bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 396: Acute Hepatic ACAT2 Knockdown Transiently Increases Plasma and Hepatic Free Cholesterol and Fecal Neutral Sterol Excretion
Authors: Marshall Stephanie M , Wilson Martha D , Kelley Kathryn L , Lee Richard G , Crooke Rosanne M , Graham Mark J , Rudel Lawrence L , Brown J M , Temel Ryan E
Published: July 2021

In spite of the advent of statins, atherosclerotic coronary vascular disease (ASCVD) remains the number one killer of Americans. A way to reduce LDL cholesterol, the primary risk factor of ASCVD, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both biliary and non-biliary pathways. The lipoprotein that delivers cholesterol from the liver through the plasma to the small intestine for transintestinal cholesterol excretion (TICE) is not yet known. We have previously shown that chronic knockdown in mice of hepatic acyl-CoA cholesterol acyltransferase 2 (ACAT2), a cellular enzyme that converts free cholesterol (FC) into cholesteryl ester (CE), appeared to cause the formation of hepatic apoB-containing lipoproteins that preferentially trafficked cholesterol to the small intestine for TICE. We tested the hypothesis that the plasma concentration of TICE-competent, apoB-containing lipoproteins, could be increased by preloading the liver with cholesterol and then acutely depleting the cholesterol by knocking down hepatic ACAT2 with antisense oligonucleotides (ASO). After feeding a high cholesterol (0.2% wt/wt) diet for six weeks, C57BL/6 mice were treated with control non-targeting ASO or ACAT2 ASO for one or two weeks. After only one week of ACAT2 knockdown (ACAT2KD) hepatic ACAT2 protein expression was decreased nearly 80%. This translated into a 50% decrease in hepatic CE concentration in conjunction with a rarely seen 2-fold increase in hepatic FC concentration. Acute hepatic ACAT2KD increased plasma FC levels by 25%, which subsided after two weeks of treatment. The increased plasma FC was primarily associated with large and small LDL. After one week of hepatic ACAT2KD mice had a minor, non-significant increase in biliary cholesterol levels but had a 2-fold increase in fecal neutral sterol (FNS) excretion. In summary our data show that when CE is rapidly cleared from the liver, FNS levels dramatically increase with a transient increase in hepatic and plasma FC levels. We believe that in the ACAT2KD mice the increased plasma FC associated with LDL is feeding into TICE thus resulting in increased FNS.
Abstract 428: Peptides Enrichment by Hydrophobic Interaction Chromatography for Detection of Oxidized Phospholipid-modified Protein Adducts
Authors: Gao Detao , Willard Belinda , Podrez Eugene
Published: July 2021

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury, and ischemia. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interaction between oxPLs and proteins could provide important information about the type of proteins preferentially modified by oxPLs and on the mechanism of interaction between proteins and oxPLs. These studies are very challenging due to low levels of oxPLs protein adducts. We have developed a highly efficient enrichment method for oxPLs-modified peptides that employs C18 solid phase extraction columns. This method takes advantage of the difference in polarity between oxPLs modified peptides and the corresponding unmodified peptides as well as other interfering compounds. Using this enrichment method, we have been able to detect very low levels of oxPLs modified peptides (< 2 ppm) from tryptic digestion mixture of cell lysate using LC-MS. To demonstrate the performance of the method, we studied the interaction of apolipoprotein A-I (apoA-I) in high density lipoprotein with a specific oxPL (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine, POVPC) at pathophysiological concentrations. We found that Lys 94, Lys 96 and Lys 195 are covalently modified by POVPC. Thus, the present study is the first to detected oxPL adducts of apoA-I in HDL.
Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles
Authors: Athar Humra , Jiang Zhenghui G , McKnight Christopher J
Published: July 2021

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.
Abstract 457: CD36, but not G2A, Modulates Efferocytosis, Inflammation and Fibrosis Following Bleomycin-induced Lung Injury
Authors: Black Leland L , Parks Brian W , Zimmerman Kurt A , Metz Allison E , Steele Chad , Murphy-Ullrich Joanne E , Kabarowski Janusz H
Published: July 2021

Oxidatively modified lipids and their by-products presented or released by cells undergoing apoptosis are thought to regulate phagocytic uptake of apoptotic cells by macrophages (efferocytosis) through CD36 scavenger and G2A chemotactic receptors. Although the ability of these receptors to mediate clearance of apoptotic cells in the context of atherosclerosis may have significant impact on lesion development and progression/stability, an overabundance of the lipid ligands for these receptors (CD36: oxidized phospholipids, G2A: lysophosphatidylcholine) as a result of oxidative processes associated with atherogenesis could conceivably impair these clearance mechanisms. Because the indolent nature of atherosclerotic lesion development precludes an accurate assessment of the spatial and kinetic features of lesional efferocytosis, we employed a bleomycin-induced model of lung injury to assess the requirement for G2A and CD36 in efferocytosis. This is a model which, similarly to atherogenesis, is associated with oxidative stress, but in which local apoptosis can be induced and efferocytosis subsequently measured over time. As there is substantial
in vivo
evidence that ApoE has a role in efferocytosis, we included ApoE knock-out mice as controls. Loss of CD36 (similarly to ApoE deficiency) delayed the clearance of apoptotic alveolar cells, potentiated inflammation (increase in lung neutrophils, lung KC [CXCL1] levels, and lung macrophages) and reduced lung fibrosis following bleomycin-induced lung injury. Reduced fibrosis in CD36-/- mice was associated with lower levels of pro-fibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1 and reduced interstitial myofibroblasts. Despite the widely held notion that G2A mediates an LPC dependent “find me” signal in macrophages facilitating apoptotic cell clearance, G2A deficiency had no significant effect on the clearance of apoptotic cells in the bleomycin-induced lung injury model. Our results show that CD36 and ApoE (but not G2A) are important for apoptotic cell clearance following lung injury and subsequently modulate inflammatory and fibrotic processes that could impact not only inflammatory lung disease but also atherosclerosis.
Abstract 462: Ascl3 Protects Vascular Smooth Muscle Cells Against Oxidative Stress
Authors: Biswas Swarajit K , Hernandez Liliana , Xiao Yan , Ahmad Mushtaq , Garcia-Barrio Minerva T
Published: July 2021

