Adenovirus-mediated overexpression of catalase attenuates oxLDL-induced apoptosis in human aortic endothelial cells via AP-1 and C-Jun N-terminal kinase/extracellular signal-regulated kinase mitogen-activated protein kinase pathways

Abstract

In a variety of vascular disorders, endothelial cells (ECs) are exposed to high levels of reactive oxygen species (ROS) generated intercellularly. Recently, several anti-oxidants, including catalase, have been suggested to be cytoprotective against the development of atherosclerosis. The object of this study was to investigate whether adenovirus-mediated gene transfer of catalase in ECs can attenuate ROS production and cell apoptosis under oxidized low density lipoprotein (oxLDL) stimulation. Adenovirus-mediated gene transfer of human catalase gene (Ad-Cat) resulted in a high level of catalase overexpression in human arterial EC (HAEC), which manifested a time-dependent increase in cell viability under the exposure of oxLDL and decreased oxLDL-induced apoptosis. Phosphorylation studies of ERK1/2, JNK, and p38, three subgroups of mitogen activator protein kinase demonstrated that catalase overexpression suppressed JNK phosphorylation and increased ERK1/2 phosphorylation. NF-κB and AP-1 were induced after the exposure of HAECs to oxLDL. While catalase overexpression was found to inactivate AP-1, it had no effect on NF-κB activity. These results provide the evidence that overexpression of catalase in ECs attenuates ROS production and cell apoptosis under oxLDL stimulation. The protective effect is mediated through the downregulation of JNK and the upregulation of ERK1/2 phosphorylation as well as AP-1 inactivation. This observation supports the feasibility of catalase gene transfer to human endothelium to protect against oxidant injury.

Introduction

Disruption of the delicate balance between pro-oxidants and anti-oxidants has been implicated in the pathophysiology of cardiovascular disorders [1]. A plethora of information suggests that oxidants, such as hydrogen peroxides (H₂O₂) and lipid peroxide, induce a number of proatherogenic changes in vascular cells that are present in the atherosclerotic artery [2]. Most of these effects could be prevented by the addition of extracellular anti-oxidants, suggesting further propagation of oxidation [3]. Studies based on anti-oxidant gene expression to examine the effects of oxidants on cells also corroborate the premise that intracellular oxidative stress might play an important role in the transduction of oxidative stress from external sources to intracellular sites [4]. Previously, Shingu et al. [5] showed that endothelial cells (ECs) and smooth muscle cells (SMCs) have very low levels of catalase activity and, therefore, are more susceptible to damage by H₂O₂. In contrast, transient overexpression of catalase in human umbilical vein ECs has been found to confer protection against H₂O₂-mediated oxidative stress [6]. Moreover, overexpression of human catalase gene has been shown to decrease oxidized lipid-induced cytotoxicity in vascular SMCs [7]. These results suggest that catalase is a key enzyme in the protection of cells against oxidative injury. Several studies have attempted to overexpress the catalase enzyme in various cell types and have examined its effect in preventing oxidation-related damage [6], [7]. However, the regulatory mechanism of catalase protein on human ECs exposed to oxidative stress still remains unclear.

Low density lipoprotein (LDL) is among the major risk factors and predictors for the development of atherosclerosis. Manifested as an increase in free radicals, oxidized LDL (oxLDL) perturbs endothelial functions via several routes, including a decrease in NO production, an increase in plasminogen activator inhibitor-1, recruitment of monocytes, and through the promotion of cell apoptosis [8], [9]. It is thought that oxLDL-induced signal transmission requires the binding to specific cellular receptors to activate and stimulate a wide spectrum of host responsive systems [10]. This requires the activation of multiple signaling molecules in transduction pathways, for example protein-tyrosine kinase (PTK), oxLDL receptor-associated serine/threonine kinase, Ras, Raf-1, IκB kinase, MEK, mitogen-activated protein kinases (MAPKs), etc., [11], [12], [13]. These molecules may converge or diverge and often have “cross-talk” properties, which result in a complicated signaling network that exert mutual influence. Subsequently, the signals further transduce to downstream pathways and activate numerous transcriptional factors, including AP-1, NF-κB, and ATF-2 [14], which trigger a large amount of genes encoded for inflammatory mediators and cytokines [10]. These elicited that cytokines are believed to be responsible for cell proliferation, differentiation, immunoregulation, and cytotoxicity [11]. Currently, the effects of oxLDL and catalase overexpression on the signaling pathways and the events involved in the transduction of programmed death signal into the nucleus of human arterial ECs (HAECs) have not been well-defined.

In this study, adenovirus-mediated gene transfer was used to overexpress catalase in HAECs to obviate concerns regarding the purity of the enzyme and preparation or fluctuations in catalase protein delivery. Our results demonstrate that HAECs enriched in catalase are resistant to the damaging effects of oxLDL. The AP-1 and JNK/ERK MAPK pathway is the major regulatory mechanism leading to the protective effects of catalase overexpression.