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

Methods

We generated dual lineage-tracing Apoe-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 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: Dual lineage tracing simultaneously labeled SMC- and EC-derived cells in healthy and atherosclerotic vessels. Irradiation induced divergent responses: SMC-derived cells failed to invest in lesions and upregulated stress-activated inflammatory genes, whereas EC-derived cells expanded and upregulated SMC-associated genes.

However, EC-derived cells within lesions failed to induce extracellular matrix genes, and lesions from irradiated mice exhibited reduced collagen content and fewer ACTA2+ cells within the fibrous cap, consistent with reduced plaque stability. Conclusions: Dual lineage-tracing of SMCs and ECs demonstrated that irradiation-induced loss of lesional SMC and expansion of EC-derived ACTA2+ 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 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. Key Words: Atherosclerosis; smooth muscle cells; endothelial cells; lineage tracing; radiation; extracellular matrix; cellular plasticity