Immunohistochemical (IHC) and immunofluorecence (IF) staining
Primary antibodies that were used for IHC and IF are listed in Table S2. Heat- induced antigen retrieval was performed with Antigen Unmasking Solution, Tris-Based (Vector, H-3301) for all antibodies. For IHC staining, endogenous peroxidase activity was blocked in 0.3% H2O2 in deionised water. Nonspecific binding was reduced by incubation in 2.5% goat serum. Sections were treated with mouse Ig blocking reagent (Vector, MKB-2213-1) before application of the primary mouse antibody. Rabbit primary antibody binding was detected with goat anti-rabbit ImmPRESS HRP goat anti-rabbit IgG (Vector, MP-741) and mouse primary antibody binding was detected with Mouse-on-Mouse ImmPRESS anti-mouse Ig reagent (Vector, MP-2400). Colour was developed with DAB-substrate chromogen system (Vector, SK-4100). Images were acquired with a DM2500 Leica microscope (Leica Microsystems).
For IF staining of aortic sections, rabbit primary antibody binding was detected with goat anti-rabbit IgG (Alexa Fluor-488), mouse primary antibody binding was detected with goat anti-mouse IgG (Alexa Fluor-647), and goat primary antibody was detected with donkey anti-goat IgG (Alexa Fluor-594). DAPI was used for nuclei visualization.  Images were acquired using an Olympus FV1000 confocal laser scanning microscope with images analysed using Fiji54.
iVSMCs or HUVECs were grown on µ-Slide 8 well chamber slides (Thistle Scientific) and fixed in 4% PFA. SVEP1, Integrin α4 and α9 staining was performed on non-permeabilised cells. For all other staining, cells were permeabilised in 0.5% Triton-X. Non-specific binding was reduced by incubation in 1% bovine serum albumin (BSA), 22.5 mg ml-1 glycine, 0.1% tween-20 PBS solution, with additional blocking in a 10% goat serum PBS solution. Cells were incubated overnight at 4°C in primary antibody (listed in Table S2) diluted in 10% goat serum. After washing, cells were incubated in 10% goat serum containing complementary secondary antibodies. Nuclei were visualised by DAPI counterstaining. Images were acquired using an Olympus FV1000 confocal laser scanning microscope with images analysed using Fiji.