bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 322: Balanced, pH-Buffered Preservation Solutions Improved Physiologic Response in Human Saphenous Vein Graft
Authors: Wise Eric S , Hocking Kyle M , Cheung-Flynn Joyce , Brophy Colleen M
Published: July 2021

Introduction:
Human saphenous vein (HSV) autografting into the coronary or peripheral arterial system is a widely practiced surgical approach. The primary mechanism of early graft failure is neointimal hyperplasia. Unfortunately, standard practice entails vein preservation in harmful solutions, notably, normal saline (NS). We hypothesized a balanced, euosmolar, pH-buffered solution would promote enhanced vasoreactivity, cellular viability and ultimately, improved patency.

Methods:
Unmanipulated samples of HSV tissue were obtained from patients undergoing CABG. Rings were cut and placed into University of Wisconsin (UW) solution, low-potassium UW, Celsior, PlasmaLyte (HP) and NS (all heparinized) for one hour, and hung on a muscle bath with unmanipulated control (UM). KCl-dependent contractility, as well as endothelial dependent (EDR) and independent (EIR) relaxation were assessed.

Results:
One-way ANOVA demonstrated KCl-induced contractility was reduced in NS-preserved HSV; significantly so relative to UW (n=9, p<0.05, Graph A). UW, low-potassium UW and Celsior produced a phenylephrine (PE)-induced contraction significantly greater than that seen in NS (n=9, p<0.05, Graph B), while UM, UW and HP vein show enhanced EDR relative to NS (n=11, p<0.05, Graph C). No EIDR differences were observed (n=7, Graph D).

Conclusions:
Using physiologic function to reflect graft viability, NS is toxic to HSV. KCl and PE-induced contractility, and EDR were all impaired. Thus, a buffered, balanced solution such as HP or UW is advocated for graft preservation.
Abstract 330: Endothelial JNK Is Critically Required for Native Collateral Artery Development
Authors: Ramo Kasmir , Sugamura Koichi , Keaney John F , Davis Roger J
Published: July 2021

Arterial occlusive diseases are major causes of morbidity and mortality in industrialized countries and represent a huge economic burden. The extent of the native collateral circulation is considered to be the most important determinant of the magnitude of tissue damage and subsequent functional impairment that ensues following an arterial occlusive event. Therefore, understanding the mechanisms responsible for collateral artery development may provide avenues for the design of strategies to enhance the collateral circulation in patients at risk for arterial occlusive diseases. Here, we identify a critical requirement for cJun-NH2-Terminal Kinase (JNK) in collateral artery development. Indeed, using immunofluorescence, microfil perfusion and three dimensional high resolution micro computed tomography (μCT) imaging techniques, we demonstrate that mice with compound JNK-deficiency in the vascular endothelium display abnormal collateral arteries, which are unable to restore blood perfusion following occlusion of a major artery leading to severe tissue necrosis in animal models of femoral and coronary artery occlusion. Furthermore, employing constitutive and inducible conditional deletion strategies, we demonstrate that JNK acts during the initial development of collateral arteries to ensure proper patterning and connectivity, but is, for the most part, dispensable for angiogenic and arteriogenic responses in adult mice. The defects in collateral artery development may reflect an enhanced sensitivity of JNK-deficient endothelial cells to cell death in specific contexts. This study introduces JNK as a major regulator of vascular development and highlights the crucial importance of the collateral circulation as the main defining factor in reducing the severity of tissue damage and functional impairment following arterial occlusive events.
Abstract 345: Inhibitor Kappa B Kinase Regulates Nitric Oxide Synthase Activity in Vascular Endothelial Cells
Authors: Natarajan Mohan , Krishnan Manikcam , Konopinski Ryzad , Gaudette Colleen , Janardhanan Preethi , Mohan Sumathy
Published: July 2021

The generation of nitric oxide (NO) in endothelial cells is predominantly regulated by endothelial nitric oxide synthase (eNOS) activity. The role of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been established. Earlier, we demonstrated an increased interaction of Hsp-90 with inhibitor kappa B kinase β (IKKβ) in aortic endothelial cells (ECs) under conditions of high glucose (HG). These studies implicated the possibility of a site-specific competition between eNOS and IKKβ to bind to Hsp-90. In this study, the binding site of IKKβ on Hsp-90β is demonstrated by generating 5’deletional mutants of Flag tagged Hsp-90 and full length enhanced yellow fluorescent protein tagged IKKβ (EYFP-IKKβ). Forceful co-expression of the Hsp-90 deletion constructs with EYFP-IKKβ plasmid followed by immunoprecipitation/immunoblotting revealed the binding site of IKKβ to be between 307-711aa on Hsp-90, the middle domain (440-600 aa) where eNOS has been shown to bind to Hsp-90. Co-immunoprecipitation experiments performed in lysates of human aortic endothelial cells incubated with 25mM high glucose exhibited enhanced IKK beta interaction with both Hsp-90 α and β isoforms. Independently blocking Hsp-90α or β expression by siRNA transfection significantly blocked VEGF-induced NO production implicating the possible mechanism that IKK beta regulates NO production by competing with eNOS for both the isoforms of Hsp-90. These results confirm the previously unknown participation of IKKβ in the downregulation of eNOS activity under the influence of HG and provide a novel therapeutic strategy of using IKKβ as an effective target to improve NO generation in diabetic arteries.
Abstract 360: Early Life Stress Increases Hematocrit and Endothelin-1 in Mice
Authors: Ho Dao H , Pollock Jennifer S
Published: July 2021

