Article
The unique angiogenic response of endothelial cells derived from human CCMs (CCM-ECs) to silencing CCM1-3 genes
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Authors
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Yuan Zhu
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany
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Qun Wu
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany; Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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Jin-Fang Xu
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany; Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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I. Erol Sandalcioglu
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany
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Ulrich Sure
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany
| Published: | May 21, 2013 |
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Outline
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Objective: Loss-of-function mutations in CCM genes are frequently detected in familial cerebral cavernous malformations (CCMs). However, the current functional studies on CCM genes have been performed mostly in commercial purchased cell lines and the results appeared discrepancy. We assumed that silencing CCM genes in the endothelia derived from human CCMs (CCM-EC) serves as a unique and valuable model for investigating the function of the CCM genes in the pathogenesis of CCMs. To this end, we investigated the role of CCM1-3 in angiogenesis using CCM-EC.
Method: CCM-ECs were isolated, purified and cultured from the fresh operative specimens of sporadic CCMs. CCM1-3 genes were respectively silenced by the specific siRNAs in CCM-ECs and in control cultures (HBMEC and HUVEC). The efficiency of gene silencing was proven by real-time PCR. Cell proliferation and apoptosis, migration, sprouting and tube formation were analyzed after silencing individual CCM genes.
Results: Silencing CCM1 inhibited endothelial proliferation in an order of HUVEC < HBMEC < CCM-EC, whereas the most significant increase in cell migration was observed in CCM-EC after CCM1 silencing. CCM3 deletion significantly promoted proliferation, reduced apoptosis in all three types of endothelium, but accelerated cell migration exclusively in CCM-EC. Interestingly, CCM2 siRNA influenced neither cell proliferation nor migration. Silencing CCM3 and, to a lesser extent, CCM1 and CCM2 stimulated the growth and extension of sprouts selectively in CCM-EC. Loss of CCM1, CCM2 or CCM3 did not significantly influence the formation of the tube-like structure. However, the maintenance of tube stability was distinctly different in CCM-EC and in HUVEC after silencing CCM1, CCM2 or CCM3.
Conclusions: The unique response of CCM-ECs to CCM1-3 silence indicated a noteworthy importance of pathological background underlying CCM-ECs. Silencing CCM genes in CCM-EC therefore provided a valuable in vitro model for further studies on the pathogenesis of CCMs.
