Short-term pharmacological activation of Nrf2 ameliorates vascular dysfunction in aged rats and in pathological human vasculature. A potential target for therapeutic intervention

Abstract

Oxidative stress contributes to endothelial dysfunction, a key step in cardiovascular disease development. Ageing-related vascular dysfunction involves defective antioxidant response. Nuclear factor erythroid 2-like-2 (Nrf2), orchestrates cellular response to oxidative stress. We evaluated the impact of Nrf2-activation on endothelium-dependent and H₂O₂-mediated vasodilations in: aorta (RA), mesenteric artery (RMA), coronary artery (RCA) and corpus cavernosum (RCC) from ageing rats and in human penile arteries (HPRA) and corpus cavernosum (HCC) from erectile dysfunction (ED) patients. Relaxant responses were evaluated in organ chambers and wire myographs. Nrf2 content and heme oxygenase-1 (HO-1) were determined by ELISA. Superoxide and Nrf2 were detected by immunofluorescence. Pharmacological activation of Nrf2 with sulforaphane (SFN) improved NO- and endothelium-derived hyperpolarizing factor-mediated endothelium-dependent vasodilation and H₂O₂-induced relaxation in vascular beds from aging rats. SFN-induced effects were associated with increased Nrf2 (RMA, RCA) and reduced superoxide detection in RCA. Improvement of vascular function was confirmed in HPRA and HCC from ED patients and mimicked by another Nrf2 activator, oltipraz. Nrf2 increase and superoxide reduction together with HO-1 increase by Nrf2 activation was evidenced in HCC from ED patients. PDE5 inhibitor-induced relaxations of HPRA and HCC from ED patients were enhanced by SFN. Nrf2 short-term pharmacological activation attenuates age-related impairment of endothelium-dependent and reactive oxygen species (ROS)-induced vasodilation in different rat and human vascular territories by upregulation of Nrf2-related signaling and decreased oxidative stress. In ED patients target tissues, Nrf2 potentiates the functional effect of ED conventional pharmacological therapy suggesting potential therapeutic implication.

Keywords: Ageing; Endothelial dysfunction; Erectile dysfunction; Human vasculature; Nrf2; Oxidative stress.

Fig. 1

Sulforaphane improves endothelium-dependent vasodilation in aorta (RA), mesenteric arteries (RMA), coronary arteries (RCA), and corpus cavernosum (RCC) from aged rats. Endothelium-dependent relaxation to acetylcholine (ACh) in RA (A) and RMA (B) precontracted with norepinephrine (NE) and in RCA (C) precontracted with serotonin (5-HT), and to carbachol (CCh) in RCC (D) precontracted with phenylephrine (PE) obtained from young (three months old, 3 M) and aged (20 months old, 20 M) rats and effects of sulforaphane (SFN, 10 μM) or vehicle (0.1% DMSO) on such responses in aged rats. Panel E shows the effects of the Nrf2 inhibitor, trigonelline (TRIG, 30 μM) on SFN-induced potentiation of endothelial vasodilations in RCA from aged rats (20 M). Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM) at the end of the experiment. n indicates the number of animals. ***p < 0.001 vs. data obtained in 3 M rats, †††p < 0.001 vs. 20 M rats and §§§p < 0.001 vs. 20 M + SFN by a two-factors ANOVA test.

Fig. 2

Sulforaphane improves hydrogen peroxide (H₂O₂)-induced vasodilation in mesenteric arteries (RMA), coronary arteries (RCA), and corpus cavernosum (RCC) from aged rats. Relaxation induced by H₂O₂ in RA (A) and RMA (B) precontracted with norepinephrine (NE), in RCA (C) precontracted with serotonin (5-HT), and in RCC (D) precontracted with phenylephrine (PE) obtained from young (three months old, 3 M) and aged (20 months old, 20 M) rats and effects of sulforaphane (SFN, 10 μM) or vehicle (0.1% DMSO) on such responses in aged rats. Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM) at the end of the experiment. n indicates the number of animals. ***p < 0.001 vs. data obtained in 3 M rats, †††p < 0.001 vs. 20 M rats by two-factors ANOVA test.

Fig. 3

Relevance of endothelium in sulforaphane-induced effects on H₂O₂-induced relaxations. Relaxation induced by H₂O₂ in mesenteric arteries (RMA, A-B) precontracted with norepinephrine (NE) and in coronary arteries (RCA, C-D) precontracted with serotonin (5-HT) obtained from old (20 months old, 20 M) rats. The influence of adding sulforaphane (SFN, 10 μM) or vehicle (0.1% DMSO) on such responses in intact arteries (E+) (A, C) and in those where endothelium was mechanically removed (E−) (B, D) is shown. Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM). n indicates the number of animals. ***p < 0.001 by two-factors ANOVA test vs E+.

