Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

doi:10.1038/s42255-020-0214-9

. 2020 Jun;2(6):514-531.

doi: 10.1038/s42255-020-0214-9. Epub 2020 Jun 8.

Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

Michele Vacca^{1

2}, Jack Leslie³, Samuel Virtue⁴, Brian Y H Lam⁵, Olivier Govaere⁶, Dina Tiniakos^{6

7}, Sophie Snow⁸, Susan Davies⁹, Kasparas Petkevicius⁴, Zhen Tong¹⁰, Vivian Peirce⁴, Mette Juul Nielsen¹¹, Zsuzsanna Ament¹², Wei Li¹⁰, Tomasz Kostrzewski⁸, Diana Julie Leeming¹¹, Vlad Ratziu¹³, Michael E D Allison⁹, Quentin M Anstee^{6

14}, Julian L Griffin^{12

15}, Fiona Oakley³, Antonio Vidal-Puig^{16

17

18}

Affiliations

¹ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK. [email protected].
² Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK. [email protected].
³ Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
⁴ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK.
⁵ Yeo Group and Genomics and Transcriptomics Core, WT/MRC Institute of Metabolic Science, University of Cambridge, Cambridge, UK.
⁶ Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
⁷ Department of Pathology, Aretaieion Hospital, Medical School, National & Kapodistrian University of Athens, Athens, Greece.
⁸ CN Bio Innovations Limited, Cambridge, UK.
⁹ Liver Unit, Department of Medicine, Cambridge Biomedical Research Centre, Cambridge University Hospitals, Cambridge, UK.
¹⁰ Department of Medicine, University of Cambridge, Cambridge, UK.
¹¹ Nordic Bioscience A/S, Herlev, Denmark.
¹² Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK.
¹³ Sorbonne Université, Institute for Cardiometabolism and Nutrition (ICAN), Hôpital Pitié-Salpêtrière, Paris, France.
¹⁴ Newcastle NIHR Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
¹⁵ Biomolecular Medicine, Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK.
¹⁶ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK. [email protected].
¹⁷ Welcome Trust Sanger Institute, Hinxton, UK. [email protected].
¹⁸ Cambridge University Nanjing Centre of Technology and Innovation, Jiangbei Area, Nanjing, P R China. [email protected].

PMID: 32694734
PMCID: PMC7617436
DOI: 10.1038/s42255-020-0214-9

Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

Michele Vacca et al. Nat Metab. 2020 Jun.

. 2020 Jun;2(6):514-531.

doi: 10.1038/s42255-020-0214-9. Epub 2020 Jun 8.

Authors

Affiliations

¹ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK. [email protected].
² Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK. [email protected].
³ Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
⁴ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK.
⁵ Yeo Group and Genomics and Transcriptomics Core, WT/MRC Institute of Metabolic Science, University of Cambridge, Cambridge, UK.
⁶ Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
⁷ Department of Pathology, Aretaieion Hospital, Medical School, National & Kapodistrian University of Athens, Athens, Greece.
⁸ CN Bio Innovations Limited, Cambridge, UK.
⁹ Liver Unit, Department of Medicine, Cambridge Biomedical Research Centre, Cambridge University Hospitals, Cambridge, UK.
¹⁰ Department of Medicine, University of Cambridge, Cambridge, UK.
¹¹ Nordic Bioscience A/S, Herlev, Denmark.
¹² Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK.
¹³ Sorbonne Université, Institute for Cardiometabolism and Nutrition (ICAN), Hôpital Pitié-Salpêtrière, Paris, France.
¹⁴ Newcastle NIHR Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
¹⁵ Biomolecular Medicine, Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK.
¹⁶ TVP Lab, WT/MRC Institute of Metabolic Science, MRC Metabolic Diseases Unit - Metabolic Research Laboratories, University of Cambridge, Cambridge, UK. [email protected].
¹⁷ Welcome Trust Sanger Institute, Hinxton, UK. [email protected].
¹⁸ Cambridge University Nanjing Centre of Technology and Innovation, Jiangbei Area, Nanjing, P R China. [email protected].

