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Matrix metalloproteinase-9 contributes to parenchymal hemorrhage and necrosis in the remnant liver after extended hepatectomy in mice.

AIM
To investigate the effect of matrix metalloproteinase-9 (MMP-9) on the remnant liver after massive hepatectomy in the mouse.


METHODS
Age-matched, C57BL/6 wild-type (WT), MMP-9(-/-), and tissue inhibitors of metalloproteinases (TIMP)-1(-/-) mice were used. The mice received 80%-partial hepatectomy (PH). Samples were obtained at 6 h after 80%-PH, and we used histology, immunohistochemical staining, western blotting analysis and zymography to investigate the effect of PH on MMP-9. The role of MMP-9 after PH was investigated using a monoclonal antibody and MMP inhibitor.


RESULTS
We examined the remnant liver 6 h after 80%-PH and found that MMP-9 deficiency attenuated the formation of hemorrhage and necrosis. There were significantly fewer and smaller hemorrhagic and necrotic lesions in MMP-9(-/-) remnant livers compared with WT and TIMP-1(-/-) livers (P < 0.01), with no difference between WT and TIMP-1(-/-) mice. Serum alanine aminotransaminase levels were significantly lower in MMP-9(-/-) mice compared with those in TIMP-1(-/-) mice (WT: 476 ± 83 IU/L, MMP-9(-/-): 392 ± 30 IU/L, TIMP-1(-/-): 673 ± 73 IU/L, P < 0.01). Western blotting and gelatin zymography demonstrated a lack of MMP-9 expression and activity in MMP-9(-/-) mice, which was in contrast to WT and TIMP-1(-/-) mice. No change in MMP-2 expression was observed in any of the study groups. Similar to MMP-9(-/-) mice, when WT mice were treated with MMP-9 monoclonal antibody or the synthetic inhibitor GM6001, hemorrhagic and necrotic lesions were significantly smaller and fewer than in control mice (P < 0.05). These results suggest that MMP-9 plays an important role in the development of parenchymal hemorrhage and necrosis in the small remnant liver.


CONCLUSION
Successful MMP-9 inhibition attenuates the formation of hemorrhage and necrosis and might be a potential therapy to ameliorate liver injury after massive hepatectomy.


INTRODUCTION
In the field of hepatobiliary surgery, liver resection is considered the standard treatment for primary liver tumor and colorectal liver metastases [1][2][3] . Major liver resections have been associated with an increased morbidity and mortality compared with more limited resections. In particular, the outcomes are worsened when a chronic underlying liver disease is present [4] . Recent studies have shown that the volume of the remnant liver is correlated with perioperative morbidity and mortality [5][6][7] , and an insufficient hepatic remnant (IHR) after extended hepatectomy is a critical issue.
On the other hand, liver transplantation (LT) is the treatment of choice for end-stage liver diseases. However, in the LT field, donor shortage and small-for-size grafts (SFSGs) are critical issues. Although split-liver transplantation (SOLT) is promising, failure of SFSGs in adult LT recipients [8,9] and after extensive hepatectomy for metastatic cancer hinders its application [10] . SFSG failure results in postoperative coagulopathy, cholestasis, ascites, encephalopathy, and death [11,12] . The mechanisms of SFSG failure remain incompletely understood.
One mechanism that has been implicated in the early development of SFSG failure is the formation of parenchymal hemorrhage and necrosis [13,14] . Previous investigators used electron microscopic studies to demonstrate sinusoidal breakdown leading to hemorrhage and necrosis after chemically or endotoxin-induced liver failure [15][16][17] , hepatic ischemia-reperfusion injury [18][19] , extensive hepatectomy [18,20,21] , and LT with SFSG or SOLT [22][23][24][25] . These findings suggested that although the inciting factors might have different causes, the pathogenesis of sinusoidal breakdown leading to hemorrhage and necrosis might be similar.
Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent neutral proteases that can degrade the extracellular matrix and basement membrane. MMPs play significant roles in cellular regulation, cell-cell communication, and tumor progression [26,27] . MMP-2 and MMP-9 have been implicated in liver injury and remodeling. Knocking out MMP-9 and other MMPs attenuate liver injury associated with interferon treatment [28] . MMP-9 contributes to the pathogenesis of experimental acute liver failure [29] . Specifically, MMP-9 has been implicated in sinusoidal breakdown leading to extravasation of circulating cells and hemorrhage [14,30] . MMP-9 plays a pivotal role in ischemia-reperfusion injury [31][32][33] . When MMP-9 is blocked with a synthetic inhibitor or by MMP-9 gene deletion, it successfully reverses ischemia-reperfusion injury [34,35] and cold preservation-warm reperfusion injury after LT [35] . Collectively, these data show that MMP-9 is involved in sinusoidal injury in liver failure. However, the role of MMP-9 in the pathogenesis of failure in IHR or SFSGs is not well known. We hypothesized that MMP-9 plays an important role in the development of liver parenchymal hemorrhage and necrosis in IHR/SFSG failure. Tissue inhibitors of metalloproteinases (TIMP)-1 are physiological tissue inhibitors of MMP-9, which increase MMP-9 activity when deleted [36,37] . We hypothesized that a deletion or decrease in MMP-9 has a beneficial effect after hepatectomy. In the current study, we examined this hypothesis in IHR after 80%-partial hepatectomy (PH) in wild-type (WT), MMP-9(-/-), and TIMP-1(-/-) mice. We also examined the efficacy of MMP-9 blockade with a monoclonal antibody and synthetic MMP-9 inhibitor.

