Abstract 11559: Acute and Chronic Effect of Heme on Vascular Function and Pulmonary Hypertension

Introduction: Patients with hemolytic anemia are at high risk of developing lung disease. We recently reported that free heme in circulation causes acute lung endothelial barrier dysfunction via activation of MKK3/p38 signaling. However, we did not evaluate the effects of chronic heme infusion in the lungs. Hypothesis: Chronic free heme infusion will cause sustained activation of the MKK3/p38 pathway leading to inflammation and vascular remodeling in the lungs. Methods: Mitogen-activated protein kinase kinase-3 knockout (MKK3-KO) mice were used for this study. Acute group mice received heme 250μM for 24-hours by i.v injection as a single dose, and the chronic group mice received 2-weeks of continuous heme i.v injection using an osmotic minipump. Human pulmonary artery endothelial cells (HPAEC) were studied in endothelial cell dysfunction experiments. Results: Heme treatment (50μM) disrupts HPAEC endothelial barrier, tube formation and decreases HPAEC migration capacity suggesting direct heme involvement in the endothelial dysfunction. Chronic heme exposure of WT mice (250uM, 2wks) increased right ventricular (RV) systolic pressure (SP) (31.89±0.88mmHg), RV-hypertrophy (0.38±0.02), and pulmonary vascular remodeling (18.47±1.15 μm) compared to the MKK3-KO group (RVSP-29.33±0.69mmHg, Fulton index-0.33±0.01, and wall thickness-13.69±1.13μm, p<0.05). Loss of tight junction proteins and activation of MKK3/p38/HSP27 axis was noticed in heme-treated WT. MKK3-KO significantly attenuated downstream targets of p38 such as Akt, STAT3, and ERK1/2. Furthermore, the MKK3-KO attenuated endothelial barrier dysfunction by maintaining the tight-junction protein, zona occludens-1 and reducing lung inflammatory cell infiltration. Cytokine multiplex analysis of plasma exhibited an inflammatory cytokine storm with a surge of 21-cytokines in WT mice, but the MKK3-KO mice were substantially protected. This includes IFN-g (1502 vs. 113 Fold Control (FC)), IL-1b (6.8 vs. 2.2 FC), IL-6 (221 vs. 6.9 FC), etc. Conclusion: Our findings suggest that free-heme triggers MKK3/p38 cascade activation resulting in the cytokines storm, endothelial barrier dysfunction, and proliferative signaling activation, all contributing to PH manifestation.