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A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury.

Santamaría B, Benito-Martin A, Ucero AC, Aroeira LS, Reyero A, Vicent MJ, Orzáez M, Celdrán A, Esteban J, Selgas R, Ruíz-Ortega M, Cabrera ML, Egido J, Pérez-Payá E, Ortiz A - PLoS ONE (2009)

Bottom Line: Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells.Specific caspase-8 and caspase-3 inhibitors were similarly effective.Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration.

View Article: PubMed Central - PubMed

Affiliation: Dialysis Unit, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Instituto Reina Sofía de Investigación Nefrológica, Madrid, Spain.

ABSTRACT

Background: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

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A nanoconjugate Apaf-1 inhibitor (PGA-peptoid QM56) inhibits caspase activation and apoptosis.A) Quantification of apoptosis in HOMC by flow cytometry. HOMC were preincubated with different concentrations of inhibitors or vehicle and cultured with TNFα and IFNγ for 48 hours. Mean±SEM of 3 different experiments *p<0.001vs TNFα/IFNγ. B) QM56 prevents procaspase-3 processing and the appearance of active p17 and p19 caspase-3 fragments 24 h following exposure to TNFα/IFNγ in HOMC. zVAD stalled the process of caspase activation at the level of the p21 precursor as previously described [73], [74]. Binding of zVAD.fmk to this p21 intermediate blocks its activity [74]. Western blot, representative of 3 independent experiments. C) Representative flow cytometry of DNA content diagrams. Cells were preincubated with QM56 and then cultured with TNFα/IFNγ for 48 h. Note the decreased number of hypodiploid apoptotic HPMC in TNFα/IFNγ/QM56 treated cells (black arrow). Inset: nuclear apoptotic morphology in cells exposed to TNFα/IFNγ and stained with DAPI (white arrows). D) Quantification of apoptosis by flow cytometry of DNA content in HPMC. Mean±SEM of 6 different experiments. *p<0.002 vs cells treated with TNFα/IFNγ/QM56.
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pone-0006634-g004: A nanoconjugate Apaf-1 inhibitor (PGA-peptoid QM56) inhibits caspase activation and apoptosis.A) Quantification of apoptosis in HOMC by flow cytometry. HOMC were preincubated with different concentrations of inhibitors or vehicle and cultured with TNFα and IFNγ for 48 hours. Mean±SEM of 3 different experiments *p<0.001vs TNFα/IFNγ. B) QM56 prevents procaspase-3 processing and the appearance of active p17 and p19 caspase-3 fragments 24 h following exposure to TNFα/IFNγ in HOMC. zVAD stalled the process of caspase activation at the level of the p21 precursor as previously described [73], [74]. Binding of zVAD.fmk to this p21 intermediate blocks its activity [74]. Western blot, representative of 3 independent experiments. C) Representative flow cytometry of DNA content diagrams. Cells were preincubated with QM56 and then cultured with TNFα/IFNγ for 48 h. Note the decreased number of hypodiploid apoptotic HPMC in TNFα/IFNγ/QM56 treated cells (black arrow). Inset: nuclear apoptotic morphology in cells exposed to TNFα/IFNγ and stained with DAPI (white arrows). D) Quantification of apoptosis by flow cytometry of DNA content in HPMC. Mean±SEM of 6 different experiments. *p<0.002 vs cells treated with TNFα/IFNγ/QM56.

Mentions: Formation of the apoptosome is a key event in the apoptotic signaling pathway. First-in-class polyglutamic acid (PGA)-based Apaf-1 inhibitor nanoconjugates (PGA-peptoids) are well suited for in vivo applications [30]–[32]. The PGA-peptoid QM56 prevented, in a dose-dependent manner, mesothelial cell apoptosis induced by a combination of cytokines, as shown for HOMC (Fig. 4.A). The QM56 concentration chosen for the rest of the experiments (10 µM) had an antiapoptotic activity similar to 200 µM zVAD (Fig. 4.A). In cultured mesothelial cells QM56 prevented caspase-3 processing induced by cytokines, as shown for HOMC (Fig. 4.B) and completely prevented the increase in caspase 3 activity induced by TNFα/IFNγ in these cells (TNFα/IFNγ 1,7±0.4 vs TNFα/IFNγ/QM56 0,56±0.18 fold over control, p<0.03, not shown). Similar to observations with caspase inhibitors, QM56 prevented apoptosis induced by TNFα and IFNγ in mesothelial cells, as shown for HPMC (Fig. 4.C,D). QM56 did not modify the percentage of cells in the S+M phases of the cell cycle (not shown). By contrast to classical caspase inhibitors, QM56 restored the wound healing capacity of mesothelial cells in the presence of cytokines, as shown for HPMC (Fig. 5.A, B).


A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury.