Animals
Male C57BL/6 mice (10-14 wk old) were obtained from Jackson Laboratory (Bar Harbor, ME), housed with a 12-h light/dark cycle, and given food and water ad libitum. MMP-9(-/-) mice, obtained from Dr. Robert Senior (Washington University, St. Louis, MO), and TIMP-1(-/-) mice, purchased from Jackson Laboratory, both with a C57BL/6 background, were bred in our facility. The study was institutionally approved in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals".

PH in mice
Our institutional procedures for PH and postoperative care have been described elsewhere in detail [38] . Left and right posterior and left and right anterior segments were resected for an 80%-PH, similar to that described in a previous report [39] . In brief, under general anesthesia, liver lobes were mobilized. A hemostatic clip (Teleflex Medical, Triangle Park, NC) was applied across the pedicle, and the liver lobes were removed, without any obstruction of portal flow and hepatic venous drainage [38] . Laparotomy alone was performed for sham operations. Before closure, 2 mL of warm saline was administered intraperitoneally. Cephalexin (30 mg/kg) and buprenorphine (0.1 mg/kg) were given subcutaneously. Postoperatively, the mice were kept in a temperature-controlled environment with free access to food and water. All mice were sacrificed 6 h after 80%-PH. The remnant livers were harvested, and blood samples were collected. We chose the extended hepatectomy model in mice because we hypothesized that MMP-9 plays an important role in the development of liver parenchymal hemorrhage and necrosis in IHR/SFSG failure.

Biochemical analysis
Serum levels of aspartate aminotransferase (AST) and alanine aminotransaminase (ALT) were determined by using a kinetic detection kit (Pointe Scientific, Inc, Canton, MI), and total bilirubin was determined by using the QuantiChrom™ Bilirubin Assay Kit (BioAssay Systems, Hayward, CA).

