Publications

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Native LDL/HDL

Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation.

Khan, N., Datta, G., Geiger, J. D., & Chen, X. (2018). Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation. Journal of neuroinflammation, 15(1), 91.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Kraehling, J. R., Chidlow, J. H., Rajagopal, C., Sugiyama, M. G., Fowler, J. W., Lee, M. Y., ... & Chen, K. (2016). Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells. Nature Communications, 7.

Complement Protein C1q Enhances Macrophage Foam Cell Survival and Efferocytosis

In the atherosclerotic lesion, macrophages ingest high levels of damaged modified low-density lipoproteins (LDLs), generating macrophage foam cells. Foam cells undergo apoptosis and, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, leading to plaque instability and rupture. As a component of the innate immune complement cascade, C1q recognizes and opsonizes modified forms of LDL, such as oxidized or acetylated LDL, and promotes ingestion by macrophages in vitro. C1q was shown to be protective in an atherosclerosis model in vivo. Therefore, this study aimed to investigate whether ingestion of modified LDL in the presence of C1q alters macrophage foam cell survival or function. In an unbiased transcriptome analysis, C1q was shown to modulate expression of clusters of genes involved in cell death and apoptosis pathways in human monocyte–derived macrophages ingesting modified LDL; this was validated by quantitative PCR in human and murine macrophages. C1q downregulated levels and activity of active caspase-3 and PARP-1 in human and mouse macrophages during ingestion of modified LDL. This led to a measurable increase in survival and decrease in cell death, as measured by alamarBlue and propidium iodide assays, respectively. C1q opsonization also increased phagocytosis and efferocytosis in macrophage foam cells. These data suggest that C1q promotes macrophage survival during ingestion of excess cholesterol, as well as improves foam cell efferocytic function. This may be important in slowing disease progression and provides insight into the protective role of C1q in early atherosclerosis.

Pulanco, M. C., Cosman, J., Ho, M. M., Huynh, J., Fing, K., Turcu, J., & Fraser, D. A. (2016). Complement Protein C1q Enhances Macrophage Foam Cell Survival and Efferocytosis. The Journal of Immunology, 1601445.

Identification of Oxidized Phosphatidylinositols Present in OxLDL and Human Atherosclerotic Plaque

Oxidized low-density lipoprotein (OxLDL) plays an important role in initiation and progression of atherosclerosis. Proatherogenic effects of OxLDL have been attributed to bioactive phospholipids generated during LDL oxidation. It is unknown what effect oxidation has on the phosphatidylinositol (PtdIns) molecules in LDL, even though PtdIns is 6% of the total LDL phospholipid pool. We sought to identify and quantitate oxidized phosphatidylinositol (OxPtdIns) species in OxLDL and human atherosclerotic plaque. Bovine liver PtdIns was subjected to non-enzymatic and lipoxygenase-catalyzed oxidation. Reversed-phase liquid chromatography with negative ESI–MS identified and confirmed compounds by fragmentation pattern analysis from which an OxPtdIns library was generated. Twenty-three OxPtdIns molecules were identified in copper-oxidized human LDL at 0, 6, 12, 24, 30, and 48 h, and in human atherosclerotic plaque. In OxLDL, OxPtdIns species containing aldehydes and carboxylates comprised 17.3 ± 0.1 and 0.9 ± 0.2%, respectively, of total OxPtdIns in OxLDL at 48 h. Hydroperoxides and isoprostanes at 24 h (68.5 ± 0.2 and 22.8 ± 0.2%) were significantly greater than 12 h (P ‹ 0.01) without additional changes thereafter. Hydroxides decreased with increased oxidation achieving a minimum at 24 h (5.2 ± 0.3%). Human atherosclerotic plaques contained OxPtdIns species including aldehydes, carboxylates, hydroxides, hydroperoxides and isoprostanes, comprising 18.6 ± 4.7, 1.5 ± 0.7, 16.5 ± 7.4, 33.3 ± 1.1 and 30.2 ± 3.3% of total OxPtdIns compounds. This is the first identification of OxPtdIns molecules in human OxLDL and atherosclerotic plaque. With these novel molecules identified we can now investigate their potential role in atherosclerosis.

Hasanally, D., Edel, A., Chaudhary, R., & Ravandi, A. (2016). Identification of Oxidized Phosphatidylinositols Present in OxLDL and Human Atherosclerotic Plaque. Lipids, 1-16.

Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons

Elevated levels of circulating cholesterol are extrinsic factors contributing to the pathogenesis of sporadic Alzheimer's disease (AD). We showed previously that rabbits fed a cholesterol-enriched diet exhibited blood–brain barrier (BBB) dysfunction, increased accumulation of apolipoprotein B (ApoB) in brain neurons, and endolysosomes in brain had disturbed structures and functions. These effects were linked to increased amyloid beta (Aβ) production, increased tau-pathology, and disrupted synaptic integrity. Because pathological changes to endolysosomes represent a very early event in sporadic AD, we determined here the extent to which ApoB-containing LDL cholesterol altered the structure and function of endolysosomes and contributed to the development of AD-like pathology in primary cultured neurons.

Hui, L., Chen, X., & Geiger, J. D. (2012). Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons. Life sciences, 91(23), 1159-1168.

Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis.

Zhu, X., Ng, H. P., Lai, Y. C., Craigo, J. K., Nagilla, P. S., Raghani, P., & Nagarajan, S. (2014). Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice. The Journal of Immunology, 193(5), 2483-2495. Chicago

Immune Activation Resulting From NKG2D/Ligand Interaction Promotes Atherosclerosis

Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune-activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed with the use of mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortas and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using apolipoprotein E–deficient mouse models, we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions.

Xia, M., Guerra, N., Sukhova, G. K., Yang, K., Miller, C. K., Shi, G. P., ... & Xiong, N. (2011). Immune activation resulting from NKG2D/ligand interaction promotes atherosclerosis. Circulation, 124(25), 2933-2943.

Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction

Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune-activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed with the use of mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortas and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using apolipoprotein E–deficient mouse models, we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions.

Lee, S. K., Chung, J. I., Park, M. S., Joo, H. K., Lee, E. J., Cho, E. J., ... & Jeon, B. H. (2011). Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction. Cardiovascular research, 91(3), 502-509.

Oxidized Low-Density Lipoprotein Contributes to Atherogenesis via Co-activation of Macrophages and Mast Cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor for atherosclerosis, due to its role in endothelial dysfunction and foam cell formation. Tissue-resident cells such as macrophages and mast cells release inflammatory mediators upon activation that in turn cause endothelial activation and monocyte adhesion. Two of these mediators are tumor necrosis factor (TNF)-α, produced by macrophages, and histamine, produced by mast cells. Static and microfluidic flow experiments were conducted to determine the number of adherent monocytes on vascular endothelium activated by supernatants of oxLDL-treated macrophages and mast cells or directly by oxLDL. The expression of adhesion molecules on activated endothelial cells and the concentration of TNF-α and histamine in the supernatants were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. A low dose of oxLDL (8 μg/ml), below the threshold for the clinical presentation of coronary artery disease, was sufficient to activate both macrophages and mast cells and synergistically increase monocyte-endothelium adhesion via released TNF-α and histamine. The direct exposure of endothelial cells to a much higher dose of oxLDL (80 μg/ml) had less effect on monocyte adhesion than the indirect activation via oxLDL-treated macrophages and mast cells. The results of this work indicate that the co-activation of macrophages and mast cells by oxLDL is an important mechanism for the endothelial dysfunction and atherogenesis. The observed synergistic effect suggests that both macrophages and mast cells play a significant role in early stages of atherosclerosis. Allergic patients with a lipid-rich diet may be at high risk for cardiovascular events due to high concentration of low-density lipoprotein and histamine in arterial vessel walls.

Chen, C., & Khismatullin, D. B. (2015). Oxidized low-density lipoprotein contributes to atherogenesis via co-activation of macrophages and mast cells. PloS one, 10(3), e0123088.

Insulin-like growth factor-1 regulates glutathione peroxidase expression and activity in vascular endothelial cells: Implications for atheroprotective actions of insulin-like growth factor-1

Oxidative stress promotes endothelial cell senescence and endothelial dysfunction, important early steps in atherogenesis. To investigate potential antioxidant effects of IGF-1 we treated human aortic endothelial cells (hAECs) with 0–100 ng/mL IGF-1 prior to exposure to native or oxidized low-density lipoprotein (oxLDL). IGF-1 dose- and time- dependently reduced basal- and oxLDL-induced ROS generation. IGF-1 did not alter superoxide dismutase or catalase activity but markedly increased activity of glutathione peroxidase (GPX), a crucial antioxidant enzyme, via a phosphoinositide-3 kinase dependent pathway. IGF-1 did not increase GPX1 mRNA levels but increased GPX1 protein levels by 2.6-fold at 24 h, and altered selenocysteine-incorporation complex formation on GPX1 mRNA. Furthermore, IGF-1 blocked hydrogen peroxide induced premature cell senescence in hAECs. In conclusion, IGF-1 upregulates GPX1 expression in hAECs via a translational mechanism, which may play an important role in the ability of IGF-1 to reduce endothelial cell oxidative stress and premature senescence. Our findings have major implications for understanding vasculoprotective effects of IGF-1.

Higashi, Y., Pandey, A., Goodwin, B., & Delafontaine, P. (2013). Insulin-like growth factor-1 regulates glutathione peroxidase expression and activity in vascular endothelial cells: Implications for atheroprotective actions of insulin-like growth factor-1. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1832(3), 391-399.

MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis

We over expressed and knocked down miR-27a in HepG2 cells to assess its effect on the expression of key players in the LDLR pathway using PCR Arrays, Elisas, and Western blots. We found that miR-27a decreases LDLR levels by 40% not only through a direct binding to its 3′ untranslated region but also indirectly by inducing a 3-fold increase in PCSK9, which enhances LDLR degradation. Interestingly, miR-27a also directly decreases LRP6 and LDLRAP1, two other key players in the LDLR pathway that are required for efficient endocytosis of the LDLR-LDL-C complex in the liver. The inhibition of miR-27a using lock nucleic acids induced a 70% increase in LDLR levels and, therefore, it would be a more efficient treatment for hypercholesterolemia because of its desirable effects not only on LDLR but also on PCSK9.

Alvarez, M. L., Khosroheidari, M., Eddy, E., & Done, S. C. (2015). MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis. Atherosclerosis, 242(2), 595-604.

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR−/−) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR−/− osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR−/− preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V0 subunit d2 and dendritic cell-specific transmembrane protein in LDLR−/− plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR−/− mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

Okayasu, M., Nakayachi, M., Hayashida, C., Ito, J., Kaneda, T., Masuhara, M., ... & Hakeda, Y. (2012). Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion. Journal of Biological Chemistry, 287(23), 19229-19241.

Potent antioxidant dendrimers lacking pro-oxidant activity

It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants, including polyphenols with potent antioxidant activities, may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical-scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens, and electron-donating ring substituents that contribute to their potent free radical-quenching properties. To minimize their pro-oxidant effects, the dendrimers were designed with a metal-chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical-scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme, and DNA against 2,2′-azobis(2-amidinopropane) dihydrochloride-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity toward Chinese hamster ovary cells.

Lee, C. Y., Sharma, A., Uzarski, R. L., Cheong, J. E., Xu, H., Held, R. A., ... & Nelson, J. L. (2011). Potent antioxidant dendrimers lacking pro-oxidant activity. Free Radical Biology and Medicine, 50(8), 918-925.

Oxidized Low-Density Lipoprotein Is a Novel Predictor of Interferon Responsiveness in Chronic Hepatitis C Infection

The unexplained association between interferon responsiveness and serum low-density lipoprotein (LDL) in chronic hepatitis C is likely due to oxidized LDL, a subfraction that blocks viral cell entry by perturbing the interaction between hepatitis C virus and its primary receptor.

Solbach, P., Westhaus, S., Deest, M., Herrmann, E., Berg, T., Manns, M. P., ... & von Hahn, T. (2015). Oxidized low-density lipoprotein is a novel predictor of interferon responsiveness in chronic hepatitis C infection. CMGH Cellular and Molecular Gastroenterology and Hepatology, 1(3), 285-294.

Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation

Alternative (M2) macrophage activation driven through interleukin 4 receptor α (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of fatty acids to support this metabolic program has not been clear. We show that the uptake of triacylglycerol substrates via CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation (OXPHOS), enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth, and blocked protective responses against this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

Abumrad, N. A., Artyomov, M. N., Pearce, E. J., Lam, W. Y., Everts, B., Yan, C., ... & O'Sullivan, D. (2014). Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation.

