bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 426: The Remodeling of HDL-SAA by Macrophage Surface Heparan Sulfate is Necessary for Efficient Cholesterol Efflux
Authors: Ancsin John B , Davidson Michael H
Published: July 2021

Serum amyloid A (SAA) is an acute-phase protein that circulates associated with high density lipoprotein (HDL) and can replace apoA-I as the predominant apoprotein. SAA can increase HDL’s binding affinity for macrophages and we provide evidence that this targeting process is dependent on cell surface heparan sulfate (HS) and facilitated by at least one of two HS binding sites that we have been previously identified in SAA. Additional studies utilizing various assays including native-PAGE, turbidity, thioflavin T fluorescence and surface plasmon resonance have demonstrated that HDL-SAA binding to HS (or heparin) causes SAA to dissociate from the HDL particle, leaving behind novel HDL species with enhanced cholesterol carrying capacities. In the present study we explore this remodeling phenomenon further and assess its potential importance for cholesterol efflux from macrophages. The chemical disruption of HDL-SAA with chaotropic agents, temperatures below 37°C and lipid oxidation with copper all interfere with HDL-SAA/heparin remodeling. The oxidation of HDL-SAA also significantly reduces its cholesterol efflux potential and SAA’s ability to be converted into AA-amyloid fibrils in cell culture, a process that requires the dissociation of SAA from HDL. Finally, we demonstrate that the removal of cell surface HS chains, by either heparinase treatment or by preventing HS biosynthesis with sodium chlorate, impairs cholesterol efflux rates from macrophages to HDL-SAA. Our interpretation of these findings is that the reduction in HDL-lipid fluidity (low temperatures or conjugated dienes) significantly reduces the ability of HS:SAA interactions to dissociate SAA from HDL. We postulate that HS normally plays an important role in separating SAA from HDL but during chronic inflammatory diseases with elevated lipoprotein oxidation, HDL-SAA species are generated in which SAA is more tightly associated with HDL, thus preventing its dissociation from HDL and impairing atheroprotective functions.
Abstract 427: Plasma PCSK9 Levels Are Positively Correlated With LDL-Cholesterol Concentrations in Familial Hypercholesterolemia
Authors: Drouin-Chartier Jean-Philippe , Tremblay André J , Hogue Jean-Charles , Ooi Teik C , Lamarche Benoît , Couture Patrick
Published: July 2021

Autosomal dominant familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR), its ligand apoB or proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. PCSK9 regulates LDL-C levels by binding to LDLR, thereby enhancing its intracellular degradation. Although PCSK9 levels have been shown to be elevated in FH subjects, the extent to which PCSK9 levels correlate with LDL-C concentrations in FH has not been examined. Therefore, the objective of the present study was to assess the relationship between PCSK9 and LDL-C levels in a large cohort of genetically defined French-Canadian (FC) FH subjects. A total of 292 FH heterozygotes (heFH) carrying one of the nine FC mutations in the LDLR gene were recruited. 226 subjects were carriers of a negative receptor (NR) mutation in the LDLR gene, while the other 66 were carriers of a defective (DR) LDLR gene mutation. 56 control subjects matched for gender and body mass index (BMI) were also recruited. Fasting blood samples were collected after a 6-week period without lipid lowering medication. PCSK9 levels were significantly higher in the heFH group than in the control group (317.9 ng/ml vs 203.3 ng/ml; P<0.001). In the heFH group, PCSK9 levels were positively and independently correlated with LDL-C concentrations and predicted 8.1% (P<0.001) of the variability in LDL-C levels. Age, the type of LDLR mutation (NR vs DR), and BMI were also significantly associated with the variability in LDL-C levels, predicting 13.6% (P<0.001), 3.9% (P<0.001), and 1.8% (P=0.02) of the LDL-C variance, respectively. Interestingly, PCSK9 levels were also positively correlated with lipoprotein(a) levels in heFH (r=0.20; P<0.001). These results indicate that PCSK9 levels are positively correlated with LDL-C and lipoprotein(a) concentrations in heFH.
Abstract 447: Acrolein-Modified HDL Impairs Cholesterol Transport and HDL Function
Authors: Chadwick Alexandra C , Holme Rebecca L , Pritchard Kirkwood A , Sahoo Daisy
Published: July 2021

