Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Abstract

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2^+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2^+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β₁-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2^+/G702D mutation in patient-derived cells.

Keywords: Cell adhesion; Cerebrovascular disease; Collagen IV; Disease mechanism; Extracellular matrix; Protein folding.

Figure 1.. Molecular organisation of FN after adsorption on PEA and PMA polymer substrates.

(A) Structure of PEA and PMA, and amount of adsorbed FN from a solution concentration of 20 μg/mL for 1 hour. (B) Immunostaining of FN. Insert: high magnification images. Scale bars: 50 μm. (C) AFM height images of FN. Scale bar: 250 nm.

Figure 2.. Deposition of Col4A2 (green) and LM (red) by control (A) and mutant fibroblasts (B) on PEA and PMA coated with FN.

Cells were grown on PEA and PMA substrates-coated with FN 20 μg/ml and on glass for 2 h under serum free conditions; then with serum before fixation at different time points (1 and 7 days). Cells were also simultaneously stained with DAPI (Blue). Scale bars: 50 μm (for all micrographs). Quantification of expressed Col4A2 (C) and LM (D) using integrated density per cell. Fractal dimension analysis of secreted Col4A2 (E). Data presented as mean ± SD, N ≥10; and analysed with an ANOVA test; *p < 0.05; ***p<0.001. Important statistical significance differences between the WT and the MT cells are indicated, including between the substrates for the MT. WT, wild type control fibroblast cells; MT, COL4A2^+/G702D mutant fibroblast cells.

Figure 3.. Protein folding capacity of COL4A2^+/G702D fibroblast cells.

Staining of COL4A2 (green) and PDI (red) in WT (top row) and MT fibroblasts (bottom row), also simultaneously stained with DAPI (Blue). Cells were cultured on FN-coated PEA and PMA substrates for 7 days. Scale bars: 50 μm (for all micrographs) (A). Quantification of expressed PDI and Col4A2 integrated density measurements per cell (B). Western blot analysis of ER stress markers Calnexin (90 kDa) and BIP (78 kDa) levels in cell lysates from WT and MT cells cultured for 7 day (C); Densitometry of western blots shown in arbitrary units (AU) (D). Data presented as mean ± SD, N =4; *p<0.05, **p<0.01, ***p<0.001; N-number: 12. WT, wild type; MT, COL4A2^+/G702D fibroblasts.

Figure 4.. Elastic modulus analysis of mutant cells and secreted ECMs.

Young’s modulus of WT and MT cells on the different substrates measured via AFM force mapping; cells were cultured for 7 days and then indented with a cantilever mounted with a 4.83 μm silica bead (A). Young’s modulus of ECMs obtained using the Hertz model on at least 20 measurements (N ≥20) taken from the points indicated by the yellow arrows in the AFM images C and D (B). AFM height images (first column) and 3D reconstruction (second column) of ECMs after decellularization of WT (C) and MT (D) cells; cells were cultured on FN-coated PEA and PMA for 7 days and then decellularized using 20 mM ammonium hydroxide (NH₄OH) solution, leaving the ECMs intact; then, AFM quantitative imaging was carried out in DPBS using a pyramidal tip. The colour scale of the 3D reconstruction represents the local Young’s modulus of the ECMs, calculated using the Hertz model. All data are presented as mean ± SD, and analysed with an ANOVA test; **p<0.01, ***p<0.001, ****p<0.0001. WT, wild type; MT, COL4A2^+/G702D cells.

Figure 5.. Focal adhesion assembly, β₁ integrin expression and cell adhesion strength measurements on FN-coated PEA and PMA substrates.

Immunofluorescence images of focal adhesions and β₁ integrin of WT (A) and MT (B) fibroblast cells on glass, and on PEA and PMA coated with 20 μg/ml of FN. Cells were cultured for 2h in media under serum-free conditions, then fixed and stained for paxillin (red), β₁ integrin (blue) and actin (green). Further images, including cells treated with blebbistatin, are provided in supplementary data (Supplementary Figure 9B). Size of WT and MT cells (C); number of FAs per cell (D); size of FAs (E). Schematic of spinning disk assay (F). Characteristic detachment profile (G) showing fraction of adherent cells (f) as a function of surface shear stress (τ). Adhesion strength measurements for WT and MT cells (H). Quantification of integrated β₁ integrin cluster size (I), area (J) and count (K). Data presented as mean ± SD, N ≥12 for images C-H and N ≥3 for images I-K, and analysed with an ANOVA test; *p<0.05, **p<0.01, ***p<0.001. Important statistical significance differences between the WT and the MT cells are indicated, including between the substrates for the MT. WT, wild type; MT, COL4A2^+/G702D fibroblasts.

Figure 6.. Deposition of COL4A2 by MT fibroblasts on FN nanonetworks is regulated by integrin binding and cell contractility.

COL4A2 staining (green) of cells grown on PEA substrates coated with either FN, FN without the RGD domain (ΔRGD FN), FN with a mutation on the synergy binding site (syn FN), or in the presence of blebbistatin (BB) in the culture medium for 7 days; LM (red) and nuclei (blue) were also stained (A). Quantification of the integrated density of COL4A2 staining after variance filtering (B). Controls are WT fibroblasts and MT fibroblasts cultured on glass. Data presented as mean ± SD, N ≥12, and analysed with an ANOVA test; *p<0.05, **p<0.01, ***p<0.001. WT, wild type; MT, COL4A2^+/G702D fibroblasts.

Figure 7.. Conceptual scheme.

Illustration of the effect of the fibrillar FN nanonetworks on PEA, which induces increased deposition of Col4A2 by COL4A2^+/G702D mutant cells.