Histological analysis
The HE-stained sections were scanned with a Scanscope XT system and analyzed with Aperio Imagescope software (Aperio Technologies, Inc., Vista, CA). Histologically positive areas were counted and measured by investigators blinded to the study group in three randomly

Parenchymal hepatic hemorrhage and necrosis occurs soon after 80%-PH
First, 80%-PH was performed in WT mice. Six hours after 80%-PH, the animals were killed and divided into two groups according to the status of parenchymal hemorrhage and necrosis in the remnant livers. Murine behavior in hepatic failure has been well described [42,43] . The animals with hemorrhage and necrosis were observed to be sick and inactive, whereas the mice without such injury were asymptomatic and active. As shown in Figure  1A and B, the groups consisted of mice with minimal or no hemorrhage and necrosis (group Ⅰ, n = 6) or those with multifocal hemorrhage and necrosis (group Ⅱ, n = 5). Group Ⅰ had a significantly greater area of necrosis (2.97% ± 0.92% vs 0.11% ± 0.08%, P < 0.05) ( Figure 1C) and number of necrotic foci compared with group Ⅱ (3.60 ± 0.87 vs 0.26 ± 0.17, P < 0.01) ( Figure 1D). Group Ⅱ had significantly higher AST (893 ± 72 IU/L vs 28 ± 12 IU/L, P < 0.05) ( Figure 1E), ALT (744 ± 35 IU/L vs 32 ± 77 IU/L, P < 0.05) ( Figure 1F) and total bilirubin levels than those in group Ⅰ (4.45 ± 0.63 mg/dL vs 1.41 ± 0.19 mg/dL, P < 0.01) ( Figure 1G). These results suggested that group Ⅱ mice were in the process of liver failure, and these results are consistent with previous reports [13,[44][45][46] .

MMP-9 is upregulated in the remnant liver with hemorrhage and necrosis
MMP-9 has been implicated in acute liver failure [14,29] . We investigated MMP-9 expression in the remnant livers of both groups I and Ⅱ 6 h after 80%-PH. Western blotting analysis demonstrated that MMP-9 was upregulated in group Ⅱ compared with group Ⅰ (P < 0.01) ( Figure  2A and B). When we examined immunohistochemical staining for MMP-9, hepatocytes showed no expression of MMP-9. However, MMP-9 was mainly observed in round-shaped cells and in stellate-shaped cells ( Figure  3A and B). To evaluate the association between necrosis and MMP-9, we compared the expression of MMP-9 in 10 randomly selected fields of 3-mm 2 in area with and without hemorrhage and necrosis. MMP-9 expression in the foci of hemorrhage and necrosis was higher than that in normal liver parenchyma (P < 0.05) ( Figure 3C). These results suggest that MMP-9 expression is associated with the development of hemorrhage and necrosis in the remnant liver after 80%-PH, which is consistent with previous reports [14,29] .

CD11b-positive neutrophils are the main source of MMP-9 in the remnant liver
To determine the source of upregulated MMP-9 in the selected 3-mm 2 fields. DAB-positive staining of MMP-9 was calculated using the positive-pixel-count function of Imagescope software in 10 randomly selected fields. The value was quantified as the percentage of positive pixels per total pixels. The number of infiltrated neutrophils in the remnant liver was evaluated in five randomly captured 40 × high-power fields (hpf) with an Olympus BX50 fluorescence microscope (Olympus Optical, Tokyo, Japan).

Preliminary study
In our institution, 90%-PH (10% of the liver remnant with an omental segment), 80%-PH (20% of the liver remnant with right middle and omental segments) and 75%-PH (25% of the liver remnant with right middle and posterior segments) are available [38] . Initially, we performed 90%-PH in WT, MMP-9(-/-) and TIMP-1(-/-) mice, as a preliminary study. A survival study after 90%-PH was performed in each murine strain. Moreover, liver damage scores in HE staining [41] , serum AST and ALT levels, and the ratios of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei/all nuclei per mm 2 and caspase 3-positive nuclei/all nuclei per mm 2 in immunohistological staining were assessed in serum or liver samples 6 h after 90%-PH in each murine strain. The results for each factor did not reach significant differences between WT and MMP-9(-/-) mice and between WT and TIMP-1(-/-) mice. Even in WT mice (control group), all mice received 90%-PH without any surgical issues but showed considerable damage and subsequently died in the early postoperative period. We therefore considered that 90%-PH is not suitable to investigate the effect of MMP-9 and TIMP-1 on the hepatic remnant after hepatectomy. To obtain relevant data according to clinical background [1][2][3][4][5][6][7][8][9][10] , we finally employed 80%-PH in each strain for further experiments in this study. We decided to evaluate 80%-PH because mice could survive for a long time after surgery.

