Anti-atherogenic effect of hydrogen sulfide by over-expression of cystathionine gamma-lyase (CSE) gene

Abstract

Hydrogen sulfide (H2S) is an important gaseous signaling molecule that functions in physiological and pathological conditions, such as atherosclerosis. H2S dilates vessels and therefore has been suggested as an anti-atherogenic molecule. Since cystathionine gamma-lyase (CSE) enzyme is responsible for producing H2S in the cardiovascular system, we hypothesized that up-regulation of CSE expression in vivo with preservation of H2S bioactivity can slow down plaque formation and, can serve as a therapeutic strategy against atherosclerosis. In this study, C57BL/6 wild type mice (WT), ApoE knockout mice (KO) and transgenic ApoE knockout mice overexpressing CSE (Tg/KO) at four weeks of age were weaned. They were then fed with either normal or atherogenic diet for 12 weeks. At week 16, serial plasma lipid levels, body weight, and blood pressure were measured prior to euthanization of the mice and the size of atherosclerotic plaques at their aortic roots was measured. Tg/KO mice showed an increase in endogenous H2S production in aortic tissue, reduced atherosclerotic plaque sizes and attenuation in plasma lipid profiles. We also showed an up-regulation in plasma glutathionine peroxidase that could indicate reduced oxidative stress. Furthermore, there was an increase in expression of p-p53 and down regulation of inflammatory nuclear factor-kappa B (NF-κB) in aorta. To conclude, alteration of endogenous H2S by CSE gene activation was associated with reduced atherosclerosis in ApoE-deficient mice. Up-regulation of CSE/H2S pathway attenuates atherosclerosis and this would be a potential target for therapeutic intervention against its formation.

Figure 1. CSE expression in heart and aorta.

CSE mRNA levels of (A) heart tissue (n = 6) and (B) aorta (n = 6) in Tg/KO mice were increased when compared to KO mice. (C) The relationship between the heart CSE mRNA level and aortic mRNA level was significantly positive correlated. (D) Besides, protein level (n = 6) was significantly increased in aorta homogenates of Tg/KO mice on either normal or atherogenic diet. (E) Representative immunohistochemical staining also indicated higher CSE expression in aorta root in Tg/KO mice as compared to the KO mice in both diets (Magnification 4×; Insert magnification 20×).

Figure 2. Direct relationship between aortic CSE expression and H₂S production.

(A) With overexpression of CSE gene in Tg/KO mice, H₂S production in heart was considerably up-regulated (n = 6). (B) Direct relationship between aorta CSE expression and heart H₂S generation was also revealed, showing significant positive correlation between them.

Figure 3. Body weight and systolic blood pressure in CSE transgenic ApoE-KO (Tg/KO) mice.

(A) Body weight gain changes during the feeding periods with different diets. Both KO and Tg/KO mice (n = 10) showed increase of body weights with same diet but the weight gain was significantly different. Mice fed with normal diet had higher weight gain than that with atherogenic diet. Moreover, it also showed that Tg/KO mice had fewer weight gains in both normal and atherogenic diets as compared to KO mice. (B) Daily food intake was similar in both KO and Tg/KO (n = 6). (C) Both KO and Tg/KO mice (n = 10) did not have significantly change with the systolic blood pressure, indicating that Tg/KO mice did not cause hypotension after insertion of CSE gene.

Figure 4. Anti-atherogenic effect of CSE gene activation with higher H₂S production.

(A) Plasma total cholesterol (n = 8 to 10), (B) LDL/VLDL cholesterol (n = 8 to 10) and (C) triglyceride level (n = 6) were significantly reduced in Tg/KO mice after feeding with 12 weeks of normal diet or atherogenic diet when comparing to KO mice. (D) Relationship between aortic CSE mRNA expression level and levels of plasma lipids. There was a significant negative relationship between CSE mRNA expression and levels of total cholesterol and LDL/VLDL cholesterol. However, its relationship with triglyceride was not statistically significant.

Figure 5. Increased plasma level of adipinectin in CSE transgenic ApoE-KO (Tg/KO) mice.

(A) Plasma adiponectin level (n = 6) was higher in Tg/KO mice when compared to the KO mice in both diets. (B) Relationship between plasma level of adiponectin and H₂S production rate was positively correlated.

Figure 6. Reduced level of oxidative stress by activation of CSE gene.

Improved plasmid lipid levels in Tg/KO mice exhibits reduced level of oxidative stress as indication by higher level of (A) glutathionine peroxidase (GPx) in plasma (n = 6), (B) blood glutathionine (GSH) (n = 6) and (C) total glutathionine (both GSH and GSSG) (n = 6).

Figure 7. Overexpression of CSE inhibits ApoE deficient mice from atherosclerosis.

(A) Atherosclerotic lesion damages in Tg/KO mice improved in both diets when compared to KO mice (Magnification 4×). (B) Quantitative comparisons of the mean percentage of plaque areas in aortic root between KO and Tg/KO mice were calculated using ImageJ software (n = 6). (C) There was a significant negative correlation between aortic CSE mRNA expression and lesion size from aortic root in mice who received both normal diet and atherogenic diet.

Figure 8. Quantitative analysis of protein expression levels in the aorta of KO and Tg/KO mice.

(A) Representative immunoblot showed that p-p53 increased in Tg/KO mice when compared to those in KO mice (n = 6). (B) Immunoblot showed the expression levels of NF-κB pathway including (C) IKK, (D) IκBα and (E) NF-κB p65 (n = 6). Correlation of each protein expression in (B) with H₂S production was shown in (F).