bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 557: The Effect of Dietary Fat and Carbohydrate on the Metabolism of HDL Subspecies Containing ApoE in Humans
Authors: Morton Allyson , Mendivil Carlos , Furtado Jeremy , Sacks Frank M
Published: March 2019

Background:
The effect of diet on the metabolism of HDL subspecies defined by protein content remains undetermined. ApoE forms subspecies on HDL with unique metabolic properties. HDL containing apoE have dramatically increased clearance from plasma compared to HDL not containing apoE, and also participate in size expansion from small to large HDL (a marker of cholesterol uptake).

Methods:
We enrolled ten adults who were overweight or obese and had low HDL-C (
<
45 mg/dl for men,
<
55 mg/dl for women). Each participant received two diets in a random-crossover design: a high-unsaturated fat diet (40% fat [10% sat, 23% MUFA, 7% PUFA], 45% carbohydrate) and a low fat diet (65% carbohydrate, 20% fat [10% sat, 8% MUFA, 7% PUFA]), each for 28 days. At the end of each diet period, participants received a bolus infusion of tri-deuterated (D3-) leucine, a stable isotope tracer, to label apoA1, and plasma was collected at 18 time points up to 94 hours post-tracer infusion. We isolated from plasma HDL subspecies containing or not containing apoE, and used compartmental modeling of apoA1 tracer enrichment and pool size to study the kinetic behavior of each HDL subspecies during each diet.

Results:
HDL containing apoE comprises about 5% of apoA1 HDL mass in plasma. On both diets, apoA1 in HDL containing apoE turns over about ten times more quickly than in HDL not containing apoE. The high-unsaturated fat diet increased the synthesis and clearance of apoA-I on HDL containing apoE by about 250% compared to the low fat diet. There was also greater evidence of HDL size interconversion in this subspecies on the high fat diet. In contrast, the high-unsaturated fat diet did not significantly affect the metabolism of apoA1 of HDL not containing apoE.

Conclusions:
Dietary unsaturated fat compared to carbohydrate increases the secretion of HDL subspecies containing apoE, and renders them more biologically active in metabolic pathways that involve reverse cholesterol transport.
Abstract 561: Reduced Serum Efflux Capacity Associates With Elevated Plasma Lp(a) Levels
Authors: Fenton Alexandra M , Minnier Jessica , Plubell Deanna L , Bergstrom Paige , Tavori Hagai , Fazio Sergio , Pamir Nathalie
Published: March 2019

Background:
Plasminogen, is a potent acceptor of cholesterol by the ABCA1 transporter in peripheral cells, and this function is inhibited by lipoprotein (a) [Lp(a)]. Patients with high Lp(a) have increased cardiovascular disease risk, but the mechanism for this effect is not known. The interplay of plasminogen and Lp(a) in contributing to the total sterol efflux capacity of whole serum (TSEC) could explain the influence of Lp(a) on vascular health. We investigated TSEC in patients with low and high plasma Lp(a) levels.

Methods:
TSEC (measured in cAMP-stimulated J774 murine macrophages), lipid profile, Lp(a), and plasminogen levels were measured in a cohort of patients (N=58) followed for standard-of-care in our lipid clinic. We used a linear regression model with sex, age, Lp(a), plasminogen, LDL and HDL cholesterol levels, and use of lipid-lowering drugs to understand the predictors of TSEC. The interaction between plasminogen, ABCA1, and Lp(a) was further characterized by biochemical studies of Lp(a) isoforms, cell based assays, and label free protein-protein interaction assays.

Results:
TSEC was 30% lower (p=0.002) in patients with plasma Lp(a) levels >50mg/dl compared with those below 50mg/dl. Plasminogen, Lp(a), Lp(a)/plasminogen interaction, and LDL-C were significant predictors of TSEC (adjusted R
2
=0.48, p<0.01). The regression model revealed varying associations of Lp(a) with TSEC for different values of plasminogen: At low plasminogen, Lp(a) showed a negative association, whereas for higher values of plasminogen, Lp(a) showed no association. Additionally, sterol efflux to increasing plasminogen concentrations was reduced when samples were co-treated with plasma from patients with Lp(a) levels >20mg/dl when compared to samples co-treated with plasma from patients with lower Lp(a) levels. Furthermore, two distinct Lp(a) isoforms isolated from different patients inhibited plasminogen mediated sterol efflux. Finally, label free protein-protein interaction assays showed that plasminogen binds to ABCA1 in a process inhibited by the presence of Lp(a).

Conclusion:
Our results support an interaction between plasminogen and Lp(a) that contribute to the total sterol efflux capacity of the serum, an atheroprotective process.
Abstract 579: Anti-apolipoprotein A-I Antibody Profile Correlates With Cardiovascular Disease Outcomes
Authors: Henson David , Samman Tahhan Ayman , Schlicht Sierra , Temel Ryan , Quyyumi Arshed , Venditto Vincent
Published: March 2019