Statistical analysis
Data are presented as the mean ± SE. Statistical comparisons were performed using analysis of variance followed by a two-sample t-test with Bonferroni adjustment. A P value less than 0.05 was considered statistically significant. remnant liver, we performed double immunofluorescent analysis with MMP-9 and cell marker antibodies. CD68 and desmin were used for Kupffer cells and hepatic stellate cells, respectively. MMP-9 was not observed in CD68-positive Kupffer cells ( Figure 4A-C). Weak immunoreactivity to MMP-9 was observed in desmin-positive hepatic stellate cells ( Figure 4D-F). In contrast, we consistently found MMP-9 expression in CD11b-positive cells ( Figure 4G-I). These results suggest that infiltrating neutrophils are the source of MMP-9 found in the foci of hemorrhage and necrosis in the remnant liver after 80%-PH, which is consistent with previous reports [28,35] .

MMP-9 deletion reduces parenchymal hemorrhage and necrosis
To further confirm the role of MMP-9 in the development of liver hemorrhage and necrosis after 80%-PH, we performed liver resections in WT, MMP-9(-/-), and Representative images of remnant liver histology demonstrating significant multiple foci of hemorrhage and necrosis in group Ⅱ mice; C: Quantitation of the size (area percentage) of hemorrhage and necrosis: Significantly larger areas and more hemorrhagic and necrotic foci were observed in group Ⅱ mice than in group Ⅰ mice ( a P < 0.05); D: Quantitation of the number of foci of hemorrhage and necrosis per mm 2 : Significantly larger areas and more hemorrhagic and necrotic foci were observed in group Ⅱ mice than in group Ⅰ mice ( b P < 0.01); E: Aspartate aminotransferase (AST) levels were significantly higher in group Ⅱ mice than those in group Ⅰ mice ( a P < 0.05); F: Alanine aminotransaminase (ALT) levels were significantly higher in group Ⅱ mice than those in group Ⅰ mice ( a P < 0.05); G: Total bilirubin levels were significantly higher in group Ⅱ mice than those in group Ⅰ mice ( b P < 0.01).

Inhibition of MMP-9 with MMP-9 mAb or GM6001 ameliorates liver hemorrhage and necrosis
We performed an MMP-9 inhibition experiment with a

B
A C specific MMP-9 monoclonal antibody (MMP-9 mAb) and the broad-spectrum MMP inhibitor GM6001, as previously described [40] . There were significantly smaller and fewer necrotic areas in the remnant liver in mice treated with MMP-9 mAb than in control IgG mice (necrotic area: 0.17% ± 0.15% vs 1.81% ± 0.66%, P < 0.05; number of necrotic foci: 0.23 ± 0.16 vs 1.23 ± 0.44, P < 0.05) ( Figure 8A and B). We showed inhibition of gelatinolytic activity with in situ gelatin zymography [40] , which demonstrated significantly reduced MMP-9 activity in the MMP-9 mAb-treated group compared with that in the IgG-treated group ( Figure 8C and D). Similarly, the number of infiltrated neutrophils was significantly lower in the group treated with MMP-9 mAb than that in the group treated with IgG (4.3 ± 1.1 vs 9.1 ± 1.9 per hpf, P < 0.05) ( Figure 8E-G).
We observed similar results with GM6001. Treatment with GM6001 showed significant suppression of liver necrosis compared with the DMSO vehicle (necrotic area: 0.19% ± 0.13% vs 1.04% ± 0.36%, P < 0.05; number of necrotic foci: 0.35 ± 0.19 vs 1.12 ± 0.31, P < 0.05). Neutrophil infiltration was significantly suppressed in the GM6001 group compared with that in the DMSO group (5.5 ± 1.0 vs 9.2 ± 1.3 per hpf, P < 0.05). In situ zymographic analysis showed a significant reduction in gelatinolytic activity in the GM6001 group compared with that in the DMSO group (data not shown). Collectively, these results demonstrate that MMP-9 inhibition plays an important role in protecting mice from developing parenchymal hemorrhage and necrosis after 80%-PH.

