Atorvastatin Modulates HMGB1 and IL-8 in LPS-Treated HUVECs

HUVEC (Lonza, Breda, The Netherlands) were cultured in EBM-2 medium supplemented with EGM-2 MV Single Quot Kit Supplements & Growth Factors (cat No. CC-3202, Lonza) and used when confluent. Three groups were evaluated: (1) HUVEC pre-incubated for 2 hours with 5 µM atorvastatin (Sigma Aldrich, Saint Louis, USA) and treated with LPS (100 ng/ml) (Sigma Aldrich, Saint Louis, USA), (2) HUVEC treated only with LPS, and (3) unstimulated HUVEC. Supernatants were collected for measuring HMGB1 and interleukin (IL)-8 at baseline, 4 hours and 24 hours. All in vitro experiments were performed twice and in duplicate. Cell viability of HUVEC was checked with 0.2% trypan blue dye (Invitrogen, Carlsbad, USA) on HUVEC treated with 0 µM, 0.1 µM, 1.0 µM, 5.0 µM and 10.0 µM atorvastatin; percentages of living cells were 94%, 95%, 95%, 92% and 91% at 36 hours, respectively. Western blot was used to measure HMGB1 in supernatants as previously described [13] (link) while IL-8 levels were measured by ELISA (R&D Systems, Minneapolis, USA).