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. 2014 Apr 7;9(4):e92273.
doi: 10.1371/journal.pone.0092273. eCollection 2014.

Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling

Ikuo Nakamura  1 , Kais Zakharia  1 , Bubu A Banini  1 , Dalia S Mikhail  1 , Tae Hyo Kim  1 , Ju Dong Yang  1 , Catherine D Moser  1 , Hassan M Shaleh  1 , Sarah R Thornburgh  1 , Ian Walters  2 , Lewis R Roberts  1
Affiliations

Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling

Ikuo Nakamura et al. PLoS One. .

Erratum in

Abstract

Background and aims: Brivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.

Methods: In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.

Results: After in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.

Conclusion: Brivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

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Conflict of interest statement

Competing Interests: Dr. Ian Walters is employed by Bristol Myers Squibb, the funder of this study, and has stock holdings in the company. He played a role in study design and in review/editing of the manuscript, but had no role in data collection and analysis or in the decision to publish. Brivanib is under development by Bristol Myers Squibb. This does not alter the authors' adherence to the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Brivanib inhibits liver fibrosis induced by bile duct ligation.
(A) Histological findings at 14 days after BDL. The Sirius red and trichrome images are taken at 100×. (B) The number of bands of bridging fibrosis per high power field were counted in images obtained at 100× magnification after Sirius red staining. (C) Hepatic levels of collagen Iα1 mRNA were measured by real time PCR in placebo, 25 mg/kg and 50 mg/kg brivanib groups at 14 days after BDL (n = 6 per group). (D) Western immunoblotting for α-SMA showing decreased whole liver α-SMA after treatment with brivanib. The expression of α-SMA in lysates extracted from liver tissue following sham ligation of the bile duct was measured by Western immunoblotting. β-actin is shown to control for loading. The sham controls confirm that brivanib does not induce liver fibrosis.
Figure 2
Figure 2. Brivanib stimulates the transcription of growth factors and their receptors in bile duct ligated mice.
Hepatic levels of growth factors and growth factor receptor mRNA were measured by real time PCR in sham and BDL mice treated with no brivanib or with brivanib 25/kg or 50 mg/kg. In all of the sham experiments, higher concentrations of brivanib decreased the mRNA levels of the growth factors and their receptors. (A, B) BDL mice treated with brivanib show a dose-dependent increase in mRNA levels of PDGFB and PDGFRB. (C, D) mRNA levels of TGFB1 increases as the concentration of brivanib increases in BDL mice; mRNA levels of TGFBR2 are higher in BDL mice treated with 25 mg/kg and 50 mg/kg of brivanib, compared to the no brivanib group. (E, F) mRNA levels of FGF2 and FGFR2 are slightly higher with brivanib compared to no brivanib in BDL mice. (G, H) the mRNA levels of VEGFA and VEGFR2 are not affected by brivanib treatment in BDL mice.
Figure 3
Figure 3. Brivanib inhibits liver fibrosis induced by carbon tetrachloride.
(A) Histological analysis of livers from placebo and brivanib (25, 50, and 100 mg/kg) groups at 4 weeks after the initiation of carbon tetrachloride (CCl4). Pictures of Sirius red and Masson's trichrome are taken at 100× magnification. (B) The number of bands of bridging fibrosis per high power field were counted in images obtained at 100× magnification after Sirius red staining. (C) Hepatic levels of collagen 1α1 mRNA were measured by real time PCR in placebo, 25 mg/kg, 50 mg/kg, and 100 mg/kg brivanib groups (n = 6 per group) at 4 weeks after the initiation of CCl4 administration. (D) Western immunoblotting for α-SMA showing decreased whole liver α-SMA after treatment with brivanib.
Figure 4
Figure 4. Brivanib decreases the transcription of growth factors and their receptors in carbon tetrachloride.
Hepatic levels of growth factors and growth factor receptor mRNA were measured by real time PCR in sham and CCl4 mice treated with no brivanib or with brivanib 25 mg/kg, 50 mg/kg, or 100 mg/kg. In all of the sham experiments, higher concentrations of brivanib decreased the mRNA levels of the growth factors and their receptors. (A) mRNA levels of PDGFB are not affected by brivanib treatment in CCl4. (B) mRNA levels of PDGFRB decrease as the concentration of brivanib increases in CCl4. (C) mRNA levels of TGFB1 decrease as the concentration of brivanib increases in CCl4. (D) mRNA levels of TGFBR2 are lower in brivanib-treated CCl4 mice compared to no brivanib. (E) mRNA levels of FGF2 are not affected by brivanib treatment in CCl4. (F) mRNA levels of FGFR2 are lower in brivanib-treated CCl4 mice compared to no brivanib. (G, H) mRNA levels of VEGFA and VEGFR2 decrease as the concentration of brivanib increases in CCl4 mice.
Figure 5
Figure 5. Brivanib inhibits liver fibrosis induced by thioacetamide.
