Hypoxia Induces Netrin-1 and Unc5b in Atherosclerotic Plaques

Abstract

Objective Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue, netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor Unc5b in plaque macrophages, and its functional consequences on these immune cells. Approach and Results We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized LDL or inducers of oxidative stress (CoCL2, DMOG, 1% O2). These responses are abrograted by inhibiting HIF-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxLDL and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression, and reduced migratory capacity compared to control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α-dependent manner. Conclusions These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques, and implicate hypoxia and HIF-1α-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

Association of Neuroimmune Guidance Cue Netrin-1 and Its Chemorepulsive Receptor UNC5B With Atherosclerotic Plaque Expression Signatures and Stability in Human(s)

“…Although recently NTN1 and UNC5B protein have been demonstrated in human atherosclerotic plaques, 15 their association with plaque type and different arterial bed in vivo is lacking.…”

“…15 Recently, it was shown that coronary endothelial cells normally express NTN1, which potently inhibits migration of monocytes in vitro. 26 Our results of downregulation of NTN1 in advanced plaques are supported by the finding that proatherogenic factors (oscillatory shear stress, proinflammatory cytokines) downregulate NTN1 expression in vitro.…”

Macrophages and Dendritic Cells

“…113 The production and secretion of repulsive neuroimmune guidance cues such as netrin-1, ephrin-B, and semaphorins 3A and 3E may contribute to macrophage retention. [114][115][116][117] Hypercholesterolemia also impairs the migration of dermal DCs to regional lymph nodes through generation of platelet-activating factor. 118 Oxidized, but not native LDL, inhibits TLR4-induced peritoneal macrophage efflux into lymphatics and in vitro migration through a process that involves CD36, inactivation of Src homology 2-containing phosphotyrosine phosphatase, sustained activation of focal adhesion kinase, and alteration of cytoskeletal dynamics.…”

HDL and Atherothrombotic Vascular Disease

“…Since the deep intimal region is distant from the circulating blood, it easily becomes hypoxic (Hulten and Levin 2009). Although survival mechanisms are activated in macrophages trapped in such hypoxic areas (Ramkhelawon et al 2013), promotion of lipid accumulation, ATP depletion, and immobilization of the macrophage foam cells is likely to ultimately lead to their death. Of note, death of a foam cell is bound to liberation of its cytoplasmic cholesteryl ester droplets into the extracellular space, where the droplets mix with the preexisting extracellular lipid droplets.…”