Hmvec d

Primary dermal human microvascular endothelial cells (HMVEC-D) were purchased from Lonza at passage 0 (p0), grown in EGM2-MV (Lonza) and experiments were performed from p4-p7 in EBM2 supplemented with 0.1% BSA.

Primary cultures of human endothelial cells derived from multiple vascular beds were used in this study. Human cerebral microvascular endothelial cells (HCMEC/D3) were purchased from Sigma-Aldrich (Sigma-Aldrich, Millipore, St. Louis, MO, USA), and cells were maintained following the manufacturer’s instructions. The cells were grown in cultured media of EndoGRO™-MV Complete Media Kit (Cat. No. SCME004, Sigma-Aldrich) supplemented with 1 ng/mL FGF-2 (Cat. No. GF003, Sigma-Aldrich). Human coronary artery endothelial cells (HCAEC), human iliac artery endothelial cells (HIAEC, Lonza, Walkersville, MD, USA), human dermal microvascular endothelial cells (hMVEC-d), human cardiac microvascular endothelial cells (HMVEC-C), and human lung microvascular endothelial cells (HMVEC-L) were purchased from Lonza (Walkersville, MD, USA), and cells were maintained following the manufacturer’s instructions. For cell culture, the procedures consisted in use of endothelial growth medium (EGM™-2MV BulletKit™; Clonetics) supplemented with 5% fetal bovine serum (FBS; Clonetics) and cells were incubated at 37 °C and 5% CO₂. For serial passaging (always before passage 4), the cells were dissociated by trypsinization, centrifuged at 220× g for 7 min, and replated at the correct density.

Human dermal microvascular endothelial cells (CC-2543), HMVEC-D (purchased from Lonza Walkersville, Inc.; Allendale, NJ, USA), were maintained, according to the manufacturer’s instructions in endothelial growth medium (EGM-2MV Bullet Kit; Lonza) and incubated at 37 °C, 5% CO₂ in a cell culture incubator. All experiments were performed at passage 10.

Human dermal microvascular ECs from neonatal foreskin (HMVEC-D; Lonza, Walkersville Inc., Walkersville, MD, USA) were cultured according to the manufacturer’s instructions in basic EC growth medium 2 (EGM-2-MV, Lonza, Switzerland) supplemented with fetal bovine serum (FBS), fibroblast growth factor (rhFGF), vascular endothelial growth factor (VEGF), vitamin C, GA-1000 (gentamicin/amphotericin B), and hydrocortisone. Human juvenile foreskin fibroblasts (FB) were isolated from residual tissue of circumcision surgery (with patient’s ethical consent and permission from Charité, Berlin, Germany EA1/081/13) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 1% l-glutamin, and 1% penicillin/streptomycin (10,000 U/mL; all from Sigma-Aldrich, Taufkirchen, Germany). All cells were maintained at 37 °C in a humidified atmosphere (37 °C, 5% CO₂). The cell culture medium was changed every 2–3 days. For each experiment, HMVEC-D were used at passage 5, and FBs were used at passages 9–10. The bottom of the culture wells was not coated with gelatin or other substances.