bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 275: Association Between Pulse Pressure and A1c
Authors: Negussie Melaku G , Hamil Sonya , Kwayaya John , Retta Tamrat M. , Ketete Muluemebet , Xu Shichen , Aqabein Samah , Rahman Sadaf , Randall Otelio S
Published: July 2021

Background:
Increased pulse pressure (PP) is an independent risk factor for cardiovascular disease (CVD) morbidity and mortality in men and women. Increased PP has also been shown to be a stronger predictor of CVD mortality, than systolic (S), diastolic (D), or mean blood pressure (BP). Changes in PP amplitude are also associated with other metabolic syndrome components. PP is increased in obesity and increases at a greater rate in patients with diabetes mellitus (DM); and may contribute to the risk and "opinion" that labels DM-CVD-risk as equivalent to previous myocardial infarction. The purpose of this study is to determine whether the metabolic marker, Hemoglobin A1c (A1c), during the early stage of DM development is also associated with a readily available clinical vascular marker, PP. Such an observation may provide an opportunity to detect and start preventive treatment earlier and provide a possible path to begin investigating the mechanisms of hypertensive-diabetic CVD.

Methodology:
Complete baseline metabolic panels and 24-hour ambulatory BP (AMBP) recordings were obtained on 102 African Americans, 65.9% females, aged 40-90 years, and with Stage-I hypertension. Participants were categorized in two groups according to A1c < 5.7 or A1c ≥5.7. T-test was used to examine difference in BP levels between the two groups. In addition, we examine the independent effect of A1c levels on PP in a linear regression model that adjusts for age and BMI.

Results:
There were 61 non-diabetic subjects with A1c <5.7, and 41 pre-diabetics or diabetics with A1c ≥5.7. Results showed no significant difference in SBP between the groups (126.2±15.2 mmHg versus 127±16.6mmHg, p=0.70). However, DBP was significantly lower in the group with an HbA1c ≥ 5.7, (75.3±11.3 versus 67.9±10.4mmHg, p<0.03). This resulted in a significantly higher PP in the A1c≥5.7 group, (50.9±10.6 versus 59.0±14.8, p<0.03). Regression analysis showed independent association of PP with A1c (β=5.84, p=0.026) after adjusting for age and BMI.

Conclusion:
There was an association between A1c levels and changes in PP even in the early stages of DM. This suggests that changes in vascular health, as reflected in increase in PP, may start earlier than previously appreciated.
Abstract 28: Mechanistic Studies of HDL Mimetic Peptide ATI-5261 Reveal Aspects of Class A Alpha-helix Structure that Induce Cytotoxicity and Hypertriglyceridemia in vivo: Design of Safe Analogs with Potent Anti-atherosclerosis and Anti-diabetic Actions
Authors: Bielicki John K , Narayanaswami Vasanthy , Hura Greg , Bittner Stefanie , Tabassum Juveria , Hafiane Anouar , Genest Jacques , Johansson Jan , Azhar Salman
Published: July 2021

HDL mimetic peptides hold promise for the treatment of cardio-metabolic diseases. Transient increases in plasma TG and/or safety concerns may limit their therapeutic applications however, particularly in high risk patients with hypertriglyceridemia and diabetes. Presently, we sought to identify peptide structural features responsible for these adverse effects. The single helix peptide ATI-5261 was used as a model, since it forms a class A α-helix like many HDL mimetic peptides and has exceptional secondary structure and solubility. High dose ip injection (300 mg/kg) of ATI-5261 induced muscle toxicity in C57Bl/6 mice vs. vehicle (serum CPK@4 h = 32,314±2359 vs 143±44 IU/L), increased ALT and AST activities (233±10 & 4595±933 vs 26±6 & 84±21 IU/L) and elevated plasma TG (1951±77 vs 37±7 mg/dl). A majority (~90%) of the cytotoxicity and TG increase was eliminated by removing phenylalanine residues from the apolar face of ATI-5261. Similar results were obtained by removing cationic arginine residues from the lipid-water interface. Thus both features were necessary, but each not sufficient, to induce cytotoxicity and TG elevations. Based on this, a peptide (CS6253) was created that was non-toxic (no adverse effect level >500 mg/kg; t1/2 =7.0±0.6 h in rats), retained secondary structure (75±5% α-helicity), high solubility and ability to stimulate ABCA1-dependent cholesterol efflux efficiently (Km=0.80±0.40 vs. 0.86±0.25 μg/ml ATI-5261). In vivo CS6253 was TG neutral, promoted macrophage RCT, and reduced (32%) substantial atherosclerosis in apoE KO mice fed western diet 10 wks (lesion area = 22±4 vs. 15±2% with 30 mg CS6253/kg, ip injection every 48 h for 6 wks, p<0.01) and improved glucose - and insulin- tolerance tests with lowering of HbA1c levels in C57Bl/6 mice fed high-fat diet (DIO model). In summary, the cytotoxic and TG elevating effects of high-dose ATI-5261 were dependent on class A α-helix structure. CS6253 with its improved safety margin represents a novel ABCA1 ligand peptide with potential to treat diabetes and associated cardiovascular disease.
Abstract 289: Abnormal Gait Pattern at Comfortable and Fast Walking Speed in Patients With Peripheral Arterial Disease
Authors: Kakihana Takaaki , Ito Osamu , Sekiguchi Yusuke , Ito Daisuke , Matsumoto Yasuharu , Kawamura Keiichiro , Goto Hitoshi , Ishihara Tetsuro , Kohzuki Masahiro
Published: July 2021

Background:
Characteristics of gait in peripheral arterial disease (PAD) patients are short step length and slow walking speed. It was reported that reduced walking speed was associated with increasing mortality in PAD patients. However, the detail of gait in PAD patients has not been fully clarified. We hypothesized that PAD patients show abnormal gait pattern, in particular in late stance phase (propulsion phase), at comfortable and fast walking speed.

