01.01 Single Cell RNA-Seq Reveals the Heterogeneity of Aortic Fibroblast During Aortic Disease Development

C. Zhang^1,2, Y. Li^1,2, A. Dawson^1,2, P. Ren^1,2, L. Zhang^1,2, Y. Li^1,2, W. Luo^1,2, Y. H. Shen^1,2,3, S. A. LeMaire^{1,2,3 1}Baylor College Of Medicine,Division Of Cardiothoracic Surgery, Michael E. DeBakey Department Of Surgery,Houston, TX, USA ²Texas Heart Institute,Department Of Cardiovascular Surgery,Houston, TX, USA ³Baylor College Of Medicine,Cardiovascular Research Institute,Houston, TX, USA

Introduction:
Aortic fibroblasts are highly dynamic cell populations that play critical roles in repair and remodeling in aortic aneurysm and dissection (AAD). However, their molecular and cellular changes during AAD development remain poorly understood. In this study, we examined aortic fibroblast heterogeneity in a sporadic AAD mouse model using single-cell RNA sequencing (scRNA-seq).

Methods:
C57BL/6 wild-type (WT) mice were either unchallenged (chow diet, saline infusion; n=3) or challenged (n=3) with 4 weeks of high-fat diet followed by 7 days of angiotensin II infusion. Thoracic aortas were excised and digested to generate single-cell suspensions for scRNA-seq. In all,16,187 cells were analyzed and categorized by using the Seurat package in R to perform cluster identification. Differentially expressed genes (DEGs) were identified using edgeR. Trajectory pseudotime analyses were performed using Monocle2.

Results:
We identified 7 major aortic cell populations including one cluster of Pdgfra^high fibroblasts. We further divided this cluster into 7 subclusters including extracellular matrix (ECM)-producing fibroblasts (with enrichment in Dcn, Lum, and Col1a2), Acta2⁺ myofibroblasts, Tnfrsf11b^high fibroblasts, mesenchymal progenitor cells (with enrichment in Cd34, Ly6a, and Ly6c1), pro-inflammatory fibroblasts (with enrichment in Col8a1, Thbs1, and Tgfb3), and Cd14⁺ macrophage-like fibroblasts (expressing myeloid-specific markers Lyz2, Cd68, and Pf4). DEGs and Gene Ontology enrichment analyses revealed specific gene expression patterns distinguishing these fibroblast subsets and uncovered their putative functions, such as increased collagen synthesis and ECM organization in ECM-producing and pro-inflammatory fibroblasts, increased cell proliferation and migration features in Tnfrsf11b^high and pro-inflammatory fibroblasts, and increased cell adhesion and cell junction organization in myofibroblasts and mesenchymal progenitor cells. Notably, pseudotime analyses suggest an important role of Tnfrsf11b in fibroblast trajectory differentiation. Interestingly, while ECM-producing fibroblasts, Acta2⁺ myofibroblasts, Tnfrsf11b^high fibroblasts, and mesenchymal progenitor cells were detected in both unchallenged and challenged mice, pro-inflammatory fibroblasts and macrophage-like fibroblasts were almost exclusively detected in challenged mice.

Conclusion:
 This study revealed marked phenotypic heterogeneity of aortic fibroblasts and identified changes in fibroblast gene expression during aortic stress, suggesting specialized functions of fibroblast subclusters. Our findings will open up novel opportunities to understand the role of fibroblasts in AAD formation and progression.