Cystathionine β-synthase inhibition is a potential therapeutic approach to treatment of ischemic injury

Abstract

Hydrogen sulfide (H2S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H2S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H2S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H2S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H2S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.

Keywords: cystathioine β-synthase; cysteine; homocysteine; hydrogen sulfide; oxygen glucose deprivation; stroke.

Figure 1.

Lentiviral vector transduced CBS-overexpressing (CBSOE) SH-SY5Y cells. (a) Fluorescence (left) and phase contrast (right) micrographs of SH-SY5Y CBSOE cells. Fluorescence indicates CBS-EGFP expression. (b) Western blot results confirming that CBS was markedly expressed compared with nontransduced control (C) cells. Only the full-length CBS (63 kDa) was detected but not the truncated CBS (45 kDa). (c) H₂S synthesizing activity of SH-SY5Y CBSOE cells measured at varying concentrations of cysteine (Cys) and homocysteine (Hcy). Data are presented as mean ± SEM, n = 3. ANOVA: F(5, 12) = 25.125, p < .05; ***p < .001 against no substrate control by Bonferroni. CBS = cystathionine β-synthase; H₂S = hydrogen sulfide; ANOVA = analysis of variance; CBSOE = CBS-overexpressing; Cys = cysteine; Hcy = homocysteine.

Figure 2.

The effects of OGD on cell viability of CBSOE cells under various conditions that affected H₂S production. (a and b) MTT assay and LDH release results are used as indicators of cell viability. CBSOE or control SH-SY5Y cells were exposed to either no, low (0.1 mM Cys + 0.01 mM Hcy), or high (1 mM Cys + 0.1 mM Hcy) substrate concentrations with or without 24 hr OGD. Control cells without OGD (a) or cells treated with 2% Triton (b) were used as 100%. ANOVA for CBSOE cells: F(2, 9) = 6.327, p < .05 (a) and F(2, 6) = 15.000, p < .01 (b). *p < .05, **p < .001 against CBSOE cells subjected to OGD without substrates by Bonferroni; #p < .05 against control cells subjected to OGD in the presence of high substrates by independent t test. Data are mean ± SEM, n = 3–4. (c and d) CBS inhibition by AOAA reversed the enhanced cell death in CBSOE cells subjected to OGD in the presence of high substrates. Cell viability is expressed as fraction to control without OGD. ANOVA for high substrate conditions: F(3, 8) = 9.799 (c) and F(3, 12) = 7.322 (d), p < .01. *p < .05, **p < .001 against without AOAA by Bonferroni. Data are mean ± SEM, n = 3–4. OGD = oxygen and glucose deprivation; CBSOE = CBS-overexpressing; H₂S = hydrogen sulfide; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH = lactate dehydrogenase; ANOVA = analysis of variance; CBS = cystathionine β-synthase; AOAA = aminooxyacetic acid; Cys = cysteine; Hcy = homocysteine.

Figure 3.

Comparison of endogenously produced and exogenous H₂S. (a and b) MTT test and LDH release results are used as indicators of cell viability. Primary cortical astrocyte were exposed to either no, low (0.1 mM Cys + 0.01 mM Hcy), or high (1 mM Cys + 0.1 mM Hcy) substrate concentrations with or without 8 hr OGD. Control cells without OGD (a) or cells treated with 2% Triton (b) were used as 100%. Data are presented as mean ± SEM, n = 3–4. ANOVA for 8 hr OGD: F(2, 9) = 5.013, p < .05 (a) and F(2, 9) = 12.107, p < .01 (b). *p < .05, **p < .01 against no substrates by Bonferroni. (c and d) Primary cortical astrocytes were exposed to either 100 or 300 µM NaHS and then subjected to 8 hr OGD. Data are presented as mean ± SEM, n = 4–6 (c) or 8 (d). ANOVA: F(2, 12) = 8.065 (c) and F(2, 21) = 6.988 (d), p < .01. **p < .01 against without NaHS by Bonferroni. H₂S = hydrogen sulfide; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH = lactate dehydrogenase; OGD = oxygen and glucose deprivation; ANOVA = analysis of variance; Cys = cysteine; Hcy = homocysteine.

