Bcr and Abr Regulate PH Figure 1. Hypoxia-treated bcr2/2 and abr2/2 mice have higher RVSP and more severe right ventricular hypertrophy. A, RVSP from bcr2/2, abr2/2 and wt mice with exposure to normoxia or hypoxia. B, Ratio of RV/LV+S calculated using the weight of RV, LV+S from the hearts of normoxic and hypoxic bcr2/2, abr2/2 and wt mice. *p,0.05 compared to the values of mice with the same genotype at normoxia. # p,0.05 when compared to wt mice in the same exposure condition. Bars represent mean 6SD; n = 6 mice per group. doi:10.1371/journal.pone.0049756.g001 expressed in PASMCs. As shown in Figure 5A and 5B, rac1 is highly expressed, rac2 mRNA can not be detected, and rac3 expression is very low in this cell type. Regulation of Rac expression levels can be one mechanism to control the effect of Rac, but it is generally accepted that the most important mode of regulation is on its GTP- or GDP-bound state. In macrophages, astroglia and neutrophils, loss of Abr and Bcr function leads to increased levels of activated Rac [7–9,11]. Therefore, we examined the activation state of Rac1 protein in lungs from normoxic and hypoxic mice and in PASMCs. As shown in Figure 5C, the baseline levels of GTP-Rac1 (active Rac1) were similar in the lungs of normoxic bcr2/2, abr2/2 and wt mice. Hypoxia increased the level of GTP-Rac1 in all lung samples. Interestingly, the level of GTP-Rac1 was higher in the lungs from hypoxic Bcr2/2 or Abr2/2 mice than in those of the WT mice (Figure 5C and D). In PASMCs, a similar basal level of GTP-Rac1 in different genotypes in normoxia was measured (Figure 5E, left 3 lanes). Hypoxia increased the levels GTP-Rac1 in PASMCs and this was higher in bcr2/2 or abr2/2 PASMCs than wt controls (Figure 5E, 5F). We also measured active Rac in hypoxia-treated PASMCs exposed to the Rac inhibitor Z62954982, and found that it significantly inhibited activation of Rac1. To investigate the possible application of this compound in vivo, we treated abr2/2 mice with intraperitoneal injections of 10 mg/ kg, every second day for the duration of the hypoxia exposure. Analysis of RV/LV+S revealed that the right ventricle hypertro- phy index was decreased in Z62954982-treated abr2/2 mice under hypoxia but this was not statistically significant. The trend was more obvious in an experiment in which 20 mg/kg/d was used (p = 0.096) (Figure S2A). There was no evidence for hepatic toxicity (10 mg/kg, Figure S3A) or myelosuppression at 20 mg/kg (Figure S3B). Lung lysates isolated 60 minutes after application of the final lower dose of Z62954982 showed that Rac activation was inhibited at least temporarily in this target tissue (Figure S2B, C). Lack of Bcr or Abr Promotes Phosphorylation of p38 MAPK and Increased IL-6 in Hypoxia Among the various intracellular pathways that can be activated by Rac, the p38 MAPK pathway was reported to be specially relevant to the pathological changes that occur in pulmonary hypertension [25–28]. Also, p38 activation was found to be associated with increased IL-6 production [29–37]. To investigate whether the p38 kinase pathway and concomitant IL-6 production are involved in the Bcr or Abr deficiency-promoted pulmonary hypertension, activation of the p38 pathway in the lungs and IL-6 levels in the serum and lungs of these mice were assessed. Under PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49756