bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 542: Effects of 1-Methylnicotinamide on Inflammatory and Lipid Biomarkers
Authors: Tardif Jean-claude , Fikry Sameh , Girard Ginette , Discenza Tania , Fortier Annik , Cefali Eugenio A , Guertin Marie-Claude
Published: July 2021

Background:
1-methylnicotinamide, a nicotinic acid metabolite, is TRIA-662’s active component. As part of a pilot study, we assessed TRIA-662’s effects on inflammatory biomarkers and blood lipids.

Methods:
Patients aged 18-80 years with mean serum triglycerides (TG) 2.26-5.65 mmol/L, low-density lipoprotein-cholesterol levels not needing drug therapy, and stable diet and exercise regimens were randomized to TRIA-662 or placebo (PBO, 3:1) up-titrating to 6 g daily orally for 14 weeks. Outcomes were treatment compliance and changes from baseline in inflammatory biomarkers and blood lipids.

Results:
The 71 randomized patients were 54±12 years old, 54% were male, 48% were obese, 37% had hypertension, 25% dyslipidemia, and 7% diabetes. Treatment compliance and study completion (95% CI) were reached in 87.1% (79.3, 95.0%) and 87.3% (79.6, 95.1%) of patients overall, respectively. Adjusted geometric mean percent change (95% CI) from baseline in high-sensitivity C-reactive protein was -15.66% (-28.40, -0.66%) with TRIA-662 (p=0.0419 vs baseline) and 1.21% (-13.95, 19.05%) with PBO. The between-group difference of -16.67% (-33.59%, 4.57%) in favor of TRIA-662 did not reach significance (p=0.1130). The effect of TRIA-662 on tumor necrosis factor (TNF) alpha was influenced by the baseline value: For a baseline TNF value of 3.30 pg/mL (third quartile), the adjusted mean change (95% CI) was -0.65 (-0.92, -0.38) pg/mL, corresponding to a 20% decrease, with TRIA-662 and 0.31 (-0.32, 0.95) pg/ml, corresponding to a 9% increase with PBO, with a between-group difference of -0.97 (-1.65, -0.29) pg/mL (p=0.0076). The adjusted mean changes from baseline in adiponectin were 0.55 μg/mL (0.23, 0.86 μg/mL) corresponding to a 6% increase for TRIA-662 and -0.06 μg/mL (-0.53, 0.42 μg/mL) corresponding to a 0.7% decrease for PBO (p=0.0391 between groups). Geometric mean TG value at baseline was 3.36 mmol/L. The adjusted geometric mean percent change (95% CI) in TG was -9.01% (-15.91%, -1.54%) for TRIA-662 and -2.09% (-13.03, 10.24%) for PBO (p=0.3088 between groups). Baseline HDL-C was 1.01 ± 0.28 mmol/L, and the difference in change over time was not significant between groups (p=0.8242).

Conclusion:
TRIA-662 favourably affected inflammatory biomarkers in this pilot study.
Abstract 543: Myeloperoxidase Induces Endothelial Dysfunction via Activation of Calpain
Authors: Etwebi Zienab , Landesberg Gavin , Scalia Rosario
Published: July 2021

Myeloperoxidase (MPO) is a peroxidase enzyme secreted by activated leukocytes, which has been associated with endothelial dysfunction and insulin resistance. The calcium dependent protease calpain has also been linked to vascular disease in insulin resistance and type 2 diabetes. Accordingly, we tested the hypothesis that endothelial expressed calpains play a role in MPO-induced endothelial dysfunction and vascular inflammation. Mouse lung microvascular endothelial cells (MMVEC) were stimulated with 10 nM MPO for 30, 60, 120, 180, and 240 minutes. Expression levels of Vascular Cell Adhesion Molecule 1 (VCAM-1), 5’ AMP Activated Protein Kinase (AMPK), Protein Kinase B (PKB) (Akt), endothelial Nitric Oxide (eNOS) phosphorylation at serine 1177 (Se1177), Protein Phosphatase 2 (PP2A), and calpains were measured by immunoblot analyses. MPO time dependently activated μ-calpain (P<0.0001 vs control) but not m-calpain. MPO also significantly increased PP2A protein levels (P<0.001), decreased AMPK phosphorylation (P<0.01), AKT phosphorylation (P<0.05), and eNOS phosphorylation at (Se1177) (P<0.01), while significantly increased VCAM-1 protein levels (P<0.01). Pretreatment of MMVECs with the selective calpain inhibitor ZLLal (100 μM) prevent MPO-induced calpain activation (P<0.0001 vs MPO alone). Inhibition of calpain activity also preserved AMPK phosphorylation, eNOS phosphorylation, and PP2A levels, in the face of MPO (P<0.001 versus MPO alone). Calpain inhibition also prevented upregulation of VCAM-1. Using an
ex vivo
leukocyte adhesion assay we also found leukocytes failed to adhere to the vascular endothelium of MPO treated aortas isolated from μ-calpain deficient mice (p<0.001 versus aortas isolated from WT mice). Taken together, our data first demonstrate a primary role for μ-calpain in endothelial dysfunction induced by leukocyte derived MPO.
Abstract 55: Disruption of
PCSK9
Using a Targeted Base Editing Strategy
Authors: Chadwick Alexandra C , Musunuru Kiran
Published: July 2021

