cellularity and myeloid cell percentages of mice exposed to hypoxia and treated every day with 20 mg/kg drug or with vehicle. *p,0.05, n = 3 mice/group. (TIF) Acknowledgments We thank Donna Foster for excellent care of the mice. Monica Wong and Sun Ju Yi are acknowledged for instruction on the RVSP measurements and the Rac activation assays, respectively. Dr. Bellusci kindly provided access to the hypoxia chamber and gas modulator. We thank Esteban References 1. Heisterkamp N, Stam K, Groffen J, de Klein A, Grosveld G (1985) Structural organization of the bcr gene and its role in the Ph’ translocation. Nature 315: 758–761. 2. Chuang TH, Xu X, Kaartinen V, Heisterkamp N, Groffen J, et al. (1995) Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc Natl Acad Sci U S A 92: 10282–10286. 3. Diekmann D, Brill S, Garrett MD, Totty N, Hsuan J, et al. (1991) Bcr encodes a GTPase-activating protein for p21rac. Nature 351: 400–402. 4. Bernards A, Settleman J (2004) GAP control: regulating the regulators of small GTPases. Trends Cell Biol 14: 377–385. 5. Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9: 690–701. 6. Van Aelst L, DSouza-Schorey C (1997) Rho GTPases and signaling networks. Genes Dev 11: 2295–2322. 7. Cho YJ, Cunnick JM, Yi SJ, Kaartinen V, Groffen J, et al. (2007) Abr and Bcr, two homologous Rac GTPase-activating proteins, control multiple cellular functions of murine macrophages. Mol Cell Biol 27: 899–911. 8. Cunnick JM, Schmidhuber S, Chen G, Yu M, Yi SJ, et al. (2009) Bcr and Abr cooperate in negatively regulating acute inflammatory responses. Mol Cell Biol 29: 5742–5750. 9. Kaartinen V, Gonzalez-Gomez I, Voncken JW, Haataja L, Faure E, et al. (2001) Abnormal function of astroglia lacking Abr and Bcr RacGAPs. Development 128: 4217–4227. 10. Oh D, Han S, Seo J, Lee JR, Choi J, et al. (2010) Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins. J Neurosci 30: 14134–14144. 11. Voncken JW, van Schaick H, Kaartinen V, Deemer K, Coates T, et al. (1995) Increased neutrophil respiratory burst in bcr-null mutants. Cell 80: 719–728. 12. Macchia A, Marchioli R, Marfisi R, Scarano M, Levantesi G, et al. (2007) A meta-analysis of trials of pulmonary hypertension: a clinical condition looking for drugs and research methodology. Am Heart J 153: 1037–1047. 13. Janssens SP, Thompson BT, Spence CR, Hales CA (1991) Polycythemia and vascular remodeling in chronic hypoxic pulmonary hypertension in guinea pigs. J Appl Physiol 71: 2218–2223. 14. Rabinovitch M, Gamble W, Nadas AS, Miettinen OS, Reid L (1979) Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol 236: H818–827. 15. Crosswhite P, Sun Z (2010) Nitric oxide, oxidative stress and inflammation in pulmonary arterial hypertension. J Hypertens 28: 201–212. 16. Price LC, Wort SJ, Perros F, Dorfmuller P, Huertas A, et al. (2012) Inflammation in pulmonary arterial hypertension. Chest 141: 210–221. 17. Sakao S, Tatsumi K (2011) The effects of antiangiogenic compound SU5416 in a rat model of pulmonary arterial hypertension. Respiration 81: 253–261. 18. Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L, Kasahara Y, et al. (2003) Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade. Am J Respir Cell Mol Biol 29: 88–97. 19. Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, et al. (2011) A novel murine model of severe pulmonary arterial hypertension. Am J Respir Crit Care Med 184: 1171–1182. 20. Yu L, Hales CA (2011) Hypoxia does neither stimulate pulmonary artery endothelial cell proliferation in mice and rats with pulmonary hypertension and vascular remodeling nor in human pulmonary artery endothelial cells. J Vasc Res 48: 465–475. 21. Stenmark KR, Fagan KA, Frid MG (2006) Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 99: 675–691. 22. Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, et al. (2004) Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 43: 13S–24S. 23. Stenmark KR, Mecham RP (1997) Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89–144. 24. Ferri N, Corsini A, Bottino P, Clerici F, Contini A (2009) Virtual screening approach for the identification of new Rac1 inhibitors. J Med Chem 52: 4087– 4090. 