Vascular dysfunction in experimental diabetes is improved by pentaerithrityl tetranitrate but not isosorbide-5-mononitrate therapy

Abstract

Objective: Diabetes is associated with vascular oxidative stress, activation of NADPH oxidase, and uncoupling of nitric oxide (NO) synthase (endothelial NO synthase [eNOS]). Pentaerithrityl tetranitrate (PETN) is an organic nitrate with potent antioxidant properties via induction of heme oxygenase-1 (HO-1). We tested whether treatment with PETN improves vascular dysfunction in the setting of experimental diabetes.

Research design and methods: After induction of hyperglycemia by streptozotocin (STZ) injection (60 mg/kg i.v.), PETN (15 mg/kg/day p.o.) or isosorbide-5-mononitrate (ISMN; 75 mg/kg/day p.o.) was fed to Wistar rats for 7 weeks. Oxidative stress was assessed by optical methods and oxidative protein modifications, vascular function was determined by isometric tension recordings, protein expression was measured by Western blotting, RNA expression was assessed by quantitative RT-PCR, and HO-1 promoter activity in stable transfected cells was determined by luciferase assays.

Results: PETN, but not ISMN, improved endothelial dysfunction. NADPH oxidase and serum xanthine oxidase activities were significantly reduced by PETN but not by ISMN. Both organic nitrates had minor effects on the expression of NADPH oxidase subunits, eNOS and dihydrofolate reductase (Western blotting). PETN, but not ISMN, normalized the expression of GTP cyclohydrolase-1, extracellular superoxide dismutase, and S-glutathionylation of eNOS, thereby preventing eNOS uncoupling. The expression of the antioxidant enzyme, HO-1, was increased by STZ treatment and further upregulated by PETN, but not ISMN, via activation of the transcription factor NRF2.

Conclusions: In contrast to ISMN, the organic nitrate, PETN, improves endothelial dysfunction in diabetes by preventing eNOS uncoupling and NADPH oxidase activation, thereby reducing oxidative stress. Thus, PETN therapy may be suited to treat patients with cardiovascular complications of diabetes.

FIG. 1.

Weight gain and blood glucose levels in control and diabetic rats 8 weeks after STZ injection and 7 weeks of organic nitrate treatment. A: Weight gain was calculated from the difference of values before and after STZ injection. B: Blood glucose was determined on the day the animals were killed (8 weeks after STZ injection). Data are the means ± SEM of 10–11 (+PETN/+ISMN groups) or 20–22 (Ctr/STZ groups) animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ-injected group.

FIG. 2.

Effects of organic nitrate treatment on endothelium-dependent and -independent vasodilation in diabetic rats. Vascular function was determined by isometric tension studies and relaxation in response to endothelium-dependent (ACh) (A) and endothelium-independent with requirement of bioactivation (GTN) (B) vasodilators. Data are shown for control (Ctr) (circles), diabetic (STZ) (squares), STZ+PETN (triangles), and STZ+ISMN (inverted triangles) animals. Data are the means ± SEM of 37–77 (ACh) and 40–88 (GTN) aortic rings from 10–22 animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ.

FIG. 3.

Effects of organic nitrate treatment on cardiac and serum oxidative stress in diabetic rats. A: Quantification of cardiac NADPH oxidase activity in membranous preparations by ECL using the superoxide-specific dye, lucigenin (5 µmol/L). B: Quantification of serum XO activity by superoxide-dependent cytochrome c (50 µmol/L) reduction traced at 550 nm. C: Detection of serum antioxidant capacity by low-molecular weight antioxidant-dependent 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) (50 µmol/L) reduction traced at 517 nm. Serum was deproteinized by the addition of 50% acetonitrile. Data are the means ± SEM of heart tissues and serum samples from 10–20 (A), 6–10 (B), and 6–12 (C) animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ.

FIG. 4.

