threatening disorder [12]. Pulmonary vascular remodeling is a critical pathological feature of PH, and this is associated with the dysfunction and uncontrolled proliferation of the pulmonary arterial vascular smooth muscle cells (PASMCs) in the vascular wall. This in turn leads to increased thickening and musculariza- tion of the small pulmonary arteries [13,14]. We therefore considered the possibility that Abr and Bcr could regulate Rac activity in this cell type and through this mechanism affect the pathology of hypoxia-generated pulmonary hypertension. Using mice deficient in Abr or Bcr function, we here demonstrate for the first time that endogenous Rac1 activity is elevated by hypoxia- induced PH and that this is controlled by Bcr and Abr. Results Increased Hypoxia-induced Pulmonary Hypertension (PH) and Right Ventricle Hypertrophy in bcr2/2 or abr2/ 2 Mice To examine the impact of chronic hypoxia on pulmonary hemodynamics in mice lacking Abr or Bcr, we measured right ventricular systolic pressure (RVSP). As illustrated in Figure 1A, the RVSP of bcr2/2 or abr2/2 mice was similar to that of wt mice in normoxia. After 3 weeks of exposure to hypoxia, all three genotypes developed pulmonary hypertension, with a significant increase in RVSP, compared with their littermates in normoxia. In the hypoxic bcr2/2 or abr2/2 mice, the RVSP was significantly higher than that in the hypoxic wild type (wt) mice (Figure 1A). We next calculated the ratio of RV to LV+S weight, to assess the impact of changes of pulmonary pressure on cardiac mass (Figure 1B). There was no significant difference in (RV/LV+S) between normoxic bcr2/2, abr2/2 and wt mice. Consistent with the RVSP, hypoxia increased the value of (RV/LV+S) in all mice, indicating the development of right ventricular hypertrophy. Compared with the hypoxic wt mice, the (RV/LV+S) value of hypoxic bcr2/2 or abr2/2 mice was higher, implying that the right ventricular hypertrophy was greater in these mice. Enhanced Hypoxia-induced Pulmonary Vascular Remodeling in bcr2/2 or abr2/2 Mice To investigate the pathologic changes in hypoxia-induced PH, we performed histological analysis on the lungs of these animals. In normoxia, the percent wall thickness was similar for the bcr2/2, abr2/2 and wt mice. However, bcr2/2 and abr2/2 mice exposed to hypoxia showed significantly greater increase in the wall thickness and larger percent wall thickness, compared with the wt mice (Figure 2A and B). Immunostaining with a-SMA antibodies on lung specimens from the mice showed, as expected, that hypoxia-challenged mice showed pulmonary vascular remod- eling, characterized by increased areas of a-SMA–immunoreactive PASMCs in the pulmonary arteries. After chronic hypoxia exposure, the bcr2/2 and abr2/2 mice developed visibly more severe vascular remodeling, with greater areas of a-SMA–positive cells in remodeled arteries than the wt mice (Figure 2C and D). Contribution of Inflammation to Hypoxia-generated Pulmonary Pathology in bcr2/2 and abr2/2 Mice Inflammation and oxidative stress are thought to contribute to the pathology of PAH [15,16]. Since Abr and Bcr negatively regulate Rac in innate immune cells [8], we quantitated the numbers of perivascular leukocytes in H&E stained lung sections. Interestingly, pulmonary extravasation of leukocytes under hyp- oxia was significantly increased in the mice lacking Abr or Bcr function as compared to wt mice (Figure 3A). To examine if Bcr and Abr Regulate PH reactive oxygen species (ROS) production could contribute to the exacerbated pathology in hypoxia-treated bcr2/2 and abr2/2 animals, we exposed macrophages from these mice to hypoxia and normoxia, and compared ROS production in their innate immune cells. We found that lack of these GAPs for Rac resulted in significantly increased ROS production under hypoxic conditions (Figure 3B). Excess ROS production by inflammatory cells can result in collateral damage to surrounding tissues including the endothelium. To address whether loss of Abr or Bcr function results in vascular injury, we exposed mice to hypoxia and then measured pulmonary endothelial dysfunction using Evans blue. Figure 3C shows that chronic hypoxia promoted loss of endothelial barrier function and, moreover, mice lacking Abr or Bcr were clearly more severely affected. The VEGFR inhibitor SU5416 can cause emphysema in rodents and, when combined with chronic hypoxia, generates PAH [17,18]. Pathological features of combined treatment with SU5416 and chronic hypoxia include increased endothelial cell proliferation and moderately increased emphysema compared to mice exposed to hypoxia alone [19]. However, mice and rats with PAH induced by hypoxia alone did not show increased proliferation of pulmonary artery endothelial cells [20]. We therefore examined lungs of mice lacking Abr or Bcr for signs of emphysema. No obvious differences in airway hypertrophy between genotypes exposed to hypoxia were noted (Figure S1A). The mean linear intercept and average alveolar area in bcr2/2 and abr2/2 mice was increased upon chronic hypoxia exposure, but this was not significantly different from that of mice at normoxia, or of wt mice at hypoxia (Figure S1B, C). Proliferation of bcr2/2 and abr2/2 PASMCs is Stimulated by Hypoxia The progress of pulmonary vascular remodeling not only involves endothelial dysfunction, but also adventitial fibroblast growth, matrix deposition, acquisition of new mural cells and PASMCs proliferation [21]. These result in the narrowing of the lumen and decreased compliance of the small pulmonary arteries, constituted mainly by PASMCs [22,23]. To examine if Bcr and Abr are expressed in PASMCs, we performed Western Blot analysis. As shown in Figure 4A, both are expressed in this cell type. To explore the underlying mechanism of the enhanced pulmonary vascular remodeling in bcr2/2 and abr2/2 mice, we investigated the in vitro proliferation of PASMCs from these animals. As shown in Figure 4B, PASMCs from the different genotypes had similar proliferation in normoxia. The proliferation of all PASMCs increased under hypoxic conditions; furthermore, the proliferation of bcr2/2 or abr2/2 PASMCs was significantly higher than that of the wt cells. We also investigated the effect of a novel Rac inhibitor, Z62954982, on the proliferation of the PASMCs. This compound has been shown to inhibit Rac but not RhoA or CDC42 activation in SMC [24]. Interestingly, Z62954982 clearly suppressed the proliferation of hypoxia-treated PASMCs, reducing it to that measured for normoxia. Flow cytometry cell cycle analysis confirmed that the hypoxic condition induced an increase in the PASMC proliferation as quantified by the proliferation index, which was more significantly elevated in PASMCs that lacked Bcr or Abr, and could be decreased by Z62954982 (Figure 4C). Deficiency of Bcr or Abr Enhances Rac Activity during Hypoxia Because Abr and Bcr can regulate all three Racs we used quantitative real-time PCR to investigate which Racs are PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49756