oxLDL Uptake Research Articles

Functional analysis of a biflavone, as a novel inhibitor of TRPV4‐dependent macrophages foam cell formation

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory disease, is the most dominant underlying pathology. Macrophages, an essential cell type in atherogenesis, are known to directly respond to changes to their surrounding mechanical environment. As TRPV4 (transient receptor potential channel of the vanilloid subfamily) channels can be sensitized by changes in matrix stiffness, we hypothesize that, TRPV4 may upregulate inflammatory/atherogenic macrophage functions. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay; we identified ginkgetin, a biflavone, as a novel antagonist to TRPV4. Primary and secondary screening was performed with normal human dermal fibroblasts and mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐elicited Ca2+ influx after TRPV4 activation with its selective agonist GSK1016790. In our hand's, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐elicited Ca2+ influx. We show that antagonism of TRPV4 channel by ginkgetin blocks oxidized LDL (oxLDL)‐induced macrophage foam cell formation in a concentration dependent manner. Mechanistically, we show that ginkgetin attenuated oxLDL uptake but not its cell surface binding in BMDMs. Altogether, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik O RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Naoxintong Retards Atherosclerosis by Inhibiting Foam Cell Formation Through Activating Pparα Pathway.

Backgrounds: We recently reported that Naoxintong (NXT), a China Food and Drug Administration (FDA)-approved cardiac medicine, could reduce the plaque size, but the underlying mechanism remains elusive now.Objective: In this study, we investigated the effects of NXT on foam cell accumulation both in vivo and in vitro and explored related mechanisms.Method: THP-1 cells and bone marrow-derived macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) with/without Naoxintong. ApoE-/- mice fed an atherogenic diet were administered to receive NXT for eight weeks. Macrophage-derived foam cell formation in plaques was measured by immunohistochemical staining. Expression of proteins was evaluated by Western blot. Lentivirus was used to knockdown PPARα in THP-1 cells.Results: After NXT treatment, foam cell accumulation was significantly reduced in atherosclerotic plaques. Further investigation revealed that oxidized low-density lipoprotein (ox-LDL) uptake was significantly decreased and expression of scavenger receptor class A (SR-A) and class B (SR-B and CD36) was significantly downregulated post-NXT treatment. On the other hand, NXT increased cholesterol efflux and upregulated ATP-binding cassette (ABC) transporters (ABCA-1 and ABCG-1) in macrophages. Above beneficial effects of NXT were partly abolished after lentiviral knockdown of PPARα.Conclusion: Our findings suggest that NXT could retard atherosclerosis by inhibiting foam cell formation through reducing ox-LDL uptake and enhancing cholesterol efflux and above beneficial effects are partly mediated through PPARα pathway.

A simple and rapid-acting approach for the reduction of C-reactive protein

C-reactive protein (CRP) is an acute-phase protein which can bind to and aggregate oxidized low-density lipoprotein (ox-LDL) particles, thereby enhancing the uptake of oxLDL by macrophages. This finally leads to the formation of foam cells that are a typical characteristic of atherosclerotic plaques. Serum CRP has been shown to bind to phospholipids such as phophatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylserine (PS). Owing to the rapid and efficient clearance of nanoliposomes from the circulation by the liver, we hypothesized that nanoliposomes composed of the mentioned phospholipids can serve as a potential tool to lower elevated serum CRP levels following acute inflammation. To evaluate this hypothesis, nanoliposomal formulations containing hydrogenated soy phosphatidylcholine (HSPC), a combination of HSPC and 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), and a combination of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) were prepared using lipid film hydration method followed by extrusion at the final concentration of 20 mM. To elevate circulating CRP levels in mice, 0.1 ml of Freund’s complete adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis was subcutaneously injected into the hind paw of the mice. CFA-challenged mice were intravenously treated with nanoliposomal formulations at the dose of 250 μmol/kg 16 h after CFA challenge that is coincided with peak serum CRP level. After 2 h, the blood was collected and serum level of CRP was measured using a quantitative sandwich enzyme-linked immunosorbent assay. All nanoliposomal formulations showed a size range from 100 to 150 nm in diameter and a polydispersity index of < 0.1. Results showed that all nanoliposomal formulations including DOPC/DOPS, HSPC and HSPC/DSPG could significantly decrease serum levels of CRP by 82.76% (74.44–86.92%, p = 0.0001), 44.41% (35.79–50.21%, p = 0.0001) and 38.47% (17.21–43.52%, p=0.0002) [Median (interquartile range)], respectively, when compared with the control group. Dexamethasone as a standard could decrease serum CRP level by 27.47% (16.32–31.63%, p = 0.0025) which was a smaller effect compared with the nanoliposomal preparations. In conclusion, negatively charged nanoliposomes could efficiently reduce the elevated serum levels of CRP in CFA-challenged mice.