Methods:
Sixteen symptomatic PAD patients (71±9 years old), who were 11 bilateral and 5 unilateral intermittent claudication (27 claudication limbs), and 7 healthy controls (14 healthy limbs) were recruited. With a 3-dimensional motion analysis system, peak hip joint generated power (HJP: pull-off power) and ankle joint generated power (AJP: push-off power) in late stance were analyzed at comfortable and fast walking speed. Ankle/hip (A/P) peak power ratio was calculated to determine whether hip or ankle power was dominantly used in late stance during walking.

Results:
Mean ankle-brachial index was 0.63±0.20 in claudication limbs. Pain lesions were buttock, thigh and calf in 8, 3 and 16 limbs, respectively. Compared with healthy controls, the PAD patients showed significant slower walking speed and shorter step length at comfortable and fast walking speed (p< 0.001). The peak HJP and AJP in late stance were significantly lower in the PAD patients at both walking speed. However, when gait speed adjusted, only peak HJP in late stance was significantly lower in the PAD patients at comfortable walking speed (p=0.025). The A/P peak power ratio was higher in the PAD patients at comfortable and fast walking speed (p=0.020, p=0.045, respectively). Among the PAD patients, the HJP, AJP and A/P peak power ratio were not significantly different between buttock pain and calf pain.

Conclusions:
PAD patients used relatively ankle planter flexors to compensate a muscle weakness of hip flexor at comfortable and fast walking speed. These results suggest that exercise training for hip flexor muscle may increase walking distance and mitigate calf pain during walking in PAD patients.
Abstract 295: Magnetic Resonance Imaging Characterization of Peripheral Arterial Chronic Total Occlusions With MicroCT and Histologic Validation
Authors: Roy Trisha , Liu Garry , Qi Xiuling , Dueck Andrew , Wright Graham
Published: July 2021

Objectives:
Guidelines recommend surgical bypass for peripheral chronic total occlusions (CTOs). Endovascular revascularization, however, offers improved morbidity and shorter length of hospitalization. Not all lesions are amenable to this technique but predicting crossability is difficult due to limitations in characterizing CTOs with current imaging techniques. This study demonstrates the ability of MRI to characterize peripheral CTO components with microCT and histologic validation.

Methods:
MRI was performed on 15 excised human peripheral arterial CTO segments from 4 patients. Each sample was imaged at 7 Tesla at high resolution (75μm3 voxels) to produce T2- and T2*-maps using ultrashort echo (UTE) sequences with echo times: {20μs, 500μs, 1ms}. A T2* difference image was produced by subtracting the UTE images and a phase map was constructed. The T2, UTE 20μs and T2* difference images were used together to differentiate CTO components. MicroCT and histology were used to validate regions of interest (ROIs).

Results:
3 independent reviewers identified 47 ROIs. There was excellent agreement between MRI and microCT for calcium (sensitivity 87%, specificity 99%). There was also good agreement between MRI and histology for adipose tissue (100%, 100%), soft tissue (97%, 97%), thrombus (78%, 100%), collagen (83%, 94%) and open lumen (95%, 98%).

Conclusions:
These results demonstrate the potential of high-resolution T2 and T2* imaging using UTE, to characterize lesion components in human peripheral CTOs. Further work is required to better differentiate thrombus from collagen. This study provides the foundation for future studies in determining the lesion crossability in CTOs.
Abstract 297: SPECT/CT Imaging of Regional Foot Perfusion Provides a Quantitative Index for Evaluation of Targeted Revascularization in the Diabetic Foot
Authors: Stacy Mitchel R , Papademetris Xenophon , Sumpio Brandon , Sumpio Bauer E , Mena Carlos , Sinusas Albert J
Published: July 2021

Introduction:
No standard quantitative imaging approach exists to evaluate volumetric changes in tissue perfusion in the lower extremities following medical treatment. In this study, we develop and apply a three-dimensional model of the foot for evaluation of regional changes in perfusion following revascularization in diabetic patients with non-healing foot ulcers. We hypothesize that SPECT/CT imaging will permit quantification of regional improvements in tissue perfusion in territories of the foot that contain non-healing ulcers, allowing for quantitative evaluation of revascularization procedures.

Methods:
Resting
99m
Tc-tetrofosmin (dose 554.0 ± 26.6 MBq) SPECT/CT was performed on diabetic patients (n=5; 64 ± 15 yrs) before and 1-3 days after lower extremity angioplasty and/or stenting. The CT attenuation scans were used to define five regions of interest (ROIs) in the foot and for quantification of relative changes in regional perfusion with
99m
Tc-tetrofosmin SPECT (Fig. 1A). Radiotracer uptake for each ROI was normalized to injected dose and ROI volume, and expressed as a percent change from baseline value.

Results:
SPECT/CT imaging demonstrated quantitative improvements in regional tissue perfusion in ROIs containing non-healing ulcers for 4 out of 5 patients following revascularization (Fig. 1B). The single patient demonstrating a negative response (11.7% decrease in perfusion) underwent eventual amputation.

