bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 207: Interesterified Fat Rich in Stearic Acid Increases Liver Neutrophil and M1 Macrophage Infiltration and Induces Nonalcoholic Steatohepatitis
Authors: Lavrador Maria Silvia F , Afonso Milessa S , Koike Marcia , Cintra Dennys E , Bombo Renata P , Nunes Valeria S , Catanozi Sergio , Nakandakare Edna R , Lottenberg Ana Maria
Published: July 2021

Introduction:
interesterified fats, rich in saturated fatty acid, have been used by food industry to replace trans. Palmitic acid is the most common dietary fat with known deleterious effects. On the hand stearic acid which is consumed in a low amount, has neutral effect on plasma lipids. Unlike vegetable oils, interesterified fats contain palmitic or stearic acid in the sn-2 position and the positional compositon of triglicerides may regulate its metabolic fate. Thus, the aim of this study was to evaluate the effect on the liver metabolism of the interesterified fats rich in stearic or palmitic fatty acids.

Methods:
Weaning LDLr-KO male mice were randomly distributed into five groups fed a high fat diet (40% of energy as fat) containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER) during 16 weeks. Total plasma cholesterol and triglyceride concentrations, liver total cholesterol, triglycerides and weight, neutrophil (neutr/area) and classically activated/inflammatory M1(CD11b) infiltrated macrophage were determined. Histological markers of NAFLD activity were assessed.

Results:
There were no differences in dietary intakes among all groups. The interesterification process did not alter plasma lipids concentrations (mg/dL±SD): PALM vs PALM INTER (499.2 ± 81.7 vs 526.8 ± 92.6) and STEAR vs STEAR INTER (342.6 ± 89.9 vs 363.9 ± 90.0). However, PALM and PALM INTER presented higher total plasma cholesterol, and STEAR and STEAR INTER showed total cholesterol concentrations similar to POLI (320.7±47.4). Liver weight, cholesterol and TG contents did not differ among the groups. POLI and STEARIC groups do not elicit NASH (score <3), PALM and PALM INTER presented intermediate stage (score ≤4) between STEARIC/POLI and STEARIC INTER. Nevertheless, STEAR INTER developed NASH (score ≥5), and induce higher neutrophil infiltration (5.53 ±4.1) as compared to all other groups. The M1 macrophage classically activated infiltration was also higher in STEAR INTER group than in all groups as demonstrated by imunofluorescence.

Conclusion:
STEAR INTER brings on NASH lesion, increased neutrophil and M1 macrophage classically activated/inflammatory infiltration in the liver.
Abstract 21: The Calcium-Binding Protein S100A1 Negatively Regulates Collagen-Dependent Platelet Activation and Thrombosis in Mice
Authors: Boulaftali Yacine , Paul David S , Piatt Raymond , Feng Dengmin , Cooley Brian C , Most Patrick , Bergmeier Wolfgang
Published: July 2021

Introduction:
S100A1 is a member of the S100 family of calcium-binding proteins. S100A1 controls Ca2+ dynamics in cardiomyocytes and plays an important role in heart failure. S100A1 is also strongly expressed in mouse platelets, but its role in platelet biology has not been investigated.

Goal:
To determine the role of S100A1 in platelet activation and thrombosis.

Methods and Results:
Platelet activation in response to threshold levels of convulxin, a specific agonist for the collagen receptor GPVI, showed significantly increased activation of αIIbβ3 integrin and α-granule release in S100A1-deficient (SKO) platelets compared with wild-type (WT) platelets. Consistently, SKO platelets also showed a more robust aggregation response to convulxin and collagen. In contrast, SKO platelets responded normally to stimulation with PAR4 receptor-activating peptide or ADP. Adhesion of SKO platelets to collagen under flow conditions was not significantly different to that of WT platelets. However, we observed a ~3-fold increase in phosphatidylserine (PS)-positive SKO platelets bound to the collagen surface (p<0.001, n=9 mice/group), suggesting that S100A1 also regulates the procoagulant response in platelets. Consistent with this hypothesis, we observed increased coated platelet formation and more sustained calcium transients in SKO platelets compared to controls. The increased reactivity of SKO platelets to GPVI agonists is explained by a ~1.5-fold increase in GPVI receptors expressed on the surface of these cells (p<0.001, n=20 mice/group). A similar increase in GPVI expression was also found in bone marrow-derived megakaryocytes. When subjected to the FeCl3 carotid artery thrombosis model, the time to vessel occlusion was significantly shorter in SKO mice compared to WT controls (4.55 ± 0.27 min vs 7.00 ± 0.62 min, respectively; p<0.01; n=8 mice/group). Furthermore, by using a collagen-rich adventitial surface inserted intralumenally in the carotid artery, we observed that thrombus formation in SKO mice is significantly more stable compared to WT mice (p=0.01, n=7 WT, n=8 SKO)

Conclusions:
We here identify S100A1 as a negative regulator of GPVI expression and collagen-dependent platelet activation and thrombosis in mice.
Abstract 224: Formation of Cholesterol-Enriched Nascent HDL Requires Helices 5 and 7
Authors: Pollard Ricquita D , Fulp Brian , Samuel Michael P , Sorci-Thomas Mary G , Thomas Michael J
Published: July 2021