In humans, early life stress (ELS) is an independent risk factor for adult cardiovascular disease (CVD). We have shown that mice subjected to ELS by maternal separation with early weaning (MSEW), develop vascular endothelial dysfunction in adulthood. A marker of endothelial dysfunction and CVD is high hematocrit, an abnormally elevated level of circulating red blood cells. Hematocrit is largely regulated by erythropoietin (EPO), a protein that is released predominantly from the kidney under conditions of hypoxia. We hypothesized that MSEW increases circulating EPO and hematocrit in adult male mice. We used the MSEW model in C57BL6J mice to study the mechanisms of ELS-mediated alteration in hematocrit. MSEW litters were subjected to maternal separation 4h/day (postnatal day (PD) 2-5) and 8h/day (PD6-16), and weaned at PD17. Control (CON) litters were undisturbed until weaning at PD21. At 13 weeks of age, blood was collected from CON and MSEW male mice by cardiac puncture and lung tissue was excised. Hematocrit of MSEW mice was significantly higher than CON mice (46.2 ± 0.03 vs 43.3 ± 0.03%, p = 0.004). Plasma EPO, as measured by ELISA, was elevated in MSEW mice, however not significantly (112.89 ± 51.32 vs 61.62 ± 20.73 pg/ml, p = 0.06). We further hypothesized that MSEW enhances circulating endothelin-1 (ET-1) levels, a vasoactive peptide regulated by hypoxia and EPO. We found that plasma ET-1 was significantly increased in MSEW mice compared to CON (1.55 ± 0.41 vs 1.26 ± 0.23 pg/ml, p = 0.02). Endothelin receptor type A and B density and binding in lung, as measured by radioligand binding, was not different between groups, suggesting that increased circulating ET-1 in MSEW mice was not due to decreased ET-1 clearance in the lungs and most likely is due to increased production of ET-1. Taken together, our data suggest that MSEW-induced endothelial dysfunction may be mediated by an interplay of increased circulating red blood cells and elevated ET-1 production. Further studies are necessary to determine the exact role of these factors in this phenomenon.
Abstract 397: Correlation Between the Whole Blood Omega-Score and Red Blood Cell Membrane Omega-3 Index in a Patient Population with Cardiovascular Disease Risk Factors Treated With a Unique Omega-3 Formulation
Authors: Jackowski George , Yantha Jason , Kupchak Petro , Shaikh Nisar A
Published: July 2021

Background:
Growing evidence supports the value of maintaining a diet rich in omega-3 polyunsaturated fatty acids (PUFA) to achieve longevity of cardiovascular health, and prevent sudden death. Omega-3 deficiency (OM3D) is highly prevalent in the US and contributes to 84,000 deaths/year. To identify patients at risk with OM3D, two laboratory tests are available, including the Omega-Score (OS - whole blood), as well as the Omega-3 Index (OI - red blood cell membrane), that measure omega-3 blood levels. Omega-Score or OI levels <6.1% suggest OM3D. Here we report on the dietary omega-3 status of Canadians, including those with CVD risk factors, through a comparative analysis of the OS vs. OI both pre- and post-treatment with a unique omega-3 formulation.

Methods:
Open label study of 143 study subjects enrolled for baseline omega-3 deficiency assessment, of which 63 subjects were scheduled to receive a 4 g/day regimen of a highly purified (>90%) omega-3 formulation (2720 mg/day of EPA + 440 mg/day of DHA), a ratio of 6:1 EPA:DHA (6:1 OM-3) for two weeks, and 31 subjects received 6:1 OM-3 for six weeks. Diagnostic evaluation of the OS, and OI was carried out at baseline, and every two weeks until the study completion.

Results:
The majority (84.5%) of subjects tested at baseline had OS levels <6.1%. The baseline, mean OS and OI levels were 3.4% and 4.4% respectively (N=143). After six weeks of treatment, a significant improvement in the OS (120.6% increase, p<0.0001), and OI (72.7% increase, p<0.0001) was observed (N=31). Comparative analysis of the OS vs. the OI resulted in comparable values, with a greater baseline OI, but with near-identical resultant OS an OI levels after six weeks of treatment (7.5%, vs. 7.6% respectively).

Conclusions:
This study shows that both the OS and OI provide highly correlative results in the assessment of nutritional omega-3 deficiency, and response to treatment with 6:1 OM-3.
Abstract 445: Increasing Macrophage Survival Delays Progression of Advanced Atherosclerotic Lesions Through Macrophage-Derived ApoE
Authors: Bouchareychas Laura , Pirault John , Saint Charles Flora , Deswaerte Virginie , Giral Philippe , Gautier Emmanuel , Huby Thierry , Lesnik Philippe
Published: July 2021

Introduction/Hypothesis:
We previously demonstrated that increasing macrophage survival delayed atherosclerotic plaque progression towards advanced stages. However, whether cell death-protected macrophages would still be efficient to hinder the progression and favor the resolution of already advanced atherosclerotic lesions, and thus prove therapeutic potential, remains unknown.

Methods:
We used a transgenic mouse model in which macrophage lifespan is enhanced through specific overexpression of the antiapoptotic gene hBcl-2 under the control of the macrophage specific CD68 promoter (Mø-hBcl2).
Apoe
-/-
or
Ldlr
-/-
recipient mice with advanced atherosclerotic lesions were irradiated and then transplanted with bone marrow cells isolated from
Apo
e
-/-
Mø-h
Bcl2
or
Apo
e
+/+
Mø-hBcl2 mice respectively and their appropriate controls.