Effect of electron donating groups on polyphenol-based antioxidant dendrimers

Numerous studies have reported the beneficial effects of antioxidants in human diseases. Among their biological effects, a majority of antioxidants scavenge reactive radicals in the body, thereby reducing oxidative stress that is associated with the pathogenesis of many diseases. Antioxidant dendrimers are a new class of potent antioxidant compounds reported recently. In this study, six polyphenol-based antioxidant dendrimers with or without electron donating groups (methoxy group) were synthesized in order to elucidate the influence of electron donating groups (EDG) on their antioxidant activities. Syringaldehyde (2 ortho methoxy groups), vanillin (1 ortho methoxy group), and 4-hydroxybenzaldehyde (0 methoxy group) were derivatized with propargylamine to form building blocks for the dendrimers. All the six dendrimers contain polyether cores, which were synthesized by attaching pentaerythritol and methyl α-d-glucopyranoside to in-house prepared spacer units. To prepare generation 1 antioxidant dendrimers, microwave energy and granulated metallic copper catalyst were used to link the cores and building blocks together via alkyne-azide 1,3-cycloaddition click chemistry. These reaction conditions resulted in high yields of the target dendrimers that were free from copper contamination. Based on DPPH antioxidant assay, antioxidant dendrimers decorated with syringaldehyde and vanillin exhibited over 70- and 170-fold increase in antioxidant activity compared to syringaldehyde and vanillin, respectively. The antioxidant activity of dendrimers increased with increasing number of EDG groups. Similar results were obtained when the dendrimers were used to protect DNA and human LDL against organic carbon and nitrogen-based free radicals. In addition, the antioxidant dendrimers did not show any pro-oxidant activity on DNA in the presence of physiological amounts of copper. Although the dendrimers showed potent antioxidant activities against carbon and nitrogen free radicals, EPR and DNA protection studies revealed lack of effectiveness of these dendrimers against hydroxyl radicals. The dendrimers were not cytotoxic to CHO-K1 cells.

Lee, C. Y., Nanah, C. N., Held, R. A., Clark, A. R., Huynh, U. G., Maraskine, M. C., ... & Sharma, A. (2015). Effect of electron donating groups on polyphenol-based antioxidant dendrimers. Biochimie, 111, 125-134.

Oxidized LDL

The SCFFBXO3 ubiquitin E3 ligase regulates inflammation in atherosclerosis

Inflammation is critical in the pathobiology of atherosclerosis. An essential player in the inflammatory process in atherosclerosis are macrophages that scavenge oxidatively modified low-density lipoproteins (OxLDL) deposited in the subendothelium of systemic arteries that secrete a myriad of pro-inflammatory mediators. Here, we identified that a subunit of the Skp-Cullin-F-box ubiquitin E3 ligase apparatus, termed FBXO3, modulates the inflammatory response in atherosclerosis. Specifically, individuals with a hypofunctioning genetic variant of FBXO3 develop less atherosclerosis. FBXO3 protein is present in cells of monocytic lineage within carotid plaques and its levels increase in those with symptomatic compared with asymptomatic atherosclerosis. Further, cellular depletion or small molecule inhibition of FBXO3 significantly reduced the inflammatory response to OxLDL by macrophages without altering OxLDL uptake. Thus, FBXO3 potentiates vascular inflammation and atherosclerosis that can be effectively mitigated by a small molecule inhibitor.

Chandra, D., Londino, J., Alexander, S., Bednash, J. S., Zhang, Y., Friedlander, R. M., ... & Ross, M. (2019). The SCFFBXO3 ubiquitin E3 ligase regulates inflammation in atherosclerosis. Journal of molecular and cellular cardiology, 126, 50-59.

A positive feedback loop between IL-1β, LPS and NEU1 may promote atherosclerosis by enhancing a pro-inflammatory state in monocytes and macrophages

Inflammation plays an important role in atherosclerosis, a notion supported by the beneficial effects of the IL-1β inhibitor canakinumab in the CANTOS trial. Sialic acids (Sias), components of the surface glycocalyx, regulate intercellular and intermolecular interactions. We investigated the expression of the Sia cleaving enzyme neuraminidase-1 (NEU1) in atherosclerotic plaques and its potential role in inflammatory processes. In isolated mononuclear blood cells from patients with myocardial infarction, NEU1 expression was increased compared to healthy controls. High expression of NEU1 in macrophages located on the intima layer, in calcified regions and the adventitia of the plaque was observed in human carotid arteries' atherectomies. IL-1β and LPS induced NEU1 expression in THP-1 monocytic cells. Lentiviral NEU1-overexpression in THP-1-cells enhanced expression of CD80, TNF-α, IL-1β, number of multinuclear cells, phagocytosis and chemotaxis indicative for M1 monocyte/macrophage polarization. CRISPR/Cas9-mediated knock-out of NEU1 in THP-1-cells did not affect differentiation of monocytes to macrophages but attenuated LPS- and IL-1β -induced TNF-α and IL-1β expression. SiRNA-mediated knock-down of NEU1 in M1-macrophages differentiated from primary human CD14+ monocytes reduced the expression of TNF-α and IL-1β. Thus, in monocytes/macrophages, LPS, NEU1 and IL-1β act in a positive feedback loop as enhancers of inflammation and may therefore promote atherosclerosis and plaque instability.

Sieve, I., Ricke-Hoch, M., Kasten, M., Battmer, K., Stapel, B., Falk, C. S., ... & Hilfiker-Kleiner, D. (2018). A positive feedback loop between IL-1β, LPS and NEU1 may promote atherosclerosis by enhancing a pro-inflammatory state in monocytes and macrophages. Vascular pharmacology, 103, 16-28.

Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model.

Jackisch, L., Kumsaiyai, W., Moore, J. D., Al-Daghri, N., Kyrou, I., Barber, T. M., ... & McTernan, P. G. (2018). Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids. Diabetologia, 61(5), 1155-1166.

Anti inflammatory effect of asiaticoside on human umbilical vein endothelial cells induced by ox-LDL

Early diagnosis and changes associated with atherosclerosis are crucial in clinical medicine. However, atherosclerosis is a multifactorial disease. Asiaticoside (AA), a triterpenoid derived from Centella asiatica, has anti-inflammatory activity. Endothelium-derived nitric oxide is important in modulating vascular tone in a distinct vessel size-dependent manner; it plays a dominant role in conduit arteries and endothelium-dependent hyperpolarisation in resistance vessels. This study evaluated the effects of AA administration on human umbilical endothelial cells with oxidised low-density lipoprotein-induced inflammation. We measured the levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).Taken together, our data suggest that AA inhibits the augmentation of endothelial permeability, thus preventing the early events of atherosclerosis.

Jing, L., Haitao, W., Qiong, W., Fu, Z., Nan, Z., & Xuezheng, Z. (2018). Anti inflammatory effect of asiaticoside on human umbilical vein endothelial cells induced by ox-LDL. Cytotechnology, 70(2), 855-864.

Vasoactive Intestinal Peptide Ameliorates Acute Myocarditis and Atherosclerosis by Regulating Inflammatory and Autoimmune Responses

Vasoactive intestinal peptide (VIP) is a neuropeptide that exerts various vascular and cardioprotective functions and regulates immune function and inflammatory response at multiple levels. However, its role in inflammatory cardiovascular disorders is largely unknown. Myocarditis and atherosclerosis are two inflammatory and autoimmune cardiovascular diseases that cause important adverse circulatory events. In this study, we investigate the therapeutic effects of VIP in various well-established preclinical models of experimental autoimmune myocarditis and atherosclerosis. Intraperitoneal injection of VIP during the effector phase of experimental autoimmune myocarditis in susceptible BALB/c mice significantly reduced its prevalence, ameliorated signs of heart hypertrophy and injury, attenuated myocardial inflammatory infiltration, and avoided subsequent profibrotic cardiac remodeling. This effect was accompanied by a reduction of Th17-driven cardiomyogenic responses in peripheral lymphoid organs and in the levels of myocardial autoantibodies. In contrast, acute and chronic atherosclerosis was induced in apolipoprotein E–deficient mice fed a hyperlipidemic diet and subjected to partial carotid ligation. Systemic VIP treatment reduced the number and size of atherosclerotic plaques in carotid, aorta, and sinus in hypercholesterolemic mice. VIP reduced Th1-driven inflammatory responses and increased regulatory T cells in atherosclerotic arteries and their draining lymph nodes. VIP also regulated cholesterol efflux in macrophages and reduced the formation of foam cells and their presence in atherosclerotic plaques. Finally, VIP inhibited proliferation and migration of smooth muscle cells and neointima formation in a mouse model of complete carotid ligation. These findings encourage further studies aimed to assess whether VIP can be used as a pharmaceutical agent to treat heart inflammation and atherosclerosis.

Benitez, R., Delgado-Maroto, V., Caro, M., Forte-Lago, I., Duran-Prado, M., O’Valle, F., ... & Delgado, M. (2018). Vasoactive Intestinal Peptide Ameliorates Acute Myocarditis and Atherosclerosis by Regulating Inflammatory and Autoimmune Responses. The Journal of Immunology, 200(11), 3697-3710.

Impaired antioxidant activity of high-density lipoprotein in chronic kidney disease

Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and increased mortality from cardiovascular disease. CKD results in oxidative stress, inflammation, and high-density lipoprotein (HDL) deficiency, which work in concert to promote atherosclerosis. Normal HDL confers protection against atherosclerosis by inhibiting the oxidation of lipids and lipoproteins and by retrieving surplus cholesterol and phospholipids from lipid-laden cells in the artery wall for disposal in the liver (reverse cholesterol transport). The plasma level of oxidized low-density lipoprotein (LDL) is increased, plasma HDL-cholesterol is reduced, and HDL maturation is impaired in CKD. This study was designed to examine the antioxidant properties of HDL in patients with CKD. In all, 32 stable hemodialysis-dependent patients and 13 age-matched controls were studied. HDL was isolated and used for determination of in vitro antioxidant activity. In addition, the plasma level of key components of HDL, namely paraoxonase (PON), glutathione peroxidase (GPX), platelet activating factor acetylhydrolase (PAF-AH), lecithin cholesterol acyltransferase (LCAT), and apolipoprotein A-I (ApoA-I), were measured. The end-stage renal disease (ESRD) patients exhibited significant reductions of HDL-cholesterol, ApoA-I (–41%), GPX (–50%), and LCAT (–60%) concentrations, and a decrease in PON (–30%) and GPX (–50%) activities. These results were accompanied by a marked reduction of antioxidant activity of HDL (–127%), which was unaffected by the hemodialysis procedure. Thus, in addition to diminished plasma HDL concentration, the composition and antioxidant activity of HDL are altered in CKD; these events can contribute to a heightened risk of atherosclerosis.

Moradi, H., Pahl, M. V., Elahimehr, R., & Vaziri, N. D. (2009). Impaired antioxidant activity of high-density lipoprotein in chronic kidney disease. Translational Research, 153(2), 77-85.

Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR) activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries.

Wei, Y. M., Li, X., Xu, M., Abais, J. M., Chen, Y., Riebling, C. R., ... & Zhang, Y. (2013). Enhancement of autophagy by simvastatin through inhibition of Rac1-mTOR signaling pathway in coronary arterial myocytes. Cellular Physiology and Biochemistry, 31(6), 925-937.

Attenuation by Statins of Membrane Raft-Redox Signaling in Coronary Arterial Endothelium

Membrane raft (MR)–redox signaling platforms associated with NADPH oxidase are involved in coronary endothelial dysfunction. Here, we studied whether statins interfere with the formation of MR-redox signaling platforms to protect the coronary arterial endothelium from oxidized low-density lipoprotein (OxLDL)–induced injury and from acute hypercholesterolemia. In cultured human coronary arterial endothelial cells, confocal microscopy detected the formation of an MRs clustering when they were exposed to OxLDL, and such MR platform formation was inhibited markedly by statins, including pravastatin and simvastatin. In these MR clusters, NADPH oxidase subunits gp91phox and p47phox were aggregated and were markedly blocked by both statins. In addition, colocalization of acid sphingomyelinase (ASM) and ceramide was induced by OxLDL, which was blocked by statins. Electron spin resonance spectrometry showed that OxLDL-induced superoxide (O2.−) production in the MR fractions was substantially reduced by statins. In coronary artery intima of mice with acute hypercholesterolemia, confocal microscopy revealed a colocalization of gp91phox, p47phox, ASM, or ceramide in MR clusters. Such colocalization was rarely observed in the arteries of normal mice or significantly reduced by pretreatment of hypercholesterolemic mice with statins. Furthermore, O2.− production in situ was 3-fold higher in the coronary arteries from hypercholesterolemic mice than in those from normal mice, and such increase was inhibited by statins. Our results indicate that blockade of MR-redox signaling platform formation in endothelial cell membrane may be another important therapeutic mechanism of statins in preventing endothelial injury and atherosclerosis and may be associated with their direct action on membrane cholesterol structure and function.

Xia, M., Guerra, N., Sukhova, G. K., Yang, K., Miller, C. K., Shi, G. P., ... & Xiong, N. (2011). Immune activation resulting from NKG2D/ligand interaction promotes atherosclerosis. Circulation, 124(25), 2933-2943.

Tauroursodeoxycholic Acid Attenuates Lipid Accumulation in Endoplasmic Reticulum-Stressed Macrophages

Treatment of cultured human macrophages with the ER-stressor tunicamycin caused an increase in the protein levels of CD-36, and augmentation of lipid-uptake both of which were inhibited by TUDCA. TUDCA-treatment inhibited tunicamycin-induced ER-stress as evidenced by the attenuation of phosphorylation of eukaryotic translation initiation factor-2α and glucose reactive protein-78. In addition, TUDCA improved insulin signaling in macrophages by augmenting Akt-phosphorylation and blunting c-Jun N-terminal kinase activity.

