Ras-Mek-Erk Signaling Regulates Nf1 Heterozygous Neointima Formation

Abstract

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes the protein neurofibromin. Neurofibromin negatively regulates Ras activity in multiple cell types by accelerating the hydrolysis of active Ras-GTP to its inactive diphosphate conformation.1Chang T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar These loss-of-function mutations accelerate Ras signaling and sensitize vessel wall cells and circulating hematopoietic cells, particularly myeloid progenitors and their differentiated progeny, to growth factors implicated in maintaining vascular wall homeostasis and disease pathogenesis.1Chang T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 2Lasater E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 3Li F. Munchhof A.M. White H.A. Mead L.E. Krier T.R. Fenoglio A. Chen S. Wu X. Cai S. Yang F.C. Ingram D.A. Neurofibromin is a novel regulator of RAS-induced signals in primary vascular smooth muscle cells.Hum Mol Genet. 2006; 15: 1921-1930Crossref PubMed Scopus (67) Google Scholar, 4Munchhof A.M. Li F. White H.A. Mead L.E. Krier T.R. Fenoglio A. Li X. Yuan J. Yang F.C. Ingram D.A. Neurofibroma-associated growth factors activate a distinct signaling network to alter the function of neurofibromin-deficient endothelial cells.Hum Mol Genet. 2006; 15: 1858-1869Crossref PubMed Scopus (51) Google Scholar Some patients with NF1 are predisposed to intimal proliferation, termed neointima, leading to debilitating arterial stenosis and tissue ischemia that contribute significantly to the premature mortality observed in this population.5Friedman J.M. Arbiser J. Epstein J.A. Gutmann D.H. Huot S.J. Lin A.E. McManus B. Korf B.R. Cardiovascular disease in neurofibromatosis 1: report of the NF1 Cardiovascular Task Force.Genet Med. 2002; 4: 105-111Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Nf1 heterozygous (Nf1+/−) mice display increased neointima formation, characterized by proliferating vascular smooth muscle cells (VSMCs) and infiltration of bone marrow–derived macrophages after arterial ligation, which is reminiscent of patients with NF1.5Friedman J.M. Arbiser J. Epstein J.A. Gutmann D.H. Huot S.J. Lin A.E. McManus B. Korf B.R. Cardiovascular disease in neurofibromatosis 1: report of the NF1 Cardiovascular Task Force.Genet Med. 2002; 4: 105-111Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 6Lasater E.A. Bessler W.K. Mead L.E. Horn W.E. Clapp D.W. Conway S.J. Ingram D.A. Li F. Nf1+/- mice have increased neointima formation via hyperactivation of a Gleevec sensitive molecular pathway.Hum Mol Genet. 2008; 17: 2336-2344Crossref PubMed Scopus (57) Google Scholar Neurofibromin-deficient endothelial cells, VSMCs, and bone marrow–derived myeloid cells demonstrate preferential activation of the Ras-Erk signaling pathway, without corresponding alterations in Ras–phosphatidylinositol 3-kinase signaling, in response to multiple growth factors in vitro.2Lasater E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 3Li F. Munchhof A.M. White H.A. Mead L.E. Krier T.R. Fenoglio A. Chen S. Wu X. Cai S. Yang F.C. Ingram D.A. Neurofibromin is a novel regulator of RAS-induced signals in primary vascular smooth muscle cells.Hum Mol Genet. 2006; 15: 1921-1930Crossref PubMed Scopus (67) Google Scholar, 4Munchhof A.M. Li F. White H.A. Mead L.E. Krier T.R. Fenoglio A. Li X. Yuan J. Yang F.C. Ingram D.A. Neurofibroma-associated growth factors activate a distinct signaling network to alter the function of neurofibromin-deficient endothelial cells.Hum Mol Genet. 2006; 15: 1858-1869Crossref PubMed Scopus (51) Google Scholar, 7Zhang Y.Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. Nf1 regulates hematopoietic progenitor cell growth and ras signaling in response to multiple cytokines.J Exp Med. 1998; 187: 1893-1902Crossref PubMed Scopus (133) Google Scholar This is an interesting observation because lineage-restricted inactivation of a single Nf1 gene in endothelial cells and/or VSMCs does not replicate the striking neointima observed in Nf1 heterozygous mice. However, we recently demonstrated that lineage-specific inactivation of a single Nf1 gene copy in monocytes/macrophages is sufficient to reproduce the enhanced neointima formation observed in Nf1 heterozygous mice compared with wild-type (WT) mice.8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar Based on these observations, we used in vitro and in vivo systems of macrophage function to test the hypothesis that Nf1 heterozygous macrophage function and mobilization to sites of inflammation are directly controlled by Ras-Erk signaling and that use of a specific and long-acting inhibitor of Ras-Erk signaling, under evaluation in multiple phase 1 and 2 clinical trials for cancer and preclinical models of NF1 malignancy,1Chang T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 9Jessen W.J. Miller S.J. Jousma E. Wu J. Rizvi T.A. Brundage M.E. Eaves D. Widemann B. Kim M.O. Dombi E. Sabo J. Hardiman Dudley A. Niwa-Kawakita M. Page G.P. Giovannini M. Aronow B.J. Cripe T.P. Ratner N. MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors.J Clin Invest. 