Proteome analysis of cultivated vascular smooth muscle cells from a CADASIL patient

Abstract

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementing disease caused by mutations in the NOTCH3 gene, most which are missense mutations leading to an uneven number of cysteine residues in epidermal growth factor-like repeats in the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on the VSMCs of small and middle-sized arteries. Recent studies have demonstrated that impairment of Notch3 signaling is not the primary cause of the disease. In the present study we used proteomic analysis to characterize the protein expression pattern of a unique material of genetically genuine cultured human CADASIL VSMCs. We identified 11 differentially expressed proteins, which are involved in protein degradation and folding, contraction of VSMCs, and cellular stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic reticulum stress and activation of unfolded protein response, leading to increased reactive oxygen species and inhibition of cell proliferation. In addition, upregulation of contractile proteins suggests an alteration in the signaling system of VSMC contraction. The accumulation of N3ECD on the cell surface possibly upregulates the angiotensin II regulatory feedback loop and thereby enhances the readiness of the cells to respond to angiotensin II stimulation.

Figure 1

Two-dimensional (2-D) gel images of the CADASIL and control samples and the spot intensities from the selected spots. (A) The partial images of the silver stained 2-D gels of human CADASIL and control VSMCs. The targeted protein analysis revealed 11 spots with a statistically significant difference (P < 0.05) in spot intensity between CADASIL and control groups (3 replicates). (B) The silver stained 2-D gels were analyzed with a PDQuest analyzing program. The statistical significance of the differences was calculated using the Student t test and the one-way ANOVA. The spot intensities in the image are mean values of three replicates in the group and ± SEMs are shown in each bar.

Figure 2

Collagen gel contraction (CGC) by control and CADASIL VSMCs. (A) Representative images of contracted collagen gels by control and CADASIL VSMCs. The gels contained 12.5 × 10³ cells/well and were cultured for 24 h before assessing gel contraction. Control VSMCs contracted spontaneously collagen gel markedly more than CADASIL VSMCs. (B) Quantification of CGC by control and CADASIL VSMCs. Columns indicate the mean of 4 replicated assays and capped bars indicate standard deviation (SD). Statistically significant difference between normal and CADASIL VSMCs is indicated by asterisk (**P < 0.01, unpaired Student t test).