Hua, Y., Kandadi, M. R., Zhu, M., Ren, J., & Sreejayan, N. (2010). Tauroursodeoxycholic acid attenuates lipid accumulation in endoplasmic reticulum-stressed macrophages. Journal of cardiovascular pharmacology, 55(1), 49.

Regulation of AMPK Activation by CD36 Links Fatty Acid Uptake to β-Oxidation

Increases in muscle energy needs activate AMPK and induce sarcolemmal recruitment of the fatty acid (FA) translocase CD36. The resulting rises in FA uptake and FA oxidation are tightly correlated, suggesting coordinated regulation. We explored the possibility that membrane CD36 signaling might influence AMPK activation. We show, using several cell types, including myocytes, that CD36 expression suppresses AMPK, keeping it quiescent, while it mediates AMPK activation by FA. These dual effects reflect the presence of CD36 in a protein complex with the AMPK kinase LKB1 (liver kinase B1) and the src kinase Fyn. This complex promotes Fyn phosphorylation of LKB1 and its nuclear sequestration, hindering LKB1 activation of AMPK. FA interaction with CD36 dissociates Fyn from the protein complex, allowing LKB1 to remain cytosolic and activate AMPK. Consistent with this, CD36−/− mice have constitutively active muscle and heart AMPK and enhanced FA oxidation of endogenous triglyceride stores. The molecular mechanism described, whereby CD36 suppresses AMPK, with FA binding to CD36 releasing this suppression, couples AMPK activation to FA availability and would be important for the maintenance of cellular FA homeostasis. Its dysfunction might contribute to the reported association of CD36 variants with metabolic complications of obesity in humans.

Samovski, D., Sun, J., Pietka, T., Gross, R. W., Eckel, R. H., Su, X., ... & Abumrad, N. A. (2015). Regulation of AMPK activation by CD36 links fatty acid uptake to β-oxidation. Diabetes, 64(2), 353-359.

The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis

Interleukin-10 (IL-10) may have therapeutic potential in various inflammatory diseases, including atherosclerosis, as it can inhibit oxLDL-induced foam cell formation and apoptosis in macrophages. This study investigated the effect of IL-10 on mitogen-activated protein kinase (MAPK) activation, and apoptosis induced by oxidized low-density lipoprotein (oxLDL) in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that IL-10 significantly blocked the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and apoptosis induced by oxLDL. The inhibitory effect of IL-10 on oxLDL-induced apoptosis was partially dependent on reduced p38, but not JNK, phosphorylation. This study also discovered a linkage between IL-10 and p38 MAPK signaling in oxLDL-induced endothelial cell apoptosis. Interestingly, this study found that lectin-like oxidized LDL receptor-1 (LOX-1) was the only scavenger receptor, on the surface of HUVEC, that was upregulated by oxLDL and the increase in LOX-1 was not suppressed by IL-10. This study confirmed that IL-10 significantly upregulated the expression of suppressor of cytokine signaling-3 (SOCS3), whereas SOCS3 knockdown by siRNA effectively blocked the inhibitory effect of IL-10 on p38 MAPK-dependent apoptosis induced by oxLDL. These results showed for the first time, that IL-10 modulated oxLDL-induced apoptosis by upregulating SOCS3, which then interrupted p38 MAPK activation in endothelial cells. These findings support the essential role of p38 MAPK in the interplay of oxLDL and IL-10 in endothelial apoptosis.

Yin, Y., Liu, W., Ji, G., & Dai, Y. (2013). The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis. European journal of cell biology, 92(4), 150-159.

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.

Bakthavatsalam, D., Soung, R. H., Tweardy, D. J., Chiu, W., Dixon, R. A., & Woodside, D. G. (2014). Chaperonin‐containing TCP‐1 complex directly binds to the cytoplasmic domain of the LOX‐1 receptor. FEBS letters, 588(13), 2133-2140.

MiR-490-3p modulates proliferation of vascular smooth muscle cells induced by ox-LDL through targeting PAPP-A

Using miRNA microarray, real-time PCR and luciferase reporter assay, our study showed that miR-490-3p and IGF1 expression was down-regulated, while PAPP-A and IGF2 expression was up-regulated significantly in hCASMC treated by ox-LDL and/or in human atherosclerotic plaque. Further studies indicated PAPP-A was the target gene of miR-490-3p. The miR-490-3p mimic could inhibit the up-regulation of PAPP-A induced by ox-LDL in hCASMC, which resulted in the suppression of the metalloprotease effect of PAPP-A on IGFBP4. All these effects leaded to the inhibition of the proliferation of hCASMC induced by ox-LDL.

Sun, Y., Chen, D., Cao, L., Zhang, R., Zhou, J., Chen, H., ... & Wang, Z. (2013). MiR-490-3p modulates proliferation of vascular smooth muscle cells induced by ox-LDL through targeting PAPP-A. Cardiovascular research, cvt172.

Ox-LDL-induced TGF-β1 production in human alveolar epithelial cells: Involvement of the Ras/ERK/PLTP pathway

Oxidized-low density lipoprotein (Ox-LDL) has been shown to play an important role in impaired surfactant metabolism and transforming growth factor-β1 (TGF-β1) is a critical mediator in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In this study, we investigated whether Ox-LDL can induce TGF-β1 protein production, and if so, how it achieves this induction in human alveolar epithelial cells (A549). We show here that Ox-LDL not only caused a dose- and time-dependent up-regulation of TGF-β1 production, but also increased Smad3 phosphorylation, Ras/extracellular signal-regulated kinase (ERK) activity and phospholipid transfer protein (PLTP) expression in A549 cells. The inhibition of Ras/ERK activity with specific inhibitors significantly suppressed Ox-LDL-induced TGF-β1 production, Smad3 phosphorylation and PLTP expression. Furthermore, treatment of cells with PLTP siRNA suppressed both TGF-β1 release and Smad3 activation induced by Ox-LDL, but not the activation of Ras/ERK cascade. Taken together, we provide evidences that induction of TGF-β1 production and Smad3 phosphorylation by Ox-LDL is mediated by Ras/ERK/PLTP pathway in human alveolar epithelial cells. J. Cell. Physiol. 227: 3185–3191, 2012. © 2011 Wiley Periodicals, Inc.

Guo, L. L., Chen, Y. J., Wang, T., An, J., Wang, C. N., Shen, Y. C., ... & Xu, D. (2012). Ox‐LDL‐induced TGF‐β1 production in human alveolar epithelial cells: Involvement of the Ras/ERK/PLTP pathway. Journal of cellular physiology, 227(9), 3185-3191.

Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation.

Quinn, K. L., Henriques, M., Tabuchi, A., Han, B., Yang, H., Cheng, W. E., ... & Tullis, E. (2011). Human neutrophil peptides mediate endothelial-monocyte interaction, foam cell formation, and platelet activation. Arteriosclerosis, thrombosis, and vascular biology, 31(9), 2070-2079.

B-1a Lymphocytes Attenuate Insulin Resistance

Obesity-associated insulin resistance, a common precursor of type 2 diabetes, is characterized by chronic inflammation of tissues, including visceral adipose tissue (VAT). Here we show that B-1a cells, a subpopulation of B lymphocytes, are novel and important regulators of this process. B-1a cells are reduced in frequency in obese high-fat diet (HFD)-fed mice, and EGFP interleukin-10 (IL-10) reporter mice show marked reductions in anti-inflammatory IL-10 production by B cells in vivo during obesity. In VAT, B-1a cells are the dominant producers of B cell–derived IL-10, contributing nearly half of the expressed IL-10 in vivo. Adoptive transfer of B-1a cells into HFD-fed B cell–deficient mice rapidly improves insulin resistance and glucose tolerance through IL-10 and polyclonal IgM-dependent mechanisms, whereas transfer of B-2 cells worsens metabolic disease. Genetic knockdown of B cell–activating factor (BAFF) in HFD-fed mice or treatment with a B-2 cell–depleting, B-1a cell–sparing anti-BAFF antibody attenuates insulin resistance. These findings establish B-1a cells as a new class of immune regulators that maintain metabolic homeostasis and suggest manipulation of these cells as a potential therapy for insulin resistance.

Shen, L., Chng, M. H. Y., Alonso, M. N., Yuan, R., Winer, D. A., & Engleman, E. G. (2015). B-1a lymphocytes attenuate insulin resistance. Diabetes, 64(2), 593-603.

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

Osteoclastogenesis in LDLR-deficient mice was reduced because of impaired cell fusion in preosteoclasts consistent with reduced Atp6v0d2 and DC-STAMP proteins, both of which regulate the fusion in the plasma membrane.

Okayasu, M., Nakayachi, M., Hayashida, C., Ito, J., Kaneda, T., Masuhara, M., ... & Hakeda, Y. (2012). Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion. Journal of Biological Chemistry, 287(23), 19229-19241.

Interleukin-10 increases reverse cholesterol transport in macrophages through its bidirectional interaction with liver X receptor α

Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL)2 and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation.

Halvorsen, B., Holm, S., Yndestad, A., Scholz, H., Sagen, E. L., Nebb, H., ... & Aukrust, P. (2014). Interleukin-10 increases reverse cholesterol transport in macrophages through its bidirectional interaction with liver X receptor α. Biochemical and biophysical research communications, 450(4), 1525-1530.

The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor

The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3′-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.

Choe, N., Kwon, J. S., Kim, Y. S., Eom, G. H., Ahn, Y. K., Baik, Y. H., ... & Kook, H. (2015). The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor. Cellular signalling, 27(6), 1056-1065.

Cerebrovascular dysfunction and blood-brain barrier permeability induced by oxidized LDL are prevented by apocynin and magnesium sulfate in female rats

Oxidized low-density lipoprotein (oxLDL) is elevated during several neurologic conditions that involve cerebral edema formation, including severe preeclampsia and eclampsia; however, our understanding of its effect on the cerebral vasculature is limited. We hypothesized that oxLDL induced blood-brain barrier (BBB) disruption and changes in cerebrovascular reactivity occurs through NADPH oxidase-derived superoxide. We also investigated the effect of MgSO4 on oxLDL-induced changes in the cerebral vasculature as this is commonly used in preventing cerebral edema formation. Posterior cerebral arteries (PCA) from female rats were perfused with 5μg/mL oxLDL in rat serum with or without 50μM apocynin or 16mM MgSO4 and BBB permeability and vascular reactivity were compared. oxLDL increased BBB permeability and decreased myogenic tone that were prevented by apocynin. oxLDL increased constriction to the nitric oxide synthase inhibitor L-NNA that was unaffected by apocynin. oxLDL enhanced dilation to the NO donor sodium nitroprusside that was prevented by apocynin. MgSO4 prevented oxLDL-induced BBB permeability without affecting oxLDL-induced changes in myogenic tone. Thus, oxLDL appears to cause BBB disruption and vascular tone dysregulation through NADPH oxidase-derived superoxide. These results highlight oxLDL and NADPH oxidase as potentially important therapeutic targets in neurologic conditions that involve elevated oxLDL.

Schreurs, M. P., & Cipolla, M. J. (2014). Cerebrovascular dysfunction and blood-brain barrier permeability induced by oxidized LDL are prevented by apocynin and magnesium sulfate in female rats. Journal of cardiovascular pharmacology, 63(1), 33.

Oxidized Low-Density Lipoprotein Is a Novel Predictor of Interferon Responsiveness in Chronic Hepatitis C Infection

In the multivariate analysis, oxLDL was found to be an independent predictor of SVR. Oxidized LDL did not correlate with markers of inflammation (alanine transaminase, ferritin), nor was serum oxLDL affected by exogenous interferon administration. Also, oxLDL did not alter the sensitivity of HCV replication to interferon. However, oxLDL was found to be a potent inhibitor of cell-to-cell spread of HCV between adjacent cells in vitro. It could thus reduce the rate at which new cells are infected by HCV through either the cell-free or cell-to-cell route. Finally, serum oxLDL was significantly associated with the estimated infected cell loss rate under treatment.

Solbach, P., Westhaus, S., Deest, M., Herrmann, E., Berg, T., Manns, M. P., ... & von Hahn, T. (2015). Oxidized low-density lipoprotein is a novel predictor of interferon responsiveness in chronic hepatitis C infection. CMGH Cellular and Molecular Gastroenterology and Hepatology, 1(3), 285-294.

MicroRNA-150 Regulates Lipid Metabolism and Inflammatory Response

We stimulated human THP-1 macrophages with oxLDL and found that one of the miRNAs, miR-150, strongly responded to the lipid accumulation and inflammatory response in these cells. Overexpression of miR-150 in macrophage cells resulted in an increase in lipid accumulation, accompanying with a high expression of several pro-inflammatory cytokines. Conversely, when miR-150 knockout mice were challenged with a high fat diet, these mice presented reduced whole body weight with less fat accumulation, improved systemic glucose tolerance and insulin sensitivity. The expression of pro-inflammatory cytokines in the insulin target adipose tissues was reduced in miR-150 null mice. We identified Adiponectin receptor 2 (AdipoR2) as a potential miR-150 target gene and suggested it may play an important role in miR-150-mediated lipid metabolism and inflammatory response.

