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Loss of CCM3 impairs DLL4-Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations.

You C, Sandalcioglu IE, Dammann P, Felbor U, Sure U, Zhu Y - J. Cell. Mol. Med. (2013)

Bottom Line: Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways.Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression.CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany.

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Silencing CCM3 affected multiple signalling components in cultured endothelial cells, which was reversed by rhDLL4. (A) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in CCMEC. CCMECs were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C). Total RNA was extracted at 48 and 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (B) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in HBMEC. HBMEC received the transfection with either 70 nM of siCCM3 or Neg. C. Total RNA was extracted 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (C) The treatment of rhDLL4 reversed the down-regulation of VEGF-R1 and the up-regulation of VEGF-R2, but did not influenced the expression of VEGF, in CCM3-silenced HUVEC. Cells were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C) in the presence or the absence of rhDLL4 (1 μg/ml). Total RNA was extracted 72 hrs after the transfection for RT2-PCR. (D) The treatment with rhDLL4 restored DLL4 protein expression, elevated the levels of cleaved Notch4 and target protein Hey1, and reversed increase in p-Erk1/2 expression mediated by silencing CCM3. Total protein was extracted 72 hrs after the transfection in the presence or the absence of rhDLL4 (1 μg/ml). The data presented in A–D were representative of at least three independent experiments. *P < 0.05, **P < 0.001 and ***P < 0.001, compared with Neg. C; +P < 0.05, ++P < 0.01 and +++P < 0.001, compared with siCCM3 alone.
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fig05: Silencing CCM3 affected multiple signalling components in cultured endothelial cells, which was reversed by rhDLL4. (A) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in CCMEC. CCMECs were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C). Total RNA was extracted at 48 and 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (B) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in HBMEC. HBMEC received the transfection with either 70 nM of siCCM3 or Neg. C. Total RNA was extracted 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (C) The treatment of rhDLL4 reversed the down-regulation of VEGF-R1 and the up-regulation of VEGF-R2, but did not influenced the expression of VEGF, in CCM3-silenced HUVEC. Cells were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C) in the presence or the absence of rhDLL4 (1 μg/ml). Total RNA was extracted 72 hrs after the transfection for RT2-PCR. (D) The treatment with rhDLL4 restored DLL4 protein expression, elevated the levels of cleaved Notch4 and target protein Hey1, and reversed increase in p-Erk1/2 expression mediated by silencing CCM3. Total protein was extracted 72 hrs after the transfection in the presence or the absence of rhDLL4 (1 μg/ml). The data presented in A–D were representative of at least three independent experiments. *P < 0.05, **P < 0.001 and ***P < 0.001, compared with Neg. C; +P < 0.05, ++P < 0.01 and +++P < 0.001, compared with siCCM3 alone.

Mentions: To study the downstream pathway(s) influenced by CCM3-silence-mediated inactivation of DLL4-Notch signalling, we examined the expression of VEGF, VEGF-R1 and VEGF-R2 in different types of cultured endothelial cells. In CCMEC, CCM3 siRNA transfection resulted in an efficient down-regulation of CCM3 level to 30% and 20% of the control at 48 hrs (P < 0.001) and 72 hrs (P < 0.001) respectively. Under these silence conditions, the level of VEGF elevated by 76% (P < 0.01) and 55% (P < 0.01), and more interestingly, the expression of VEGF-R2 increased by 82% and 187% of control at 48 hrs (P < 0.01) and 72 hrs (P < 0.001), respectively, after the transfection. In contrast, the VEGF-R1 mRNA level was moderately down-regulated at 72 hrs after siCCM3 transfection (P < 0.05; Fig. 5A). A similar tendency of change in the expression of VEGF, and VEGF-R1 and VEGF-R2 was observed in CCM3-silenced HUVEC (Fig. 5C), whereas CCM3 silencing in HBMEC resulted in only minor elevation of VEGF-R2 level (Fig. 5B).


Loss of CCM3 impairs DLL4-Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations.

