Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p<0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p<0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets

Raw and processed microscope images of fixed cells at baseline and following various experimental perturbations

The data included in this article comprise raw and processed images of fixed cells at baseline and subjected to various experimental perturbations. This dataset includes images of HUVEC cells fixed and subsequently incubated at either 37 °C or room temperature, primary rat vascular smooth muscle cells exposed to 25 mM glucose, and SH-SY5Y neurons exposed to hydrogen peroxide. Raw images appear exactly as they were captured on the microscope, while processed images show the binarization provided by software used for measurements of mitochondrial morphology. For in-depth discussion of the experiments and computational methods pertaining to this data, please refer to the corresponding research article titled “Fully automated software for quantitative measurements of mitochondrial morphology” (McClatchey et al., in press) [1]

(–)-Epicatechin Modulates Mitochondrial Redox in Vascular Cell Models of Oxidative Stress

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

(&ndash;)-Epicatechin Improves Vasoreactivity and Mitochondrial Respiration in Thermoneutral-Housed Wistar Rat Vasculature

Cardiovascular disease (CVD) is a global health concern. Vascular dysfunction is an aspect of CVD, and novel treatments targeting vascular physiology are necessary. In the endothelium, eNOS regulates vasodilation and mitochondrial function; both are disrupted in CVD. (&ndash;)-Epicatechin, a botanical compound known for its vasodilatory, eNOS, and mitochondrial-stimulating properties, is a potential therapy in those with CVD. We hypothesized that (&ndash;)-epicatechin would support eNOS activity and mitochondrial respiration, leading to improved vasoreactivity in a thermoneutral-derived rat model of vascular dysfunction. We housed Wistar rats at room temperature or in thermoneutral conditions for a total of 16 week and treated them with 1mg/kg body weight (&ndash;)-epicatechin for 15 day. Vasoreactivity, eNOS activity, and mitochondrial respiration were measured, in addition to the protein expression of upstream cellular signaling molecules including AMPK and CaMKII. We observed a significant improvement of vasodilation in those housed in thermoneutrality and treated with (&ndash;)-epicatechin (p &lt; 0.05), as well as dampened mitochondrial respiration (p &lt; 0.05). AMPK and CaMKII&alpha; and &beta; expression were lessened with (&ndash;)-epicatechin treatment in those housed at thermoneutrality (p &lt; 0.05). The opposite was observed with animals housed at room temperature supplemented with (&ndash;)-epicatechin. These data illustrate a context-dependent vascular response to (&ndash;)-epicatechin, a candidate for CVD therapeutic development