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases blood low-density lipoprotein (LDL) cholesterol by acting as an LDL receptor antagonist, thereby impairing LDL particle clearance. Since genetic disruption of
PCSK9
is linked to reduced risk of coronary heart disease (CHD), our recent work has sought to permanently knock out the gene by using new genome editing technology. Recently reported “base editors” introduce point mutations at specific locations in the genome without the need for DNA double-strand breaks and, thus, with a lowered incidence of off-target effects. These base editors build on the CRISPR-Cas9 system by tethering an RNA-editing domain to a nickase version of Cas9, allowing for specific CT and/or GA base alterations. In this study, we used the “BE3” base editor (which uses the APOBEC-1 RNA-editing domain) to specifically target codons encoding tryptophans (TGG) or glutamines (CAG or CAA) to introduce nonsense mutations (producing stop codons TAG, TGA, or TAA) into human
PCSK9
. The number of testable targets was increased by introducing specific point mutations into the BE3 construct (D1135V, R1335Q, T1337R in the Cas9 portion of BE3) to alter the protospacer adjacent motif (PAM) from NGG to NGA. Using HEK293 cells, we individually targeted a number of codons spanning the first seven exons in
PCSK9
and identified several efficient targets. Most notably, the codons encoding glutamine 278 and glutamine 302 could be altered to stop codons in ~50% of alleles, as determined by the CEL-I nuclease mismatch assay and Sanger sequencing. We then targeted glutamine 278 in human induced pluripotent stem cells (iPSCs) and demonstrated base editing to introduce nonsense mutations into
PCSK9
, albeit at a lower efficiency than in HEK293 cells. As a next step towards translation to human patients, we are targeting
Pcsk9
in the mouse liver
in vivo
with BE3. Base editing may prove to be an efficient, safer strategy than standard CRISPR-Cas9 genome editing and holds promise as a strategy for the prevention of CHD.
Abstract 550: Alteration of Glycolysis Metabolite Levels and Impaired Hypoxic Response in Diabetic Atherosclerosis
Authors: Yamashita Atsushi , Matsuura Yunosuke , Zhao Yan , Iwakiri Takashi , Yamasaki Kazuaki , Sugita Chihiro , Koshimoto Chihiro , Kitamura Kazuo , Kawai Keiichi , Tamaki Nagara , Zhao Songji , Kuge Yuji , Asada Yujiro
Published: July 2021

Objective:
Diabetes mellitus accelerates atherosclerosis that causes most cardiovascular events. Several metabolic pathways are considered to contribute to the development of atherosclerosis, but comprehensive metabolic alterations to atherosclerotic arterial cells remain unknown. The present study investigated metabolic status and their relationship to vascular histopathological changes in the atherosclerotic arteries of rabbits with alloxan-induced diabetes.

Approach and Results:
Diabetic atherosclerosis was induced in rabbits ilio-femoral arteries by injecting alloxan (100 mg/kg), injuring the arteries using a balloon, and feeding with a 0.5% cholesterol diet. Plaque burden, macrophage content, and hypoxic areas were more prevalent in arteries with diabetic, than non-diabetic atherosclerosis. Metabolomic analyses highlighted 12 metabolites that were significantly altered between diabetic and non-diabetic atherosclerosis. A half of them were associated with glycolysis metabolites, and their levels were decreased in diabetic atherosclerosis. The uptake of glucose evaluated as
18
F-fluorodeoxyglucose in atherosclerotic lesions increased according to increased macrophage content or hypoxic areas in non-diabetic, but not diabetic rabbits. Despite profound hypoxic areas, the nuclear localization of hypoxia-inducible factor-1α decreased and the number of apoptotic cells increased in diabetic atherosclerotic lesions.

Conclusions:
Altered glycolysis metabolism and an impaired response to hypoxia in atherosclerotic lesions under conditions of insulin-dependent diabetes might be involved in the development of diabetic atherosclerosis.
Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1
Authors: Bi Xin , McParland James , Wang Jian , McIntyre Adam D , Hegele Robert A , Rader Daniel J , Cuchel Marina
Published: July 2021

Objectives:
ABCA1
encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I).
ABCA1
variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known.

Methods:
In silico
analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of
ABCA1
missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel
ABCA1
variants were generated
in vitro
through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods.

Results:
A total of 32 mutations in
ABCA1
were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors.

Conclusions:
These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.
Abstract 558: PCPE2 Promotes Adipocyte Maturation via Regulation of SR-BI Activity
Authors: Xu Hao , Kaul Sushma , Maruko Elisa , Fatema Kaniz , Glastonbury Craig A , Small Kerrin , Thomas Michael J , Sorci-Thomas Mary
Published: July 2021

Procollagen C-endopeptidase enhancer protein 2 (PCPE2) is an enhancer protein that enhances the cleavage of the C-termini of procollagen by bone morphogenetic protein 1. But the function of PCPE2 is not limited to collagen maturation. Recently our lab reported that PCPE2 increased HDL-associated cholesteryl ester uptake via scavenger receptor class B type 1 (SR-BI), an HDL cholesterol receptor highly expressed in adipose tissue. Interestingly, TwinsUK study in human provided data showing that PCPE2 mRNA is highly correlated with adipose tissue distribution. Further, our lab observed a reduced size of visceral fat pad in PCPE2 deficient mice despite its indifference in body weight compared to the wild type. To study the molecular and cellular mechanisms of how PCPE2 regulates SR-BI function and how it affects adipose tissue formation, we generated PCPE2 knockout (PCPE2
-/-
) cell line from murine preadipocyte 3T3-L1 cell using CRISPR-Cas9 technology. Induction of 3T3-L1 cell to differentiate into mature adipocyte increases the expression of both PCPE2 and SR-BI hand in hand around 3 fold, which parallels the process of lipid droplet formation. Immunofluorescence studies showed that PCPE2 is distributed all over the cell in mature adipocyte, while SR-BI is mostly distributed along the cytoplasmic and lipid droplet membranes, in addition to the perinuclear region. More interestingly, PCPE2
-/-
3T3-L1 cell shows an impairment in lipid droplet formation during the induction of differentiation, with less than 10% of cells generating lipid droplets, and the size of lipid droplet being only about 50% of that in wild type. Although PCPE2
-/-
3T3-L1 cell expresses SR-BI at a higher level compared with wild type cell, nearly 50% of the SR-BI in wild type cell are in homodimer structure while PCPE2
-/-
3T3-L1 cell has almost none SR-BI multimer, indicating that SR-BI in PCPE2
-/-
3T3-L1 cell is inactive. Taken together, we concluded that PCPE2 plays a critical role in keeping SR-BI in active conformation, which, in turn, controls adipocyte maturation. Whether this effect is associated with collagen maturation will be assessed in our future studies.
Abstract 564: Structure-Function Studies of ApoA-I Mimetic Peptides for ABCA1-Dependent Cholesterol Efflux and HDL Formation
Authors: Islam Rafique M , Permousa Mohsen , Gordon Scott , Sviridov Denis , Pastor Richard W , Remaley Alan T
Published: July 2021

