Endothelial-specific ACKR1 overexpression attenuates macrophage infiltration and blood–brain barrier disruption after ischemic stroke

Abstract

Ischemic stroke triggers a profound inflammatory response that exacerbates secondary brain injury, with infiltrating macrophages serving as major pathological mediators. Although previous studies have largely focused on immune cell–intrinsic mechanisms, the immunomodulatory role of cerebral endothelial cells in post-stroke inflammation remains poorly understood. In the current study, using single-cell RNA sequencing, we identified extensive inflammatory reprogramming in cerebral endothelial cells after stroke and discovered atypical chemokine receptor 1 (ACKR1) as a prominently upregulated regulator associated with chemokine sequestration and leukocyte recruitment. To determine its functional significance, we selectively overexpressed ACKR1 in cerebral endothelial cells using endothelial-targeted AAV-X1.1 in a mouse model of ischemic stroke. At 1 day after transient middle cerebral artery occlusion, endothelial ACKR1 overexpression preserved blood–brain barrier integrity, evidenced by maintained ZO-1 continuity, enhanced pericyte and astrocytic endfoot coverage, and reduced leakage of both exogenous tracers and endogenous IgG. By day 3, ACKR1 overexpression markedly reduced macrophage infiltration, decreased infarct volume, and improved neurological outcomes. These protective effects persisted through day 28, accompanied by improved sensorimotor recovery and attenuated brain atrophy. Collectively, our findings identify cerebral endothelial cells as active regulators of post-stroke neuroinflammation and reveal endothelial ACKR1 signaling as a potential therapeutic strategy for ischemic stroke.