The interaction between scavenger receptor BI (SR-BI) and HDL reduces the risk for cardiovascular disease (CVD) by playing key roles in the reverse cholesterol transport pathway (RCT). In the beginning of RCT, SR-BI stimulates release of free cholesterol (FC) from peripheral macrophages to HDL, whereas in the final step of RCT, HDL binds to hepatic SR-BI for delivery of excess cholesteryl esters (CE) into the liver for excretion via bile. While “functioning” HDL is crucial in protection against CVD, environmental factors such as acrolein, a highly reactive aldehyde found in tobacco smoke, can induce oxidative stress that promotes HDL modifications and renders the particle “dysfunctional”. Although there is a strong correlation between cigarette smoking and CVD risk, much less is known about the effects of smoking on RCT. We hypothesized that acrolein modifications to HDL (acro-HDL) generate a dysfunctional particle by: (i) impairing SR-BI-mediated selective uptake of HDL-CE and efflux of FC and (ii) upregulating inflammatory pathways in macrophages that promote atherogenesis. To test this hypothesis, human HDL was modified with 250μM acrolein and acrolein adducts on HDL were confirmed by immunoblot analysis and mass spectrometry. To evaluate acro-HDL’s functionality at the beginning of RCT, SR-BI-mediated FC release to native- or acro-HDL was measured in SR-BI-transfected COS-7 cells. Our data revealed that there was 30% less efflux of FC in the presence of acro-HDL as compared to native HDL. To assess acro-HDL functionality at the end of RCT, HDL binding and HDL-CE uptake assays were performed and acro-HDL (0-50μg/mL) was 10-25% less efficient at delivering CE to cells via SR-BI. Finally, to determine whether acro-HDL was rendered atherogenic, we used qRT-PCR to demonstrate that acro-HDL, but not native HDL, paralleled the effects of oxidized LDL by upregulating mRNA scavenger receptor expression (CD36 and LOX-1) and inflammatory marker expression (MCP-1, IL-6, ICAM-1) in THP-1 macrophages. Altogether, our data reveal that acrolein modification produces a dysfunctional HDL particle that upregulates inflammatory pathways. More detailed investigations into the impact of acrolein adducts on HDL function may help prevent smoking-related CVD deaths.
Abstract 449: Increased Risk of Coronary Artery Disease in Patients with Persistent Type of Nonvalvular Atrial Fibrillation
Authors: Chaikriangkrai Kongkiat , Alchalabi Sama , Khaleel bala Sayf , Chang Su Min
Published: July 2021

Background:
This study is to examine relationship between coronary artery disease (CAD) and types of atrial fibrillation (AF)

Methods:
A total of 403 nonvalvular atrial fibrillation patients without known history of CAD underwent coronary artery calcium score (CACS) evaluation by multi-detector cardiac computed tomography. Clinical characteristics and CACS were compared between patients with persistent type of AF and paroxysmal type of AF.

Results:
The cohort comprised of 65% (279 of 430) male with a mean (SD) age of 63(10) years. Prevalence of persistent AF was 60% (259 of 430). Mean (SD) 10-year risk of CAD by Framingham score was 14(7)%. Median CACS was 22 (range 0-5402) with 75% CACS>0 (321 of 430). Compared to paroxysmal type, those with persistent type had higher prevalence of CAC>0 as shown in Figure1 and more history of hypertension (p<0.001) but less history of smoking (p0.004), statins use (p0.018) and warfarin use (p<0.001). There was no statistically significant difference in mean age (p0.783) and CAD risk by Framingham score (p0.477) between two groups. In multivariate analysis, persistent type is an independent predictor for CACS>0 (OR 1.938; 95%CI 1.197, 3.138; p0.007).

Conclusion:
In patients with AF, persistent type of AF is independently associated with CACS>0. Our findings suggest potential benefit from evaluation of CAD in this population.
Abstract 451: PNS-R1 Attenuates the Development of Atherosclerosis in ApoE-Deficient Mouse Model
Authors: Chen Yu , Jia Chenglin , Xiong Minqi , Cui Jingang , Yang Qinbo , Zhang Teng
Published: July 2021

Atherosclerosis is a chronic pathological condition featured by accumulation of lipids and fibrous elements in the artery. Atherosclerosis remains as the primary cause of cardiovascular diseases and agents that effectively intervenes the development of atherosclerosis are still needed. Panax notoginseng has been extensively used as therapeutic agent in Asia to treat cardiovascular disorders. Panax notoginseng saponins (PNS) is the major class of active components of Panax notoginseng. PNS has been reported to possess anti-atherosclerotic effect. However, which component of PNS is responsible for this effect remains unknown. The current study evaluated the effects of a component unique to PNS, PNS-R1, on atherosclerosis in ApoE-/- mouse model. Histological examination revealed that the extent of atherosclerotic lesion was significantly alleviated in PNS-R1-treated ApoE-/- mice compared to that from the ApoE-/- controls. Meanwhile, PNS-R1 treatment significantly reduced the level of oxidative stress in the atherosclerotic lesion, which was prominently enhanced in ApoE-/- controls. This effect on oxidative stress was corroborated by increased serum level of GSH and SOD and decreased level of MDH in PNS-treated mice. Furthermore, PNS-R1 treatment significantly decreased the levels of total cholesterol, triglycerides and increased the level of HDL without affecting the level of LDL, suggesting an effect of PNS-R1 on lipid metabolism, which could in part contribute to its action in attenuating atherosclerosis. Additionally, the levels of inflammatory factors including IL-2, IL-6, TNF-α, γ-IFN and ox-LDL were markedly reduced in PNS-R1-treated mice compared to that from the ApoE-/- controls, demonstrating a significant anti-inflammatory effect of PNS-R1 in the development of atherosclerosis. Expression of microRNAs known to be involved in the pathogenesis of atherosclerosis was further evaluated and showed that PNS-R1 treatment led to a significant reduction in the expression of miR-122, miR-132 and miR-155. Collectively, our results provided for the first time the experimental evidence supporting the anti-atherosclerotic effects of PNS-R1, which could be a promising therapeutic agent treating atherosclerosis.
Abstract 460: Characterization of Glycated Albumin Isolated From Poorly Controlled Diabetic Patients and Its Role in Macrophage Cholesterol Efflux
Authors: Machado-Lima Adriana , Oliveira Erika R , Iborra Rodrigo T , Castilho Gabriela , Nakandakare Edna R , Corrêa-Giannella Maria Lucia C , Traldi Pietro , Porcu Simona , Roverso Marco , Lapolla Annunziata , Passarelli Marisa
Published: July 2021

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. In vitro produced AGE-albumin induces oxidative stress that is linked to the reduction in ABCA-1 levels and cholesterol efflux mediated by apo A-I and HDL-subfractions, leading to macrophage cholesterol accumulation.

