Research ArticleFibrosis

Targeted apoptosis of myofibroblasts with the BH3 mimetic ABT-263 reverses established fibrosis

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Science Translational Medicine  13 Dec 2017:
Vol. 9, Issue 420, eaal3765
DOI: 10.1126/scitranslmed.aal3765
  • Fig. 1. Matrix stiffness sensitizes myofibroblasts to apoptosis induced by inhibition of their mechanotransduction pathways.

    (A) Schematic diagram of atomic force microscopy (AFM) for microindentation on skin tissues. AFM was applied to map local elastic properties of thin slices of fresh mouse skin samples, harvested after 28 daily subcutaneous (sc) injections of either saline or bleomycin (BLM). (B) Representative hematoxylin and eosin (H&E)–stained images from 28-day saline- versus bleomycin-treated mice. Scale bars, 100 μm. n = 6 for all groups. (C) Representative elastographs from AFM microindentation of tissue stiffness in normal and fibrotic skin. The color bar indicates Young’s modulus, which is quantified in (D). Maps were made from tissue in the respective regions of interest (boxes) identified in (B). Data are means ± SD of stiffness measurements pooled from five animals each for normal and fibrotic groups in two independent bleomycin injection experiments. P value was determined by Student’s t test. (E and F) Effect of matrix stiffness on α-smooth muscle actin (α-SMA) protein expression as assessed by immunofluorescence. Human dermal fibroblasts (HDFs) were cultured on collagen-coated polyacrylamide hydrogels that recapitulated the stiffness of normal (4 kPa) (E) or densely fibrotic (50 kPa) (F) skin for 24 hours. Myofibroblasts were identified by staining for α-SMA (green). Fibroblasts were costained with phalloidin (red) to visualize F-actin and 4′,6-diamidino-2-phenylindole (DAPI; blue) to visualize nuclei. Scale bars, 50 μm. (G to L) Effect of matrix stiffness on the susceptibility of primary HDFs to apoptosis induced by inhibition of mechanotransduction pathways. HDFs were cultured on collagen-coated polyacrylamide hydrogels that recapitulated the stiffness of normal (4 kPa) or densely fibrotic (50 kPa) skin for 24 hours and treated with or without the agents indicated for an additional 48 hours. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test.

  • Fig. 2. Increased mitochondrial apoptotic priming in stiffness-induced myofibroblasts.

    (A) Schematic diagram of BH3 profiling assay. HDFs were cultured on matrices with different stiffnesses, permeabilized, and exposed to an array of peptides derived from different proapoptotic BH3-only proteins, and MOMP was measured indirectly using the mitochondrial JC-1 dye (fluorescent dye sensitive to the electropotential gradient across the inner mitochondrial membrane). The decay of ΔΨm was measured as fluorescence at 590 nm over 180 min. (B) Schematic illustrating how BCL-2 family interactions govern MOMP. The terminal execution phase of apoptosis is controlled by BCL-2 family members BAX and BAK, which are required to initiate apoptosis by causing MOMP through their pore-forming activities. MOMP releases caspase-activating factors (cytochrome c) into the cytoplasm, which results in collapse of the ΔΨm and caspase activation. MOMP activation is directly guarded by prosurvival proteins [BCL-2, BCL-XL, BCL-W, MCL-1, and A1 (Bfl-1)], which directly bind and block proapoptotic BAX/BAK. BH3-only activator proteins convey signals to initiate apoptosis by direct activation of BAX/BAK (BIM and BID) or indirectly by binding and inhibiting their prosurvival relatives (PUMA, BAD, NOXA, and HRK), which are known as sensitizers. (C) Interaction map for BH3 peptides and the antiapoptotic BCL-2 members. Red boxes indicate tight binding with a Kd (dissociation constant) of less than 100 nM; green boxes indicate weak or absent interaction, as determined by fluorescence polarization. Diagrams from (A) to (C) are adapted with permission from (23). (D) Heat map of mitochondrial depolarization induced by peptides derived from different proapoptotic BH3-only proteins. HDFs cultured for 48 hours on matrices with stiffness of 1, 4, 8, 25, and 50 kPa were subjected to BH3 profiling assay using BIM and BID (activate proapoptotic effectors BAX and BAK), PUMA (block prosurvival proteins), BAD (selective BCL-2, BCL-XL, and BCL-W sensitizer), and HRK (binds BCL-XL and MS1 and blocks MCL-1) peptides. The concentration of peptides in the assays was 100 μM except for MS1 (10 μM). Data are means ± SD of three replicate wells for each peptide. (E to H) Mitochondrial depolarization caused by the PUMA (E), BAD (F), HRK (G), and MS1 (H) peptides in HDFs cultured on matrices with increasing matrix stiffness. (I) Working model. Matrix stiffness–induced fibroblast-to-myofibroblast transformation increases mitochondrial priming.

