Broad-spectrum matrix metalloproteinase inhibition curbs inflammation and liver injury but aggravates experimental liver fibrosis in mice

Abstract

Background: Liver fibrosis is characterized by excessive synthesis of extracellular matrix proteins, which prevails over their enzymatic degradation, primarily by matrix metalloproteinases (MMPs). The effect of pharmacological MMP inhibition on fibrogenesis, however, is largely unexplored. Inflammation is considered a prerequisite and important co-contributor to fibrosis and is, in part, mediated by tumor necrosis factor (TNF)-alpha-converting enzyme (TACE). We hypothesized that treatment with a broad-spectrum MMP and TACE-inhibitor (Marimastat) would ameliorate injury and inflammation, leading to decreased fibrogenesis during repeated hepatotoxin-induced liver injury.

Methodology/principal findings: Liver fibrosis was induced in mice by repeated carbon tetrachloride (CCl4) administration, during which the mice received either Marimastat or vehicle twice daily. A single dose of CCl4 was administered to investigate acute liver injury in mice pretreated with Marimastat, mice deficient in Mmp9, or mice deficient in both TNF-alpha receptors. Liver injury was quantified by alanine aminotransferase (ALT) levels and confirmed by histology. Hepatic collagen was determined as hydroxyproline, and expression of fibrogenesis and fibrolysis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. Marimastat-treated animals demonstrated significantly attenuated liver injury and inflammation but a 25% increase in collagen deposition. Transcripts related to fibrogenesis were significantly less upregulated compared to vehicle-treated animals, while MMP expression and activity analysis revealed efficient pharmacologic MMP-inhibition and decreased fibrolysis following Marimastat treatment. Marimastat pre-treatment significantly attenuated liver injury following acute CCl4-administration, whereas Mmp9 deficient animals demonstrated no protection. Mice deficient in both TNF-alpha receptors exhibited an 80% reduction of serum ALT, confirming the hepatoprotective effects of Marimastat via the TNF-signaling pathway.

Conclusions/significance: Inhibition of MMP and TACE activity with Marimastat during chronic CCl4 administration counterbalanced any beneficial anti-inflammatory effect, resulting in a positive balance of collagen deposition. Since effective inhibition of MMPs accelerates fibrosis progression, MMP inhibitors should be used with caution in patients with chronic liver diseases.

Figure 1. Marimastat treatment reduced liver injury, necrosis, and inflammation following repeated carbon tetrachloride (CCl4) administration.

Chronic CCl4 administration resulted in liver enlargement and fibrosis (A). Hematoxylin and eosin staining of liver sections revealed decreased steatosis and inflammation (yellow arrows), and no evidence of necrosis (black arrows) in the Marimastat treated mice (B). On liver sections scored by a blinded pathologist and compared to vehicle treated controls, Marimastat treated animals showed a significantly lower steatosis score (C), less inflammatory foci per 200× field (D) and essentially no evidence of necrosis was observed (E); despite body weight loss (F). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error. Original magnification: 200×.

Figure 2. Marimastat treatment ameliorated hepatic injury and the inflammatory response following repeated carbon tetrachloride (CCl4) administration.

Marimastat treatment significantly reduced serum alkaline phosphatase levels (A), and resulted in a 14-fold decrease of serum ALT (B), indicating decreased hepatic injury. Serum TNF-α receptor II (p75) levels as measured by ELISA decreased following Marimastat treatment, suggesting successful inhibition of TNF-α converting enzyme (TACE) and an ameliorated inflammatory response. IL-6 serum levels as measured by ELISA increased following Marimastat treatment, suggesting hepatoprotection and stimulated liver regeneration (D). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; ALT, alanine aminotransferase; TNF, tumor necrosis factor; IL, interleukin; *, P<0.05; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error.

Figure 3. Marimastat altered the hepatic fibrosis-related gene expression profile by downregulation of multiple pro-fibrogenic transcripts following repeated carbon tetrachloride (CCl4) administration.

Hepatic procollagen α1(I) (A), β6 integrin (B), TGF-β1 (C), TGF-β2 (D), α-SMA (E) and TIMP-1 (F) expression as quantified by real-time RT-PCR in total liver RNA. Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; TGF, transforming growth factor; SMA, smooth muscle actin; TIMP, tissue inhibitor of metalloproteinases; *, P<0.05; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error and in arbitrary units relative to non-fibrotic vehicle treated control.

Figure 4. Marimastat treatment increased hepatic fibrosis following repeated carbon tetrachloride (CCl4) administration.

In mice treated with Marimastat, the liver to body weight ratio (A), as well as the spleen to body weight ratio (B) were increased. Masson trichrome staining of liver sections for collagen (blue; C) revealed bridging portal fibrosis. Livers from Marimastat treated animals showed occasional focal cirrhosis; however, advanced fibrosis was predominant (C). Livers from Marimastat treated mice exhibited increased collagen deposition as determined biochemically as relative hydroxyproline content and total hydroxyproline content in liver samples from two different lobes (D, E). Relative fibrotic area was increased in livers from mice treated with Marimastat, as quantified using morphometric analysis of Sirius Red stained liver sections (F). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; *, P<0.05; **, P<0.01; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error.

