Single-Cell RNA Sequencing Suggests Roles For CellMatrix Interactions And Inflammation In The Pathogenesis Of Aortopathy In Loeys-Dietz Syndrome

Background: Humans with Loeys-Dietz syndrome (LDS; caused by heterozygous loss-of-function mutations in TGFBR2 or other TGF-β signaling molecules) develop proximal aortic aneurysms and dissections. The mechanisms through which heterozygosity for these mutations causes aortic disease are poorly understood. Roles for decreased and increased TGF-β signaling in SMC have been suggested. Methods: To investigate the pathogenesis of aortopathy caused by heterozygosity for a TGFBR2 loss-of-function variant found in humans with LDS, we used mice with an orthologous variant ( Tgfbr2 G357W/+ ). We performed single-cell RNA sequencing with pathway analysis on proximal aortas of 17-20-wk-old mice and histology on aortic roots of 30–50-wk-old mice. Results: ScRNA-seq showed either mildly decreased (less than 50%) or equivalent expression of TGF-β signaling targets ( Serpine1, Ctgf, Col1a1, Col1a2 ) in proximal aortic SMC of Tgfbr2 G357W/+ mice vs WT littermate controls. Moreover, Tgfbr2 G357W/+ SMC had no significant changes in expression of transcripts encoding any of the three TGF-β ligands. Pathway analysis of SMC transcriptomes showed upregulation of genes involved in focal adhesion and extracellular matrix (ECM) interactions (FDR<0.05 for both) in Tgfbr2 G357W/+ mice. ScRNA-seq also identified a unique cluster of fibroblast-like cells present only in Tgfbr2 G357W/+ aortas. These cells had increased expression of genes related to immune activation and macrophage recruitment (Saa3, Ccl2, Cxcl12 ; ~2-fold change and p<0.001 for all) and increased mRNA encoding Mmp3 (>3-fold change, p<0.001). The percentage of immune cells identified by scRNA-seq was ~3-fold higher in Tgfbr2 G357W/+ versus WT proximal aortas (p=0.03). These immune cells included macrophages that express Ccr2 and Cxcr4 (receptors for CCL2 and CXCL12). Aortic root sections of older Tgfbr2 G357W/+ mice had significant adventitial inflammation. Conclusions: We found evidence for decreased—but not increased—TGF-β signaling in proximal aortas of Tgfbr2 G357W/+ mice. Emergence of a proinflammatory and potentially destructive fibroblast-like cell type and infiltration of inflammatory cells may play roles in aortopathy caused by a heterozygous loss-of-function variant of Tgfbr2 .