IL-34 promotes foam cell formation by enhancing CD36 expression through p38 MAPK pathway

Atherosclerosis is characterized as a chronic inflammatory disease and macrophage-derived foam cells play a central role during the pathologic processes. A newly discovered cytokine interleukin-34 (IL-34) is closely associated with various inflammatory and autoimmune diseases. Expression of IL-34 in obesity, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), lupus nephritis and coronary artery diseases (CAD) are significantly elevated. However, the role of IL-34 in atherosclerosis remains unknown. In our present study, we found that IL-34 treatment markedly increased the uptake of oxLDL, intracellular total and esterified cholesterol content but not cholesterol efflux, subsequently promoted foam cell formation through up-regulating CD36 expression via p38 MAPK signal pathway in bone marrow derived macrophages (BMDMs). Furthermore, treatment with IL-34 significantly elevated the oxLDL-induced up-regulation of pro-inflammatory cytokines. Our results conclude that IL-34 facilitates foam cell formation by increasing CD36-mediated lipid uptake and suggest a potential new risk biomarker for atherosclerosis.

Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice

The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim−/−) and wild-type (Vim+/+) mice. Atherosclerosis was induced in Ldlr−/− mice transplanted with Vim−/− and Vim+/+ bone marrow, and in Vim−/− and Vim+/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12–15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim−/− macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim−/− macrophages. Vim−/− macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim−/− mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim−/− mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis.

S100/RAGE-Mediated Inflammation and Modified Cholesterol Lipoproteins as Mediators of Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

Arterial calcification is a feature of atherosclerosis and shares many risk factors including diabetes, dyslipidemia, chronic kidney disease, hypertension, and age. Although there is overlap in risk factors, anti-atherosclerotic therapies, including statins, fail to reduce arterial, and aortic valve calcifications. This suggests that low density lipoprotein (LDL) may not be the main driver for aortic valve disease and arterial calcification. This review focuses on modified LDLs and their role in mediating foam cell formation in smooth muscle cells (SMCs), with special emphasis on enzyme modified non-oxidized LDL (ELDL). In vivo, ELDL represents one of the many forms of modified LDLs present in the atherosclerotic vessel. Phenotypic changes of macrophages and SMCs brought about by the uptake of modified LDLs overlap significantly in an atherosclerotic milieu, making it practically impossible to differentiate between the effects from oxidized LDL, ELDL, and other LDL modification. By studying in vitro-generated modifications of LDL, we were able to demonstrate marked differences in the transcriptome of human coronary artery SMCs (HCASMCs) upon uptake of ELDL, OxLDL, and native LDL, indicating that specific modifications of LDL in atherosclerotic plaques may determine the biology and functional consequences in vasculature. Enzyme-modified non-oxidized LDL (ELDL) induces calcification of SMCs and this is associated with reduced mRNA levels for genes protective for calcification (ENPP1, MGP) and upregulation of osteoblastic genes. A second focus of this review is on the synergy between hyperlipidemia and accelerated calcification In vivo in a mouse models with transgenic expression of human S100A12. We summarize mechanisms of S100A12/RAGE mediated vascular inflammation promoting vascular and valve calcification in vivo.

