bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 428: An Unexpected Role for ACC in Lipid Droplet Formation in Macrophages in Response to Acellular Adipocyte Fat
Authors: Meher Akshaya K , Serbulea Vlad , Upchurch Clint M , Osinski Victoria , Sahu Srabani , Hargett Stefan , Hoehn Kyle L , Harris Thurl E , Klibanov Alexander L , McNamara Coleen A , Leitinger Norbert
Published: March 2019

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the
in vivo
scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages
in vivo
. Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel
in vitro
model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and
de novo
lipogenesis-mediated lipid droplet formation in macrophages.
Abstract 435: 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 the glycolytic 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 compounds with 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 level by real-time PCR.

Results:
PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary 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 therapeutic approach to promote plaque stability.
Abstract 437: Large-scale Validation of Zebrafish Larvae as a Model System for Systematic Genetic Screens in Dyslipidemia, Atherosclerosis and Coronary Artery Disease
Authors: Bandaru Manoj , den Hoed Marcel
Published: March 2019

Background:
Genome-wide association studies have identified hundreds of loci that are robustly associated with circulating lipids, atherosclerosis and coronary artery disease (CAD). In all but a few of these loci the causal genes and mechanisms remain unknown. Small-scale studies suggest that zebrafish larvae represent a promising model system for genetic screens in dyslipidemia, early-stage atherosclerosis and CAD. We expanded the phenotypic pipeline, increased the throughput, and performed a large-scale validation study.

Methods:
We used a three-tiered approach to validate the zebrafish model system: 1) a dietary intervention to examine the effect of overfeeding and cholesterol supplementation (N=1956); 2) treatment with atorvastatin and ezetimibe (N=835); and 3) a genetic screen for zebrafish orthologues of
APOE, APOB
and
LDLR
using a multiplex CRISPR-Cas9 approach (N=330). After imaging of vascular atherogenic traits and body size, whole-body lipid and glucose levels were assessed using enzymatic assays, and CRISPR-Cas9 target sites were sequenced on a MiSeq.

Results:
Overfeeding and cholesterol supplementation have independent pro-atherogenic effects, including higher LDLc and triglyceride levels, more vascular infiltration by lipids and oxidized LDLc, and more vascular co-localization of lipids with macrophages and neutrophils. Treatment with atorvastatin and ezetimibe can largely prevent this diet-induced pro-atherogenic state, but results in higher glucose levels. Finally, mutations in
apoea
and
apoeb
result in higher whole-body LDLc levels; mutations in
apobb.1
result in more vascular co-localization of lipids and neutrophils; and mutations in
ldlra
result in more vascular infiltration by lipids, and in more vascular co-localization of lipids and macrophages. Data from all larvae combined show that atherosclerosis in 10-day-old zebrafish larvae is mainly driven by higher triglyceride levels.

Conclusion:
Systematic genetic screens in zebrafish larvae will likely help increase our understanding of disease aetiology and identify new targets that can be translated into efficient medication for prevention and treatment.
Abstract 441: Zebrafish Larvae as a Model System for High-throughput, Image-based Genetic Screens in Dyslipidemia and Atherosclerosis
Authors: Bandaru Manoj , von der Heyde Benedikt , Emmanouilidou Anastasia , Ranefall Petter , Allalou Amin , Brooke Hannah , Larsson Anders , Wählby Carolina , Ingelsson Erik , den Hoed Marcel
Published: March 2019

Background:
Genome-wide association (GWAS) have identified hundreds of loci that are robustly associated with the risk of cardiometabolic risk factors and diseases. With few exceptions, the causal genes through which these loci influence disease risk remain uncharacterized. Since murine model systems are not suitable for systematic characterization of candidate genes in hundreds of loci, novel
in vivo
model systems are desirable.

Methods:
My group has developed and validated zebrafish model systems that make optimal use of: 1) the zebrafish’ well-annotated genome, with orthologues of at least 71.4% of human genes; 2) recent developments in multiplex CRISPR-Cas9-based mutagenesis; 3) advances in automated positioning of non-embedded zebrafish larvae; 4) fluorescent transgenes and dyes; and 5) custom-written image-quantification pipelines.

Results:
Five days of overfeeding, dietary cholesterol supplementation and/or exposure to glucose induce atherogenic and insulin resistant phenotypes (higher/more whole-body LDL cholesterol (LDLc) and triglyceride levels; vascular foam cell formation and inflammation; beta-cell number and volume; subcutaneous and hepatic accumulation of fat) that can largely be prevented by concomitant treatment with lipid-lowering or diabetes medication (N>4000). In line with recent results in humans, treatment with atorvastatin and ezetimibe results in higher whole-body glucose levels (N~835). Each additional mutated allele in zebrafish orthologues of
APOE
results in higher LDLc levels, while mutations in
LDLR
affect vascular infiltration by lipids and macrophages (N~330). Ongoing characterization of 37 prioritized genes in GWAS-identified loci for triglyceride levels identified genes that influence triglycerides, LDLc, and/or vascular inflammatory and atherogenic traits, while characterizing prioritized genes in loci associated with heart rate variability yielded genes that influence cardiac rate and rhythm.

