Faculty Bibliography

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

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 uncoordinated-5-B 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 low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1alpha, 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 oxidized low-density lipoprotein and require HIF-1alpha. Correspondingly, J774 macrophages overexpressing active HIF-1alpha show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with 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-1alpha-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-1alpha-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-C(hi) monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/S100A9, and its subsequent interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions, and promotes regression. In patients with type 1 diabetes, plasma S100A8/S100A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and promotes atherogenesis in diabetes.