Dual Lineage Tracing Identifies Cellular Mechanisms Underlying Radiation-Associated Changes in Atherosclerotic Lesion Composition

Background: fate-map both cell types within the same lesion, with most studies mapping one lineage while inferring the other using unreliable dynamically changing marker genes, risking false-positive and false-negative assignment. Methods: -deficient mice, enabling simultaneous fate mapping of SMCs and ECs. This model was used to extend prior findings from single lineage-tracing models demonstrating irradiation-induced loss of SMC lesion investment and expansion of EC-derived cells. Dual lineage-tracing mice were subjected to irradiation and bone marrow transplantation, followed by Western diet feeding to induce atherosclerosis. Lineage tracing, immunostaining and scRNA-seq analysis were used to define coordinated SMC and EC responses and identify changes relevant to plaque instability. Results: cells within the fibrous cap, consistent with reduced plaque stability. Conclusions: cells are not artifacts of false lineage assignment. By resolving SMC and EC fate within the same lesion, we identify irradiation-induced cell dynamics including stress-activated inflammatory reprogramming of SMCs, EC phenotypic modulation, impaired extracellular matrix organization, and reduced ACTA2⁺ fibrous cap cellularity that may contribute to radiotherapy-associated increased atherosclerotic cardiovascular disease risk. Clinical Perspective: Cancer therapies involving radiotherapy are associated with increased long-term risk of atherosclerotic cardiovascular disease.Our findings identify a potential cellular mechanism underlying this risk, in which irradiation-induced smooth muscle cell loss is not functionally compensated by endothelial-to-mesenchymal transition toward a SMC-like state.This dual lineage-tracing model provides a tool to evaluate how cancer therapies and other vascular stressors may alter arterial wall cell fate and indices of plaque stability in atherosclerosis and other vascular diseases.