Crl 4052

EC from human aorta (TeloHAEC, CRL-4052, ATCC, Manassas, VA, USA) and primary human umbilical vein (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were grown as previously reported [50 (link)]. To mimic septic inflammatory condition, EC were treated up to 24 h with increasing concentrations (0.5–10 µg/mL) of lipopolysaccharides (LPS, L5543, Sigma-Aldrich, St. Louis, MO, USA). To selectively inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), EC were incubated with 100 µg/mL of evolocumab (i-PCSK9, Repatha, Amgen Europe B.V) along with LPS 10 µg/mL for 24 h. TeloHAEC were transfected with 2 µg antagomiR hsa-miR-15b-5p (i-miR-15b, customized by Aurogene, Rome, Italy) or with corresponding antagomir Negative Control (NC, Aurogene, Rome, Italy) using the vehicle Lullaby (LL70500, OZ Biosciences, Marseille, France) in medium without serum and antibiotic. After 6 h, fetal bovine serum was added to the culture medium and incubated for further 12 h before treatments with LPS and/or i-PCSK9. The miR-15b-5p inhibition efficiency after transfection was assessed by quantitative real-time PCR (qRT–PCR). Control cells (Ctr) were grown as previously reported [11 (link)].

Human umbilical vein endothelial cells were isolated from human umbilical veins using type I collagenase, and human amniotic epithelial cells (HAECs) were purchased from ATCC (CRL-4052, RRID: CVCL_Z065; Manassas, VA, United States). Mouse brain vascular endothelial cells (BECs) were isolated from mouse brain tissue using type II collagenase. The cells were cultured in M199 (31100035; Gibco, Amarillo, TX, United States) containing 20% fetal bovine serum (FBS) (Hyclone, Logan, UT, United States), recombinant human fibroblast growth factor (Sigma-Aldrich, St. Louis, MO, United States), and heparin (Sigma-Aldrich, St. Louis, MO, United States). Two hundred ninety-three T cells (ATCC, CRL-3216, RRID: CVCL_0063) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS. The cells were passaged by 0.05% trypsin digestion and maintained in a humidified incubator at 37°C with 5% CO₂.

We purchased pooled human umbilical vein endothelial cells (HUVEC; C2519A), human aortic endothelial cells (HAEC; CC-2535), and human coronary artery endothelial cells (HCAEC; CC-2585; Lonza) and cultured them in EBM-2 media supplemented with EGM-2 or EGM2-MV SingleQuot kit. We purchased immortalized HAEC (teloHAEC; CRL-4052; ATCC) and cultured them in vascular cell basal medium supplemented with vascular endothelial cell growth kit and puromycin at 0.3 μg/mL. Human coronary artery smooth muscle cells (HCASMC) and human aortic smooth muscle cells (HASMC) were obtained from Dr. Tardif’s lab and cultured in medium 231 (M-231-500) supplemented with smooth muscle growth supplement (S-007-25; Gibco). Monocytes were obtained from Dr. Rioux’s lab and we cultured them in RPMI with 10% fetal bovine serum. RNA from tissues was extracted either with the Ribopure Kit (Ambion) or using EZ1-XL Advance and Qiagen EZ1 RNA Tissue Mini Kit. hCA were obtained from the “Réseau d’Échanges de Tissus et d’Échantillons Biologiques” (RÉTEB) biorepository at the Montreal Heart Institute and Quebec Heart and Lung Institute. We purchased adult brain total RNA (540005–41, lot#6048990) and adult heart total RNA (540011–41, lot#6056165; Agilent Technologies).