The orphan nuclear receptor Nur77 inhibits low shear stress-induced carotid artery remodeling in mice

Abstract

Shear stress, particularly low and oscillatory shear stress, plays a critical pathophysiological role in vascular remodeling-related cardiovascular diseases. Growing evidence suggests that the orphan nuclear receptor Nur77 [also known as TR3 or nuclear receptor subfamily 4, group A, member 1 (NR4A1)] is expressed in diseased human vascular tissue and plays an important role in vascular physiology and pathology. In the present study, we used a mouse model of flow-dependent remodeling by partial ligation of the left common carotid artery (LCCA) to define the exact role of Nur77 in vascular remodeling induced by low shear stress. Following vascular remodeling, Nur77 was highly expressed in neointimal vascular smooth muscle cells (VSMCs) in the ligated carotid arteries. The reactive oxygen species (ROS) levels were elevated in the remodeled arteries in vivo and in primary rat VSMCs in vitro following stimulation with platelet-derived growth factor (PDGF). Further in vitro experiments revealed that Nur77 expression was rapidly increased in the VSMCs following stimulation with PDGF and H2O2, whereas treatment with N-acetyl cysteine (NAC, a ROS scavenger) reversed the increase in the protein level of Nur77 induced by H2O2. Moreover, Nur77 overexpression markedly inhibited the proliferation and migration of VSMCs, induced by PDGF. Finally, to determine the in vivo role of Nur77 in low shear stress-induced vascular remodeling, wild-type (WT) and Nur77-deficient mice were subjected to partial ligation of the LCCA. Four weeks following surgery, in the LCCAs of the Nur77‑deficient mice, a significant increase in the intima-media area and carotid intima-media thickness was noted, as well as more severe elastin disruption and collagen deposition compared to the WT mice. Immunofluorescence staining revealed an increase in VSMC proliferation [determined by the expression of proliferating cell nuclear antigen (PCNA)] and matrix metalloproteinase 9 (MMP-9) production in the Nur77-deficient mice. There was no difference in the number of intimal apoptotic cells between the groups. Taken together, our results indicate that Nur77 may be a sensor of oxidative stress and an inhibitor of vascular remodeling induced by low shear stress. Nur77, as well as its downstream cell signals, may thus be a potential therapeutic target for the suppression of vascular remodeling.

Figure 1

Expression of Nur77 in neointimal vascular smooth muscle cells (VSMCs) following vascular remodeling induced by low shear stress (LSS). (A) Representative images of H&E staining (scale bar, 100 µm) and quantification of the intima-media area and intima-media thickness in the control (Con) and LSS groups (^*p<0.05, n=8). (B) Representative images of Verhoeff-Van Gieson staining (VVG) and picrosirius red staining (PSR) (scale bar, 100 µm) and quantification of the collagen area (^*p<0.05, n=8). (C) Representative images of dual immunofluorescence staining of Nur77 and smooth muscle (SM)-α-actin, and quantification of percentage of Nur77-positive cells and the ratio of Nur77-positive area to the α-actin-positive area (^*p<0.05, n=6). Values represent the means ± SEM.

Figure 2

Platelet-derived growth factor (PDGF) induces Nur77 expression in primary rat vascular smooth muscle cells (VSMCs). (A and B) Primary rat VSMCs were serum-starved for 24 h and then stimulated with PDGF (20 ng/ml) for the indicated periods of time. (A) mRNA and (B) protein levels of Nur77 were quantified by RT-qPCR and western blot analysis, respectively (^*p<0.05). (C) Primary rat VSMCs were treated with increasing concentrations of PDGF for 1 h. Protein expression of Nur77 was examined by western blot analysis. (D) Representative confocal images of rat VSMCs treated with the vehicle or PDGF (20 ng/ml) for 1 h and stained for Nur77 (green) and nuclei (blue). Values represent the means ± SEM. Con, control.

Figure 3

Upregulation of Nur77 is reactive oxygen species (ROS)-dependent in primary rat vascular smooth muscle cells (VSMCs). (A) Primary rat VSMCs were treated with platelet-derived growth factor (PDGF) (20 ng/ml) for 30 min. ROS levels were detected by flow cytometry following DCFH-DA staining. (B) Representative images of dihydroethidium (DHE) staining (scale bar, 100 µm) and quantification of DHE staining intensity in control (Con) and low shear stress (LSS) groups (^*p<0.05, n=6). (C) Primary rat VSMCs were treated with increasing concentrations of H₂O₂ for 1 h. And protein expression of Nur77 was examined by western blot analysis. (D) Primary rat VSMCs were stimulated with H₂O₂ (100 µM) for the indicated periods of time. Protein expression of Nur77 was examined by western blot analysis. (E) Primary rat VSMCs were treated with vehicle or 5 mM N-acetyl cysteine (NAC) for 6 h prior to H₂O₂ stimulation for another 1 h. Protein expression of Nur77 was examined by western blot analysis (^*p<0.05). Values represent the means ± SEM.

Figure 4

Nur77 inhibits platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) proliferation and migration in primary VSMCs in vitro. (A) Representative images of green fluorescent protein (GFP)-Nur77 adenovirus (Ad-GFP-Nur77) and GFP control adenovirus (Ad-GFP) infection in VSMCs in vitro. (B and C) Representative images of (B) cell density and quantification, as measured by cell count and (C) cell counting kit-8 (CCK-8) assay in Ad-GFP-Nur77-infected cells compared with Ad-GFP-infected cells upon PDGF (20 ng/ml) stimulation (^*p<0.05). (D) Western blot analysis measuring levels of GFP-Nur77, cyclin D1 and proliferating cell nuclear antigen (PCNA). β-actin was used as an internal control. (E) Representative images of the in vitro scratch-wound assay and quantification of number of migrated cells (^*p<0.05). Values represent the means ± SEM. Con, control.

Figure 5

Nur77 deletion enhances vascular remodeling induced by low shear stress (LSS) in vivo. (A) Representative images of H&E staining (scale bar, 100 µm) and quantification of the intima-media area and intima-media thickness in wild-type (WT) and Nur77-deficent mice (^*p<0.05, n=8). (B) Representative images of Verhoeff-Van Gieson (VVG) staining (scale bar, 25 µm), picrosirius red staining (PSR) (scale bar, 100 µm), and quantification of collagen area in WT and Nur77-deficent mice (^*p<0.05, n=8). Arrows point to disrupted elastin structure. Values represent the means ± SEM.Con, control.

Figure 6

Nur77 deletion enhances vascular smooth muscle cells (VSMCs) proliferation and matrix metalloproteinase (MMP) expression. (A) Representative images of dual immunofluorescence staining of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM)-α-actin (scale bar, 100 µm) and quantification of the ratio of PCNA-positive area to α-actin-positive area in wild-type (WT) and Nur77-deficent mice (^*p<0.05, n=6). (B and C) Representative images of (B) TUNEL staining (scale bar, 100 µm) and (C) quantification of the percentage of TUNEL-positive nuclei (^*p<0.05, n=6). (D and E) Representative images of (D) immunofluorescence staining of MMP-9 (scale bar, 100 µm) and (E) quantification of ratio of the MMP-9-positive area to intima area (^*p<0.05, n=6). Values represent the means ± SEM.