We characterized the glycation level/profile of human serum albumin (HSA) isolated by fast protein liquid chromatography from poorly controlled type 1 (DM1) and type 2 (DM2) diabetes mellitus patients (HbA1c > 8%) in comparison to control (C) individuals, and how these AGE-albumin can interfere in macrophage lipid accumulation.

The glycation level of HSA from C, DM1 and DM2 was analyzed by MALDI mass spectrometry and was similar between DM1 and DM2-HSA. An increased mean mass was observed in DM1-HSA (68,544 ± 192 Da; n=6) and DM2-HSA (68,547 ± 132 Da; n=6) compared to C-HSA (67,846 ± 301 Da; n=6), reflecting the condensation of at least 8 and 5 units of glucose, respectively. The tryptic digestion of C-HSA generated a number of peptide species higher than those originated from DM1 and DM2-HSA.

Macrophages isolated from peritoneal wild-type mice were treated for 18 h with C, DM1 or DM2-HSA in order to measure the 14C-cholesterol efflux and the mRNA expression of NOX-4 (NADPHoxidase4), ABCA-1 (Abca1) and ABCG-1 (Abcg1). Data were compared by one-way ANOVA and Dunnet′s post test.

In comparison to cells treated with C-HSA the expression of NADPHoxidase4 (p<0.05; n=3) mRNA was increased after cell treatment with DM1 (3.2x) and DM2-HSA (0.7x), confirming oxidative stress. Abcg1 mRNA was reduced by DM2-HSA (26%; p<0.05; n=3); Abca1 mRNA was unchanged but ABCA-1 protein content was greatly reduced (82 and 25%, respectively in DM1 and DM2-HAS; p<0.05; n=12). The % of apo A-I mediated cholesterol efflux was impaired in DM1 (1.3 ± 0.3) and DM2-HSA-treated cells (2.4 ± 0.5) as compared to C-HSA (4.4 ± 0.5; n= 5; p<0.05).

The level of advanced glycation that takes place in vivo was similar between DM1 and DM2-HSA and induced macrophage oxidative stress and impairment in cholesterol efflux that may contribute to atherogenesis in DM.

Funding:
FAPESP, Brazil (2012/19112-0)
Abstract 462: Effect of Coronary Revascularization on Plasma Level of Asymmetric Dimethylarginine
Authors: Nemeth Adam , Lenkey Zsofia , Ajtay Zeno , Cziraki Attila , Sulyok Endre , Horvath Ivan , Nemeth Balazs , Szabados Sandor , Nasri Alotti , Lobenhoffer Jens-Martens , Bode-Böger Stefanie M
Published: July 2021

Background:
Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase. Increased ADMA plasma levels are associated with atherosclerosis and cardiovascular disease. In this study we investigated the plasma levels of ADMA in patients who underwent different type of coronary revascularization.

Methods:
Concentrations of ADMA, l-arginine, symmetric dimethylarginine (SDMA) were measured by liquid chromatography-tandem mass spectrometry in four groups: group I consisted of 16 patients with ST-elevation myocardial infarction (STEMI), and group II included 24 patients who underwent elective percutaneous coronary intervention (PCI). Before PCI and at 1 hour, 5 days and 30 days after reperfusion blood samples were taken for measurement of l-arginine, ADMA and SDMA. Group III consisted of 21 patients undergoing off-pump and group IV included 20 patients undergoing on-pump coronary artery bypass graft (CABG) surgery. The measurements were performed 24 h before, 3 times during the operation (via coronary sinus catheter), and on the 1st and 5th postoperative day.

Results:
In patients with elective stent implantation stenting induced a prompt and sustained depression of ADMA (F=9.594, p<0.001), however ADMA remained constant for STEMI patients after stent placement (F=2.982, p=0.069). The differences in the time-course for ADMA (F=9.431, p<0.001) proved to be significant between the two groups. We did not find significant change in the plasma concentration of ADMA during off-pump CABG surgery (F=0.416, p=0.68), however ADMA concentration increased significantly in patients who underwent on-pump surgery both in the coronary sinus (F=14.751, p<0.001) and peripheral blood (F=30.738, p<0.001). Therefore, the intersubject time effect, was significant (F=6.990, p=0.002).

Conclusions:
The main finding of the present study is that the response pattern of the new cardiovascular risk factor, ADMA was significantly different between the two PCI and the CABG groups. Its long-term follow-up may be suitable to monitor the improvement of coronary function after revascularisation. This method should be feasible to monitor of the ADMA metabolism during coronary revascularization.
Abstract 466: Stigmasterol Accumulation in ABCG8 Knockout Mice Does Not Account for Xenosterol Toxicity
Authors: Patel Shailendra B
Published: July 2021

Xenosterol accumulation mice deficient in sterolin function leads to significant toxicity, with infertility, decreased body fat accumulation, macrothrombocytopenia, cardiac fibrosis and premature death1, 2. The predominant xenosterols are phytosterols derived from the diet and are a mixture of sitosterol (typically 70%), campesterol (~20%) and stigmasterol (~5-10%). Although sitosterol has been shown to have some biological effect in tissue culture, only stigmasterol has been shown to have a potent biological effect, by activating the transcriptional factor, Lxr3. To delineate whether all of the toxic biological effects were mediated by stigmasterol accumulation, we designed a diet that was supplemented with highly enriched stigmasterol (>80% purity) and fed this to Abcg8 knockout mice. Stigmasterol accumulation in the plasma and tissues was comparable to the levels observed in mice fed a mixed phytosterol-enriched diet that had been shown to result in toxicity.

