bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 552: Enhancement of Rat Lymphatic Lipid Transport by Glucose or Amino Acids Ingestion
Authors: Hayashi Hiroshi
Published: March 2019

Objectives:
Dietary lipid absorption usually occurs with absorption of other nutrients because lipid is contained in foods fed as a meal. The effect of simultaneously fed carbohydrate and protein on lipid absorption is not clear. In this study, lymphatic lipid transports in the rat intestine were observed during intraduodenal lipid feeding with or without simultaneous feeding of glucose or amino acids.

Methods:
Mesenteric lymph duct was cannulated in male SD rats and a feeding tube was inserted into the duodenum through the fundus of the stomach. Postoperatively, the rats were intraduodenally infused with saline. On the day after surgery, the saline infusion was replaced by a lipid infusion containing 40 μmol/h (35.4 mg/h) of triolein, 2.74 μmol/h (1.06 mg/h) of cholesterol, 7.8 μmol/h ( 6.08 mg/h) of egg lecithin with 300 mg/h of glucose (the Glucose group), with 150 mg/h of amino acids derived from medical amino acid injections (the Amino Acids group), or without any additive (the Control group). The lipid emulsion was infused at 3mL/h for 8 hours.

Results:
The amounts of triglyceride transported in lymph for 8 hours were 185 ± 12 (mean ± SE) mg in the Amino Acids group (n=4), 175 ± 3 mg in the Glucose group (n=5), and 147 ± 7 mg in the Control group (n=4), respectively, with a statistically significant difference (p < 0.05) among the groups. The amount of cholesterol transported in lymph for 8 hours were 7.30 ± 0.63 mg in the Amino Acids group, 6.82 ± 0.46 mg in the Glucose group, and 5.96 ± 0.17 mg in the Control group, respectively. The amount of phosphatidylcholine transported in lymph for 8 hours were 16.4 ± 1.0 mg in the Amino Acids group, 15.7 ± 0.4 mg in the Glucose group, and 12.4 ± 0.3 mg in the Control group, respectively, with a statistically significant difference (p < 0.01) among the groups.

Conclusions:
Simultaneous glucose or amino acids feeding enhanced lymphatic lipid transport in the rat intestine during lipid feeding.
Abstract 555: Investigation of 4-oxo-2-nonenal Mediated HDL Dysfunction and the Use of Lipid Dicarbonyl Scavengers in Preserving HDL Function
Authors: May-Zhang Linda S , Borja Mark S , Pleasent Tiffany , Venkataraman Amarnath , Yancey Patricia G , Linton MacRae F , Davies Sean S
Published: March 2019

Background:
Lipid peroxidation products such as hydroxy-2-nonenal (HNE) are elevated in atherosclerosis and cause HDL dysfunction. Generated in parallel to HNE is 4-oxo-2-nonenal (ONE), a less studied lipid dicarbonyl possibly due to its far greater reactivity. The consequences of ONE modification of HDL have not been studied. We have recently found that scavengers of lipid dicarbonyls such as salicylamine (SAM) and pentylpyridoxamine (PPM) prevent HDL dysfunction induced by malondialdehyde and isolevuglandin (IsoLG). In this study, we examine the impact of ONE adduct formation on HDL structure-function, and the scavenging abilities of various small molecules in preventing ONE modification.

Methods and Results:
By Western blot analysis, ONE crosslinked apoA-I on HDL at a concentration of 3 ONE molecules for every 10 apoA-I proteins (0.3 molar equivalence, eq.), which is 100 fold lower than HNE but comparable to IsoLG. ONE-mediated crosslinking of HDL proteins preferentially produced a 39 kDa band on SDS-PAGE, likely an apoA-I/apoA-II heterodimer. ONE-modified HDL partially inhibited the ability of HDL to protect against the inflammatory response of macrophages (as shown in TNFα and IL-1β mRNA expression), but did not render HDL pro-inflammatory. At 3 eq., ONE dramatically decreased the ability of apoA-I to exchange among HDLs, from ~46.5% to only ~18.4% (P<0.001). HDL-mediated macrophage cholesterol efflux was decreased by ~70.6% (P<0.005) and 56.1% (P<0.001) by HDL modified by 0.3 and 3 eq. ONE, respectively. Investigation of various scavenger analogues in protecting HDL from ONE modification showed that while SAM and its fluoro- and chloro- analogues partially prevented ONE-mediated HDL protein crosslinking, PPM nearly completely blocked crosslinking. PPM pretreatment of HDL prior to 3 eq. ONE modification was also able to restore HDL-mediated macrophage cholesterol efflux, from 56.1~ to ~83.0% (P<0.01) while the inactive analogue pentylpyridoxine had no effect.

Conclusions:
Our study is the first to show that ONE causes HDL dysfunction, and demonstrates that not all modified HDLs result in the same “dysfunction”. We also demonstrate the use of PPM in preferentially scavenging ONE in biological systems.
Abstract 563: Working Out the Kinks of SR-BI-mediated Cholesterol Transport
Authors: Proudfoot Sarah C , Chadwick Alexandra C , Sahoo Daisy
Published: March 2019