60 - Aegeline, targets LOX1, the receptor for oxidized LDL to mitigate hypercholesterolemia: a new perspective in its anti-atherosclerotic action

Overview Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the major receptors for oxidized low-density lipoprotein (Ox-LDL) in the aorta of aged rats. Ox-LDL initiates LOX-1 activation in the endothelium of lipid accumulating sites of both animal and human subjects of hypercholesterolemia. This study has explored the binding affinity of Aegeline to LOX1 for the first time. Targeting LOX-1 may provide a novel diagnostic strategy for hypercholesterolemia and vascular diseases. Objective This study was planned to address whether an Aegeline (AG) could bind to LOX-1 with a higher affinity and modulate the uptake of ox-LDL in hypercholesterolemia. Methods Thirty-six Wistar rats were divided into six groups. The pathology group rats were fed with High Cholesterol Diet (HCD) for 45 days and treatment group rats were fed with HCD and AG/AV for last 30 days. In vivo and in vitro experiments were carried out to assay the markers of atherosclerosis like Ox-LDL and LOX1 levels. Histopathological examination of aorta tissue sections was performed. Oil red O staining was carried out in the mouse peritoneal macrophage cell line IC21. Docking studies were performed to find out the binding affinity of AG and AV to LOX1. Results AG administration effectively decreased the lipid levels induced by HCD as evidenced by lipid profile. Further, the lowered levels of Ox-LDL and LOX1 in AG administered rats deem it to be a potent anti-hypercholesterolemic agent. Conclusion This study thus validates AG to be an effective stratagem in bringing down the lipid stress induced by HCD and by down-regulating LOX1 mediated uptake of Ox-LDL.

Primary prevention of atherosclerosis by pretreatment of low-density lipoprotein receptor knockout mice with sesame oil and its aqueous components

Pharmacological intervention using statins and PCSK9 inhibitors have become first-line therapy in the prevention of hypercholesterolemia and atherosclerosis. Currently, no agent is available for the primary prevention of atherosclerosis. However, there is an emerging hypothesis that atherosclerosis could be driven by inflammation. In this study, we tested whether pretreatment with an aqueous extract from sesame oil (SOAE), which showed potent anti-inflammatory properties without hypocholesterolemic actions, would prevent subsequent atherosclerosis development in a mouse model. RAW 264.7 macrophages and female low-density lipoprotein receptor knockout (LDLR−/−) mice were used for in vitro and in vivo studies, respectively. Plasma lipids, cytokines and atherosclerotic lesions were quantified at the end of the study. RNA was extracted from the liver and aortic tissues and used for gene analysis. Pre-treatment of SOAE prevented Ox-LDL uptake by RAW macrophages and further inflammation in vitro. SOAE pre-treatment significantly reduced atherosclerotic lesions and pro-inflammatory gene expressions in LDLR−/− mice as compared to control mice. No significant change in plasma cholesterol levels was observed. A significant reduction in plasma levels of TNF-α, IL-6, MCP-1 and VCAM1 was observed in the SOAE pre-treated animals. This is the first study that demonstrates that pre-treatment with anti-inflammatory agents, could delay/decrease atherosclerosis.

Inhibition of the CD36 receptor reduces visceral fat accumulation and improves insulin resistance in obese mice carrying the BDNF-Val66Met variant

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor (BDNF) gene that results in the substitution of a valine with a methionine at codon 66 (Val66Met) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of cluster of differentiation 36 (CD36), a multifunctional receptor implicated in obesity and insulin resistance, in WT mice and mice with the BDNF Val66Met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese WT mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was abrogated in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the Val66Met variant in both alleles (BDNFM/M) fed a high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNFM/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extremely obese BDNFM/M mice compared with the WT mice. The study demonstrated a link between BDNF Val66Met and elevated CD36 expression and suggested that CD36 inhibition may be a potential strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population.