You C, Sandalcioglu IE, Dammann P, Felbor U, Sure U, Zhu Y - J. Cell. Mol. Med. (2013)

Silencing CCM3 affected multiple signalling components in cultured endothelial cells, which was reversed by rhDLL4. (A) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in CCMEC. CCMECs were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C). Total RNA was extracted at 48 and 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (B) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in HBMEC. HBMEC received the transfection with either 70 nM of siCCM3 or Neg. C. Total RNA was extracted 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (C) The treatment of rhDLL4 reversed the down-regulation of VEGF-R1 and the up-regulation of VEGF-R2, but did not influenced the expression of VEGF, in CCM3-silenced HUVEC. Cells were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C) in the presence or the absence of rhDLL4 (1 μg/ml). Total RNA was extracted 72 hrs after the transfection for RT2-PCR. (D) The treatment with rhDLL4 restored DLL4 protein expression, elevated the levels of cleaved Notch4 and target protein Hey1, and reversed increase in p-Erk1/2 expression mediated by silencing CCM3. Total protein was extracted 72 hrs after the transfection in the presence or the absence of rhDLL4 (1 μg/ml). The data presented in A–D were representative of at least three independent experiments. *P < 0.05, **P < 0.001 and ***P < 0.001, compared with Neg. C; +P < 0.05, ++P < 0.01 and +++P < 0.001, compared with siCCM3 alone.
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Related In: Results  -  Collection

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fig05: Silencing CCM3 affected multiple signalling components in cultured endothelial cells, which was reversed by rhDLL4. (A) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in CCMEC. CCMECs were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C). Total RNA was extracted at 48 and 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (B) The expression of VEGF, VEGF-R1 and VEGF-R2 after siCCM3 transfection in HBMEC. HBMEC received the transfection with either 70 nM of siCCM3 or Neg. C. Total RNA was extracted 72 hrs after the transfection for RT2-PCR. CCM3 was detected for controlling an efficient silence induced by siRNA transfection. (C) The treatment of rhDLL4 reversed the down-regulation of VEGF-R1 and the up-regulation of VEGF-R2, but did not influenced the expression of VEGF, in CCM3-silenced HUVEC. Cells were transfected with either 70 nM of specific CCM3 siRNA (siCCM3) or control siRNA (Neg. C) in the presence or the absence of rhDLL4 (1 μg/ml). Total RNA was extracted 72 hrs after the transfection for RT2-PCR. (D) The treatment with rhDLL4 restored DLL4 protein expression, elevated the levels of cleaved Notch4 and target protein Hey1, and reversed increase in p-Erk1/2 expression mediated by silencing CCM3. Total protein was extracted 72 hrs after the transfection in the presence or the absence of rhDLL4 (1 μg/ml). The data presented in A–D were representative of at least three independent experiments. *P < 0.05, **P < 0.001 and ***P < 0.001, compared with Neg. C; +P < 0.05, ++P < 0.01 and +++P < 0.001, compared with siCCM3 alone.
Mentions: To study the downstream pathway(s) influenced by CCM3-silence-mediated inactivation of DLL4-Notch signalling, we examined the expression of VEGF, VEGF-R1 and VEGF-R2 in different types of cultured endothelial cells. In CCMEC, CCM3 siRNA transfection resulted in an efficient down-regulation of CCM3 level to 30% and 20% of the control at 48 hrs (P < 0.001) and 72 hrs (P < 0.001) respectively. Under these silence conditions, the level of VEGF elevated by 76% (P < 0.01) and 55% (P < 0.01), and more interestingly, the expression of VEGF-R2 increased by 82% and 187% of control at 48 hrs (P < 0.01) and 72 hrs (P < 0.001), respectively, after the transfection. In contrast, the VEGF-R1 mRNA level was moderately down-regulated at 72 hrs after siCCM3 transfection (P < 0.05; Fig. 5A). A similar tendency of change in the expression of VEGF, and VEGF-R1 and VEGF-R2 was observed in CCM3-silenced HUVEC (Fig. 5C), whereas CCM3 silencing in HBMEC resulted in only minor elevation of VEGF-R2 level (Fig. 5B).

Bottom Line: Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways.Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression.CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany.

Show MeSH
Related in: MedlinePlus