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. 2010 Jul 13;107(28):12640-5.
doi: 10.1073/pnas.1000132107. Epub 2010 Jun 24.

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

Joycelyn Wüstehube  1 , Arne Bartol, Sven S Liebler, René Brütsch, Yuan Zhu, Ute Felbor, Ulrich Sure, Hellmut G Augustin, Andreas Fischer
Affiliations

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

Joycelyn Wüstehube et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CCM1 inhibits sprouting angiogenesis. (A–C) HUVEC were adenovirally transduced with CCM1 or GFP as control leading to an about 5-fold overexpression of CCM1 mRNA as determined by qPCR (A). (B) CCM1 inhibited tube formation and branching on a Matrigel matrix. (Scale bars, 2,000 μm.) (C) CCM1 inhibited VEGF (25 ng/mL) or FGF2 (25 ng/mL) stimulated sprouting angiogenesis in a collagen matrix. (Scale bar, 200 μm.) (D) Down-regulation of CCM1 mRNA expression by two siRNAs (Ambion) led to about 80% reduction of mRNA levels. (E) Small interfering RNA-mediated CCM1 silencing strongly enhanced endothelial sprouting in collagen gels under basal conditions and after VEGF or FGF2 stimulation. (Scale bar, 200 μm.) (F and G) Control HUVEC were labeled with the red fluorescent membrane dye PKH26 and mixed with CCM1 shRNA (F) or cDNA (G) expressing HUVEC stained with the green membrane dye PKH67. After sprouting in collagen beds (25 ng/mL VEGF), spheroids were imaged with confocal laser microscopy and 3D images were calculated. (F) CCM1 depletion caused more and larger sprouts. (Scale bar, 100 μm.) (G) CCM1-expressing HUVEC formed few regular sprouts compared with control. *P < 0.05. (Scale bar, 100 μm.) Error bars are means ± SD of n = 10 in C and E.
Fig. 2.
Fig. 2.
CCM1 inhibits endothelial migration and proliferation. (A) Confluent HUVEC were wounded and migration was assayed. Short hairpin RNA against CCM1 enhanced closure of the gap, whereas lentiviral CCM1 expression inhibited cell migration (arrows). Because shRNA vectors also expressed GFP, fluorescence pictures are shown, whereas CCM1 over expression is shown as bright field image. (Scale bars, 200 μm.) (B) Transmigration through a collagen coated filter (8-μm pore size) toward VEGF (25 ng/mL) or FGF2 (25 ng/mL) was inhibited by CCM1 and enhanced after CCM1 knockdown. (C) Antibody staining against focal adhesion kinase (FAK) revealed a higher number and more polarized focal adhesion contacts at the cell periphery after CCM1 knockdown. (Scale bar, 50 μm.) (D–G) CCM1 inhibited endothelial proliferation as shown by reduced BrdU incorporation (D), decreased fraction of cells in S-phase as determined by FACS (E), and up-regulation of the cell cycle inhibitors p21 and p27 mRNA (F). (G) Western blot analysis 48 h after CCM1 adenovirus transduction showed that CCM1 reduced phosphorylation of ERK1/2 proteins, whereas siRNA treatment elevated phospho-ERK1/2 levels. (H) Protein lysates of human CCM lesion exhibit high phospho-ERK1/2 amounts. (I) Apoptosis of HUVEC under normal culture conditions and 2 h after addition of 250 nmol/L staurosporine was measured by detection of caspase-3 and -7 activities with a luminescent substrate. CCM1 expression reduced the rate of staurosporine-induced cell death. (J and K) CCM1 expression increased the amount of phosphorylated AKT protein at serine 473. *P < 0.05. Error bars are means ± SD of n = 5.
Fig. 3.
Fig. 3.
