Background: In brain, microvascular endothelial cells are exposed to various forces, including shear stress (SS). However, little is known about the effects of high shear stress (HSS) on human brain microvascular endothelial cells (HBMECs) and the underlying mechanism. The cholesterol efflux regulator ATP-binding cassette subfamily A member 1 (ABCA1) has been demonstrated to exert protective effect on HBMECs. However, whether ABCA1 is involved in the mechanism underneath the effect of HSS on HBMECs remains obscure. In the present study, a series of experiments were performed to better understand the effect of HSS on cellular processes of HBMECs and the possible involvement of ABCA1 and PI3K/Akt/eNOS in the underlying mechanisms. Results: HBMECs were subjected to physiological SS (PSS) or high SS (HSS). Cell migration was evaluated using Transwell assay. Apoptotic HBMECs were detected by flow cytometry or caspase3/7 activity. IL-1β, IL-6, MCP-1 and TNF-α levels were measured by ELISA. RT-qPCR and western blotting were used for mRNA and protein expression detection, respectively. ROS and NO levels were detected using specific detection kits. Compared to PSS, HBMECs exhibited decreased cell viability and migration and increased cell apoptosis, increased levels of inflammatory cytokines, and improved ROS and NO productions after HSS treatment. Moreover, HSS downregulated ABCA1 but upregulated the cholesterol efflux-related proteins MMP9, AQP4, and CYP46 and activated PI3K/Akt/eNOS pathway. Overexpression of ABCA1 in HBMECS inhibited PI3K/Akt/eNOS pathway and counteracted the deleterious effects of HSS. Contrary effects were observed by ABCA1 silencing. Inhibiting PI3K/Akt/eNOS pathway mimicked ABCA1 effects, suggesting that ABCA1 protects HBMECs from HSS via PI3K/Akt/eNOS signaling. Conclusion: These results advanced our understanding on the mechanisms of HSS on HBMECs and potentiated ABCA1/PI3K/Akt/eNOS pathway as therapeutic target for cerebrovascular diseases.

Hemodynamic shear stress is a critical mechanical determinant of endothelial homeostasis, yet the mechanosensitive pathways governing metabolic reprogramming remain incompletely understood. Apolipoprotein A-I binding protein (AIBP) regulates cholesterol metabolism, but its role in endothelial mechanotransduction is unknown. Here, we investigated how shear stress modulates AIBP expression to direct human brain microvascular endothelial cell (HBMEC) function via vascular endothelial growth factor (VEGF)-dependent glycolytic reprogramming. HBMECs were subjected to laminar shear stress (1 Pa) with or without the mechanosensitive channel inhibitor GsMTx4. We utilized loss-of-function approaches (shRNA targeting AIBP and VEGF), metabolic assays (metabolite quantification, Western blotting of glycolytic enzymes), and functional assays (EdU proliferation, Transwell migration, tube formation, and apoptosis analysis). Laminar shear stress significantly downregulated AIBP levels via mechanosensitive channel activation. Downregulation or genetic silencing of AIBP stabilized HIF-1α, relieving constitutive repression of VEGF and activating downstream Akt and ERK1/2 signaling. This axis drove glycolytic reprogramming—evidenced by coordinated upregulation of GLUT1, HK2, PFK-A, PKM2, PTBP1, and LDHA, and accumulation of L(+)-lactate and pyruvate—which enhanced proliferation, migration, and tube formation while attenuating apoptosis. Pharmacological inhibition with GsMTx4 prevented shear-induced AIBP downregulation and subsequent metabolic activation. Crucially, simultaneous knockdown of VEGF abolished the glycolytic and phenotypic shifts induced by AIBP deficiency. AIBP functions as a novel mechanosensitive effector in endothelial cells. Shear stress-induced, channel-dependent downregulation of AIBP orchestrates endothelial activation strictly through VEGF-mediated glycolytic reprogramming, identifying a key mechano-metabolic coupling mechanism in vascular regulation.