bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 557: Gene Polymorphisms in IL-10, INF-γ and TGF-β are Associated with Early-Onset Coronary Artery Disease and Microvascular Function.
Authors: Faffe Débora S , De Lorenzo Andrea , Coutinho Jorge Luiz A , Souza Elaine G , Oliveira Glaucia M , Tibiriça Eduardo , Rondinelli Edson
Published: March 2022

The influence of genetics on atherosclerosis development remains unclear. Association of single-nucleotide polymorphisms (SNPs) with early-onset coronary artery disease (EOCAD), as well as with carotid intima-media thickness (CIMT), and systemic microvascular reactivity was examined in 52 patients with EOCAD (45 ±3 years old) and 35 age-matched controls. SNPs in ACE, TNF-α (-308G/A, -238 G/A), IFN-γ (+874 A/T), MMP-9 (-1562 C/T), IL-10 (-1082 A/G, -819 C/T, 592 C/A), NOS3 (894 G/T), ApoA1 (rs964184), and TGF-β (codon 10 T/C) genes were genotyped by automatic sequencing. CIMT was determined by ultrasound and capillary density by intravital video-microscopy. Microvascular reactivity was evaluated using laser speckle contrast imaging, cutaneous blood flow was assessed at rest and during post-occlusive reactive hyperemia (PORH), and acetylcholine (Ach) or sodium nitroprusside (SNP) iontophoresis. Genotype distribution was: ACE: 31 DD, 12 II, 43 DI; TNF-α: 56 GG, 20 GA, 3 AA at position -308, and 76 GG, 3 GA at position -238; IFN-γ: 8 subjects with high producer TT genotype; MMP-9: 62 CC, 23 CT; IL-10: 8 high producer haplotype, 35 intermediate, and 43 low; NOS3: 41 GG, 34 GT, 11 TT; ApoA1: 47 CC, 29 CG, 10 GG; TGF-β (cd 25): 75 GG, 10 GC; TGF-β (cd 10): 66 high producer genotype, 19 low producer. IFN-γ, IL-10 haplotype, and TGF-β were significantly related to EOCAD. CIMT was increased (0.93 ±0.02 mm, p<0.0001), while capillary density (87 ±3 capillaries/mm2, p=0.029) was reduced in EOCAD. CC genotype of TGF-β showed higher CIMT (p=0.004) and lower capillary density (p=0.019) than TT. High IL-10 haplotype reduced vasodilator response after PORH (0.32 APU/mmHg, p=0.0316), while high IFN-γ genotype reduced the response induced by both Ach (0.19 APU/mmHg) and SNP (0.13 APU/mmHg) in relation to low producer (0.44, 0.31, and 0.24 APU/mmHg, respectively). TT genotype of TGF-β showed lower vasodilator response after SNP than CC. In conclusion, IFN-γ, IL-10 and TGF-β genotypes were associated with EOCAD, as well as with systemic microvascular response, CIMT, and capillary rarefaction. The results suggest that IFN-γ, IL-10 and TGF-β genotypes could influence EOCAD, affecting microvascular reactivity through endothelium-dependent and -independent pathways.
Abstract 564: CD146 Deficiency Leads to Accelerated Atherosclerosis in Mice
Authors: Blin Muriel G , Bachelier Richard , Fernandez Samantha , Guillet Benjamin , Fallague Karim , Robert Stéphane , Bardin Nathalie , Blot-Chabaud Marcel , Dignat-George Françoise , Leroyer Aurelie S
Published: March 2022

Atherosclerosis is a chronic inflammatory disease of the large vessels and its progression is based in part on the continued recruitment of macrophages in the inflammatory wall. The well described role of CD146 in leukocyte infiltration in vitro suggests a role for this endothelial junction molecule in the early stages of the formation of atherosclerotic plaques. However, the involvement of CD146 in atherogenesis has not been elucidated.

To investigate the role of CD146 deficiency in atherosclerosis, CD146 knocked-out (KO) mice were bred with ApoE KO mice. Mice were fed a high-fat Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. After 24 weeks of diet, the arterial wall thickness was significantly higher in CD146 deficient mice (0.19±0.01mm vs 0.27±0.01mm; p=0.012) and there was a significant 47 % increase (p=0.015) in total aortic lesion area, evaluated by histological oil red O coloration. However, mice weights and total cholesterol plasma levels were similar between the two groups of mice. We detected a 2-fold increase in macrophage recruitment to the atherosclerotic plaque of the double KO mice compared with ApoE KO mice, suggesting that the atherosclerotic lesions in these mice were more inflammatory. In addition, CD146 deficient plaques contained significantly less smooth muscle cells (p=0.036) and could be more vulnerable. We observed a maladaptive immune response in double KO mice since the circulating neutrophils and monocytes were significantly increased (p=0.008 and p=0.015 respectively) in the absence of CD146 whereas circulating T lymphocytes were significantly decreased (p=0.026). In a similar way, splenocytes isolated from double KO mice contained significantly lower levels of T cells (p=0.002), suggesting a default of adaptive immunity in the absence of CD146 during atherosclerosis.

Our results demonstrated the involvement of CD146 in the progression of atherosclerosis in mice. Our data also suggest that CD146 is involved in the regulation of adaptive immune response and its absence lead to an inflammatory phenotype, which could influence atherosclerosis plaque fate. Thus, CD146 should be considered as a potential target for protection against atherosclerosis.
Abstract 567: Atg16l1 Expression in Carotid Atherosclerotic Plaques Is Associated With Plaque Vulnerability
Authors: Magné Joelle , Gustafsson Peter , Jin Hong , Maegdefessel Lars , Hultenby Kjell , Wernerson Annika , Eriksson Per , Franco-Cereceda Anders , Kovanen Petri , Gonçalves Isabel , Ehrenborg Ewa
Published: March 2022

Objective:
Autophagy has emerged as a cell survival mechanism critical for cellular homeostasis, which may play a protective role in atherosclerosis. ATG16L1, a protein essential for early stages of autophagy, has been implicated in the pathogenesis of inflammatory diseases such as Crohn’s disease. However, it is unknown whether ATG16L1 is involved in atherosclerosis. Our aim was to analyze ATG16L1 expression in carotid atherosclerotic plaques in relation to markers of plaque vulnerability.

Approach and results:
Histological analysis of 143 endarterectomized human carotid atherosclerotic plaques revealed that ATG16L1 was expressed in areas surrounding the necrotic core and the shoulder regions. Double immunofluorescence labelling for ATG16L1 and cell type markers CD68, α-actin-smooth muscle cell, CD31 or tryptase revealed that ATG16L1 was abundantly expressed in phagocytic cells, endothelial cells, as well as mast cells in human advanced carotid plaques. ATG16L1 immunogold labelling was predominantly observed in endothelial cells and foamy smooth muscle cells of the plaques. ATG16L1 protein expression correlated with plaque content of pro-inflammatory cytokines and matrix metalloproteinases. Analysis of Atg16L1 at two distinct stages of the atherothrombotic process in a murine model of plaque rupture by incomplete ligation and cuff placement in carotid arteries of apolipoprotein-E-deficient mice, revealed a strong colocalization of Atg16L1 and smooth muscle cells only in early atherosclerotic lesions developing intimal hyperplasia.

Conclusions:
Taken together, this study shows that ATG16L1 protein expression is associated with a rupture-prone and inflamed plaque phenotype, and could also contribute to the development of plaque vulnerability already at earlier stages of the atherogenic process.
Abstract 576: Gender Differences in Carotid Artery Plaque Composition Do Not Exist in Response to Lipid Lowering Therapy at Two Years Among Men and Women with Carotid or Coronary Artery Disease and Elevated ApoB Levels
Authors: Paramsothy Pathmaja , Hong Jaekyoung , Isquith Daniel , Hulphers Elizabeth , Bai Hua , Shadzi Pey , Neradilek Moni , Gill Edward A , Zhao Xue-Qiao
Published: March 2022

Introduction:
Recent, age-adjusted stroke death rates declined greater in men than women. Whether this is related to gender differences in the atherosclerotic plaque response to therapy is not known. Postmortem coronary artery histopathology and post-surgical carotid endarterectomy studies demonstrate that women have less calcification and inflammatory cells, but more smooth muscle cells than men.

Hypothesis:
We hypothesized that gender differences exist in carotid plaque composition (CPC) in response to lipid lowering therapy (LLT) comparing living men and women.

Methods:
The CPC study is a prospective, randomized study evaluating the effect of LLT: 1) atorvastatin + placebo + placebo vs 2) atorvastatin + niacin ER + placebo 3) atorvastatin + niacin ER + colsevelam on CPC. Participants had coronary or carotid artery disease and ApoB levels ≥120 mg/dL. CPC was evaluated using MRI. The change over two years in % wall volume (PWV) [(wall volume/total vessel volume) х 100%], a measure of plaque burden that adjusts for variation in artery size, and % lipid rich necrotic core (LRNC) volume among slices with LRNC present were evaluated. Statistical analysis used Wilcoxon rank sum test, chi-square, and multivariate linear regression.

Results:
There were 40 women and 73 men in the study with both baseline and 2 year MRI scans. Women vs. men were older, mean±SD age 58±9 vs. 54±8 yrs. (p=0.009), had higher HDL-C, 49±14 vs. 40±11 mg/dL (p=0.002), and higher ApoA1 145±26 vs. 126±20 mg/dL (p<0.001). ApoB levels were not significantly different, 127±31 vs. 121±25 mg/dL (p=0.2). Adjusted for age, HDL-C (strongly correlated with ApoA1, r=0.89), ApoE, prevalence of MI and metabolic syndrome (statistically significant in the full baseline cohort), there were no statistically significant gender differences at 2 years with LLT in change in PWV, [[Unable to Display Character: ∆]] -0.1 (95% CI: -0.7, 0.6%) (p=0.8) or %LRNC among participants with LRNC, [[Unable to Display Character: ∆]] 0.5 (95% CI: -2.5, 3.5%) (p=0.7).

Conclusions:
We did not detect statistically significant gender differences in change in PWV and %LRNC in response to LLT. Although the results are consistent with no gender differences they remain inconclusive due to the small sample size. Further gender studies in the biology and treatment of arterial atherosclerosis are needed.
Abstract 589: Macrophage SR-BI Binds GST-Pi and Suppresses Cell Apoptosis in Atherosclerosis
Authors: Tao Huan , Yancey Patricia G , Blakemore John L , Zhang Youmin , Ding Lei , Davies Sean S , Vickers Kasey C , Linton MacRae F
Published: March 2022

Background:
Oxidative stress accelerates atherosclerosis by promoting cell death. We hypothesize that macrophage scavenger receptor class B I (SR-BI) inhibits apoptosis by reducing oxidative stress in atherosclerosis. Methods and Results: Macrophage SR-BI deficiency led to 2.1-, 1.9-, and 2.7-fold more apoptosis after treatment with OxLDL, H2O2, or isolevugladin-phosphatidylethanolamine (IsoLG-PE) versus WT cells (P<0.05), respectively. To examine the impact of SR-BI deficiency in atherosclerosis, bone marrow (BM) transplantation studies were performed using apoE-/- and LDLr-/- recipient mice fed a Western diet for 8 or 16 weeks, respectively. Apoptosis as measured by active Caspase 3 was increased 2.6-fold in atherosclerotic lesions of LDLr-/- recipients of SR-BI-/- versus WT BM (P<0.01). Consistent with SR-BI reducing oxidative stress, the lesions of ApoE-/- mice reconstituted with SR-BI-/-ApoE-/- versus ApoE-/- BM showed 5.1-fold higher immunostaining for 4-HNE (P<0.05) and 70.4% lower GSH/GSSG ratio as determined by an enzymatic assay of aortic tissue lysates (P<0.01). Similarly, OxLDL treatment resulted in a 64.5% decrease in GSH/GSSG ratio in SR-BI-/- versus WT macrophages. ER stress was significantly increased in SR-BI-/- versus WT macrophages treated with OxLDL as evaluated by CHOP and BiP (P<0.05). Similarly, ER stress was increased in atherosclerotic lesions of ApoE-/- recipients of SR-BI-/-ApoE-/- versus ApoE-/- BM. Importantly, an antibody-based proteomic assay revealed that macrophage SR-BI interacts with glutathione S-transferase pi (GST-pi). SR-BI deficiency caused a 48.7% reduction of GST-pi expression in atherosclerotic lesions (P<0.05) and a 51.3% decrease in macrophage GST activity (P<0.05). Forced expression of GST-pi rescued OxLDL-induced apoptosis in SR-BI-/- macrophages. A lipid-protein overlap assay showed SR-BI binds IsoLG-PE, but not native PE, suggesting that SR-BI recognizes oxidized lipid for clearance via a GST-pi detoxification pathway. Conclusion: Macrophage SR-BI interacts with GST-pi and suppresses oxidative stress-induced apoptosis in atherosclerosis. This novel finding of SR-BI GST-pi interaction provides a new target for prevention and treatment of atherosclerosis.
Abstract 591: Increased Retention of LDL From Type 1 Diabetic Patients in an Atherosclerosis-prone Area of the Murine Arterial Wall
Authors: Hagensen Mette K , Mortensen Martin B , Kjolby Mads , Bentzon Jacob F , Gregersen Soeren
Published: March 2022

Background:
Individuals with type 1 diabetes mellitus (T1DM) are at high risk of developing atherosclerotic cardiovascular disease but the exact mechanisms by which T1DM accelerates atherosclerosis remain unknown. In the present study, we investigated whether modifications of low density lipoprotein (LDL) in T1DM patients causes an increased retention of LDL in the vessel wall.

Methods and Results:
Fluorescently-labeled human LDL from either T1DM patients (n=10) or healthy non-diabetic subjects (control) (n=7) was injected into mice with T1DM. After 24 hours, when LDL was completely cleared from the circulation, cryosections of the inner curvature of the aortic arch (i.e. atherosclerosis prone area) was analyzed for retained LDL by fluorescence microscopy (Red in figure A). We found significantly more retained T1DM LDL (n=10) compared to LDL from non-diabetic subjects (n=7) with a fold change of 4.35 (p < 0.05). Nuclear magnetic resonance (NMR) spectroscopy analysis of LDL revealed no differences in the level of the atherogenic small dense LDL between T1DM LDL (274.4±47.8 nmol/L) and non-diabetic LDL (263.8±55.3 nmol/L). Using LiCor Odyssey infrared imaging scanning of the whole aorta en face, we found that in vitro glycation of LDL from a non-diabetic subject significantly increased retention (n=8) compared to LDL prepared similarly but without glucose (n=8), with a fold change of 8.87 (p < 0.001) (Figure B).

Conclusion:
LDL from patients with T1DM as well as ex vivo glycated LDL showed increased retention at atherosclerosis-prone sites in the arterial wall of mice. This may contribute to the accelerated development of atherosclerosis in T1DM.
Abstract 594: Characterization of Anti-thrombotic and Anti-inflammatory Properties of New Synthetic, Protamine Reversible Low Molecular Weight Heparin
Authors: Chandarajoti Kasemsiri , Sparkenbaugh Erica M , Key Nigel S , Xu Yongmei , Cooley Brian , Liu Jian , Pawlinski Rafal
Published: March 2022

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders; however, lack of full reversibility with protamine and clearance by kidney limit treatment options. Recently we have developed a chemoenzymatic method to synthesize a new synthetic dodecasaccharide (12mer) with high anti-factor Xa (FXa) activity which was protamine reversible in vitro and in vivo.

In the current study, we further characterized the pharmacokinetic, anti-thrombotic and anti-inflammatory properties of this compound using various mouse models. First, time course (0.5, 1, 2, 4, 8 hours, n=4) and dose response (0.5, 1, 1.5, 2.0 mg/kg, n=4) studies in C57Bl/6J mice showed that FXa activity was inhibited by 12mer in vivo and the half-life of the compound injected s.c. at the dose of 1.5 mg/kg was 3 hours. 12mer was cleared from the circulation by 8 hours.

Furthermore, fibrin accumulation observed in the femoral vein 60 minutes after electrolytic injury (2.99 ± 1.16; p<0.05; normalized relative fluorescent intensity to control mouse, mean ± SD), was similarly reduced by 12mer (0.6 mg/kg, 1.63± 0.48), UFH (150 U/kg, 1.26 ± 0.35) and enoxaparin (1 mg/kg, 1.39 ± 0.85, p<0.05 for all). Importantly, protamine reversed the anti-thrombotic effect of 12mer and returned fibrin accumulation to normal levels (2.69 ± 0.87; p < 0.05 vs.12mer alone).

Finally, we investigated if 12mer can inhibit thrombotic and inflammatory effect of FXa, previously demonstrated by us in the Berkeley mouse model of sickle cell disease. Sickle mice received subcutaneous injection of saline (n=6) or 12-mer (n=6; 2.0 mg/kg) every 8 hours for 7 days. Compared to saline, 12mer reduced plasma levels of thrombin anti-thrombin complexes (8.2±1.4 vs 4.4±1.3 ng/mL; p=0.07, mean ± S.E.M), IL-6 (5.5±1.5 vs 1.1±0.4 pg/mL, p<0.05) and sVCAM1 (1.1±0.2 vs 0.6±0.07 μg/mL, p< 0.05) in sickle mice. Together, our data demonstrates that chemoenzymatic synthesis offers a promising approach toward obtaining new, protamine-reversible LMWHs with excellent anti-coagulant and anti-inflammatory properties.
Abstract 601: Circulating Platelet Aggregate Damages Endothelial Cells in Culture
Authors: Aluganti Narasimhulu Chandrakala , Nandave Mukesh , Bonilla Diana , Singaravelu Janani , Ravi Yazhini , Parthasarathy Sampath , Chittoor Bhaskar Sai-Sudhakar
Published: March 2022

Background:
The presence of circulating endothelial cells (CECs) in systemic circulation might be an indicator of endothelial damage and/or denudation, and thus the body’s response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt and denude the endothelium, and contribute to the presence of CEC and EC-derived particles (ECDP). In this study, we used endothelial cells and sheep carotid artery segments.

Methods:
Endothelial cells (sheep aortic and human umbilical vein) were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes as well as sheep carotid artery segments were connected to a Mini-Pump Variable Flow System to study the effect of circulating AgPlts on the endothelium. ECs in glass tube/artery segment were exposed to medium alone, non-aggregated platelets (NAgPlts), AgPlts and 90 micron polystyrene beads at a flow rate of 20-85 ml/minute for various time intervals (0, 30 seconds, 1, and 5 min). Collected effluents were cultured and incubated for 72 hrs to analyze the growth potential of dislodged but intact ECs. Inflammatory genes of the damaged endothelium were analyzed by RT-PCR and the presence of ECDP was further assessed by western blot analysis using an antibody to human von Willebrand factor (VWF).

Results:
No ECs and ECDP were observed in effluents collected after injecting medium alone or medium and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs and released ECs and ECDP in effluents. There were no ECs and ECDP in samples collected at 0 and 30 seconds. However, the number of ECs and EDCP in samples collected at 1 and 5 minutes was proportionately increased. Effluents collected when endothelial cell damage was seen showed an increased presence of VWF as compared to control effluents.

Conclusion:
Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelial damage. Moreover, a major source of the CECs in the blood circulation could be the damaged endothelium.
Abstract 610: Microrna-146a Accumulates in the Atherosclerotic Plaque but Decreases in Circulation of Patients With ST Segment Elevation MI
Authors: Gray Warren D , Corrigan Frank E , Mitchell Adam , Alexy Tamas , Rooney Kim , Garcia-Bengochea Youssef , Devireddy Chandan , Jaber Wissam , Liberman Henry , Finn Aloke , Searles Charles , Kumar Gautam
Published: March 2022

MicroRNAs (miRNAs) function as regulators of gene expression intracellularly or as paracrine signals extracellularly. Their role in modulation of cardiovascular disease is emerging. Changes in miRNA profiles have been characterized in the circulation of patients with myocardial infarction (MI), but little is known of the correlation between circulating and plaque miRNAs during atherosclerosis progression and ST-elevated MI (STEMI). During percutaneous interventions (PCI), angioplasty catheter balloons can retain plaque debris post-inflation and may provide insight into plaque miRNA expression in disease.

Our objective was to assess the correlation between miRNA levels in circulation and plaques of patients with coronary artery disease (CAD), including those with ST segment elevation MI (STEMI).

We collected balloons and plasma from patients undergoing PCI for STEMI (n=6) or elective cardiac catheterization (n=2). Plasma was collected from age and sex-matched patients at low risk for CAD (n=5) or with stable CAD (n=6). RNA was extracted from balloon-adherent debris and patient plasma and sequenced. miRNA levels were validated by qRT-PCR.

Deep sequencing of patient plasma suggested a decrease in levels of circulating miR-146a-5p in STEMI patients (fig A). This miRNA is found in several cell types (endothelial cells, immune cells, and macrophages), and targets inflammatory signaling pathways. Via PCR, we found that levels of circulating miR-146a dropped markedly both in patients with stable CAD and STEMI (fig B). However, the trend was reversed in plaques; miR-146a levels increased 15-fold from stable to ruptured plaques (fig C). We postulate that miR-146a accumulation in plaques is a compensatory response to inflammation, but that the drop in circulating miR-146a levels may contribute to atherosclerotic plaque instability. These findings have implications for development of miRNA-based strategies for diagnosis and management of vulnerable plaques.
Abstract 628: Circulating Microparticles From Patients With Valvular Heart Disease and Cardiac Surgery Inhibit Endothelium Dependent Vasodilation.
Authors: Ou Jingsong , Fu Li , Hu Xiao-xia , Lin Ze-Bang , Chang Feng-Jun , Ou Zhi-jun , Wang Zhi-ping
Published: March 2022

Objective:
Circulating microparticles (MPs) from some diseases play important roles on inflammation, coagulation and vascular injury. The vascular function is quite important for maintaining the circulation after cardiac surgery. However, the impact of MPs generated from valvular heart disease (VHD) and cardiac surgery on vascular function remains unknown. The present study is to investigate the impact of such MPs on vasodilation.

Methods:
MPs were isolated from age-match healthy subjects and patients with VHD before and at 12 h, 72 h after cardiac surgery. Effects of MPs on vasodilation of mice aorta, the phosphorylation and expression of Akt, protein kinase C βII (PKC-βII), p70S6K, endothelial nitric oxide synthase (eNOS) and caveolin-1, the association of eNOS with heat shock protein 90 (HSP90), generation of nitric oxide (NO) and superoxide anion (O2•–) of human umbilical vein endothelial cells (HUVECs) were determined.

Results:
Compared with MPs from healthy subjects, MPs from VHD patients before cardiac surgery can impair endothelium-dependent vasodilation, inhibit phosphorylation of Akt and eNOS, enhance expression of caveolin-1, and activation of PKC-βII, p70S6K, and reduced association of HSP90 with eNOS, decrease NO production and increase O2•– generation. Those effects were even stronger when treated with MPs from post- cardiac surgery.

Conclusion:
Our data demonstrated that circulating MPs generated before and after cardiac surgery in VHD patients contributed to endothelial dysfunction by inhibiting and uncoupling of eNOS activity. Circulating MPs may be a potential therapeutic target for maintenance of vascular function post-cardiac surgery.
Abstract 633: B Cells in Artery Tertiary Lymphoid Organs of Aged Apolipoprotein-e Deficient Mice
Authors: Srikakulapu Prasad , Bontha Sai Vineela , Hu Desheng , McNamara Coleen , Habenicht Andreas
Published: March 2022

Background:
Atherosclerosis is recognized as a chronic in[[Unable to Display Character: ﬂ]]ammatory disease of arteries characterized by accumulation of in[[Unable to Display Character: ﬂ]]ammatory cells in the inner layer of the arterial wall, i.e. the intima. Though adaptive immune responses play important roles in all stages of atherosclerosis, the role of B cells is controversial as both atheroprotective and atherogenic effects have been reported. Moreover, information on B cells in the arterial wall is limited. We observed well-structured T and B cell aggregates in the adventitia of aorta segments adjacent to atherosclerotic lesions of aged Apolipoprotein-E Deficient (ApoE-/-) mice and referred to these structures as artery tertiary lymphoid organs (ATLOs).

Methods and Results:
To understand atherosclerosis-associated B cell responses better, we undertook to delineate the B cell response in ATLOs versus secondary lymphoid organs. In ATLOs, marked numbers of germinal centre (GC) B cells, mantle zone B cells, and plasma cells were observed. Surprisingly, high numbers of B-1 B cells (20% per total B cells in ATLOs) were identified with B-1b cells making up 80% per total B-1 cells. The accumulation of B-1b cells in the diseased aorta was atherosclerosis-specific as the B-1b/B-1a ratio in ATLOs was increased when compared with the ratio in the peritoneal cavity (the major location of B-1 cells in mice), the spleen, and renal lymph nodes of Wt and ApoE-/- mice. ATLOs contained ~10% of GL-7+, PNA+ GC B cells per total IgD- B cells. The IgG1+ memory B cell percentage among total IgD- B cells was significantly increased, the IgM+/IgD+ (naïve B cells) percentage was significantly decreased, and the IgM-/IgD- percentage was significantly increased in ATLOs when compared with spleen, renal lymph nodes, and blood of aged Wt and ApoE-/- mice. Adoptive B-2 B cell transfer experiments revealed that B cell recruitment into ATLOs was specific, rapid, and highly regulated. Constitutively IgM and IgG antibody secreting cells were observed in ATLOs by ELISPOT.

Conclusion:
Adventitial B cell aggregates were associated with marked expression of the B lymphocyte chemoattractant CXCL13. Our data indicate that ATLOs are a major site where adaptive and innate B cell immune responses are carried out in atherosclerosis.
Abstract 646: Locked Nucleic Acid miR-143 Ameliorates Aortic Valve Functions in a ROS-Mediated Murine Model of Aortic Valve Sclerosis
Authors: Branchetti Emanuela , Lee Suengwon , Grau Juan B , Hazen Stanley L , Levy Robert J , Ferrari Giovanni
Published: March 2022

Background:
Calcific Aortic Valve Disease (CAVD) is the leading cause of valve replacement in the US. While the final stages (Aortic Stenosis -AS) are well described, subclinical stages (Aortic Sclerosis -AVSc) remain understudied. We reported accumulation of Reactive Oxygen Species (ROS) in subclinical stages. Adeno-delivery of antioxidant enzymes rescues Valve Interstitial Cells (VICs) from calcification and osteogenic activation. Furthermore we described OPN-CD44 axis as a calcium-derepression system in AVSc and developed a ROS-mediated murine model based on Angiotensin II infusion that mimic AV remodeling as in AVSc. Here we aim to identify oxidative products able to mediate the transition from AVSc to AS and to test them in vivo.

Methods:
Oxidized amino-acids were quantified by mass spectrometry (LC MS/MS) on human AVs. Osteogenic-induced VICs and explanted tissues were tested for miR143 expression. C57BL/6J mice were fed an enriched diet and infused with saline (n=24) or AngII (1,000 ng/kg/min) (n=40) for 8 weeks and injected with LNA-miR143 after the first 4 weeks. Echo data were recorded.

Results:
LC MS/MS revels prdicution of dityrosine in AS only (221±41 μmol/mol), while other oxidized products were undetectable. VICs were then isolated from patients with AVSc and induced to further differentiate into osteogenic VICs by TGF-β1 (10ng/ml for 12 days); In silico target prediction revels miR143 as a possible regulator of CD44. miR143 increases in stimulated cells (4.1 fold vs. untreated, p<0.05) as well as in AV human explants (6.2 fold vs controls, p<0.01). After 8 weeks of Ang II we found sustained activation of VICs (SMA+, OPN+) and ECM remodeling. Mice injected with LNA-miR143 after the development of AV thickening have reduced AV peak gradient, peak velocity, and velocity-time-interval.

Conclusions:
It is concluded that a) oxidative damage drives the transition between early subclinical stages to calcification and osteogenic differentiation; b) oxidized amino-acid di-tyrosine and miR143 are associated with end-stage diseases c) miR143 mediates the pathological remodeling of the AV in a murine model of subclinical CAVD. Altogether, these data suggest that oxidative damage control VIC activation and AV remodeling via miR143.
Abstract 648: Reduced Expression of Phosphoprotein Enriched in Astrocytes-15 (PEA-15) Increases Neointimal Hyperplasia
Authors: Greig Fiona H , Kennedy Simon , Ramos Joe W , Nixon Graeme F
Published: March 2022

Neointimal hyperplasia from increased vascular smooth muscle cell (VSMC) proliferation remains a significant clinical problem. Phosphoprotein enriched in astrocytes-15 (PEA-15) is an extracellular signal-regulated kinases 1/2 (ERK1/2) binding protein. We have previously demonstrated in vitro that PEA-15 acts as a cytoplasmic “anchor” in VSMCs and prevents ERK1/2 translocation to the nucleus, decreasing proliferation. We now assess the hypothesis that expression of PEA-15 regulates the development of neointimal hyperplasia. Arterial injury was induced by wire denudation with ligation of the carotid artery in C57BL/6 mice and examined 3, 7 and 14 days after surgery. Neointima formation was significantly increased in injured arteries from day 7 compared to control arteries. PEA-15 protein expression was significantly decreased in injured arteries in comparison to control vessels 3 days after arterial injury (ratio to GAPDH - 0.29±0.10 vs. 0.91±0.1, n=3), preceding significant neointima formation at day 7. A similar decrease in PEA-15 protein and mRNA levels was observed in human saphenous vein rings incubated ex vivo in 30% serum for 14 days to induce neointimal hyperplasia (protein ratio to GAPDH - 0.36±0.05 vs. freshly isolated veins 1.34±0.20, n=5). This coincided with increases in proliferative markers. To determine a causal relationship between decreased PEA-15 expression and neointimal formation, we induced arterial injury in mice deficient in PEA-15 (PEA-15-/-). There was a significantly exaggerated neointima formation in PEA-15-/- mice 7 days after surgery compared to C57BL/6 mice at the same time point (intima-to-media ratio - 0.95±0.26 vs. 0.35±0.09, n=3-5). The mechanisms involved were investigated. Immunolocalization of ERK1/2 was assessed in primary cultured VSMCs isolated from aortae of C57BL/6 and PEA-15-/- mice. In unstimulated VSMCs from PEA-15-/- mice, there was a significant increase in nuclear staining of ERK1/2 compared with cells from C57BL/6 mice. In conclusion, decreased PEA-15 expression occurs in VSMCs during the development of neointima hyperplasia which may increase proliferation. Targeting this pathway could be beneficial in the treatment of restenosis.
Abstract 655: Increased Nadph-Oxidase Activity Is Associated With Reduced Telomere Length in the Human Vascular Wall: The Influence of Oxidative Stress on Biological Aging
Authors: Margaritis Marios , Patel Sheena , Antonopoulos Alexios S , Herdman Laura , Sanna Fabio , Ananthan Kiruthika , Petrou Mario , Krasopoulos George , Channon Keith M , Antoniades Charalambos
Published: March 2022

Background:
Accelerated telomere shortening is a hallmark of cellular senescence and has been associated with cardiovascular disease (CVD). Oxidative stress has been proposed as a cause of telomere damage. We explored the association between vascular redox state and telomere length in patients with advanced atherosclerosis.

Methods:
We recruited xx patients undergoing bypass surgery. Saphenous vein (SV) samples were used to measure superoxide (O2-) generation using lucigenin chemiluminescence (+NADPH 100 uM to estimate NADPH-oxidase activity). DNA was extracted from whole-blood and SV samples and used to measure relative telomere length (TL) using qPCR (which determines T/S ratio using albumin as a single-copy gene). DNA was also used to genotype for two functional single nucleotide polymorphisms (SNPs rs4673 and rs1049255) in the CYBA locus, which encodes for the p22phox membrane subunit of NADPH-oxidase and is associated with activity of the enzyme.

Results:
Shorter vascular TL was associated with increased NADPH-stimulated O2- generation in SV tissue (A). The additive effect of the rs4673C and rs1049255G alleles was linked to increased vascular NADPH-oxidase activity (B). In addition, the same combination of SNP alleles was linked to reduced T/S ratio in whole blood DNA (C), suggesting a causal association between high oxidative stress and accelerated telomere shortening.

Conclusions:
We demonstrate for the first time in humans that genetically determined high NADPH-oxidase activity could be causally linked to reduced TL in vascular and blood cells. These novel findings highlight the importance of oxidative stress on cellular senescence and provide an important link between aging and cardiovascular disease.
Abstract 657: Resolvin D1 Attenuates Vascular Smooth Muscle Cell Migration via the cAMP Pathway
Authors: Mottola Giorgio , Wu Bian , Chatterjee Anuran , Chen Mian , Komshian Sevan R , Conte Michael S
Published: March 2022

Introduction:
Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), attenuates the migratory phenotype in vascular smooth muscle cells (VSMC), which is critical to the development of atherosclerosis and neointimal hyperplasia. SPM are known to interact with G-protein coupled receptors (GPCR). We sought to investigate the biochemical pathways by which RvD1 influences VSMC migration.

Methods:
VSMC were harvested from human saphenous veins. cAMP levels were measured via ELISA, in the absence or presence of WRW4, a blocking peptide for the RvD1 receptor ALX, a GPCR. PDGF-BB (10ng/ml), thrombin (1U/ml) and angiotensin (AT II; 1μM) were used as agonists in a VSMC scratch assay. PDGF-BB-induced cytoskeletal changes were measured as length to width ratio after actin-phalloidin staining. Rp-8-Br-cAMP (10μM), a selective PKA inhibitor, was used in the phenotypic assays.

Results:
RvD1 treatment (10nM) of VSMC induced a significant acute flux in cAMP levels, causing a dramatic increase at 5 minutes (Fig. 1A; n≥3); this increase was abolished by WRW4. RvD1 (10nM) attenuated VSMC migration stimulated by PDGF-BB, thrombin and AT II (Fig. 1B; n≥3) and cytoskeletal rearrangements stimulated by PDGF-BB (n=4). RvD1’s effects were negated by the addition of Rp-8-Br-cAMP, suggesting a central role for the cAMP pathway.

Conclusion:
Our results suggest that RvD1 attenuates VSMC migration by increasing levels of cAMP through ALX.
Abstract 662: X-box Binding Protein 1 Splicing Promotes Wound Healing via Modulating Endothelial Nitric Oxide Synthase
Authors: Yang Junyao , Zeng Lingfang , Wang Wen , Li Yi
Published: March 2022

Instruction:
X-box binding protein 1 (XBP1) is a pivotal transcription factor in the pathogenesis of the immune system and in the endoplasmic reticulum stress response. Our previous studies have proved that XBP1 is involved in VEGF-mediated endothelial cell (EC) proliferation and angiogenesis. However, whether XBP1 can influence EC migration is unknown.

Hypothesis:
We assessed the hypothesis that XBP1 splicing is involved in endothelial migration contributing to wound healing and angiogenesis.

Method and results:
In this study, we used EC monolayer wound healing, tube formation and transwell migration models to study the involvement of XBP1 splicing in EC migration. We found that scratching on EC monolayer triggered XBP1 splicing, which was attenuated by the presence of LY294002, FAK-14 and L-NAME, suggesting that PI3K/Akt, FAK and eNOS may be involved in scratching-mediated upregulation of XBP1 splicing. Over-expression of spliced XBP1 (XBP1s) via Ad-XBP1s gene transfer increased EC migration, while knockdown of IRE1α or XBP1 by ShRNA lentiviruses suppressed EC migration. Over-expression of XBP1s upregulated eNOS at mRNA and protein levels leading to the increase of nitric oxide production. The eNOS promoter reporter analysis revealed that XBP1s down-regulated eNOS transcription. The pSI-Check2-eNOS-3’UTR reporter analysis indicated that XBP1s might upregulate eNOS mRNA via 3’UTR-mediated mRNA stabilization. The microRNA array assays found that XBP1s down-regulated microRNAs mir-24, mir-125 and mir-214. Transfection of EC with the miRNA mimics of those three miRNAs attenuated XBP1s-induced eNOS mRNA upregulation and EC migration.

Conclusion:
These results suggest that XBP1 splicing may increase eNOS mRNA stabilization and nitric oxide production via down-regulating miRNAs that target eNOS mRNA 3’-UTR, leading to EC migration and contributing to wound healing and angiogenesis.

Significance:
XBP1 splicing may provide a new therapeutic strategy to tackle endothelium injury-related cardiovascular disease.
Abstract 675: Abdominal Aortic Aneurysm Rupture Often Occur Outside the Maximal Diameter Area and is Preceded by Rapid Local Growth
Authors: Siika Antti , Lindquist Liljeqvist Moritz , Hultgren Rebecka , Gasser T. Christian , Roy Joy
Published: March 2022

Objectives:
Surveillance of abdominal aortic aneurysms (AAA) currently relies on maximal diameter (Dmax) measurements. However, the annual risk of rupture in AAAs with a Dmax smaller than 55 mm is over 1.5 percent and many large AAAs remain stable. It was recently shown that significant structural changes may occur locally in the aneurysm without affecting the Dmax. Our objective was to quantify ruptures of small AAAs and to relate the location and growth of the local rupture site to the Dmax.

Methods:
Cases were included from a retrospective cohort of patients who presented with ruptured AAA in our catchment area between the years 2009-2013. Dmax was measured orthogonally to the centerline in the 50 aneurysms that had available computed tomography (CT) images. Local diameter at the rupture site and its centerline distance from the Dmax region was measured in 30 AAAs that had an identifiable rupture site, defined as active contrast extravasation or wall discontinuity adjacent to a hematoma. In nine patients who had undergone CT within two years before rupture and at the time of rupture, diameter growth rates at Dmax and rupture site were compared. All measurements were made from CT images using multi-planar reconstruction in a Sectra PACS workstation.

Results:
Median diameter at rupture CT was 77 mm (range 46-120 mm). Two ruptured AAAs (4%) had a Dmax < 55 mm and five (10%) had a Dmax < 60 mm. Fifteen (50%) of the 30 identified rupture sites were located outside the Dmax region, nine (30%) at a centerline distance of 20 mm or greater. Six (20%) occurred at a local diameter smaller than 60 mm. The diameter growth rate was in six cases out of nine (67%) higher in the rupture site than in the Dmax region between the pre-rupture and rupture CTs but there was no general difference (medians 4.7 vs 4.9 mm/y, p = 0.89).

Conclusions:
Four to ten percent of the ruptured aneurysms had small diameters. Ruptures often occurred outside the maximal diameter region, sometimes in segments with relatively small local diameters, and were preceded by rapid local diameter growth. Our results support the notion of considering the entire structure of an aneurysm, rather than sole reliance on maximal diameter measurements.
Abstract 680: What Matters Most, Statin Intensity or Achieved LDL? - Evaluating Concordance of AHA/ACC Guidelines for Statin Use with Practice Outcomes at Stanford Hospital & Clinics
Authors: Gyang Elsie , Shah Nigam , Leeper Nicholas
Published: March 2022

Introduction:
Recommendations from the 2013 American College of Cardiology/American Heart Association Guidelines on the Assessment of Cardiovascular Risk have had a mixed reception. The goal of our study was to evaluate practice-based evidence from Stanford Hospital using structured and unstructured bioinformatics data to determine whether achieved LDL or statin intensity was more predictive of major adverse cardiac events (MACE).

Methods:
We performed a retrospective cohort analysis of all adult patients >= 21 years of age prescribed statin therapy between 1996 and 2015. Demographic and clinical variables were extracted from coded data and unstructured clinical text using a validated data extraction pipeline. To account for possible treatment selection bias we performed 1:1 propensity score matching to produce a patient cohort with similar demographic, comorbid disease, and laboratory value distributions between treatment groups. Mcnemar’s Chi-squared and conditional logistic regression analyses were performed to identify variables predictive of MACE.

Results:
We identified 7,378 adults on statin therapy with complete data. Patients were followed on average for 3.3 years, 56% percent were male, 55% were Caucasian, and mean age was 64 years. Patients had a high burden of comorbidities (85% with HTN, 64% with Type II Diabetes, 58% with CAD, 42% with CHF, and 32% with PAD). Patients on high-intensity statins were matched with those on low-/moderate-intensity statins, producing a cohort of 2,056 patients with equal distributions of comorbidities, co-prescriptions and LDL measurements. In this well-balanced cohort, statin intensity did not predict MACE (P=0.29) while achieved LDL was a significant predictor of outcomes (P=0.006). OR for MACE was 0.73 (95% CI 0.65-0.82) in patients with LDL of <=70 versus those with LDL >100. OR for MACE for LDL <=70 versus LDL >= 130 was 0.32 (95% CI 0.28-0.38).

Conclusions:
Using a retrospective matched cohort design we found that achieved LDL was a significant predictor of MACE outcome, while statin intensity was not. This finding implies that despite recent guidelines, statin therapy should continue to be aimed at achieving specific LDL targets rather than arbitrarily selecting statins based on intensity alone.
Abstract 701: Differential Expression of Epicardial Fat MiRNA Targets in Patients with Coronary Artery Disease
Authors: Santanam Nalini , Davis Mary , Primerano Don , Gress Todd , Wehner Paulette , Chowdhury Nepal
Published: March 2022

Background and Objectives:
Adipose dysfunction is a major player in cardiometabolic diseases. Epicardial fat (EF) plays an important role in the pathophysiology of coronary-artery disease (CAD). EF factors have a paracrine and endocrine effect on heart and vasculature. We earlier showed differences in EF-derived biomarkers in patients with and without CAD. The objective of this study was to assess EF miRNA and its target mRNA expression profiles in patients with and without CAD.

Methods:
EF was obtained from men and women undergoing coronary-artery-bypass-graft or valve-type surgery (IRB approved study). Total RNA and miRNA were isolated from EF. Whole RNA sequencing (Next-generation sequencing) and whole-genome miRNA array of the EF from all patients was performed.

Results:
31 miRNAs were differentially expressed (24 up and 7 down-regulated) in EF from CAD compared to non-CAD patients. Cross-referencing the target mRNAs of these miRNAs using Ingenuity Pathway Analysis and RNA-seq data, identified genes that played a role in CAD: GRIN1, DUSP4 (ion transport); APOB, LDLR (lipoprotein homeostasis); TUBB1 (platelet function); and FOXO3 (vascular homeostasis).

Conclusion:
Our results identified miRNA-mRNA pairs differentially expressed in EF that play a role in CAD. Our future research will focus on validating the functional importance of these miRNA-mRNA pairs in relevance to epicardial fat-vascular cross talk
Abstract 708: Changes in Carotid Plaque Lipid Content in Subjects Who Continued and Discontinued Statin Therapy
Authors: Du Ruixue , Zhao Xue-Qiao , Cai Jianming , Cui Bao , Ye Ping
Published: March 2022

Objective:
Changes in carotid plaque lipid-rich necrotic core (LRNC) as assessed by magnetic resonance imaging (MRI) were investigated in subjects who continued and discontinued statin therapy for 2 years after a prospective study.

Methods:
The Rosuvastatin Evaluation of Atherosclerotic Chinese Patients (REACH) study in 32 lipid treatment naïve subjects with LRNC showed a significant reduction in LRNC during 24 months (M) of rosuvastatin therapy. All subjects received a clinical follow-up (F/U) visit and a repeat carotid MRI scan at 48 M as planned REACH-F/U. Despite receiving a strong recommendation to continue the statin therapy at 24 M when REACH was completed, only 15 subjects continued taking statins (rosuva.=9, simva.=4 and atorva.=2) in REACH-F/U and 17 discontinued. Lipids and LRNC, both in volume (V) and % (LRNC-V/Wall Vх100%), were compared between the statin-continued and -discontinued groups at 48 M.

Results:
There were no significant differences in demographic, clinical characteristics, lipids and plaque changes during 24 M in REACH between the statin-continued and -discontinued groups in REACH-F/U. Not surprisingly, at 48 M, total-Cholesterol (C), LDL-C and triglycerides were significantly lower in subjects who continued statin than those discontinued (163±43 vs. 207±30 mg/dl, p=0.002), (93±36 vs. 131±22 mg/dl, p=0.001) and (85±27 vs. 143±65 mg/dl, p=0.003), while HDL-C levels were similar. LRNC-V and %LRNC decreased significantly from 24 M in the statin-continued group (101±76 mm
3
at 24 M vs. 76±65 mm
3
at 48 M, p=0.001) and (17.3±11.9% at 24 M vs. 12.6±7.6% at 48 M, p=0.04). By contrast, subjects who discontinued statin showed non-statistically significant increase in LRNC-V and %LRNC (103±93 mm
3
at 24 M vs. 112±106 mm
3
at 48 M, p=0.4) and (15.4±11.3% at 24 M vs. 16.7±11.4% at 48 M, p=0.07). Furthermore, the changes in LRNC-V and %LRNC from 24 to 48 M were significantly different between the statin-continued and -discontinued groups in REACH-F/U (-25±18 vs. 9±14 mm
3
, p<0.001) and (-4.6±8.2 vs. 1.3±2.8%, p=0.009).

Conclusion:
Continued statin therapy in REACH-F/U resulted in continued decrease in LRNC, which indicates improved plaque stability. These findings provided vascular biological evidence supporting long-term statin therapy.
Abstract 716: Echocardiographic Evidence of Systemic Atherosclerosis and Long-Term Outcomes in Patients with Non-Hemorrhagic Stroke
Authors: Pieper Justin , Ashamalla Michael , Yager Neil , Sedhom Daniel , Ghate Ketan , Nguyen Veronica , Shkolnik Boris , Torosoff Mikhail
Published: March 2022

Background:
Echocardiography is routinely performed for evaluation of cardiac embolic causes in patients with non-hemorrhagic stroke. However, added value of echocardiographic evidence for systemic atherosclerosis in patients with known peripheral disease and non-hemorrhagic stroke has not been well established.

Material and Methods:
Retrospective chart review was performed in 517 consecutive patients treated for non-hemorrhagic stroke at a single academic medical center. Complete transthoracic echocardiograms were reviewed for presence of mitral annular calcifications, valve sclerosis, or aortic atherosclerosis. Long-term outcomes were ascertained through the National Death Index. This study was approved by the institutional IRB.

Results:
The study cohort included 48% females. Coronary artery disease (CAD) was present in 27%, hypertension in 62%, diabetes in 20%, dyslipidemia in 38%, peripheral vascular disease (PVD) in 4%, and end-stage renal disease in 5%. Echocardiographic evidence of systemic atherosclerosis was noted in 43%.

We observed 28% mortality at the mean follow-up length of 46+/-20 months. Mortality was significantly increased in older patients, and in patients with histories of end-stage renal disease (p=0.048), PVD (p<0.001), diabetes (p=0.05), hypertension (p=0.027), and CAD (p=0.012). Echocardiographic evidence of systemic atherosclerosis was associated with a 2-fold increase in mortality (20 vs. 40%, p<0.0001).

In multivariate logistic regression analysis, when adjusted for recorded end-points, the echocardiographic evidence of systemic atherosclerosis was not predictive of adverse outcomes. Only age (HR 1.9, 95%CI 1.6-2.4, p<0.0001), diabetes (HR 1.9, 95% CI 1.1-3.3, p=0.029), and PVD (HR 5.1, 95%CI 1.8-14.4, p=0.0024) were associated with increased mortality.

Conclusions:
In patients with non-hemorrhagic stroke, systemic evidence of atherosclerosis suggested by transthoracic echocardiography is not associated with worse long-term prognosis. This should not preclude providers from performing an echocardiographic evaluation if clinically indicated. The question of diagnostic utility for post stroke transthoracic echocardiography is an important topic and warrants further study.