Microregional RNA sequencing reveals layer-specific smooth muscle cell heterogeneity in human bicuspid aortic valve-associated aortic dilatation

Abstract

Objective

Smooth muscle cells in the human ascending aorta exhibit layer-specific heterogeneity; however, their molecular profiles and contribution to aortic diseases remain poorly understood. In this study, we applied microregional RNA sequencing to characterize layer-specific transcriptomic signatures and investigate the differences between bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV) aortopathy.

Methods

Dilated ascending aortic wall samples were obtained from nine patients who underwent ascending aorta replacement concomitant with aortic valve replacement: five with BAV and four with TAV. Microdissected tissues were collected from four distinct layers in media using a micro-punching device. RNA was extracted and sequenced, and the transcriptomic differences across the layers and between BAV- and TAV-dilated aortas were analyzed.

Results

Microregional RNA sequencing revealed significant upregulation of several extracellular matrix remodeling genes in the innermost layer, including FN1, VCAN, MGP, and POSTN. Among them, POSTN was further validated at the protein level, confirming its upregulation in the innermost layer, supporting the validity of this analysis. Next, the transcriptional differences between BAV- and TAV-dilated aortas across individual layers were analyzed. The gene expression differences were the most prominent in the innermost layer. Gene set enrichment analysis revealed that epithelial-mesenchymal transition and transforming growth factor β signaling-related genes were enriched in BAV-dilated aortas. In contrast, inflammation-related genes were enriched in TAV-dilated aortas. We also evaluated the pathways associated with the genes implicated in BAV formation to identify potential upstream mechanisms, revealing that mechanosensing-related genes are coordinately suppressed in BAV-dilated aortas.

Conclusions

The present study demonstrated the application of microregional RNA sequencing to human aortic tissue, revealing layer-specific transcriptomic differences in aortic diseases. The differences in gene-set-level findings indicate distinct biological contexts and mechanisms in BAV- and TAV-dilated aortas, particularly in the innermost media layer. This approach is useful for assessing layer-specific profiles in aortic diseases.

Clinical relevance

This study identified layer-specific heterogeneity within the human aorta, with molecular alterations in the innermost medial layer potentially contributing to differences between TAV- and BAV-associated aortopathy. The findings suggest that layer-specific transcriptome profiles differ across disease types. Accordingly, diagnostic approaches and therapeutic strategies may be more effective when tailored to specific aortic regions and their underlying molecular characteristics.