Astaxanthin alleviates oxidative stress insults-related derangements in human vascular endothelial cells exposed to glucose fluctuations

Abstract

Glycemic fluctuations may play a critical role in the pathogenesis of diabetic complications, such as cardiovascular disease. We investigated whether the oxycarotenoid astaxanthin can reduce the detrimental effects of fluctuating glucose on vascular endothelial cells. Human umbilical venous endothelial cells were incubated for 3 days in media containing 5.5 mM glucose, 22 mM glucose, or 5.5 mM glucose alternating with 22 mM glucose in the absence or presence of astaxanthin or N-acetyl-L-cysteine (NAC). Constant high glucose increased reactive oxygen species (ROS) generation, but such an effect was more pronounced in fluctuating glucose. This was associated with up-regulated p22^phox expression and down-regulated peroxisome proliferator activated receptor-γ coactivator (PGC-1α) expression. Astaxanthin inhibited ROS generation, p22^phox up-regulation, and PGC-1α down-regulation by the stimuli of glucose fluctuation. Fluctuating glucose, but not constant high glucose, significantly decreased the endothelial nitric oxide synthase (eNOS) phosphorylation level at Ser-1177 without affecting total eNOS expression, which was prevented by astaxanthin as well as by the anti-oxidant NAC. Transferase-mediated dUTP nick end labeling (TUNEL) showed increased cell apoptosis in fluctuating glucose. Glucose fluctuation also resulted in up-regulating gene expression of pro-inflammatory mediators, interleukin-6 and intercellular adhesion molecule-1. These adverse changes were subdued by astaxanthin. The phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 were significantly increased by glucose fluctuations, and astaxanthin significantly inhibited the increase in JNK and p38 phosphorylation. Taken together, our results suggest that astaxanthin can protect vascular endothelial cells against glucose fluctuation by reducing ROS generation.

Introduction

Individuals with diabetes mellitus are at an increased risk for cardiovascular disease which can eventually be disabling or even life-threatening [1]. Chronic hyperglycemia, which is contributed by fasting and postprandial hyperglycemia, plays a central role in developing diabetic cardiovascular complications, and the benefit of glycemic control in reducing the risk for cardiovascular disease has been established [2]. However, the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial has revealed that intensive glycemic control can increase all-cause mortality more than standard therapy [3], and the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) trial has demonstrated that intensive glucose-lowering strategy has no significant effect in reducing macrovascular disease [4]. In the meantime, fluctuations in blood glucose levels that can potentially occur in inadequate glycemic management with insulin or oral antidiabetics have also been identified as a risk for cardiovascular events [5], [6]. Furthermore, diabetic individuals may experience fluctuations in blood glucose levels with a habitual diet that is high in concentrated sweets [7].

Vascular endothelial cells have emerged as a key regulator of vascular homeostasis, in that they have not merely a barrier function but also produce and secrete mediators that regulate a wide range of physiological and pathological processes, including vasomotor tone, angiogenesis, inflammation, and coagulation [8], [9], [10]. A growing body of substantial evidence demonstrates that endothelial dysfunction underlies the basic pathophysiology of diabetic vascular disorders [10], [11], [12]. In clinical practice, vascular endothelial function has been portrayed to be impaired by fluctuations in blood glucose levels in type 2 diabetic patients [13]. Intriguingly, intermittent high glucose appears to be more harmful than constant high glucose medium for human vascular endothelial cells. It has been shown that periodic exposure of human vascular endothelial cells to high glucose results in elevated apoptosis [14], [15], [16], up-regulated expression of adhesion molecules [17], [18], and accelerated senescence [19], [20].

Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione) is a non-provitamin A carotenoid classified as a xanthophyll and is found in high amounts in the red pigment of crustacean shells (e.g., crabs, shrimp), salmon, trout, and asteroidean [21], [22]. It has been demonstrated that astaxanthin displays a wide variety of biological activities, including anti-oxidative, anticancer, and anti-inflammatory effects [22]. Given such biological features of this naturally occurring carotenoid, astaxanthin may serve as a vascular endothelial protecting agent. Recent studies using animal models of diabetes and hypertension suggest that astaxanthin can alleviate endothelial dysfunction under these pathological conditions [23], [24].

In the present study, we tested the hypothesis that astaxanthin can reduce the detrimental effects of fluctuating glucose on vascular endothelial cells. We observed that fluctuating glucose led to highly increased generation of reactive oxygen species (ROS), decreased basal activity of endothelial nitric oxide synthase (eNOS), increased endothelial cell apoptosis, and up-regulated gene expression of pro-inflammatory mediators in human vascular endothelial cells. We found that treatment with astaxanthin protected endothelial cells from the detrimental changes by glucose fluctuation.