Human pulmonary artery endothelial cells

Human pulmonary artery endothelial cells were obtained from Lonza (Allendale, NJ). Cells were maintained according to the manufacturer’s recommendations and used for experiments at passages 5–7. Bacterial lipopolysaccharide (LPS) (Escherichia coli O127:B8) was obtained from Sigma-Aldrich (St. Louis, MO). Antibodies to ICAM1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to GEF-H1 were from Cell Signaling (Beverly, MA). All reagents for immunofluorescence were purchased from Molecular Probes (Eugene, OR). Unless specified, reagents were obtained from Sigma (St. Louis, MO).

All procedures were approved by the Institutional Animal Care and Use Committee of Boston University (Protocol # 12–031) and the experiments were performed in accordance with relevant guidelines and regulations. One male Wistar-Kyoto rat (age: 10 weeks) was anesthetized with a mixture solution of Xylazin^® (10 mg/kg) and Ketamin^® (90 mg/kg) and the thoracic aorta was harvested. An abdominal aortomy was performed to excise aorta. The aorta was then washed with PBS, cleaned from residual tissue on the outside, cut open and sectioned into pieces of about 2 × 5 mm. The edges of the sample were glued to a cover slip and labeled with beads for flow experiments.
Human pulmonary artery endothelial cells were purchased from Lonza Biologics Inc (Hopkinton, MA). Vascular smooth muscle cells were isolated from bovine aortae using the explant method as previously described^{36 (link)} and experiments were performed at the first passage. Cells were seeded in the middle section of collagen type I coated cover slips immersed in cell culture media and were allowed to attach for 24 h and subsequently labeled with beads described below.