Single-cell RNA sequencing reveals altered aortic cellular composition and the emergence of a proliferative, pro-inflammatory fibroblast population in a mouse model of IPO8-related thoracic aortic ane

Abstract Bi-allelic pathogenic loss-of-function variants in IPO8, which encodes the cytosol-to-nucleus transport receptor Importin-8, cause thoracic aortic aneurysm (TAA). While vascular smooth muscle cells (VSMCs) have been the primary focus of pathophysiological TAA research, the contribution of other aortic cell types remains poorly understood and is often overlooked. To explore the involvement of different aortic cell populations in IPO8-related TAA, we performed single-cell RNA sequencing (scRNA-seq) on aortic (ascending aorta and root) samples of TAA-presenting C57BI6N Ipo8-/- mice and wild-type (WT) littermates. Samples were collected at two timepoints: prior to aneurysm development and after the aneurysm formation. ScRNA-seq analysis was carried out using Seurat, with clustering performed using the shared nearest-neighbour algorithm, implemented through the FindNeighbors() and FindClusters() functions. Differential gene expression was evaluated with the FindMarkers() function using the MAST method. Immunostaining for the VSMC marker Myh11 was performed to validate alterations in VSMC abundance. ScRNA-seq data analysis revealed an increase in relative fibroblast and immune cell abundance (macrophages and T cells) in Ipo8-/- dilated aortas, alongside a decrease in VSMC proportion. Clustering analysis identified three fibroblast subpopulations: classical fibroblasts, myofibroblasts, and a distinct subpopulation found nearly exclusively in dilated Ipo8-/- aortas (20% in aneurysmal Ipo8-/- aortas vs. ~1% in all other groups). This subpopulation is characterized by high Spp1 expression, a cytokine profile and dysregulation of proliferation-associated pathways, including the Hippo, Ras, and MAPK signalling pathways. Its unique occurrence in aneurysmal mutant aortas suggests association with the aneurysmal phenotype. Pro-inflammatory fibroblast activation is not limited to this subcluster, since a subset of the classical fibroblasts in Ipo8-/- aneurysmal aortas also display elevated cytokine expression compared to WT. VSMC quantification in Ipo8-/- aortic sections by immunofluorescence confirmed the reduction in VSMC proportion, particularly in samples exhibiting a severe phenotype. This decrease in VSMC fraction is primarily driven by an increase in adventitial cells, where fibroblasts are the predominant cell type, alongside thinning of the VSMC layer in certain areas. To conclude, loss of Importin-8 leads to alterations in aortic cellular composition in severely affected Ipo8-/- mice, including a relative increase in fibroblasts and immune cells. The latter matches the thickening of the adventitia observed in Ipo8-/- aneurysmal aortic tissue sections. This adventitial expansion may be driven by the emergence of a proliferative and pro-inflammatory fibroblast subpopulation. Further experiments will be done to validate the expression of inflammatory markers in fibroblasts and to pinpoint the location of the aneurysm-specific fibroblast subpopulation.