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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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Concomitant down-regulation of CCM3 expression and inactivation of DLL4-Notch signalling in human cavernous tissue derived from a CCM3 mutation carrier in comparison with the controls (sporadic CCMs). (A) Down-regulation of mRNA levels of CCM3 and the core components of DLL4-Notch signalling in a familial CCM harbouring CCM3 mutation. Total RNA was extracted from operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM3 mutation carrier (Mu-CCM3). The expression of the genes was quantified by RT2-PCR. The relative gene expression was calculated when the gene level in familial CCM was normalized as 1. (B) Down-regulation of the protein levels of CCM3, DLL4 and cleaved Notch4 accompanied with an up-regulation of VEGF and p-Erk1/2 in human CCM3-mutated tissue. The total protein was extracted from the operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM harbouring CCM3 mutation (Mu-CCM3). Semi-quantification of the blot was performed by measuring integrate optical density (IOD) of each band followed by calculation of the ratio of IOD from the interested protein to that from the housekeeping protein GAPDH. The data were presented as the percentage of the Mu-CCM3. (C) Pathological morphology of CCM revealed by haematoxylin and eosin staining on the section from a sporadic CCM. The lesion consists of immature capillary-like vessels without normal interposed brain parenchyma. These vessels are dilated and often thrombosed (asterisks). (D–J) Immunofluorescent staining of CCM3, DLL4 and vWF on the sections of human CCM lesions. The immunofluorescent staining was simultaneously performed on the adjacent paraffin sections prepared from a CCM harbouring CCM3 mutation (Mu3-CCM; D–F) and from a sporadic CCM (Sp-CCM; G–J). In Mu3-CCM, vWF staining revealed the endothelial layer (arrows) in the thrombosed cavern (F), whereas the immunoreactivity of CCM3 (D) and DLL4 (E) was absent in these endothelial cells. In Sp-CCM, CCM3 (H), DLL4 (I) and vWF (J) were concomitantly detected in the endothelial layer (arrows) of the adjacent sections. Negative control omitting the primary antibody (Neg; G) did not show specific staining, and revealed only unspecific signal (red) in thrombosis and in erythrocytes (arrows) which appeared nuclear staining (blue) negative. The original magnification was 200× in main graphs and 400× in white box.
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fig03: Concomitant down-regulation of CCM3 expression and inactivation of DLL4-Notch signalling in human cavernous tissue derived from a CCM3 mutation carrier in comparison with the controls (sporadic CCMs). (A) Down-regulation of mRNA levels of CCM3 and the core components of DLL4-Notch signalling in a familial CCM harbouring CCM3 mutation. Total RNA was extracted from operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM3 mutation carrier (Mu-CCM3). The expression of the genes was quantified by RT2-PCR. The relative gene expression was calculated when the gene level in familial CCM was normalized as 1. (B) Down-regulation of the protein levels of CCM3, DLL4 and cleaved Notch4 accompanied with an up-regulation of VEGF and p-Erk1/2 in human CCM3-mutated tissue. The total protein was extracted from the operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM harbouring CCM3 mutation (Mu-CCM3). Semi-quantification of the blot was performed by measuring integrate optical density (IOD) of each band followed by calculation of the ratio of IOD from the interested protein to that from the housekeeping protein GAPDH. The data were presented as the percentage of the Mu-CCM3. (C) Pathological morphology of CCM revealed by haematoxylin and eosin staining on the section from a sporadic CCM. The lesion consists of immature capillary-like vessels without normal interposed brain parenchyma. These vessels are dilated and often thrombosed (asterisks). (D–J) Immunofluorescent staining of CCM3, DLL4 and vWF on the sections of human CCM lesions. The immunofluorescent staining was simultaneously performed on the adjacent paraffin sections prepared from a CCM harbouring CCM3 mutation (Mu3-CCM; D–F) and from a sporadic CCM (Sp-CCM; G–J). In Mu3-CCM, vWF staining revealed the endothelial layer (arrows) in the thrombosed cavern (F), whereas the immunoreactivity of CCM3 (D) and DLL4 (E) was absent in these endothelial cells. In Sp-CCM, CCM3 (H), DLL4 (I) and vWF (J) were concomitantly detected in the endothelial layer (arrows) of the adjacent sections. Negative control omitting the primary antibody (Neg; G) did not show specific staining, and revealed only unspecific signal (red) in thrombosis and in erythrocytes (arrows) which appeared nuclear staining (blue) negative. The original magnification was 200× in main graphs and 400× in white box.

Mentions: Next, we examined DLL4-Notch signalling in human CCMs. Because of the extremely rare availability, only one human surgical specimen of a CCM3 germline mutation carrier could be obtained during this study. To draw a more convinced comparison between familial and sporadic CCMs, we used six surgical specimens from sporadic CCM as control. RT2-PCR detected around 5-, 38- and 35-fold decrease in CCM3, DLL4 and Notch4 expression, respectively, in the CCM3-deficient cavernous lesion (Mu-CCM3) in comparison with the human sporadic CCMs (Sp-CCM). A dramatic down-regulation of the target gene HEY2 and HES1 was also observed in Mu-CCM3, indicating an impaired DLL4-Notch signalling in familial CCM with germline mutation in CCM3. Interestingly, the level of Notch1 was similarly detected in Mu-CCM3 and in Sp-CCMs (Fig. 3A). These data drawn from CCM patients were entirely consistent with the results obtained from CCM3-silenced HUVEC (Fig. 2A) and CCMEC (Fig. 2D).


