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Role of plasminogen in macrophage accumulation during liver repair
Thrombosis Research 125 (2010) e214–e221
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Role of plasminogen in macrophage accumulation during liver repair Naoyuki Kawao a, Nobuo Nagai a, Kiyotaka Okada a, Katsumi Okumoto b, Shigeru Ueshima a,c, Osamu Matsuo a,⁎ a b c
Department of Physiology, Kinki University School of Medicine, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan Center for Morphological Analyses in Central Research Facilities, Kinki University School of Medicine, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan Department of Food Science and Nutrition, Kinki University School of Agriculture, 3327-204 Nakamachi, Nara 631-8505, Japan
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Article history: Received 11 August 2009 Received in revised form 17 November 2009 Accepted 9 December 2009 Available online 10 January 2010 Keywords: Plasminogen Macrophage accumulation Traumatic liver injury Hepatic ischemia-reperfusion injury Liver repair
a b s t r a c t Introduction: Although the involvement of plasminogen in liver repair has been reported, its roles are still poorly understood. Here, we investigated the role of plasminogen in accumulations of macrophages and neutrophils after liver injury in mice with gene deficient of plasminogen (Plg−/−) or its wild type (Plg+/+). Materials and Methods: Mice received traumatic liver injury caused by stabbing on the lobe or hepatic ischemia-reperfusion, and the damaged sites were histologically analyzed. Results: After the traumatic liver injury, both the stab wound and the damaged tissue were decreased until day 7 in the Plg+/+ mice. In contrast, both the stab wound and the damaged tissue were still remained until day 7 in the Plg−/− mice. On day 4 after traumatic liver injury, macrophages were abundant at the surrounding area of the damaged site in the Plg+/+ mice. However, the macrophage accumulation was impaired in the Plg−/− mice. After hepatic ischemia-reperfusion injury, macrophage accumulation and decrease in the damaged tissue were also observed in the Plg+/+ mice until day 7. In contrast, these responses were also impaired in the Plg−/− mice. Furthermore, neutrophil accumulation at the surrounding area of the damaged site was also impaired in the Plg−/− mice on day 4 after both liver traumatic liver injury and hepatic ischemia-reperfusion injury. Conclusions: Our data indicate that plasminogen plays a crucial role in macrophage accumulation together with the neutrophil accumulation after liver injury in both models, which may be essential for triggering the subsequent healing responses including decrease in the damaged tissue. © 2009 Elsevier Ltd. All rights reserved.
Liver injury is caused by several harmful stimuli including virus infection, alcohol, drugs, chemicals, trauma, and ischemia-reperfusion. Among them, traumatic liver injury often arises in both physical surgery and accidents following the ischemia-induced hepatocellular death by vascular damage [1]. Liver injury by ischemia together with subsequent reperfusion occurs in diverse pathophysiological circumstances, including liver surgery, liver transplantation, hemorrhagic shock with fluid resuscitation, veno-occlusive disease, and heart failure [1,2]. Hepatic ischemia-reperfusion injury is caused biphasically, oxidant-dependent injury by reactive oxygen species produced by Kupffer cells in the early phase and an intense inflammatory response in the late phase [2]. Although different mechanisms contribute to liver injury caused by trauma and ischemia-reperfusion [1], general wound healing responses are triggered in the damaged
Abbreviations: ECM, extracellular matrix; PA, plasminogen activator; MMP, matrix metalloproteinase; Plg, plasminogen; Plg−/−, plasminogen gene deficient; Plg+/+, wild-type mice for Plg−/− mice; H&E, hematoxylin-eosin; MCP-1, monocyte chemoattractant protein-1. ⁎ Corresponding author. Tel.: +81 72 366 0221x3165; fax: +81 72 366 0206. E-mail address: [email protected] (O. Matsuo). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.12.009
sites of these injuries [3]. Especially, macrophages are recruited to the damaged sites following inflammation and participate in wound healing responses through both the release of growth factors and cytokines, which stimulate production of extracellular matrix (ECM), angiogenesis and the clearance of the damaged tissue through phagocytosis [4]. Critical roles of these macrophages on liver repair were previously demonstrated by using the selective macrophage depletion during the recovery phase [5]. Other cells including neutrophils also are recruited to the damaged site, which is considered to be involved in the healing responses after liver injury [3,4]. Plasminogen is an inactive proenzyme, which is converted to active serine protease plasmin by plasminogen activators (PAs) including tissue-type PA as well as urokinase [6]. Previous studies have shown that the PA/plasmin system regulates recruitment of monocytes/macrophages and neutrophils during inflammation [7–9]. Plasmin activates matrix-sequestered matrix metalloproteinases (MMPs), which contributes to cell migration [6] and tissue remodeling [10]. The PA/plasmin system is also involved in liver repair through proteolysis of ECM and clearance of cellular debris [11–13]. Furthermore, plasminogen/plasmin suppresses hepatocyte apoptosis
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through activation of ERK1/2 signaling [14] and induces hepatocyte proliferation via binding to hepatocytes and exert pericellular proteolysis [15]. Recently, it has been shown that plasmin causes proteolytic activation of hepatocyte growth factor [16]. Although plasminogen/plasmin plays critical roles in liver repair, little is known about the in vivo evidence that plasminogen/plasmin contributes to macrophage recruitment accompanied with recovery responses. In this study, we examined accumulation of macrophages in mice deficient of the plasminogen gene (Plg−/−) together with their wildtype counterparts (Plg+/+) during liver repair after either traumatic liver injury or hepatic ischemia-reperfusion injury. We also studied the accumulation of neutrophils in the Plg−/− mice and Plg+/+ mice in both models.
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lobe was stabbed with a sterile surgical blade (Feather Safety Razor, Osaka, Japan). The size of stab wound was 3 mm in length and 2 mm in depth from surface of the lobe. After hemostasis of the injured lobe, the incised muscle and skin were sterilely sutured, and the anesthesia was discontinued. Body temperature was kept on a heating pad at 37 °C during the surgery. On day 1, 4, or 7 after the surgery, mice were anesthetized with pentobarbital (50 mg/kg, i.p.), and the injured lobe was exposed and photographed using a digital camera (Nikon D70, Nikon, Tokyo, Japan). Then, the area of damage was quantified by planimetry. After perfusion with 4% paraformaldehyde, the liver was removed, embedded in paraffin, and utilized for histological analysis.