2013; 123: 340-347Crossref PubMed Scopus (258) Google Scholar, 10Lyubynska N. Gorman M.F. Lauchle J.O. Hong W.X. Akutagawa J.K. Shannon K. Braun B.S. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice.Sci Transl Med. 2011; 3: 76ra27Crossref PubMed Scopus (80) Google Scholar, 11Boasberg P.D. Redfern C.H. Daniels G.A. Bodkin D. Garrett C.R. Ricart A.D. Pilot study of PD-0325901 in previously treated patients with advanced melanoma, breast cancer, and colon cancer.Cancer Chemother Pharmacol. 2011; 68: 547-552Crossref PubMed Scopus (58) Google Scholar, 12Haura E.B. Ricart A.D. Larson T.G. Stella P.J. Bazhenova L. Miller V.A. Cohen R.B. Eisenberg P.D. Selaru P. Wilner K.D. Gadgeel S.M. A phase II study of PD-0325901, an oral MEK inhibitor, in previously treated patients with advanced non-small cell lung cancer.Clin Cancer Res. 2010; 16: 2450-2457Crossref PubMed Scopus (210) Google Scholar will reduce neointima formation after mechanical injury. Protocols were approved by the Institutional Animal Care and Use Committee at Indiana University (Indianapolis, IN). Nf1+/− mice were obtained from Tyler Jacks, Ph.D., (Massachusetts Institute of Technology, Cambridge, MA) and backcrossed 13 generations into the C57BL/6J strain. Mice were genotyped by PCR, as previously described.2Lasater E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar Nf1+/− and WT mice (C57BL/6J) were crossed to generate experimental progeny. Male mice, between 12 and 15 weeks of age, were used for experiments. In five independent experiments, the carotid arteries of Nf1+/− and WT mice were mechanically injured via ligation of the right common carotid artery proximal to the bifurcation, as previously described.2Lasater E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar Briefly, experimental and control animals were anesthetized by inhalation of a 2% isoflurane and 98% oxygen admixture. Under a dissecting microscope, the proximal arterial tree was exposed through a midline neck incision and the right common carotid artery was ligated using a 6-0 silk suture. The contralateral carotid artery was sham ligated as a control. Buprenorphine, 15 μg (Reckitt, Richmond, VA), was administered via i.p. injection and mice recovered for 28 days without complication. Nf1+/− and WT mice were administered 5 or 10 mg PD0325901/kg once daily via oral gavage. Treatment days arterial and tissue was 28 days after arterial In some experiments, was days after arterial and through tissue at 28 days after injury. Nf1+/− and WT control mice were administered an of as a control. After a mice were anesthetized via inhalation of a 2% isoflurane and 98% oxygen admixture. arteries were in with 10 of for 10 by as previously described.2Lasater E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar By using a dissecting microscope, injured and control carotid arteries were in at for and of the carotid bifurcation, arterial were at the of the was to standard and arterial at and proximal to the ligation were for neointima formation using the and the were neointima and in were and at were from of mice not significantly to and/or Nf1+/− and WT mice were with μg or by i.p. In experiments, mice were at and after and cells were via in 5 of and cells were in cell for 10 by inactivation with 1 of were and in of in The of cells was using a were on with murine for 10 with and cell for and in 2% in were on a with a a and a were for were and using and macrophage cell were as the of or as a of cells and the cell and as of In some experiments, mice were administered 5 mg PD0325901/kg via oral days injection and was at macrophages were from of to WT and Nf1+/− mice. Briefly, cells were into a using a and were by at for 5 at and cells were with cell for 5 at The cells were and in 5 cells were by using cells were in of with and macrophage factor were used for between and were in in without growth factors for to A of 5 macrophages were in a in in the presence or of the of were for and with for were using an cell and was as cells were with the of PD0325901 or for 1 The of MA) was with for 2 at and with 2% The were in the with or without A of macrophages were in and to the of the After of at cells in the were with a The cells that to the of the were with in and 2% for 5 at The of cells was in five with an using a cells were with the of PD0325901 or for 1 were with in for 1 at were once with with for 1 at to and with of cell on the 1 cells were to and at for the of the cells were by and were with cells were with and with The was with and was at using a cells