SignificanceThe limited understanding of the molecular mechanisms governing cerebral microvascular dysfunction in stroke has been a major obstacle in the development of novel therapeutic approaches. Herein, we elucidated the stroke-induced transcriptomic changes in the mouse cerebral microvasculature and compared them with the alterations observed in human brain stroke lesions. Our study revealed the presence of shared alterations in microvessel-enriched, vascular disease-associated, druggable targets, highlighting the relevance of cerebral microvascular dysfunction in human stroke pathophysiology. We have also identified molecular alterations in the sphingolipid metabolism and signaling pathway in the cerebral microvasculature, which were significantly altered in human stroke. Our work provides a knowledge platform for future investigation of novel endothelial-enriched therapeutic candidates in stroke.AbstractStroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.