Figure 4.

Effects of OGD on CBS expression in primary cultures of cortical astrocytes. (a) Representative immunocytochemical staining of primary cortical astrocytes after OGD (0.5 to 3 hr) for CBS (green) and GFAP (red). (b) Representative Western blot images showing the increased expression of both the full length CBS (63 kDa) and truncated CBS (45 kDa) after OGD. (c) Densitometry measurement of full length (63 kDa) CBS and truncated (45 kDa) CBS expression after OGD. Data are presented as mean ± SEM, n = 3. ANOVA for CBS 63 kDa: F(3, 8) = 12.193, p < .01. *p < .05; **p < .01 against without OGD by Bonferroni. ANOVA for CBS 45 kDa: F(3, 8) = 10.808, p < .05; *p < .05; **p < .01 against without OGD by Bonferroni. OGD = oxygen and glucose deprivation; CBS = cystathionine β-synthase; ANOVA = analysis of variance.

Figure 5.

CBS expression in the cerebral cortex after pMCAO. (a) Representative Western blot results on CBS expression at 3 to 24 hr post-pMCAO in the cerebral cortex. (b) Densitometry measurement of CBS expression over 24 hr after pMCAO. Protein expression is expressed relative to the ipsilateral control C. Data are presented as mean ± SEM, n = 3–4. ANOVA for CBS 63 kDa on the ipsilateral side: F(3, 12) = 0.608, p = .623. ANOVA for CBS 45 kDa on the ipsilateral side: F(3, 8) = 6.702, p < .05; #p < .05 against ipsilateral control by Bonferroni; **p < .005 against the contralateral side by independent t test. (c) Immunofluorescent staining of CBS showed increased CBS expression in the cortex 8 hr after pMCAO (a) compared with sham-control rats (b). Scale bar: 200 µm. (c) shows the location where the CBS immunofluorescent photomicrographs were taken. (d) Colocalization of CBS (green) and GFAP (red, top panel) and lack of colocalization of CBS (green) and NeuN (red, bottom panel) in the cortex at 8 hr after pMCAO. Scale bar: 50 µm. CBS = cystathionine β-synthase; pMCAO = permanent middle cerebral artery occlusion; ANOVA = analysis of variance.

Figure 6.

CBS expression in the striatum after pMCAO. (a) Representative Western blot results on CBS expression at 3 to 24 hr post-pMCAO in the striatum. (b) Densitometry measurement of CBS expression over 24 hr after pMCAO. Protein expression is expressed relative to the ipsilateral control C. Data are presented as mean ± SEM, n = 3–4. ANOVA for CBS 63 kDa: F(3, 8) = 3.524, p = .068. ANOVA for CBS 45 kDa: F(3, 8) = 13.637, p < .05; #p < .05 against the ipsilateral control by Bonferroni; *p < .05 against the contralateral by independent t test. (c) Immunofluorescent staining of CBS showed increased CBS expression in the striatum 24 hr after pMCAO (a) compared with sham-control rats (b). Scale bar: 200 µm. (c) shows the location where the CBS immunofluorescent photomicrographs were taken. (d) Colocalization of CBS (green) and GFAP (red, top panel) and lack of colocalization of CBS (green) and NeuN (red, bottom panel) in the striatum at 24 hr after pMCAO. Scale bar: 50 µm. CBS = cystathionine β-synthase; pMCAO = permanent middle cerebral artery occlusion; ANOVA = analysis of variance.

Figure 7.

Infarct volumes at 8 and 24 hr post-pMCAO. (a) Representative TTC-stained sections showing unstained infarct areas. (b) Infarct volumes are calculated as % of total volume of the brain region after correction for edema using the contralateral hemisphere as control and presented as mean ± SEM, n = 3. *p < .01 by independent t test. TTC = 2,3,5-triphenyltetrazolium chloride; pMCAO = permanent middle cerebral artery occlusion.