Article
Loss of CCM2, but not CCM3, impairs the integrity of endothelial tube structure – An association with the incidence of intracerebal haemorrhage of inherited cerebral cavernous malformations (CCMs)?
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Published: | September 16, 2010 |
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Objective: Mutations in CCM genes may cause autosomal dominant cerebral cavernous malformations (CCMs). Indeed, a different intracranial hemorrhage rate has been observed in CCMs carrying individual CCM gene mutations. We proposed a differential regulation of angiogenesis by these disease genes. The present study thus investigated the function of CCM2 and CCM3 in tube formation, a key step involved in angiogenesis.
Methods: CCM2 and CCM3 were silenced by the specific siRNAs in endothelial cells (HUVEC). The time course of the gene silencing was established by detecting the efficiency of knockdown using real-time PCR. For tube formation assay, the transfected cells were pooled in matrigel-coated 96-well plate followed by 6 h, 24 h and 48 h of incubation. The tube formation was analyzed under a microscope.
Results: Real-time PCR showed that CCM2 expression declined by more that 80% at 48 h and 72 h, and returned to the control level at 96 h after the transfection, whereas CCM3 gene levels were consistently reduced around 80% from 48 h to 96 h after siRNA. Under these efficient knockdown conditions, we observed that the tube was similarly formed in the CCM2- and CCM3-silenced cells and in control at 6h after the incubation. However, the maintenance of tube integrity was largely impaired by CCM2, but not CCM3, silencing at 24 h and 48 h after pooling cells on matrigel. Quantification of the number of tubes revealed that CCM2 silencing resulted in a 50% of reduction of tubes at 24 h and a complete loss of the tube structure at 48h after the incubation.
Conclusions: Our results demonstrated a distinct role of CCM2 and CCM3 in maintenance of tube stability. The severe impairment of the integrity of tube structure after endothelial CCM2 silencing may be associated with the clinical observation that CCM2-mutated CCMs present higher incidence of haemorrhage in comparison to CCM3-mutated cases.