Methods: Using human umbilical vein cell line (HuVEC), we individually knocked down each CCM protein with shRNA. We then applied a label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) to examine the expressed proteomic profile for each knockdown cell-line (CCM1, CCM2, and CCM3) as well as two control cell lines; mock shRNA and wild type cell-lines. Differentially expressed proteins were identified (ANOVA; p<0.05, and p<0.01). Principle component analysis (PCA) and cluster analysis were used to analyze identified proteins.
Results: 290 and 192 proteins (at p<0.05 and p<0.01, respectively) were found to be differentially expressed amongst the five cell-lines. Similar to the mouse cell line analysis, PCA and cluster analysis results demonstrate the effects of individual CCM knockdown and a unique proteomic profile for each cell line. Cytoskeletal proteins, similar to the mouse system, are among the most affected proteins by CCM gene knockdown.
Conclusions: Proteomic analysis suggests that cytoskeletal development is disrupted by CCM knockdown and further analysis of cellular signaling in cytoskeletal development may provide the key to treat CCM condition.