To examine the mechanism of action of ApoA-I mimetics, we designed 4 peptides with a variable number of E, L, K and A residues and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. They were named based on a unique physical property: N=ELK-neutral (EKLKELLEKLLEKLKELL), H=ELK-hydrophobic (EKLLELLKKLLELLKELL), P=ELK-positive (EKLKALLEKLKAKLKELL), Neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed that H and P were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment, all peptides showed greater than 40% helicity. In DMPC vesicle solubilization assay, we observed following order: P>N>H>>Neg. By non-denaturing gel electrophoresis, N, H, P formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC. However, Neg did not form any detectable particle. A mixture of natural lipids (PC:SM:LysoPC:PE:PS:PI; mol% 55:12:5:10:8:10), intended to simulate ABCA1 lipid membrane microdomain, yielded similar results . Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: H>N>>P, whereas Neg peptide was inactive. All-atom MD simulation of N carried out on a 12–nm disc containing POPC:cholest (200:20), 26 peptides, and initially placed in belt-like configuration as head-to-tail dimers, the starting structure was maintained for at least 3 μ-sec, indicated a strong preference for this configuration. However, when N was initially oriented in a picket-fence configuration, it distorted to a belt-like pattern within 1 μ-sec. For Neg, both starting configurations were much less stable with dimers losing their connectivity and monomers migrating to the top and bottom of the disc, leaving large hydrophobic patches of acyl chains exposed. Cross-linking studies were consistent with the ability of H and N to form dimers to stabilize the HDL structure, and thereby, were also consistent with simulation. Together, we show that a net neutral charge, a relatively large hydrophobic moment (0.78), and a broad hydrophobic face (180 degrees) are optimum features for an apoA-I mimetic peptide to promote cholesterol efflux and to stabilize a nascent discoidal HDL structure.
Abstract 567: Genetic and Clinical Heterogeneity of Marked High Density Lipoprotein Deficiency
Authors: Polisecki Eliana , Geller Andrew S , Sacco Taylor , Diffenderfer Margaret R , Asztalos Bela F , Karathanasis Sotirios K , Hegele Robert A , Schaefer Ernst J
Published: July 2021

Aim:
Our goal was to assess the population prevalence, genetics and clinical phenotypes of subjects with marked HDL deficiency.

Methods:
200 subjects (26% female, mean age 52 years) with serum HDL-C < 20 mg/dL, fasting triglycerides (TG) < 600 mg/dL, C reactive protein (CRP) < 10 mg/L, myeloperoxidase (MPO) < 1000 pmoles/L, HbA1c < 8.0%, liver transaminases < 120 U/L, and not taking anabolic steroids were studied. Lipids, inflammation markers, liver enzymes, and HDL particles were assessed; and sequencing of 31 lipid metabolism genes including
ABCA1, APOA1, LCAT
, and
LPL
was done.

Results:
HDL-C < 20 mg/dl was observed in 473 (0.35%) men (n=135,912) and 140 (0.089%) women (n=160,964) in our population over one year. In this low HDL group, 6.4% had elevated CRP, 4.4% had abnormal liver function, 4.2% had elevated HbA1c, 1.0% had elevated MPO, 0.5% had elevated TG, and 11.5% of men were taking anabolic steroids. These subjects were excluded. The 200 subjects studied had plasma values of LDL-C 102, HDL-C 15, TG 239, and apoA-I 76 mg/dL, with a marked deficiency of very large and large α-1 and α-2 HDL.
ABCA1
mutations were found in 38 subjects (19.0%; 29 heterozygotes, 6 compound heterozygotes, 3 homozygotes).
APOA1
mutations were found in 10 subjects (5.0%; 9 heterozygotes, 1 homozygote).
LCAT
mutations were found in 17 subjects (8.5%; 13 heterozygotes, 3 compound heterozygotes, 1 homozygote). In addition, 11 subjects (5.5%) had the
LPL
N318S variant, and 13 subjects (6.5%) had the
ABCA1
c.-279 C>G variant. Premature coronary heart disease (CHD) was observed in some subjects with
ABCA1 and APOA1
mutations. Neuropathy was observed in some subjects with
ABCA1
mutations, and kidney failure was observed in the subject with the homozygous
LCAT
mutation.