Comparative analysis of endothelial cell and sub-endothelial cell elastic moduli in young and aged mice: Role of CD36

ObjectiveTo perform comparative analysis of the role of scavenger receptor CD36 on endothelial vs. sub-endothelial elastic modulus (stiffness) in the aortas of young and aged mice. Approaches and ResultsElastic moduli of endothelial and sub-endothelial layers of freshly isolated mouse aortas were quantified using atomic force microscopy. In young mice (4–6 months old), we found that while endothelial stiffness is markedly reduced in aortas of CD36−/−mice, as compared to WT controls, no difference between CD36−/− and WT aortas is observed in the stiffness of the sub-endothelial layer in denuded arteries. Additionally, inhibition of myosin phosphorylation also decreases the elastic modulus in the EC, but not the sub-EC layer in WT mice. Moreover, inhibiting CD36 mediated uptake of oxLDL in intact WT aortas abrogated oxLDL-induced endothelial stiffening. Further analysis of aged mice (22–25 months) revealed that aging resulted not only in significant stiffening of the denuded arteries, as was previously known, but also a comparable increase in the elastic modulus of the endothelial layer. Most significantly, this stiffening in the EC layer is dependent on CD36, whereas the denuded layer is not affected. ConclusionsOur results show that the role CD36 in stiffening of cellular components of intact aortas is endothelial-specific and that genetic deficiency of CD36 protects against endothelial stiffening in aged mice. Moreover, these data suggest that endothelial stiffness in intact mouse aortas depends more on the expression of CD36 than on the stiffness of the sub-endothelial layer.

Dan-Lou Prescription Inhibits Foam Cell Formation Induced by ox-LDL via the TLR4/NF-κB and PPARγ Signaling Pathways.

Atherosclerosis is the major worldwide cause of mortality for patients with coronary heart disease. Many traditional Chinese medicine compound prescriptions for atherosclerosis treatment have been tried in patients. Dan-Lou prescription, which is improved from Gualou-Xiebai-Banxia decoction, has been used to treat chest discomfort (coronary atherosclerosis) for approximately 2,000 years in China. Although the anti-inflammatory activities of Dan-Lou prescription have been proposed previously, the mechanism remains to be explored. Based on the interaction between inflammation and atherosclerosis, we further investigated the effect of Dan-Lou prescription on macrophage-derived foam cell formation and disclosed the underlying mechanisms. In the oxidative low-density lipoprotein (ox-LDL) induced foam cells model using murine macrophage RAW 264.7 cells, the ethanol extract from Dan-Lou prescription (EEDL) reduced ox-LDL uptake and lipid deposition by inhibiting the protein and mRNA expression of Toll-like receptor (TLR)4 and scavenger receptor (SR)B1. After stimulation with ox-LDL, the metabolic profile of macrophages was also changed, while the intervention of the EEDL mainly regulated the metabolism of isovalerylcarnitine, arachidonic acid, cholesterol, aspartic acid, arginine, lysine, L-glutamine and phosphatidylethanolamine (36:3), which participated in the regulation of the inflammatory response, lipid accumulation and cell apoptosis. In total, 27 inflammation-related gene targets were screened, and the biological mechanisms, pathways and biological functions of the EEDL on macrophage-derived foam cells were systemically analyzed by Ingenuity Pathway Analysis system (IPA). After verification, we found that EEDL alleviated ox-LDL induced macrophage foam cell formation by antagonizing the mRNA and protein over-expression of PPARγ, blocking the phosphorylation of IKKα/β, IκBα and NF-κB p65 and maintaining the expression balance between Bax and Bcl-2. In conclusion, we provided evidences that Dan-Lou prescription effectively attenuated macrophage foam cell formation via the TLR4/NF-κB and PPARγ signaling pathways.

Ox-LDL induces endothelial cell apoptosis and macrophage migration by regulating caveolin-1 phosphorylation.