Results:
Both
Apoe
-/-
Mø-h
Bcl2
→
Apoe
-/-
and
Apoe
+/+
Mø-h
Bcl2
→
Ldl
r
-/-
mice presented a significant decrease in lesional apoptotic cells content (-30%, P<0.05) as compared to their respective controls. Additionally, hBcl2 expression in macrophages was associated with a larger pool of tissue macrophages in vivo, including Küppfer cells in the liver, in both Apoe
-/-
(+40% P<0.05) and
Ldlr
-/-
(+36% P<0.05) recipients. By contrast, only
Ldlr
-/-
recipient mice showed reduction of lesional necrotic areas (-37%, P<0.05), plasma cholesterol levels (-15%, P<0.05) and atherosclerotic lesions (-30%, P<0.05). As those reductions were not significant in the context of ApoE deficiency, these findings supported that Mø-derived ApoE was key in regulating plasma cholesterol levels, lesional necrosis and advanced plaque progression in the context of increased macrophage pool. Indeed, increased liver Küpffer cells content in the liver of
Ldlr
-/-
recipient mice was associated with elevated ApoE mRNA levels (+30%, P<0.05), which is likely to promote reverse cholesterol transport.

Conclusions:
Collectively, these data suggest that macrophage survival hindered advanced lesion progression. One potential mechanistic explanation lied to the increased Küpffer cells content, which could modulate directly or indirectly cholesterol homeostasis.
Abstract 459: Vascular Neuropeptide Y Contributes to Atherosclerotic Plaque Progression and Perivascular Mast Cell Activation
Authors: Lagraauw H Maxime , Westra Marijke M , Bot Martine , Wezel Anouk , Santbrink Peter J , Pasterkamp Gerard , Biessen Erik A , Kuiper Johan , Bot Ilze
Published: July 2021

Aim:
Neuropeptide Y is an abundantly expressed neurotransmitter capable of modulating both immune and metabolic responses related to the development of atherosclerosis. NPY receptors are expressed by a number of vascular wall cell types, among which mast cells. However, the direct effects of NPY on perivascular inflammation and atherosclerotic plaque progression remain to be investigated. In this study we thus aimed to determine whether NPY is expressed in atherosclerotic plaques and to establish its role in atherosclerotic plaque development.

Methods and Results:
NPY expression was seen to be increased up to 2-fold in unstable human endarterectomy plaques, as compared to stable plaques (p<0.05, n=9-12), and to be significantly upregulated during lesion progression in apoE
-/-
mice (p<0.001, n=4 per timepoint). In apoE
-/-
mice overexpression of NPY in the carotid artery by means of local application of a lentiviral vector significantly increased atherosclerotic plaque size compared to controls (54 ± 9 *10
3
μm
2
versus 31 ± 6 *10
3
μm
2
, P<0.05, n=12), while plaque composition was unaffected. Interestingly, perivascular mast cell activation was significantly higher in the NPY-overexpressing mice (48.1 ± 4.0 % versus 30.2 ± 6.0 %, P<0.05), suggesting that NPY may impact plaque progression in part via mast cell activation. Furthermore, in vitro NPY-induced murine mast cell activation resulted in the release of pro-atherogenic mediators including IL-6 (515.0 ± 12.5 pg/ml vs. 87.5 ± 5.0 pg/ml) and tryptase.

Conclusions:
Our data show that NPY expression is increased during atherogenesis and in particular in unstable plaques. Furthermore, perivascular overexpression of NPY promoted plaque development and perivascular mast cell activation, suggestive of a role for NPY-induced mast cell activation in lesion progression.
Abstract 492: Resolvin D2 Inhibits Murine Abdominal Aortic Aneurysm and Increases M2 Macrophage Differentiation
Authors: Pope Nicolas H , Salmon Morgan , Conte Michael S , Ailawadi Gorav , Upchurch Gilbert R
Published: July 2021

Objectives:
Macrophages are critical to abdominal aortic aneurysm (AAA) formation; however, the role of anti-inflammatory M2 macrophages is not known. Resolvins have been shown to play a protective role in neointimal hyperplasia; however, their role in AAA has not been established. We hypothesized that treatment with Resolvin D2 (RvD2) attenuates murine AAA formation through alterations in macrophage polarization and cytokine expression.

Methods:
Male C57/B6 mice (n=9/group) of 8-12 weeks of age received RvD2 (100 ng/kg/treatment) or vehicle only every third day beginning three days prior to abdominal aortic perfusion with elastase. Aortas were harvested 14 days following elastase perfusion. Cytokine analysis (n=5/group) or confocal microscopy (n=4/group) was performed. Cytokine profiles were analyzed using a murine antibody array. To determine the effect of RvD2 on macrophage polarization, confocal staining for macrophages (Mac2), M1 (MCP-1) and M2 (Arg-1) macrophage subtypes, α-actin and 4',6-diamidino-2-phenylindole (DAPI) was utilized.

Results:
Mean aortic dilation was 96.23 % (± 13.07 %) for vehicle treated and 56.58% (± 9.69%) for RvD2 treated mice (p<0.0001). Pro-inflammatory cytokines CXCL-10, IL-1β, TIMP-1 and MCP-1 were significantly elevated in control as compared to RvD2 treated animals. Confocal histology demonstrated a prevalence of M2 macrophages within the aortic media in mice treated with RvD2 (Figure 1).

Conclusions:
Resolvin D2 exhibits a potent protective effect against experimental AAA formation. Treatment with RvD2 significantly influences macrophage polarization and decreases several important pro-inflammatory cytokines. Resolvins and the alteration of macrophage polarization represent potential future targets for prevention of AAA.
Abstract 498: A Single Nucleotide Polymorphism in the CDKN1B Gene is Associated With Carotid Plaque Progression
Authors: Hatsukami Thomas S , Zhao Xue-Qiao , Rader Daniel , Hippe Daniel S , Tuteja-Stevens Sony , Yuan Chun , Clowes Alexander
Published: July 2021

Objective:
Studies suggest that a SNP in the promoter region of the C
DKN1B
gene (-838 C>A; rs36228499), a cell cycle inhibitor, may be associated with decreased vascular SMC proliferation and increased risk of MI. Our objective was to test the hypothesis that the -838A allele is associated with decreased carotid plaque progression.