  • Fig. 3. Pharmacological or genetic inhibition of BCL-XL induces apoptosis of stiffness-primed myofibroblasts.

    (A) Effect of matrix stiffness on gene expression of BCL-2 family members in HDFs (n = 4 for each condition). Gene expression was analyzed by quantitative polymerase chain reaction (qPCR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (B) Effect of matrix stiffness on BCL-2, BCL-XL, MCL-1, BIM, BID, BAX, and BAK protein expression as demonstrated by Western blotting. GAPDH was used as a loading control. (C and D) Effect of small interfering RNA (siRNA)–mediated BCL-2 or BCL-XL knockdown on mitochondrial priming (C) and apoptosis (D) of HDFs exposed to 4 or 50 kPa matrix stiffness. Nontargeting siRNA was used as control. Mitochondrial priming was determined by BH3 profiling using the PUMA-derived peptide (10 μM). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used as a positive control for mitochondrial depolarization. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (E) Effect of siRNA-mediated BIM knockdown on BCL-XL knockdown–induced apoptosis of HDFs. Nontargeting siRNA was used as a control. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (F and G) Effect of BH3 mimetic drugs ABT-199 (BCL-2 inhibitor) and ABT-263 (BCL-XL inhibitor) on mitochondrial priming (F) and apoptosis (G) of HDFs exposed to 4 or 50 kPa matrix stiffness. Dimethyl sulfoxide (DMSO) was used as a control. Mitochondrial priming was determined by BH3 profiling using the PUMA-derived peptide (10 μM). FCCP is used as a positive control for mitochondrial depolarization. Apoptosis was assessed by annexin V staining. Data are means ± SD of three replicate wells for each peptide. P value was determined by Student’s t test. (H) BIM is sequestered by BCL-XL in stiffness-exposed myofibroblasts and displaced by antagonism of BCL-XL by ABT-263. Anti-BIM immunoprecipitation of whole-cell lysates (50 μg) from HDFs exposed to 4 or 50 kPa matrix stiffness for 24 hours and then treated with or without ABT-263 (1 μm) for 6 hours. BIM and BCL-XL were immunoblotted. (I) Effect of siRNA-mediated BIM knockdown on ABT-263–induced apoptosis of HDFs exposed to 50 kPa matrix stiffness. Nontargeting siRNA was used as a control. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (J) Model of ABT-263–induced myofibroblast apoptosis. Prosurvival BCL-XL is sequestered by pro-death BIM in stiffness-exposed myofibroblasts. Upon treatment with ABT-263, BIM is displaced and BCL-XL becomes occupied by ABT-263. Freed BIM then interacts with BAX or BAK, which initiates apoptosis in myofibroblasts.

  • Fig. 4. Pharmacological BCL-XL inhibition attenuates bleomycin-induced skin fibrosis.

    (A and B) Paraffin-embedded skin sections from mice treated with saline or bleomycin stained for BCL-2 or BCL-XL (red), α-SMA (green), and DAPI (blue) to visualize the expression of BCL-2 or BCL-XL, myofibroblasts, and nuclei, respectively. Arrows in higher-magnification views (insets, enlarged fivefold) denote overexpression of BCL-2 and BCL-XL in α-SMA + myofibroblasts in vivo. Scale bars, 100 μm. n = 4 for all groups. (C) Viability of mouse dermal fibroblasts (MDFs) explanted from saline- or bleomycin-treated mice (n = 3 per condition) and exposed to serial dilutions of either ABT-263 or ABT-199 for 48 hours. Data are means ± SD. P value was determined by Student’s t test. IC50, median inhibitory concentration. (D) Gene expression analysis of prosurvival and proapoptotic members in MDFs explanted from saline- or bleomycin-treated mice (n = 4 for each cell line). Gene expression was analyzed by qPCR, and GAPDH was used as a housekeeping gene. P value was determined by Student’s t test. (E) Schematic showing BH3 mimetics (small-molecule inhibitors that mimic the activity of proapoptotic BH3-only proteins, counteracting prosurvival proteins). ABT-737 and its orally bioavailable derivate ABT-263 (navitoclax) bind and neutralize the prosurvival proteins BCL-2 and BCL-XL but not MCL-1 or A1. ABT-199 is a highly potent, orally bioavailable BCL-2–selective inhibitor. (F to I) Therapeutic effects of BH3 mimetic drugs ABT-263 and ABT-199 on bleomycin-induced dermal fibrosis in vivo as assessed by Masson’s trichrome stain (G), dermal thickness (H), and hydroxyproline content (I). n = 8 for all groups. Graphs are means ± SD. P value was determined by two-way analysis of variance (ANOVA). Scale bar, 100 μm. (J and K) Paraffin-embedded skin sections from mice treated with bleomycin in combination with saline vehicle (J) or ABT-263 (K) stained for cleaved (active) caspase (red), α-SMA (green), and DAPI (blue) to visualize apoptotic cells, myofibroblasts, and nuclei, respectively. Arrows in higher-magnification views (enlarged fivefold) indicate dual cleaved caspase-3+ α-SMA+ myofibroblasts in vivo. n = 4 for all groups. Scale bars, 50 μm. (L and M) Quantification of number of total α-SMA+ myofibroblasts (MF) and apoptotic myofibroblasts (α-SMA+ caspase-3+) in skin sections from mice treated with bleomycin in combination with saline vehicle (L) or ABT-263 (M). Three hundred cells were counted per condition. Data are means ± SD (n = 4 for all groups). P value was determined by two-way ANOVA. (N to Q) Effect of ABT-263 or ABT-199 treatment on immune cell populations in mice treated with bleomycin or saline control, (N) total number of leukocytes, (O) neutrophils, (P) inflammatory monocytes, and (Q) dendritic cells. n = 4 for all groups. Graphs are means ± SD. P value was determined by two-way ANOVA.