Oxidative low-density lipoprotein (ox-LDL) is a risk factor for atherosclerosis. Ox-LDL leads to endothelial injury in the initial stage of atherosclerosis. In this study, we investigated the role of ox-LDL in endothelial injury and macrophage recruitment. We demonstrated that ox-LDL promoted a dose-dependent phosphorylation of caveolin-1 in human umbilical vein endothelial cells. Phosphorylated caveolin-1 increased ox-LDL uptake. Intracellular accumulation of ox-LDL induced NF-κB p65 phosphorylation, promoted HMGB1 translocation from nucleus to cytoplasm and cytochrome C release from mitochondria to cytoplasm, and activated caspase 3, resulting in cell apoptosis. NF-κB activation also facilitated cavolin-1 phosphorylation and HMGB1 expression. In addition, caveolin-1 phosphorylation favored HMGB1 release and nuclear translocation of EGR1. Nuclear translocation of EGR1 contributed to cytoplasmic translocation of HMGB1. The extracellular HMGB1 induced the migration of PMBC-derived macrophages toward HUVECs in a TLR4-dependent manner. Our results suggested that ox-LDL promoted HUVECs apoptosis and macrophage recruitment by regulating caveolin-1 phosphorylation.

Abstract 629: The (Pro)renin Receptor Regulates Scavenger Receptor CD36 Protein Degradation and Functions

Background: Accumulation of oxidized lipids in monocyte-derived macrophages results in foam cell formation and induces chronic inflammation in the vascular wall, thus contributing to the onset and progression of atherosclerosis. Scavenger receptor CD36 is the most important clearance receptor for ox-LDL, and has been implicated in triggering inflammatory responses in macrophages by ox-LDL. Genetically deleting CD36 reduced foam cell formation and lesion size in ApoE -/- mice. Additionally, circulating macrophages isolated from patients that are deficient of CD36 show much less ox-LDL uptake and accumulation than those from normal subjects. Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as a regulator of CD36 protein degradation. Interestingly, we found that inhibiting the hepatic (pro)renin receptor [(P)RR] dramatically increased plasma PCSK9 levels in C57BL/6J mice. Thus, we wondered if the (P)RR could regulate CD36 protein degradation and functions by increasing PCSK9 levels. Methods and Results: We inhibited hepatic (P)RR expression in PCSK9 -/- mice and wildtype mice, using GalNAc-modified anti-sense oligos targeting the (P)RR. Hepatic (P)RR inhibition reduced hepatic CD36 protein abundance by ~40% in WT mice, without affecting CD36 transcriptional levels. Unexpectedly, inhibiting hepatic (P)RR in PCSK9 -/- mice showed a similar reduction in hepatic CD36 protein abundance, suggesting that such reduction in CD36 is unlikely to be mediated by increased plasma PCSK9 levels. To clarify this, we exogenously expressed CD36 in HEK293T cells and studied whether silencing the (P)RR could directly affect CD36 protein abundance. Indeed, silencing the (P)RR reduced exogenously expressed CD36 by almost 50%, confirming that the (P)RR directly controls CD36 degradation. Functional studies revealed that inhibiting/deleting the (P)RR attenuated fatty acid uptake in differentiated 3T3-L1 adipocytes, and ox-LDL uptake in circulating mouse macrophages in a CD36-dependent manner. Conclusion: The (P)RR acts as a post-transcriptional regulator of CD36, and this occurs in a PCSK9-independent manner. Macrophagic (P)RR may play a role in the development of atherosclerosis by interacting with CD36.

Abstract 440: Targeting LOX-1 Scavenger Receptor and oxLDL Uptake With Affimers

Introduction: Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1, SREC-1, OLR1) is a class E scavenger receptor which binds oxidized low density lipoprotein (oxLDL) particles implicated in promoting atherosclerosis. Affimers are non-antibody synthetic protein-based scaffolds that enable recognition of a wide range of molecular targets. Aims: To investigate the ability of Affimers to bind LOX-1 and modulate oxLDL accumulation and effects on cellular responses. Methods: Recombinant soluble human LOX-1 was screened with an Affimer library and LOX-1-specific Affimers were isolated and characterized. Human embryonic kidney (HEK293T) cells inducibly expressing human LOX-1 were incubated with labeled oxLDL and Affimers to modulate LOX-1 and oxLDL uptake and trafficking. Furthermore, Affimer-mediated effects on oxLDL-regulated metabolism and apoptosis was evaluated. Results: Five Affimers specific for human LOX-1 were identified. Fluorescent labelled Affimers demonstrate specific binding to LOX-1 expressing HEK293T cells and modulate LOX-1 and oxLDL trafficking and accumulation in endosomes and lysosomes. LOX-1-specific Affimers also show effects in modulating oxLDL-mediated effects in specific cellular responses. Conclusion: LOX-1-specific Affimers are a new class of synthetic molecules that have therapeutic potential in targeting LOX-1, a key mediator of inflammation and arterial dysfunction in cardiovascular disease. Membrane specific binding is demonstrated only in induced LOX-1 expressing cells (LOX+). Increasing concentration of affimer prior to incubation with DiI labelled oxLDL.

