Preliminary biomarkers for identification of human ascending thoracic aortic aneurysm

Abstract

Background: Human ascending thoracic aortic aneurysms (ATAAs) are life threatening and constitute a leading cause of mortality in the United States. Previously, we demonstrated that collagens α2(V) and α1(XI) mRNA and protein expression levels are significantly increased in ATAAs.

Methods and results: In this report, the authors extended these preliminary studies using high-throughput proteomic analysis to identify additional biomarkers for use in whole blood real-time RT-PCR analysis to allow for the identification of ATAAs before dissection or rupture. Human ATAA samples were obtained from male and female patients aged 65 ± 14 years. Both bicuspid and tricuspid aortic valve patients were included and compared with nonaneurysmal aortas (mean diameter 2.3 cm). Five biomarkers were identified as being suitable for detection and identification of ATAAs using qRT-PCR analysis of whole blood. Analysis of 41 samples (19 small, 13 medium-sized, and 9 large ATAAs) demonstrated the overexpression of 3 of these transcript biomarkers correctly identified 79.4% of patients with ATAA of ≥4.0 cm (P<0.001, sensitivity 0.79, CI=0.62 to 0.91; specificity 1.00, 95% CI=0.42 to 1.00).

Conclusion: A preliminary transcript biomarker panel for the identification of ATAAs using whole blood qRT-PCR analysis in men and women is presented.

Keywords: FHL1; ascending thoracic aortic aneurysm; collagen.

Figure 1.

A, Principal component analysis. B, Hierarchical cluster analysis of differentially expressed proteins. C, Western blot analysis. D, Immunoblot analysis. E, Coomassie gel. A, Principal component analysis (PCA) indicated no difference in differential protein expression patterns between BAV vs TAV or male vs female (identified with gender icon). B, Differentially expressed proteins compared with control were identified by supervised analysis on the basis of P value <0.01 in each group. The differentially expressed proteins are identified with short descriptions obtained from the SWISS‐PROT database. The log‐fold change (LFC) in protein expression is shown with pseudocolor scale (−1 to 1) with red denoting up‐regulation and green denoting down‐regulation. The columns represent LFC comparisons and the rows represent the proteins. Dendrograms are found on the left side, experimental groups are found on the top, and protein names are found on the right side of the figure. C, Representative 10% Novex Tris‐glycine gel. Protein samples (25 μg) from full‐thickness aortic tissue from the site of the maximal diameter containing all 3 layers were fractionated on 10% Novex Tris‐glycine gel, Validation of proteomic results via Western blot (WB) analysis. Results displayed as fold changes compared with controls. 1, Control; 2, small ATAAs; 3, medium‐sized ATAAs; 4, large ATAAs. D, Image of immunoblot, performed using mouse monoclonal four and a half LIM domains protein 1 (FHL1) antibody (1:2000 dilution, Abcam, Inc). 1, Control; 2, small ATAAs; 3, medium‐sized ATAAs; 4, large ATAAs. E, Image of Coomassie blue–stained gel. MW, Molecular weight marker. 1, Control; 2, small ATAAs; 3, medium‐sized ATAAs; 4, large ATAAs. ATAA indicates ascending thoracic aortic aneurysm; BAV, bicuspid aortic valve; PC1, principal component 1; TAV, tricuspid aortic valve.

Figure 2.

Pathway analysis based on size. A, Small aneurysms (4.0 to 4.9 cm); B, medium‐sized aneurysms (5.0 to 5.5 cm); C, large aneurysms (>5.5 cm). Pathway analysis was performed by identifying the over‐represented GO categories in differentially expressed proteins: this was done using the Biological Processes and Molecular Functions Enrichment Analysis available from the Database for Annotation, Visualization and Integrated Discovery (DAVID). Functional pathways are labeled on the abscissa and the −log (P value) is on the ordinate. On the right side is the ratio of proteins in pathway over total proteins and the yellow line shows the ratio of each pathway. The red line is the threshold at P=0.05. Any pathway that passes the red line is significantly enriched. ILK indicates integrin linked kinase; NRF, nuclear respiratory factor; RA, rheumatoid arthritis; TR/RXR, thyroid hormone receptor/retinoid X receptor.