Activation of Nrf2 inhibits atherosclerosis in ApoE-/- mice through suppressing endothelial cell inflammation and lipid peroxidation

Abstract

Background: Nuclear erythroid 2-related factor 2 (Nrf2), a transcription factor, is critically involved in the regulation of oxidative stress and inflammation. However, the role of endothelial Nrf2 in atherogenesis has yet to be defined. In addition, how endothelial Nrf2 is activated and whether Nrf2 can be targeted for the prevention and treatment of atherosclerosis is not explored.

Methods: RNA-sequencing and single-cell RNA sequencing analysis of mouse atherosclerotic aortas were used to identify the differentially expressed genes. In vivo endothelial cell (EC)-specific activation of Nrf2 was achieved by injecting adeno-associated viruses into ApoE^-/- mice, while EC-specific knockdown of Nrf2 was generated in Cdh5^CreCas9^floxed-stopApoE^-/- mice.

Results: Endothelial inflammation appeared as early as on day 3 after feeding of a high cholesterol diet (HCD) in ApoE^-/- mice, as reflected by mRNA levels, immunostaining and global mRNA profiling, while the immunosignal of the end-product of lipid peroxidation (LPO), 4-hydroxynonenal (4-HNE), started to increase on day 10. TNF-α, 4-HNE, and erastin (LPO inducer), activated Nrf2 signaling in human ECs by increasing the mRNA and protein expression of Nrf2 target genes. Knockdown of endothelial Nrf2 resulted in augmented endothelial inflammation and LPO, and accelerated atherosclerosis in Cdh5^CreCas9^floxed-stopApoE^-/- mice. By contrast, both EC-specific and pharmacological activation of Nrf2 inhibited endothelial inflammation, LPO, and atherogenesis.

Conclusions: Upon HCD feeding in ApoE^-/- mice, endothelial inflammation is an earliest event, followed by the appearance of LPO. EC-specific activation of Nrf2 inhibits atherosclerosis while EC-specific knockdown of Nrf2 results in the opposite effect. Pharmacological activators of endothelial Nrf2 may represent a novel therapeutic strategy for the treatment of atherosclerosis.

Keywords: Atherosclerosis; Endothelial cells; Inflammation; Lipid peroxidation; Nuclear erythroid 2-related factor 2.

