Cell-Free Hemoglobin Induces Vascular and Mitochondrial Dysfunction in the Pulmonary Endothelium in an Oxidation-State Dependent Manner

Background: Sepsis results in significant organ damage due in part to a breakdown in endothelial vascular function. Elevated levels of cell-free hemoglobin (CFH) drive endothelial dysfunction in the lungs. CFH generates reactive oxygen species (ROS) via the iron present in the heme moiety, which can be oxidized from ferrous (CFH2+) to ferric (CFH3+). Recent studies suggest mitochondrial dysfunction may be a key pathway leading to microvascular dysfunction. We hypothesize that CFH induces mitochondrial dysfunction and microvascular endothelial barrier disruption in the lung in an oxidative-state dependent manner. Methods: CFH was converted from CFH2+ to CFH3+ by UV irradiation (validated by spectrophotometry). Primary human lung microvascular endothelial cells (HLMVECs) were treated with 1.0 mg/mL CFH2+, CFH3+, or vehicle control. Barrier dysfunction was measured via electric cell-substrate impedance sensing (ECIS) and express permeability test (XPerT) at 24h. We quantified mitochondrial superoxide (MitoSOX Red; 5 uM) by flow cytometry (1, 6, 24h) and total cellular ROS production (CellROX Deep Red; 5 uM) by fluorescence microscopy (6 and 24 h). Mitochondrial morphology (electron density, roundness, area, circularity, and aspect ratio) was assessed by transmission electron micrographs at 6h and quantified by a blinded reviewer (ImageJ). Activation of the mitochondrial permeability transition pore (mPTP) was assessed by flow cytometry using calcein-AM with cobalt chloride. DNA was purified from patient plasma and the copy number of mitochondrial genes ND4L and CYB was quantified by digital droplet PCR. Results: In HLMVECs, CFH generated excess ROS (CellROX and MitoSOX) at 6h compared to vehicle (522 vs 265 MFI, p < 0.01). Morphology analysis demonstrated increased electron density and aspect ratio and decreased circularity and roundness. mPTP activity was increased by CFH3+ (1295 vs 2197 MFI, p < 0.01) compared to vehicle or CFH2+. Barrier dysfunction caused by CFH3+ measured by ECIS peaked at 6h (-1270 vs 262 max TER drop, p < 0.0001), while XPerT confirmed dysfunction at 24h (9.41e+06 vs 6.53e+05 total fluorescent area, p < 0.01). Quantification of mtDNA copy number in patient plasma revealed that circulating mt-ND4L copies correlate with CFH (r = 0.44, p < 0.01). Conclusions: The results demonstrate significant perturbations to mitochondrial function and network dynamics caused by oxidized CFH (3+). The novel findings point towards the ferric (3+) form of hemoglobin as the primary driver of endothelial dysfunction, and the underlying mechanism is directly related to disruption of the mitochondrial network. The association of mitochondrial and vascular biomarkers demonstrates an urgent need to better understand how mitochondrial dysfunction can be prevented to limit vascular damage. This work was funded by the National Institutes of Health NHLBI 5R35HL150783 and 1F31HL167471. This is the full abstract presented at the American Physiology Summit 2024 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.