Over a 12-week period, both male and female Abcg8 knockout mice gained normal amounts of weight, body fat, showed no disturbances in tail-cuff measured blood pressure, and plasma analyses showed no abnormalities of platelet counts or volumes, blood glucose, plasma cholesterol, despite accumulation of stigmasterol in the plasma and tissues. Fertility testing showed no abnormalities. Gene expression analyses of livers did not show any consistent patterns, although Lxr target genes were not up-regulated.

These data do not support the concept that stigmasterol accumulation, at levels of 5-10mg/dL in plasma, account for the xenosterol-mediated toxicity observed.

1. McDaniel, A.L., H.M. Alger, J.K. Sawyer, K.L. Kelley, N.D. Kock, J.M. Brown, R.E. Temel, and L.L. Rudel, Phytosterol feeding causes toxicity in ABCG5/G8 knockout mice. Am J Pathol, 2013. 182(4): p. 1131-8.

2. Solca, C., G.S. Tint, and S.B. Patel, Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice. J Lipid Res, 2013. 54(2): p. 397-409.

3. Yang, C., L. Yu, W. Li, F. Xu, J.C. Cohen, and H.H. Hobbs, Disruption of cholesterol homeostasis by plant sterols. J. Clin. Invest., 2004. 114(6): p. 813-22.
Abstract 471: Receptor for Advanced Glycation End Products, Reverse Transmigration and Impaired Regression of Diabetic Atherosclerosis
Authors: Senatus Laura M , Shen Xiaoping , Li Huilin Li , Daffu Gurdip , Rosario Rosa , Schmidt Ann Marie
Published: July 2021

Cellular migration is fundamental processes linked to diverse pathological states including atherosclerosis and diabetes. RAGE is a multiligand cell surface macromolecule, which binds ligands such as advanced glycation endproducts. In evaluating the relationship between impaired regression of atherosclerosis in diabetes and RAGE, we tested the hypothesis that RAGE contributes to impaired regression by suppression of reverse transmigration of macrophages in an AGE-enriched environment. We evaluated if the expression of RAGE modulates influx and/or egress of macrophages from the atherosclerotic lesions in the non-diabetic and diabetic states. We used a monocyte bead-tracking model in an aorta transplantation study with LDL-/-diabetic mice fed high-fat diet as a baseline (donors), and non-diabetic RAGE-/- or WT mice as recipient. Migration patterns evaluated by labeled monocytes showed a decrease in bead number in RAGE null recipients (23±0.05p<0.05) compared to WT (30±0.05p<0.05) indicating that monocyte-derived cell content in the diabetic state was lower in the absence of RAGE. In our surgical aorta transplant model, we also examined the morphologic changes in plaques found in RAGE-/- or WT recipient mice. After lipid normalization into RAGE-/- recipient mice we observed reduced plaque cells positive for macrophage marker CD68+ (-58%), but increased plaque collagen (+51%), compared to WT recipients. Our data indicate Oil Red O stains for fatty deposit in plaque suggest a decrease (-53%) in plaque area stained and lower AGEs staining (-22%) in RAGE-/- recipient mice compared to WT recipient. Therefore, the presence of RAGE hindered plaque regression in our atherosclerotic mouse model.

We performed our studies
in vitro,
a reverse transmigration assay, using primary murine aortic endothelial cells from wild-type C57BL6 mice and wild-type or RAGE deficient bone marrow derived macrophages. Our data show RAGE-AGEs impact macrophage-endothelial transmigration. Our studies suggest RAGE-AGEs action delays reverse transmigration of macrophages and augments pro-inflammatory gene expression. Therefore blockade of RAGE may be able to limit pathological acceleration of vascular diseases.
Abstract 472: Effect of Different Doses of Angiotensin II Receptor Blockers on Adipokine Levels in Hypertensive Patients
Authors: Shargorodsky Marina
Published: July 2021

