β-PDGF receptor expressed by hepatic stellate cells regulates fibrosis in murine liver injury, but not carcinogenesis

Abstract

Background & aims: Rapid induction of β-PDGF receptor (β-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of β-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent β-PDGFR activity, and assessed its prognostic implications in human cirrhosis.

Methods: The impact of either loss or constitutive activation of β-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked β-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis.

Results: Depletion of β-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-κB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of β-PDGFR in stellate cells. In the human cohort, the β-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis.

Conclusions: β-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Keywords: Cirrhosis; Gene expression signatures; HCC; Pathway analysis; Receptor tyrosine kinase.

Fig. 1. β-PDGFR expression correlates with activation of mouse hepatic stellate cells in vivo and in vitro

(A) Mice were injected three times with either oil or CCl₄ to induce acute liver injury and were sacrificed two days after the last injection. Immunoblot of whole liver lysates demonstrating increased expression of β-PDGFR, phospho-β-PDGFR and αSMA upon injury. (B, C) Δβ-PDGFR mice and control littermates were injected once with CCl₄ followed by isolation of HSCs 48 hours thereafter. Primary HSCs were kept in culture for 6 days. (B) Immunoblot demonstrates phosphorylation of the receptor in control animals upon ligand exposure and lack of receptor activation in HSCs of Δβ-PDGFR animals. (C) Immunoblot showing decreased HSC activation of Δβ-PDGFR mice in culture compared to wild type β-PDGFR mice, with reduced expression of Collagen I and αSMA. Graph indicating densitometric analysis of each band, verifying significant knock down of β-PDGFR in primary HSCs of Δβ-PDGFR mice and decreased expression of HSC activation markers. Data represent the mean value of at least 3 separate experiments (*p<0.05, error bars indicate SEM). 3 animals per condition were used in each experiment. Protein ratios (normalized to calnexin) were used to quantify the fold change relative to control, and are shown below each blot.

Fig. 2. Loss of β-PDGFR on HSCs leads to decreased collagen deposition in vivo

(A–E) Δβ-PDGFR and control mice were injected with CCl₄ over either one or six weeks to induce acute or chronic liver injury. (A) Sirius Red staining of paraffin embedded liver sections following acute or chronic liver injury depicts significantly lower collagen deposition after chronic injury (magnification 200×). (B) Graph displays the percentage of liver area positive for Sirius Red staining measured by morphometric analysis. The area of fibrotic tissue is significantly reduced within Δβ-PDGFR animals compared to controls. (C) Measurement of hydroxyproline content per gram of whole liver after 6 weeks of CCl₄ reflects reduced hydroxyproline content in livers of Δβ-PDGFR versus controls. (D) Levels of serum AST and ALT during acute and chronic injury. (E) Whole liver mRNA expression of Collagen α1(I), αSMA, β-PDGFR after 6 weeks of CCl₄ treatment confirm increased expression of stellate cell activation genes within control animals upon injury, as well as lack of increase within the Δβ-PDGFR group. All figures represent the mean of at least n=3 animals per experimental group. mRNA is expressed normalized to Gapdh (*p < 0.05, **p< 0.001; error bars indicate SEM).

Fig. 3. The ‘βJ’ constitutively activating mutant of β-PDGFR on stellate cells leads to increased collagen deposition upon injury in vivo

(A–D) βJ and control mice were injected with CCl₄ over either one or six weeks to induce acute or chronic liver injury. (A) Sirius Red staining of paraffin embedded liver sections following acute or chronic liver injury depicts significantly higher collagen deposition after acute and chronic injury (magnification 200×). (B) Graph shows the percentage of liver area positive for Sirius Red staining measured by morphometry. The area of fibrotic tissue is significantly increased in livers of βJ animals compared to controls. (C) Increased hydroxyproline content per gram of whole liver after 6 weeks of CCl₄ in livers of βJ mice compared to controls. (D) Whole liver mRNA expression of αSMA and Collagen α1(I) after 6 weeks of CCl₄ treatment confirms increased expression of stellate cell activation genes in βJ animals upon liver injury. (E) Desmin staining of paraffin embedded liver sections following acute liver injury depicts increased stellate cell expansion within livers of βJ mice versus controls. (F) Graph shows percentage of tissue area positive for desmin measured by morphometry. All figures represent the mean of at least n=5 animals per experimental group. mRNA is expressed normalized to Gapdh (*p < 0.05, **p< 0.001; error bars indicate SEM).

Fig. 4. Deletion of β-PDGFR reduces fibrosis but not tumor burden in mice treated with DEN and chronic CCl₄, and is linked to better outcome in patients with cirrhosis

Mice were treated with a single dose of DEN at day 15, followed by weekly injections with CCl₄ and sacrificed 48h after the last of 22 injections with CCl₄. (A) Paraffin embedded liver sections were stained with Sirius Red to assess fibrotic tissue content (magnification 200×). (B) Decreased collagen area measured by morphometry in livers of Δβ-PDGFR mice. The bar graph represents the mean of n=5 animals per experimental group. (**p< 0.001; error bars indicate SEM). (C) GSEA plots demonstrate β-PDGFR-dependent association with gene signatures for either a good or poor prognosis in an HCV cirrhotic patient cohort. Gene array samples of primary hepatic stellate cells isolated from either β-PDGFRfl/fl GFAP-Cre negative (indicated as WT) or β-PDGFRfl/fl GFAP-Cre positive (indicated as KO) mice were correlated with gene signatures for good or poor overall prognosis of a human HCV cirrhosis cohort. (a) Association of the β-PGDFR-knockout gene signature with a good outcome in liver cirrhosis. Enrichment of the β-PGDFR-knockout gene signature was evaluated in association with the risk of overall mortality in 216 HCV-related cirrhosis patients with early-stage cirrhosis (n=216). NES=−1.14, nominal p=0.21, FDR=0.22. (b) Association of the β-PGDFR gene signature with a poor outcome in HCV cirrhosis. Enrichment of the β-PGDFR gene signature was evaluated in association with the risk of overall mortality in 216 HCV-related cirrhosis patients with early-stage cirrhosis (n=216). NES=1.11, nominal p=0.25, FDR=0.23. Genes were evaluated using GSEA. (D) Comparison of β-PDGFR knockout-mediated differential gene expression between DNA microarray and RT-qPCR was performed choosing five of the top differentially expressed genes (Anxa1, Dab2, Ergic2, Lpp, and Prdx5) between wild type and β-PDGFR-knockout mice as selected from DNA microarray data. To verify the differential expression in RT-qPCR, 7 pairs of primers (2 pairs for Ergic and Prdx5) were designed and the same RNA aliquots were assayed in triplicate.