A Transcriptomic Comparative Study of Cranial Vasculature

Abstract

In genetic studies of cerebrovascular diseases, the optimal vessels to use as controls remain unclear. Our goal is to compare the transcriptomic profiles among 3 different types of control vessels: superficial temporal artery (STA), middle cerebral arteries (MCA), and arteries from the circle of Willis obtained from autopsies (AU). We examined the transcriptomic profiles of STA, MCA, and AU using RNAseq. We also investigated the effects of using these control groups on the results of the comparisons between aneurysms and the control arteries. Our study showed that when comparing pathological cerebral arteries to control groups, all control groups presented similar responses in the activation of immunological processes, the regulation of intracellular signaling pathways, and extracellular matrix productions, despite their intrinsic biological differences. When compared to STA, AU exhibited upregulation of stress and apoptosis genes, whereas MCA showed upregulation of genes associated with tRNA/rRNA processing. Moreover, our results suggest that the matched case-control study design, which involves control STA samples collected from the same subjects of matched aneurysm samples in our study, can improve the identification of non-inherited disease-associated genes. Given the challenges associated with obtaining fresh intracranial arteries from healthy individuals, our study suggests that using MCA, AU, or paired STA samples as controls are feasible strategies for future large-scale studies investigating cerebral vasculopathies. However, the intrinsic differences of each type of control should be taken into consideration when interpreting the results. With the limitations of each control type, it may be most optimal to use multiple tissues as controls.

Keywords: Cerebral vasculature; Intracerebral artery; Intracranial aneurysm; Superficial temporal artery.

Figure 1.

An overview of carotid artery sites and sample relationships at the transcriptome level. (A) Diagrams of the artery sites for sampling (left panel) and the number of samples for each group in the study (right panel). (B) PCA visualization of the distribution of transcriptomic samples in the first two principal components (PC). Each dot represents an individual sample. Samples are colored by group. The percentage of variance explained by each PC is indicated in the titles of axes. The ellipses with group colors represent the 95% confident intervals of sample distribution for each group. (C) Boxplots of expression level of the top 9 contributing genes in the aneurysm and control artery groups. Mean ± SEM, Welch’s ANOVA test with Dunnett’s T3 post hoc tests. ****, adjusted P < 0.0001. In (B, C), ANEU (n = 11): aneurysm; MCA (n = 5): middle cerebral artery; AU (n = 4): Arteries of the circle of Willis from autopsies; STA (n = 6): superficial temporal artery.

Figure 2.

Pairwise comparisons of transcriptomic profiles among AU, STA, MCA groups. (A) Pearson correlation heatmap of control artery samples. Color scale: white, low PCC; red, high PCC. PCC: Pearson correlation coefficient. (B) Venn diagram showing the overlapped gene sets among the DEGs identified by pairwise comparisons among MCA, AU, and STA, with AU vs. MCA, STA vs. MCA, and STA vs. AU, respectively. (C) Gene expression heatmap showing DEGs with consistent low/high expression in any control group. The clustered row annotation represents the sample group. The clustered column annotation represents the gene set group.

Figure 3.

Comparisons of transcriptomic profiles between aneurysm and control artery groups. (A) Intersection plot of the number of DEGs identified between the aneurysm (ANEU, n = 11) and each control artery group (MCA, n = 5; AU, n = 4; STA, n = 6). The horizontal bars on the left-hand side represent the number of DEGs detected in each comparison. The vertical bars at the top show the number of intersections between each combination of DEG groups. The dots on the right-hand side represent the presence of DEGs in each gene set. Vertical bar colors represent the direction of gene expression change. (B) Density plot of the coefficient of variation (CV) of gene expression in ANEU, MCA, AU, and STA groups. The x-axis representing the CV. The y-axis representing the density distribution of CV. (C) Scatter plot of all known genes ranked by robust rank aggregation (RRA) P values. Dot color: red, high expression in ANEU; blue, low expression in ANEU; Grey, unstable expression in ANEU. The x-axis represents the gene’s rank order in RRA analysis, and the y-axis represents −Log₁₀P of the gene in RRA analysis. (D) Enriched gene ontology biological process (GOBP) gene sets and enriched Reatome pathways and reactions (REACTOME) of robust DEGs (rDEGs, genes with P < 0.05 determined by RRA analysis) shown as a dot plot, with top 5 enriched GOBP and REACTOME terms highlighted, respectively. The x-axis showing the normalized enrichment score (NES). The y-axis showing the −Log₁₀FDR. (E) Heatmap showing the presence/absent of genes across the top enriched terms highlighted in (D). Rows representing all aneurysm-associated rDEGs involved in these terms. Cell color: red, presence; blue, absent.

Figure 4.

Differences and similarities of DEGs between the ANEU and STA groups using both paired and unpaired comparisons. (A) Intersection plot of the number of DEGs using paired and unpaired linear regression models. The horizontal bars on the left-hand side represent the number of DEGs detected in each comparison. The vertical bars at the top show the number of intersections between each combination of DEG groups. The dots on the right-hand side represent the presence of DEGs in each gene set. Vertical bar colors represent the direction of gene expression change. (B) Scatter plot of rank-ordered aneurysm rDEGs (P < 0.05) that were also identified by paired or unpaired comparisons. rDEGs with name highlighted contain SNPs significantly associated with cerebral aneurysm in the PheWAS database (P < 1e-5 and allele frequency > 0.01, phenocode: 433.5) ^,. Dot shapes represent the comparison group. Dot colors represent the direction of gene expression change in aneurysm samples. The x-axis represents the gene rank order in RRA analysis. The y-axis represents −Log₁₀P of the genes in RRA analysis. (C) Gene expression heatmap of the top 25 upregulated and top 25 downregulated rDEGs that were also identified by paired comparison and unpaired comparison between ANEU and STA groups. The row annotation on the left-hand side displays the sample groups. The column annotation at the top displays the RRA rank orders.

Figure 5.

Integrated network analysis of DEGs between ANEU and STA and associated functions in Reactome prior-knowledge network. (A) Gene-gene interaction network modules that are connected by Reactome co-occurrence relationships. Nodes represent DEGs. Edges represent the known connections between genes in the Reactome Knowledgebase. (B) Gene functional network of enriched Reactome pathways and reactions with high-level grouped functional themes highlighted using colored clusters. Node colors of diamond nodes represent the functional group of the enriched term. Edges represent the relationship between enriched terms or between terms and genes. Diamond node: Reactome term; Circular node: gene.

Figure 6.

Estimated cell proportion of human cerebral aneurysm and control arteries using mRNA transcriptome profiling. (A) The percentages of each cell type in samples from different artery groups (ANEU, MCA, AU, STA) are shown in the barplot. (B) Barplots of CIBERSORTx scores of SMC, EC, and FBMC in each artery group. Mean ± SEM, Welch’s ANOVA test with Dunnett’s T3 post hoc tests. *, adjusted P < 0.05; **, adjusted P < 0.01; ***, adjusted P < 0.001; ns, not statistically significant. (A, B) EC, endothelial cell; SMC, smooth muscle cell; PC, pericyte; FB, fibroblast; FBMC, fibromyocyte; MG/Mac, microglia/macrophage; BC, B cell; TC, T cell; Mono, monocyte; DC, dendritic cell. ANEU, n = 11; MCA, n = 5; AU, n = 4; STA, n = 6.