Hepatic ischemia-reperfusion injury model Materials and Methods Animals Plg−/− mice and their Plg+/+ littermates (control), each weighing 18 to 25 g in 12 to 16 weeks old were used [17]. All experiments were approved by the Institutional Animal Care and Use Committee at the Kinki University School of Medicine. Traumatic liver injury model Traumatic liver injury was induced by stabbing on the surface of the lobe using a surgical blade. Briefly, left lateral liver lobe was exposed after laparotomy under isoflurane anaesthesia, and then the
Hepatic ischemia-reperfusion protocol has been described previously [18]. Briefly, a midline laparotomy incision was performed to expose the liver under isoflurane anaesthesia. A microaneurysm clamp was applied to the hepatic artery and portal vein resulting in ischemia of the left lateral and median lobes of the liver. After 90 min of partial hepatic ischemia, the clamp was removed to initiate hepatic reperfusion. The incised muscle and skin were sterilely sutured, and the anesthesia was discontinued. Body temperature was kept on a heating pad at 37 °C during the surgery. On day 1, 4 or 7 after the surgery, mice were perfused with 4% paraformaldehyde. Then the liver was removed, embedded in paraffin, and utilized for histological analysis. To determine the degree of hepatocyte injury, the plasma level of alanine aminotransferase (ALT) was measured on day 0, 1, 4,
Fig. 1. Deficiency of plasminogen gene impaired the recovery of the damaged site after traumatic liver injury. (A) Photographs of liver surface at the damaged site after the injury in the Plg+/+ mice (top) and Plg−/− mice (bottom) from day 1 to day 7. Similar results were obtained from 4 mice in each group. (B) Quantified results of the damaged area on the liver surface. The pale surface area was measured by NIH Image. The data represent the mean ± SEM of 4 mice in each group. *p b 0.05 and **p b 0.01 versus Plg+/+ mice.
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and 7 by using a colorimetric assay kit (Transaminase CII-test Wako, Wako Pure Chem., Osaka, Japan). Histological analysis Immunostaining for F4/80 (a macrophage marker), neutrophilspecific maker, and fibrinogen was performed as described elsewhere [19]. The 4-µm sections, which was obtained from the middle of the damaged site in each mouse, prepared from paraffin-embedded liver were incubated with rat monoclonal anti F4/80 antibody (AbD Serotec, Raleigh, NC, USA) at a dilution of 1:1000, rat monoclonal antineutrophil antibody (NIMP-R14, Abcam, Cambridge, UK) at a dilution of 1:100, or biotin-conjugated goat polyclonal anti-fibrinogen antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) at a dilution of 1:1000. The sections were then incubated with histofine simple stein mouse MAX-PO (Rat) (Nichirei Biosciences Inc., Tokyo, Japan) for anti-F4/80 antibody and anti-neutrophil antibody or streptavidin conjugated with peroxidase for anti-fibrinogen. The F4/ 80- and neutrophil-positive signals were visualized using a tyramide signal amplification system (PerkinElmer Waltham, MS, USA). The fibrinogen-positive signals were visualized by diaminobenzidine coloration. The sections were photographed under a microscope (E800, Canon, Tokyo, Japan) with the CCD camera. The adjacent sections of immunostaining for F4/80 were stained with hematoxylineosin (H&E). The broadness of the condensed F4/80-immunoreactivity at the surrounding area of the damaged site was measured as the mean of the area in 3 individual microscopic fields in the sections by an image
process program (Mac SCOPE, Mitani Co., Fukui, Japan) in a blinded evaluation. The number of neutrophils was also measured as the mean of the number of immunoreactive cells for neutrophil-specific marker in 5 individual microscopic fields at the surrounding area of the damaged site in the sections. Statistical analysis All data are expressed as the mean ± SEM. Statistical significance was evaluated by Student's t-test and was set at the p b 0.05 level. Results Deficiency of plasminogen gene impaired the recovery of the damaged site after traumatic liver injury On day 1 after traumatic liver injury, the damaged site was observed as a pale area on the surface in both genotypes (Fig. 1A, left). The area of damaged site on the surface was decreased until day 7 in the Plg+/+ mice (Fig. 1A, top and B). In contrast, the damaged site remained until day 7 in the Plg−/− mice (Fig 1A, bottom and B). The area of damaged site in the Plg−/− mice was significantly larger than that in the Plg+/+ mice on days 4 and 7 after traumatic liver injury (Fig. 1B). The damaged site in hepatic parenchyma was observed at the surroundings of the stab wound in the H&E-stained vertical sections on day 1 in both genotypes (Fig. 2A, D, G, J). In the Plg+/+ mice, both the stab wound and the surrounding damaged site were enormously diminished on day 4 and day 7, respectively (Fig. 2B, C,
Fig. 2. H&E-stained vertical sections of the damaged site after traumatic liver injury in the Plg+/+ mice (A-C, G-I) and Plg−/− mice (D-F, J-L). The dotted line indicates the border of damaged tissue (A-F). The asterisk indicates the damaged tissue (G-L). Arrowhead indicates a stab wound caused by traumatic injury (G-L). Panels G, H, I, J, K, and L were enlarged photographs of the squares in panels A, B, C, D, E, and F, respectively. Scale bars indicate 200 µm (A-F) and 100 µm (G-L). Similar results were obtained from 4 mice in each group.