Methods:
214 AIM-HIGH participants underwent annual carotid MRI over 2 years using a standardized protocol. Images were analyzed by Core Lab reviewers blinded to the genotyping results. Plaques with ulceration or hemorrhage were excluded as these local features may contribute excess variability or override genotype. Changes in wall thickness (WT) and lumen, wall, fibrous tissue and total vessel volumes were measured. Associations between number of A alleles and remodeling patterns were tested using linear regression.

Results:
Of the 214 subjects, 161 had suitable MRI and genotype data available for analysis. There was a significant trend toward less growth (more regression) in mean WT for each A allele (p=0.043) (Figure 1). Other plaque variables were not significantly associated with genotype but directionally showed weak trends toward more expansive remodeling for each C allele.

Conclusions:
These data suggest that -838 C>A SNP of the C
DKN1B
gene is significantly associated with carotid mean WT progression among subjects with established vascular disease well treated for LDL-C and blood pressure.
Abstract 567: A Successful Use of Tissue Plasminogen Activator and Argatroban for Acute Therapy of a Submassive Pulmonary Embolism Caused by Heparin-Induced Thrombocytopenia
Authors: Assaad Murad , Ataya Samy , Ha Linda
Published: July 2021

The field of anticoagulation has become a highly relevant and expanding division of medicine. Clinicians must maintain an adequate knowledge of the indications, risks, and benefits of each particular anticoagulant before utilizing them in patient care. Despite great advancements in anticoagulate therapies, areas of uncertainty exist when applying combined use of anticoagulants. This vignette illustrates a unique circumstance of initial therapy combining argatroban with tissue plasminogen activator (tPA) and subsequent transition to warfarin in the management of submassive pulmonary embolism (PE) caused by heparin-induced thrombocytopenia (HIT). There are currently no other published cases or guidelines that have demonstrated this treatment strategy.

We present the case of a 56-year-old African American female who was started on argatroban therapy for submassive bilateral PE secondary to HIT. She required tPA after becoming progressively unstable. The argatroban drip was continued without any hemorrhagic complications. This combination of therapy led to her stabilization, and she was ultimately safely transitioned to warfarin therapy for long term maintenance anticoagulation.

Argatroban is effective in the prevention and treatment of life-threatening complications of HIT. Very little research has been conducted in patients receiving tPA followed by anticoagulation therapy. This case highlights the successful therapy with combined use of tPA and argatroban followed by warfarin transition in the treatment of submassive PE caused by HIT. We find that tPA was safe and effective when the initial argatroban failed to stabilize our patient. The continued use of argatroban infusion after tPA did not cause any hemorrhagic complications. Furthermore, the overlap use of warfarin with argatroban until an INR greater than 4 was achieved for maintenance therapy did not contribute to any hemorrhagic complications. We conclude that the combination of tPA and argatroban is effective in the acute management of hemodynamically unstable PE caused by HIT without significant hemorrhagic complications.
Abstract 627: A Deleterious Mutation in SCARB1 Significantly Alters High-Density Lipoprotein Level, Composition and Biological Function in Humans
Authors: Hancock-Cerutti William , Zanoni Paolo , Lhomme Marie , Lecocq Sora , Dauteuille Carolane , Larach Daniel , Chapman John , Cuchel Marina , Rader Daniel , Kontush Anatol
Published: July 2021

The membrane protein Scavenger Receptor Class B, Type 1 (SR-BI), coded by the gene SCARB1, facilitates the selective uptake of cholesteryl esters from HDL in the liver. Its impact on human lipoprotein metabolism however is not fully understood. Our laboratory has recently identified two homozygotes and 16 heterozygotes for a coding mutation in SCARB1 (c.1127C>T, p.Pro376Leu). We assessed the hypothesis that this SCARB1 mutation significantly increases HDL-C and alters HDL composition and biological function. Plasma from subjects homozygous (n=2) or heterozygous (n=8) for the SR-BI Pro376Leu mutation was compared with that of normolipidemic controls. Five major HDL subfractions were isolated from EDTA-treated plasma using isopycnic density gradient ultracentrifugation and their chemical composition was assayed using commercial enzymatic assay kits. Quantification of >160 molecular species of HDL phospho- and sphingolipids was accomplished via a novel LC-ESI/MS/MS based analysis. Cholesterol efflux capacity, measured from THP-1 macrophages, was determined in total HDL. We identified a >2-fold increase in HDL-C in homozygotes and a >1.5-fold increase in heterozygotes compared to controls, owing mainly to an increase in large, light HDL2 particles. The ApoA-I/ApoA-II ratio was increased in a gene-dose dependent fashion. HDL isolated from heterozygotes mediated 13.5% less efflux when compared to particles from control subjects. Analysis of the HDL phosphosphingolipidome revealed a 23% decrease in phosphatidylinositol content between carriers and controls. A gene-dose dependent trend towards diminishment of phosphatidylserine and increased ceramide was noticeable. In conclusion, the SR-BI Pro376Leu significantly increases HDL levels, but potentially reduces the capacity of HDL to mediate cellular cholesterol efflux. This may be due in part to a decrease in minor, negatively charged phospholipid subclasses including PI and PS, which have previously been shown to play a functional role in stimulating cellular cholesterol efflux.
Abstract 629: Niacin Beneficially Alters the Secretion of Intestinal HDL in a Rat Model of Insulin Resistance by Opposing Effects on PPAR Alpha and TNF Alpha
Authors: Mangat Rabban , Borthwick Faye , Ullrich Tina , Jacome Miriam , Vine Donna F , Proctor Spencer D
Published: July 2021

INTRODUCTION:
Niacin is the oldest hypolipidemic drug that effectively lowers plasma triglycerides whilst increasing HDL-C concentration. However the mechanism of action of niacin still remains elusive. Emerging evidence shows that in addition to the liver, the intestine also contributes to whole body HDL metabolism. The effect of insulin resistance and niacin on intestinal HDL secretion is unclear.