PMID: 32694734
PMCID: PMC7617436
DOI: 10.1038/s42255-020-0214-9

Abstract

Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)-BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ-BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.

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Conflict of interest statement

Competing Interests Statement: At the time of this study, TK and SS were employees of CN Bio Innovations Ltd; MJN and DJL of Nordic Bioscience and are among original inventors and patent holders for the PRO-C3, C3M and MMP9 assays. F.O is a director of Fibrofind limited. J.L and F.O are shareholders in Fibrofind limited. The other authors declare no competing interest.

Figures

**Extended Data Fig. 1. In liver disease, BMP8b is overexpressed in Albumin and αSma positive cells**
A) Relative mRNA expression levels of Bmp8b measured by quantitative real-time polymerase chain reaction (RTqPCR) in murine livers. The levels of BMP8b mRNA transcript increase in Western Diet (WD)-induced NASH [(CTRL) n: 8; (WD) n:8], but not in High Fat Diet (HFD)-induced NAFL [(CTRL) n:5; (HFD) n:6], and in acute [3 days; (CTRL) n: 6; (CCl₄) n:4] (3 days; n:4-5/group) and chronic [8 weeks; (CTRL) n: 5; (CCl₄) n:5] carbon tetrachloride (CCl₄) -induced liver damage. All the results are shown as mean ± standard error; expression data of biological replicates represented as dot plots; statistical significance (vs. control treatment) assessed by two-sided Student’s t-test. **B-C**) Representative IF of Bmp8b and Albumin (Alb) or αSma protein expression: in Western Diet (WD)-induced NASH and following acute (3 days) CCl₄ challenge Bmp8b is expressed in the liver, and co-localizes with both αSma and Albumin thus suggesting that Bmp8b is expressed in hepatocytes and activated HSC following hepatic damage (2 replicates / condition; staining repeated twice).

Extended Data Fig. 2. Bmp8b is overexpressed by primary hepatocytes when cultured *in vitro*
A) Relative mRNA gene expression levels of Bmp8b measured by quantitative real-time polymerase chain reaction (RTqPCR) in PH cultured at low (20,000 cells/cm²) or high (100,000 cells/cm²) confluence to model a highly proliferative (B; Cyclin E1, Ccne1) or differentiated (C; Albumin, Alb) behavior, respectively. Bmp8b mRNA expression levels are very low at baseline, and induced from 24h after culturing. All the results are shown as mean ± standard error. Statistical significance was assessed by Multivariate Analysis of Variance (MANOVA; 5 replicates/group). D) Microarray data from publicly available database GSE122660 of human primary hepatocytes (PH) and human hepatocytes cell lines (HepG2 and Huh7) cultured in 2D and challenged for 72h with DMSO or a mixture of oleic (OA) and palmitic (PA) fatty acids (with/without TNFα) suggest that also human hepatocytes express BMP8B and that the challenges with fatty acids and/or pro-inflammatory factors do not influence BMP8B expression. Expression data were retrieved using the tool Geo2R from NCBI, and statistical significance was assessed using One-Way Analysis of Variance (ANOVA; 3 replicates/group).