Apolipoprotein A-I (ApoA-I) is a target of IgG autoantibody induction in patients, but the role of these antibodies has not been fully elucidated. Previous research has characterized anti-ApoA-I IgG antibodies targeting delipidated ApoA-I as a biomarker of cardiovascular progression, but only a moderate association was observed. We hypothesize that free anti-ApoA-I IgG is a single component of the anti-ApoA-I response and characterization of anti-ApoA-I antibody profiles will be more predictive of adverse cardiovascular outcomes. Given the relative concentrations of ApoA-I and anti-ApoA-I antibodies, we examined sera samples from 375 patients with coronary artery disease (CAD) to quantify soluble ApoA-I/IgG immune complexes (ICs). We found a range of ApoA-I/IgG IC concentrations in patients, irrespective of free anti-ApoA-I antibodies. While free antibodies failed to predict outcomes in this CAD cohort, a median Cox regression analysis over 6 years of follow-up determined a hazard ratio of 1.5 (95% CI: 1.03-2.18,
p
= 0.03) for patients with below median ApoA-I/IgG ICs levels after adjusting for 11 traditional cardiovascular risk factors. In comparison, a cohort of healthy subjects exhibited significantly higher ApoA-I/IgG ICs. Pearson correlation analysis between ApoA-I/IgG ICs in the 375 patients with CAD and 25 patient characteristics found that only hypertension showed a significant association with ApoA-I/IgG ICs (r = -0.154, p = 0.003). In addition, no significant relationship between ApoA-I/IgG ICs and total ApoA-I concentration (r = -0.0601, p = 0.51) or total IgG concentration (r = 0.134, p = 0.137) was observed. Continued characterization of the molecular characteristics and immunologic sequelae associated with the immune complexes will help determine the functional implications of ApoA-I/IgG ICs
in vivo
. Current data using affinity column purification shows co-elution of ApoA-I and IgG in patients with elevated levels of ApoA-I/IgG ICs. Furthermore, ApoA-I/IgG ICs elute through size exclusion chromatography in the HDL fraction. The identification and characterization of ApoA-I/IgG ICs has the potential to guide clinical diagnosis and intervention strategies in patients with atherosclerotic cardiovascular disease.
Abstract 595: Transcriptional Control of Intestinal Cholesterol Absorption, Adipose Energy Expenditure and Lipid Handling by Sortilin
Authors: Hagita Sumihiko , Rogers Maximillian , Pham Tan , Wen Jennifer , Mlynarchik Andrew , Aikawa Masanori , Aikawa Elena
Published: March 2019

Objective:
The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear.

Approach and Results:
Here we show that in the low-density lipoprotein receptor-deficient (Ldlr
-/-
) atherosclerosis model, Sortilin deficiency (
Sort1
-/-
) in female mice inhibits intestinal Niemann-Pick type C1-Like 1 (Npc1l1) expression (-60.6 %, p<0.01), reduces body (-17.2 %, p<0.01) and white adipose tissue weight (-35.2 %, p<0.05), and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor (Figure). Female
Ldlr
-/-
Sort1
-/-
mice on a high fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 (+89.1 %, p<0.05) and Adiponectin (+37.7 %, p<0.01), an adipokine that when reduced is associated with obesity and cardiovascular disease related factors. Additionally, Sortilin deficiency suppressed cholesterol absorption in both human colon Caco-2 cells (-16.5 %, p<0.05) and mouse ex vivo intestinal tissue (-16.9 %, p<0.05) in a similar manner to treatment with the Npc1l1 inhibitor - ezetimibe.

Conclusions:
Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.
Abstract 605: Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Angiogenesis
Authors: Abdali Anahita , Corsini Alberto , Baci Denisa , De Dominicis Carlo , Zanda Matteo , Gelmi Maria Luisa , Bellosta Stefano
Published: March 2019

Aim:
Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of theglycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compoundswith a potential to efficiently modulate (inhibit) angiogenesis.

Methods:
Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA levelby real-time PCR.

Results:
PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2suppressedcapillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds.

Conclusions:
These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeuticapproach to promote plaque stability.
Abstract 608: Integrated Omics and Network Analysis Identify Drivers of Calcific Bicuspid Aortic Valve Disease
Authors: Blaser Mark C , Schlotter Florian , Lee Lang H , Halu Arda , Zhou Wunan , Passos Livia S , Higashi Hideyuki , Pham Tan H , Sharma Amitabh , Nunes Maria C , Blankstein Ron , DiCarli Marcelo F , Aikawa Masanori , Singh Sasha A , Body Simon C , Aikawa Elena
Published: March 2019

Objectives:
Calcific aortic valve disease (CAVD) shares risk factors with atherosclerosis but has no pharmacotherapy. Bicuspid aortic valve (BAV) is the most common congenital cardiac defect (2% of humans), and individuals with BAVs acquire CAVD at a rate >15-25x of those with tricuspid aortic valves (TAVs). The causes and mechanisms of accelerated BAV-CAVD remain unclear. This study demonstrates the integrated transcriptome and proteome of BAV-CAVD.

Methods:
Quantification of gated chest CTs assessed aortic valve calcification. We performed transcriptomics using Illumina sequencing and global unlabeled proteomics by LC-MS/MS in a total of 55 human aortic valves (24 stenotic TAVs and 25 stenotic BAVs from valve replacement surgeries segmented into fibrotic and calcific portions, and 6 non-diseased aortic valves from heart transplants).

Results:
Calcific burden did not differ between BAVs and TAVs (Agatston score: p = 0.88), but BAV mineralization was significantly accelerated as BAVs underwent surgery 11.5±1.74 years earlier than TAVs (p < 0.01). Transcriptomics sequenced 11,223 genes and proteomics identified 1,798 proteins. Of these, 174 genes (fibrotic = 121, calcified = 53) and 98 proteins (fibrotic = 24, calcified = 74) were differentially expressed between BAV- and TAV-CAVD. Pathway analysis of proteins enriched in BAVs vs. TAVs found significant enhancement of iron metabolism and proteoglycan function in BAV-CAVD, while complement activation and collagen biosynthesis distinguished TAVs. When overrepresented proteins were mapped to the protein-protein interactome, network analysis identified disease stage- and valve type-specific subnetworks. While numerous protein-protein interactions existed between the fibrosis and calcification subnetworks of TAVs, a disconnection between these two pathological processes was identified in BAV subnetworks.

