The mouse model construction of TAA dependent on BAPN combined with Ang II

We constructed an animal model of TAA with BAPN plus Ang II treatment to explore the roles of serum exosomes in TAA. In this study, we found that the hair of the mice became dry and dull, and the activity level, diet, and drinking water consumed decreased in the model group. However, this phenomenon was not observed in the control and sham groups.

The results showed that administration of BAPN (1 g/d/kg) plus Ang II (1 μg/kg/min) induced TAA in C57BL/6 mice. After forty-eight hours of pumping Ang II, an echocardiogram showed that the inner diameter difference of the thoracic aorta in the model group was 1.5 times larger than that in the control group; this was defined as successful model construction. Approximately 25% of mice developed TAA, and the incidence of thoracoabdominal aortic aneurysm (TAAA) was 2.78% (Fig. 1A). In this group, 44.44% of the mice died of aortic dissection or rupture. However, no mice in the control group or sham group died. The aortic diameter was measured by echocardiography on the third day and the 30th day for the control group, sham operation group, and model group. There was no local tumour-like dilation of the thoracic aorta in the control group or sham group. The difference in the inner diameter of the thoracic aorta measured by echocardiography on the third day and the 30th day of control group and sham group mice was 0.156 ± 0.028 mm and 0.156 ± 0.043 mm, respectively. However, the inner diameter of the thoracic aorta of the model group mice was 0.569 ± 0.023 mm. The changes in the internal diameter of the abdominal aorta for the control group, sham group, and model group were 0.261 ± 0.024 mm, 0.217 ± 0.058 mm, and 0.237 ± 0.027 mm, respectively (Fig. 1B). The echocardiogram indicated that the thoracic aorta was globularly dilated in C57BL/6 mice in the BAPN combined with Ang II group (Fig. 1C).

Fig. 1

The construction of a TAA animal model in C57BL/6 mice. A The incidence of aortic aneurysm in the BAPN plus Ang II group. B The differences in aortic diameter among the three groups. The yellow lines indicate the measurement position. C The diameter of the thoracic aorta in the three groups was measured by echocardiography. D Anatomical images of the thoracic aorta of mice in the three groups; the white arrow indicates a TAA site. E HE staining of the thoracic aorta in the three groups of mice. TAA, Thoracic aortic aneurysm, AAA Abdominal aortic aneurysm, TAAA Thoracoabdominal aortic aneurysm

We dissected the aortic vessels of mice using a stereomicroscope and found that the vessels of the ascending aorta and the aortic arch were obviously dilated and spherical. Intramural haematomas (IMHs) were observed in the lesions. However, the aortic wall was smooth without dilation in the control group and sham group (Fig. 1D). Haematoxylin and eosin staining showed that the vascular intima of the model group was damaged and discontinuous. In the control group and the sham group, the intima of the aortic wall was intact, and the vascular smooth muscle cells were arranged in a ring-like shape (Fig. 1E). Therefore, we generated a reliable and convenient TAA model in C57BL/6 mice to study the pathological process of TAA and explore therapeutic targets.

Characteristics of serum exosomes from mice with TAA

To identify the characteristics of serum exosomes in mice with TAA induced by BAPN combined with Ang II, we isolated serum exosomes from C57BL/6 mice via ultracentrifugation. Transmission electron microscopy revealed similar morphological characteristics of exosomes, with round or elliptical vesicles and a double-layer membrane structure, among the control group, the sham group, and the model group (Fig. 2A). NTA of the isolated exosomes showed that the main peak of the vesicle size distribution was 122 ± 55.6 nm (Fig. 2B). Western blotting showed that the exosome markers CD63, CD9, GM130, and ALIX were all expressed (Fig. 2C). The above results indicated that exosomes were successfully isolated for further proteomic analysis.

Fig. 2

Characterization of exosomes. (A) TEM image of exosomes. (B) NTA of exosomes. (C) Western blot detection of CD63, CD9, GM130 and ALIX was performed after exosome protein extraction

Mass spectrometry-based identification of proteins in serum exosomes from mice with TAA

A mass spectrometry-based proteomics study was used to characterize the protein constituents of exosomes during TAA development induced by BAPN plus Ang II. A total of 399 proteins were identified by proteome analysis of serum exosomes from mice in the sham group and the model group. To further illustrate the differences in protein expression, we generated volcano plots for the sham and model groups by considering proteins with p < 0.05 and a fold change (FC) greater than 1.5 to be significantly dysregulated (up- or downregulated) compared to the sham group. Compared with that in the sham group, the expression of 196 proteins significantly differed in serum exosomes of mice with TAA. There were 122 significantly upregulated DEPs and 74 significantly downregulated DEPs (Fig. 3A). Clustering analysis revealed the relationships among the proteins in the sham group and the model group. In this study, we found that in the serum exosomes of mice in the sham group and the model group, there were five representative patterns of differential protein expression (Fig. 3B). GO enrichment analysis was carried out to identify exosome proteins based on biological process and molecular function. In cluster I, we found that the proteins were mainly involved in biological processes such as the protein activation cascade, complement activation, humoral immune response, and immunoglobulin-mediated immune response. The biological process GO terms negative regulation of peptidase activity, negative regulation of proteolysis, and blood coagulation were mainly enriched in cluster II. Cluster III proteins were involved in biological processes such as cellular response to insulin stimulus and negative regulation of cell migration. In cluster IV, the most markedly enriched GO terms for biological process were leukocyte migration and positive regulation of endocytosis. DNA replication and negative regulation of megakaryocyte differentiation was the most significantly enriched GO term for the biological process in the cluster V proteins (Fig. 3C). Therefore, exosomal proteins are likely to be involved in the pathophysiological process associated with aortic aneurysm.