The present study was designed to determine the effect of different doses of the angiotensin II receptor blocker, candesartan, on circulating adiponectin and leptin levels as well as plasma leptin adiponectin ratio (LAR) in hypertensive patients with multiple cardiovascular risk factors. 69 hypertensive patients were randomized to 3 groups: group 1 included patients treated with high doses of Candesartan (32mg), group 2 included patients treated with conventional doses of Candesartan (16mg), group 3 included patients that received antihypertensive treatment other that ARBs or ACEIs. Patients were evaluated for lipid profile, HbA1C, insulin, C-peptide, CRP, aldosterone, renin,HOMA-IR, leptin , adiponectin and LAR. Baseline adiponectin, leptin and LAR levels did not differ significantly between the three groups. After six months of treatment LAR was significantly higher in group 3 than group 1 (p=0.007) or group 2 (p=0.023). Marginal across-group differences were detected for post-treatment circulating adiponectin level (p=0.064). Univariate GLM analysis of posttreatment LAR detected significant by-group differences even after adjustment for age, sex, baseline values of LAR and blood pressure. In this model, group was the only significant predictor of LAR after controlling for these variables. Treatment with high doses of angiotensin II receptor blocker, candesartan, is associated with significantly reduced LAR and marginally increased circulating adiponectin levels in hypertensive patients with multiple cardiovascular risk factors.
Abstract 476: Mast Cell--Mediated Neutrophil Influx Enhances Plaque Progression
Authors: Wezel Anouk , Lagraauw H Maxime , van der Velden Daniël , de Jager Saskia C , Quax Paul H , Kuiper Johan , Bot Ilze
Published: July 2021

Rationale:
Activated mast cells have been identified in atherosclerotic plaques. As mast cells have the ability to release chemokines that mediate leukocyte fluxes, we propose that activated mast cells play a pivotal role in leukocyte recruitment during atherosclerosis.

Methods and Results:
Diet fed B-cell deficient apoE-/-muChain mice, which lack endogenous IgE, received 6 IgE or PBS injections over a period of 8 weeks. Mast cells in the aortic root were significantly more activated after IgE treatment and concomitantly, we detected an increase in plaque size (P=0.050). Intriguingly, a striking increase in the amount of perivascular neutrophils was observed in IgE treated mice (control: 57.6 ± 10.6 neutrophils/mm2 tissue; IgE: 183.0 ± 38.7 neutrophils/mm2 tissue; P=0.01). In order to investigate if activated mast cells can directly attract neutrophils, we injected C57Bl/6 or mast cell deficient Kit(W-sh/W-sh) mice intra-peritoneal with the mast cell activator 48/80. Interestingly, mast cell activation led to a massive neutrophil influx into the peritoneal cavity, while neutrophil numbers in mast cell deficient mice remained unaffected. To confirm these findings, we performed an in vitro migration assay. Indeed, supernatant of activated mast cells caused a 3-fold increase in neutrophil migration (P=0.007). Moreover, addition of anti-CXCR2 to the neutrophils resulted in a major reduction of migration (P=0.03) whereas anti-CXCR4 did not reduce migration significantly.

Conclusions:
Chemokines released from activated perivascular mast cells induce neutrophil recruitment to the atherosclerotic plaque, thereby aggravating the inflammatory response.
Abstract 484: Uptake of 18F-Fluorodeoxyglucose Detected by Positron Emission Tomography in the Abdominal Aortic Aneurysm is Correlated With Endoleaks and Predicts the Adverse Outcome of Aneurysm After Endovascular Aortic Repair
Authors: Courtois Audrey , Namur Gauthier , El Hachemi Mounia , Cremmers Etienne , Nusgens Betty , Hustinx Roland , Allaire Eric , Defraigne Jean-Olivier , Colige Alain , Sakalihasan Natzi
Published: July 2021

Endovascular aortic repair (EVAR) has been applied to abdominal aortic aneurysm (AAA) to decrease and prevent morbidity and mortality due to open surgery. However, this therapeutic approach may lead to complications such as occurrence of endoleaks which may result in rupture of the aneurysm. Decisions for complementary treatment after EVAR to prevent aneurysm rupture are based on endoleak characterization and aortic diameter evolution. The presence of endoleak or enlarging diameter is however not always predictive of rupture. Recent reports support the view that 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) provides unique information on cellular metabolic activity thought to drive aneurysm expansion and rupture.

We evaluated if FDG/PET could predict the outcome of aneurysm repaired by endoprosthesis. A cohort of 56 AAA patients (55 male; mean age of 74.6 years), treated by EVAR, underwent one or several PET/CT before and/or after surgery with a total number of 108 examinations. The quantification analysis of FDG uptake was not correlated to the AAA diameter but was strongly correlated with the presence of endoleak (55% of endoleak in patients with significant FDG uptake (PET+) versus 25% in PET0). Moreover, the PET+ AAA presented symptoms such as increasing diameter of aneurysmal sac (64% in PET+ versus 33% in PET0). On the other hand, two PET+ patients underwent conversion for open surgery for rapid growth and rupture with leaking. These outcomes did not occur among the PET0 patients. Increased circulating levels of fibrinolytic factors (D-dimers, thrombin/anti-thrombin and plasmin/anti-plasmin complexes) and inflammatory cytokines IL6 and IL8, were found in patients with endoleak while the collagenase MMP1 was largely decreased. Finally, two other MMPs, MMP12 and MMP13 were correlated with the uptake level of FDG.

Altogether, these results suggest that the presence of endoleak is associated with an activation of the fibrinolytic pathway and PET positivity induces factors involved in the extracellular matrix remodeling leading to instability of the wall and its rupture. This study suggests that PET/CT might predict the negative outcome of EVAR and represent a beneficial tool for patient management.
Abstract 496: Extracellular Matrix Secretion by Vascular Smooth Muscle Cells: Role of MiR-195 and MiR-29b
Authors: Zampetaki Anna , Yin Xiaoke , Mayr Ursula , Gomes Renata , Langley Sarah , Willeit Peter , Waltham Mark , Abdulkareem Nada , Jahangiri Marjan , Smith Alberto , Mayr Manuel
Published: July 2021

Rationale:
Extracellular matrix (ECM) remodeling is a key function of vascular smooth muscle cells (SMCs). MicroRNAs (miRNAs), in particular the miR-29 family and miR-195, have been implicated in the control of ECM secretion.