Conclusions:
Integrated multi-omics of CAVD stratified by disease stage and valve type identify unique contributors to BAV pathogenesis and shed light on molecular drivers of disease. Network analysis finds that while fibrocalcific responses are linked in TAVs, fibrosis and calcification may develop independently in BAVs, with vital implications for targeting of therapeutics.
Abstract 611: Association Between Thoracic Irradiation and Increased Progression of Coronary Artery Calcium
Authors: Davison Mark A , Yakupovich Anel , Kharouta Michael Z , Turian Julius , Seder Christopher W , Batus Marta , Kalra Dinesh , Kosinski Mark , Taskesen Tuncay , Okwuosa Tochukwu M
Published: March 2019

Introduction:
Thoracic irradiation (TIR) is associated with increased risk of coronary artery disease (CAD) and coronary death. Coronary artery calcium (CAC) is the result of coronary plaque accumulation and has been shown to predict CAD and overall cardiovascular mortality. We hypothesized that TIR in lung cancer patients receiving radiotherapy would be associated with CAC progression.

Methods:
We evaluated CAC progression (pre- and post-TIR) from chest CT scans of lung cancer patients identified from a cancer registry at an urban academic medical center. A 2:1 matched control population was established controlling for age, gender, race, and CT scan interval. Vessel-specific CAC progression and extension in pre- and post-interval CT studies was evaluated by 2 independent reviewers using existing standard methodologies. Whole heart and the left anterior descending (LAD) coronary artery were retrospectively segmented on the CT study used for treatment planning. The volume of each structure and associated dose metrics were obtained using the standard tools available in the Pinnacle Treatment Planning software. Chi squared tests were used to compare vessel-specific CAC progression (increase in CAC volume) and extension (CAC lengthening within a vessel) between groups. Pearson correlation analysis explored associations between radiation volume and CAC progression.

Results:
We included 35 patients and 65 controls (50% female). Mean and max whole heart TIR doses: 13.5 Gy (95% CI 10.3-16.7 Gy) and 52.1 Gy (95% CI 46.2 – 58.0 Gy); LAD: 21.4 Gy (95% CI 16.0 – 26.8 Gy) and 34.9 Gy (95% CI 28.7 – 41.1 Gy), respectively. CAC progression and extension in LAD and left circumflex coronary artery (LCx) were significantly greater in patients vs. controls (
p
<0.03 for all). There was statistically significant correlation between LAD radiation volume and CAC progression in the left main coronary artery (LM) (r =0.33,
p
=0.05).

Conclusions:
TIR is associated with CAC progression in the LAD and LCx. For LAD and LM, the CAC progression correlated with the irradiated volume of these structures although neither a dose nor a volume threshold could be established. Future studies examining the utility of CAC screening for radiation-induced CAD and cardiovascular mortality are required.
Abstract 612: Low Expression of Lysosomal Acid Lipase in Smooth Muscle Cells Relative to Macrophages Provides New Insights into Foam Cell Lipid Accumulation
Authors: Dubland Joshua A , Boukais Kamel , Wang Ying , Allahverdian Sima , Pryma Collin S , Chan Teddy , Francis Gordon A
Published: March 2019

Background and Hypothesis:
Smooth muscle cells (SMCs) are the predominant cell type within the intima of human atherosclerosis-prone arteries and promote initial retention of atherogenic lipoproteins in the deep intima. We previously found that ≥50% of foam cells in intermediate coronary atheromas are of SMC origin and that intimal SMCs have reduced expression of the ATP-binding cassette transporter A1 (ABCA1). Previously we also found that ABCA1 expression is acutely dependent on the flux of cholesterol out of lysosomes and subsequent generation of oxysterols for promotion of gene transcription by the nuclear liver X receptor (LXR).

Hypothesis:
In the present studies we tested the hypothesis that SMCs have lysosomal dysfunction that contributes to foam cell formation.

Methods and Results:
Human monocyte-derived macrophages (HMMs) and arterial SMCs were treated with aggregated LDL (agLDL) to increase intracellular cholesterol. Unlike HMMs, agLDL treatment failed to upregulate ABCA1 expression in SMCs and did not significantly increase 27-hydroxycholesterol levels. Also in contrast to HMMs, SMCs did not downregulate new cholesterol synthesis and displayed minimal increases in cholesteryl ester formation with agLDL loading. This data suggested retention of lipids within lysosomal compartments. Indeed, confocal microscopy revealed retention of lipids identified by the neutral lipid dye BODIPY within lysosomal compartments stained with LAMP1 in SMCs, while HMMs showed mostly cytosolic lipid droplets [Pearson correlation of colocalization of +0.3197±0.0123 in SMCs and -0.0897±0.0143 in HMMs (p<0.0001, avg±SEM, n>100 cells)].
LIPA
mRNA levels and LAL protein were markedly reduced in SMCs relative to HMMs, with LAL activity being 23.4-times higher in HMMs compared to SMCs (p<0.0001, n=4). Similarly, we found reduced LAL levels in mouse SMCs relative to macrophages. Incubation of SMCs with medium containing LAL reduced lysosomal lipid accumulation, resulting in decreased new cholesterol synthesis and increased cholesterol efflux.

