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

Abstract

Integrins are transmembrane heterodimeric receptors that contribute to diverse biological functions and play critical roles in many human diseases. Studies using integrin subunit knockout mice and inhibitory antibodies have identified important roles for nearly every integrin heterodimer and led to the development of a number of potentially useful therapeutics. One notable exception is the αvβ1 integrin. αv and β1 subunits are individually present in numerous dimer pairs, making it challenging to infer specific roles for αvβ1 by genetic inactivation of individual subunits, and αvβ1 complex-specific blocking antibodies do not yet exist. We therefore developed a potent and highly specific small-molecule inhibitor of αvβ1 to probe the function of this understudied integrin. We found that αvβ1, which is highly expressed on activated fibroblasts, directly binds to the latency-associated peptide of transforming growth factor-β1 (TGFβ1) and mediates TGFβ1 activation. Therapeutic delivery of this αvβ1 inhibitor attenuated bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis, suggesting that drugs based on this lead compound could be broadly useful for treatment of diseases characterized by excessive tissue fibrosis.

Fig. 1. α_vβ₁ integrin inhibitor design and demonstration of potency and specificity

(A) Design principle of α_vβ₁ integrin inhibitor by combining a positively charged guanidine moiety in α_vβ₃ integrin inhibitor (blue shading) and a sulfonamidoproline moiety in α₂β₁ integrin inhibitor (green shading). (B) Structures of α_vβ₁ integrin–specific inhibitor c8. (C) Docking model of α_vβ₁ integrin inhibitor c8 bound to α_vβ₁ 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 α_v 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. α_vβ₁ 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 α_vβ₁ heterodimers in human and murine cell lines from the liver and lung. Cell lysates were immunoprecipated with antibodies to α_v (antibody RMV-7 for murine cells or L230 for all other cells), followed by Western blotting for either α_v (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 α_vβ₁); CHO (α_v) (α₅-deficient CHO cells engineered to express the α_vβ₁); hAT2 (human alveolar type 2 cells, which lack expression of α_vβ₁); hPAEC (human pulmonary artery endothelial cells, which lack expression of α_vβ₁). IgG, immunoglobulin G. (B) WT CHO cells (lacking α_vβ₁) adhere poorly, whereas CHO cells with forced expression of α_vβ₁ (CHO α_v) 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 α_vβ₁ (IC₅₀ range, 0.35 to 0.50nM). 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 (plateletderived 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.