Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Dec 31;14(12):e0226854.
doi: 10.1371/journal.pone.0226854. eCollection 2019.

Thrombospondin-I is a critical modulator in non-alcoholic steatohepatitis (NASH)

Jessica Min-DeBartolo  1   2 , Franklin Schlerman  3 , Sandeep Akare  4 , Ju Wang  3 , James McMahon  3 , Yutian Zhan  4 , Jameel Syed  4 , Wen He  5 , Baohong Zhang  5 , Robert V Martinez  3
Affiliations

Thrombospondin-I is a critical modulator in non-alcoholic steatohepatitis (NASH)

Jessica Min-DeBartolo et al. PLoS One. .

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease characterized by dysregulated lipid metabolism and chronic inflammation ultimately resulting in fibrosis. Untreated, NAFLD may progress to non-alcoholic steatohepatitis (NASH), cirrhosis and death. However, currently there are no FDA approved therapies that treat NAFLD/NASH. Thrombospondin-I (TSP-1) is a large glycoprotein in the extracellular matrix that regulates numerous cellular pathways including transforming growth factor beta 1 (TGF-β1) activation, angiogenesis, inflammation and cellular adhesion. Increased expression of TSP-1 has been reported in various liver diseases; however, its role in NAFLD/NASH is not well understood. We first examined TSP-1 modulation in hepatic stellate cell activation, a critical initiating step in hepatic fibrosis. Knockdown or inhibition of TSP-1 attenuated HSC activation measured by alpha smooth muscle actin (α-SMA) and Collagen I expression. To investigate the impact of TSP-1 modulation in context of NAFLD/NASH, we examined the effect of TSP-1 deficiency in the choline deficient L-amino acid defined high fat diet (CDAHFD) model of NASH in mice by assessing total body and liver weight, serum liver enzyme levels, serum lipid levels, liver steatosis, liver fibrosis and liver gene expression in wild type (WT) and TSP-1 null mice. CDAHFD fed mice, regardless of genotype, developed phenotypes of NASH, including significant increase in liver weight and liver enzymes, steatosis and fibrosis. However, in comparison to WT, CDAHFD-fed TSP-1 deficient mice were protected against numerous NASH phenotypes. TSP-1 null mice exhibited a decrease in serum lipid levels, inflammation markers and hepatic fibrosis. RNA-seq based transcriptomic profiles from the liver of CDAHFD fed mice determined that both WT and TSP-1 null mice exhibited similar gene expression signatures following CDAHFD, similar to biophysical and histological assessment comparison. Comparison of transcriptomic profiles based on genotype suggested that peroxisome proliferator activated receptor alpha (PPARα) pathway and amino acid metabolism pathways are differentially expressed in TSP-1 null mice. Activation of PPARα pathway was supported by observed decrease in serum lipid levels. Our findings provide important insights into the role of TSP-1 in context of NAFLD/NASH and TSP-1 may be a target of interest to develop anti-fibrotic therapeutics for NAFLD/NASH.

