Oxidative stress and vascular function

Many drug-induced complications and diseases are known to be associated with or even based on a dysequilibrium between the formation of reactive oxygen or nitrogen species (RONS) and the expression/activity of antioxidant enzymes that catalyze the breakdown of these harmful reactive species. The “kindling radical” concept is based on the initial formation of RONS that in turn activate additional sources of RONS in certain pathological conditions. Recently, we and others have demonstrated such “cross-talk” between NADPH oxidases and mitochondria in the setting of nitroglycerin-induced nitrate tolerance, the aging process and angiotensin-II triggered arterial hypertension via redox pathways compromising the mitochondrial, ATP-sensitive potassium channel (mKATP), the mitochondrial permeability transition pore (mPTP), cSrc and protein kinases and the NADPH oxidase isoform Nox2 (and eventually Nox1). This review will focus on the uncoupling of endothelial nitric oxide synthase (eNOS) by initially formed “kindling radicals” (RONS) and on the different “redox switches” that are involved in the uncoupling process of eNOS. S-glutathionylation of the eNOS reductase domain, adverse phosphorylation of eNOS, and of course the oxidative depletion of tetrahydrobiopterin (BH4) will be highlighted as potential “redox switches” in eNOS. In addition, RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) and L-arginine depletion will be discussed as alternative reasons for dysfunctional eNOS. Finally, we present the clinical perspectives of eNOS uncoupling (and dysfunction) for the development and progression of cardiovascular disease and discuss the important prognostic value of the measurement of endothelial function (e.g. by flow-mediated dilation or forearm plethysmography) for patients with cardiovascular disease

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 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

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 vascular parameters in diabetic rats.

<p>Microscopic determination of wall thickness (grey) and collagen content (black) by sirius red staining of paraffinated aortic sections (<b>A</b>). Representative microscope images are shown along with the densitometric quantification. Effects of SGLT2i therapy on endothelium-dependent and independent vascular relaxation by the vasodilators acetylcholine (ACh, <b>B</b>) and nitroglycerin (GTN, <b>C</b>), respectively. Data are the means±SEM from 6–7 (<b>A</b>) and 9–12 (<b>B,C</b>) animals/group. Each single value for an animal corresponds to the means of 4 individual aortic rings from this animal. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected group. For the vascular function data the significance levels were determined by two-way-ANOVA and significances for the entire curves are indicated when at least one compared concentration condition showed significant differences. Vasodilator potency (EC₅₀, pD₂) and efficacy (max. relaxation) were also calculated and subjected to statistical analysis using one-way-ANOVA. The data and results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone-0112394-t001" target="_blank">Table 1</a>.</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

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