Abstract 036: Functionalized Nanoparticle Based “Two-pronged” Approach to Attenuate/regress Atherosclerosis by Simultaneous Knock Down OfSR-A and LXR-mediated Stimulation of Macrophage Cholesterol Efflux

Imbalance between lipoprotein uptake and cholesterol efflux from macrophages underlies the formation of foam cells and development of fatty streaks, a process that starts early in life. While currently available lipid lowering interventions attenuate the progression of atherosclerotic plaques, no targeted therapy is presently available for reduction of existing plaques, a process dependent not only on limiting further accumulation of cholesterol but also on stimulation of cholesterol efflux. We earlier reported the development of a mannose functionalized dendrimeric nanoparticles (mDNP)-based platform for targeted delivery of therapeutics to arterial plaque associated macrophages. In this study, we used the optimized mDNP platform to simultaneously deliver SR-A siRNA (to knock down SR-A and limit modified LDL uptake) and LXR ligand (LXR-L, to stimulate macrophage cholesterol efflux by inducing expression of ABCA1/G1) - a “ Two Pronged ” approach. Compared to non-specific NS-siRNA, mDNP mediated delivery of SR-A siRNA led to significant reduction in expression of SR-A (A&amp;B) with a corresponding decrease in uptake of DiI-labeled oxLDL (C). Delivery of LXR-L increased expression of ABCA1/G1 (D) as well as cholesterol efflux (E) and simultaneous delivery of SR-A siRNA did not alter these LXR-L effects. However, combined delivery of siRNA and LXR-L led to significantly higher decrease in macrophage cholesterol content compared to either treatment alone (F). Administration of this in vitro optimized formulation of mDNP complexed with SR-A siRNA and LXR-L to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to a dramatic reduction in lesion area compared to single treatment alone (G). In conclusion, targeted delivery of multiple therapeutics using mDNP platform represents a novel strategy to not only reduce lesion size but it can also be used to simultaneously alter many lesion characteristics such as reducing inflammation or enhancing efferocytosis.

Pseudolaric acid B attenuates atherosclerosis progression and inflammation by suppressing PPARγ-mediated NF-κB activation

Aims/objectiveAtherosclerosis is a progressive disease of large arteries characterized with chronic inflammation and aberrant immune response. Pseudolaric acid B (PB) has been found to exert multiple effects by inhibiting inflammatory response. However, there is no comprehensive assessment of the effects of PB on atherosclerosis using relevant in vivo and in vitro models. Material and methodsMale ApoE−/− mice were treated with PB orally with a high fat diet (HFD) to clarify its anti-atherosclerotic activities. RAW264.7 macrophage line, a well-accepted cell model of atherosclerosis, was used to investigate anti-inflammatory effects and molecular mechanisms of PB. ResultsPB significantly attenuated atherosclerotic lesions by modulating plasma lipid profiles as well as inhibiting inflammatory responses in macrophages of atherosclerotic mice. Meanwhile, PB markedly suppressed the expression of pro-inflammatory cytokines, and regulated cholesterol efflux related genes in oxidative low density lipoprotein (ox-LDL)-loaded macrophages. The cellular uptake of Dil-labeled ox-LDL was significantly inhibited by PB either. Moreover, the ability of PB to suppress nuclear factor kappa B (NF-κB) and activate peroxisome proliferator-activated receptor gamma (PPARγ) was confirmed using luciferase reporter assays. Conversely, the selective PPARγ antagonist GW9662 reversed the influence of PB in macrophages. ConclusionTogether, these findings indicate that PB exerts its protective effects on atherosclerosis by inhibiting macrophage-mediated inflammatory response and cellular ox-LDL uptake, and promoting cholesterol efflux by suppressing NF-κB activation PPARγ-dependently. Therefore, PB may be a promising agent for inflammatory and atherosclerotic diseases.

