bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 230: Stimulation of Cholesterol Efflux from Human Macrophage by Liver X Receptor Agonists is a 2-Step Mechanism Requiring ARL7 and ABCA1
Authors: Superville Alexandre , Frisdal Eric , Quinn Carmel M , Kim Mi-Jurng , Jessup Wendy , Lesnik Philippe , Guérin Maryse , Le Goff Wilfried 
Published: March 2022

Nuclear Liver X Receptors activation by synthetic agonists was proven to be atheroprotective in mice; an effect likely based on stimulation of cellular cholesterol efflux from arterial macrophages. However, mechanisms involved in free cholesterol efflux from mouse macrophages appear distinct from those operating in human macrophages.
The objective of this study was to decipher precise cellular mechanisms controlling free cholesterol efflux from human macrophages upon LXR stimulation.
In THP-1 and human monocyte-derived macrophages (HMDM), treatment with the LXR agonist GW3965 efficiently induced ARL7 expression (6-fold, p<0.05), an effect associated with an increased amount of plasma membrane free cholesterol available for efflux (+25%, p<0.05) and a higher lipid rafts formation (+10%, p<0.05). Both effects were abolished in ARL7 Knockdown (KD) macrophages, leading to a lack of stimulation of cholesterol efflux by GW3965.
Specific targeting of each LXR isoforms, LXRα and LXRβ, by RNAi revealed that LXRα silencing in THP-1 and HMDM reduced significantly expression of cholesterol transporters ABCA1, ABCG1 and receptor SR-BI/Cla-1 mRNA levels, as well as free cholesterol efflux to apoA1 (-30%, p<0.05) and to HDL (-20%, p<0.05) upon stimulation with LXR, whereas LXRβ silencing has no impact. Interestingly, stimulation of cholesterol efflux to HDL by GW3965 was significantly reduced (-50%, p<0.05) in ABCA1 KD THP-1 macrophages; those cells being incapable to promote cholesterol efflux to apoA1. However, silencing of ABCG1 or SR-B1/Cla-1 had no impact on cholesterol efflux to HDL from either control or ABCA1 KD THP-1 macrophages treated or not with LXR agonist. By contrast stimulation of cholesterol efflux to HDL by GW3965 was completely abolished in LXRα/ABCA1 double KD macrophages, highlighting the major contribution of ABCA1 in cholesterol efflux from human macrophage.
We conclude that LXR-mediated stimulation of cholesterol efflux from human macrophages is a two-steps mechanism. First, LXR activation promotes ARL7-dependent free cholesterol transport to plasma membrane, mostly in lipid raft domains. Then, membrane free cholesterol is exported to apoA1 and HDL acceptors through ABCA1; this latter step being controlled selectively by LXRα.
Abstract 246: Anacetrapib Adds to the Antiatherogenic Effect of Atorvastatin, Mainly by Increasing the Clearance of Non-HDL Cholesterol
Authors: van der Tuin Sam J , Kuhnast Susan , Van der Hoorn José W , van Klinken Jan B , Simic Branko , Pieterman Elsbet J , Havekes Louis M , Landmesser Ulf , Luscher Thomas F , Willems-van Dijk Ko , Rensen Patrick C , Jukema J W , Princen Hans M 
Published: March 2022

Introduction:
The residual risk that remains after statin treatment has stimulated the search for secondary treatment targets. Epidemiological studies propose HDL-C as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. The CETP inhibitor anacetrapib decreases (V)LDL-C by ~15-40% and increases HDL-C by ~40-140% in clinical trials.

We evaluated the effects of anacetrapib on lipid metabolism, HDL function and atherosclerosis, and examined possible additive/synergistic effects of anacetrapib on top of atorvastatin in APOE*3Leiden.CETP mice.

Methods and results:
Mice were fed a diet without or with ascending dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/d), atorvastatin (2.4 mg/kg/d) alone or in combination with anacetrapib (0.3 mg/kg/d) for 21 weeks.

Anacetrapib dose-dependently reduced CETP activity (-59% to -100%, P<0.01), thereby decreasing nonHDL-C (-24% to -45%, P<0.001) and increasing HDL-C (+30% to +86%, P<0.001). Anacetrapib did not affect HDL function. Additionally, we showed that anacetrapib reduced nonHDL-C mainly by increasing the clearance by the liver.
Anacetrapib dose-dependently reduced atherosclerotic lesion area (-41% to -92%, P<0.01) and severity, increased the plaque stability index and added to the effects of atorvastatin by further decreasing lesion size (-95%, P<0.001) and severity. Analysis of covariance showed that both anacetrapib (P<0.05) and nonHDL-C (P<0.001), but not HDL-C (P=0.76), independently decreased lesion size.