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H, I), which were comparable with the damaged site on the surface. On the other hand, both the stab wound and the damaged tissue were still remained until day 7 in the Plg−/− mice (Fig. 2F, L). These results indicate that plasminogen played a critical role in liver repair after traumatic injury. Deficiency of plasminogen gene impaired the accumulation of F4/80immunoreactive cells at the surrounding area of the damaged site after traumatic liver injury The H&E-stained sections indicated that granular cells were abundant in the region between the normal and the damage sites in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3A). F4/80immunoreactive cells were accumulated in this region (Fig. 3C). The number of the granular cells between the normal and the damage sites was less in the Plg−/− mice than in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3B), which was consistent with few accumulation of F4/80-immunoreactive cells at the surrounding area of the damaged site (Fig. 3D). The area of F4/80-immunoreactive cells
Fig. 4. Plasma ALT levels in the Plg+/+ mice and Plg−/− mice after hepatic ischemiareperfusion injury. The data represent the mean ± SEM of 6-8 mice in each time point.
in the Plg−/− mice was significantly smaller than that in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3E). These results indicate that plasminogen contributed to the accumulation of F4/80-
Fig. 3. Involvement of plasminogen on the accumulation of F4/80-immunoreactive cells at the surrounding area of the damaged site after traumatic injury. (A, B) Microphotographs of the H&E-stained vertical sections of the damaged site on day 4 in the Plg+/+ mice (A) and Plg−/− mice (B). (C, D) Microphotographs of F4/80-immunoreactive cells (red) in the Plg+/+ mice (C) and Plg−/− mice (D) on day 4 in the adjacent section of (A) and (B), respectively. The arrowheads indicate the accumulation of granular cells (A, B) and F4/80immunoreactive cells (C, D) between normal and damaged sites. Scale bars indicate 100 µm. Similar results were obtained from 4 mice in each group. (E) Quantification of the area of the accumulated F4/80-immunoreactive cells in a section obtained from the middle of the damage in the Plg+/+ and Plg−/− mice on day 4 after traumatic liver injury. The data represent the mean ± SEM of 4 mice in each group.
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immunoreactive cells at the surrounding area of the damaged site after traumatic liver injury. Deficiency in the plasminogen gene impaired the accumulation of F4/80immunoreactive cells at the surrounding area of the damaged site after hepatic ischemia-reperfusion injury In the Plg+/+ mice, the plasma ALT levels was increased on day 1 after hepatic ischemia-reperfusion injury and returned to basal levels on day 4 (Fig. 4). The transient increased was also observed in the Plg−/− mice, in which both the peak level and returned basal level were
comparable with that in the Plg+/+ mice (Fig. 4). Although the damage was observed extensively at the liver on day 1 after hepatic ischemiareperfusion injury in both genotypes, the accumulation of F4/80immunnoreactive cells was not observed at the surrounding area of the damaged site (Fig. 5A, D, G, J). On day 4 after hepatic ischemiareperfusion injury, F4/80-immunoreactive cells were observed at the surrounding area of the damaged site in the Plg+/+ mice (Fig. 5B, H). On day 7, the damaged tissue was disappeared near the accumulated F4/80immunoreactive cells in the Plg+/+ mice (Fig. 5C, I). In contrast, a massive damaged tissue was also remained until day 7 after hepatic ischemia-reperfusion injury in the Plg−/− mice (Fig. 5E, F, K, L). The
Fig. 5. Involvement of plasminogen on the recovery of the damaged site and the accumulation of macrophages after hepatic ischemia-reperfusion injury. (A-F) Microphotographs of the H&E-stained sections in the Plg+/+ (A-C) and Plg−/− mice (D-F). (G-L) Microphotographs of F4/80-immunostained sections in the Plg+/+ mice (G-I) and Plg−/− mice (J-L) in the adjacent sections of (A-C) and (D-F), respectively. The arrowheads indicate the accumulation of granular cells (A-F) and F4/80-immunoreactive cells (G-L) between normal and damaged sites. Scale bars indicate 100 µm. Similar results were obtained from 4 mice in each group. (M) Quantification of the area of the condensed F4/80-immunoreactivity at the surrounding area of the damaged site in the Plg+/+ mice and Plg−/− mice after hepatic ischemia-reperfusion injury. The data represent the mean ± SEM of 4 mice in each group. n.s., not significant.
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number of accumulated F4/80-immunoreactive cells was significantly less in the Plg−/− mice than that in the Plg+/+ on day 4 (Fig. 5M).
Fibrin deposited at the damaged site after both traumatic liver injury and hepatic ischemia-reperfusion injury
Deficiency in the plasminogen gene impaired the accumulation of neutrophils at the surrounding area of the damaged site after traumatic liver injury or hepatic ischemia-reperfusion injury
Fibrinogen-immunoreactivity was observed at the damaged site, especially at the peripheral region of the damaged site, in both genotypes on day 1 after traumatic liver injury (Fig. 7A, B). Same tendency was observed on day 1 after hepatic ischemia-reperfusion injury in both genotypes (Fig. 7C, D).
Neutrophils were observed mostly at the surrounding area of the damaged site on day 4 after traumatic liver injury in the Plg+/+ mice (Fig. 6A). In contrast, neutrophils were scarce at the surrounding area of the damaged site in the Plg−/− mice on day 4 after traumatic liver injury (Fig. 6B). The number of recruited neutrophils was significantly less in the Plg−/− mice than in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 6C). The same tendency was observed when the number of neutrophils was quantitatively estimated on day 4 after hepatic ischemia-reperfusion injury in both genotypes (Fig. 6D-F). These results suggest that plasminogen also contributed to the accumulation of neutrophils at the surrounding area of the damaged site on day 4 after both traumatic liver injury and hepatic ischemia-reperfusion injury.
Discussion The present study shows that deficiency of plasminogen leads to impairment of liver repair together with delayed clearance of the damaged tissue. Furthermore, our data demonstrate that plasminogen plays a critical role in accumulations of both macrophages and neutrophils at the surrounding area of the damaged site after both traumatic liver injury and hepatic ischemia-reperfusion injury. The wound healing responses are triggered in various organs including the liver after an injury [3], which can generally be divided into three phases: acute inflammation, granulation tissue formation,
Fig. 6. Involvement of plasminogen on the accumulation of neutrophils on day 4 after both traumatic liver injury and hepatic ischemia-reperfusion injury. (A, B, D, E) Microphotographs of neutrophil marker immunoreactive cells (red) in the Plg+/+ mice (A, D) and the Plg−/− mice (B, E) on day 4 after traumatic liver injury (A, B) and hepatic ischemia-reperfusion injury (D, E). The sections were counterstained with DAPI (blue). The arrowheads indicate the accumulation of granular cells between the normal and the damaged sites. Scale bars indicate 50 µm. Similar results were obtained from 4 mice in each group. (C, F) Quantification of the number of the immunoreactive neutrophils at the surrounding area of the damaged site in the Plg+/+ mice and Plg−/− mice on day 4 after traumatic liver injury (C) and hepatic ischemia-reperfusion injury (F). The data represent the mean ± SEM of 4 mice in each group.