were with the of PD0325901 or for 1 cells from Nf1+/− and WT mice treated with PD0325901 or control were via at after stimulation with μg were in and of protein were on a to The protein was a and was using and macrophages from WT and mice were and in and growth and with for 5 in the presence or of were and as and was using and are as and for were by of with a test using was and macrophage response to growth factors with arterial we used a well-established model of in Nf1+/− and WT mice. After i.p. injection of μg Nf1 heterozygous mice hematopoietic cells to the at compared with WT mice of the demonstrated a of and macrophages to the of Nf1+/− mice, compared with WT mice and The preferential mobilization of Nf1+/− and macrophages was with daily administration of PD0325901 a specific and inhibitor of Ras-Erk T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar of WT and macrophages was by PD0325901, demonstrating that the Ras-Erk signaling pathway regulates the of monocytes/macrophages to sites of inflammation in the of Ras-Erk signaling in macrophage macrophages were with a growth factor implicated in disease and arterial M. T. N. A. K. K. formation in the phase after vascular in the of the PubMed Scopus Google Scholar in the presence or of Neurofibromin-deficient macrophages demonstrated increased proliferation, and in response to single growth factor stimulation via Ras-Erk activation compared with WT macrophages which is with E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar In the presence of of PD0325901, Nf1+/− macrophage function was significantly reduced to levels of WT macrophages in vitro WT macrophages demonstrated an response to in the presence of this response was compared with Nf1+/− macrophages the of Ras-Erk signaling in Nf1+/− and WT neointima formation, Nf1+/− and WT mice were administered daily oral 5 or 10 mg PD0325901/kg for 1 arterial this was arteries were This downstream signaling for in multiple bone as demonstrated in T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 10Lyubynska N. Gorman M.F. Lauchle J.O. Hong W.X. Akutagawa J.K. Shannon K. Braun B.S. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice.Sci Transl Med. 2011; 3: 76ra27Crossref PubMed Scopus (80) Google Scholar After arterial ligation of the right common carotid Nf1+/− mice demonstrated enhanced neointima formation compared with WT mice, which is with E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar, 6Lasater E.A. Bessler W.K. Mead L.E. Horn W.E. Clapp D.W. Conway S.J. Ingram D.A. Li F. Nf1+/- mice have increased neointima formation via hyperactivation of a Gleevec sensitive molecular pathway.Hum Mol Genet. 2008; 17: 2336-2344Crossref PubMed Scopus (57) Google Scholar, 8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar Daily administration of PD0325901 significantly reduced Nf1+/− neointima formation in a arterial stenosis and to levels observed in WT mice. with in vitro and in vivo that Erk signaling is in neurofibromin-deficient myeloid cells, vascular wall cells, and T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 10Lyubynska N. Gorman M.F. Lauchle J.O. Hong W.X. Akutagawa J.K. Shannon K. Braun B.S. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice.Sci Transl Med. 2011; 3: 76ra27Crossref PubMed Scopus (80) Google Scholar, N. Lauchle J.O. A. Braun B.S. E. Kogan L. Shannon inactivation of Nf1 in hematopoietic cells results in a myeloproliferative PubMed Scopus Google Scholar Nf1+/− compared with WT neointimas, of demonstrating the of PD0325901 not The observation that PD0325901 reduced neointima formation in WT mice signaling the vascular wall in and regulates the response of the vascular wall to injury. Recent using a murine model of myeloproliferative have demonstrated that PD0325901 aberrant and observed in this T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 10Lyubynska N. Gorman M.F. Lauchle J.O. Hong W.X. Akutagawa J.K. Shannon K. Braun B.S. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice.Sci Transl Med. 2011; 3: 76ra27Crossref PubMed Scopus (80) Google Scholar However, of the bone from Nf1+/− and WT mice in not in or that the of neointima observed in is to on differentiated macrophage function the bone through alterations in not test inhibition of the signaling is in neointima formation after arterial Nf1+/− and WT mice were administered daily 5 mg PD0325901/kg or on after arterial and through 28 after injury. of injured and control arteries from Nf1+/− and WT mice demonstrated that of days after arterial significantly reduced neointima and in Nf1+/− and WT mice compared with mice neointima formation in Nf1+/− and WT mice was increased in the of Nf1+/− and WT neointima formation was was after arterial injury. In Nf1+/− from mice administered PD0325901 demonstrated macrophages compared with mice evidence that Ras signaling