Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2-/- ) mice

Abstract

Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2^-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis.

Conclusion: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).

Figure 1

Senescent cholangiocytes upregulate Bcl-xL and exhibit enhanced apoptotic sensitivity to the specific Bcl-xL inhibitor A-1331852. Normal human cholangiocytes (NHC) were induced to senesce with ionizing radiation (IR; 10Gy). (A) Protein lysates were subjected to Western blot for quantification of selected Bcl-2 family proteins as well as the senescence marker p16^Ink4a. (B) Treatment of NHC with A-1331852 resulted in a reduction of cell viability in senescent but not in non-senescent cholangiocytes, as determined by MTT assay. (C) A-1331852 treatment also significantly increased caspase-3/7 activity in senescent cholangiocytes as compared to non-senescent cells. (D) In addition, cleavage of PARP was increased in A-1331852-treated senescent cholangiocytes. Data in B and C are presented as mean ± S.D. of at least three independent experiments, performed in triplicate.

Figure 2

PDGF-activated fibroblasts are sensitive to navitoclax-induced apoptosis. Human and mouse fibroblasts were activated in vitro with 50 ng/ml PDGF-BB for 24 h. Fibroblasts were then treated with the pro-apoptotic BH3 mimetic navitoclax for an additional 24 h to induce apoptosis. (A) Apoptosis induction in human HFF-1 fibroblasts and primary fibroblasts (hFB) was quantified biochemically by measuring caspase-3/7 activity using the fluorogenic Ac-DEVD-AMC substrate and morphologically using DAPI staining. The number of apoptotic nuclei was quantified in at least four high power fields and is expressed as a percentage of total. (B) Apoptosis induction in murine 3T3 fibroblasts and human primary fibroblasts from PSC (hFB-PSC) was quantified biochemically by measuring caspase-3/7 activity. Data are presented as mean ± S.D. of at least three independent experiments, performed in triplicate. Abbreviations: ns, not significant; PDGF, platelet-derived growth factor

Figure 3

Activation of fibroblasts results in reduction of Bcl-2 protein. HFF-1 (left panel) and primary human fibroblasts (hFB, right panel) were activated in vitro with 50 ng/ml PDGF-BB for 24 h. (A) Gene expression of the Bcl-2 family was assessed by quantitative real time PCR. Changes following PDGF treatment are shown as fold change relative to vehicle. Data are presented as mean ± S.D. of at least three independent experiments, performed in triplicate. (B) Whole cell lysates were subjected to Western blot for quantification of selected pro- and anti-apoptotic Bcl-2 family proteins. Diagrams represent data ± S.E.M. of at least three independent experiments. Abbreviations: ns, not significant; PDGF, platelet-derived growth factor

Figure 4

Survival of activated fibroblasts is Bcl-xL-dependent. (A) Human HFF-1 and mouse 3T3 fibroblasts were activated in vitro with 50 ng/ml PDGF-BB for 24 h. Cells were then treated with pro-apoptotic BH3 mimetics specific for different Bcl-2 proteins for additional 24 h. Apoptosis was quantified biochemically by measuring caspase-3/7 activity using the fluorogenic Ac-DEVD-AMC substrate. Treatment of activated fibroblasts with 1 μM A-1331852, a specific Bcl-xL inhibitor, leads to a significant increase of caspase-3/7 activity, whereas specific inhibition of Bcl-2 using 1 μM ABT-199 (A-1195425) and Mcl1 using 10 μM A-1210477 shows no apoptotic effect. (B,C) PDGF-activated fibroblasts were treated with 1 μM Bcl-xL inhibitor A-1331852 for 24 h. Apoptosis induction was quantified biochemically by measuring caspase-3/7 activity using the fluorogenic Ac-DEVD-AMC substrate and morphologically using DAPI staining. The number of apoptotic nuclei was quantified in at least four high power fields and is expressed as a percentage of total. (D, E) PDGF induces apoptosis in fibroblasts after Bcl-xL knockdown. Fibroblasts were treated with 10 nM Bcl-xL-specific siRNA or nonsense siRNA for 48 h. Cells were then activated with 50 ng/ml PDGF-BB for 24 h. (D) Induction of apoptosis was quantified biochemically by measuring caspase-3/7 activity using the fluorogenic Ac-DEVD-AMC substrate. (E) Knockdown of Bcl-xL was confirmed by Western blot. Western blot for cleaved caspase-3 shows an increase only in fibroblasts with Bcl-xL-knockdown confirming induction of apoptosis by PDGF in these cells. Caspase activity is presented as mean ± S.D. of at least three independent experiments, performed in triplicate. Abbreviations: ns, not significant; PDGF, platelet-derived growth factor