OBJECTIVE:
To determine the effect of niacin on the secretion and composition of intestinal lymphatic HDL in a model of insulin resistance (JCR:LA-cp rat).

METHODS:
Insulin resistant (IR) rats were fed control chow or chow supplemented with niacin (1% w/w) for 6 weeks. The mesenteric lymph duct was cannulated and lymph sampled following an intra-gastric intralipid (fed) infusion for 6 hrs. The lymphatic HDL (1.063-1.21g/ml) fraction was separated by density ultracentrifugation; associated lipid and protein composition was analyzed. Primary enterocytes were isolated by an adaptation of the Wieser method. Jejunal explants from IR rats were treated with 5mg/ml niacin or nicotinamide and mRNA expression was assessed.

RESULTS:
In IR rats, apo-AI lymphatic HDL secretion was reduced (-47%), but associated TG content enriched (86%), compared to non-IR rats. Interestingly, niacin was found to stimulate the secretion of lymph HDL (>60%) compared to non-treated IR rats. Niacin treatment also normalized the TG content of lymphatic HDL particles. In parallel experiments, niacin was also shown to beneficially reduce lymphatic apoB secretion, suggesting concomitant pathways. Primary enterocytes isolated from niacin treated animals had increased ABCA1 and PPAR alpha mRNA compared to untreated control. Further, niacin (but not nicotinamide) significantly increased PPAR alpha and CPT1a mRNA, while annulling TNF alpha mRNA in treated jejunal explants. Conversely, there was no effect of niacin or nicotinamide treatment on ATGL, PLIN2 or ATG5.

CONCLUSION:
Insulin resistance may reduce the secretion of apoAI HDL into mesenteric lymph, potentially contributing to particle dysfunction. Niacin may contribute to improving CVD risk by restoring the number and TG enrichment of intestinal apoAI particles.
Abstract 630: Lipoprotein Subclasses by Ion Mobility and First Cardiovascular Events: An Analysis of 11,227 Participants From the JUPITER Trial
Authors: Mora Samia , Caulfield Michael P , Wohlgemuth Jay , Chen Zhihong , Superko H R , Glynn Robert J , Ridker Paul M , Krauss Ronald M
Published: July 2021

Background:
Ion mobility (IM) measures lipoprotein subclasses based on physically separating particles by their size. It is uncertain how IM lipoproteins compare with standard lipids and apolipoproteins in predicting CVD, and whether they relate to residual risk after statin therapy.

Methods:
JUPITER recruited participants based on LDL-C <130 mg/dL and hsCRP ≥2 mg/L. In 11,227 participants, baseline and on-treatment lipoproteins were measured by IM, and lipids and apolipoproteins were chemically assayed before and after randomization to rosuvastatin or placebo. Levels were examined with the primary trial endpoint of first CVD (n=309) and an expanded endpoint of CVD/all-cause death (n=524).

Results:
In placebo-allocated participants, baseline LDL-C was not associated with CVD (risk-factor adjusted hazard ratio [HR] per 1-SD, 1.04, 95% CI 0.90-1.20), in contrast to significant associations for baseline non-HDL-C, apoB, and IM-measured non-HDL particles (non-HDL-P), LDL particles (LDL-P) and most LDL subclasses. Significant associations were also noted for baseline triglycerides, medium and large VLDL, and HDL-C, but not IM IDL or HDL subclasses. In addition, IM measures a region (size range between very small LDL and large HDL) with two fractions that were significantly associated with CVD: Mid-Zone 1 (190-180 Angstrom) and Mid-Zone 2 (180-145 Angstrom). In separate models that adjusted for risk factors plus standard lipids, significant associations were noted for apoB (adjusted HR per 1-SD 1.26, 1.02-1.56), non-HDL-P (1.19, 1.03-1.39), LDL-P (1.18, 1.03-1.36), Mid-Zones 1 (1.20, 1.04-1.38) and 2 (1.15, 1.04-1.28), several LDL subclasses, and small IDL. In statin-allocated participants, risk of CVD was associated with on-treatment LDL-C, non-HDL-C, and apoB but not on-treatment IM lipoproteins although some, such as Mid-Zone 2, were related to risk of CVD/all-cause death.

Conclusion:
In placebo-allocated JUPITER participants, LDL particle-based measures were more strongly related to CVD than LDL-C, while in statin-allocated participants, residual risk was related to on-treatment LDL-C, non-HDL-C and apoB. Furthermore, this is the first study to identify two novel fractions that signified increased risk and warrant future investigation.
Abstract 638: Urotensin II Promotes Calcification in Vascular Smooth Muscle Cells
Authors: Albanese Isabella , You Zhipeng , Yu Bin , Barratt Bianca , Shum-Tim Dominique , Al Kindi Hamood , Alreshidan Mohammed , Schwertani Adel
Published: July 2021

Introduction:
Atherosclerosis is a leading cause of death in Western societies. Vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis and several other cardiovascular diseases however further research is required to develop a complete understanding of UII’s role in the pathogenesis of atherosclerosis.