High density lipoproteins (HDL) facilitate reverse cholesterol transport (RCT), the process by which cholesterol effluxed from peripheral tissues is delivered via selective uptake to the liver for excretion. These processes are mediated by HDL's high-affinity receptor, scavenger receptor BI (SR-BI). SR-BI is an 82 kDa glycoprotein with two transmembrane domains (TMD), two short cytoplasmic N/C-terminal domains, and a large extracellular domain. Our laboratory solved the NMR structure of the C-terminal TMD and nearby extracellular domain. This peptide contains four conserved proline residues, an amino acid that confers structural flexibility. In our structure, P408 and P412 are in an extracellular alpha-helix, P438 lies at a helix-loop junction, and P459 introduces a kink into the TMD helix. We hypothesized that these proline residues support SR-BI-mediated cholesterol transport. To test this hypothesis, we mutated each proline to alanine and transiently expressed wild-type (WT), P408A-, P412A-, P438A-, or P459A-SR-BI in COS-7 cells. Total lysate and cell surface expression of SR-BI was examined by immunoblot analysis and flow cytometry, respectively. All mutants expressed on the cell surface, except P408A-SR-BI, which was absent from whole cell lysates. P412A- and P438A-SR-BI were significantly impaired in their ability to efflux cell-associated [
3
H]cholesterol to HDL, as compared to WT-SR-BI. Likewise, cellular binding of [
125
I]HDL and selective uptake of [
3
H]cholesteryl oleyl ether were reduced with P412A- and P438A-SR-BI. We assessed SR-BI’s ability to modulate membrane cholesterol pools by cholesterol oxidase treatment. SR-BI enhances the accessibility of cholesterol to the exogenous enzyme, and a proportional increase in cholestenone is detected by thin layer chromatography. Cholestenone was increased with WT-SR-BI but not P412A- or P438A-SR-BI, suggesting defective membrane cholesterol distribution. Taken together, key proline residues in the near-C-TMD extracellular domain support efficient HDL binding, selective uptake, cholesterol efflux, and membrane cholesterol organization. These critical residues appear to support SR-BI in a conformation that allows for efficient RCT.
Abstract 565: Effects of PCSK9 Inhibition on Plasma PCSK9 Concentration and Hepatic
PCSK9
Expression
Authors: Shapiro Michael D , Tavori Hagai , Oleaga Carlota , Miles Joshua , Fazio Sergio
Published: March 2019

Background:
We previously reported a marked (~7-fold) increase in plasma PCSK9 concentrations in patients treated with monoclonal antibodies blocking PCSK9 action. It is unclear whether this pronounced increase in plasma PCSK9 is due to increased hepatic production of PCSK9, slow plasma clearance of the antibody/PCSK9 complex, or both.

Objectives:
To determine whether hepatic
PCSK9
gene expression increases after exposure to a therapeutic monoclonal antibody targeted to PCSK9.

Methods:
C57Bl/6 wild-type (WT) mice and human PCSK9 transgenic (PCSK9tg) mice were treated with a single injection (10 mg/kg) of a monoclonal antibody targeted to PCSK9. Plasma samples were collected at baseline and then at 1, 2, 4, 8, 24, 48, 72, 192 and 336 hours after injection. Livers were sampled from age-matched mice (n=4) at baseline and 24 hours post injection for gene expression analyses.

Results:
In WT mice (n=8), mean baseline plasma PCSK9 level was 69±28 ng/mL and mean peak plasma PCSK9 level was 719±239 ng/mL (a 10.4-fold increase). Peak PCSK9 levels were reached at 24 hours after injection of the monoclonal antibody. At 24 hours after injection a statistically significant 3-fold increase in hepatic
PCSK9
gene expression was observed, compared with baseline, in WT mice. On the other hand, treatment with the antibody increased human PCSK9 levels only by 2-fold in our transgenic mice, whose human PCSK9 is expressed by extrahepatic tissues and in a stable and unregulated fashion.

Conclusion:
We observed a much larger increase in plasma PCSK9 levels in WT vs. PCSK9tg mice 24 hours after injection of a monoclonal antibody that blocks plasma PCSK9. This finding suggests that the striking increase in plasma PCSK9 in WT mice, which mirrors the effects seen in patients under treatment with PCSK9 blocking antibodies, is mostly due to increased hepatic production and only modestly due to reduced plasma clearance of the antibody/PCSK9 complex.
Abstract 566: High-density Lipoprotein Inhibits Serum Amyloid a -Mediated Inflammasome Activation
Authors: Shridas Preetha , de Beer Maria C , Webb Nancy R
Published: March 2019

Objectives:
Interleukin-1beta (IL-1β) has been implicated in inflammatory diseases, including atherosclerosis and abdominal aortic aneurysm (AAA). Production of bioactive IL-1β is controlled by the inflammasome, a multi-protein complex that regulates caspase-1 activity. Serum Amyloid A (SAA) is an acute-phase protein whose levels in circulation is elevated in individuals with chronic inflammation. We previously reported that deficiency of SAA protects mice from angiotensin II (AngII)-induced AAA. Here we report that reduced AngII-induced AAA in SAA-deficient mice is accompanied by significant reductions in plasma IL-1β, indicating that SAA is required for inflammasome activation in AngII-infused mice. The objective of this study is to investigate mechanisms involved in SAA-mediated inflammasome activation.

Methods/Results:
SAA dose-dependently induced both caspase-1 activation and IL-1β secretion in J774 macrophage-like cells incubated with 0-25 μg/ml purified mouse SAA. The ability of SAA to induce IL-1β secretion was significantly reduced in bone marrow-derived macrophages deficient in NLRP3. A caspase-1inhibitor, Z-YVAD-FMK, significantly suppressed IL-1β secretion induced by SAA, whereas the P2X7-receptor antagonist, AA38079, had no effect. Inhibition of reactive oxygen species (ROS), cathepsin-B activation, and cellular potassium efflux by N-acetyl-L-cysteine, CA-074, and glyburide, respectively, blocked NLRP3 inflammasome activation by SAA. Pre-incubating SAA with HDL prior to cell treatments completely abrogated SAA-mediated inflammasome activation. In contrast, HDL did not alter inflammasome activation triggered by ATP.