Understanding the role of pancreatic β‐cell CD36 in the development of Type 2 Diabetes

CD36 is a transmembrane scavenger receptor for oxidized LDL (oxLDL). Expression of CD36 and subsequent uptake of oxLDL into macrophages is central to foam cell formation in atherosclerotic plaques. CD36 is also reported to play a pivotal role in glucotoxicity‐induced β‐cell dysfunction in Type 2 Diabetes (T2D). The aim of this study is to investigate mechanistic functions of CD36 that contribute to β‐cell dysfunction and the onset of T2D. For this study, rat INS‐1 pancreatic β‐cells were cultured in either low (5 mM) or high (11 mM) glucose conditions. Cells grown in high glucose exhibited a left shift in glucose stimulated insulin secretion (GSIS), showing that the cells are more sensitive to lower glucose levels. These cells also contained more lipid droplets, as shown by Nile Red staining. Using immunofluorescence and Western blotting, we demonstrated that CD36 expression increased with increasing glucose concentrations. When cells were incubated with DiI‐labeled oxLDL to investigate uptake, cells that were grown in high glucose exhibited increased labeling. Interestingly, oxLDL uptake by INS‐1 cells grown in 11 mM glucose can be suppressed by the addition of the ω‐3 fatty acid, docosahexaenoic acid (DHA). Studies investigating the effect of DHA and additional fatty acids on uptake of oxLDL by INS‐1 cells and effects on GSIS are ongoing. We propose that CD36 plays a role in how β‐cells respond to high glucose levels, similarly found in T2D. The increase in oxLDL uptake via CD36 in these cells may contribute to glucolipotoxic effects in β‐cells that results in aberrant insulin secretion in T2D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P. gingivalis lipopolysaccharide‐induced exacerbation of oxidized LDL‐mediated macrophage foam cell formation is reliant on TRPV4 channels

Although P. gingivalis, a predominant causative agent of periodontitis, has been linked to lipidladen macrophage foam cell formation, a critical process in atherogenesis, the mechanism remains elusive. Emerging data support a role for both a biochemical factor, e.g., lipopolysaccharides (LPS), and a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family, and a known mechanosensor, is the likely mediator of P. gingivalis lipopolysaccharide (PgLPS)‐induced exacerbation of oxidized low‐density lipoprotein (oxLDL)‐mediated macrophage foam cell formation. Specifically, we found that: i) genetic deletion of TRPV4 or antagonism of its function by a chemical inhibitor blocked PgLPS‐induced exacerbation of oxLDL‐induced macrophage foam cell formation; ii) macrophage TRPV4 activity was increased in response to both PgLPS and pathophysiological range matrix stiffness, and iii) TRPV4 deficiency blocked PgLPS‐induced exacerbation of matrix stiffness‐induced macrophage foam cell formation. Mechanistically, we show that TRPV4 regulates PgLPS‐induced oxLDL uptake but not its cell surface binding in macrophages. Altogether, these findings identify a critical role for TRPV4 in regulating PgLPS‐induced exacerbation of macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA‐146a Deficiency Promotes Atherosclerosis by Dysregulating Cholesterol Homeostasis in Macrophages

