bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 462: Reductions of ApoB-containing Lipoproteins Prevented the Progression of Established Abdominal Aortic Aneurysms in AngII Infused Mice
Authors: Liu Jing , Lu Hong , Moorleghen Jessica , Howatt Deborah , Balakrishnan Anju , Cassia Lisa , Daugherty Alan
Published: July 2021

Background and Objective:
We have demonstrated that increased plasma apolipoprotein B (apoB)-containing lipoproteins augment development of AngII-induced abdominal aortic aneurysms (AAAs) in male mice. The purpose of this study was to determine whether plasma apoB-containing lipoproteins also influenced progression of established AAAs during chronic AngII-infusion.

Methods and Results:
Male LDL receptor -/- mice fed either a normal (Group 1) or Western (42% calories from saturated fat) diet were infused with AngII (1,000 ng/kg/min) for 28 days to induced AAAs. Maximal luminal diameters of the suprarenal region were compared with baseline after 4 weeks of AngII infusion to determine AAA presence. Mice fed Western diet were stratified by aortic lumen diameter into either a group maintained on Western diet (Group 2) or switched to a normal laboratory diet (Group 3). AngII infusion was continued for another 8 weeks in all 3 groups. Plasma cholesterol concentrations were determined at 4, 6, and 12 weeks of AngII infusion. Mice fed a Western diet for the initial 4 weeks were hypercholesterolemic (260 ± 13 to 1,469 ± 59 mg/dl). Two weeks after Group 3 were returned to normal laboratory diet, plasma cholesterol concentrations were profoundly reduced to 382 ± 22 mg/dl which was maintained for the remaining interval. These reductions were due to decreases in plasma cholesterol concentrations apoB-containing lipoproteins. Luminal diameters of suprarenal aortas were measured at 6 and 12 weeks of AngII infusion by ultrasonography. While luminal expansion progressed in mice fed Western diet, no continuous expansion was detected in mice with established AAAs that were fed normal laboratory diet. Ultrasonic measurements were confirmed at study termination by ex vivo measurements of maximal widths of suprarenal aortas (Group 1 verus 2 versus 3: 1.35 ± 0.04 mm, 3.32 ±0.34 mm, 1.91 ± 0.11 mm, respectively).

Conclusions:
Plasma apoB-containing lipoproteins contribute to the progressive expansion of established AAAs in AngII-infused mice.
Abstract 47: High-Density Lipoproteins Induce Neutrophil-miRNA Transcription and Export: Contribution to HDL-miRNA Cell to Cell Communication
Authors: Tabet Fatiha , Cuesta Torres Luisa F , Wiese Carrie B , Öhrling Gustav , Larsson Rasmus , Barter Philip J , Vickers Kasey C , Rye Kerry-Anne
Published: July 2021

Background:
We have recently shown that high-density lipoproteins (HDL)-associated miR-223 is exported from HDL to endothelial cells in a key pathway mechanism by which HDL mediate the suppression of endothelial cell adhesion molecule expression. Interestingly, miR-223 was found to be the most abundant miRNA in polymorphonuclear neutrophils (PMNs) and miR-223 is critical to PMNs activation. Here, we sought to determine if PMNs export miR-223 to HDL and if PMNs are a significant contributor to HDL-miR-223 cell-to-cell communication, which we previously reported.

Results:
To determine if PMNs export miR-223 to native HDL, PMNs and HDL were collected from 8 healthy donors by polymorph preps and density-gradient ultracentrifugation; respectively. HDL (1 mg/mL) were incubated with PMNs for 4 h and HDL were then isolated from culture media using apoA-I immunoprecipitation columns. Total RNA was isolated from HDL, and miR-223 levels were measured by TaqMan assays. miRNA export assays showed a significant increase in miR-223 on HDL post-PMN incubation compared to pre-incubation. To determine if other myeloid cells also export miR-223 to HDL, we isolated human monocytes by elutriation and differentiated them to macrophages (HMDM) over 7 days. Similar to PMNs, HMDM were also found to export miR-223 to HDL after 16 h. As a consequence of miR-223 export, we predicted that PMN cellular levels of mature miR-223 would decrease. Strikingly, we found no differences in cellular mature miR-223 levels in PMNs or HMDM treated with HDL. To determine if cellular miR-223 levels are supported by increased miR-223 transcription in response to HDL induction and miRNA export, we quantified primary (pri)-miR-223 levels using real-time PCR and TaqMan assays. We found that HDL induce a significant 3.75-fold increase in PMN pri-miR-223 levels after 4 h. Likewise; we found a significant 2.24-fold increase in HMDM pri-miR-223 levels after 16 h of HDL treatment

Conclusion:
PMNs export miR-223 to HDL; however, miRNA export does not reduce cellular mature miR-223 levels likely due to HDL-induced pri-miR-223 transcription. This suggests that HDL induce the transcription of miRNAs selected for export and indicates that cells can satisfy demand for export and maintain cellular miRNA levels.
Abstract 472: Prevalence of Fredrickson-Levy Dyslipidemia Phenotypes at Extreme HDL-C Levels: The Very Large Database of Lipids (VLDL-9B)
Authors: Quispe Renato , Al-Hijji Mohammed , Swiger Kristopher J , Martin Seth S , Elshazly Mohamed , Blaha Michael J , Joshi Parag H , Blumenthal Roger S , Sniderman Allan , Toth Peter P , Jones Steven R
Published: July 2021

Introduction:
Big data techniques offer a novel opportunity to characterize individuals with extreme phenotypes. In this context, we aimed to describe the prevalence of classical Fredrickson-Levy dyslipidemia phenotypes at the extremes of HDL-C levels in a cross-sectional big data study.