Extended Data Fig. 3. Bmp8b is overexpressed by Primary Hepatic Stellate Cells when cultured *in vitro*
Relative mRNA gene expression levels measured by RNA sequencing (**A-C**) or quantitative real-time polymerase chain reaction (RTqPCR; D) in murine primary hepatic stellate cells cultured at a density of 35,000 cells/cm² and harvested before (day 0) and after culture (days 1, 4, 8, 12). Average mRNA abundance (Log2CPM) of BMP/TGFβ receptors and effectors (A), and of TGFβ/BMP family members (B): Artn, Bmp 2/8a, and Inhb b/c/e were suppressed; Bmp3/5/7/9/10 and Gdf9/10 were suppressed after a transient upregulation at day 1 of culture; Tgfβ2/3, Gdf6 and Inhba were upregulated (Nodal and Gdf3 were not expressed by HSC). C) IPA “upstream regulator” analysis based on time-dependent GE changes (NGS) in HSC at different stages of the trans-activation program compared to Day 1 (D1) of culturing shows activation of multiple TGFβ-related effectors Detailed NGS analysis (significantly modulated genes, IPA analysis) available in Supplementary Table 5; statistical significance was assessed by GLM likelihood ratio (edgeR) and then adjusted by the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). “Upstream Regulators” shown are all significantly enriched (P<0.05 - and with a 2≤Z-Score≥2 as defined by IPA) in at least one comparison. D) Time-dependent changes of Bmp8b expression in HSC treated with drivers of HSC activation (PDGFB 10ng/mL; Oleic Acid 100 μM; Palmitic Acid 100 μM; TNFα 30ng/mL; LPS 50 ng/mL). Bmp8b mRNA expression levels are very low at baseline and induced 24h after culturing (Area Under the Curve, AUC, in the small panel); Palmitic Acid significantly induces Bmp8b, while LPS reduces its expression over the time. All the results are shown as mean ± standard error or in a heatmap format (representing gene abundance expressed as Log2CPM, or the degree of “Upstream Regulator” activation -Z-score- at the IPA “Upstream Regulator” Analysis). Sample Size: 4 biological replicates/group for panels A-C (each biological replicate is a pool of 3 livers); 3 replicates/time point for panel D. Statistical significance was assessed by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference test (D). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

Extended Data Fig. 4. Recombinant BMP8 rescues the defect in HSC activation observed in Bmp8b KO HSC cultured *in vitro*
Freshly isolated HSC from Bmp8b KO mice and WT littermates were cultured for 4 and 6 days at a density of 35K cells/cm² in presence/absence of recombinant BMP8 (30 mg/mL) to study the effect of BMP8b gain of function/recovery in both KO and WT cells. αSMA protein expression (A&B; IF; staining repeated twice in each biological replicate; magnification: 10X), and gene expression (RTqPCR) of multiple markers of HSC transactivation (C), inflammation (D) and TGFβ/BMP targets (E) were checked to study HSC’s activation status (4 biological replicates/group; each biological replicate is a pool of three livers). All the results are shown as mean ± standard error. Statistical significance was assessed by Multivariate Analysis of Variance (MANOVA).

**Extended Data Fig. 5. BMP8 stimulates TGFβ/BMP signaling in a human 3D *in vitro* NASH model promoting inflammation and proliferation (continues from Figure 6)**
A) BMP8b mRNA expression (RTqPCR) in the cells cultured without/with medium containing a mixture of saturated and unsaturated FFAs (Lean vs. Fat; n: 6 replicates/group). B) Targeted phospho-proteomics data of cells studied 30 min after BMP8 challenge (vs. Control; 3 biological replicates/group) in cells cultured in “Fat” medium; C) TGFβ/BMP targets studied by BMP8b mRNA expression (RTqPCR) studied after 2 challenges (every 24h) of BMP8, TGFβ, or BMP7 (in “Fat” medium; cells and media were harvested 48h after commencing the challenges; n: 4 replicates/group). D) Secreted proteins quantified in the culturing media of 48h treated cells (n: 4 replicates/group); E) IPA “upstream regulator” analysis of the RNA sequencing of cells treated with recombinant BMP8 for 5h vs 48h (4 replicates/group; full list of genes differentially regulated and statistical design are provided in Supplementary Table 7). All the results are shown as mean ± standard error (expression data of biological replicates are represented as dot plots), or in a heatmap format [representing the degree of “Upstream Regulator” activation (-2 ≤ Z-score ≥2) at the IPA “Upstream Regulator” Analysis]. To provide a framework of interpretation of the sequencing data, we clustered the results in “Early response” regulators (modulated at 5h; not modulated at 48h), “Persistent Response” regulators (modulated both at 5h and 48h with the same direction), “late response” regulators (mildly/not regulated at 5h and modulated at 48h), “biphasic” regulators (showing opposite direction of regulation between 5h and 48h data). Statistical significance was assessed by two-sided Student T-Test (A, B), or by One-Way analysis of variance (ANOVA; C, D) plus Fisher’s least significant difference test (n: 4 replicates/group). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Extended Data Fig. 6. Bmp8b KO mice challenged with CDHFD model (NASH F2 fibrosis)**
BMP8b KO and wild-type littermates mice were treated for 14 weeks with a choline deficient high-fat diet (CDHFD – N: 9WT & 6KO). CDHFD -treated Bmp8b KO mice show no difference in BW (A), Liver to body weight percent ratio (B: LW/BW%), glucose and lipid metabolism (C), ALT (D), NASH activity (E, H&E; F, NASH activity score) and Fibrosis (G, Picro-Sirius Red, PSR; H, “Kleiner” Fibrosis Stage; I, PSR quantification: % of stained area quantified using HALO imaging software, Indica Lab, on the whole-tissue scanned slide; J, Procollagen C3, PRO-C3). However, the relative mRNA expression levels of key genes measured by RTqPCR in the livers (K), and in freshly isolated HSC (L; Sample size: 6WT & 3 KO HSC pools) show impaired activation of TGFβ/BMP signaling, reduced inflammation, and defective HSC activation in Bmp8b KO mice compared to WT littermates. **A-L**) All the results are shown as mean ± standard error (biological replicates are represented as dot plots). Statistical significance was assessed by two-sided Student T-Test.

