Hydrogen Sulfide Attenuates Atherosclerosis in a Partially Ligated Carotid Artery Mouse model via Regulating Angiotensin Converting Enzyme 2 Expression

Figure 1. (A) Schematic diagram of low and oscillatory flow-induced atherosclerosis by partial ligation of LCA (PLA). Three branches of LCA, external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA) were ligated with 6-0 silk suture, while leaving the superior thyroid artery (STA) intact. (B) Representative light microscopy images of hematoxylin and eosin-stained LCA cross sections were taken from high-fat fed apoE−/− mice 1 or 4 weeks after PLA or sham operation. Scale bar for histological images = 100 μm. (C–E) Alterations in H2S biosynthesis during PLA induced atherosclerosis in high-fat fed apoE−/− mice. CSE expression (C) and activity (D) in LCA, and plasma H2S level (E) were assayed at indicated time points (1 or 4 weeks after PLA or sham operation). Results shown are the mean ± SEM (n = 6 animals in each group). *P < 0.05 for the comparison between mice 4 weeks after sham operation and mice 4 weeks after PLA. #P < 0.05 for the comparison between mice 1 week after PLA and mice 4 weeks after PLA.

Figure 2. Alterations in ACE2 expression during PLA induced atherosclerosis in high-fat fed apoE−/− mice. (A) Representative immunohistochemical images of LCA cross sections were taken from high-fat fed apoE−/− mice 1 or 4 weeks after PLA or sham operation. Scale bar for histological images = 50 μm. (B) LCA sections from mice 1 or 4 weeks after PLA or sham operation were quantified immunohistochemically for ACE2 positive staining. Levels of Ang-(1-7) (C) and Ang II (D) in LCA were assayed by ELISA at indicated time points (1 or 4 weeks after PLA or sham operation). Results shown are the mean ± SEM (n = 6 animals in each group). Arrowheads represented positive staining for ACE2 in endothelial cells. *P < 0.05 for the comparison between mice 1 week after sham operation and mice 1 week after PLA. **P < 0.05 for the comparison between mice 4 weeks after sham operation and mice 4 weeks after PLA. #P < 0.05 for the comparison between mice 1 week after PLA and mice 4 weeks after PLA.

Figure 3. Effect of NaHS or PAG on PLA-induced atherosclerosis in LCA. Representative light microscopy images of hematoxylin and eosin-stained (A) or Masson trichrome-stained (D) LCA cross sections were taken from sham-operated mice with saline, PLA mice with NaHS, PLA mice with PAG, or PLA mice with NaHS and MLN (intervention with MLN-4760 for 14 days). Scale bar for histological images = 100 μm. Lesion area of intima (B), intima/media ratio (C) and collagen deposits (E) in the neointimal hyperplasia in LCA were measured as described in Materials and Methods. Results shown are the mean ± SEM (n = 6 animals in each group). *P < 0.05 for the comparison between sham+saline and PLA+saline. **P < 0.05 for the comparison between PLA+saline and PLA+NaHS. ***P < 0.05 for the comparison between PLA+saline and PLA+PAG. #P < 0.05 for the comparison between PLA+NaHS and PLA+NaHS+MLN.

Figure 4. Effect of NaHS or PAG on ACE2 expression in LCA from mice with PLA-induced atherosclerosis. (A) Representative immunohistochemical images of LCA cross sections were taken from sham-operated mice with saline, PLA mice with NaHS, PLA mice with PAG, or PLA mice with NaHS and MLN (intervention with MLN-4760 for 14 days). Scale bar for histological images = 50 μm. (B) LCA sections were quantified immunohistochemically for ACE2 positive staining. (C) ACE2 expression in LCA was measured by Western blot as described in Materials and Methods. Levels of Ang (1-7) (D) and Ang II (E) in LCA were assayed by ELISA. Results shown are the mean ± SEM (n = 6 animals in each group). Arrowheads represented positive staining for ACE2 in endothelial cells. *P < 0.05 for the comparison between sham+saline and PLA+saline. **P < 0.05 for the comparison between PLA+saline and PLA+NaHS. ***P < 0.05 for the comparison between PLA+saline and PLA+PAG. #P < 0.05 for the comparison between PLA+NaHS and PLA+NaHS+MLN.

