αvβ3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis

Abstract

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvβ3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvβ3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvβ3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvβ3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Keywords: Cell Biology; Extracellular matrix; Fibrosis; Integrins; Pulmonology.

Figure 1. Characterization of microscale IPF tissue rigidity and elasticity.

(A) Experimental setup of atomic force microscope (AFM) mechanical measurements, depicting the cantilever (red dotted line) overlying lung tissue. Fluorescence images were acquired using an inverted optical microscope in combination with AFM. DAPI (cell nuclei), tissue autofluorescence (mainly elastin microfibrils), and phase-contrast images are shown. Scale bar: 100 μm. (B) Example force indentation and Young’s modulus (E) indentation curves of fibroblastic foci (FF, blue) and mature fibrosis (MF, red) regions; the low indentation regime, E_low, is highlighted (pink dotted line, gray background) and the linearly elastic regime indentation limit, δ_L, is demarked (black dotted line, bottom). The equation to calculate Young’s modulus from force indentation is shown. (C) H&E staining of IPF tissue. Scale bar: 200 μm. (D) Magnified views of the region in C (green; zoom in region) stained for H&E, Masson’s trichrome, and fibronectin-EDA (FN-EDA, with regions of interest, including FF (blue box) and MF (red box), indicated. Scale bar: 100 μm. (E) AFM force maps with E (“black-red-white” heatmap, range 0–4 kPa for NL and FF; 0–10 kPa for MF) and elasticity (L; “rainbow” heatmap) shown for NL (data not shown), FF (blue box), and MF (red box), with regions of interest depicted in D. (F) Histogram of E values are shown for normal lung (NL, black; n = 5), FF (blue; n = 8), and MF (red; n = 6) regions from 2 patients, and Gaussian functions were fit to the distributions. (G) E and L values for the number of regions (N) and measurements (n). (H) Dot plots and the mean ± SD of L for the complete data set is shown.

Figure 2. Fibroblast activation is modulated by extracellular matrix type, modulus, and elasticity.

(A) Extracellular matrix (ECM) Young’s modulus (E) and elasticity (L) of type I collagen (Col1) gels, cell-derived matrices (CDMs), or high-density fibrillar collagen (HDFC) matrices, as measured by AFM. Individual data points and mean ± SD are shown. (B) E and L values (mean ± SD) for the number of measurements (n). Approximate values of E and L for FN-gl are listed. (C) Immunofluorescence images of normal or IPF lung fibroblasts cultured on the indicated substrates stained for MRTF-A (gray; red, overlay), vinculin (gray), F-actin (green, overlay), and DAPI (blue, overlay). Nuclear (pink arrows), nuclear and cytoplasmic (yellow arrowheads), and cytoplasmic (green arrowheads) MRTF-A staining is denoted. (D) Box-and-whisker plots (10th–90th percentiles, with outliers) of cell area from 2 independent experiments of normal and IPF lung fibroblasts cultured on Col1 gels (black outline), CDMs (blue outline), HDFC (magenta outline), or FN-gl (red outline) substrates. (E) Fraction of cells with nuclear (Nuc, black fill), nuclear and cytoplasmic (N/C, gray fill), and cytoplasmic (Cyto, white fill) MRTF-A localization (mean ± SEM) in the same conditions as in D. Scale bar: 100 μm.

Figure 3. α_vβ₃ Integrin engagement of soft provisional ECM enhances fibroblast activation.

(A) Immunofluorescence images of normal lung fibroblasts cultured on the indicated substrates stained for α_vβ₃ (green, overlay; gray), β₁ integrin (red, overlay; gray), and DAPI (blue, overlay). Zoomed regions (yellow box) are shown (inverted). FN staining (purple) is shown for CDM and FN-gl. Original magnification, ×60. (B) Integrin engagement was quantified for α_vβ₃ and β₁ integrins within segmented FAs by ratiometric pixel intensity. All identified FAs were averaged for a single cell; mean ± SD is shown for a minimum of n = 15 cells per group from 2 independent experiments. (C) Immunofluorescence images of normal lung fibroblasts on CDMs treated with anti-α_vβ₃ integrin antibody or IgG control stained for MRTF-A (gray; red, overlay), F-actin (green, overlay), and DAPI (blue, overlay). Nuclear (pink arrows), nuclear and cytoplasmic (yellow arrowheads), and cytoplasmic (green arrowheads) MRTF-A staining is denoted. (D) The fraction of cells with nuclear (Nuc, black fill), nuclear and cytoplasmic (N/C, gray fill), and cytoplasmic (Cyto, white fill) MRTF-A localization (mean ± SEM) in conditions the same conditions as in C. One-way ANOVA and Newman-Keuls multiple comparisons post hoc test was used to calculate statistical significance. **P < 0.01; ***P < 0.001 between indicated groups. Scale bar: 100 μm.

