Human mfg e8

Mouse VSMCs were enzymatically isolated as described in the literature [33 (link)]. In brief, the thoracic aortas of the young and aged mice were isolated, and the adventitia and intima were removed from the vessels. The aortae were cut into 1–2-mm pieces and placed into tubes containing 2 mg/mL collagenase II (Worthington Biochemical, Lakewood, NJ, USA) at 37 °C for 3–5 h. The isolated cells were washed with and plated in complete Dulbecco’s Modified Eagle’s Medium (DMEM). To ensure the purity of the isolated cells, > 95% of the cells had to be positive for the two specific smooth muscle cell markers: smooth muscle α-actin and smooth muscle myosin heavy chain for them to be used [34 ]. Early passage VSMCs were treated with Ang II (1 μM, R&D Systems), tumor necrosis factor-α (TNF-α, 20 ng/mL, R&D Systems), or mouse MFG-E8 (250 ng/mL, R&D Systems) for 24 h prior to the subsequent experiments. Human aortic smooth muscle cells (hAoSMCs) were purchased from Lonza (Basel, Switzerland) and cultured in SmGM-2 medium (Lonza) for 24 h in either the presence or absence of human MFG-E8 (250 ng/mL, R&D systems).