Evidence for anti-angiogenic and pro-survival functions of the cerebral cavernous malformation protein 3
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- Schleider, E., Stahl, S., Wüstehube, J. et al. Neurogenetics (2011) 12: 83. doi:10.1007/s10048-010-0261-6
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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.
KeywordsCerebral cavernous malformationCCM3PDCD10
Cerebral cavernous malformations (CCM) have a prevalence of about one in 200 individuals and can occur sporadically or in an autosomal dominantly inherited manner with 60% disease penetrance. Affected patients present with chronic headaches, epilepsy, or hemorrhagic stroke. Second hit somatic loss-of-function mutations in CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10) have been identified within cavernous lesions .
The gene products of CCM1 and CCM2 were shown to be important for endothelial cell–cell junction formation and maintenance of endothelial barrier function [2, 3]. Most recently, it could be demonstrated that CCM1 also inhibits endothelial proliferation, apoptosis, migration, lumen formation, and sprouting angiogenesis in primary human endothelial cells . In contrast, CCM3 remains without precise endothelial cell function description. Originally, CCM3 mRNA was found to be upregulated in an apoptotic human myeloid cell line . An apoptosis-inducing role was suggested based on CCM3 overexpression experiments in HeLa cells and siRNA-mediated inhibition of endogenously increased CCM3 in serum-deprived human umbilical vein endothelial cells (HUVECs) . In contrast, CCM3 was reported to promote cell proliferation in a human prostate cancer cell line . Endothelial cell-specific disruption of ccm3 in mice demonstrated that CCM3 is essential for early embryonic vascular development and acts through stabilization of VEGFR2 signaling .
The aim of this study was to characterize CCM3 function in primary human endothelial cells. The isolation of highly pure endothelial cells from CCM lesions has been hampered by calcification of the resected lesions and the presence of diverse cell types within CCM specimen including not only cavernous but also normal neoangiogenic endothelial cells . Therefore, an adenoviral approach with almost 100% transduction efficiency was used for overexpression of human CCM3 in HUVECs (details are provided in Supplementary Information (ESM 1)).
Similarly, it was recently shown that CCM1 shifts the balance from ERK-mediated proliferation to Akt-mediated cell survival and endothelial quiescence . Accordingly, CCM3-transduced endothelial cells showed decreased metabolic activity which we interpret to be a sign of endothelial quiescence rather than cell death (Fig. 1f).
As observed for CCM1 , adenoviral CCM3 expression per se did not induce apoptosis under normal endothelial cell culture conditions (Fig. 1g). However, similar to CCM1, CCM3 reduced the rate of staurosporine-induced cell death: induction of apoptosis with 250 nmol/l staurosporine for 2 h resulted in more than fourfold increased caspase 3/7 activities in GFP-overexpressing HUVECs. In contrast, CCM3-overexpressing HUVECs showed only a less than twofold increase in caspase 3/7 activities (Fig. 1g). These data are in line with the observation by Chen and coworkers that endogenous CCM3 expression is increased in HUVECs 30 min after induction of apoptosis by serum deprivation prior to the increase of cleaved caspase three levels seen after 3 h . However, our data demonstrate that upregulation of CCM3 protects endothelial cells from staurosporine-induced cell death rather than inducing it.
Tyrosine kinase activity profiling represents an unbiased novel approach to dissect the mechanisms behind the observed negative effects of CCM3 overexpression on endothelial cell proliferation, migration, tube formation, and apoptosis. Details on tyrosine kinase activity profiling are provided in Supplementary Information (ESM 1). HUVECs transduced with CCM3 demonstrated significantly increased tyrosine 9-specific phosphorylation for 3-phosphoinositide-dependent protein kinase 1 (PDPK-1, data not shown). Elevated levels of PDPK-1 phosphorylated at its tyrosine-9 residue could be confirmed with phosphospecific antibodies against PDPK-1 in HUVECs overexpressing CCM3 (Fig. 1h). PDPK-1 had been shown to be important for endothelial cell migration  and formation of a circulatory system in embryonic mice  and plays a central role in Akt activation upon growth factor stimulation . In accordance with the observation that embryonic CCM3 knockout tissues showed decreased Akt phosphorylation , CCM3 overexpression led to increased phosphorylation of the potent inhibitor of apoptosis Akt at threonine 308 and serine 473 (Fig. 1h) . Again, our data are in line with the fact that CCM1 expression increased the amount of phosphorylated Akt at serine 473 . Thus, CCM3 expression resulted in altered phosphorylation patterns in endothelial cells including activation of Akt and its upstream activator PDPK-1.
Taken together, we show that CCM3 inhibits endothelial cell migration, proliferation, and tube formation and supports PDPK-1/Akt-mediated endothelial cell survival. Our data suggest that CCM3 plays a crucial role in maintaining endothelial integrity and protection from cell death.
The authors thank D. Pijnenburg and A. Brilleman from PamGene International B.V. for performing PamChip kinase arrays and bioinformatics. This work was supported by the Bavarian Genome Network. E.S. received a stipend from the Graduiertenkolleg 1048. A.F. receives funding from the Deutsche Forschungsgemeinschaft DFG (SFB-Transregio 23 and Graduiertenkolleg 880).
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