bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 019: Anti-MicroRNA-144 Therapy Attenuates Progression and Promotes Regression of Atherosclerosis
Authors: Tarling Elizabeth J , Cheng Joan , Cheng Angela , Pamir Nathalie , Sallam Tamer , de Aguiar Vallim Thomas Q
Published: March 2019

Rationale:
Removal of cholesterol from atherosclerotic lesions remains an unmet therapeutic need. The ATP Binding Cassette Transporter A1 (ABCA1) plays a key role in the efflux of cholesterol from macrophages, therefore strategies that increase macrophage ABCA1 levels are likely to be atheroprotective.

Objective:
To determine the potential of miR-144 antagonism as an interventional therapeutic in high-fat, high-cholesterol-fed low-density lipoprotein receptor null (Ldlr
-/-
) mice.

Methods:
Ldlr
-/-
mice were treated with saline, control anti-miR, or anti-miR-144 oligonucleotides once a week for 16 weeks to assess the efficacy of long-term silencing of miR-144 on atherosclerosis progression. Second, Ldlr
-/-
mice with established atherosclerotic plaques were treated with anti-miR-144 for 4 weeks, in a model of atherosclerosis regression. All anti-miR-144-treated mice showed increased circulating high-density lipoprotein (HDL) levels. Anti-miR-144-treated mice also showed enhanced reverse cholesterol transport to the plasma, liver, and feces in the regression model. Consistent with these observations, all anti-miR-144-treated mice showed reductions in atherosclerotic burden. Notably, miR-144 antagonism also significantly remodeled the HDL particle proteome in both progression and regression models of atherosclerosis.

Conclusions:
These studies suggest anti-miR-144 treatment may be a promising interventional therapeutic strategy to treat atherosclerotic disease.
Abstract 020: Regression of Atherosclerosis Through Manipulation of Vascular Macrophages; a Novel Gene-therapy Approach
Authors: Howard Courtney , Ammi Azzadine , Muelle Paul , Huynh Katherine , Moccetti Federico , Latifi Yllka , Nelson Jonathan , Xie Aris , Lindner Jonathan R , Tavori Hagai
Published: March 2019

Aggressive lipid lowering halts atherosclerotic plaque progression but does not lead to bulk plaque regression in humans. We have previously reported that gene transfection of the HDL protein apoAI into macrophages (MΦ-apoAI) decrease the rate of plaque development in atherosclerosis-prone mice. In this study, we hypothesized that MΦ-apoAI can promote regression of atherosclerosis synergistically with lipid-lowering.

Atherogenic mice (LDLR
-/-
and LDLR
-/-
/MΦ-apoAI) were fed a high-fat diet to promote stage II/III atherosclerotic lesions (as baseline comparator), and then switched to an extreme lipid-lowering intervention (chow with MTTP inhibitor). After 3 weeks on the lipid-lowering diet, both groups showed reduced systemic and lesion inflammation, and reduced macrophage content on histology when compared to baseline. Upon lipid-lowering, vascular ultrasound showed that LDLR
-/-
/MΦ-apoAI mice has 21% improvement in aortic pulse transit time, and a 37.1% improvement in instantaneous aortic compliance (a surrogate marker for arterial elastic modulus), compared to LDLR
-/-
mice. Histologic evidence showed that LDLR
-/-
/MΦ-apoAI mice on lipid-lowering diet also had reduced lesion size (-24%), reduced macrophage content (-27.5%), increased M2/M1 ratio (+51%) and reduced necrotic core area (-28.6%), compared to LDLR
-/-
mice. Using transplantation of bone marrow cells from MΦ-apoAI mice into recipient mice with established lesions, we show that newly recruited cells are not major contributors to the regression afforded by MΦ-apoAI. Thus, to study the kinetics of pre-existing cells in the lesion during regression, we developed a contrast ultrasound-mediated gene delivery system to tag and trace lesion cells in LDLR
-/-
mice, which revealed that lesion macrophages expressing apoAI specifically migrate to mediastinal LN in a CCR7-dependent manner during regression.