PubMed Disclaimer

Conflict of interest statement

The authors have commercial affiliations to Pfizer Worldwide Research and Development, Biogen and Wave Life Sciences and these affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. TGF-β1 dose response with α-SMA and Collagen I expression in primary human HSCs.
HSCs were treated for 48 hours with 9–0.001 ng/ml change to lowest to highest TGF-β1 and then fixed and stained for α-SMA and Collagen I. Percent value is based on maximal response from addition of TGF-β1. EC50 α-SMA expression: 200 pg/ml TGF-β1; EC50 Collagen I expression: 50 pg/ml TGF-β1. % expression values were calculated by defining zero as the smallest value in each data set and one hundred as the largest value in each data set.
Fig 2
Fig 2. Peptide-mediated inhibition or knockdown of TSP-1, attenuates markers of primary human HSC activation into myofibroblasts.
A. Primary human HSCs activated with 1 μg/ml TGF-β1 for 48 hours show increased expression of TSP-1 by Western Blot analysis. Bar graphs represent densitometry measurements, error bars show standard error of the mean (SEM). TSP-1/GAPDH expression was normalized to values from cells treated with vehicle (0.01% DMSO). B. Expression of TSP-1 is significantly reduced in TGF-β1 activated primary human HSCs transduced with adenoviral particles carrying shRNA against transcribed gene for TSP-1 (shTHBS1) compared to HSCs transduced with control shRNA adenovirus. Cells were transduced at multiplicity of infection (MOI) of 300 for 48 hours prior to treatment with TGFβ then harvested after 48 hours. Bar graph represent densitometry measurements, error bars show SEM. TSP-1/GAPDH was normalized to cells transduced with adenovirus carrying control shRNA. C. TSP-1 knockdown by shRNA transduction in primary human HSCs attenuate TGF-β1 mediated expression of markers of fibrosis, alpha smooth muscle actin (α-SMA) and Collagen I measured by immunofluorescence. HSCs were transduced with adenoviral shTHBS1 (gene for TSP-1) for 48 hours then treated with TGF-β1 for 48 hours prior to fixation for immunofluorescence, n = 3 per treatment, per experiment. Error bars represent SEM; % expression is normalized to cells transduced with control shRNA. D. Treatment with inhibitory peptide against TGF-β1 activating motif (LSKL) in TSP-1 results in attenuation of TGF-β1 mediated expression of markers of fibrosis, α-SMA and Collagen I. n = 3 per treatment, per experiment. Peptide with scrambled sequence (SLLK) does not significantly (n.s) impact expression of TGF-β1-induced α-SMA or Collagen I. Error bars represent SEM; % expression is normalized to cells treated with vehicle (0.1% DMSO). All presented data are representative of at least 3 separate experiments. Statistical analysis was performed using GraphPad Prism v 7.04 with either student’s t test or oneway ANOVA Tukey’s multiple comparison tests. P value was considered significant at <0.05.
Fig 3
Fig 3. Study design and body weight, liver weight and liver function comparison of CDAHFD vs normal diet fed TSP-1 KO and Wild type mice.
A. Study design outline. B. Both wild type (WT) and TSP-1 null mice (TSP-1 KO) mice fed choline deficient l-amino acid defined diet (CDAHFD) did not show increase in body weight with increase in time. Both WT and TSP-1 null mice fed control diet increased similarly in body weight with increase in time with heaviest weight at week 12. Both TSP-1 null and WT mice fed CDAHFD had significantly increased liver:body weight ratio and liver weight compared to mice fed control diet. C. Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) of CDAHFD vs. control diet fed mice were significantly increased in both WT and TSP-1 null groups. No significant difference was observed between TSP-1 null and WT groups fed control diet for ALT, AST and GLDH. In CDAHFD fed mice, TSP-1 null mice had significantly higher GLDH than WT. Each point represents a single animal. Statistical analysis are for each genotype group (TSP-1 KO or WT) or for each diet group (CDAHFD or control), using one way ANOVA Tukey’s multiple comparison tests. Error bars represent standard error of the mean (SEM). P value was considered significant at <0.05. **** p<0.0001, ***p = 0.0003, *p = 0.0325.
Fig 4
Fig 4. Lipid profile comparison of CDAHFD vs Normal diet fed TSP-1 null (TSP-1 KO) and Wild type mice at 12 weeks.
Comparison of serum levels of A. total cholesterol, B. Low density lipoprotein (LDL) cholesterol C. High density lipoprotein (HDL) cholesterol and D. Triglycerides measurements at week 12 of CDAHFD or control diet fed mice in both TSP-1 null and WT mice. Comparison between WT vs TSP-1 null animals demonstrate significantly reduced serum cholesterol (total, LDL and HDL) levels in TSP-1 null vs WT in both control diet and CDAHFD fed cohorts. Each point represents a single animal. Error bars represent standard error of mean (SEM). Statistical analysis are for each genotype group (TSP-1 KO or WT) or for each diet group (CDAHFD or control), using one way ANOVA Tukey’s multiple comparison tests ****p <0.0001 for all graphs, *p = 0.0115 for cholesterol, ** p = 0.0018, p = 0.0241 for LDL cholesterol, *** p = 0.0007 for triglycerides, ns = no significance between compared groups.