Luo, N., Garvey, W. T., Wang, D. Z., & Fu, Y. (2014). MicroRNA-150 Regulates Lipid Metabolism and Inflammatory Response. Journal of Metabolic Syndrome, 2014.

Lectin-Like Oxidized Low-Density Lipoprotein 1 Receptor in a Reduced Uteroplacental Perfusion Pressure Rat Model of Preeclampsia

Preeclampsia is a major cause of maternal and fetal morbidity and mortality that has been associated with endothelial dysfunction attributed, in part, to dyslipidemia, an imbalance in angiogenic factors and oxidative stress. One of the many factors that have been shown to be elevated in women with preeclampsia is low-density lipoprotein (LDL) and the more oxidizable, small dense LDL, which can lead to increased vascular oxidative stress and decreased bioavailability of NO. Lectin-like oxidized LDL-1 receptor (LOX-1) is a specific receptor for oxidized LDL. We hypothesized that a reduction of placental perfusion using a rat model of reduced uteroplacental perfusion pressure would result in enhanced LOX-1 expression in the maternal vasculature causing impaired vascular endothelial function through the actions of increased superoxide production and decreased NO-mediated vasodilation. We demonstrated a significant increase in the expression of the LOX-1 receptor (4.3-fold; P=0.002), endothelial NO synthase (2.7-fold; P=0.001), and superoxide (P=0.02) in thoracic aorta of the reduced uteroplacental perfusion pressure model, whereas maximal vasodilator function was modestly decreased (P< 0.05). Endothelial-dependent vasodilator function was unaffected by either oxidized LDL or an LOX-1 receptor inhibitor in controls but was modestly increased in the presence of both oxidized LDL and the LOX-1 receptor inhibitor in reduced uteroplacental perfusion pressure (P=0.03). In summary, we have shown that, in a rat model of preeclampsia, there is a dramatic increase in the expression levels of both the LOX-1 receptor and the endothelial NO synthase enzyme, along with evidence of increased superoxide production and subsequent modestly decreased endothelial function.

Morton, J. S., Abdalvand, A., Jiang, Y., Sawamura, T., Uwiera, R. R., & Davidge, S. T. (2012). Lectin-like oxidized low-density lipoprotein 1 receptor in a reduced uteroplacental perfusion pressure rat model of preeclampsia. Hypertension, 59(5), 1014-1020.

Digested protein isolate from fresh and stored Carioca beans reduced markers of atherosclerosis in oxidized LDL-induced THP-1 macrophages

Oxidized LDL (ox-LDL) and its interaction with lectin type ox-LDL receptor (LOX-1) determine atherosclerosis progression. The potential of peptides from digested protein isolate (DPI) derived from fresh and stored Carioca bean (Phaseolus vulgaris L.) to prevent ox-LDL induced inflammation in human THP-1 macrophage-like cells was examined here. BRS Pontal (PO) and BRSMG Madreperola (MP) fresh and stored for 6 months were cooked, and protein isolates were digested by pepsin–pancreatin. Peptides from stored Carioca beans decreased prostaglandin E-2 (PGE-2) (from 17.7 to 47%) and tumour necrosis factor-α (TNF-α) (12.3 to 82%). Stored MP (0.01 mg/mL) inhibited reactive oxygen species (ROS). LOX-1 and matrix metalloproteinase-9 (MMP-9) expression were reduced by peptides from all beans. Stored PO and MP digested protein isolates reduced expression of 10 cytokines related to atherosclerosis process. Peptides from Carioca beans presented anti-atherosclerotic properties comparable to simvastatin, which were related to inhibition of LOX-1, MMP-9 and ICAM-1.

Alves, N. E., Vasconcelos, C. M., Bassinello, P. Z., de Mejia, E. G., & Martino, H. S. (2016). Digested protein isolate from fresh and stored Carioca beans reduced markers of atherosclerosis in oxidized LDL-induced THP-1 macrophages. Journal of Functional Foods, 24, 97-111.

The role of macrophage transcription factor MafB in atherosclerotic plaque stability

There was no significant difference in advanced atherosclerotic lesion area between DN-MafB/ApoE KO mice and littermate control ApoE KO mice 9 weeks after high-cholesterol diet. However, DN-MafB/ApoE KO mice showed significantly larger necrotic cores and lower collagen content in atherosclerotic plaques than ApoE KO mice. Although there was no difference in intraplaque macrophage infiltration and efferocytosis, DN-MafB/ApoE KO mice showed significantly more apoptotic macrophages at the plaque edges than did ApoE KO mice. Real-time PCR analysis revealed that peritoneal macrophages of DN-MafB/ApoE KO mice had a greater increase in matrix metalloproteinase-9 and mRNA expression of inflammatory/M1 macrophage markers (tissue necrosis factor-α, interleukin-6, CD11c, and p47phox) after lipopolysaccharide stimulation than those of ApoE KO mice.

Hasegawa, H., Watanabe, T., Kato, S., Toshima, T., Yokoyama, M., Aida, Y., ... & Otaki, Y. (2016). The role of macrophage transcription factor MafB in atherosclerotic plaque stability. Atherosclerosis, 250, 133-143.

Platelet bound oxLDL shows an inverse correlation with plasma anaphylatoxin C5a in patients with coronary artery disease

Two hundred and seven patients with coronary artery disease (CAD) were analyzed in this study. Using enzyme-linked immunosorbent assays, plasma levels of oxLDL, C3a, and C5a were measured. Moreover, we assessed platelet bound oxLDL by flow cytometry. The overall level of C5a in the troponin negative group (stable angina (SA) and unstable angina (UA)) compared to the troponin positive group (non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI)) did not differ significantly (62.7 ± 32.4 ng/ml versus 65.8 ± 40.3 ng/ml). While C5a and C3a showed a significant correlation with each other (r = 0.25, p < 0.001), there was no statistically significant relationship between C3a and platelet bound oxLDL (r = 0.06, p = 0.37). Furthermore, plasma oxLDL did not correlate with either C3a or C5a. However, we observed a moderate, yet significant negative correlation between plasma C5a and platelet bound oxLDL (r = −0.15, p = 0.04). Partial correlation analysis correcting for the presence of acute coronary syndrome (ACS), troponin status or the subgroups SA, UA, NSTEMI, or STEMI did not alter this correlation substantially. Interestingly, flow cytometric analysis of human platelets showed increased expression of C5aR and P-selectin after in vitro stimulation with oxLDL.

Nording, H., Giesser, A., Patzelt, J., Sauter, R., Emschermann, F., Stellos, K., ... & Langer, H. F. (2016). Platelet bound oxLDL shows an inverse correlation with plasma anaphylatoxin C5a in patients with coronary artery disease. Platelets, 1-5.

Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

BER gene expression levels were analyzed in 162 carotid plaques, 8 disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls.

Skarpengland, T., Dahl, T. B., Skjelland, M., Scheffler, K., de Sousa, M. M. L., Gregersen, I., ... & Segers, F. M. (2016). Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA. Free Radical Biology and Medicine.

Established atherosclerosis might be a prerequisite for chicory and its constituent protocatechuic acid to promote endothelium-dependent vasodilation in mice

Apolipoprotein E-deficient (ApoE−/−) mice with established atherosclerosis and C57BL/6J mice without atherosclerosis were fed an AIN-93G diet, or AIN-93G plus 0.5% freeze-dried chicory or 0.003% PCA for 1 wk. In ApoE−/− mice, both chicory and PCA consumption increased endothelium-dependent vasodilation and endothelial nitric oxide synthase (eNOS) activity independent of eNOS and phospho-eNOS Ser1177 and Thr495 protein expression. Chicory- or PCA-induced eNOS activities were associated with increased vascular tetrahydrobiopterin (BH4) levels that result from reduced BH4 oxidation partially through preventing eNOS uncoupling. In C57BL/6J mice, neither chicory nor PCA consumption affected endothelium-dependent vasodilation and eNOS activity. Notably, in vitro studies showed that PCA increases eNOS activity in mouse aortic endothelial cells in co-culture with macrophage foam cells, but not in aortic endothelial cells alone.

Liu, C., Wang, W., Lin, W., Ling, W., & Wang, D. (2016). Established atherosclerosis might be a perquisite for chicory and its constituent protocatechuic acid to promote endothelium‐dependent vasodilation in mice. Molecular nutrition & food research.

The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor

The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3′-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.

Baik, Y. H., Park, H. Y., & Kook, H. (2015). The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor.

Cryptotanshinone Attenuates In Vitro oxLDL-Induced Pre-Lesional Atherosclerotic Events

Development of early stage atherosclerosis involves the activation of endothelial cells by oxidized low-density lipoprotein (oxLDL) with subsequent increases in endothelial permeability and expression of adhesion molecules favoring the adherence of monocytes to the endothelium. Cryptotanshinone (CTS), a major compound derived from the Chinese herb Salvia miltiorrhiza, is known for its protective effects against cardiovascular diseases. The aim of this study was to determine whether CTS could prevent the oxLDL-induced early atherosclerotic events. OxLDL (100 µg/mL) was used to increase endothelial permeability and induce monocyte-endothelial cell adhesion in human umbilical vein endothelial cells (HUVECs). Endothelial nitric oxide (NO) concentrations, a permeability-regulating molecule, and expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured. Results show that a) endothelial hyperpermeability was suppressed by 94 % (p < 0.005), b) monocyte adhesion by 105 % (p < 0.01), and c) ICAM-1 and VCAM-1 expressions by 90 % (p < 0.01) and 150 % (p < 0.005), respectively, when CTS was applied. In contrast, CTS increased NO levels by 129 % (p < 0.01) and was found to be noncytotoxic in the concentrations between 1–10 µM. These findings indicate that CTS suppresses an increase in endothelial permeability, likely due to the restoration of NO bioavailability in endothelial cells. They also indicate that CTS may attenuate monocyte adhesion to endothelial cells through the inhibition of adhesion molecules' expression. Thus, CTS may play an important role in the prevention of early or pre-lesional stage of atherosclerosis.

Ang, K. P., Tan, H. K., Selvaraja, M., Kadir, A. A., Somchit, M. N., Akim, A. M., ... & Ahmad, Z. (2011). Cryptotanshinone attenuates in vitro oxLDL-induced pre-lesional atherosclerotic events. Planta medica, 77(16), 1782-1787.

Increased oxidized low-density lipoprotein causes blood-brain barrier disruption in early-onset preeclampsia through LOX-1

Early-onset preeclampsia (EPE) is a severe form of preeclampsia that involves life-threatening neurological complications. However, the underlying mechanism by which EPE affects the maternal brain is not known. We hypothesized that plasma from women with EPE increases blood-brain barrier (BBB) permeability vs. plasma from women with late-onset preeclampsia (LPE) or normal pregnancy (NP) and investigated its underlying mechanism by perfusing cerebral veins from nonpregnant rats (n=6–7/group) with human plasma from women with EPE, LPE, or NP and measuring permeability. We show that plasma from women with EPE significantly increased BBB permeability vs. plasma from women with LPE or NP (P<0.001). BBB disruption in response to EPE plasma was due to a 260% increase of circulating oxidized LDL (oxLDL) binding to its receptor, LOX-1, and subsequent generation of peroxynitrite (P<0.001). A rat model with pathologically high lipid levels in pregnancy showed symptoms of preeclampsia, including elevated blood pressure, growth-restricted fetuses, and LOX-1-dependent BBB disruption, similar to EPE (P<0.05). Thus, we have identified LOX-1 activation by oxLDL and subsequent peroxynitrite generation as a novel mechanism by which disruption of the BBB occurs in EPE. As increased BBB permeability is a primary means by which seizure and other neurological symptoms ensue, our findings highlight oxLDL, LOX-1, and peroxynitrite as important therapeutic targets in EPE.—Schreurs, M. P. H., Hubel, C. A., Bernstein, I. M., Jeyabalan, A., and Cipolla, M. J. Increased oxidized low-density lipoprotein causes blood-brain barrier disruption in early-onset preeclampsia through LOX-1.

Schreurs, M. P., Hubel, C. A., Bernstein, I. M., Jeyabalan, A., & Cipolla, M. J. (2013). Increased oxidized low-density lipoprotein causes blood-brain barrier disruption in early-onset preeclampsia through LOX-1. The FASEB Journal, 27(3), 1254-1263.

N-acetyl cysteine suppresses the foam cell formation that is induced by oxidized low density lipoprotein via regulation of gene expression

Foam cells derived from macrophages have been implicated as markers of early stage atherosclerosis development. In this study, we found that N-acetyl cysteine (NAC), a well-known inhibitor of reactive oxygen species (ROS), decreased the generation of ROS and suppressed foam cell formation in the presence of oxidized low density lipoprotein through down-regulation of cluster of differentiation 36 expression. We investigated gene expression profiles in order to determine the effects of NAC on foam cell formation using a microarray analysis. The level of apolipoprotein E, which is involved in lipid efflux, was increased and the levels of the antioxidant genes glutathione peroxidase 1 and 3 were also increased. The expression levels of the oxidative stress response and the DNA repair genes were decreased. These results were confirmed using quantitative real-time PCR. Our results indicate that oxidative stress plays an important role in foam cell formation, and that regulation of oxidation using antioxidants is a potential therapeutic method for blocking atherosclerosis development.