25. Das M, Bouchey DM, Moore MJ, Hopkins DC, Nemenoff RA, et al. (2001) Hypoxia-induced proliferative response of vascular adventitial fibroblasts is Bcr and Abr Regulate PH Fernandez of the Imaging Core of the Saban Research Institute, Children’s Hospital Los Angeles for help with quantification of the mean linear intercept, the alveolar size and of areas of a-SMA-positive cells. We thank Dr. Stefan M. Lee, who generously allowed us to use the BP-1 machine for invasive BP measurements. Author Contributions Conceived and designed the experiments: MY DG ML AA JG NH. Performed the experiments: MY DG ML AA. Analyzed the data: MY DG AA JG NH. Wrote the paper: MY DG AA JG NH. dependent on g protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 15631–15640. 26. Karakiulakis G, Papakonstantinou E, Aletras AJ, Tamm M, Roth M (2007) Cell type-specific effect of hypoxia and platelet-derived growth factor-BB on extracellular matrix turnover and its consequences for lung remodeling. J Biol Chem 282: 908–915. 27. Lu J, Shimpo H, Shimamoto A, Chong AJ, Hampton CR, et al. (2004) Specific inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates vascular proliferation in monocrotaline-induced pulmonary hypertension in rats. J Thorac Cardiovasc Surg 128: 850–859. 28. Sodhi CP, Phadke SA, Batlle D, Sahai A (2001) Hypoxia stimulates osteopontin expression and proliferation of cultured vascular smooth muscle cells: potentiation by high glucose. Diabetes 50: 1482–1490. 29. Hattori Y, Suzuki M, Hattori S, Kasai K (2002) Vascular smooth muscle cell activation by glycated albumin (Amadori adducts). Hypertension 39: 22–28. 30. Tokunou T, Ichiki T, Takeda K, Funakoshi Y, Iino N, et al. (2001) Thrombin induces interleukin-6 expression through the cAMP response element in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21: 1759–1763. 31. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, et al. (2000) Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology 47: 185–201. 32. Sano M, Fukuda K, Sato T, Kawaguchi H, Suematsu M, et al. (2001) ERK and p38 MAPK, but not NF-kappaB, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res 89: 661–669. 33. Selimovic N, Bergh CH, Andersson B, Sakiniene E, Carlsten H, et al. (2009) Growth factors and interleukin-6 across the lung circulation in pulmonary hypertension. Eur Respir J 34: 662–668. 34. Humbert M, Monti G, Brenot F, Sitbon O, Portier A, et al. (1995) Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 151: 1628–1631. 35. Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, et al. (2009) Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104: 236–244, 228p following 244. 36. Berk BC (2001) Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81: 999–1030. 37. Soon E, Holmes AM, Treacy CM, Doughty NJ, Southgate L, et al. (2010) Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension. Circulation 122: 920–927. 38. Galie N, Manes A, Negro L, Palazzini M, Bacchi-Reggiani ML, et al. (2009) A meta-analysis of randomized controlled trials in pulmonary arterial hyperten- sion. Eur Heart J 30: 394–403. 39. Diebold I, Petry A, Djordjevic T, Belaiba RS, Fineman J, et al. (2010) Reciprocal regulation of Rac1 and PAK-1 by HIF-1alpha: a positive-feedback loop promoting pulmonary vascular remodeling. Antioxid Redox Signal 13: 399–412. 40. Grobe AC, Wells SM, Benavidez E, Oishi P, Azakie A, et al. (2006) Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase. Am J Physiol Lung Cell Mol Physiol 290: L1069–1077. 41. Teng RJ, Eis A, Bakhutashvili I, Arul N, Konduri GG (2009) Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 297: L184–195. 42. Diebold I, Djordjevic T, Hess J, Gorlach A (2008) Rac-1 promotes pulmonary artery smooth muscle cell proliferation by upregulation of plasminogen activator inhibitor-1: role of NFkappaB-dependent hypoxia-inducible factor-1alpha transcription. Thromb Haemost 100: 1021–1028. 43. Tian Y, Autieri MV (2007) Cytokine expression and AIF-1-mediated activation of Rac2 in vascular smooth muscle cells: a role for Rac2 in VSMC activation. Am J Physiol Cell Physiol 292: C841–849. 44. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, et al. (1999) Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo. J Biol Chem 274: 19814–19822. PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49756