Dissecting Neurofibromatosis Type 1 Vasculopathy

A genetic predisposition for premature and severe arterial stenosis has been identified in persons with neurofibromatosis type 1 (NF1). NF1 results from mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, a negative regulator of the p21 (Ras) signaling pathway. Previously, we utilized lineage-restricted mutant mice to show that heterozygous inactivation of Nf1 (Nf1+/-) in myeloid cells is sufficient to enhance arterial stenosis after carotid artery ligation and mobilize inflammatory Ly6Chi CCR2+ monocytes in mice. We provide novel data to show that Nf1+/- macrophages produce excessive quantities of monocyte chemotactic protein-1 (MCP-1), the primary ligand for CCR2, which induces Nf1+/- macrophage chemotaxis and Nf1+/- smooth muscle cell (SMC) proliferation. To verify the importance of MCP-1/CCR2 signaling in Nf1 arterial stenosis, we demonstrate that genetic deletion of MCP-1 reduces Nf1+/- arterial stenosis. As MCP-1/CCR2 signaling modulates reactive oxygen species (ROS) production in leukocytes, we show that Nf1+/- macrophages exhibit enhanced generation of ROS and that incubation of Nf1+/- SMC with hydrogen peroxide induces Nf1+/- SMC proliferation and Ras-Erk activation. To interrogate the contribution of Nf1+/- macrophage ROS in vivo, we show that apocynin, an inhibitor of NADPH oxidase (i.e. Nox2) and ROS in leukocytes, completely abrogates neointima formation in Nf1+/- mice. We propose two specific aims to test our overarching hypothesis that loss of neurofibromin induces an inflammatory macrophage phenotype and sensitizes macrophages and SMC to cytokines (i.e. MCP-1) and oxidative stress resulting in severe arterial stenosis. In Aim 1, we will generate compound mutant Nf1+/- mice with genetic deletion of MCP-1 or CCR2 and perform carotid artery ligation to induce arterial stenosis. We will perform primary cell and co-culture experiments with Nf1+/- and WT macrophages and SMC to comprehensively interrogate MCP-1/CCR2 signaling. In Aim 2, we seek to elucidate the mechanism underlying enhanced ROS production in Nf1+/- macrophages by intercrossing Nf1+/- and WT mice with p47phox knockout mice (a cytosolic component of active Nox2). We will also seek to understand how neurofibromin regulates macrophage ROS and elucidate the mechanisms of enhanced Nf1+/- SMC proliferation in response to ROS. Our studies will provide key insights into the pathogenesis of NF1 arterial stenosis and lead to novel therapies for NF1 patients. (AHA Program: Scientist Development Grant)