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Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice.

Bréchot N, Gomez E, Bignon M, Khallou-Laschet J, Dussiot M, Cazes A, Alanio-Bréchot C, Durand M, Philippe J, Silvestre JS, Van Rooijen N, Corvol P, Nicoletti A, Chazaud B, Germain S - PLoS ONE (2008)

Bottom Line: Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration.Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries.Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U833, Paris, France.

ABSTRACT

Background: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Methods and findings: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice.

Conclusion: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

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Depletion of circulating monocytes reverses tissue protection in tsp-1−/− mice.Histological analysis of gastrocnemius muscle sections at d4 after ischemia under Clo-Lip treatment. (A–D) show H&E staining. (E–H) and (I–L) show immunostainings of adjacent sections for macrophages using Mac-3 Ab, and endothelial cells using CD31 Ab, respectively. (A, B; E, F; I, J) scale bar = 500 µm; (C, D; G, H; K, L) scale bar = 200 µm. (M) Quantification of histologically different area types ratio; mean±S.E.M. are shown, n = 5. (N) Proportion of mice exhibiting macroscopic necrosis under Clo-Lip treatment, d4 after ischemia. * = p<0.05, n = 10. (O) Quantification of regenerating basic myofibers in necrotic areas, mean±S.E.M. are shown, * = p<0.05, n = 10. (P) Quantification of capillary density under Clo-Lip treatment in the different area types in gastrocnemius muscles, d4 after ischemia; Pbs-Lip injected mice served as control. Mean±SEM are shown. * = p<0.05; n = 5.
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pone-0003950-g008: Depletion of circulating monocytes reverses tissue protection in tsp-1−/− mice.Histological analysis of gastrocnemius muscle sections at d4 after ischemia under Clo-Lip treatment. (A–D) show H&E staining. (E–H) and (I–L) show immunostainings of adjacent sections for macrophages using Mac-3 Ab, and endothelial cells using CD31 Ab, respectively. (A, B; E, F; I, J) scale bar = 500 µm; (C, D; G, H; K, L) scale bar = 200 µm. (M) Quantification of histologically different area types ratio; mean±S.E.M. are shown, n = 5. (N) Proportion of mice exhibiting macroscopic necrosis under Clo-Lip treatment, d4 after ischemia. * = p<0.05, n = 10. (O) Quantification of regenerating basic myofibers in necrotic areas, mean±S.E.M. are shown, * = p<0.05, n = 10. (P) Quantification of capillary density under Clo-Lip treatment in the different area types in gastrocnemius muscles, d4 after ischemia; Pbs-Lip injected mice served as control. Mean±SEM are shown. * = p<0.05; n = 5.

Mentions: As macrophages in ischemic tissues exhibited distinct phenotypes in wt and tsp-1−/− animals, we next sought to evaluate their potential role in the tissue protection described in tsp-1−/− mice. We selectively depleted circulating monocytes in tsp-1−/− and wt mice using clodronate-containing liposomes (Clo-Lip) [35]. White blood cells counts on blood samples demonstrated that monocyte depletion was selective and equally efficient in both groups (Table S1). As expected, macrophage infiltrate almost disappeared (fig. 8F&8H) as well as phagocyted myofibers in ischemic muscles of wt mice (fig. 8B&8D). Regenerating basic myofibers were almost completely absent (fig. 8D&8O) and capillary density decreased (fig. 8L&8P). Taken together, these data highlight the crucial role played by macrophages in muscle healing from ischemia, both promoting tissue regeneration and angiogenesis.


Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice.

Bréchot N, Gomez E, Bignon M, Khallou-Laschet J, Dussiot M, Cazes A, Alanio-Bréchot C, Durand M, Philippe J, Silvestre JS, Van Rooijen N, Corvol P, Nicoletti A, Chazaud B, Germain S - PLoS ONE (2008)

Depletion of circulating monocytes reverses tissue protection in tsp-1−/− mice.Histological analysis of gastrocnemius muscle sections at d4 after ischemia under Clo-Lip treatment. (A–D) show H&E staining. (E–H) and (I–L) show immunostainings of adjacent sections for macrophages using Mac-3 Ab, and endothelial cells using CD31 Ab, respectively. (A, B; E, F; I, J) scale bar = 500 µm; (C, D; G, H; K, L) scale bar = 200 µm. (M) Quantification of histologically different area types ratio; mean±S.E.M. are shown, n = 5. (N) Proportion of mice exhibiting macroscopic necrosis under Clo-Lip treatment, d4 after ischemia. * = p<0.05, n = 10. (O) Quantification of regenerating basic myofibers in necrotic areas, mean±S.E.M. are shown, * = p<0.05, n = 10. (P) Quantification of capillary density under Clo-Lip treatment in the different area types in gastrocnemius muscles, d4 after ischemia; Pbs-Lip injected mice served as control. Mean±SEM are shown. * = p<0.05; n = 5.
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Related In: Results  -  Collection

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pone-0003950-g008: Depletion of circulating monocytes reverses tissue protection in tsp-1−/− mice.Histological analysis of gastrocnemius muscle sections at d4 after ischemia under Clo-Lip treatment. (A–D) show H&E staining. (E–H) and (I–L) show immunostainings of adjacent sections for macrophages using Mac-3 Ab, and endothelial cells using CD31 Ab, respectively. (A, B; E, F; I, J) scale bar = 500 µm; (C, D; G, H; K, L) scale bar = 200 µm. (M) Quantification of histologically different area types ratio; mean±S.E.M. are shown, n = 5. (N) Proportion of mice exhibiting macroscopic necrosis under Clo-Lip treatment, d4 after ischemia. * = p<0.05, n = 10. (O) Quantification of regenerating basic myofibers in necrotic areas, mean±S.E.M. are shown, * = p<0.05, n = 10. (P) Quantification of capillary density under Clo-Lip treatment in the different area types in gastrocnemius muscles, d4 after ischemia; Pbs-Lip injected mice served as control. Mean±SEM are shown. * = p<0.05; n = 5.
Mentions: As macrophages in ischemic tissues exhibited distinct phenotypes in wt and tsp-1−/− animals, we next sought to evaluate their potential role in the tissue protection described in tsp-1−/− mice. We selectively depleted circulating monocytes in tsp-1−/− and wt mice using clodronate-containing liposomes (Clo-Lip) [35]. White blood cells counts on blood samples demonstrated that monocyte depletion was selective and equally efficient in both groups (Table S1). As expected, macrophage infiltrate almost disappeared (fig. 8F&8H) as well as phagocyted myofibers in ischemic muscles of wt mice (fig. 8B&8D). Regenerating basic myofibers were almost completely absent (fig. 8D&8O) and capillary density decreased (fig. 8L&8P). Taken together, these data highlight the crucial role played by macrophages in muscle healing from ischemia, both promoting tissue regeneration and angiogenesis.

Bottom Line: Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration.Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries.Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U833, Paris, France.

ABSTRACT

Background: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Methods and findings: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice.

Conclusion: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Show MeSH
Related in: MedlinePlus