Atherosclerosis initiation and progression depends on multiple risk factors and is sustained by self-perpetuating inflammatory cellular and molecular events that ultimately translate into increased oxidative stress inducing endothelial dysfunction, macrophage activation and altering vascular smooth muscle cell (VSMC) homeostasis. Our initial work identified the under-characterized bHLH achaete-scute like 3, ASCL3, as a pro-proliferative anti-apoptotic factor in VSMC and prompted the hypothesis that ASCL3 plays a protective role against oxidative stress in VSMC. We determined that ASCL3 expression is up-regulated in the media and lesions of the atherosclerotic aorta in ApoE-mice fed a high fat diet.
In vitro
, ASCL3 expression is consistent with an adaptive role in oxidative stress. In primary RASMC, time- and dose-response to H
2
O
2
(0.5-2mM, 4-24h) result in marked ASCL3 mRNA increase (~10-fold) at low concentrations or shorter times and is reduced as oxidative stress increases. siRNA knockdown of ASCL3 increases susceptibility of RASMC to 0.25mM H
2
O
2
(low dose) as determined by caspase-3 activation. Stable over-expression of ASCL3 in A7r5 cells results in enhanced protection at higher doses of H
2
O
2
, and is associated with higher AKT and ERK1/2 phosporylation. This protective effect involves the mitochondrial pathway since ASCL3 up-regulates Bcl-2/Bax ratio in basal and induced conditions (0.4mM, 4h), inhibits mitochondrial depolarization, prevents mitochondrial fragmentation and preserves mitochondrial DNA. ROS levels are significantly reduced in ASCL3 over-expressing cells (0.2mM, 2h). ASCL3 over-expression appears to increase mitochondrial density and results in altered mitochondrial and cristae morphology as determined by TEM. Surprisingly, subcellular localization studies indicate that ASCL3 localizes primarily to the mitochondria. In summary, low oxidative stress in VSMC promotes survival, with enhanced proliferation, and as it increases, induces adaptation associated with an antioxidant gene expression profile. Eventually high oxidative stress leads to cell death. Altogether, our data indicate that ASCL3 plays a protective role in the survival and adaptation of vascular smooth muscle cells to oxidative stress.
Abstract 483: Clot Degradation Assay for the Analysis of Vascular Bed Fibrinolytic Reactivity
Authors: Clark Amanda L , Kumar Neil , Roztocil Elisa , Gillespie David , Cullen John P
Published: July 2021

Introduction

Pulmonary emboli cause significant morbidities and risk of mortality to affected patients, yet the mechanism behind their formation is still elusive. A greater understanding of clot formation and fibrinolysis in the pulmonary artery (PA) and iliac vein (IV) vascular beds has become an important area of research in order to find better therapeutics for the treatment of deep vein thrombi and pulmonary emboli. The aim of this study was to develop a novel
in vitro
clot degradation assay for the comparison of fibrinolytic reactivity of different vascular beds.

Methods

Thrombin was added to fibrinogen at 37
°
C for 24 hours and the resulting fibrin clot was centrifuged, dried and weighed. Human PA and IV endothelial cells were incubated for 24 hours and cellular extract and media samples were collected and evaluated for their ability to degrade fibrin clots in the presence of plasminogen. Fibrinolysis, stimulated by plasmin formed by endothelial cell derived plasminogen activator, was measured as percent clot degradation by weight.

Results

Fibrinogen and thrombin dose responses were completed to find the appropriate concentrations of each to use for clot formation: fibrinogen (6.04x10
-6
M) and thrombin (0.0005 NIH units). To validate the clot degradation assay, a uPA dose response was conducted from 150fM to 15nM uPA and demonstrated that each ten-fold increase in uPA concentration corresponded to an average of 14% degradation (p<0.05, n=3) up to 72.5% degradation in the presence of 15 nM uPA. While PA cell extracts showed no effect on clot degradation, IV cell extracts increased degradation by 30%. However, when clots were exposed to PA media there was a 14% increase in clot degradation (p<0.05, n=3), whereas the corresponding IV media samples had no effect on clot weight.

Conclusions

This study demonstrates the development of a novel and reproducible methodology to examine fibrinolytic reactivity in cell culture samples. Endothelial cells from the pulmonary artery circulation exhibit greater ability to extrinsically degrade fibrin clots when compared to endothelial cells from the iliac venous bed which may explain, in part, why more clots are found in the deep veins and not the pulmonary vasculature.
Abstract 530: Diurnal and Day-to-Day Variability of Endothelial Progenitor Cells
Authors: Campia Umberto , Saber Rana , Liao Yihua , Liu Kiang , MDermott Mary , Vaughan Douglas , Lloyd-Jones Donald
Published: July 2021

Background

Circulating endothelial progenitor cells (EPCs) may contribute to vascular repair. Whether a diurnal and/or day-to-day variability of circulating EPCs is physiologically present in healthy adults has not been investigated.