Atherosclerosis is a chronic inflammatory disease that results from imbalance of high‐density lipoprotein (HDL) cholesterol metabolism and a maladaptive immune response driven by the accumulation of cholesterol‐laden macrophages in the artery wall. MicroRNA‐146a (miR‐146a) serves as anti‐inflammatory function in a variety of cell types, but the underlying mechanisms of miR‐146a in atherosclerosis still remain unclear. In the present study, we assessed the impact of the genetic deficiency of miR‐146a in a mouse model of atherosclerosis and determined the miR‐146a‐mediated balance of cholesterol metabolism in macrophages. In comparison with miR‐146a+/+ApoE−/− mice fed with high‐cholesterol diet for 10 weeks, miR‐146a−/−ApoE−/− mice showed an increase number and area of atherosclerotic plaques, lipid content, pro‐inflammatory cytokines and abundant macrophages accumulated in the plaques. In addition, miR‐146a−/−ApoE−/− mice also showed decrease in HDL levels. Bone marrow chimera experiments indicated a major effect of miR‐146a deficiency on lesional macrophages. Bone marrow derived‐macrophages (BMDMs) were isolated from miR‐146a−/−ApoE−/− and miR‐146a+/+ApoE−/− mice to stimulated with oxidized low‐density lipoprotein (oxLDL). In comparison with BMDMs of miR‐146a+/+ApoE−/−, BMDMs of miR‐146a−/−ApoE−/− showed a significant oxLDL uptake and reduction in cholesterol efflux capacity. The expression of cholesterol transporters, including the ATP‐binding cassette sub‐family A1 (ABCA1) and sub‐family G1 (ABCG1) were significantly decreased in BMDMs of miR‐146a−/−ApoE−/−. By using the global gene expression analysis and argonaute‐2 immunoprecipitation, we demonstrated that toll‐like receptor 4 (TLR4) is directly targeted by miR‐146a. Overexpression of miR‐146a and silence of TLR4 not only increased the expression of ABCA1 and ABCG1, but also increased the cholesterol efflux capacity and reduced the oxidized LDL uptake. These data demonstrated that miR‐146a is involved in regulation of ABCA1 and ABCG1 by targeting TLR4, and that maintains the balance of HDL cholesterol metabolism to protect against the atherosclerosis. MiR‐146a is a potential target in treatment of atherosclerotic vascular disease.Support or Funding InformationMOST 104‐2321‐B‐400‐020‐MY3 (to L.‐J. C.) and MOST 104‐2321‐B‐400‐017 (to J.‐J. C.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metformin attenuates effects of cyclophilin A on macrophages, reduces lipid uptake and secretion of cytokines by repressing decreased AMPK activity.

Growing evidence implicates cyclophilin A secreted by vascular wall cells and monocytes as a key mediator in atherosclerosis. Cyclophilin A in addition to its proliferative effects, during hyperglycemic conditions, increases lipid uptake in macrophages by increasing scavenger receptors on the cell's surface. It also promotes macrophage migration across endothelial cells and conversion of macrophages into foam cells. Given the known effects of metformin in reducing vascular complications of diabetes, we investigated the effect of metformin on cyclophilin A action in macrophages. Using an ex vivo model of cultured macrophages isolated from patients with type 2 diabetes with and without coronary artery disease (CAD), we measured the effect of metformin on cyclophilin A expression, lipid accumulation, expression of scavenger receptors, plasma cytokine levels and AMP-activated protein kinase (AMPK) activity in macrophages. In addition, the effects of metformin on migration of monocytes, reactive oxygen species (ROS) formation, lipid uptake in the presence of cyclophilin A inhibitors and comparison with pioglitazone were studied using THP-1 monocytes. Metformin reduced cyclophilin A expression in human monocyte-derived macrophages. Metformin also decreased the effects of cyclophilin A on macrophages such as oxidized low-density lipoprotein (oxLDL) uptake, scavenger receptor expression, ROS formation and secretion of inflammatory cytokines in high-glucose conditions. Metformin reversed cyclophilin A-induced decrease in AMPK-1α activity in macrophages. These effects of metformin were similar to those of cyclophilin A inhibitors. Metformin can thus function as a suppressor of pro-inflammatory effects of cyclophilin A in high-glucose conditions by attenuating its expression and repressing cyclophilin A-induced decrease in AMPK-1α activity in macrophages.