Methods:
We examined 848,801 U.S. adults and children from the Very Large Database of Lipids 1.0 who were referred for lipoprotein testing from 2009 to 2011. We categorized patients into HDL-C percentile categories (<0.1th, 0.1th to 99th to 99.9th, and >99.9th). We examined the prevalence of Fredrickson-Levy dyslipidemia phenotypes (I, IIa, IIb, III, IV and V) within these categories. We identified those who did not meet criteria for any classical dyslipidemia phenotype as the continuum group.

Results:
Type I and V were mostly present at extremely low HDL-C levels. Type IIa was more prevalent in high vs. low HDL-C levels. Type III was 2-fold more prevalent in extremely low vs. high HDL-C levels. Type IV was the most prevalent classical dyslipidemia phenotype in our population, and was the most frequent at low HDL-C percentiles. About 50% of the extremely low and 90% of the extremely high HDL-C levels were classified into the continuum group.

Conclusion:
In our cross-sectional big data analysis, there was a significantly higher prevalence of most classical dyslipidemia phenotypes at extremely low HDL-C compared with extremely high HDL-C levels. Only types I and V were more prevalent in extreme groups than general population. To some extent, very low HDL-C levels may be determined by inheritable, highly atherogenic dyslipidemias.
Abstract 491: Detection of Subclinical Coronary Artery Disease by Calcium Score in Patients With Atrial Fibrillation: Potential Clinical Implications
Authors: Chaikriangkrai Kongkiat , Valderrabano Miguel , Khaleel Bala Sayf , Alchalabi Sama , Graviss Edward , Nabi Faisal , Mahmarian John , Chang Su Min
Published: July 2021

Background:
Clinical implications of detecting subclinical coronary artery disease (CAD) in patients with atrial fibrillation (AF) are unclear.

Methods:
A total of 430 AF patients (age 63 ± 10 y, 65% male, 62% hypertensive, 16% diabetic, 42% dyslipidemic) without known CAD undergoing pre-procedural CT for catheter ablation were included. We evaluated the change in:

1) numbers of patients with CACS-diagnosed CAD who could potentially be on statin.

2) CHA2DS2-VASc score after incorporating CACS>100 (related to increased risk of stroke) into the original definition of vascular diseases who could potentially be on anticoagulants.

Results:
1) Prevalence of subclinical CAD (CACS>0) was 74% (319/430) and 25% (106/430) had CACS>100. There were 62% (267/430) who were not on statin. Of these patients, 71% (190/267) had subclinical CAD while 21% (34/163) of statin users had CACS of 0.

2) The median original CHA2DS2-VASc score was 2. After incorporating CACS>100 into the original score, 24% (18/75) with the original score of 0 had the score changed to 1 (7/35 in persistent AF [PST-AF] and 11/40 in paroxysmal AF [PRX-AF]) (figure A) and 17% (22/131) with the original score of 1 had the score changed to ≥ 2 (10/83 in PST-AF and 12/48 in PRX-AF) (figure B). PRX-AF had more frequent increase in CHA2DS2-VASc score than PST-AF (p=0.035)(figure C).

Conclusion:
In AF patients without known history of CAD, detecting subclinical CAD by CACS potentially has important therapeutic implications for prevention forprogression of CAD and stroke.
Abstract 500: Vitamin D Supplementation Attenuates ADAM-12-mediated Proliferation of Carotid Artery Smooth Muscle Cells of Hypercholesterolemic Swine
Authors: Kansal Vikash , Patterson Eric B
Published: July 2021

Introduction:
Risk of hypertension, peripheral artery disease, myocardial infarction and development of human atherosclerosis has been linked to vitamin D deficiency. Atheromatous cytokines, including IL-6, TNF-α and epidermal growth factor receptor (EGFR) family growth factors are released at the site of atherosclerosis and boost the activity of proteolytic enzymes such as ADAMs (a disintegrin and metalloproteinases). ADAM-12 cleaves proHB-EGF (Heparin-binding EGF-like growth factor) activating EGFR, resulting in increased proliferation of smooth muscle cells (SMCs). The aim of this study was to examine the effect of vitamin D on IL-6 and TNF-α-induced ADAM-12, pEGFR expression, HB-EGF release and SMC proliferation.

Methods:
Micro-swine were fed with vitamin D-deficient high cholesterol diet, high cholesterol diet containing 900 IU of vitamin D, and high cholesterol diet containing 3000 IU of vitamin D for total of 12 months. After six months, serum cholesterol levels of 500-600 mg/dL were achieved in all the three groups. The protein expression of ADAM-12 & pEGFR, and HB-EGF release, in presence or absence of IL-6, TNF-α and Calcitriol, in SMCs was quantified by western Blot. HB-EGF release was measured by ELISA. The proliferation was assayed by [3H]-Thymidine incorporation and cell counting method.

Results:
The protein expression of ADAM-12 & pEGFR, HB-EGF release were significantly reduced in carotid artery SMCs isolated from Vitamin D-supplemented swine. IL-6 and TNF-α treatment increased the protein expression of ADAM-12 & pEGFR and HB-EGF release in carotid artery SMCs. Proliferation capacity was higher in SMCs isolated from Vitamin D-deficient swine carotid artery, potentiated by IL-6 and TNF-α. Calcitriol inhibited the ADAM-12, pEGFR expression and HB-EGF released in SMCs of hypercholesterolemic swine. Calcitriol also inhibited the proliferation of carotid artery SMCs isolated from Vit D-deficient, D-sufficient and D-supplemented swine.

