Decoding the senescent endothelial landscape in ischemic stroke through single-cell and bulk RNA sequencing

Ischemic stroke is a major cause of disability and mortality worldwide, but its underlying pathogenic mechanisms remain elusive. Although cellular senescence has been implicated in a wide range of age-related diseases, its specific contribution through endothelial cells to ischemic stroke pathogenesis remains unclear. To address this gap, the present study adopted an integrative strategy, combining single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (RNA-seq), to systematically characterize the senescent endothelial landscape in ischemic stroke. We identified a distinct senescence-associated gene signature in endothelial cells and highlighted four key genes-- Arhgap31, Tm4sf1, Itm2a, and Vwa1--that were closely associated with ischemic stroke pathogenesis. These genes showed differential expression and apparent discriminatory potential in the discovery bulk RNA-seq dataset, and external validation using independent scRNA-seq and cerebral microvessel bulk RNA-seq datasets provided additional support for the signature, particularly for Arhgap31, Tm4sf1, and Vwa1. Pathway enrichment and transcription factor analyses suggested potential pathways and upstream regulators associated with these candidate genes. In vitro experiments confirmed the differential expression of Arhgap31, Tm4sf1, Itm2a, and Vwa1 in senescent endothelial cells. Reduced expression of Arhgap31 attenuated endothelial cell senescence while enhancing migration and vasculogenesis capabilities. Arhgap31 may emerge as a potential target for ameliorating endothelial cell senescence and promoting tissue repair. Moreover, Connectivity Map analysis predicted several candidate compounds with expression profiles opposing the disease-associated signature, providing hypotheses for future pharmacological validation. Our findings highlight the contribution of endothelial cell senescence to post-ischemic endothelial dysfunction and inflammatory remodeling, supporting further investigation of this process in ischemic stroke pathogenesis. This work provides crucial insights into the interplay between cellular senescence and cerebrovascular disease, opening new avenues for precision medicine interventions in ischemic stroke management.