Lumen-occluding FVIII-r.ag.-positive cells were used to identify plexiform lesions. Some of the cells in the plexiform lesion also stained positive for a-SMA. Twenty-eight plexiform lesions were examined for caveolin expression. All FVIII-r.ag and a-SMA-positive cells in the plexiform lesions were counted (total of 5,479 cells) and graded as either positive or negative for caveolin 1 expression. Images were obtained with a digital camera (Axiocam-HRC; Zeiss; Thornwood, NY) connected to a personal computer (TEC; Knoxville, TN). Image analysis was performed using software (KS 300 3.0 Image; Zeiss). Plexiform lesions in lung tissue sections from the same 14 patients with severe PH were also examined for the expression of HO-1. canadian neighbor pharmacy
Frozen human lung tissue was homogenized in homogenization buffer containing 50 mM hydroxyethyl piperazine-ethanesulfonic acid, pH 7.55, 1 mM dithiothreitol, 10% glycerol, and 0.1% Triton X-100 using homogenizer (model PT10/35; Polytron; Westbury, NY). The lysates were centrifuged twice in a centrifuge (Eppendorf Hamburg, Germany) at 14,000 revolutions per minute for 10 min. The protein concentration in the supernatant was measured using the Bradford assay. Equal amount of proteins were loaded on NuPAGE 4 to 12% Bis-Tris gels (Novex; Invitrogen Corporation; Carlsbad, CA) and transferred to PolyScreen PVDF transfer membrane (NEN Life Science Products; Foster City, CA) in NuPAGE transfer buffer containing 10% methanol.
Prestained molecular mass marker proteins (Bio-Rad Laboratories; Hercules, CA) were used as standards. Rabbit polyclonal anti-caveolin antibodies (BD Transduction Laboratories, San Diego, CA) were diluted 1:1000. Western blots were visualized using a reagent (Renaissance Western Blot Chemiluminescence Reagent; NEN Life Science Product, Piscataway, NJ). Intensity of experimental bands was measured by the ImageQuant program (Molecular Dynamics). Results are expressed as mean ± SEM. Statistical analysis was performed using Student t test to determine the significance of change in the densitometric measurements. A significant difference was considered at p < 0.05.