Conclusions:
SAA-mediated NLRP3 inflammasome activation in macrophages is dependent on ROS generation, release of cathepsin-B, and potassium efflux, and is independent of the P2X7 receptor. Moreover, our data identify a novel mechanism by which HDL may exert cardioprotective effects.
Abstract 567: Insulin Reverses Mcp-1 Suppressed Cholesterol Efflux to Hdl3/apoa1 Through Up-regulation of Abca1, Abcg1 and Sr-bi in Differentiated 3t3-l1 Adipocytes
Authors: Sun Runlu , Huang Canxia , Jiang Jieyu , Bao Jinlan , Zhang Yuling
Published: March 2019

Aims:
Insulin has been reported to influence cholesterol removal from different cells, but the results have been controversial. Based on our previous study, which we found that insulin promote cholesterol efflux from HepG2 cells and can reverse the decreased cholesterol efflux to HDL though PI3K-Akt pathway, we further investigate the effects of insulin on damaged cholesterol efflux by MCP-1 in this report.

Methods:
3T3-L1 preadipocytes were differentiated into adipocytes as described previously. Fully differentiated adipocytes (day 13) were labeled with
3
H-cholesterol (1 Ci/ml) for 24 h. Cellular cholesterol efflux was initiated by 20μg/ml human apoA1 or 50 μg/ml HDL
3
with the indicated dose of MCP-1 for the indicated period of time in the presence or absence of insulin. After incubation, the radioactivity of the medium and cells was measured using a liquid scintillation counter. Cholesterol efflux was calculated as the percentage of total [
3
H]-cholesterol released into the medium after subtraction of the values obtained in the absence of a cholesterol acceptor. 3T3-L1 adipocytes were harvested for PCR , western blotting, cell-surface protein assays and Confocal microscopy.

Results:
1. MCP-1 reduced cholesterol efflux to HDL3 and apoA1 in differentiated 3T3-L1 adipocytes. 2. In differentiated 3T3-L1 adipocytes, MCP-1 reduced cholesterol efflux to HDL
3
mainly by inhibiting SR-BI and ABCG1 and to apoA1 mainly by inhibiting ABCA1. 3. MCP-1 decreased ABCA1 and SR-BI mRNA expression but not ABCG1. 4. MCP-1 decreased total and cell surface ABCA1, ABCG1, and SR-BI protein expression as shown by Western blotting and confocal microscopy in differentiated 3T3-L1 adipocytes. 5. Insulin increased MCP-1 suppressed cholesterol efflux to HDL
3
and apoA1 depending on Akt phosphorylation. 6. Insulin reversed MCP-1 suppressed ABCA1 and SR-BI mRNA expression, and ABCA1, ABCG1 and SR-BI protein expression via PI3K/Akt pathway.

Conclusions:
Insulin reverses the suppressed cholesterol efflux to HDL3 and apoA1 by MCP-1 through up-regulation of ABCA1, ABCG1 and SR-BI through PI3K/Akt pathway in 3T3-L1 adipocytes, which provides evidences that insulin may improve the MCP-1-induced adipocyte cholesterol imbalance to exert the anti-inflammatory effect.
Abstract 569: Pnpla2 Influences Secretion of Triglyceride-rich Lipoproteins but Does Not Affect Lipid-droplet Metabolism of Human Hepatoma Cells
Authors: Taxiarchis Apostolos , Mahdessian Hovsep , Silveira Angela , Eriksson Per , Fisher Rachel , van ’t Hooft Ferdinand M.
Published: March 2019

Background:
Increased hepatic secretion of triglyceride (TG)-rich lipoproteins and cellular TG-accumulation are associated with enhanced risk for cardiovascular disease and diabetes mellitus but the factors regulating these processes are largely unknown. KO studies demonstrated that patatin-domain containing protein 2 (PNPLA2), also known as ATGL, is largely responsible for hepatic TG-hydrolysis in mice but the physiological role of PNPLA2 in human liver metabolism has thus far not been evaluated.

Aim:
To investigate the role of PNPLA2 in the secretion of TG-rich lipoproteins and TG-accumulation in human hepatoma Huh7 and HepG2 cell-lines.

Results:
Gene-specific siRNA silencing of PNPLA2 generated mRNA reductions of 88±9% and 82±5% in Huh7 and HepG2 cells, respectively. PNPLA2-silencing was associated with 48±5% and 45±12% decreases in cellular TG-hydrolysis in both hepatoma cell-lines as measured by an
in vivo
assay. Secretion of TG-rich lipoproteins was analyzed using an ELISA for apolipoprotein B (APOB) and quantification of
14
C -TG following incubation with
14
C-TG labelled glycerol. It was found that PNPLA2-silencing reduced the secretion of APOB and TG by 35±3% and 37±10% in Huh7 cells and by 23±13% and 29±6% in HepG2 cells, respectively. Surprisingly, no changes in cellular TG-accumulation were observed following PNPLA2-inhibition in Huh7 and HepG2 cells. Confocal microscopy studies confirmed that PNPLA2 inhibition did not change the lipid-droplet content of the hepatoma cells. Subcellular localization studies with a monoclonal PNPLA2 antibody found no evidence for the colocalization of PNPLA2 with lipid droplets. In contrast, considerable overlap was observed between PNPLA2 and the endoplasmic reticulum (ER) marker protein disulphide isomerase (PDI). The colocalization was quantified using Pearson correlation analysis (Rcoloc). The Rcoloc value for PNPLA2 and PDI was 0.78 ±0.06, compatible with predominant localization of PNPLA2 in the ER in human hepatoma cells.

