bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 281: Nogo-B, a Novel Regulator of Macrophage Foam Cell Formation
Authors: Zhang Xinbo , Yu Jun
Published: March 2022

Nogo-B, a novel regulator of macrophage foam cell formation Macrophage-derived foam cell formation has long been considered a requisite and initiating event in the development and progression of atherosclerosis. Nogo-B belongs to the reticulon family of proteins and plays important roles in vascular remodeling and angiogenesis
in vivo
. Previously, we have shown that Nogo
-/-
mice on an ApoE
-/-
background develop larger atherosclerotic lesions and increased lipid content compared to ApoE
-/-
mice
in vivo
. In the present study, we will define the role of Nogo-B in foam cell formation and its underlying mechanisms. Our results show that both a high-fat regiment and modified LDL stimulation upregulate Nogo-B in macrophages
in vivo
and in culture, respectively. Loss of Nogo in macrophages increased cholesterol binding, uptake and foam cell formation. Moreover, the loss of Nogo-B specifically increased the expression of type I scavenger receptor A (SR-AI) in modified LDL treated macrophages and in the laser capture microdissected macrophages from atherosclerotic lesions
in vivo
. Mechanistically, we show that Nogo-B directly binds to MEKK3, and loss of Nogo-B enhanced JNK2 phosphorylation and its downstream c-Jun/AP-1 phosphorylation and nuclear translocation. ChIP assays show c-Jun/AP-1 directly binds to the AP-1 binding sites in the promoter region of Nogo-B and SR-AI and promotes transcription of Nogo-B and SR-AI. Collectively, we have identified Nogo-B as a novel regulator of foam cell formation. Our results suggest that 1) modified LDL activates MEKK3/JNK2/c-Jun signaling pathway, and c-Jun directly activates Nogo-B and SR-AI; 2) upregulated Nogo-B interacts MEKK3 and modulate foam cell formation through negative feedback on the MEKK3/JNK2 pathway.
Abstract 286: Concordance Between Ankle-Brachial Index Measurement Methods (Palpatory Versus Doppler)
Authors: Orellana-Barrios Menfil A , Penagos-Cordon Jose C , Cifuentes-Escobar Luis M , Rodriguez-De Leon Edgar R , Perez-Jordan Adolfo E , Oliva-Gonzalez Edgar A
Published: March 2022

Peripheral arterial disease (PAD) is one of the manifestations of atherosclerosis and its detection is commonly made by ultrasound Doppler screening to estimate the ankle brachial index (ABI). However, this method may be unavailable in developing countries with low diagnostic resources. Measurement of the ABI via a palpatory method offers an inexpensive, readily available alternative approach for early disease detection of PAD.We measured the ABI in 186 patients (48% male, mean age 65 SD 12.5), prospectively hospitalized in an Internal Medicine ward (HGSJDD in Guatemala City, Guatemala) for unrelated reasons and no previous diagnosis of PAD. Two independent observers then performed ABI measurement via either a palpatory or Doppler method on all patients. The palpatory method involved digital palpation of foot arteries and use of a calibrated mercury sphygmomanometer. The Doppler technique was performed using a 8 MHz probe and the same calibrated sphygmomanometer. PAD cases were identified as having an ABI < 0.9. A total of 36 cases of abnormally low ABI (positive for PAD) were identified by Doppler method (19.4% prevalence). Of these, 30 were also appropriately identified as positive cases by the palpatory method. A total of 150 normal range ABI's were identified by Doppler method, and of these, 145 were concomitantly also categorized as normal by the palpatory method. Overall, we found a 94.1 % concordance rate (175 of 186 paired categorizations). The Cohen's kappa coefficient was determined to be 0.808, and in comparison to the gold standard Doppler method, the sensitivity and specificity of the palpatory method was 83% and 96%, respectively. We conclude that the palpatory method is a reliable method to measuring ABI in medical patients, particularly in resource restrained settings.
Abstract 293: ApoB-Containing Lipoproteins Contribute to Augmentation of AngII-Induced Abdominal Aortic Aneurysms
Authors: Liu Jing , Lu Hong , Howatt Deborah , Balakrishnan Anju , Moorleghen Jessica , Sorci-Thomas Mary , Cassis Lisa , Daugherty Alan
Published: March 2022

Background and objective:
Presence of abdominal aortic aneurysms (AAA) is associated with dyslipidemia in humans. Hypercholesterolemic mice having a predominance of ApoB-containing lipoproteins are more susceptible to Angiotensin (Ang)II-induced AAA formation. A recent publication demonstrated that exogenously delivered high-density lipoprotein (HDL) prevented AngII-induced AAAs. ApoAI is the major apolipoprotein of HDL. The purpose of this study was to determine whether manipulations of ApoAI or ApoB-containing lipoproteins influence the development of AngII-induced AAA.