Figure 5. Effect of NaHS on ACE2 and Ang-(1-7) expression in HUVECs. In the time course study, cells were pre-incubated with saline or NaHS (100 μM) for 0, 6, 24 or 48 h (A–C,G,H). In the dose ranging study, cells were pre-incubated with saline or NaHS (50, 100, and 200 μM) for 24 h (D–F,I,J). ACE2 mRNA (A,C,D,F) and protein expression (B,C,E,F) were analyzed by real time PCR and Western blot respectively. Levels of Ang (1-7) (G,I) and Ang II (H,J) in culture medium were assayed by ELISA. The data are means ± SEM of at least three independent experiments. *P < 0.05 for the comparison between control at baseline and HUVECs treated with NaHS for 24 h. **P < 0.05 for the comparison between control at baseline and HUVECs treated with NaHS for 48 h. #P < 0.05 for the comparison between HUVECs treated with NaHS for 6 h and HUVECs treated with NaHS for 24 h. §P < 0.05 for the comparison between HUVECs treated with NaHS for 6 h and HUVECs treated with NaHS for 48 h. &P < 0.05 for the comparison between control at baseline and HUVECs treated with NaHS at a concentration of 100 μM. †P < 0.05 for the comparison between HUVECs treated with NaHS at a concentration of 50 μM and HUVECs treated with NaHS…

Figure 6. Effect of NaHS on ACE2 and Ang-(1-7) expression in HUVECs stimulated with LPS (100 ng/ml). HUVECs were pre-incubated with saline or NaHS (50, 100, and 200 μM) for 24 h and subsequently stimulated with saline or LPS (100 ng/ml) for 24 h. ACE2 mRNA (A,C) and protein (B,C) expression were analyzed by real time PCR and Western blot respectively. Levels of Ang (1-7) (D) and Ang II (E) in culture medium were assayed by ELISA. The data are means ± SEM of at least three independent experiments. *P < 0.05 for the comparison between control at baseline and LPS-stimulated HUVECs treated with saline. #P < 0.05 for the comparison between LPS-stimulated HUVECs treated with saline and LPS-stimulated HUVECs treated with NaHS at a concentration of 100 μM. &P < 0.05 for the comparison between LPS-stimulated HUVECs treated with saline and LPS-stimulated HUVECs treated with NaHS at a concentration of 200 μM. **P < 0.05 for the comparison between LPS-stimulated HUVECs treated with NaHS at a concentration of 50 μM and LPS-stimulated HUVECs treated with NaHS at a concentration of 200 μM.

Figure 7. Effect of NaHS on cytokine and chemokine production in unstimulated or LPS-stimulated HUVECs. HUVECs were pre-incubated with saline or NaHS (50, 100, and 200 μM) for 24 h and subsequently stimulated with saline or LPS (100 ng/ml) for 24 h. Production of cytokines (TNF-α, IL-6) (A,B) and chemokine (MCP-1) (C) was assayed by ELISA. The data are means ± SEM of at least three independent experiments. *P < 0.05 for the comparison between unstimulated HUVECs treated with saline and unstimulated HUVECs treated with NaHS at a concentration of 100 μM. #P < 0.05 for the comparison between unstimulated HUVECs treated with NaHS at a concentration of 50 μM and unstimulated HUVECs treated with NaHS at a concentration of 100 μM. †P < 0.05 for the comparison between unstimulated HUVECs treated with saline and unstimulated HUVECs treated with NaHS at a concentration of 200 μM. **P < 0.05 for the comparison between unstimulated HUVECs treated with saline and LPS-stimulated HUVECs treated with saline. &P < 0.05 for the comparison between LPS-stimulated HUVECs treated with saline and LPS-stimulated HUVECs treated with NaHS at a concentration of 100 μM. §P < 0.05 for the comparison between…

Figure 8. Effect of pretreatment with DX600, a selective ACE2 antagonist on suppressing the production of cytokines and chemokine induced by NaHS in unstimulated or LPS-stimulated HUVECs. Before pre-incubation with NaHS (100 μM) for 24 h, HUVECs were pre-treated with DX600 (1 μM) for 2 h. Then the cells were stimulated with saline or LPS (100 ng/ml) for 24 h. Production of cytokines (TNF-α, IL-6) (A,B) and chemokine (MCP-1) (C) was assayed by ELISA. The data are means ± SEM of at least three independent experiments. *P < 0.05 for the comparison between control at baseline and unstimulated HUVECs treated with NaHS. #P < 0.05 for the comparison between unstimulated HUVECs treated with NaHS and unstimulated HUVECs treated with NaHS and DX600. §P < 0.05 for the comparison between control at baseline and LPS-stimulated HUVECs treated with saline. **P < 0.05 for the comparison between LPS-stimulated HUVECs treated with saline and LPS-stimulated HUVECs treated with NaHS. &P < 0.05 for the comparison between LPS-stimulated HUVECs treated with NaHS and LPS-stimulated HUVECs treated with NaHS and DX600.