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Fig. 7. Microphotographs of fibrinogen-immunoreactivity at the damaged site in the Plg+/+ mice (A, C) and Plg−/− mice (B, D) on day 1 after traumatic live injury (A, B) and hepatic ischemia-reperfusion injury (C, D). The fibrinogen-immunoreactivity was visualized by diaminobenzidine coloration (brown). The sections were counterstained with hematoxylin (blue). Scale bars indicate 50 µm. Similar results were obtained from 4 mice in each group.
and tissue remodeling [4]. Granulation tissue mainly consists of numerous macrophages and ECM-producing cells together with newly generated blood vessels [4]. It is well known that macrophages play a central role in the healing responses after liver injury through release of cytokines and growth factors, degradation of ECM proteins, and phagocytosis of cellular debris [3–5]. In this study, we observed that the damaged tissue which was surrounded by numerous macrophages on day 4 disappeared on day 7 after both traumatic liver injury and ischemia-reperfusion injury in the Plg+/+ mice. These results suggest that the damaged tissue is cleared by the accumulated macrophages. We also found the impairment of liver repair and the decrease in the damaged tissue in the Plg−/− mice, which were in line with previous findings [11–13]. Furthermore, the accumulation of macrophages was impaired in the Plg−/− mice after both traumatic liver injury and hepatic ischemia-reperfusion injury. Since the previous in vitro evidence indicated that phagocytic function of macrophages is comparable between Plg+/+ and Plg−/− mice [8], the remaining of the damaged tissue in the Plg−/− mice is thought to be responsible for the impairment of macrophage accumulation. Collectively, plasminogen most likely plays a critical role in facilitating recruitment of macrophages and triggering a cascade of subsequent recovery responses including clearance of the damaged tissue. Although the importance of PA/plasminogen system on macrophage recruitment has been demonstrated in distinct inflammatory models using Plg−/− mice [7–9,20], the involvement in macrophage accumulation after liver injury was not reported. Here our findings clearly show the involvement of plasminogen in macrophage accumulation at the surrounding area of the damaged site after liver injury. The several mechanisms are considered to be responsible for the plasminogen-dependent accumulation of macrophages. The previous studies indicate that plasminogen is involved in monocytes/macrophages migration. Plasminogen binds to their surface through its receptors including α-enolase [21], annexin 2 [22], and histone H2B [20], and degrades ECM proteins through MMP-9 activation [9]. In addition, plasmin triggers the expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for
macrophages, in primary monocytes through activation of mitogenactivated protein kinase signaling [23] and activates MCP-1 by its also proteolytic action [24]. Furthermore, fibrinogen-derived peptides synthesized by degradation of fibrinogen by plasmin possess chemotactic activity for leukocytes [25]. These mechanisms may explain that macrophages fail to be attracted to the surrounding area of the damaged site in the Plg−/− mice. We found that the neutrophil accumulation was also impaired in the Plg−/− mice at the surrounding area of the damaged site in both traumatic liver injury and hepatic ischemia-reperfusion injury models, indicating that plasminogen contributes to the neutrophil accumulation at the area. However, the involvement of the accumulated neutrophils in liver repair is still remained as a future subject. The impairment of liver repair in the Plg−/− mice might be attributed to the decrease in the activation of MMP-9 at the damaged site, because plasmin regulates MMPs activity including MMP-9 [9,26], which is known to be important for liver regeneration after partial hepatectomy [27]. In addition, since fibrin deposition was observed at the surroundings of the damaged tissue in both genotypes after traumatic liver injury and hepatic ischemia-reperfusion injury, a possibility that the deposited fibrin which was escaped from degradation in the Plg−/− mice might interrupt the phagocytic reaction of macrophages at the damaged sites can not be ruled out. Liver injury was caused by stabbing on the lobe or hepatic ischemia-reperfusion in this study. The trauma-induced hepatocellular death may be caused by ischemia followed by vascular damage [1]. The hepatic ischemia-reperfusion injury is caused by release of reactive oxygen species from activated Kupffer cells, and subsequently an intense inflammatory response including recruitment of neutrophils to the damaged sites [1]. Although the mechanisms underlying hepatocellular injury are different in these models, the accumulation of macrophages at the surrounding area of the damaged sites accompanied by clearance of the damaged tissue was comparable. Therefore, recovery responses including the macrophage accumulation after liver injury are likely to be close in each model. Our data also show that the accumulation of macrophages was impaired in the Plg−/−
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mice both after traumatic liver injury and hepatic ischemia-reperfusion injury. Therefore, plasminogen is considered to be essential for accumulation of macrophages during liver repair regardless of whether liver injury was caused by trauma or ischemia-reperfusion. Conflict of interest statement No conflicts of interest exist. Acknowledgements The authors thank Kanako Takenishi for supporting the histological analysis. This study was partly supported by a Grant-in-Aid for Young Scientists (B: 20790182) from The Ministry of Education, Culture, Sports, Science, and Technology of Japan and a grant from the High-Tech Research Center at Kinki University, Graduate School of Medicine from the Japan Society for the Promotion of Science (JSPS). References [1] Bilzer M, Gerbes AL. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J Hepatol 2000;32:508–15. [2] Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/ reperfusion injury. Gastroenterology 2003;125:1246–57. [3] Wallace K, Burt AD, Wright MC. Liver fibrosis. Biochem J 2008;411:1–18. [4] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738–46. [5] Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 2005;115:56–65. [6] Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost 2005;93:647–54. [7] Busuttil SJ, Ploplis VA, Castellino FJ, Tang L, Eaton JW, Plow EF. A central role for plasminogen in the inflammatory response to biomaterials. J Thromb Haemost 2004;2:1798–805. [8] Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF. Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood 1998;91:2005–9. [9] Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 2008;118:3012–24. [10] Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007;8:221–33. [11] Bezerra JA, Bugge TH, Melin-Aldana H, Sabla G, Kombrinck KW, Witte DP, Degen JL. Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver. Proc Natl Acad Sci U S A 1999;96:15143–8.