Santamaría B, Benito-Martin A, Ucero AC, Aroeira LS, Reyero A, Vicent MJ, Orzáez M, Celdrán A, Esteban J, Selgas R, Ruíz-Ortega M, Cabrera ML, Egido J, Pérez-Payá E, Ortiz A - PLoS ONE (2009)

A nanoconjugate Apaf-1 inhibitor (PGA-peptoid QM56) inhibits caspase activation and apoptosis.A) Quantification of apoptosis in HOMC by flow cytometry. HOMC were preincubated with different concentrations of inhibitors or vehicle and cultured with TNFα and IFNγ for 48 hours. Mean±SEM of 3 different experiments *p<0.001vs TNFα/IFNγ. B) QM56 prevents procaspase-3 processing and the appearance of active p17 and p19 caspase-3 fragments 24 h following exposure to TNFα/IFNγ in HOMC. zVAD stalled the process of caspase activation at the level of the p21 precursor as previously described [73], [74]. Binding of zVAD.fmk to this p21 intermediate blocks its activity [74]. Western blot, representative of 3 independent experiments. C) Representative flow cytometry of DNA content diagrams. Cells were preincubated with QM56 and then cultured with TNFα/IFNγ for 48 h. Note the decreased number of hypodiploid apoptotic HPMC in TNFα/IFNγ/QM56 treated cells (black arrow). Inset: nuclear apoptotic morphology in cells exposed to TNFα/IFNγ and stained with DAPI (white arrows). D) Quantification of apoptosis by flow cytometry of DNA content in HPMC. Mean±SEM of 6 different experiments. *p<0.002 vs cells treated with TNFα/IFNγ/QM56.
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getmorefigures.php?uid=PMC2722088&req=5

pone-0006634-g004: A nanoconjugate Apaf-1 inhibitor (PGA-peptoid QM56) inhibits caspase activation and apoptosis.A) Quantification of apoptosis in HOMC by flow cytometry. HOMC were preincubated with different concentrations of inhibitors or vehicle and cultured with TNFα and IFNγ for 48 hours. Mean±SEM of 3 different experiments *p<0.001vs TNFα/IFNγ. B) QM56 prevents procaspase-3 processing and the appearance of active p17 and p19 caspase-3 fragments 24 h following exposure to TNFα/IFNγ in HOMC. zVAD stalled the process of caspase activation at the level of the p21 precursor as previously described [73], [74]. Binding of zVAD.fmk to this p21 intermediate blocks its activity [74]. Western blot, representative of 3 independent experiments. C) Representative flow cytometry of DNA content diagrams. Cells were preincubated with QM56 and then cultured with TNFα/IFNγ for 48 h. Note the decreased number of hypodiploid apoptotic HPMC in TNFα/IFNγ/QM56 treated cells (black arrow). Inset: nuclear apoptotic morphology in cells exposed to TNFα/IFNγ and stained with DAPI (white arrows). D) Quantification of apoptosis by flow cytometry of DNA content in HPMC. Mean±SEM of 6 different experiments. *p<0.002 vs cells treated with TNFα/IFNγ/QM56.
Mentions: Formation of the apoptosome is a key event in the apoptotic signaling pathway. First-in-class polyglutamic acid (PGA)-based Apaf-1 inhibitor nanoconjugates (PGA-peptoids) are well suited for in vivo applications [30]–[32]. The PGA-peptoid QM56 prevented, in a dose-dependent manner, mesothelial cell apoptosis induced by a combination of cytokines, as shown for HOMC (Fig. 4.A). The QM56 concentration chosen for the rest of the experiments (10 µM) had an antiapoptotic activity similar to 200 µM zVAD (Fig. 4.A). In cultured mesothelial cells QM56 prevented caspase-3 processing induced by cytokines, as shown for HOMC (Fig. 4.B) and completely prevented the increase in caspase 3 activity induced by TNFα/IFNγ in these cells (TNFα/IFNγ 1,7±0.4 vs TNFα/IFNγ/QM56 0,56±0.18 fold over control, p<0.03, not shown). Similar to observations with caspase inhibitors, QM56 prevented apoptosis induced by TNFα and IFNγ in mesothelial cells, as shown for HPMC (Fig. 4.C,D). QM56 did not modify the percentage of cells in the S+M phases of the cell cycle (not shown). By contrast to classical caspase inhibitors, QM56 restored the wound healing capacity of mesothelial cells in the presence of cytokines, as shown for HPMC (Fig. 5.A, B).

Bottom Line: Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells.Specific caspase-8 and caspase-3 inhibitors were similarly effective.Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration.

View Article: PubMed Central - PubMed

Affiliation: Dialysis Unit, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Instituto Reina Sofía de Investigación Nefrológica, Madrid, Spain.

ABSTRACT

Background: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

Show MeSH
Related in: MedlinePlus