Evidence for anti-angiogenic and pro-survival functions of the cerebral cavernous malformation protein 3.

Mutations in CCM1, CCM2, or CCM3 lead to cerebral cavernous malformations, one of the most common hereditary vascular diseases of the brain. Endothelial cells within these lesions are the main disease compartments. Here, we show that adenoviral CCM3 expression inhibits endothelial cell migration, proliferation, and tube formation while downregulation of endogenous CCM3 results in increased formation of tube-like structures. Adenoviral CCM3 expression does not induce apoptosis under normal endothelial cell culture conditions but protects endothelial cells from staurosporine-induced cell death. Tyrosine kinase activity profiling suggests that CCM3 supports PDPK-1/Akt-mediated endothelial cell quiescence and survival.

Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling.

Cerebral cavernous malformations (CCM) are frequent vascular abnormalities caused by mutations in one of the CCM genes. CCM1 (also known as KRIT1) stabilizes endothelial junctions and is essential for vascular morphogenesis in mouse embryos. However, cellular functions of CCM1 during the early steps of the CCM pathogenesis remain unknown. We show here that CCM1 represents an antiangiogenic protein to keep the human endothelium quiescent. CCM1 inhibits endothelial proliferation, apoptosis, migration, lumen formation, and sprouting angiogenesis in primary human endothelial cells. CCM1 strongly induces DLL4-NOTCH signaling, which promotes AKT phosphorylation but reduces phosphorylation of the mitogen-activated protein kinase ERK. Consistently, blocking of NOTCH activity alleviates CCM1 effects. ERK phosphorylation is increased in human CCM lesions. Transplantation of CCM1-silenced human endothelial cells into SCID mice recapitulates hallmarks of the CCM pathology and serves as a unique CCM model system. In this setting, the multikinase inhibitor Sorafenib can ameliorate loss of CCM1-induced excessive microvascular growth, reducing the microvessel density to levels of normal wild-type endothelial cells. Collectively, our data suggest that the origin of CCM lesions is caused by perturbed Notch signaling-induced excessive capillary sprouting, which can be therapeutically targeted.

Integrin cytoplasmic domain-associated protein-1 attenuates sprouting angiogenesis.

RATIONALE: The ICAP1 (integrin cytoplasmic domain-associated protein-1) is a specific intracellular binding protein of beta1-integrins and the cerebral cavernous malformation (CCM) protein CCM1. ICAP1 recruits CCM1 to the cell membrane and activates CCM1 by changing its conformation. Because CCM1 plays a critical role for cardiovascular development, we hypothesized that its activator ICAP1 is involved in vascular differentiation. OBJECTIVE: The objective of this study was to define the role of ICAP1 in endothelial cells. METHODS AND RESULTS: Loss of ICAP1 in primary human endothelial cells causes excessive angiogenic branching and network formation in vitro (3D sprouting angiogenesis) and in vivo (xenotransplantation of ICAP1-silenced human endothelial cells). ICAP1 increases cell motility and the initial formation of capillary sprouts but prevents vessel outgrowth. ICAP1 inhibits Rho kinase activity and ERK (extracellular signal-regulated kinase) phosphorylation and induces expression of the cell cycle inhibitors p21 and p27, leading to less endothelial proliferation. However, ICAP1 promotes endothelial survival and AKT phosphorylation. Global gene expression analyses revealed that the ICAP1 effects are mediated by strong activation of DELTA-NOTCH signaling. Active NOTCH1 or silencing of the NOTCH ligand DLL4 phenocopy the ICAP1 effects and blockade of NOTCH cleavage rescues the ICAP1-mediated defects in endothelial cells. Both ICAP1 and NOTCH1 reduce the expression of ESM1 (endothelial cell-specific molecule-1), and silencing of ESM1 disturbs vascular endothelial growth factor- or fibroblast growth factor 2-induced sprouting angiogenesis. CONCLUSIONS: In this study, we identified ICAP1 as a novel regulator to prevent excessive sprouting angiogenesis.

Regulation of mitochondrial dynamics--characterization of fusion and fission genes in the ascomycete Podospora anserina.

The filamentous ascomycete Podospora anserina is a model system for studying aging, a complex process that is regulated by multiple factors. Among these, mitochondria were shown to be of crucial importance. Recently, it was shown that the morphology of these organelles, which is dependent on dynamic fusion and fission processes, has profound effects on P. anserina aging. To further analyze this phenomenon, we characterized molecular components of the machinery regulating the dynamic behavior of mitochondria by utilizing transgenic strains in which fission genes (PaDnm1, PaFis1 and PaMdv1) and a fusion gene (PaFzo1) are overexpressed. While overexpression of PaFis1 has no phenotypic effects in the genetic background of the wild type, it surprisingly promotes mitochondrial fusion and decreases the life span in a mutant overexpressing PaDnm1. Remarkably, when grown on synthetic medium, overexpression of PaDnm1 leads to a decreased life span compared to the wild type. Increased expression of PaMdv1 results in the formation of ring-shaped mitochondria, a morphology of these organelles that has not been previously observed in P. anserina. Transformants with elevated PaFzo1 transcript levels show no altered life span, although the age-dependent fragmentation of mitochondria is impaired.