Identification of Transcriptional Variation in Aortic Remodeling Using a Murine Transverse Aortic Constriction (TAC) Model

Abstract

Arterial remodeling is a major pathological consequence of hypertension, which is recognized as the most common chronic non-communicable disease. However, the detailed mechanism of how arterial remodeling is induced by hypertension has not yet been fully elucidated. Evaluating the transcriptional changes in arterial tissue in response to elevated blood pressure at an early stage may provide new insights and identify novel therapeutic candidates in preventing arterial remodeling. Here, we used the ascending aorta of the transverse aortic constriction (TAC) model to induce arterial remodeling in C57BL/6 male mice. Age-matched mice were subjected to sham surgery as controls. The TAC model was only considered successful if the mice conformed to the criteria (RC/LC blood flow velocity with 5-10-fold change) 1 week after the surgery. Two weeks after surgery, the ascending aorta developed severe remodeling in TAC mice as compared to the sham group. High throughput sequencing was then applied to identify differentially expressed (DE) transcripts. In silicon analysis were then performed to systematically network transcriptional changes. A total of 1,019 mRNAs were significantly changed between TAC and the sham group at the transcriptional level. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that stress/stimulus/immune-related biological processes played a crucial role during arterial remodeling. Our data provide a comprehensive understanding of global gene expression changes in the TAC model, which suggests that targeting inflammation and vascular smooth cell transformation are potential therapeutic strategies to interfere with the aortic remodeling at an early stage in the development of hypertension.

Keywords: arterial remodeling; bioinformatics analysis; hypertension; transcriptional changes; transverse aortic constriction (TAC).

Figure 1

Increased blood pressure and remodeling of the aortic wall in established TAC model. (A) TAC was constructed by ligation between the brachiocephalic trunk and LCCA. Tissues used for all analysis are indicated by the arrow and the red circle; (B) Blood flow velocity measurement at RCCA and LCCA of the sham or TAC groups, respectively; (C) Right carotid /left carotid (RC/LC) flow ratio of TAC and sham group; (D–F) Representative images and quantification analysis of hematoxylin and eosin (H&E) staining (D), Weigert's staining (E), and Sirius red staining (F); n = 6 per group. Original magnification: ×400. ****P < 0.0001.

Figure 2

Transcriptional changes in TAC models. (A) An overview of DEGs in TAC models; (B) Volcano plot illustrating DEGs between the TAC and sham groups. Up-regulated mRNAs are labeled in red, down-regulated in blue, and the rest in gray; (C) Hierarchically clustered heat map illustrating DEGs between TAC and sham groups, fold change >2.0, P < 0.05 and FDR < 0.05, n = 4 per group.

Figure 3

Functional enrichment assay of transcripts in the TAC model. (A,B) The top GO enriched biological functions of up (A) and down-regulated DEGs (B), respectively; (C,D) the top KEGG enrichment items of up (C) and down-regulated DEGs (D), respectively.

Figure 4

The Network analysis of DEGs and predicted PPI networks in the TAC group. (A) KEGG analysis suggested that differentially mRNAs were mainly targeted to the ECM-receptor interaction and Complement and coagulation cascades. Circle size represented degree; (B) The network showed the potential relationship between the top 200 differentially up-regulated and down-regulated expressed mRNAs, Up-regulated mRNAs are displayed in the red circle, down-regulated in the green circle.

Figure 5

Protein level validation of DEGs revealed by RNA-seq. (A–G) Western blot analysis of α-SMA, COL-I, Angptl1, TGF-β1, and PCNA in the TAC and sham groups. GAPDH and β-Tubulin were used as endogenous loading controls. Lysates were prepared from ascending aortas of sham-operated or TAC mice, n = 5 per group. The experiments were repeated 3 times independently; (H–L) Immunohistochemistry and quantification analysis of α-SMA, COL-I, Angptl1, TGF-β1, and PCNA in ascending aortas derived from TAC or sham controlled mice. n = 6 per group. Original magnification: ×400. Scale bars: 50 μm. **P < 0.01, ****P < 0.0001.