Methods and Results:
Eight week old male ApoAI +/+ or -/- mice with either wild type or LDL receptor -/- background were fed normal diet and infused with AngII (1 mg/kg/min) for 28 days. Plasma lipoprotein distributions were resolved by size exclusion gel chromatography. Deficiency of ApoAI led to profound reductions of plasma HDL-C concentrations in both strains. However, ApoAI deficiency did not exacerbate AngII-induced AAA as determined by maximal outer widths of suprarenal aortas (ApoAI +/+ versus -/-:1.52 ± 0.25 versus 1.22 ± 0.18 mm, P= 0.2 for C57BL/6 strain;1.21 ± 0.18 versus 1.11 ± 0.17 mm, P= 0.2 for LDLR -/- strain). Ezetimibe, an intestinal cholesterol absorption inhibitor, was administered to eight week old male ApoE -/- mice fed normal diet. Ezetimibe decreased plasma cholesterol concentrations from 451 ± 8 to 204 ± 18 mg/dl (P<0.001). This reduction was due to reductions in ApoB-containing lipoproteins. Administration of ezetimibe decreased AngII-induced AAA (control versus ezetimibe: 2.25 ± 0.28 mm versus 1.37 ± 0.1 mm, P=0.009). The same dose of ezetimibe was also administered to ApoE -/- mice fed western diet. Plasma cholesterol concentrations were reduced (from 980 ± 86 to 546 ± 11 mg/dl, P<0.001) attributed to reductions of apoB-containing lipoproteins. Administration of ezetimibe led to a profound reduction of atherosclerosis in western diet fed apoE -/- mice, but did not attenuate AngII-induced AAAs.

Conclusion:
ApoB-containing lipoproteins, rather than HDL, contribute to the augmentation of AngII-induced AAAs. However, substantial reductions in apoB-containing lipoproteins were required to attenuate AngII-induced AAA.
Abstract 31: A Novel Fluorescence-Based Assay is Used to Investigate the Triglyceride Hydrolytic Activity of Lipases and to Identify Synthetic Peptides With Strong Lipase-Stimulating Activities
Authors: Janizek Joseph D , Munro Kim , Davidson Michael H , Ancsin John B
Published: March 2022

Hypertriglyceridemia is estimated to affect about one third of Americans and is an independent risk factor for cardiovascular disease, pancreatitis and xanthomatosis. Lipoprotein lipase (LPL) is a key enzyme involved in the hydrolysis and clearance of triglycerides from plasma and its enzymatic activity is regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. We have developed a fluorescence-based lipase assay that utilizes a fluorogenic triglyceride analog (EnzChek®, Molecular Probes) and facilitates the rapid analysis of lipase enzyme kinetics in a microplate format. This assay has made it possible for us to undertake the screening of the Prestwick (1,200 FDA-approved drugs) and Natural Product (~3,000) libraries at the University of Illinois high-throughput screening facility, in order to identify lipase activating compounds. To validate the assay we have characterized the influence of heparin, different metals and apolipoproteins on the hydrolytic activities of LPL in vitro, and hepatic lipase in HepG2 cell culture. As expected, enzyme activities were supported by heparin and calcium, and both apoC-II and apoC-III when complexed with VLDL, stimulated and inhibited lipase activity, respectively. Interestingly, the heparin/Fe2+ complex was a strong inhibitor of lipase activity, possibly explaining the high incidence of hypertriglyceridemia associated with iron supplementation to treat iron-deficiency. Finally, using a synthetic peptide approach we have localized apoA-V’s lipase stimulating and heparin binding domains to a short amphipathic, alpha-helical peptide. When included in the assays this peptide could increase lipase hydrolytic rates by 3-6 fold both in vitro, and in cell culture. Given that on a molar basis apoA-V is about 1200-fold less abundant than apoC-II, we anticipate that apoA-V will provide valuable sequence information for the development of potent triglyceride lowering therapeutics.
Abstract 32: Liver-Specific Transgenic Expression of Cholesteryl Ester Hydrolase Reduces Atherosclerosis in LDLR-/- Mice
Authors: Bie Jinghua , Wang Jing , Yuan Quan , Kakiyama Genta , Ghosh Siddhartha S , Ghosh Shobha
Published: March 2022

Liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC) and lipoprotein cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol mainly as cholesteryl esters (CE) from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human cholesteryl ester hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on SR-BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver led to significant increase in flux of HDL-CE to bile acids. In LDLR-/- background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis in LDLR-/-LCEH2 mice (Figure) with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.
Abstract 370: Interferon Regulatory Factors 3 and 7 Regulate Vein Graft Remodeling and Vascular Inflammation
Authors: de Vries Margreet R , Karper Jacco C , Peters Erna A , van der Pouw Kraan Tineke C , de Jong Rob C , Hamming Jaap F , Quax Paul H , Horrevoets Anton J
Published: March 2022

Introduction:
Type I interferons (IFN) are implicated in the development of vascular proliferative diseases. Pathway analysis by gene set enrichment analysis of hypercholesterolemic ApoE3*Leiden murine vein grafts revealed that the type I IFN pathway belonged to the top 15 of significant regulated pathways. The transcriptional regulators of type I IFN and type I IFN responsive genes are the interferon regulatory factors (IRF). Activation of TLR3 results in activation of type I IFN in a IRF3 and IRF7 dependent manner. TLR3 has been shown to be protective in vascular remodeling. The aim of this study was to investigate the role of IRF3 and IRF7 on vein graft remodeling.

Methods and Results:
The importance of IRFs in vein graft remodeling is illustrated by the increase in vein graft thickening in

Irf3
-/-

and

Irf7
-/-

mice in comparison to control mice (n=9/group,

Irf3
-/-

; 39%, p=0.185,

Irf7
-/-

; 68% p=0.003). Also an increase in outward remodeling (

Irf3
-/-

; 26%, p=0.081,

Irf7
-/-

; 42%, p=0.049) was observed comparable to that of

Tlr3
-/-

(n=8, 52%, p<0.001) Immunohistochemical analysis revealed that both

Irf3
-/-

and

Irf7
-/-

mice showed a significant higher influx of macrophages in the vessel wall than the control mice whereas the

Irf7
-/-

mice also showed a significant decrease in collagen content. RNA levels of typical type I IFN responsive genes such as Mx1, Ifit1-3 and Oas2 were down regulated in the knockout vein grafts in comparison to control vein grafts. Activation of both

Irf3
-/-

and

Irf7
-/-

bone marrow derived macrophages with LPS and poly:(IC) resulted in a significant increase in TNFα production.

