Background and Purpose— Because brain endothelial cells exist at the neurovascular interface, they may serve as cellular reporters of brain dysfunction by releasing biomarkers into the circulation. Methods— We used proteomic techniques to screen conditioned media from human brain endothelial cultures subjected to oxidative stress induced by nitric oxide over 24 hours. Plasma samples from human stroke patients were analyzed by enzyme-linked immunosorbent assay. Results— In healthy endothelial cells, interaction mapping demonstrated cross-talk involving secreted factors, membrane receptors, and matrix components. In oxidatively challenged endothelial cells, networks of interacting proteins failed to emerge. Instead, inflammatory markers increased, secreted factors oscillated over time, and endothelial injury repair was manifested as changes in factors related to matrix integrity. Elevated inflammatory markers included heat shock protein, chemokine ligand-1, serum amyloid-A1, annexin-A5, and thrombospondin-1. Neurotrophic factors (prosaposin, nucleobindin-1, and tachykinin precursors) peaked at 12 hours, then rapidly decreased by 24 hours. Basement membrane components (fibronectin, desomoglein, profiling-1) were decreased. Cytoskeletal markers (actin, vimentin, nidogen, and filamin B) increased over time. From this initial analysis, the high-ranking candidate thrombospondin-1 was further explored in human plasma. Acute ischemic stroke patients had significantly higher thrombospondin-1 levels within 8 hours of symptom onset compared to controls with similar clinical risk factors (659±81 vs 1132±98 ng/mL; P <0.05; n=20). Conclusions— Screening of simplified cell culture systems may aid the discovery of novel biomarkers in clinical neurovascular injury. Further collaborative efforts are warranted to discover and validate more candidates of interest.