Fibrillar Collagen

Bovine dermal collagen was obtained as acidified, pepsinized solution (stock concentration 3.1 mg/ml; PureCol, Advanced BioMatrix); non-pepsinized (telopeptide intact) rat tail collagen was purified by acid extraction, stored in acidified lyophilized form (Hotary et al., 2000 (link)), and re-solubilized before use in 0.2% acetic acid. Fibrillar collagen matrices were reconstituted in vitro by raising the pH to 7.4 using either final concentrations of 0.345 N NaOH and buffering by 25 µM Hepes (Sigma-Aldrich) for rat tail matrices, or 0.75% Na-bicarbonate solution (Gibco) for bovine dermal lattices, together with mimimum essential Eagle’s medium (Sigma-Aldrich), and polymerized at 37°C, unless indicated otherwise (Hotary et al., 2000 (link); Wolf et al., 2007 (link)). High-density matrices of bovine dermal collagen were reconstituted after concentration of the collagen solution (20 mg/ml; speed vac concentrator; Savant). Delayed polymerization speed of rat tail collagen was achieved by lowering the temperature during polymerization to 20, 14, or 9°C. Cell-free collagen lattices were used for bright-field microscopy (vertical invasion studies; Fig. S1 A), 3D confocal and electron microscopy, and atomic force microscopy. For cell migration assays, cells (20,000/100 µl) after detachment with EDTA (2 mM) or multicellular spheroids (2–7 spheroids/100 µl) were suspended in neutralized collagen solution before polymerization and incorporated into a self-constructed chamber (see Fig. S1 A). After addition of medium (containing 10% FCS) spontaneous migration was monitored, except for PMNs which were stimulated by 100 ng/ml IL-8. Inhibition experiments were performed using 20 µM GM6001, a five-component protease inhibitor cocktail, function-perturbing anti-β1 mAb 4B4, or Y-27632. Inhibitors were added to both the cell–collagen mixture and supernatant. For collagen contraction assays, cells (8 or 16 × 10⁴, for incubation with Y-27632 or 4B4, respectively) were incorporated into non-anchored bovine collagen lattices (300 µl) in 48-well plates.

Histological studies were performed on carotid arteries fixed with 10% buffered formalin under pressure (100 mmHg) in vivo for 2 h. Fixed specimens were embedded in paraffin following regular procedures. Five micrometre sections were stained with Sirius Red and Picrosirius Red (PSR) for collagen. PSR sections were imaged under cross-polarized light (darkfield) to observe collagen birefringence. Images were acquired on an Olympus BX/51 microscope using an Olympus DP70 digital camera (cellSens Dimension) under a × 40 magnification objective. The area fraction of collagen was based on total fibrillar collagen. A custom MATLAB script was used to extract layer-specific (media or adventitia) wall percentages as well as the proportions of thick (red) and thin (orange–yellow–green) birefringent collagen fibres.^{16 (link),17 (link)}For integrin α_v subunit immunostaining, rabbit polyclonal antibodies against integrin α_v subunit, biotinylated goat anti-rabbit immunoglobulins, HRP-conjugated streptavidin, and 3,3′-diaminobenzidine (DAB) substrate (see Supplementary material online, Tables S7 and S8) were used. Composition of the arterial wall and the media cross-sectional area (MCSA) were determined using a Nikon NIS-Elements Basic Research microscope imaging software as described previously.^{18 (link)}Immunofluorescence staining on 8 µm cryo-sections fixed with 4% paraformaldehyde or cooled acetone for 5 min was performed with specific antibodies as described previously.^{14 (link)} Briefly, sections were incubated with primary antibodies at 4°C overnight after permeabilization with 0.2% Triton X100 for 10 min and blocking with 5% bovine serum albumin (BSA) for 1 h. After washing in PBS-Tween 20, sections were incubated with fluorescent-conjugated secondary antibodies. A complete list of antibodies is provided in Supplementary material online, Table S8. Image acquisition was on a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany) with the same depth of field and with identical settings for laser, gain, and offset intensity.

BP strips with dimensions of 20 mm by 4 mm by 0.3 mm were cut from the membrane using a laser cutter. The strips were tested using a SAXS machine (SAXSpoint 2.0). The obtained diffraction patterns were analyzed by the software Fit2D. The diffraction maxima occurring at angles 2θ to the x-ray beam (Bragg reflections) are given by the equation: 2sin(θ) = Λn/d, where n is the order of the reflection, Λ is the wavelength of x-ray, and d is the spacing between the d-bands on collagen fibrils.