Conclusions:
Early changes in tissue perfusion following revascularization can be non-invasively evaluated in specific vascular territories of the foot using SPECT/CT imaging and may be associated with wound healing and limb salvage outcomes.
Abstract 32: Coagulation Factor XI and Thrombin Mediate Angiotensin II-induced Vascular Dysfunction
Authors: Ehlken Moritz , Kossmann Sabine , Jäckel Sven , Jurk Kerstin , Schönfelder Tanja , Walter Ulrich , Renné Thomas , Ruf Wolfram , Münzel Thomas , Wenzel Philip
Published: July 2021

Background:
Multicellular interactions of platelets, coagulation factors, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating arterial and venous thrombosis. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring inflammatory leukocyte recruitment and reactive oxygen species (ROS) production within the vessel wall coupled to vascular dysfunction. How platelets, coagulation factors, leukocytes and the vessel wall cooperate to promote vascular inflammation is unclear.

Methods and results:
Here we delineate a novel non-thrombotic, FXI-dependent, pro-inflammatory coagulation pathway that substantially regulates vascular tone and endothelial function. We can demonstrate that pharmacological inhibition of the synthesis of FXI by an antisense-oligonucleotide (ASO) attenuates ATII-induced vascular dysfunction in acetylcholine-induced vascular relaxation. ATII-induced arterial hypertension, vascular inflammation and leukocyte infiltration are equally diminished by FXI ASO treatment. In addition, vascular ROS formation and fibrotic remodeling is blocked by FXI ASO treatment in ATII-infused mice. Accordingly, intravital microscopy video imaging (IVM) shows that ATII-induced leukocyte rolling as well as adhesion in the carotid artery is reduced by inhibition of the synthesis of FXI, whereas acute infusion of recombinant FXI is not able to restore ATII-driven leukocyte adhesion. Hence FXI is required continuously for the ATII-induced vascular pathology. We show that this pathway does not depend on FXII-dependent activation and that FXII-deficiency does not prevent from ATII-induced vascular dysfunction and inflammation.

Conclusion:
Our results provide novel insight into coagulation-inflammation circuits and point to broader utility of specific FXI-targeted anticoagulants beyond indications as antithrombitic agents in cardiovascular diseases.
Abstract 322: HDL Dynamics in Circulation: Complexity of Protein Distribution and Metabolism Across HDL Size
Authors: Singh Sasha , Andraski Allison , Pieper Brett , Goh Wilson , Sacks Frank M , Aikawa Masanori
Published: July 2021

Introduction:
The composition of specific apolipoproteins may determine HDL functions. We present novel mass spectrometry (MS)-based methods that capture the absolute quantities and kinetics of 7 apolipoproteins in 5 HDL size fractions.

Methods and Results:
Three participants were recruited, infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-containing HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0 by ND-PAGE, and in-gel trypsinized for MS. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE and apoM. Each protein pool size had a distinct distribution across the HDL sizes. ApoE and apoM were enriched in larger HDL, whereas apoC-III and apoA-IV were enriched in smaller HDL sizes. We evaluated the tracer enrichment curves of these 7 proteins in the 5 fractions using high resolution parallel reaction monitoring performed on a quadrupole Orbitrap (Thermo). The enrichment curves for each protein varied from each other by slope and time of peak enrichment (Fig. 1a). In contrast, the enrichment curves across HDL sizes for a single protein showed smaller, but likely meaningful, differences in either slope or time of peak enrichment. Irrespective of the HDL size on which it resides, apoE had the fastest FCR, followed by apoA-IV, and apoC-III. ApoA-I/A-II, apoM, and apoD had slower but similar FCRs (Fig. 1b).

Conclusions:
This study showed distinct distribution and kinetic behaviors of 7 HDL proteins across 5 HDL size fractions that were conserved in the three participants. These findings may help elucidate the functional role of these proteins and the HDL particles that contain them.
Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase
Authors: Wang Shuhui , Brubaker Gregory , Smith Jonathan D
Published: July 2021

Objective:
We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation.

Methods and Results:
We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH < 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37
o
C led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21
o
C or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V
1
domain and an embedded V
0
domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V
0
A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V
0
A1 subunit.

Conclusion:
Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.
Abstract 330: Ezetimibe Prevents Atherogenesis Through Increased Catabolism and Fecal Excretion of LDL-cholesterol and Reduced Atherosclerotic Plaque Inflammation in Apolipoprotein E Knock-out Mice Fed a Paigen Diet
Authors: Briand Francois , Dumas Laurent , Broisat Alexis , Ahmadi Mitra , Bacot Sandrine , Devoogdt Nick , Riou Laurent , Ghezzi Catherine , Sulpice Thierry
Published: July 2021

Background:
Intestinal cholesterol absorption inhibitor ezetimibe (EZE) added to a statin therapy has demonstrated benefits in the IMPROVE-IT trial by further reducing LDL-cholesterol levels than statin therapy alone. We investigated the mechanisms by which EZE could contribute to cardiovascular events reduction in apolipoprotein E knock-out (apoE ko) mice.