Heart disease claims nearly 400,000 individuals in the US per year. Large population studies have repeatedly demonstrated an inverse relationship between HDL concentration and risk. Despite the strong correlation, pharmacological trials that raise plasma HDL have shown little efficacy, suggesting that HDL function should be monitored. Studies were initiated into the mechanism of cholesterol efflux leading to nascent HDL (nHDL) formation. The most productive cholesterol removal occurs when lipid-poor apoA-I, the main structural protein in nHDL, forms particles after interacting with ABCA1. ApoA-I acquires cholesterol and phospholipid at the cell surface forming 10-12 nm diameter nHDL whose composition resembles lipid rafts. Because lipidated apoA-I has a significantly different 3-D conformation compared to its lipid-poor counterpart, the structural modifications leading to nHDL formation were investigated. First, we completed a 3-D solution structure of lipid-poor apoA-I using lysine specific chemical cross-linking in conjunction with specifically engineered double cysteine-containing apoA-I mutants. Strategically placed cysteines form disulfide bonds (locked) when residues are within ~0.5 nm. If no disulfide bond formed (unlocked) then the two cysteines were separated by more than ~0.5 nm. Both methods rely on mass spectrometry to identify amino acid sequence and distance constraints within each 10 repeating helices comprising 80% of full-length apoA-I. By comparing lipid-poor apoA-I conformation to our previously described 3-D structure of apoA-I on 10-12 nm nHDL, we formulated a series of hypothetical helical domain transitions that might drive protein-lipid interaction and particle formation. To test, 10 different double cysteine mutants were employed in either their “locked” (oxidized) or “unlocked” (reduced) state by evaluating their ability to form recombinant HDL using synthetic phospholipid or to form nHDL using HEK cells. After determining HDL particle yield, size and composition from all locked and unlocked double cysteine apoA-I mutants we conclude that both the N- and C-terminal ends of apoA-I are essential for the first steps in lipid acquisition, but that the central helices 5, 6 and 7 are essential for nHDL formation.
Abstract 235: Atorvastatin Has No Impact on the Effects of CETP Inhibitors on HDL Structure and Cholesterol Efflux Capacity in Chow-Fed Rabbits
Authors: Brodeur Mathieu R , Rhainds David , Charpentier Daniel , Mihalache-Avram Téodora , Mecteau Mélanie , Brand Geneviève , Maugeais Cyrille , Niesor Eric J , Rhéaume Eric , Tardif Jean-Claude
Published: July 2021

Introduction:
In the dal-OUTCOMES trial, cholesteryl ester transfer protein (CETP) inhibition by dalcetrapib increased HDL-C but had a neutral effect on CV risk. However, these patients were also treated with standard-of-care therapies including statins, a drug class shown to reduce ABCA1 mRNA expression in human peripheral blood mononuclear cells (PBMC).

Hypothesis:
Given the role of ABCA1 in cholesterol efflux and HDL biogenesis, we determined the impact of atorvastatin on the potential beneficial effects provided by dalcetrapib and anacetrapib, two CETP inhibitors.

Methods:
Dalcetrapib (300 mg/kg) or anacetrapib (30 mg/kg) were administered to New Zealand White rabbits receiving or not atorvastatin (2.5 mg/kg) for 14 days. Lipid profiles were measured biochemically. ApoA-I distribution in HDL subclasses was evaluated by 1D-non-denaturing gradient gel electrophoresis (1D-NDGGE). BHK cells with inducible expression of human ABCA1 and HepG2 hepatocytes were used to measure ABCA1- and SR-BI-dépendent cholesterol efflux. Human THP-1 macrophages and PBMC isolated from rabbits were also used for efflux assays.

Results:
Dalcetrapib increased HDL-C by +81% (p<0.01) and total apoA-I (1D-NDGGE) by 1.5-fold (p<0.01). This was associated with an increase in both large and small α-migrating HDL (+52%, p<0.05; +74%, p<0.05). Cholesterol efflux showed that ABCA1- and SR-BI-dependent effluxes to apoB-depleted serum were increased in dalcetrapib-treated rabbits (+31%, p<0.01; +38%, p<0.001). Atorvastatin had no impact on all these parameters. Moreover, treatment of THP-1 with atorvastatin did not inhibit the increase of cholesterol efflux (+24%, p<0.01) induced by depleted serum from dalcetrapib-treated rabbits. Efflux capacity of PBMC isolated from rabbits treated with atorvastatin was also unchanged compared to the other groups. The absence of effect of atorvastatin on cholesterol efflux was also observed with anacetrapib.

Conclusion:
CETP inhibition increased HDL-C, apoA-I levels and cholesterol efflux from different cell types. Atorvastatin had no effect on these parameters, suggesting that the increased cholesterol efflux capacity induced by CETPi is not hampered by statins.
Abstract 236: Lipoprotein Metabolism in APOB L343V Familial Hypobetalipoproteinemia
Authors: Hooper Amanda J , Robertson Ken , Heeks Liesl V , Champain Danie , Barrett P Hugh R , Parhofer Klaus G , van Bockxmeer Frank M , Burnett John R
Published: July 2021

Familial hypobetalipoproteinemia (FHBL) is a codominant disorder of lipoprotein metabolism characterized by decreased plasma concentrations of LDL-cholesterol and apolipoprotein (apo) B. We examined the effect of heterozygous
APOB
L343V FHBL on fasting and postprandial lipoprotein metabolism. VLDL, IDL-, and LDL-apoB kinetics were determined in the fasting state using stable isotope methods and compartmental modeling. VLDL-apoB concentrations in FHBL subjects (n=2) were reduced by more than 75% compared to healthy, normolipidemic control subjects (
P
<0.01). VLDL-apoB fractional catabolic rate (FCR) was more than 5-fold higher in the FHBL subjects (
P
=0.07). ApoB production rates and IDL- and LDL-apoB FCRs were not different between FHBL subjects and controls. To assess postprandial lipoprotein metabolism, a standardized oral fat load was given after a 12 h fast to heterozygous
APOB
L343V FHBL subjects (n=3) and normolipidemic controls. The postprandial incremental area under the curve (0-10 h) in FHBL subjects was decreased for large TRL-triglyceride (-77%;
P
<0.0001), small TRL-cholesterol (-83%;
P
<0.001), small TRL-triglyceride (-88%;
P
<0.0.001) and plasma apoB (-63%;
P
<0.0001) compared with controls. Compartmental modeling analysis showed that apoB-48 production was decreased (-91%;
P
<0.05) compared with controls. We conclude that when compared to controls,
APOB
L343V FHBL heterozygotes show decreased TRL production with normal postprandial TRL particle clearance. In contrast, VLDL-apoB production was normal, while the FCR was higher in heterozygotes compared with lean control subjects. These mechanisms account for the marked hypolipidemic state observed in these FHBL subjects.
Abstract 240: Effect of Co-Administration of Simvastatin and Pioglitazone on Lipid Profile and Insulin Resistance in Insulin-Resistant Rats
Authors: Mohammadi Abbas , Danesh Behnaz , Fallah Hossein
Published: July 2021

