Comparative bioinformatics analysis between proteomes of rabbit aneurysm model and human intracranial aneurysm with label-free quantitative proteomics

Abstract

Aims: This study aimed to find critical proteins involved in the development of intracranial aneurysm by comparing proteomes of rabbit aneurysm model and human aneurysms.

Methods: Five human intracranial aneurysm samples and 5 superficial temporal artery samples, and 4 rabbit aneurysm samples and 4 control samples were collected for protein mass spectrometry. Four human intracranial aneurysm samples and 4 superficial temporal artery samples, and 6 rabbit aneurysm samples and 6 control samples were used for immunochemistry.

Results: Proteomic analysis revealed 180 significantly differentially expressed proteins in human intracranial aneurysms and 716 significantly differentially expressed proteins in rabbit aneurysms. Among them, 57 proteins were differentially expressed in both species, in which 24 were increased and 33 were decreased in aneurysms compared to the control groups. Proteins were involved in focal adhesion and extracellular matrix-receptor interaction pathways. We found that COL4A2, MYLK, VCL, and TAGLN may be related to aneurysm development.

Conclusion: Proteomics analysis provided fundamental insights into the pathogenesis of aneurysm. Proteins related to focal adhesion and extracellular matrix-receptor interaction pathways play an important role in the occurrence and development of intracranial aneurysm.

Keywords: bioinformatics; focal adhesion; intracranial aneurysm; proteomics; rabbit elastase-induced aneurysm.

FIGURE 1

Proteome analysis of the rabbit elastase‐induced aneurysm model. (A) Volcano plot shows differential protein expression between the aneurysm group and the control group. Red dots represent up‐regulated proteins (n = 476) with p‐values <0.05. Down‐regulated proteins (n = 240) are labeled in green (p < 0.05). Black dots represent proteins whose expression levels are not significantly different. (B) Proteins with significantly different expressions between the aneurysm group and the control group. (C) Heatmap showing the comparison of the differentially expressed genes. Hierarchical clustering is shown on the left

FIGURE 2

Gene Ontology (GO) classification of the 57 common proteins differentially expressed in both human and rabbit aneurysms. A, Molecular function. B, Cellular component. C, Biological process. D, Protein‐protein interaction (PPI) network by STRING. Proteins are color‐coded according to their involvement in focal adhesion pathway (red), smooth muscle contraction (blue), ECM‐receptor interaction (yellow), and ECM organization (green)

FIGURE 3

Proteins involved in the focal adhesion pathway were mapped onto KEGG pathway. Proteins that participate in both focal adhesion and ECM‐receptor interaction are highlighted in yellow. Proteins marked in purple are the ones involved in both focal adhesion and smooth muscle contraction. Proteins with significantly different expression levels in both human and rabbit aneurysms that are involved in focal adhesion are highlighted in pink

FIGURE 4

Vascular wall analysis. (A) human IA/STA vascular wall analysis: (a‐d) H&E staining on the vessel wall of STA and IA samples. (e‐h) EVG staining on the vessel wall of STA and IA samples. Bar =250 μm (a, c, e, g) or 50 μm (b, d, f, h). (B) Rabbit aneurysm and normal CCA vascular wall analysis: (a‐d) H&E staining on the vessel wall of aneurysm and control samples. (e‐h) EVG staining on the vessel wall of aneurysm and control samples. Bar =250 μm (a, c, e, g) or 50 μm (b, d, f, h)

FIGURE 5

$\alpha$‐SMA, VCL, and COL4A2 expression. A, $\alpha$‐SMA, VCL, and COL4A2 expression in human IA/STA samples: (a‐d) $\alpha$‐SMA expression in STA and IA samples. (e‐h) VCL expression in STA and IA samples. (i‐l) COL4A2 expression in STA and IA samples. Bar =250 μm (a, c, e, g, i, k) or 50 μm (b, d, f, h, j, l). (B) $\alpha$‐SMA, VCL and COL4A2 expression in rabbit aneurysm and normal CCA: (a‐d) $\alpha$‐SMA expression in aneurysm and control tissues. (e‐h) VCL expression in aneurysm and control groups. (i‐l) COL4A2 expression in aneurysm and control groups. Bar =250 μm (a, c, e, g, i, k) or 50 μm (b, d, f, h, j, l)

FIGURE 6

Protein expression levels in the TNF‐$\alpha$‐induced SMC phenotypic modulation model. A‐G, Protein expression levels of genes of interest that were expressed differentially in both human and rabbit aneurysms, expressed as fold changes