Vascular dysfunction heralds the onset of diabetic cardiovascular disease. We have reported that vascular mitochondrial dysfunction contributes to impaired vasoreactivity in diabetes. The botanical compound -(-) epicatechin (EPICAT) is a known vasodilator. We hypothesized that EPICAT restores vasodilation by bolstering mitochondrial function in the context of high glucose via support of endothelial nitric oxide synthase (eNOS) activity and mitigation of reactive oxygen species (ROS). To test this hypothesis, we examined the impact of EPICAT on vasoreactivity ex situ and endothelial cell function in vitro. EPICAT significantly increased endothelium dependent acetylcholine-stimulated vasodilation (64.74%, p=0.002). HUVEC cells were incubated with 7 mM (normal glucose, NG) or 30 mM glucose (HG) with or without 0.1 µM EPICAT for 30 min, 2, 4-6 hours, and 24 hours. Mitochondrial superoxide was detected via electron spin resonance spectroscopy, and respiration and hydrogen peroxide were measured using Oroboros. Mitochondrial superoxide production was elevated in HG cells (2 hours=1.63 ± 0.17 µM/mg (HG) vs. 1.18 ± 0.07 µM/mg (NG), p=0.017 and 4-6 hours=1.46 ± 0.16 µM/mg (HG) vs. 1.18 ± 0.07 µM/mg (NG), p=0.09). Mitochondrial respiration was greater in cells exposed to HG for 2 hours in state 3 (23.8%) and uncoupled (14.5%), and hydrogen peroxide was higher in HG treated cells, while both metrics were unchanged after 6 hours, and respiration was decreased after 24 hours. Cells pretreated with EPICAT had elevated rates of respiration (state 3=73.4% and uncoupled=55.2%) without a commensurate increase in superoxide, and EPICAT dampened HG superoxide generation after 2 hours by 17.0%. Cells pretreated with EPICAT and exposed to HG had significantly elevated eNOS protein expression (p=0.001). Our data suggest that EPICAT potentiates vasodilation by acutely activating eNOS, leading to an increase in mitochondrial respiration while mitigating excess ROS production.
A.C. Keller: None. S.E. Hull: None. L. Knaub: None. A. Johnston: None. J.E. Reusch: Research Support; Self; Merck & Co., Inc.. Board Member; Self; American Diabetes Association. Research Support; Self; AstraZeneca. Other Relationship; Self; Sanofi-Aventis.