8‐oxoguanine glycosylase (OGG1) is a determinant of endothelial dysfunction via calcium and nitric oxide signaling

Abstract

Diabetes mellitus is a chronic disease that hinders the body's ability to produce or respond to the hormone insulin, resulting in abnormal metabolism and elevated glucose levels. Type II diabetes has been characterized by a two-to-four-fold increase in cardiovascular disease, such as carotid artery stenosis, generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. With it being a very common and significant health complication, understanding the pathology and the mechanisms by which diabetes-induced oxidative stress serves as a precursor to several notable vascular diseases is paramount, and is the overall objective of this study. Endothelial dysfunction (ED) is a vascular phenomenon attributed to a reduction in nitric oxide (NO) bioactivity and an increase in oxygen free radical formation with subsequent oxidative stress, thus leading to impaired vasodilation, and compromised vascular function. ED tends to be a consistent finding in diabetes. A major source of oxidative stress is the abnormal production of reactive oxygen species (ROS) in the context of diabetic inflammation. Increased ROS production leads to mitochondrial DNA (mt DNA) damage and aberrant cell signaling. Typically, mt DNA damage is repaired through the base excision repair (BER) pathway. However, during chronic inflammation in diabetes, this repairing capability is ineffective due to downregulation of key BER enzymes, namely 8-oxoguanine glycosylase (OGG1). Given this rational, we hypothesized that the BER pathway is an important determinant of endothelial dysfunction via impaired calcium and NO signaling. To test this hypothesis, we examined the effect of BER defects on endothelial calcium signaling and NO production using carotid arteries isolated from mice deficient in OGG1. Opened artery measurements of endothelial calcium and NO signaling were performed using a custom-designed parallel plate flow chamber. Briefly, segments of carotid arteries (~200 µm in diameter) were isolated from OGG1 deficient (KO) and wild type (WT) mice and cut open lengthwise to expose the endothelial surface. The isolated vessels were then stained with a loading solution composed of Pluronic acid (Fischer), Cal-520 (AAT bio) calcium indicator dye and DAR-4M (Calbiochem), a fluorescent probe for NO. Time lapse signal measurements were collected via confocal microscopy and were then analyzed and processed with S8, a custom automated solution to signal analysis based on noise filtering and object tracking, which computed dynamic amplitude, frequency, duration, and spatial spread. We discovered that although the number of calcium signals detected within the measurement interval were similar between KO and WT (10 ± 3 vs 9 ± 3, µ ± SEM, respectively), the spatial spread of signals within the KO (19.95 ± 3.78 µm2, µ ± SEM) was greater than WT (9.65 ± 2.7 µm2, p = 0.057 via Welch's unpaired t-test). These data suggest that OGG1 may be a determinant of normal endothelial calcium signaling and might serve as a therapeutic target for preventing the adverse effects of endothelial dysfunction present in diabetes.