Objective:
To perform a proteomics comparison of miRNA effects on ECM production by vascular SMCs.

Methods and Results:
Murine SMCs were transfected with miRNA mimics and antimiRs of miR-29b and miR-195, and their conditioned medium was analyzed by mass spectrometry. Both miRNAs targeted a cadre of ECM proteins, including proteoglycans, collagens, proteases, elastin and proteins associated with elastic microfibrils, albeit miR-29 showed a stronger effect. The proteomics findings were subsequently validated at the transcription level using quantitative polymerase chain reaction. Similar to miR-29, in vivo inhibition of miR-195 by intraperitoneal injection of cholesterol bound antagomiRs led to significant alterations of elastin expression in murine aortas. Since elastin degradation is a key event in aortic aneurysm formation, we investigated miR-195 expression in patients. In human aortic aneurysmal tissue, miR-195 expression was reduced compared to non-aneurysmal tissue. In plasma, a comparison between male patients with abdominal aortic aneurysms and controls matched for diabetes and hypertension returned a panel of five highly correlated miRNAs: miR-195, miR-125b, miR-148a, miR-20a and miR-340 showed significant inverse associations with the presence of abdominal aortic aneurysms and aortic diameter, with miR-195 dominating in terms of association strength.

Conclusions:
Using proteomic analysis, we compared the effect of miR-29 and miR-195 on ECM secretion by vascular SMCs and identified novel miRNA targets. Findings in patients support an important role for miR-195 in vascular remodeling as evidenced by reduced miR-195 expression in human aneurysmal tissue and an inverse correlation between plasma miR-195 levels and aortic diameter.
Abstract 502: Weekend Effect on Subarachnoid Hemorrhage: A Stroke of Bad Luck
Authors: Khoshchehreh Mahdi , Yu Katherine , Tran Joseph , Malik Shaista
Published: July 2021

Background and Purpose:
Recently, there has been a significant emergence of literature suggesting patients who present with time-sensitive medical conditions such as myocardial infarction or pulmonary embolism on the weekend have higher mortality and poorer outcomes compared to those admitted on weekdays, otherwise known as the “weekend effect.” The literature is sparse concerning the presence of the “weekend effect” in patients admitted with a primary diagnosis of subarachnoid hemorrhage (SAH).

Methods:
Using discharge data for patients with a primary diagnosis of SAH from the 2009 Nationwide Inpatient Sample, we compared the mortality rates and timing of proper diagnostic procedures between weekend and weekday admissions. All analyses were adjusted for patient demographics and existing comorbidities.

Results:
830,739 admissions of SAH were identified of which 26.5% were weekend admissions. After adjusting for patient demographics and comorbidities, the odds ratio for weekend admissions was 1.11 (95% CI; 1.03-1.19). We found significant fewer diagnostic/therapeutic procedures performed on first day of admission among weekend admitted patients. (OR 0.80, 95% CI, 0.74-0.87).

Conclusion:
Patients presenting with SAH on the weekend have a higher mortality rate when compared to those admitted on the weekdays, and have a longer time to diagnostic and therapeutic procedures.
Abstract 516: Altered Autophagy Contributes to Endothelial Dysfunction in Human Diabetes Mellitus
Authors: Fetterman Jessica L , Flint Nir , Holbrook Monica , Linder Erika A , Berk Brittany D , Feng Bihua , Duess Mai A , Farb Melissa G , Gokce Noyan , Shirihai Orian S , Hamburg Naomi M , Vita Joseph A
Published: July 2021

Diabetes is associated with increased oxidative stress and decreased nitric oxide (NO) bioactivity in the vasculature. Autophagy is a critical multistep pathway that eliminates oxidatively damaged proteins and organelles from the cell. Emerging evidence implicates impaired autophagy in a variety of non-vascular tissues that contributes to the pathogenesis of diabetes in experimental models. We hypothesized that impaired autophagy contributes to endothelial dysfunction associated with type 2 diabetes mellitus in humans. We measured vascular function and markers of autophagy in freshly isolated endothelial cells from patients with diabetes (n=41) and non-diabetic controls (n=41). Diabetes was associated with endothelial dysfunction characterized by lower brachial artery flow-mediated dilation. Endothelial cells from diabetic patients displayed no differences in LC3 puncta compared to those from controls but had higher levels of endothelial p62 (p=0.004), a protein that accumulates with reduced autophagic flux. In endothelial cells from controls, the autophagy inhibitor bafilomycin impaired eNOS activation, confirming that intact autophagy is necessary for NO signaling. Global activation of autophagy with spermidine reversed endothelial dysfunction in cultured endothelial cells exposed to high glucose (p=0.007). In regard to mechanism of impairment, the autophagy initiator rapamycin failed to improve eNOS activation in endothelial cells from diabetics. Further, expression of beclin-1, a key contributor to autophagosome formation, was similar in diabetics and controls, arguing against impaired initiation of autophagy. In endothelial cells from diabetics, blocking the terminal step of autophagy with bafilomycin led to a further accumulation of p62 (P=0.01), suggesting intact but insufficient levels of autophagy. Lamp2a, which facilitates the merger of autophagosomes and lysosomes, was higher in diabetic cells, possibly reflecting a compensatory response to reduced flux. Collectively, these findings provide evidence for inadequate autophagic flux in endothelial cells from diabetic patients that contributes to endothelial dysfunction and may be a target for therapy of diabetic vascular disease.
Abstract 522: Shotgun Proteomics Implicates Matrix Proteolysis, Cell Adhesion and Cell Death in Urokinase-Accelerated Vascular Disease
Authors: Hu Jie Hong , Vaisar Tomas , Kinsella Michael G , Heinecke Jay W , Dichek David
Published: July 2021