  • Fig. 5. Forced expression of α-SMA increases mitochondrial priming and sensitivity of dermal myofibroblasts to ABT-263–induced apoptosis.

    (A) Schematic diagram of adenoviral vector design for overexpression of human α-SMA fused to red fluorescent protein (Ad-CMV-αSMA-RFP). (B and C) α-SMA mRNA (B) and protein (C) expression in HDFs transduced with either control null adenovirus or Ad-CMV-αSMA-RFP with increasing multiplicities of infection (MOIs). GAPDH was used as a housekeeping gene and loading control. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (D and E) Gene expression analysis of BCL-XL and BIM in HDFs transduced with either control null adenovirus or Ad-CMV-αSMA-RFP with increasing MOI. GAPDH was used as a housekeeping gene. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (F) Effect of ABT-263 (BCL-XL inhibitor; 300 nM) on apoptosis of HDFs transduced with Ad-Null or Ad-CMV-αSMA-RFP. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (G) Schematic diagram of adenoviral vector design for overexpression of green fluorescent protein (GFP) and shRNA against human α-SMA (Ad-U6-shRNA-αSMA-CMV-GFP). (H and I) α-SMA mRNA and protein expression in HDFs transduced with either control null adenovirus or Ad-U6-shRNA-αSMA-CMV-GFP with increasing MOI. α-SMA expression was quantified by qPCR and Western blotting. GAPDH was used as a housekeeping gene and loading control. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (J and K) Gene expression analysis of BCL-XL (J) and BIM (K) in HDFs transduced with either control null adenovirus or Ad-U6-shRNA-αSMA-CMV-GFP with increasing MOI. GAPDH was used as a housekeeping gene. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (L) Effect of ABT-263 (300 nM) on apoptosis of HDFs transduced with Ad-Null or Ad-CMV-αSMA-RFP. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (M) Schematic showing experimental design. HDFs were transduced with Ad-CMV-αSMA-RFP or Ad-U6-shRNA-αSMA-CMV-GFP, and then cells were mixed at a 1:1 ratio and plated on 50 kPa hydrogels for 24 hours. Cells were treated with ABT-263 for 12 hours before assessment of cell apoptosis. (N) Table showing calculation of ABT-263 IC50 for each cell line. Data are means ± SD from three independent experiments. (O) Representative image of ABT-263–induced apoptosis of fibroblast transduced with either Ad-CMV-αSMA-RFP or Ad-U6-shRNA-αSMA-CMV-GFP as assessed by fluorescence live cell imaging. White arrows indicate apoptotic cells. (P) Quantification of cell apoptosis in (O) by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test.

  • Fig. 6. Mitochondrial priming predicts response to ABT-263 in scleroderma fibroblasts.

    (A) Schematic diagram of BH3 profiling in scleroderma clinical practice. Single-cell suspension of fibroblasts obtained from scleroderma cell lines (passage 2 to 4) or from fresh skin primary biopsy from scleroderma patients is subjected to BH3 profiling assays. Diagram adapted with permission from (27). RFU, relative fluorescence units. (B) Heat map of mitochondrial depolarization caused by the BIM, BID, and PUMA peptides (measurement of overall “priming”) in dermal fibroblasts isolated from six patients with scleroderma (SSc) compared to six healthy controls (N). Individual patient codes are shown along the x axis, and samples are ordered according to increasing depolarization by peptide. The concentration of peptides in the assays was 100 μM except for MS1 (10 μM). PUMA2a peptide was used as a negative control, and FCCP was used as a positive control for mitochondrial depolarization. (C) Mitochondrial depolarization caused by the HRK peptide in scleroderma patient samples compared with their responses to ABT-263–induced cell apoptosis. Plot showing correlation between Δ% priming induced by HRK peptide and Δ% cell death at 48 hours upon ABT-263 treatment. Data are means ± SD from three independent experiments. P value was determined by two-tailed Spearman rank test.