Expression of apoAI in pre-existing lesion macrophages, promotes regression of atherosclerosis beyond lipid-lowering alone, and increases CCR7-dependent egress of macrophages to adjacent lymph nodes. Ultrasound-mediated gene delivery can be a useful device to study the kinetics of cell in the artery wall and also represent a novel approach with application to human therapy.
Abstract 025: Anti-Apolipoprotein A-I Antibody Profiles Predict Cardiovascular Disease Outcomes in Patients and in a Mouse Model of Atherosclerosis
Authors: Kline Robert H , Henson David , Venditto Vincent J
Published: March 2019

Apolipoprotein A-I (ApoA-I) is the target of antibody induction in patients suffering from chronic inflammation associated with obesity, autoimmunity and cardiovascular disease (CVD). Anti-ApoA-I antibodies are considered serum markers for CVD, but the exact immunologic stimuli and role of these antibodies in disease progression are poorly understood. Our efforts in characterizing the anti-ApoA-I antibody profiles in 375 patients with coronary artery disease resulted in a hazard ratio of 1.52 (95% CI: 1.03-2.18;
p
= 0.02) when adjusted for 12 traditional markers of cardiovascular disease. We then aimed to recapitulate these findings in mice using an immunostimulatory liposomal formulation decorated with peptides derived from ApoA-I to induce epitope-specific antibody responses in mice. Intriguingly, acute inflammation associated with adjuvant exposure and high fat diet alone induced an anti-ApoA-I antibody response in mice. Immunization shifted the antibody profile to a pro-atherogenic phenotype, which accelerated atherosclerosis as measured by
en face
lesion area. Protective antibody profiles were also observed in mice, which recapitulate the phenotype observed in patients protected from major adverse cardiovascular events. Comparison of mice exhibiting a pro-atherogenic antibody profile with mice exhibiting an anti-inflammatory antibody profile result in lesion areas of 20% and 5% (
p
= 0.005), respectively. Continued evaluation of the immunologic mechanisms that lead to pro-atherogenic and anti-atherogenic antibody responses and the immunologic sequelae associated with specific antibody profiles will promote novel therapeutic strategies and guide interventions to improve patient outcomes.
Abstract 027: A Micropeptide Concealed in a Putative Long Non-coding RNA Directs Inflammation
Authors: van Solingen Coen , Sharma Monika , Bijkerk Roel , Afonso Milessa S , Koelwyn Graeme J , Scacalossi Kaitlyn R , van Zonneveld Anton Jan , Moore Kathryn J
Published: March 2019

Long non-coding RNAs (lncRNAs), once considered ‘genomic junk’, have been found to regulate diverse biological processes and their study continues to reveal novel insights into lncRNA functions. Recent studies revealed that some lncRNAs may harbor small open reading frames (ORFs) that code for functional micropeptides. While investigating an unannotated primate-specific lncRNA, lncVLDLR, that is altered in patients with type II diabetes and cardiovascular disease, we discovered a previously unrecognized ORF encoding a 44 amino acid micropeptide.
In vitro
transcription and translation of the IMP coding sequence in the presence of
35
S-methionine produced a single 8 kDa peptide, which we have named Inflammation-modulating MicroPeptide (IMP). To dissect IMP function, we focused on its amino acid sequence and putative structure. These analyses revealed high sequence homology between IMP and transcription factors such as NFκB, c-myb and zinc finger proteins, and the presence of a hydrophobic region with an LxxLL motif often found in transcriptional regulators. Circular dichroism spectroscopy of synthesized IMP predicted an intrinsically disordered peptide, which is a common characteristic of transcriptional coactivators. To investigate a potential role of IMP in regulating gene transcription, we cloned a MYC-epitope tag in-frame with IMP within the full-length transcript of lncVLDLR and expressed it in HEK293 cells. Immunofluorescence staining, and cell fractionation combined with western blotting, confirmed nuclear localization of IMP. RNA-seq analysis of THP1 macrophages overexpressing IMP revealed an increase in inflammatory genes, including cytokines and chemokines. Moreover, analysis of upstream regulators of these genes suggests that IMP may interact with KIX domain-containing transcriptional coactivators to regulate inflammatory gene expression. Together our data identify a novel human micropeptide, encoded within a putative lncRNA that is dysregulated in diabetes and cardiovascular disease, that regulates inflammatory gene transcription. Further characterization of IMP and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular and other inflammatory diseases.
Abstract 035: Recombinant LCAT Restores Defective HDL mediated Endothelial Protection in Acute Coronary Syndrome
Authors: Ossoli Alice , Simonelli Sara , Varrenti Marisa , Morici Nuccia , Oliva Fabrizio , Stucchi Miriam , Gomaraschi Monica , Arnaboldi Lorenzo , Corsini Alberto , Thomas Michael , Karathanasis Sotirios , Veglia Fabrizio , Calabresi Laura
Published: March 2019