Introduction:
Elevated activity of urokinase plasminogen activator (uPA) in human arteries associates with atherosclerosis, aneurysms, and plaque rupture. However, the underlying mechanisms remain unknown. ApoE null mice with macrophage-specific uPA overexpression (SR-uPA mice) have elevated vascular uPA activity, accelerated atherosclerosis, dilated aortas, and histologic features of plaque rupture. Thus, SR-uPA mice may be a useful model of uPA-accelerated human vascular disease.

Hypothesis:
We hypothesized that mechanisms of uPA-accelerated vascular disease would be revealed by shotgun proteomics of aortas of SR-uPA and nontransgenic (ntg) ApoE null mice. We focused our studies on extracellular matrix (ECM) because previous studies implicate uPA in alteration of matrix-associated proteins.

Methods:
Using aortas, we developed a method for generating tissue extracts (TE) that selectively isolate ECM proteins. We extracted proteins from aortic arches of SR-uPA and ntg ApoE null mice. We also collected conditioned medium (CM) from their explanted aortas.

Results:
Mass spectrometry-based proteomics demonstrated that aortic TE were greatly enriched in ECM proteins as determined by Gene Ontology (GO) analysis. These TE contained all major and several minor vascular collagens, and many proteoglycans. CM contained a similar number of ECM proteins as TE; however, TE were enriched in collagens, proteoglycans, elastin, and laminins. Using stringent dual statistical criteria and a FDR of 5%, 107 of 1125 proteins were differentially present in SR-uPA aorta. MMP-2, -3, and -10 were increased; laminins, collagen I, fibronectin, and elastin were decreased. CM analysis revealed 1161 proteins; 57 were differentially present in SR-uPA. NuMa1, nuclear lamins, histones, actin, and myosins were increased; PAI-1, Plg, and A2m were decreased. Unbiased GO analysis helped identify proteolysis, ECM, cell adhesion, and cell death as processes that differed significantly between SR-uPA and ntg aortas.

Conclusions:
Elevated aortic uPA activity alters abundance of a limited number of proteins and affects specific biological processes, implicating ECM proteolysis, altered cell adhesion, and cell death in the pathogenesis of uPA-mediated vascular disease.
Abstract 531: Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development
Authors: El-Rass Suzan , Eisa-Beygi Shahram , Liu Xiao-hua , Mauro Antonio , Wang Youdong , Plant Pamela , Zhang Haibo , Clark Karl , Ekker Stephen , Wen Xiao-Yan
Published: July 2021

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.
Abstract 535: Screening Mutations of MYBPC3 in 114 Unrelated Patients With Hypertrophic Cardiomyopathy Using Targeted Capture and Next-Generation Sequencing
Authors: Liu Xuxia , Jiang Tengyong , Piao Chunmei , Cai Tao , Du Jie
Published: July 2021

Background and Purpose:
Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease and a major cause of sudden cardiac death. Mutations in the gene encoding the myosin binding protein C (MYBPC3) represent the cause of HCM in ~35% of all cases of HCM. However, genetic testing for MYBPC3 in clinic setting has been limited due to the cost and relatively time-consuming Sanger sequencing. Therefore, we developed a HCM gene diagnostic kit enabling ultra-low-cost candidate gene targeted resequencing in a large cohort.

Methods:
Clinical evaluations, electrocardiography and echocardiography data of all patients with HCM were collected in Anzhen Hospital, Beijing, from 2012 to 2013. Exomes were captured with MYBPC3-containing panel using biotinylatedoligo-probes and sequenced by Illumina HiSeq2000.

Results:
In a cohort of 114 patients with HCM, a total of 21 different mutations in the MYBPC3 gene, including 3 nonsense, 6 indels and 12 missense mutations, were identified from 29 patients. These patients were presented in a broad range of phenotypes with mean age of 36.6 ± 12.5 years at onset. Phenotype-genotype analyses showed that clinical manifestations (NYHA class 3~4) were more severe in the double-mutation group (n=3, 100%) as well as the premature termination codon (PTC) group (n=12, 50%), compared with the missense mutation group (n=14, 21.4%) (p=0.03). Interestingly, the p.Y842X mutation was detected in four unrelated cases (4/29, 13.79%) with severe phenotypes, such as syncope, arrhythmias, and history of receiving invasive therapies.