Conclusion:
Together, these results suggest that vitamin D deficiency enhances proliferation of SMCs, which is potentiated by atheromatous cytokines. Whereas, vitamin D supplementation regulates ADAM-12-mediated cleavage of proHB-EGF and activation of EGFR inhibiting SMC proliferation.
Abstract 502: Smokers Have Significantly Increased Rates of Intraoperative Microembolization Detected on Transcranial Doppler with Carotid Angioplasty/Stenting Compared to Endarterectomy Procedure in Preliminary Study
Authors: Li Jennifer , Pina Christian , Alicea Daniel , Giannarelli Chiara , Mani Venkatesh , Vouyouka Ageliki , Krishnan Prakash , Tadros Rami , Badimon Juan , Fayad Zahi , Wiley Jose , Faries Peter
Published: July 2021

Background:
The benefits of revascularization must be balanced against the risks of peri/post-operative cerebrovascular events in determining the management of carotid stenosis. Higher rates of intra-operative microembolization during elective carotid revascularization are associated with increased risks of clinically detectable cerebrovascular accidents, and the identification of specific patient demographics that predispose to vulnerable plaque formation can be useful in guiding intervention and decreasing embolic potential during procedures.

Methods:
150 patients (symptomatic, stenosis>50%; asymptomatic, stenosis>70%) undergoing either CAS or CEA will be enrolled. A pre-operative Mini-mental Status Exam, carotid MRI, and cerebral diffusion-weighted MRI are obtained one week prior to intervention. All MRI are performed with 3T MR system, carotid plaque are classified according to AHA criteria. Intraoperatively, transcranial Doppler is performed to monitor for microembolic signals (MES), detected as high-intensity unidirectional transient signals. Plaque specimen and filter debris are analyzed histologically for high-risk characteristics and correlated with MRI. A post-operative DW-MRI and MMSE are performed within 72 hours to examine for microinfarcts and changes in neurocognitive function. Demographic analysis for cardiovascular risk factors, current and former smokers, was performed retrospectively.

Results:
Preliminary results with 33 patients demonstrate an average of 46 total MES generated during carotid angioplasty/stenting in 4 nonsmokers, compared to 19.9 total MES in 9 nonsmoking patients undergoing endarterectomy (p = 0.091). In the smoker population, an average of 63.4 total MES was generated in 13 patients undergoing CAS compared to 21 total MES in 7 patients undergoing CEA (p = 0.024). Procedural phases with the highest production of microemboli include dissection phase during CEA (35.5% of total MES) and filter placement in CAS (22.2% of total MES).

Conclusions:
Preliminary results suggest significantly increased microembolic potential and risk of clinically detectable CVA with carotid angioplasty/stenting compared to endarterectomy in smokers.
Abstract 507: Plasma Cyclophilin A as a Useful Biomarker for Effective Percutaneous Transluminal Pulmonary Angioplasty in Chronic Thromboembolic Pulmonary Hypertension
Authors: Satoh Kimio , Sugimura Koichiro , Aoki Tatsuo , Tatebe Shunsuke , Shimizu Toru , Ikeda Shohei , Yaoita Nobuhiro , Kudo Shun , Suzuki Kota , Omura Junichi , Kikuchi Nobuhiro , Satoh Taijyu , Kurosawa Ryo , Sunamura Shinichiro , Otsuki Tomohiro , Shimokawa Hiroaki
Published: July 2021

Background:
Cyclophilin A (CyPA, encoded by Ppia) is secreted from pulmonary vascular smooth muscle cells in response to several stimuli including mechanical stretch and hypoxia. Recently, we demonstrated that extracellular CyPA and vascular Basigin (Bsg, encoded by Bsg) are crucial for hypoxia-induced PH by inducing growth factor secretion, inflammatory cell recruitment and VSMC proliferation in mice. Moreover, we demonstrated that high plasma CyPA levels are significantly associated with poor outcome (death or lung transplantation) in patients with pulmonary arterial hypertension (PAH). In this study, we examined plasma levels of CyPA in patients with chronic thromboembolic pulmonary hypertension (CTEPH) before and after percutaneous transluminal pulmonary angioplasty (PTPA), which is recently developed as a catheter-based therapy.

Methods and Results:
In consecutive 86 PTPA sessions in 24 patients, we examined the relationship between plasma CyPA levels, the severity of CTEPH and hemodynamic improvements after PTPA. We measured plasma CyPA levels by an immunoassay based on the sandwich technique. Plasma CyPA levels (ng/mL) were significantly elevated in patients with CTEPH (13.8±6.0, n=24) compared with those without PH (4.9±4.0, n=7) or healthy controls (4.6±2.8, n=18) (both P<0.001). Moreover, the severity of CTEPH assessed by pulmonary vascular resistance (PVR) correlated with plasma levels of CyPA, IFN-α2, stem cell factor and adipsin (all P<0.05). PTPA significantly reduced mean pulmonary artery pressure (P<0.05), PVR (P<0.01) and improved long-term prognosis compared with historical controls. Importantly, PTPA significantly reduced plasma CyPA levels in patients with CTEPH (5.4±4.1, P<0.001), suggesting that plasma CyPA is useful for evaluation of therapeutic effects of PTPA. Finally, we observed significant increase in anti-inflammatory cytokines (interleukin-4, 7, 13) and reduction in stem cell growth factor-β and adipsin (all P<0.05) after PTPA, suggesting improved metabolic and inflammatory state in CTEPH patients.

