Early hypoxia-induced secretome remodeling reveals adaptive mechanisms and biomarkers of blood-brain barrier dysfunction in ischemic stroke

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Abstract Reversible disruption of the blood-brain barrier (BBB) occurs within hours after the onset of ischemic stroke (IS), offering a critical window for therapeutic intervention. However, the molecular characteristics and their potential as circulating biomarkers associated with this transient phase of BBB dysfunction remain poorly defined. To elucidate these mechanisms, we employed an oxygen-glucose deprivation (OGD) model in human cerebral microvascular endothelial cells (hCMEC/D3) to simulate early ischemic stress, and systematically profiled their secreted proteome and metabolome. By comparing with non-brain-derived human umbilical vein endothelial cells (HUVECs), we identified brain endothelium-specific hypoxic response signatures. These molecules were significantly enriched in pathways related to metabolic reprogramming, antioxidant defense, and epigenetic regulation pathways, indicating a coordinated adaptive response to preserve BBB homeostasis. Furthermore, integrative multi-omics analysis revealed 14 protein-metabolite pairs with potential functional synergy. Based on a multi-criteria screening strategy including brain specificity, functional relevance, and secretory potential, we prioritized 10 candidate circulating biomarkers: ALDH2, ITGA5, KYNU, TFRC, CD44, COL1A2, HEXB, HSPG2, THBS4, and DLD. Preliminary validation using serum from acute IS (AIS) patients and healthy controls showed significantly altered levels of ALDH2, ITGA5, KYNU, and TFRC, with TFRC exhibiting promising diagnostic performance both individually (AUC = 0.816) and in combination with the other three biomarkers (AUC = 0.876). Moreover, multivariate logistic regression analysis revealed that elevated TFRC was independently associated with poor 90-day outcomes (OR = 1.02, 95% CI 1.00–1.04, P = 0.031), while higher DLD levels were correlated with good prognosis (OR = 0.68, 95% CI 0.39–0.90, P = 0.047). Notably, TFRC expression was upregulated in hCMEC/D3 cells under early hypoxic stress, while its extracellular secretion was reduced. This observation suggests a potential early cellular adaptation to preserve iron homeostasis. In summary, these findings uncover early molecular adaptations of brain microvascular endothelial cells to ischemic stress and propose a panel of secreted biomarkers with translational potential for early diagnosis and outcome prediction in IS, potentially guiding the development of time-sensitive therapeutic strategies.

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