Conclusions:
IRFs regulates vein graft remodeling since

Irf3
-/-

and especially

Irf7
-/-

vein grafts show increased vessel wall thickening and outward remodeling. This increased remodeling is the result of a pro-inflammatory response as reflected by the increase in macrophages in the vein graft wall.
Abstract 375: Mast Cell-Specific Regulation of Cardiomyocyte Contractility: Reevaluation of Role in Cardiac Function Following Myocardial Infarction
Authors: Ngkelo Anta , Richart Adele , Vilar Jose , Marck Pauline , Heymes Christophe , Rodewald Hans-Reimer , Silvestre Jean-Sebastien
Published: March 2022

Inflammatory cells orchestrate post-ischemic cardiac remodeling after myocardial infarction (MI). Studies in Kit mutant mice suggest key roles for mast cells in post-ischemic tissue remodeling. However, Kit mutations affect multiple cell types of both immune and non-immune origin. The aim of this study was to address the impact of mast cells on cardiac function following MI, using selectively mast cell-deficient mice (Cpa3Cre/+ mice).

The number of mast cell progenitor (Lin-CD45+CD34+FcγRII/III+β7+) increased in the bone marrow and the white adipose tissue on day 3 after MI, infiltrated the heart on day 5 and differentiated to mature mast cells (Cd117+FcεRI+Sca1+) by day 7 in cardiac tissue. To assess the functional effect of mast cell infiltration, the cardiac function 14 days following MI was assessed in wild-type (WT) and 3 mast cells deficient mice models, the kitW/Wv mice, the Cpa3Cre/+ mice and mice treated with the degranulation inhibitor DSCG (50μg/g, once a day). There was a significant decrease of the left ventricular shortening fraction in all 3 mast cell deficient models compared to WT mice (1.4-, 1.6- and 2-fold respectively) that correlated with increased cardiomyocyte apoptosis in the kitW/Wv mice; and increased fibrosis in the DSCG-treated mice (n=8-14 per group, p<0.01 vs WT). Interestingly, in the Cpa3Cre/+ mice there were no differences on post-MI cardiac remodeling, but instead we observed a significant reduction in cardiomyocyte contractility (both cell shortening and maximal contracting velocity) (p<0.01) with no effect on Ca2+ transient peak or sarcoplasmic reticulum load.

This work re-evaluates existing mast-cell deficient models on their effect on cardiac remodeling and identifies a novel function for mast cells on modulation of cardiomyocyte contractility, Ca2+ cycling independently. Identification of the molecular regulators involved in the cardiac effect of mast cell will provide a new platform of regulators with potential therapeutic properties.
Abstract 379: Modulation of Inflammatory Signaling by PPARα and PPARγ in Acrolein-Treated gp91phox Knockout Mice
Authors: Yousefipour Zivar , Newaz Mohammad A
Published: March 2022

Acrolein, a copious environmental pollutant plays a role in development of inflammation in vasculature. Oxidative stress & inflammation are important in acrolein-induced toxicity. Peroxisome Proliferator Activated receptor alpha and gamma (PPARα and PPARγ) and their ability to influence oxidative stress (OS) has been the focus of inflammatory signaling. NAD(P)H oxidase is a source of free radical generation. This study investigated the influence of PPARα and γ ligands (clofibrate & GW1929) in attenuating oxidative stress in acrolein-induced insult. gp91phox knockout (KO) mice and age/weight matched wild type (WT) were treated with acrolein (0.5 μg/kg; i.p.; 7 days) with/without clofibrate (Clof: 250 mg/kg; i.p; 10 days) or GW1929 (5 mg/kg, gavage, 10 days). KO mice (1.44±0.14 pg/μg prt) had higher (97%) 8-Isoprostane in response to acrolein than WT (2.19±0.11 pg/μg prt). Clof had no effect on 8-Isoprostane but GW1929 reduced it in both WT and KO mice by 35% and 43% (p<0.05) respectively. There was a significant reduction in Total Antioxidant Status (TAS) which was more prominent in WT (69%) than KO (53%). Clof and GW1929 improved TAS in WT (21% and 19%). Xanthine Oxidase (XO) enzyme activity was higher in KO (0.53±0.05) than WT (0.35+0.02). Acrolein and clof increased XO activity in WT while GW1929 had no effect. Acrolein increased cycloxygenase (COX) in WT (4.58±0.3) and remained at basal level in KO. Clof and GW1929 reduced COX in WT (54% and 48% respectively). Clof reduced Cox only in KO mice (31%). NFκB activity was twice in acrolein treated WT and remained unchanged in KO. Both GW1929 and clof blunted this increase. Acrolein increased CD36 activity, an inflammatory marker, in KO (56%). Clof attenuated this increase in KO but increased it in acrolein treated WT. GW1929 blunted CD36 increase in KO mice but did not affect WT. These results suggest that in absence of a functioning NAD(P)H oxidase other oxygenase contributes in acrolein-induced oxidative stress and PPARα ligand, clofibrate, is beneficial via attenuating inflammatory signaling of NFκB and COX. We also suggest that beneficial effect of PPARγ ligand involves inhibition of NAD(P)H oxidase as in absence of NAD(P)H this effects are minimal.
Abstract 380: PDI and ERp57 Localization in Platelets Is Regulated by Actin Polymerization
Authors: Crescente Marilena , Pluthero Fred G , Schenk Michael P , Kahr Walter H , Gibbins Jonathan M
Published: March 2022

