Cytoskeletal-related genes function as checkpoints for the maintenance of VSMC contractile phenotype and prevent pathological remodeling in arterial diseases

• The study makes a significant contribution to the field of cardiovascular research by uncovering the critical role of cytoskeleton-related genes in vascular pathological remodeling. • The study utilizes an integrative cross-species single-cell RNA-seq model with published public datasets and plots the conserved trajectory of VSMCs phenotypic switching. This may indicate the potential function importance of trajectory-related genes that benefit the research area. • The study finds a checkpoint state during the transition which have significant modulation of cytoskeleton-related gene expression. • The inhibition of Fblim1 , Tns1 and Synpo2 discriminated the contractile phenotypic function in primary VSMCs under pathological stress. • Both the in vivo experiments and in vitro animal model experiments validate a promising treatment potential of the cytoskeleton-related genes in preventing vascular pathological remodeling. Arterial pathological remodeling, central to arterial diseases including atherosclerosis and aortic aneurysms, is characterized by vascular smooth muscle cell (VSMC) phenotypic switching with concomitant loss of contractile markers. Uncovering the molecular changes initiating phenotypic transition may advance the understanding of vascular pathogenesis and provide new therapeutic strategies. To construct a cross-species integrative model of VSMC transition in arterial diseases including atherosclerosis and aortic aneurysm, identify key genes regulating phenotypic switching in the trajectory from contractile to other phenotypes, and further validate their function in arterial remodeling models. Public single-cell RNA-seq datasets were analyzed to map VSMC transcriptional dynamics and identify regulated gene expression patterns during transition. The changes were further checked using experimental animal aneurysm samples and PDGF-BB treated VSMCs. Functional validation included in vitro siRNA-mediated knockdown using primary VSMCs and in vivo gene-manipulated (AAv-shRNA/Adv-overexpression) wire-injury models. Dysregulation of cytoskeletal-related genes ( Fblim1 , Tns1 , and Synpo2 ) may cause disarrangement of actin cytoskeleton, and were identified as checkpoint process before VSMCs transition initiation. Knockdown of target genes suppressed contractile markers, enhanced proliferation, migration, and disrupted cytoskeleton architecture in VSMCs. In animal models, gene down-regulation exacerbated pathological remodeling while over-expression partially reverted these effects. The findings highlight the critical role of cytoskeleton-related genes in arterial diseases that function as a critical checkpoint in preventing VSMC pathological phenotypic switching.