  • Fig. 7. Mitochondrial priming and expression of BCL-2 family proteins is controlled by mechanotransduction pathways in scleroderma fibroblasts.

    (A) BH3 profiling. Heat map of mitochondrial depolarization caused by the BIM, PUMA, BAD, HRK, and MS1 peptides in dermal fibroblasts isolated from six patients with scleroderma compared to six healthy controls. (B) Gene expression analysis of prosurvival and proapoptotic members in HDFs from healthy volunteer “Normal 3” and from patients with scleroderma: “Scleroderma 4” and “Scleroderma 6.” Gene expression was analyzed by qPCR, and GAPDH was used as a housekeeping gene. Data are means ± SD from three independent experiments. (C and D) Effect of siRNA-mediated knockdown of MCL-1 (C) or S63845 (D), an MCL-1–selective BH3 mimetic, on ABT-263–induced apoptosis in Scleroderma 6 fibroblasts. Nontargeting siRNA or vehicle was used as a control. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (E) Effect of inhibition of mechanotransduction pathways on the expression of pro- and antiapoptotic family members in Scleroderma 6 fibroblasts. Cells were treated for 24 hours with the FAK inhibitor PF-562,271 or the ROCK inhibitor Y-27632, and gene expression was analyzed by qPCR. GAPDH was used as a housekeeping gene. Data are means ± SD from three independent experiments. P value was determined by Student’s t test. (F) Effect of PF-562,271 before treatment for 24 hours on ABT-263–induced mitochondrial depolarization in Scleroderma 6 fibroblasts. DMSO was used as a vehicle control. Data are means ± SD from three independent experiments. (G) Effect of PF-562,271 before treatment for 24 hours on ABT-263–induced apoptosis in Scleroderma 6 fibroblasts. DMSO was used as a vehicle control. Data are means ± SD from three independent experiments. P value was determined by Student’s t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/420/eaal3765/DC1

    Materials and Methods

    Fig. S1. Effect of pan-caspase inhibitor Z-VAD and caspase-8 inhibitor emricasan on myofibroblast apoptosis induced by FAK inhibition.

    Fig. S2. siRNA transfection efficiency and gene expression of BCL-2, BCL-XL, and MCL-1.

    Fig. S3. Effect of YAP/TAZ knockdown and overexpression on fibroblast mitochondrial priming and survival.

    Fig. S4. BH3 profiling of Normal 3, Scleroderma 4, and Scleroderma 6 fibroblasts.

    Fig. S5. Effect of FAK and ROCK inhibition on mitochondrial priming and antiapoptotic BCL-2 members.

    Fig. S6. Targeted apoptosis of myofibroblast reverses established fibrosis.

    Table S1. Reverse transcription PCR primer and siRNA sequences.

  • Supplementary Material for:

    Targeted apoptosis of myofibroblasts with the BH3 mimetic ABT-263 reverses established fibrosis

    David Lagares,* Alba Santos, Paula E. Grasberger, Fei Liu, Clemens K. Probst, Rod A. Rahimi, Norihiko Sakai, Tobias Kuehl, Jeremy Ryan, Patrick Bhola, Joan Montero, Mohit Kapoor, Murray Baron, Xaralabos Varelas, Daniel J. Tschumperlin, Anthony Letai, Andrew M. Tager

    *Corresponding author. Email: dlagares{at}mgh.harvard.edu

    Published 13 December 2017, Sci. Transl. Med. 9, eaal3765 (2017)
    DOI: 10.1126/scitranslmed.aal3765

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Effect of pan-caspase inhibitor Z-VAD and caspase-8 inhibitor emricasan on myofibroblast apoptosis induced by FAK inhibition.
    • Fig. S2. siRNA transfection efficiency and gene expression of BCL-2, BCL-XL, and MCL-1.
    • Fig. S3. Effect of YAP/TAZ knockdown and overexpression on fibroblastmitochondrial priming and survival.
    • Fig. S4. BH3 profiling of Normal 3, Scleroderma 4, and Scleroderma 6 fibroblasts.
    • Fig. S5. Effect of FAK and ROCK inhibition on mitochondrial priming and antiapoptotic BCL-2 members.
    • Fig. S6. Targeted apoptosis of myofibroblast reverses established fibrosis.
    • Table S1. Reverse transcription PCR primer and siRNA sequences.

    [Download PDF]

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