Methods:
ApoE ko mice were fed a Paigen diet without (control) or with EZE (7mg/kg/day) for 6 weeks. To evaluate the effects of EZE on LDL-cholesterol metabolism and excretion, a first set of mice was injected intravenously with
3
H-cholesteryl oleate labeled human LDL. A second set of mice was used for in vivo SPECT/CT imaging of
99m
Tc-cAbVCAM1-5, a single domain antibody directed against the Vascular Cell Adhesion Molecule-1 (VCAM-1), which was used as a marker of inflamed atherosclerotic plaques. The same mice were sacrificed for autoradiography and histology of aortic atherosclerotic plaques.

Results:
Compared with control, EZE treatment for 6 weeks induced a significant 41% and 65% reduction in plasma total cholesterol levels and atherosclerotic plaque area, respectively. After injection of
3
H-cholesteryl oleate labeled human LDL, mice treated with EZE showed a 173% higher LDL-cholesteryl ester catabolism (p<0.001 vs. control). At time 96 hours after radiolabeled LDL injection,
3
H-tracer hepatic recovery was reduced by 61% with EZE (p<0.001). Meanwhile, LDL-derived
3
H-tracer excretion in the feces was increased by 107% in the fecal cholesterol fraction (p<0.001). Similar trends were observed for hepatic cholesterol levels and fecal cholesterol mass excretion, with a 75% reduction and 99% increase with EZE, respectively (both p<0.001).

After intravenous injection of
99m
Tc-cAbVCAM1-5, mice treated with EZE also showed a significant 52% reduction in aortic uptake, which was confirmed by significant reduction in tracer uptake in ex vivo biodistribution and autoradiography analysis.

Conclusion:
EZE promotes anti-atherosclerotic effects through increased LDL-cholesterol catabolism and LDL-derived cholesterol fecal excretion, and reduced inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding EZE to a statin therapy.
Abstract 336: Increased Circulating Betatrophin Concentrations in Patients with Coronary Heart Disease
Authors: Qin Yanwen , Wang Yi , Wang Yuan , Tang Chaoshu , Du Jie
Published: July 2021

Objective:
Betatrophin has recently been described as a key hormone to stimulate beta-cell mass expansion in response to insulin resistance and obesity in mice. It is reported that betatrophin can regulate the plasma levels of triglyceride. Triglyceride elevations are a highly significant independent risk factor for coronary heart disease (CHD). However, the circulating levels of betatrophin in patients with CHD are not known. This study measured serum betatrophin levels in patients with CHD and explored the correlations between its serum levels and various parameters in CHD.

Methods and Results:
We analyzed the concentrations of betatrophin by ELISA in blood samples of 40 diagnosed patients with CHD by coronary angiography and 40 health controls. Routine anthropometric and serologic data were collected. Serum betatrophin, fasting glucose, lipid protein profiles, homocysteine and high-sensitivity C-reactive protein were measured. Serum betatrophin levels were significantly higher in CHD patients than in healthy control subjects (569.09 (444.52-798.85) versus 410.46 (339.02-526.41) pg/ml; p < 0.01). Importantly, serum betatrophin associated positively with the severity of coronary branch luminal narrowings (R = 0.304, p <0.05), homocysteine (R = 0.278, p <0.01) and high-sensitivity C-reactive protein (R = 0.179, p <0.05) in CHD patients, whereas there was no significant connection with blood lipids and glucose. In CHD subjects, multivariate regression analyses showed that the homocysteine was independent factors influencing serum betatrophin levels.

Conclusions:
Serum betatrophin levels are significantly increased and associated positively with the severity of coronary branch luminal narrowings in patients with CHD. Our results suggest that betatrophin may play a role in the pathogenesis of CHD.
Abstract 348: TNFα-induced Expression of Adamts7 Affects Primary Mouse Smooth Muscle Cell Migration ex vivo
Authors: Bauer Robert C , Zhang Xuan , Tohyama Junichiro , Cui Jian , Smith Mikhaila A , Ou Kristy , Zheng X. Long , Parmacek Michael S , Rader Daniel J , Reilly Muredach P
Published: July 2021

ADAMTS7 has recently been identified as a novel genomic locus associated with coronary artery disease (CAD) in humans. We have recently shown that deficiency of Adamts7 in hyperlipidemic mouse models reduces atherosclerosis following prolonged western diet feeding, however the mechanism of this reduction remains unknown. Using a β-galactosidase (β-gal) reporter, we observed that vascular Adamts7 expression is induced in response to both mechanical injury and western diet feeding in a transient manner, and this expression predominantly colocalizes to vascular smooth muscle cells (VSMCs). We investigated the ability of multiple cytokines and growth factors (PDGF-BB, TNFα, IFNγ, and Angiotensin II) to stimulate Adamts7 expression in primary mouse VSMCs, and found that of these only TNFα induced Adamts7 expression (5.5-fold, p=0.0007). We determined the expression of Adamts7 and TNFα in whole aortas and aortic roots of Apoe-/- mice at various timepoints of western diet feeding, and found trends towards increased Adamts7 expression while TNFα expression consistently increased over the course of the experiment (4.4-fold increase, p=0.002). We next examined proliferation and migration of primary VSMCs from Adamts7 WT and KO animals. Unstimulated Adamts7 KO VSMCs had no observable difference in migration as compared to WT controls. However, in the setting of TNFα stimulation we observed a decrease in migration of Adamts7 KO VSMCs plated on collagen I (50% reduction, p=0.02) and laminin (60% reduction, p=0.0002). Finally, using both public and private data sets, we assessed if associations exist between SNPs in and around the human ADAMTS7 locus and the expression of ADAMTS7 in relevant tissues. In small data sets we find that SNPs with genome-wide association with CAD also associate with ADAMTS7 expression, and the direction of this association is consistent with our in vivo reduction of atherosclerosis in Adamts7 KO animals. In summary, Adamts7 expression in mice is induced in response to vascular injury, perhaps mediated by TNFα expression, and this induction can modulate VSMC migration. These observations present a novel mechanistic paradigm for ADAMTS7 modulation of atherogenesis and suggest its inhibition might reduce CHD in clinic.
Abstract 354: Lower Circulating Levels of Tumor Necrosis Factor Related Apoptosis-inducing Ligand (TRAIL) are Associated with Increased Sub-clinical Coronary Atherosclerosis among Smokers
Authors: Gupta Aman , Chandra Divay , Zhang Yingze , Reis Steven , Sciurba Frank
Published: July 2021

