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Background: During sepsis, red blood cells lyse and release cell-free hemoglobin (CFH) into the circulation causing vascular dysfunction and ROS production. The heme moiety of CFH is normally in the CFH2+ oxidative state but can be oxidized to CFH3+ and CFH4+. Our knowledge of which oxidation state of CFH causes vascular injury remains unknown. One potential mechanism of ROS generation may be due to CFH-induced mitochondrial dysfunction. This mechanism is supported by clinical observations that sepsis patients have elevated levels of both CFH and markers of mitochondrial dysfunction including lactate and mitochondrial DNA (mtDNA). To further explore this mechanism, we tested the hypothesis that oxidized CFH3+ would cause changes to mitochondrial morphology and ROS homeostasis in primary cultured human lung microvascular endothelial cells (HLMVECs). Methods: HLMVECs from donors of both sexes were treated with CFH (2+ or 3+, 1 mg/ml for 6h). Mitochondrial morphology was analyzed by both confocal microscopy and high-resolution TEM. Mitochondrial superoxide production was quantified by MitoSOX staining followed by flow cytometry analysis. Relative levels of 8-hydroxyguanine (8OHG) were determined via immunofluorescence using an antibody against 8OHG and imaged by confocal microscopy. DNA was extracted from critically ill patient plasma and mtDNA was quantified via ddPCR using probes specific to mt-CO3 and mt-ND4L. A non-parametric Spearman correlation coefficient was calculated using previously measured CFH levels in the patient plasma and the absolute quantification values of circulating mtDNA. Results: Treatment of HLMVECs with CFH3+ decreased total mitochondrial footprint (p<0.05) compared to vehicle control. TEM imaging of CFH3+ treated cells showed a decrease in mitochondrial roundness (p<0.001) and increases in mean electron density (p<0.001) and aspect ratio (p<0.001) compared to control cells. Treatment of cells with unoxidized CFH2+ did not alter mitochondrial footprint, circularity, and other morphological parameters. Exposure to CFH3+ in comparison to control cells caused a significant increase in both mitochondrial superoxide production (608 vs 283 adjusted MFI, p<0.01) and nuclear 8-OHG in HLMVECs (7.4 vs 5.1 MFI, p<0.0001). In critically ill patients, circulating mtDNA levels correlated with CFH in plasma (R=0.44, p<0.01). Discussion: In endothelial cells, CFH3+, but not CFH2+, caused profound remodeling of the mitochondrial network resulting in a highly fused network with higher levels of mitochondrial superoxide production. In critically ill patients, we found a significant correlation between circulating mtDNA and CFH opening the way for mtDNA as a potential biomarker of disease severity. These studies reveal a novel mechanism of oxidized CFH3+ causing microvascular endothelial dysfunction via induction of mitochondrial morphological changes, generation of ROS, and DNA damage. Research was supported by NHLBI of the NIH under award number HL150783, HL158906, and HL167471. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.