Recent evidence suggests that the vasoprotective effects of HDL are impaired in patients during an acute coronary syndrome (ACS). In particular, HDL from ACS patients are defective in promoting nitric oxide (NO) release from cultured endothelial cells (ECs). Lecithin:cholesterol acyltransferase (LCAT) plays a key role in HDL maturation and remodeling, and it is the enzyme responsible for cholesterol esterification in plasma. Very few studies have suggested that LCAT activity is decreased during an acute myocardial infarction, but the possible link between LCAT mass/activity and the HDL dysfunction observed in ACS has never been tested. To test this hypothesis, plasma from 30 STEMI patients was collected at admission, 48 and 72 hours and 30 days after event. LCAT concentration and activity significantly decreased 48 hours after event (-0.49±0.11 μg/ml, P<0.001, and -3.15±1.05 nmol/ml/h, P=0.006) and remained reduced at 72 hours. HDL isolated from STEMI patients lose the capacity to promote NO production in ECs (-0.13 ± 0.06 fold, P=0.04) and the reduction is significantly correlated to decreased LCAT activity (R= 0.52, P=0.003).
In vitro
studies were performed in which STEMI patients’ plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in ECs.
In vitro
incubation of STEMI patients’ plasma with rhLCAT remodels phospholipids and protein content in HDL and restores HDL ability to promote endothelial NO production (+25%, P=0.03). Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during ACS. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in ACS.
Abstract 041: Neutrophil Extracellular Traps Enhance Venous Thrombosis in Mice Bearing Human Pancreatic Tumors
Authors: Hisada Yohei , Houston Reaves , Maqsood Anaum , Thalin Charlotte , Noubouossie Denis F , Wallen Hakan , Kolev Krasimir , Cooley Brian C , Key Nigel S , Mackman Nigel
Published: March 2019

Introduction:
Cancer patients have an increased risk of venous thromboembolism (VTE) compared with healthy individuals. Pancreatic cancer has one of the highest rates of VTE. We are interested in elucidating the pathways that contribute to cancer-associated thrombosis (CAT) particularly in pancreatic cancer. A recent study reported an association between plasma levels of citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), and VTE in a population of patients with a variety of cancer-types. Importantly, subgroup analysis showed that increased levels of H3Cit are associate with VTE in patients with pancreatic and lung cancer but not in patients with other types of cancer. We use mouse models to investigate the role of different pathways in CAT.

Aim:
To test the hypothesis that NETs enhance thrombosis in mice bearing human pancreatic tumors.

Methods:
We recently established a CAT model in which venous thrombosis is induced by infrarenal vena cava (IVC) stasis in nude mice bearing human pancreatic tumors (BxPc-3).

Results:
We found that tumor-bearing mice had increased levels of plasma granulocyte-colony stimulating factor and neutrophilia. In addition, levels of plasma cell-free DNA are increased, which is used as a marker of NET formation. Tumor-bearing mice also had larger thrombi in an IVC stasis model compared with controls. Thrombi from tumor-bearing mice had increased levels of neutrophils and decreased levels of red blood cells compared with thrombi from control mice. Importantly, administration of DNAse I reduced thrombus size in tumor-bearing mice but not in control mice.