Conclusions:
Marked HDL deficiency occurred in 0.2% of our population and can be associated with liver disease, inflammation, diabetes, severe hypertriglyceridemia, and the use of anabolic steroids. In the absence of these conditions, mutations in
ABCA1, APOA1, or LCAT
were found in 32% of subjects studied. Premature CHD was seen in some subjects with
ABCA1 or APOA1
mutations, neuropathy was seen in some subjects with
ABCA1
mutations, and kidney failure was observed in some subjects with
LCAT
mutations.
Abstract 568: Lipidomic Comparison of Hyperglycemic and Normal Subjects Using Absolute Quantitation of >700 Lipid Species
Authors: Rees Jon , Kuklenyik Susan , Gardner Michael , Schieltz David , Parks Bryan A , Jones Jeffrey , Toth Christopher , McWilliams Lisa , Andrews Michael , Barr John R
Published: July 2021

The presence of diabetes mellitus in patients significantly increases the risk of cardiovascular disease, among many negative health outcomes. Several research groups have reported lipid species anomalies in subjects showing insulin resistance, including higher concentrations of sphingomyelin species relative to normal controls. Advances in lipidomic methodologies has allowed for the profiling of numerous lipid species in a single extraction and analytical run. We used the SCIEX Lipidyzer platform to determine the absolute concentration of 770 distinct lipid species from 52 subjects categorized into hyperglycemic (n=30) and normal subjects (n=22). Lipid species were determined from the following classes: cholesterol esters (CE), ceramides (CER), diacylglycerols (DAG), dihydroceramide (DCER), free fatty acids (FFA), hexosylceramides (HCER), lactosylceramides (LCER), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (LPE), sphingomyelins (SM), and triacylglycerols (TAG).

After normalizing by the sum of total apolipoprotein A and apolipoprotein B (to account for the possibility of reduced particle number due to medicinal intervention), lipids both by class and species were compared using one way analysis of variance. On a class level, relative to normal subjects, hyperglycemic patients showed increased levels of CER and FFA. Individual lipid species of these classes that were higher in hyperglycemic patients include CER(18:0), CER(20:0), CER(24:1), FFA(16:0), FFA(16:1), FFA(18:0), FFA(18:1), FFA(18:2), FFA(18:3), and FFA(20:3) Differences were also found in SM concentrations between hyperglycemic and normal patients resulting in a higher the SM/PC ratio, indicating changes in the lipid fluidity of the shell in lipoprotein particles of hyperglycemic patients. On a species level, SM(16:0), SM(18:1), and SM(24.1), were elevated in hyperglycemic subjects relative to normal subjects. This finding of elevated unsaturated SM species in hyperglycemic patients is at odds with other studies where it was found that the saturated SM moieties were elevated in insulin resistant subjects.
Abstract 57: The Epigenetic Enzyme Kdm6b Controls the Pro-Fibrotic Transcriptome Signature of Foam Cells
Authors: Neele Annette E , Prange Koen H , Hoeksema Marten A , van der Velden Saskia , Lucas Tina , Dimmeler Stefanie , Lutgens Esther , Van den Bossche Jan , de Winther Menno P
Published: July 2021

Aim:
Foam cells are a key hallmark of atherosclerotic lesion formation. Within the atherosclerotic lesion macrophages scavenge modified lipoproteins and thereby acquire their foam cell characteristics. Besides their foam cell phenotype, macrophages can have specific inflammation regulatory functions in atherosclerotic lesions. Epigenetic pathways are crucial for monocyte to macrophage differentiation and activation. The H3K27 demethylase Kdm6b (also known as Jmjd3) is regulated in response to various triggers and regulates several modes of macrophage activation. Given the crucial role of macrophage foam cells in atherosclerosis, we here studied Kdm6b in peritoneal foam cells in order to identify regulated pathways.

Material and Methods:
A myeloid deficient Kdm6b mice (LysMCre-Kdm6b
fl/fl
) was generated and bone marrow of Kdm6b
wt
or Kdm6b
del
mice was transplanted to irradiated Ldlr
-/-
mice which were fed a high fat diet for 9 weeks to induce foam cell formation. Peritoneal foam cells from Kdm6b
del
or Kdm6b
wt
mice were isolated and used for RNA-sequencing analysis.

Results:
Among the list of downregulated genes many genes involving fibrosis were affected in Kdm6b deficient foam cells including
Collagen
genes (
Col1a1
,
Col1a2
),
Alpha smooth muscle actin
(
Acta2
) and
Fibronectin-1
(
Fn1
). Pathway analysis on downregulated genes (
P
-value < 0.05) indicated that pathways involved in epithelial to mesenchymaltransition (EMT) (
q-
value=10
-13
) and extracellular matrix organization (
q-
value=10
-4
) were significantly downregulated. Pro-fibrotic pathways were thus strongly suppressed in Kdm6b deleted foam cells. Analysis of published datasets of foam cells showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both data sets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner.

Conclusion:
We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.
Abstract 570: Macrophage Catabolism of Aggregated Lipoproteins Using a Novel Extracellular Compartment Regulates Lipid Accumulation During Atherosclerosis
Authors: Singh Rajesh K , Haka Abigail S , Barbosa-Lorenzi Valeria C , Asmal Arky , Lund Frederik , Xiong Yuquan , Chin Harvey F , Grosheva Inna , Hla Timothy , Maxfield Frederick R
Published: July 2021

Despite impressive advances in research, prevention, and treatment, atherosclerotic vascular disease remains the leading cause of death in the developed world. Mechanisms of cholesterol accumulation in the arteries have been studied intensively, but the
in vivo
contributions of different pathways leading to lipid accumulation and foam cell formation are not understood. In the arteries, low-density lipoprotein (LDL) is aggregated and bound to the extracellular matrix. When such aggregated LDL is presented to macrophages, they form a novel acidic, hydrolytic compartment that is topologically extracellular, to which lysosomal enzymes are secreted. Such compartments are observed
in vivo
in murine atherosclerotic plaque macrophages interacting with cholesterol rich deposits. Using state-of-the-art quantitative and high resolution microscopy techniques, characterization of compartment morphology reveals how macrophages use local actin polymerization to drive plasma membrane remodeling at the interface with aggregated LDL. This leads to sequestration of aggregated LDL into topologically convoluted structures that allow acidification, catabolism and internalization of LDL. We find that a TLR4/MyD88/Syk/PI3 kinase/Akt dependent signaling pathway in macrophages regulates the formation of such catabolic compartments. Consistent with this, deficiency of TLR4
in vivo
can protect macrophages from lipid accumulation in murine atherosclerotic plaques. Herein, we provide compelling evidence for a novel form of catabolism that macrophages use to degrade aggregated LDL
in vivo
during atherosclerosis and this process leads to foam cell formation, cell death and promotes disease progression.
Abstract 577: MicroRNA-30c Reduces Plasma Cholesterol in Different Hypercholesterolemic and Hyperglycemic Mouse Models
Authors: Irani Sara , Iqbal Jahangir , Hussain M.Mahmood
Published: July 2021