Conclusions:
Systematic characterization of candidate genes for cardiometabolic traits in zebrafish model systems will help increase our understanding of human disease and identify novel targets that can be translated into efficient therapeutics.
Abstract 443: Cushioned-Density Gradient Ultracentrifugation (C-DGUC) Improves the Isolation Efficiency of Exosomes for their use in Atherosclerosis Research
Authors: Duong Phat , Chung Allen , Wong David K , Li Kang , Hong King Y , Bouchareychas Laura , Raffai Robert L
Published: March 2019

Background and Purpose:
Ultracentrifugation (UC) is recognized as a robust approach for the isolation of exosomes especially when combined with a second step that involves density gradient ultracentrifugation (DGUC). However, recent studies have highlighted limitations associated with the use of UC including low recovery efficiencies and possible aggregation of exosomes. Such effects could subsequently impact on downstream assessments of exosome function in biological systems.

Methods:
We tested the benefit of using a liquid cushion of iodixanol during the first UC step to improve the yield of exosomes that are concentrated from the conditioned media (CM) of J774.1 murine macrophages in a method we recently termed Cushioned(C)-DGUC. We also compared the yield and purity of exosomes isolated by C-DGUC with those isolated by first subjecting CM to two other forms of concentration that included: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation prior to DGUC.

Results:
Our data show that the concentration step largely determines the yield and purity of exosomes isolated following the second DGUC step. The use of a high-density iodixanol cushion in cushioned-UC (C-UC) led to a threefold improvement in exosome yield over conventional UC. Although subjecting the CM to UF resulted in a similar exosome recovery efficiency, it retained eight-fold more soluble proteins than C-UC method. Strikingly, PEG precipitation of the CM generated a substantial number of non-exosomal nanoparticles, which could not be efficiently eliminated by the DGUC step. Western blot analysis reproducibly detected exosome markers CD-81, TSG101 and Alix in fractions 6 and 7. Finally, an
in vitro
assay of exosome-mediated microRNA delivery confirmed that C-DGUC provided the highest yield of functional exosomes.

Conclusions:
Collectively, our data demonstrate that the use of a high-density liquid cushion of iodixanol during the concentration step of C-DGUC substantially improves the yield and purity of exosomes derived from cell culture media. This approach can therefore facilitate functional studies of cell-derived exosomes in atherosclerosis.
Abstract 444: Role of CaMKII in Atherosclerotic Plaque Progression in ApoE-Deficient Mice
Authors: Ebenebe Obialunanma V , Heather Alison K , Erickson Jeffrey R
Published: March 2019

Non-obstructive mixed atherosclerotic plaques are an independent risk factor in predicting mortality in coronary artery disease patients. The multifactorial process of plaque progression and complication is a result of proliferation and migration of VSMCs and macrophages, apoptosis and calcification. A number of osteogenic regulators and inflammatory stimuli driving these processes in plaque advancement have been identified. Yet rupture, a result of complex mixed plaques, remains the leading cause of fatal cardiovascular events. Further understanding of the mechanisms underlying plaque progression is required to identify potential targets for therapies. Key signalling factors that modulate cell proliferation and migration, which potentially underlie plaque complication include the nodal multifunctional calcium/calmodulin dependent protein kinase II (CaMKII). Thus we hypothesized that, as plaques progress in a mouse model of atherosclerosis (ApoE
-/-
), CaMKII activity would increase. We also hypothesized that systemic inhibition of CaMKII would impede plaque progression. Our data show a trend towards increased CaMKII phosphorylation, an indirect measure of kinase activity, in plaques as mice advance from 26 to 30 weeks. Pharmacological inhibition of CaMKII with KN-93, on alternate days for 4 weeks, in 30 week old mice reduced the incidence of calcification from 50% (KN-92 control: 4 out of 8) to 25% (KN-93: 2 out of 8). This was associated with a trend towards reduced calcification area (KN-92 control: 0.43mm
2
vs. KN-93: 0.06mm
2
). These results suggest that CaMKII is activated during atherosclerosis and may be involved with plaque progression through calcification.
Abstract 450: Vascular Smooth Muscle Cell-expressed Tie2 Controls Atherosclerosis Progression
Authors: Kapel Stephanie S , Shi Jingjing , Hasanov Zulfiyya , Appak-Baskoy Sila , Singhal Mahak , Wojtarowicz Jessica , Hertel Stella , Krunic Damir , Korn Claudia , Hu Junhao , Arnold Caroline , Robciuc Marius , Korff Thomas , Augustin Hellmut G
Published: March 2019