Rationale:
There is significant in vitro evidence demonstrating anti-atherogenic effect of circulating Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Also, decreased circulating TRAIL levels have been reported in patients with acute myocardial infarction and in those undergoing coronary catheterization due to suspected coronary atherosclerosis. However, it remains unknown if TRAIL levels are associated with sub-clinical coronary atherosclerosis.

Methods:
The study included 460 current and former smokers enrolled in the Pittsburgh COPD SCCOR study. Serum TRAIL levels were measured by electrochemiluminescence immunoassay, according to the manufacture’s protocol (Meso Scale Discovery, Gaithersburg, Maryland). Coronary atherosclerosis was assessed by a validated visual coronary artery calcium scoring system using non-EKG gated chest CT scans (Weston score). Ordinal logistic regression models were used to identify significant associations between categories of CAC score (0, 1-3, 4-8, and 9-12) and TRAIL level, and to adjust for cardiovascular risk factors.

Results:
The mean age of the 460 participants was 65.7 ± 6.3 years, 52.2% were male, and the mean pack years of smoking was 55.0 ± 30.8 years. In univariate analyses, each standard deviation decrease in TRAIL levels was associated with 1.42-fold increase in the odds of having calcium scores in one higher category (p<0.001). This association persisted despite adjustment for age, gender, race, body mass index, hypertension, diabetes, hyperlipidemia, pack years of smoking, and current smoking status (adjusted OR for higher category of calcium score per SD decrease in TRAIL level 1.22, p=0.04).

Conclusions:
Our results expand on the in vitro and in vivo data linking decreased TRAIL levels with increased atherosclerosis by demonstrating a novel association between lower circulating TRAIL and increased subclinical coronary atherosclerosis.
Abstract 356: IKK-Beta Deletion Alleviates the Atherogenetic Effect of Intermittent Hypoxia Induced Macrophage Foam Cell Formation
Authors: Imamura Toshihiro , Hartley Iain S , Massri Abdull J , Poulsen Orit , Zhou Dan , Haddad Gabriel G
Published: July 2021

Background:
Obstructive sleep apnea syndrome (OSAS) is a common sleeping disorder characterized by intermittent hypoxia (IH). Clinical studies have previously shown an independent association between obstructive sleep apnea and atherosclerosis. Furthermore, it has been previously shown that such a predisposition to atherosclerosis in OSAS patient can be caused by various inflammatory mediators, particularly the NF-kappa B (NF-kB) pathway. Foam cells or lipid-laden macrophages in the atherosclerotic lesion have been well documented as a hallmark of atherosclerosis; however, the contribution of IH, such as in OSAS, to foam cell formation is not yet fully understood. Previous observations have led us to hypothesized that IH induces macrophage foam cell formation due to the activation of NF-kappa B pathway.

Methods:
Myeloid restricted IKK-beta deleted mice were generated by a Cre/lox recombination system to inactivate the NF-kB pathway in macrophages. Thioglycollate-elicited peritoneal macrophages were incubated with 200 μg/ml of low-density lipoprotein and simultaneously exposed to either IH (Normoxia: 8min, 0.5% O2: 10min) or normoxia for 24 hours. After exposure, the extent of foam cell formation was assessed by quantification of intracellular cholesterol. Finally, we compared the differences in gene expression using RNA-seq between wild type and IKK-beta deleted macrophages exposed to either IH or normoxia for 24 hours.

Results:
IH significantly increased total cholesterol in wild type macrophages (63.4±3.3 μg/mg of cellular protein, n=9) in comparison to normoxia (51.2±1.6). Interestingly, such increase in intracellular cholesterol in response to IH-exposure was abolished by IKK-beta deletion (IH 52.4±1.1; normoxia 50.0±1.6 n=8), suggesting that NF-kB pathway regulated gene expression is critical for IH-induced foam cell formation. Indeed, we have found that NF-kB knockout abolished IH-induced expressional alterations in 364 genes, which are potential candidates for regulating intracellular cholesterol.