Conclusion:
These studies demonstrate that PNPLA2 acts as a TG-hydrolase in human hepatoma cells and it is involved in the secretion of TG-rich lipoproteins. However, PNPLA2 does not influence hepatic lipid-droplet homeostasis and is not co-localized with large lipid-droplets.
Abstract 577: NF-kB Inhibition via Celastrol-Loaded Nanocarriers Induces Distinct Changes in Atheroma-Resident Immune Cell Composition in Male vs. Female ldlr -/- Mice
Authors: Allen Sean D , Liu Yugang , Scott Evan A
Published: March 2019

The immune component of atherosclerosis is strongly influenced by inflammatory signaling. Although NF-kB signaling is a key regulator of cytokine expression and activation of atheroma-resident macrophages and dendritic cells, previous attempts at its therapeutic inhibition have generated inconsistent results. These difficulties may arise from nonspecific systemic administration of inhibitors that have pleiotropic effects in both immune and non-immune cells in multiple organs.

To address these concerns, we have developed a nanocarrier delivery system (NCs) capable of transporting both hydrophobic and hydrophilic therapeutics to the cytosol of specific immune cell subsets. Following I.V. administration, our NCs are readily endocytosed by macrophages and dendritic cells within atheromas for modulation of signaling pathways. We hypothesized that delivery of the NF-kB pathway inhibitor celastrol to these cells
via
celastrol-loaded NCs (Cel-NCs) may alleviate atherosclerotic inflammation while minimizing off-target effects. Celastrol has potent anti-inflammatory effects, but also modulates unrelated signaling pathways in a range of cells and is cytotoxic near its effective inhibitory concentration (EIC). The low water solubility of celastrol is not amenable to I.V. injection, and oral administration achieves poor bioavailability and specificity for atheroma-resident cells.

We have found that loading celastrol into Cel-NCs expands its EIC, reduces cytotoxicity, and allows it to be administered
via
I.V. injection for improved targeting of atheroma. When injected into ldlr -/- mice, Cel-NCs modulated the immune composition within the aorta and spleen. Notably, these changes differed between male and female mice, possibly due to immuno-endocrine related mechanisms. Our work introduces a nanotherapeutic to reduce atherosclerotic inflammation and highlights the need to identify the effects of anti-inflammatory strategies in both sexes.
Abstract 578: Macrophage Cholesterol Levels Modulate Ion Channel Activity: Effect(s) on Ion Flux Dependent Inflammatory Events
Authors: He Hongliang , Wang Jing , Yannie Paul , Park Sung , Baumgarten Clive , Ghosh Shobha
Published: March 2019

Accumulation of cholesterol loaded macrophage foam cells is the hallmark of atherosclerotic lesions where high cellular cholesterol content not only contributes to the plaque volume but is also responsible for the inflammatory milieu. Causal relationship exist between macrophage cholesterol content and inflammatory status involving TLR4, NFκB or cholesterol crystal mediated activation of inflammasome and IL1β secretion. Since K+ efflux is central to inflammasome activation, in this study we examined the hypothesis that cellular cholesterol content directly affects K+ channel activity as well as K+ efflux. K+ currents were monitored in mouse peritoneal macrophages (MPMs) from chow or western diet (WD) fed LDLR-/- or LDLR-/-CEHTg mice. WD feeding led to a significant increase in the K+ currents in MPMs from LDLR-/- mice (A, red). In contrast, WD feeding did not affect K+ currents in MPMs from LDLR-/-CEHTg mice; these MPMs have reduced cellular cholesterol accumulation due to increased efflux. Consistently, cholesterol loading with AcLDL led to an increase in K+ currents in THP1 macrophages (B, red) and this increase was attenuated following cholesterol efflux. The physiological effect of changes in K+ channel activity or K+ efflux on inflammasome activation in MPMs by different activators (K+ ionophore Nigericin, N or ALUM crystals) was examined. Increase in extracellular K+ (+KCl) or inhibition of K+ efflux by glyburide significantly reduced IL1β secretion (C&D). Consistently, cellular K+ levels after LPS+N or LPS+ALUM treatment were significantly reduced (E). While in MPMs only K
ATP
channel inhibitors reduced IL1β secretion (F), in human THP1 macrophages inhibition of K
ATP
as well as Na/K pump inhibitors significantly reduced IL1β secretion (G). These data demonstrate a novel mechanism for cholesterol mediated regulation of inflammatory pathways by regulation of K+ channel activity and also illustrate species specific differences in K+ channels involved
Abstract 581: Identification on Anti-Atherosclerotic Mechanism of SGLT-2 Inhibitor in Diabetic Rabbit Model
Authors: Lee Seulgee , Kim Jung-Sun , Oh Jaewon , Bong Sung-Kyung , lee jung-jae
Published: March 2019

Aim:
The sodium-glucose cotransporter 2 inhibitor (SGLT-2i) suggested a possible anti-atherosclerotic and cardioprotective effects beyond glucose lowering effect. However, further study is needed to prove underlying mechanisms of improve on cardiovascular outcomes by SGLT-2i. Therefore, we investigated possible underlying anti-atherosclerotic effect of SGLT-2i ‘Dapagliflozin’ with diabetic rabbit model by inducing atherosclerosis.

Methods:
Rabbit divided into two groups (each group/n=10); DA: Diabetic atherosclerosis, DAD: Diabetic atherosclerosis + Dapagliflozin. Dapagliflozin was given for a total of 8 weeks. Atherosclerotic plaques were induced with a high cholesterol (HC) diet and balloon inflation. In vivo intravascular imaging and histological assessment was performed.

