Atorvastatin

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).

Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood samples by standard gradient centrifugation over Ficoll-Hypaque (GE Healthcare Bio-Sciences, Piscataway, NJ). CD4⁺T-cells were isolated by magnetic micro-beads (CD4+T-cell isolation kit MACS, Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Pure atorvastatin, kindly provided by Pfizer (New York, NY), was dissolved in 2% dimethilsulfoxide (DMSO) solution at final concentration of 50mM. CD4⁺T-cells were incubated for 24 hours, under sterile conditions at 37°C in an atmosphere containing 5% CO2, at a density of 1 × 10⁶/ml in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovin serum (Invitrogen, Carlsbad, CA) with and without increasing doses of atorvastatin: 3-10-26 μg/ml. These doses were chosen on the basis of the observation that comparable plasmatic concentrations correspond to in vivo doses of 10-40-80 mg of atorvastatin [61 (link)].

Antibodies Eculizumab (HCA312), Bevacizumab (HCA182) and CD126/IL-6R (AHP2449) were obtained from Biorad.
Dipyridamole (06–915–00), Ciclesonide (63–575), FTY720 (61–761–0), Pirfenidone (P1871100MG), Atorvastatin (A24761G), Indomethacin (1065525G), Bromhexine HCl (B405425G), Maraviroc (37–561–0), Darunavir (67–101–0), Danoprevir (50–190–4862), Azithromycin (37–715–0), Nitazoxanide (N1031200MG) and Tiecoplanin (AC455400010) were obtained from ThermoFisher Scientific.
Valganciclovir (S4050), Rimantadine (S5484), Letermovir (S8873), and Baloxavir (S5952) were obtained from Selleckchem.