Fig. 4

Sulforaphane increases Nrf2 content and decreases oxidative stress in arteries from aged rats. Panel A shows the effect of sulforaphane (SFN 10 μM) on Nrf2 content in mesenteric arteries (RMA) from young (three months old, 3 M) and old (20 months old, 20 M) rats exposed to H₂O₂. Data are expressed as mean ± SEM of ng of Nrf2 per mg of tissue protein. n indicates the number of animals. †p < 0.05 vs. H₂O₂ alone by paired t-test. Panels B and C show representative images (x200) of Nrf2 detection by immunofluorescence (green) in RCA from aged (20 M) rats treated with vehicle (0.1% DMSO) (B) or SFN 10 μM (C) for 60 min and then exposed to H₂O₂ (300 μM) for 10 min. Panels E and F show representative images (x200) of dihydroethidium (DHE) detection (red) in RCA from 20 M rats treated vehicle (E) or SFN (F) as above described. Counterstaining of nuclei with DAPI in blue is merged into all images. Panels D and G show quantification of Nrf2 and DHE-induced fluorescence, respectively, relative to number of nuclei. Data are expressed as mean ± SEM of the percentage with respect to H₂O₂ alone. n indicates the number of animals. *p < 0.05 vs. H₂O₂ by Student t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Sulforaphane improves endothelium-dependent and H₂O₂-induced relaxations in human vascular tissue. Relaxation induced by acetylcholine (ACh, A-B) and H₂O₂ (C–D) in human penile resistance arteries (HPRA, A, C) and human corpus cavernosum (HCC, B, D) from patients with erectile dysfunction (ED) and from organ donors without erectile dysfunction (No ED). The influence of adding sulforaphane (SFN, 10 μM) on such responses in HPRA and HCC from ED patients is shown. Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM). n indicates the number of patients. ***p < 0.001 vs. No ED and ††† vs. ED by two-factors ANOVA test.

Fig. 6

Sulforaphane improves both endothelium-derived hyperpolarizing factor (EDHF)- and NO-mediated components of endothelial vasodilation in arteries from aged rats and human patients. Effects of sulforaphane (SFN, 10 μM) or the vehicle (0.1% DMSO) on relaxation induced by acetylcholine (ACh) in mesenteric arteries (RMA) (A, D) and coronary arteries (RCA) (B, E) from aged (20 M) rats and in human penile resistance arteries (HPRA) (C, F) from patients with erectile dysfunction (ED). Upper panels show ACh-induced relaxations in the presence of N^G-nitro l-arginine methyl ester (l-NAME, 100 μM) and indomethacin (INDO, 10 μM) to isolate the EDHF component of endothelial vasodilation. Lower panels show ACh-induced relaxations in arteries contracted with KCl (25–50 mM) in the presence of INDO to isolate the NO component of endothelial vasodilation. Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM). n indicates the number of animals (A, B, D, E) or patients (C, F). ***p < 0.001 vs. l-NAME + INDO or vs K⁺+ INDO by two-factors ANOVA test.

Fig. 7

Nrf2 activators increase Nrf2 content, decrease oxidative stress content in vascular tissues from patients with erectile dysfunction (ED). Representative images (x200) of immunodetection of Nrf2 (green) (A, B) and on superoxide content detected by dihydroethidium (DHE)-induced fluorescence (red) (D, E) in corpus cavernosum from organ donors without erectile dysfunction (No ED) (A, D) and from patients with erectile dysfunction (ED) (B, E). Nuclei staining with DAPI (blue) are merged in all images. Reduced Nrf2 content and increased DHE staining in ED is confirmed by fluorescence quantification relative to number of nuclei (C and F, respectively). Data are expressed as mean ± SEM of the percentage of the average value obtained in No ED. n indicates the number of patients. **p < 0.01 vs. No ED by Student t-test. Other panels show representative images (x200) of the effects of treatment with vehicle (0.1% DMSO) (G, K), sulforaphane (SFN 10 μM) (H, L), or oltipraz (OLT, 30 μM) (I, M) on Nrf2 immunodetection (G–I) and DHE-induced fluorescence (K–M) in cavernosal tissue from patients with ED. Tissues were treated with SFN, OLT or vehicle for 60 min and then exposed to H₂O₂ (300 μM) for 10 min. Panels J and N shows quantification of Nrf2 and DHE fluorescence, respectively, relative to number of nuclei. Data are expressed as mean ± SEM of the percentage of the value obtained in control conditions (without H₂O₂). n indicates the number of patients. †p < 0.05, ††p < 0.01 vs. H₂O₂ by one-factor ANOVA followed by Student-Newmann-Keuls test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Nrf2 activation increases heme oxygenase expression in human vascular tissues from ED patients. Panels A and B show the effects of treatment with vehicle (0.1% DMSO), sulforaphane (SFN, 10 μM) (A) or oltipraz (OLT, 10 μM) (B) on heme oxygenase-1 (HO-1) content determined by ELISA in cavernosal tissue from patients with ED. Tissues were treated with SFN, OLT or vehicle for 60 min and then exposed to H₂O₂ (300 μM) for 10 min. Data are expressed as mean ± SEM of the percentage of the value obtained in control conditions (without H₂O₂). n indicates the number of patients. *p < 0.05 vs. H₂O₂ by Student t-test.

Fig. 9

Nrf2 activation enhances relaxant capacity of the type 5 phosphodiesterase inhibitor, tadalafil, in human vascular tissues from ED patients. Effects of the treatment with SFN (10 μM) or vehicle on relaxation induced by tadalafil in human penile resistance arteries (HPRA) (A) and human's corpus cavernosum (HCC) (B) from patients with ED. Data are expressed as mean ± SEM of the percentage of maximal relaxation induced by papaverine (0.1 mM). n indicates the number of patients. ††p < 0.01, †††p < 0.001 vs. ED by two-factors ANOVA test.