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

ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis.

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis

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

ANKS1B promotes endothelial cell permeability.

<p>(A) Representative graphs of TER and Ccl measurement after ANKS1B- and ANKS1A-silencing via siRNA transfection, alone or in combination. Measurements with a second siRNA against ANKS1B showed comparable results. (B) Representative TER/Ccl measurement comparing ANKS1B overexpressing endothelial cells to GFP expressing +/- 5 μM cyclic AMP treated cells. Adenoviral overexpression of ANKS1B leads to a significantly increased electrical resistance in HUVEC comparable to values reached with cyclic AMP treatment. (C) Representative graphs of TER and Ccl measurement after ROCK1-, KRIT1- and ANKS1B-silencing via siRNA transfection, Knockdown of ROCK1 or addition of 8-pCPT-2′-O-Me-cAMP could not rescue the drop of electrical resistance due to ANKS1B silencing. In contrast silencing of ROCK1 lead to an rescue of almost 100% in KRIT1-silenced endothelial monolayers. (D) Representative graph of a transendothelial electrical resistance (TER) and the corresponding capacity (Ccl) measurement. Endothelial cells deficient of KRIT1 were treated with an adenovirus overexpressing ANKS1B. The increase of electrical resistance is comparable with KRIT1-deficient endothelial cells silenced for ROCK1. *, p < 0.05; **, p < 0.01, ***, p < 0.001, n = 3 experiments. Bar graphs show mean values, error bars indicate SD.</p

ANKS1B has no effect on endothelial sprouting.

<p>(A) HUVEC spheroids were embedded in collagen and stimulated with 25 ng/ml VEGF or FGF2. Capillary formation was measured 24 h later. Silencing of ANKS1B had no effect on cumulative sprout length (B), individual sprout length (C) and sprout number (D). N = 5 experiments. Scale bar equals 100 μm. Bar graphs show mean values, error bars indicate SD.</p

Silencing of ANKS1B does not affect endothelial proliferation and migration.

<p>(A) Representative Western blot for HUVEC protein lysates probed with anti-ANKS1B antibody after transfection with non-silencing control siRNA or two different single ANKS1B siRNAs. VCP served as protein loading control. (B) Transfection of HUVEC with two independent siRNA duplexes led to approximately 80% mRNA knockdown rates compared to non-silencing control siRNA. (C) BrdU incorporation into DNA was only slightly increased after by silencing ANKS1B expression. (D) ANKS1B knockdown did not alter the migration speed of HUVEC after wounding a monolayer. ***, p < 0.001, n = 3 experiments. Scale bar equals 100 μm. Bar graphs show mean values, error bars indicate SD.</p

Combined silencing of ANKS1A and ANKS1B does not affect endothelial migration and angiogenesis.

<p>(A) Representative Western blot for HUVEC protein lysates probed with anti-ANKS1A and anti-ANKS1B after transfection with non-silencing control siRNA or simultaneous transfection with ANKS1B and a pool of ANKS1A siRNAs. GAPDH served as protein loading control. (B) Quantitative realtime analysis of ANKS1A and ANKS1B mRNA expression in HUVEC treated with the indicated siRNAs. (C) Proliferation and (D) wound closure of HUVEC was not impaired after simultaneous silencing of ANKS1A and ANKS1B. (E) Sprouting angiogenesis under basal conditions, VEGF (25 ng/ml) or FGF2 (25 ng/ml) stimulation in collagen gels was not significantly changed after silencing of ANKS1A and ANKS1B. *, p < 0.05; **, p < 0.01, n = 3 experiments. Bar graphs show mean values, error bars indicate SD.</p