Conclusions:
We demonstrated that the power of targeted resequencing in a cohort of HCM patients, and found that MYBPC3 is a common HCM-causing gene in Chinese patients (~25.4%). We also confirmed that truncation mutations or double mutations in MYBPC3 could lead to relatively severe phenotypes, and propose that the p.Y842X mutation might be correlated with severe phenotypes in Chinese patients with HCM.
Abstract 537: Higher Prevalence of Risk Alleles for Hyperuricemia Parallels Elevated Incidence of Gout and Growing Risk for Cardiovascular Diseases in a Hmong Population
Authors: Roman Youssef M , Xiong Jenny D , Menk Jeremiah S , Culhane-Pera Kathleen , Straka Robert J
Published: July 2021

Introduction:
Hyperuricemia (HU) is the strongest predictor of gout and highly associated with major CVD including hypertension, HF, and CKD. The Hmong, a unique Asian population numbering > 60,000 in Minnesota, have a two-fold increased risk of gout compared to non-Hmong, rising prevalence of CVD, and may differ from other Asian populations in these regards. A genetic predisposition to elevated Serum Uric Acid (SUA) may help identify individuals at risk for gout and CVD. We quantified the Minor Allele Frequencies (MAFs) of known genetic variants (SNPs) associated with HU and compared the MAFs between our Hmong sample and both a reference (HapMap) population of Caucasian (CEU) as well as Han-Chinese (CHB).

Methods:
Salivary DNA from 235 self-identified Hmong was genotyped using either a Sequenom (iPLEX Gold) or TaqMan approach. MAFs for seven SNPs within candidate genes (
SLC2A9, SLC22A12, PDZK1, and ABCG2
) identified by GWAS, were determined in our Hmong sample. Associations between HU and genotype were examined for 57/235 Hmong with known SUA levels. A Chi-Square or Fisher exact test with a Bonferroni corrected significance level (<0.007) was used to evaluate MAF differences. Mean SUA concentrations were compared by genotype using one-way ANOVA.

Results:
Our Hmong participant's age [mean (±SD)] was 30.2 (15.4) years, with >61% overweight or obese and a mean (±SD) SUA of 6.3 (1.7) mg/dL.

Although the frequency of risk alleles in the Hmong were significantly higher compared to CEU (6/7) and CHB (3/7) populations, independent SNP by SNP analysis did not show a clear association with SUA. Risk allele frequencies were always more frequent in the Hmong versus comparator groups.

Conclusion:
MAFs of selected SNPs for HU are not independent of race. The higher prevalence of risk alleles for HU in the Hmong versus CEU and CHB populations may partly explain the clinically observed higher prevalence of gout and CVD risk in the Hmong. Although sample size precludes a robust assessment of an association between genotype and SUA, to our knowledge this is the first study to examine the genetic basis of HU in the Hmong.
Abstract 54: PSI Domain of ß3 Integrin Has Endogenous Thiol Isomerase Function and is a Novel Antithrombotic Therapeutic Target
Authors: Reddy Emily C , Zhu Guangheng , Chen Pingguo , Reheman Adili , Lei Xi , Li June , Xu Xiaohong , Freedman John , Ni Heyu
Published: July 2021

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI).

Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential.

Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation
in vitro
and
ex vivo
and inhibited murine thrombus formation
in vivo
without significantly changing bleeding time or platelet count.

In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.
Abstract 542: Leukocyte TLR5 Deficiency Inhibits Atherosclerosis by Reduced Macrophage Recruitment and Defective T-Cell Responsiveness
Authors: de Jager Saskia C , van Puijvelde Gijs H , Anas Adam A , Bot Martine , Asbach Miriam , Habets Kim L , Foks Amanda C , Pasterkamp Gerard , van der Poll Tom , Kuiper Johan
Published: July 2021

Background:
Toll-like-receptors (TLRs) provide a critical link between innate and adaptive immune responses. It has been shown that TLR5 ligand Flagellin can enhance the suppressive capacity of regulatory T-cells (Treg), but can also functions as an adjuvant. The immune response in atherosclerosis is characterized by an imbalance of pro- and anti-atherogenic T-cells. We aimed to establish if the TLR5/Flagellin axis is involved in the immune response of atherosclerosis.

Methods:
We first assessed the effect of Flagellin exposure on macrophage maturation and T-cell polarization. Next, we created TLR5-/-/LDLr-/- chimeras to study the TLR5/Flagellin axis in atherosclerosis.

Results:
Flagellin exposure to primary macrophages did not result in clear polarization differences, but we did observe a less migratory phenotype (decreased MCP-1, CCR2 expression) in TLR5-/- macrophages. Interestingly, expression of the T-cell polarizing cytokine IL-6 was induced by Flagellin exposure, a phenomenon not observed in TLR5-/- macrophages. Next, we assessed potential T cell polarizing properties of Flagellin. Flagellin can induce expansion of regulatory T-cells, however this induction is completely overruled when Flagellin is used as an adjuvant. Hematopoietic absence of TLR5 significantly attenuates atherosclerotic lesion formation by 25% (1.03±0.06x10^6 μm2 vs 0.79±0.06 x10^6 μm2 in TLR5-/-, p = 0.01). This was accompanied by a decrease in macrophage area (-46% ,p=0.01) and necrotic core size (-32%, p<0.05) while collagen content was similar between groups. Interestingly, plasma levels of IL-6 were significantly lower in TLR5-/- chimeras (40.2±6.3 in WT vs. 15.1±2.7 pg/ml in TLR5-/-, p=0.003). Concomitantly, TLR5-/- chimeras displayed defective T-cell responsiveness, as seen by impaired proliferation and decreased splenic T cell content. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T cell responsiveness.