Nitroglycerin induces DNA damage and vascular cell death in the setting of nitrate tolerance

Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10–1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O 6-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O 6-methylguanine-DNA methyltransferase displayed more vascular O 6-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O 6-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction

The SGLT2 inhibitor empagliflozin improves the primary diabetic complications in ZDF rats

Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, β-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF‐Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial)

The Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin Improves Diabetes-Induced Vascular Dysfunction in the Streptozotocin Diabetes Rat Model by Interfering with Oxidative Stress and Glucotoxicity

<div><p>Objective</p><p>In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.</p><p>Methods</p><p>Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).</p><p>Results</p><p>Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.</p><p>Conclusions</p><p>Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.</p></div

Summary of beneficial effects of SGLT2i treatment on diabetes induced vascular dysfunction and other adverse effects of hyperglycemia in rats.

<p>Summary of beneficial effects of SGLT2i treatment on diabetes induced vascular dysfunction and other adverse effects of hyperglycemia in rats.</p

Effects of SGLT2i treatment on aortic protein expression of the NO/cGMP signaling cascade as well as oxidative stress and inflammatory pathways in diabetic rats.

<p>Expression of endothelial nitric oxide synthase (eNOS, <b>A</b>), serine1177 phosphorylated eNOS (<b>B</b>), dihydrofolate reductase (DHFR, <b>C</b>), ratio of cGK-I and serine239 phosphorylated VASP (<b>D</b>) were assessed by Western blotting analysis and specific antibodies. Expression of NADPH oxidases Nox1 (<b>E</b>) and Nox2 (<b>F</b>), heme oxygenase-1 (HO-1) (<b>G</b>) and monocyte-chemoattractant-protein-1 (MCP-1 or CCL-2, <b>H</b>) were assessed by Western blotting analysis and specific antibodies. Representative blots for all proteins are shown in supplemental Figure S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone.0112394.s001" target="_blank">File S1</a>. The data are expressed as % of control and are the means ± SEM from 8–9 (<b>A</b>), 5–6 (<b>B</b>), 7 (<b>C</b>), 4 (<b>D</b>), 6–7 (<b>E</b>), 7–9 (<b>F</b>), 7–9 (<b>G</b>) and 4–6 (<b>H</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^$, p<0.05 vs. low dose SGLT2i treated.</p

Weight gain, and blood and serum parameters in controls and diabetic rats.

a<p>Weight gain was calculated from the difference of values prior and 8 weeks post STZ injection. Blood glucose was determined three days after STZ injection without SGLT2i treatment; fasting and non-fasting blood glucose as well as HbA1c levels were measured 8 weeks post STZ injection. The data are the means ± SEM of the indicated number of animals/group; n.d. means not detectable.</p><p>*, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^§, p<0.05 vs. low dose SGLT2i treated.</p>b<p>Separation of HDL and LDL using HF5 (Hollow Fiber Flow Field Flow Fractionation).</p><p>Weight gain, and blood and serum parameters in controls and diabetic rats.</p

Effects of SGLT2i treatment on oxidative stress parameters in diabetic rats.

<p>Leukocyte-derived ROS (oxidative burst) in whole blood at 30 min upon zymosan A (<b>A</b>) stimulation along with the effects of the NADPH oxidase isoform 2 (Nox2) inhibitor VAS2870 and the intracellular calcium chelator BAPTA-AM (<b>B</b>) and upon PDBu stimulation (<b>C</b>). <b>(D)</b> Quantification of cardiac NADPH oxidase activity in membrane preparations by lucigenin (5 µM)-derived chemiluminescence. Dihydroethidium (DHE, 1 µM)-fluorescence microtopography was used to assess the effects of SGLT2i treatment on vascular (<b>E</b>) and endothelial (<b>F</b>) ROS production with and without incubation with the eNOS inhibitor L-NAME. Representative microscope images are shown along with the densitometric quantification. Red fluorescence indicates ROS formation whereas green fluorescence represents basal laminae autofluorescence. Data are the means±SEM from 6–7 (<b>A,B</b>), 5 (<b>C</b>), 7–8 (<b>D</b>), 9 (<b>E</b>) or 5–6 (<b>F</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^§, p<0.05 vs. low dose SGLT2i treated and ^$, p<0.05 vs. w/o L-NAME.</p

Effects of SGLT2i treatment on AGE/RAGE signaling in diabetic rats.

<p>Quantification of AGE-positive proteins by dot blot analysis (<b>A</b>) and RAGE expression was assessed by Western blotting analysis with specific antibodies (<b>B</b>) and quantitative RT-PCR analysis (<b>C</b>). Representative blots are shown at the bottom of the densitometric quantifications. Serum methylglyoxal levels were assessed by HPLC-based quantification (<b>D</b>). Representative chromatograms are shown in supplemental Figure S7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone.0112394.s001" target="_blank">File S1</a>. The data are expressed as % of control and are the means ±SEM from 7 (<b>A</b>), 6–7 (<b>B</b>) and 8–11 (<b>C,D</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ–injected.</p