Bcr and Abr Regulate PH Figure 2. Hypoxia-induced pulmonary vascular remodeling in bcr2/2 and abr2/2 mice. A, Hematoxylin and eosin staining on representative lung specimens from bcr2/2, abr2/2 and wt mice under normoxia or hypoxia. Note that the walls of the pulmonary arteries of the bcr2/2 and abr2/2 mice are remarkably thicker than those of the wt mice after hypoxia. Magnification, 2006. B, Quantification of changes in the pulmonary artery walls. Percent wall thickness was determined on H&E stained sections as described in Methods on nine vessels of comparable size per mouse, with 6 mice per genotype per condition. *p,0.05 compared with the same genotype at normoxia. # p,0.05 compared with WT mice in the same exposure condition. Bars, mean 6 SD. C, Immunostaining with a-SMA antibodies on pulmonary vessels from representative normoxia or hypoxia-treated mice. D, Quantification of areas for a-SMA-positive cells. Areas of a-SMA-positive cells were calculated using ImageJ software as described in the Materials and Methods. doi:10.1371/journal.pone.0049756.g002 conditions of normoxia, the phosphorylation of p38 was minimal (Figure 6A, B) and IL-6 levels were low in serum and in lungs (Figure 6C, D). Exposure to hypoxia remarkably elevated the levels of p-p38 in lungs and this was increased significantly more in bcr2/2 or abr2/2 mice than in the wt mice (Figure 6A, B). This was accompanied by a significantly larger increase in IL-6 levels in lung and serum in the mutant mice (Figure 6C, D). We also evaluated p38 activation and IL-6 production in the PASMCs. When present at normoxia, PASMC had very low levels of p-p38 and produced little IL-6, but after hypoxia exposure this was significantly increased (Figure 6E–G), especially in bcr2/2 or abr2/2 PASMCs. To explore whether the Rac activation was correlated with the p38 MAPK phosphorylation and IL-6 production, we assessed p-p38 and IL-6 production in PASMCs treated with the Rac inhibitor Z62954982. Our results show that Z62954982 decreased both phosphorylation of p38 as well as secreted IL-6 in PASMCs in response to hypoxia (Figure 6E–G). Discussion Pulmonary hypertension is a devastating disease with poor prognosis. Because no validated therapy has been reported that can prevent it or affect its development [12,38], the identification of potential targets for treatment remains a crucial first step. A number of studies indirectly suggest that Rac may play a role in PH and form such a potential target [39–42], but the possible contribution of endogenous Rac protein to PH has not been adequately examined. Firstly, since there are three distinct Rac proteins, Rac1, Rac2 and Rac3, there is a controversy regarding which of these are expressed in PASMCs, a critical cell type involved in the pathogenesis of PH. Although Rac2 is regarded as an isoform that is only expressed in hematopoietic cells, Rac2 was reported to also be present in aortic smooth muscle cells [43,44]. However Rac2 expression in PASMCs had not been reported. In the current study, using quantitative real-time RT/PCR, PASMCs cultured in standard culture medium [45] were found to contain virtually no rac2 mRNA. Our results additionally showed low rac3 PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49756