Angiopoietin (Angpt)/Tie signaling in microvascular endothelial cells (EC) controls vascular development, remodeling and maturation. Biomarker studies also imply a role of macrovascular Angpt/Tie signaling. The model of Angpt1/stimulation versus Angpt2/destabilization would imply an anti-atherosclerotic function of Angpt1 and a pro-atherosclerotic function of Angpt2. Yet, experimental studies on the role of the Angpt ligands and the Tie receptors in atherosclerosis have yielded conflicting results suggesting spatiotemporally context-dependent pro- and anti-atherosclerotic functions in different experimental settings. The endotheliocentric view of Angpt/Tie signaling is not sufficient to mechanistically explain the divergent roles of Angpt/Tie signaling during atherosclerosis. We hypothesized that vascular smooth muscle cell (VSMC)-expressed Tie2 may contribute to the pathogenesis of atherosclerosis. Employing genetic models, the present study was aimed at elucidating the role of VSMC-expressed Tie2 during atherosclerosis. Compared to EC, VSMC express lower, but consistently detectable levels of functional Tie2. We therefore generated VSMC-specific Tie2
-
deficient mice (
Tie2
SMC-KO
), using a mural cell-specific
Sm22α-Cre
driver line. These were crossed with atherosclerosis-prone
ApoE-
deficient mice (
ApoE
KO
Tie2
SMC-KO
).
ApoE
KO
Tie2
SMC-KO
mice, fed a Western-type diet for 14 weeks, showed significantly reduced atherosclerotic lesion progression with less VSMC content. Transcriptionally, Tie2 controlled the phenotypic switch of VSMC with increased contractile and reduced synthetic phenotype-specific gene expression in isolated Tie2-deficient VSMC. Correspondingly, migration and proliferation was significantly reduced in Tie2-deficient cultured VSMC. Serum Angpt2 as well as the Angpt2/Angpt1 ratio were significantly increased in
ApoE
KO
Tie2
SMC-KO
mice. Collectively, the data expand and revise the endotheliocentric Tie2 signaling concept to show that mural cell-expressed Tie2 is involved in regulating macrovascular functions related to atherosclerosis. VSMC-expressed Tie2 acts pro-atherosclerotic to control the phenotypic switch towards a proliferative and migratory synthetic VSMC phenotype.
Abstract 453: Sestrin1 is Required to Maintain Plasma Cholesterol Homeostasis During Cholesterol Feeding
Authors: Li Zhonggang , Ly Sophia M , Belisle Sabrina L , Leyva Jaimes Fernanda B , Parks Brian W
Published: March 2019

Elevated plasma cholesterol is an established risk factor in the development of heart disease; however, the detailed mechanisms controlling plasma cholesterol levels are complex and under active investigation. Integrating mouse liver global co-expression networks along with human lipid genome-wide association study (GWAS) data, we identified
Sestrin1
as a candidate gene associated with cholesterol in mice and humans. To test the role of
Sestrin1
in cholesterol metabolism, we performed
in vitro
and
in vivo
studies to determine its sensitivity to cholesterol and influence on cellular and plasma cholesterol levels.
In vitro
,
Sestrin1
mRNA is upregulated following cellular cholesterol depletion with methyl-β-cyclodextrin for 4 hours in AML12 hepatocyte cells. This upregulation is blocked when cholesterol complexes or Low-Density Lipoprotein (LDL)-cholesterol is supplemented back into the media.
In vivo
, hepatic
Sestrin1
mRNA is downregulated when mice after feeding a 0.2% cholesterol diet for 8 weeks. In contrast, treatment of mice with 0.02% Lovastatin for 1 week results in upregulation of hepatic
Sestrin1
mRNA. Consistent with transcriptional regulation by cholesterol
in vitro
and
in vivo
, siRNA knockdown of Sterol regulatory element-binding protein 2 (Srebp2) represses
Sestrin1
transcription and prevents the transcriptional regulation of
Sestrin1
by cholesterol. Knockdown of
Sestrin1
with siRNA in AML12 cells results in cellular cholesterol accumulation along with altered cholesterol related gene expression, indicating
Sestrin1
can regulate cholesterol metabolism
in vitro
.
In vivo
, homozygote or heterozygote
Sestrin1
knockout mice fed a 0.2% cholesterol diet for 4 weeks show elevated plasma cholesterol levels compared to littermate control mice. Our results establish
Sestrin1
as a
Srebp2
target gene that is capable of modulating cellular cholesterol levels
in vitro
and regulating plasma cholesterol homeostasis
in vivo
.
Abstract 460: Modulation of the SUMOylation of Fish Oil Receptor G-protein Coupled Receptor (GPR) 120 by AMP-activated Protein Kinase α2 Controls the Anti-atherosclerotic Effects of Fish Oils in vivo
Authors: Yan Chenghui , Lu Qulun , Song Ping , Han Yaling , Zou Ming-hui
Published: March 2019

Background:
Fish oils including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have cardiovascular benefits in general population but fail to show therapeutic effects in patients with cardiovascular diseases (CVD). The aim of this study was to determine how fish oils are ineffective in CVD patients.

