Abstract Body (Do not enter title and authors here): Introduction:
Thoracic aortic aneurysms (TAAs) affect up to 1% of the population. Transcriptional regulation governs vascular smooth muscle cell (VSMC) phenotypic plasticity, yet upstream epigenomic mechanisms remain poorly defined. Here, we characterize the epigenomic cis-regulatory landscape of sporadic TAAs to uncover regulatory features driving VSMC dysfunction.

Methods:
We prospectively collected fresh-frozen aortic tissue from patients with TAA and donor controls across anatomically distinct regions of the thoracic aorta: root, proximal, mid, and distal ascending. Patients were classified into root- or ascending-dominant phenotypes based on maximally dilatied regions. Bulk ATAC-seq was used to profile chromatin accessibility in TAAs, bulk methylation to assess CpG methylation, and snRNA-seq to compare dilated vs. non-dilated segments in ascending-dominant TAAs. DESeq2 identified differentially accessible peaks and methylated regions (q < 0.05, log2FC > 0), while HOMER assessed transcription factor (TF) motif enrichment.

Results:
We analyzed 23 segments from 12 individuals (20 segments from 11 TAA patients, 3 segments from 1 donor control). All samples were profiled using ATAC-seq. DNA methylation profiling was performed on 6 samples from 3 patients, and snRNA-seq was conducted on 4 samples from 2 patients. Principal component analysis of 158,420 ATAC-seq peaks clearly separated TAA and control samples, including TAA subtypes. We identified 731 differentially accessible peaks in TAA samples linked to genes implicated in TAA pathogenesis, including KLF4 (log2FC=1.1, q=0.02). Motif analysis revealed enrichment of over 200 TF binding sites that were distinct between TAA phenotypes. Chromatin accessibility and DNA methylation scores were inversely correlated in promoter regions (r = –0.31, p < 2.2e-16), consistent with regulatory activity. From 41,791 nuclei profiled by snRNA-seq, we identified 10 distinct cell clusters, with 4 VSMC clusters comprising the majority (80.9%). TF motifs from differentially accessible peaks had higher expression in VSMCs from dilated samples.

Conclusion:
Our integrative analysis reveals a distinct coupling between chromatin accessibility and gene expression in TAAs, suggesting a stable and unique cis-regulatory architecture in VSMCs. We identify chromatin accessibility changes and transcriptional programs associated with VSMC dysfunction that warrant further exploration.