Iron Overload Damages the Endothelial Mitochondria via the ROS/ADMA/DDAHII/eNOS/NO Pathway.

Dan Liu,Huan He,Qing Zhou,Yang Qiao,Dong Yin,Xuepiao Chen,Ming He,Zhiqing Wang

doi:10.1155/2019/2340392

Abstract

It has been recognized that iron overload may harm the body's health. Vascular endothelial cells (VECs) are one of the main targets of iron overload injury, and the mechanism involved was thought to be related to the excessive generation of reactive oxygen species (ROS). However, the subcellular and temporal characteristics of ROS generation, potential downstream mechanisms, and target organelles in VECs injured by iron overload have not been expounded yet. In this study, we elucidated the abovementioned issues through both in vivo and in vitro experiments. Mice were fed pellet diets that were supplemented with iron for 4 consecutive months. Results showed that the thoracic aortic strips' endothelium-dependent dilation was significantly impaired and associated with inflammatory changes, noticeable under brown TUNEL-positive staining in microscopy analysis. In addition, the serum content of asymmetric dimethylarginine (ADMA) increased, whereas nitric oxide (NO) levels decreased. Furthermore, the dimethylarginine dimethylaminohydrolase II (DDAHII) expression and activity, as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) in aortic tissue, were inhibited. Human umbilical vein endothelial cells were treated with 50 μM iron dextran for 48 hours, after which the cell viability, NO content, DDAHII expression and activity, and phosphorylation of eNOS decreased and lactate dehydrogenase and caspase-3 activity, ADMA content, and apoptotic cells significantly increased. After the addition of L-arginine (L-Arg) or pAD/DDAHII, the abovementioned changes were reversed. By dynamically detecting the changes of ROS generation in the cytoplasm and mitochondria and interfering with different aspects of signaling pathways, we have confirmed for the first time that excessive ROS originates from the cytoplasm and activates the ROS-induced ROS release (RIRR) mechanism, leading to mitochondrial dysfunction. Together, our data suggested that excessive free iron ions produced excess ROS in the cytoplasm. Thus, excess ROS create one vicious circle by activating the ADMA/eNOS/DDAHII/NO pathway and another vicious circle by activation of the RIRR mechanism, which, when combined, induce a ROS burst, resulting in mitochondrial dysfunction and damaged VECs.

Highlights

In recent years, the damage caused by iron overload has attracted increasing attention [1]
We found that Human umbilical vein endothelial cells (HUVECs) was treated with Iron dextran (iron-D) after the addition of 100 μM Eda; intracellular reactive oxygen species (ROS) generation was slowly and persistently increased with the duration of iron-D treatment, but mitochondrial ROS generation was stable at baseline from the beginning of the experiment to 16 hours (see Figure 5(c))
We found that in HUVECs treated with ironD after the addition of pAD/dimethylarginine dimethylaminohydrolase II (DDAHII)-shRNA or pAD/DDAHII and 10 μM l-NAME, a specific inhibitor of endothelial nitric oxide synthase (eNOS), the cell viability decreased and the lactate dehydrogenase (LDH) activity in the culture medium increased (P < 0:01, Supplementary Materials, Figure S6 A-B)

Summary

Introduction

The damage caused by iron overload has attracted increasing attention [1]. Excessive iron intake may cause damage to cells, organs, and even the entire body, thereby leading to variety of diseases, including cardiovascular events. Many clinical studies have shown that the patients of hereditary/hemolytic/hemorrhagic diseases or hemodialysis might suffer from VEC injury caused by either iron overload or iron accumulation [4,5,6,7,8,9,10,11,12,13]. An iron chelator was effective in clinical treatment [14]. Iron dextran can cause serious injury in mice [15, 16] or VECs [17,18,19]

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