Methods:
The effects of fish oils on atherosclerosis, the expression of AMP-activated protein kinase (AMPK), fish oil receptor G-protein coupled receptor (GPR) 120, and inflammation were assessed in CVD patients, atherosclerosis prone LDLR
-/-
mice with or without AMPKα2, lipid reversal atherosclerotic mouse model (LDLR
-/-
ApoB100
+/+
) and cultured cells.

Results:
Healthy individuals had significant higher levels of EPA and DHA in blood than CVD patients. The levels of EPA or DHA were negatively correlated with SYTNAX scores in CVD patients with high AMPKα2 expression but had no correlation in those patients with low AMPKα2 expression. Administration of fish oils (5%, 12 weeks) significantly reduced aortic lesions and inflammation in both LDLR
-/-
mice and LDLR
-/-
ApoB100
+/+
but not in LDLR
-/-
/AMPKα2
-/-
mice. In both human and mouse arteries, AMPKα2 was inversely correlated with GPR120. Further, AMPKα2 suppresses GPR120 SUMOylation resulting in an increase of GPR120 internalization. Consistently, GPR120 inhibition abolished the anti-atherosclerotic effects of fish oils in cultured cells. Moreover, selective inhibition of AMPKα2 but not AMPKα1 increased both SUMOylated protein UBC9 and its transcriptional factor c-myc expression. Furthermore, inhibition of c-myc ablated UBC9 transcription and GPR120 SUMOylation. Mechanistically, we found that AMPKα2 controls c-myc stability by phosphorylating c-myc at serine 67, a key site for its degradation.

Conclusion:
We conclude that AMPK α2 mediates the anti-atherosclerotic effects of fish oils by modulating GPR120 SUMOylation and internalization.
Abstract 461: Enhanced Glycolysis in Endothelial Cells Protects Against Atherosclerosis
Authors: Yang Qiuhua , Xu Jiean , Liu Zhiping , Sudhahar Varadarajan , Ma Qian , Cao Yapeng , Wang Lina , Zeng Xianqiu , Zhou Yaqi , Zhang Min , Xu Yiming , Wang Yong , Weintraub Neal , Zhang Chunxiang , Fukai Tohru , Wu Chaodong , Hong Mei , David Fulton , Huo Yuqing
Published: March 2019

Background:
Increased aerobic glycolysis in endothelial cells (ECs) of atheroprone areas of blood vessels is hypothesized to drive increased inflammation and lesion burden but the direct links are not yet established. The goal of this study was to determine how metabolic adaptation in ECs impacts atherosclerosis.

Methods:
The immunostaining, Real-time PCR, Western blots, Seahorse assayes are used to analyze the expression and activity of PRKA/AMPKs and metabolic glycolysis in the arterial endothelium of mice and cultured endothelial cells under disturbed flows. Staining of Edu, TUNNEL and Evans blue were utilized to evaluate endothelial turnover and endothelium integrity. Oil Red O staining and histological cellular characterization were performed to evaluate atherosclerosis.

Results:
ECs exposed to disturbed flow in vivo and in vitro exhibit increased levels of PRKA/AMPKs (protein kinase AMP-activated; AMP-activated protein kinases) and glycolysis. Selective deletion of endothelial Prkaa1 (protein kinase AMP-activated catalytic subunit alpha1) reduced glycolysis, compromised EC proliferation and EC monolayer integrity, and accelerated the formation of atherosclerotic lesions in hyperlipidemic mice. Rescue of impaired glycolysis in Prkaa1-deficient ECs through Slc2a1 transfection enhanced EC viability and integrity of the EC barrier and reversed susceptibility to atherosclerosis. These results suggest that increased glycolysis in the endothelium of atheroprone arteries is a protective mechanism.

Conclusions:
Endothelial glycolysis plays a vital role in EC proliferation, repair of the EC monolayer, and protects mice from atherosclerosis. Thus, approaches such as activating AMPK activity or increasing endothelial glycolysis are preventive and therapeutic for arterial diseases.
Abstract 466: Carotid Atherosclerosis on the Association Between Hypothyroidism and Renal Dysfunction in a Healthy Japanese Population
Authors: Furusyo Norihiro , Tanaka Yuuki , Ikezaki Hiroaki , Murata Masayuki , Hayashi Jun
Published: March 2019

Aim:
This large-scale Japanese population study was done to evaluate the relation between the serum thyroid stimulating hormone (TSH) level and renal function.

