Endothelial growth supplements

Primary human umbilical vein endothelial cells (HUVECs) were purchased and maintained with EGM-2 medium that contained endothelial growth supplements (Lonza, Seoul, Korea) with 2% fetal bovine serum (FBS) at 37 °C in a retained 5% CO₂ incubator. The human leukemia THP-1 cells were cultured in an RPMI 1640 medium with 0.05 mM 2-mercaptoethanol with 10% inactivated FBS. To treat with the glycer-AGEs, the HUVECs were co-treated with different concentrations of PM and 100 μg/mL of AGEs using FBS-free media. In specific inhibitor-treated experiments, the inhibitors were pre-treated for 1 h and removed before the sample treatments.

For our cell experiments, we used human umbilical vein endothelial cells (HUV-EC-C [HUVEC], ATCC CRL-1730^™, Manassas, VA) that were cultured in endothelial basal media with endothelial growth supplements (Lonza, Hopkinton, MA). This particular HUVEC line is cultured minimally commercially to maintain consistency; these HUVECs are a non-continuous line being pooled from many individuals but are not immortalized and only survive for 10 passages in culture. Cells were used between passage 2–8. Cells were passaged onto glass slides (VWR International, Radnor, PA) in 35 mm tissue culture dishes (Corning Inc., Corning, NY). For transfection experiments, cells were transfected using the endothelial cell-specific Lipofectin transfection reagent (Life Technologies, Grand Island, NY) in Opti-MEM I Reduced Serum Medium (Life Technologies, Grand Island, NY). For these experiments we used rDNA of GFP-Fibrillarin (kind gift from D. Discher, University of Pennsylvania). Cells were incubated for transfection for five hours, at which time we changed to normal growth media and incubated the cells for an additional 24–72 hours prior to experiments to allow for adequate expression and proliferation to confluency.