Introduction:
PDI and ERp57 are members of the thiol isomerase family of enzymes that are released to the platelet surface where they contribute to a range of platelet responses. PDI has been reported to be present in subcellular structures in resting platelets, although it is excluded from α - and δ -granules, and lysosomes. It was, however, recently shown to co-localise with TLR9 in T-granules, intracellular bodies underlying the plasma membrane. The subcellular localisation of other platelet thiol isomerases or their mechanisms of translocation to the cell surface have not been established.

Hypothesis and Methods:
Using spinning disk confocal microscopy we sought to explore whether platelet thiol isomerases ERp57 and PDI are co-localised in resting and activated platelets and to test the hypothesis that translocation of thiol isomerases to the platelet surface is dependent on actin polymerization.

Results and Conclusions:
In resting platelets, PDI and ERp57 were organized in punctate structures both on the platelet surface and in the cytoplasm. They did not colocalise with P-selectin consistent with them residing outside α -granules. In contrast to expectations, TLR9 did not colocalize with PDI and ERp57, suggesting their exclusion from T-granules. Partial colocalization was observed in resting platelets between PDI and ERp57 in granule-like structures. Upon platelet activation, this degree of co-distribution persisted with PDI and ERp57 migrating to the platelet surface. Latrunculin A, an inhibitor of actin polymerization, decreased P-selectin exposure on the platelet surface, as measured by flow cytometry, and agonist-stimulated platelets treated with this agent retained a rounded shape, as shown by tubulin staining. Importantly, latrunculin A also blocked the translocation of PDI and ERp57 from internal granule-like structures to the platelet surface. We conclude that in resting platelets PDI and ERp57 are organized, and partially co-localised, in punctuate structures unlikely to represent α- or T-granules. The polymerization of actin during platelet activation exerts a fundamental role in the relocalization of PDI and ERp57 from these cytoplasmic structures to the platelet surface.
Abstract 388: Regular Aerobic Exercise Enhances Endothelium tPA Release in Adults With HIV-1
Authors: Diehl Kyle J , Stauffer Brian L , Greiner Jared J , Connick Liz , DeSouza Christopher A
Published: March 2022

HIV-1 infection is associated with vascular endothelial dysfunction and increased risk of cardiovascular disease. We have previously demonstrated that the capacity of the endothelium to release tissue-type plasminogen activator (t-PA), the primary activator of endogenous fibrinolysis, is markedly impaired in HIV-1-seropositive compared with seronegative adults, regardless of antiretroviral therapy. Endothelial fibrinolytic dysfunction underlies thrombogenesis and is thought to contribute to the increased risk of atherothrombotic events in HIV-1-infected adults. Regular aerobic exercise can beneficially affect vascular health and reduce cardiovascular risk. However, whether aerobic exercise training can alleviate HIV-1-related endothelial fibrinolytic dysfunction is unknown. In an ongoing study, we are testing the hypothesis that regular aerobic exercise improves endothelial t-PA release in HIV-1 seropositive adults. Net endothelial release of t-PA was determined, in vivo, in response to intrabrachial infusions of bradykinin (BK: 125-500 ng/min) and sodium nitroprusside (SNP: 2-8 μg/min) before and after a 3-month aerobic exercise intervention. To date, 10 HIV-1 seropositive adults (age: 38+2 yr; 8 M/2 F) have completed the exercise intervention (walking ~5.1 d/wk, ~50 min/d @ ~70% of maximal heart rate). There were no changes in body weight, % body fat, or maximal oxygen consumption after exercise training, however, treadmill exercise time to exhaustion increased 25% (from 9.0+0.4 to 11.2+0.4 min; p<0.01). The capacity of the endothelium to release t-PA in response to BK was significantly higher after (-0.58+1.5 to 102.3+15.5 ng/100 mL tissue/min) vs before (-0.8+1.0 to 48.7+10.1 ng/100 mL tissue/min) exercise training. In addition, total amount of t-PA released (area under the BK curve) was increased more than two-fold (329+73 vs 728+108 ng/100 mL tisuue) in response to the exercise intervention. There was no effect of exercise training on either t-PA release to SNP or endothelial release of plasminogen activator inhibitor-1. These initial results indicate that habitual aerobic exercise is an effective lifestyle intervention strategy for improving endothelial fibrinolytic capacity in HIV-1 infected adults.
Abstract 42: Haptoglobin Genotype as a Marker of Vascular Intraplaque Hemorrhage
Authors: Binesh Marvasti Tina , Singh Navneet , Tyrrell Pascal , Wong Betty , Cole David , Moody Alan
Published: March 2022

Background:
Intraplaque hemorrhage (IPH), a component of late stage atherosclerosis, is a critical factor in plaque destabilization leading to stroke and myocardial infarction. Magnetic resonance imaging (MRI) is able to identify IPH and allows for identification of high risk patients. Currently however, no routinely available tests are conducted to identify individuals at risk of IPH.

