The functional analysis of iron induced microparticles derived from endothelial cells and macrophages

Abstract

Iron is an essential element involved in a wide variety of metabolic processes in the body. Accumulation of excessive iron in tissues causes overproduction of superoxide (O2•-) and hydroxyl (OH-) radicals via Fenton and Haber-Weis reactions. Such increased production of reactive oxygen species (ROS) may lead to oxidative stress resulting in cell damage. Clinically, such conditions of iron overload can be found in thalassemia patients with continuous blood transfusions and increased intestinal iron absorption secondary to anemia. It is well known that that iron-overload-induced myocardial damage and systemic arterial dysfunction secondary to endothelial cell damage is a major long term complication in thalassemia patients. When cells undergo activation, injury or apoptosis, cell membrane blebs are formed. Eventually such blebs are evolved into microparticles (MPs) and released. The mechanism of biogenesis of endothelial microparticles (EMPs) by iron overload remains unknown. I found that iron increased the production of ROS and induced mitochondrial damage, as shown by increased calcium levels in mitochondria [〖Ca〗^(2+)]m and loss of mitochondrial membrane potential (ΔΨm). Apoptotic cells were significantly increased under iron treatment. This was associated with increase in activated caspase-3 suggesting that apoptosis was induced via a mitochondria-mediated caspase dependent pathway. My findings suggest that iron overload induced endothelial cells to release EMPs and such release is closely associated with increased oxidative stress, increased [〖Ca〗^(2+)]m, loss of mitochondrial membrane potential and apoptosis of endothelial cells. MPs can serve as vehicle for biological soluble factors involving in the regulation of a wide spectrum of biological functions. Iron overload enhanced tissue factor expression and induced tissue factor-bearing EMPs released from endothelial cells. Furthermore, iron-induced EMPs contain various upregulated miRNA which are associated with cellular senescence, apoptosis, angiogenesis and vascular inflammation. It is interesting that these miRNA-rich EMPs can be taking up by other normal endothelial cell and induced them into apoptosis. It is also interesting that different cells under the same iron overload treatment secrete MPs with different soluble factors which have distinct target cell function. Excessive iron induced MPs releasing from macrophages and they contain tumour necrotic factor- (TNF-). Such TNF- rich MPs enhanced mesenchymal stem cells to differentiate into osteoblasts. This finding explains the possible mechanism of ectopic bone formation at heterotopic ossification site in seriously wounded patients. Finally, I investigated the potential role of interventional agents such as L1, carvedilol in preventing iron-overload-induced cell apoptosis. We found that both L1 and carvedilol inhibited endothelial cell ROS generation, apoptosis, and EMP release. Whether they can prevent iron-overload-induced macrophage secretion of TNF--bearing MPs in the setting of trauma-induced heterotopic ossification remains to be explored. These findings in my project provide new insights to the pathogenesis and potential management of iron-overload-induced thrombosis, cardiovascular disease, and trauma-induced heterotopic ossification.