H2S attenuates oxidative stress via Nrf2/NF-κB signaling to regulate restenosis after percutaneous transluminal angioplasty

Abstract

Restenosis after angioplasty of peripheral arteries is a clinical problem involving oxidative stress. Hydrogen sulfide (H₂S) participates in oxidative stress regulation and activates nuclear factor erythroid 2-related factor 2 (Nrf2). This study investigated the effect of H₂S and Nrf2 on restenosis-induced arterial injury. Using an in vivo rat model of restenosis, we investigated whether H₂S inhibits restenosis after percutaneous transluminal angioplasty (PTA) and the oxidative stress-related mechanisms implicated therein. The involvement of Nrf2 was explored using Nrf2-shRNA. Neointimal formation and the deposition of elastic fibers were assessed histologically. Inflammatory cytokine secretion and the expression of proteins associated with oxidative stress and inflammation were evaluated. The artery of rats subjected to restenosis showed increased arterial intimal thickness, with prominent elastic fiber deposition. Sodium hydrosulfide (NaHS), an H₂S donor, counteracted these changes in vivo. Restenosis caused a decrease in anti-oxidative stress signaling. This phenomenon was inhibited by NaHS, but Nrf2-shRNA counteracted the effects of NaHS. In terms of inflammation, inflammatory cytokines were upregulated, whereas NaHS suppressed the induced inflammatory reaction. Similarly, Nrf2 downregulation blocked the effect of NaHS. In vitro studies using aortic endothelial and vascular smooth muscle cells isolated from experimental animals showed consistent results as those of in vivo studies, and the participation of the nuclear factor-kappa B signaling pathway was demonstrated. Collectively, H₂S played a role in regulating post-PTA restenosis by alleviating oxidative stress, modulating anti-oxidant defense, and targeting Nrf2-related pathways via nuclear factor-kappa B signaling.

Keywords: Hydrogen sulfide; anti-oxidant defense; inflammation; neointimal hyperplasia; nuclear factor erythroid 2-related factor 2.

Figure 1.

Outline of treatments and groupings for in vivo post-PTA restenosis modeling in male Sprague Dawley rats. C-4W: control rats sacrificed at four weeks; RS-4W: post-PTA restenosis modeling for four weeks.

Figure 2.

Histological evaluation of rat arteries after percutaneous transluminal angioplasty. (a) HE staining of arterial morphology. The analysis was performed after induction of restenosis, and the arterial intima was stained in rose-pink. Restenosis caused an obvious increase in the intimal thickness, whereas NaHS counteracted this effect. Magnified areas show clear distinction between the intimal and medial layers. Scale bar, 100 μm. (b) Verhoeff’s staining of elastic fiber deposition in arterial tissues. The analysis was performed after induction of restenosis, and elastic fibers were stained in blue-black. Scale bar = 50 μm. (c) Intimal and (d) medial thicknesses were measured by selecting 10 areas from each cross-sectional HE image while ensuring that the distribution of thicknesses was taken into consideration, and the (e) intimal/medial thickness ratio was calculated. The data are presented as the mean ± standard deviation (n = 10). *P < 0.05 (Tukey’s post-hoc multiple comparisons test). C-4W: control rats sacrificed at four weeks; RS-4W: post-PTA restenosis modeling for four weeks; HE: hematoxylin and eosin. (A color version of this figure is available in the online journal.)

Figure 3.

Western blot characterization of Nrf2 and anti-oxidant factors. (a) The expression of Nrf2 in rats after Nrf2-shRNA transfection. Four shRNAs were applied and successful transfection is indicated by the downregulation of Nrf2 expression. (b) Quantification of the results in (a). Nrf2-shRNA 2 was chosen as it showed the highest transfection efficiency. (c) Effect of NaHS and Nrf2 interference on redox balance in rats subjected to post-PTA restenosis. The expression of Nrf2 and the anti-oxidant factors HO-1, GSH, CSE, SOD, and CAT in rats subjected to restenosis and treated with NaHS, with or without Nrf2-shRNA transfection, was measured and quantified. In each case, the level of the anti-oxidant factor was decreased by restenosis, whereas the administration of NaHS significantly upregulated its expression. In addition, the effect of NaHS was attenuated to some degree by the presence of Nrf2-shRNA. (d) Quantification of the protein band gray values. The data are presented as the mean ± standard deviation (n = 10). *P < 0.05 (Tukey’s post-hoc multiple comparisons test). C-4W: control rats sacrificed at four weeks; RS-4W: post-PTA restenosis modeling for four weeks; HO-1: heme oxygenase-1; GSH: glutathione; CSE: cystathionine-γ-lyase; SOD: superoxide dismutase; CAT: catalase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Figure 4.