Figure 5. Marimastat decreases hepatic stellate cell (HSC) activation, but increases recruitment of inflammatory cells.

Chronic carbon tetrachloride (CCl4) administration in animals treated with Marimastat resulted in a decreased activation of HSCs, as identified by alpha-smooth muscle actin (α-SMA) staining (A). Quantification revealed that following chronic CCl4 administration, Marimastat treated animals had a 74% decrease of activated HSCs, compared to controls (B). Liver sections from animals that were chronically challenged with CCl4 showed that resident T cells (CD3, C,D) and macrophages (F4/80, E,F) counts increased up to 2-fold upon Marimastat treatment. Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; α-SMA, alpha-smooth muscle actin; *, P<0.05; **, P<0.01; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error. Original magnification: 200×.

Figure 6. Marimastat treatment downregulates matrix metalloproteinase (MMP) gene expression and MMP-activities.

Following repeated carbon tetrachloride (CCl4) administration, Marimastat did not affect hepatic transcript levels of MMP-2 (A), or MMP-3 (B) as quantified by real-time RT-PCR. Hepatic MMP-8 (C) and MMP-9 (D) transcripts were significantly upregulated in livers from Marimastat treated animals, whereas hepatic MMP-13 mRNA was significantly higher in the vehicle treated animals (E). Marimastat inhibits interstitial collagenolytic and gelatinolytic activity in a dose-dependent matter (F). Relative gelatinase and interstitial collagenase activities after 4 hours in liver homogenates supplemented with increasing concentrations of Marimastat, as determined by degradation of DQ-gelatin and collagen (black and grey squares, respectively). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; *, P<0.05; **, P<0.01; ***, P<0.001 vs. vehicle alone. Data are expressed as means ± standard error and in arbitrary units.

Figure 7. Marimastat reduces necroinflammatory injury following a single dose of carbon tetrachloride (CCl4) via a TNF-dependent pathway.

Liver sections from Marimastat-treated animals that had been administered a single dose of CCl4 revealed decreased necroinflammatory injury after 24 h (A). Marimastat treatment resulted in a 57% decrease of serum ALT levels (B). Liver sections from Mmp9−/− and wild type (Mmp9+/+) animals that were challenged with a single dose of CCl4 showed comparable, extensive necroinflammatory changes around the central veins after 24 h (C). Serum ALT levels of Mmp9−/− animals were similar to those of wild type controls (D). 24 h after a single dose of CCl4, liver sections from wild type (TNF p55+/+ p75+/+) animals revealed extensive necrosis and inflammation around the central veins, whereas TNF p55−/− p75−/− animals showed markedly reduced hepatic injury (E). Serum ALT levels in TNF p55−/− p75−/− animals were 5-fold lower compared to wild type controls, corroborating the findings on histology and indicating decreased hepatic injury in animals lacking both TNF-receptors (F). Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; +/+, wild type controls; −/−, knock out animals; ALT, alanine aminotransferase; TNF, tumor necrosis factor; **, P<0.01 vs. vehicle alone. Data are expressed as means ± standard error. Original magnification: 300×.

Figure 8. Broad-spectrum matrix metalloproteinase (MMP)-inhibition results in increased hepatic fibrosis following chronic hepatic injury.

In the absence of liver injury a physiological balance exists between extracellular matrix synthesis and its degradation (A). Chronic hepatic injury causes excessive synthesis of extracellular matrix proteins including collagen, which prevails over their enzymatic degradation resulting in liver fibrosis (B). Despite a significant attenuation of fibrogenesis and inflammation, efficient inhibition of fibrolytic matrix metalloproteinases by a broad spectrum MMP-inhibitor has profound effects on collagen degradation, tilting the balance towards net extracellular matrix deposition and scar tissue formation (C).

Figure 9. Design of the studies.

Two groups of C57Bl/6J mice were randomized to two subgroups to receive either Marimastat or methylcellulose vehicle twice daily (A). Liver fibrosis was induced by repeated carbon tetrachloride (CCl4) administration for 6 weeks in one group of animals, whereas a second group of control animals received the mineral oil vehicle alone (A). The protective effects of Marimastat were further evaluated in a model of acute CCl4-induced hepatotoxicity (B). C57Bl/6J mice received either Marimastat or methylcellulose vehicle twice daily for 1 week, after which they were subjected to a single dose of CCl4 or mineral oil as control (B). The mechanism was further elucidated by subjecting Mmp9−/− mice and their wild type (WT) littermates (C), or TNF p55−/−p75−/− mice and their WT littermates (D) to either a single dose of CCl4, or mineral oil. In all acute CCl4-experiments, animals were sacrificed after 24 hours. Oil, non-fibrotic control group; CCl4, fibrotic mice; VEH, vehicle treated control group; MAR, Marimastat treated experimental group; WT, wild type.