Conclusions:
These results indicate that plasma levels of CyPA are significantly increased in CTEPH patients and could be considered as an effective biomarker for assessment of the effects of PTPA.
Abstract 535: Lipid Phosphate Phosphatase 3: A Novel Mediator of Heart Failure Development and Progression
Authors: Panchatcharam Manikandan , Maxey Benjamin , Day Alicia , Escalante-Alcalde Diana , Miriyala Sumitra
Published: July 2021

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that is found in abundance in atherosclerotic plaques and whose production is accelerated by activated platelets. Two fold rise in serum LPA concentration in acute myocardial infarct patients (6.4±1.3 micro mol/L vs 2.5 ± 1.1 micro mol/L, P = 0.0001) and its effect to cause cardiomyocyte hypertrophy suggests, a critical role for LPA regulation in the myocardium. The actions of local and circulating LPA may be terminated by enzymatic dephosphorylation of the lipid by a family of hexahelical membrane spanning proteins, termed lipid phosphate phosphatases (LPP). Of the three LPP enzymes with preference for LPA, LPP3 appears the most likely to play a biologic role in regulating LPA levels, and deficiency of LPP3 results in embyronic lethality in mice due to vascular and neural tube defects. We have generated mice that specifically lack LPP3 in cardiomyocytes. These mice are viable and fertile but showed increased mortality ~8 months (Fig1). Blood pressure was similar in Ppap2b
fl/fl
(96 ± 9 mmHg; n = 19) and Myh6-Ppap2b
Δ
mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6-Ppap2b
Δ
3 month old mice (642 ± 21 bpm, compared to Ppap2b
fl/fl
with 600± 17 bpm; P<0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiomyocyte hypertrophy induced by LPA.
Abstract 54: Syndesome-Based Dressings for Enhanced Wound Healing in Diabetic Ulcers
Authors: Das Subhamoy , Singh Gunjan , Monteforte Anthony , Martinez Matthew , Wright Catherine , Martin Patricia , Dunn Andrew , Baker Aaron
Published: July 2021

Incidence of chronic non-healing wounds has significantly increased over the last decade due to a rising epidemic in type-II diabetes and peripheral arterial disease (PAD). Previous research has attempted to use growth factor proteins or genes to enhance the healing of cutaneous wounds but have achieved only limited success in healing chronic wounds in the long-term. Our previous work has demonstrated a significant reduction of syndecan-4 protein due to long-term diabetic condition and co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 enhanced angiogenesis. In this study, we tested the efficacy of a novel wound dressing that delivered FGF-2 with syndesomes in ob/ob mice.

To recapitulate the human disease state, we used ob/ob mice and fed them a high fat diet for 15 weeks. We utilized a splinted, excisional wound model and implanted 2% alginate disks containing treatments into the wound, which were fabricated using a custom made high throughput mold. We monitored the perfusion of the wounds over time using laser speckle imaging.

At day 14, wounds treated with syndesomes (S4PL) and FGF-2 healed the wound significantly more than all other groups (A, B). Histological analysis demonstrated increased re-epithelialization of the wounds treated with S4PL with FGF-2 (C, D). Laser speckle imaging of the wound showed increased perfusion in the S4PL with FGF-2 treated group. Furthermore, immunostaining for the M1 macrophage marker (CD86) showed significantly reduced inflammatory macrophages in both S4PL+FGF-2 and S4PL groups (E, F). Staining for an M2 macrophage marker (CD163) revealed enhanced levels when the syndesomes were delivered, compared to control and FGF-2 groups (G, H).

Taken together, our studies support that syndesomes significantly enhance FGF-2 activity in wound healing in diabetic mice. Thus, syndesome-containing wound dressings may be useful in treating chronic wounds and restoring growth factor activity in diseased states.
Abstract 540: Correlation of Improved Cholesterol Efflux Capacity of High Density Lipoprotein With Survival and Allograft Vasculopathy in Cardiac Transplant Recipients
Authors: Javaheri Ali , Zamani Payman , Molina Maria , Rodrigues Amrith , Chambers Susan , Stutman Patricia , Maslanek Wilhelmina , Williams Mary , Lukmanova Daniya , Picataggi Antonino , Lilly Scott , Heeger Peter S , Sayegh Mohamed H , Chandraker Anil K , Sarling Randall C , Briscoe David M , Daly Kevin P , Stehlik Joseph , Ikle David , Christie Jason , Woo Y. Joseph , Goldberg Lee R , Billheimer Jeffrey , Rader Daniel J
Published: July 2021

Background:
Coronary allograft vasculopathy (CAV) is an important cause of mortality after cardiac transplantation. High density lipoprotein (HDL) cholesterol efflux capacity has been inversely associated with coronary artery disease and is impaired in cardiac transplant recipients. We performed a single center case-cohort study to test the hypothesis that reduced efflux capacity is a risk factor for mortality and a second, multi-center retrospective study to test if efflux capacity is associated with CAV progression.

Methods:
We designed a single center case-cohort study in which we identified cardiac transplant patients who died between 2009-2012 (cases, n=34) and controls as cardiac transplant patients who were alive as of the fourth quarter of 2013 (n=57). Efflux capacity was measured by incubating apolipoprotein B-depleted serum with macrophages in a validated ex vivo system. In a second study, we utilized pre-transplant samples from the Clinical Trials in Organ Transplantation 5 (CTOT5) study to determine the association between ATP-binding-cassette (ABC) A1-dependent cholesterol efflux and CAV progression at 1 year.

Results:
In our single center study, the average time from transplant to study entry was well-matched between cases and controls (7.6±1.0 vs 7.7±0.8 years, respectively, p=0.48). Multivariable Cox proportional hazard ratios demonstrated that higher levels of HDL cholesterol efflux capacity were associated with survival (HR 0.61, 95% CI 0.43-0.85), even after adjustment for HDL cholesterol mass. To determine whether excess mortality observed in subjects with reduced efflux could be attributable to CAV progression, we tested the relationship between intravascular ultrasound (IVUS) progression of CAV and cholesterol efflux capacity using linear regression. ABCA1-dependent efflux and IVUS progression were significantly associated (β = -0.90, 95% CI [-1.73 - -0.07], p = 0.037, R2 = 0.37).

