Abstract 4370228: Targeting Mitochondrial ROS Restores Migration and Angiogenesis in Brain Endothelial Cells with Complex I Inhibition

Background: Brain microvascular endothelial cells (BMECs) are essential for maintaining blood-brain barrier (BBB) integrity and cerebrovascular homeostasis. Mitochondrial dysfunction is increasingly recognized as a contributor to vascular pathology in neurodegenerative diseases. While rotenone, a mitochondrial complex I inhibitor, is known to induce oxidative stress in neurons, its effects on BMEC function remain poorly defined. Objective: To investigate how mitochondrial oxidative stress affects BMEC migration and angiogenic capacity, and whether targeting mitochondrial ROS with mitoTEMPO can reverse these effects. Methods: Human brain endothelial cells (HBECs; passage 8) were treated with 0.5 µM rotenone in the presence or absence of 10 µM mitoTEMPO for twelve hours. Cell migration was assessed using a wound healing assay, and angiogenic function was evaluated via Matrigel tube formation. Mitochondrial membrane potential (ΔΨm) was measured using TMRE uptake. Wound closure and tube length were quantified using ImageJ. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test. Results: Rotenone significantly impaired HBEC migration, reducing wound closure by 4.39-fold compared to control (Rotenone: 20.9 ± 4.4%; Control: 4.75 ± 2.45%; p = 0.023, n = 3–4 wells). MitoTEMPO co-treatment significantly improved migration ( p < 0.05 vs. rotenone). In the tube formation assay, rotenone reduced total tube length by 45% ( p = 0.000). Co-treatment with mitoTEMPO partially rescued tube formation, showing a 31% improvement (trend-level, p = 0.058). Notably, mitoTEMPO did not restore the rotenone-induced loss of mitochondrial membrane potential, suggesting that its protective effects are independent of ΔΨm. Conclusions: Mitochondrial ROS impairs endothelial migration and angiogenesis in brain endothelial cells. MitoTEMPO partially rescues these functions, despite persistent mitochondrial membrane depolarization. These findings suggest that mitochondrial oxidative stress, rather than loss of ΔΨm, plays a primary role in BMEC dysfunction and may represent a therapeutic target in cerebrovascular disease.