Roy Y. Kim

ApoE Suppresses Atherosclerosis by Reducing Lipid Accumulation in Circulating Monocytes and the Expression of Inflammatory Molecules on Monocytes and Vascular Endothelium

Objective—We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results—We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr−/−) were compared to Apoe−/−Ldlr−/− mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr−/− mice displayed similar plasma cholesterol as Apoe−/− Ldlr−/− mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr−/− mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr−/− mice had less circulating leukocytes and proinflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr−/− mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions—Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

A screen in mice uncovers repression of lipoprotein lipase by microRNA‐29a as a mechanism for lipid distribution away from the liver

Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy‐dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR‐29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR‐29a levels causes lipids to accumulate in mouse livers. Conclusion: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR‐29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR‐29as known antifibrotic effect, suggest miR‐29a is a therapeutic target in fatty liver disease. (Hepatology 2015;61:141–152)

The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction.

Abstract: FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61h/h/SRB1−/− mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg−1·d−1) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61h/h/SRB1−/− mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg−1·d−1). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-&bgr; and reduced IFN-&ggr; expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-&bgr; and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

Apolipoprotein E4 Domain Interaction Accelerates Diet-Induced Atherosclerosis in Hypomorphic Arg-61 Apoe Mice

Objectives—Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. Methods and Results—We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE (ApoeTh/h or ApoeRh/hmice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, ApoeRh/h mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with ApoeTh/h mice. In addition, evidence of cellular dysfunction was identified in peritoneal ApoeRh/h macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. Conclusions—These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

Inducible Apoe Gene Repair in Hypomorphic ApoE Mice Deficient in the Low-Density Lipoprotein Receptor Promotes Atheroma Stabilization with a Human-Like Lipoprotein Profile

Objective—To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)–lipoprotein levels. Approach and Results—Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoeh/hLdlr–/–Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non–high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6Chigh monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions—Restoring apoE expression in Apoeh/hLdlr–/–Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non–high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6Chigh monocytes levels and genetic reprogramming of lesional macrophages.

Inducible Apoe Gene Repair in Hypomorphic ApoE Mice Deficient in the LDL Receptor Promotes Atheroma Stabilization with a Human-like Lipoprotein Profile

Objective—To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)–lipoprotein levels. Approach and Results—Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoeh/hLdlr–/–Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non–high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6Chigh monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions—Restoring apoE expression in Apoeh/hLdlr–/–Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non–high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6Chigh monocytes levels and genetic reprogramming of lesional macrophages.

Immunosuppression With FTY720 Reverses Cardiac Dysfunction in Hypomorphic ApoE Mice Deficient in SR-BI Expression That Survive Myocardial Infarction Caused by Coronary Atherosclerosis.

Aims: We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI–/– mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI–/– mice that survive MI and subsequently develop chronic heart failure. Methods/Results: HypoE/SR-BI–/– mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. Conclusions: Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

Isolation of Plasma Lipoproteins as a Source of Extracellular RNA

Plasma lipoproteins are essential vehicles of lipid distribution for cellular energy and structural requirements as well as for excretion of lipid excess. Imbalances in lipoprotein metabolism are known to contribute to metabolic diseases ranging from vascular inflammation and atherosclerosis to obesity and diabetes. The lipid and protein cargo carried by lipoprotein subclasses have long been the focus of studies exploring the contribution of plasma lipoproteins in health and in metabolic disorders. More recent studies have revealed the presence of noncoding RNA as a new form of cargo carried by plasma lipoproteins. Lipoprotein-associated microRNAs have been identified to distribute differentially among plasma lipoprotein subclasses and contribute to cellular signaling. These findings highlight plasma lipoprotein-associated RNA as a potential source of biological signaling and warrant a renewed interest in the study of plasma lipoprotein biology. This chapter describes principles and methods based on density ultracentrifugation and size exclusion chromatography for the isolation of plasma lipoproteins as a source of extracellular RNA.