Conclusions:
We find that arterial SMCs have a relative deficiency in LAL expression resulting in defects in downstream sterol regulatory events compared to macrophages. Our results provide a novel reason for SMC foam cell formation and a potential therapeutic target.
Abstract 623: Activating Liver X Receptors Alters Endothelial Progenitor Cell Differentiation and Enhances the Release of Factors that Reduce Atherosclerosis
Authors: Rasheed Adil , Tsai Ricky , Rayner Katey J , Cummins Carolyn L
Published: March 2019

The liver x receptors (LXRα/β) are nuclear receptors that are highly expressed in macrophages and endothelial progenitor cells (EPCs). EPCs are bone marrow (BM) derived cells that facilitate endothelial repair through the secretion of factors. The importance of LXRs in preventing atherosclerosis has been attributed to promoting cholesterol efflux from macrophages. However, a role for EPCs in the LXR response has not been explored. We hypothesized that LXR activation in EPCs contributes to their anti-atherogenic effects by preventing endothelial activation during atherogenesis. In the
Lxr
-/- mice, circulating EPCs were decreased by 27% compared to WT, while myeloid populations were increased. Isolated hematopoietic stem cells treated with LXR agonist (1μM GW3965)
ex vivo
showed a 36% increase in the number of EPC colonies formed, while myeloid colonies were decreased by 25%. GW3965 treatment during EPC differentiation resulted in a 76% decrease in endothelial lineage marker (
Cd144
and
Vegfr2
) expression. To investigate endothelial cell activation, activated human umbilical vein endothelial cells were incubated with the conditioned media (CM) from GW3965-treated WT EPCs, which reduced THP-1 monocyte adhesion by 36% compared to the CM from vehicle treatment. To determine the effects of the EPC-derived CM
in vivo
,
Ldlr
-/- mice were injected with concentrated CM bi-weekly over an 8-week period, which decreased aortic sinus lesion area by 52% compared to the media control injections. Taken together, the loss of LXRs perturbed hematopoietic balance
in vivo
, while the activation of LXRs were associated with enhanced
ex vivo
EPC formation. Furthermore, LXR activation altered EPC differentiation and secretome resulting in decreased
in vitro
endothelial-monocyte adhesion. When administered to atherosclerotic-prone mice, these secreted factors reduced lesion formation in the aortic sinus. These data suggest that LXRs reduce the development of atherosclerosis in part through the release of EPC-derived factors that can reduce endothelial activation.
Abstract 635: Osteopontin Targeted Theranostic Nanomedicine for Dual-modality Imaging Guided Photodynamic Therapy for Vulnerable Atherosclerotic Plaque
Authors: Xu Mengqi , GUO Weisheng , Wang Yabin , Liang Xingjie , Cao Feng
Published: March 2019

Background:
Macrophage infiltration plays a vital role in vulnerable atherosclerotic plaque (VASP) progression. Our previous work has demonstrated that osteopontin (OPN), overexpressed in foamy macrophages, could be a target for VASP detection. Photodynamic therapy (PDT) can kill foamy macrophages selectively via combining photosensitizer and illumination to generate reactive oxygen species (ROS). Herein, we constructed human serum albumin (HSA)-based nanomedicine (ICG/TPZ/HSA-Ce6-OPN) to achieve precise identification of VASP by optical/MR imaging as well as specific therapy against atherosclerosis (AS).

Methods:
Nanomedicines (NMs) were prepared by targeting OPN peptide, conjugating MRI contrast agent Ce6-Mn
2+
, encapsulating fluorescent dye (ICG) and hypoxia-activated drug tirapazamine (TPZ) (A). Characterization, toxicity, sensitivity and therapeutic effect were evaluated
in vitro
. For
in vivo
study, ApoE
–/–
mice were fed with a high-fat diet and a perivascular cuff was placed around carotid artery to establish VASP model. NMs were intravenously injected to obtain optical/MR imaging signal. The size and vulnerability of plaques were evaluated after 2 weeks administration of NMs.

Results:
The transmission electron microscopy image (B), fluorescence (FL) spectra and UV absorption spectra manifested the discoid-shaped NMs were well-prepared (C, D). Good binding specificity were confirmed according to cellular uptake analysis. CCK-8 assay showed excellent bio-compatibility and therapeutic effect during PDT.
In vivo
study revealed the NMs were accumulated in VASP (F, G).

Conclusion:
OPN targeted NMs indicates great potentials for precise identification and therapeutic effect on AS. However, PDT is constrained by the limited light-penetration depth in tissue, so plaques in deep-seated arteries can hardly be treated. Thus, further study is required to overcome light-penetration limitation.
Abstract 636: Angiotensinogen and Megalin Interaction Contribute to Renal Angiotensin II Production and Hypercholesterolemia-induced Atherosclerosis
Authors: Ye Feiming , Wu Chia-Hua , Howatt Deborah A , Mullick Adam , Graham Mark , Vander Kooi Craig , Danser A.H. J , Wang Jian-An , Daugherty Alan , Lu Hong
Published: March 2019

Objective:
Hepatocyte-derived angiotensinogen (AGT) contributes to atherosclerosis through its cleavage to angiotensin II (AngII) by renin and angiotensin-converting enzyme (ACE). Molecular mechanisms of this process and the source of AngII to promote atherosclerosis are unknown. In this study, we used hepatocyte-specific AGT (hepAGT)-/- mice and megalin antisense oligonucleotides (ASO) to determine AGT and megalin interaction and their contributions to renal AngII production and atherosclerosis.

