The Effect and Mechanism of Action of Aged Packed Red Blood Cell-Derived Microparticles on Pulmonary Microthrombus Formation

Abstract

Background: Aged packed red blood cells (pRBCs) cause pulmonary thrombosis, during which P-Selectin and peroxisome proliferator-activated receptor-gamma (PPARγ) are abnormally expressed. This study explored the effect and mechanism of action of aged pRBC on pulmonary microthrombus formation. Methods: Microparticles were collected from murine pRBCs and cultured with murine lung microvascular endothelial cells (MLECs), which had been pretreated with pioglitazone (a PPARγ agonist) or not. P-selection and PPARγ levels in MLECs were determined using ELISA (enzyme linked immunosorbent assay), qRT-PCR (quantitative reverse transcription polymerase chain reaction) or western blot. MLEC viability and apoptosis were assessed using CCK-8 (cell counting kit-8) assay and flow cytometry. Male C57BL/6 mice were injected with pRBC-derived microparticles labeled by 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Pulmonary micro-thrombosis was checked for with hematoxylin-eosin staining and immunohistochemistry, and the microparticles distribution was checked for with a fluorescence microscope. Results: pRBC-derived microparticle-processed MLECs showed significantly upregulated P-selection expression and significantly downregulated PPARγ, with significantly increased viability and significantly inhibited apoptosis. Pioglitazone reversed the effect of pRBC-derived microparticle on the PPARγ and P-selection expressions, viability and apoptosis in MLECs. Injection of pRBC-derived microparticles significantly increased the number of microthrombi in each high-powered field and significantly promoted the aggregation of platelets and the microparticles in murine lung, but injection of pioglitazone significantly alleviated pRBC-derived microparticle-delivered effects on murine lung. Conclusions: Aged pRBC-derived microparticles significantly promoted pulmonary micro-thrombosis through PPARγ inhibition-requiring P-selectin upregulation.