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Role of cytoskeletal proteins in cerebral cavernous malformation signaling pathways: a proteomic analysis.

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

ABSTRACT
Three genetic mutations were found to cause cerebral cavernous malformation (CCM), a vascular anomaly predisposing affected individuals to hemorrhagic stroke. These CCM proteins function together as a protein complex in the cell. Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to explore the proteomic profile for loss of each CCM gene expression in mouse endothelial stem cells (MEES) compared to mock shRNA and no shRNA control cell-lines. Differentially expressed proteins were identified (p < 0.05). 120 proteins were differentially expressed among the cell-lines. Principal component analysis and cluster analysis show the effects of individual knockdown. In all knockdown cell-lines, altered expression of cytoskeletal proteins is the most common. While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

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In vitro tube formation assays in MEES cell lines; (A) knockdown CCM1 (cell line 1), (B) knockdown CCM2 (cell line 2), (C) knockdown CCM2 (cell line 3), (D) wild-type (cell line 4), and (E) mock knockdown (cell line 5).
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fig1: In vitro tube formation assays in MEES cell lines; (A) knockdown CCM1 (cell line 1), (B) knockdown CCM2 (cell line 2), (C) knockdown CCM2 (cell line 3), (D) wild-type (cell line 4), and (E) mock knockdown (cell line 5).

Mentions: We compared each CCM knockdown cell line with a wild type and a mock knockdown to first look for proteins affected by the CCM knockdown. Then we compared the proteomic profiles among three CCM knockdowns. Expression of each of the CCM proteins in MEES cell lines was selectively inhibited by shRNA. A wild-type MEES cell line and a mock shRNA (pLKO.1-empty vector) cell line were used as controls. The levels of expression of each CCM gene after the knockdown were evaluated using Western analysis similar to our previous work.31 An in vitro tube formation assay was used to determine the functional deficiency of each of the cell lines after the knockdown (Fig. 1A–E). Note that all figures here use the following assignments: cell line 1 (knockdown CCM1), cell line 2 (knockdown CCM2), cell line 3 (knockdown CCM3), cell line 4 (wild-type MEES), and cell line 5 (mock knockdown). A knockdown of each CCM gene results in the inability to form endothelial tubes. This in vitro phenotype was believed to mimic the clinical CCM lesion in the brain.31 While the clinical and in vitro phenotypes look similar for all CCM mutations. Previous work from our group and others suggested that while there are certain common signaling pathways, there may be a unique pathway mediated by each individual CCM protein.


Role of cytoskeletal proteins in cerebral cavernous malformation signaling pathways: a proteomic analysis.

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

In vitro tube formation assays in MEES cell lines; (A) knockdown CCM1 (cell line 1), (B) knockdown CCM2 (cell line 2), (C) knockdown CCM2 (cell line 3), (D) wild-type (cell line 4), and (E) mock knockdown (cell line 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043921&req=5

fig1: In vitro tube formation assays in MEES cell lines; (A) knockdown CCM1 (cell line 1), (B) knockdown CCM2 (cell line 2), (C) knockdown CCM2 (cell line 3), (D) wild-type (cell line 4), and (E) mock knockdown (cell line 5).
Mentions: We compared each CCM knockdown cell line with a wild type and a mock knockdown to first look for proteins affected by the CCM knockdown. Then we compared the proteomic profiles among three CCM knockdowns. Expression of each of the CCM proteins in MEES cell lines was selectively inhibited by shRNA. A wild-type MEES cell line and a mock shRNA (pLKO.1-empty vector) cell line were used as controls. The levels of expression of each CCM gene after the knockdown were evaluated using Western analysis similar to our previous work.31 An in vitro tube formation assay was used to determine the functional deficiency of each of the cell lines after the knockdown (Fig. 1A–E). Note that all figures here use the following assignments: cell line 1 (knockdown CCM1), cell line 2 (knockdown CCM2), cell line 3 (knockdown CCM3), cell line 4 (wild-type MEES), and cell line 5 (mock knockdown). A knockdown of each CCM gene results in the inability to form endothelial tubes. This in vitro phenotype was believed to mimic the clinical CCM lesion in the brain.31 While the clinical and in vitro phenotypes look similar for all CCM mutations. Previous work from our group and others suggested that while there are certain common signaling pathways, there may be a unique pathway mediated by each individual CCM protein.

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

ABSTRACT
Three genetic mutations were found to cause cerebral cavernous malformation (CCM), a vascular anomaly predisposing affected individuals to hemorrhagic stroke. These CCM proteins function together as a protein complex in the cell. Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to explore the proteomic profile for loss of each CCM gene expression in mouse endothelial stem cells (MEES) compared to mock shRNA and no shRNA control cell-lines. Differentially expressed proteins were identified (p < 0.05). 120 proteins were differentially expressed among the cell-lines. Principal component analysis and cluster analysis show the effects of individual knockdown. In all knockdown cell-lines, altered expression of cytoskeletal proteins is the most common. While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

Show MeSH
Related in: MedlinePlus