Objective:
Insulin resistance is characterized by hyperglycemia, dyslipidemia, hyperinsulinemia and hypertension. Also, in obesity, decreased plasma adiponectin and increased free fatty acids, are the main factors that correlate with insulin resistance. Thiazolidinediones, like pioglitazone are used to improve insulin sensitivity and ATPIII guidelines suggest statin therapy for correction of dyslipidemia in metabolic syndrome. In this study we evaluated co-administration of simvastatin and pioglitazone on insulin resistance in rats.

Materials and Methods:
Rats were randomly divided into five groups. After 6 weeks on a high-fructose diet, one group received simvastatin, one group received pioglitazone, and one received pioglitazone and simvastatin. After 2 weeks of treatment, animals were anesthetized with ether. Blood was collected from their heart. Liver, visceral adipose, and muscle tissues were collected and were immediately frozen. Serum glucose, TGs, cholesterol, insulin, adiponectin, and free fatty acids were measured. Expression of PPAR.γ and GLUT4 genes was checked by real-time PCR and Western blotting.

Results:
Only the results that showed a significant difference are shown. Blood glucose: pioglitazone group (129.1±5.8mg/dl), simvastatin-pioglitazone treated group (137.1±9.9 mg/dl); TG: simvastatin group (123.6±16.6 mg/dl), simvastatin-pioglitazone group (101.5±7.5 mg/dl); Insulin: pioglitazone group (40.27±2.75 p mol/l), simvastatin group (70.07±10.35 pmol/l), simvastatin-pioglitazone (47.62±2.8 pmol/l); Adiponectin: pioglitazone (5.90±0.29 μg/ml), simvastatin-pioglitazone (5.89±0.41μg/ml); HOMA-IR: pioglitazone (2.11±0.13), simvastatin (4.76±0.37), simvastatin-pioglitazone (2.70±0.29). In pioglitazone group, PPAR.γ expression at both mRNA and protein levels was significantly different from insulin resistant group.

Conclusion:
As the results show, simvastatin has beneficial effects on insulin resistance in rats. Pioglitazone did not show any synergistic effect with simvastatin, however, when used in combination with simvastatin, pioglitazone shows a synergistic effects in lowering TG and HOMAIR that potentially can lower diabetes complications.
Abstract 242: Functional Characterization of a Cis-eQTL Locus for Plasma Cholesterol Using CRISPR/Cas Genome Editing in Human Pluripotent Stem Cells
Authors: Raghavan Avanthi , Peters Derek , Kuperwasser Nicolas , Melnikov Alexandre , Rogov Peter , Wang Li , Zhang Xiaolan , Mikkelsen Tarjei , Musunuru Kiran
Published: July 2021

Genome-wide association studies (GWASs) have discovered many novel genetic loci linked to serum lipid levels, yet pinpointing the causal variants remains a major challenge. Expression quantitative trait locus (eQTL) analysis of liver and adipose indicates that many lipid-associated variants influence gene expression in a cis-regulatory manner. To identify causal variants at 57 lipid-associated eQTL loci, we performed a massively parallel reporter assay (MPRA) in which the genomic region surrounding each candidate SNP was coupled to a reporter gene with a unique barcode identifier. This construct pool was transfected into 3T3-L1 adipocytes, and the copy number of each expressed barcode determined by RNAseq. Variants were prioritized according to allele-specific regulatory activity.

The top-ranked variant, rs2277862, is associated with total cholesterol and is flanked by the ERGIC3, CPNE1, and CEP250 genes, none of which have previous connections to lipid metabolism. We hypothesized that rs2277862 is causal for the eQTL at the 20q11 locus, and that it lies within a transcriptional regulatory site to influence local gene expression. We identified human pluripotent stem cell (hPSC) lines with different genotypes at rs2277862 and, using CRISPR/Cas genome-editing technology, either deleted the putative regulatory site encompassing rs2277862 or knocked in the alternate SNP allele. Analysis of undifferentiated rs2277862 homozygous major deletion mutants revealed diminished expression of ERGIC3, CPNE1, and CEP250; conversely, heterozygous deletion mutants had increased expression of the same three genes. Our results suggest that in hPSCs the major and minor alleles of rs2277862 have opposing effects on gene expression.

In ongoing work, we are validating these findings in differentiated adipocytes and seek to identify the factor(s) that binds the putative regulatory site to mediate allele-specific gene expression. We anticipate that this work will offer fresh insight into the mechanisms by which GWAS-implicated SNPs influence expression of causal genes for lipid metabolism and, more broadly, establish a novel methodology for finding causal variants for any phenotype of interest.
Abstract 243: Canadian Familial Hypercholesterolemia Registry
Authors: Ruel Isabelle , Gagnon Marie-Hélène , Brophy James , Gaudet Daniel , Frohlich Jiri , Hegele Robert A , Genest Jacques
Published: July 2021

Familial hypercholesterolemia (FH) is the most frequent genetic lipoprotein disorder associated with premature CAD. In Canada, the burden of disease is estimated to be approximately 83,500 patients.

