Prevention of reactive oxygen species-induced oxidative stress in human microvascular endothelial cells by green tea polyphenol

Abstract

The potential protective roles played by green tea polyphenol (GTP) against the injurious effects of reactive oxygen species in human microvascular endothelial cells (HUMVECs) were investigated. Oxidative stress was induced in cultured HUMVECs, either by adding 10 mM H₂O₂ or by the action of 10 U/l xanthine oxidase (XO) in the presence of xanthine (250 μM). Both treatments produced a significant reduction (to 68% and 71%, respectively) in HUMVEC viability, as assessed by fluorescence double staining method followed by flow cytometric analysis. On the microscopic observations, the morphological changes and necrotic detachment were appreciably induced by both treatments. The H₂O₂-induced alterations were completely prevented by pre-incubating the ECs with 10 μg/ml GTP for 1 h. When the oxidative stress was induced by XO, the cell viability and morphology were also significantly maintained at the same GTP concentration. These results demonstrate that GTP can act as a biological antioxidant in a cell culture experimental model and prevent oxidative stress-induced cytotoxicity in ECs.

Introduction

Reactive oxygen species (ROS) induce a number of molecular alterations in cellular components, which lead to changes in cell morphology, viability and function (Halliwell, 1994). Increased oxidative stress impairs endothelial function and is thought to mediate vascular diseases. Several pathological conditions increase the production of ROS in the vascular wall, including hypercholesterolemia (Stokes et al., 2002), diabetes (Basta et al., 2004) and hypertension (Touyz and Schiffrin, 2004), and these conditions are associated with endothelial dysfunction and cardiovascular disease. ROS generators such as H₂O₂ can be produced by activated neutrophils and macrophages, and one of the major biological targets of H₂O₂ are endothelial cells (ECs), in which it increases the liberation of nitric oxide (Rubanyi and Vanhoutte, 1986). Moreover, H₂O₂ is an important mediator of acute oxidative injury to vascular ECs (Ager and Gordon, 1984). Similarly, several reports have shown a marked decrease in the number of antioxidant defense mechanisms in atherosclerosis (Tesfamariam, 1994). One possible way of preventing ROS-mediated cellular injury is to augment the endogenous oxidative defenses by the dietary intake of antioxidants such as vitamins A, C and E (Block et al., 1992, Di Mascio et al., 1991). Recently, a great deal of attention has been focused on a variety of non-vitamin antioxidants such as phenolic compounds, which may also contribute to the cellular antioxidative defense mechanisms, and which can be found in many plant species, such as green tea, fruits and vegetables (Decker, 1995, Manna et al., 1997, Gupta et al., 2000). These polyphenols, including catechin and its derivatives, resveratrol, curcumin, etc., have attracted attention as a functional food with various bioactivities, for example anticancer, antimutagenic, antimicrobial and antiviral activities (Nakayama et al., 1993, Hsieh and Wu, 1999, Pal et al., 2001). It has been reported that Morin, a bioactive pigment found in yellow Brazil wood, could prolong the survival time of cells from the human circulatory system (ventricular myocytes, saphenous vein ECs and erythrocytes) in the presence of ROS generators in vitro (Wu et al., 1994). In this study, the effect of ROS injury on the viability of human microvascular ECs (HUMVECs) and the possible protective role of green tea polyphenols (GTPs) against ROS were investigated. It is well known that vascular ECs play an important role in physiological hemostasis and blood vessel permeability and that they express immune-related functions in monocytes and macrophages. The viability of ECs is essential in the prediction of the post-operative function and durability of a transplanted vessel (Miossec et al., 1986). Therefore, HUMVECs are a suitable model for evaluating the physiological response of ECs to oxidative stress.