Pentaerythritol Tetranitrate In Vivo Treatment Improves Oxidative Stress and Vascular Dysfunction by Suppression of Endothelin‐1 Signaling in Monocrotaline‐Induced Pulmonary Hypertension

Figures

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  Figure 1. Characterization of morphological changes and protein nitration in lung tissue and serum antioxidant capacity of monocrotaline-treated rats. (a) Trichrome staining of paraffinated lung tissue (magnification of 100x, 200x, and 400x). (b) Levels of 3-NT positive proteins in lung tissue were assessed by dot blot analysis and specific antibodies. Representative blots are shown below the densitometric quantification. (c) Antioxidant capacity was determined by DPPH• assay (ΔE 517 nm). The data are mean ± SEM from 3–8 (a, b) and 3–6 (c) animals/group. ∗p < 0.05 versus control and #p < 0.05 versus MCT30.

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  Figure 2. Characterization of vascular function in the model of monocrotaline-induced pulmonary hypertension. Endothelium-dependent (ACh) maximum relaxation was determined by isometric tension studies in aortic ring segments (a, c) and pulmonary ring segments (b, d) from rats treated with different doses of MCT and different time protocols for the induction of pulmonary hypertension. A total number of 4–22 (a, b) and 6–14 (c) and 6 (d) aortic and pulmonary artery ring segments from at least 3 male rats were used. ∗p < 0.05 versus control (a–d); #p < 0.05 versus MCT30 (a, b) or 2 weeks (c, d); §p < 0.05 versus MCT40 (a, b).

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  Figure 3. Effects of PETN therapy on heart/body and lung/body weight ratio, pulmonary artery dilation, and pulmonary arterial pressure in monocrotaline-treated rats (MCT40, 4 weeks). (a) Chemical structure of PETN. (b) Lung/body and (c) heart/body weight ratio were determined. (d) Pulmonary artery dilation was qualitatively envisaged by photographic images. (e) Echocardiography was used to measure pulmonary arterial pressure. The data are mean ± SEM of 4 (b, c) and 3-4 (d, e) animals per group ∗p < 0.05 versus control and #p < 0.05 versus MCT.

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  Figure 4. Effects of PETN therapy on endothelial function, oxidative stress, and protein tyrosine nitration of aorta and pulmonary artery in monocrotaline-treated rats (MCT40 4 weeks). (a, b) Endothelium-dependent (ACh) relaxation was determined by isometric tension studies in rat aortic ring segments and pulmonary ring segments. (c) DHE (1 µM) oxidative fluorescence microtopography was used to assess vascular oxidative stress. (d) Levels of 3-NT positive proteins in lung tissue were assessed by dot blot analysis and specific antibodies. Representative blots are shown below the densitometric quantification. A total number of 6–22 aortic (a) and 6–17 pulmonary tissue (b–d) ring segments from male rats were used. ∗p < 0.05 versus control and #p < 0.05 versus MCT40.

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  Figure 5. Effects of PETN therapy on prooxidative protein activity (Nox, XO), oxidative burst in whole blood, and antioxidant HO-1 mRNA expression in pulmonary hypertension (MCT40, 4 weeks). (a) Cardiac NADPH oxidase (Nox) activity was measured by the chemiluminescence probe lucigenin (5 µM) in the presence of NADPH (200 µM). (b) Leukocyte-derived oxidative burst in whole blood was examined by the chemiluminescence probe L-012 (100 µM) upon stimulation with PDBu (10 µM). (c) Xanthine oxidase (XO) activity was assessed by a photometric assay using cytochrome c (change in absorbance: ΔA 550 nm). qRT-PCR was used to determine mRNA expression levels of the antioxidant enzyme (d) heme oxygenase-1 (HO-1) and (e) vascular adhesion molecule-1 (VCAM-1) in lung tissue. (f) NADPH oxidase 2 (Nox2) protein expression was determined by Western blot analysis. The data are mean ± SEM from 3–6 animals/group. ∗p < 0.05 versus control; #p < 0.05 versus MCT40.

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  Figure 6. Effects of PETN therapy on endothelin-1 signaling (ET-1, ECE-1, and ET-1a/b receptor) and oxidative burst in whole blood. qRT-PCR was used to determine mRNA expression levels of (a) endothelin-1, (b) endothelin-1 converting enzyme-1, (c) endothelin-1 a receptor, and (d) endothelin-1 b receptor in lung tissue. (e) Leukocyte-derived oxidative burst in whole blood was examined by the chemiluminescence probe L-012 (100 µM) upon stimulation with the endothelin-1 agonist BQ-3020 (0.05–5 µM). (f) Effect of PETN (100 µM) on BQ-3020 (5 µM)/zymosan A (0.5 µg/mL) stimulated whole blood oxidative burst. (g) qRT-PCR was used to determine mRNA expression levels of ICAM-1 in EA.hy cells upon stimulation with BQ-3020 and PETN. The data are mean ± SEM from 6 animals/group (a–d) and 16 (e-f) or 3–7 (g) independent experiments. ∗p < 0.05 versus control; #p < 0.05 versus MCT40.