The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Nephrolog

Abstract 825: Impaired ABCA-1 Mediated Cholesterol Efflux In Hypoxic Macrophages

Atherosclerotic plaques contain large numbers of metabolically active cells that can be deprived of normal oxygen (0 2 ) tension, with levels documented below 10mm Hg (~1% O 2 ) in rabbit and human plaques (vs. normal tissue; 20 –70mmHg, 2.5%–9% O 2 ). Hypoxia has been assumed, but not documented in mouse plaques. Hypoxia stabilizes transcription factor HIF-1α, the master regulator of the cellular response to hypoxia and also involved in reduction-oxidation and inflammatory pathways. Atherosclerotic plaques of apoE knockout mice were immunostained and areas + for HIF-1α as well as two downstream targets (VEGF and Glut-1) were present in increased levels in deeper plaque regions. We next investigated the effect of hypoxia on macrophage function in vitro by incubating murine J774 macrophages in 1% 0 2 in DMEM containing 15% FBS for 24 h and found a 30% increase in cellular total cholesterol (TC) associated with hypoxia. In cells loaded by cyclodextrin-cholesterol, a ~400% increase in TC was observed in hypoxic relative to normoxic cells. Assays using apoAI as a cholesterol acceptor showed that in 1% 0 2 there was a ~90% decrease in ABCA-1 mediated efflux relative to normoxia. In spite of this, ABCA-1 mRNA was increased ~2.5 fold in hypoxic vs. normoxic macrophages. ABCA-1 protein levels by Western analysis remained equal in the 2 conditions. However, by immunofluorescence and confocal microscopy, in sharp distinction to the plasma membrane and cytoplasmic distribution seen in normoxic cells, ABCA-1 localized mainly in a juxtanuclear location in hypoxic macrophages, suggesting impaired trafficking to the membrane. Conclusions: HIF-1α is present in murine plaques, suggesting that they have true hypoxic areas; Hypoxia in vitro promotes macrophage cholesterol accumulation concurrent with ABCA-1 dysfunction; and Hypoxia redistributes ABCA1 from the plasma membrane to the juxtanuclear region. </jats:p

Abstract 78: Inhibition of Mir-33 Overcomes the Deleterious Effects of Diabetes on Atherosclerosis Regression.

Objective Diabetes increases the risk of cardiovascular morbidity and mortality which remains elevated with conventional therapies, such as statins. We have shown that diabetes also impairs plaque regression following LDL reduction in mice, as evidenced by a higher macrophage (CD68+ cells) plaque content and a higher inflammatory state of these cells compared to non-diabetic mice. Type-2 diabetics have dyslipidemia characterized by elevated triglyceride levels and low HDL, the latter is thought to contribute to their risk, based on epidemiologic studies. In non-diabetic atherosclerotic mice, inhibition of miR-33, an intronic micro-RNA located within the SREBF2 gene, increases the plasma level of apoAI and HDL-cholesterol (HDL-C) and promotes plaque regression. We hypothesize that elevation of HDL through the inhibition of miR-33 will overcome the deleterious effect of diabetes on macrophage content and inflammatory phenotype in plaques in a regression evironment. Methods and Results Diabetic (STZ-injected) and non-diabetic Reversa (LdLr-/-Apob100/100Mttpfl/flMx1-Cre+/+) mice, a model in which diet-induced hypercholesterolemia can be quickly and efficiently reversed, were fed a western diet for 16 weeks and then treated with anti-miR-33 oligonucleotide or control anti-miR for 4 weeks. Treatment with anti-miR-33 increased HDL-cholesterol in both diabetic (17%) and non-diabetic mice (30%). Anti-miR-33 treated diabetic mice showed an improvement in plaque regression as evidenced by a 26% reduction in CD68 content compared to diabetic mice receiving control anti-miR, and an enrichment in anti-inflammatory M2 macrophages (assessed by MR 1 and YM1). Monocyte tracking with latex beads suggests that the reduction of macrophage content is due to a decrease in monocyte recruitment to the plaque, rather than an increase in macrophage egress. This reduction in recruitment correlated with a decrease in the monocytosis associated with diabetes in the anti-miR-33 treated diabetic mice. Conclusion These findings suggest that miR-33 inhibition is able to overcome the deleterious effects of diabetes on atherosclerosis regression, by decreasing monocytosis, monocyte recruitment and improving the inflammatory status of plaque macrophages. </jats:sec

Scavenger Receptor Class B Type I Is a Plasma Membrane Cholesterol Sensor

Rationale: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. Objective: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. Methods and Results: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-β-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusions: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells. </jats:sec