Fig 5
Fig 5. TSP-1 null mice are protected against CDAHFD induced liver fibrosis.
A. Representative images of mouse liver sections stained with Picrosirius red (PSR). B. TSP-1 KO mice fed CDAHFD had significantly lower PSR+ area compared to WT mice fed CDAHFD (**** p<0.0001). The graphs were plotted as ratio of control group mean (using group mean of concurrent age matched control chow values for PSR. Statistical analysis was done using ordinary one-way ANOVA using multiple comparison (Sidak’s multiple comparison test) of selected pairs to compare control diet and CDAHFD groups from WT and TSP-1 null genotypes (total 2 selected pairs). C. Relative qRT-PCR analysis of collagen and inflammation markers from liver mRNA. Error bars represent SEM. Significance at p<0.05 NDWT: Control diet fed wild type mice. HFWT: CDAHFD fed wild type mice. NDKO: control diet fed TSP-1 null mice. HFKO: CDAHFD fed TSP-1 null mice. Relative fold change normalized to equivalent genotype fed control diet. Statistical analysis was done using ordinary one-way ANOVA using Tukey’s multiple comparison test with GraphPad Prism v. 7.04. D. Representative H&E stains of mouse liver sections. Bar represents 500 μm. E. Principal Component Analysis (PCA) of liver RNA-seq gene expression data. Each data point represents individual mice. Difference in genotype is represented by shape (circle: TSP-1 null or KO, triangle: wild type or WT). Difference in diet is represented by color (pink: CDAHFD fed or HF, blue: control diet fed, or ND). PCA analysis was performed by build-in R function prcomp() on all genes and the plot was generated by R packages ggplot2 and ggrepel.
Fig 6
Fig 6. Transcriptomic analysis to compare the impact of CDAHFD vs. control diet in gene expression profiles of TSP-1 KO and WT mice.
A. Venn diagram of differentially expressed genes (DEGs) from CDAHFD fed vs control diet fed TSP-1 null mice and WT mice. B. Heat map of fold change (log2FC) in DEGs (red: upregulated, green: downregulated) from genes in Fig 5A. C. Heat map with top 10 upregulated (in red) and downregulated (in blue) pathways by WikiPathways. DEGs were defined by FDR>0.05 and >1.5 fold change comparing CDAHFD vs. control diet.
Fig 7
Fig 7. Transcriptomic analysis to compare the impact of TSP-1 KO vs. wild type genotype in CDAHFD vs. control diet fed mice.
A. Venn diagram of differentially expressed genes (DEGs) from TSP-1 KO vs WT mice fed CDAHFD or control diet. B. Hierarchical clustering and heat map of DEGs based on log2FC values C. Heat map with top 10 upregulated (in red) and downregulated (in blue) pathways by WikiPathways. DEGs were defined by FDR<0.05 and fold change >1.5 (log2FC>0.58) comparing TSP-1 null vs. WT mice.
Fig 8
Fig 8. Visualization of log2FC values of genes identified in the four comparisons in amino acid metabolism pathway by WikiPathways.
Comparison 1 is WT: CDAHFD/control. Comparison 2 is TSP-1 null: CDAHFD/control. Comparison 3 is control diet: TSP-1 null/WT. Comparison 4 is CDAHFD: TSP-1 null/WT.
Fig 9
Fig 9. Visualization of log2FC values of genes identified in the four comparisons in PPARα pathway by WikiPathways.
Comparison 1 is WT: CDAHFD/control. Comparison 2 is TSP-1 null: CDAHFD/control. Comparison 3 is control diet: TSP-1 null/WT. Comparison 4 is CDAHFD: TSP-1 null/WT. FC: fold change.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mortality GBD, Causes of Death C. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;385(9963):117–71. Epub 2014/12/23. 10.1016/S0140-6736(14)61682-2 - DOI - PMC - PubMed
    1. Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol. 2014;14(3):181–94. Epub 2014/02/26. 10.1038/nri3623 . - DOI - PubMed
    1. Ellis EL, Mann DA. Clinical evidence for the regression of liver fibrosis. J Hepatol. 2012;56(5):1171–80. Epub 2012/01/17. 10.1016/j.jhep.2011.09.024 . - DOI - PubMed
    1. Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, et al. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology. 2011;140(1):124–31. Epub 2010/09/23. 10.1053/j.gastro.2010.09.038 . - DOI - PubMed
    1. Rinella ME. Nonalcoholic fatty liver disease: a systematic review. JAMA. 2015;313(22):2263–73. Epub 2015/06/10. 10.1001/jama.2015.5370 . - DOI - PubMed

Grants and funding

J.M-D, F.S, J.W, J.M,Y.Z, W.H, J.S. and R.V.M are employed by the commercial entity Pfizer Worldwide Research and Development. B.Z is employed by the commercial entity Biogen Inc. S.A is employed by the commercial entity Wave Life Sciences. The funders provided support in the form of salaries for authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
-