Methods

Eighteen healthy adults underwent EPCs count on three different time points: two in the same day between 7:00 and 8:30 and between 12:30 and 14:00; and one in another day between 7:00 and 8:30. EPCs (CD34+CD45lo; CD34+CD45loCD133+; and CD34+CD45loCD31+) were quantified using multi-parameter flow cytometry and an ISHAGE based gating strategy. Variability of EPCs was assessed by coefficient of variation (CV) between time points in the same day and in different days.

Results

Baseline participant characteristics are reported in table 1. EPCs measurements and CVs are reported in table 2.

Conclusions

In healthy middle-aged adults, circulating EPCs appear to have a marked diurnal variability but a low day-to-day variability. Further study is needed to understand the mechanisms of EPCs variability and its biological significance.
Abstract 69: Anacetrapib Dose-dependently Decreases Atherosclerosis Development and Adds to the Beneficial Effects of Atorvastatin in APOE*3Leiden.CETP Mice
Authors: Kühnast Susan , van der Tuin Sam J , Havekes Louis M , Willems van Dijk Ko , Rensen Patrick C , van der Hoorn José W , Jukema J W , Princen Hans M
Published: July 2021

Introduction

The residual risk of cardiovascular disease that remains after statin treatment has triggered the search for a secondary treatment target. Epidemiological studies propose HDL-cholesterol (HDL-C) as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. In human intervention trials, the CETP inhibitor anacetrapib decreases (V)LDL-C by 30-40% and increases HDL-C by 40-140%.

Hypothesis

Complete inhibition of CETP activity may result in adverse effects as compared to partial inhibition due to the appearance of a dysfunctional HDL. We, therefore, evaluated the effect of a broad treatment window of anacetrapib-induced CETP inhibition with partial to full inhibition, as well as the combination of atorvastatin and anacetrapib on atherosclerosis development in APOE*3Leiden.CETP mice.

Methods

Female mice were fed a Western-type diet containing 0.1% cholesterol without or with incremental dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/d), atorvastatin (2.4 mg/kg/d) or a combination of anacetrapib (0.3 mg/kg/d) and atorvastatin (2.4 mg/kg/d) for 20 weeks. Effects on plasma lipids, CETP activity and levels, as well as atherosclerotic lesion size and severity were assessed.

Results

Anacetrapib dose-dependently reduced CETP activity (-60% to -100%, P<0.001) and increased CETP levels (+13% to +31%, P<0.05), thereby decreasing nonHDL-C (-23% to -44%, P<0.001) and increasing HDL-C (+32% to +88%, P<0.001). Atorvastatin decreased CETP activity (-29%, P<0.001) and nonHDL-C (-36%, P<0.001). Anacetrapib dose-dependently decreased atherosclerotic lesion size (-36%, P<0.05 to -92%, P<0.001) and the percentage severe lesions. Anacetrapib added to the effects of atorvastatin by further reducing nonHDL-C (-36%, P<0.001), increasing HDL-C (+72%, P<0.001) and decreasing lesion size (-86%, P<0.001) and severity. Results on lesion composition are pending.

Conclusions

Anacetrapib dose-dependently decreases atherosclerosis development and adds to the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. Total blockage of CETP activity does not reveal adverse effects as compared to partial blockage.
Abstract 114: Macrophages Mediate the Ability of the Receptor for Advanced Glycation End Products to Prevent Formation of Abdominal Aortic Aneurysm in a Murine Model
Authors: Kell Pamela , Ennis Terri , Chang Katherine , Hotchkiss Richard , Thompson Robert W , Schmidt Ann Marie , Raman Kathleen G
Published: July 2021

INTRODUCTION:
The pathophysiology of AAAs is multifactorial, involving chronic inflammation, increased MMP expression/activity, and medial elastic fiber destruction. RAGE transduces the biological impact of ligand families including AGEs, S100, and HMGB1 to upregulate inflammatory and tissue injury-provoking genes including MMPs. We previously identified increased RAGE expression in human AAAs and have shown that the absence of RAGE, whether by genetic deletion or pharmacologic blockade with sRAGE, prevents AAA formation in a mouse model. We sought to characterize the cellular mediators of this effect.

METHODS:
C57BL/6 wild-type (WT) and RAGE null (KO) mice underwent transient elastase perfusion to induce AAAs. Mice were sacrificed at day 14 and flow cytometry performed to characterize differences in activation (CD69+) of lymphocytes (CD4/8), neutrophils (GR1+/CD11b+), macrophages (F480), NK cells (NK1.1) (n=24/group). Immunohistochemistry was performed for Mac-3 and Arg 1 (M2 marker) or Arg 2 (M1 marker). Primary BMDMs were isolated and skewed towards M1 (LPS/IFN ¡) or M2 (LPS, IL-4, IL-13) with the addition of S100b or HMGB1 to determine the ligands' effect upon macrophage skewing by ELISA.