IPH is a rich source of free hemoglobin (Hb), a potent oxidant. The plasma protein haptoglobin (Hp) binds Hb forming a Hb-Hp complex, that can be engulfed by tissue macrophages.The reduction in oxidative stress decreases vascular inflammation. In humans, the Hp gene has three different genotypes: Hp1-1, Hp2-2 and Hp1-2. The Hb-Hp2-2 complex has a lower binding affinity to macrophages, resulting in higher oxidative burden. Numerous studies have demonstrated a higher risk of cardiovascular events in Hp2-2 individuals. We therefore hypothesized that patients of a Hp2-2 genotype have a greater risk of MR-IPH.

Methods:
From 2010 to 2014, patients with non-surgical carotid artery disease (30-95% stenosis) underwent 3T carotid MRI that included a MR-IPH sequence. A radiologist with 25 years of expertise in MR-IPH imaging determined the presence of MR-IPH using widely accepted methods. Patients’ charts were blindly reviewed for demographics and medical history. Patients were genotyped for Hp1/2 using an established PCR genotyping method. Statistical association analysis was performed using logistic regression.

Results and Conclusions:
Out of the 80 recruited patients (mean age, 72.8 years; range 52-100), those with Hp2-2 genotype vs. Hp1-1/Hp1-2 had a prevalence of MR-IPH of 65% compared to 41%(p=0.09). Non-diabetics (HbA1c <6.5%) with Hp2-2 genotype had an increased risk of IPH compared to non-diabetics with Hp1-1/Hp 1-2 genotype (OR=3, 95% CI 1.04-8.73, p=0.03). Males were also at an increased risk of IPH (OR=4.05, 95% CI 1.53-10.70, p=0.004) and the majority (71%) of males with IPH were of the Hp2-2 genotype. In conclusion, non-diabetic Hp2-2 male patients with history of vascular disease are the population with the highest risk of developing IPH and they can be identified using simple haptoglobin genotype testing.
Abstract 430: A Novel Nonradioactive Method Documents Strong Elevation of the Cholesterol Esterification Rate by CSL112, a Novel Formulation of Human Apolipoprotein A-I
Authors: Boerema David J , Martin Kirstee L , Meikle Peter J , Wright Samuel D , Gille Andreas
Published: March 2022

Background:
CSL112 was designed as an infusible formulation of human apoA-I to rapidly remove plaque cholesterol and reduce risk after an ACS. Following the initial efflux of unesterified cholesterol from cells, the second step of reverse cholesterol transport is the esterification of cholesterol by LCAT. Unfortunately, the current methods for measuring cholesterol esterification rate (CER) require radioactive labeling, are impractical for clinical laboratories and are unable to quantify individual cholesteryl esters (CE). In this study, we developed a novel non-radioactive method using D7-cholesterol as tracer and liquid chromatography-mass spectrometry (LC-MS) to detect and quantify seven different CE formed by LCAT esterification in plasma samples.

Methods:
Plasma from normal healthy subjects was spiked with a range of concentrations (0 - 2.8 mg/mL) and formulations of human apoA-I (purified HDL; CSL112 (formulated with soy phosphatidyl choline (PC)); or formulated with sphingomyelin (SM). Dithionitrobenzoic acid was added to block esterification, and D7-cholesterol tracer was allowed to diffuse into the lipoproteins by 24 h, 4°C incubation with filter disks containing hydrous D7-cholesterol crystals. The esterification reaction was conducted at 37°C for 1 h in the presence of β-mercaptoethanol to reactivate LCAT. Newly formed D7-CE were quantitated by LC-MS following extraction from the plasma.

Results:
As expected, native HDL did not increase CER and was used as control. The addition of CSL112 elevated CER in a concentration dependent manner. CSL112 is formulated with soy PC, a good substrate for LCAT, and the fatty acids appearing in the CE reflect the fatty acid composition of the soy PC. To confirm a role for the added PC in elevation of CER we showed that substitution of SM for PC caused CER to decrease rather than increase.

Conclusion:
A sensitive non-radioactive method for CER has been developed that allows the quantification of LCAT-mediated formation of seven individual CE. CSL112, an apoA-I formulation containing soy PC supported concentration-dependent increases in CER, while a formulation of apoA-I with SM inhibited CER in a concentration-dependent manner.
Abstract 433: Examination of Factors Affecting the Association of PCSK9 With Low-Density Lipoprotein Particles in Human Plasma
Authors: Golder Mia , Sarkar Samantha , Kosenko Tanja , McPherson Ruth , Lagace Thomas A
Published: March 2022

Rationale:
We have previously shown that a substantial proportion of plasma PCSK9 (30-40%) is associated with LDL particles in normolipidemic subjects. Cellular assays show that LDL-bound PCSK9 is less active for binding to cell surface LDLRs. Therefore, the ability of circulating PCSK9 to direct LDLR degradation in liver could be regulated by plasma LDL levels. In addition, LDL subspecies may have altered abilities in binding PCSK9. We have mapped the LDL binding region to a short stretch of amino acids (aa 31-52) in the PCSK9 prodomain. It is unknown whether a common loss-of-function PCSK9 mutation (R46L) within this region affects LDL binding.

Objective:
To determine whether plasma PCSK9 distribution (LDL-bound versus unbound) is affected in hypercholesterolemic subjects. To further characterize the interaction of PCSK9 and LDL, we investigated the interaction of PCSK9 with two subspecies of LDL - large, buoyant LDL (LBLDL; d=1.019-1.044 g/ml) and small, dense LDL (SDLDL; d=1.044-1.063 g/ml). Additionally, we investigated the effect of the R46L PCSK9 mutation on the LDL binding affinity of PCSK9.