Results:
From the histologic evaluation, atheromatous plaque and lipid accumulation were significantly less developed in the Diabetic atherosclerosis + Dapagliflozin group compared to the Diabetic atherosclerosis group. Significantly less macrophage infiltration and inflammatory proteins expression level was observed in the Diabetic atherosclerosis + Dapagliflozin group. The mRNA and protein level of pro-inflammatory markers such as HMGB1, TNF-α, iNOS, and RAGE were significantly lower in the Diabetic atherosclerosis + Dapagliflozin group. The polarization of M1 macrophages was also significantly lower in the Dapagliflozin treated group. The fibronectin and type IV collagen were less observed in the glomerulus of kidney in the Dapagliflozin treated group. The lipid droplet area and size in the liver of Dapagliflozin treated group was smaller and fewer than those in the non-treated group.

Conclusion:
These results suggest that dapagliflozin may prevent an acceleration atherosclerosis by reducing a vascular inflammation and modifying the macrophage polarization in diabetic animal model. This preliminary clinical observation demonstrates the therapeutic potential of dapagliflozin in diabetic patients with cardiovascular disease.
Abstract 584: Neuroimmune Guidance Cues Important for Monocyte-Endothelial Cell Interaction and Monocyte to Macrophage Differentiation
Authors: Vreeken Dianne , Bruikman Caroline S. , Zhang Huayu , Sol Wendy M.P.J. , van Zonneveld Anton Jan , Hovingh G. Kees , van Gils Janine M.
Published: March 2019

Introduction:
Atherosclerosis is a systemic inflammatory disease, characterized by the accumulation of macrophages in the vascular wall. Studies in mice showed that neuroimmune guidance cues (NGCs) are involved in atherosclerosis-related processes. In this study we aimed to determine the NGCs involved in monocyte-endothelium adhesion and transmigration, and subsequent macrophage differentiation.

Methods and Results:
Combining publically available gene expression data of >600 endothelial, monocytes or macrophage samples we determined the specific NGCs expressed by these cells involved in the onset and progression of atherosclerosis. Next, the mRNA levels of the expressed NGCs were analyzed in primary human endothelial cells and monocytes upon TNFα, IL1β or oxidized LDL stimulation (5 or 24 hours), as wells as in human monocytes differentiated into macrophages.

In endothelial cells a significant (P<0.05, abs(logFC)>1) downregulation was observed for
NTN4, SEMA6C
and
PLXNA4
while a significant upregulation was observed for
EFNA1, EFNB1, UNC5B, ROBO1, SEMA6D
and
SEMA7A
upon stimulation. In monocytes a significant downregulation was detected for
SEMA6B, PLXNC1, NRP1, NRP2
and
EPHB6
, while
SEMA7A
and
EPHB2
were significantly upregulated upon stimulation. These findings combined resulted in potentially interesting concurrent changes in the NGC ligand-receptor combinations; (1) endothelial
PLXNA4
receptor with monocyte
SEMA3A
and (2) endothelial
EFNB1
ligand with monocyte
EPHB2
receptor. These changes seen at mRNA were validated at protein level. Remarkably, monocyte to macrophage differentiation induced a major increase and change in NGC expression levels, mainly in the SEMA family of ligands and receptors (including
SEMA3G
,
SEMA7A, NRP1, and NRP2
), as well as in
EPHB2
as seen in monocyte stimulation.

Conclusion:
In conclusion, in our current study we observed a differential expression of NGC ligands and receptors in endothelial cells, monocytes and macrophages, culprit cell types in atherosclerosis, once subjected to pro-atherogenic stimuli. Our findings confirm a potential role for NGCs in human atherosclerosis. The next step is to further investigate the underlying mechanism of these NGCs and their role in atherosclerosis.
Abstract 599: Exposure to Biodiesel Exhaust Triggers Atherosclerotic Plaque Destabilisation through Increased Apoptosis of Vascular Smooth Muscle Cells in the Arterial Vessel Wall
Authors: Posma Jens J , Kilinç Evrin , Borissoff Julian I , Haegens Astrid , Jedynska Aleksandra D , Ten Cate Hugo , Kooter Ingeborg M , Spronk Henri M
Published: March 2019

Introduction:
Long-term exposure to air pollutants increases cardiovascular morbidity and mortality. Particulate matter (PM) derived from diesel exhaust has been documented to be pro-atherogenic in animal studies. Biodiesels are widely introduced as new fuels with improved emission characteristics. However, biodiesel contains relatively more polycyclic aromatic carbohydrates (PAH) compared to diesel. Therefore, we hypothesise that exposure to biodiesel as compared to diesel exhaust results in increased atherosclerosis.

Methods and Results:
In a carotid cuff atherosclerosis model, the effects of exposure to exhaust PM of biodiesel vs. diesel PM vs. saline (control) on atherosclerosis were evaluated. Both common carotid arteries in LDLR
-/-
mice were cuffed at week 2 in the course of an 8-week high-fat diet regimen. All mice were intratracheally instilled with saline, PM biodiesel or biodiesel once-weekly for 5 times. Immunohistochemistry and primary human vascular smooth muscle cells (hVSMC) were used for evaluation.

Results:
Exposure to both biodiesel and diesel didn’t exacerbate atherosclerosis development, however, biodiesel affected plaque composition towards a more vulnerable phenotype as compared to diesel with decreased total collagen content and hVSMC (respectively -100%, p=0.09 and -339%, p<0.01 and tend to increase necrotic core volumes (37.5% vs 28.9 % p=0.2). Exposure to biodiesel PM triggered loss of hVSMC in tunica-media, strongly correlating with apoptosis in the vessel wall (Pearson r=0.7, p<0.01). These findings were supported by dose-dependent apoptosis of hVSMCs upon 2-hour PAH treatment.