Fig. 3

Comparison of DEPs in serum exosomes from mice with TAA induced by BAPN combined with Ang II. A Volcano plots were generated to show the DEPs in serum exosomes from mice with TAA induced by BAPN combined with Ang II. Red and green dots represent up- and downregulated proteins, respectively. B Heatmap analysis was performed to show the significant DEPs in mouse serum exosomes between the sham group and BAPN plus Ang II group. Red represents high expression, while green represents low expression. KEGG pathway analysis was performed to investigate the pathways significantly enriched in expressed proteins. C Functional classification of the dysregulated proteins was performed by GO enrichment analysis

Functional analysis of upregulated DEPs in TAA

GO enrichment analysis was employed to analyse differentially upregulated proteins in the serum exosomes of mice with TAA induced by BAPN plus Ang II. The results indicated that the molecular functions of the upregulated proteins were mainly enriched in signaling receptor binding, molecular function regulation, and antioxidant activity. These proteins were closely related to the stress response, protein metabolism process regulation, inflammatory response, and regulation of the immune system process. After performing reactome pathway analysis of serum exosome proteins of mice with TAA, we observed that platelet activation, signaling and aggregation signaling pathways, plasma lipoprotein assembly, remodeling and clearance signaling pathways, and smooth muscle contraction signaling pathways were distinctly enriched. The KEGG pathway enrichment analysis indicated that the complement and coagulation cascades were significantly enriched in the upregulated proteins (Table 1). We constructed a protein‒protein interaction (PPI) network for 122 significantly upregulated proteins in serum exosomes from TAA model mice in comparison with the sham group and screened the core cluster MCODE I-IV in the PPI network based on the Cytoscape app with the MCODE plugin (Fig. 4A). By performing functional analysis of the core clusters, we found that multiple process terms were related to the development of TAA (Fig. 4B, C). In these interaction networks, the upregulated proteins of the MCODE I complex were involved in oxidative stress (Cat, Gpx1, Sod3) (Fig. 4B). The upregulated proteins of the MCODE II complex participated in blood vessel development (Ecm1, Myh9, Postn, Flna, Tpm4) and supramolecular fibre organization (Col1a1, Col1a2, Dcn, Clec3b) (Fig. 4B). Our analysis showed that four proteins were involved in the humoral immune response (B2m, Cfp) and regulation of peptidase activity (Vcp, Grn) (Fig. 4B). Most proteins were involved in fibrinolysis (Trf, Plg, Clu, F13b, Sap, Fga, Fgb, Fgg) and acute-phase response (Hp, Hpx, Rbp4, Orm1, Orm2, Itih4, Ttr, Gc) and were highly connected within the MCODE IV complex (Fig. 4C). This indicates that TAA formation is regulated by multiple biological processes associated with upregulated exosomal proteins.

Table 1 Functional enrichment analysis of up-regulated proteinsFig. 4

Protein network analysis of upregulated DEPs in mouse serum exosomes between the sham group and BAPN plus Ang II group. (A) STRING analysis revealed the protein–protein interaction (PPI) network of the 122 upregulated proteins. (B), (C) Functional analysis based on Metascape was performed to investigate significantly enriched terms by the upregulated proteins in the MCODE I-III (B) and MCODE IV (C), respectively

Functional analysis of downregulated DEPs in TAA

In this study, we constructed a PPI network for 74 serum exosome proteins that were significantly downregulated compared with the sham group and screened core cluster MCODE I-IV by bioinformatics analysis. At the same time, we performed functional analysis of these four core clusters based on Metascape (http://metascape.org/gp/index.html#/main/step1). The analysis demonstrated that the significantly downregulated proteins in MCODE I-IV were involved in multiple biological processes that were related to the TAA process, such as complement and coagulation cascades (C8a, C2, C1s2, C9, C8b, C8g, C1qc), blood coagulation (F9, Serpina10, Serpina6, Pzp, Masp1, Hc, Cpb2, Pltp, Pla2g7, Serpina1b, Thbs1, Pros1, F13a1) and regulation of plasma lipoprotein particle levels (Apob, Apoc3, Apom) (Fig. 5A). Functional and pathway enrichment analyses were implemented to assess downregulated DEPs in serum exosomes from mice with TAA (Fig. 5B). Enrichment analysis indicated that the molecular function of the downregulated DEPs was related to enzyme regulator activity and protein-containing complex binding activity and was significantly involved in biological processes, such as regulation of endopeptidase activity, regulation of proteolysis and blood coagulation. Reactome pathway analysis demonstrated that these DEPs were mainly enriched in the immune system, regulation of complement cascade, platelet activation, signaling and aggregation, MAPK6/MAPK4 signaling, and vesicle-mediated transport. KEGG pathway analysis was also performed to investigate the pathways significantly enriched for these downregulated proteins. We found that the most enriched KEGG pathways were mainly the complement and coagulation cascades (Fig. 5B). Interestingly, downregulated exosomal proteins in multiple MCODE complexes were involved in the biological process complement and coagulation cascade, which suggests that the complement and coagulation cascade may play an important role in the development of aortic aneurysm.

Fig. 5

Bioinformatics analysis of downregulated DEPs. A STRING analysis revealed the protein–protein interaction (PPI) network of the 74 downregulated proteins. B GO enrichment, KEGG pathway and Reactome pathway analyses were used to elucidate the potential biological functions of all downregulated proteins in serum exosomes of mice with TAA