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A2888 - Hemin causes Endothelial Barrier Dysfunction via Disruption of Tight Junctions
Author Block: A. Srivastava1, C. Eccles1, R. Rafikov2; 1University of Arizona, Tucson, AZ, United States, 2Medicine, University of Arizona, Tucson, AZ, United States.
RATIONALE: Pulmonary arterial hypertension (PAH) is a multifaceted disease characterized by vascular remodeling, vasoconstriction, inflammation, fibrosis and right heart hypertrophy. There is a clear need to better understand the underlying molecular mechanisms of PAH. An increasing number of studies have demonstrated that endothelial cells (EC) have an important role in PAH pathogenesis. Further, hemolytic anemias are implicated to be associated with PAH. In this study we investigated the role of free hemin on endothelial barrier integrity and studied mechanisms of hemin-mediated intracellular signaling.
METHODS: Human Lung Micro Vascular Endothelial Cells (HLMVEC) (Sciencell) were cultured using endothelium media specific for HLMVECs and 10% FBS (Sciencell). Cells were used from passages 3-8. All experiments were performed on 100% confluent cells. The effect of free hemin on HLMVEC barrier function was assessed using Transendothelial electrical resistance (TEER) measurements. Junction proteins after hemin treatment were analyzed by either confocal microscopy or western blotting. Mitochondrial respiration and glycolytic rates were analyzed using Seahorse Extracellular Flux Analyzer (Agilent).
RESULTS: Hemin induced a rapid drop in the EC barrier integrity which correlated with the dose. We used 50μM hemin as the highest dose which is an accepted physiological dose. Sulfasalazine, an inhibitor of Heme Carrier Protein-1, prevented the hemin induced decline in the barrier function, indicating that intracellular levels of hemin affect the barrier integrity. Further investigation into the barrier proteins revealed that hemin primarily affects the tight junction proteins. The levels of zona occludens-1, claudin-1, and claudin-5 were significantly attenuated after hemin exposure. The levels of heat shock protein 27 (HSP27), which is involved in the regulation of the endothelial cytoskeleton and junction, were altered after hemin treatment. Hemin caused a decline in glycolysis within 15 min of treatment and mitochondrial respiration was decreased after 3h. Interestingly, we observed restoration of initial glycolytic rate at 24h post hemin treatment.
CONCLUSIONS: Hemin disrupts endothelial barrier function through disruption of tight junction and possibly involves derangement of HSP27 dependent cytoskeleton organization. Hemin affects glycolysis and mitochondrial function to cause an altered metabolic state of the endothelium. Further studies are needed to understand the mechanistic basis of hemin induced endothelial barrier disruption. Our studies highlight the previously under-appreciated role of hemin in the development of endothelial dysfunction which could potentially lead to the development of PAH.
Funding: ALA Biomedical Research Award, RG 415350, Arizona Area Health and Education Centers Program Grant RG 2017-11, AHA 14SDG20480354 and NHLBI R01 HL133085