Neurofibromin is a novel regulator of Ras-induced reactive oxygen species production in mice and humans

Neurofibromatosis type 1 (NF1) predisposes individuals to early and debilitating cardiovascular disease. Loss of function mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, leads to accelerated p21(Ras) activity and phosphorylation of multiple downstream kinases, including Erk and Akt. Nf1 heterozygous (Nf1(+/-)) mice develop a robust neointima that mimics human disease. Monocytes/macrophages play a central role in NF1 arterial stenosis as Nf1 mutations in myeloid cells alone are sufficient to reproduce the enhanced neointima observed in Nf1(+/-) mice. Though the molecular mechanisms underlying NF1 arterial stenosis remain elusive, macrophages are important producers of reactive oxygen species (ROS) and Ras activity directly regulates ROS production. Here, we use compound mutant and lineage-restricted mice to demonstrate that Nf1(+/-) macrophages produce excessive ROS, which enhance Nf1(+/-) smooth muscle cell proliferation in vitro and in vivo. Further, use of a specific NADPH oxidase-2 inhibitor to limit ROS production prevents neointima formation in Nf1(+/-) mice. Finally, mononuclear cells from asymptomatic NF1 patients have increased oxidative DNA damage, an indicator of chronic exposure to oxidative stress. These data provide genetic and pharmacologic evidence that excessive exposure to oxidant species underlie NF1 arterial stenosis and provide a platform for designing novels therapies and interventions

Neurofibromin Deficiency Induces Endothelial Cell Proliferation and Retinal Neovascularization

Purpose: Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization. Methods: Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching. Results: Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas. Conclusions: These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial

Nf1+/- monocytes/macrophages induce neointima formation via CCR2 activation

Persons with neurofibromatosis type 1 (NF1) have a predisposition for premature and severe arterial stenosis. Mutations in the NF1 gene result in decreased expression of neurofibromin, a negative regulator of p21(Ras), and increases Ras signaling. Heterozygous Nf1 (Nf1(+/-)) mice develop a marked arterial stenosis characterized by proliferating smooth muscle cells (SMCs) and a predominance of infiltrating macrophages, which closely resembles arterial lesions from NF1 patients. Interestingly, lineage-restricted inactivation of a single Nf1 allele in monocytes/macrophages is sufficient to recapitulate the phenotype observed in Nf1(+/-) mice and to mobilize proinflammatory CCR2+ monocytes into the peripheral blood. Therefore, we hypothesized that CCR2 receptor activation by its primary ligand monocyte chemotactic protein-1 (MCP-1) is critical for monocyte infiltration into the arterial wall and neointima formation in Nf1(+/-) mice. MCP-1 induces a dose-responsive increase in Nf1(+/-) macrophage migration and proliferation that corresponds with activation of multiple Ras kinases. In addition, Nf1(+/-) SMCs, which express CCR2, demonstrate an enhanced proliferative response to MCP-1 when compared with WT SMCs. To interrogate the role of CCR2 activation on Nf1(+/-) neointima formation, we induced neointima formation by carotid artery ligation in Nf1(+/-) and WT mice with genetic deletion of either MCP1 or CCR2. Loss of MCP-1 or CCR2 expression effectively inhibited Nf1(+/-) neointima formation and reduced macrophage content in the arterial wall. Finally, administration of a CCR2 antagonist significantly reduced Nf1(+/-) neointima formation. These studies identify MCP-1 as a potent chemokine for Nf1(+/-) monocytes/macrophages and CCR2 as a viable therapeutic target for NF1 arterial stenosis