Abstract 508: Heme-Loaded Microparticles May Connect Subclinical Intravascular Hemolysis to Endothelial Cell Injury in Type 2 Diabetes

Abstract

Rational: Type 2 Diabetes induces heavy hemoglobin glycation and discrete red blood cell (RBC) fragility. Intravascular hemolysis in type 2 diabetes is not a paramount clinical feature of the disease and it receives little attention. But hyperglycemia favors hemolysis and diabetes is associated to raised circulating levels of submicrometric cell membrane fragments from RBCs, called microparticles (MPs). We hypothesized that circulating diabetic MPs could be loaded with heme and participate in oxidative vascular injury. Material and Methods: A type 2 diabetic blood collection was assembled. Diabetic and healthy RBCs were separated from platelet-free plasma (PFP) by centrifugation. We purified MPs from native PFP, or from RBCs made to vesiculate in vitro with the thrombospondin-1-derived peptide 4N1-1. We characterized the size of MPs by dynamic light scattering, and quantified the heme contained in MPs by spectrophotometry, before and after MP depletion by ultracentrifugation. We exposed vascular endothelial cells to these different MP preparations and determined radical oxygen species (ROS) production with a fluorescent probe, as well as apoptosis by fluorescence microscopy and flow cytometry. Results: Purified diabetic and healthy RBCs released MPs expressing surface phosphatidylserine. MPs from type 2 diabetic RBCs were generally larger than healthy RBC MPs and contained significantly more heme. In cultured endothelial cells, MPs from diabetic RBCs stimulated the production of ROS in a dose- and time-dependant fashion, at concentrations observed in diabetic plasma. In contrast, healthy RBC MPs and hemoglobin or heme alone had little effects at such concentrations. Moreover, overproduction of ROS was followed by endothelial apoptosis after 24h, revealed by pyknotic nuclei and degraded chromatine, as well as reduced DNA contents by flow cytometry. Conclusion: Diabetic RBC can release MPs that are loaded with heme and trigger oxydative cell injury and apoptosis in cultured endothelial cells. These results identify for the first time the fact that heme-loaded MPs vector oxidative cell injury in type 2 diabetes. Diabetic RBC MPs may thus form a novel bridge between RBC glycation, sub-clinical intravascular hemolysis and diabetic vasculopathy.