Methods:
Among 1,374 residents who attended a free public physical examination between 2010 and 2011, we evaluated the data of 888 for whom the serum TSH level and estimated glomerular filtration rate (eGFR) were successfully measured. The participants were divided into three groups by TSH level (normal TSH, ≤2.4; high-normal TSH, 2.5-4.4; subclinical hypothyroid, ≥4.5 μIU/L). Multiple linear regression analysis adjusted for cardiovascular risk factors was done to determine the relation between serum TSH level and renal function.

Results:
The mean ± SD TSH level was 2.0±1.4 μIU/mL. Of the participants, 75.9% (n=674) had a normal TSH level, 17.9% (n=159) were high-normal TSH, and 6.2% (n=55) were subclinical hypothyroid. The mean eGFR significantly decreased with increased TSH level (normal TSH group, 79.3±14.1; high-normal TSH group, 77.4±13.0; subclinical hypothyroid group; 72.3±12.2 mL/min/1.73m2: P for trend <0.01). Multiple linear regression analysis extracted log-transformed TSH level as an independent factor correlated with eGFR in the high-normal TSH group (beta=-0.18, P=0.02).

Conclusions:
The eGFR level significantly decreased with increased TSH level. Significant correlation between TSH and eGFR levels was found only in high-normal TSH individuals. Even in healthy individuals, increase risk of chronic kidney disease could be started from high-normal TSH level.
Abstract 478: Resident Multipotent Vascular Stem Cells Isolated From Susceptible and Non-susceptible Arteriosclerotic Regions of the Mouse Aorta Are Sca1/s100β/nestin
+
and Respond Similarly to the Same Myogenic Inductive Stimulus
Authors: Di Luca Mariana , Fitzpatrick Emma , Liu Weimin , Morrow David , Redmond Eileen M , Cahill Paul A
Published: March 2019

Vascular remodeling leading to arterial obstruction is a hallmark of arteriosclerosis and in-stent restenosis and is due in part to the accumulation of vascular smooth muscle (SMC)-like cells within the vessel wall. The source of these vascular cells has been controversial with many studies providing compelling evidence for a putative role for stem cell-derived myogenic progeny. It is known that neuroectoderm-derived vascular regions (ascending aorta, aortic arch, carotid artery) are more susceptible to arteriosclerotic lesion formation in comparison with mesoderm derived regions (descending and abdominal aorta, femoral artery). Our aim was to isolate and characterize stem cells from arteriosclerotic-susceptible and non-susceptible regions and determine their differential responsiveness to discrete myogenic inductive stimuli. A population of myosin heavy chain (Myh11
-
) negative, Sca1/S100β/Nestin
+
multipotent vascular stem cells (MVSCs) was first shown to accumulate within the intima of murine carotid arteries following injury using Sca1 and S100β e-GFP transgenic mice. Resident MVSCs were isolated from mouse aorta arch (susceptible) and descending aorta (non-susceptible) by enzymatic dissociation and initial seeding on non-adherent plates for 48 h before suspended cells were re-seeded on adherent plates and grown in B27 supplemented maintenance media. Cells were characterized by fluorescent immunocytochemistry using stem (Sca1/S100β/Nestin) and vascular SMC cell markers (CNN1 and Myh11). MVSCs from both aortic arch and descending aorta were positive for stem cell markers but negative for Myh11 and Cnn1. Treatment of both cell populations with TGF-β1 or the Notch ligand, Jagged-1 for 7 days promoted myogenic differentiation by increasing the number of cells expressing SMC markers concomitant with increased Myh11 and CNN1 mRNA levels, respectively. There was no difference in the responsiveness of stem cells from either arteriosclerotic-prone and non-prone regions. We conclude that the aortic arch and descending aortic region both house a neuroectoderm-derived Sca1/S100β/Nestin
+
stem cell population that responds similarly to myogenic inductive stimulation.
Abstract 479: Dickkopf-3 Binds to CXCR7 of Vascular Progenitor Cells to Enhance Degradable Graft Regeneration
Authors: Issa Bhaloo Shirin , Wu Yifan , Le Bras Alexandra , Hu Yanhua , Xu Qingbo , Zhao Qiang
Published: March 2019

Background:
Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown.

Methods:
Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested
in vitro
using transwell and wound healing assays and
ex vivo
by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess
in vivo
the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta.