Conclusions:
This study demonstrates that exposure to biodiesel exhaust PM dramatically modulates atherosclerotic plaque composition, resulting in an unstable plaque phenotype through enhanced pro-apoptotic mechanisms.
Abstract 602: The Role of Interleukin-19 in Adipose Tissue Homeostasis
Authors: Vrakas Christine N , Herman Allison B , Ray Mitali , Landesberg Gavin , Kelemen Sheri E , Autieri Michael V , Scalia Rosario
Published: March 2019

Expanding adipose depots experience hypoxia and inflammation, a phenomenon considered pathogenic of metabolic and cardiovascular complications. Currently very little is known about the potential for endogenously expressed anti-inflammatory cytokines to attenuate inflammation and also provide pro-angiogenic effects. Interleukin-19 is a uniquely anti-inflammatory, pro-angiogenic cytokine expressed in adipose tissue. We hypothesized that in the inflamed adipose depots of the overweight-obese organism IL-19 promotes a compensatory, anti-inflammatory and pro-angiogenic action to restore adipocyte homeostasis in the face of excess energy intake. We report that IL-19 is constitutively expressed in visceral and subcutaneous adipose depots of lean wild-type mice. We also found evidence of significantly increased IL-19 expression in the visceral adipose depots of diet-induced obese mice (p<0.001 vs lean mice). Using

Il19
-/-

mice, we first demonstrate that the loss of IL-19 leads to a reduction in the metabolically protective and anti-inflammatory genes PPARγ and adiponectin. Simultaneously, pro-inflammatory factors including TNFα and IL-6 are increased, and pro-angiogenic factors such as VEGF are down regulated.

Il19
-/-

mice display elevated fasted blood glucose levels relative to WT controls (p<0.05). Insulin tolerance tests revealed that

Il19
-/-

mice exhibit insulin resistance compared to WT controls. The metabolic profile of

Il19
-/-

mice appears to be related to IL-19 actions on adipose tissue as suggested by the fact that

Il19
-/-

mice fed high fat diet experience significant down-regulation of IL-19 receptor (IL-20Rα/IL-20Rβ) expression in the visceral adipose tissue, but not in the liver and skeletal muscle. Mechanistic studies in differentiated 3T3-L1 adipocytes demonstrated that addition of rIL-19 increases PPARγ and adiponectin at both the transcript and protein level. These data suggest that potentiation of endogenous IL-19 activity may represent a novel therapeutic strategy to avert the inflammatory and metabolic complications associated with the adipose tissue dysfunction of diet-induced obesity.
Abstract 607: Increased Vitamin D
3-
induced Kidney Dysfunction Associates With Intrarenal VascularCalcification in Type 2 Diabetes Mellitus Murine Model
Authors: Almeida Youri E , Fessel Melissa R , Carmo Luciana S , Farias-Silva Elisangela , Alves Luciana P , Jorgetti Vanda , Gamarra Lionel F , Rangel Erika B , Liberman Marcel
Published: March 2019

Vascular calcification associates with increased cardiovascular morbidity and mortality in patients with diabetes mellitus and end-stage renal disease. We evaluated intrarenal vascular calcification and renal dysfunction after either vitamin D
3
(VitD
3
4.4-6.4x104 IU/day i.p. for 21 days) or saline in ob/ob mice (n=12), a murine model of type 2 diabetes mellitus (T2DM), in comparison to paired C57BL/6 mice (n=12). The animal protocol #2242-14 was approved by Institutional ethics committee. VitD3 increased serum calcium levels in both animal strains. Serum creatinine increased in VitD3-treated mice (92% and 43% in ob/ob and C57BL/6 respectively), while serum urea levels decreased in ob/ob and increased in C57BL/6 after calcifying protocol. Both VitD
3
-treated ob/ob and C57BL/6 decreased 24-hour urine volume, which was more pronounced in diabetic animals (~5 times and ~1.5 reduction in ob/ob and in C57BL/6 respectively). Moreover, creatinine clearance decreased in VitD
3
-treated mice, especially in ob/ob animals (~7 and ~3 fold reduction in ob/ob and C57BL/6, respectively). Additionally, urinary albumin/creatinine ratio increased after VitD
3
administration in ob/ob mice only (~13 times in comparison to control ob/ob). Alizarin Red S, Von Kossa and Osteosense
TM
revealed that only ob/ob mice developed extensive intrarenal vascular calcification after VitD
3
. Interestingly, histomorphometric analysis of the femur showed that VitD
3
induced similar osteomalacia in both ob/ob and C57BL/6 mice. Coincidently with increased intrarenal vascular mineralization in ob/ob, confocal immunofluorescence analysis demonstrated that Bone Morphogenetic Protein-2 (BMP-2) was highly expressed in these arteries exclusively in ob/ob. These data suggest a greater susceptibility of T2DM murine model to VitD
3
, in comparison to paired C57BL/6 littermates to induce kidney dysfunction associated with increased intrarenal vascular calcification, together with BMP-2 signaling pathway activation. These findings were independent of bone remodeling and serum calcium levels. In conclusion, this study unfolded novel mechanisms of progressive renal dysfunction in T2DM after VitD
3
in vivo associated with increased intrarenal vascular calcification.
Abstract 610: Functional Relationship of The
CARS2
Locus to Coronary Artery Disease
Authors: Dang AnhThu , Turner Adam , Lau Paulina , Soubeyrand Sébastien , McPherson Ruth
Published: March 2019

