Bcr and Abr Regulate PH Figure 3. Lack of Abr and Bcr function exacerbates hypoxia-induced leukocyte involvement in PH. A, Numbers of perivascular leukocytes surrounding terminal arterioles. Data are expressed as total leukocyte counts per high-power field (2006 amplification). Bars, mean+SEM. n = 3/group. B, Peritoneal-elicited macrophages exposed to normoxia or hypoxia for 24 hours activated with 1 mM PMA. ROS production was measured by FACS 1 hr later. Controls, cells without PMA activation. Bars, mean+SEM. n = 5 mice/group. *, p,0.05. MFI, mean fluorescent intensity. C, Vascular leakage. Evans blue was injected when mice had been exposed to normoxia or hypoxia for 3 weeks. Bars, mean+SEM. n = 3–4 mice/group. A and C, *p,0.05 comparison between animals of the same genotype at normoxia. #p,0.05 compared to WT mice exposed to the same condition. doi:10.1371/journal.pone.0049756.g003 and high rac1 mRNA levels in PASMC, indicating that Rac1 is likely to be the most important Rac for this cell type. Secondly, previous reports that suggest Rac1 regulates processes relevant to pulmonary hypertension based their conclusions on the detection of increased levels of total Rac1 protein using Western blotting, or on the overexpression of constitutively active V12Rac or dominant negative N17Rac in PASMCs [40,46]. Although the latter studies obviously are important, under physiological conditions Rac proteins are never permanently frozen into a GTP- or GDP-bound state, but instead participate in a cycle of activation and deactivation. Also, in our study, no evidence was found for increased total Rac1 protein levels upon exposure of mouse lungs or PASMC to hypoxia (Figure 5E). Therefore, we here analyzed the levels of endogenously activated, GTP-bound Rac1 as a physiologically relevant readout for the putative involvement of Rac in PH. We were able to detect not only the activation of endogenous Rac1 in the lungs in a mouse model of PH, but we also found enhanced hypoxia-induced activation of pulmonary Rac1 in mice deficient of Bcr or Abr. Consistent with the in vivo results, we confirmed that Rac1 becomes activated in PASMCs that are subjected to hypoxia. Moreover, the hypoxia-induced activation of Rac1 was significantly more notable in PASMCs lacking Bcr or Abr. We conclude that Bcr and Abr are very important components of a regulatory pathway that connects PH caused by hypoxia to Rac activation and the proliferation of PASMC. Our data also connect Bcr and Abr to the pathology of PH in a different cell type, namely by suppression of excessive ROS production in macrophages exposed to hypoxia. The activation of Rac1 in PASMC can lead to a number of essential signaling events. The phosphorylation of p38 has been reported as a critical factor in the development of pulmonary hypertension [47,48]. Our results also showed for the first time that the suppression of endogenous Rac1 activation with the Rac inhibitor Z62954982 led to inhibition of phosphorylation of p38 in hypoxic PASMCs. This result is consistent with a previous study which reported that knock-down of Rac1 with siRNA suppresses the phosphorylation of p38 in aortic smooth muscle cells exposed to cyclic strain [49]. Of the various cytokines that are generated downstream of the phosphorylation and activation of the p38 pathway, IL-6 was recently highlighted because of its important role in pulmonary vascular remodeling and pulmonary hypertension [29,30,32]. In agreement with previous reports, our study showed increased IL-6 in mouse serum and lung homogenates in hypoxia-induced PH. We also observed further elevated IL-6 in hypoxic Bcr- or Abr- deficient mice. IL-6 can be secreted by vascular smooth muscle cells, and can also promote their growth [36,50]. Moreover, hypoxia is a factor that induces transcription and translation of IL- 6 in human PASMCs [51]. Our results are consistent with these reports. We confirmed that hypoxia can increase IL-6 levels in the culture supernatant of PASMCs. Interestingly, we showed that decreasing activated Rac1 levels with Z62954982 can decrease IL- 6 produced by hypoxia-exposed PASMC, suggesting that IL-6 is regulated by Rac1 activation, and is possibly related with phosphorylation of p38. Figure 7 summarizes our data that link Abr and Bcr to the Rac cycle and downstream events in hypoxia-treated PASMCs. Which GEF is upstream of Rac in promoting PASMC proliferation, and how that GEF is activated in PH, is currently unknown. We noted that different GEFs for Rac including DOCK1, TIAM1, TRIO, VAV1, VAV2 are highly expressed both in human control lung as well as PH RNA samples (GSE15197) This does not support the possibility that the GEFs are regulated at the level of gene transcription. Instead, we suggest that the GEF may be activated through tyrosine phosphorylation. In particular, PDGF-R signal- ing is known to play a role in pulmonary hypertension [52]. Since hypoxia induces increased PDGF-BB mRNA in human primary VSMCs, which also express the PDGF-R, paracrine signaling may contribute to VSMC cell proliferation under hypoxic conditions [53]. PDGF signaling in VSMC also generates reactive oxygen species, leading to the activation of the Src and Abl tyrosine kinases and promoting growth of these cells [54]. In fibroblasts, Abl is needed for the PDGF-R signaling that leads to Rac activation and mitogenesis [55]. Dock1 (DOCK180), a GEF for Rac, becomes tyrosine phosphorylated by Src downstream of PDGFRa activation in glioblastoma cells [56]. Thus, we speculate that one mechanism through which hypoxia could result in Rac activation is by paracrine PDGF-R signaling, that in turn leads to ROS production, Src/Abl activation and tyrosine phosphoryla- tion/activation of a GEF for Rac. This mechanism would be consistent with the activity of the Abl and PDGF-R inhibitor Imatinib currently in clinical trials for PAH. There are very few small molecule inhibitors available that decrease levels of activated Rac [24]. Z62954982 is a new compound that was identified through pharmacophore virtual screening, with the commonly used Rac inhibitor NSC23766 as reference compound [24]. Z62954982 does not inhibit Rac directly, but interferes with the interaction of Rac with a GEF, which activates Rac by catalyzing the exchange of the GDP bound to it for GTP. Considering the fact that there are 69 GEFs in mammals that act on various GTPases with a probable tissue specific pattern of expression [57], the targets of such inhibitors of the GEF-Rac interaction are not well-defined and need to be empirically discovered. Z62954982 was tested in human vascular smooth muscle cells, in which it interfered with the binding of Rac1 to Tiam1 and was more efficient in reducing Rac1-GTP levels than the chemically unrelated Rac inhibitor NSC23766 [24]. However, Z62954982 has not been used in animals. In our experiments, we measured reduction in levels of activated Rac and found evidence for a beneficial effect on the pathology of hypoxia-induced PH in mice. The relatively moderate extent of this effect could be caused by, for example, a rapid clearance of the drug, and further pharmacokinetic and pharmacodynamic studies will be needed to determine the utility of this particular chemical compound for treatment of PAH. Our results do establish that lack of Abr and Bcr function, resulting in elevated levels of activated Rac, affects macrophages and PASMC, two distinct cell types that are involved in the pathology of PH. Thus treatment with Rac inhibitors remains a viable approach to suppress hypoxia-induced pulmonary hyper- PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49756