Conclusions:
Anacetrapib reduces atherosclerosis, and adds to the anti-atherogenic effect of atorvastatin. The anti-atherogenic effect is mainly ascribed to a reduction in nonHDL-C, as a consequence of an increased clearance. In addition, anacetrapib improves lesion stability.
Abstract 302: Generation of Patient-Specific Tissue-Engineered Blood Vessels From Nonintegrated Induced Pluripotent Stem Cells
Authors: Wang Yongyu , Hu Jiang , Jiao Jiao , Chen Y Eugene , Ma Peter , Yang Bo 
Published: March 2022

Cell resourcing remains one of the major problems in the development of patient-specific TEBVs. The iPSC derived cardiovascular cells provide a promising source for TEBVs and cell therapy. The goal of this study is to construct TEBVs using patient-specific iPSCs.
Using a non-integrated episomal vector based reprogramming system, we generated human iPSCs from peripheral blood mononuclear cells (PBMCs) isolated from the blood harvested from aneurysm patients with bicuspid aortic valves and coronary artery bypass patients with normal aortas and aortic valves. These iPSCs showed the expression of pluripotent markers and could differentiate into 3 layers of cells in vitro. We directed the iPSCs differentiation into vascular smooth muscle cells (VSMCs) in vitro. We documented the expression of VSMC markers, including smooth muscle α-actin (α-SMA), calponin and SM22α by quantitative real-time-PCR, immunocytochemistry, as well as Western-blotting. Furthermore, we confirmed that the iPSC-derived VSMCs could contract in response to carbachol, a muscarinic agonist. The treatment of iPSC-derived VSMCs with IL-1β, an atherogenic cytokine, changed MMPs (matrix metallopeptidases) activity. Finally, we found that iPSC-derived VSMCs could survive well and secrete collagen when transferred and cultured on PLLA scaffolds in vitro, suggesting a great cell sourcing for tissue-engineered blood vessels.
Our data suggests that by using non-integrated iPSCs, we could efficiently differentiate patient-specific iPSCs into functional VSMCs, which may provide great cell sourcing for patient-specific TEBVs.
Abstract 461: Compositional Changes to High-Density Lipoproteins That Are Related to Increased Cardiocascular Disease Burden
Authors: McEneny Jane , McKavanagh Peter , York Edmund , Nadeem Nida , Lusk Lisa , Ball P , Trinick Tom , Stevenson Michael , Young Ian , Donnelly Patrick 
Published: March 2022

Atherosclerosis is a vascular disease characterized by the build up of lipid derived plaques, and an individual’s risk of acute coronary syndrome is related to plaque burden and composition. Multiple risk markers for atherosclerosis and CVD act in a synergistic way through inflammatory pathways. One such inflammatory marker is serum amyloid A (SAA), which associates with HDL, negating its antiatherogeinc properties, which may have a causal role in atherosclerosis development.
This study examined if SAA was related to CVD burden, and if this influenced the antiatherogenic properties of HDL.

Subjects (n=240) referred to the rapid chest pain clinic at the Ulster Hospital UK, had atherosclerotic burden assessed by cardiac computerised tomography (CCT) and were classified as no CVD; mild CVD stenosis <50% and moderate/severe CVD stenosis >50%. HDL
2
and HDL
3
were isolated from serum by rapid ultracentrifugation. SAA was measured by an ELISA procedure, lipids by a colorimetric method and CETP activity by a fluorimetric assay.

Results:
Although lipids were similar in HDL
2
across the groups, lipids decreased in HDL
3
with increasing CVD burden. In addition, the concentration of SAA and the activity of CETP increased with increasing CVD burden (see table). Additionally, ordinal regression analysis illustrated that HDL
3
-SAA and HDL
2
-CETP where independently related to CVD burden (p=0.001).

Conclusions:
This study has shown that SAA was associated with increased CVD burden and that HDL had altered lipid composition and increased CETP activity. Thus, SAA and CETP may be useful biomarkers for the detection and assessment of CVD.