Objective:
The goal of this initiative is to create a registry of subjects with FH across Canada. Rare diseases of lipoprotein metabolism will also be included (SMASH initiative).

Methods and Results:
Using a “hub and spoke” model, the registry will be extended in various communities to link primary care physicians with provincial academic centers. The registry will include clinical, biochemical and demographic information. Specimens (plasma/serum and DNA) will be collected for local biobanking. We propose a three-tier registry: local, provincial and Canada-wide, which will be completely anonymized. The registry will be made available for clinicians to manage patient care, identify relatives for screening and treatment (cascade screening), to provide advice to general practitioners and to support collaborative studies in biomedical, clinical, health outcomes and health economics research. The data extracted for the provincial portion of the database will allow administrative database research that will provide important information to key stakeholders and permit allocation of resources. It will also allow a sound and uniform rationale for the use of novel therapeutic agents and provide expert advice to regulatory agencies. At the Canadian level, the database will allow clinicians and researchers to determine the burden of disease and the long-term effects of treatment.

Conclusion:
Through the creation of a Canada-wide network of academic clinics, integrating lipid specialists, endocrinologists and cardiologists, the Canadian FH registry will lead to significant benefits for FH patients, clinicians and researchers, biopharmaceutical industry and government.
Abstract 245: Intravenously Injected Human Apolipoprotein A-I Rapidly Enters the Central Nervous System via the Choroid Plexus in Mice
Authors: Stukas Sophie , Robert Jerome , Lee Michael , Kulic Iva , DeValle Nicole , Carr Michael , Fan Jianjia , Namjoshi Dhananjay , Lemke Kalistyne , Oda Michael N , Wellington Cheryl L
Published: July 2021

Background:
Lipoprotein metabolism in the brain is based on particles that resemble high-density lipoproteins (HDL) that use apolipoprotein (apo) E as opposed to apoA-I as their primary protein component. Although apoA-I is not synthesized by astrocytes or microglia, which secrete apoE, it is abundant in cerebrospinal fluid (CSF) and is readily detectable in brain tissue lysates.However, the mechanisms by which plasma apoA-I enters and is metabolized within the central nervous system (CNS) are unknown.

Methods and Results:
Western blot analysis shows that steady state levels of endogenous apoA-I in CSF and brain are approximately 0.01% and 10-15% of its levels in plasma and liver, respectively. Recombinant, fluorescently tagged, lipid-free human (h) apoA-I injected into the tail vein of wild-type mice localizes to the choroid plexus within 0.5h and accumulates in a saturable, dose-dependent manner in brain. hApoA-I accumulates in the brain for up to 2h, after which it is turned over with a half life of ~133 minutes, 3 times longer than the relatively quick turnover 40-45 minutes found in plasma, liver, and kidney. In vitro, hApoA-I is taken up and actively transported across confluent monolayers of primary human choroid epithelial cells.

Conclusions:
Following intravenous injection, hApoA-I rapidly and strongly localizes to the choroid plexus, suggesting it gains access to the CNS primarily via the blood CSF barrier. Further, apoA-I found in the CNS of mice is exclusively derived from the circulation as apoA-I mRNA is not detectable in murine brain. These results suggest that apoA-I based HDL may primarily play a role in CSF lipoprotein metabolism in addition to potentially impacting cerebrovasculature health and function from the lumen of the vessel.
Abstract 258: A Pharmacological Approach to Promote Shear Stress-Dependent Anti-inflammatory Mechanisms in Arteries
Authors: Luong Le , Duckles Hayley , Schenkel Torsten , Arnold Nadine , Gsell Willy , Lungu Angela , Spencer Tim , Hose Rodney , Halliday Ian , Evans Paul
Published: July 2021

Atherosclerosis is influenced by blood flow whichS exert wall shear stress (WSS) on endothelial cells (EC). Low oscillatory WSS promotes atherosclerosis by inducing inflammatory molecules (e.g. VCAM-1), whereas high uniform WSS induces anti-inflammatory genes (e.g. eNOS). Ivabradine decreases heart rate by inhibiting the If current in the sinus node, without affecting blood pressure or cardiac contractility. Besides cardio-protection, ivabradine protects arteries from atherosclerosis via unknown mechanisms. We hypothesised that ivabradine reduces arterial inflammation by increasing blood flow-induced WSS.

Mice (C57BL/6, male, aged 10 weeks) were treated with ivabradine for 1 week (162 mg/L in drinking water) or remained untreated. WSS was mapped at atherosusceptible (inner curvature) and protected (outer curvature) sites of the aorta by defining the geometry (CT angiography; Siemens) and flow characteristics (ultrasound; Visualsonics). Segmentation and smoothing were performed to reconstruct the volume of interest (Amira, VMTK) and a tetrahedral mesh with prismatic boundary layer was generated (IcemCFD). Fluid behavior was modeled over the cardiac cycle using Fluent 14.5 (ANSYS). Computational fluid dynamics revealed that WSS at the inner curvature of the aorta had lower magnitude and higher oscillatory index compared to the outer curvature. Ivabradine treatment reduced heart rate by 20% (p<0.05; n=5 per group) and altered WSS at the inner curvature by increasing magnitude and reducing oscillatory index. En face immunostaining and confocal microscopy demonstrated that treatment with ivabradine led to reduced VCAM-1 expression (mean fluorescence intensities (MFIs): 710.6 (untreated) vs 4.4 (treated); p<0.01; n=5 per group) and enhanced eNOS expression (MFIs: 3.48 (untreated) and 6.25 (treated); p=0.05; n=5 per group) at the inner curvature.