Methods and Results:
Immunostaining of mouse kidney sections demonstrated that AGT, renin, and ACE co-localized with megalin in proximal tubules. Hepatocyte-specific AGT deficiency diminished AGT protein and AngII production in mouse kidney and increased renal renin abundance without changes in plasma AngII concentrations. Inhibition of megalin by administering an ASO eliminated AGT and renin in proximal convoluted tubules and led to profound augmentations of their concentrations in urine, accompanied by 70% reduction of renal, but not plasma, AngII concentrations. To determine whether megalin contributes to atherosclerosis, male LDL receptor -/- mice were fed Western diet and administered two different sequences of megalin ASOs. Megalin inhibition by either ASO resulted in pronounced reductions of atherosclerosis compared to their control groups. These findings were also confirmed in female mice. LDL receptor -/- mice fed Western diet exhibited extensive vacuolization derived from enlarged lysosomes in proximal convoluted tubules. This pathological characteristic was absent in mice with either hepatocyte-specific AGT depletion or megalin inhibition.

Conclusion:
Our study demonstrated that megalin regulates AGT homeostasis, and AngII produced in kidney is a contributor to atherosclerosis through AGT and megalin interaction in proximal tubules.
Abstract 650: Stem Cell-derived SMCs Are Useful for Vascular Tissue Engineering
Authors: Gu Wendou , Hong Xuechong , Le Bras Alexandra , Xu Qingbo , Hu Yanhua
Published: March 2019

Objectives:
Tissue engineered vascular grafts with long-term patency are in great need in the clinics, for which smooth muscle cells (SMCs) are essential. Recently several laboratories have established methods to obtain SMCs from embryonic stem cells or iPS cells. However, the mechanisms of stem cell differentiation are not fully understood. And also it is hard to obtain a large number of SMCs with higher purity. To solve these issues, we take the advantage of mesenchymal stem cells (MSCs) from human umbilical cord that were cultivated and differentiated in vitro.

Methods:
MSCs from human umbilical cord were cultivated and differentiated to vascular lineages. Signal pathways involved in stem cell differentiation were studied using Western blot, siRNA, PCR, immunoprecipation and transfection. For vascular tissue engineering, stem cell-derived SMCs were applied to decelularized scaffold to create the vessels in a Bioreactor.

Results:
In response to TGFβ1 stimulation, MSCs were abundantly expressing SMC markers including αSMA, SM22, calponin and SMMHC at the gene and protein levels. Functionally, differentiated SMCs displayed a contraction ability in vitro and formed vessels in matrigel plug assay SCID mice. Micro-RNA (miR) array analysis showed the upregulation of miR-503 and the downregulation of miR-222-5p at early time points after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. Mechanistically, miR-503 was demonstrated to promote SMC differentiation through directly targeting Smad7, a negative regulator of TGFβ1 Smad-related signaling pathways. The expression of miR-503 was Smad4-dependent and Smad4 was enriched at the promoter region of miR-503. In addition, miR-222-5p inhibited SMC differentiation through targeting ROCK2 and αSMA 3’-UTR. Finally, SMCs differentiated from stem cells exhibited the ability to migrate into decellularized mouse aorta and gave rise to vascular graft with smooth muscle layer.

Conclusions:
We established a method to produce a large number of human SMCs from MSCs, which demonstrated the potential of utilizing the cells to generate vascular grafts. And we provide the mechanistic data demonstrating the impact of miRNA-503/222-5p on TGFβ1-mediated signaling in SMC differentiation from stem cells.
Abstract 655: Notch Inhibition Reverses the Polarization of Pro-Inflammatory (M1) to Anti-Inflammatory-like (M2) Macrophages
Authors: Dev Rishabh , Mahajan Advitiya , Sharma Neekun , Hans Chetan P
Published: March 2019

Introduction:
Our previous studies demonstrated that loss of Notch1 signaling reduces the incidence of abdominal aortic aneurysm (AAA) by preventing the influx of pro-inflammatory M1 macrophages and that Notch1 deficiency promotes M2-polarization. In the present study, we determined if inhibition of Notch signaling could reverse the pre-existing pro-inflammatory M1 macrophages to anti-inflammatory M2 phenotype.

Methods and Results:
Raw cells (264.7 murine macrophage cell line) were treated with various concentrations of LPS (5, 10, 100 ng/ml) and IFN-γ (10 ng/ml) for 3h to polarize naïve macrophages into M1-phenotype. After washing, these cells were treated with either vehicle or DAPT (10 ng/ml or 25 ng/ml); a potent Notch inhibitor for 6, 12, 24 or 48h and later used for gene expression studies or functional assays. LPS pretreatment significantly increased the gene expression of M1-genes;
Il6
,
Il12, Cd38, Fpr2
and
iNOS
, whereas the expression of M2 genes;
Egr2
and
c-Myc
was significantly decreased. Replacement of LPS with vehicle alone lowered the expression of
Il6
and
Il12
within 6-12h, but remained significantly higher than basal levels. Expression of
iNOS
,
Cd38
and
Fpr2
remained unchanged in response to vehicle in the LPS-pretreated macrophages. DAPT treatment further reduced the gene expression of
Il6
and
Il12
significantly and
iNOS
moderately as compared to vehicle-treated macrophages. Interestingly, Notch inhibition increased the expression of
Egr2
and
c-Myc
genes at 24 and 48h. Similar trends with regard to M1 and M2 genes were observed in bone marrow derived macrophages from

Apoe
-/-

and
WT
mice treated with LPS followed by vehicle or DAPT. Phagocytosis assay showed increased uptake of zymosan bioparticles in the macrophages treated with LPS. Replacement of LPS with vehicle for 24h did not affect the phagocytosis significantly. DAPT treatment decreased phagocytic activity of these macrophages as compared to vehicle-treated macrophages.