High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein assembly and secretion; however, this is associated with steatosis. We have shown that microRNA-30c (miR-30c) reduces Western diet-induced hypercholesterolemia and atherosclerosis in C57BL/6J and
Apoe

-/-

mice with no obvious adverse effects by reducing hepatic lipoprotein production and lipid synthesis. Here, we tested the effect of miR-30c on plasma lipids, transaminases and hepatic lipids in five different mouse models. Hepatic delivery of miR-30c reduced MTP activity but did not affect plasma cholesterol, triglyceride and glucose in chow-fed C57Bl6J and streptozotocin-induced diabetic, normolipidemic mice. However, hepatic delivery of miR-30c to chow fed leptin deficient (
ob/ob
) and leptin receptor deficient (
db/db
) hypercholesterolemic and hyperglycemic type 2 diabetic mice reduced cholesterol in total plasma and VLDL/LDL by ~ 28%and ~ 25%, respectively, without affecting phospholipid, triglyceride and glucose levels. Interestingly, these mice had lower plasma transaminases and creatine kinases indicating possible beneficial effects. Mechanistic studies showed that miR-30c reduced hepatic MTP activity and lipid synthesis. Moreover, miR-30c significantly lowered plasma cholesterol and atherosclerosis in Western-diet fed low density lipoprotein receptor knockout mice with no effect on plasma triglyceride, glucose and transaminases, suggesting that miR-30c can be a potential therapeutic agent for homozygous familial hypercholesterolemia. In all these studies, hepatic lipid levels were similar in control and miR-30c injected mice. These studies indicate that miR-30c reduces plasma cholesterol in diet-induced and diabetic hyperholesterolemic mice but not in normocholesterolemic mice. Thus, miR-30c may be beneficial in lowering plasma cholesterol in different metabolic disorders independent of the origin of hypercholesterolemia.
Abstract 58: Hypercholesterolemia Accelerates the Ageing of Hematopoietic Stem Cells by a Tet1 Dependent Pathway
Authors: Tie Guodong , Yan Jinglian , Khair Lyne , Tutto Amanda , Messina Louis
Published: July 2021

Rationale:
Hypercholesterolemia, a major risk factor for cardiovascular disease, also increases all-cause mortality. Previous work from our lab and others has shown that hypercholesterolemia induces an oxidant stress-dependent accelerated ageing of hematopoietic stem cells (HSCs) that shortens the telomeres of both HSCs and WBCs. However, little is known about the underlying epigenetic mechanism.

Objective:
We found that hypercholesterolemia causes an oxidant stress-dependent downregulation of Tet1 and so we hypothesize that hypercholesterolemia accelerates the ageing of HSCs through a Tet1 dependent epigenetic pathway. Identifying the mechanisms by which hypercholesterolemia accelerates the ageing of HSCs may provide clues to understanding how hypercholesterolemia increases all-cause mortality in divergent clinical studies.

Methods and Results:
RT-PCR showed that the expression of Tet1 was specifically downregulated in HSCs isolated from ApoE
-/-
mice. FACS analysis showed that the frequency of total HSCs was significantly higher in Tet1
-/-
mice than those in WT control, while the long-term and side populations of HSCs in Tet1
-/-
mice were significantly fewer than those in WT mice. The reconstitution capacity of total HSCs in Tet1
-/-
mice was significantly less than that of WT mice. In contrast, long term HSCs from Tet1
-/-
mice showed higher reconstitution capacity than that of long term HSCs from WT mice. Restoration of the Tet1 expression in Tet1
-/-
HSCs effectively restored the LT-HSC population as well as the reconstitution capacity of total HSCs. RT-PCR showed that the expression of p19 and p21 were upregulated in HSCs from Tet1
-/-
mice. DNA pyrosequencing did not show significant change in DNA methylation status in the promoter region of p19 and p21, while ChIP-PCR indicated that Tet1 deficiency facilitated the accumulation of the H3K4m3 modifications of p19 and p21.

Conclusion:
Tet1-dependent epigenetic regulation is critical to maintain a healthy HSC compartment. Hypercholesterolemia induced downregulation of Tet1. Tet1 increased the expression of p19 and p21, and thereby reduces the long-term HSC population, resulting accelerated ageing in HSCs.
Abstract 585: Mathematical Modelling of Monocytes and Monocyte-Derived Cells in Inflammation Shows Cholesterol Biomagnification as a Determinant of Atherosclerotic Plaque Fate
Authors: Ford Hugh
Published: July 2021

Efferocytosis recycles substances, such as cholesterol, from apoptotic monocytes and monocyte-derived macrophages (Mo/Mphi) back into the Mo/Mphi population. Without net removal, these substances will concentrate within successive generations of Mo/Mphis --- a biomagnification process seldom explored in inflammation. We propose that biomagnification of cholesterol within the Mo/Mphi population is an important feature of atherosclerotic plaque progression by gradually enhancing inflammation and necrosis over time.