CCM1 inhibits angiogenesis in transplanted endothelial cells. (A) Scheme of the spheroid-based angiogenesis assay. (B) Representative sections through plugs stained against human CD34 (green) for endothelial cells. HUVEC expressing control shRNA formed a regular capillary vascular network. CCM1 silenced HUVEC formed a denser network with larger vessels. (Scale bar, 50 μm.) (C) Quantification of microvessel density determined by the number of vessels per square milimeter. n = 7 plugs of control and CCM1 shRNA. (D) Thick sections (50 μm) were stained with human specific anti-CD31 to stain the endothelial network and assessed by confocal laser microscopy (LSM710; Zeiss). shRNA against CCM1 led to formation of a denser network with larger vessels compared with control.
Fig. 4.
Fig. 4.
CCM1 activates DELTA-NOTCH signaling. (A) Quantitative RT-PCR showing significantly elevated mRNA levels of the NOTCH ligand DLL4 and the target genes HEY1 and HEY2 48 h after adenoviral CCM1 transduction. Small interfering RNA treatment against CCM1 down-regulated expression of DLL4 and NOTCH target genes. (B) CCM1 expression in HUVEC increased the amounts of cleaved NOTCH receptor proteins as detected by a cleavage-specific antibody. (C) Western blotting revealed strongly increased levels of phospho-AKT after 36 h of adenoviral CCM1 transduction. The NOTCH inhibitor DAPT (25 μM) could not prevent AKT phosphorylation. Inhibition of PI3K activity by Wortmannin (2 μM) prevented AKT phosphorylation independent of CCM1 expression. (D) Adenoviral expression of constitutive active NOTCH1 (intracellular domain, ICD) increased phospho-AKT levels. (E) Adenoviral CCM1 expression decreased ERK1/2 phosphorylation and this could fully be prevented with the NOTCH inhibitor DAPT (25 μM). (F) Active NOTCH1 strongly inhibited ERK1/2 phosphorylation. (G) Proposed scheme of CCM1-mediated regulation of endothelial functions. *P < 0.05.
Fig. 5.
Fig. 5.
Rescue of the CCM1 phenotype. (A and B) CCM1 expression inhibited sprouting angiogenesis of HUVEC after stimulation with VEGF (25 ng/mL). The NOTCH cleavage inhibitor DAPT (25 μM) could almost completely counteract this defect. (Scale bar in A, 200 μm.) (C and D) Sorafenib (10 μM) could block VEGF-induced sprouting in control and CCM1 siRNA silenced HUVEC. (Scale bar in C, 200 μm.) (E–G) The spheroid-based angiogenesis assay was employed to test Sorafenib in vivo. Twenty-eight days after implantation, mice were treated with Sorafenib or solvent for 7 d. (E) Quantification of microvascular density. (F) Representative sections show a hyperdense vascular network of CCM1-silenced endothelial cells which can be reverted by Sorafenib. (Scale bar, 50 μm.) (G) Three-dimensional reconstruction of the vascular networks by confocal microscopy (LSM710, Zeiss). Sorafenib normalizes the CCM vasculature. Error bars are means ± SD of n = 3 plugs; *P < 0.05.
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References

    1. Moriarity JL, et al. The natural history of cavernous malformations: A prospective study of 68 patients. Neurosurgery. 1999;44:1166–1171. - PubMed
    1. Labauge P, Denier C, Bergametti F, Tournier-Lasserve E. Genetics of cavernous angiomas. Lancet Neurol. 2007;6:237–244. - PubMed
    1. Akers AL, Johnson E, Steinberg GK, Zabramski JM, Marchuk DA. Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): Evidence for a two-hit mechanism of CCM pathogenesis. Hum Mol Genet. 2009;18:919–930. - PMC - PubMed
    1. Pagenstecher A, Stahl S, Sure U, Felbor U. A two-hit mechanism causes cerebral cavernous malformations: Complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells. Hum Mol Genet. 2009;18:911–918. - PMC - PubMed
    1. Glading A, Han J, Stockton RA, Ginsberg MH. KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell cell junctions. J Cell Biol. 2007;179:247–254. - PMC - PubMed

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