We conclude that ivabradine treatment altered hemodynamics in the murine aorta by increasing the magnitude and uniformity of shear stress. These changes were accompanied by induction of a mechanosensitive protective gene (eNOS) and suppression of inflammatory VCAM-1. Thus ivabradine may protect arteries by altering local mechanical conditions to trigger an anti-inflammatory response.
Abstract 267: Histomorphometric Evaluation of Estrogen Effects on Coronary Artery Atherosclerosis: Inhibition of Plaque Formation, Reduction in Plaque Size and Promotion of Positive Remodeling Independent of Plaque Complications
Authors: Melendez Giselle C , Clarkson Thomas B
Published: July 2021

Introduction:
Whether and to what extent postmenopausal estrogen therapy has beneficial effects on progression of coronary artery atherosclerosis is controversial. In previous studies with monkey models we have shown that the stage of plaque progression modulates estrogen benefits and have led to the “timing hypothesis”. In this study, we used a histomorphometric approach to understand better the effects of estrogen therapy on critical components of atherosclerosis pathogenesis. Our hypothesis was that estrogen therapy would inhibit the initial development of atherosclerotic plaques, result in smaller but not less complicated plaques and among subjects that developed plaques, it would promote positive arterial remodeling.

Methods:
Forty-eight surgically postmenopausal monkeys (Macaca fascicularis) were fed a moderately atherogenic diet and randomly assigned to receive no treatment (CTL) or a women’s equivalent dose of conjugated equine estrogen (CEE) (0.45 mg/day). The experimental period lasted for 20 months (equivalent to 5 years in humans). At the end of the experiment, prevalence of atherosclerotic plaques was determined. From those monkeys with affected arteries, plaque extent, severity and prevalence of stenosis were determined.

Results:
Prevalence: The CEE treated group had significantly fewer monkeys with coronary artery atherosclerosis compared to the CTL group (CTL: 57 % vs. CEE: 33 % p=0.005). Extent: Among affected monkeys, the mean plaque size was significantly smaller in the CEE treated group compared to the CTL group (CTL: 0.30 ± 0.048 mm2 vs. CEE: 0.153 ± 0.028 mm2 p=0.015). Remodeling: The proportion of stenotic plaques was lower in the CEE group compared to the CTL group (CTL: 11.14 % vs. CEE: 1.34 % p= 0.008). Severity: CEE treatment did not have a beneficial effect on coronary artery severity (% of complicated [AHA grade≥ III] plaques- CTL: 52.34 % vs. 49.61 %, p=0.397).

Conclusions:
These histomorphometric findings support previous evidence that beginning of estrogen therapy in the perimenopausal transition prevents plaque initiation. Among affected cases, estrogen treated monkeys had smaller plaques but were equally complicated. Notably, estrogen also promoted positive arterial remodeling among affected arteries.
Abstract 273: Shear Stress--Induced Atherosclerotic Plaque Regression Explained by Increased Macrophage Efferocytosis and Migration
Authors: Simeone Stefania , Ebrahimian Talin , Michaud Veronique , Lehoux Stephanie
Published: July 2021

Atherosclerotic plaques form in regions of low blood flow, whereas vessels exposed to high shear stress remain lesion-free. Using a model of arteriovenous fistula (AVF) in mice, we have previously shown that exposing established atherosclerotic plaques to elevated shear stress leads to lesion regression and increased matrix metalloproteinase (MMP) activity. MMP inhibition abolished shear stress-induced plaque regression and macrophage migration, suggesting that facilitating inflammatory cell movement within the plaque contributes to regression. We hypothesized that increased shear stress also leads to more efficient efferocytosis, another important hallmark of regression. LDLR-/- mice were placed on a high-fat diet. Sham and AVF surgery was performed at week 12 and mice were kept on a high-fat diet for a further 4 weeks (wk16). Control mice were sacrificed at wk12. The AVF procedure increases the shear stress in the brachiocephalic artery (BCA) but does not alter serum lipid levels. Using 3D echocardiography between wk12 and wk16, we observed that plaques progressed in the BCA of sham mice, whereas the AVF plaques regressed. Furthermore, the size of the necrotic core was significantly smaller in the AVF plaques than sham and control (P<0.05). This could be due to increased efferocytosis in the AVF, as verified using an in vitro model of the plaque environment. Endothelial cells (EC) were co-cultured with macrophages in a system wherein ECs are exposed to high or no shear stress and macrophages are exposed to the EC effluent. Uptake of apoptotic cells by macrophages was 50% higher in the high shear vs. static conditions (P<0.01). The MMP inhibitor, GM6001, had no effect on efferocytosis. However, a cytokine array on the co-culture effluent revealed interesting candidates. CCL3 and GMCSF, both of which have been associated with enhanced efferocytosis, were more highly expressed in high shear conditions vs. static. Our findings suggest that shear stress increases efferocytosis, leading to smaller necrotic cores through a mechanism that probably involves altered cytokine production. The combination of more efficient cell migration and efferocytosis in the presence of increased shear stress likely leads to plaque regression.
Abstract 276: SR-BI Suppresses Apoptosis by Reducing Endoplasmic Reticulum Stress and Promoting Akt Activity in Macrophages
Authors: Tao Huan , Yancey Patricia G , Davies Sean S , Roberts L Jackson , Blakemore John L , Zhang Youmin , Ding Lei , Fazio Sergio , Linton MacRae F
Published: July 2021

Objective:
Macrophage apoptosis contributes to atherosclerotic plaque necrosis, inflammation, development and rupture. Scavenger receptor class B type I (SR-BI) is a key regulator of HDL metabolism and cellular cholesterol homeostasis. Here we examined the hypothesis that macrophage SR-BI modulates lipid-associated cellular stress and apoptosis.