Methods and Results:
We used flotation ultracentrifugation in Optiprep density gradients to fractionate human plasma samples followed by immunoprecipitation and western blot to quantify PCSK9 distribution in LDL and non-LDL fractions. In a pilot study, the proportion of total plasma PCSK9 in the LDL fraction was increased from 38±5% to 57±3% (N=6) in hypercholesterolemic subjects (LDL>4.9mM, TG<2.3mM) versus normal controls (LDL<3 mM, TG<2.3mM). Saturation binding assays showed that SDLDL bound PCSK9 with lower affinity (Kd = 361.9 nM) than LDLDL (Kd = 263.9 nM). Competition binding assays determined that recombinant purified PCSK9-R46L secreted from HEK293 cells did not bind to isolated LDL with significantly altered affinity compared to wild-type PCSK9.

Conclusion:
Our preliminary results indicate that plasma PCSK9 distribution is altered in hypercholesterolemia, with an increased proportion of total PCSK9 bound to LDL particles. Our in vitro results suggest that circulating small, dense LDL may bind more poorly to PCSK9 than larger LDL subspecies.
Abstract 458: Poor Progression of in-Stent Neoatherosclerosis in Patients With Everolimus-Eluting Stent Implantation: Coronary Angioscopic Observation
Authors: Ichikawa Minoru , Yoshima Shohei , Takei Yoshizumi , Kawai Kenji , Oshita Takeshi , Fukuda Kenji , Takagi Takashi , Kijima Yoshiyuki
Published: March 2022

Very late stent thrombosis (VLST) is one of the most serious issues in the era of drug-eluting stents (DES). VLST has been attributed to delayed neointimal stent coverage (NSC) due to bio-suppressive drugs and vulnerability of in-stent neoatherosclerosis. The latter was recently proposed by pathologists as in-stent atherosclerotic lesions at chronic phase of coronary stenting. The aim of this study was angioscopic observation of in-stent vessel wall at 3 years after implantation of various generations of DES. Enrolled were 37 stented segments in 26 patients with stable angina (25 Cypher sirolimus-eluting stents, SES; 5 Endeavor zotarolimus-eluting stents, ZES; and 7 Xience V everolimus eluting stents, EES). Dual anti-platelet therapy, aspirin plus clopidogrel, had been continued since the stenting procedure. Coronary angiography showed no in-stent restenosis at chronic phase. Coronary angioscopy revealed the in-stent appearance; that is, grades of NSC [between score 0 (no coverage) and score 3 (full coverage)], presence of yellow plaque (YP), and subclinical stent thrombosis (ST). There was no significant difference in grades of NSC among SES, ZES, and EES (1.40±0.57, 1.71±0.77, and 1.80±0.75, respectively). YP and ST were not found in EES. YP was 24% and 20% in SES and ZES, respectively. ST was 20% both in SES and ZES. In conclusion, in-stent neoatherosclerosis appears not to progress at least for 3 years in EES-stented coronary segments, a representative of the second generation of DES.
Abstract 478: Single-Cell Isolation and Analysis of Viable Proliferating Macrophages From Atherosclerotic Plaques
Authors: Zavitz Caleb C , Ensan Sherine , Robbins Clinton S
Published: March 2022

INTRODUCTION:
We have recently shown that the proliferation of macrophages within the atherosclerotic plaque, rather than the recruitment of new monocytes from the blood is the key driver of plaque growth in established atherosclerosis. The study of proliferating macrophages has been hampered by the lack of a technique for identifying proliferating macrophages and isolating them from the atherosclerotic plaque in a viable state.

OBJECTIVE:
Develop and validate a process for identifying proliferating macrophages in atherosclerotic lesions, and isolating them as single, viable cells, then perform a molecular characterization of these cells.

METHODS:
ApoE-/- and LDLR-/- mice were fed a diet high in fat and cholesterol. Atherosclerotic aortas were collected, and cells were isolated by mechanical and enzymatic dissociation. Cells were stained using fluorescent markers and DNA-binding dyes, then analyzed and isolated by fluorescence-activated cell sorting.

RESULTS:
Mincing and enzymatic digestion in collagenase I, collagenase XI, DNAse, and hyaluronidase optimally isolates macrophages from aortic explants. Cell-surface staining with fluorescent antibodies against B220, F4/80, Ly6c, MHC II, and CD11b allows identification of lesional macrophages. DNA staining with Vybrant DyeCycle Violet allows identification of proliferating cells. Combined, these processes allow for the FACS-based sorting of viable proliferating macrophages from atherosclerotic plaque, and subsequent RNA or protein analysis, or culture.

CONCLUSIONS:
The process described here allows the first-ever viable isolation of proliferating macrophages from atherosclerotic plaques. This will permit the investigation of molecular targets to interfere with the pathogenesis of atherosclerosis.
Abstract 519: Consumption of Energy Beverage is Associated With an Attenuation of Arterial Endothelial Flow--Mediated Dilatation
Authors: Higgins John , Yang Benjamin , Ortiz Brandon , Herrin Nikki , Doolittle Johnathan , Kahlden Kyle , Dayah Tariq , Cassel David , Ali Asif
Published: March 2022

Introduction:
Energy drinks are being consumed more and more worldwide, and have been associated with some deaths in adolescents and young adults, especially when consumed while exercising. The consumption of energy drinks before or during exercise might be linked to an increased risk for myocardial ischemia as a symptom of endothelial dysfunction. Energy beverage consumption and its acute effect on endothelial function is not well-studied.