Results:
We demonstrated that Dkk3 induced the chemotaxis of Sca-1+ cells
in vitro
, and
ex vivo
in culture of aortic rings derived from Sca-1-GFP transgenic mice. Flow cytometry and WB analysis revealed that Sca-1+ cells expressed CXCR7, while overexpression or knockdown of CXCR7 resulted in alterations in cell migration. Interestingly, Co-IP experiments showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high affinity Dkk3-receptor binding with a dissociation constant of 14.14 nM. Activation of CXCR7 by Dkk3 triggered the ERK1/2, PI3K/AKT, Rac1 and RhoA signalling pathways. Furthermore, when Dkk3-loaded tissue-engineered vessels were grafted to the abdominal artery of rats, the grafts showed efficient endothelization and recruitment of VPCs, which had acquired characteristics of mature SMCs. CXCR7 blocking using specific antibodies in our vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodelling.

Conclusions:
We provide novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced VPCs migration
in vitro
and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.
Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death
Authors: Franco Sarah R , Stranz Amelia , Ljumani Fiona , Urabe Go , Stewart Danielle , Chaudhary Mirnal , Liu Bo
Published: March 2019

Objectives:
Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury.

Methods & Results:
The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern
in vitro
. Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation.

Conclusions:
In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.
Abstract 514: Netrin 4 Deficiency Leads to Endothelial Cell Senescence
Authors: ZHANG HUAYU , Vreeken Dianne , de Bruin Ruben G , Leuning Danielle G , Maanaoui Mehdi , Sol Wendy M , van der Veer Eric P , Rabelink Ton J , van Zonneveld Anton Jan , van Gils Janine M
Published: March 2019

Senescence phenotype of endothelial cells (ECs) has pathophysiological consequences, such as decreased regeneration capacity, pro-atherogenic tendency and dysregulated vessel tune. We find that netrin 4 (NTN4), recognized in neural and vascular development, is highly expressed by mature ECs. Remarkably, little is known about its role in vascular biology after development. NTN4 is deposited in the extracellular matrix. Using human decellularized kidney extracelluar matrix scaffolds, we found that pre-treatment of the scaffolds with NTN4 increased numbers of EC adhesion to the matrix, showing a pro-survival effect of NTN4.

Subsequently we explored the regulation of NTN4 expression in ECs. We found a 1.8-fold (±0.3; p<0.05) upregulation in NTN4 expression in ECs cultured under laminar flow conditions compared to static culture conditions. In contrast, ECs stimulated with TNFα resulted in decreased NTN4 expression (0.17 ± 0.06 fold; p<0.05), indicating a role for NTN4 in quiescent healthy endothelium. Silencing of NTN4 in ECs, to investigate the necessity of NTN4 in ECs, markedly resulted in more senescent associated β-galactosidase activity (20-50%; p<0.05) that could be rescued by NTN4 protein coating. Consistent with increased senescence, NTN4 reduction is accompanied with increased expression of senescence-associated transcription factors, CDKN1A and CDKN2A, as well as decreased ability to proliferate. Importantly, ECs with reduced levels of NTN4 have also increased expression of ICAM-1 and VCAM-1, are more prone to adhesion of human monocyte and have impaired barrier function, measured in an electric cell-substrate impedance sensing system as well as ‘organ-on-a-chip’ microfluidic system.

In conclusion, our results identified the anti-senescence function of NTN4 and thereby provides novel insights in the role of NTN4 in EC function. In situations like acute inflammation and unfavourable hemodynamic conditions, NTN4 expression decreases, so that there is a possible window for us to improve EC function by normalizing NTN4 expression.
Abstract 521: Pharmacological Profile of JNJ-64179375: a Novel, Long Acting Exosite-1 Thrombin Inhibitor
Authors: Huang Devine Zheng , Li Qiu , Chintala Madhu
Published: March 2019

Background:
Thrombin is a key serine protease involved in hemostasis and thrombosis by mediating the conversion of soluble fibrinogen to insoluble fibrin that forms the meshwork of a clot. In addition, it is the most potent activator of platelets. Hence thrombin inhibition has been a target for the development of anticoagulant drugs. Recently, an IgA that binds to the exosite 1 region of thrombin was identified in a patient that was profoundly anticoagulated but without any abnormal bleeding episodes over a prolonged follow-up. JNJ-64179375 (JNJ-9375) is a monoclonal antibody derivative of the IgA which by selectively inhibiting exosite 1 may allow for some of thrombin functions mediated by exosite 2 and the active site which might be important for hemostasis. JNJ-9375 is being developed as a new long-acting anti-coagulant with a potential for better efficacy with a reduced bleeding liability.

Objectives:
To assess the in vitro effects of spiked JNJ-9375 on various clotting assays in human and animal plasma and to evaluate the antithrombotic activity in a rat A-V shunt model of thrombosis.

