Il 1a

Human and rat cardiac fibroblasts were isolated and cultured as described previously^{26 (link),27} . In short, cardiac tissue was washed and digested using collagenase (Worthington type II). Fibroblasts were separated from other cardiac cells by adherence for 1 hour to cell culture flasks. Primary fibroblasts were used up to passage 3. To determine regulation and effects of CILP1, cells were serum-starved for 24 hours before incubation with TGFβ1 (1 ng/ml), IL-1a (1 ng/ml), IGF1 (100 ng/ml) and CILP1 (100 ng/ml) for the indicated time (All obtained from R&D Systems). Adult and neonatal cardiomyocytes were isolated and cultured as described previously^{28 (link),29 (link)}. To inhibit TGFβ type 1 receptor, cells were incubated in serum-containing culture medium for 24 hrs with SB431542 (10 µM, Sigma).

Human umbilical cord endothelial cells (HUVECs) and human microvascular cells (HMEC-1) were purchased from Lonza (Basel, Switzerland) and cultured in their EGM2 and EGM2-MV media respectively. IL8 and IL1a (R&D Systems, Minneapolis, MN, USA) were added to cells at 10 ng/mL. IL1-receptor antagonist (Sigma, St. Louis, MO, USA) was added at 1 μg/mL. The endogenous sequence of ELTD1 is poorly expressed therefore the cDNA was codon optimised using the JCat bioinformatics tool (http://www.jcat.de/) (accessed on 31 November 2017) without altering the amino acid coding sequence. Codon optimised ELTD1 (coELTD1) and HA-tagged coELTD1 were cloned into pLenti6.2V5DEST (ThermoFisher, Waltham, MA, USA) and the vector alone was used as a control. Virus was produced in 293T and concentrated by ultracentrifugation using standard techniques. The viruses were titred using blasticidin resistance and endothelial cells infected at MOI 5 for all experiments.

DPBS-washed astrocytes were seeded into new culture flasks (107 cells/flask) and cultured in a medium containing FBS without exosomes (SBI System Biosciences, Palo Alto, CA, USA). Some 12 h later, the cells were stimulated with a cocktail of proinflammatory cytokines: IL-1a (10 ng/mL, R&D Systems, Ixonia, WI, USA), TNF-α (30 ng/mL, R&D Systems), C1q (400 ng/mL, R&D Systems). Alternative phenotypes of astrocytes were induced by IL-10 (10 ng/mL) or TGF-β (10 ng/mL) stimulation (both cytokines from R&D Systems). Astrocytes cultured without cytokines were also included. In our previous work, we characterized the secretory activity of astrocytes in response to these stimuli [81 (link)]. After a six-day culture, the media were collected and centrifuged (300× g, 10 min, 20 °C) and the supernatants were centrifuged again (16,000× g, 30 min, 20 °C). Next, the exosomes were precipitated (12 h, 4 °C) with exosome precipitation reagent (Exo-spin Buffer, CellGS, Cambridge, UK) and centrifuged (16,000× g, 1.5 h, 20 °C). The supernatants were removed and the pellets were resuspended in PBS and purified on exosome size-exclusion columns (Exo-spin, Cell Guidence Systems, Cambridge, UK) according to a protocol provided by the assay manufacturer. After purification, the exosomes were aliquoted and frozen for further procedures.