(A) Histological analysis of livers from placebo and brivanib-treated (25 and 50 mg/kg) groups at 4 weeks after initiation of thioacetamide (TAA). Pictures of Sirius red and Masson's trichrome staining are taken at 100×, magnification. (B) The number of bands of bridging fibrosis per high power field were counted in images obtained at 100× magnification after Sirius red staining. There was a significant decrease in the number of bands in both the 25 mg/kg and 50 mg/kg brivanib groups, compared to placebo. (C) The hepatic level of collagen Iα1 mRNA was measured by real time PCR in placebo, 25 mg/kg and 50 mg/kg brivanib groups (n = 6 per group) at 4 weeks after the initiation of TAA. There was a substantial reduction in collagen Iα1 mRNA at both brivanib dose levels. (D) Western immunoblotting for α-SMA showing decreased whole liver α-SMA after treatment with brivanib.
Figure 6
Figure 6. Brivanib does not affect the transcription of growth factors and their receptors in thioacetamide treated mice.
Hepatic levels of growth factors and growth factor receptor mRNA were measured by real time PCR in sham and TAA mice treated with no brivanib or with brivanib 25/kg, 50 mg/kg, or 100 mg/kg. In all of the sham experiments, higher concentrations of brivanib decreased the mRNA levels of the growth factors and their receptors. mRNA levels of PDGF, PDGFBR, TGFB1, TGFBR2, FGF2, FGFR2, VEGFA, and VEGFR2 are not affected by brivanib treatment of TAA mice.
Figure 7
Figure 7. PDGF, VEGF and FGF2 all induce proliferation of human LX-2 hepatic stellate cells.
Relative proliferation of LX-2 cells as assessed by BrdU incorporation after addition of TGF-β1 (A); PDGF (B); VEGF (C); or FGF (D). The LX-2 HSCs were starved for 24 hours and then treated with the respective cytokine or growth factor. BrdU incorporation was measured 72 hours later. Data shown are representative of four samples per group and are presented as mean ± SEM. *, P<0.05 (normalized to BrdU incorporation in the absence of the growth factor).
Figure 8
Figure 8. Brivanib inhibits unstimulated and PDGF, VEGF, or FGF2-stimulated LX-2 cell proliferation.
The effect of brivanib on cell proliferation of LX-2 HSCs without growth factor (A). The effect of brivanib on LX-2 cell proliferation induced by 50 ng/ml PDGF (B); 1 ng/ml VEGF (C); or 10 ng/ml FGF (D). LX-2 HSCs were starved for 24 hours, brivanib was added at the indicated concentrations, and 2 hours later, the respective growth factor was added. BrdU incorporation was measured at 72 hours after the administration of growth factor. Data shown are representative of four samples per treatment group and are presented as mean ± SEM. ∞,P<0.05 (vs. without growth factor and without brivanib) and *, P<0.05 (vs. with growth factor and without brivanib).
Figure 9
Figure 9. Brivanib decreases viability of PDGF-BB treated LX-2 cells.
(A) The effect of increasing concentrations of brivanib on the viability of LX-2 HSCs cultured in DMEM/10% FBS was assessed using Cell Counting Kit-8 (CCK-8). Cells showed a brivanib dose-dependent decrease in viability, with half maximal inhibitory concentration (IC50) of 16.98 µM (95% CI 13.95 µM–20.67 µM). (B) Cells stimulated with 5 ng/ml PDGF-BB in serum-free medium showed a similar trend albeit more pronounced decrease in viability compared to cells cultured in 10% FBS. The IC50 of LX-2 cells cultured in serum-free media supplemented with PDGF-BB was 8.79 µM (95% CI 7.80 µM–9.99 µM).
Figure 10
Figure 10. Brivanib does not inhibit TGF-β1-induced α-SMA expression in human LX-2 hepatic stellate cells.
(A) The expression of α-SMA in LX-2 HSCs after TGF-β1 treatment was assessed by Western immunoblotting. HSCs were incubated with 10% or 1% FBS for 24 hours, and TGF-β1 was added at different concentrations. The lysate was extracted 24 hours after addition of TGF-β1. Peak α-SMA expression was seen after treatment with 2 ng/ml of TGF-β1. (B) To determine the effect of brivanib on TGF-β1-induced activation of HSCs as assessed by α-SMA expression, LX-2 HSCs were partially serum-starved by incubation with 1% FBS for 24 hours. Brivanib was then added at increasing concentrations 2 hours before addition of 2 ng/ml TGF-β1. Cell lysates were prepared 24 hours after adding TGF-β1 and analyzed by Western immunoblotting.
Figure 11
Figure 11. Brivanib inhibits PDGF-BB induced phosphorylation of PDGFRβ in human LX-2 hepatic stellate cells.
(A) The expression of phosphorylated (P-PDGFRβ) and total PDGFRβ (T-PDGFRβ) in HSCs after PDGF-BB treatment was assessed by Western immunoblotting. HSCs were incubated in 10% FBS-supplemented DMEM for 24 hours, followed by starvation in serum-free medium for 24 hours. PDGF-BB was added at 5 or 10 ng/ml, followed by extraction of protein lysate 1 or 5 minutes after induction with PDGF-BB. Both time- and dose-dependent increase in phosphorylated PDGFRβ were seen, with minimal change in total PDGFRβ. β-actin was used as a loading control. (B) To determine the effect of brivanib on PDGF-BB induced phosphorylation of PDGFRβ, LX-2 cells were serum starved, followed by treatment with 5, 10 or 20 µM of brivanib. Two hours after brivanib treatment, 5 ng/ml PDGF-BB was added, and protein lysates prepared after 5 minutes of PDGF-BB exposure. All doses of brivanib tested inhibited PDGF-BB induced phosphorylation of PDGFRβ. (C) Ratio of phosphorylated PDGFRβ relative to total PDGFRβ. Prior to calculation of the P-PDGFRβ/T-PDGFRβ ratio, protein levels were first normalized to β-actin, and then to the P-PDGFRβ or T-PDGFRβ level in their respective control studies. Data represents the mean of four replicate studies ± SEM.
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