Funded by Heart-Research UK
Abstract 463: The Effect of Intrauterine Inflammation on the Early Development of Atherosclerosis
Authors: Nguyen Maria , Wallace Megan , Menheniott Trevelyan , Pepe Salvatore , Gaspari Tracey , Widdop Robert , Moss Timothy , Burgner David 
Published: March 2022

Atherosclerosis is a chronic condition that may begin prior to birth, particularly in the presence of factors that act through inflammatory pathways. Inflammation may alter epigenetic marks, and changes in methylation are observed in atherosclerotic lesions. We hypothesised that intrauterine inflammation (chorioamnionitis, which complicates 1-4% of all births in the US) would alter development of early atherosclerosis. We report preliminary findings from a novel mouse model of chorioamnionitis and atherosclerosis.
We aim to investigate:
1. the effect of intrauterine inflammation on the development of early atherosclerosis in mice
2. molecular and epigenetic mechanisms

Methods:
Apolipoprotein E deficient pregnant mice (n=15-19 per group) received LPS (0.1 ng in 5 μl saline) or saline (5 μl) into each amniotic sac on gestational day 15. Offspring were weaned onto a high fat (21% v/v) diet at 4 weeks. Aortae, heart and plasma were collected from 6- and 12-week-old offspring. Aortae were analysed following Haematoxylin and Eosin (H&E) and Oil Red O staining. Heart and aortae cholesterol concentrations were quantified using colorimetric assay. Gene expression (Real-Time PCR) of candidate genes was measured.

Results:
Intra-amniotic LPS injection induced histological chorioamnionitis. In 6-week-old mice, intrauterine inflammation increased cholesterol concentrations in aorta and cardiac tissue (p<0.05), and increased aortic Dnmt1 mRNA levels (p=0.02; a key regulator of DNA methylation). No differences between LPS- and saline-treated animals were found in H&E stained aortic sinus. In 12-week-old mice, reduced mean intimal area (p=0.02) and mean intimal thickness index (p=0.02) were found in H&E stained brachiocephalic artery sections from LPS-treated offspring. No differences were found between groups in H&E stained aortic sinus or in an en face analysis of Oil Red O-stained aorta.

Conclusions:
Exposure to intrauterine inflammation has complex effects in this model. Contrary to our hypothesis, 12-week-old LPS-treated mice showed decreased intimal area with no other apparent histological differences, however early cholesterol and molecular biology changes exist. Differential epigenetic regulation may be an early underlying mechanism.
Abstract 475: Circulating Immunoglobulins Are Not Associated With Intraplaque Mast Cell Number and Other Vulnerable Plaque Characteristics in Patients With Carotid Artery Stenosis
Authors: van der Velden Daniël , Willems Sanne , Quax P H , de Borst G J , de Vries J.P.P.M. , Moll F L , Kuiper J , Toes R E , de Jager S C , de Kleijn D P , Hoefer I E , Pasterkamp G , Bot I 
Published: March 2022

Background:
Atherosclerosis is a chronic systemic inflammatory disease in which many immune cells participate. Recently we have shown that mast cell numbers are associated with atherosclerotic plaque vulnerability and interestingly with future cardiovascular events (S. Willems et.al. 2013). The endogenous trigger that activates mast cells within the lesion remains unidentified. We hypothesized that mast cells are activated via receptors for IgG and/or IgE.

In this study we aimed to establish whether specific immunoglobulin levels in plasma of atherosclerotic patients were related to intraplaque mast cell numbers. In addition, the levels were related to other vulnerable plaque characteristics and baseline clinical data.

Results:
Serum levels of total IgG, total IgE and oxLDL specific IgG were determined in 135 patients that underwent carotid endarterectomy. The studied patient population with a mean age of 67 and a male prevalence (71%) reflects a relatively typical population of patients with vascular occlusive diseases. The majority of patients were symptomatic (74%) as illustrated by the incidence of amaurosis fugax, a TIA or a stroke.

No associations were observed between either the serum immunoglobulin levels or the total mast cell numbers in atherosclerotic plaques (total IgG vs. total mast cells r= -0,038, p=0.664) (tIgE vs total mast cells r=-0.038, p=0.664) (oxLDL-IgG vs total mast cells r=0.137 p=0.114). Furthermore, no associations were found between immunoglobulin levels and the following plaque characteristics: lipid core size, degree of calcification, number of macrophages or smooth muscle cells, amount of collagen and number of microvessels.
Interestingly, we observed a negative association between statin use and total IgE (97.5 [43.1-276.8] vs. 157.3 [75.2-545.4] ng/mL, p=0.012) and statin use and oxLDL-IgG (288 [224-406] vs. 399 [282-584] mU/mL, p=0.004).