In a recent meta-analysis of GWAS for cardiovascular disease (CAD) with imputation from 1000 Genomes (Nat Genet 2015), we identified 202 significant CAD associated variants (FDR q< 0.05) in 129 loci. Seven independent association signals were identified in a gene cluster at chromosome 13q34 including rs61969072. This SNP is located in an intergenic region proximal to the
ING1
,
CARKD
and
CARS2
genes. Expression data (eQTL) obtained from the GTEx Project demonstrate a strong association of rs61969072 with
CARS2
mRNA abundance in artery with decreased
CARS2
expression in carriers of the risk G allele, hinting that
CARS2
may be a causal CAD gene.
CARS2
encodes a putative mitochondrial cysteinyl t-RNA synthetase. Bioinformatics data (BioGPS) indicate high gene expression of
CARS2
in monocytes, a precursor of macrophages. Having then demonstrated that
CARS2
mRNA expression is decreased in M1 macrophages compared to M0 and M2 macrophages, we examined the role of
CARS2
in the anti-inflammatory pathway in macrophages using THP-1 cells, a macrophage model system. Gene expression profiling of 84 key players of the inflammatory response was performed on siRNA mediated knockdown of
CARS2
samples. Comparisons between knockdown and control samples (n=4) identified 12 upregulated and 4 down regulated genes with ≥ 1.25 or ≤0.75 fold difference in expression (p<0.05). Gene Set Enrichment Analysis (GSEA), using inflammatory response PCR array data, identified the interleukin-10 signaling pathway as an enriched category (FDR q<0.05). IL-10 has an atheroprotective effect against plaque rupture and thrombus formation by influencing the local inflammatory process in the lesion. IL-10 activates the STAT3 cascade, where phosphorylated STAT3 homodimers translocate to the nucleus to activate specific target effector genes which repress the proinflammatory genes at the transcriptional level. IL-10 induced STAT3 phosphorylation was measured by Western blotting in THP-1 cells. In response to IL-10,
CARS2
knockdown samples showed dampened STAT3 phosphorylation compared to control suggesting that
CARS2
serves an anti-inflammatory function within the IL-10 signaling pathway.
Abstract 615: Absence of RIP3 Reduces Atherosclerotic Progression in Type 1 Diabetes
Authors: Karunakaran Denuja , Geoffrion Michele , Susser Leah , Rayner Katey
Published: March 2019

Introduction:
Patients suffering from Type 1 diabetes (T1D) have increased risk of developing atherosclerosis. T1D is a chronic autoimmune metabolic disease characterized by a deficiency in insulin secretion and hyperglycemia, promoting oxidative stress and low-grade inflammation in part via advanced glycation end products (AGE). Recently, we and others showed that necroptosis, a newly defined pro-inflammatory programmed cell death, drives advanced atherosclerosis. Given that diabetes accelerates inflammation and atherosclerosis, we hypothesized that hyperglycemia may exacerbate macrophage necroptosis to drive atherosclerotic lesion formation.

Methods/Results:
Male
Ldlr-/-
mice were lethally irradiated, transplanted with bone-marrow from WT or

Rip3
-/-

mice and then administered streptozotocin (STZ) or vehicle daily for 5 days to induce T1D, prior to being fed a western diet for 6 wks. As expected, STZ-treated WT BMT

Ldlr
-/-

mice had a marked increased in atherosclerotic aortic lesion area compared to non-diabetic mice (1.08%±0.30 vs 2.13%±0.45). which was blunted in

Rip3
-/-

BMT

Ldr
-/-

mice (arch: 49% reduction, sinus: 54% reduction, p<0.05). Bone marrow derived macrophages (BMDMs) cultured in high glucose (HG) increased cell death in response to necroptotic stimuli (oxidized LDL ± caspase inhibitor zVAD) relative to those cultured in low glucose (LG). Further, the addition of recombinant s100A8/9, a soluble AGE, markedly amplified oxLDL-induced death in HG cultured BMDMs with minimal effects on LG cultured BMDMs, suggesting that pro-inflammatory AGEs released in diabetes can augment macrophage cell death. Further, the increased cell death induced by oxLDL+S100A8/9 was inhibited by a necroptotic inhibitor, Nec-1, or in
Rip3

-/-

BMDMs cultured in HG. Mechanistically, treatment of BMDMs cultured in HG with oxLDL+s100A8/9 increased the expression of Rip3 and Mlkl (~5-fold and ~2-fold respectively), which may drive necroptotic cell death during hyperglycemia.

Conclusion:
Deletion of Rip3 markedly halts atherosclerotic progression in a STZ-induced T1D mouse model by inhibiting macrophage necroptosis induced by both atherogenic ligands (oxLDL) and soluble AGE products (s100A8/9).
Abstract 621: Vascular Smooth Muscle EphA2 Signaling Regulates Fibrillar Adhesion Formation to Drive Fibronectin Deposition
Authors: Finney Alexandra C , Green Jonette M , Alexander J. S , Traylor James G , Orr Anthony W
Published: March 2019

