Figure 4. Bcr2/2 and abr2/2 PASMC show increased prolifer- ation when exposed to hypoxia in vitro. A, Western blot analysis on PASMC lysates from wt, bcr2/2 or abr2/2 mice with anti-Bcr N20 antibodies or Abr antiserum. GAPDH, loading control. B, Third passage primary PASMC isolated from the intrapulmonary arteries of 5 different mice per genotype (16104 cells/well) were synchronized by serum free medium for 24 hrs, then cultured in medium with 10% FBS for 5 days, after which cells were counted. The Rac inhibitor Z62954982 was added to the indicated samples. * p,0.05 compared with the outcomes from the same genotype PASMCs in normoxia. # p,0.05 compared with wt PASMCs exposed to the same condition. Bars, mean 6SD of triplicate wells. C, Proliferation index of wt, bcr2/2 and abr2/2 PASMCs was calculated as described in Methods. Flow cytometry analysis was done Bcr and Abr Regulate PH based on 10,000 PASMCs per sample, 3 samples per genotype per condition. * p,0.05 compared with the outcomes from the same genotype PASMCs in normoxia. # p,0.05 compared with wt PASMCs exposed to the same condition. Bars are shown as mean 6SD of triplicate wells. doi:10.1371/journal.pone.0049756.g004 tension, in particular if a combination of GEF inhibitors, or a single Rac inhibitor can be identified that would target Rac activation in PASMC as well as in innate immune cells. Materials and Methods Ethics Statement Animal experiments were approved by the Children’s Hospital Los Angeles Institutional Animal Care and Use Committee, and were conducted in compliance with the NIH guide for the Care and Use of Laboratory Animals. Animals and Chronic Hypoxia Model Bcr2/2 and abr2/2 mice were generated as previously reported [9,11] and were on an f6 FVB/J inbred genetic background. Male bcr2/2, abr2/2 and wt mice (8 to 10-week-old littermates) were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. For in vivo Rac1 inhibitor treatment experiments, Z62954982 was administered intraperitoneally (i.p.) at 10 mg/kg every other day or 20 mg/kg daily. Control mice were injected with the same volume of vehicle (DMSO : corn oil = 1: 9). During and at the end of the treatment, we monitored normoxia-exposed vehicle and Z62954982-treated wild type mice for possible obvious signs of toxicity of the drug. However there was no evidence for toxicity. Numbers of myeloid cells in the bone marrows were comparable and kidney (not shown) and liver appeared normal in both groups (Figure S3). Mice were anesthetized with intraperitoneal ketamine hydro- chloride (60 mg/kg) and xylazine (8 mg/kg). A catheter connected to a pressure transducer was inserted into the right ventricle (RV) through the right jugular vein. Right ventricular systolic pressure (RVSP) was measured by BP-1 (World Precision Instruments, FL, USA). The mice were then sacrificed with CO2. The serum, hearts and lungs were immediately collected. Pathology and Image Analysis Percent wall thickness was calculated based on the analysis of the arterial area of pulmonary terminal arterioles, according to the following formula: wall thickness (%) = (areaext–areaint)/area- extx100 (areaext is the area bounded by external wall of the vessel, and areaint is the area bounded by internal wall of the vessel). Nine vessels of comparable size (25–100 mm) per mouse were measured (6 mice per group). In total, 324 vessels were included. PASMCs were detected by immunohistochemistry with a mouse monoclonal antibody to a-smooth muscle actin (a-SMA; 1:1000, Sigma). For assessment of right ventricle hypertrophy, right ventricles (RV), left ventricles and septae (LV+S) were isolated and weighed. The ratio of the weight of RV to LV+S (RV/LV+S) was analyzed as an index of right ventricle hypertrophy (RVH). For examination of the lungs, the right primary bronchi of the lungs were ligated. The left lobes were inflated through the trachea with 10% formalin at a perfusion pressure of 20 cm H2O. Excised inflated lung tissue was fixed in 10% formalin, processed and embedded with paraffin. Hematoxylin and eosin staining was performed on 5 mm sections. The images of the pulmonary terminal arterioles were captured with a Zeiss microscope digital PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49756