Conclusion:
Reduced efflux capacity is an important mediator of CAV progression and mortality in cardiac transplant recipients. This finding suggests that interventions to increase HDL cholesterol efflux capacity may provide clinical benefit in cardiac transplant recipients.
Abstract 551: Deficiency of Hepatic Nuclear Factor 4 Alpha Results in Impaired Metabolism of Endogenous Methylarginines and Beta-aminoisobutiric Acid - A Novel Mechanism of Cardiovascular Complications in Patients With Type 2 Diabetes?
Authors: Burdin Dmitrii V , Kolobov Alexey A , Demyanov Anton V , Soshnev Alexey A , Broker Chad N , Samusik Nikolay , Brilloff Silke , Martens-Lobenhoffer Jens , Reetz Theresa , Maas Renke , Bode-Böger Stefanie M , Gonzalez Frank , Weiss Norbert , Rodionov Roman N
Published: July 2021

Introduction:
Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only known enzyme capable of degradation of all three endogenous methylarginines, which serve as markers and potentially mediators of cardiovascular disease. Recent studies also suggest that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) play important role in lipid metabolism. The predicted core promoter region of mammalian AGXT2 promoter contains a highly conserved putative binding site for hepatic nuclear factor 4 alpha (HNF4A). Patients with severe deficiency in HNF4a develop maturity onset diabetes of young 1. Furthermore, polymorphisms of HNF4A are associated with increased risk of diabetes type 2. The aim of this study was to test the hypothesis that HNF4A is a major regulator of AGXT2 expression and activity.

Methods and results:
We demonstrated direct binding of HNF4A to the Agxt2 promoter region in hepatic cell line Hepa 1-6 using chromatin immunoprecipitation assays. Then we showed that mutations of the predicted HNF4A binding site in the Agxt2 core promoter result in up to 80% decrease in the promoter activity as assessed by luciferase reporter assays (p<0.001). We used siRNA-mediated knockdown of HNF4A to determine whether this factor is required for basal Agxt2 expression in Hepa 1-6 cells. Knockdown of HNF4A led to almost 50% reduction in Agxt2 mRNA levels compared to controls (p<0.01). We took advantage of the previously characterized inducible liver-specific Hnf4a knockout (KO) mice to determine whether HNF4A regulates Agxt2 expression in vivo and showed a 90% (p<0.001) decrease in liver Agxt2 expression and a 85% (p<0.01) decrease in liver AGXT2 activity towards methylarginines in Hnf4a KO mice compared with the wild-type littermates. Finaly, on a functional level, Hnf4a KO mice had significant amounts of BAIB present in plasma, whereas BAIB was not detectable in the plasma of the wild-type littermates.

Conclusions:
In our study we identified HNF4A as the major regulator of Agxt2 gene expression. This finding suggests that diabetic patients with HNF4A deficiency might have a unique mechanism for development of cardiovascular complication via AGXT2-dependent impairment of lipid metabolism and methylarginines-mediated vascular dysfunction.
Abstract 561: A Novel Integrin Modulating Arg-Gly-Asp(RGD)-Peptide-Coated Stent Reduces Neoatherosclerosis in a Rabbit Model
Authors: Guo Liang , Koppara Tobias , Cheng Qi , Acampado Eduardo , Kolodgie Frank D , Davis Harry R , Virmani Renu , Joner Michael
Published: July 2021

Objective:
While drug-eluting stents have dramatically reduced restenosis, they are often associated with increased risk of late stent thrombosis and neointimal atherosclerosis (“neoatherosclerosis”). In-stent neoatherosclerosis represents a further accelerated and possibly more unstable manifestation of atherosclerotic progression in nascent neointimal tissue after stent implantation. We hypothesized that poor arterial re-endothelialization accompanied by leaky endothelial junctions is a primary mechanism contributing to neoatherosclerosis. In the present study, we tested the ability of a RGD-peptidomimetic-coating of bare metal stents to reduce the formation of in-stent neoatherosclerosis by promoting integrin-mediated adhesion of endothelial cells, and improving their functional integrity.

Approach and Results:
Established iliofemoral plaques were created in male New Zealand white rabbits (n=10) by balloon injury and feeding a high cholesterol (1.0%) diet for 5 wks, which was then switched to a low cholesterol diet (0.025%) for the remainder of the study. At 3 wks after diet switch, an RGD-coated stent was implanted together with a drug-eluting stent in the contralateral iliac artery, and animals were continued on the low cholesterol diet for 7 wks (serum cholesterol between 400-1,000 mg/dl). Endothelial permeability was assessed by FITC-dextran (250/500 kDa, approx. LDL radius) injected intravenously 1 h before termination. Oil red O and Filipin staining demonstrated neutral lipid and free cholesterol accumulation in the developing neointimal of stented arteries.
En face
confocal microscopy of the luminal surface following immunostaining against endothelial markers followed by scanning electron microscopy showed greater endothelial coverage of luminal surfaces for RGD-coated stents. Stents were further quantified by histomorphometric analysis for vascular healing, thrombus formation and inflammation.

Conclusion:
A rabbit neoatherosclerosis model was established to study the formation and progression of this novel disease manifestation. RGD-coated stents showed reduced formation of neoatherosclerosis in association with improved vessel healing and re-endothelialization compared to drug-eluting stents.
Abstract 570: Deficiency of Inhibitory Fcgamma Receptor II Does Not Exacerbate Atherosclerosis
Authors: Nagarajan Shanmugam , Zhu Xinmei , Ng Hangpong
Published: July 2021

Objective:
Functionally, Fcgamma receptors (FcgRs) can be classified as activating (FcgRI, III, and IV) and inhibitory (FcgRII, CD32b) receptors. We have reported that deletion of activating FcgRs in apoE knockout mice decreased atherosclerosis. In this report we investigated the hypothesis that the deficiency of inhibitory CD32b exacerbates atherosclerosis in a hypercholesterolemic mouse model.