HYPOTHESIS

Consumption of an energy drink will acutely impair endothelial function in a young healthy adult.

Methods:
After fasting from caffeine for at least 24 h and food for at least 8 h prior, eleven healthy non-smoker medical students (9 males), average age 24.5 years (range 23-27 years), average BMI 22.8, received an ECG, blood pressure & pulse check, and underwent baseline testing (BL) of endothelial function using the technique of endothelium-dependent flow-mediated dilatation (FMD) with high-resolution ultrasound (according to recommended guidelines of the University of Wisconsin Atherosclerosis Imaging Research Program Core Laboratory). The subjects then drank an energy beverage (EB), a 24-oz can of Monster Energy Drink®, and the above was repeated at 90 min after consumption. The FMD was calculated as the ratio between the post-cuff release diameter and the baseline diameter. Each FMD image was checked for quality control, and each artery diameter was measured as the distance between opposing tunica medias by two experts. Three measurements were taken at the QRS complex, repeated on three separate beats, and then all were averaged.

Results:
The hemodynamics were as follows, BL vs EB group respectively (mean+/-SD): Heart rate 65.2+/-11.3 vs 68.2+/-11.8 beats per minute, Systolic Blood pressure 114.0+/-10.4 vs. 114.1+/-10.4 mmHg, Diastolic blood pressure 68.8+/-9.3 vs. 70.6+/-7.1 mmHg; all were not significantly different. However, consumption of the EB resulted in a significantly attenuated peak FMD response (mean+/-SD): BL group 5.9+/-4.6% vs. EB group 1.9+/-2.1%; P=0.03).

Conclusions:
Acute exposure to an energy beverage impairs arterial endothelial function in healthy young adults. Given the increased use of these beverages associated with exercise in young adults, more research is needed.
Abstract 521: Endothelial Cell Forward Migration in a Disturbed Wall Shear Stress Environment is Promoted by Rho-Kinase Inhibition
Authors: Hsiao Sarah , Tovar-Lopez Francisco , Gunn Julian , Spencer Timothy , Halliday Ian , Perrault Cecile , Evans Paul
Published: March 2022

Stent deployment to treat coronary artery disease causes damage and loss of endothelial cells (EC). Repair of injured arteries by EC migration is co-ordinated by Rho family GTPases. It is also regulated by wall shear stress (WSS), a mechanical force exerted by flowing blood on the vessel wall, via mechanisms that are poorly understood. It was hypothesised that stent struts may impede repair of injured endothelium by inducing localised disturbances in WSS.

To simulate a stented artery in vitro, chamber slides were fabricated with ridges (100 μm high) positioned perpendicularly to the flow direction. Confluent EC monolayers seeded on one side of either ridged or non-ridged (control) chambers were exposed to flowing culture medium (Ibidi® system). Flow patterns were determined by computational fluid dynamic (CFD) modelling and particle velocimetry. Live cell imaging and analysis using ImageJ® software enabled quantification of EC migration velocity and directional persistence (DP).

EC on the non-ridged chamber slide were exposed to a uniform WSS of 13 dyn/cm2 and migrated relatively uniformly in parallel with the flow direction (average velocity 1.13 μm/min; DP 0.59). By contrast, CFD and particle velocimetry revealed spatial WSS differences over the ridged chamber, with significant spikes above physiological levels (>70 dyn/cm2) at the corners of the ridges and distinctive flow recirculation zone immediately downstream from ridges (-4 dyn/cm2). While EC were able to migrate over the ridges, cells that reached the recirculation zone displayed non-uniform directionality (DP 0.25) and reduced velocity (0.78 μm/min). Inhibition of the RhoA signalling pathway with ROCK inhibitors promoted EC forward migration within the recirculation zone by elevating DP (0.41) and velocity (1.16 μm/min).

It was concluded that disturbed WSS downstream from stent strut-like ridges prevented the forward migration of EC. Inhibition of the RhoA/ROCK signaling pathway promoted EC migration and re-population of these sites. Our data suggest that treatment using a ROCK inhibitor may promote re-endothelialisation of stented arteries.
Abstract 528: MicroRNA-30 Family Inhibits Neointima Hyperplasia by Targeting CaMKIIδ
Authors: Liu Yongfeng , Singer Harold
Published: March 2022

miRNA is one class of small and non-coding RNAs, which regulate coding gene expressions. miR-30 family members are abundantly expressed in the vascular smooth muscle layer in the artery walls. However, the roles of miR-30 in vascular smooth muscle and vascular diseases are little unknown. Here, we report that the expression of miR-30 family members is dramatically reduced in the vascular walls upon vascular injury. Rescuing down-regulated miR-30 by overexpression using lenti-vitral delivery in injured rat carotid artery significantly inhibited injury-induced neointima formation. In vitro, the expression of miR-30 family members in the dedifferentiated cultured vascular smooth muscle cells (VSMCs) was approximately 50% lower than it in the intact vascular smooth muscle. Western blot analysis and luciferase assay showed that miR-30 directly targeted 3' UTR of calcium calmodulin dependent protein kinase II δ isoform (CaMKIIδ) and inhibited CaMKIIδ expression in the culture VSMCs. VSMC proliferation was significantly inhibited by overexpressing miR-30 and this inhibitory effect was partially recovered by CaMKIIδ rescue. These data suggest that miR-30 is a strong and novel regulatory factor in neointima formation, and may be developed to be a new therapeutic target to prevent or reduce vascular diseases.
Abstract 546: Systemic Transcriptional Alterations of Innate-Adaptive Immune Signaling Pathways in Atherosclerosis, Ischemic Stroke and Myocardial Infarction
Authors: Huang Chiang-Ching , Chantler Paul , VanGilder Reyna , Barr Taura
Published: March 2022

Objective:
The immune system regulates inflammation associated with chronic cardiovascular disease. The primary aim of this study was to compare gene expression profiles of three innate and adaptive immune signaling pathways --the toll-like receptor (TLR), T-cell receptor (TCR), and B-cell receptor (BCR) signaling pathways in patients with subclinical atherosclerosis, acute ischemic stroke (IS), and myocardial infarction (MI).

