Single-Cell RNA Sequencing Reveals the Pathogenic Relevance of Intracranial Atherosclerosis in Blood Blister-Like Aneurysms

Abstract

Background: Intracranial non-branching site blood blister-like aneurysms (BBA) are extremely rare and vicious. Their etiology remains elusive, and no molecular study has been carried out to reveal its pathogenic relevance to intracranial atherosclerosis. To investigate its transcriptomic landscape and underlying potential pathogenesis, we performed single-cell RNA sequencing with extensive pathological validation.

Methods: In total, 12,245 cells were recovered for single-cell RNA sequencing analysis from 1 BBA and 2 saccular intracranial aneurysms (IAs). Unbiased clustering using Seurat-based pipeline was used for cellular landscape profiling. Cellchat was used to understand intracellular communications. Furthermore, 10 BBAs and 30 IAs were retrospectively collected for pathological validations like scanning electron microscopy, H&E stain, Masson stain, Verhoeff Van Gielson stain, and immunofluorescence.

Results: Single-cell transcriptome profiled 14 total subclusters in 6 major groups, namely, 6 monocyte/macrophage clusters, 2 T&NK clusters, 3 vascular smooth muscle cell (VSMC) clusters, 1 dendritic cell, 1 B cell, and 1 endothelial cell cluster. The only mural cell identified in BBAs was VSMC-2 cluster, while mural cells in IAs comprise most clusters of VSMCs and endothelial cells. Upregulated genes in BBA-derived VSMCs are related to arterial mineralization and atherosclerosis, such as PTX3, SPP1, LOX, etc., whereas vasodilation and physiological regulatory genes such as MGP, ACTA2, and MYL9 were conversely enriched in conventional IA-derived VSMCs. Immune cells in the BBA were predominantly macrophages, with a low fraction of T&NK cells, while conventional IAs had a higher percentage of T&NK. Gene enrichment analysis suggested that macrophages in BBA were highly enriched in lipid metabolism as well as atherosclerosis. Ligand-receptor interaction suggested that secretory phosphoprotein 1 (also known as osteopontin) played a major role in mediating the intracellular communication between VSMC and macrophages, especially in BBA. Pathological experiments corroborate with the bioinformatic findings and further characterized BBAs as a thin-walled thrombotic aneurysm with severe atherosclerotic lesions, where ApoE+ macrophages and OPN+ mural cells are intimately involved in the inflammation process.

Conclusions: The preexisting intracranial atherosclerosis might predispose the parent artery to the pathogenic occurrence of BBAs. These data shed light on the pathophysiology of intracranial aneurysms and might assist in the further resolution of the complexity in aneurysm pathogenesis.

Keywords: atherosclerosis; blood blister-like aneurysm; pathogenesis; pathology; single-cell RNA sequencing.

Figure 1

Single-cell RNA sequencing of 2 saccular intracranial aneurysms (IA) and a blood blister-like aneurysm (BBA). (A) Experiment design of scRNA-seq. (B) Seurat clustering of all cells in aneurysms. (C) Featured genes used for classification. (D) Major cell types and subclusters of cells. (E) Heat map showing the gene enrichment of different clusters. (F) Different distributions of cells in BBA and IA. (G, H) Distinct distribution of cell clusters in cell atlas.

Figure 2

Distinct distribution of vascular smooth muscle cells (VSMCs) in blood blister-like aneurysm (BBA) and intracranial aneurysms (IA). (A) The different distributions of VSMCs in uniform manifold approximation and projection and differentially expressed genes. (B) Transcriptional features and enriched cellular signaling pathway of BBA-derived VSMCs. (C) Transcriptional features and enriched cellular signaling pathway of IA-derived VSMCs. (D, E) Upregulated signaling pathways in BBA and IAs profiled in gene set enrichment analysis.

Figure 3

Analysis of macrophages in aneurysms. (A) Enriched gene plot of monocyte/macrophage clusters 1 to 6. (B) Different distributions of macrophages in uniform manifold approximation and projection grouped by different identities. (C) Differentially expressed genes in blood blister-like aneurysm (BBA)-derived macrophages and intracranial aneurysm (IA)-derived macrophages. (D) Kyoto Encyclopedia of Genes and Genomes plot showing enriched cellular pathways in macrophages derived from BBA and IAs.

Figure 4

Intracellular analysis between vascular smooth muscle cells (VSMCs) and macrophages in blood blister-like aneurysm (BBA). (A) Net plot showing the interaction number and strength. (B) River plot showing the communicating and working patterns in cells. (C) Network plot showing the specific communicating pathways and their strength. (D, E) SPP1 signaling pathways between VSMC subclusters and macrophages in BBA.

Figure 5

Intracellular analysis between vascular smooth muscle cells (VSMCs) and macrophages in intracranial aneurysms (IAs). (A) Net plot showing the interaction number and strength. (B) River plot showing the communicating and working patterns in cells. (C) Network plot showing the specific communicating pathways and their strength. (D, E) SPP1 signaling pathways between VSMC subclusters and macrophages in IAs.

Figure 6

Pathological validations of blood blister-like aneurysm (BBA) and intracranial aneurysm (IA) differences. (A) Pathological differences between IAs and BBAs, with statistical analysis. All scale bars are noted in the figures. (B, C) Immunofluorescence validation between IAs and BBAs; scale bars are noted in the figures. *Statistically significant; p-values were also noted in the plots, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001.