Hpaec

Human pulmonary artery endothelial cells (HPAEC) and human monocyte cell line (THP1) were obtained from American Type Culture Collection (ATCC). 2 × 10⁵ cells were seeded in 24-well plates and cultured in RPMI-1640 (10% fetal bovine serum, 100 U ml⁻¹ penicillin/streptomycin, 2 mM glutamine) in a 5% CO₂ humidified atmosphere at 37 °C. After the cells reached 80–90% confluence, they were incubated with 50% of the sera samples (approximately 400 μl) for 24 h, which were pooled from the patients before or within 24 h after transplantation, respectively. To verify the impact of extracellular histones in the sera, 20 μg ml⁻¹ of anti-histone H4 antibody or 200 U ml⁻¹ of heparin (Sigma-Aldrich, USA) was added to the cultured cells, respectively.
For the cytotoxicity assay, the cultured HPAEC cells were detached and washed with PBS and incubated with PI dye solution (10 μg ml⁻¹, Sigma-Aldrich, USA) in the dark for 10 min at room temperature. The cells were then subjected to flow cytometry analysis. In addition, the supernatants were collected and analyzed for LDH levels. To test the influence of extracellular histones in sera samples on THP1 cells, the supernatants (approximately 100 μl) were collected and analyzed for the production of multiple cytokines (IL-1β, IL-6, IL-10, IL-17A, IL-18, TNF-α) using a Multiplex Immunoassay kit from Affymetrix eBioscience.

All cell lines including human pulmonary artery endothelial cells (HPAECs), human lung fibroblasts (HLFs), and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC), 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), cholesterol, 25-hydroxycholesterol (25HC), and soy L-α-phosphatidylinositol (PI) were purchased from Avanti Polar Lipids.1,2-Dipalmitoyl derivatives of PIP₂ and PI(4)P were from Cayman Chemical Co. (Cat no. 10008115). Fibronectin for the cell culture plate coating was purchased from Millipore Sigma (Cat no. 341631, lot no. 3273650). Fibroblast basal medium and fibroblast growth kit-low serum for HLF cells were purchased from ATCC. A transfection reagent JetPRIME was from Polyplus transfection. Mitochondria marker MitoTracker™ Deep Red FM was purchased from Invitrogen. Human and mouse Cav1 siRNAs were purchased from Qiagen and Integrated DNA Technologies, respectively.

HPAECs (ATCC American Type Culture Collection) in the growth phase were divided into four groups: a blank normoxia control, a hypoxia, a normoxia+HMGB1, and a hypoxia+anti-HMGB1 group. After the cells were passaged and adhered to the plate for 6 h, they were incubated under normoxic or hypoxic conditions and subjected to corresponding treatments (1 μg/mL HMGB1 or 5 μg/mL anti-HMGB1) depending on the group for 24 h. After treatment, the cell supernatant and cellular proteins were collected and used to detect the expression of IL-1β and IL-6 and of HMGB1 and RAGE, respectively.

The formation of cord-like structures by human pulmonary artery endothelial cells (HPAECs [ATCC, Manassas, VA]) was assessed in Matrigel-coated wells [8] (link). HPAECs (40,000 cells/well) were seeded into 48-well plates coated with Matrigel (BD Biosciences, Mississauga, ON) into groups of triplicates: (1) room air, (2) room air+GYY4137 (100 microM) (3) hyperoxia (95% O₂), (4) hyperoxia+GYY4137 (100 microM) and incubated at 37°C for 8 h. GYY4137 (morpholin-4-ium-4-methoxyphenyl (morpholino) phosphinodithioate) is a recently described slow-releasing H₂S donor (Cayman chemical, Ann Arbor, Michigan). Cord-like structures were observed using an inverted phase contrast microscope (Leica, Richmond Hill, ON, Canada) and quantified by measuring the number of intersections and the length of structures in random fields from each well using OpenLab (Quorum Technologies Inc, ON, Canada).