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Regular Article
Role of plasminogen in macrophage accumulation during liver repair Naoyuki Kawao a, Nobuo Nagai a, Kiyotaka Okada a, Katsumi Okumoto b, Shigeru Ueshima a,c, Osamu Matsuo a,⁎ a b c
Department of Physiology, Kinki University School of Medicine, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan Center for Morphological Analyses in Central Research Facilities, Kinki University School of Medicine, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan Department of Food Science and Nutrition, Kinki University School of Agriculture, 3327-204 Nakamachi, Nara 631-8505, Japan
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Article history: Received 11 August 2009 Received in revised form 17 November 2009 Accepted 9 December 2009 Available online 10 January 2010 Keywords: Plasminogen Macrophage accumulation Traumatic liver injury Hepatic ischemia-reperfusion injury Liver repair
a b s t r a c t Introduction: Although the involvement of plasminogen in liver repair has been reported, its roles are still poorly understood. Here, we investigated the role of plasminogen in accumulations of macrophages and neutrophils after liver injury in mice with gene deficient of plasminogen (Plg−/−) or its wild type (Plg+/+). Materials and Methods: Mice received traumatic liver injury caused by stabbing on the lobe or hepatic ischemia-reperfusion, and the damaged sites were histologically analyzed. Results: After the traumatic liver injury, both the stab wound and the damaged tissue were decreased until day 7 in the Plg+/+ mice. In contrast, both the stab wound and the damaged tissue were still remained until day 7 in the Plg−/− mice. On day 4 after traumatic liver injury, macrophages were abundant at the surrounding area of the damaged site in the Plg+/+ mice. However, the macrophage accumulation was impaired in the Plg−/− mice. After hepatic ischemia-reperfusion injury, macrophage accumulation and decrease in the damaged tissue were also observed in the Plg+/+ mice until day 7. In contrast, these responses were also impaired in the Plg−/− mice. Furthermore, neutrophil accumulation at the surrounding area of the damaged site was also impaired in the Plg−/− mice on day 4 after both liver traumatic liver injury and hepatic ischemia-reperfusion injury. Conclusions: Our data indicate that plasminogen plays a crucial role in macrophage accumulation together with the neutrophil accumulation after liver injury in both models, which may be essential for triggering the subsequent healing responses including decrease in the damaged tissue. © 2009 Elsevier Ltd. All rights reserved.
Liver injury is caused by several harmful stimuli including virus infection, alcohol, drugs, chemicals, trauma, and ischemia-reperfusion. Among them, traumatic liver injury often arises in both physical surgery and accidents following the ischemia-induced hepatocellular death by vascular damage [1]. Liver injury by ischemia together with subsequent reperfusion occurs in diverse pathophysiological circumstances, including liver surgery, liver transplantation, hemorrhagic shock with fluid resuscitation, veno-occlusive disease, and heart failure [1,2]. Hepatic ischemia-reperfusion injury is caused biphasically, oxidant-dependent injury by reactive oxygen species produced by Kupffer cells in the early phase and an intense inflammatory response in the late phase [2]. Although different mechanisms contribute to liver injury caused by trauma and ischemia-reperfusion [1], general wound healing responses are triggered in the damaged
Abbreviations: ECM, extracellular matrix; PA, plasminogen activator; MMP, matrix metalloproteinase; Plg, plasminogen; Plg−/−, plasminogen gene deficient; Plg+/+, wild-type mice for Plg−/− mice; H&E, hematoxylin-eosin; MCP-1, monocyte chemoattractant protein-1. ⁎ Corresponding author. Tel.: +81 72 366 0221x3165; fax: +81 72 366 0206. E-mail address: [email protected] (O. Matsuo). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.12.009
sites of these injuries [3]. Especially, macrophages are recruited to the damaged sites following inflammation and participate in wound healing responses through both the release of growth factors and cytokines, which stimulate production of extracellular matrix (ECM), angiogenesis and the clearance of the damaged tissue through phagocytosis [4]. Critical roles of these macrophages on liver repair were previously demonstrated by using the selective macrophage depletion during the recovery phase [5]. Other cells including neutrophils also are recruited to the damaged site, which is considered to be involved in the healing responses after liver injury [3,4]. Plasminogen is an inactive proenzyme, which is converted to active serine protease plasmin by plasminogen activators (PAs) including tissue-type PA as well as urokinase [6]. Previous studies have shown that the PA/plasmin system regulates recruitment of monocytes/macrophages and neutrophils during inflammation [7–9]. Plasmin activates matrix-sequestered matrix metalloproteinases (MMPs), which contributes to cell migration [6] and tissue remodeling [10]. The PA/plasmin system is also involved in liver repair through proteolysis of ECM and clearance of cellular debris [11–13]. Furthermore, plasminogen/plasmin suppresses hepatocyte apoptosis
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through activation of ERK1/2 signaling [14] and induces hepatocyte proliferation via binding to hepatocytes and exert pericellular proteolysis [15]. Recently, it has been shown that plasmin causes proteolytic activation of hepatocyte growth factor [16]. Although plasminogen/plasmin plays critical roles in liver repair, little is known about the in vivo evidence that plasminogen/plasmin contributes to macrophage recruitment accompanied with recovery responses. In this study, we examined accumulation of macrophages in mice deficient of the plasminogen gene (Plg−/−) together with their wildtype counterparts (Plg+/+) during liver repair after either traumatic liver injury or hepatic ischemia-reperfusion injury. We also studied the accumulation of neutrophils in the Plg−/− mice and Plg+/+ mice in both models.
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lobe was stabbed with a sterile surgical blade (Feather Safety Razor, Osaka, Japan). The size of stab wound was 3 mm in length and 2 mm in depth from surface of the lobe. After hemostasis of the injured lobe, the incised muscle and skin were sterilely sutured, and the anesthesia was discontinued. Body temperature was kept on a heating pad at 37 °C during the surgery. On day 1, 4, or 7 after the surgery, mice were anesthetized with pentobarbital (50 mg/kg, i.p.), and the injured lobe was exposed and photographed using a digital camera (Nikon D70, Nikon, Tokyo, Japan). Then, the area of damage was quantified by planimetry. After perfusion with 4% paraformaldehyde, the liver was removed, embedded in paraffin, and utilized for histological analysis.
