Proteomic Analysis for Cerebral Cavernous Malformations

Objectives: Cerebral cavernous malformation (CCM) is a vascular anomaly which predisposes affected individuals to hemorrhagic stroke. The etiology of CCM has been traced to three genetic mutations. These CCM genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3), interact as a protein complex within the cell. The subsequent loss of expression of each CCM gene results in disruption of in vivo endothelial tube formation as well as reduction in actin fiber formation. However, the global effects of CCM gene knockdown are not known. There lies the additional question of whether any of these knockdowns will reveal novel signaling pathways related to the in vitro and clinical phenotypes. Here we used a proteomic approach to define the signaling pathways involved in CCM.

Methods: Using mouse endothelial stem cells (MEES), we examined the global proteomic effects of the loss of each CCM gene expression. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to examine the expressed proteomic profile for each knockdown cell-line (CCM1, CCM2, and CCM3) compared to mock shRNA and no shRNA control cell-lines. Those differentially expressed proteins were identified (ANOVA; p<0.05) and principle component analysis (PCA) as well as cluster analysis were used to analyze identified proteins. 

Results: 122 proteins were found to be differentially expressed amongst the five cell-lines. PCA and cluster analysis results demonstrate the effects of individual CCM knockdown and suggests a unique proteomic profile for each cell line. In each of the three CCM knockdown cell-lines, overexpression of cytoskeletal proteins is the most common. 

Conclusions: The results suggest that these CCM genes play essential role in cytoskeletal development and that proteomic analysis is proven to be a useful tool for future examination of CCM-related signaling pathways.