DISCUSSION
IHR after extended liver resection or a SFSG after SOLT is prone to develop parenchymal hemorrhage and necrosis. Uncontrolled progression of the foci of hemorrhage and necrosis leads to liver failure. The role of MMP-9 in liver injury in the hepatic remnant has not been investigated. In this study, we investigated the role of MMP-9 in the development of hemorrhage and necrosis in IHR  after 80%-PH in mice. We demonstrated that in IHR 6 h postoperatively, MMP-9 was upregulated in infiltrating neutrophils associated with the foci of hemorrhage and necrosis. Blocking MMP-9 expression by using a synthetic inhibitor, a specific monoclonal antibody, or gene deletion significantly ameliorated the formation of parenchymal hemorrhage and necrosis.
In the current study, we found that the number and percentage of necrotic foci were decreased in MMP-9(-/-) mice compared with WT mice. While TIMP-1(-/-) mice appeared to show slightly increased necrosis, there were no statistical differences between WT and TIMP-1(-/-) mice. TIMP-1 is a physiological tissue inhibitor of MMP-9 [36,37] , and therefore, MMP-9 activity is increased under the absence of TIMP-1 [36,37] . Our results demonstrated that a decrease or absence of MMP-9 prevented liver injury, and that a physiological inhibitor of MMP-9, i.e., TIMP-1, caused similar liver damage in WT mice with IHR after 80%-PH. One possible explanation for the similar damage between WT and TIMP-1(-/-) mice is that liver injury after 80%-PH is very severe in these mice.
In our study of IHR after 80%-PH in mice, we found that infiltrating neutrophils carry MMP-9, which appears to be responsible for the development of hemorrhage and necrosis. This is consistent with previous reports [14,28,35] . Wielockx and colleagues showed that MMPs, including MMP-9, play a central role in the destruction of sinusoidal integrity, resulting in hemorrhage and necrosis [28] . They speculated that MMPs could be derived from infiltrating neutrophils, resident hepatic stellate cells, and Kupffer cells. Recently, hepatic stellate cells have been shown to play a critical role in acute liver failure induced by lipopolysaccharide and beta-galactosamine or carbon tetrachloride by releasing MMP-9, which disrupts sinusoid integrity leading to sinusoidal collapse and liver failure [29] . Similarly, in hepatic warm ischemia-reperfusion injury, hepatic MMP-9 and MMP-2 induce sinusoidal injury in a manner that is independent of neutrophil cytotoxicity [35] . Together, these data suggest that decreasing MMP-9 expression is useful to reduce early sinusoidal injury in IHR and SFSGs.
Inhibition of MMP-9 appears to be a potential therapeutic option in ischemic liver injury. In a previous study in rats, 70% of the liver was rendered ischemic for 90 minutes followed by reperfusion; a phosphinic MMP inhibitor attenuated the liver injury and necrosis [49] . Gene deletion of MMP-9 mitigates liver injury after 90 minutes of warm ischemia and reperfusion in mice [34,35] . Inhibition of MMP-9 has been shown to protect rat livers from cold preservation-warm reperfusion injury [34,35] . Interestingly, after cold ischemic preservation and warm reperfusion, it was found that MMP-9 and MMP-2 were not of hepatocellular origin but from an extrahepatic source [34,35] . Therefore, collectively, these data support MMP-9 reduction as a potential pathway to improve parenchymal integrity and function in IHR after extended liver resection or a SFSG after SOLT. Although MMP-9 can cause liver injury in IHR after extended liver resection or SFSGs after SOLT, MMP-9 is also essential for liver regeneration [50] . It has been shown that MMP-9 releases hepatic growth factor from the hepatic extracellular matrix during the priming phase of liver regeneration [51,52] . Together with its physiological tissue inhibitor, TIMP-1, MMP-9 participates in regulating the hepatocellular cell cycle and proliferation [51] . MMP-9 is temporally correlated to angiogenesis and regeneration in the liver. Therefore, prolonged inhibition of MMP-9 might be clinically undesirable. Recent studies have suggested that inhibition of MMP-9 to ameliorate the early formation of parenchymal hemorrhage and necrosis should probably be started within and be limited to the first 2 d after extended liver resection or SOLT with SF-SGs [50,52] . However, the use of MMP-9 inhibition in the clinical management of IHR or SFSGs requires further investigation and understanding to optimize its therapeutic effectiveness.