Methods and results:
JNJ-9375 produced a concentration-dependent prolongation of thrombin time (TT), ecarin clotting time (ECT), prothrombin time (PT), and activated partial thromboplastin time (aPTT) when added to normal human plasma. The concentrations required to prolong clotting time by twofold (EC2Xs) were 4.4, 12.4, 202.7, and 172.6 μg/ml for TT, ECT, aPTT, and PT respectively. Therefore, TT was the most sensitive assay to show pharmacodynamic activity of JNJ-9375. Similar effects were observed in other species studied including monkey, rat, and mouse. In the rat A-V shunt model of thrombosis, JNJ-9375 dose dependently inhibited thrombus weight with an efficacious dose of 0.3 mg/kg, which was accompanied by prolongation of TT, ECT, PT, and aPTT from ex vivo plasma samples.

Conclusion:
Results from these studies demonstrate that selective inhibition of exosite 1 on thrombin leads to robust anticoagulation and antithrombotic efficacy. JNJ-9375 is currently in early clinical development.
Abstract 522: VWF Inhibition by an RNA Aptamer Improves Functional Outcome Compared to rTPA in Ferric Chloride-induced Carotid Artery Thrombosis in Mice
Authors: Wilson Jenna , Wheeler Debra , Huttinger Allyson , Anderson Cole , Talentino Spencer , Balch Maria , Rink Cameron , Nimjee Shahid
Published: March 2019

Objective:
To compare the functional effects of von Willebrand Factor (VWF) inhibition by RNA aptamer (T79) vs rTPA utilizing
in vivo
arterial thrombosis in mice.

Approach and Results:
We previously demonstrated that inhibition of VWF by a targeted RNA aptamer (T79) both prevents thrombosis and thrombolyses stabilized clots in a murine model of ferric chloride (FeCl
3
)-induced vascular injury suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesized that T79 treatment, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA, would result in improved behavioral outcome with 7 day survival after vascular injury. Baseline locomotor testing in an open field was performed on both male and female, 8-16 week old, wild-type C57BL/6J mice. Occlusive arterial thrombus formation was induced by a 3 minute exposure to 10% FeCl
3
on the right common carotid monitored by Doppler flow and time to occlusion (blood flow of 0 ml/min) was measured. Twenty minutes after thrombus stabilization either T79 (0.1 mg/kg or 0.5 mg/kg bolus), rTPA (10 mg/kg 45 min infusion) or saline vehicle (45 min infusion) was introduced via a saphenous catheter. Animals were recovered and locomotor testing was repeated 48 hours and 7 days after injury with baseline set as 100%. All groups including vehicle control resulted in a statistically significant decrease in distance traveled (meters) and speed (meters/sec) at 48 hours after injury. At 7 days, 0.1 mg/kg T79 increased 29.89% from 48 hours, 0.5 mg/kg T79 increased 22.50% from 48 hours but rTPA treatment showed no significant improvement (2.79% increase) (n=4, p<0.001). In addition, 0.1 mg/kg and 0.5 mg/kg T79 resulted in a 28.46% and 22.44% increase in speed, respectively, at 7 days compared to 48 hours whereas rTPA did not (2.94% increase) (n=4, p<0.001). Interestingly, males scored lower on most behavioral parameters at 48 hours, but recovered to the same level as females by 7 days. No significant differences in time to occlusion or baseline locomotor testing were observed in any group.

Conclusion:
Inhibition of VWF by T79 aptamer markedly improves behavioral outcomes compared to rTPA after survival carotid artery occlusion following ferric chloride-induced injury in mice.
Abstract 524: Fibrinogen Depletion Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm
Authors: Russell Hannah M , Saum Keith , Sundermann Alexandra C , Jones Shannon M , Wanhainen Anders , Edwards Todd L , Holle Lori A , Wolberg Alisa S , Owens A. Phillip
Published: March 2019

Background:
Fibrinogen and fibrin provide physical and biochemical support to a developing clot and is defined as one of the most crucial independent risk factors for cardiovascular diseases (CVDs). In addition to clot formation, fibrinogen promotes wound healing and powerful inflammatory and immune responses by engagement of leukocytes. Increased circulating fibrinogen and fibrin degradation products are correlated with increased diameter and progression of abdominal aortic aneurysm (AAA). However, a causal link between fibrinogen and AAA has not yet been established. The objective of this study was to determine the role of fibrinogen depletion in a mouse model of AAA.