**Figure 1. BMP8B is overexpressed in Human NASH according to disease stage and is expressed in both hepatocytes and stellate cells (HSC).**
Relative mRNA expression levels of *BMP8B* measured by quantitative real-time polymerase chain reaction (**A-E**; RTqPCR; **F-J:** Nanostring) in the RNA extracted from liver biopsies of two independent cohorts of NASH patients. The levels of *BMP8B* transcript increase with disease progression according to the NASH activity score (NAS; **A & F**). The components of the NAS score show that the main drivers of *BMP8B* expression are hepatocellular ballooning (**C & H**), fibrosis stage (**E & J**), gender, and the diagnosis/treatment of T2D. K) Representative IF of BMP8B, K18 (mainly hepatocytes) and αSMA (activated HSC/pericytes) protein expression in FFPE liver biopsies of NASH subjects at different stages of the disease (N: 6; 2-3 needle biopsies specimens/patient stained; Magnification: 20x). BMP8B is expressed in NASH fibrosis, and co-localizes with both K18 and αSMA (White arrows indicate co-localization with αSMA). Results are shown as mean ± standard error (n: 40 Cambridge Cohort; n: 113 Newcastle/Paris cohort); expression data of biological replicates represented as dot plots. Statistical significance (p<0.05) was assessed by Multivariate Analysis of Variance (MANOVA) with histological variables, gender and T2D as covariates. Full details of the population in Supplementary Table 2.

