Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a PI3K/Akt-dependent pathway in human endothelial cells

Abstract

Aims

Advanced glycation end products (AGEs) trigger an oxidative reaction which then accelerates endothelial cell apoptosis; this is a critical event in the process of diabetic vascular complications. We previously demonstrated that hepatocyte growth factor (HGF) protects human endothelial cells against AGE-induced injury. The present study was designed to investigate the possible involvement of MAPK and PI3K/Akt signaling in the action of HGF.

Main methods

HUVECs were treated with AGEs in the presence or absence of HGF. For detection of apoptosis, the morphological Acridine Orange staining, flow cytometry, and caspase-3 activity assay were used. Generation of reactive oxygen species (ROS) and the change in mitochondrial membrane potential were measured using flow cytometry and fluorescence immune analysis. The activation of MAPK and Akt was assayed by Western blot.

Key findings

HGF exerted its prosurvival effect by inhibiting the overproduction of intracellular ROS and the depolarization of mitochondrial membrane, induced by AGEs. HGF-induced survival correlated with Akt activity and was inhibited by the specific PI3K inhibitor. ERK also was activated by HGF and rescued cells from apoptosis, although the cytoprotective effect was less marked than for PI3K/Akt. HGF-mediated survival was independent of JNK and p38MAPK pathways. Furthermore, blocking the PI3K and Akt activities with PI3K inhibitors or transfection of HUVECs with the dominant-negative p85 or Akt effectively abolished the inhibition of the intracellular ROS production and mitochondrial damage.

Significance

Our studies suggest that HGF, via PI3K/Akt signaling, prevents AGE-induced apoptosis and oxidative stress through the inhibition of mitochondrial damage in HUVECs.

Introduction

Accumulating evidence suggests that endothelial dysfunction and injury present early steps in the pathophysiology of vascular complications in diabetes mellitus. The formation and accumulation of advanced glycation end products (AGEs) in various tissues are known to accelerate in diabetes mellitus (Bierhaus et al., 1998, Yamagishi et al., 2003). Increased AGEs have been implicated as one of the major factors responsible for the pathogenesis of various diabetic vascular complications (Ahmed 2005). Evidence suggests that, in endothelial cells, AGEs induce adverse effects on mitochondrial function, with elevated production of reactive oxygen species (ROS), and consequently increased oxidative stress leading to cellular dysfunction and even cell death. AGEs increase the formation of intracellular ROS, NO, and ceramides as well as the MAPK cascade, which, through intermediate molecules, activates different targets including transcription factors such as NF-κB and activator protein 1 (AP-1) (Cellek, 2004, Denis et al., 2002, Ramasamy et al., 2005, Alikhani et al., 2007). Thus prevention of AGE-triggered endothelial cell injury may have important implications for preventing diabetes-associated vascular complications.

We have previously shown that hepatocyte growth factor (HGF) protects human endothelial cells against AGE-induced apoptosis (Zhou et al. 2006). HGF can act as a direct antioxidant that scavenges or quenches oxygen free radicals intracellularly to block ROS-mediated programmed cell death. HGF protects adult cardiac myocytes against apoptosis induced by hydrogen peroxide, serum deprivation, and chemotherapeutic agents inducing oxidative stress. Upon HGF binding to its receptor c-Met, specific signaling pathways are activated, including PI3K-dependent Akt phosphorylation and MAPK pathways. Different studies suggest that the anti-apoptotic effects of HGF occur through several different pathways. HGF protects podocytes from cyclosporin A-induced apoptosis by regulation of Bcl-xL in a PI3K dependent but MEK-1 independent manner (Fornoni et al. 2001). HGF inhibits FFA-induced oxidative stress and apoptosis in RIN5mF cells by Bcl-2 and PI3K/Akt pathways (Santangelo et al. 2007). It has also been reported that in lung endothelial cells, the inhibition of hypoxia/reoxygenation-induced cell death by HGF primarily involved p38 MAPK and in part Akt-dependent pathways, but not ERK1/2 (Wang et al. 2004). Other studies demonstrated that the prosurvival effect of HGF is dependent on activation of PI3K and ERK1/2 signal transduction pathway in various types of cells, but not the p38 MAPK pathway (Ma et al., 2002, Thompson et al., 2004, He et al., 2008). HGF has multifunctional activities and exerts its prosurvival action by different mechanisms depending on the various apoptotic stimuli and in different cell types. We demonstrated that the upregulation of Bcl-2, NF-κB expression and inhibition of caspase-3 and -9 activation by HGF could be responsible for its protection against AGE-induced apoptosis in the endothelial cells. Recent reports suggest that Bcl-2 might prevent apoptosis by regulating the cellular antioxidant defense mechanisms, thus acting as free radical scavenger. However, the precise molecular mechanisms by which HGF inhibits AGE-induced injury in endothelial cells have not yet been thoroughly elucidated. The purpose of our current study was to determine the intracellular signaling in anti-apoptotic action of HGF from AGEs in human endothelial cells.