Fig. 1

Endothelial Nrf2 signaling was activated during atherogenesis. (A & B) Representative immunofluorescence images and statistical analysis of Nrf2 (red) in human aortas with intimal thickening. The EC marker vWF (green) was used. n = 6. (C) Dot plots showed the signatures of Nrf2 target genes from scRNA sequencing data in aortas of ApoE^−/− mice after 20-week high cholesterol diet (HCD) feeding. (D) A schematic diagram of the protocol for feeding mice with normal chow diet (NCD) or HCD for 3, 7, 10, 14 and 28 days was included. (E) ECs were isolated for qPCR to profile the expression of Nrf2 targeted genes (n = 6–8). (F) GESA analysis of the RNA sequencing results of EC components showed significant enrichment of Nrf2 signaling. (G) Representative immunofluorescence images of Nrf2 (in red) and CD31 (in green) in the ascending aorta of ApoE^−/− mice were shown. (H) Statistical analysis of the results in (G), n = 6–7. Results are means ± SD. Statistical analysis was performed using an unpaired two-tailed Student's t-test for (B), and one-way ANOVA followed by Tukey's test for (E) and (H). L, lumen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Endothelial inflammation and lipid peroxidation contributed to Nrf2 activation during atherogenesis. (A) The KEGG analysis results revealed the enriched signaling pathways of the aortic EC and non-EC components in ApoE^−/− mice after being fed with an HCD for 3, 7, 10, 14, and 28 days. (B) Representative immunofluorescence images of VCAM-1, TNF-α and 4-HNE in the aortas of ApoE^−/− mice. The yellow arrowhead denotes VCAM-1 (+), TNF-α (+) and 4-HNE (+) ECs in aortas. (C) Statistical analysis results for (B). (D & E) Representative immunofluorescence images of 4-HNE (red) and TFRC (red) in the carotid arteries of human atherosclerotic lesions. vWF (green) is an EC marker. (F) Statistical analysis results for (D & E). (G & H) HUVECs were exposed to 10 ng/mL TNF-α for 12 h, the expression of Nrf2 and target genes were measured using Western blot and qPCR. (I) HAECs were incubated with 50 μM 4-HNE for the indicated time, western blotting was used to determine the protein levels of Nrf2 and HO-1. (J) HUVECs were treated with erastin (20 μM) and the mRNA levels of Nrf2 targeted genes were detected by qPCR, n = 4–6. (K) HUVECs were pretreated with Fer (2 μM) for 0.5 h before being exposed to 75 μg/mL ox-LDL for 12 h, the mRNA levels of Nrf2 target genes were measured using qPCR, n = 4. (L) The schematic model illustrates the timing of EC inflammation, LPO, and Nrf2 activation. Results are means ± SD. Statistical analysis was performed using an unpaired two-tailed Student's t-test for (F), (G) and (K) and a one-way ANOVA followed by Tukey's test for (C) and (J). L, lumen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Knockdown Nrf2 increased lipid peroxidation and inflammation in endothelial cells. HUVECs were infected with lentivirus encoding Nrf2 shRNA and underwent RNA sequencing. (A) KEGG enrichment analysis revealed alterations in signaling pathways. GSEA was used to analyze the changes in the inflammatory response (B) and glutathione metabolism (C) pathways. (D) SCR and shNrf2-treated HUVECs were incubated with ox-LDL (75 μg/mL) for 36 h or H₂O₂ (100 μM) for 3 h. The liperfluo staining was used to measure the lipid peroxidation state. (E) Statistical analysis result for (D), n = 8–12. (F) SCR and shNrf2-treated HUVECs were pretreated with DFO (100 μM) for 0.5 h and then incubated with TNF-α (10 ng/mL) for 12 h. The representative images and statistical summary of liperfluo staining are shown, n = 9–11. (G) SCR- and shNrf2-treated HUVECs were pretreated with deferoxamine (DFO, 100 μM) for 0.5 h and then incubated with TNF-α (10 ng/mL) for 12 h. The mRNA expression of inflammatory genes was measured using qPCR, n = 4. (H) The THP1 monocyte adhesion to HUVECs, n = 6. (I) Statistical analysis for (H). Results are means ± SD. Statistical analysis was performed using an unpaired two-tailed Student's t-test for (E) and a one-way ANOVA followed by Tukey's test for (F), (G), and (I).