regulates vascular wall homeostasis and that in Ras signaling results in a of and disease J.K. R. B. M. of the kinase pathway is in smooth muscle of vascular smooth muscle cell proliferation and neointima 2010; PubMed Scopus (67) Google Scholar, L. F. Lin A.E. D.A. from the of the from to and A. 2010; PubMed Scopus Google Scholar Recent studies demonstrate that amplification of Ras signaling enhances neointima formation, inhibition of the pathway neointima formation and proliferation and migration in J.K. R. B. M. of the kinase pathway is in smooth muscle of vascular smooth muscle cell proliferation and neointima 2010; PubMed Scopus (67) Google Scholar, Wu J. Li S. of active and negative of Ras on arterial neointima Res. Full Text PDF PubMed Scopus Google Scholar the protein of the NF1 gene, the hydrolysis of Ras-GTP to its inactive to negatively Ras of to Ras and increased cell proliferation and in and In of this arterial from neurofibromin-deficient mice increased Erk signaling the neointima compared with WT The signaling is with administration of an inhibitor of multiple signaling the growth after arterial E.A. Bessler W.K. Mead L.E. Horn W.E. Clapp D.W. Conway S.J. Ingram D.A. Li F. Nf1+/- mice have increased neointima formation via hyperactivation of a Gleevec sensitive molecular pathway.Hum Mol Genet. 2008; 17: 2336-2344Crossref PubMed Scopus (57) Google Scholar are and cellular of and neointima M. T. N. A. K. K. formation in the phase after vascular in the of the PubMed Scopus Google Scholar, A. A. E.A. P. S. of the in after arterial in mice and on Res. PubMed Scopus Google Scholar, Epstein J. E. J. J. R. of macrophages by formation in the carotid Pharmacol. PubMed Scopus Google Scholar growth factors and that vascular smooth muscle cell proliferation and and are implicated in disease M. T. N. A. K. K. formation in the phase after vascular in the of the PubMed Scopus Google Scholar, Epstein J. E. J. J. R. of macrophages by formation in the carotid Pharmacol. PubMed Scopus Google Scholar macrophages a of the cell Nf1+/− and WT neointimas, neurofibromin-deficient macrophages significantly the response of Nf1+/− and WT VSMCs via the signaling compared with WT E.A. Li F. Bessler W.K. Estes M.L. Vemula S. Hingtgen C.M. Dinauer M.C. Kapur R. Conway S.J. Ingram Jr, D.A. Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.J Clin Invest. 2010; 120: 859-870Crossref PubMed Scopus (64) Google Scholar These report demonstrating that heterozygous inactivation of the Nf1 gene in monocytes/macrophages the neointima phenotype observed in Nf1+/− mice.8Stansfield B.K. Bessler W.K. Mali R. Mund J.A. Downing B. Li F. Sarchet K.N. DiStasi M.R. Conway S.J. Kapur R. Ingram Jr., D.A. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.Hum Mol Genet. 2013; 22: 977-988Crossref PubMed Scopus (16) Google Scholar we that neurofibromin-deficient and macrophages are to inflammatory and growth factor which is via the signaling we that daily administration of PD0325901 to signaling significantly reduced Nf1+/− in a and in a of WT of PD0325901 at 1 after arterial reduced neointima formation in Nf1+/− and WT mice, a PD0325901 is a inhibitor and used as an experimental in preclinical for multiple and phase 1 and 2 clinical trials for W.J. Miller S.J. Jousma E. Wu J. Rizvi T.A. Brundage M.E. Eaves D. Widemann B. Kim M.O. Dombi E. Sabo J. Hardiman Dudley A. Niwa-Kawakita M. Page G.P. Giovannini M. Aronow B.J. Cripe T.P. Ratner N. MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors.J Clin Invest. 2013; 123: 340-347Crossref PubMed Scopus (258) Google Scholar, 11Boasberg P.D. Redfern C.H. Daniels G.A. Bodkin D. Garrett C.R. Ricart A.D. Pilot study of PD-0325901 in previously treated patients with advanced melanoma, breast cancer, and colon cancer.Cancer Chemother Pharmacol. 2011; 68: 547-552Crossref PubMed Scopus (58) Google Scholar, 12Haura E.B. Ricart A.D. Larson T.G. Stella P.J. Bazhenova L. Miller V.A. Cohen R.B. Eisenberg P.D. Selaru P. Wilner K.D. Gadgeel S.M. A phase II study of PD-0325901, an oral MEK inhibitor, in previously treated patients with advanced non-small cell lung cancer.Clin Cancer Res. 2010; 16: 2450-2457Crossref PubMed Scopus (210) Google Scholar In PD0325901 efficacy in preclinical models of a common with Ras mutations in myeloid progenitor T. Krisman K. Theobald E.H. Xu J. Akutagawa J. Lauchle J.O. Kogan S. Braun B.S. Shannon K. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice.J Clin Invest. 2013; 123: 335-339Crossref PubMed Scopus (108) Google Scholar, 10Lyubynska N. Gorman M.F. Lauchle J.O. Hong W.X. Akutagawa J.K. Shannon K. Braun B.S. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice.Sci Transl Med. 2011; 3: 76ra27Crossref PubMed Scopus (80) Google Scholar Ras-Erk signaling in neurofibromin-deficient monocytes/macrophages an molecular for the of novel to or NF1