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. 2013 Feb;12(2):407-25.
doi: 10.1074/mcp.M112.021873. Epub 2012 Nov 26.

A combined proteomic and transcriptomic approach shows diverging molecular mechanisms in thoracic aortic aneurysm development in patients with tricuspid- and bicuspid aortic valve

Sanela Kjellqvist  1 , Shohreh Maleki, Therese Olsson, Maggy Chwastyniak, Rui Miguel Mamede Branca, Janne Lehtiö, Florence Pinet, Anders Franco-Cereceda, Per Eriksson
Affiliations

A combined proteomic and transcriptomic approach shows diverging molecular mechanisms in thoracic aortic aneurysm development in patients with tricuspid- and bicuspid aortic valve

Sanela Kjellqvist et al. Mol Cell Proteomics. 2013 Feb.

Abstract

Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.

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Figures

Fig. 1.
Fig. 1.
Representative gel figure showing internal standard. Spots chosen for identification are marked in circles and numbers. The isoelectric point range is 3–10 nonlinear, as indicated on top of the figure. Statistically significant protein spots are color coded red, blue, and green according to Figure 3.
Fig. 2.
Fig. 2.
PCA (A, B) and OPLS-DA (C, D) of protein spot expression data. The analyses were performed on 44 patients and 302 protein spots. The Hotelling's T2 (based on 95% confidence level) tolerance ellipsoid and ellipse are shown in the scores plots of PCA and OPLS-DA, respectively. Three-dimensional scores plot showing the PC1-PC3 plane of nondilated (black) and dilated (red) thoracic aorta tissue samples with TAV (A) and BAV (B) patients separately. Two-dimensional scores plot of an OPLS-DA showing the first predictive component (tp1) and orthogal component (to1) plane of nondilated (black) and dilated (red) thoracic aorta tissue samples with TAV (C) and BAV (D) separately.
Fig. 3.
Fig. 3.
Venn diagram and OPLS-DA of protein spot expression data. The significantly differentially expressed protein spots have been calculated with OPLS-DA where ABS (loadings)—ABS (jack-knife confidence interval) if positive indicates significance. The model has been applied on 302 protein spots of dilated versus nondilated samples (44 patients with and without TAA) in TAV and BAV separately. The bar plot of loadings and jack-knife confidence intervals contributing to the first PC for significant spots common for TAV and BAV models, red bars (A), statistically significant only in TAV, blue bars, and BAV, green bars (B) and (C) respectively. D, Venn diagram based on ABS (loading)—ABS (jack-knife confidence interval) significant protein spots of dilated versus nondilated samples in TAV and BAV patients separately (derived from Supplemental Table 2), color coded according to (A)–(C). E, Combined model scatter plot where loadings from TAV model of dilated versus nondilated aorta samples is plotted against loadings from BAV model of dilated versus nondilated aorta samples, color coded according to (A)–(C). Proteins that are significantly differentially expressed only in TAV and BAV are marked in blue and green respectively, whereas proteins that show statistically significant differential expression in both TAV and BAV are marked in red. 109 protein spots chosen for identification by MALDI-TOF are marked with numbers in the combined model scatter plot. The black diagonal is aimed for the interpretation purposes.
Fig. 4.
Fig. 4.
Western blot validation of differentially expressed protein LDH. Eight patients with TAV, four with dilated and four with nondilated aorta tissues, were chosen for Western blot validation. A, Western blot gel showing LDH and the internal standard TF. B, Box-plot of TF normalized LDH expression (y axis) in four dilated and four nondilated aorta tissue samples from (A).
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