L1 protects endothelial cells from iron induced oxidative stress and mitochondrial injury

Abstract

Introduction: Iron-overload induced cardiac disease is an important cause of morbidity and mortality in patients with b-thalassaemia major. Arterial stiffening and endothelial dysfunction is another major concern in this group of patients (Cheung et al, Circulation 2002). Excessive iron load is hence associated with endothelial dysfunction, while L1, an iron chelator with antioxidant effect, has been shown to improve endothelial function in these patients clinically. Therefore in this study, we investigated how excessive iron induced endothelial damage. We further explored the protective effect of iron chelator L1 on iron-overloaded endothelial cells.

Methods and results: Using human umbilical vein endothelial cells (HUVECs), we showed that iron-overload induced endothelial microparticles (MPs) generation in a dose dependent manner. Iron increased reactive oxygen species (ROS) production (75-300 mM) (n=3) and increased calcium influx into mitochondria [Ca2+]m (300 mM, 24 hrs) in HUVECs as evidenced by increased rhod-2 AM fluorescent intensity using flow cytometry. These led to loss of mitochondrial membrane potential (DYm) (300 mM, 24 hrs) as shown by JC-1 assay (n=3). Apoptotic cells were found to be significantly increased under iron treatment (300 mM, 48 hrs) by annexin V/PI assay (n=3). The expression of caspase dependent apoptotic marker active caspase-3 significantly increased in iron treated cells (n=4). Co-incubation with L1 (10 mM) showed an inhibitory effect on iron-induced generation of EMPs by HUVECs (n=3). L1 (100 mM) decreased iron-induced calcium influx into mitochondria and L1 (10 mM) delayed DYm derangement of HUVECs (n=3). Furthermore, L1 (10 mM) protected endothelial cells from iron-induced ROS generation and apoptosis. ROS determination, dot-plot analysis of annexin V/PI staining and active caspase-3 assay demonstrated that L1 significantly reduced ROS generation, apoptotic cells and active caspase-3 expression.

Conclusions: Our findings suggest that excessive iron induces endothelial cells injury via increased oxidative stress, increased [Ca2+]m, loss of mitochondrial membrane potential and apoptosis. L1 could protect endothelial cells from the above phenomenon and may have therapeutic potential for iron-induced endothelial damage.