Ephb4

As previously reported, tracheal segments devoid of large veins from C57BL/6 mice were dissected, minced and digested [9] (link). The cellular digest was filtered and centrifuged, the cell pellet was resuspended and placed on a gelatin-coated 35 mm dish. After 5–7 days, cells positively labeled with fluorescent Dil-ac-LDL (Molecular Probes, Eugene, OR) as an endothelial cell marker and labeled EphB4 (Santa-Cruz, Santa Cruz, CA) as a marker of venous endothelial cells were selected using the FACSAria cell-sorting system (BD Biosciences, San Jose, CA) and replated (0.2% gelatin-coated T-25 flask). Depending on outcome variable and prior to challenge, TvEC were incubated (2% FBS DMEM; 2 h) and treated with vehicle, N^G-nitro-L-arginine methyl ester (L-NAME; 10⁻⁵ M), N-acetylcysteine amide (NACA; 5×10⁻³ M), superoxide dismutase (SOD; 10⁻⁵ M) or excess BH₄ (10⁻⁴ M [17] for 30 min prior to challenge, TvEC were distended with the Flexercell tension Plus System (FX-4000T; Flexcell Int, Hillsborough, NC) using 5% elongation representing physiological levels of ventilatory stretch, 18% elongation representing pathophysiological overdistension [9] (link), [18] or treated with thrombin (5 Units/ml) all for 5 min exposures.

Andrographolide (purity > 98%) was purchased from the National Institutes for Food and Drug Control, China. Arsenic trioxide (As₂O₃), trypsin, phosphate buffer saline (PBS), ethylene diamine tetraacetic acid (EDTA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), monodansylcadaverine (MDC), propidium iodide (PI), Annexin V, and rapamycin were purchased from Sigma Chemical Corp. (St. Louis, MO, USA). Primary antibodies against caspase-3, LC3, Atg-5, EphB4 and VEGFR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the other chemical reagents in the study were of analytical reagent grade.