A Novel Humanized Model of NASH and Its Treatment With META4, A Potent Agonist of MET

Abstract

Background & aims: Nonalcoholic fatty liver disease is a frequent cause of hepatic dysfunction and is now a global epidemic. This ailment can progress to an advanced form called nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, the molecular basis of NASH pathogenesis is poorly understood, and no effective therapies exist to treat NASH. These shortcomings are due to the paucity of experimental NASH models directly relevant to humans.

Methods: We used chimeric mice with humanized liver to investigate nonalcoholic fatty liver disease in a relevant model. We carried out histologic, biochemical, and molecular approaches including RNA-Seq. For comparison, we used side-by-side human NASH samples.

Results: Herein, we describe a "humanized" model of NASH using transplantation of human hepatocytes into fumarylacetoacetate hydrolase-deficient mice. Once fed a high-fat diet, these mice develop NAFLD faithfully, recapitulating human NASH at the histologic, cellular, biochemical, and molecular levels. Our RNA-Seq analyses uncovered that a variety of important signaling pathways that govern liver homeostasis are profoundly deregulated in both humanized and human NASH livers. Notably, we made the novel discovery that hepatocyte growth factor (HGF) function is compromised in human and humanized NASH at several levels including a significant increase in the expression of the HGF antagonists known as NK1/NK2 and marked decrease in HGF activator. Based on these observations, we generated a potent, human-specific, and stable agonist of human MET that we have named META4 (Metaphor) and used it in the humanized NASH model to restore HGF function.

Conclusions: Our studies revealed that the humanized NASH model recapitulates human NASH and uncovered that HGF-MET function is impaired in this disease. We show that restoring HGF-MET function by META4 therapy ameliorates NASH and reinstates normal liver function in the humanized NASH model. Our results show that the HGF-MET signaling pathway is a dominant regulator of hepatic homeostasis.

Keywords: FAH Mice; Fatty Liver Disease; HGF; HGF antagonist; Hepatocyte Growth Factor; High-fat Diet; Humanized Liver; Liver Cancer; MET; Metabolic Syndrome; NAFLD; NASH; NK1; NK2; Type 2 Diabetes.

Graphical abstract

Figure 1

Mice with humanized liver develop NAFLD if placed on an HFD.A, Images of liver sections from humanized liver stained with hematoxylin and eosin (H&E), Oil-Red-O, FAH, and TUNEL as indicated. Arrows points to fat-laden hepatocytes. B, Liver and serum triglyceride level. N = 4–6 mice per group. Bar graphs depict the relative expression. ∗∗∗P = .001 and ∗∗P = .01, respectively. C and D, FAH immunostain. FAH-positive human hepatocytes are marked by filled arrows and FAH-negative mouse hepatocytes are marked by unfilled arrows. In D, note the foci of inflammatory cells surrounding the human hepatocytes. E, TUNEL stain. Arrow points to the same region positive for FAH. Scale: 100 mm in panels A, C, E and 30 mm in panels B and D, respectively.

Figure 2

Humanized fatty liver phenocopies human NASH at the histologic, cellular, and biochemical levels. Results shown are from analyses performed side-by-side on the humanized (A) and human NASH livers (B), and nontransplanted livers for the indicated markers as determined by immunohistochemistry. Scale: 100 mm for left and 30 mm for right images in each column. C, Depicts higher magnification image of humanized liver stained with trichrome for collagen.

Figure 3

Quantification of the results shown inFigure 2. Graphs in (A) and (B) depict indicated markers shown in Figure 2 as determined by image analysis. C, Illustrates quantification of collagen content in the liver by measuring hydroxyproline a component of collagen. Nontransplanted FRGN and wild type CD1 mice are also included for comparison. Asterisks denote P < .05. See text for details.

Figure 4

Humanized NASH recapitulates human NASH as determined by RNA-Seq analyses. Shown are examples of the top 10 pathways that are significantly down-regulated (A) or upregulated (B) in human and humanized NASH livers as compared with their corresponding normal livers. Pathway names and number of genes impacted are indicated in the graphs. Pathways are ordered from top to bottom by P values. Bars with blue and red colors denote identical pathways that are affected in both human and humanized NASH.

Figure 5

Pathway of cell death is upregulated in human and humanized NASH. Shown are heat maps of Pathway of Necroptosis [KEGG hsa04217]. Red and blue colors indicate up- or down-regulated expression, respectively.

Figure 6

Pathways of viral infection is regulated in human and humanized NASH. Shown are the heatmaps of the hepatitis C [KEGG hsa05160]. Red and blue colors indicate up- or down-regulated expression, respectively.

Figure 7

Human NASH and humanized NASH co-cluster as determined by RNA-Seq and principal component analysis (PCA). Shown is the PCA graph. PCA was performed with genes that have the analysis of variance P value of .05 or less on FPKM abundance estimations. The Figure is an overview of samples clustering. The result from PCA shows a distinguishable gene expression profiling among the samples. A, Normal human liver samples (labeled NHL) co-cluster with each other and human liver samples with NASH (labeled FHL) co-cluster with each other; n = 3 for human non-fatty; n = 3 for human NASH. B, Similarly, humanized NASH co-cluster with each other and humanized normal co-cluster together; n = 6 per group. C, Human and humanized NASH co-cluster with each other, and human normal and humanized normal group together; n = 3–6 per group.