Sung, H. J., Kim, J., Kim, Y., Jang, S. W., & Ko, J. (2012). N-acetyl cysteine suppresses the foam cell formation that is induced by oxidized low density lipoprotein via regulation of gene expression. Molecular biology reports, 39(3), 3001-3007.

Pep19 drives epitope spreading in periodontitis and periodontitis-associated autoimmune diseases

Epitope spreading is one of valid mechanisms operating in immunopathological processes of infection-induced autoimmune diseases. We hypothesized that the peptide 19 from Porphyromonas gingivalis heat shock protein (HSP) 60 (Pep19) may be the dominant epitope from which epitope-specific immune response to subdominant epitopes may diversify sequentially into autoimmune responses directed at human neoepitopes in P. gingivalis-induced periodontitis and autoimmune diseases. However, the exact feature and mechanism on how Pep19 may drive epitope spreading into human autoantigens in chronic periodontitis or P. gingivalis-induced experimental periodontitis has not been clarified. The present study was performed with the following specific aims: (i) to delineate retrospectively the features of epitope spreading by human cross-sectional analysis; (ii) to demonstrate prospectively the epitope spreading into new antigenic determinants in an ordered, predictable and sequential manner in experimental periodontitis; and (iii) to clarify the mechanism on how immunization with Pep19 may mobilize helper T cells or elicit B-cell responses to human autoantigens and neoantigen.

Kwon, E. Y., Cha, G. S., Jeong, E., Lee, J. Y., Kim, S. J., Surh, C. D., & Choi, J. (2015). Pep19 drives epitope spreading in periodontitis and periodontitis‐associated autoimmune diseases. Journal of periodontal research.

Intake of cooked tomato sauce preserves coronary endothelial function and improves apolipoprotein A-I and apolipoprotein J protein profile in high-density lipoproteins

Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remains unknown whether tomato intake exerts protective effects on the vasculature. The aim of this study was to determine whether medium-term supplementation with cooked tomato sauce (CTS) Mediterranean style (sofrito) attenuates diet-induced coronary endothelial dysfunction in an animal model with clinical impact and explore the mechanisms behind the effects. Pigs (N = 18) were fed a 10-day hypercholesterolemic diet. Half of the animals were given a supplement of 100 g/d of CTS (21.5 mg lycopene per day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide synthase [eNOS] inhibitor) were measured using flow Doppler. In the coronary arteries, we investigated eNOS gene expression and activation, monocyte chemoattractant protein 1 (MCP-1) expression, and oxidative DNA damage. In the circulation, we investigated lipoprotein resistance to oxidation and the differential proteomic protein profile. In dyslipidemic animals, CTS intake prevented diet-induced impairment of receptor-operated and nonreceptor-operated endothelial-dependent coronary vasodilation. These beneficial effects were associated with enhanced eNOS transcription and activation and diminished DNA damage in the coronary arteries. CTS-fed animals showed lower lipid peroxidation, higher high-density lipoprotein (HDL) antioxidant potential and plasma lycopene levels of 0.16 mg/L. Interestingly, improved HDL functionality was associated with protein profile changes in apolipoprotein A-I and apolipoprotein J. Lipids levels and MCP-1 expression were not affected by CTS. We report that CTS intake protects against low-density lipoprotein–induced coronary endothelial dysfunction by reducing oxidative damage, enhancing eNOS expression and activity, and improving HDL functionality.

Vilahur, G., Cubedo, J., Padró, T., Casaní, L., Mendieta, G., González, A., & Badimon, L. (2015). Intake of cooked tomato sauce preserves coronary endothelial function and improves apolipoprotein AI and apolipoprotein J protein profile in high-density lipoproteins. Translational Research, 166(1), 44-56.

Effect of sodium tanshinone IIA sulfonate treatment in a rat model of preeclampsia

Preeclampsia is a disorder of pregnancy with a significant impact on maternal and fetal health. The complexity of this multifactorial condition has precluded development of effective therapies and, although many potential pathways have been investigated, the etiology still requires clarification. Our group has investigated the scavenger lectin-like oxidized LDL (LOX-1) receptor, which may respond to factors released from the distressed placenta that contribute to the vascular pathologies observed in preeclampsia. Given the known beneficial effects of sodium tanshinone IIA sulfonate (STS; a component of Salvia miltiorrhiza) on vasodilation, reduction of oxidative stress, and lipid profiles, we have investigated its role as a potential treatment strategy. We hypothesized that STS would improve vascular endothelial function and, combined with a reduction in oxidative stress, would improve pregnancy outcomes in a rat model of preeclampsia (reduced uteroplacental perfusion pressure, RUPP). We further hypothesized this may occur via the action of STS on the LOX-1 and/or platelet-activating factor (PAF) receptor axes. The RUPP model increased maternal blood pressure, vascular oxidative stress, and involvement of the vascular PAF receptor. Treatment with STS during pregnancy decreased both oxidative stress and involvement of the PAF receptor; however, it also increased involvement of the LOX-1 receptor, which is in line with the concept that scavenger receptors, such as LOX-1 and PAF, are upregulated in response to ligand binding and/or under pathological conditions. In this model of preeclampsia, however, the vascular actions of STS did not lead to improvements in pregnancy outcome such as fetal biometrics or maternal blood pressure.

Morton, J. S., Quon, A., Cheung, P. Y., Sawamura, T., & Davidge, S. T. (2015). Effect of sodium tanshinone IIA sulfonate treatment in a rat model of preeclampsia. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 308(3), R163-R172.

OxLDL induces endothelial dysfunction and death via TRAF3IP2: Inhibition by HDL3 and AMPK activators

Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-κB and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-κB and JNK/AP-1. Here we show that oxLDL-induced death in human primary coronary artery endothelial cells (ECs) was markedly attenuated by the knockdown of TRAF3IP2 or the lectin-like oxLDL receptor 1 (LOX-1). Further, oxLDL induced Nox2/superoxide-dependent TRAF3IP2 expression, IKK/p65 and JNK/c-Jun activation, and LOX-1 upregulation, suggesting a reinforcing mechanism. Similarly, the lysolipids present in oxLDL (16:0-LPC and 18:0-LPC) and minimally modified LDL also upregulated TRAF3IP2 expression. Notably, whereas native HDL3 reversed oxLDL-induced TRAF3IP2 expression and cell death, 15-lipoxygenase-modified HDL3 potentiated its proapoptotic effects. The activators of the AMPK/Akt pathway, adiponectin, AICAR, and metformin, attenuated superoxide generation, TRAF3IP2 expression, and oxLDL/TRAF3IP2-mediated EC death. Further, both HDL3 and adiponectin reversed oxLDL/TRAF3IP2-dependent monocyte adhesion to endothelial cells in vitro. Importantly, TRAF3IP2 gene deletion and the AMPK activators reversed oxLDL-induced impaired vasorelaxation ex vivo. These results indicate that oxLDL-induced endothelial cell death and dysfunction are mediated via TRAF3IP2 and that native HDL3 and the AMPK activators inhibit this response. Targeting TRAF3IP2 could potentially inhibit progression of atherosclerotic vascular diseases.

Valente, A. J., Irimpen, A. M., Siebenlist, U., & Chandrasekar, B. (2014). OxLDL induces endothelial dysfunction and death via TRAF3IP2: inhibition by HDL3 and AMPK activators. Free Radical Biology and Medicine, 70, 117-128.

Selective activation of CB2 receptor improves efferocytosis in cultured macrophages

Recent evidence indicates that the defective ability to clear apoptotic cells by macrophages (efferocytosis) and the resultant apoptotic cells accumulation in atherosclerotic plaques play an important role during the progression of unstable plaques. The cannabinoid type 2 receptor (CB2), has recently been emerging as a new target to reduce vulnerability and promote stability of plaques, however, the underlying mechanisms have not been studied in detail. In the present study, we investigated whether selective activation of CB2 improves efferocytosis of macrophages.

Jiang, L., Chen, Y., Huang, X., Yuan, A., Shao, Q., Pu, J., & He, B. (2016). Selective activation of CB2 receptor improves efferocytosis in cultured macrophages. Life Sciences.

Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion

Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice.

Oberoi, R., Schuett, J., Schuett, H., Koch, A. K., Luchtefeld, M., Grote, K., & Schieffer, B. (2016). Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion. PloS one, 11(7), e0160145.

Labeled LDL (Our DiI and DiO products)

Nanotherapeutics Containing Lithocholic Acid-Based Amphiphilic Scorpion-Like Macromolecules Reduce In Vitro Inflammation in Macrophages: Implications for Atherosclerosis

Previously-designed amphiphilic scorpion-like macromolecule (AScM) nanoparticles (NPs) showed elevated potency to counteract oxidized low-density lipoprotein (oxLDL) uptake in atherosclerotic macrophages, but failed to ameliorate oxLDL-induced inflammation. We designed a new class of composite AScMs incorporating lithocholic acid (LCA), a natural agonist for the TGR5 receptor that is known to counteract atherosclerotic inflammation, with two complementary goals: to simultaneously decrease lipid uptake and inhibit pro-inflammatory cytokine secretion by macrophages. LCA was conjugated to AScMs for favorable interaction with TGR5 and was also hydrophobically modified to enable encapsulation in the core of AScM-based NPs. Conjugates were formulated into negatively charged NPs with different core/shell combinations, inspired by the negative charge on oxLDL to enable competitive interaction with scavenger receptors (SRs). NPs with LCA-containing shells exhibited reduced sizes, and all NPs lowered oxLDL uptake to <30% of untreated, human derived macrophages in vitro, while slightly downregulating SR expression. Pro-inflammatory cytokine expression, including IL-1β, IL-8, and IL-10, is known to be modulated by TGR5, and was dependent on NP composition, with LCA-modified cores downregulating inflammation. Our studies indicate that LCA-conjugated AScM NPs offer a unique approach to minimize atherogenesis and counteract inflammation.

Moretti, A., Li, Q., Chmielowski, R., Joseph, L. B., Moghe, P. V., & Uhrich, K. E. (2018). Nanotherapeutics Containing Lithocholic Acid-Based Amphiphilic Scorpion-Like Macromolecules Reduce In Vitro Inflammation in Macrophages: Implications for Atherosclerosis. Nanomaterials, 8(2), 84.

AMP-activated Protein Kinase α1 Promotes Atherogenesis by Increasing Monocyte-to-macrophage Differentiation

Monocyte-to-macrophage differentiation, which can be initiated by physiological or atherogenic factors, is a pivotal process in atherogenesis, a disorder in which monocytes adhere to endothelial cells and subsequently migrate into the sub-endothelial spaces, where they differentiate into macrophages and macrophage-derived foam cells and cause atherosclerotic lesions. However, the monocyte-differentiation signaling pathways that are activated by atherogenic factors are poorly defined. Here we report that the AMP- activated protein kinase (AMPK) α1 in monocytes promotes atherosclerosis by increasing monocyte differentiation and survival. Exposure of monocytes to oxidized-low-density lipoprotein, 7-ketocholesterol, phorbol-12-myristate-13-acetat or macrophage colony-stimulated factor (M-CSF) significantly activated AMPK and promoted monocyte to macrophage differentiation. M-CSF activated AMPK is via M-CSF receptor dependent reactive oxygen species production. Consistently, genetic deletion of AMPKα1 or pharmacological inhibition of AMPK blunted monocyte to macrophage differentiation and promoted monocyte/macrophage apoptosis. Compared with apolipoprotein E knockout (ApoE-/-) mice, which show impaired clearing of plasma lipoproteins and spontaneously develop atherosclerosis, ApoE-/-/AMPKα1-/- mice showed less atherosclerotic lesion sizes and fewer macrophage numbers in the lesions. Furthermore, aortic lesion were decreased in ApoE-/- mice transplanted with ApoE-/-/AMPKα1-/- bone marrow and in myeloid-specific AMPKα1-deficient ApoE-/- mice. Finally, rapamycin treatment, which abolished delayed monocytes differentiation in ApoE-/-/AMPKα1-/- mice, lost its atherosclerosis-lowering effects in these mice. Mechanistically, we found that AMPK α1 regulates FoxO3-dependent expression of both LC3 and ULK1, which are two important autophagy-related markers. Rapamycin treatment increased FoxO3 activity as well as LC3 and ULK1 expressions in macrophages from AMPKα1-/- mice. Our results reveal that AMPKα1 deficiency impairs autophagy-mediated monocytes differentiation and declines monocyte/macrophage survival, which attenuates atherosclerosis in ApoE-/- mice in vivo.

Zhang, M., Zhu, H., Ding, Y., Liu, Z., Cai, Z., & Zou, M. H. (2017). AMP-activated Protein Kinase α1 Promotes Atherogenesis by Increasing Monocyte-to-macrophage Differentiation. Journal of Biological Chemistry, jbc-M117.

Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P‹0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

Sarkar, S., Bailey, E., Go, Y. Y., Cook, R. F., Kalbfleisch, T., Eberth, J., ... & Balasuriya, U. B. (2016). Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion. PLOS Genetics, 12(12), e1006467.