Conclusion:
NF-kB activation plays a critical role in IH-induced macrophage foam cell formation.
Abstract 378: Contribution of Unfolded Protein Response in Arsenic-Induced Endothelial Activation and Atherosclerois
Authors: Sithu Srinivas D , Wickramasinghe Nalinie S , Vladykovskaya Elena , Haberzettl Petra , Agarwal Abhinav , Winner Millicent , D’Souza Stanley E , Srivastava Sanjay
Published: July 2021

Epidemiological and animal studies suggest that exposure to arsenic contaminated water exacerbates atherosclerosis. However, the mechanisms by which arsenic exerts its atherogenic effects are not known. We observed that chronic (1 μM; 4 days) or acute (20 μM; 2-6 hours) exposure of sodium arsenite to human umbilical vein endothelial cells (HUVEC) increased the surface expression of adhesion molecules ICAM-1, VCAM-1 and E-Selectin by 1.2-1.5-fold; leukocyte adhesion by 1.5-3.5 fold; leukocyte trans-endothelial migration by 1.7-2.5-fold; and mRNA expression of IL-8 by 5-25-fold. Similarly, exposure of human aortic endothelial cells and mouse aortic endothelial cells to arsenic also caused endothelial activation. Arsenic also enhanced the unfolded protein response (UPR) in endothelial cells as evident by the phosphorylation of IRE-1 and activation of its downstream proteins JNK and NF-kappaB; nuclear translocation of ATF6, and increased expression of molecular chaperones GRP78 and HERP. SiRNA-mediated knockdown of IRE-1 attenuated arsenic-induced NF-kappaB activation and IL-8 expression, but did not affect JNK phosphorylation. Adenoviral transfection with ATF-6 upregulated GRP78 and decreased the expression of IL-8. Similarly, pre-incubation of endothelial cells with phenyl butyric acid (PBA; 5 mM, 16h), a chemical chaperone of protein folding, significantly (P<0.05) prevented arsenic-induced leukocyte adhesion, trans-endothelial migration and IL-8 expression. Feeding of apoE-null mice with PBA for 16 weeks inhibited the arsenic-induced UPR in atherosclerotic lesions; expression of adhesion molecules on endothelial cells lining the atherosclerotic lesions; lesional and systemic inflammation; and prevented the arsenic-induced exacerbation of lesion formation by 90% (P<0.05). Together, these data suggest that arsenic causes endothelial activation and exacerbates atherosclerosis by triggering UPR and chemical chaperones of protein folding prevent arsenic-induced exacerbation of atherogenesis.
Abstract 379: Introducing Stress-Based Quantitative Plaque Vulnerability Index for Patients with Coronary Artery Disease
Authors: Tang Dalin , Wang Liang , Zheng Jie , Wu Zheyang , Maehara Akiko , Yang Chun , Bach Richard G , Muccigrosso David , Mintz Gary S
Published: July 2021

Introduction:
Mechanical forces play an important role in plaque progression and rupture process. It is of interest to introduce stress-based plaque vulnerability indices and to study their associations with morphology-based vulnerability indices. Stress-based indices include both mechanical and morphological conditions and may improve over image-only assessment schemes.

Method:
In vivo intravascular ultrasound (IVUS) data were acquired from 14 patients (11M, 3F, mean age: 59) after voluntary informed consent. IVUS-based 3D fluid-structure interaction (FSI) models were constructed to obtain critical plaque wall stress (CPWS), strain (CPWSn), flow shear stress (CFSS), cap thickness and lipid percentage following our established procedures. 617 slices from the 14 plaques were obtained for analysis. Analogous to AHA histology-based classifications, three morphological indices with values from 0 to 4 were introduced for each slice. A lipid index was assigned depending on slice lipid percentage. A cap index was assigned according to slice minimal cap thickness. The morphological index was defined as the maximum of lipid index and cap index. Each slice was assigned a stress index (0, 1, 2, 3, or 4) according to its CPWS value by using five stress intervals with optimal match rates with morphological index. Correlations between mechanical and morphological quantities were investigated.

Results:
Based on data from the 617 slices, CPWS had negative correlation with cap thickness (r = -0.6570, p<0.001) and positive correlation with lipid percentage (r=0.2209, p<0.001). CPWS correlated with morphological index positively (r=0.7725, p<0.001). CFSS correlated with cap thickness positively (r = 0.1336, p<0.001) and lipid percentage negatively (r = -0.1569, p=0.0025). CFSS correlations were weaker. The stress index correlated positively with morphological index (r=0.7939, p<0.001). The match rate between the two indices was 66.8%.

Conclusion:
The results demonstrated that stress index had good agreement with morphological features closely related to plaque vulnerability. Stress index provides an opportunity for improved assessment for the 33.2% cases where two indices differ. Final validation of those indices requires large-scale patient studies.
Abstract 385: Factor XII Knockout or Knockdown in Rat Produces Robust Antithrombotic Efficacy With No Bleeding
Authors: Chen Zhu , Cai Tian-Quan , Tadin-Strapps Marija , Shin Myung , Wu Weizhen , Xu Yiming , Hoos Lizbeth , Zhou Yuchen , Jochnowitz Nina , Seiffert Dietmar
Published: July 2021