Figure 4. Disinhibition of α_vβ₃ integrin enables fibroblasts to strain-stiffen soft, nonlinearly elastic provisional ECMs.

(A) Immunofluorescence images of NLFs transduced cont.shRNA or Thy-1.shRNA NLFs cultured on CDMs and FN-gl; FN (purple, overlay), α_vβ₃ (green, overlay; gray), and β₁ integrin (red, overlay; gray) are shown. (B) Quantification of α_vβ₃ versus β₁ integrin engagement. All identified FAs were averaged for a single cell; data are shown for a minimum of n = 15 cells from 2 independent experiments. (C) Immunofluorescence images of cont.shRNA and Thy-1.shRNA fibroblasts cultured on CDMs and FN-gl stained for MRTF-A (red, overlay; gray, right) and F-actin (green, overlay), representative of 2 independent experiments. Nuclear (pink arrows), nuclear/cytoplasmic (yellow arrowheads), and cytoplasmic (yellow arrowheads) MRTF-A staining is denoted. (D) Fraction of cells with nuclear (Nuc, black fill), nuclear and cytoplasmic (N/C, gray fill), and cytoplasmic (Cyto, white fill) MRTF-A localization (mean ± SEM) in the same conditions as in C. (E) Cont.shRNA or Thy-1.shRNA fibroblasts cultured on CDMs or FN-gl; F-actin (green), vinculin (purple), and nuclei (blue) are overlaid for the entire viewing field; FN (red) is overlaid for the corresponding area (inset, top left); and a magnified view (yellow box) of vinculin is shown (inverted, right). (F) Single-cell stiffness measurements for cont.shRNA and Thy-1.shRNA fibroblasts, labeled as in C. Data shown are pooled from 3 independent experiments. (G) Box-and-whisker plots of FA area for a minimum of n = 10 cells from 2 independent experiments are shown. One-way ANOVA and Newman-Keuls multiple comparisons tests were used to calculate statistical significance. **P < 0.01; ***P < 0.001 between indicated groups. Scale bar: 100 μm. Error bars are SEM.

Figure 5. α_vβ₃ Integrin engagement potentiates fibroblasts contractility and strain stiffening of soft, nonlinearly elastic provisional ECM.

(A) Immunofluorescence images of cont.shRNA or Thy-1.shRNA fibroblasts cultured on CDMs; actin (red), FN matrix (gray), or FN matrix only (gray) are shown. (B) AFM stiffness measurements of the ECM were taken either within 10 μm (proximal, purple outline) or greater than 10 μm (distal, black outline) from the cell body. ECM Young’s modulus (E) and elasticity (L) of distal versus proximal measurements for each shRNA treatment and Thy-1.shRNA fibroblasts treated with anti-_v3 blocking antibody are shown. Dot plots and the mean ± SEM are shown, and statistical significance was calculated using a Kruskal-Wallis nonparametric test with Dunn’s multiple comparison. *P < 0.05; **P < 0.01; ***P < 0.001 between indicated groups. Scale bar: 100 μm.

Figure 6. Thy-1 loss elevates α_vβ₃ integrin activity and causes progressive fibrosis in a model of lung fibrosis.

(A) Immunofluorescence images of active α_vβ₃ (WOW-1 Ab, green), α-SMA (red), and nuclei (blue) in lung tissue sections in WT and Thy-1^–/– mice at 0, 14, and 42 days after intratracheal bleomycin treatment to induce fibrosis. A magnified image (yellow box) of active α_vβ₃ is shown (gray, right). Original magnification, ×20. (B) Quantification of WOW-1 staining intensity in WT (black outline) and Thy-1^–/– (red outline) lungs at 14, 28, 42, and 56 days following bleomycin treatment. (C) H&E and Mason’s trichrome staining of WT and Thy-1^–/– mice 14 and 42 days after bleomycin treatment. Note sustained alveolar destruction and connective tissue deposition in Thy-1^–/– lungs 42 days after bleomycin, while WT lungs are largely normal. Original magnification, ×10. (D) Quantification of pulmonary elastic resistance from whole-lung forced oscillation maneuvers in WT and Thy-1^–/– mice over time after bleomycin treatment. (E) Kaplan-Meyer survival curve for WT and Thy-1^–/– mice after bleomycin treatment. Error bars are SEM.