Hpaec

Human pulmonary artery endothelial cells (hPAECs) were purchased from Lonza, Inc. (Allendale, NJ, USA). hPAECs were cultured in EBM-2 medium supplemented with 5% fetal bovine serum (FBS) supplemented with EGM-2 SingleQuots (Lonza). hPAECs were characterized by immunofluorescence with antibodies specific to vWF/Factor VIII and CD31 (PECAM). Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and were cultured in SmBM medium supplemented with 5% FBS and SmGM-2 SingleQuots (Lonza). hPASMCs were characterized by immunofluorescence with antibodies specific to α-smooth muscle actin. Additionally, hPAECs and hPASMCs were further characterized by morphological observation throughout the serial passages and were only used between passages 2 through 7 for this study. Cells were grown in 5% CO₂ at 37 °C and were passaged at the confluence. All of the cells tested negative for mycoplasma, bacteria, yeast, fungi, HIV-1, hepatitis B, and hepatitis C before use.

Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza Clonetics (Walkersville, MD). HPAEC were grown in EGM-2 medium (Lonza), which contains basic growth medium (EBM-2), fetal bovine serum (FBS), and antibiotics, ascorbic acid, vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF-B), hydrocortisone, human epidermal growth factor (hEGF), R³-IGF-1 (insulin-like growth factor), GA-1000 (gentamicin, amphotericin B), and heparin. Unless otherwise stated, cells were maintained in a 37°C incubator at 5% CO₂.

An alveolar bilayer model was constructed as previously described (Hope et al., 2007 (link); Hope, 2009 (link)). Briefly, human pulmonary artery endothelial cells (HPAEC, Lonza) and adenocarcinoma human alveolar basal epithelial cells (A549, Lonza) were cultured in 75 cm² flasks in HPAEC medium + 0.1% GA-1000 at 37°C, 5% CO₂. 1 × 10⁵ HPAECs were seeded on the lower side of a 6.5 mm diameter 3 μm pore Transwell^® membrane (Corning). After 2 h incubation, inserts were placed in HPAEC medium in a 24 well plate overnight. Subsequently, 5 × 10⁴ A549 cells suspended in 100 μl HPAEC medium were added to the upper compartment. Incubation of the assembly was performed in 24 well plates containing 600 μl HPAEC medium per well at 37°C, 5% CO₂. Media changes were performed every other day. 50 μl moDC solution (2.5 × 10⁵ cells in HPAEC medium) or plain medium were added on day 5. Afterwards, inserts were infected by adding 50 μl fungal solution (2.5 × 10⁵ spores in HPAEC medium), followed by another 30 h incubation period at 37°C, 5% CO₂. For selected experiments, 50 μg/ml gefitinib (Santa Cruz Biotechnology), 8 mg/ml D-(+)-glucose (Sigma-Aldrich), or 1 mg/ml beta-hydroxybutyrate (Sigma-Aldrich) were added to the endothelial compartment.

A humidified tissue culture incubator was filled with 5% CO₂ and 95% air and set at 37 °C for cell culture. Primary human umbilical vein endothelial cells (HUVEC), human pulmonary artery endothelial cells (HPAEC), and human lung microvascular endothelial cells (HMVEC-L) were purchased from Lonza (Walkersville, MD, USA). HUVEC and HPAEC were cultured in endothelial cell growth medium 2 (EGM-2), while HMVEC-L was grown in EGM-2-MV medium (Lonza, Walkersville, MD, USA). HUVECs stably expressing the MSCV-IRES-GFP (HUVEC/Vector), MSCV-huGPR4-IRES-GFP (HUVEC/GPR4), MSCV-huGPR4 R115A-IRES-GFP (HUVEC/GPR4-R115A), Flink-control shRNA (HUVEC/Control-shRNA), or Flink-huGPR4 shRNA (HUVEC/GPR4-shRNA) were obtained by cell transduction with construct-containing retroviral/lentiviral particles and then by fluorescence-activated cell sorting (FACS), based on green fluorescence signals as previously described [1 (link)].

Primary HMVEC-L and HPAEC (fourth passage) were obtained from Lonza (Cat No. CC-2527 and CC-2530, respectively) and maintained in endothelial basal medium-2 (EBM-2) supplemented with endothelial growth medium (EGM-2) SingleQuots™ Supplements (CC-4176; HPAEC) or MV SingleQuots (CC-4147; HMVEC-L) plus heat-inactivated fetal bovine serum up to 10% at 37°C and 5% CO₂ until they reached 90%–100% confluence. Confluent cells were treated with S. mansoni egg antigen p40 (Sm-p40; Biomatik; Cat #RPC20108) for short-term signaling pathway analysis (0 min, 15 min, 30 min, 60 min, 120 min, and 240 min) or long-term analysis (24 h and 48 h). Cell morphology was assessed daily using brightfield contrast microscopy, and cell lysates were used for protein quantification. Cells were used up to the maximum level in the seventh passage.

HPAECs (Lonza, Wokingham, UK) were maintained in endothelial growth medium (EGM-2; Lonza) with 2% FBS according to the instructions supplied. Human aortic endothelial cells (HAECs) were purchased from PromoCell (Heidelberg, Germany) and maintained in EGM2-mv (Lonza) with 5% FBS (Invitrogen, Carlsbad, CA). Endothelial cells were cultured at 37°C in a 5% CO₂ humidified atmosphere and used in experiments at passages 4-6. Cells were confirmed negative for mycoplasma. For studies, endothelial cells were seeded in 24-well or 6-well plates and grown to confluence. Cells were serum-restricted by washing once with 0.1% FBS [EBM2 (Lonza) containing 0.1% FBS and antibiotic/antimycotic (Invitrogen)], followed by incubation in 0.1% FBS. Cells were then treated as described. Cells were regularly tested and confirmed to be mycoplasma-free.