Sesn2 attenuates the damage of endothelial progenitor cells induced by angiotensin II through regulating the Keap1/Nrf2 signal pathway

Abstract

Endothelial progenitor cell (EPC) dysfunction is an important physiopathological mechanism in the dynamics of the formation of atherosclerosis. It has been reported that angiotensin II (Ang-II) damages the function of EPCs in atherosclerotic plaque through induction of oxidative stress. Sestrin 2 (Sesn2) serves as an antioxidant role in oxidative stress, however, the exact mechanisms underlying the dynamics of how Sesn2 may factor into EPCs after Ang-II treatments needs to be illustrated. We isolated EPCs from human umbilical cord blood samples and treated with Ang-II. Western blotting, qRT-PCR, transwell assays, immunofluorescence and so on were used to investigate the mechanisms underlying the roles of Sesn2 in EPCs treated with Ang-II. Ang-II was found to promote the apoptosis of EPCs as well as inhibited the mRNA and protein expression of Sesn2. Upregulation of Sesn2 attenuated the negative effect of Ang-II. Sesn2 increased the protein expression of Nrf2 by enhancing P62-dependent autophagy. Silencing of Nrf2 enhanced the degree of apoptosis of EPCs as well as resulted in the impairment of EPC functions through inducing the promotion of (reactive oxygen species) ROS production. Our study results indicated that Sesn2 facilitated the viability of EPCs After treatment with Ang-II, as well as provided a potential therapeutic target to alleviate the progression of atherosclerosis.

Keywords: Nrf2; angiotensins; atherosclerosis; endothelial progenitor cells; sestrin 2.

Figure 1

Identification of EPCs and Ang-II increased Sesn2 and Nrf2 protein expression in EPCs. Identification and exposures of EPCs to Ang-II for prolonged periods of time. (A) EPCs (passage (P) 1) from human umbilical cord. Scale bars = 5 μm and Scale bars = 40 μm. (B) We used immunofluorescence to detect the measures of CD31, CD34, CD134, and VEGFR2. (C) The treatments with Ang-II caused an increased levels of Sesn2 mRNA in EPCs. (D) The levels of Nrf2 mRNA were unchanged in EPCs exposed to Ang-II. (E) The treatments with Ang-II increased the levels of Sesn2 and Nrf2 proteins in EPCs. (F) We used Hoechst 33258 to detect the apoptosis of EPCs and found that the treatments with Ang-II caused an increase in the apoptosis of EPCs. Scale bars = 5 μm. All experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM. (G) We used Hoechst 33258 staining to detect the apoptosis of EPCs. Scale bars = 5 μm.

Figure 2

Overexpression of Sesn2 promoted the survival of EPCs after Ang-II treatment. EPCs were transfected with Lenti-Sesn2 or NC before the treatments with Ang-II. (A) The effects of Lenti-Sesn2 were confirmed by using qRT-PCR. (B) The levels of Sesn2 proteins were upregulated significantly by the applications of Lenti-Sesn2. (C) The levels of Keap1, Nrf2, Bax, Bcl-2, Mit cyt-c, Cyto cyt-c, Cleaved-caspase 3 and GAPDH related proteins in EPCs were detected by using Western blotting. (D) The levels of Keap1 proteins in EPCs were detected by using Immunofluorescence. Scale bars = 2 μm. All experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Mit cyt-c: Mitochondria cytochrome C; Cyto cyt-c: Cytoplasm cytochrome C. Data are represented as mean +/- SEM.

Figure 3

Knockdown of Nrf2 could reverse the protective effect of Sesn2 in EPCs treated with Ang-II. EPCs were divided into treatment groups including for a control, Ang-II, Ang-II + Lenti-Sesn2, and Ang-II+ Lenti-Sesn2 + si-Nrf2 treatment groups. (A) The levels of Nrf2 mRNA were found to have decreased in EPCs transfected with siNrf2. (B) The levels of Nrf2 proteins were found to have been reduced in EPCs transfected with si-Nrf2. (C) Measures for Sesn2, Nrf2, Cleaved-caspase3, Bcl-2, Bax, and GAPDH protein expression in EPCs were determined by using Western blotting. (D) We used immunofluorescence to detect levels of caspase3 protein in ECPs. Scale bars = 2 μm. (E) We determined the migration of EPCs by using Transwell assays. Scale bars = 5 μm. All experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM.

Figure 4

Repression of autophagy inhibited the protective effect of Sesn2 in EPCs. EPCs were divided into treatment groups representing the control, CQ, Lenti-Sesn2, and Lenti-Sesn2 + CQ cohorts. (A) The levels of expression of proteins of Keap1, Nrf2, Cleaved-caspase3, Cleaved-caspase9, P62, LC-3, and GAPDH in EPCs were detected by use of Western blotting. (B) The levels of expression of proteins of Nrf2 in EPCs were determined by using Immunofluorescence. Scale bars = 2 μm. (C) Measures of the migration of EPCs were determined by using Transwell assays. All experiments were performed in triplicate. Scale bars = 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM.

Figure 5

Sesn2 protected the EPCs through regulating the autophagic flux. EPCs were divided into treatment groups representing the control, si-Sesn2, and si-Sesn2 + CQ. (A) The levels of P62, LC-3, Cleaved-caspase3, Keap1, Nrf-2, and of GAPDH proteins in EPCs. (B) Measures of ROS production in EPCs were assessed by the use of fluorescent dye H2DCFDA. Scale bars = 5 μm. (C) Measures of the migration of EPCs were determined by using Transwell assays. Scale bars = 20 μm. All experiments were performed in triplicate. Scale bars = 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM.

Figure 6

Upregulation of Nrf2 inhibited Ang-II-induced apoptosis and dysfunction of EPCs. EPCs were divided into treatment groups representative of the control, Ang-II, and Ang-II + Lenti-Nrf2. (A) The levels of Nrf2 mRNA were found to have increased significantly in EPCs transfected with Lenti-Nrf2. (B) The levels of Nrf2 proteins were found to have increased significantly in the Nrf2 samples transfected with Lenti-Nrf2. (C) The levels of for Bax, Bcl-2, Mit cyt-c, Cyto cyt-c, Cleaved-caspase 3, and GAPDH proteins in EPCs were detected by using Western blotting. (D) ROS production in EPCs was detected by the use of fluorescent dye H2DCFDA. Scale bars = 5 μm. (E) Tube formation abilities were assessed on Matrigel. All experiments were performed in triplicate. Scale bars = 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM.

Figure 7

NAC facilitated the survival and migration of EPCs after silencing of Nrf2. EPCs were divided into treatment groups representative of the control, si-Nrf2 and, si-Nrf2 + NAC. (A) The levels of Cleaved-caspase3, Bax, Bcl-2, Beclin-1, LC-3, and GAPDH proteins in EPCs were determined by using Western blotting. (B) We used Hoechst 33258 staining to detect the apoptosis of EPCs. Scale bars = 5 μm. (C) Migration of EPCs was determined by the application of wound healing assays (details given in methods). All experiments were performed in triplicate. Scale bars = 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control. Data are represented as mean +/- SEM.

Figure 8

Sesn2 serves as antioxidants by improving P62-dependent autophagic degradation of Keap1 and thereby promoting Nrf2 expression and activity. When Sesn2 binds to P62 and Keap1, P62 binds to LC3 simultaneously at autophagosomes and thereby promote Keap1 degradation. The degradation of Keap1 was found to have led to Nrf2 expression and activation, and then promoted the transcription of genes for various antioxidant enzymes.