RESULTS:
Flow cytometry revealed increased aortic expression of activated CD8+ cells (p=0.002), macrophages (p-0.002), and NK cells (p=0.03). No differences were noted in cellular activation from spleens from the same animals. Immunohistochemistry revealed increased macrophage expression in WT vs KO aortae, highest at day 5 (p=0.04). Arg 1 and 2 expression on macrophages was elevated in WT vs KO animals (p=0.03). Skewing BMDMs from WT and KO towards M1 led to elevated TNFα with addition of either S100b (p=0.02) or HMGB1 (p=0.03). Skewing BMDMs towards M2 led to elevated IL-10 in KO vs WT cells with the addition of S100b (p=0.01). HMGB1 has no effect upon skewing towards M2.

CONCLUSIONS:
The effect of RAGE in the development of murine AAA appears to be mediated by macrophages. Differential macrophage skewing towards an M1 by both S100b and HMGB1 likely plays a role in this effect. Skewing towards M2 is mediated by S100b and not HMGB1. Further studies will seek to identify at which stage in AAA formation activation of the RAGE-ligand axis becomes functionally important.
Abstract 117: Hypertension Modulates Sexual Dimorphism of Aortic Aneurysm in TGF-ß--Deficient Mice via ERK Activation
Authors: Schmit Bradley M , Fu Chunhua , Yang Pu , DeSart Kenneth , Shalit Bernie , Berceli Scott A , Jiang Zhihua
Published: July 2021

INTRO:
Hypertension and gender are established risk factors for aortic aneurysm (AA) development, yet recent evidence suggests that alterations in TGF-beta signaling is one of the key initiators of AA. We have previously shown that loss of the Alk5 TGF-B type 1 receptor in male but not female mice leads to AA (100% penetrance). Here, we explore the interaction of gender, biomechanical forces, and TGF-B biology. We hypothesize that the female Alk-5 deficient mice are a subclinical phenotype that can be unmasked by the biologic alterations that accompany systemic hypertension.

METHODS:
SMC specific Alk5 deletion (Alk5-/-) was achieved via an inducible Cre-loxP system. Wild-type (Alk5f/f) and Alk5-/- females received continuous infusion of Norepinephrine (NE) to increase blood pressure. Non-infused Alk5f/f and Alk5-/- females were included as controls. Aortic pathology was examined grossly and histologically at 4 wks. Production of p-Erk, an index for activation of the non-canonical TGF-B signaling pathway, was quantified on aortic tissue before and after 2wks of treatment.

RESULTS:
NE elevated SBP in both groups by 35 mmHg. The combination of Alk5-/- and HTN (Alk5-/-/HTN) in female mice increased the penetrance of AA from zero to 57% (p< .05). In contrast, no pathology was detected in the wild-type/HTN or ALK5-/- mice, where animals were only affected by a single factor. Alk5-/-/HTN aortas showed significant upregulation (2.1x, p<.05) of pErk, while a single factor, either Alk5-/- or HTN, was unable to enhance early Erk signaling in affected aortic tissue.

CONCLUSION:
Hypertension breaks the sex-protective effects on AA formation induced by SMC specific Alk5 deletion in females. The combination of hypertension and Alk5 deficiency is required to intensify an early Erk signaling and subsequently induce aortic pathology. This strong association suggests Erk serves as the biological mediator for hemodynamic stress modulated sexual dimorphism during AA development.
Abstract 123: Recently Ruptured Carotid Plaques Have Increased Levels of Circular RNA-16, Which Negatively Regulates the Proproliferative and Antiapoptotic MicroRNA-221: A Novel Mediator of Carotid Plaque Rupture
Authors: Bazan Hernan A , Lightell Daniel , Sternbergh W Charles , Woods T Cooper
Published: July 2021

Objectives:
Circular RNAs (circRNAs) are dynamically expressed during development and possess binding sites for microRNAs (miRs), small RNAs that negatively regulate gene expression. We recently demonstrated that miR-221, which is associated with VSMC proliferation and inhibition of apoptosis, is decreased in acutely symptomatic carotid plaques. As circRNA-16 possesses binding sites for miR-221 thru seed sequences found within, we hypothesized that circRNA-16 is increased in acutely symptomatic compared to asymptomatic carotid plaques.

Methods:
Relative changes in gene expression levels of circRNA-16 were compared using a real-time PCR assay and the ΔΔCt method. All samples were run in duplicate; mean/standard error were calculated. One-way ANOVA with Tukey’s test was used to determine significance between groups.

Results:
Expression of circRNA-16 was confirmed in human VSMC using PCR and resistance to RNAse H. To investigate its role in carotid plaque rupture, levels of circRNA-16 and miR-221 were quantified in patients undergoing urgent CEA for acute neurologic symptoms (n=27), with a mean duration of symptom onset to CEA of 2.4 days, compared to asymptomatic carotid plaques (n=19). In contrast to miR-221, circRNA-16 is increased in the urgent group compared to the asymptomatic carotid plaque group (Figure; 1.51±.26 vs. 1.00±.10, p=.03).