**Figure 2. BMP8B activates both Smad 2/3 and Smad 1/5/9 signaling pathways in Bmp8b KO HSC.**
Primary murine Bmp8b KO HSC (confluence 35,000 cells/cm²) activated in vitro (Day 7) were treated, after 3h FBS starvation, with/without recombinant human BMP8 protein 30-75 ng/mL, and/or ALK 1/2/3/6 inhibitor (K02288, 1µM) or ALK 4/5/7 inhibitor (A-8301, 5µM) to study Smad 2 (**A, C**) or Smad 1/5/9 (**B, D**) phosphorylation after 30 min incubation. TGF-β (5ng/mL) or BMP7 (5ng/mL) were used as positive controls. Immunofluorescence (Magnification: 20x) was used to study phosphorylated Smads (in green); αSMA (in red) was used to stain the cytoplasm, Hoechst 33342 (blue) to stain the nucleus. The % of positive nuclei was assessed by Harmony High Content Imaging and Analysis Software (PerkinElmer). Gene expression (E; Relative mRNA expression levels of TGFβ/BMP targets measured by RTqPCR) was assessed after 5h of incubation with ligands and inhibitors. All the results are shown as mean ± standard error; expression data of biological replicates are represented as dot plots. The one-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test was used to estimate the statistical significance among treatments (4 biological replicates/group; each biological replicate is a pool of three livers). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Figure 3. Genetic ablation of Bmp8b attenuates HSC transactivation *in vitro*.**
Freshly isolated HSC from Bmp8b KO mice and WT littermate mice were cultured for 4, 6 and 8 days at a density of 35K cells/cm². Gene expression (A; RTqPCR) and αSMA protein expression (**B&C**; IF; Magnification: 10X) were studied to investigate HSC’s activation status (4 biological replicates/group; each biological replicate is a pool of three livers). Prediction of “Canonical Pathways” and “Upstream Regulators” (D) significantly enriched and predicted as activated (red) or inhibited (blue) according to IPA/URA in fully activated (Day 8) HSC (Details the NGS analysis in Supplementary Table 6); E) graphical representation in a network format of the changes of TGFβ targets levels suppressed in the NGS dataset in KO (vs. WT) cells. (F) Profiling of the culturing media (WT: 3 biological replicates/group; KO: 4 biological replicates/group; each biological replicate is a pool of cells from three murine livers): collagen 3 secretion and processing [proposed as marker of NASH-Fibrosis ] was assessed studying PRO-C3, MMP9 and C3M assays; secretion of pro-inflammatory mediators was assessed quantifying Il6 and Mcp1 (Rantes was below detectability) in the culturing media at multiple time points (Day 4, 8, 11 of culturing). Relative mRNA expression of genes measured by RTqPCR or NGS; protein quantification was performed by immunofluorescence and quantified using ImageJ 1.8.0. Sequencing data are shown in a heatmap format representing the “activation” (Z) score [inhibition (blue) or activation (red)] of pathways/upstream regulators predicted by IPA/URA. All the other results are shown as mean ± standard error; biological replicates are represented by dots. Statistical significance was assessed by Multivariate Analysis of Variance (MANOVA) using time-point and genotype as covariates.

