GO analysis of DEPs was performed using Fun-Rich software.
The results revealed that most DEPs in the biological process (BP) category were enriched in 'cell communication', 'cell growth and/or maintenance', 'signal transduction', 'protein metabolism', 'metabolic metabolism', 'energy pathways', 'regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism', and 'ion transport' (Fig. 1A). GO cell component (CC) enrichment analysis revealed that most DEPs in this category were enriched in 'cytoplasm', 'exosomes', 'extracellular space', 'actin cytoskeleton', 'extracellular matrix', 'muscle thin filament tropomyosin', 'Z disc', 'Bleb, 'acrosomal vesicle', and 'mitochondrial proton transporting ATP synthase complex, catalytic core F (1)' (Fig. 1B). In the molecular function (MF) category, most DEPs were associated with 'cytoskeletal protein binding', 'catalytic activity', 'Transcription regulator activity', 'transferase activity, transfer of aldehyde or ketonic groups', 'growth factor binding', 'Heat shock protein activity', 'signal transducer activity, 'Isomerase activity', 'protease inhibitor activity', 'ATPase activity'. (Fig. 1C). As shown in Fig. 1D, KEGG analysis revealed that these differentially enriched proteins were mainly enriched in 'glycolysis/gluconeogenesis', 'biosynthesis of amino acids', 'HIF-1 signaling pathway', and 'carbon metabolism'. The differential expression of TPI1, LDHA, ENO2, ADH1B, TKT, and SERPINE1 are closely related to these pathways (Fig. 1D).
Establishment of an AA model by Ang-II infusion in C57bl/6J mice
Based on the results of the KEGG analysis and the upregulation of TPI1 in human AA, we sought to confirm this association in vivo by developing a mouse model. Hypertension is an independent inducer of AA; thus, we developed our animal model by infusing Ang-II to mice for 28 days. We found that Ang-II led to AA from the first week after Ang-II treatment (Fig. 2A, B), moreover, half of the mice experienced AA, in contrast to none of the mice in the control group (Fig. 2C). Dissection of the whole artery demonstrated the presence of AA in the aorta of the Ang-II-treated mice (Fig. 2A). Ang-II triggered the AA onset not only from blood pressure but also independently (20). H&E, EVG, and Masson staining confirmed Ang-II infusion to mice contributing to the occurrence of AA.
Expression of TPI1 protein in aortic tissues from human samples and mice
TPI1 is a key enzyme for glycolysis, and the KEGG analysis derived from proteomic analysis of proteins from human aortic tissues suggested that overexpression of TPI1 and the glycolysis pathway was associated with aortic aneurysms. Thus, we verified these results by western blotting analysis. The expression of TPI1 in the aortic tissue of AA patients is upregulated (Fig. 3A), and the same phenomenon was observed in the aortic tissue of mice treated with Ang-II (Fig. 3B), and the results were further confirmed by immunohistochemistry assays (Fig. 3C, D).
Expression of SM22, α-SMA, OPN, MMP2 and MMP9 proteins in mouse aortic tissues
The function of VSMCs is reflected in their phenotypes; the phenotypic switches in VSMCs are a feature of the aortic aneurysm. At the same time, the up-regulating MMP2/9 are high-risk factor for the aortic aneurysm. Western Blot demonstrated that the contractile markers α-SMA, and SM22 levels decreased in the Ang-II induced mouse(Fig. 4A, B), while changes in the synthetic marker osteopontin (OPN) levels showed the opposite patern(Fig. 4C).The MMP2 and MMP9 were upregulated in the Ang-II induced group mouse too(Fig. 4D, E). Representative images of immunoblotting for the five protein expression in aortic tissues, densitometric data were normalized to β-actin and expressed as relative levels compared with control group(Fig. 4F).
Ang-II induced the change of the genes TPI1 SM22, α-SMA, OPN, MMP2 and MMP9 expression in HA-VSMCs
The Ang II-induced the changes expression of TPI1 and phenotypic-related genes in VSMCs, which were analyzed by RT-qPCR and Western Blot. we found that the mRNA expression of TPI1, SM22, α-SMA, MMP2, and MMP9 were down regulated in cells treated with Ang-II compared to the control (Ctrl)(Figs. 5A, B, C, E, F); while changes in OPN level showed the opposite pattern (Fig. 5D); the similar changes tendency demonstrated on each proteins (Fig. 5G, H, I, J, K, L), Representative images of immunoblotting for the five protein expression in aortic tissues, densitometric data were normalized to β-actin and expressed as relative levels compared with control group(Fig. 5M).
mRNA-TPI induced HA-VASMC phenotypic switching and contributed to MMP2/MMP9 secreting
The function of VSMCs is reflected in their phenotypes; the phenotypic switches in VSMCs are a feature of the aortic aneurysm. To investigate whether TPI1 is responsible for the phenotypic change and MMP2/9 secreting, mRNA-TPI was transfected into T/G HA-VSMC cells, RT-qPCR and Western blot respectively demonstrated that mRNA-TPI were effective in inducing overexpression, compared to negative control (NC) (Fig. 6A, G). The expression of the contractile markers α-SMA, SM22, and the synthetic marker osteopontin (OPN), were analyzed by RT-qPCR and western blotting (Fig. 4E). we found that SM22 and α-SMA levels decreased in cells transfected with mRNA TPI compared to the negative control (NC) (Figs. 6B,C, H, I); while changes in OPN level showed the opposite pattern (Fig. 6D, J). at the same time, the gene expression of MMP2 and MMP9 were upregulated in in cells transfected with mRNA Tpi compared to the negative control (NC) (Figs. 6E, F, K, L), Representative images of immunoblotting for the five protein expression in aortic tissues, densitometric data were normalized to β-actin and expressed as relative levels compared with control group(Fig. 6M).