Methods and Results:
In vitro cell apoptosis assays were performed in primary macrophages, and for in vivo evidence, we examined TUNEL staining of atherosclerotic lesions of LDLR
-/-
mice that were reconstituted with SR-BI
-/-
or WT bone marrow after 16 weeks on a Western diet. We found that SR-BI deficiency led to ~64.3% more apoptotic cells induced by oxidized LDL or free cholesterol in primary macrophages, and 6-fold more lesional apoptotic cells in SR-BI
-/-
→LDLR
-/-
mice compared to WT recipient mice. In macrophages, SR-BI deficiency caused significant accumulations of cellular free cholesterol and elevated markers of endoplasmic reticulum (ER) stress. These were exacerbated by feeding mice a high-cholesterol diet or inactivating the apolipoprotein E gene. Peroxidation of lipoproteins and cell membranes leads to modification of phosphatidylethanolamine by lipid aldehydes including isolevuglandins (IsoLG-PE). Treatment of macrophages with IsoLG-PE induced 52.6% more apoptotic cells in SR-BI
-/-
macrophages compared to WT. Transgenic expression of SR-BI by transfection of SR-BI
-/-
macrophages rescued oxidative stress-induced ER stress and cell apoptosis. SR-BI deficiency inhibited the Akt pathway compromising macrophage survival and increasing lesion necrosis. Moreover, Akt Activator was able to rescue SR-BI deficiency associated apoptosis in macrophages. Apolipoprotein E interacts with SR-BI in macrophages, co-operating for cellular lipid homeostasis and cell survival signaling.

Conclusion:
SR-BI protects against cell apoptosis induced by lipid stress in macrophages and atherosclerotic lesions. The underlying mechanisms are, at least in part, through reducing lipid-associated ER stress and promoting Akt activity in macrophages. Thus, we identify macrophage SR-BI-mediated apoptosis pathways as molecular targets for the prevention of atherosclerotic cardiovascular events.
Abstract 29: MiR-33 Coordinately Regulates Macrophage Autophagy
Authors: Ouimet Mireille , Ediriweera Hasini , Ramkhelawon Bhama , Hennessy Elizabeth , Karunakaran Denuja , Liao Xianghai , Esau Christine , Tabas Ira , Marcel Yves , Rayner Katey , Moore Kathryn
Published: July 2021

Macrophage autophagy is thought to be essential for protecting from atherosclerosis, and compromised autophagy in macrophages of the artery wall leads to a number of pathologic processes including activation of the inflammasome, defective efferocytosis, and impaired cholesterol metabolism. Autophagy of lipid droplets (LDs) or “lipophagy” catabolizes stored lipids to maintain cellular energy homeostasis and plays a key role in cholesterol efflux by regulating LD-cholesterol mobilization, a rate-limiting step in macrophage reverse cholesterol transport (RCT). MicroRNA-33 (miR-33) is a well-established post-transcriptional RCT regulator, yet the complete mechanisms by which anti-miR33 exerts its beneficial effects on cholesterol metabolism are not known. Notably, microRNA target prediction algorithms identify a number of essential autophagy-related proteins (ATG5, ATG7) and lysosomal effectors (lysosomal-associated membrane protein 1 [LAMP1], lysosomal acid lipase [LAL]) as putative miR-33 targets. Quantitative PCR array profiling in mouse peritoneal macrophages revealed that a high proportion of autophagy genes are reciprocally regulated by miR-33 overexpression and inhibition. We validated a subset of genes in the autophagy pathway as bona fide miR-33 targets using 3′UTR luciferase assays and confirmed regulation of these targets by miR-33 using quantitative PCR and western blot analysis. Furthermore, we show that miR-33 indirectly regulates the expression of two master regulators of autophagy and lysosomal biogenesis gene programs: forkhead box O (FOXO) 3 and transcription factor EB (TFEB), via targeting of 5' AMP-activated protein kinase (AMPK). Inhibition of miR-33 in peritoneal macrophage in vitro enhanced cellular autophagic flux, as observed by fluorescence microscopy and western blot analysis, and autophagy was required for anti-miR33 promotion of cholesterol efflux. Furthermore, anti-miR33 treatment of atherosclerotic Ldlr-/- mice enhanced autophagy in plaque macrophages and triggered atherosclerosis regression. These data describe a novel role for miR-33 in the regulation of autophagy and identify additional mechanisms by which anti-miR33 therapy protects against atherosclerosis.
Abstract 3: HDL-MicroRNA-92a and Interleukin-33 Axis Underlies Endothelial Dysfunction Associated With Atherosclerosis and Chronic Kidney Disease
Authors: Wiese Carrie B , Toth Cynthia L , Tabet Fatiha , Taylor Robert C , Landstreet Stuart R , Rye Kerry-Anne , Hofmeister Lucas H , Harrison David G , Kon Valentina , Vickers Kasey C
Published: July 2021