LOX-1 expression and oxidized LDL uptake and toxicity in the HN33 neuronal cell line

Cardiovascular risk factors appear to influence the risk and progression of neurodegenerative disease, but the mechanisms involved are poorly understood. We investigated the possible involvement of oxidized low-density lipoprotein receptor (LOX-1) and oxidized low-density lipoprotein (Ox-LDL) in neurodegeneration by studying the expression of LOX-1 and the effects of Ox-LDL in HN33 cells, a neuronal cell line of central nervous system origin. HN33 cells showed LOX-1 protein expression, hypoxic induction of LOX-1, Ox-LDL uptake and Ox-LDL toxicity. LOX-1/Ox-LDL signaling may contribute to the association between cardiovascular risk factors and neurodegenerative disease.

Zhu, X., Ng, H. P., Lai, Y. C., Craigo, J. K., Nagilla, P. S., Raghani, P., & Nagarajan, S. (2014). Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice. The Journal of Immunology, 193(5), 2483-2495.

Impact of ionizing radiation on physicochemical and biological properties of an amphiphilic macromolecule

An amphiphilic macromolecule (AM) was exposed to ionizing radiation (both electron beam and gamma) at doses of 25 kGy and 50 kGy to study the impact of these sterilization methods on the physicochemical properties and bioactivity of the AM. Proton nuclear magnetic resonance and gel permeation chromatography were used to determine the chemical structure and molecular weight, respectively. Size and zeta potential of the micelles formed from AMs in aqueous media were evaluated by dynamic light scattering. Bioactivity of irradiated AMs was evaluated by measuring inhibition of oxidized low-density lipoprotein uptake in macrophages. From these studies, no significant changes in the physicochemical properties or bioactivity were observed after the irradiation, demonstrating that the AMs can withstand typical radiation doses used to sterilize materials.

Gu, L., Zablocki, K., Lavelle, L., Bodnar, S., Halperin, F., Harper, I., ... & Uhrich, K. E. (2012). Impact of ionizing radiation on physicochemical and biological properties of an amphiphilic macromolecule. Polymer degradation and stability, 97(9), 1686-1689.

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis.

Hossain, E., Ota, A., Karnan, S., Damdindorj, L., Takahashi, M., Konishi, Y., ... & Hosokawa, Y. (2013). Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells. Toxicology and applied pharmacology, 273(3), 651-658.

Flow-induced regulation of brain endothelial cells in vitro

Endothelial cell (EC) function and susceptibility to vascular disease are regulated by flow; this relationship has been modeled in systemic, but not cerebrovascular, EC culture. We studied the effects of unidirectional flow of medium, produced by orbital rotation of cultures, on morphology and protein expression in bEnd.3 mouse brain ECs. Flow altered the expression of key transcription factors and gasotransmitter-synthesizing enzymes, and increased NO production. Statins and angiotensin receptor blockers reproduced the effect of flow on endothelial nitric oxide synthase expression. Thus, flow modified brain EC properties and function in vitro, with similarities and possible differences compared to previous studies on systemic ECs. Thus, the effect of flow on brain ECs can be modeled in vitro and may assist the investigation of mechanisms of cerebrovascular disease.

Mao, X., Xie, L., Greenberg, R. B., Greenberg, J. B., Peng, B., Mieling, I., ... & Greenberg, D. A. (2014). Flow-induced regulation of brain endothelial cells in vitro. Vascular pharmacology, 62(2), 82-87.

Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.

Mao, X., Xie, L., & Greenberg, D. A. (2015). Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures. Free Radical Biology and Medicine, 89, 638-641.

Function modification of SR-PSOX by point mutations of basic amino acids

A cell model to study the functions of SR-PSOX was successfully established. Based on the cell model, a series of mutants of human SR-PSOX were constructed by replacing the single basic amino acid residue in the non-conservative region of the chemokine domain (arginine 62, arginine 78, histidine 80, arginine 82, histidine 85, lysine 105, lysine 119, histidine 123) with alanine (designated as R62A, R78A, H80A, R82A, H85A, K105A, K119A and H123A, respectively). Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX. In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.

Liu, W., Yin, L., Chen, C., & Dai, Y. (2011). Function modification of SR-PSOX by point mutations of basic amino acids. Lipids in health and disease, 10(1), 1.

IL-19 Halts Progression of Atherosclerotic Plaque, Polarizes, and Increases Cholesterol Uptake and Efflux in Macrophages

Atherosclerosis regression is an important clinical goal, and treatments that can reverse atherosclerotic plaque formation are actively being sought. Our aim was to determine whether administration of exogenous IL-19, a Th2 cytokine, could attenuate progression of preformed atherosclerotic plaque and to identify molecular mechanisms. LDLR−/− mice were fed a Western diet for 12 weeks, then administered rIL-19 or phosphate-buffered saline concomitant with Western diet for an additional 8 weeks. Analysis of atherosclerosis burden showed that IL-19–treated mice were similar to baseline, in contrast to control mice which showed a 54% increase in plaque, suggesting that IL-19 halted the progression of atherosclerosis. Plaque characterization showed that IL-19–treated mice had key features of atherosclerosis regression, including a reduction in macrophage content and an enrichment in markers of M2 macrophages. Mechanistic studies revealed that IL-19 promotes the activation of key pathways leading to M2 macrophage polarization, including STAT3, STAT6, Kruppel-like factor 4, and peroxisome proliferator-activated receptor γ, and can reduce cytokine-induced inflammation in vivo. We identified a novel role for IL-19 in regulating macrophage lipid metabolism through peroxisome proliferator-activated receptor γ-dependent regulation of scavenger receptor–mediated cholesterol uptake and ABCA1-mediated cholesterol efflux. These data show that IL-19 can halt progression of preformed atherosclerotic plaques by regulating both macrophage inflammation and cholesterol homeostasis and implicate IL-19 as a link between inflammation and macrophage cholesterol metabolism.

Gabunia, K., Ellison, S., Kelemen, S., Kako, F., Cornwell, W. D., Rogers, T. J., ... & Autieri, M. V. (2016). IL-19 halts progression of atherosclerotic plaque, polarizes, and increases cholesterol uptake and efflux in macrophages. The American journal of pathology, 186(5), 1361-1374.

Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164

FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane-impermeable sulfo-N-hydroxysuccinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA-binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated, and the protein was pulled down, deglycosylated, and resolved by electrophoresis. The CD36 band was extracted from the gel and digested for analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of Lys-164. Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated Lys-164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting the importance of Lys-164 for both FA effects. Furthermore, consistent with the importance of Lys-164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses Lys-164 in the FA-binding site on CD36, and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket.

Kuda, O., Pietka, T. A., Demianova, Z., Kudova, E., Cvacka, J., Kopecky, J., & Abumrad, N. A. (2013). Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164 SSO ALSO INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN UPTAKE BY MACROPHAGES. Journal of Biological Chemistry, 288(22), 15547-15555.

Modification of SR-PSOX functions by multi-point mutations of basic amino acid residues

SR-PSOX can function as a scavenger receptor, a chemokine and an adhesion molecule, and it could be an interesting player in the formation of atherosclerotic lesions. Our previous studies demonstrated that basic amino acid residues in the chemokine domain of SR-PSOX are critical for its functions. In this study the combinations of the key basic amino acids in the chemokine domain of SR-PSOX have been identified. Five combinations of basic amino acid residues that may form conformational motif for SR-PSOX functions were selected for multi-point mutants. The double mutants of K61AR62A, R76AK79A, R82AH85A, and treble mutants of R76AR78AK79A, R78AR82AH85A were successfully constructed by replacing the combinations of two or three basic amino acid residues with alanine. After successful expression of these mutants on the cells, the functional studies showed that the cells expressing R76AK79A and R82AH85A mutants significantly increased the activity of oxLDL uptake compared with that of wild-type SR-PSOX. Meanwhile, the cells expressing R76AK79A mutant also dramatically enhanced the phagocytotic activity of SR-PSOX. However, the cells expressing the construct of combination of R78A mutation in R76AK79A or R82AH85A could abolish these effects. More interestingly, the adhesive activities were remarkably down regulated in the cells expressing the multi-point mutants respectively. This study revealed that some conformational motifs of basic amino acid residues, especially R76 with K79 in SR-PSOX, may form a common functional motif for its critical functions. R78 in SR-PSOX has the potential action to stabilize the function of oxLDL uptake and bacterial phagocytosis. The results obtained may provide new insight for the development of drug target of atherosclerosis.

Liu, W., Yin, L., & Dai, Y. (2013). Modification of SR-PSOX functions by multi-point mutations of basic amino acid residues. Biochimie, 95(2), 224-230.

Increased atherosclerosis and vascular smooth muscle cell activation in AIF-1 transgenic mice fed a high-fat diet

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, scaffold signal transduction protein constitutively expressed in inflammatory cells, but inducible in vascular smooth muscle cells (VSMCs) in response to injury or inflammatory stimuli. Although several basic science and population studies have reported increased AIF-1 expression in human and experimental atherosclerosis, a direct causal effect of AIF-1 expression on development of atherosclerosis has not been reported. The purpose of this study is to establish a direct relationship between AIF-1 expression and development of atherosclerosis. AIF-1 expression is detected VSMC in atherosclerotic lesions from ApoE−/− mice, but not normal arteries from wild-type mice. AIF-1 expression can be induced in cultured VSMC by stimulation with oxidized LDL (ox-LDL). Transgenic mice in which AIF-1 expression is driven by the G/C modified SM22 alpha promoter to restrict AIF-1 expression to VSMC develop significantly increased atherosclerosis compared with wild-type control mice when fed a high-fat diet (P = 0.022). Cultured VSMC isolated from Tg mice demonstrated significantly increased migration in response to ox-LDL compared with matched controls (P < 0.001). VSMC isolated from Tg mice and cultured human VSMC which over express AIF-1 demonstrated increased expression of MMP-2 and MMP-9 mRNA and protein and increased NF-κB activation in response to ox-LDL as compared with wild-type control mice. VSMC which over express AIF-1 have significantly increased uptake of ox-LDL, and increased CD36 expression. Together, these data suggest a strong association between AIF-1 expression, NF-κB activation, and development of experimental atherosclerosis.

Sommerville, L. J., Kelemen, S. E., Ellison, S. P., England, R. N., & Autieri, M. V. (2012). Increased atherosclerosis and vascular smooth muscle cell activation in AIF-1 transgenic mice fed a high-fat diet. Atherosclerosis, 220(1), 45-52.

Nanoscale Amphiphilic Macromolecules with Variable Lipophilicity and Stereochemistry Modulate Inhibition of Oxidized Low-Density Lipoprotein Uptake

Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) and counteract foam cell formation, a key characteristic of early atherogenesis. To investigate the influence of lipophilicity and stereochemistry on the AMs’ physicochemical and biological properties, mucic acid-based AMs bearing four aliphatic chains and tartaric acid-based AMs bearing tw and four aliphatic chains were synthesized and evaluated. Solution aggregation studies suggested that both the number of hydrophobic arms and the length of the hydrophobic domain impact AM micelle sizes, whereas stereochemistry impacts micelle stability. 2l, the meso analogue of 2b, elicited the highest reported oxLDL uptake inhibition values (89%), highlighting the crucial effect of stereochemistry on biological properties. This study suggests that stereochemistry plays a critical role in modulating oxLDL uptake and must be considered when designing biomaterials for potential cardiovascular therapies.

Poree, D. E., Zablocki, K., Faig, A., Moghe, P. V., & Uhrich, K. E. (2013). Nanoscale amphiphilic macromolecules with variable lipophilicity and stereochemistry modulate inhibition of oxidized low-density lipoprotein uptake. Biomacromolecules, 14(8), 2463-2469.

Lipin-1 contributes to modified low-density lipoprotein-elicited macrophage pro-inflammatory responses

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and the underlying cause of cardiovascular disease, a major cause of mortality worldwide. The over-accumulation of modified cholesterol-containing low-density lipoproteins (e.g. oxLDL) in the artery wall and the subsequent recruitment and activation of macrophages contributes to the development of atherosclerosis. The excessive uptake of modified-LDL by macrophages leads to a lipid-laden “foamy” phenotype and pro-inflammatory cytokine production. Modified-LDLs promote foam cell formation in part by stimulating de novo lipid biosynthesis. However, it is unknown if lipid biosynthesis directly regulates foam cell pro-inflammatory mediator production. Lipin-1, a phosphatidate phosphohydrolase required for the generation of diacylglycerol during glycerolipid synthesis has recently been demonstrated to contribute to bacterial-induced pro-inflammatory responses by macrophages. In this study we present evidence demonstrating the presence of lipin-1 within macrophages in human atherosclerotic plaques. Additionally, reducing lipin-1 levels in macrophages significantly inhibits both modified-LDL-induced foam cell formation in vitro, as observed by smaller/fewer intracellular lipid inclusions, and ablates modified-LDL-elicited production of the pro-atherogenic mediators tumor necrosis factor-α, interleukin-6, and prostaglandin E2. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to modified-LDL. These data begin to link the processes of foam cell formation and pro-inflammatory cytokine production within macrophages

Navratil, A. R., Vozenilek, A. E., Cardelli, J. A., Green, J. M., Thomas, M. J., Sorci-Thomas, M. G., ... & Woolard, M. D. (2015). Lipin-1 contributes to modified low-density lipoprotein-elicited macrophage pro-inflammatory responses. Atherosclerosis, 242(2), 424-432.

A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO4 and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18–oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.

Vance, D. T., Dufresne, J., Florentinus-Mefailoski, A., Tucholska, M., Trimble, W., Grinstein, S., & Marshall, J. G. (2016). A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages. Analytical biochemistry, 500, 24-34.

A common polymorphism in the LDL receptor gene has multiple effects on LDL receptor function

A common synonymous single nucleotide polymorphism in exon 12 of the low-density lipoprotein receptor (LDLR) gene, rs688, has been associated with increased plasma total and LDL cholesterol in several populations. Using immortalized lymphoblastoid cell lines from a healthy study population, we confirmed an earlier report that the minor allele of rs688 is associated with increased exon 12 alternative splicing (P < 0.05) and showed that this triggered nonsense-mediated decay (NMD) of the alternatively spliced LDLR mRNA. However, since synonymous single nucleotide polymorphisms may influence structure and function of the encoded proteins by co-translational effects, we sought to test whether rs688 was also functional in the full-length mRNA. In HepG2 cells expressing LDLR cDNA constructs engineered to contain the major or minor allele of rs688, the latter was associated with a smaller amount of LDLR protein at the cell surface (−21.8 ± 0.6%, P = 0.012), a higher amount in the lysosome fraction (+25.7 ± 0.3%, P = 0.037) and reduced uptake of fluorescently labeled LDL (−24.3 ± 0.7%, P < 0.01). Moreover, in the presence of exogenous proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that reduces cellular LDL uptake by promoting lysosomal degradation of LDLR, the minor allele resulted in reduced capacity of a PCSK9 monoclonal antibody to increase LDL uptake. These findings are consistent with the hypothesis that rs688, which is located in the β-propeller region of LDLR, has effects on LDLR activity beyond its role in alternative splicing due to impairment of LDLR endosomal recycling and/or PCSK9 binding, processes in which the β-propeller is critically involved.

Gao, F., Ihn, H. E., Medina, M. W., & Krauss, R. M. (2013). A common polymorphism in the LDL receptor gene has multiple effects on LDL receptor function. Human molecular genetics, 22(7), 1424-1431.

Amphiphilic macromolecule nanoassemblies suppress smooth muscle cell proliferation and platelet adhesion

While the development of second- and third-generation drug-eluting stents (DES) have significantly improved patient outcomes by reducing smooth muscle cell (SMC) proliferation, DES have also been associated with an increased risk of late-stent thrombosis due to delayed re-endothelialization and hypersensitivity reactions from the drug-polymer coating. Furthermore, DES anti-proliferative agents do not counteract the upstream oxidative stress that triggers the SMC proliferation cascade. In this study, we investigate biocompatible amphiphilic macromolecules (AMs) that address high oxidative lipoprotein microenvironments by competitively binding oxidized lipid receptors and suppressing SMC proliferation with minimal cytotoxicity. To determine the influence of nanoscale assembly on proliferation, micelles and nanoparticles were fabricated from AM unimers containing a phosphonate or carboxylate end-group, a sugar-based hydrophobic domain, and a hydrophilic poly(ethylene glycol) domain. The results indicate that when SMCs are exposed to high levels of oxidized lipid stimuli, nanotherapeutics inhibit lipid uptake, downregulate scavenger receptor expression, and attenuate scavenger receptor gene transcription in SMCs, and thus significantly suppress proliferation. Although both functional end-groups were similarly efficacious, nanoparticles suppressed oxidized lipid uptake and scavenger receptor expression more effectively compared to micelles, indicating the relative importance of formulation characteristics (e.g., higher localized AM concentrations and nanotherapeutic stability) in scavenger receptor binding as compared to AM end-group functionality. Furthermore, AM coatings significantly prevented platelet adhesion to metal, demonstrating its potential as an anti-platelet therapy to treat thrombosis. Thus, AM micelles and NPs can effectively repress early stage SMC proliferation and thrombosis through non-cytotoxic mechanisms, highlighting the promise of nanomedicine for next-generation cardiovascular therapeutics.

Chan, J. W., Lewis, D. R., Petersen, L. K., Moghe, P. V., & Uhrich, K. E. (2016). Amphiphilic macromolecule nanoassemblies suppress smooth muscle cell proliferation and platelet adhesion. Biomaterials, 84, 219-229.

In silico design of anti-atherogenic biomaterials

Atherogenesis, the uncontrolled deposition of modified lipoproteins in inflamed arteries, serves as a focal trigger of cardiovascular disease (CVD). Polymeric biomaterials have been envisioned to counteract atherogenesis based on their ability to repress scavenger mediated uptake of oxidized lipoprotein (oxLDL) in macrophages. Following the conceptualization in our laboratories of a new library of amphiphilic macromolecules (AMs), assembled from sugar backbones, aliphatic chains and poly(ethylene glycol) tails, a more rational approach is necessary to parse the diverse features such as charge, hydrophobicity, sugar composition and stereochemistry. In this study, we advance a computational biomaterials design approach to screen and elucidate anti-atherogenic biomaterials with high efficacy. AMs were quantified in terms of not only 1D (molecular formula) and 2D (molecular connectivity) descriptors, but also new 3D (molecular geometry) descriptors of AMs modeled by coarse-grained molecular dynamics (MD) followed by all-atom MD simulations. Quantitative structure-activity relationship (QSAR) models for anti-atherogenic activity were then constructed by screening a total of 1164 descriptors against the corresponding, experimentally measured potency of AM inhibition of oxLDL uptake in human monocyte-derived macrophages. Five key descriptors were identified to provide a strong linear correlation between the predicted and observed anti-atherogenic activity values, and were then used to correctly forecast the efficacy of three newly designed AMs. Thus, a new ligand-based drug design framework was successfully adapted to computationally screen and design biomaterials with cardiovascular therapeutic properties.

Lewis, D. R., Kholodovych, V., Tomasini, M. D., Abdelhamid, D., Petersen, L. K., Welsh, W. J., ... & Moghe, P. V. (2013). In silico design of anti-atherogenic biomaterials. Biomaterials, 34(32), 7950-7959.

Amphiphilic Nanoparticles Repress Macrophage Atherogenesis: Novel Core/Shell Designs for Scavenger Receptor Targeting and Down-Regulation

Atherosclerosis, an inflammatory lipid-rich plaque disease is perpetuated by the unregulated scavenger-receptor-mediated uptake of oxidized lipoproteins (oxLDL) in macrophages. Current treatments lack the ability to directly inhibit oxLDL accumulation and foam cell conversion within diseased arteries. In this work, we harness nanotechnology to design and fabricate a new class of nanoparticles (NPs) based on hydrophobic mucic acid cores and amphiphilic shells with the ability to inhibit the uncontrolled uptake of modified lipids in human macrophages. Our results indicate that tailored NP core and shell formulations repress oxLDL internalization via dual complementary mechanisms. Specifically, the most atheroprotective molecules in the NP cores competitively reduced NP-mediated uptake to scavenger receptor A (SRA) and also down-regulated the surface expression of SRA and CD36. Thus, nanoparticles can be designed to switch activated, lipid-scavenging macrophages to antiatherogenic phenotypes, which could be the basis for future antiatherosclerotic therapeutics.

Petersen, L. K., York, A. W., Lewis, D. R., Ahuja, S., Uhrich, K. E., Prud’homme, R. K., & Moghe, P. V. (2014). Amphiphilic nanoparticles repress macrophage atherogenesis: novel core/shell designs for scavenger receptor targeting and down-regulation. Molecular pharmaceutics, 11(8), 2815-2824.

Tartaric acid-based amphiphilic macromolecules with ether linkages exhibit enhanced repression of oxidized low density lipoprotein uptake

Cardiovascular disease initiates with the atherogenic cascade of scavenger receptor- (SR-) mediated oxidized low-density lipoprotein (oxLDL) uptake. Resulting foam cell formation leads to lipid-rich lesions within arteries. We designed amphiphilic macromolecules (AMs) to inhibit these processes by competitively blocking oxLDL uptake via SRs, potentially arresting atherosclerotic development. In this study, we investigated the impact of replacing ester linkages with ether linkages in the AM hydrophobic domain. We hypothesized that ether linkages would impart flexibility for orientation to improve binding to SR binding pockets, enhancing anti-atherogenic activity. A series of tartaric acid-based AMs with varying hydrophobic chain lengths and conjugation chemistries were synthesized, characterized, and evaluated for bioactivity. 3-D conformations of AMs in aqueous conditions may have significant effects on anti-atherogenic potency and were simulated by molecular modeling. Notably, ether-linked AMs exhibited significantly higher levels of inhibition of oxLDL uptake than their corresponding ester analogues, indicating a dominant effect of linkage flexibility on pharmacological activity. The degradation stability was also enhanced for ether-linked AMs. These studies further suggested that alkyl chain length (i.e., relative hydrophobicity), conformation (i.e., orientation), and chemical stability play a critical role in modulating oxLDL uptake, and guide the design of innovative cardiovascular therapies.

Abdelhamid, D. S., Zhang, Y., Lewis, D. R., Moghe, P. V., Welsh, W. J., & Uhrich, K. E. (2015). Tartaric acid-based amphiphilic macromolecules with ether linkages exhibit enhanced repression of oxidized low density lipoprotein uptake. Biomaterials, 53, 32-39.

Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages

There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis.

Hossain, E., Ota, A., Karnan, S., Takahashi, M., Mannan, S. B., Konishi, H., & Hosokawa, Y. (2015). Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages. Molecular and cellular biochemistry, 400(1-2), 29-40.

Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6Chigh monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe−/− mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6Chigh monocytes and macrophages. SYK inhibition limited Ly6Chigh monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe−/− mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.

Lindau, A., Härdtner, C., Hergeth, S. P., Blanz, K. D., Dufner, B., Hoppe, N., ... & Heidt, T. (2016). Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression. Basic research in cardiology, 111(2), 1-11.

Micellar and structural stability of nanoscale amphiphilic polymers: Implications for anti-atherosclerotic bioactivity

Atherosclerosis, a leading cause of mortality in developed countries, is characterized by the buildup of oxidized low-density lipoprotein (oxLDL) within the vascular intima, unregulated oxLDL uptake by macrophages, and ensuing formation of arterial plaque. Amphiphilic polymers (AMPs) comprised of a branched hydrophobic domain and a hydrophilic poly(ethylene glycol) (PEG) tail have shown promising anti-atherogenic effects through direct inhibition of oxLDL uptake by macrophages. In this study, five AMPs with controlled variations were evaluated for their micellar and structural stability in the presence of serum and lipase, respectively, to develop underlying structure-atheroprotective activity relations. In parallel, molecular dynamics simulations were performed to explore the AMP conformational preferences within an aqueous environment. Notably, AMPs with ether linkages between the hydrophobic arms and sugar backbones demonstrated enhanced degradation stability and storage stability, and also elicited enhanced anti-atherogenic bioactivity. Additionally, AMPs with increased hydrophobicity elicited increased atheroprotective bioactivity in the presence of serum. These studies provide key insights for designing more serum-stable polymeric micelles as prospective cardiovascular nanotherapies.

Zhang, Y., Li, Q., Welsh, W. J., Moghe, P. V., & Uhrich, K. E. (2016). Micellar and structural stability of nanoscale amphiphilic polymers: Implications for anti-atherosclerotic bioactivity. Biomaterials, 84, 230-240.

High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts

High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24 h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24 h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs.

Angius, F., Spolitu, S., Uda, S., Deligia, S., Frau, A., Banni, S., ... & Batetta, B. (2015). High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts. Scientific reports, 5.

Impact of Hydrophobic Chain Composition on Amphiphilic Macromolecule Antiatherogenic Bioactivity

Amphiphilic macromolecules (AMs) composed of sugar backbones modified with branched aliphatic chains and a poly(ethylene glycol) (PEG) tail can inhibit macrophage uptake of oxidized low-density lipoproteins (oxLDL), a major event underlying atherosclerosis development. Previous studies indicate that AM hydrophobic domains influence this bioactivity through interacting with macrophage scavenger receptors, which can contain basic and/or hydrophobic residues within their binding pockets. In this study, we compare two classes of AMs to investigate their ability to promote athero-protective potency via hydrogen-bonding or hydrophobic interactions with scavenger receptors. A series of ether-AMs, containing methoxy-terminated aliphatic arms capable of hydrogen-bonding, was synthesized. Compared to analogous AMs containing no ether moieties (alkyl-AMs), ether-AMs showed improved cytotoxicity profiles. Increasing AM hydrophobicity via incorporation of longer and/or alkyl-terminated hydrophobic chains yielded macromolecules with enhanced oxLDL uptake inhibition. These findings indicate that hydrophobic interactions and the length of AM aliphatic arms more significantly influence AM bioactivity than hydrogen-bonding.

Faig, A., Petersen, L. K., Moghe, P. V., & Uhrich, K. E. (2014). Impact of hydrophobic chain composition on amphiphilic macromolecule antiatherogenic bioactivity. Biomacromolecules, 15(9), 3328-3337.

Acetylated LDL

Rat and human HARE/stabilin-2 are clearance receptors for high- and low-molecular-weight heparins

The human hyaluronic acid (HA) receptor for endocytosis (HARE/stabilin-2) is the primary clearance receptor for systemic HA, chondroitin sulfates, and heparin, but not for heparan sulfate or keratan sulfate (Harris EN, Weigel JA, Weigel PH. J Biol Chem 283: 17341–17350, 2008). HARE is expressed in the sinusoidal endothelial cells (SECs) of liver and lymph nodes where it acts as a scavenger for uptake and degradation of glycosaminoglycans, both as free chains and proteoglycan fragments. Unfractionated heparin (UFH; ∼14 kDa) and low-molecular-weight heparin (LMWH; ∼4 kDa) are commonly used in treatments for thrombosis and cancer and in surgical and dialysis procedures. The reported half-lives of UFH and LMWH in the blood are ∼1 h and 2–6 h, respectively. In this study, we demonstrate that anti-HARE antibodies specifically block the uptake of LMWH and UFH by isolated rat liver SECs and by human 293 cells expressing recombinant human HARE (hHARE). hHARE has a significant affinity (Kd = 10 μM) for LMWH, and higher affinity (Kd = 0.06 μM) for the larger UFH. Rat liver SECs or cells expressing the recombinant 190-kDa HARE isoform internalized both UFH and LMWH, and both heparins cross-compete with each other, suggesting that they share the same binding sites. These cellular results were confirmed in ELISA-like assays using purified soluble 190-hHARE ectodomain. We conclude that both UFH and LMWH are cleared by HARE/Stab2 and that the differences in the affinities of HARE binding to LMWH and UFH likely explain the longer in vivo circulating half-life of LMWH compared with UFH.

Harris, E. N., Baggenstoss, B. A., & Weigel, P. H. (2009). Rat and human HARE/stabilin-2 are clearance receptors for high-and low-molecular-weight heparins. American Journal of Physiology-Gastrointestinal and Liver Physiology, 296(6), G1191-G1199.

Interleukin 8 Inhibition Enhanced Cholesterol Efflux in Acetylated Low-Density Lipoprotein–Stimulated THP-1 Macrophages

Cholesterol efflux plays a major role in antiatherogenesis, and modification of this process may provide a new therapeutic approach to cardiovascular disease. Interleukin 8 (IL-8) is implicated in various aspects of atherosclerosis. However, the effect of IL-8 on cholesterol efflux is still unclear. Here, we used human IL-8–neutralizing antibody to inhibit IL-8 and analyze the function of IL-8 in cholesterol efflux from acetylated low-density lipoprotein–loaded THP-1 macrophages. Acetylated low-density lipoprotein loading resulted in an approximately 2.5-fold increase in both the mRNA and protein levels of IL-8 in THP-1 macrophages, when compared with nonloaded THP-1 macrophages (P < 0.01). Five and 10 µg/mL of human IL-8–neutralizing antibody enhanced cholesterol efflux from THP-1–derived macrophages by 1.2- and 1.4-fold, respectively. Moreover, anti–IL-8–treated cells showed increased expression of peroxisome proliferator-activated γ, liver X receptor alpha, and ATP-binding cassette transporter A1 at both the mRNA and protein levels. Ten micromoles of SB203580, an inhibitor of p38, almost completely suppressed the production of IL-8 from acetylated low-density lipoprotein–loaded THP-1 macrophages and accelerated cholesterol efflux. Taken together, our results indicate that IL-8 exerts negative regulatory effects on cholesterol efflux from THP-1 cells and may thus represent a potential target for prevention and treatment of atherosclerosis.

Chen, Y., Wang, Z., & Zhou, L. (2014). Interleukin 8 Inhibition Enhanced Cholesterol Efflux in Acetylated Low-Density Lipoprotein–Stimulated THP-1 Macrophages. Journal of Investigative Medicine, 62(3), 615-620.

Inhibiting DNA Methylation by 5-Aza-2′-deoxycytidine Ameliorates Atherosclerosis Through Suppressing Macrophage Inflammation

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr−/−) mice. Ldlr−/− mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr−/− mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis.

Cao, Q., Wang, X., Jia, L., Mondal, A. K., Diallo, A., Hawkins, G. A., ... & Shi, H. (2014). Inhibiting DNA Methylation by 5-aza-2′-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation. Endocrinology, 155(12), 4925-4938. Chicago

Gremlin-1 inhibits macrophage migration inhibitory factor-dependent monocyte function and survival

We found that Gremlin-1 inhibited MIF-dependent monocyte migration and adhesion to activated endothelial cells in flow chamber perfusion assay in vitro and to the injured carotid artery of WT and ApoE−/− mice in vivo as deciphered by intravital microscopy. Intravenous administration of Gremlin-1, but not of control protein, significantly reduced leukocyte recruitment towards the inflamed carotid artery of ApoE−/− mice. Besides, leukocytes from MIF−/− when administered into ApoE−/− mice showed lesser adhesion as compared to wild type. In the presence of Gremlin-1 however, adhesion of wild type, but not of MIF−/− leukocytes, to the carotid artery was significantly inhibited as compared to control. Gremlin-1 also inhibited the MIF-induced differentiation of monocytes into macrophages. Gremlin-1 substantially inhibited the anti-apoptotic impact of MIF on monocytes against BH3 mimetic ABT-737-induced apoptosis as verified by Annexin V-binding, caspase 3 activity, and mitochondrial depolarization.

Müller, I. I., Chatterjee, M., Schneider, M., Borst, O., Seizer, P., Schönberger, T., ... & Langer, H. (2014). Gremlin-1 inhibits macrophage migration inhibitory factor-dependent monocyte function and survival. International journal of cardiology, 176(3), 923-929.

VLDL

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles

Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA).

Mørk, M., Handberg, A., Pedersen, S., Jørgensen, M. M., Bæk, R., Nielsen, M. K., & Kristensen, S. R. (2017). Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles. Journal of Extracellular Vesicles, 6(1), 1308779.

Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic in vitro the physiological conditions under which LPL acts. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recordings of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or post-heparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride-poor. Addition of angiopoietin-like proteins 3 or 4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system.

Reimund, M., Kovrov, O., Olivecrona, G., & Lookene, A. (2016). Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry. Journal of Lipid Research, jlr-D071787.

Lipoprotein Lipase Links Dietary Fat to Solid Tumor Cell Proliferation

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA.

Kuemmerle, N. B., Rysman, E., Lombardo, P. S., Flanagan, A. J., Lipe, B. C., Wells, W. A., ... & Swinnen, J. V. (2011). Lipoprotein lipase links dietary fat to solid tumor cell proliferation. Molecular cancer therapeutics, 10(3), 427-436.

Phospholipase D Activity Underlies Very-Low-Density Lipoprotein (VLDL)-induced Aldosterone Production in Adrenal Glomerulosa Cells

Aldosterone is the mineralocorticoid responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure as well as renal disease, stroke, and visual loss via both direct effects and effects on blood pressure. Weight gain is often associated with increased blood pressure, but it remains unclear how obesity increases blood pressure. Obese patients typically have higher lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very-low-density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Although previous studies have demonstrated that VLDL can stimulate aldosterone production, the mechanisms underlying this effect are largely unclear. Here we show for the first time that phospholipase D (PLD) is involved in VLDL-induced aldosterone production in both a human adrenocortical cell line (HAC15) and primary cultures of bovine zona glomerulosa cells. Our data also reveal that PLD mediates steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression via increasing the phosphorylation (activation) of their regulatory transcription factors. Finally, by using selective PLD inhibitors, our studies suggest that both PLD1 and PLD2 isoforms play an important role in VLDL-induced aldosterone production.

Tsai, Y. Y., Rainey, W. E., Pan, Z. Q., Frohman, M. A., Choudhary, V., & Bollag, W. B. (2014). Phospholipase D activity underlies very-low-density lipoprotein (VLDL)-induced aldosterone production in adrenal glomerulosa cells. Endocrinology, 155(9), 3550-3560.

Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies.

Ma, W., Paik, D. C., & Barile, G. R. (2014). Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical PathwayBioactive Lysophospholipids Complement Activation. Investigative ophthalmology & visual science, 55(10), 6187-6193.

VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production

Aldosterone plays an important role in regulating ion and fluid homeostasis and thus blood pressure, and hyperaldosteronism results in hypertension. Hypertension is also observed with obesity, which is associated with additional health risks, including cardiovascular disease. Obese individuals have high serum levels of very low-density lipoprotein (VLDL), which has been shown to stimulate aldosterone production; however, the mechanisms underlying VLDL-induced aldosterone production are still unclear. Here we demonstrate in human adrenocortical carcinoma (HAC15) cells that submaximal concentrations of angiotensin II and VLDL stimulate aldosterone production in an additive fashion, suggesting the possibility of common mechanisms of action. We show using inhibitors that VLDL-induced aldosterone production is mediated by the PLC/IP3/PKC signaling pathway. Our results suggest that PKC is upstream of the extracellular signal-regulated kinase (ERK) activation previously observed with VLDL. An understanding of the mechanisms mediating VLDL-induced aldosterone production may provide insights into therapies to treat obesity-associated hypertension.

Tsai, Y. Y., Rainey, W. E., Johnson, M. H., & Bollag, W. B. (2016). VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production. Molecular and cellular endocrinology.

Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions.

Tiaden, A. N., Bahrenberg, G., Mirsaidi, A., Glanz, S., Blüher, M., & Richards, P. J. (2016). Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase‐Mediated MMP Upregulation. STEM CELLS, 34(6), 1601-1614.

Fetal Bovine Lipoprotein Depleted Serum

An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer

Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.

Chen, M., Zhang, J., Sampieri, K., Clohessy, J. G., Mendez, L., Gonzalez-Billalabeitia, E., ... & Menon, A. V. (2018). An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer. Nature genetics, 1.

Ganoderic Acid A improves high fat diet-induced obesity, lipid accumulation and insulin sensitivity through regulating SREBP pathway

Obesity and its major co-morbidity, type 2 diabetes, have been an alarming epidemic prevalence without an effective treatment available. Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. Therefore, inhibition of SREBP pathway may be a useful strategy to treat obesity with type 2 diabetes. Here, we identify a small molecule, Ganoderic Acid A (GAA), inhibits the SREBP expression and decreases the cellular levels of cholesterol and fatty acid in vitro. GAA also ameliorates body weight gain and fat accumulation in liver or adipose tissues, and improves serum lipid levels and insulin sensitivity in high fat diet (HFD)-induced obese mice. Consistently, GAA regulates SREBPs target genes and metabolism associated genes in liver or adipose tissues, which may directly contribute to the lower lipid level and improvement of insulin resistance. Taken together, GAA could be a potential leading compound for development of drugs for the prevention of obesity and insulin resistance.

Zhu, J., Jin, J., Ding, J., Li, S., Cen, P., Wang, K., ... & Xia, J. (2018). Ganoderic Acid A improves high fat diet-induced obesity, lipid accumulation and insulin sensitivity through regulating SREBP pathway. Chemico-Biological Interactions.

Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity.

Breen, M. R., Camps, M., Carvalho-Simoes, F., Zorzano, A., & Pilch, P. F. (2012). Cholesterol depletion in adipocytes causes caveolae collapse concomitant with proteosomal degradation of cavin-2 in a switch-like fashion. PloS one, 7(4), e34516.

Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei

Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro.

Pérez-Moreno, G., Sealey-Cardona, M., Rodrigues-Poveda, C., Gelb, M. H., Ruiz-Pérez, L. M., Castillo-Acosta, V., ... & González-Pacanowska, D. (2012). Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei. International journal for parasitology, 42(11), 975-989.

Anhydroicaritin improves diet-induced obesity and hyperlipidemia and alleviates insulin resistance by suppressing SREBPs activation

SREBPs play important roles in the regulation of lipid metabolism, and are closely related to the occurrence and development of many metabolic diseases. Small molecular inhibitors of SERBPs are important tools in developing efficient treatment of metabolic diseases. However, there are no listing drug targeting SREBPs. Therefore, there is an urgent need to develop highly specific small molecules that inhibit SREBPs. In this study, using a hepatocyte-based high-throughput screening, we identified anhydroicaritin (AHI) as a novel inhibitor of SREBPs. HepG2, HL-7702, and human primary hepatocytes were used to verify the effects of AHI. We explored the mechanism by which AHI blocks the binding of SCAP/SREBPs complex with Sec23α/24D via regulating LKB1/AMPK/mTOR pathway. AHI reduced liver cell lipid level by preventing de novo lipogenesis. In diet induced obese mice, AHI ameliorated obesity, insulin resistance, fatty accumulation in liver and hyperlipemia. In conclusion, AHI improves diet-induced obesity and alleviates insulin resistance by suppressing SREBPs maturation which is dependent on LKB1/AMPK/mTOR pathway. Thus, AHI can serve as a leading compound for pharmacological control of metabolic diseases.

Zheng, Z. G., Zhou, Y. P., Zhang, X., Thu, P. M., Xie, Z. S., Lu, C., ... & Chen, X. W. (2016). Anhydroicaritin improves diet-induced obesity and hyperlipidemia and alleviates insulin resistance by suppressing SREBPs activation. Biochemical Pharmacology, 122, 42-61.