Conclusions:
We demonstrate circRNA-16 levels are increased and miR-221 levels decreased in acutely ruptured carotid plaques. Furthermore, our data suggest that a circRNA-16/miR-221 axis may be important in fibrous cap degradation and rupture during the transition from a stable to an unstable carotid atherosclerotic plaque.
Abstract 141: Coronary Heart Disease-Associated Variation in TCF21 Disrupts a MicroRNA-224 Binding Site and miRNA-Mediated Regulation
Authors: Miller Clint L , Haas Ulrike , Diaz Roxanne , Leeper Nicholas J , Kundu Ramendra K , Patlolla Bhagat , Assimes Themistocles L , Kaiser Frank J , Perisic Ljubica , Hedin Ulf , Maegdefessel Lars , Schunkert Heribert , Erdmann Jeanette , Sczakiel Georg , Quertermous Thomas
Published: July 2021

Introduction:
Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated variant in this region, rs12190287, resides in the 3’ untranslated region of the basic-helix-loop-helix transcription factor,
TCF21
, and is predicted to alter the seed binding site for miR-224. Allelic imbalance studies demonstrated significant imbalance of the
TCF21
transcript that correlated with genotype at rs12190287.

Hypothesis:
We hypothesize that genetic variation at rs12190287 contributes to allele-specific
TCF21
expression imbalance via miR-224 regulation, thus altering CHD risk.

Methods and Results:
Reporter assays in human coronary artery smooth muscle cells (HCASMC) showed that the disease-associated C allele is selectively targeted by miR-224, leading to reduced expression compared to the protective G allele. Kinetic analyses
in vitro
revealed faster RNA-RNA complex formation and greater binding of miR-224 with the
TCF21
C allelic transcript. In addition,
in vitro
probing with Pb
2+
and RNase T1 revealed structural differences between the
TCF21
variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and
TCF21
expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of
TCF21
to transforming growth factor- and platelet derived growth factor signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis
in vivo
.

Conclusions:
Together, these data suggest that miR-224 interaction with the
TCF21
transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for CHD at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of
TCF21
, in conjunction with previous studies showing that this variant modulates transcriptional regulation through AP-1, suggests a unique bimodal level of complexity previously unreported for disease-associated variants.
Abstract 146: Expression of Axl, a Receptor Tyrosine Kinase, Regulates Vein Graft Remodeling
Authors: Korshunov Vyacheslav A , Ko Kyung A , Donlon Kathy , Mickelsen Deanne , Doyley Marvin M , Morrell Craig N , Batchu Sri N
Published: July 2021

Introduction:
We recently found that expression of Axl, a receptor tyrosine kinase, in non-bone marrow derived cells is important for carotid thickening in response to low flood flow. We hypothesize that Axl plays a role in smooth muscle cell functions during vein graft remodeling.

Methods and Results:
A segment of inferior vena cava was harvested from male Axl wild type (Axl+/+) or knockout (Axl-/-) mice and anastomosed to a carotid artery in female Axl+/+ mice under isoflurane anesthesia. Ultrasound Vevo2100 system was used to evaluate successful engraftment of the veins. Initial assessments of the blood flow through the graft were done at 1week after the surgery. A 3-dimensional high-resolution imaging was performed in mice after 3weeks after surgery. We found similar mean blood flow velocity ~110mm/s in the vein grafts between Axl+/+ >Axl+/+ and Axl-/- >Axl+/+ mice. There were no differences in the length of the graft between the groups in reconstruction of 3D-Mode imaging. However, graft volumes had a tendency (p~0.1) to be lower in Axl-/- >Axl+/+ vs. Axl+/+ >Axl+/+ mice (5.3±0.2 vs. 5.8±0.4mm
3
). We also observed ~20% reduction of graft wall thickness in Axl-/- >Axl+/+ mice. Finally, pulsatility and resistive indexes of vein grafts were ~10% lower in Axl-/- >Axl+/+ mice.

Conclusions:
This is the first study to demonstrate contribution of Axl to vein graft remodeling. Although, to confirm ultrasound differences between experimental groups vein graft wall changes are being evaluated by histology.
Abstract 149: Regulator of G-Protein Signaling-1 Modulates Leukocyte Trafficking in Atherosclerosis and Aortic Aneurysm Formation Through Chemokine Receptor Desensitization
Authors: Patel Jyoti , McNeill Eileen , Douglas Gillian , Hale Ashley , de Bono Joseph , Greaves David R , Channon Keith M
Published: July 2021

The regulation of macrophage recruitment and retention into the vascular wall is critical in the progression of atherosclerosis and aortic aneurysm formation. This can be mediated by chemokine activation of multiple G-protein coupled receptors. The Regulator of G-Protein Signaling-1 (RGS1) acts to deactivate the intracellular response to sustained chemokine stimulation. We have found that RGS1 is upregulated with atherosclerotic plaque progression and with monocyte-macrophage activation but its role is unknown.

Rgs1-/- macrophages have significantly enhanced migratory responses to atherogenic chemokines and have impaired desensitization to chemokine re-stimulation (p<0.001). In vivo, RGS1 has a role in the accumulation of macrophages in atherosclerotic lesions and during Angiotensin II (AngII) aortic aneurysm rupture. In the absence of RGS1, atherosclerosis and macrophage accumulation is attenuated in early lesions in the aortic root and aortas of ApoE-/- mice (p<0.001). Rgs1-/- mice are protected from AngII induced aneurysm rupture compared to ApoE-/- mice with 94% survival vs. 56%. Rgs1-/- mice have significantly fewer CD11b+ myeloid cells and CD14+ macrophages in aortas than ApoE-/- mice (p<0.05) after 5 days of AngII infusion.