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)

Concomitant down-regulation of CCM3 expression and inactivation of DLL4-Notch signalling in human cavernous tissue derived from a CCM3 mutation carrier in comparison with the controls (sporadic CCMs). (A) Down-regulation of mRNA levels of CCM3 and the core components of DLL4-Notch signalling in a familial CCM harbouring CCM3 mutation. Total RNA was extracted from operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM3 mutation carrier (Mu-CCM3). The expression of the genes was quantified by RT2-PCR. The relative gene expression was calculated when the gene level in familial CCM was normalized as 1. (B) Down-regulation of the protein levels of CCM3, DLL4 and cleaved Notch4 accompanied with an up-regulation of VEGF and p-Erk1/2 in human CCM3-mutated tissue. The total protein was extracted from the operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM harbouring CCM3 mutation (Mu-CCM3). Semi-quantification of the blot was performed by measuring integrate optical density (IOD) of each band followed by calculation of the ratio of IOD from the interested protein to that from the housekeeping protein GAPDH. The data were presented as the percentage of the Mu-CCM3. (C) Pathological morphology of CCM revealed by haematoxylin and eosin staining on the section from a sporadic CCM. The lesion consists of immature capillary-like vessels without normal interposed brain parenchyma. These vessels are dilated and often thrombosed (asterisks). (D–J) Immunofluorescent staining of CCM3, DLL4 and vWF on the sections of human CCM lesions. The immunofluorescent staining was simultaneously performed on the adjacent paraffin sections prepared from a CCM harbouring CCM3 mutation (Mu3-CCM; D–F) and from a sporadic CCM (Sp-CCM; G–J). In Mu3-CCM, vWF staining revealed the endothelial layer (arrows) in the thrombosed cavern (F), whereas the immunoreactivity of CCM3 (D) and DLL4 (E) was absent in these endothelial cells. In Sp-CCM, CCM3 (H), DLL4 (I) and vWF (J) were concomitantly detected in the endothelial layer (arrows) of the adjacent sections. Negative control omitting the primary antibody (Neg; G) did not show specific staining, and revealed only unspecific signal (red) in thrombosis and in erythrocytes (arrows) which appeared nuclear staining (blue) negative. The original magnification was 200× in main graphs and 400× in white box.
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fig03: Concomitant down-regulation of CCM3 expression and inactivation of DLL4-Notch signalling in human cavernous tissue derived from a CCM3 mutation carrier in comparison with the controls (sporadic CCMs). (A) Down-regulation of mRNA levels of CCM3 and the core components of DLL4-Notch signalling in a familial CCM harbouring CCM3 mutation. Total RNA was extracted from operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM3 mutation carrier (Mu-CCM3). The expression of the genes was quantified by RT2-PCR. The relative gene expression was calculated when the gene level in familial CCM was normalized as 1. (B) Down-regulation of the protein levels of CCM3, DLL4 and cleaved Notch4 accompanied with an up-regulation of VEGF and p-Erk1/2 in human CCM3-mutated tissue. The total protein was extracted from the operative specimens of six sporadic CCMs (Sp-CCM) and a familial CCM harbouring CCM3 mutation (Mu-CCM3). Semi-quantification of the blot was performed by measuring integrate optical density (IOD) of each band followed by calculation of the ratio of IOD from the interested protein to that from the housekeeping protein GAPDH. The data were presented as the percentage of the Mu-CCM3. (C) Pathological morphology of CCM revealed by haematoxylin and eosin staining on the section from a sporadic CCM. The lesion consists of immature capillary-like vessels without normal interposed brain parenchyma. These vessels are dilated and often thrombosed (asterisks). (D–J) Immunofluorescent staining of CCM3, DLL4 and vWF on the sections of human CCM lesions. The immunofluorescent staining was simultaneously performed on the adjacent paraffin sections prepared from a CCM harbouring CCM3 mutation (Mu3-CCM; D–F) and from a sporadic CCM (Sp-CCM; G–J). In Mu3-CCM, vWF staining revealed the endothelial layer (arrows) in the thrombosed cavern (F), whereas the immunoreactivity of CCM3 (D) and DLL4 (E) was absent in these endothelial cells. In Sp-CCM, CCM3 (H), DLL4 (I) and vWF (J) were concomitantly detected in the endothelial layer (arrows) of the adjacent sections. Negative control omitting the primary antibody (Neg; G) did not show specific staining, and revealed only unspecific signal (red) in thrombosis and in erythrocytes (arrows) which appeared nuclear staining (blue) negative. The original magnification was 200× in main graphs and 400× in white box.
Mentions: Next, we examined DLL4-Notch signalling in human CCMs. Because of the extremely rare availability, only one human surgical specimen of a CCM3 germline mutation carrier could be obtained during this study. To draw a more convinced comparison between familial and sporadic CCMs, we used six surgical specimens from sporadic CCM as control. RT2-PCR detected around 5-, 38- and 35-fold decrease in CCM3, DLL4 and Notch4 expression, respectively, in the CCM3-deficient cavernous lesion (Mu-CCM3) in comparison with the human sporadic CCMs (Sp-CCM). A dramatic down-regulation of the target gene HEY2 and HES1 was also observed in Mu-CCM3, indicating an impaired DLL4-Notch signalling in familial CCM with germline mutation in CCM3. Interestingly, the level of Notch1 was similarly detected in Mu-CCM3 and in Sp-CCMs (Fig. 3A). These data drawn from CCM patients were entirely consistent with the results obtained from CCM3-silenced HUVEC (Fig. 2A) and CCMEC (Fig. 2D).

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