Conclusions:
In patients suffering from carotid artery disease, total IgG or IgE and oxLDL-IgG levels do not associate with plaque mast cell numbers or vulnerable plaque histopathological characteristics These data may indicate that other endogenous factors activate mast cells within the atherosclerotic plaque.
Abstract 603: O-GlcNAcylation of Runx2 is Essential for Vascular Calcification
Authors: Heath Jack , Mao Xia , Huang Lu , Sun Yong , Deng Liang , Chen Yabing 
Published: March 2022

Osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to vascular calcification, a characteristic of advanced atherosclerosis, chronic kidney disease, and diabetes. Using Runx2 shRNA and SMC-specific Runx2 deletion mice, we have demonstrated an essential role of Runx2 in regulating vascular calcification in vitro and in vivo. The present studies aim to pinpoint the Runx2 functional domain that is critical for its osteogenic function in VSMC and to elucidate the underlying molecular mechanisms.
A series of Runx2 deletion mutants were generated and stably transfected into VSMC. Full-length Runx2 protein and Runx2 deletion mutants containing amino acids 1-495 or 1-432, but not Runx2 1-391, induced VMC calcification, indicating that the Runx2 osteogenic functional domain is located between amino acids 391-432. Site-directed mutagenesis analysis identified that mutations at the amino acids 412, 413, 425 and 426-428 markedly reduced Runx2 transactivity and inhibited Runx2-induced VSMC calcification. Mutations at amino acids 412, 413 and 425 were found to inhibit Runx2 post-translational modification by O-GlcNAcylation. Immunoprecipitation revealed that Runx2 O-GlcNAcylation at 412, at 413, and proximal to 425 was critical for its binding to Smad 1/5/8 and Smad 4, but not Smad 2/3. Using lentivirus-mediated shRNA, we determined that knockdown of Smad1/5/8, but not Smad 2/3, inhibited Runx2-induced VSMC calcification, suggesting that BMP but not TGF-beta signaling is critical for the osteogenic function of Runx2 in VSMC. Taken together, these data demonstrate that Runx2 amino acids 412, 413, 425 and 426-428 are key for its osteogenic function in VSMC, and Runx2 O-GlcNAcylation at 412, 413 and 425 is essential for its interaction with Smad proteins to determine Runx2 transactivity and VSMC calcification.
We have identified a novel Runx2 post-translational modification by O-GlcNAcylation at key residues that are essential for its osteogenic function. Our studies provide molecular insights into the function of Runx2 in regulating VSMC calcification, which may shed light on novel targets that are amenable to drug discovery for vascular calcification.
Abstract 623: Robust and High-Throughput Cholesterol Efflux Assay
Authors: Dixit Mallikarjun N 
Published: March 2022

Background:
The objective of this study was to develop a robust high-throughput assay for ABCA1 specific cholesterol efflux supporting its use in large clinical trials, which require consistent robust performance and cost-efficiency.

Methods:
J774 cells were seeded on a 48-well plate and pulsed with
3
H Cholesterol in a media with ACAT inhibitor. ABCA1 expression was induced by 8-bromo cAMP. Test serum (HDL) isolated by PEG precipitation was added to each well and efflux capacity determined as the ratio of radioactive counts in growth medium versus total counts when comparing cells expressing ABCA1 with those not expressing this transporter. Precision, linearity, limit of detection, specificity, reference range, robustness, and short-and long term stability were assessed.

Results:
The overall intra-assay precision varied from 8.5% to 12%, whereas the inter-assay precision was about 20% based on fifteen individual runs on different days by three different analysts. The linear range of the assay was 0.81%-6.5% and 0.43%-13% cholesterol efflux (CE) for PEGylated and non-PEGylated serum, respectively. The LOD was 1.08% CE based on mean of negative control from all 16 runs plus 3 SD. Specificity was determined as the relative increase (11.3 fold) of cholesterol efflux by ApoA1 between cAMP and non-cAMP treated cells. Verification of reference range was determined as 2.1%-7.0% CE (mean±2SD). Consistency of the assay determined by %CV was not affected when different lots of cAMP (CV:6.4%) or ApoA1 (10.3%) were used. The CE values were not appreciably affected when identical samples were run by different analysts (CV:3.2%). Based on average of three data sets samples were stable for up to three days at 2-8°C (mean difference 9.6%), 10 days at -20°C (7.8%), and 7 months at -80°C (8.1%). About 3,000 samples were run over 4 weeks equaling 750 samples /week in a high-throughput format with consistent performance as assessed by each of the five QCs run in each run.