Hypothesis:
We hypothesized that UII stimulates calcification in vascular smooth muscle cells and that UII, urotensin II related peptide (URP) and UT receptor expression are upregulated in calcified aortic valves.

Methods and Results:
Human aortic smooth muscle cells (HASMC) were cultured in phosphate media (2.6mmol/L) for 13 days in the presence of varying concentrations of UII (0, 10, 50, 100nm) and the amount of calcium was measured with a calcium assay kit. Protein was extracted and measured with a protein assay kit. HASMC calcification was assessed as the ratio of calcium (μg)/protein (mg). HASMC calcification increased with increasing UII concentration and was significantly elevated in 100nm of UII (N=6, P<0.05) 13 days after incubation. We also examined UII, URP and UT protein expression in 90 carotid endarterectomies and 87 mitral, non-calcified and calcified aortic valves by immunohistochemistry. Multivariant Spearman correlation analyses in carotids revealed significant positive correlations between UII, URP and UT overall staining with calcification, remodeling and inflammation (P<0.05). In valves there was significant positive correlations between UII, URP and UT overall staining with calcification, fibrosis, remodeling, inflammation, lipid score and microvessels (P<0.05).

Conclusion:
The stimulatory effect of UII on vascular smooth muscle cell calcification as well as the upregulated expression of UII, URP and UT in calcified aortic valves suggests that the UT receptor system plays a key role in the pathogenesis of atherosclerosis and valve calcification.
Abstract 653: Endothelial ASC Modulates Early Atherosclerosis by Limiting Monocyte Recruitment
Authors: Lau Andrew C , Hyduk Sharon J , Jongstra-Bilen Jenny , Cybulsky Myron I
Published: July 2021

The inflammasome consists of an innate immune receptor, the adaptor molecule ASC, and the effector Caspase-1 that is responsible for processing signature cytokines such as IL-1beta. The exact role of the inflammasome in atherosclerosis is not clear since both pathogenic and dispensable roles have been reported. Notably, ASC has functions that may impact atherosclerosis but are unrelated to the inflammasome, for example the induction of apoptosis and the regulation of pro-inflammatory transcription factors. The various roles of ASC (i.e. IL-1beta processing, cell death, NF-kappaB and AP-1 regulation) may modulate the progression of atherosclerosis depending on lesion stage and explain the divergent findings highlighted above.

To examine the role of ASC in lesion progression, we bred
ASC
-deficient mice onto the
Ldlr
-/-
background and initiated a cholesterol-rich diet (CRD). In the early stages of atherogenesis (2 weeks of CRD),

ASC
-/-
Ldlr
-/-

mice had a significantly increased lesion surface area, leukocyte content, and rate of Ly6C
hi
monocyte recruitment in the ascending aorta. Bone marrow transplant experiments suggested that
ASC
deficiency in the vessel wall was responsible for this phenotype. Knockdown of ASC by siRNA in cultured human umbilical vein endothelial cells lead to increased TNF-alpha-induced expression of
VCAM1
and monocyte adhesion in parallel-plate flow chamber assays. Monocyte adhesion was not affected by silencing Caspase-1, suggesting that the effect of ASC was independent of the inflammasome. Preliminary findings implicate MAPK family members in ASC-mediated regulation of endothelial adhesion molecules.

At 16 weeks of a CRD

ASC
-/-
Ldlr
-/-

mice had a significantly decreased lesion size compared with controls. Lesions from

ASC
-/-
Ldlr
-/-

mice had a smaller necrotic core area accompanied by decreased numbers of apoptotic leukocytes as determined by active Caspase-3 and TUNEL positivity. These data are in accordance with the pro-atherogenic role of ASC in later stages of atherosclerosis. However our findings demonstrate a novel protective role for ASC in the early stage, which represents a non-inflammasome function via modulation of endothelial-associated adhesion molecules.
Abstract 658: Macrophage Dynamics in Inflammation: Tree Ring--Like Growth of Macrophages in Layers Within Atherosclerotic Plaques
Authors: Martel Catherine , Li Wenjun , Potteaux Stephane , Kreisel Daniel , Randolph Gwendalyn
Published: July 2021

The mechanisms that control macrophage accumulation and retraction from plaques are fervently debated. We had earlier wondered if monocyte-derived cells might be driven to become motile cells resembling dendritic cells, capable of leaving plaque, under conditions when macrophage content in plaques waned. Thus, our laboratory developed a technique that allows the use of fluorescent phagocytic tracers to quantitatively monitor macrophage influx into plaques as well as the potential emigration of macrophages out of plaques. The phagocytic tracer was not removed from plaques when plaque macrophage content receded in conjunction with a stark lowering of plasma cholesterol, arguing that macrophage loss does not result from robust emigration of macrophages out of plaques. Nonetheless, here we show that the distribution of the phagocytic tracer was very different under conditions when macrophage content wanes, as the tracer is distributed near the arterial lumen in this case, versus conditions when macrophages increase, when the tracer is located deep within plaques. Under all conditions and time points, the tracer remained equidistant from the internal elastic lamina that defined the border of the inflammatory plaque, indicating that tracer-bearing macrophages underwent no net displacement. Indeed, we could generate a tree-ring like appearance in plaques by labeling a second wave of monocytes with fluorescent particles of a different color than the first and show that they created a second layer, resembling growth of rings on an aging tree. Preliminary data from multiphoton imaging in atherosclerotic plaques supports the concept of restricted territories for plaque macrophages following recruitment. Under conditions when monocyte recruitment continues, previously recruited macrophages that get buried would likely be subjected to increasing hypoxia and related signals that are pro-inflammatory. This layering of macrophages on macrophages may itself drive a vicious cycle that perpetuates inflammation beyond through signals other than the signal that initiated plaque development in the first place.
Abstract 662: Maternal Soy Protein Diet Ameliorates Atherosclerotic Lesions in ApoE-Deficient Mice Offspring by Promoting Anti-inflammatory Responses Through Epigenetic Changes
Authors: Nagarajan Shanmugam , Burris Ramona , Wick Sara
Published: July 2021