Conclusions:
Our study indicates that the presence of human pancreatic tumors in mice induces neutrophilia and increases NET formation that enhances venous thrombosis. These results suggest that neutrophils and/or NETs represent new targets to prevent VTE in pancreatic cancer patients.
Abstract 043: Endothelial Adenosine Kinase Deficiency Ameliorates Diet-induced Insulin Resistance
Authors: Xu Jiean , Yang Qiuhua , Zhang Xiaoyu , Liu Zhiping , Cao Yapeng , Wang Lina , Zhou Yaqi , Zeng Xianqiu , Zhang Min , Ma Qian , Xu Yiming , Wang Yong , Bagi Zsolt , Fulton David J. , Hong Mei , Huo Yuqing
Published: March 2019

Background:
Obesity is associated with endothelial dysfunction characterized by reduced bioavailability of nitric oxide (NO). It is well known that adenosine regulates endothelial nitric oxide release and vasodilation. However, it is unclear whether intracellular adenosine is able to modulate endothelial nitric oxide production and diet-induced metabolic disorders. This study is to investigate the effect of elevated intracellular adenosine caused by adenosine kinase (ADK) deletion or knockdown on endothelial nitric oxide production and diet-induced insulin resistance as well as the pathways associated to this effect.

Methods and results:
Endothelial-specific ADK knockout mice fed a high-fat diet show decreased fasting blood glucose and insulin, suppressed adipose tissue inflammation and hepatic steatosis, increased skeletal muscle arteriole vasodilation in an eNOS dependent manner. Mechanistically, ADK knockdown in Human Umbilical Vein Endothelial Cells (HUVECs) with adenoviral ADK shRNA elevated both intracellular and extracellular adenosine, and consequently increased endothelial nitric oxide synthase (eNOS) expression and activation, resulting in an increase in NO production, insulin uptake and vasodilatory-stimulated phosphoprotein (VASP) phosphorylation. Treatment of HUVECs with adenosine A2b receptor antagonist MRS 1754 abolished ADK-knockdown induced eNOS expression. eNOS phosphorylation in ADK-knockdown HUVECs were not interfered with adenosine receptors antagonists.

Conclusion:
ADK knockdown-mediated elevation of intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders. This is attributed to an enhancement of NO production caused by increased eNOS activation and expression, the latter is regulated via endothelial adenosine A2b receptor.
Abstract 044: Platelet-Endothelial Interactions in Atherosclerosis-Prone Arteries in a Non-Human Primate Model of Obesity and Insulin Resistance
Authors: Brown Eran I , Belcik J. Todd , Hodovan James M , Moccetti Federico , Ozawa Koya , Bader Lindsay A , Kievit Paul , Lindner Jonathan R
Published: March 2019

Background:
Platelet interactions with the vascular endothelium contribute to early atherogenesis, and occur in part from oxidative stress and secondary dysregulation of endothelial-associated von Willebrand factor (VWF). We used in vivo contrast enhanced ultrasound (CEU) molecular imaging of the carotid endothelium in a non-human primate model of diet-induced obesity to test whether platelet adhesion is present in atherosclerosis-susceptible arteries prior to plaque development, and whether it can be modified through NADPH-oxidase (Nox) inhibition.

Methods:
Six adult rhesus macaques fed a high-fat diet (HFD), with 30% of calories from fat, for >2 years were studied at baseline and after 8 weeks of therapy with the Nox inhibitor apocynin (50 mg/kg/d). Six lean chow-fed controls were also studied. Intravenous glucose tolerance tests (IVGTT) and body composition were assessed at each interval. CEU molecular imaging of VCAM-1 and platelet GPIbα of the carotid arteries bilaterally was used to assess endothelial activation and platelet adhesion. Carotid intimal thickening (IMT) and brachial flow-mediated dilation (FMD) were assessed by ultrasound.

Results:
Before therapy, animals on HFD compared to controls were obese (16.0 vs 9.3 kg, p=0.003), had increased visceral adiposity (49% vs 25% truncal fat, p=0.002), and were insulin resistant (4-fold higher insulin AUC on IVGTT, p=0.002). FMD and IMT were similar between cohorts, although mild plaque was seen in the carotid bulb in 3 HFD macaques. HFD animals had greater (p<0.01 vs. controls) carotid CEU signal for VCAM-1 (20.0 vs -2.7 IU) and GPIbα (12.7 vs -0.4 IU). There was a linear correlation between VCAM-1 and GP1ba signal (r
2
=0.50, p=0.0001). Apocynin significantly reduced (p<0.01 vs pre-treatment) signal for VCAM-1 (3.96 IU) and GPIbα (0.19 IU); but did not alter IMT, FMD, visceral adiposity, or insulin resistance on IVGTT.

