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Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Fox B, Schantz JT, Haigh R, Wood ME, Moore PK, Viner N, Spencer JP, Winyard PG, Whiteman M - J. Cell. Mol. Med. (2012)

Bottom Line: Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment.These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs.H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

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Affiliation: Peninsula Medical School, University of Exeter, St. Luke's Campus, Exeter, Devon, UK.

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Effect of H2S on oxidative stress induced mitochondrial dysfunction. (A–C) Mitochondrial membrane potential (DCm) and assessment of (D) ATP and (E) cytochrome c levels. MPCs were exposed to (A) SIN-1 (500 μmol/l), (B) H2O2 (200 μmol/l) and 4-HNE (25 μmol/l) for 18 hrs and mitochondrial membrane potential (DCm) determined by flow cytometry using tetramethylrhodamine methyl ester (TMRM; 50 nmol/l) as described in Materials and Methods. PAG (1 mmol/l) and AOAA (1 mmol/l) were used to inhibit endogenous CSE and CBS activity, respectively. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added as a positive control. (C) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of mitochondrial DCm in MPCs. (D, E) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of ATP (D) and cytoplasmic levels of cytochrome c (E). Cells were treated with CSE or CBS siRNA or non-coding controls (NCC-1 and NCC-2) as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. GYY4137 *P < 0.05, **P < 0.01, ***P < 0.001, cf. oxidant-treated cells.
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fig08: Effect of H2S on oxidative stress induced mitochondrial dysfunction. (A–C) Mitochondrial membrane potential (DCm) and assessment of (D) ATP and (E) cytochrome c levels. MPCs were exposed to (A) SIN-1 (500 μmol/l), (B) H2O2 (200 μmol/l) and 4-HNE (25 μmol/l) for 18 hrs and mitochondrial membrane potential (DCm) determined by flow cytometry using tetramethylrhodamine methyl ester (TMRM; 50 nmol/l) as described in Materials and Methods. PAG (1 mmol/l) and AOAA (1 mmol/l) were used to inhibit endogenous CSE and CBS activity, respectively. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added as a positive control. (C) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of mitochondrial DCm in MPCs. (D, E) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of ATP (D) and cytoplasmic levels of cytochrome c (E). Cells were treated with CSE or CBS siRNA or non-coding controls (NCC-1 and NCC-2) as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. GYY4137 *P < 0.05, **P < 0.01, ***P < 0.001, cf. oxidant-treated cells.

Mentions: There is increasing evidence for perturbed mitochondrial function in inflammatory and degenerative joint disease [29,43,58] and that mitochondrial dysfunction may potentiate inflammation [29]. Because SIN-1 [26], 4-HNE [42] and H2O2 [59] have previously been shown to collapse mitochondrial DCm in human chondrocytes, we next investigated whether pharmacological H2S could protect mitochondria in situ from oxidative injury by examining mitochondrial DCm, mitochondrial ATP and cytoplasmic cytochrome c. In MPCs, inhibition of CSE (with PAG) or CBS (with AOAA) significantly increased the loss of mitochondrial DCm induced by SIN-1 (Fig. 8A), H2O2 and 4-HNE (Fig. 8B), whereas GYY4137 added at a final concentration of 200 μmol/l or 500 μmol/l significantly inhibited mitochondrial toxicity. In agreement with these observations, treatment of chondrocytes with CSE-siRNA or CBS-siRNA but not non-coding controls (see figure legend) similarly increased oxidative stress-induced collapse of mitochondrial DCm. In these additional experiments, GYY4137 (500 μmol/l) also inhibited SIN-1-, H2O2- and 4-HNE-induced mitochondrial toxicity in CSE-siRNA and CBS-siRNA-treated chondrocytes (Fig. 8C).


Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Fox B, Schantz JT, Haigh R, Wood ME, Moore PK, Viner N, Spencer JP, Winyard PG, Whiteman M - J. Cell. Mol. Med. (2012)

Effect of H2S on oxidative stress induced mitochondrial dysfunction. (A–C) Mitochondrial membrane potential (DCm) and assessment of (D) ATP and (E) cytochrome c levels. MPCs were exposed to (A) SIN-1 (500 μmol/l), (B) H2O2 (200 μmol/l) and 4-HNE (25 μmol/l) for 18 hrs and mitochondrial membrane potential (DCm) determined by flow cytometry using tetramethylrhodamine methyl ester (TMRM; 50 nmol/l) as described in Materials and Methods. PAG (1 mmol/l) and AOAA (1 mmol/l) were used to inhibit endogenous CSE and CBS activity, respectively. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added as a positive control. (C) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of mitochondrial DCm in MPCs. (D, E) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of ATP (D) and cytoplasmic levels of cytochrome c (E). Cells were treated with CSE or CBS siRNA or non-coding controls (NCC-1 and NCC-2) as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. GYY4137 *P < 0.05, **P < 0.01, ***P < 0.001, cf. oxidant-treated cells.
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fig08: Effect of H2S on oxidative stress induced mitochondrial dysfunction. (A–C) Mitochondrial membrane potential (DCm) and assessment of (D) ATP and (E) cytochrome c levels. MPCs were exposed to (A) SIN-1 (500 μmol/l), (B) H2O2 (200 μmol/l) and 4-HNE (25 μmol/l) for 18 hrs and mitochondrial membrane potential (DCm) determined by flow cytometry using tetramethylrhodamine methyl ester (TMRM; 50 nmol/l) as described in Materials and Methods. PAG (1 mmol/l) and AOAA (1 mmol/l) were used to inhibit endogenous CSE and CBS activity, respectively. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added as a positive control. (C) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of mitochondrial DCm in MPCs. (D, E) Effect of CSE and CBS-siRNA treatment on oxidant induced loss of ATP (D) and cytoplasmic levels of cytochrome c (E). Cells were treated with CSE or CBS siRNA or non-coding controls (NCC-1 and NCC-2) as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. GYY4137 *P < 0.05, **P < 0.01, ***P < 0.001, cf. oxidant-treated cells.
Mentions: There is increasing evidence for perturbed mitochondrial function in inflammatory and degenerative joint disease [29,43,58] and that mitochondrial dysfunction may potentiate inflammation [29]. Because SIN-1 [26], 4-HNE [42] and H2O2 [59] have previously been shown to collapse mitochondrial DCm in human chondrocytes, we next investigated whether pharmacological H2S could protect mitochondria in situ from oxidative injury by examining mitochondrial DCm, mitochondrial ATP and cytoplasmic cytochrome c. In MPCs, inhibition of CSE (with PAG) or CBS (with AOAA) significantly increased the loss of mitochondrial DCm induced by SIN-1 (Fig. 8A), H2O2 and 4-HNE (Fig. 8B), whereas GYY4137 added at a final concentration of 200 μmol/l or 500 μmol/l significantly inhibited mitochondrial toxicity. In agreement with these observations, treatment of chondrocytes with CSE-siRNA or CBS-siRNA but not non-coding controls (see figure legend) similarly increased oxidative stress-induced collapse of mitochondrial DCm. In these additional experiments, GYY4137 (500 μmol/l) also inhibited SIN-1-, H2O2- and 4-HNE-induced mitochondrial toxicity in CSE-siRNA and CBS-siRNA-treated chondrocytes (Fig. 8C).

Bottom Line: Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment.These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs.H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Peninsula Medical School, University of Exeter, St. Luke's Campus, Exeter, Devon, UK.

Show MeSH
Related in: MedlinePlus