Abstract 349: Hypoxia Induces the Expression of the Neuroimmune Guidance Cue Netrin-1 in Atherosclerotic Plaques: A Mechanism for Macrophage Retention

Hypoxia is intimately linked to atherosclerosis and promotes its progression by increasing lipid accumulation, inflammation and angiogenesis. We recently demonstrated that the neuroimmune guidance cue, netrin-1 (Ntn1) is expressed in atherosclerotic lesions where it inactivates macrophage (Mφ) migration to chemokines implicated in the egress of these inflammatory cells from plaques, including MCP-1 and CCL19. However, the mechanisms governing Ntn1 expression in atherosclerosis are not known. Immunostaining of mouse atherosclerotic plaques revealed that Ntn1 colocalized with a hypoxia probe in Mφ-rich regions of the plaque. We hypothesized that oxidative stress in the plaque provokes Ntn1 expression in Mφ, leading to local secretion of this negative regulator of Mφ migration. In vitro, stimulation of Mφ with oxidized LDL induced mRNA levels of Ntn1 and its receptor Unc5b by 3- and 6-fold respectively (p<0.01), as well as the HIF-1α target gene, Vegf (3-fold). Similarly, hypoxia mimetics such as CoCL2 (0.1 mM) and DMOG (1 mM), or exposure of Mφ to 4% O2 also upregulated Mφ expression of Ntn1 and Unc5b. Notably, these responses were abrograted by inhibiting HIF1α suggesting a causative role for this transcription factor in regulating Ntn1 and Unc5b expression in Mφ. Indeed, oxLDL-induced hypoxia increased Ntn1 and Unc5b promoter-luciferase activity by 2-5-fold, which was lost upon treatment with a HIF1α inhibitor. Furthermore, gene expression profiling of J774 macrophages overexpressing a stable form of Hif-1α (J774Hif) showed that both Ntn1 and Unc5b mRNAs were upregulated compared to control J744 cells (J774Con). Moreover, J774Hif Mφ showed reduced migration to MCP-1 compared to J774Con, consistent with the secretion of Netrin1 by these cells, and this was reversed by pretreating J774Hif with recombinant Unc5b-Fc to block the effects of Netrin1. Finally, using a bone marrow transplant model we show that targeted deletion of Ntn1 in Mφ dramatically diminishes atherosclerosis progression in Ldlr -/- mice, and promotes Mφ emigration from plaques. Together, these data suggest a causative role for hypoxia in Mφ retention and chronic inflammation in atherosclerotic lesions though its induction of this negative regulator of Mφ migration.