Chronic kidney disease (CKD) is associated with endothelial dysfunction and atherosclerosis. High-density lipoproteins (HDL) serve as a general cargo carrier for a wide-variety of proteins, nucleic acids, and small molecules which likely confer many of HDL’s alternative functions. Human coronary artery endothelial cells (HCAEC) treated with native HDL were found to have increased intracellular levels of miR-92a, a miRNA recently found to protect against endothelial dysfunction and atherosclerosis. Moreover, HDL delivery of miR-92a was inhibited using SR-BI blocking antibodies in endothelial cells. Likewise, aortic endothelial miR-92a levels were found to be significantly decreased (50%) in Apoe-/- mice compared to wild-type controls, which may be a function of the absence of HDL in these mice. Using photoactivatable-ribonucleoside-crosslinked-immunoprecipitation-high-throughput-sequencing, we found that HDL transfer multiple miR-92a isomiR variants from macrophages to HCAECs, which were found in association with Argonaute2 RNA-induced silencing complexes. Using whole-genome arrays, we found that HDL alters the expression of many genes in HCAECs, including the significant down-regulation of 18 putative miR-92a targets. Strikingly, miR-92a was found to be significantly increased 8.5-fold on HDL from CKD subjects. Interleukin-33 (IL-33) is a cytokine and nuclear transcription factor largely expressed in endothelial cells. HDL from both normal and CKD subjects significantly decreased IL-33 expression in HCAECs, as determined by real-time PCR and microarrays. In a mouse model of CKD (Apoe-/-; 5/6 nephrectomy), IL-33 expression was increased in aortic endothelial cells; however, a single HDL injection (4mg) reduced IL-33 mRNA back to sham levels at 7d post-injection. Most interestingly, HDL loaded with locked-nucleic acid miR-92a inhibitors blocked HDL’s ability to suppress CKD induced IL-33 expression in the aortic endothelium. IL-33 may represent a novel marker of endothelial dysfunction, as IL-33 mRNA levels were also found to be increased in HCAECs under disturbed flow conditions. Results from this study suggest that HDL suppression of IL-33 in CKD requires miR-92a, which is also transferred to endothelial cells by HDL.
Abstract 30: Localized Immune Response to Oxidized Lipids in ABCG1-/- Mice: A New Model for Lipid-Driven Autoimmunity
Authors: Baldan Angel , Gonen Ayelet , Choung Christina , Marquart Tyler , Que Xuchu , Diehl Cody , Witztum Joseph L , Tarling Elizabeth J
Published: July 2021

Many metabolic diseases, including atherosclerosis, type 2 diabetes, pulmonary alveolar proteinosis (PAP), and obesity, have a chronic inflammatory component involving both innate and adaptive immunity. Mice lacking the ATP binding cassette (ABC) transporter ABCG1, develop chronic inflammation in the lungs, associated with lipid accumulation (cholesterol, cholesterol ester, phospholipid, oxidized lipids) and cholesterol crystal deposition, characteristic of atherosclerotic lesions and PAP. Here we demonstrate that specific lipids, likely oxidized (Ox) phospholipids and/or sterols, elicit a lung-specific immune response in Abcg1-/- mice. Loss of ABCG1 results in increased levels of specific oxysterols, phosphatidylcholines and oxidized phospholipids, including 1-palmitoyl-2-(5’-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), in the lungs. Further, we identify a niche-specific increase in natural antibody (NAb)-secreting B-1 B cells in response to this lipid accumulation that is paralleled by increased titers of IgM, IgA and IgG against oxidation specific epitopes such as those on OxLDL and malondialdehyde-modified LDL (MDA-LDL). Finally, we identify a cytokine/chemokine signature in the lungs of Abcg1-/- mice reflective of increased B cell activation, antibody secretion and homing. Collectively, these data demonstrate that the accumulation of lipids in Abcg1-/- mice induces the specific expansion and localization of B-1 B cells, which secrete NAbs that may help protect against the development of atherosclerosis. Indeed, despite chronic lipid accumulation and inflammation, hyperlipidemic mice lacking ABCG1 develop smaller atherosclerotic lesions compared to controls. These data also suggest that Abcg1-/- mice may represent a new model in which to study the protective functions of B-1 B cells/NAbs, and may provide novel targets for pharmacologic intervention and treatment of disease.
Abstract 327: ETS Factors Regulate the VEGF-Dependent, Arterial-Specific Expression of Dll4
Authors: Dang Lan T , Wythe Joshua D , Devine Patrick W , Boudreau Emilie , Artap Stanley T , He Daniel , Schachterle William , Stainier Didier Y , Oettgen Peter , Black Brian L , Bruneau Benoit G , Fish Jason E
Published: July 2021

Development of the vascular system begins with vasculogenesis, followed by the specification of endothelial cells into arterial and venous fates. Establishing and maintaining these separate fates is critical for the proper functioning of circulatory networks in the embryo as well as the mature adult. In fact, the congenital condition, Arteriovenous Malformation (AVM), arises due to defects during arterial-venous (AV) specification of endothelial cells, resulting in abnormal AV shunts that may burst leading to vascular lesions and stroke. To date, the molecular processes that control early AV specification remain incompletely understood. In the mouse, Delta-like 4 (Dll4) is a critical mediator of AV identity as loss of only one copy of Dll4 produces AV specification defects and embryonic lethality. Although previous studies have shown that Dll4 is the first Notch ligand expressed in the arterial endothelium and that its expression is induced by Vascular endothelial growth factor, the transcriptional pathways that direct its arterial-specific expression are unknown. Here we report the identification of an arterial-specific enhancer of the Dll4 gene. We show through deletion analyses as well as gain-of-function and loss-of-function studies that Vegf signaling activates ETS factors in the arterial endothelium to drive expression of Dll4. Surprisingly, Notch signaling is not required for the initiation of Dll4 expression but rather functions as a maintenance factor in the arterial endothelium. These studies provide novel mechanistic insight into the transcriptional program that mediates arterial specification and may provide insight into the underlying defects that occur during the development of AVMs.
Abstract 335: Carbon Nanoparticles Cause Loss of Gap Junctional Intercellular Communication and Nuclear Translocation of ß-Catenin in Endothelial Cells
Authors: Ale-Agha Niloofar , Unfried Klaus , Altschmied Joachim , Haendeler Judith
Published: July 2021

Introduction:
Environmental factors like industrial or consumer derived pollution are known to increase the risk for cardiovascular diseases. Previous studies demonstrated that senescent endothelial cells contribute to the onset of atherosclerosis. We have recently shown that non-toxic and non-inflammatory concentrations of carbon nanoparticles (CNP), a major component of industrial pollution, induce senescence in human primary endothelial cells (EC). However, the underlying molecular mechanisms leading to senescent EC are far from understood. Gap junctional intercellular communication (GJIC), which depends on Connexin 43 (Cx43) and β-catenin, is important for endothelial function and seems to be dysregulated during aging. Phosphorylation of Cx43 on serine 368 reduces the trafficking of Cx43 to the plasma membrane and assembly of Cx43 into gap junctional structures.

