Glutathione Peroxidase-1 Deficiency Potentiates Dysregulatory Modifications of Endothelial Nitric Oxide Synthase and Vascular Dysfunction in Aging

Abstract

390T he incidence and frequency of cardiovascular diseases, such as hypertension, diabetes mellitus, and myocardial infarction, increase substantially with age.1 Increased oxidative stress from mitochondria and other enzymatic sources as well as vascular dysfunction manifest in aged animals. 2 This observation points to a strong correlation between aging, oxidative stress, and, as a consequence, development of vascular/endothelial dysfunction.3 In 1954, Harman 4 expressed for the first time the free radical theory of aging. The prevailing hypothesis is that an age-dependent increase in We have previously shown that genetic deletion of the mitochondrial antioxidant proteins manganese superoxide dismutase and aldehyde dehydrogenase-2 contributes to aging-dependent vascular dysfunction and mitochondrial oxidative stress.2 It is worth noting and of high clinical importance that antioxidant enzymes have a significant effect on the healthspan of animals during normal aging (eg, indicated by less aging-associated cardiovascular complications during the sunset years) and also on the resistance to stress conditions. 6 Previous studies have revealed increased mitochondrial and systemic oxidative stress in glutathione peroxidase-1 (GPx-1)-ablated mice 7 and synergistic negative effects on vascular function in the setting of hyperlipidemia, 8 diabetes mellitus, 9 and hypertension. 10 Moreover, increased senescence was demonstrated for fibroblasts from GPx-1 −/− mice. 7 Most importantly, a correlation between GPx-1 activity in blood cells andAbstract-Recently, we demonstrated that gene ablation of mitochondrial manganese superoxide dismutase and aldehyde dehydrogenase-2 markedly contributed to age-related vascular dysfunction and mitochondrial oxidative stress. The present study has sought to investigate the extent of vascular dysfunction and oxidant formation in glutathione peroxidase-1-deficient (GPx-1 −/− ) mice during the aging process with special emphasis on dysregulation (uncoupling) of the endothelial NO synthase. GPx-1 −/− mice on a C57 black 6 (C57BL/6) background at 2, 6, and 12 months of age were used. Vascular function was significantly impaired in 12-month-old GPx-1 −/− -mice as compared with age-matched controls. Oxidant formation, detected by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was increased in the aged GPx-1 −/− mice. Aging per se caused a substantial protein kinase C-and protein tyrosine kinase-dependent phosphorylation as well as S-glutathionylation of endothelial NO synthase associated with uncoupling, a phenomenon that was more pronounced in aged GPx-1 −/− mice. GPx-1 ablation increased adhesion of leukocytes to cultured endothelial cells and CD68 and F4/80 staining in cardiac tissue. Aged GPx-1 −/− mice displayed increased oxidant formation as compared with their wild-type littermates, triggering redox-signaling pathways associated with endothelial NO synthase dysfunction and uncoupling. Thus, our data demonstrate that aging leads to decreased NO bioavailabi...

Taking up the cudgels for the traditional reactive oxygen and nitrogen species detection assays and their use in the cardiovascular system

“…These are only selected references since in total we have successfully used the DHE (cryo) staining assay in more than 70 original publications with all kinds of pharmacological interventions of genetic manipulation of redox pathways (for summary see [1,10,34]. The DHE (cryo) staining intensity always nicely correlated with the severity of the respective phenotype, was reduced by pharmacological therapy [22,23,36,43,45,48,53] or genetic deletion of the Nox subunit p47 phox [54] and aggravated by genetic deletion of protective antioxidant enzymes such as heme oxygenase-1, manganese superoxide dismutase or glutathione peroxidase-1 [49,55,56] and eliminated by ex vivo incubation with specific superoxide quenchers such as PEG-SOD [53]. It would be a quite surprising coincidence if in all these models, interventions and studies DHE (cryo) staining always would have identified the diseased group where higher oxidative stress levels were shown by other widely accepted methods for ROS and RNS detection, in particular since in all these models reduction in superoxide leels went along with an increase in vascular…”