Coagulation Factor XII (FXII) is an attractive novel target for antithrombotic therapies as studies with FXIIa inhibitors and FXII knockout mouse suggest FXII is critically involved in pathological thrombus formation but is dispensable for hemostasis. To further assess the role of FXII in thrombosis and hemostasis, FXII knockout or knockdown models in rat were established and characterized. FXII knockout in rat was generated via the Zinc Finger nuclease (ZFN) technology and confirmed by lack of detectable zymogen in circulation. Titratable FXII knockdown in rat was achieved with a siRNA reagent that is potent on FXII mRNA in liver and selective against all other coagulation factors. FXII knockout in rats produced marked antithrombotic efficacy in both the arteriovenous shunt model (~100% clot weight reduction), and the 10% FeCl
3
-induced carotid artery thrombosis model (no reduction in blood flow post injury), without any increase in the cuticle bleeding time compared to the WT control rats. Ex vivo aPTT and ellagic acid-triggered thrombin generation assay (TGA) exhibited corresponding changes whereas ex vivo PT or TF (20 pM)-triggered TGA was indistinguishable from WT rats. Rats that received a single dose of FXII siRNA (at 0 (non-targeting siRNA control), or 0.01, or 0.03, or 0.1, or 0.3 mg/kg) exhibited dose-dependent knockdown in FXII mRNA in the liver and FXII zymogen in plasma (90% and 97%, respectively, for the 0.3 mg/kg dose) at day 7 post dosing. FXII knockdown was associated with dose-dependent antithrombotic efficacy (with the 0.3 mg/kg dose delivering near maximal efficacy in both models) and minimal increase in cuticle bleeding times (<50% across all doses). Ex vivo pharmacodynamic markers (aPTT and TGA) tracked with the knockdown levels and efficacy. In summary, our findings in both the knockout model and the knockdown model in rats confirmed and extended literature observations with regard to the highly desirable benefit-to-risk profile of targeting FXII in preclinical species; the siRNA tool will allow detailed studies on level of target engagement needed for translatable efficacy; the KO rat will be valuable for addressing any mechanism-based safety concerns as well as off-target effects of putative FXIIa inhibitors.
Abstract 408: Identifying the Molecular Basis of Monocyte Development Using Enhancer Profiling
Authors: Thomas Graham D , Hanna Richard N , Glass Christopher K , Hedrick Catherine C
Published: July 2021

Intro:
Monocytes (Mo) are key drivers of atherosclerosis. In mouse the two main monocyte populations are distinguished based on Ly6C expression. Classical Ly6Chi Mo drive atherosclerosis while Ly6Clow Mo help to maintain vascular integrity. Indirect evidence also suggests that Ly6Clow Mo protect against atherosclerosis. Mo develop in the bone marrow (BM) from the macrophage dendritic cell precursor (MDP) and common monocyte progenitor (cMoP), although precise ontological relationships are unclear. We have recently described an obligate cell-intrinsic role for the transcription factor Nr4a1 in Ly6Clow monocyte development.

Hypothesis:
Enhancers drive transcription factor-dependent programs of gene expression in a cell-specific manner. We hypothesize that a functional analysis of Nr4a1-associated enhancers will provide insight into mechanisms regulating Ly6Clow Mo development. Furthermore, a genome-wide analysis of Mo and their progenitor enhancers will consolidate our understanding of lineage relationships between these cells.

Methods:
Using H3K4me2 and H3K27ac to define enhancers and enhancer activity respectively we have performed ChIP-Seq on primary mouse MDP, cMoP, Ly6Chi and Ly6Clow Mo.

Results:
Of 114,755 enhancers 10,070 were differentially regulated. Hierarchical cluster analysis of these regions supports the consensus view that Ly6Clow Mo arise from Ly6Chi Mo. A 20kb ‘super-enhancer’ spanning Nr4a1 (Nr4a1se) is selectively induced in Ly6Clo Mo. We have begun to dissect Nr4a1se to identify regulators of Ly6Clow monocyte development. Three Nr4a1se regions have in vitro enhancer activity and show high H3K27ac in CD14dimCD16+ Mo, the proposed orthologue of mouse Ly6Clow Mo. This is evidence that Nr4a1 expression is conserved between humans and mice.

Conclusion:
We have identified candidate enhancers regulating Ly6Clow monocyte development. We are currently knocking out these regions using the CRISPR-Cas9 system to test their role in Ly6Clow Mo development in vivo. We aim to present preliminary data from these experiments at this meeting.
Abstract 420: Exploring the Role of TNFα in TLR4-/NLPR3 Inflammasome-driven Inflammation in Humans
Authors: Moerland Matthijs , Malone Karen , Dillingh Marlous , Grievink Wieke , Reijers Joannes , Burggraaf Koos
Published: July 2021

The role of TNFα in the pathogenesis of atherosclerosis is incompletely understood. TNFα blockade reduces the severity of various autoimmune diseases and the often related atherosclerosis. However, excessively released TNFα is only one component of the hyperactive innate immune system in such diseases. To provide more insight into the role of TNFα in the induction of inflammation, we explored the effects of TNFα blockade in human whole blood.

TLR4/NLPR3 inflammasome challenges were applied to induce an inflammatory response. For this purpose, whole blood was incubated 4 hours with LPS and aluminium hydroxide (Alhydrogel). TNFα blockade was evaluated in vitro (LPS/Alhydrogel challenge in whole blood of 4 healthy human subjects, +concentration range of adalimumab) and ex vivo (LPS/Alhydrogel challenge in whole blood of 13 healthy human subjects receiving a single subcutaneous (sc) dose of 40 mg adalimumab). Cytokine release was evaluated in culture supernatants.

In vitro, TNFα blockade strongly reduced TNFα levels detected; -97±1% at the lowest adalimumab concentration (0.3125 μg/mL). TNFα blockade did not affect LPS/Alhydrogel-induced IL-6, IL-1β and IL-18 release, but reduced IFNγ release; maximally -93±4% at 5 μg/mL adalimumab. A single sc adalimumab dose in healthy subjects reduced LPS/Alhydrogel-induced TNFα levels (maximally -98±1% on day 4, and still -58±59% on day 64; versus baseline). IL-6, IL-1β and IL-8 release were not reduced after anti-TNFα treatment. The effect of TNFα blockade on IFNγ release could not be reliably estimated due to highly variable IFNγ levels, especially between genders (baseline IFNγ levels 1248±1771 and 140±283 pg/mL, males vs females).