Conclusions:
Platelet- endothelial interactions occur early in atherosclerosis-prone carotid arteries of obese and insulin resistant non-human primates, and correlates with the degree of endothelial adhesion molecule expression. Inhibition of Nox suppresses platelet adhesion and reduces VCAM-1 expression independent of any effects on insulin resistance and obesity.
Abstract 050: The Vascular Ecto-enzyme, CD39 Protects from Venous Thrombogenesis by inhibiting Innate Immune Activation
Authors: Kanthi Yogendra , Jacobs Benjamin N , Chi Liguo , Yadav Vinita , Zhao Raymond , Banka Alison , Knight Jason S , Pinsky David J
Published: March 2019

Background:
Venous thrombosis (DVT) is a serious health concern, with growing incidence in an aging, comorbid population. DVT is marked by sterile inflammation and innate immune activation. Early participants in DVT include platelets, and neutrophils (PMN) which extrude their DNA to form thrombus-potentiating extracellular traps (NETs). To identify improved therapeutic targets at the nexus of inflammation and coagulation, we focused on the vascular ectonucleotidase CD39, found on leukocytes and the endothelium. CD39 dissipates extracellular ATP & ADP, thrombo-inflammatory “danger” signals. We hypothesized that CD39 is a critical enzyme in venous homeostasis, restraining unchecked inflammation and thrombosis in DVT.

Methods:
DVT was induced in
Cd39
+/-
and WT control mice using a “flow-restriction” model of inferior vena cava (IVC) stenosis. Thrombus frequency and size were assessed 48h after DVT induction. A novel flow cytometry method, immunoblot, immunostaining were used to examine thrombus cellular content, fibrin, NETs, and inflammasome activation.

Results:
Cd39
+/-
mice had a significantly higher thrombus frequency (2.5-fold) and clot size (3-fold), with more fibrin content by immunoblot. Flow cytometry revealed exaggerated PMN recruitment to the growing thrombus in
Cd39
+/-
mice, with more NETs
in vitro
, and within DVT compared with WT mice.
Cd39
+/-
mice also had amplified DVT inflammasome activity
in vivo
, measured by increased NFkB phosphorylation and mature IL-1 beta compared with WT DVT. Flow cytometry of the thrombus revealed an increase in activated platelet-PMN heteroaggregates within
Cd39
+/-
DVTs, indicating enhanced innate-coagulation system crosstalk.

Conclusion:
CD39 is a critical vasculoprotective ecto-enzyme in venous thrombogenesis.
Cd39
+/-
mice have increased DVT burden, fibrin deposition, activated platelet-PMN interactions, and exaggerated inflammasome activation compared with WT mice. By enrolling NET formation and inflammasome activation, CD39 posits a previously unexplored link between venous inflammation and thrombosis. Studies are underway to modulate inflammasome activation in
Cd39
+/-
mice, and to delineate the relative contributions of leukocyte/endothelial CD39 in venous thrombosis.
Abstract 057: Adenosine Kinase Epigenetically Regulates Abdominal Aortic Aneurysm Formation
Authors: Liu Zhiping , Xu Jiean , Wang Jiaojiao , Xu Yiming , Wang Yong , Wang Lina , Cao Yapeng , Yang Qiuhua , Kim Ha Won , Weintraub Neal L , Huo Yuqing
Published: March 2019

Objectives:
Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality worldwide; however, the molecular mechanisms involved in AAA formation are not well understood. The nucleoside adenosine plays important roles in modulating vascular homeostasis. In this study, we sought to determine whether adenosine kinase (ADK), the adenosine-metabolizing enzyme, modulates AAA formation via control of intracellular adenosine level and investigate the involvement of ADK-mediated epigenetic mechanism in this aortic disease.

