Cross-Species Multi-Omics Analysis Reveals Myeloid-Driven Endothelial Oxidative Stress in Ischemic Stroke.

Background: Ischemic stroke is a leading cause of mortality and disability worldwide, yet the interplay between peripheral and central immune responses is still only partially understood. Emerging evidence suggests that myeloid cells, when activated in the periphery, infiltrate the ischemic brain and contribute to the disruption of the blood-brain barrier (BBB) through both inflammatory and metabolic mechanisms.

Methods: In this study, we integrated bulk RNA-sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), spatial transcriptomics, and flow cytometry data from human and mouse models of ischemic stroke. Mouse stroke models were induced by transient middle cerebral artery occlusion (tMCAO), and brain tissues were later collected at specified time points for analysis. We examined time-dependent transcriptional changes in the peripheral blood, delineated cell-type-specific responses by single-cell profiling, and validated myeloid infiltration into the ischemic brain. We also investigated endothelial metabolic reprogramming and oxidative stress by combining scMetabolism analyses (a computational R package for inferring metabolic pathway activity at the single-cell level) with in vitro oxygen-glucose deprivation/reperfusion (OGD/R) experiments.

Results: Cross-species bulk RNA-seq revealed a modest early immune shift at 3 h post-stroke, escalating significantly by 24 h, with robust myeloid-centric gene signatures conserved in humans and mice. Single-cell analyses confirmed a pronounced expansion of neutrophils, monocytes, and megakaryocytes in peripheral blood, coupled with a decrease in T and B lymphocytes. Spatial transcriptomics and flow cytometry demonstrated substantial infiltration of CD11b⁺ myeloid cells into the infarct core, which showed extensive interaction with endothelial cells. Endothelial scRNA-seq data showed reductions in the oxidative phosphorylation, glutathione, and nicotinate metabolic pathways, together with elevated pentose phosphate pathway activity, suggestive of oxidative stress and compromised antioxidant capacity. Functional scoring further indicated diminished endothelial inflammation/repair potential, while in vitro OGD/R experiments revealed morphological disruption, CD31 downregulation, and increased 4-hydroxynonenal (4-HNE), underscoring the importance of endothelial oxidative damage in BBB breakdown.

Conclusions: These multi-omics findings highlight the existence of a coordinated peripheral-central immune axis in ischemic stroke, wherein myeloid cell recruitment and endothelial metabolic vulnerability jointly exacerbate inflammation and oxidative stress. The targeting of endothelial oxidative injury and myeloid-endothelial crosstalk may represent a promising strategy to mitigate secondary brain injury in ischemic stroke.