Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states

Background Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. Methods and results We generated a novel Spp1 -rSTOPr-Cre (Spp1 Cre ) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5′ junction, 3′ junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1 Cre mice were crossed with Myh11 DreER T2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, GFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, GFP-positive cells markedly expanded within atherosclerotic lesions induced by PCSK9-AAV and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of GFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. Conclusion We report the generation and validation of a novel Myh11 Dre -Spp1 Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1 -associated VSMC populations during vascular remodeling and cardiovascular disease.