Hepatic ischemia-reperfusion injury model Materials and Methods Animals Plg−/− mice and their Plg+/+ littermates (control), each weighing 18 to 25 g in 12 to 16 weeks old were used [17]. All experiments were approved by the Institutional Animal Care and Use Committee at the Kinki University School of Medicine. Traumatic liver injury model Traumatic liver injury was induced by stabbing on the surface of the lobe using a surgical blade. Briefly, left lateral liver lobe was exposed after laparotomy under isoflurane anaesthesia, and then the
Hepatic ischemia-reperfusion protocol has been described previously [18]. Briefly, a midline laparotomy incision was performed to expose the liver under isoflurane anaesthesia. A microaneurysm clamp was applied to the hepatic artery and portal vein resulting in ischemia of the left lateral and median lobes of the liver. After 90 min of partial hepatic ischemia, the clamp was removed to initiate hepatic reperfusion. The incised muscle and skin were sterilely sutured, and the anesthesia was discontinued. Body temperature was kept on a heating pad at 37 °C during the surgery. On day 1, 4 or 7 after the surgery, mice were perfused with 4% paraformaldehyde. Then the liver was removed, embedded in paraffin, and utilized for histological analysis. To determine the degree of hepatocyte injury, the plasma level of alanine aminotransferase (ALT) was measured on day 0, 1, 4,
Fig. 1. Deficiency of plasminogen gene impaired the recovery of the damaged site after traumatic liver injury. (A) Photographs of liver surface at the damaged site after the injury in the Plg+/+ mice (top) and Plg−/− mice (bottom) from day 1 to day 7. Similar results were obtained from 4 mice in each group. (B) Quantified results of the damaged area on the liver surface. The pale surface area was measured by NIH Image. The data represent the mean ± SEM of 4 mice in each group. *p b 0.05 and **p b 0.01 versus Plg+/+ mice.
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and 7 by using a colorimetric assay kit (Transaminase CII-test Wako, Wako Pure Chem., Osaka, Japan). Histological analysis Immunostaining for F4/80 (a macrophage marker), neutrophilspecific maker, and fibrinogen was performed as described elsewhere [19]. The 4-µm sections, which was obtained from the middle of the damaged site in each mouse, prepared from paraffin-embedded liver were incubated with rat monoclonal anti F4/80 antibody (AbD Serotec, Raleigh, NC, USA) at a dilution of 1:1000, rat monoclonal antineutrophil antibody (NIMP-R14, Abcam, Cambridge, UK) at a dilution of 1:100, or biotin-conjugated goat polyclonal anti-fibrinogen antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) at a dilution of 1:1000. The sections were then incubated with histofine simple stein mouse MAX-PO (Rat) (Nichirei Biosciences Inc., Tokyo, Japan) for anti-F4/80 antibody and anti-neutrophil antibody or streptavidin conjugated with peroxidase for anti-fibrinogen. The F4/ 80- and neutrophil-positive signals were visualized using a tyramide signal amplification system (PerkinElmer Waltham, MS, USA). The fibrinogen-positive signals were visualized by diaminobenzidine coloration. The sections were photographed under a microscope (E800, Canon, Tokyo, Japan) with the CCD camera. The adjacent sections of immunostaining for F4/80 were stained with hematoxylineosin (H&E). The broadness of the condensed F4/80-immunoreactivity at the surrounding area of the damaged site was measured as the mean of the area in 3 individual microscopic fields in the sections by an image
process program (Mac SCOPE, Mitani Co., Fukui, Japan) in a blinded evaluation. The number of neutrophils was also measured as the mean of the number of immunoreactive cells for neutrophil-specific marker in 5 individual microscopic fields at the surrounding area of the damaged site in the sections. Statistical analysis All data are expressed as the mean ± SEM. Statistical significance was evaluated by Student's t-test and was set at the p b 0.05 level. Results Deficiency of plasminogen gene impaired the recovery of the damaged site after traumatic liver injury On day 1 after traumatic liver injury, the damaged site was observed as a pale area on the surface in both genotypes (Fig. 1A, left). The area of damaged site on the surface was decreased until day 7 in the Plg+/+ mice (Fig. 1A, top and B). In contrast, the damaged site remained until day 7 in the Plg−/− mice (Fig 1A, bottom and B). The area of damaged site in the Plg−/− mice was significantly larger than that in the Plg+/+ mice on days 4 and 7 after traumatic liver injury (Fig. 1B). The damaged site in hepatic parenchyma was observed at the surroundings of the stab wound in the H&E-stained vertical sections on day 1 in both genotypes (Fig. 2A, D, G, J). In the Plg+/+ mice, both the stab wound and the surrounding damaged site were enormously diminished on day 4 and day 7, respectively (Fig. 2B, C,
Fig. 2. H&E-stained vertical sections of the damaged site after traumatic liver injury in the Plg+/+ mice (A-C, G-I) and Plg−/− mice (D-F, J-L). The dotted line indicates the border of damaged tissue (A-F). The asterisk indicates the damaged tissue (G-L). Arrowhead indicates a stab wound caused by traumatic injury (G-L). Panels G, H, I, J, K, and L were enlarged photographs of the squares in panels A, B, C, D, E, and F, respectively. Scale bars indicate 200 µm (A-F) and 100 µm (G-L). Similar results were obtained from 4 mice in each group.