Conclusions:
Our data suggest that Notch inhibition reverses the M1-polarized macrophages towards M2-like macrophages. These novel insights in the functions of Notch signaling may have potential implications in chronic inflammatory diseases including AAA and atherosclerosis.
Abstract 673: Discovering and Dissecting the Function of Conserved NF-κB Binding Events in the Human Genome
Authors: Alizada Azad , Khyzha Nadiya , Khor Melvin , Antounians Lina , Wang Liangxi , Medina-Rivera Alejandra , Liang Minggao , Hou Huayun , Wilson Michael D , Fish Jason E
Published: March 2019

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor plays a prominent role in inflammation and contributes to the development of atherosclerosis. Genome-wide DNA binding assays of the human NF-κB subunit RELA (p65) have revealed tens of thousands of NF-κB binding sites and hundreds of target genes. However, the function of individual RELA binding sites and the extent to which NF-κB occupancy and function is conserved across mammals are not well understood. To better understand the function of NF-κB we characterized the genome-wide binding of RELA in primary vascular endothelial cells (ECs) isolated from the aortas of human, mouse and cow. ECs were stimulated acutely with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA) and we profiled RELA occupancy, open chromatin, select histone modifications, and RNA expression. We found ~5000 RELA binding events conserved across all three species and these highly conserved human binding events were enriched for genes controlling vascular development, apoptosis, and pro-inflammatory responses. Approximately 2000 of these highly conserved RELA binding events were also shared across multiple human cell types, revealing a conserved core of robustly bound NF-κB sites. These NF-κB binding sites were also prominent components of ~40 inflammation-induced super-enhancers (SE) common to several tissues. To gain insight into the function of individual conserved NF-κB binding sites we focused on the inflammation-induced SE proximal to the monocyte recruiting chemokine
CCL2
, which we detected as a SE in all three species and across multiple cell types. We tested the functional significance of six conserved RELA binding sites comprising this SE using CRISPR/Cas9 genome editing. We found that only deletion of the most proximal upstream RELA binding site could abolish the induction of
CCL2
upon TNFA treatment. This site also contains a disease associated variant that can modulate
CCL2
induction. Overall, our comparative genomics assessment of NF-κB binding gives new insight into NF-κB biology and the function of conserved transcription factor binding events within mammalian super-enhancers.
Abstract 700: Adipocyte-Specific Calpain-2 Deficiency Attenuates Obesity-accelerated Abdominal Aortic Aneurysms in Mice
Authors: Javidan Aida , Jiang Weihua , Moorleghen Jessica J. , Subramanian Venkateswaran
Published: March 2019

Background and Objective:
Recent clinical studies demonstrated that abdominal adiposity is associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are non-lysosomal calcium dependent cysteine proteases that are highly expressed in human and experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we identified that calpain-2 (a major ubiquitous isoform) plays a critical role in Angiotensin II (AngII)-induced AAA formation in obese mice. In addition, we demonstrated that calpain inhibition strongly suppressed adipose tissue inflammation in obese mice. The purpose of this study was to determine the role of adipocyte-specific calpain-2 on obesity-accelerated AAA.

Methods and Results:
Calpain-2 floxed mice that were hemizygous for Adiponectin Cre (Cre+/0) were produced by breeding male Cre+/0 to female calpain-2 floxed mice. Littermates that were homozygous for the floxed calpain-2 gene, but without the Cre transgene (Cre0/0), were used as control mice. Western blot analyses showed that calpain-2 protein is depleted in various fat tissues including periaortic adipose, from Cre+/0 mice, while not influencing abundance in aorta and other tissues. Male Cre+/0 and Cre0/0 mice were fed a high fat diet (60% Kcal) for 20 weeks. After 16 weeks of diet feeding, mice (n=20-22) were infused with either saline or AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. Depletion of calpain-2 in adipocytes had no effect on high fat diet-induced body weight gain, fat mass, glucose and insulin tolerance. Interestingly, adipocyte-specific calpain-2 depletion significantly attenuated AngII-induced expansion of ex-vivo maximal diameter of abdominal aortas in obese mice (Saline- Cre0/0: 0.95 ± 0.03; Cre+/0: 0.98 ± 0.02 mm; AngII - Cre0/0: 1.95 ± 0.20; Cre+/0: 1.25 ± 0.08 mm P<0.001). In addition, calpain-2 depletion also reduced the incidence of AngII-induced AAAs in mice (Cre0/0: 76% versus Cre+/0: 27%).

Conclusion:
These findings suggest that adipocyte-derived calpain-2 plays a critical role in AngII-induced AAA development in diet-induced obese mice.
Abstract 710: Deficiency of IL12p40 Promotes Angiotensin II-Induced Abdominal Aortic Aneurysm
Authors: Sharma Neekun , Belenchia Anthony , Dev Rishabh , Toedebusch Ryan , Aroor Annayya , Pulakat Lakshmi , Hans Chetan
Published: March 2019

Introduction:
Abdominal aortic aneurysm (AAA) is caused by the immune and inflammatory cell accumulation of the aortic wall, leading to enhanced inflammation and aneurysm formation. Our recent studies demonstrate that inhibition of Notch signaling attenuates AAA formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40 knockout (KO) mice, we investigated the direct effects of M2-like macrophages in the development of AAA.