We have developed a mathematical model of Mo/Mphi in inflammation where a population of Mo/Mphi is modelled with a distribution of internalised lipids in the population. We use this to explore the relationship between cholesterol accumulation and plaque fate. Although the number of Mo/Mphis in the plaque remains stable when the rates of recruitment and proliferation are balanced by apoptosis and egress, lipid dynamics (such as cholesterol bioaccumulation) within this system may change. The model can be used to investigate the conditions when lipids are and are not stably trafficked between Mo/Mphi and apoptotic Mo/Mphi. Destabilisation of lipid dynamics results in a continuous throughput of material from Mo/Mphi and apoptotic Mo/Mphi to the necrotic core. These conditions include the amount of modified LDL relative to HDL, the propensity of Mo/Mphi to replicate and to leave the plaque. Surprisingly Mo/Mphi proliferation provides a highly protective mechanism by halving accumulated cholesterol between two daughter cells and doubling the phagocytic capacity of the parent cell. Overall the technique of mathematical modelling offers novel insights into both plaque pro- and regression. We will conclude with a discussion on how the theory of biomagnification extends to all innate inflammatory systems.
Abstract 586: CD40L on T Cells is a Major Contributor to Atherosclerosis in Hyperlipidemic Mice
Authors: Gerdes Norbert , Buerger Christina , Winkels Holger , Weber Christian , Lutgens Esther
Published: July 2021

Atherosclerosis is a lipid driven chronic inflammatory disease of the arterial wall, involving both innate and adaptive immune responses. Specialized immune cells such as monocytes, B cells, T cells and dendritic cells (DCs) contribute to disease progression or control the inflammatory responses. The CD40-CD40L dyad was identified as an efficient modulator of cellular immune responses. CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is activated by CD40 ligand (CD40L). CD40 and CD40L both are expressed on the majority of immune and non-immune cells associated with atherosclerosis. However, the specific contribution of CD40-CD40L signaling on the different single cell types towards atherosclerosis progression remains undefined. Here, we aimed to investigate the cell type-specific mechanisms of CD40-CD40L interactions in atherosclerosis by generating mice with a conditional ablation of CD40L on T cells. Hyperlipidemic mice with a T cell-specific deficiency of CD40L developed significantly smaller atherosclerotic lesions in the ascending after 28 weeks of chow diet, and following 6 weeks of a cholesterol-enriched diet when compared to their littermate controls. Changes in lesion size were accompanied by a modified anti-inflammatory plaque phenotype, characterized by an increased proportion of smooth muscle cells and a reduced number of pro-inflammatory immune cells, such as macrophages and T cells. T cell CD40L-deficient mice displayed systematically reduced expression of pro-inflammatory cytokines such as IL-1β, IL-2, IL-12, and IFNγ, and increased expression of anti-inflammatory cytokines IL-10 and TGFβ. This anti-inflammatory milieu was paralleled a change in the development and activation status of the T cells with mice lacking CD40L on T cells displaying a reduction in the expression of cytokines and gene markers associated with the activation of T cells (e.g., IL-2, CD69). This change was also reflected within the T cell populations which had a reduced proportion of activated effector T cells and an increased ratio of naïve T cells. Thus, our study ascribes CD40L on T cells a central role in atherosclerosis.
Abstract 587: Absence of Coronary Calcium on Non-contrast Enhanced, Non ECG-gated CT Correlates with Non-obstructive Coronary Artery Disease. A Retrospective Data Analysis of Liver Transplant Recipients
Authors: Gonzalez-Bonilla Hilda M , Thakkar Akanksha , Duran Antonio , Senapati Alpana , Gupta Nakul , Alvarez Paulino , Chang Su Min
Published: July 2021

Background:
Coronary angiography (CAG) remains the gold standard to diagnose coronary artery disease (CAD). However, it is associated with multiple risks and its utility is not well defined in the liver transplant population. Alternatives to evaluate for CAD such as coronary artery calcium score (CACS) are being increasingly investigated.

Hypothesis:
To determine if the absence of coronary arterial calcium (CACS=0) on non-contrast, non-ECG gated chest CT scan can exclude obstructive CAD in liver transplant patients.

Methods:
We performed a retrospective analysis of data collected from liver transplant recipients. We included patients who had a CT chest without contrast and CAG less than one year apart. Agatston score was derived from non-IV contrast, non-ECG gated chest CT’s utilizing the syngo.via platform (Siemens Healthcare). CACS was compared against CAG. Patients with coronary stents were excluded. We determined NPV, PPV, sensitivity and specificity of using CACS = 0 as predictor of the absence of obstructive CAD.

Results:
Mean age at date of transplant was 59.03 and males accounted for 68.8% of our population. The negative predictive value for CACS=0 as a predictor of non-obstructive CAD was 100%. Positive predictive value for CACS≥1 was 6.8%. Sensitivity and specificity for the correlation between CACS and CAD were 100% and 33% respectively (Figure 1). CACS was stratified into four subgroups based severity, and we found that all patients with obstructive CAD had scores >400 (Figure 2).

Conclusion:
The absence of coronary arterial calcium (CACS=0) on non-contrast, non ECG gated chest CT has a high negative predictive value and can exclude the presence of obstructive CAD.
Abstract 598: Carotid Artery Ultrasound Texture, Cardiovascular Risk Factors, and Subclinical Arterial Disease: The Multi-Ethnic Study of Atherosclerosis (Mesa)
Authors: Mitchell Carol , Korcarz Claudia E , Tattersall Matthew , Gepner Adam D , Post Wendy S , Kaufman Joel D , McClelland Robyn L , Stein James H
Published: July 2021

Background:
Ultrasound texture “contrast” can be used to characterize the arterial wall non-invasively. Texture contrast provides information about the distribution and gray level differences of the pixels in the arterial wall and may describe early changes related to arterial injury. We determined if ultrasound texture contrast was associated with cardiovascular disease (CVD) risk factors and subclinical arterial disease.