Approach and Results:
ApoE-CD32b double knockout mice congenic to the C57BL/6 (apoE-CD32b
B6
-/-) were generated and atherosclerotic lesions were assessed. Contrary to our hypothesis, arterial lesions were significantly decreased in chow or high fat diet fed apoE-CD32b
B6
-/- male and female mice, relative to apoE-/- mice
.
Bone marrow chimera approach using apoE-/- mice transplanted with apoE-CD32b
B6
-/- marrow also showed significantly reduced arterial lesions. ApoE-CD32b
B6
-/- mice had increased levels of IL-10 and TGF-b by CD4+ T cells, while IFN-g and IL-17 was decreased. As our findings conflict with a previous report using apoE-CD32b
129/6
-/- mixed background, we investigated if strain differences contributed to the anti-inflammatory response. Macrophages from mixed apoE-CD32b
129/B6
-/- mice showed more IL-1b, IL-6 and MCP-1 in response to immune complexes, while congenic CD32b
B6
-/- mice showed more IL-10 and significantly reduced IL-1b. Interestingly expression of lupus-associated slam genes, located in close proximity to
cd32b
in mouse chromosome 1, is upregulated only in mixed CD32b
129/B6
-/- mice.

Conclusions:
Our findings demonstrate a detrimental role for CD32b signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-CD32b
B6
-/- mice. As 129/sv genome derived lupus associated genes have been implicated in lupus phenotype in CD32b
129/B6
-/- mice our findings suggest possible epistatic mechanism contributing to the decreased lesions.
Abstract 571: Atherogenic Macrophages Transfer microRNA-containing Exosomes to Naïve Cells and Promote Macrophage Migration
Authors: Nguyen My-Anh , Karunakaran Denuja , Rayner Katey
Published: July 2021

Atherosclerosis, a maladaptive inflammatory disease, is driven by the interplay between excess cholesterol accumulation in the vessel wall and the immune system. Macrophages are fundamental to the propagation of atherosclerosis due to their capacity to engulf modified lipoproteins and induce a pro-inflammatory state. Macrophages and other immune cells can secrete miRNAs in exosomes as a form of extracellular communication during inflammation and infection. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs and can repress gene expression in recipient cells when transferred via exosomes or other vesicles. Here, we hypothesize that atherogenic macrophages secrete miRNAs in exosomes to mediate cell-cell communication, which regulates the atherogenic response. Results: We examined the miRNA expression profile of exosomes from control and cholesterol-loaded (oxLDL-treated) mouse macrophages. Among the 88 differentially expressed miRNAs, 68 were down-regulated whereas 20 were up-regulated in exosomes isolated from cholesterol-loaded macrophages relative to control. The enrichment of several miRNAs such as miR-146a, miR-128, miR-185 and miR-503 in exosomes from atherogenic macrophages was confirmed by qPCR. Bioinformatic pathway analysis suggests that atherogenic exosomal miRNAs regulate cell migration and adhesion pathways via targeting the 3’UTR of migration/adhesion genes (i.e. integrin α3, VE-cadherin, CXCR4). Interestingly, live cell imaging demonstrated that fluorescent-labeled exosomes secreted from control or oxLDL-loaded macrophages were internalized by naïve macrophages and exogenous miRNA (from C.elegans) can transfer from normal/atherogenic macrophage exosomes to naïve cells. Further, naïve macrophages treated with extracellular components from atherogenic but not control macrophages promoted the migration of naïve cells towards a chemokine stimulus (1.5-fold increase in migration relative to control). Thus, miRNAs secreted from atherogenic macrophages may promote atherosclerosis by regulating macrophage migration in the vessel wall and we are now testing the function of these exo-miRNAs in vitro and in vivo.
Abstract 583: Receptor for Advanced Glycation End Products (
Ager
) and Diaphanous-1 (
Drf1)
Suppress Macrophage Reverse Transendothelial Migration and Regression of Diabetic Atherosclerosis
Authors: Senatus Laura M , Li Qing , Rosario Rosa , Liu Jianhua , Li Huilin , Vengrenyuk Yuliya , Distel Emilie , Barrett Tessa , Daffu Gurdip , Shen Xiaoping , Ramasamy Ravichandran , Fisher Edward , Schmidt Ann Marie
Published: July 2021

Macrophages display complex trafficking properties
in vivo
, involving interaction with vascular endothelial cells. Macrophage migration is an important mechanism linked to the progression and regression of atherosclerosis. The underlying mechanisms involved in these processes are not fully defined. RAGE is a multiligand cell surface macromolecule, which binds distinct ligands. The RAGE cytoplasmic domain interacts with Diaphanous-1 (
Drf1
), both
in vitro
and
in vivo
and this interaction is essential for RAGE ligand-mediated signaling in macrophages. We tested the hypothesis that RAGE and Diaphanous-1 suppress regression of diabetic atherosclerosis; at least in part by impaired reduction of plaque macrophage content in an AGE-enriched diabetic environment. In a aorta transplantation model, we examined the morphologic changes in LDLr-/- donor plaques found in
Ager
-/-,
Drf1
-/- or WT recipient diabetic mice. After lipid normalization into
Ager
-/- recipient diabetic mice, we observed reduced plaque macrophage density (CD68 -46%;p<0.05), but increased plaque collagen (PSR+43%;p<0.06), compared to WT diabetic recipients. Oil Red O staining for fatty deposit suggests a decrease (-51%;p<0.05) in plaque area stained and lower AGE staining (-32%;p<0.05) in diabetic
Drf1
-/- recipient mice compared to WT diabetic recipients. We employed a monocyte bead-tracking model in this
in vivo
aorta transplantation study. In WT recipient mice, when the donors were LDLr-/- mice fed a western diet, bead frequency per plaque section was reduced (22%;p< 0.05) on day 5 post-transplant compared with baseline. In contrast, bead frequency per plaque section was significantly reduced by (41%;p< 0.05) compared with baseline in the absence of
Ager
(gene encoding RAGE).
In vitro,
in a reverse transendothelial migration assay using primary aortic endothelial cells and
Ager or Drf1
deficient bone marrow derived macrophages (BMDMs), revealed in the setting of treatment with RAGE ligand AGEs, deletion of
Ager
or
Drf1
in BMDMs facilitated macrophage reverse migration vs. that observed in WT BMDMs. Therefore, we propose that deletion of
Ager
or
Drf1
in recipient diabetic mice accelerates atherosclerotic plaque regression, via reduction in lesional macrophage content.
Abstract 599: Lighting Up P2Y12 Oligomers: Insights into the Role of Oligomerization in P2Y12 Function
Authors: Khan Aasma , Caplan Jeffrey L , Woulfe Donna
Published: July 2021

Introduction:
Little is known about the role of P2Y12 oligomerization in receptor function and whether P2Y12 receptor mutations associated with human bleeding disorders may be explained by alterations in oligomerization.