Maternal hypercholesterolemia has been implicated with an earlier onset of atherosclerotic lesions in neonatal offspring. In this study, we investigated whether gestational exposure to soy protein isolate (SPI) diet (maternal diet) primes the vessel wall to attenuate development of atherosclerosis in adult F1 offspring. Pregnant apolipoprotein E knockout (apoE-/-) female mice were fed SPI diet until postnatal day 21 (PND21) of the offspring (SPI-offspring). At PND21, SPI-offspring was switched to casein (CAS) diet until PND140. Mice fed CAS throughout the lifetime (gestation to adult) were used as control (CAS-offspring). Atherosclerotic lesions in aortic sinus were reduced in SPI-offspring compared with CAS-offspring. Total cholesterol levels in CAS- or SPI-offspring, and CAS- or SPI-fed dams were not different suggesting that alternative mechanism(s) contributes to the athero-protective effect of gestational SPI diet. Aortic VCAM-1, MCP-1 and TNF-α mRNA and protein expression were elevated in CAS offspring, while expression of these molecules were 50% reduced in SPI-offspring. Interestingly, compared to CAS-offspring IFN-γ expression (Th1) was reduced, while expression of IL-10 (Th2/Treg), and IL-13 (Th2) expression was increased in SPI-offspring, suggesting that Th2/Treg bias could have contributed to the athero-protective effect. DNA methylation analyses revealed gata3 and il13 promoter regions were hypomethylated in SPI-offspring. We conclude that in utero soy protein exposure inhibits susceptibility to atherosclerotic lesion formation by promoting anti-inflammatory responses through epigenetic regulation.
Abstract 663: Identification of 2 Novel Candidate Genes of Atherosclerosis Susceptibility on Mouse Chromosome 3: Pla2g12a and Elovl6
Authors: Nicolaou Alexandros , Sass Kristina , Northoff Bernd H , Teupser Daniel , Holdt Lesca M
Published: July 2021

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3
B6/B6
mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility.

We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3
B6/B6
and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3
B6/B6
and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C
is
-regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi.

Pla2g12a
and
Elovl6
were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244.
Pla2g12a
mRNA expression was inversely correlated (r
2
=0.2, p=0.002) with atherosclerotic lesion size in F2 mice while
Elovl6
expression was positively correlated (r
2
=0.18, p=0.002). qRT-PCR revealed a strong expression of
Pla2g12a
in muscle and fat tissues whereas
Elovl6
was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of
Elovl6
was associated with reduced adhesion and increased apoptosis.

In conclusion, we identified
Elovl6
and
Pla2g12a
as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.
Abstract 77: Endothelial LOX-1 Protects Against Arterial Thrombosis via Activation of the Oct-1/SIRT1 Pathway
Authors: Akhmedov Alexander , Camici Giovanni G , Paneni Francesco , Costantino Sarah , Holy Erik W , Stivala Simona , Speer Thimoteus , Breitenstein Alexander , Reiner Martin F , Lohmann Christine , Beer Juerg-Hans , Lüscher Thomas F
Published: July 2021

Background:
The lectin-like oxLDL receptor-1 (LOX-1) promotes the endothelial uptake of oxidized low-density lipoprotein (oxLDL). However, LOX-1 is involved in several other biological processes and its role in arterial thrombus formation remains unknown. The present study was designed to investigate whether LOX-1 activation plays a role in thrombus formation in vivo.

Methods and Results:
Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). While plasma levels of oxLDL were comparable, carotid tissue oxLDL content was markedly increased in LOX-1TG as compared to wild type (WT). Arterial thrombus formation was assessed using an in vivo photochemical injury model. Time to arterial occlusion was prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity were reduced by 50% in the carotid arteries of LOX-1TG mice. This effect was mediated by the activation of the transcription factor Oct-1 leading to upregulation of mammalian deacetylase SIRT1 via binding to its promoter and subsequent inhibition of NF-κB signaling as demonstrated by siRNA experiments. This was further confirmed in LOX-1TG endothelial cells (EC) where expression of Oct-1 and SIRT1 was increased upon exposure to oxLDL. Increased expression of SIRT1 was further associated with decreased DNA-binding of RelA/p65 subunit of NF-κB.

Conclusions:
LOX-1 activates a novel compensatory pathway which protects against arterial thrombus formation in vivo. These unexpected findings suggest that Oct-1/SIRT1 signaling may represent a novel target for the prevention of arterial thrombus formation in the setting of hyperlipidemia and atherosclerosis.
Abstract 111: HDL-apolipoprotein A-I Exchange Is Impaired in the Metabolic Syndrome
Authors: Borja Mark S , Hammerson Bradley , Savinova Olga V , Shearer Gregory C , Oda Michael N
Published: July 2021

Objective:
Metabolic syndrome (MetSyn) is associated with a host of cardiovascular risk factors including increased waist circumference, triglycerides, blood pressure, fasting glucose, and reduced high density lipoprotein cholesterol (HDL-C). Changes in HDL function, especially the ability to participate in reverse cholesterol transport, are more indicative of the atheroprotective properties of HDL compared to HDL-C levels alone. In this study, we determine the association of HDL-apolipoproteinA-I (apoA-I) exchange, a measure of HDL remodeling/apoA-I exchange, with MetSyn and its individual components.