Following bone marrow transplantation, recipient mice receiving ApoE-/- bone marrow were more susceptible to aortic aneurysm rupture (p=0.0124), indicating bone marrow-derived RGS1 is required for aneurysm rupture. Furthermore, AngII treatment increased systolic blood pressure to a greater extent in Rgs1-/- mice than ApoE-/- mice suggesting aneurysm formation in these mice is independent of AngII induced hypertension and this is mediated by vascular-derived RGS1. To gain insight into the mechanism by which RGS1 regulates trafficking, we selectively labelled inflammatory monocytes in vivo to track their movement into aortas following AngII infusion. We found an accumulation of labelled CD45+ cells in the aortas of ApoE-/- mice from day 3 to day 5 but not in Rgs1-/- mice indicating RGS1 as a regulator of macrophage retention in aortic aneurysms.

These findings identify a novel role for RGS1 in leukocyte retention in vascular inflammation, highlighting RGS1 as a potential target in cardiovascular disease.
Abstract 186: Use of Optical Coherence Tomography to Study Arteriogenesis After Hind-Limb Ischemia
Authors: Tang Gale L , Shi Lei , Eliahoo Galit , Li Yuandong J , Wang Ruikang
Published: July 2021

Introduction:
Because of their small size, direct measurement of collateral artery flow and diameter is difficult, so laser Doppler perfusion is used as a surrogate with micro-computerized tomography (microCT) performed to assess collateral networks and diameters after sacrifice. We applied optical coherence tomography (OCT) to measure artery diameter, flow, and velocity serially after hindlimb ischemia.

Hypothesis:
OCT is superior to microCT for measuring arteriogenesis.

Methods:
OCT was performed on the hindlimb musculature of male C57BL/6 mice, ages 3-5 months (n=5) bilaterally prior to unilateral femoral artery ligation, immediately after, and weekly for four weeks. Femoral artery and collateral artery diameter, flow, and axial velocity were measured. On postoperative day 28, mice were sacrificed by pressure perfusion fixation with formalin and injected with contrast. MicroCT was then performed at 18 micron resolution. Collateral artery diameter and vessel density from 3-d reconstructed datasets were analyzed using Matlab.

Results:
Figure 1 shows representative OCT (1A) and microCT (1B) images. Femoral artery flow and axial velocity decreased significantly after ligation (0.046 ± 0.040 from 2.1 ± 0.5 mm
3
/sec,
p
=0.006 and 1.1 ± 1.0 from 16.0 ± 1.1 mm/sec,
p
<0.001). The velocity of the ligated femoral was still significantly slower at 28 days (5.0 ± 1.9 vs 11.9 ± 1.0 mm/sec). Collateral artery flow reversed direction after ligation. At 28 days, the velocity in the collateral artery had increased significantly (12.4 ± 2.1 from 1.7 ± 1.0 mm/sec at baseline). MicroCT tended to overestimate the unligated collateral diameter (95 ± 9 vs 63 ± 8 microns by OCT,
p
=0.0537), but was accurate for the ligated side collaterals.

Conclusions:
Optical coherence tomography provides important physiologic and structural data regarding arteriogenesis following hindlimb ischemia and is superior to microCT for assessment of arteriogenesis.
Abstract 213: Phospholipid Transfer Protein Deficiency Attenuates High-Fat Diet--Induced Obesity and Insulin Resistance
Authors: Song Guohua , Zong Chuanlong , Shao Mingzhu , Yu Yang , Guo Shoudong , Liu Qian , Zhao Hui , Lin Quanqiang , Wang Xinnong , Lee Phoebe , Luo Yi , Jiang Xian-Cheng , Qin Shucun
Published: July 2021

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with diabetes, obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover we found that the deficiency induced significantly more GLUT4 immunostaining in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP deficient hepatocytes had less sphingomyelin and free cholesterol in the plasma membrane and lipid raft than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increasing in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity in high-fat-diet induced insulin resistance mice model.

Foundation:National Natural Science Foundation of China (# 81070247, 81170785) and Taishan Scholar Foundation of Shandong Province.
Abstract 226: Serum Amyloid A Isoforms and the Capacity to Mediate Cholesterol Efflux
Authors: Yassine Hussein , Trenchevska Olgica , Borges Chad , Nedelkov Dobrin , Nelson Randall W , Reaven Peter D
Published: July 2021

Serum Amyloid A (SAA) is an acute phase reactant protein that exists in multiple isoforms, can form HDL, and participates in cholesterol efflux. In vitro studies suggest that the SAA 2.1 isoform has an increased capacity to mediate cholesterol efflux compared to the other isoforms. We examined SAA isoforms using a novel mass spectrometric immunoassay (MSIA) and HDL’s cholesterol efflux capacity (via ABCA-1 and SR-BI) in samples from 59 subjects with (n=33) and without type 2 diabetes (n=26). SAA 1.1 levels were detectable in 58, SAA 2.1 in 14 and SAA 2.2 in 36 of the 59 subjects. SAA 2.1 levels significantly correlated with SR-BI cholesterol efflux (r=0.71, p=0.01, n=14), but not ABCA-1 mediated efflux (r=0.1, P=0.1). This correlation was not explained by changes in HDL phospholipids, Apo A-I or HDL cholesterol levels. In contrast, SAA 2.2 or 1.1 levels did not correlate with changes in SR-BI or ABCA-1 mediated efflux. Although the SAA 2.1 isoform is less frequently detected in plasma, our data confirm that it is closely linked with HDL mediated cholesterol efflux, particularly that is SR-BI mediated.
Abstract 232: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal ß-Sheet in the Lipid Transfer and ApoB Secretion Activities of Microsomal Triglyceride Transfer Protein
Authors: Walsh Meghan T , Di Leo Enza , Ozaydin Eda , Tarugi Patrizia , Hussain Mahmood
Published: July 2021

