The hydrogen sulfide donor, GYY4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E(-/-) mice

Abstract

Background and purpose: Atherosclerosis is associated with reduced vascular hydrogen sulfide (H2 S) biosynthesis. GYY4137 is a novel slow-releasing H2 S compound that may effectively mimic the time course of H2 S release in vivo. However, it is not known whether GYY4137 affects atherosclerosis.

Experimental approach: RAW 264.7 cells and human blood monocyte-derived macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with/without GYY4137. ApoE(-/-) mice were fed a high-fat diet for 4 weeks and administered GYY4137 for 30 days. Lipid and atherosclerotic lesions were measured by oil red O staining. Endothelium-dependent relaxation was assessed in response to acetylcholine. Superoxide production was detected by dihydroethidium staining. Expression of mRNA and protein were evaluated by quantitative real-time PCR and Western blot.

Key results: GYY4137 inhibited ox-LDL-induced foam cell formation and cholesterol esterification in cultured cells. GYY4137 decreased the expression of lectin-like ox-LDL receptor-1, iNOS, phosphorylated IκBα, NF-κB, ICAM-1, VCAM-1 and chemokines, including CXCL2, CXCR4, CXCL10 and CCL17, but increased the scavenger protein CD36, in ox-LDL-treated RAW 264.7 cells. In vivo, GYY4137 decreased aortic atherosclerotic plaque formation and partially restored aortic endothelium-dependent relaxation in apoE(-/-) mice. GYY4137 decreased ICAM-1, TNF-α and IL-6 mRNA expression as well as superoxide (O2 (-) ) generation in aorta. In addition, GYY4137 increased aortic eNOS phosphorylation and expression of PI3K, enhanced Akt Ser(473) phosphorylation and down-regulated the expression of LOX-1.

Conclusion and implications: GYY4137 inhibits lipid accumulation induced by ox-LDL in RAW 264.7 cells. In vivo, GYY4137 decreased vascular inflammation and oxidative stress, improved endothelial function and reduced atherosclerotic plaque formation in apoE(-/-) mice.

Keywords: arteriosclerosis; endothelial dysfunction; hydrogen sulfide; inflammation; oxidative stress.

Figure 1

Effect of GYY4137 on lipid accumulation and H₂S in RAW264.7 cells. RAW 264.7 cells were exposed to ox-LDL (100 μg·mL⁻¹) in the presence or absence of GYY4137 (50–400 μmol·L⁻¹) or ZYJ1122 (200 μmol·L⁻¹) for 24 h. Representative photographs showing RAW 264.7 cells stained with Oil Red O (A and B). Measurement of CE in RAW 264.7 cells (C and D). H₂S concentration in culture medium (E). Western blot analysis and quantification of CSE protein expression (F and G). ⁺P < 0.05, compared with no treatment, *P < 0.05, compared with treatment with ox-LDL (n = 4).

Figure 2

Effect of GYY4137 on lipid accumulation in human macrophages in vitro; the macrophages were exposed to ox-LDL (50 μg·mL⁻¹) for 48 h. Monocyte-derived macrophages from healthy donors (A) and patients with coronary heart disease (B) stained with Oil Red O. Measurement of CE in monocyte-derived macrophages from healthy donors (C) and patients with coronary heart disease (D). ⁺P < 0.05, compared with no treatment, *P < 0.05, compared with treatment with ox-LDL (n = 4–9).

Figure 3

Effect of GYY4137 on LOX-1, iNOS expression and chemokine release in RAW264.7 cells. RAW 264.7 cells were exposed to ox-LDL (100 μg·mL⁻¹) in the presence or absence of GYY4137 (50–400 μmol·L⁻¹) or ZYJ1122 (400 μmol·L⁻¹) for 24 h. Western blot analysis and quantification of LOX-1 protein expression (A). Quantification of iNOS mRNA by real-time PCR (B). Western blot analysis and quantification of iNOS protein expression (C). Quantification of CXCL2 (D), CXCR-4 (E), CXCL10 (F) and CD36 (G) as carried out by real-time PCR. ⁺P < 0.05, compared with no treatment, *P < 0.05, compared with treatment with ox-LDL (n = 4).

Figure 4

Effect of GYY4137 on NF-κB activation in RAW 264.7 cells. RAW 264.7 cells were exposed to ox-LDL (100 μg·mL⁻¹) in the presence or absence of GYY4137 (50–400 μmol·L⁻¹) or ZYJ1122 (200 μmol·L⁻¹) for 24 h. Western blots and quantification of NF-κB p65 protein expression (A). Representative examples of Western blots and quantification of IκBα phosphorylation (B), IκBα degradation (C) and ratio of IκBα phosphorylation to IκBα (D). NF-κB binding activity was evaluated by EMSA: (E-a) 1. Control, 2. ox-LDL alone, 3. ox-LDL+GYY4137 (50 μmol·L⁻¹), 4. ox-LDL+GYY4137 (100 μmol·L⁻¹), 5. ox-LDL+GYY4137 (200 μmol·L⁻¹), 6. ox-LDL+GYY4137 (400 μmol·L⁻¹). (E-b) Bands quantified by densitometric analysis. (F-a) 1. Control, 2. ox-LDL alone, 3. ox-LDL+GYY4137 (200 μmol·L⁻¹), 4. ox-LDL+ZYJ1122 (200 μmol·L⁻¹). Bands quantified by densitometric analysis (F-b). Quantification of ICAM-1 and VCAM-1 mRNA by real-time PCR (G-H). Western blot analysis and quantification of ICAM-1 and VCAM-1 protein expression (I-J). ⁺P < 0.05, compared with no treatment, *P < 0.05, compared with treatment with ox-LDL (n = 3–5).

Figure 5

Effect of GYY4137 on atherosclerotic lesion formation and CSE expression in aorta of apoE^−/− mice. Effect of GYY4137 (133 μmol·kg⁻¹·day⁻¹) on atherosclerotic lesion formation determined by Oil-Red O-staining of the aortic sinus (A, 40 × magnification). Quantitation of lesion area in Oil Red O-stained aortic sections by Image-Pro Plus software (B). Western blot analysis and quantification of CSE protein expression (C). ^‡P < 0.05, compared with WT mice, ^§P < 0.05, compared with apoE^−/− + NS (n = 5).

Figure 6

Effect of GYY4137 on superoxide production, LOX-1 expression, cytokines and ICAM-1 in aortas of apoE^−/− mice. Dihydroethidium (DHE) staining of aortic sections for superoxide production (A-a) and quantification of superoxide production(A-b). Western blot analyses and quantification of LOX-1 protein expression (B). Quantification of aortic TNF-α mRNA (C), IL-6 mRNA (D) and ICAM-1mRNA (E) was carried out by real-time PCR. ^‡P < 0.05, compared with WT mice, ^§P < 0.05, compared with apoE^−/− + NS (n = 5).

Figure 7

Effect of GYY4137 on aortic endothelium-dependent relaxation in intact apoE^−/− mice. Endothelium-dependent vasodilatation of aortic segments induced by acetylcholine (A). Western blots and quantification of phospho-eNOS protein (B), p85α subunit of PI3K (C) and phospho-Akt (D) protein. ^‡P < 0.05, compared with WT mice, ^§P < 0.05, compared with apoE^−/− + NS. n = 5.