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H, I), which were comparable with the damaged site on the surface. On the other hand, both the stab wound and the damaged tissue were still remained until day 7 in the Plg−/− mice (Fig. 2F, L). These results indicate that plasminogen played a critical role in liver repair after traumatic injury. Deficiency of plasminogen gene impaired the accumulation of F4/80immunoreactive cells at the surrounding area of the damaged site after traumatic liver injury The H&E-stained sections indicated that granular cells were abundant in the region between the normal and the damage sites in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3A). F4/80immunoreactive cells were accumulated in this region (Fig. 3C). The number of the granular cells between the normal and the damage sites was less in the Plg−/− mice than in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3B), which was consistent with few accumulation of F4/80-immunoreactive cells at the surrounding area of the damaged site (Fig. 3D). The area of F4/80-immunoreactive cells
Fig. 4. Plasma ALT levels in the Plg+/+ mice and Plg−/− mice after hepatic ischemiareperfusion injury. The data represent the mean ± SEM of 6-8 mice in each time point.
in the Plg−/− mice was significantly smaller than that in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 3E). These results indicate that plasminogen contributed to the accumulation of F4/80-
Fig. 3. Involvement of plasminogen on the accumulation of F4/80-immunoreactive cells at the surrounding area of the damaged site after traumatic injury. (A, B) Microphotographs of the H&E-stained vertical sections of the damaged site on day 4 in the Plg+/+ mice (A) and Plg−/− mice (B). (C, D) Microphotographs of F4/80-immunoreactive cells (red) in the Plg+/+ mice (C) and Plg−/− mice (D) on day 4 in the adjacent section of (A) and (B), respectively. The arrowheads indicate the accumulation of granular cells (A, B) and F4/80immunoreactive cells (C, D) between normal and damaged sites. Scale bars indicate 100 µm. Similar results were obtained from 4 mice in each group. (E) Quantification of the area of the accumulated F4/80-immunoreactive cells in a section obtained from the middle of the damage in the Plg+/+ and Plg−/− mice on day 4 after traumatic liver injury. The data represent the mean ± SEM of 4 mice in each group.
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immunoreactive cells at the surrounding area of the damaged site after traumatic liver injury. Deficiency in the plasminogen gene impaired the accumulation of F4/80immunoreactive cells at the surrounding area of the damaged site after hepatic ischemia-reperfusion injury In the Plg+/+ mice, the plasma ALT levels was increased on day 1 after hepatic ischemia-reperfusion injury and returned to basal levels on day 4 (Fig. 4). The transient increased was also observed in the Plg−/− mice, in which both the peak level and returned basal level were
comparable with that in the Plg+/+ mice (Fig. 4). Although the damage was observed extensively at the liver on day 1 after hepatic ischemiareperfusion injury in both genotypes, the accumulation of F4/80immunnoreactive cells was not observed at the surrounding area of the damaged site (Fig. 5A, D, G, J). On day 4 after hepatic ischemiareperfusion injury, F4/80-immunoreactive cells were observed at the surrounding area of the damaged site in the Plg+/+ mice (Fig. 5B, H). On day 7, the damaged tissue was disappeared near the accumulated F4/80immunoreactive cells in the Plg+/+ mice (Fig. 5C, I). In contrast, a massive damaged tissue was also remained until day 7 after hepatic ischemia-reperfusion injury in the Plg−/− mice (Fig. 5E, F, K, L). The
Fig. 5. Involvement of plasminogen on the recovery of the damaged site and the accumulation of macrophages after hepatic ischemia-reperfusion injury. (A-F) Microphotographs of the H&E-stained sections in the Plg+/+ (A-C) and Plg−/− mice (D-F). (G-L) Microphotographs of F4/80-immunostained sections in the Plg+/+ mice (G-I) and Plg−/− mice (J-L) in the adjacent sections of (A-C) and (D-F), respectively. The arrowheads indicate the accumulation of granular cells (A-F) and F4/80-immunoreactive cells (G-L) between normal and damaged sites. Scale bars indicate 100 µm. Similar results were obtained from 4 mice in each group. (M) Quantification of the area of the condensed F4/80-immunoreactivity at the surrounding area of the damaged site in the Plg+/+ mice and Plg−/− mice after hepatic ischemia-reperfusion injury. The data represent the mean ± SEM of 4 mice in each group. n.s., not significant.
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number of accumulated F4/80-immunoreactive cells was significantly less in the Plg−/− mice than that in the Plg+/+ on day 4 (Fig. 5M).
Fibrin deposited at the damaged site after both traumatic liver injury and hepatic ischemia-reperfusion injury
Deficiency in the plasminogen gene impaired the accumulation of neutrophils at the surrounding area of the damaged site after traumatic liver injury or hepatic ischemia-reperfusion injury
Fibrinogen-immunoreactivity was observed at the damaged site, especially at the peripheral region of the damaged site, in both genotypes on day 1 after traumatic liver injury (Fig. 7A, B). Same tendency was observed on day 1 after hepatic ischemia-reperfusion injury in both genotypes (Fig. 7C, D).
Neutrophils were observed mostly at the surrounding area of the damaged site on day 4 after traumatic liver injury in the Plg+/+ mice (Fig. 6A). In contrast, neutrophils were scarce at the surrounding area of the damaged site in the Plg−/− mice on day 4 after traumatic liver injury (Fig. 6B). The number of recruited neutrophils was significantly less in the Plg−/− mice than in the Plg+/+ mice on day 4 after traumatic liver injury (Fig. 6C). The same tendency was observed when the number of neutrophils was quantitatively estimated on day 4 after hepatic ischemia-reperfusion injury in both genotypes (Fig. 6D-F). These results suggest that plasminogen also contributed to the accumulation of neutrophils at the surrounding area of the damaged site on day 4 after both traumatic liver injury and hepatic ischemia-reperfusion injury.
Discussion The present study shows that deficiency of plasminogen leads to impairment of liver repair together with delayed clearance of the damaged tissue. Furthermore, our data demonstrate that plasminogen plays a critical role in accumulations of both macrophages and neutrophils at the surrounding area of the damaged site after both traumatic liver injury and hepatic ischemia-reperfusion injury. The wound healing responses are triggered in various organs including the liver after an injury [3], which can generally be divided into three phases: acute inflammation, granulation tissue formation,
Fig. 6. Involvement of plasminogen on the accumulation of neutrophils on day 4 after both traumatic liver injury and hepatic ischemia-reperfusion injury. (A, B, D, E) Microphotographs of neutrophil marker immunoreactive cells (red) in the Plg+/+ mice (A, D) and the Plg−/− mice (B, E) on day 4 after traumatic liver injury (A, B) and hepatic ischemia-reperfusion injury (D, E). The sections were counterstained with DAPI (blue). The arrowheads indicate the accumulation of granular cells between the normal and the damaged sites. Scale bars indicate 50 µm. Similar results were obtained from 4 mice in each group. (C, F) Quantification of the number of the immunoreactive neutrophils at the surrounding area of the damaged site in the Plg+/+ mice and Plg−/− mice on day 4 after traumatic liver injury (C) and hepatic ischemia-reperfusion injury (F). The data represent the mean ± SEM of 4 mice in each group.