Figure 5

Senescent cholangiocytes activate stellate cells in a PDGF-mediated manner. Normal human cholangiocytes (NHC) were induced to senescence with ionizing radiation (IR; 10Gy). (A) Cell culture supernatant of normal and senescent NHCs was analyzed for the cytokines and chemokines indicated using a membrane-based antibody array. (B) Normal and senescent NHCs were co-cultured with human hepatic stellate cells (LX-2) for 24 h. Whole cell lysates of LX-2 were analyzed for fibroblast activation marker αSMA. Addition of anti-PDGF antibodies prior co-culture completely blocked LX-2 activation. Diagrams represent mean ± S.D. of at least three independent experiments. Abbreviations: CXCL1, (C-X-C motif) chemokine; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte/macrophage colony stimulating factor; IL, interleukin; αSMA, alpha smooth muscle actin

Figure 6

Bcl-xL inhibition eliminates senescent cholangiocytes and decreases pro-fibrotic factors in Mdr2^−/− mice. Female BALB/cJ wild type and Mdr2^−/− mice were treated with vehicle or 25 mg/kg/d Bcl-xL inhibitor A-1331852 for 14 days by daily oral gavage starting at week 12 of age. (A) Co-staining of p16^Ink4a mRNA and Cytokeratin 19 protein was performed in formalin-fixed paraffin-embedded mouse liver tissue. Representative images (p16^Ink4A probe = green; CK19 = red; DAPI = blue) and semi-quantitative analysis of fluorescence intensity demonstrate a significant reduction of senescent cholangiocytes in Bcl-xL inhibitor-treated Mdr2^−/− mice compared to vehicle-treated animals. Bar = 50μm (B) Expression of pro-fibrotic cytokines and growth factors was assessed by quantitative real-time PCR. Gene expression is shown as fold change relative to vehicle. Data are presented as mean ± S.D. of at least three independent experiments, performed in triplicate. (C) Expression of pro-fibrotic cytokines and growth factors in cholangiocytes was investigated by chromogenic RNAscope^® in situ hybridization. Representative images and semi-quantitative assessment of RNAscope® stainings are shown. Abbreviations: FISH, fluorescence in situ hybridization; ns, not significant

Figure 7

Treatment with Bcl-xL inhibitors reduces liver fibrosis and expression of pro-fibrotic genes in Mdr2^−/− mice. Female BALB/cJ wild type and Mdr2^−/− mice were treated with 25 mg/kg/d Bcl-xL inhibitors A-1331852 for 14 days by daily oral gavage starting at week 12 of age. (A) Liver fibrosis was visualized by Sirius red collagen staining. (B) Morphometric analysis revealed a significant reduction of liver fibrosis after A-1331852 treatment. (C) Hydroxyproline quantification of 100 mg liver tissue confirmed the reduction of liver fibrosis. (D) Gene expression analysis of Collagen1A1 (Col1A1) and Tenascin c (TNC) by real-time PCR demonstrated a reduction in A-1331852-treated animals. (E) Quantitative PCR for αSMA, a marker for activated fibroblasts, shows a reduction in mice treated with Bcl-xL inhibitor. Data in D and E are presented as mean ± S.D. of at least three independent experiments, performed in triplicate.

Figure 8

Proposed mechanism of liver fibrosis reduction by Bcl-xL inhibition. Persistent injury of cholangiocytes in PSC leads to a pro-inflammatory response, which further results in chronic inflammation, cholestasis, and fibrosis. A large number of these cholangiocytes become senescent and upregulate Bcl-xL for survival. Although senescent cells do not proliferate, they remain metabolically active and produce increased levels of cytokines and growth factors. Pro-fibrotic growth factors such as PDGF are key mediators of fibroblast activation. Persistent fibroblast activation is a hallmark of liver fibrosis. Activated fibroblasts show a reduction of pro-survival Bcl-2 and their survival becomes Bcl-xL-dependent. Since Bcl-xL is pivotal for the survival of both cell types, treatment of liver fibrosis with specific Bcl-xL inhibitors represents a potential dual therapeutic strategy. Abbreviations: bd, bile duct; cv, central vein; ECM, extracellular matrix ha, hepatic artery; PDGF, platelet-derived growth factor; pv, portal vein; PSC, primary sclerosing cholangitis; ns, not significant