Effects of organic nitrate treatment on vascular and cardiac production of RONS in diabetic rats. A: DHE (1 µmol/L) fluorescence microtopography was used to assess vascular RONS formation in aortic cryosections. Five representative microscope images are shown below. B: Dot-blot analysis with a 3NT-specific antibody was used to assess 3-nitrotyrosine content in cardiac proteins. Three representative dot-blot results are shown below. C: Dot-blot analysis with a malondialdehyde (MDA)-specific antibody was used to assess malondialdehyde content in cardiac proteins. Three representative dot-blot results are shown below. Data are the means ± SEM of samples from 10–20 (A) and 6–15 (B and C) animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ; $P < 0.05 vs. PETN-treated group. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Effects of organic nitrate treatment on the expression of vascular Nox1 and Nox2 as well as antioxidant proteins in diabetic rats. Nox1 (A), Nox2 (B), HO-1 (C), and ecSOD (D) were assessed using the Western blotting technique and specific antibodies. Representative blots are shown at the bottom of each densitometric quantification. Data are the means ± SEM of aortic protein preparations from 6–15 (A–C) and 5 (D) animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ; $P < 0.05 vs. PETN-treated group.

FIG. 6.

Effects of organic nitrate treatment on the expression of vascular eNOS, GCH-I, and DHFR and phosphorylation, as well as the S-glutathionylation state of eNOS in diabetic rats. eNOS (A), Ser1177-phospho-eNOS (B), the ratio of Ser1177-phospho-eNOS to eNOS (C), GCH-I (D), and DHFR (E) were assessed using the Western blotting technique and specific antibodies. F: S-glutathionylation of eNOS was determined by eNOS immunoprecipitation, followed by anti-glutathione staining. After stripping the membrane, the bands were stained for eNOS to allow normalization of the signals. As a control for the specificity of the antibody for GSH-positive proteins, diabetic samples were treated with β-mercaptoethanol. Representative blots are shown at the bottom of each densitometric quantification. Data are the means ± SEM of aortic rings from 6–15 (A, D, and E) and 5–8 (B, C, and F) animals per group. *P < 0.05 vs. control; #P < 0.05 vs. STZ; $P < 0.05 vs. PETN.

FIG. 7.

Effect of organic nitrates on the activity of the 11-kb human HO-1 promoter in the dependence of NRF2 and induction of GCH-I by HO-1 products. A: DLD-1-HO-1-prom cells were washed with PBS and incubated with Dulbecco’s modified Eagle’s medium containing 2 mmol/L l-glutamine in the absence of serum and phenol red. After 16 h, cells were incubated with 50 μmol/L PETN (or DMSO as a solvent control) or 50 μmol/L ISMN (or water as a solvent control) for 8 h, lyzed in 1× passive lysis buffer, and protein concentrations were measured using the Bradford reagent. Luciferase activity in the extracts was determined using the luciferase assay system. The light units of the firefly luciferase were normalized to the protein concentration of the cell extracts. The relative luciferase activity level of cells treated with the solvent control was set to 100%. B: DLD-1-HO-1-prom cells were transiently transfected with an anti-NRF2 siRNA (siNRF2), which is shown to downregulate NRF2 expression, or a negative control siRNA (siCtr) by lipofection with HiPerFect HTS Reagent according to the manufacturer’s recommendations. After 48 h, cells were treated as described above to analyze PETN-induced human HO-1-11kb promoter activity. The relative luciferase activity level of cells treated with DMSO and siCtr was set to 100%. C: Confluent human endothelial cells (EA.hy 926) were treated in six-well plates with solvent (0.1% DMSO = Ctr), bilirubin (BR; 10 μmol/L), the carbon monoxide–releasing compound (CORM = CO; 50 μmol/L), or PETN (P; 50 μmol/L) for 15 h in the incubator. Two wells were pooled for GCH-I protein analysis (1:2,500; Dr. E. Werner, Innsbruck, Austria). Data are the means ± SEM of 6–10 (A and B) and 3–9 (C) independent experiments. *P < 0.05 vs. control; #P < 0.05 vs. PETN (siCtr).