**Figure 4. Inflammation, stellate cells activation and hepatocyte proliferation are attenuated in Bmp8b KO livers injured with CCl₄.**
Bmp8b KO and littermate mice received a single olive oil (OO) or CCl₄ IP injection, and were culled 3 and 5 days later [n/group (refers to all the comparisons): (OO-WT) 6; (CCL4-WT3D) 4; (CCL4-KO3D) 6; (CCL4-WT5D) 7; (CCL4-KO5D) 5]. A) Relative *Bmp8b* mRNA expression measured by RTqPCR (whole liver). B) Relative TGFβ/BMP family ligands mRNA expression (3 days post injection) measured by NGS (the heatmap represents relative basal expression level - blue<yellow<orange -, the dotted graph represents Log2FC; Details in Supplementary Table 8). C) *In situ* hybridization (2 replicates; staining repeated twice) reveals that, upon CCl₄ challenge, Bmp8b mRNA is expressed by K18+ cells (mainly hepatocytes) and αSma+ cells (mainly activated HSC and pericytes) – Magnification: 40X. D) Liver to body weight ratio (LW/BW%) is increased by CCl₄; Bmp8b KO mice show less pronounced liver enlargement. CCl₄-treated Bmp8b KO mice show a trend in reduced serum alanine aminotransferase (ALT; E) levels three days after the challenge. HALO imaging software analysis on the whole-tissue (H&E) suggests that CCl₄ induces a substantial increase of hepatocellular damage (F; HALO quantification) and inflammation (G; HALO quantification) peaking 3 days after the injection; Bmp8b KO mice showed reduced inflammation at the same time point. Further IHC immune cell profiling (H; Day 3) suggests a reduced infiltration in the Bmp8b KO livers from CD3+ (mostly lymphocytes) and of Ly6CG+ (mostly myeloid) cells in the KO liver. This result is also confirmed by the “immune cell-type deconvolution analysis” performed on the NGS data (I). We also profiled by IHC α Smooth Muscle Actin staining (αSMA; J) and Proliferating Cell Nuclear Antigen IHC staining (PCNA; K) that appeared reduced in Bmp8b KO mice compared to WT littermates, especially at Day 5. Furthermore, in light with decreased compensatory proliferation, also circulating levels of Afp (L) appeared reduced in the Bmp8b KO mice; **M & N**) Relative mRNA expression (whole liver RNA) levels of genes measured by RTqPCR and IPA on the NGS data (Day 3) confirm reduced inflammation, stellate cells activation and proliferation in Bmp8b KO mice. Representative images of H&E and IHC in Supplementary Figure 5. Supplementary Table 4 shows that the lipid composition of the livers did not differ between WT and KO mice. All the results are shown as mean ± standard error; biological replicates are represented as dot plots. Statistical significance was assessed either by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test, or by two-sided Student T-Test (and FDR correction for NGS data). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Figure 5. Absence of Bmp8b leads to defective liver regeneration in the Partial Hepatectomy (PHx) model.**
Bmp8b KO and littermate mice received PHx and were culled 3 days later [n/group (for all the comparisons): D0-WT: 5; D0-KO: 4; D3-WT: 5; D3-KO: 4]. A) Relative *Bmp8b* mRNA expression measured by RTqPCR. B) Relative TGFβ family ligands mRNA expression (3 days post injection) measured by NGS (the heatmap represents relative basal expression level - blue<yellow<orange -, the dotted graph represents Log2FC; Details in Supplementary Table 8). **C-D**) FISH (Magnification: 40X; 2 replicates; staining repeated twice) and IF (2 replicates; staining repeated twice) reveal that after PHx, *Bmp8b* mRNA and protein are expressed by K18+ cells (mainly hepatocytes) and αSMA+ cells (mainly activated HSC and pericytes). E) Liver to body weight ratio (LW/BW %) shows that Bmp8b KO mice have reduced regenerative potential compared to WT littermates (resected liver in red as reference). Defective hepatocyte proliferation was also confirmed by reduced Proliferating Cell Nuclear Antigen IHC staining (PCNA quantified by HALO imaging software analysis on the whole-tissue; staining repeated once; Magnification: 10X; **F & H**) and reduced number of mitotic figures (H&E staining repeated once; Magnification: 20X; **G & I**). Relative mRNA expression levels (whole liver RNA; resected livers from the same mice were used as D0 reference) of genes measured by RTqPCR (J) and IPA pathway analysis (K; NGS) confirm reduced inflammation and compensatory proliferation in Bmp8b KO mice during the proliferative phase of liver regeneration. All the results are shown as mean ± standard error; replicates are represented as dot plots. Statistical significance was assessed either by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test, or by two-sided Student T-Test (and FDR correction for NGS data). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Figure 6. BMP8 stimulates TGFβ/BMP signaling in a human 3D *in vitro* NASH model promoting inflammation and proliferation.**
Human primary liver cells (PH, KC, and HSC) were cultured in an *in vitro* NASH model (LiverChip™ - CN Bio Innovations) consisting of 3D perfused micro-tissues of primary human cells challenged with a medium containing a mixture of FFAs, in the presence of physiologically relevant quantities of insulin and sugars to induce a “NASH-like” phenotype. (A) Comparison of the transcriptomic data (NGS) of this *in vitro* model with the *in vivo* WD model of NASH (in Figures 7&8) shows high homology (∼80% overlapping genes); details on differentially expressed genes in Supplementary Table 7: this model expresses a plethora of TGFβ/BMP effectors (B) and ligands (C), comparable to *in vivo* NASH models, and develops lipid droplets as a consequence of FFA challenge as observed through Oil Red O staining (example picture; Magnification: 10X; staining repeated multiple times giving similar results) (D). One week after fat challenge, cells were treated with/without recombinant Human BMP8 protein (75 ng/mL), with/without ALK 1/2/3/6 inhibitor (K02288, 1 µM) or ALK 4/5/7 inhibitor (A-8301, 5 µM) to study TGFβ/BMP-related gene expression changes at 5h (E: NGS data). F: RTqPCR confirmation of TGFβ/BMP targets (long-term consequences) at 48h (G: RTqPCR; NGS in Extended Data Figure 5E). Treatment with TGFβ (5 ng/mL) or BMP7 (5 ng/mL) were used as positive controls. All the results are shown as mean ± standard error (biological replicates are represented as dot plots) or in a heatmap matrix representing the “activation” Z-score of “Canonical Pathways” and “Upstream Regulators” significantly (p<0.05) enriched and predicted as activated (red) or inhibited (blue) according to IPA. To provide a framework of interpretation, the data have been clustered in “BMP-like” (modulated –significantly enriched and with a -2<Z-score>2 - by both BMP8 and BMP7; modulation reduced/prevented by K02288), “TGFβ-like” (modulated by both BMP8 and TGFβ; modulation reduced/prevented by A-8301) and “BMP/TGFβ-like” (modulated by BMP8, BMP7 and TGFβ; modulation reduced/prevented by both ALK inhibitors). Two days after culturing with challenges, we also studied the cell secretome in the culturing media (**H, I**, Inflammatory factors and markers of collagen deposition and remodeling; additional data in Extended Data Figure 5D; PRO-C3 was undetectable). Statistical significance (4 biological replicates/group) was assessed by two-sided T-test (E; each treatment vs control) for the NGS data, or by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test (F-I). Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Figure 7. Attenuated inflammation and fibrosis in Bmp8b KO mice challenged with WD to model NASH/F1.**
Bmp8b KO and wild-type littermate mice were treated for 32 weeks with WD (9WT & 8KO) or Low-Fat Diet (LFD; 8WT & 6KO) control. Relative mRNA expression levels of *Bmp8b* and other TGFβ ligands was measured by RTqPCR (A) or NGS (B; the heatmap represents relative basal expression level – blue<yellow<orange -; the dotted graph represents Log2FC; detailed NGS analysis in Supplementary Table 8) in murine livers. The levels of Bmp8b mRNA transcript are almost absent in the normal liver and highly expressed in WD-induced NASH in K18+ cells (mainly hepatocytes) and αSma+ cells (mainly activated HSC and pericytes) by *FISH* (C; 2 replicates; staining repeated twice; magnification: 40X) as also observed at protein level (Extended Data Figure 1). D) Liver to body weight % ratio (LW/BW%) is increased by WD with no genotype-associated effects. WD-treated Bmp8b KO mice show reduced serum alanine aminotransferase (E; ALT), SAF activity score (F&G; details in Supplementary Figure 8A & 8B), and Fibrosis (H&I; PSR stain quantified using “Indica Lab” HALO imaging software on the whole-tissue scanned slide). (J) A trend in reduced circulating PRO-C3, a non-invasive biomarker of fibrosis, is also observed in WD-challenged Bmp8b KO mice. (**K & L**) IHC immune cell profiling (WD) suggests a reduced infiltration in the KO livers from CD3+ (mostly lymphocytes), Ly6CG+ (mostly myeloid) cells, and CD45R+ (mostly B) cells in WD-challenged KO livers; this result is also confirmed by the “immune cell-type deconvolution analysis” (M) performed on the NGS data. Relative mRNA expression levels (whole liver RNA) of genes involved measured by RTqPCR (N) and IPA analysis (O; NGS) confirm reduced inflammation, fibrosis and stellate cells activation in WD-challenged Bmp8b KO mice. All the results are shown as mean ± standard error; biological replicates are represented as dot plots. Statistical significance was assessed by either two-tailed Student T-Test (plus FDR for NGS), one-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test, or by Kruskal–Wallis one-way analysis of variance on ranks plus Dunn’s multiple-comparison test (G), when relevant. Lowercase letters indicate post-hoc analysis significance: “a” means reference group; when groups show different letters, they should be considered statistically different at the post-hoc comparison; groups showing the same letter are statistically not-significant at the post-hoc comparison.