Methods and results:
ADK expression and activity were increased in murine AAA models, including calcium chloride-stimulated AAA and angiotensin II-induced AAA. Using a loss-of-function genetic mouse model, we demonstrated that genetic ADK knockout mice led to increased Intracellular adenosine level in macrophages and vascular cells and developed significantly smaller aortic expansion when subjected to AAA inductions. Notably, knockout of ADK alleviated arterial inflammation and diminished infiltration of macrophages, as well as suppressed arterial extracellular matrix degradation, which was associated with down-regulation of CXCL12, MMP2/9 and adhesion molecule expression. Mechanistically, elevation of intracellular adenosine by genetic inactivation of ADK or exogenous adenosine reduces activation of the transmethylation pathway via inactivation of SAH hydrolase (SAHH) and histone H3K4 methyltransferase (HMT), thus down-regulating expression and signaling of mitogen-activated protein kinases in activated macrophages and vascular cells with pro-inflammatory stimuli. Blocking of the H3K4 transmethylation pathway by knockdown of WDR5, a core subunit of HMT complex, abrogated the beneficial effects of ADK inactivation.

Conclusions:
Our findings reveal a previously unrecognized role of ADK mediated epigenetic pathways in the pathogenesis of AAA and provides a therapeutic target for the prevention of AAA.
Abstract 059: Defining the Mechanisms of Autologous Bone Marrow Cell Therapy in Critical Limb Ischemia
Authors: Kenyon Bianca , Gutwein Ashley , Wang S. Keisin , Green Linden , Murphy Michael
Published: March 2019

Introduction:
Here we present a composite of proteomic, cellular, and radiological analyses that define the mechanisms by which autologous concentrated bone marrow mononuclear cells (cBMNC) promote limb preservation in patients with critical limb ischemia.

Methods:
CD45
+
, CD34
+
, CD105
+
, and VEGFR-2
+
cells were enumerated using fluorescent activated cell sorting (FACS) from aliquots of the cBMNC from each patient enrolled in the Phase III MOBILE TRIAL. Direct limb perfusion was measured with Positron Emission Tomography/Computed Tomography (PET/CT) with radiolabeled water (
15
H20). Anterior tibialis muscle (ATM) into which cBMNC was injected prior to below knee amputation in the Phase I CHAMP trial were collected for capillary density and proteomic analyses.

Results:
There were no differences in the number of CD45
+
(636 ± 388 vs. 868 ± 699 x 10
6
, p= 0.279), VEGFR-2
+
(0.4 ± 0.8 vs. 0.3 ± 0.6 x10
6
, p=0.757) and CD34
+
(21 ± 13 vs. 35 ± 30 x 10
6
, p= 0.156) cells in the cBMNC product injected in those patients undergoing amputation and those with a preserved limb (n=90). There was a significant association between CD105
+
(7 ± 4 vs. 16 ± 13 x 10
6
, p= 0.05) cells in patients and freedom from amputation. A Blood Perfusion Index (
BPI
) was calculated by comparing the ratio of H2O
15
peak tracer uptake level of the untreated: treated leg with an increase from 0.38 at baseline to 0.54 (42%) at 12 weeks (n=4, p< 0.05). There was an increase in CD31
+
capillaries in the ATM after injection of cBMNC. ATM specimen also showed increases in VEGF-A, angiopoietin-2, and MMP-9 compared to the untreated specimen.

Conclusion:
This first in man analyses provides conclusive evidence that cBMNC improves limb perfusion via capillary formation. This study suggests that bone marrow cell mediated angiogenesis may be dependent on CD105
+
mesenchymal progenitor cells.
Abstract 087: Hepatic Sortilin Regulates Apolipoprotein B Secretion only Under Conditions of Secretory StressDependent of the Presence of Secretory Stress
Authors: Conlon Donna M , Rodrigues Amrith , Guo Kathy , Hand Nicholas , Rader Daniel
Published: March 2019

SORT1
is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.
Abstract 088: Fibronectin Promotes EphA2 Expression through RGD Integrin Signaling in Vascular Smooth Muscle Cells
Authors: Finney Alexandra C , Green Jonette M , Traylor James G , Orr A. Wayne
Published: March 2019

