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Young and aging pAVECs were grown to confluency on plastic 24 well plates (for RNA extraction) or on Matrigen Softwell Custom collagen I coated, 8Kpa, hydrogel plates (for immunocytochemistry). Cells were treated with fresh medium, supplemented with TGF-β1 (2ng/ml) or with equal volumes of 0.1% BSA every 2–3 days for 7 days. Following culture, cells were fixed in 4% PFA for 30 minutes at room temperature and stored in PBS until immunocytochemistry or lysed in Trizol (Invitrogen) RNA extraction respectively (n=8). For immunostaining, cells were 4% PFA- (SMA) or methanol-fixed (VE-cadherin) for 20 minutes, blocked and incubated with primary antibodies against VE-cadherin (Santa Cruz SC-6458, 1:100) or SMA (Sigma A2546, 1:100) for two hours at room temperature. Secondary antibodies were applied as described above (2.1.1).

Eight hundred thousand B2m^−/−Ciita^−/− Cd47 tg miECs, miSMCs or miCMs in 1:1 diluted Matrigel (Corning) were injected into allogeneic BALB/c mice. Matrigel plugs were recovered after 1, 2, 3, 4, 5, 6 and 8 weeks and fixed in 4% paraformaldehyde in PBS with 1% glutaraldehyde for 24 h. Samples were dehydrated, embedded in paraffin and cut into sections of 5 µm thickness. For histopathology, sections were stained with hematoxylin and eosin (Carl Roth) and images taken with an inverted light microscope. Origin of cells was demonstrated with immunofluorescence staining. Sections were rehydrated, and underwent antigen retrieval and blocking. Samples were incubated with antibodies against luciferase (ab21176), SMA (ab21027, Abcam), VE-Cadherin (SC-6458) or α-sarcomeric actinin (EA-53, Abcam) and a corresponding secondary antibody conjugated with AF488 or AF555 (Invitrogen). Cell nuclei were counterstained with DAPI and images taken with a Leica SP5 laser confocal microscope (Leica).
For co-staining experiments of miECs and immune cells, primary antibodies were used against VE-Cadherin (SC-6458, Sigma) and CD3 (ab16669, Abcam), followed by the corresponding secondary antibody conjugated with AF488 or AF555 (Invitrogen).