Oxidation of LDL by Hemoglobin Alters Heme- and Lipid-Processing, Cytoskeletal, Adhesion, and Chemotactic Pathways in Human Pulmonary Microvascular Endothelial Cells

Introduction: Hemoglobin (Hb)-mediated pulmonary microvascular endothelial barrier dysfunction is a critical driver of acute lung injury in several pathologies, including sepsis, sickle-cell disease, pulmonary arterial hypertension, and transfusion-related acute lung injury. The precise signaling mechanisms of Hb-mediated endothelial injury are incompletely understood. A potential mechanism by which Hb might disrupt the microvascular endothelial barrier is through its known ability to oxidize lipids and lipoproteins, including low-density lipoprotein (LDL). We recently discovered that oxidation of LDL by Hb (Hb-oxLDL) disrupted the human pulmonary microvascular endothelial cell (HPMEC) barrier through the lectin-like oxidized LDL receptor 1 (LOX-1); however, the downstream signaling mechanisms leading to barrier dysfunction are currently unknown. Therefore, we used an unbiased proteomics approach to analyze changes in HPMEC signaling in response to Hb-oxLDL. We hypothesized that Hb-oxLDL stimulation of endothelium would lead to differential expression of proteins related to heme- and lipid-processing, oxidative stress, inflammation, and cell adhesion. Methods: LDL was oxidized by combining purified human LDL with Hb overnight at 37°C. HPMEC protein lysate was collected after cells were stimulated with Hb-oxLDL or PBS control for 1, 3, or 6 hours. Label-free differential expression discovery proteomics was performed by the Vanderbilt Mass Spectrometry Research Center. Two separate analyses were performed using Spectronaut (Biognosys, Inc.) and MSStats in R to identify potential protein targets. Comparisons between treatment groups and time-matched controls were performed and significant differences were determined by adjusted p-values less than 0.05. Pathway analysis was performed using STRING. Results: We identified 57 unique differentially expressed proteins across the three timepoints investigated. As expected, several proteins related to oxidative stress and heme- and iron-processing (HBA1, HBB, HBD, HGBG1, HGBG2, CAT, TF, BLVRB, HPX, HMOX1, HIGD2A, FTH1, FTL), as well as lipid-processing (APOM, APOB, APOA1), were increased with Hb-oxLDL stimulation. Iron-processing protein IREB2 was the only significantly downregulated protein at 6 hours. These changes may indicate the involvement of ferroptosis in the cellular response. Additionally, proteins related to cytoskeletal (RAP1GDS1, RELL1, TUBA4A) and adhesion (DSC3, FBLN1, TJP2) pathways were increased with Hb-oxLDL stimulation compared to control. Concomitantly, proteins that may be associated with barrier regulation (DOCK9, SYNPO) were decreased in cells stimulated with Hb-oxLDL. These changes indicate that there may be an important interplay between cytoskeletal and adhesion regulation in the barrier response to Hb-oxLDL. Furthermore, proteins related to chemotactic (CCL2, CX3CL1) pathways were also upregulated in response to Hb-oxLDL, indicating a potential role for endothelial activation and pro-inflammatory immune cell adhesion in this process. KEGG and Reactome analyses revealed pathways related to ferroptosis, mineral absorption, binding and uptake of ligands by scavenger receptors, heme signaling, iron uptake and transport, and vesicle-mediated transport. Conclusions: Oxidation of LDL by Hb contributes to HPMEC barrier dysfunction, potentially via pathways related to oxidative stress, heme and iron processing, cytoskeletal changes, cell-cell or cell-matrix adhesion, and chemotaxis/inflammation. Future studies will determine the relevance of these specific signaling pathways in Hb-oxLDL-mediated pulmonary microvascular endothelial barrier dysfunction. Parker B Francis Fellowship (JEM); NIH R00HL166865 (JEM), R01HL148069 (TS), R01HL167997 (TS), P01HL066299 (TS), R01HL158906 (LBW), R35HL150783 (JAB), RF1AG075341 (JAB) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.