Introduction: Heme-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. We recently implicated the lectin-like oxidized LDL receptor 1 (LOX-1) in mediating human pulmonary microvascular endothelial cell (HPMEC) barrier dysfunction in response to heme-mediated oxidation of LDL (Meegan AJP-Lung 2025). An unbiased proteomics study pointed to iron dysregulation as a possible mechanism linking LOX-1 to barrier dysfunction in this context (Meegan, APS 2025). This is not surprising given that each heme molecule contains a central iron atom. Additionally, evidence is accumulating for implicating LOX-1 beyond oxLDL-dependent mechanisms. Taken together, we hypothesized that heme may disrupt the HPMEC barrier by inducing iron overload through a LOX-1 dependent mechanism. Methods: HPMEC (Promocell) were cultured two days past confluence to form a tight monolayer. Intracellular iron was measured in HPMEC using FerroOrange immunofluorescence microscopy after 1h of heme (50 µg/mL) stimulation. HPMEC were treated over a time course with ferric ammonium citrate (FAC; 200 µM) to induce iron overload. LOX-1 cell surface expression (rabbit anti-LOX-1 primary antibody, 1:200; Cat. No. PA5-95750, Invitrogen) was assessed using immunofluorescence microscopy in non-permeabilized cells. Barrier dysfunction was assessed using XPerT assay. LOX-1 small molecule inhibitor (10 µM; Cat. No. BI-0115, Boehringer Ingelheim) was used to block LOX-1 receptor. Statistical analysis was performed using GraphPad Prism with significance set at α = 0.05. Data were analyzed using Mann–Whitney to compare two groups or Kruskal–Wallis with Dunn’s multiple comparisons post hoc analysis or One-way ANOVA with Tukey’s multiple comparisons post hoc analysis to compare more than two groups. Results: Heme stimulation of HPMEC significantly increased intracellular iron compared to PBS control after 1h (p=0.008). FAC stimulation of HPMEC significantly increased LOX-1 cell surface expression at 24h (p=0.04) and 48h (p=0.002) and barrier dysfunction at 48h (p=0.04). Inhibiting LOX-1 reduced barrier dysfunction induced by FAC (p=0.0001). Conclusions: Heme increases barrier dysfunction and intracellular iron in HPMEC. Direct iron stimulation of HPMEC increases LOX-1 cell surface expression and barrier dysfunction. Inhibiting LOX-1 with a small molecule inhibitor prevents barrier dysfunction induced by iron overload. Together, these data provide evidence for a possible LOX-1-dependent mechanism of barrier dysfunction through intracellular iron dysregulation. Future studies will determine if the intracellular iron increase by heme is dependent on LOX-1, investigate the signaling pathways by which iron dysregulation causes barrier dysfunction, and the potential implications of this pathway in a preclinical model of murine sepsis. Funding: NIH R00HL166865 (NHLBI) and Parker B. Francis Fellowship to JEM This abstract was presented at the American Physiology Summit 2026 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.
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