After extended liver resection or SOLT with SFSGs, portal hyperperfusion is likely to occur. Although increased portal hypertension is important in liver regeneration, uncontrolled acute portal hypertension with portal hyperperfusion can be detrimental [21,25,53] . In SOLT with SFSGs, portal hypertension and hyperperfusion are known to contribute to SFSL failure [25,54,55] . In experimental models [25,53,56] and in clinical LT with SFSGs [8,9,25,54,55] , decompression of portal hyperperfusion has been shown to significantly attenuate hepatic hemorrhage and necrosis [57,58] . Evidence suggests that surgical and anatomical reduction of portal hyperperfusion is important in the management of IHR and SFSGs.
The molecular mechanisms by which portal hyperperfusion affects the outcome of IHR after extended hepatectomy and SFSGs after SOLT are poorly understood. Shear stress has been shown to induce nitric oxide production in microvascular endothelial cells [59] , which might upregulate MMP-9 synthesis and production [60] . Reduction of portal hyperperfusion could thus indirectly mitigate the effect of MMP-9 production. However, it is not known how portal hyperperfusion relates to MMP-9 with respect to sinusoidal injury leading to parenchymal hemorrhage and necrosis. Other potential important mediators in the injury of IHR or SFSGs include oxidative stress [61] and tumor necrosis factor [62] . Understanding the role of MMP-9 and other signaling pathways will increase our ability to develop therapeutic options to heal and prevent liver injury associated with IHR and SFSGs.
Neutrophils have been consistently implicated in ischemia-reperfusion injury [19,35] . Neutrophils might produce and release MMPs that contribute to initiating sinusoidal injury [14] . Conversely, hepatic MMP-9 might be derived from hepatic stellate cells or sinusoidal cells, including endothelial cells and Kupffer cells [29] . Hepatic-derived MMP-9 might then attract neutrophils [30] into the liver after an injury. Neutrophils could then initiate and/or propagate sinusoidal hemorrhage and progressive necrosis [35] . Therefore, targeting neutrophils for treatment could be impor-tant in some diseases and conditions.
The limitations of this study include the following. Although MMP-9 is implicated in the formation of parenchymal hemorrhage and necrosis in IHR after 80%-PH, our study did not address how and where MMP-9 acts on the sinusoidal elements. The cascade of nitric oxide synthetase, which is important in upregulating MMP-9, was not examined [59,60] . Even though we did not observe constitutive MMP-9 in association with parenchymal hemorrhage and necrosis, we could not rule out the possibility that hepatic cells, including sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells [30] , produced MMP-9 at a minute level that was adequate to initiate liver injury with subsequent recruitment of circulating neutrophils. The potential role of T cells was not evaluated [63] . The possibility of myeloperoxidase-associated oxidative inactivation of TIMP-1, thus activating MMP-9, was not considered in this study [64] . We consider that the effects of upregulation of MMP-9 on sinusoidal and endothelial cells should be investigated in detail.
Initially, we hypothesized that MMP-9 plays an important role in parenchymal hemorrhage and necrosis in the small remnant liver. We found significantly less hemorrhagic and necrotic lesions in MMP-9(-/-) remnant livers than in WT and TIMP-1(-/-) remnant livers. Similar results were observed with MMP-9 monoclonal antibody or the synthetic inhibitor GM6001. In conclusion, we demonstrated that MMP-9 plays an important role in the development of hepatic hemorrhage and necrosis in the small liver remnant 6 h after extensive partial hepatectomy. Moreover, we showed that infiltrating neutrophils are critical to the process of liver injury. Targeting MMP-9 could provide a clinically useful tool to optimize the function of a small liver volume, i.e., IHR after extended hepatectomy or SFSGs after SOLT.