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. Mutations in the MTTP gene cause abetalipoproteinemia (ABL). Missense mutations in ABL have revealed that the central α-helical and C-terminal β-sheet domains are important for the lipid transfer activity of MTP and for the assembly and secretion of apoB-containing lipoproteins. The N-terminal domain, on the other hand, has mainly been implicated in apoB- and membrane-binding. Here, we describe a novel ABL missense mutation (D169V) in the N-terminal β-sheet of MTP. Although this mutant MTP (MTPD169V) is expressed and localized to the endoplasmic reticulum, it is unable to transfer triglycerides and phospholipids. Further, MTPD169V does not support the assembly and secretion of apoB-containing lipoproteins. Computational molecular modeling suggests that D169 could form an internal salt bridge with K187 and K189. Indeed, mutagenesis of these lysine residues to leucine abolishes triglyceride transfer and apoB secretion activities of MTP. Furthermore, conservative mutagenesis that preserves charges on these residues partially restores triglyceride transfer and apoB secretion activities of MTP. Therefore, D169 is probably involved in an internal salt bridge with K187 and K189. Disruption of this internal salt bridge in the N-terminal region affects the lipid transfer activity present in the C-terminal end of the MTP molecule. We speculate that this salt bridge, although away from the speculated lipid transfer site, might be important in providing structural integrity necessary for the lipid transfer activity of MTP.
Abstract 27: Role of Sirtuin 6 in the Initiation and Progression of Calcific Aortic Valve Disease
Authors: Roos Carolyn M , Zhang Bin , Verzosa Grace , Oehler Elise A , Hagler Michael A , Zhang Heyu , Miller Jordan D
Published: July 2021

Increasing age is a major risk factor for calcific aortic valve disease (CAVD). Interestingly, SIRT6 knockout mice have a marked progeroid phenotype, and we recently reported that sirtuin enzyme expression is dramatically reduced with aging and that SIRT6 expression is reduced further in valves from patients with end-stage CAVD. It is unknown, however, whether experimentally reducing SIRT6 promotes osteogenic signaling in the valve and ultimately accelerates progression of CAVD. Thus, we used cultured mouse aortic valve interstitial cells and ldlr-deficient, apolipoprotein B100-only mice (LA) that were SIRT6 wild-type (LA-SIRT6
+/+
) or heterozygous (LA-SIRT6
+/-
) and fed a Western diet for 3 or 12 months to determine the role of SIRT6 in valve calcification.
In vitro
, reduction of SIRT6 increased histone acetylation and significantly increased mRNA and protein levels of the osteogenic genes Runx2 and Sp7 in response to bone morphogenetic protein 2 (100ng/ml BMP2 for 18 hours), and siRNA knockdown of SIRT6 increased mRNA levels of Sp7 even in the absence of exogenous bone morphogens. Using high-resolution ultrasound to evaluate aortic valve function
in vivo,
we found that 3 month old LA-SIRT6
+/-
mice did not have significant impairments in valve function compared to LA-SIRT6
+/+
mice. In contrast, 12 month old LA-SIRT6
+/-
had dramatically worsened aortic valve dysfunction and stenosis compared to LA-SIRT6
+/+
mice, which was also associated with reductions in left ventricular ejection fraction. Collectively, our data strongly suggest SIRT6 plays a critical role in the tonic repression of osteogenic signaling in the aortic valve, and that age-related reductions in SIRT6 are likely to increase susceptibility to valve calcification in response to risk factors for CAVD such as hypercholesterolemia. Collectively, increasing activity of SIRT6 or reducing acetylation of its targets may serve as viable therapeutic strategies to slow progression of age-related valvular calcification and stenosis.
Abstract 304: Chemokine Receptor CCR2 Mediates Monocyte Mobilization and Migration in Experimental Aneurysms
Authors: Xu Baohui , Fujimura Naoki , Xuan Haojun , Dalman Jackson , Furisho Yuko , Tanaka Hiroki , Glover Keith , Rouer Martin , Aoyama Kohji , Michie Sara , Dalman Ronald
Published: July 2021

Abdominal aortic aneurysm (AAA) is a macrophage-driven vascular inflammatory disease. Chemokine receptor CCR2, a critical mediator of monocyte mobilization and migration, promotes AAA pathogenesis in hyperlipidemic mice following angiotensin II infusion. This study examined the role o CCR2 signaling in AAA formation in a mechanistically distinct experimental aneurysm model. AAAs were created in 10 week old male CCR2 knockout (KO) and wild type C57BL/6J mice via transient intra-aortic porcine pancreatic elastase (PPE) infusion. Aneurysm progression and aortic cellularity were evaluated using ultrasonography, histology and FACS analyses. In CCR2 KO mice, minimal aneurysmal enlargement was apparent following PPE infusion. Histologically, aortic medial elastin and smooth muscle cellularity were minimally degraded, and mural macrophage accumulation and angiogenesis were also significantly reduced compared to WT mice. Significantly fewer circulating and splenic inflammatory monocytes were identified via FACS analysis in KO mice, with a reciprocal increase in bone marrow monocyte cellularity. In in vivo monocyte migration assays, CCR2 deficiency was associated with a 60 -70% reduction in migration of intravenously transferred monocytes into aneurysmal aortae. In conclusion, targeted deletion of CCR2 in mice profoundly reduces mobilization of inflammatory monocytes from the bone marrow, while suppressing aneurysmal degeneration following PPE infusion. CCR2 signaling may represent an attractive target for therapeutic inhibition in medical management of AAA disease.
Abstract 310: Do Biologic Variations in Plasminogen Levels Affect Endogenous Fibrinolysis and Outcomes in Acute Thrombotic Disease Such as Ischemic Stroke?
Authors: Singh Satish , Houng Aiilyan , Reed Guy L
Published: July 2021