Methods:
We evaluated ultrasound images of the distal right common carotid artery from a convenience sample of 151 participants from the first examination of the Multi-Ethnic Study of Atherosclerosis, a population-based cohort of individuals without clinical CVD. Images were digitized, normalized, and standardized to a pixel density of 20/mm. Plaque texture analysis software (LifeQ Medical, Cyprus) used the gray level difference statistics method to determine the contrast of the far wall of the carotid intima-media complex. Multivariable linear regression models (adjusted for age, sex and race/ethnicity) were used to examine relationships between contrast, CVD risk factors (age, BMI, total cholesterol, high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol, triglycerides, hypertension, diabetes mellitus, smoking, glomerular filtration rate, C-reactive protein, interleukin-6, D-dimer, fibrinogen, alcohol consumption, education level, physical activity level, statin use), carotid intima-media thickness (IMT), coronary artery calcium (CAC), and CVD risk score.

Results:
The 151 participants were mean (standard deviation) 68.1 (9.2) years old (54% female; 31% Hispanic, 28% Black, 10% Chinese, and 31% White). In models that included age, sex, and race, contrast was associated independently with age (beta [standard error] -0.9 [0.4] per year; p=0.02), HDL-C (0.6 [0.2] per mg/dL; p=0.02), C-reactive protein (-2.3 [0.9] per mg/L; p=0.02), and carotid IMT (-1.3 [0.4] microns; p=0.001). Other CVD risk factors and CAC were not associated independently with contrast.

Conclusions:
Lower contrast, an ultrasound texture feature, is associated with increasing age, lower HDL-C, higher CRP, and higher carotid IMT, supporting its potential use for evaluating arterial injury and CVD risk.
Abstract 612: High Dimensional Single-Cell Intracellular Signaling Identifies CCL5 as a Potential Key Driver of Myeloid Cell Reprogramming in Atherosclerotic Patients
Authors: Rahman Adeeb , Amadori Letizia , Kim-Schulze Seunghee , Mury Pauline , Moss Noah , Li Jennifer , Pina Christian , Faries Peter , Merad Miriam , Giannarelli Chiara
Published: July 2021

Atherosclerosis is a disease characterized by chronic inflammation. However, identifying new molecularly targeted interventions in human atherosclerosis has proven difficult. In this study, we used phosphoCyTOF to comprehensively characterize functional responses of peripheral immune populations in human atherosclerosis at the single-cell level. Healthy PBMCs were exposed to plasma of patients with atherosclerosis (n=20) or healthy donors (n=10) and the activation of major signaling pathways across all major immune subsets using 18 surface markers and 10 intracellular phospho-proteins. Using viSNE we visualized and defined 10 major immune cell populations: B cells, Basophils, CD1c DCs, CD4 T cells, CD8 T cells, CD14 and CD16 monocytes, NK cells, NKT cells, and pDCs. Next, we evaluated the relative expression of 10 phospho-proteins (IkBa, pCREB, pERK1/2, pMAPKAP2, pp38, pPLCg2, pS6, pSTAT1, pSTAT3, and pSTAT5) across this immunological map and visualized functional pathways that were differentially induced in response to plasma of atherosclerotic patients vs. healthy donors. Monocytes exhibited the greatest immune activation, showing increased phosphorylation of p38, MAPKAP2, ERK1/2, CREB and S6 in response to atherosclerotic plasma. To identify plasma cytokines responsible for this effect, we measured a panel of 41 cytokines and identified CCL5, CXCL10 (IP-10), CXCL1 (GRO), PDGF-AA and PDGF-BB as the most differentially expressed in atherosclerotic vs. healthy plasma. PhosphoCyTOF analysis showed that only CCL5 reproduced the phosphorylation pattern induced by atherosclerotic plasma in monocytes, an effect that was completely inhibited by a CCL5 blocking antibody (Fig. 1). These results suggest that CCL5 in plasma of patients with atherosclerotic disease drives specific innate immune cell signaling functions that mark the inflammatory response in atherosclerotic disease.
Abstract 614: Shear Stress-Induced Glycolytic Metabolites Promote Vascular Repair
Authors: Baek Kyung In , Li Rongsong , Liem David , Beebe Tyler , Ma Jianguo , Choi Howard , Jen Nelson , Yen Hilary , Ping Peipei , Hsiai Tzung K
Published: July 2021

Introduction:
Hemodynamic shear stress is intimately linked with transcriptomic and epigenomic changes to maintain endothelial homeostasis. Metabolomics studies have led to emergent metabolic biomarkers and therapeutic targets. Whether shear stress modulates metabolomic pathway to promote vascular repair remains to be investigated.

Hypothesis:
We hypothesized that shear stress regulates VEGF receptor-PKCε-PFKFB3 signaling-mediated glycolytic metabolites to promote vascular repair.

Method and Results:
Both pulsatile (PSS: 23 ± 8 dyn·cm
-2
at 1 Hz) and oscillatory shear stress (OSS: 0.1 ± 3 dyn·cm
-2
at 1 Hz) up-regulated PKCε expressions and the activity (*
P
< 0.05,
n
=3), whereas silencing VEGFR2 with siRNA, or treating with VEGFR inhibitor, Cediranib, attenuated shear stress-mediated PKCε expression in human aortic endothelial cells(HAEC). Constitutively active (CA)-PKCε adenovirus infection enhanced tube formation assessed by Matrigel as well as significantly increased PFKFB3 expressions promoting glycolysis, whereas the dominant negative(DN) PKCε resulted in opposite effects. Co-localization of PKCε and PFKFB3 expression was demonstrated in the endothelium of aortic arch and thoracic aorta in a New Zealand White rabbit model. In the zebrafish tail amputation model, reduction of shear stress via
GATA-1a
morpholino oligonucleotide(MO) injection and inhibition of PKCε expression via PKCε MO impaired vascular repair between the dorsal aorta and the dorsal longitudinal anastomotic vessel at 3 days post amputation(dpa). PKCε mRNA rescued
GATA-1a
MO-mediated impairment of vascular repair (*
P
< 0.01,
n
=20, **
P
< 0.05,
n
=5). Metabolomic analysis in HAEC applied to PSS and OSS revealed modulation of a number of metabolites including increased glycolytic metabolite dihydroxyacetone, which was blocked by PKCε siRNA. Treatment with dihydroxyacetone rescued PKCε-impaired vascular repair.

