Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause stroke and seizures in younger individuals1. CCMs arise from endothelial cell loss of KRIT1, CCM2, or PDCD10, non-homologous proteins that form an adaptor complex2. How disruption of the CCM complex results in disease remains controversial, with numerous signaling pathways (including Rho3,4, SMAD5 and Wnt/β-catenin6) and processes such as endothelial-mesenchymal transition (EndMT)5 proposed to play causal roles. CCM2 binds MEKK37–11, and we have recently demonstrated that CCM complex regulation of MEKK3 is essential during vertebrate heart development12. Here, we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we find that expression of the MEKK3 target genes KLF2 and KLF4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. In contrast, we find no evidence of EndMT or increased SMAD or Wnt signaling during early CCM formation. Endothelial-specific loss of Mekk3, Klf2, or Klf4 dramatically prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we demonstrate that endothelial expression of KLF2 and KLF4 is elevated in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signaling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

SignificancePIEZOs are mechanically activated cation channels. Recently, loss-of-function mutations of human PIEZO1 were found among patients with familial lymphedema, suggesting a requirement of PIEZO1 in the lymphatic system. In this paper, utilizing mouse models lacking PIEZO1 in endothelial cells, we show that this ion channel is required for the formation of lymphatic valves, a key structure for proper circulation of lymph in the body. The requirement of PIEZO1 in valve formation provides mechanistic insight on how PIEZO1 variants cause lymphatic dysfunction in patients. This study also extends the relevance of PIEZOs beyond acute signaling molecules (e.g., touch sensation) and highlights the importance of these ion channels in controlling morphological/structural specification during development.

SUMMARY The cerebral cavernous malformation (CCM) pathway is required in endothelial cells for normal cardiovascular development and to prevent postnatal vascular malformations, but its molecular effectors are not well defined. Here we show that loss of CCM signaling in endocardial cells results in mid-gestation heart failure associated with premature degradation of cardiac jelly. CCM deficiency dramatically alters endocardial and endothelial gene expression, including increased expression of the Klf2 and Klf4 transcription factors and the Adamts4 and Adamts5 proteases that degrade cardiac jelly. These changes in gene expression result from increased activity of MEKK3, a mitogen-activated protein kinase that binds CCM2 in endothelial cells. MEKK3 is both necessary and sufficient for expression of these genes, and partial loss of MEKK3 rescues cardiac defects in CCM-deficient embryos. These findings reveal a molecular mechanism by which CCM signaling controls endothelial gene expression during cardiovascular development that may also underlie CCM formation.

Summary Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b. Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development, and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway.

Summary Vascular permeability is a highly coordinated process that integrates vesicular trafficking, complex junctional rearrangements, and refined cytoskeletal dynamics. In response to the extracellular environment, these three cellular activities have been previously assumed to work in parallel to regulate the passage of solutes between the blood and tissues. New developments in the area of vascular permeability, however have highlighted the interdependence between trans- and para-cellular pathways, the cross-communication between adherens and tight junctions, and the instructional role of pericytes on endothelial expression of barrier-related genes. Additionally, significant effort has been placed in understanding the molecular underpinings that contribute to barrier restoration following acute permeability events and in clarifying the importance of context-dependent signaling initiated by permeability mediators. Finally, recent findings have uncovered an unpredicted role for transcription factors in the coordination of vascular permeability and clarified how junctional complexes can transmit signals to the nucleus to control barrier function. The goal of this review is to provide a concise and updated view of vascular permeability, discuss the most recent advances in molecular and cellular regulation, and introduce integrated information on the central mechanisms involved in trans-endothelial transport.