**Figure 8. NGS analysis confirms on a large scale that absence of Bmp8b impacts pro-inflammatory, and pro-fibrotic, and proliferative pathways in CCl₄, PHx and WD models.**
Gene expression profiled by NGS, analyzed with two-sided T-test (sample size as defined in figures 4, 5, 7), and analyzed by IPA in Bmp8b KO mice and WT mice following CCl₄ (3 days), PHx (3 days), and WD (32 weeks) challenges. Details in Supplementary Table 8. Prediction of “Canonical Pathways” (A) and “Upstream Regulators” (B) significantly enriched and predicted as activated (red) or inhibited (blue) according to IPA/URA. Data are shown as a heatmap matrix format representing the “activation” (Z) score prediction by IPA. C) Graphical representation in networks of differentially modulated genes (green: down-regulated; red: up-regulated in the WD – KO vs. WT comparison) leading to the predicted (-2<Z-score>2) inhibition (blue) of the “upstream regulators” according to IPA. D) Comparing the genes differentially modulated in the treated Bmp8b KO mice (vs. WT), a subset of 171 genes was modulated in at least 2 of the three datasets – i.e. differentially modulated (p<0.05; -0.378 < Log2FC > 0.378); of these genes, 36 hits (E) show the same direction of regulation in all the three datasets, so being most-directly associated to Bmp8b absence independently from the challenge. Data are shown in a heat-map with a matrix format representing the regulation (Log2FC KO vs WT; yellow: up; green: down).