Methods and results:
To determine whether aneurysm resulted in a procoagulant environment, we examined plasma levels of thrombin generation by calibrated automated thrombography (CAT), thrombin anti-thrombin (TAT), and fibrinogen in control and AAA plasma from mice and humans. Patients and mice with AAA had significant elevations in thrombin generation, TAT, and fibrinogen versus saline controls (mice) and control patients (human). To determine the effect of fibrinogen, in vivo, low density lipoprotein receptor deficient (

Ldlr
-/-

) mice were injected with scrambled anti-sense oligonucleotide (ASO) or β-fibrinogen ASO (30 mg/kg) 3 weeks prior to experimentation and throughout the study. Fibrinogen ASO treatment achieved > 90% depletion of fibrinogen. After 3 weeks, mice were fed a fat and cholesterol enriched diet (42% milk fat; 0.2% cholesterol) 1 week prior to and throughout infusion with angiotensin II (AngII; 1,000 ng/kg/day) for 28 days. Fibrinogen ASO attenuated abdominal diameter (33% decrease; P = 0.001), and inflammatory cytokines (>75% decreased IL-1 and IL-6; P = 0.001) versus scrambled ASO control. Further, fibrinogen depletion significantly attenuated aneurysm incidence and rupture-induced death (P < 0.05).

Conclusions:
Our results demonstrate that AAAs augment procoagulant markers in both humans and mice. Importantly, fibrinogen depletion attenuates AAA incidence, diameter, rupture-induced death, and inflammation. Therefore, reduction of plasma fibrinogen may be a novel treatment strategy in patients with AAA.
Abstract 532: Shear-induced Release of Platelet Receptors Contributes to Bleeding Outcomes in Patients With Lvads or Exposed to Ecmo
Authors: Gardiner Elizabeth E , Casan Joshua , Montague Samantha J , Lukito Pohan , Andrews Robert K , Davis Amanda K
Published: March 2019

Despite advances in design and materials, as well as pharmacological prophylaxis, hemostatic complications continue to plague device recipients. Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. We examined blood samples from patients with heart failure or in receipt of high fluid shear mechanocirculatory support to assess whether loss of platelet receptors occurs
in vivo
, and the relationship with acquired von Willebrand syndrome (AVWS) and changes in inflammatory cytokines. Platelet counts, levels of inflammatory cytokines IL-1β, IL-6, TNFα, MCP-1, IL-17A and IFNγ, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 13 continuous flow VAD (CF-VAD), 14 ECMO, and 24 heart failure patients. Levels of platelet receptors were measured by flow cytometry or ELISA. The loss of high molecular weight VWF multimers was observed in 12 of 13 CF-VAD and 7 of 14 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Significant differences in levels of inflammatory cytokines monocyte-chemoattractant protein and tissue necrosis factor-a in a subset of patients with decompensated HF. These data link AVWS and increased platelet receptor shedding in patients with CFVADs or ECMO. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CFVAD and ECMO patients may be linked with extent of inflammation and may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.
Abstract 537: Nature’s Surfaces: a Potential Role for Arterial Wrinkles in Vascular Homeostasis
Authors: Nath Nandan , Pocacivsek Luka , Gao Ya , Salem Karim , Velankar Sachin , Tzeng Edith
Published: March 2019

Biologic interfaces play important roles in tissue function. The tissue-blood interface in blood vessels represents a dynamic interaction between the endothelium and circulating RBCs, platelets and inflammatory cells. This interface is critical in regulating vascular thrombosis. While the study of vascular homeostasis has focused on the endothelium, the arterial lumen has been shown to possess a regular wrinkled pattern that has been previously attributed to fixation artifact. We hypothesize that the arterial luminal surface undergoes phasic wrinkling and flattening driven by the cardiac cycle and this dynamic topography may represent a novel mechanism of resisting platelet adhesion. Using an
ex vivo
pulsatile pump system to actuate arteries and grafts, we examined the effect of physiologic pressures on luminal topography and the effect of dynamic topography on platelet adhesion in vascular graft function. Pig carotid arteries were distended to 40-140 mmHg, fixed, and examined histologically for luminal surface wrinkle amplitudes. Arteries at 40 mmHg had an average wrinkle amplitude of 20.52 ± 4.13 μm which decreased to 5.86 ± 1.69 μm at 140 mmHg (
p
<0.05; Figure 1A-D). We synthesized silicone grafts with a wrinkled luminal surface similar to the arteries or with a smooth surface. The grafts were placed on a pulsatile pump circuit set to a systolic pressure of 100-120 mmHg and activated platelets were circulated through the graft. Wrinkled grafts undergoing pulsatile flow exhibited an 87% reduction in platelet deposition by Giemsa stain in comparison to grafts with smooth luminal surfaces (
p
<0.05, Figure1E,F). These findings confirm that arteries do possess luminal topography that varies with physiologic ranges in blood pressure. Grafts that mimic this behavior show reduced platelet accumulation, suggesting that dynamic topography contributes to vascular homeostasis. Incorporation of this topography into vascular grafts may improve graft patency.