Primary role of superoxide anion generation in the cascade of events leading to endothelial dysfunction and damage in high glucose treated HUVEC

Abstract

Aim

The aim of the study was to elucidate the chain of events leading to oxidative damage in endothelial cells exposed to high glucose.

Method

The nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH₄), the peroxynitrite decomposition catalyst FP15, the inhibitor of mitochondrial complex II thenoyltrifluoroacetone (TTFA) and the antioxidant superoxide dismutase (SOD) mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were individually added to human umbilical vein endothelial cells (HUVEC) cultured in high glucose. This study was designed to establish the possible sequence of action of NOS, peroxynitrite and superoxide anion in the oxidative damage cascade.

Results

We found that in high glucose, nitrotyrosine, 8OHdG, NO (+40%) and O₂⁻ (+300%) production, eNOS and caspase-3 expression increased, while Bcl-2 expression decreased. MnTBAP and TTFA were able to normalize all the parameters assayed. FP15 caused an increase in NO production, did not interfere with eNOS expression and O₂⁻ generation, but was able to reduce apoptosis and to normalize nitrotyrosine and 8OHdG formation. BH₄ enrichment was able to reduce O₂⁻ generation, nitrotyrosine and 8OHdG formation and apoptosis. The addition of this cofactor did not affect eNOS expression, but increased NO formation, more than FP15.

Conclusion

These data show the starting role of superoxide anion generated at mitochondrial level in the cascade of events leading to hyperglycemia generated apoptosis.

Introduction

There is growing evidence that oxidative stress, i.e. the imbalance between free radical production and antioxidant defenses, is involved in the pathogenesis of vascular disease in diabetes [1]. It is well known that the pathologic pathway is facilitated by a loss of nitric oxide (NO) bioactivity, a simple molecule with a central role in maintaining vascular homeostasis [2]. NO is generated in the vascular wall by the endothelial isoform of NOS (eNOS), which oxidizes l-arginine to l-citrulline using molecular oxygen. Tetrahydrobiopterin is a cofactor essential for the catalytic activity of nitric oxide synthase (NOS); it has important effects on the structure of the enzyme, including the ability to shift its heme iron to a high spin state, to increase arginine binding, and to stabilize the active dimeric form of the enzyme [3]. In fact, in the early 1990s biochemical studies demonstrated that decreased availability of tetrahydrobiopterin can modify the activity of this enzyme, leading to a phenomenon called the “uncoupling of NOS”. When it happens, electron flow from the reductase domain to the oxygenase domain of the enzyme is diverted to molecular oxygen rather than to l-arginine, with subsequent formation of superoxide anions and hydrogen peroxide rather than NO [4], [5]. Beneficial effects of tetrahydrobiopterin supplementation on vascular endothelial function in the setting of diabetes mellitus have been shown in experimental and clinical studies [6], [7].

Cosentino et al. demonstrated that in human aortic endothelial cells, prolonged exposure to high glucose concentrations increases eNOS gene expression and protein expression, a phenomenon accompanied by increased production of NO and by a more marked increase of O₂⁻, suggesting a possible uncoupling state of the enzyme [8]. So, at the same time, due to the high glucose presence, there is a focused and a concentrated production of NO and O₂⁻, molecules that react to produce peroxynitrite, a potent, long-living oxidant [9]. Detrimental effects of peroxynitrite anion are many and various. It interacts with BH₄, oxidizing it in in vitro conditions [10], but peroxynitrite can also inhibit electron transport at the mitochondrial level, oxidize sulfhydryl groups in proteins, initiate lipid peroxidation without the requirement of transition metals, nitrate amino acids, affecting many signal transduction pathways [11], and it is a potent generator of DNA strand breaks [12]. The oxidized nucleoside 8-hydroxydeoxyguanosine (8OHdG) is known as a sensitive biomarker for the DNA damage [13], [14]; increased levels of 8OHdG have been found in the kidneys of diabetic rats and also in the tissues or body fluids of diabetic patients [12], [15], [16], and an augmented production of NT has been demonstrated in both in vivo and in vitro [17], [18].

But the malfunctioning of the eNOS enzyme does not seem to completely account for the high glucose induced oxidative stress inside the cell. Nishikawa et al. have recently shown that in an abnormally high glucose environment, like the one established by diabetes, there is an increased production of superoxide (O₂⁻) at the mitochondrial electron transport chain level [19], a site that many authors consider the hot spot of oxidative stress generation for the diabetic condition.

The aim of this study was to better understand the sequence of the events in the chain that leads to oxidative damage in high glucose treated endothelial cells. For this purpose we added, individually, to human umbilical vein endothelial cells (HUVEC) cultured in high glucose medium, the NOS cofactor tetrahydrobiopterin (BH₄), the novel peroxynitrite decomposition catalyst FP15 [20], [21], the inhibitor of mitochondrial complex II thenoyltrifluoroacetone (TTFA) and the SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). Measuring the oxidative stress markers NT and 8OHdG, NO and O₂⁻ production, eNOS expression and apoptosis levels through Bcl-2 and caspase-3 activity and expression, we aimed to evaluate the possible steps at which these compounds work, therefore establishing the possible sequence of action of NOS, peroxynitrite and superoxide anion.