Abstract Body: Introduction:
Thoracic aortic aneurysms (TAA) are driven by progressive vascular smooth muscle cell (VSMC) dysfunction and phenotypic switching. While recent studies in heart failure suggest that the T-box transcription factor 20 (TBX20) enhances cardiomyocyte contractility, its role in aortic pathology remains unexplored. Here, we identify TBX20 as a novel, context-dependent regulator of VSMC dedifferentiation in TAA and a novel potential therapeutic target.
Methods:
We performed comprehensive transcriptomic profiling using bulk RNA-sequencing (n=195) and single-cell RNA-seq (n=34) on ascending aortic specimens from TAA patients and healthy heart transplant donors. TAA tissues were stratified by valve phenotype (bicuspid vs. tricuspid), clinical presentation (aortic stenosis vs. regurgitation), and anatomical location (outer curvature vs. inner curvature). Datasets were integrated via deconvolution and RNA velocity analysis to map cellular trajectories. Findings were validated by immunohistochemistry (IHC) and qRT-PCR. Alterations on VSMC plasticity and function were assessed by using siRNA-mediated knockdown in patient-derived VSMCs, followed by live cell imaging analysis and collagen-based gel contraction assays.
Results:
Bulk RNA-seq revealed significant TBX20 upregulation in TAA (>2-fold, p<0.05), independent of valve morphology, clinical presentation, or anatomical location. These findings were subsequently validated by qPCR. Single-cell analysis localized this upregulation specifically to VSMCs, peaking in a pathology-associated synthetic subpopulation (>5-fold, p<0.001). This correlated with a significant loss of contractile cell identity with a concomitant expansion of the synthetic cluster. RNA velocity trajectory analysis demonstrated a unidirectional transition from contractile to synthetic states driven by TBX20. TBX20 knockdown in TAA-derived VSMCs further modulated migration and proliferation, restored contractile markers (2-fold increase for ACTA2/TAGLN; p<0.05), and significantly enhanced contractile force in functional assays.
Conclusion:
TBX20 acts as a critical molecular switch driving maladaptive VSMC dedifferentiation in TAA. Unlike its previously reported role in heart failure, aortic TBX20 suppresses contractile programs to promote synthetic transformation. Targeting this pathway offers a novel strategy to preserve aortic wall integrity across diverse TAA etiologies.