Figure 8

Pronounced changes in mRNA alternative splicing events occur in human NASH and humanized NASH livers as determined by RNA-Seq and pathway analyses. Humanized and human NASH liver was analyzed side-by-side using RNA-Seq and gene set enrichment analysis (GSEA). A, Depicted is the differential alternative splicing (AS) events summary plots for human and NASH livers as compared with their corresponding normal livers. Upregulated transcript variants are shown in red and downregulated in green colors, respectively. Splice types are: skipped exon (SE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3′SS), retain intron (RI), and mutually excluded (MXE) exons. Numbers in the plot correspond to transcript numbers involved. B, Heat maps of the spliceosome pathway (KEGG-HSA03040) impacted in human and humanized NASH livers. Upregulated transcript variants are shown in red and down-regulated in blue colors, respectively; n = 6 for human and n = 4 for humanized livers.

Figure 9

HGF antagonists NK1 and NK2 are expressed in human NASH liver.A, Results of RT-PCR (n = 3 cases per group); and B, Western immunoblot for HGF antagonist (n = 5 cases per group) using antibody to the N-terminal region of HGF. Bar graphs depict the relative expression. C, D, HGFAC expression is significantly reduced in the livers of humans with NASH. C, Shown is the relative abundance of HGF activator transcript in human liver as determined by RNA-seq. ∗P = .02. D, Depicted are the Western blot results for HGFAC in human normal and NASH livers (n = 5 and n = 6 cases per group as indicated).

Figure 10

HGF antagonist is present in the plasma of patients with NASH. Shown are the results of Western immunoblot of plasma samples (3 microliters) using antibody to the N-terminal region of HGF. Coomassie blue stain of the gel is shown below the blots. Coomasie blue stain of gel is shown for equal loading of plasma samples. Bar graphs depicts the relative expression of NK1/NK2 signals. NASH (n = 10 different cases) and normal (n = 3 different cases).

Figure 11

HGF expression is reduced in the liver of wild-type mice C57/Bl6 fed a HFD whereas that of HGF antagonist is induced.A, Western blot data for HGF; and B, RT-PCR results for NK1 expression. Animals were culled at feed or after an overnight fast as indicated. Mice were fed on HFD for 3 months.

Figure 12

Robust and rapid activation of MET and MET signaling effectors by META4. A, Activation of MET in human hepatocyte cell line HepG2; shown is the Western blot for the indicated effectors. B, META4 does not activate rodent MET. Western blot data showing that META4 activates MET in human but not mouse hepatocytes (Hepa 1-6 cell line). Cells were treated for 15 minutes and processed for MET activation (pMET 1234Y) and total MET as indicated. HGF was used as a positive control, which activates mouse and human hepatocytes. C, META4 activates MET in non-human primates Rhesus monkey kidney epithelial cell line LLC-MK2 and in human kidney epithelial cell line HEK-293. D, Production of active recombinant META4. HEK-293 ells were transfected with META4 heavy plus light chain expression vectors or by individual chains as indicated. Culture media were harvested 5 days post-transfection, and META4 was purified by protein-A chromatography. Activity was assessed by MET activation as in (A).

Figure 13

META4 activates MET and MET in humanized mice liver. META4 was injected intraperitoneally at 1 mg/g, and livers were collected at 30 and 60 minutes and assessed for MET activation as indicated.

Figure 14

Restoration of MET signaling by META4 therapy ameliorates liver inflammation and fibrosis in the humanized NASH and promotes expansion of the transplanted human hepatocytes.A, Shown are representative images of liver sections from humanized mice with NASH treated with META4 or with mIgG1 stained for the indicated markers. B-D, Confirmation of META4 effects at the protein level. A, Alpha smooth muscle actin (α-SMA); B, Vimentin; and C, IKBa. Livers from nontransplanted (non-TXP) FRGN and ob/ob mice are included for comparison (n = 4) for META4 and (n = 2) for and control.

Figure 15

META4 promotes survival and proliferation of human hepatocytes in humanized NASH model. Shown are representative images of liver sections stained for TUNEL (A) and Ki67 and FAH double staining as indicated. Scale: 100 mm in the left panel and 30 mm in the right panel, respectively. Black arrows point to FAH-positive and Ki67-negative, and white arrows point to hepatocytes positive for FAH and nuclear Ki67. Mice were on HFD for 6 weeks and then 4 weeks of META4 therapy (single intraperitoneal injection weekly). B, Results of Western blot for FAH indicating expansion (survival and proliferation) of human hepatocytes by META4.

Figure 16

META4 therapy ameliorates weight lost (A) and hepatomegaly (B) in mice with humanized liver.A, Bar graphs show gradual weight loss in control-treated mice after NTBC withdrawal. ∗P = .016. Significance was assessed by the Student t test (n = 7 per group). B, Shown are the gross appearance of livers and plots of liver to body ratios for META4- (n = 4) or mIgG1(n = 4) treated mice as indicated. ∗∗P = .01.

Figure 17

HGF-MET axis promotes down regulation of pathways involved in NAFLD, inflammation, oxidative phosphorylation, and cell death as determined by RNA-seq. Depicts the top 10 pathways that are downregulated (A) or upregulated (B) by META4 (bar graph colors are arbitrary). Pathway names and number of genes impacted are indicated in the graphs. Pathways are ordered by P values from top to bottom. C, Illustrates heat maps of the NFkB, chemokine, and NAFLD pathways and their effector genes as determined by gene set enrichment analysis (GSEA). Red and blue colors indicate induced and repressed genes, respectively. C denotes control and M indicates META4-treated, respectively. A total of 12 humanized mice were analyzed (n = 5 for control and n = 7 for META4 group).