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      Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signaling

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          Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause stroke and seizures in younger individuals 1 . CCMs arise from endothelial cell loss of KRIT1, CCM2, or PDCD10, non-homologous proteins that form an adaptor complex 2 . How disruption of the CCM complex results in disease remains controversial, with numerous signaling pathways (including Rho 3, 4 , SMAD 5 and Wnt/β-catenin 6 ) and processes such as endothelial-mesenchymal transition (EndMT) 5 proposed to play causal roles. CCM2 binds MEKK3 711 , and we have recently demonstrated that CCM complex regulation of MEKK3 is essential during vertebrate heart development 12 . 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.

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          Most cited references 39

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          Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.

          In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.
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            Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.

            Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting. Copyright 2001 Academic Press.
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              EndMT contributes to the onset and progression of cerebral cavernous malformations.

              Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-β and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.

                Author and article information

                2 February 2016
                30 March 2016
                7 April 2016
                30 September 2016
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                : 122-126
                [1 ]Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 3400 Civic Center Blvd, Philadelphia PA 19104
                [2 ]Lab of Cardiovascular Signaling, Centenary Institute, Sydney NSW 2050, Australia
                [3 ]Neurovascular Surgery Program, Section of Neurosurgery, Department of Surgery, The University of Chicago Medicine and Biological Sciences, Chicago, Illinois, USA
                [4 ]Department of Radiology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia PA 19104
                [5 ]Sydney Microscopy & Microanalysis, University of Sydney, Sydney, NSW 2050, Australia
                [6 ]Division of Cell Signaling and Immunology, University of Dundee, Dundee, United Kingdom, DD1 5EH
                [7 ]Division of Cardiovascular Medicine and the Program in Molecular Medicine, University of Utah, Salt Lake City, UT 84112
                [8 ]Faculty of Medicine, Sydney Medical School, University of Sydney, Sydney, NSW 2050, Australia
                Author notes
                Correspondence should be addressed to: M.L.K. ( markkahn@ ), Telephone: 215-898-9007 FAX: 215-573-2094

                These authors contributed equally


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