During atherosclerosis, synthetic vascular smooth muscle cells drive development of the fibrous cap through extracellular matrix deposition. Deposition of the provisional matrix protein fibronectin appears to critically regulate fibrous cap development by promoting smooth muscle recruitment and by providing a critical scaffold for assembly of fibrillar collagens. We previously showed that deletion of the receptor tyrosine kinase EphA2 in ApoE knockout mice attenuated plaque size and progression, characterized by a reduction of both smooth muscle content and fibrosis, and we further demonstrated that EphA2 depletion attenuates smooth muscle fibronectin deposition
in vitro.
Despite this, the mechanisms by which EphA2 regulates smooth muscle matrix deposition remains unknown. Fibronectin fibrillogenesis involves both the formation of α5β1 integrin-rich fibrillar adhesions and contractility-dependent fibronectin unfolding. We now show EphA2 localizes to integrin adhesions and appears to regulate matrix deposition through multiple mechanisms. Knockdown of EphA2 reduces the formation of fibrillar adhesions characterized by a loss of α5β1 integrins and tensin, whereas EphA2 overexpression is sufficient to induce fibrillar adhesion formation in contractile smooth muscle cells. In addition, vascular smooth muscle cells show diminished collagen gel contraction following EphA2 knockdown, and EphA2 depletion significantly reduces smooth muscle myosin light chain phosphorylation, suggesting that EphA2 expression is required to generate the contractive forces required for matrix remodeling in synthetic smooth muscle cells. Together these data identify a novel role for EphA2 in the regulation of smooth muscle matrix deposition through effects on both integrin adhesion structure and cytoskeletal contractility.
Abstract 624: Interactions Between Adenylate Cyclase Type 9 (ADCY9) and Cholesteryl Ester Transfer Protein (CETP) in Atherosclerosis
Authors: Rautureau Yohann , Deschambault Vanessa , Higgins Marie-Ève , Rivas Daniel , Mecteau Mélanie , Geoffroy Pascale , Miquel Géraldine , Uy Kurunradeth , Sanchez Rocio , Nault Audrey , Williams Pierre-Marc , Suarez Maria-Laura , Brand Geneviève , Lapointe Line , Duquette Natacha , Gillis Marc-Antoine , Samami Samaneh , Mayer Gaétan , Pouliot Philippe , Raignault Adeline , Maafi Foued , Brodeur Mathieu , Lévesque Sylvie , Guertin Marie-Claude , Dubé Marie-Pierre , Thorin Éric , Rhainds David , Rhéaume Éric , Tardif Jean-Claude
Published: March 2019

Pharmacogenomic studies have shown that
ADCY9
genotype determines the effects of the cholesteryl ester transfer protein (CETP) inhibitor dalcetrapib on cardiovascular events, atherosclerosis imaging and body weight variation. The underlying mechanisms responsible for the interactions between ADCY9 and CETP have not yet been determined.

Adcy9
-inactivated (
Adcy9
Gt/Gt
) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETP
Gt
and CETP
WT
), were submitted to an atherogenic protocol (injection of an AAV8 expressing a PCSK9 gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, cell adhesion, vasorelaxation, telemetry and adipose tissue MRI were evaluated.

Adcy9
Gt/Gt
mice had a 65% reduction in aortic atherosclerosis compared to WT (
P
<0.01). CD68-positive macrophage accumulation and proliferation in plaques were reduced in
Adcy9
Gt/Gt
mice compared to WT animals (
P
<0.05 for both).
Adcy9
inactivation did not change counts of blood monocytes and their subsets. Splenocytes showed reduced adhesion to native aortic endothelium from
Adcy9
Gt/Gt
mice (
P
<0.05 vs WT). Femoral artery endothelial-dependent vasorelaxation was improved in
Adcy9
Gt/Gt
mice (versus WT,
P
<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase and endothelial-dependent hyperpolarization pathways all contributed to the improvement of vasodilatation in
Adcy9
Gt/Gt
versus WT (
P
<0.01 for all).
Adcy9
Gt/Gt
mice gained more weight than WT with the atherogenic diet, and this was associated with an increase in whole body adipose tissue volume (
P
<0.05 for both). Feed efficiency was increased in
Adcy9
Gt/Gt
compared to WT mice (
P
<0.05), which was accompanied by improved nocturnal heart rate variability (
P
=0.0572) and prolonged cardiac RR interval (
P
<0.05).
Adcy9
inactivation-induced effects on atherosclerosis, endothelium-dependent vasodilation, weight gain and feed efficiency were lost in CETP
Gt
mice (
P
>0.05 vs CETP
WT
).

Adcy9
inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, improved endothelial function and autonomic tone.
Abstract 629: The (Pro)renin Receptor Regulates Scavenger Receptor CD36 Protein Degradation and Functions
Authors: Sun Yuan , Ren Liwei , Danser A.H.Jan , Lu Xifeng
Published: March 2019

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 630: Pcsk9 Deficiency Reduces Atherosclerosis, Apolipoprotein B Secretion, Lipogenesis and Endothelial Dysfunction
Authors: Sun hua , Krauss Ronald M , Chang Jeffrey T , Teng Ba-bie
Published: March 2019

Objective:
Proprotein convertase subtilisin kexin type 9 (PCSK9) interacts directly with cytoplasmic apolipoprotein B (apoB) and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis.

Approach and Results:
We backcrossed
Pcsk9-/-
mice with atherosclerosis prone LDb (
Ldlr-/-Apobec1-/-)
mice to generate LTp (
Ldlr-/-Apobec1-/-Pcsk9-/-
). Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux and autophagy signaling, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDL) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDL. Deficiency of PCSK9 affected SREBP-1c lipogenesis pathway. Moreover LTp-LDL induced lower endothelial expression of the genes encoding
TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62
, and
TRAF6
. Collectively, these effects were associated with substantially less atherosclerosis development (> 4-fold) in LTp mice.

Conclusions:
The absence of PCSK9 in
Ldlr-/-Apobec1-/-
mice results in decreased lipid and apoB levels, less atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors, and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity.