bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 318: Circulating Neutrophils and their Secreted Mediator S100A8/A9 Correlate with Carotid Artery Disease and Predict Cardiovascular Events in Healthy Individuals
Authors: Cotoi Ovidiu S , Ko Nayoung , Dunér Pontus , Björkbacka Harry , Hedblad Bo , Nilsson Jan , Schiopu Alexandru
Published: March 2022

Aims

S100A8/A9 is an inflammatory mediator secreted by activated neutrophils and monocytes/macrophages. Serum S100A8/A9 levels were previously found to correlate with the severity of coronary artery disease in diabetic patients and to predict cardiovascular (CV) events in healthy postmenopausal women. Our aim was to investigate whether S100A8/A9 correlates with carotid artery disease and whether it can predict CV risk in healthy individuals.

Methods

We measured baseline levels of S100A8/A9 in 664 individuals with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer cohort. We examined the correlation between S100A8/A9 and traditional CV risk factors, circulating cell populations, plasma cytokines, carotid intima media thickness (IMT) and area (IM area), and the incidence of CV events during a follow-up period of 16.2 years.

Results

We detected significant positive correlations between baseline S100A8/A9 and the number of circulating neutrophils, IFNγ, TNFα, IL-1β, smoking, BMI, systolic blood pressure and an inverse correlation with HDL. In a multivariate linear regression model adjusted for age and sex, S100A8/A9 and the neutrophil counts were correlated with IMT and IM area in the common carotid artery. The associations with IM area remained significant after further adjustment for CV risk factors. In a Cox regression model corrected for age and sex, S100A8/A9 and circulating neutrophils predicted the incidence of coronary events (fatal or non-fatal myocardial infarction) [HR (95%CI) per SD: 1.28 (1.03-1.59),
P
=0.026 and 1.26 (1.04-1.53),
P
=0.020, respectively] and CV death [1.34 (1.06-1.71),
P
=0.015 and 1.59 (1.33-1.90),
P
<0.001, respectively]. There was no relationship with stroke.

Conclusions

S100A8/A9 correlates strongly with circulating neutrophil numbers, but not with monocytes or monocyte sub-populations. Baseline S100A8/A9 concentrations and circulating neutrophils are significantly associated with carotid artery disease and predict coronary events and CV death in apparently healthy individuals. Our study provides further evidence for the involvement of activated neutrophils in atherogenesis and acute coronary events.
Abstract 332: Genomically Enhanced Risk Stratification in Abdominal Aortic Aneurysm Repair
Authors: Ryer Evan J , Smelser Diane T , Garvin Robert P , Yoon H. R , Bernard-Eckroth Kamell R , Franklin David P , Tromp Gerard , Elmore James R , Carey David J , Kuivaniemi S. H
Published: March 2022

OBJECTIVE

The purpose of this study was to investigate the association between selected single-nucleotide polymorphisms (SNPs), associated with abdominal aortic aneurysms (AAA) in previous genome-wide association studies, and major adverse events following AAA repair

METHODS

Genomic DNA was isolated from blood samples collected from AAA patients and used for genotyping. A genetic risk score was constructed using five SNPs known to be associated with AAA. Epidemiologic data were extracted from the electronic medical record and analyzed retrospectively. Major adverse events were defined as myocardial infarction, cardiac arrest, respiratory failure requiring tracheostomy, renal failure requiring dialysis, colonic ischemia requiring resection, limb ischemia requiring operation, multi-system organ failure or death.

RESULTS

Three hundred and twenty one patients (267 male, 83%) with complete genomic and clinical data underwent AAA repair from 2004-2012. Demographics were consistent with a standard AAA population. 204 patients (64%) underwent endovascular repair and 117 (36%) underwent conventional open surgical repair. Quality control checks were performed on the clinical and laboratory data and none of the 5 selected SNPs deviated from the Hardy-Weinberg Equilibrium. Stepwise logistic regression was then used to determine the most parsimonious model of variables associated with adverse outcomes from AAA repair. Serum creatinine, height, resting heart rate, diastolic blood pressure and the genetic risk score remained in the model. The Hosmer and Lemeshow test of deviation from goodness-of-fit (p = 0.56) indicated that the data fit the model well. The regression coefficients from this model will be used to construct a clinician-friendly risk calculator to help identify patients at increased risk for a major adverse event following AAA repair.

CONCLUSIONS

Selected SNPs help identify patients at risk for major adverse events following AAA repair. Incorporating genetic data, especially in patients with few clinical risk factors, may result in a better preoperative risk assessment.
Abstract 339: Association of the American Heart Association’s Life’s Simple 7 Metric for Cardiovascular Health with Risk of Venous Thrombosis: the Reasons for Geographic and Racial Differences in Stroke Cohort
Authors: Cushman Mary , Judd Suzanne E , McClure Leslie A , Lakoski Susan G , Folsom Aaron R , Zakai Neil A
Published: March 2022

Background

The AHA released a metric, Life’s Simple 7 (LS7), in 2010 to track cardiovascular health. The AHA 2020 Goals include improving population levels of LS7 by 20%. LS7 classifies 7 health components into poor, intermediate or ideal levels. The 7 components are physical activity, diet, smoking, body-mass index, glucose, blood pressure and cholesterol. Associations of these individual components with risk of venous thrombosis (VT) vary in the literature, but we hypothesized a decreased risk of VT with favorable LS7 summary scores.

Methods

We recruited 30,239 black and white participants age ≥45 across the US in 2003-7. With 4.6 years of follow up we validated 278 cases of first-time VT, including deep vein thrombosis and pulmonary embolus. We considered a 14 point summary score for LS7 and divided it into inadequate (0-4 points), average (5-9 points) and optimum (10-14) cardiovascular health. Cox proportional hazards models were used to relate baseline LS7 score to risk of subsequent VT, adjusting for age, sex, race, income, education and region of residence.

Results

Cases of VT were older and more likely to be male than noncases. Across categories of optimum, average and inadequate baseline health based on the summary LS7 score, the incidence rates of VT were 1.8, 2.1 and 3.1 per 1000 person years, respectively. Compared to those with inadequate health, persons with average health had an adjusted HR of VT of 0.60 (95% CI 0.42-0.84) while those with optimum health had a HR 0.52 (95% CI 0.36-0.77). The HR for a 1 point improvement in overall score (analogous to improvement of one of the 7 factors from poor–>intermediate or intermediate–>ideal) was 0.87 (95% CI 0.81-0.94). The individual factors of LS7 most strongly related to VT risk were ideal physical activity level (HR 0.61, 95% CI 0.46-0.83) and ideal body-mass index (HR 0.42, 95% CI 0.30-0.60); these associations were independent of each other. All associations were similar in blacks and whites.

Conclusions

A summary measure of cardiovascular health was strongly associated with reduced risk of VT in this US population sample. Results suggest that lifestyle interventions might be effective in reducing incidence of VT, and that achieving AHA’s 2020 Goals will also reduce VT incidence.
Abstract 340: Circulating TGF-ß1 Correlates with Increased Wall Shear Stress in a Hypercholesterolemic Murine Model of Spontaneous Aortic Valve Stenosis
Authors: Wang Wei , Vootukuri Spandana , Coller Barry S , Ahamed Jasimuddin
Published: March 2022

Aortic valve stenosis (AS) is a chronic disorder involving fibrosis and calcification of the aortic valve (AV), leading to a decrease in AV orifice area and an increase in wall shear stress (WSS) across the valves. We previously demonstrated that the pro-fibrotic cytokine TGF-β1, which is secreted as a latent complex, can be activated by shear both in vitro and in vivo. Thus, we hypothesize that shear-induced TGF-β1 activation contributes to the pathophysiology of AS. In this study, we used Ldlr
-/-
Apob
100/100
/Mttp
fl
/
fl
/Mx1Cre
+/+
mice (“Reversa”, kindly provided by Dr. Heistad), which spontaneously develop AS after 6 months of a high fat diet, to test this hypothesis.

Treating Reversa mice with a high fat diet for 6 months was associated with a significant decrease in AV cusp separation distance (median [IQR] from 0.85 [0.64-0.97] to 0.46 [0.37-0.65] mm) and significant increases in plasma cholesterol (mean±SD from 273±27 to 837±98 mg/dl), WSS across the AV (from 189 [152-235] to 308 [236-568] dyn/cm
2
), and plasma total TGF-β1 levels (from 1.24 [1.08-0.38] to 1.66 [1.34-2.10] ng/ml) (all p<0.01). In contrast, no change in AV cusp separation distance, WSS, or plasma total TGF-β1 levels were observed in mice on a chow diet (n=14, all p>0.05). The increase in plasma total TGF-β1 levels in mice on a high fat diet correlated with the increase in WSS (r=0.32, p=0.04). Immunofluorescence staining showed increased Smad2/3 phosphorylation and α-smooth muscle actin (α-SMA) expression in the stenotic valves. We tested the effect of TGF-β1 (1ng/ml) on cultured porcine AV interstitial cells and found increased Smad2/3 phosphorylation and increased expression of α-SMA after 12 or 24 hours treatment.

We conclude that AS progression is associated with both an increase in total plasma TGF-β1 levels and evidence of TGF-β1-induced cellular activation in this hypercholesterolemic murine model of spontaneous AS. These data are consistent with a model in which cellular release of TGF-β1 results in increased plasma TGF-β1, activation of latent TGF-β1 by the increased WSS across the AV, and TGF-β1 induces fibrosis and calcification of the AV. Since WSS increases with AS progression, a feed-forward mechanism may lead to accelerated AS progression.
Abstract 345: The Thrombotic Reactivity of Endothelial Cells from Deep Vein and Pulmonary Artery Venous Beds in Response to Thrombin
Authors: Caliste Xzabia A , Roztocil Elisa , Clarke Amanda L , Cullen John P , Gillespie David L
Published: March 2022

Background

Pulmonary embolism (PE) is thought to occur largely due to deep venous thrombosis from the lower extremities. A portion of PEs however may result from primary pulmonary artery thrombosis. Recent studies have shown pulmonary artery endothelial cells have increased fibrinolytic reactivity compared to iliac vein endothelial cells in response to TNFα stimulation, a cytokine known to be up-regulated in the systemic response to trauma. The goal of this study was to determine if similar thrombotic stimuli occurs in non-trauma situations.

Methods

Human iliac vein endothelial cells (HIVEC) and human pulmonary artery endothelial cells (HPAEC) were incubated at 37° C in the presence or absence of 25pM thrombin for 24 hours. Plasminogen activator inhibitor-1 (PAI-1), uPA, tPA, EPCR, PAR-1 and TM expression in cell lysates were evaluated by Western blot analysis. ICAM-1 cell surface expression was evaluated by flow cytometry.

Results

While thrombin had no effect on the expression of uPA in HPAEC, there was a ~15% decrease in expression in HIVEC. With tPA, HPAEC and HIVEC had an 11% and ~25% increase in expression, respectively. In contrast, thrombin stimulation caused a decrease in PAI-1 expression in HPAEC while having no effect on HIVEC. There was no change in EPCR expression in HPAEC, however there was a 22% decrease in HIVEC. HPAEC demonstrated decreased expression of PAR1, in contrast to an almost 40% increase in PAR1 expression in HIVEC. Thrombin produced no change in HIVEC recombinant TM expression, however, 11% increase in expression was observed in HPAEC. With ICAM-1, neither HIVEC nor HPAEC had a change in expression after thrombin stimulation.

Conclusion

This data shows that endothelium of the deep veins reacts differently from pulmonary arterial endothelium in response to non-trauma stimuli. Endothelial cells from the iliac vein and pulmonary artery venous beds differentially express markers of the fibrinolytic and coagulation pathways, and this variance in expression may play a role in the thrombotic reactivity of these vascular beds.
Abstract 349: Measurement of Platelet Cyclooxygenase-1 Acetylation Quantifies Interaction of Nonsteroidal Anti-Inflammatory Drugs with Aspirin in vivo
Authors: Li Xuanwen , Fries Susanne , Li Ruizhi , Lawson John A , Diamond Scott L , Blair Ian A , FitzGerald Garret A , Grosser Tilo
Published: March 2022

Aspirin prevents heart attack and stroke by irreversibly acetylating serine-529 in human platelet cyclooxygenase-1 (COX-1) and thereby inhibiting the biosynthesis of thromboxane (Tx). We hypothesized that precise quantification of COX-1 acetylation would allow the direct detection of drug-drug interactions between reversible COX inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), and aspirin, which may abrogate its cardio-protective effect. We developed a stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for measurement of platelet COX-1 acetylation. Serine-529 acetylation reflected faithfully aspirin pharmacology determined with conventional markers (serum TxB2 formation, urinary excretion of 11-dehydro-TxB2, and arachidonic acid induced platelet aggregation), but showed lower technical (CV% 3.7±2.7) and inter-individual variability (CV% 3.7). Oral administration of 325 mg of plain aspirin resulted in complete acetylation of platelet COX-1 within 2 hours and sustained maximal acetylation (71 ± 3% of total COX-1 amount) for up to two days in health volunteers (n=8). Recovery from acetylation, which reflects platelet turn-over, occurred at a rate of 7.2 ± 0.2 % per day. We compared the potential of five NSAIDs to block aspirin from acetylating platelet COX-1 in healthy volunteers (n=7 for each NSAID). Single doses of naproxen (500 mg), ibuprofen (600 mg), and diclofenac (100 mg) but not celecoxib (200 mg) and acetaminophen (1000 mg) prevented complete COX-1 acetylation by 325 mg aspirin administered 2 hours after the NSAID. The competition of NSAIDs with aspirin was conditioned by NSAID pharmacokinetics, potency, and COX isoform selectivity, suggesting variability in the likelihood of the drug-drug interaction between compounds and between patients. Monitoring platelet COX-1 acetylation may have utility in the identification of drug interactions that may limit the cardio-protective effect of low dose aspirin.
Abstract 352: Adeno-Associated Virus-mediated Restoration of Angiotensinogen Function in Mice With Hepatocyte-specific Deficiency
Authors: Wu Congqing , Lu Hong , Moorleghen Jessica J , Howatt Deborah A , Balakrishnan Anju , Cassis Lisa A , Daugherty Alan
Published: March 2022

Background and Objective

We have reported previously that genetic or pharmacological inhibition of angiotensinogen (AGT) leads to resistance to dietary fat-induced body weight gain. This phenotype has not been observed in mice subject to pharmacological inhibitors of other components of the renin angiotensin system including renin, ACE, and AT1 receptors. To investigate whether restoration of AGT reverses this lean phenotype, we injected an adeno-associated virus (AAV) 2/8 vector carrying wild type AGT to mice with hepatocyte-specific deficiency of AGT (hepAGT -/-).

Methods and Results

HepAGT -/- mice were generated by disrupting AGT gene in hepatocytes via albumin promoter driven Cre recombinase expressed in mice with floxed AGT alleles. An AAV AGT vector was constructed with the insertion of wild type AGT. AAV null vector was used as control. AAV expression was driven by hepatocyte-specific thyroxine-binding globulin promoter. Five groups of male LDL receptor -/- mice at age of 8 - 10 weeks were studied: wild type (hepAGT +/+) mice received either no AAV (PBS) or null AAV, and hepAGT -/- mice received low dose (1 x 1010 genome copy), high dose (3 x 1010 genome copy), or null AAV. Four weeks after AAV injection (i.p.), all mice were fed a saturated fat-enriched diet (42% calories from milk fat) for 12 weeks. Plasma samples were collected through retro-orbital bleeding at weeks 2, 4, 8, and 16 after AAV injection. Plasma AGT concentrations peaked between weeks 2 and 4, then declined modestly, irrespective of dosage. However, at the high dose, plasma AGT concentrations in hepAGT -/- mice were comparable to those in hepAGT +/+ mice. Plasma total cholesterol concentrations, body weight gains, and systolic blood pressures were not different between hepAGT +/+ and hepAGT -/- mice receiving either dose of AGT AAV. Atherosclerosis, quantified as percent lesion area covering the intima of the aortic arch, was equivalent between hepAGT -/- mice receiving either dose of AGT AAV and hepAGT +/+ mice, whereas it was nearly absent in hepAGT -/- mice receiving null AAV.

Conclusions

AGT in an AAV 2/8 vector efficiently recovers plasma AGT concentration in hepAGT -/- mice in a dose-dependent manner. This approach permits the elucidation of structural mutations on AGT function.
Abstract 36: Array-based Profiling Reveals Biomarker and Therapeutic Potential for Different microRNAs in Patients with Symptomatic Carotid Stenosis
Authors: Osterholm Cecilia , Eken Suzanne M , Chernogubova Ekaterina , Perisic Ljubica , Razuvaev Anton , Jin Hong , Roy Joy , Hedin Ulf , Maegdefessel Lars
Published: March 2022

The discovery of an entirely new method of recognition and regulation of gene expression and protein translation by microRNAs (miRs) has provided new hope for innovative disease discovery and therapy. Research in recent years has assigned miRs a key regulatory role during various pathological processes. Lately, their potential as biomarkers in disease received a lot of attention. Aim of the present study was to explore the role of miRs as potential regulators and plasma biomarkers in patients with carotid stenosis and at risk of stroke.

Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we were able to profile the regulation of miRs in patients with asymptomatic vs. symptomatic carotid stenosis undergoing carotid endarterectomy. A PCR-based microRNA array of plasma, sampled at the carotid lesion site, identified eight miRs (miRs-15b, -29c, -30c/d, -150, -191, -210 and -500) being substantially altered in symptomatic vs. asymptomatic patients. In peripheral plasma samples, the expression of miRs-150 and -191 remained significantly different. Using an in vitro cell streamer system, we subjected human carotid smooth muscle cells, as well as human carotid endothelial cells, to physiologic and pathologic flow conditions. Interestingly and in accordance with our findings from the initial plasma profiling study, laminar (physiologic) flow induced miR-210 up-regulation, whereas turbulent (pathologic) flow suppressed miR-210 expression in both cell lines. In addition, similar significant changes in miR-210 regulation were detectable in a model of vascular remodelling, balloon injury of the carotid artery in Sprague Dawley rats. Of importance, miR-210 targets several key genes and signalling pathways involved in vascular remodelling and atherosclerotic plaque stability.

The present study explores the diagnostic, prognostic and therapeutic potential of miR regulation in patients with symptomatic carotid stenosis and increased risk of stroke. We were able to identify miRs-150 and -191 as potential diagnostic plasma markers, and miR-210 as a key modulator of pathologic processes in atherosclerosis-related vascular remodelling.
Abstract 369: Cd36 Sensing of Metabolic Danger Signals is a Common Mechanism of Nlrp3 Inflammasome Activation
Authors: Sheedy Frederick J , Moore Kathryn J
Published: March 2022

Atherosclerosis and type 2 diabetes (T2D) are associated with low-grade systemic inflammation characterized by increased expression of cytokines, particularly interleukin-1β (IL-1β), yet the molecular mechanisms of immune activation remain unclear. Recent studies identified cholesterol crystals and the fibrillar proteins Aβ and IAPP as novel activators of the NLRP3 inflammasome in atherosclerosis, Alzheimer’s disease and T2D, respectively. Notably, the scavenger receptor CD36 has also been implicated in these sterile inflammatory conditions. We now show that CD36 binding of soluble endogenous ligands that accumulate in these diseases (oxLDL, soluble Aβ and IAPP) initiates cooperative signaling via the newly described TLR4/TLR6 heterodimer to prime the inflammasome, including induction of Il1α, IL1β and Nlrp3 mRNAs. Importantly, the unregulated ligand uptake characteristic of CD36 promotes the intracellular formation of inflammasome activators by transforming these soluble ligands into crystalline or fibrillar danger signals. Using combined confocal reflectance microscopy we show that whereas WT macrophages incubated with oxLDL exhibit intracellular crystal formation and lysosomal rupture, these pathologic events are absent in Cd36-/- macrophages. Furthermore, atherosclerotic Apoe-/- mice lacking Cd36 are protected from plaque cholesterol crystal accumulation, caspase-1 activation and serum IL-1β elevation. Via an analogous mechanism, CD36 uptake of soluble Aβ or IAPP leads to intracellular thioflavin-positive fibril formation, lysosomal disruption and inflammasome activation. These findings indicate a shift from the current paradigm that such endogenous crystalline/fibrillar ligands are generated extracellularly and identify CD36-mediated ligand uptake as a shared mechanism by which the NLRP3 inflammasome is triggered in metabolic diseases.
Abstract 374: Soy Supplementation Prevents Progression of Smaller but Not Larger Atherosclerotic Plaques: Results of a Postmenopausal Monkey Reverse Translational Trial
Authors: Meléndez Giselle C , Pajewski Nicholas M , Appt Susan E , Kaplan Jay R , Register Thomas C , Clarkson Thomas B
Published: March 2022

Rationale_

A recent clinical trial reported that soy supplementation reduced atherosclerosis progression in the carotid arteries of women who were <5 years postmenopausal, an effect not shared by women >5 years postmenopausal. Here we tested the hypothesis that soy atheroprotection may be influenced by the size of pre-existing atherosclerotic lesions at the initiation of treatment.

Methods_

Premenopausal female cynomolgus macaques (n=38) were fed an atherogenic diet (Casein/Lactalbumin [CL]) for 34 months. Animals were then ovariectomized (OVX) and the left iliac artery (LCI, a surrogate for the coronary arteries) was biopsied to determine the amount of atherosclerosis present at baseline. Immediately after OVX, monkeys were randomized to either continue consuming the CL diet (n = 20) or to consume a soy protein diet (SP, n = 18) for an additional 34 months. At necropsy, the right iliac (RCI) and coronary arteries were collected and atherosclerosis extent was quantified.

Results_

The CL diet group increased plaque size in the RCI compared to pretreatment (0.43 ± 0.06 mm
2
vs. 0.85 ± 0.12 mm
2
,
p
=0.019) whereas, plaques did not increase in size in the SP diet group, (LCI: 0.60 ± 0.10 mm
2
vs. RCI: 0.82 ± 0.16 mm
2
, p=0.17). Notably, SP diet did not have a beneficial effect on coronary artery plaque extent (CL: 0.73 ± 0.20 mm
2
vs. SP: 0.63 ± 0.16 mm
2
,
p
=NS). However, we observed that when animals were divided at the median based on pretreatment LCI plaque sizes (below; 0.21 ± 0.14 mm
2
: above; 0.82 ± 0.06 mm
2
), the SP diet prevented plaque progression in both the iliac (CL: 0.54 ± 0.14 mm
2
vs. SP: 0.19 ± 0.04 mm
2
,
p
=0.04) and coronary arteries (CL: 0.52 ± 0.11 vs. SP: 0.17 ± 0.06 mm
2
,
p
=0.04) only in animals with smaller pretreatment plaques. There was no atheroinhibition among soy treated monkeys with plaques that were above the median (RCI CL: 1.32 ± 0.11 mm
2
vs. SP: 1.35 ± 0.14,
p
=0.94 and coronary artery CL: 1.06 ± 0.11 vs. Soy: 1.00 ± 0.17,
p
=0.63).

Conclusions_

Pretreatment atherosclerosis stage is a major determinant of the potential atheroprotective effect of soy treatment. These data suggest that to obtain cardiovascular benefits of soy supplements, treatment should begin during the perimenopausal transition or in the very early postmenopause while plaques are usually still small.
Abstract 376: Platelet Depletion Accelerates the Vulnerability of Early Atherosclerosis in Apolipoprotein E-Deficient Mice
Authors: Li Rong , Ren Meiping , Luo Mao , Chen Ni , Xia Jiyi , Deng Xin , Wu Jianbo
Published: March 2022

Objective.

Platelets were presented in human atherosclerotic plaque. The function of platelets in the development of atherosclerosis is far poorly defined. We addressed the role of platelets in mediating atherosclerosis formation.

Methods and Results.

Apolipoprotein E-deficient mice (ApoE-/-) were fed with a high-fat Western diet for 12 weeks. Thrombocytopenia was induced by intraperitoneal (i.p.) injections every 3 days with mouse of the platelet-depleting antibody to ApoE-/- mice from 8 weeks. Until the experimental endpoint, atherosclerotic lesion in the aorta and aortic roots were quantified after staining. Size of atherosclerotic plaque in aortic root did not differ between platelet-depleted mice and controls (134±13.1 μm2 [x103] vs 124±11.4 μm2 [x103]; P>0.5). Plasma cholesterol did not differ between groups. However, atherosclerotic lesions of platelet-depleted mice exhibited thinner fibrous caps (62.5±9.2 μm vs. 323±63 μm; P<0.05) and larger necrotic cores (19.5±4.9 μm2 [x103] vs. 4.8±0.8 μm2 [x103]; P<0.05) than controls. Furthermore, compared with controls, platelet-depleted AopE-/- mice promoted significantly increased number of macrophage infiltration into the atherosclerotic lesions.

Conclusion.

Our results suggest that platelet depletion promotes atherosclerotic plaque instability in the development of early atherosclerosis, likely though the macrophage infiltration. These results offer important insights into the in vivo function of platelet, and help to explain the association between enhanced platelet deposition and thrombotic complications of atherosclerosis, such as myocardial infarction.
Abstract 383: Lack of Endogenous Acute Phase Serum Amyloid A Does Not Impact Atherosclerotic Lipid Deposition in ApoE-/- Mice.
Authors: De Beer Maria C , Wroblewski Joanne M , Rateri Debra L , Howatt Deborah , Balakrishnan Anju , Tannock Lisa R , Daugherty Alan , De Beer Frederick C , Webb Nancy R
Published: March 2022

Inflammation is involved in all stages of the development of an atherosclerotic plaque. Serum amyloid A (SAA), a marker of inflammation, is a well-known predictor of increased cardiovascular risk. Although elevated levels of plasma SAA have been associated with increased atherosclerosis, the role of SAA in the pathogenesis of atherosclerosis remains obscure. To investigate the impact of endogenous SAA in the etiology of atherosclerosis we bred mice lacking both major acute phase SAA isoforms SAA1.1 and SAA2.1 to apoE-/- mice. Both strains were in the C57BL/6 background. For ease of terminology we refer to these mice as SAAKO and apoE-/- mice. Aortic atherosclerotic lesion areas were quantified by en face analysis in male and female mice fed normal chow for 12 months. Although female mice weighed less and had lower plasma lipid levels than male mice, the absence of SAA did not affect plasma lipid concentrations or lipoprotein-cholesterol profiles or the size of aortic atherosclerotic lesions of apoE-/- mice. To accelerate lesion formation, male and female apoE-/- and SAAKO mice were fed a Western diet for 12 weeks. We observed gender-related differences in body weight, weight gain and plasma lipid concentrations. However, absence of SAA in apoE-/- mice fed a diet enriched in saturated fat had no discernable effect on plasma lipid concentrations, lipoprotein-cholesterol profiles or the size of the aortic atherosclerotic lesions. Lesions in the aortic root of male mice, quantitated by oil red O staining, was also not affected by genotype.

We conclude that absence of endogenous acute phase SAA does not impact atherosclerotic lesion development in apoE-/- mice fed normal or fat-enriched diets.
Abstract 388: Apolipoprotein A-V Deficiency in Mice Results In Increased Triglyceride and Cholesterol Absorption
Authors: Zhang Linda S , Kohan Alison B , Yang Qing , Xu Min , Ryan Robert O , Tso Patrick
Published: March 2022

Elevated plasma triglycerides constitute an independent risk factor for coronary heart disease. Discovered in 2001, apolipoprotein A-V (apoA-V) was found inversely proportional to plasma triglycerides (TG). Synthesized only in the liver, apoA-V exists in very low concentrations in the plasma (~10,000 fold lower than apoA-I). While apoA-V is proposed to enhance hydrolysis and reduce hepatic production of TG-rich lipoproteins, the role of apoA-V on intestinal lipid absorption has not been investigated. The aim of this study is to examine the effects of apoA-V deficiency on intestinal lipid absorption in chow-fed mice. Since mice lacking functional apoA-V have drastically elevated plasma TG, we hypothesize that apoA-V deficiency in mice elevates the lymphatic output of TG after a lipid meal. Using apoA-V knockout (n=8) and wildtype (n=8) control mice, we employed the conscious lymph fistula model to examine the transport of dietary TG and cholesterol into the lymph during a continuous lipid infusion. Interestingly, apoA-V knockout mice displayed a 2-fold elevation in dietary TG (P<0.001) and a 3-fold elevation in dietary cholesterol (P<0.001) transport into the lymph compared to wildtype mice, suggesting that a loss of apoA-V enhances transport across the enterocyte. However, apoA-V knockout mice displayed a 2-fold reduction (P<0.05) in dietary TG accumulated in the intestinal mucosa while there was no difference in the mucosal accumulation of dietary cholesterol between apoA-V knockout and wildtype mice, suggesting that apoA-V affected TG and cholesterol transport in the enterocyte differently. We conclude apoA-V deficiency results in increased TG and cholesterol absorption in mice, and therefore plays a previously unknown role in intestinal lipid absorption.
Abstract 390: Perilipin 1 (plin1) Genotype and Not Gpr109a Associated with Free Fatty Acid Rebound Following Niacin Administration
Authors: Tuteja Sony , Dunbar Richard , Qu Liming , Li Mingyao , Mucksavage Megan , DerOhannessian Stephanie , Reilly Muredach , Rader Daniel
Published: March 2022

Niacin has been used for decades as a lipid lowering agent. Stimulation of a G-protein coupled receptor (GPR109A) on adipocytes by niacin results in the anti-lipolytic effect of the drug and reduction in free fatty acids (FFA), a key pharmacodynamic readout of niacin action. Niacin also causes a subsequent rebound increase in FFA which may be linked to adverse glycemic effects. An evoked phenotype genetic study was undertaken to address the hypothesis that variation in
GPR109A
is associated with the FFA suppression and/or rebound with single-dose niacin.

The study recruited n=294 healthy volunteers (mean age 26 ± 7 years, 51% Female, 35% African-American (AA), 65% Caucasian (Cau)) to an inpatient immediate release 1 gm niacin challenge. The single exon gene
GPR109A
(1092 bp) was sequenced and genotyping was performed on Affymetrix 6.0 genomewide SNP array. Adipose tissue proteomics was performed for 6 subjects comparing pre-post niacin samples (Applied Biomics, Hayward, CA). Serial plasma samples were collected for FFA, insulin, and glucose determination.

The FFA nadir was 0.09 ± 0.08 meq/L, occurred at 2.2 ± 0.7 hr post niacin, and was lower in Cau (0.09 ± 0.08 vs 0.10 ± 0.07, p=0.003). FFA rebound occurred at 4.9 ± 0.7 hr and was lower in AA (1.7 ± 0.5 vs 2.0 ± 0.5, p<0.0001). Fasting insulin levels and HOMA-IR increased 94 ± 106% (p<0.0001) and 96 ± 149% (p<0.0001), respectively at 5 hr compared to baseline. The two common
GPR109A
SNPs, C931T (MAF 0.12) and G951A (MAF 0.31), were not associated with FFA nadir nor rebound. Proteomics analysis of adipose tissue samples revealed increased perilpin1 protein expression two hours after niacin. Cau individuals homozygous for the variant allele for
PLIN1
(
rs894160
) had a lower FFA rebound than those with the wild-type allele (p=0.04).

Variation in the niacin receptor
GPR109A
was not associated with FFA suppression nor rebound. Using an unbiased proteomics screen, we discovered that niacin increased protein expression of perilipin1, a lipid droplet protein that functions to restrict access of lipases to the triglyceride core. A common non-coding variant in
PLIN1
was asociated with niacin induced FFA rebound. Pharmacogenomics of niacin may serve to identify patients who may be hyper-reponders to niacin therapy.
Abstract 397: Changes in Lipoprotein Subfraction Concentrations After Treatment with the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib Alone or in Combination with Atorvastatin
Authors: Krauss Ronald M , Pinto Cathy Anne , Liu Yang , Johnson-Levonas Amy O , Dansky Hayes M
Published: March 2022

Background

Anacetrapib (ANA), a cholesteryl ester transfer protein inhibitor, has been shown to increase plasma HDL cholesterol and reduce LDL cholesterol, both as monotherapy and in combination with atorvastatin (ATORVA). The effects of these regimens on lipoprotein subfractions have not been reported.

Methods

ApoB-containing lipoprotein particle subfractions were measured by ion mobility in a post-hoc analysis of baseline and 8 week on-treatment samples from 464 dyslipidemic participants who had been randomized to placebo, ATORVA 20 mg/d, or to ANA at several doses administered with and without ATORVA (
clinicaltrials.gov
NCT00325455
).

Results

ANA (combined 150 mg/d and 300 mg/d groups) significantly reduced total LDL particle concentration by 51%, and levels of large, medium, and small LDL particles (LDL 1, 2, and 3a) by 43-72% but significantly increased levels of the less abundant cholesterol-depleted very small LDL 4a and 4b species by ~64%. In contrast, ATORVA, despite producing a similar reduction of total LDL particle concentration (50%), reduced the levels of all LDL particle subfractions, although the magnitude was smaller for LDL 4a, with a much smaller non-significant reduction for LDL 4b. Compared with ANA monotherapy, results for combination treatment were generally consistent with additive effects. Notably, for individuals with baseline triglyceride (TG) above the median (>154 mg/dL), increases of very small LDL after ANA were markedly attenuated, whereas after ATORVA there was greater reduction of LDL 4a in the higher TG group.

Conclusions

Similar reductions of LDL levels after ANA or ATORVA were not associated with comparable effects on all LDL particle subfractions, and neither drug was effective in lowering levels of the smallest LDL particles. Assessment of the clinical impact of the effects of ANA on LDL subfractions, and the differential changes in very small LDL in individuals with normal versus elevated TG, await cardiovascular outcome data from REVEAL.
Abstract 40: Neutrophil Serine Protease PR3 Promotes Platelet Adhesion and Activation on von Willebrand Factor (vWF) Expressing Endothelial Cells
Authors: Kuckleburg Christopher J , Newman Peter J
Published: March 2022

Introduction

Neutrophils play important roles in inflammation, regulating vascular permeability and promoting thrombus formation. Upon activation, neutrophils release serine proteases that can activate coagulation factors and protease activated receptors (PARs). Proteinase 3 (PR3) is the most abundant serine protease in neutrophils; however, the role of PR3 in regulating vascular function and thrombosis is not clear. In this study we have used flow chamber assays and confocal microscopy to investigate PR3 activation of endothelial cells through PAR-2 and the recruitment and activation of platelets and leukocytes under shear conditions.

Results

After endothelial cells were incubated with PR3 or a PAR-2 activating peptide, we observed significant release and surface presentation of von Willebrand (vWF) compared to untreated cells. In addition, we also found that PR3 increased endothelial cell adhesion molecule expression and ERK phosphorylation. Using flow chamber assays, we observed that PR3-treated endothelial cells expressing vWF were able to capture platelets from whole blood under venous shear conditions; however, blocking antibodies against either platelet GpIb or GpIIb/IIIa significantly reduced platelet adhesion. Using whole blood, we also found that significant numbers of leukocytes were recruited to PR3-treated endothelial cells. However, in platelet-depleted whole blood, or blood treated with antibodies against GpIb or GpIIb/IIIa, we observed a dramatic reduction in leukocyte adhesion. Therefore, leukocyte recruitment to PR3-treated endothelial cells required platelet activation and adhesion and this was mediated in part by vWF.

Conclusions

Neutrophil PR3 can promote platelet adhesion to the vascular endothelium through a process requiring endothelial cell vWF, platelet GpIb and GpIIb/IIIa. PR3 also promotes leukocyte recruitment to the endothelium through a platelet-dependant process. This data demonstrates that PR3 can be a significant contributor to thrombus formation and leukocyte recruitment to the vascular endothelium and PR3 may be an important target for disrupting thrombus formation
in vivo
.
Abstract 412: In Silico Structural and Metabolic Profiling of Non-Synonymous Single Nucleotide Polymorphisms in APOA-I Gene
Authors: Dergunov Alexander D
Published: March 2022

Mature apoA-I contains 243 residues organized in two-domains as N-terminal four-helix bundle and C-terminal two helices. Cholesterol interaction with both lipid-free and lipid-bound apoA-I may be one of the key events in amyloidosis and reverse cholesterol transport (RCT). By the SIFT score, 20 non-synonymous missense mutations in APOA-I gene, located almost exclusively in N-domain, may impair apoA-I function. A majority (4 from 5) of putative cholesterol recognition amino acid consensus (CRAC: L/V-X(1-5)-Y-X(1-5)-R/K) and a single cholesterol consensus motif (CCM: R/K-X(2-6)-I/L/V-X(3)-W/Y) aligned within/between helices in the bundle. Importantly, CRAC2 in apoA-I co-aligned with CRAC2 in apoE and CRAC4 in apoA-IV. A short four amino acid sequence L44-L47 may present disorder-to-order transitioning binding region in apoA-I as predicted by molecular recognition feature (MoRF). Indeed, CCM corresponded to unstructured region with low stability in lipid-free apoA-I. Stabilizing effect due to the direct cholesterol-peptide interaction with the involvement of CRAC1 (L14-K23) and/or CCM (K40-W50) in amyloidogenic peptide 1-93 is suggested to underlie the known effect of cholesterol on the induction of helix formation in this peptide. Cholesterol may be speculated to act in helix formation as a chemical chaperone thus triggering disorder-to-order transition within MoRF; without cholesterol, this MoRF may be involved into protein-protein interaction. Indeed, the association of W50R substitution with the accelerated mutant aggregation rate and the involvement of L44-S55 segment into cross-beta-sheet formation were demonstrated by others. Also, D102H substitution in CRAC2 (V97-K106) may be harmful in RCT. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in CRAC2 motif of apoA-I in nascent HDL. Besides, the resistance of two-domain apoA-I structure to denaturation by GuHCl is sensitive to cholesterol level in discoidal HDL. Overall, different CRACs may contribute into amyloidosis-connected apoA-I misfolding and cholesterol efflux for lipid-free apoA-I. Cholesterol binding to lipid-bound apolipoprotein may change the structural plasticity of two domains and/or their relative dimensions.
Abstract 426: Selective Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: New Insights Into sPLA2-dependent Catabolism of HDL
Authors: Jayaraman Shobini
Published: March 2022

Introduction

Secretory phospholipase A2 (sPLA2) is a pro-inflammatory enzyme that promotes atherogenesis via several mechanisms. One of these mechanisms probably involves high-density lipoproteins (HDL). Previous in vitro studies of HDL modified by sPLA2 showed increased exposure of certain domains of the major HDL protein, apoA-I, without protein fragmentation. Over-expression of sPLA2 in transgenic mice reduced plasma levels of HDL cholesterol due to rapid HDL catabolism. The origin of this enhanced HDL catabolism is unknown.

Hypothesis

In addition to hydrolysis of HDL phospholipids, sPLA2 can also hydrolyze apoA-I, leading to enhanced catabolism of sPLA2 modified HDL.

Methods

Human plasma HDL and lipid-free apoA-I were incubated with sPLA2 at 37 oC. The HDL lipids were removed, and the protein integrity was analyzed by gel electrophoresis and mass spectrometry.

Results

Consistent with previous studies, HDL hydrolysis by sPLA2 slightly reduced the particle size without release of protein. SDS PAGE of hydrolyzed HDL showed no significant changes in the protein composition. Interestingly, delipidation of sPLA2-hydrolyzed HDL showed that nearly 10% of apoA-I formed well-defined fragments of 6-15 kDa. Incubation of sPLA2 with lipid-free apoA-I produces very similar fragments, indicating direct action of sPLA2 on apoA-I. Similar incubation of sPLA2 with other HDL proteins, apoA-II and apoC-I, did not lead to proteolysis, indicating enzymatic specificity of sPLA2 for apoA-I. ApoA-I hydrolysis by sPLA2 was Ca-independent and hence, occurred via a different mechanism than the phospholipid hydrolysis by sPLA2 that is Ca-dependent. Mass spectrometry analysis of isolated proteolytic fragments indicated several N-and C-terminal fragments of apoA-I, suggesting at least two cleavage sites in the central domain of apoA-I.

Conclusion

We have identified a previously unknown proteolytic activity of sPLA2 which is specific to apoA-I and is independent of the apoA-I lipidation. This novel enzymatic activity of sPLA2 helps explain enhanced catabolism of HDL in vivo, and is expected to contribute to the pro-atherogenic action of this pro-inflammatory enzyme.
Abstract 429: HDL Protects Against Polymicrobial-induced Sepsis in Mice
Authors: Guo Ling , Zheng Zhong , Howatt Deborah , Daugherty Alan , Huang Bin , Li Xiang-an
Published: March 2022

HDL has been considered a protective factor in sepsis, however, most contributing studies were conducted using endotoxic animal model; and evidence from clinically relevant septic animal models remains limited and controversial. Furthermore, little is known about the roles of HDL in sepsis other than LPS neutralization. In this study, we employed cecal ligation and puncture (CLP), a clinically relevant septic animal model and utilized ApoA-I knockout (ApoA-I KO) mice and ApoA-I transgenic (ApoA-I-tg) mice to elucidate the roles of HDL in sepsis. ApoA-I KO mice were more susceptible to CLP-induced septic death as shown by 47.1% survival of ApoA-I KO mice versus 76.7% survival of C57BL/6J (B6) mice (p = 0.038). ApoA-I KO mice had exacerbated inflammatory cytokine production during sepsis compared with B6 mice. Further study indicated that serum from ApoA-I KO mice displays less capacity for LPS neutralization compared with serum from B6 mice. In addition, ApoA-I KO mice had moderately impaired LPS clearance and a significant reduction in corticosterone generation in sepsis. In contrast to ApoA-I KO mice, ApoA-I-tg mice were moderately resistant to CLP-induced septic death compared with B6 mice. In conclusion, our findings reveal multiple protective roles of HDL in CLP-induced sepsis. In addition to its well-established role in neutralization of LPS, HDL exerts its protection against sepsis through promoting LPS clearance and modulating corticosterone production. Our study supports efforts to raise HDL levels as a therapeutic approach for sepsis.
Abstract 430: PCPE2 a Novel Determinant in HDL Biogenesis and Metabolism
Authors: Pollard Ricquita D , Weiser Erica , Blesso Christopher , Zabalawi Manal , Fulp Brian , Thomas Michael J , Sorci-Thomas Mary G
Published: March 2022

Recent attempts to reduce cardiovascular events by pharmacologically raising plasma HDL concentration have not proven efficacious. This has lead to investigations on how to increase functional versus dysfunctional plasma HDL. Our work shows that a functional subfraction from ABCA1 mediated biogenesis of nascent HDL (nHDL) consists of 3 apoA-I, 105 phosphotidylcholine, 27 sphingomyelin and 108 free cholesterol molecules, compositionally reminiscent of lipid rafts. Our studies are now focused on procollagen C-proteinase enhancer 2 (PCPE2), first described by Francone, OL, et al. (2011), to play a significant role in the ABCA1 mediated efflux of cholesterol to apoA-I. Based on previous studies, we hypothesize that PCPE2 binds lipid-free apoA-I thereby aligning specific helices among 3 molecules of apoA-I for lipidation. To test this, we created loss-of-function apoA-I mutant proteins to identify specific helical repeats that participated in nHDL formation. The various mutations span the central helices as well as the N- and C- termini of apoA-I. Opposing cysteines were placed within 3-5 å using a composite 3-D model of lipid-free apoA-I conformation. The mutant apoA-I disulfide “locked” (oxidized) and “unlocked” (reduced) status was monitored using mass spectrometric sequencing. Using both locked and unlocked versions of each of the mutant proteins, secondary structure as well as the extent of lipidation was investigated. In addition, studies, utilizing LDL receptor, PCPE2 double knockout mice (LDLr
-/-
, PCPE2
-/-
) fed an atherogenic diet have been carried out. Despite having a higher HDL concentration LDLr
-/-
, PCPE2
-/-
mice had significantly more aortic lipid deposition compared to LDLr
-/-
mice. The absence of PCPE2 substantially reduced the ability to efflux cholesterol suggesting that the HDL was dysfunctional. These data strongly suggest that PCPE2 participates in lipidation by helping apoA-I to either open or target a site on ABCA1 that promotes apoA-I opening.
Abstract 440: Non-HDL Cholesterol, Guideline Targets, and Population Percentiles: The Very Large Database of Lipids (VLDL-2 Study)
Authors: Elshazly Mohamed B , Martin Seth S , Joshi Parag H , Blaha Michael J , Kulkarni Krishnaji R , Jones Steven R
Published: March 2022

Background

Worldwide guidelines recommend a Non-HDL-C target of <100 mg/dL (30 mg/dL above LDL-C target) in high risk patients only when triglycerides (TG) ≥200 mg/dL, although TG of 100 mg/dL is considered “optimal” and TG 150-200 mg/dL is considered “elevated”. The performance of the present guidelines-based TG criterion in identifying treatable dyslipidemia is largely unknown.

Methods

We examined 1,340,614 U.S. adults in the Very Large Database of Lipids (VLDL). Lipid testing was performed by ultracentrifugation (Atherotech, Birmingham, AL). LDL-C was estimated by the Friedewald method, excluding patients with TG ≥400 mg/dL (2.3% of sample). We calculated the proportion of individuals reclassified using a population percentile based Non-HDL-C target versus the recommended target of <100 mg/dL.

Results

The equivalent Non-HDL-C percentile to LDL-C of 70 mg/dL was 93 mg/dL. In those with LDL-C <70 mg/dL, 15% had a Non-HDL-C≥100 mg/dL while 30% had a Non-HDL-C≥90 mg/dL. Considerable reclassification occurred at TG levels of 100-199 mg/dL.

Conclusion

If a population percentile based Non-HDL-C target of 90 mg/dL were used instead of the guideline mandated 100 mg/dL, twice as many patients with LDL-C <70 mg/dL would be eligible for treatment, including a considerable proportion of patients with TG <200mg/dL. More generalized use of population percentile based Non-HDL-C targets may be superior for secondary prevention at TG levels <200 mg/dL, with implications for guideline development.