Fig. 4

Endothelium-specific knockdown Nrf2 exacerbated atherosclerosis. Cdh5^CreCas9^floxed-stopApoE^−/− mice were injected with AAV9-sgNrf2 via tail veins and maintained for 3 weeks followed by 8 weeks of HCD feeding. (A) Representative images and (B) statistical analysis of the atherosclerotic plaque stained by Oil Red O staining. n = 6–8. (C & D) The lesions of the aortic root were stained by Oil Red O staining and H&E staining. Immunofluorescence images (E & F) and statistical summary (G) of VCAM-1 and 4-HNE staining in aortas, n = 6–8. Results are means ± SD. Statistical analysis was performed using an unpaired two-tailed Student's t-test for (B), (D) and (G). L, lumen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Endothelium-specific activation of Nrf2 inhibited lipid peroxidation, inflammation and atherosclerosis. SCR- and shKeap1-infected HUVECs were incubated with TNF-α (10 ng/mL) for 12 h. (A) the mRNA levels of TNFA, VCAM1, and SELE, (B) the protein levels of VCAM-1, SELE, HO-1, NQO1, and Keap1 were measured. (C, D) SCR- and shKeap1-treated HUVECs were incubated with TNF-α (10 ng/mL) for 12 h; the THP1 monocyte adhesion to HUVECs was measured, n = 6. (E) SCR- and shKeap1-treated HUVECs were incubated with ox-LDL (75 μg/mL) for 36 h, liperfluo fluorescence intensity was measured by confocal microscopy, n = 11–12. (F) The summary statistical result for (E). Male and female ApoE^−/− mice were injected with AAV9-CMV-Cas9-U6-sgKeap1 via tail veins for 3 weeks, followed by 8 weeks of HCD feeding. (G) Representative images and (H) statistical analysis result of the atherosclerotic plaque stained by Oil Red O staining, n = 10. (I, J) The Oil Red O staining and H&E staining were performed to measure the aortic root lesions. Immunofluorescence images (K) and the statistical analysis result (L) of VCAM-1 and 4-HNE staining in aortas of ApoE^−/− mice, n = 6. Results are means ± SD. Statistical analysis was performed using two-way repeated measures ANOVA followed by Tukey's test for (A), (D), (H) and (J), unpaired two-tailed Student's t-test for (F) and (L). L, lumen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Dimethyl itaconate (DMI) suppressed endothelial inflammation through activating Nrf2. HUVECs were incubated with DMI (100 μM) for 15 h for RNA sequencing. (A) A heatmap of Nrf2 target genes was generated. (B) HUVECs were incubated with DMI for different time points to measure the protein levels of p62, Nrf2, and HO-1. (C, D) SCR and Nrf2 knockdown HUVECs were pretreated with DMI (100 μM, 3 h) followed by incubation with TNF-α (10 ng/mL) for 12 h, protein level of VCAM-1 was measured using Western blotting, n = 4. HUVECs were incubated with DMI (100 μM) for 1, 3, and 12 h H₂O₂ (100 μM, 1 h) was used as a positive control. The levels of GSH (E) and GSSG (F) were determined (n = 4–7). (G) Reactive oxygen species levels were measured by flow cytometry analysis after CM-H2DCFDA staining of HUVECs following treatment with DMI for 1, 3, and 12 h n = 9. (H) HUVECs were pretreated with DMI (100 μM) for 3 h followed by NAC (1 mM) and GSH (2 mM) for 12 h. The protein levels of Nrf2 and HO-1 were measured. (I) HUVECs were pretreated with DMI (100 μM) for 3 h followed by NAC (1 mM) and GSH (2 mM) in the presence of TNF-α (10 ng/mL) for 12 h; the protein levels of VCAM-1, SELE and HO-1 were measured. (J) Statistical summary of (I), n = 4. Treatment medium, DMEM (0.5 mM glucose, without pyruvate or glutamine). NAC, N-acetyl cysteine; GSH, glutathione. Results are means ± SD. Statistical analysis was performed using One-way ANOVA followed by Tukey's test for (D), (E–G) and (J).

Fig. 7

DMI inhibited atherosclerosis in ApoE−/− mice. (A) The protocol of DMI administration to ApoE^−/− mice. The prevention group received HCD for 4 weeks with daily DMI administration, while the treatment group received HCD for 4 weeks with daily DMI administration during the last 2 weeks. (B, C) After the 4-week treatment, plaque formation was measured in the aortas, aortic arch and descending thoracic aortas using Oil Red O staining, n = 9–10. (D, E) Representative immunofluorescence images and (F) statistical summary of VCAM-1 and 4-HNE staining in aortas of ApoE^−/− mice, n = 6. (G) A schematic diagram illustrates the sequential roles of inflammation, LPO and Nrf2 in atherogenesis. Results are means ± SD. Statistical analysis was performed using One-way ANOVA followed by Tukey's test for (C) and (F). The yellow arrowhead indicates VCAM-1 (+) or 4-HNE (+) cells in the aortic plaque. L, lumen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)