ACKNOWLEDGMENTS
We are very grateful to Dickson W Dennis, Monica Castanedes-Casey, Virginia R Phillips, Linda G Rousseau and Melissa E Murray (Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL, United States) for their diagnostic and technical support for histopathological and immunohistological assessment, and to Kathleen Norton and Lisa Maroski for their editorial assistance.

Background
In the field of hepatobiliary surgery, liver resection is considered the standard treatment for primary liver tumors and colorectal liver metastases. Major liver resections have been associated with an increased morbidity and mortality compared with more limited resections. In particular, the outcomes are worsened when a chronic underlying liver disease is present. Recent studies have shown that the volume of the remnant liver is correlated with perioperative morbidity and mortality, and an insufficient hepatic remnant (IHR) after extended hepatectomy is a critical issue.

Research frontiers
Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent neutral proteases that can degrade the extracellular matrix and basement membrane.

COMMENTS
MMPs play significant roles in cellular regulation, cell-cell communication, and tumor progression. MMP-2 and MMP-9 have been implicated in liver injury and remodeling. Knocking out MMP-9 and other MMPs attenuates liver injury associated with interferon treatments. MMP-9 contributes to the pathogenesis of experimental acute liver failure. Specifically, MMP-9 has been implicated in sinusoidal breakdown leading to extravasation of circulating cells and hemorrhage. MMP-9 plays a pivotal role in ischemia-reperfusion injury. When MMP-9 is blocked with a synthetic inhibitor or by MMP-9 gene deletion, it successfully reverses ischemia-reperfusion injury. Collectively, these data show that MMP-9 is involved in sinusoidal injury in liver failure. However, the role of MMP-9 in the pathogenesis of failure in IHR is not well understood.

Innovations and breakthroughs
Initially, the authors hypothesized that MMP-9 plays an important role in parenchymal hemorrhage and necrosis in the small remnant liver. In this study, the authors found significantly less hemorrhagic and necrotic lesions in MMP-9(-/-) remnant livers than in wild-type and tissue inhibitors of metalloproteinases (TIMP)-1(-/-) remnant livers. Similar results were observed with a MMP-9 monoclonal antibody or synthetic inhibitor GM6001.

Applications
The authors demonstrated that MMP-9 plays an important role in the development of hepatic hemorrhage and necrosis in the small remnant liver 6 h after extensive partial hepatectomy. Moreover, the authors showed that infiltrating neutrophils are critical to the process of liver injury.

Terminology
Targeting MMP-9 could provide a clinically useful tool to optimize the function of a small liver volume, i.e., IHR after extended hepatectomy.

Peer review
Using MMP-9(-/-), wild type, and TIMP-1(-/-) mice, the authors demonstrated that in IHR at 6 h postoperatively MMP-9 is upregulated in the infiltrating neutrophils associated with the foci of hemorrhage and necrosis. Blocking MMP-9 expression by using a synthetic inhibitor, specific monoclonal antibodies, or gene deletion significantly ameliorated the formation of parenchymal hemorrhage and necrosis. The authors finally proposed that MMP is tightly associated with liver parenchymal hemorrhage and necrosis for insufficient liver remnant, which always leads a lethal outcome clinically.