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Fig. 7. Microphotographs of fibrinogen-immunoreactivity at the damaged site in the Plg+/+ mice (A, C) and Plg−/− mice (B, D) on day 1 after traumatic live injury (A, B) and hepatic ischemia-reperfusion injury (C, D). The fibrinogen-immunoreactivity was visualized by diaminobenzidine coloration (brown). The sections were counterstained with hematoxylin (blue). Scale bars indicate 50 µm. Similar results were obtained from 4 mice in each group.
and tissue remodeling [4]. Granulation tissue mainly consists of numerous macrophages and ECM-producing cells together with newly generated blood vessels [4]. It is well known that macrophages play a central role in the healing responses after liver injury through release of cytokines and growth factors, degradation of ECM proteins, and phagocytosis of cellular debris [3–5]. In this study, we observed that the damaged tissue which was surrounded by numerous macrophages on day 4 disappeared on day 7 after both traumatic liver injury and ischemia-reperfusion injury in the Plg+/+ mice. These results suggest that the damaged tissue is cleared by the accumulated macrophages. We also found the impairment of liver repair and the decrease in the damaged tissue in the Plg−/− mice, which were in line with previous findings [11–13]. Furthermore, the accumulation of macrophages was impaired in the Plg−/− mice after both traumatic liver injury and hepatic ischemia-reperfusion injury. Since the previous in vitro evidence indicated that phagocytic function of macrophages is comparable between Plg+/+ and Plg−/− mice [8], the remaining of the damaged tissue in the Plg−/− mice is thought to be responsible for the impairment of macrophage accumulation. Collectively, plasminogen most likely plays a critical role in facilitating recruitment of macrophages and triggering a cascade of subsequent recovery responses including clearance of the damaged tissue. Although the importance of PA/plasminogen system on macrophage recruitment has been demonstrated in distinct inflammatory models using Plg−/− mice [7–9,20], the involvement in macrophage accumulation after liver injury was not reported. Here our findings clearly show the involvement of plasminogen in macrophage accumulation at the surrounding area of the damaged site after liver injury. The several mechanisms are considered to be responsible for the plasminogen-dependent accumulation of macrophages. The previous studies indicate that plasminogen is involved in monocytes/macrophages migration. Plasminogen binds to their surface through its receptors including α-enolase [21], annexin 2 [22], and histone H2B [20], and degrades ECM proteins through MMP-9 activation [9]. In addition, plasmin triggers the expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for
macrophages, in primary monocytes through activation of mitogenactivated protein kinase signaling [23] and activates MCP-1 by its also proteolytic action [24]. Furthermore, fibrinogen-derived peptides synthesized by degradation of fibrinogen by plasmin possess chemotactic activity for leukocytes [25]. These mechanisms may explain that macrophages fail to be attracted to the surrounding area of the damaged site in the Plg−/− mice. We found that the neutrophil accumulation was also impaired in the Plg−/− mice at the surrounding area of the damaged site in both traumatic liver injury and hepatic ischemia-reperfusion injury models, indicating that plasminogen contributes to the neutrophil accumulation at the area. However, the involvement of the accumulated neutrophils in liver repair is still remained as a future subject. The impairment of liver repair in the Plg−/− mice might be attributed to the decrease in the activation of MMP-9 at the damaged site, because plasmin regulates MMPs activity including MMP-9 [9,26], which is known to be important for liver regeneration after partial hepatectomy [27]. In addition, since fibrin deposition was observed at the surroundings of the damaged tissue in both genotypes after traumatic liver injury and hepatic ischemia-reperfusion injury, a possibility that the deposited fibrin which was escaped from degradation in the Plg−/− mice might interrupt the phagocytic reaction of macrophages at the damaged sites can not be ruled out. Liver injury was caused by stabbing on the lobe or hepatic ischemia-reperfusion in this study. The trauma-induced hepatocellular death may be caused by ischemia followed by vascular damage [1]. The hepatic ischemia-reperfusion injury is caused by release of reactive oxygen species from activated Kupffer cells, and subsequently an intense inflammatory response including recruitment of neutrophils to the damaged sites [1]. Although the mechanisms underlying hepatocellular injury are different in these models, the accumulation of macrophages at the surrounding area of the damaged sites accompanied by clearance of the damaged tissue was comparable. Therefore, recovery responses including the macrophage accumulation after liver injury are likely to be close in each model. Our data also show that the accumulation of macrophages was impaired in the Plg−/−
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mice both after traumatic liver injury and hepatic ischemia-reperfusion injury. Therefore, plasminogen is considered to be essential for accumulation of macrophages during liver repair regardless of whether liver injury was caused by trauma or ischemia-reperfusion. Conflict of interest statement No conflicts of interest exist. Acknowledgements The authors thank Kanako Takenishi for supporting the histological analysis. This study was partly supported by a Grant-in-Aid for Young Scientists (B: 20790182) from The Ministry of Education, Culture, Sports, Science, and Technology of Japan and a grant from the High-Tech Research Center at Kinki University, Graduate School of Medicine from the Japan Society for the Promotion of Science (JSPS). References [1] Bilzer M, Gerbes AL. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J Hepatol 2000;32:508–15. [2] Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/ reperfusion injury. Gastroenterology 2003;125:1246–57. [3] Wallace K, Burt AD, Wright MC. Liver fibrosis. Biochem J 2008;411:1–18. [4] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738–46. [5] Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 2005;115:56–65. [6] Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost 2005;93:647–54. [7] Busuttil SJ, Ploplis VA, Castellino FJ, Tang L, Eaton JW, Plow EF. A central role for plasminogen in the inflammatory response to biomaterials. J Thromb Haemost 2004;2:1798–805. [8] Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF. Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood 1998;91:2005–9. [9] Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 2008;118:3012–24. [10] Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007;8:221–33. [11] Bezerra JA, Bugge TH, Melin-Aldana H, Sabla G, Kombrinck KW, Witte DP, Degen JL. Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver. Proc Natl Acad Sci U S A 1999;96:15143–8.
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