SAT-175 (-)-Epicatechin Modulates Cellular Signaling and Mitochondrial Redox in Endothelium

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 vasodilation and arterial stiffness, both of which are abnormal in diabetes. Diabetes-mediated generation of excess reactive oxygen species (ROS) may be contributing to this problem. We have identified (-)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS accumulation in cells treated with high glucose (HG) or antimycin A (AA, a compound targeting complex III) could be diminished by EPICAT. We further hypothesized that reduction of ROS would correlate with improved mitochondrial dynamics and cellular signaling. To test this hypothesis, we employed human umbilical vein endothelial cells (HUVEC) treated with HG, AA, and/or 0.1 or 1.0 µM of EPICAT, and measured mitochondrial and cellular superoxide, mitochondrial dynamics, and cellular signaling upstream of mitochondrial function. AA significantly increased mitochondrial width (p=0.012) and decreased mitochondrial length (p=0.0001), but we observed no impact of EPICAT. We measured superoxide production by electron paramagnetic resonance spectroscopy using two spin probes specific to mitochondrial (Mito-TEMPO-H) and total cell (CMH) superoxide. No impact of HG treatment was detected in the total cell superoxide; however, an increase in mitochondrial superoxide was measured in cells treated with HG as compared to control (0.42 µM superoxide vs. 0.31 µM superoxide, respectively). We found that both concentrations of EPICAT significantly attenuated mitochondrial superoxide production when treated with HG (HG alone=0.44 µM superoxide, HG + 0.1 µM EPICAT=0.34 µM superoxide, HG + 1.0 µM EPICAT= 0.34 µM superoxide, p=0.02 EPICAT effect), but not with AA treatment. We found that in combination with HG, 0.1 µM EPICAT significantly increased eNOS expression (p<0.05). At 1.0 µM, EPICAT also increased peNOS expression (p<0.05) and attenuated a decrease in pAMPK (p<0.05) in response to HG. EPICAT at both concentrations significantly increased mitochondrial complex II expression (p<0.05 for both). In summary, HG-induced mitochondrial ROS is responsive to EPICAT, whereas AA-induced ROS is not, suggesting that EPICAT is useful in mitigating moderate ROS concentrations and may be modulating mitochondrial activity upstream of complex III. Our data further illustrate that EPICAT may be a tool to modulate both cellular and mitochondrial glucose toxicity through the regulation of AMPK and eNOS, upstream of mitochondrial activity, as well as bolstering endogenous antioxidant defenses.