Vascular smooth muscle cells undergo a phenotypic shift to a “synthetic” phenotype during atherosclerosis characterized by downregulation of contractile markers and augmented proliferation, migration, and deposition of extracellular matrix. While absent in contractile, medial vascular smooth muscle, the receptor tyrosine kinase EphA2 is detectable in vascular smooth muscle within the plaque and in synthetic vascular smooth muscle cells
in vitro
, and deletion of EphA2 in ApoE knockout mice attenuates plaque size and progression characterized by a loss of smooth muscle and fibrous tissue content. However, the mechanisms driving EphA2 expression in vascular smooth muscle remains unknown. Both serum treatment and plating on provisional extracellular matrix proteins (e.g. fibronectin) are sufficient to induce EphA2 protein expression and transcriptional activation of the EphA2 promoter. While multiple factors in serum could contribute to EphA2 expression, treating vascular smooth muscle cells with individual growth factors (PDGF, FGF, EGF) or insulin failed to recapitulate the increase in EphA2 expression. However, depleting fibronectin from the serum significantly attenuated expression of EphA2, suggesting fibronectin may critically regulate smooth muscle EphA2 expression. This reduction in serum-induced EphA2 expression upon fibronectin depletion was further enhanced by siRNA knockdown of smooth muscle fibronectin expression, suggesting that both serum fibronectin and smooth muscle-derived fibronectin contribute to the enhanced EphA2 expression. Fibronectin binds to a distinct subset of RGD-binding integrins, and blocking fibronectin-integrin interactions or inhibiting specific fibronectin-binding integrins both attenuate EphA2 expression. Together these data identify a novel role for fibronectin-dependent integrin signaling in the induction of smooth muscle EphA2 expression during phenotypic transition.
Abstract 103: Local Mir-29b Inhibition Using Drug-eluting Balloons Blocks Abdominal Aortic Aneurysm Expansion in Atherosclerotic Ldlr-/- Mini-pigs
Authors: Busch Albert , Maegdefessel Lars
Published: March 2019

Introduction:
Abdominal aortic aneurysm (AAA) is a major challenge in vascular surgery practice due to high morbidity and mortality, especially in case of rupture. Despite open surgery or endovascular stent implantation, no therapeutic option remains to clinicians. Micro-RNAs (miR) have emerged as novel therapeutic targets in the cardiovascular field. For translational research, animal models mimicking human disease are crucial, however, most often a compromise between size, cost and features similar to human disease limits the translational value.

Material and Methods:
We used the homozygote low-density lipoprotein receptor (LDLR) knockout (KO) Yucatan mini-pig to create a human-relevant animal model for AAA. Local application of porcine pancreatic elastase was applied to the infrarenal aorta for aneurysm inductions. Vascular ultrasound and CT was used for follow-up examination. 8 days after induction, catheter-guided anti-miR-29b coated balloon angioplasty was performed. Animals were sacrificed after 30 days. PCR and immunohistochemistry were used for further characterization of the model.

Results:
The LDLR KO is hypercholesterolemic and shows severe atherosclerosis in the infrarenal aorta. Aneurysms can be induced by local elastase application for 10 minutes and the aortic diameter increases by 60% over the course of four weeks. Despite atherosclerosis, the aneurysmatic vessel wall shows many features of human disease such as inflammation, extracellular matrix remodeling, angiogenesis and vascular smooth muscle cell phenotype switch. Targeted delivery by balloon angioplasty is feasible and anti-miR-29b penetrates through all layers of the aortic wall, eventually inducing local matrix remodeling. Anti-miR-29b treated animals had significantly smaller AAAs, while not showing any signs of off-target effects.

