Astaxanthin alleviates oxidative stress insults-related derangements in human vascular endothelial cells exposed to glucose fluctuations
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.
Section snippets
Cell culture
Human umbilical venous endothelial cells (HUVECs) were purchased from Promocell (Heidelberg, Germany) and Lonza (Basal, Switzerland) and were cultured in endothelial cell growth medium-2 (Takara Bio, Otsu, Japan) until the start of the experiment. HUVECs were cultured at 37 °C under a humidified atmosphere containing 5% CO2 and 95% air. According to our previous studies [25], [26], four- to seven-passage subconfluent cells were used in the experiments. Cells were harvested at 70–80% confluence
Effect of fluctuating glucose on ROS generation
When intracellular ROS were visualized using the fluorescence dye CM-H2DCFPA, exposure of HUVECs to constant high glucose (22 mM) for 3 days led to a significant increase in intracellular fluorescence (Fig. 1A). The increase in ROS-induced intracellular fluorescence was much more pronounced under the fluctuating-glucose condition (Fig. 1A). The presence of NAC (10 mM), a widely used thiol-containing antioxidant, strikingly eliminated ROS generation in endothelial cells exposed to intermittent high
Discussion
ROS are chemically reactive molecules containing oxygen that are generated as natural byproducts of the normal metabolism of oxygen. Accumulating evidence suggests that ROS play essential roles in cell signaling and regulation [36]. However, an excess production or decreased scavenging of ROS has been implicated in the pathogenesis of diverse diseases including diabetes. Oxidative stress plays a pivotal role in endothelial dysfunction that is closely associated with diabetic vascular disease
Disclosure of conflicts
The authors declare no potential conflicts of interest.
Acknowledgments
This work was in part supported by the Naito Foundation (No. 4288) (T.S.). We are grateful to the financial support of the Egyptian Government (KH381) for Lobna A. Abedelzaher during the research work. We are thankful to Dr. Natsumi Mizuno, Laboratory of Pharmacology of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, for providing needs of this project. We also thank Fuji Chemical Industries for providing astaxanthin used in this study. Fuji Chemical
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