The SGLT2 Inhibitor Empagliflozin Improves the Primary Diabetic Complications in ZDF Rats

“…To investigate the involvement of eNOS uncoupling in ROS production and endothelial dysfunction, aortic rings were preincubated with the NOS inhibitor L-NAME (0.5 mM) [29,31,32]. Adventitial ROS formation was measured by densitometric quantification of red fluorescence in a defined area within the adventitial tissue [33]. ROS-derived red fluorescence was detected using a Zeiss Axiovert 40 CFL microscope, Zeiss lenses and Axiocam MRm camera.…”

“…Immunoprecipitation of eNOS was performed with M-280 sheep antimouse IgG coated beads from Invitrogen (Darmstadt, Germany) along with a monoclonal mouse eNOS (Biosciences, USA) antibody [33]. The precipitates were washed and transferred to gel and subjected to SDS-PAGE followed by a standard Western blot procedure using a monoclonal rabbit phospho-Thr495-eNOS antibody (P-eNOS Thr495 , 1:1000, Upstate Biotechnology, MA, USA).…”

“…The precipitates were washed and transferred to gel and subjected to SDS-PAGE followed by a standard Western blot procedure using a monoclonal rabbit phospho-Thr495-eNOS antibody (P-eNOS Thr495 , 1:1000, Upstate Biotechnology, MA, USA). SDS-PAGE and Western blotting were performed as previously described [18,33] using monoclonal mouse α-actinin (1:10,000, Sigma-Aldrich, Munich, Germany) as a control for loading and transfer, monoclonal mouse NADPH oxidase isoform 2 (Nox2 or gp91 phox , 1:1000, BD Biosciences, USA), monoclonal mouse dihydrofolate reductase (DHFR, 1 µg/ml, Abnova Corp., Germany), monoclonal mouse GTP-cyclohydrolase-1 (GCH-1, 1 µg/ml, Abnova Corp., Germany), monoclonal mouse heme oxygenase-1 (HO-1) (4 µg/ml, Stressgen, San Diego, CA), polyclonal goat cGMP-dependent protein kinase (cGK-I, 1:200, Santa Cruz Biotechnologies, USA), vasodilator stimulated phosphoprotein (VASP) phosphorylated on serine239 (P-VASP Ser239 , clone 16C2, 1. Methylglyoxal serum levels were quantified by an HPLC-based method upon derivatization of the reactive aldehyde with 1,2-diaminobenzene as previously described [33].…”

“…SDS-PAGE and Western blotting were performed as previously described [18,33] using monoclonal mouse α-actinin (1:10,000, Sigma-Aldrich, Munich, Germany) as a control for loading and transfer, monoclonal mouse NADPH oxidase isoform 2 (Nox2 or gp91 phox , 1:1000, BD Biosciences, USA), monoclonal mouse dihydrofolate reductase (DHFR, 1 µg/ml, Abnova Corp., Germany), monoclonal mouse GTP-cyclohydrolase-1 (GCH-1, 1 µg/ml, Abnova Corp., Germany), monoclonal mouse heme oxygenase-1 (HO-1) (4 µg/ml, Stressgen, San Diego, CA), polyclonal goat cGMP-dependent protein kinase (cGK-I, 1:200, Santa Cruz Biotechnologies, USA), vasodilator stimulated phosphoprotein (VASP) phosphorylated on serine239 (P-VASP Ser239 , clone 16C2, 1. Methylglyoxal serum levels were quantified by an HPLC-based method upon derivatization of the reactive aldehyde with 1,2-diaminobenzene as previously described [33]. The activity of ALDH-2 in isolated heart mitochondria was determined by measuring the conversion of 6-methoxy-2-naphthylaldehyde (Monal 62) to the fluorescent naphthoic acid product by an HPLC-based assay in the absence and presence of the specific ALDH-2 inhibitor daidzin [33].…”