Effect of NaHS and Nrf2 interference on inflammation in rats subjected to post-PTA restenosis. Plasma levels of (a) IL-1β, (b) IL-6, (c) VCAM-1, and (d) ICAM-1 were measured by ELISA and quantified. In each case, the level of the inflammatory factor was increased by restenosis, whereas the administration of NaHS significantly downregulated its expression. In addition, the effect of NaHS was counteracted to some degree by the presence of Nrf2-shRNA. The data are presented as the mean ± standard deviation (n = 10). *P < 0.05 (Tukey’s post-hoc multiple comparisons test). C-4W: control rats sacrificed at four weeks; RS-4W: post-PTA restenosis modeling for four weeks; IL: interleukin; ICAM-1: intercellular cell adhesion molecule; VCAM-1: vascular cell adhesion molecule.

Figure 5.

Effect of NaHS and Nrf2 on the migration and proliferation of RAECs and RVSMCs. (a, e) Scratch assay to assess the migration of RAECs and RVSMCs subjected to no treatment (control), H₂S administration (100 μmol/L), Nrf2 overexpression (Nrf2), or Nrf2 interference (Nrf2-siRNA). Images were taken immediately and 48 h after scratching. (b, f) Quantification of gap closure after 48 h of scratching in RAECs and RVSMCs. (c, g) MTT assay of the relative proliferation of RAECs and RVSMCs over 96 h. Absorbance was measured at 24, 48, and 96 h. (d, h) EdU assay of the relative proliferation of RAECs and RVSMCs over 96 h. Absorbance was measured at 24, 48, 72, and 96 h. H₂S and Nrf2 promoted the migration and proliferation of RAECs but inhibited those of RVSMCs, whereas Nrf2-siRNA inhibited the migration and proliferation of RAECs and enhanced those of RVSMCs. The data are presented as the mean ± standard deviation (n = 3). *P < 0.05 (Tukey’s post-hoc multiple comparisons test); ^&P < 0.05 compared to control at the same time point (Tukey’s post-hoc multiple comparisons test); ^@P < 0.05 compared to control at 96 h (Tukey’s post-hoc multiple comparisons test). RAECs: rat aortic endothelial cells; RVSMCs: rat vascular smooth muscle cells.

Figure 6.

Effect of NaHS and Nrf2 on redox signaling in RAECs and RVSMCs. The expression of Nrf2, HO-1, GSH, CSE, SOD, and CAT after H₂S treatment (50, 100, and 200 µmol/L) or Nrf2 overexpression/interference (with corresponding negative controls, NC) in (a) RAECs and (b) RVSMCs. H₂S reinforced anti-oxidant defense by upregulating the anti-oxidant factors in a concentration-dependent manner. In addition, Nrf2 overexpression (denoted by “Nrf2”) evidently upregulated the expression of the measured anti-oxidant factors, whereas Nrf2-siRNA did the opposite compared to the control. The data are presented as the mean ± standard deviation (n = 3). *P < 0.05 (Tukey’s post-hoc multiple comparisons test). RAECs: rat aortic endothelial cells; RVSMCs: rat vascular smooth muscle cells; HO-1: heme oxygenase-1; GSH: glutathione; CSE: cystathionine-γ-lyase; SOD: superoxide dismutase; CAT: catalase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. (A color version of this figure is available in the online journal.)

Figure 7.

Effect of NaHS and Nrf2 on inflammatory signaling in RAECs and RVSMCs. The expression of NF-κB (subunits p65 and p50/105) after H₂S treatment (50, 100, and 200 µmol/L) or Nrf2 overexpression/interference (with corresponding negative controls, NC) in (a) RAECs and (b) RVSMCs. H₂S alleviated inflammatory response by downregulating p65 and p50/105 NF-κB in a concentration-dependent manner. In addition, Nrf2 overexpression (denoted by “Nrf2”) evidently downregulated the expression of p65 NF-κB, whereas Nrf2-siRNA did the opposite compared to the NC. Nrf2 overexpression or interference did not induce consistent trends in the expression of p50/105 NF-κB. The data are presented as the mean ± standard deviation (n = 3). *P < 0.05 (Tukey’s post-hoc multiple comparisons test). RAECs: rat aortic endothelial cells; RVSMCs: rat vascular smooth muscle cells; NF-κB: nuclear factor-kappa B.

Figure 8.

Proposed mechanism through which exogenous H2S mediates Nrf2-induced anti-oxidant defense and attenuates inflammatory signaling. H2S contributes to reduced oxidative stress via nuclear translocation and activation of Nrf2, resulting in the activation of downstream anti-oxidant factors including HO-1, GSH, CSE, SOD, and CAT to lower ROS production. Subsequently, the reduction in ROS levels leads to the inhibition of NF-κB signaling and suppression of pro-inflammatory genes such as interleukins. As a result, restenosis is alleviated. ARE: anti-oxidant response element; ROS: reactive oxygen species; IL: interleukin; HO-1: heme oxygenase-1; GSH: glutathione; CSE: cystathionine-γ-lyase; SOD: superoxide dismutase; CAT: catalase; NF-κB: nuclear factor-kappa B. (A color version of this figure is available in the online journal.)