Methods and Results:
We analyzed HDL-apoA-I exchange (HAE) in the plasma of 60 well-characterized subjects with MetSyn and 14 healthy control subjects. HAE measurements were obtained using electron paramagnetic resonance (EPR) following incubation of plasma with lipid-freeapoA-I containing a methanethiosulfonate spin label. HAE positively correlated with plasma HDL-C and apoA-I levels, and inversely correlated with fasting blood glucose levels, blood pressure, BMI, and triglycerides. Multiple linear regression showed that HAE is significantly correlated with MetSyn (HAE: r2 = 0.57, P<0.0001), though MetSyn subjects on statins exhibited significantly higher HAE compared to subjects not on statins.

Conclusions:
MetSyn has a significant negative impact on HDL remodelin/apoA-I exchange, as measured by HAE. HAE is a strong identifier of MetSyn status even after adjusting for individual components of MetSyn.
Abstract 127: Effects of miR-33 Antagonism on Glucose and Triglyceride Metabolism in Nonhuman Primates.
Authors: Layne Joseph D , McDaniel Allison L , Marshall Stephanie M , Kavanagh Kylie , Esau Christine C , Moore Kathryn J , Temel Ryan E
Published: July 2021

Elevated plasma low-density lipoprotein (LDL) and decreased high-density lipoprotein (HDL) cholesterol levels increase the risk of cardiovascular disease (CVD). While strategies to lower LDL cholesterol have been successful in reducing CVD-related mortality, there is still an unmet need for developing therapies to reduce the residual risk of atherosclerotic CVD. Plasma HDL cholesterol levels are inversely correlated with CVD risk and thus novel therapies to increase plasma HDL cholesterol levels have garnered much attention in recent years. MicroRNA-33a and b (miR-33a/b) are intronically-encoded microRNAs residing in the sterol response element binding protein genes
SREBF2
and
SREBF1
and suppress the expression of the genes involved in cholesterol efflux and fatty acid oxidation. Recent studies show that antagonism of miR-33 results in increased ABCA1expression and elevated plasma HDL levels in both mice and nonhuman primates. However, findings in mice suggest that long term miR-33 antagonism may lead to hepatic steatosis and increased production of very low-density lipoprotein (VLDL), the precursor to proatherogenic LDL. In the current study, cynomolgus monkeys were fed a high carbohydrate diet plus Kool Aid prior to administration of antisense oligonucleotides (ASOs) specific for miR-33a/b. As expected, miR-33 antagonism resulted in elevated plasma total cholesterol levels, predominantly due to increased HDL. No differences in plasma LDL or triglycerides were observed between treatment groups. MiR-33a/b antagonism had no effect on VLDL triglyceride secretion rates. Anti-miR-33 treatment did not promote hepatic lipid accumulation or adversely affect liver transaminase levels. Glucose tolerance tests revealed no differences in either plasma glucose or insulin levels in response to miR-33a/b antagonism. These findings suggest that anti-miR-33 therapy raises HDL cholesterol without negatively impacting either triglyceride or glucose homeostasis in nonhuman primates.
Abstract 142: Rac2 Modulates Macrophage IL-1β Production, Atherosclerotic Plaque Inflammation, and Calcific Atherosclerosis
Authors: Ceneri Nicolle M , Young Bryan D , Yarovinsky Timur O , Sadeghi Mehran , Bender Jeffrey R , Morrison Alan R
Published: July 2021

Ischemic heart disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaques is a well-described pathologic finding that has predictive value in terms of atherosclerotic burden, cardiovascular event risk, and mortality. Inflammation is critical to atherogenesis, but inflammatory mechanisms that drive atherosclerotic calcification are minimally understood. The Rho/Rac family of low molecular weight GTPases regulates the activation, recruitment, and differentiation of monocytes. Racs compete for similar regulatory elements, indicating that targeted modulation of specific family members may influence the role of other members in biologic processes. The hypothesis is that Rac2 can modulate atherosclerotic plaque inflammation and calcium composition via consequent effects on the activation of Rac1. Rac2 gene deletion results in elevated activation (GTP-binding) of Rac1 in macrophages. Increased activation of Rac1 is associated with higher macrophage IL-1β production in response to LPS-coupled cholesterol crystal exposure. In fact, macrophage IL-1β production is dependent on Rac1. When subjected to a high fat diet for 14 weeks, Rac2 -/-ApoE -/- mice demonstrate similar weight gain, serum cholesterol levels, circulating inflammatory monocyte levels, and aortic atherosclerotic lipid burden relative to Rac2 +/+ApoE -/- mice. Atherosclerotic plaques from Rac2 -/-ApoE -/- mice contain increased inflammatory cellular infiltration by histology and relative RNA expression. Ex vivo microCT imaging reveals calcified aortic plaques, and quantification of calcification by near-infrared imaging demonstrates increased calcium composition in plaques from Rac2 -/-ApoE -/- mice relative to Rac2 +/+ApoE -/- mice. Calcified aortic plaques are associated with an increase in IL-1β mRNA expression in the aortic tissue. In summary, Rac2 is a critical modulator of signaling mechanisms that regulate the activation state of Rac1, resulting in enhanced macrophage IL-1β expression in vitro and inflammatory calcification of atherosclerotic plaques in vivo. Targeting the balance of Rac regulation has therapeutic potential in modulation of atherosclerotic plaque composition.