Abstract
Diabetes mellitus affects 451 million people worldwide, and people with diabetes are 3-5 times more likely to develop cardiovascular disease. In vascular tissue, mitochondrial function is important for vasoreactivity. Diabetes-mediated generation of excess reactive oxygen species (ROS) may contribute to vascular dysfunction via damage to mitochondria and regulation of endothelial nitric oxide synthase (eNOS). We have identified (–)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS in cells treated with antimycin A (AA, a compound targeting mitochondrial complex III) or high glucose (HG, global perturbation) could be normalized by EPICAT, and correlate with improved mitochondrial dynamics and cellular signaling. Human umbilical vein endothelial cells (HUVEC) were treated with HG, AA, and/or 0.1 or 1.0 μM of EPICAT. Mitochondrial and cellular superoxide, mitochondrial respiration, and cellular signaling upstream of mitochondrial function were assessed. EPICAT at 1.0 μM significantly attenuated mitochondrial superoxide in HG-treated cells. At 0.1 μM, EPICAT nonsignificantly increased mitochondrial respiration, agreeing with previous reports. EPICAT significantly increased complex I expression in AA-treated cells, and 1.0 μM EPICAT significantly decreased mitochondrial complex V expression in HG-treated cells. No significant effects were seen on either AMPK or eNOS expression. Our study suggests that EPICAT is useful in mitigating moderate ROS concentrations from a global perturbation and may modulate mitochondrial complex activity. Our data illustrate that EPICAT acts in the cell in a dose-dependent manner, demonstrating hormesis.
Highlights
Diabetes mellitus (DM) confers an excess risk of cardiovascular disease (CVD), preceded by dysfunction in vascular reactivity [1]
For Western blot and respiration experiments, collagenase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, K-lactobionate, taurine, potassium phosphate, HEPES, digitonin, pyruvate, malic acid, glutamic acid, adenosine diphosphate, succinic acid, oligomycin, carbonyl cyanide 4phenylhydrazone (FCCP), antibody to β-actin, phenylephrine and acetylcholine, trypsin inhibitor, and cytochrome c were procured from Sigma-Aldrich (MO, USA)
Using a comprehensive suite of endpoints to pinpoint EPICAT bioactivity on mitochondrial reactive oxygen species (ROS) and content and function, we found a difference between the response of a mitochondrial poison (AA) versus nutrient
Summary
Diabetes mellitus (DM) confers an excess risk of cardiovascular disease (CVD), preceded by dysfunction in vascular reactivity [1]. Disruptions in redox regulation and elevated ROS are linked to hyperglycemia, dampened antioxidant defenses, insulin resistance, and dysfunctional cellular signaling [2, 3, 5,6,7,8]. Stochiometric approaches to excess ROS alleviation do not consider the important signaling role of ROS in cellular homeostasis [11, 12]. Studies targeting elevated ROS must approach redox from a broad perspective. Mitochondria are a central source of cellular superoxide under normal physiological conditions; excess mitochondrial-derived oxidative damage has been shown to cause age-related vascular inflammation and arterial stiffness [13,14,15,16]. As the mitochondrial function is critical to effective vasoreactivity, targeting redox homeostasis in this organelle is a promising therapeutic direction
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