, but have remained largely unexplored in the context of vascular abnormalities, partly due to a lack of suitable genetic and cellular models. One such vascular abnormality, cerebral cavernous malformation (CCM) is characterized by leaky, tumor-like vessels in the brain, where CCM mutant cells recruit wild-type cells from the surrounding endothelium to form mosaic lesions and promote lesion growth; however the mechanisms underlying this recruitment remain poorly understood. Here, we use 3D traction force microscopy in a in-vitro model of early angiogenic invasion to reveal that hyper-angiogenic CCM2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We show that mechanically hyperactive CCM2-silenced tips guide wild-type cells by exerting and transmitting pulling forces and by leaving degraded paths in the matrix as cues promoting invasion in a ROCKs-dependent manner. This transmission of forces is associated with a reinforcement of β1 integrin-dependent adhesive sites and actin cytoskeleton in the wild-type followers. We also show that during this process wild-type cells are reprogrammed into stalk cells through activation of matrisome and DNA replication programs, eventually leading to cell proliferation. These observations unveil a novel vascular lesion growth mechanism where CCM2 mutants hijack the function of wild-type cells to fuel CCM lesion growth. By integrating biophysical computational methodologies to quantify cellular forces with advanced molecular techniques, we provide new insights in the etiology of vascular malformations, and open up avenues to study the role of cell mechanics in tissue heterogeneity and disease progression.

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Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells

Cited by 7 publications

References 52 publications

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature

Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion

Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth

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