Introduction:
After activation of plasminogen (Pg), plasmin is the key enzyme that acutely dissolves thrombi. Yet, despite significant biologic variation in Pg levels between humans, there is no clear link between Pg levels (or Pg deficiency) and the development or resolution of acute thrombotic vascular diseases such as stroke.

Hypothesis:
We tested the hypothesis that plasma levels of Pg affect endogenous fibrinolysis, brain injury, hemorrhage and swelling after thromboembolic stroke.

Methods and results:
We examined male and female Pg
+/+
([Pg] =84±4.8 μg/ml), Pg
+/-
([Pg] =37±5.2 μg/ml) and Pg
-/-
([Pg] =1.3±3 μg/ml) mice, n= 10-13 per group. Ischemic stroke was induced by endovascular placement of an autologous
125
I-fibrin plasma clot at the origin of middle cerebral artery (MCA) that reduced hemispheric blood flow by ≥80%. After 6 hours of stroke, the dissolution of the culprit MCA thrombus was determined and the effects on brain injury were assessed. Data was analyzed by a two-way ANOVA with Neumann-Keul’s correction. There was a dose-dependent relationship between Pg levels and endogenous dissolution of the MCA thrombus (p=0.0019) with Pg
+/+
mice showing greater lysis (20.7±6.6%) than Pg
+/-
mice (19.0±3.8%) and Pg
-/-
mice (12.6±6.4%). There was a significant, inverse dose-response effect on brain infarction with higher Pg levels associated with less severe infarction: Pg
+/+
(24.8±4.3%) <Pg
+/-
(27.3±9.4%) <Pg
-/-
(37.4±8.9%) mice. No significant sex based differences were found in fibrinolysis and brain injury. Brain swelling showed a significant, but non-linear dose-dependence on Pg levels (p<0.01) which was affected by sex (p<0.01). There was no significant association between brain hemorrhage and Pg levels.

Conclusions:
In vivo, Pg levels have concentration-dependent effects on endogenous fibrinolysis, brain infarction and swelling after thromboembolic stroke. Further studies should be done to assess how Pg levels may affect outcomes in human thrombotic diseases.
Abstract 312: Transcriptional and Posttranscriptional Circuitry in the Brain After Transient and Permanent Ischemia
Authors: Tabet Fatiha , Lee Sandy , Cuesta Torres Luisa F , Levin Michael G , Drummond Grant R , Remaley Alan T , Rye Kerry-Anne , Sobey Christopher G , Vickers Kasey C
Published: July 2021

Background:
Stroke is a major neurovascular disease and a leading cause of mortality and long-term disability. Within cells of the brain, short non-encoding microRNAs (miRNAs) serve to modulate gene expression and likely contribute to most, neurological processes. However, miRNA changes in the brain tissue in response to stroke have not been reported.

Aim:
To investigate the functional roles of brain miRNAs and gene regulatory networks in stroke injury.

Methods:
Adult (8-12 weeks old) male C57Bl/6 mice underwent intraluminal filament-induced middle cerebral artery (MCA) occlusion. Permanent ischemia (ischemia no reperfusion, InoR; n=8) was achieved by occlusion for 24 h, and ischemia with reperfusion (IR; n=8) was completed after 30 min of MCA followed by 23.5 h of reperfusion. Sham-operated mice (n=8) were used as controls. Total RNA was isolated from mouse brains and gene arrays (Affymetrix) and miRNA arrays (TaqMan OpenArray microRNA) were performed. Validation studies were performed using RT-PCR and TaqMan Individual Assays.

Results:
Relative to the sham-operated mice, InoR significantly altered (p≤0.05; fold-change≥1.5) the levels of 471 genes (mRNA) in the brain. By contrast, IR resulted in only 114 significant changes in gene expression after 24 h. Brain miRNAs were also very sensitive to both ischemia and reperfusion. 28 miRNAs (11 down, 17 up) were significantly altered by InoR compared to the sham procedure. Likewise, 12 miRNAs (3 down, 9 up) were significantly altered with reperfusion compared to the sham procedure. Interestingly, we found 10 miRNAs to be significantly altered (5 up, 5 down) with ischemia (InoR/Sham), but were also significantly corrected towards normal Sham levels by 23.5 h reperfusion (IR/InoR). Validation studies confirmed that levels of multiple miRNAs were significantly altered with InoR. Reperfusion increased the levels of all these miRNAs. 48% (327/680) of the mRNAs that were altered were predicted targets of significantly altered miRNAs, and our results showed inverse directional changes.

Conclusion:
Results from our study show the role of miRNAs and post-transcriptional circuits in both adaptive and maladaptive responses to ischemic stroke and reperfusion.