In conclusion, the CE assay described herein is the first high-throughput and robust assay fit for use in large clinical trials where consistent performance during the development of a drug program is critical.
Confidential
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Abstract 281: Local MicroRNA Modulation Using a Novel Anti-mir-21-eluting Stent Effectively Prevents In-stent Restenosis
Authors: Chernogubova Ekaterina , Wang Dong , Deuse Tobias , Stubbendorff Mandy , Erben Reinhold G , Eken Suzanne M , Jin Hong , Heeger Christian , Behnisch Boris , Reichenpurner Hermann , Robbins Robert C , Spin Joshua M , Tsao Philip S , Schrepfer Sonja , Maegdefessel Lars 
Published: March 2022

The major limitation of vascular angioplasty remains the occurrence of in-stent restenosis (ISR). The central role of miRNAs in the pathophysiology of the proliferative response in cardiovascular disease offers attractive concepts for possible therapeutic interventions, with systemic miRNA modulation however potentially causing substantial off-target effects. Aim of the current study was to test the feasibility of local miRNA modulation using drug eluted stent (DES) technology.
Utilizing human ISR tissue samples, as well as a humanized rodent model of balloon-injured human internal mammary arteries with subsequent stent implantation in RNU rats, we were able to identify several miRNAs dys-regulated, including the well-characterized miR-21. We inhibited miR-21 (with a FAM-tagged-LNA-anti-miR-21), using either systemic or local delivery via DES.
Systemic suppression of miR-21 showed a dose-dependent reduction of luminal obliteration, while not impeding vascular re-endothelialization. However, miR-21 expression in liver, heart, lung, and kidney appeared significantly reduced, with increased levels of serum creatinine, pointing towards the expected off-target effects of systemic miRNA modulation. Local mir-21 suppression, using the anti-miR-21-DES, effectively reduced myointimal hyperplasia and ISR compared to bare metal stents, while not exerting detectable off-target effects on any other organ we investigated. Additional in vitro experiments in cultured human coronary artery smooth muscle and endothelial cells further confirmed the ability of anti_miR-21 to limit SMC proliferation and migration by repressing mRNA and protein levels of its established target PTEN, while not affecting the proliferative response in the ECs.
This is the first experimental study to demonstrate the efficacy, feasibility and suitability of an anti-miRNA-eluting stent (anti-miR-21-DES) for the reduction of ISR through dominant inhibition of SMC proliferation.
Abstract 333: Protective Role of Gut Microbiota
Akkermansia municiphila
Against Development of Atherosclerosis in Mice
Authors: LI Jin , Woo Connie , Xu Aimin 
Published: March 2022

Gut microbiota is involved in both onset and progression of obesity. Obesity is associated with several cardiovascular diseases, among which atherosclerosis is the leading cause of morbidity and mortality. However, the role of gut microbiota in obesity-related cardiovascular complication remains to be explored. We found that the fecal amount of Akkermansia municiphila (Akk), a beneficial bacterium, decreased in apolipoprotein E-deficient (apoE -/-) mice fed a Western diet. In this study, we investigated whether Akk may exert beneficial role against the development of atherosclerosis.
ApoE -/- mice were oral gavaged with either PBS or Akk daily along with feeding a Western diet for 8 weeks. Atherosclerotic lesions in these mice were analyzed by H&E staining and Oil Red O staining. Local and systemic expressions of inflammatory molecules including MCP-1, ICAM, VCAM, IL-1beta and TNF-alpha were evaluated.
Western diet substantially altered gut microbiota pattern in apoE -/- mice. However, treatment of Akk, without changing the gut microbiota composition, significantly reduced atherosclerotic lesion. Although decreased body weight and fat mass were observed in Akk treatment group, the changing levels were physiologically insignificant. Serum total cholesterol, triglyceride, HDL and LDL levels were not affected by Akk treatment. However, local expression levels of MCP-1, ICAM, VCAM and TNF-alpha along with macrophage infiltration at the lesions were diminished by Akk. Furthermore, Akk treatment ameliorated systemic inflammatory status by decreasing circulating MCP-1 and IL1-beta levels. It appeared that Akk treatment was able to decrease gut permeability by inducing the expression levels of tight junction proteins, ZO-1 and occludin. Akk-induced reduction of gut permeability led to elimination of bacterial products penetration and serum LPS level was decreased. We speculated that the protective effect of Akk against atherosclerosis was mediated by decreasing gut permeability and LPS level in circulation.
Collectively, our results demonstrated that Akk can decrease atherosclerotic lesions by ameliorating local and systemic inflammation. Probiotic Akkermansia municiphila may be a potential therapeutic target for atherosclerosis.