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Novel regulatory pathway in NASH identified.
Morris A. Morris A. Nat Rev Endocrinol. 2020 Aug;16(8):401. doi: 10.1038/s41574-020-0384-2. Nat Rev Endocrinol. 2020. PMID: 32576989 No abstract available.

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References

1. Younossi Z, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018;15:11–20. - PubMed
1. Azzu V, Vacca M, Virtue S, Allison M, Vidal-Puig A. Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in non-alcoholic fatty liver disease. Gastroenterology. 2020 - PubMed
1. Vacca M, Allison M, Griffin JL, Vidal-Puig A. Fatty Acid and Glucose Sensors in Hepatic Lipid Metabolism: Implications in NAFLD. Semin Liver Dis. 2015;35:250–261. - PubMed
1. Bedossa P, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology. 2012;56:1751–1759. - PubMed
1. Kleiner DE, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–1321. - PubMed

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1. Zhao GQ, Deng K, Labosky PA, Liaw L, Hogan BL. The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev. 1996;10:1657–1669. - PubMed
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[1] Younossi Z, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018;15:11–20. - PubMed

[2] Younossi Z, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018;15:11–20. - PubMed

[3] Azzu V, Vacca M, Virtue S, Allison M, Vidal-Puig A. Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in non-alcoholic fatty liver disease. Gastroenterology. 2020 - PubMed

[4] Azzu V, Vacca M, Virtue S, Allison M, Vidal-Puig A. Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in non-alcoholic fatty liver disease. Gastroenterology. 2020 - PubMed

[5] Vacca M, Allison M, Griffin JL, Vidal-Puig A. Fatty Acid and Glucose Sensors in Hepatic Lipid Metabolism: Implications in NAFLD. Semin Liver Dis. 2015;35:250–261. - PubMed

[6] Vacca M, Allison M, Griffin JL, Vidal-Puig A. Fatty Acid and Glucose Sensors in Hepatic Lipid Metabolism: Implications in NAFLD. Semin Liver Dis. 2015;35:250–261. - PubMed

[7] Bedossa P, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology. 2012;56:1751–1759. - PubMed

[8] Bedossa P, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology. 2012;56:1751–1759. - PubMed

[9] Kleiner DE, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–1321. - PubMed

[10] Kleiner DE, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–1321. - PubMed

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Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

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