Research ArticleFibrosis

The αvβ1 integrin plays a critical in vivo role in tissue fibrosis

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Science Translational Medicine  20 May 2015:
Vol. 7, Issue 288, pp. 288ra79
DOI: 10.1126/scitranslmed.aaa5094
  • Fig. 1. αvβ1 integrin inhibitor design and demonstration of potency and specificity.

    (A) Design principle of αvβ1 integrin inhibitor by combining a positively charged guanidine moiety in αvβ3 integrin inhibitor (blue shading) and a sulfonamidoproline moiety in α2β1 integrin inhibitor (green shading). (B) Structures of αvβ1 integrin–specific inhibitor c8. (C) Docking model of αvβ1 integrin inhibitor c8 bound to αvβ1 integrin, where α and β subunits are, respectively, in green and blue. The model predicted that a linker length of n = 3 or 4 would have the highest affinity. (D) Dose-dependent cell adhesion assay of c8 against all αv and related integrins. (E) Curve-fitted IC50 of c8 against RGD (arginine–glycine–aspartic acid)–binding integrins in cell adhesion assay. Data represent means ± SEM; n = 3 (three experimental replicates).

  • Fig. 2. αvβ1 is expressed on pulmonary and hepatic fibroblasts and mediates adhesion to TGFβ1 LAP and activation of latent TGFβ.

    (A) Coimmunoprecipitation and Western blot reveal expression of αvβ1 heterodimers in human and murine cell lines from the liver and lung. Cell lysates were immunoprecipated with antibodies to αv (antibody RMV-7 for murine cells or L230 for all other cells), followed by Western blotting for either αv (lower panels, to control for capture, antibody 611012) or β1 (upper panels, antibody 04-1109). nhLu fb control (normal human lung fibroblasts from an uninjured control subject); IPF fb (lung fibroblasts isolated from a patient with IPF); mLu fb (murine lung fibroblasts); mHSC (murine hepatic stellate cells); WI38 (diploid human lung fibroblast); CHO [WT (wild-type)] control α5-deficient CHO cells (which lack expression of αvβ1); CHO (αv) (α5-deficient CHO cells engineered to express the αvβ1); hAT2 (human alveolar type 2 cells, which lack expression of αvβ1); hPAEC (human pulmonary artery endothelial cells, which lack expression of αvβ1). IgG, immunoglobulin G. (B) WT CHO cells (lacking αvβ1) adhere poorly, whereas CHO cells with forced expression of αvβ1 (CHO αv) and WI38 cells strongly adhere to TGFβ1 LAP. (C) WI38 cell adhesion to TGFβ1 LAP (0.3 μg/ml) is inhibited by c8 (IC50 = 0.72 nM). (D) c8 treatment reduced activation of TGFβ by cells expressing αvβ1 (IC50 range, 0.35 to 0.50 nM). Fibroblasts (as indicated) were cocultured with TGFβ reporter (PAI1-luciferase) cell line in the presence of a range of concentrations of c8. Data represent means ± SEM, n = 3 (three experimental replicates).

  • Fig. 3. c8 protects from liver and lung fibrosis.

    (A) Effects of c8 or c16 on mouse fibrosis model (liver). c8 or c16 (inactive control compound) was continuously delivered by Alzet pump beginning 3 weeks after intraperitoneal administration with oil (sham) or CCl4 to induce liver fibrosis. (B and C) Treatment with c8 significantly reduced liver fibrosis, as determined by (B) Sirius red staining (collagen deposition) of liver tissue after olive oil (top panels) or CCl4 treatment (bottom panels), (C) digital image analysis quantification of collagen staining, and hydroxyproline analysis. Sirius red (liver) n = 8; P = 0.0005. Hydroxyproline (liver) n = 10; P < 0.0001. (D) Effects of c8 or c16 on murine fibrosis model (lung). c8 or c16 was continuously delivered to mice using Alzet pumps beginning 14 days after intratracheal instillation of bleomycin (Bleo) to induce pulmonary fibrosis or water (H2O). (E and F) Treatment with c8 significantly reduced lung fibrosis, as determined by (E) Sirius red staining (collagen deposition) of lung tissue after water (top panels) or bleomycin treatment (bottom panels), (F) digital image analysis quantification of collagen staining, and hydroxyproline analysis. Sirius red (lung) n = 10; P = 0.0022. Hydroxyproline (lung) n = 10; P = 0.0027. Data represent means ± SEM. Scale bars, 100 μm. P values were calculated using the unpaired Student’s t tests.

  • Fig. 4. Reduced pSmad3 in mice treated with c8.

    (A) Representative liver (top panels) and lung (bottom panels) sections from mice treated with c16 or c8 after induced fibrotic injury stained for fibroblasts [PDGFRβ (platelet-derived growth factor receptor β), green] and pSmad3 (red). (B) Quantification of pSmad3 nuclear intensity within individual PDGFRβ+ cells documents a significant reduction in fibroblast-specific pSmad3 in fibrotic mice treated with c8. Data represent means ± SEM; n = 102 (Bleo c8), n = 254 (Bleo c16), n = 262 (CCl4 c8), and n = 361(CCl4 c16). For both comparisons, P < 0.0001 (shown is a representative example of the distribution of individual pSmad3 mean fluorescence intensities in PDGFRβ+ cells, and the average of these means, for a single sample condition). Scale bar, 100 μm. P values were calculated using the unpaired Student’s t tests.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/288/288ra79/DC1

    Fig. S1. Validation of cell adhesion conditions used to determine potency and specificity of αvβ1 inhibitors.

    Fig. S2. C8 and C6 adhesion assays.

    Fig. S3. Adhesion of WI38 cells in low concentration of TGFβ1 LAP.

    Fig. S4. Adhesion and TGFβ activation assays in A549 and 293 cells.

    Fig. S5. Original Western blot images of Fig. 2A.

    Table S1. Source data for Fig. 1 (D and E).

    Table S2. Source data for Fig. 2B.

    Table S3. Source data for Fig. 2C.

    Table S4. Source data for Fig. 2D.

    Table S5. Source data for Fig. 3C.

    Table S6. Source data for Fig. 3F.

  • Supplementary Material for:

    The αvβ1 integrin plays a critical in vivo role in tissue fibrosis

    Nilgun I. Reed, Hyunil Jo, Chun Chen, Kazuyuki Tsujino, Thomas D. Arnold, William F. DeGrado,* Dean Sheppard*

    *Corresponding author. E-mail: rmazitschek{at}mgh.harvard.edu (R.M.); dfwirth{at}hsph.harvard.edu (D.F.W.)

    Published 20 May 2015, Sci. Transl. Med. 7, 288ra79 (2015)
    DOI: 10.1126/scitranslmed.aaa5094

    This PDF file includes:

    • Fig. S1. Validation of cell adhesion conditions used to determine potency and specificity of αvβ1 inhibitors.
    • Fig. S2. C8 and C6 adhesion assays.
    • Fig. S3. Adhesion of WI38 cells in low concentration of TGFβ1 LAP.
    • Fig. S4. Adhesion and TGFβ activation assays in A549 and 293 cells.
    • Fig. S5. Original Western blot images of Fig. 2A.
    • Table S1. Source data for Fig. 1 (D and E).
    • Table S2. Source data for Fig. 2B.
    • Table S3. Source data for Fig. 2C.
    • Table S4. Source data for Fig. 2D.
    • Table S5. Source data for Fig. 3C.
    • Table S6. Source data for Fig. 3F.

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