Objectives:

1) To determine whether P2Y12 homo- and hetero-oligomers are constitutive or dynamically regulated.

2) To explore whether P2Y12 mutants R256Q and R265W (previously detected in patients with abnormal bleeding, but with unaltered ADP binding) have different oligomerization affinities or kinetics and determine whether differences in P2Y12 oligomerization explain the functional defects.

Methods:
We employed a Venus-based Bimolecular Fluorescence Complementation (BiFC) approach in HEK293T cells transiently co-expressing P2Y12 or its mutant forms (R256Q or R265W) tagged with either the N-terminal (P2Y12-VN) or C terminal fragment (P2Y12-VC) of Venus, to characterize their homomeric interactions, in live cells using confocal microscopy and quantitative flow cytometry assays.

Results:
Agonist-independent formation of P2Y12 receptor homo-oligomers were detected on cell membranes. Time lapse imaging showed movement of P2Y12 receptor pairs from the endoplamic reticulum and Golgi network to the plasma membrane, suggesting that they are constitutive and required for export. Co-expression of P2Y12-VN with increasing amounts of P2Y12-VC demonstrated a dose-dependent increase in the fluorescence intensity of Venus, and reached saturation at a ratio of 1:3. Interestingly, the fluorescence intensities of homomeric P2Y12-R256Q-VN and R256Q-VC and, separately, P2Y12-R265W-VN and P2Y12R265W-VC were almost 4 times stronger than that of the wild type receptor as quantified in flow cytometry-based BiFC. Similar results were obtained in confocal microscopy. This suggests that these P2Y12 mutants form an increased number of dimers or oligomers with increased self-affinities.

Conclusion:
We demonstrate that P2Y12 forms constitutive homo-oligomers. Two mutations associated with bleeding disorders in patients have altered receptor-receptor interactions. Future investigation will explore the effect of mutations and receptor oligomers on G protein coupling and receptor: G protein stoichiometry.
Abstract 635: CD40-Filamin A Interactions Are Required for Translocation of CD40 to Lipid Rafts In Endothelial Cells and for Endothelial Cell Activation
Authors: van Tiel Claudia M , Burger Patrick , Zelcer Noam , Hordijk Peter L , van Buul Jaap D , Lutgens Esther
Published: July 2021

Background and Aim:
CD40 is a member of the co-stimulatory tumor necrosis factor receptor superfamily that is not only constitutively expressed on professional antigen presenting cells like macrophages, dendritic cells and B-cells, but also on endothelial cells. Interactions between CD40 and its ligand, CD40 ligand (CD40L) are crucial for proper immune cell activation. Activation of CD40 signaling plays a role in chronic diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. However, since CD40-CD40L interactions modulate immune reactions as well as thrombosis, blocking CD40(L) can have adverse side-effects. Therefore, in search for new therapeutic targets, we aimed to identify new CD40-binding partners.

Methods and Results:
We created a cDNA library of murine aortas containing atherosclerotic plaques at various stages and performed a yeast-two-hybrid with the C-terminal domain of CD40 as bait. We identified filamin A as a novel CD40 binding partner in atherosclerosis, which was confirmed by co-immunoprecipitation. Accordingly, using confocal microscopy we showed that, upon activation of CD40, filamin A was recruited to CD40 in endothelial cells. Site directed mutagenesis revealed that Filamin A binds to the intracellular domain of CD40, near the transmembrane domain, at a site distinct from the tumor necrosis receptor associated factor (TRAF) binding sites. In endothelial cells, CD40-signaling is dependent on intact lipid rafts. Interestingly, when we knocked-down filamin in endothelial cells using siRNAs, the translocation of CD40 to lipid rafts upon activation of CD40 signaling was inhibited, resulting in repression of CD40-mediated Akt signaling and subsequent inhibition of VCAM-1 and CCL-2, but not of CD40-mediated JNK signaling.

Conclusions:
We show that CD40 interacts with filamin A in endothelial cells. This interaction is crucial for translocation of CD40 to the lipid rafts and CD40-mediated activation of the Akt pathway. The reduced upregulation of VCAM-1 and CCL-2 makes CD40-filamin interactions an interesting target for treatment of atherosclerosis.
Abstract 640: PASMC Hyperproliferation and Metabolic Shift in a Model of Pulmonary Overcirculation
Authors: Boehme Jason , Tormos Kathryn , Fineman Jeff , Maltepe Emin
Published: July 2021

BACKGROUND:
Pulmonary arterial hypertension (PAH) is a complex disease that lacks adequate therapies. Recognition that endothelial and smooth muscle cell hyper-proliferation contribute to the pathophysiology has drawn parallels to cancer biology and highlighted shared alterations in growth signaling and metabolism. Similar to the Warburg Effect described in malignancies, smooth muscle and endothelial cells exhibit a phenotype of aerobic glycolysis in the setting of PAH.

Objective:
We hypothesize that in an animal model of increased pulmonary blood flow and early pulmonary vascular disease, smooth muscle cells from the central pulmonary arteries will exhibit metabolic alterations similar to the Warburg Effect.

