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. 2008;3(12):e3950.
doi: 10.1371/journal.pone.0003950. Epub 2008 Dec 16.

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

Nicolas Bréchot  1 , Elisa Gomez, Marine Bignon, Jamila Khallou-Laschet, Michael Dussiot, Aurélie Cazes, Cécile Alanio-Bréchot, Mélanie Durand, Josette Philippe, Jean-Sébastien Silvestre, Nico Van Rooijen, Pierre Corvol, Antonino Nicoletti, Bénédicte Chazaud, Stéphane Germain
Affiliations

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

Nicolas Bréchot et al. PLoS One. 2008.

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Thrombospondin-1 is expressed during critical hind limb ischemia in mice.
Thrombospondin-1 mRNA expression was analyzed in gastrocnemius muscle using in situ hybridization at d4 (D), d6 (F), d16 (H) and d21 (J) after ischemia. D0 (B) represents non ischemic tissue. Adjacent sections (respectively C, E, G, I and A were stained with H&E (Scale bar = 200 µm). (L) Gastrocnemius muscle analyzed at d4 for tsp-1 mRNA expression at a higher magnification (scale bar = 25 µm). (K, M) Adjacent sections were stained for macrophages (K) and endothelial cells (M), showing expression of tsp-1 mRNA in macrophages (see arrows in K&L) and endothelial cells (see arrowheads in L&M). Arrowheads in H&J show tsp-1 mRNA expression in endothelial cells at d16 and d21 respectively. (N) Western blot analysis of TSP-1 expression in gastrocnemius muscle at similar time points. Arrowhead = 150 kD.
Figure 2
Figure 2. Clinical necrosis is reduced in tsp-1−/− mice.
(A) Pictures show mice either protected from necrosis (left) or affected by necrosis (right). (B) Cumulative incidence of necrosis was followed during 21 days after ischemia in both wt and tsp-1−/− mice and represented as Kaplan-Meier estimates (n = 19; p<0.05 tsp-1−/− vs. wt).
Figure 3
Figure 3. Histological analysis showing tissue protection in tsp-1−/− mice.
(A&B) H&E staining of gastrocnemius muscle sections at d4 were performed in tsp-1−/− (A) and wt (B) mice (scale bar = 500 µm). Right panel show higher magnifications of preserved area (C), infiltrated area in tsp-1−/− (E) and in wt (G) and necrotic non-infiltrated area (I), with adjacent slides immunostained for macrophages using Mac-3 Ab (D, F, H, J, respectively); scale bar = 100 µm. (K) Quantification of histologically different area types surface (see text for definition); mean±S.E.M. are shown, * = p<0.05, n = 9. (L) Quantification of regenerating basic myofibers in necrotic areas, mean±S.E.M. are shown, ** = p<0.01, n = 9.
Figure 4
Figure 4. Postischemic angiogenesis is increased in tsp-1−/− mice.
(A&B) Capillary density of gastrocnemius muscle sections was assessed at d4 using CD31 immunostaining in tsp-1−/− (A) and wt (B) mice (scale bar = 500 µm). (C&D) CD31 immunostaining of infiltrated area in tsp-1−/− (C) and wt (D) mice; scale bar = 200 µm. (E) Quantification of capillary density in the different area types in gastrocnemius muscles, d4 after ischemia. Mean±SEM are shown. * = p<0.05; ** = p<0.01; n = 9.
Figure 5
Figure 5. Postischemic angiogenesis is increased in tsp-1−/− mice.
(A&B) Capillary density of gastrocnemius muscle sections was assessed at d4 using CD31 immunostaining in tsp-1−/− (A) and wt (B) mice (scale bar = 500 µm). (C&D) CD31 immunostaining of infiltrated area in tsp-1−/− (C) and wt (D) mice; scale bar = 200 µm. (E) Quantification of capillary density in the different area types in gastrocnemius muscles, d4 after ischemia. Mean±SEM are shown. * = p<0.05; ** = p<0.01; n = 9.
Figure 6
Figure 6. Phagocytosis of necrotic muscle cells debris is a pro-inflammatory signal for macrophages.
TNF-α, IL-6 and IL-10 secretion was quantified by ELISA in the conditioned medium of IFN-γ-activated macrophages, pre-treated or not with 10 µg/ml Colchicine (+colc) for 30 min, after phagocytosis of necrotic myogenic precursor cells (mpc). Mean±SEM of three experiments are shown. * = p<0,05 vs. control.
Figure 7
Figure 7. Thrombospondin-1−/− mice exhibit a less pro-inflammatory macrophage activation state in response to ischemia.
(A) FACS analyses of CD45+ cells isolated from ischemic muscles at d4, stained for F4/80 and Ly-6C expression, wt, left panel; tsp-1−/−, right panel; n = 5 mice per group. (B) Quantification of F4/80(lo)Ly-6C(hi), F4/80(hi)Ly-6C(hi) and F4/80(hi)Ly-6C(lo) macrophage proportions in CD45+ cells isolated from ischemic muscles at d4 in both genotypes (n = 5 mice per group in five independent experiments). * = p<0,05 vs. wt. (C) Quantitative RT-PCR analyses of IL-1β, TNF-α, IL-10, PPAR-γ, TGF-β, iNOS and Arg1 expression in F4/80(hi) vs. F4/80(lo)Ly-6C(hi) cells isolated from ischemic muscles at d4 in both genotypes.
Figure 8
Figure 8. 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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