Methods:
Peripheral blood gene expression profiles from human atherosclerotic diseases were downloaded from Gene Expression Omnibus (GEO): Atherosclerosis (GSE20129: 48 patients, 71 controls), IS (GSE16561: 39 patients - samples within 24 hours from symptom onset, 24 controls), and MI (GSE29111, 18 MI patients (7 and 30 days) samples after experiencing MI). Genes in the TLR (106 genes), TCR (104 genes), and BCR (72 genes) pathways were retrieved from the NCBI BioSystems database. Gene set enrichment analysis was used to evaluate the statistical significance of gene enrichment in each signaling pathway for each study. Weighted Gene Co-expression Network Analysis was used to identify gene modules and evaluate network connectivity across the disease states.

Results:
All three immune pathways significantly enriched differentially expressed genes in each disease state (P<0.01) - TLR (38% in atherosclerosis, 47% in IS, 30% in MI); TCR (51% in atherosclerosis, 54% in IS, and 53% in MI); BCR (57% in atherosclerosis; 60% in IS; 43% in MI). Transcriptional changes in the pathways are highly concordant across the diseases. The correlation of expression changes between any two diseases ranges from 0.53 to 0.76 (mean±SD: 0.66±0.09, P<0.00001). Although TLR genes were all up-regulated in each condition, network analysis revealed a striking down-regulation of gene modules in the TCR and BCR pathways. Of significance, the TCR-CD3 complex and CD79A/B were significantly down-regulated in patients of all three disease states.

Conclusion:
We found a strong overlap in expression between innate and adaptive immune pathways in three atherosclerotic related diseases. The down regulation of the TCR-CD3 complex suggests immune suppression is a common occurrence in either chronic or acute inflammation associated atherosclerotic disease.
Abstract 569: Neutrophil Extracellular Traps Promote Thrombin Generation Through Platelet-Dependent and -Independent Mechanisms
Authors: Gould Travis J , Vu Trang , Swystun Laura , Dwivedi Dhruva , Weitz Jeffrey , Liaw Patricia
Published: March 2022

Objective:
Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) which are comprised of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from septic patients.

Approach and Results:
NETs released from phorbyl myristate (PMA) activated neutrophils enhance thrombin generation in platelet-poor plasma (PPP). This effect was DNA-dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with FXII- and FXI-depleted plasma). In platelet-rich plasma treated with corn-trypsin inhibitor, addition of PMA-activated neutrophils increased thrombin generation and shortened the lag time in a TLR-2- and TLR-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In PPP plasma samples from septic patients, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation.

Conclusions:
These studies are the first to examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis.
Abstract 599: Cardiovascular Risk Factors and Cardiovascular Events in Patients Undergoing Liver Transplantation
Authors: Donovan Robert J , Ali Asghar , Giles Hochong , Mohmand Asad , Heuman Douglas M , Jovin Ion S
Published: March 2022

Introduction:
Patients undergoing liver transplantation often have cardiovascular risk factors and may experience cardiovascular morbidity and mortality. Understanding the role of risk factors and the benefit of treatment is important for the careful selection of liver transplantation candidates to minimize post-transplant cardiac morbidity. We investigated the prevalence of cardiovascular risk factors and their association with cardiovascular events in liver transplantation patients.

Methods:
We studied 182 consecutive patients undergoing liver transplantation in our center between 1997 and 2011 to evaluate factors associated with new cardiovascular events after liver transplantation.

Results:
The cohort consisted of 179 men and 3 women with a mean age of 58.8 ± 6.2 years, model for end-stage liver disease (MELD) score of 17.4 ±7 at initial evaluation, and Framingham risk score of 9.0 ±5.1. In this cohort 15 ( 8.2%) patients were smokers, 48 (26.4%) had hypertension, 47 (25.8%) had diabetes mellitus, 35 (19.2%) had coronary artery disease, and 27

(14.8%) had dyslipidemia prior to their transplant surgery. After a median follow-up of 5.6 years, a total of 52 (28.6%) patients died and 6 (3.3%) died of cardiac causes. New coronary events and/or revascularization procedures post-transplant occurred in 22 (12%) patients. The pre-transplant variable associated with post-transplant cardiac events and/or revascularization was smoking status at the time of evaluation (P=0.01). Post-transplant factors associated with cardiac events included use of beta-blockers (P=0.01) or statins (P=0.01).

Conclusion:
Although cardiovascular risk factors and coronary disease are very common, cardiovascular events are relatively infrequent in patients undergoing liver transplantation. Smoking at time of transplant evaluation and post-transplant medical therapy with beta-blockers and statins are significantly associated with the occurrence of post-transplant cardiac events including revascularization.