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).

The Spectrum of Reactive Cholangiocytes in Primary Sclerosing Cholangitis

“…SC SASP can also affect progression of peribiliary fibrosis. Notably, in an in vitro coculture system, SC conditioned medium activates the HSC line LX‐2, and this activation is abrogated by blocking PDGF secretion from SCs . In addition, secretin stimulation has been shown to drive cholangiocyte senescence, and HSC activation is induced when cells are incubated with cholangiocyte supernatant from secretin‐treated wild‐type mice or bile duct–ligated mice, in which cholangiocyte senescence is also increased.…”

“…Indeed, in preclinical studies, treatment with the selective Bcl‐xL inhibitors A‐1331852 or navitoclax (ABT‐263) induced apoptosis of cultured SCs but not normal cholangiocytes. More importantly, treatment of Mdr2 −/− mice with A‐1331852 or navitoclax reduced the number of p16 INK4a ‐positive cholangiocytes, in addition to decreasing the expression of activated fibroblasts, reducing levels of liver cytokine and chemokine expression and the overall level of peribiliary fibrosis . Clinical trials to test the use of navitoclax in cancer therapy have been limited by the development of severe thrombocytopenia as Bcl‐xL is required for platelet survival; however, because senescent cells do not replicate and accumulate slowly in the tissue, their elimination could be achieved by intermittent dosing, thereby limiting the toxic side effects of the treatment.…”

“…Notably, in an in vitro coculture system, SC conditioned medium activates the HSC line LX-2, and this activation is abrogated by blocking PDGF secretion from SCs. (41) In addition, secretin stimulation has been shown to drive cholangiocyte senescence, and HSC activation is induced when cells are incubated with cholangiocyte supernatant from secretin-treated wild-type mice or bile duct-ligated mice, in which cholangiocyte senescence is also increased. Secretininduced SCs modulate liver fibrosis through transforming growth factor beta 1 secretion, which also promotes cholangiocyte senescence in an autocrine loop.…”

“…(44) Experimentally induced SCs, as well as cholangiocytes from patients with PSC and Mdr2 −/− mice, overexpress the antiapoptotic protein Bcl-xL and exhibit dependence on Bcl-xL for survival, which can be exploited therapeutically. (37,41) Indeed, in preclinical studies, treatment with the selective Bcl-xL inhibitors A-1331852 or navitoclax (ABT-263) induced apoptosis of cultured SCs but not normal cholangiocytes. More importantly, treatment of Mdr2 −/− mice with A-1331852 or navitoclax reduced the number of p16 IN-…”

“…-positive cholangiocytes, in addition to decreasing the expression of activated fibroblasts, reducing levels of liver cytokine and chemokine expression and the overall level of peribiliary fibrosis. (41) Clinical trials to test the use of navitoclax in cancer therapy have been limited by the development of severe thrombocytopenia as Bcl-xL is required for platelet survival; however, because senescent cells do not replicate and accumulate slowly in the tissue, their elimination could be achieved by intermittent dosing, thereby limiting the toxic side effects of the treatment. Similarly, experimentally induced DRCs, but not normal cholangiocytes, display a selective dependence on the antiapoptotic protein Mcl-1, and pharmacologic targeting of DRCs by the Mcl-1 inhibitor S63845 effectively eliminates the parenchymal fibrosis in Mdr2 −/− Tr −/− mice, supporting the hypothesis that DRCs actively participate in the development of parenchymal fibrosis in cholestatic injury.…”