Conclusion:
In conclusion, shear stress-mediated VEGFR-PKCε-PFKFB3 signaling increased glycolytic metabolites to mediate vascular repair.
Abstract 620: Circulating miRNAs a Novel Tool to Assess BAG3 Related Dilated Cardiomyopathy
Authors: Toro Rocio , Saura Marta , De Gonzalo David , Llorente-Cortes Vicenta , Rosa-Longobardo Felix , Mangas Alipio , Zamorano Jose Luis , Zaragoza Carlos
Published: July 2021

Background:
A new familial dilated cardiomyopathy (DCM) was recently found related to mutations in the antiapoptotic BAG3 gene. MicroRNAs (miRNAs) are short non-coding RNAs playing significant roles in cardiac disease, including DCM, thus representing new potential targets of treatment. However, no previous study has evaluated the clinical association between BAG3-related DCM and circulating miRNAs.

Purpose:
We aimed to evaluate whether the clinical association between BAG3-related familial DCM and the circulating miRNA profile may represent a new tool for the diagnosis and progression assessment of the disease.

Methods:
Detailed clinical and echocardiographic information was obtained from 21 patients with familial DCM carrying the BAG3 mutation and 21 age-matched healthy subjects. RNA was isolated from peripheral blood and analysed using ultrasequencing. Bioinformatic analysis was performed to explore the potential molecular pathways related to the miRNA profile.

Results:
To determine the miRNA profile in BAG3-associated DCM, the analysis of 1759 circulating miRNA was performed in symptomatic and asymptomatic patients with BAG3 mutation, and compared to healthy age-matched subjects. The expression profiles showed significant differences between controls and BAG3 mutation carriers: miRNAs 3191-3p, 6769b-3p, 1249-ep, 154-5p, 6855-5p, and 182-5p were at least 2-fold downregulated in patients compared to healthy subjects. Endogenous gene targets of these miRNAs are now under investigation, highlighting miR-182-5p,and its target Ankyrin G.

Conclusions:
miRNAs emerge as a novel tool to differentiate healthy subjects and patients with BAG3-related DCM. Of particular interest is the downstream analysis of endogenous miRNA targets, the Ankyrin G gene. Further investigation regarding the contribution of Ankyrin G and other target genes of the miRNA profile described in BAG3-related DCM will be a key step to deeply understand the contribution of miRNAs in the pathophysiology of familial DCM.
Abstract 626: Apabetalone (Rvx-208), a Selective Bet Protein Inhibitor, Reduces Expression of Acute Phase Response Markers
in vitro
and in Patients With Cardiovascular Disease and Chronic Kidney Disease
Authors: Kulikowski Ewelina , Wasiak Sylwia , Gilham Dean , Tsujikawa Laura , Halliday Christopher , Jahagirdar Ravi , Sweeney Mike , Johansson Jan , Wong Norm
Published: July 2021

Apabetalone is an inhibitor of the epigenetic readers bromodomain and extraterminal (BET) proteins, currently in a phase 3 outcomes trial in patients with cardiovascular disease (CVD) and diabetes mellitus. A post hoc analysis of phase 2b trials demonstrated a 55% relative risk reduction in major adverse cardiac events in CVD patients. Elevated inflammatory markers correlate with CVD. Inflammation also accompanies chronic kidney disease (CKD) and CKD patients are at risk of CVD. Previous research has shown that apabetalone modulates pathways that contribute to chronic inflammation, including the acute phase response (APR). Here, pathway analysis of gene microarrays showed downregulation of APR by apabetalone in primary human hepatocytes (PHH). Real-time PCR and ELISA analysis of RVX-208 treated PHHs confirmed that APR genes that correlate with CVD are suppressed by 20 to 95%, including CRP, ceruloplasmin (CP), serum amyloid P (SAP), PAI-1, alpha 2-macroglobulin (A2M), complement C2, C3 and C5, MBL2, serum amyloid A and interleukin 18. Apabetalone decreased IL-6-induced expression of CP, SAP and A2M, with most striking effects on CRP (-75%). Apabetalone also decreased LPS-induced expression of SAP in a mouse endotoxemia model. To assess effects of apabetalone on inflammatory mediators in CVD patients, SOMAscan™ 1.3K proteomic analysis was performed on plasma from phase 2b ASSERT (12 weeks; n=25) and ASSURE (26 weeks; n=47) clinical trials. This approach identified APR as the top downregulated pathway by apabetalone in both trials. APR biomarkers are elevated in CKD patients where they correlate with disease progression. To gain insight into the pharmacodynamics of the APR response to apabetalone, stage 4 CKD patients (n=8) received a single dose of the drug followed by plasma proteomics at several time points. At 12h post dose, APR was significantly downregulated by apabetalone. Of note, CRP was decreased in CKD patients after 12h of treatment (-7%, p=0.04) versus baseline, as well as in ASSERT (-43%, p=0.01) and ASSURE (-21%, p=0.02) trials versus placebo. Downregulation of the APR pathway by apabetalone may lead to reduced chronic inflammation in CVD and CKD patients and contribute to the reduction in MACE in patients with high residual CVD risk.