Aortic Cellular Diversity and Quantitative GWAS Trait Prioritization through Single Nuclear RNA Sequencing (snRNA-Seq) of the Aneurysmal Human Aorta

In current models of ascending aortic disease, mural cells undergo phenotypic changes that promote extracellular matrix destruction and structural weakening. To explore the intersection of this biology with quantitative trait GWAS loci we analyzed the genetic features of thoracic aortic aneurysm tissue. Single nuclear RNA sequencing was performed on 13 samples from human donors, 6 with thoracic aortic aneurysm and 7 without aneurysm. Individual transcriptomes were then clustered based on transcriptional profiles. Clusters were used for between-disease differential gene expression analyses, subcluster analysis, and analyzed for intersection with genetic aortic trait data. In total, we sequenced 71,689 nuclei from human thoracic aortas without aneurysm, and with sporadic aneurysm. We identified 14 clusters, aligning with 11 cell types, predominantly VSMCs, consistent with existing histologic data, but contrary to the majority of human aortic single cell studies to-date. With unbiased methodology, we found 7 VSMC and 6 fibroblast subclusters. Differentially expressed genes (DEGs) analysis revealed a VSMC group accounting for the majority of differential gene expression. Fibroblast populations in aneurysm exhibit distinct behavior with almost complete disappearance of quiescent fibroblasts. DEGs were used to prioritize genes at aortic diameter and distensibility GWAS loci highlighting the genes JUN, LTBP4, and IL34 in fibroblasts, ENTPD1, PDLIM5, ACTN4, and GLRX in VSMCs, as well as LRP1 in macrophage populations. In conclusion, using nuclear RNA sequencing we describe the cellular diversity of healthy and aneurysmal human ascending aorta. Sporadic aortic aneurysm is characterized by differential gene expression within known cellular classes rather than by the appearance of novel cellular forms. Single nuclear RNA sequencing of aortic tissue can be used to prioritize genes at aortic trait loci. Overall design: We performed snRNA-Seq on 13 samples from human donors, 6 with thoracic aortic aneurysm and 7 without aneurysm. For patients without aneurysm, adult human ascending aortic samples were collected from deceased organ donors by MAGNet (the Myocardial Applied Genetics Network) and the MGH rapid autopsy study. These patients had tricuspid valves, no clinical diagnosis or radiographic evidence of aortic aneurysm, or family history of aneurysm disease. For patients with aneurysm, tissue collection was performed on patients who underwent open aortic reconstruction for sporadic ascending aortic aneurysm. Patients were excluded if they had bicuspid valves, aortic dissection or heritable aortopathy.