Proeryptotic Activity of 4-Hydroxynonenal: A New Potential Physiopathological Role for Lipid Peroxidation Products

Eryptosis is a physiological, apoptosis-like death of injured erythrocytes crucial to prevent premature haemolysis and the pathological sequalae generated by cell-free haemoglobin. When dysregulated, the process is associated to several inflammatory-based pathologies. 4-Hydroxy-trans-2-nonenal (HNE) is an endogenous signalling molecule at physiological levels and, at higher concentrations, is involved in the pathogenesis of several inflammatory-based diseases. This work evaluated whether HNE could induce eryptosis in human erythrocytes.

Measurements of phosphatidylserine, cell volume, intracellular oxidants, Ca⁺⁺, glutathione, ICAM-1, and ceramide were assessed by flow cytometry. Scanning electron microscopy evaluated morphological alterations of erythrocytes. Western blotting assessed caspases. PGE₂ was measured by ELISA. Adhesion of erythrocytes on endothelial cells was evaluated by gravity adherence assay.

HNE in the concentration range between 10-100 µM induces eryptosis, morphological alterations correlated to caspase-3 activation, and increased Ca⁺⁺ levels. The process is not mediated by redox-dependent mechanisms; rather, it strongly depends on PGE₂ and ceramide. Interestingly, HNE induces significant increase of erythrocytes adhesion to endothelial cells (ECs) that are in turn dysfunctionated as evident by overexpression of ICAM-1.

Our results unveil a new physiopathological role for HNE, provide mechanistic details of the HNE-induced eryptosis, and suggest a novel mechanism through which HNE could exert pro-inflammatory effects.