Methods and Results:
Male (8-10 week-old) wild-type (WT) and IL12p40-KO mice (n=10) on C57BL/6 background were infused with angiotensin-II (AngII, 1,000 ng/kg/min) by implanting osmotic pumps subcutaneously for 28 days. Comparable increase in mean, systolic and diastolic arterial blood pressure was observed between WT and IL12p40-KO mice in response to AngII. Unexpectedly, AngII significantly increased the AAA-associated mortality (2/10) and luminal expansion (7/10) in IL12p40-KO mice as determined by transabdominal ultrasound. In addition, IL12p40-deficiency significantly increased aortic stiffness in the abdominal aortas in response to AngII than WT as measured by pulse wave velocity (PWV) and atomic force microscopy (AFM). Macroscopic and histological examination also showed that IL12p40-KO mice exhibited severe abdominal aortic lesion formation and increased maximal aortic width compared to WT mice. Increased elastin fragmentation and collagen deposition was detected in aorta of AngII-infused IL12p40-KO mice compared to AngII-infused WT mice. Further, there was no significant change in the total cholesterol, triglycerides and HDL-cholesterol levels in the serum of WT and IL12p40-KO mice treated with AngII. Importantly, bone marrow derived macrophages (BMDM) from IL12p40-KO mice showed increased mRNA expression of M2-markers (TGF-β1 and IL10) and decreased expression of M1-markers (iNOS and TNF-α) at basal level compared to WT BMDM.

Conclusions:
IL12p40 deficiency may promote the development of AAA, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic-stiffness and fibrosis mediated by TGF-β1 and IL10. In summary, optimal M1/M2 balance rather than predominance of M2-phenotype may be prerequisite to prevent AAA formation.
Abstract 713: Angiotensin 1-7 Suppresses Experimental Abdominal Aortic Aneurysms
Authors: Xu Baohui , Li Gang , Deng Hongping , Cabot Anna , Chen Xiaofeng , Yuan Hai , Wu Xuejun , Michie Sara A , Dalman Ronald L
Published: March 2019

Objectives:
Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease, the pathogenesis of which remains incompletely defined. We and others have demonstrated the critical role of angiotensin (Ang) II and its receptor AT1 in experimental AAAs. Although Ang 1-7 and its receptor Mas counteract many AT1 functions, the role of the Ang 1-7/Mas pathway has not been studied in AAAs. This study evaluated the influences of Ang 1-7 and Mas on experimental AAAs.

Methods:
AAAs were created in male C57BL/6J mice via porcine pancreatic elastase (PPE) infusion. Mice were treated with Ang 1-7 (0.5 mg/kg/day via s.c) or vehicle starting immediately prior to, or 4 days following, PPE infusion. Additional mice were co-treated with Ang 1-7 and the Mas receptor antagonist A779 (0.5 mg/kg/day) beginning immediately prior to PPE infusion. Influences on AAAs were evaluated by serial ultrasound imaging and histology.

Results:
Figure summarizes major results. In ultrasound imaging, at 14 days Ang 1-7 treatment initiated immediately following aneurysm creation substantially suppressed aortic enlargement as compared to vehicle treatment. Histologically, Ang 1-7 treatment was associated with attenuation of aortic medial elastin and smooth muscle cell depletion, mural macrophage, CD4
+
, CD8
+
T and B cell density and neoangiogenesis. Co-treatment with Ang 1-7 receptor Mas antagonist A779 “rescued” the aneurysm phenotype. Additionally, Ang 1-7 treatment initiated even 4 days following AAA creation was still effective in limiting further aneurysmal enlargement and pathologic evolution.

Conclusions:
Ang 1-7 treatment, initiated either at, or up to 4 days following, AAA initiation limits further progression of experimental AAAs. This inhibitory effect seems likely related to activation of the Mas receptor. These results suggest that either Ang 1-7 or alternative Mas receptor agonists may have clinical utility in suppressing progression of early AAA disease.
Abstract 714: Does the Sickle Trait Portend Increased Cardiovascular Risks?
Authors: Okwan-Duodu Derick , Quintana Raymundo A , Joseph Giji , Hansen Laura , Liu Wenxue , Sellak Hassan , Archer David , Taylor W Robert
Published: March 2019

Objectives:
Despite the increased incidence and worse outcomes of cardiovascular diseases among African-Americans compared to the general population, the contribution of sickle cell trait (AS) to this racial disparity remains unknown. We aimed to delineate the impact of AS on the pathogenesis of cardiovascular diseases alone, and in the setting of other risk factors.

Methods:
We compared multiple cardiovascular endpoints between AS and wild-type genotype (AA) using the humanized Townes sickle cell mouse model. To determine whether AS modifies a pre-existing cardiovascular risk factor, diabetes was induced in a cohort of mice using streptozotocin (STZ). Hind limb ischemia model was employed to evaluate collateral vessel formation, and perfusion recovery was characterized with LASER Doppler perfusion imaging (LDPI). The running wheel assay assessed spontaneous motor function after ischemia. Angiotensin II (Ang II) and L-NAME were used to evaluate hypertensive response. Experimental stroke was induced by unilateral common carotid artery ligation and daily 15 min exposure to 7.5% oxygen for 3 days, and the infarct size was quantified as the ratio of the infarcted area to the total area of the uninjured contralateral hemisphere. PCSK9 gain-of-function mutation in the setting of Ang II and high-fat diet was used to evaluate atherosclerosis development.