DESIGN/METHODS:
Fetal lambs underwent surgical placement of an aortopulmonary shunt resulting in a postnatal increase in pulmonary blood flow. Smooth muscle cells (PASMCs) from the central PAs were isolated for analysis. Extracellular flux analysis was used to measure concurrent oxygen consumption and extracellular acidification. Targeted metabolites were measured using HPLC-mass spectrometry.

Results:
PASMCs from the shunted animals exhibit faster growth and a shorter calculated doubling time (29.5 vs. 45.2 hours). PASMCs derived from shunted animals had a 55% decrease in oxygen consumption rate and a 59% decrease in extracellular acidification rate. This is accompanied by a relative decrease in the mitochondrial membrane potential in the shunt cells compared to controls. Metabolic analysis shows increases in the PPP metabolites erythritol, S7P and R5P/X5P, decreases in the glycolytic metabolites Fru1,6BP and PEP, decreased malate in the TCA cycle and accumulation of acetyl-CoA in shunted cells compared to controls.

Conclusions:
PASMCs from lambs with pulmonary overcirculation and early PAH have a phenotype of hyper-proliferation and altered metabolism. The low rate of oxygen consumption and extracellular acidification suggest concurrent decreases of both mitochondrial respiration and glycolytic flux. Targeted metabolite analysis suggests increased glucose entry into the pentose phosphate shunt rather than glycolysis and relative depletion of TCA cycle intermediates may underlie these observed changes.
Abstract 677: Inhibition of Receptor-interacting Protein Kinase 1 With Necrostatin-1 and -1s Ameliorates Aneurysm Progression in the Elastase-induced Mouse Model of Abdominal Aortic Aneurysms (AAAs)
Authors: Wang Qiwei , Liu Zhenjie , Ren Jun , Morgan Stephanie , Assa Carmel , Liu Bo
Published: July 2021

Objective:
Abdominal aortic aneurysm (AAA) is a common vascular disease with a progressive nature. Currently, no pharmacological treatment is approved to effectively slow aneurysm growth or prevent rupture. We have recently demonstrated that receptor interacting protein kinase 3 (RIP3), a critical mediator of necroptosis, contributes to smooth muscle depletion and vascular inflammation associated with AAA. In this study, we tested the hypothesis that inhibition of necroptosis may mitigate aneurysm progression using Necrostatin-1 (Nec-1) or an optimized form of Nec-1 called Nec-1s (7-Cl-O-Nec-1), known inhibitors of another necroptosis mediator RIP1.

Approach and Results:
Using elastase perfusion model, we first demonstrated that Nec-1 attenuated aneurysm formation when administered daily by intraperitoneal (IP) injection started 30 min before aneurysm induction. Nec-1 also profoundly reduced elastin fragmentation, macrophage infiltration and SMC necrosis after elastase perfusion. To test whether RIP1 inhibitors can inhibit AAA progression, we randomly divided mice to four groups 7 days after elastase perfusion when aortic dilatation is small but significant. Group 1 was sacrificed to obtain a baseline aortic dilatation, while Group 2, 3, and 4 received daily IP injections of DMSO, Nec-1 (3.2 mg/kg/day) or Nec-1s (1.6mg/kg/day), respectively. 14 days after perfusion, mice in Group 2 displayed larger aneurysmal expansion as compared to Group 1 (
P
<0.05), a reflection of aneurysm growth. In contrast, mice in Group 3 and 4 showed similar aortic dilatations compared to mice in Group 1 (
P
>0.05), indicating insignificant aneurysmal growth. Furthermore, real-time PCR and histological analyses demonstrated that RIP1 inhibition significantly reduced aortic accumulation of proinflammatory cytokines and inflammatory cell infiltration.

Conclusions:
Taken together, our study suggests that necroptosis may serve as a therapeutic target for AAAs. Pharmacological inhibition of RIP1 kinase activity prevented aneurysm formation and stabilized pre-existing aneurysms in mice.
Abstract 685: Wnt5a/JNK Signaling Mediates Endothelial Insulin Resistance in Human Diabetes
Authors: Breton-Romero Rosa , Feng Bihua , Holbrook Monika , Farb Melissa G , Fetterman Jessica L , Linder Erika A , Berk Brittany D , Inagaki Elica , Gokce Noyan , Fuster Jose J , Walsh Kenneth , Vita Joseph A , Hamburg Naomi M
Published: July 2021

Diabetes mellitus (DM) is associated with endothelial dysfunction that contributes to the development of atherosclerosis and promotes cardiovascular disease. Recent work has identified the secreted Wnt5a as a pro-inflammatory mediator of metabolic dysfunction in obesity acting through a non-canonical signaling pathway involving JNK activation. In animal models, JNK activity has been widely described to be involved in systemic insulin resistance. However, the relevance of Wnt5a/JNK axis in the vasculature is unknown. Our study aimed to evaluate the involvement of Wnt5a/JNK in endothelial dysfunction, studying its potential role in altered eNOS activation and insulin signaling in patients with DM. We measured protein expression, and insulin response in freshly isolated endothelial cells (ECs) from patients with DM (n=26) and non-diabetic controls (n=32). ECs from patients with DM displayed impaired insulin-mediated eNOS activation and reduced NO release in conjunction with higher expression of Wnt5a (P=0.01). This effect was accompanied with an enhanced activation of JNK (P<0.01). In endothelial cells from non-diabetic subjects, Wnt5a treatment increased JNK activation and reduced insulin-mediated activation of eNOS (P<0.001). In ECs from patients with DM, treatment with a JNK chemical inhibitor (SP600125) restored eNOS activation and insulin response (P<0.001). In summary, our data provide evidence for the presence of altered endothelial Wnt5a and JNK activity in human diabetes, suggesting that Wnt5a/JNK signaling pathway contributes to endothelial insulin resistance in DM and support further studies of Wnt5a inhibitors to restore endothelial function.