TNFα is a major inducer of NFκB-driven cytokine gene transcription, but TNFα blocking did not reduce LPS/Alhydrogel-induced release of IL-1β, IL-6, IL-8 or IL-18 by primary human cells. This suggests that primary TLR4- and inflammasome-mediated signalling is sufficient to drive secretion of these cytokines. However, in vitro TNFα blockade did impair IFNγ release. Since IFNγ is a key factor in atherogenesis, exerting both pro- and anti-atherogenic properties, our data warrant further mechanistic investigation of the role of TNFα and anti-TNFα therapies in atherosclerosis.
Abstract 447: β-cyclodextrin Reduces Cholesterol Crystal-induced Inflammation Through Modulating Complement Activation
Authors: Espevik Terje , Bakke Siril S , Niyonzima Nathalie , Damås Jan K , Ryan Liv , Latz Eicke , Mollnes Tom E
Published: July 2021

Atherosclerosis is an inflammatory condition and the underlying cause for cardiovascular disease. Cholesterol crystals (CC) are found to be abundant in atherosclerotic plaques and we have previously shown that CC initiate an inflammatory response via the complement system and inflammasome activation. Cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (BCD) is a compound that solubilizes lipophilic substances and is commonly used in pharmaceuticals or drug delivery. BCD is reported to increase cholesterol solubility and to promote the removal of cholesterol from foam cells. However, it remains unknown whether BCD has any effect on crystalline cholesterol. We here show that BCD attenuates the CC -induced inflammatory cytokine response as well as regulates a range of CC-related genes in human peripheral blood mononuclear cells. BCD binds to CC and prevents deposition of complement factors on CC in human plasma. Furthermore, BCD also decreases the formation of soluble terminal complement complex (TCC) and the expression of complement receptor 3 in response to CC stimulation in human whole blood. Induction of TCC by mono sodium urate crystals or zymosan was not affected by BCD. Of interest, after 1 hr of incubation, BCD is starting to dissolve the CC. These data demonstrate that BCD is a strong inhibitor of CC-induced inflammation, which might be explained by BCD-mediated attenuation of complement activation. These data suggest that BCD is a potential candidate for treatment of atherosclerosis.
Abstract 458: Loss of Smooth Muscle α-actin in Mice Results in Thoracic Aortic Aneurysms via Increased Reactive Oxygen Species, Increased Nox4,and Increased Angiotensin II type 1 Receptor-Mediated Signaling
Authors: Chen Jiyuan , Kwartler Callie , Papke Christina L , Peters Andrew , Ringuette Lea-Jeanne , Cao Jiumei , Wang Shanzhi , Villamizar Carlos , Byanova Katerina L , Madonna Rosalinda , Kee Patrick , Geng Yong-Jian , Brasier Allan R , Davis Elaine C , Prakash Siddharth , Milewicz Dianna M
Published: July 2021

Objective:
ACTA2 mutations cause 10-14% of familial thoracic aortic aneurysms and dissections. Mice deficient in smooth muscle α-actin (Acta2-/-) develop root and ascending thoracic aortic enlargement associated with thickening of the aortic media and fragmentation and disarray of elastic fibers. We hypothesized that blocking AT1 activation would block the aortic pathology and prevent aortic enlargement in Acta2-/- mice.

Methods and Results:
Beginning at 4 weeks of age, Acta2-/- mice were treated with losartan or placebo (n≥10) for 6 months and echocardiograms were performed at baseline and every other month. The aortic root in Acta2-/- mice was found to undergo progressive dilatation. After 6 months of treatment, there was no difference in the diameter of the aortic root between wild-type (WT) mice and the losartan treated mice (p=0.44). Histologic analysis of Acta2-/- aortas demonstrated medial thickening and fragmentation of elastic fibers which was normalized by treatment with losartan. Gene expression of matrix metalloproteinase-2 and -9 (Mmp2, Mmp9), along with lumican and decorin, interleukin-6 (Il6) and phosphorylation of RelA (a subunit of nuclear factor κB, NF-κB) was increased in Acta2-/- aortas, and was corrected by treatment with losartan. NADPH oxidase 4 and AT1a mRNA was increased in Acta2-/- aortic smooth muscle cells (SMCs) and aortas. Increase of AT1a was blocked by lowering reactive oxygen species (ROS) with N-actetyl cysteine (NAC). Angiotensin II (AngII) dose response studies suggested Acta2-/- aortic SMCs had increased sensitivity to Ang II. Additionally, Acta2-/- SMCs had increased ROS compared to WT by flow cytometry (P<0.05). Increased ROS was also observed in aortic samples derived from Acta2-/- mice. Blocking ROS using NAC attenuated aneurysm formation by reducing the expression of Mmps and IL6 and reducing signaling through AT1 in Acta2-/- mice. Genetic deletion of AT1a attenuated ROS and expression of Mmps and IL6 levels in the Acta2-/-aortas but not to WT levels.

Conclusions:
Our results demonstrate that complete loss of α-SMA leads to aortic dilation and pathologic changes by increasing SMC ROS levels, thus increasing sensitivity to AngII, which results in NF-κB activation and increased expression of Mmps and IL6.