Results:
4 groups of mice were studied: AA, AS, AA-STZ and AS-STZ. The formation of collateral vessels was equivalent between the AA and AS (day 28, AA: 80 ± 6%, AS: 85 ± 5%, p=0.8), and in the setting of diabetes (39 ± 5% AA-STZ; 36 ± 4% AS-STZ). Systolic blood pressure at baseline (110 ± 4 mmHg vs 112 ± 5 mmHg), and in response to Ang II (148 ± 4 mmHg vs 142 ± 5 mmHg) and L-NAME (135 ± 3mmHg vs 138 ± 5 mmHg) were equivalent. Mortality from stroke was equivalent between AA and AS. After 1 month of PCKS9 gain-of-function mutation/high-fat diet, AA and AS demonstrated similar lipid profiles. Mortality and infarct volume after stroke induction were identical between AA and AS.

Conclusions:
All major indices of cardiovascular outcomes were similar between AA and AS, even in the presence of diabetes. These various experimental models suggest that the sickle trait likely does not contribute significantly to the poor cardiovascular outcomes seen in the African-American population.
Abstract 717: Sequential Inflow Occlusion and Walking Capacity in Large Animal Model of PAD
Authors: Brewster Luke P , Upchurch Walker
Published: March 2019

Introduction:
Peripheral artery disease (PAD) affects over 8 million Americans over 40 years of age. PAD patients are heavily burdened by pain and walking dysfunction. Treatment paradigms for PAD are evolving, but optimal medical therapy, risk factor modification, and supervised exercise therapy (SET) are the first line therapies for these persons. SET improves walking time and may improve skeletal muscle perfusion. However, the mechanisms by which SET and other treatments are not well understood. Translational models of PAD may be useful in developing novel therapeutics for PAD patients.

Objective:
The objective of this work is to introduce a large animal (porcine) model of PAD that includes sequential occlusion of inflow that can be used on patient-like treadmills for SET or quantification of walking distance and dysfunction.

Methods:
Two female Yorkshire swine were accommodated to our treadmill one week prior to procedure. On day 0, swine underwent endovascular placement of a covered stent (GORE® VIABAHN® endoprosthesis) into the right external iliac artery followed by placement of a vascular plug (AMPLATZER, St. Jude Medical) inside this stent was used in the initial procedure. In order to mimic the common presentation of PAD patients having one leg more severely ischemic than the other, we performed a sequential occlusion of the ipsilateral internal iliac artery and contralateral external iliac artery one week later. Vascular flow were quantified by arteriogram, a modified ankle brachial index, and duplex ultrasound. Animals were walked weekly on a large animal treadmill with a 3.5 KM/HR pace and 10 degree incline until failure (pigs would slide to end of treadmill).

Results:
There were no technical failures or complications. One and three weeks after the initial procedure, pigs walked 40% and 45% of pre-procedural maximal walking time. There was sustained depression of the blood pressure ratio of ischemic hindlimb to unaffected forelimb (hindlimb index or HLI), which was significantly inhibited out to 6 weeks (
HLI of 0.4-0.63
).

Conclusion:
We have created a pilot model of PAD that can be used for both SET testing and quantifying maximal walking time/distance. This model may be useful in testing novel therapeutics and validating promising mechanistic findings from small animals.
Abstract 724: Improved Carotid Stenoses Quantification on Novel 4D/3D-Doppler Ultrasonography Indexing to the Common Carotid Artery
Authors: Macharzina Roland R , Kocher Sascha , Hoffmann Fabian , Rastan Aljoscha , Fan Nian , Neumann Franz-Josef , Zeller Thomas
Published: March 2019

Objective:
Accuracy of internal carotid artery stenosis (ICAS) quantification depends on the method of stenosis measurement, impacting therapeutic decisions and outcomes. The NASCET-method references the stenotic to the distal ICAS lumen, the ECST-method to the local outer and the common carotid artery (CC) method to the CC-diameter. Direct morphometric stenosis measurement with four-dimensionally guided three-dimensional ultrasonography (4D/3DC-US) demonstrated good validity for the commonly used NASCET-method. The NASCET-definition has clinically relevant drawbacks. Our purpose was to investigate the validity of the ECST- and CC-method.

Approach:
4D/3DC-US percent-stenosis measures of 103 stenoses (80 patients) were compared to quantitative catheter angiography and duplex-ultrasonography (DUS) in a blinded fashion.

Results:
The 4D/3DC-US versus angiography intermethod standard deviation of differences (SDD, n = 103) was lower for the CC (5.7%) compared to the NASCET- (8.1%, p <0.001) and ECST-method (9.1%, p <0.001). Additionally, it was lower than the NASCET angiography interrater SDD of 52 stenoses (SDD 7.2%, p = 0.047) and non-inferior for the ECST-method (p = 0.065). Interobserver analysis of equivalent grading methods showed no differences for the SDDs between angiography and 4D/3DC-US observers (p >0.076). Binary comparison to angiography showed equal Kappa values >0.7 and an accuracy >85% for the NASCET- and CC-method, higher than for the ECST-method. Binary accuracy of ICAS grading did not differ from DUS for all methods.

Conclusion:
The new 4D/3DC-US CC-method is an accurate and well reproducible alternative to the NASCET- and ECST-method and offers perspectives for clinical application.