Objective:
The aim of this study was to investigate whether CNP affect GJIC and β-catenin in EC.

Material and Methods:
EC were treated with CNP at a measured size of 10 - 34 nm and GJIC was measured by dye transfer assay. The localization of Cx43 and β-catenin was assessed by immunofluorescence and phosphorylation and protein levels of Cx43 were by immunoblots.

Results:
Two weeks incubation with CNP concentrations, which we are exposed to everyday, elevated the number of senescent EC. This was determined by increased staining for senescence-associated beta-galactosidase and the cell cycle inhibitor p21 - two established markers of senescence. The same concentrations of CNP resulted in an enhanced phosphorylation of Cx43 on serine 368. Moreover, CNP induced internalization of Cx43 and led to nuclear translocation of the Cx43 interacting protein β-catenin. This resulted in reduced GJIC, which was shown by decreased dye transfer. Thus, CNP-induced senescence is accompanied by loss of cell-cell communication.

Conclusion:
These data indicate for the first time that Cx43 and β-catenin, which are essential for endothelial functionality, are affected by CNP and that loss of GJIC is involved in CNP-induced senescence. Overexpression studies will investigate whether a causal link exists between CNP-induced senescence and loss of GJIC in primary EC.
Abstract 339: Ang-(1-7) Counteracts Ang II in Regulating Cerebrovascular Endothelial Cell Function and Gene Expression
Authors: Chen Ji , Xiao Xiang , Chen Shuzhen , Zhang Cheng , Wang Jinju , Chen Yanfang
Published: July 2021

Cerebrovascular endothelial cells (cECs) play an important role in maintaining the health of cerebral vasculatures. In this study, we investigated the role of angiotensin (Ang)-(1-7) in counteracting Ang II-induced effects on cECs. The 3rd-5th passages of brain microvascular endothelial cells (BMECs, Cell system) cultured in CSC complete medium (Cell system) were used for this study. BMECs were treated with Ang II (100 nM), Ang-(1-7) (100 nM) and A-779 (100 nM, Mas receptor antagonist). After treatment for 24 hrs, cell apoptosis was determined by flow cytometery. BMEC tube formation ability was determined using a commercial kit. The levels of tumor necrosis factor- α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) in the cell culture supernatant were determined by ELISA. Gene expressions of NFκB, inhibitor of kappa B (IκB) were determined by western blot. To verify the involvement of NFκB pathway, the inhibitor (BAY11-7082, 10 uM) was applied. We found (Figure): 1) Treatment with Ang-(1-7) decreased Ang II-induced apoptosis (22 ± 1.5% vs. 14 ± 1.2%, Ang II vs. Ang II + Ang-(1-7), P<0.01). 2) Ang-(1-7) improved BMEC tube formation ability compromised by Ang II, which was accompanied by up-regulation of IκB expression, the down-regulation of NFκB, and decrease of TNF-α, MCP-1 and IL-8 production of BMECs. 3) These effects of Ang-(1-7) were totally abolished by A-779 and partially blocked by BAY 11-7082. In conclusion, Ang-(1-7)/Mas activation counteracts Ang II-induced BMEC apoptosis and dysfunction, partially via the NFκB dependent inflammation pathway.
Abstract 342: The Antiapoptotic Function of APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 in Endothelial Cells Is Linked to Changes in Transcription Factor Activities and Independent of Its DNA Repair Domain
Authors: Dyballa-Rukes Nadine , Rabanter Lothar , Goy Christine , Altschmied Joachim , Haendeler Judith
Published: July 2021

APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has both DNA repair and redox regulatory activity. Interestingly, APEX1 is also able to modulate transcription factor reduction, a prerequisite for DNA binding, independent of its intrinsic redox activity, probably by recruiting other reducing molecules like Thioredoxin-1 (TXN). APEX1 has been shown to exert anti-apoptotic properties in different cell lines and is localized in the cytosol and in the nucleus. So far it has not been studied whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and whether this is dependent on its nuclear or cytosolic localization.

After cloning human APEX1 and overexpression in EC we demonstrated that it inhibits not only basal but also H2O2-induced apoptosis. To further characterize the anti-apoptotic properties of APEX1 we generated mutants that lacked the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) (APEX1ΔN 21-318). We first analyzed the localization of the mutants by immunostaining. APEX1wt and APEX1ΔC 1-127 showed a predominant nuclear staining whereas APEX1ΔN 21-318 was mainly localized in the cytosol. With respect to the anti-apoptotic properties, only the mutant with intact NLS and redox domain APEX1ΔC 1-127 prevented apoptosis both under basal conditions as well as after H2O2 treatment similar to APEX1wt.

The association between nuclear localization of APEX1wt and its anti-apoptotic properties in EC suggests that this function is linked to changes in transcription factor activities. It is known that the p65 subunit of the transcription factor complex NF-kB is activated by pro-apoptotic stimuli in EC. Using a specific DNA binding assay we could show that overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant had no effect.

In conclusion, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity, is independent of its DNA repair domain and is at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyze the impact of APEX1 on other transcription factors in concert with TXN.