Conclusion:
Elastase perfusion in atherosclerosis-prone pigs is a feasible pre-clinical AAA model with many features similar to human disease. Locally balloon-delivered anti-miR-29b induces a pro-fibrotic response, halting aneurysm growth.
Abstract 109: Exosomes from Mesenchymal Stem Cells Suppress Aortic Elastin Induced Inflammation in Macrophages
Authors: Oskowitz Adam , Conte Michael
Published: March 2019

Introduction:
Mesenchymal Stem Cells (MSCs) from bone marrow have been shown to attenuate aortic aneurysm formation in a variety of rodent models. These effects appear to rely on modulation of systemic inflammation, including the attenuation of macrophage mediated inflammation. Elastin appears to play a significant role in inducing this inflammation and stimulating macrophages to express an M1 phenotype. Here we evaluate the effects human elastin and elastin break down products on macrophage phenotype. We also evaluate the role of human exosomes derived from MSCs on modulating the effect of elastin on macrophage phenotype.

Methods:
Human peripheral blood monocytes were collected and cultured for six days to isolate human macrophages. The cells were then treated with digested elastin from human aortic tissue or the human peptide VGVAPG in combination with human MSCs or exosomes derived from human MSCs. The phenotype of the macrophages was then analyzed using enzyme linked immune sandwich assays for tumor necrosis factor alpha (TNF-a) secretion, quantitative real time PCR and flow-cytometry for cellular proteins.

Results:
Macrophages cultured with digested elastin from aortic tissue showed a significant dose response increase in TNF-a production and cell surface markers consistent with an M1 phenotype. In addition, transcripts for pro-inflammatory cytokines significantly increased after exposure to digested elastin. Similar results were obtained using the elastin peptide sequence VGVAPG. Co-culture with MSCs significantly attenuated elastin induced macrophage TNF-a production, production of pro-inflammatory transcripts and M1 related proteins. When MSC derived exosomes were cultured with macrophages, pro-inflammatory markers and TNF-a production was reduced below baseline levels, even after stimulation with elastin peptide.

Conclusions:
Macrophages develop an M1 phenotype in response to digested aortic elastin and elastin derived peptides. This response is significantly attenuated by exosomes derived from human MSCs
Abstract 112: IL-27 Receptor Signaling Potentiates Angiotensin II Induced Myelopoiesis and Promotes Abdominal Aortic Aneurysm
Authors: Peshkova Iuliia , Peshkova Iuliia , Aghayev Turan , Fatkhullina Aliia , Makhov Petr , Sykes Stephen , Koltsova Ekaterina
Published: March 2019

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is mediated by accumulated immune cells. Several cytokines have been suggested to play role in AAA, however the role of cytokines in immune cell accumulation and AAA progression remains poorly defined. Here we report an unexpected role of IL-27R signaling in the development of AAA. We found that in an animal model of AAA, prolonged infusion of Angiotensin (Ang) II robustly induced AAA formation in hyperlipidemic

Apoe
-/-

and

Apoe
-/-
Il27ra

+/-
mice but failed to do so in

Apoe
-/-
Il27ra
-/-

mice. This mitigation of AAA formation in

Apoe
-/-
Il27ra
-/-

mice was associated with a blunted accumulation of pathogenic myeloid cells in suprarenal aortas resulting from a reduction in Ang II-induced hematopoietic stem and progenitor cell (HSPCs) expansion. We found that in the absence of IL-27R signaling HSPCs were unable to fully downregulate p21 expression in response to Ang II, which impeded the ability of

Apoe
-/-
Il27ra
-/-

HSPCs to proliferate. Collectively, these data demonstrate that IL-27R signaling influences AAA development by activating emergency hematopoiesis in response to elevated Ang II. Further, they provide new insights into how the immunoregulatory cytokine IL-27 drives a prominent lethal vascular disease by distant regulation of stress/emergency hematopoiesis.
Abstract 114: Smooth Muscle Origin-specific Effects of LRP1 Deletion on Angiotensin II-induced Ascending Aortic Aneurysm
Authors: Sawada Hisashi , Rateri Debra L , Wright Bradley C , Moorleghen Jessica J , Howatt Deborah A , Majesky Mark W , Daugherty Alan
Published: March 2019

Objective:
Low-density lipoprotein receptor-related protein 1 (LRP1) plays a critical role in maintaining aortic wall integrity. LRP1 deletion in smooth muscle cells (SMCs) augments angiotensin II (AngII)-induced ascending aortic aneurysms. SMCs in the ascending aorta originate from both the second heart field (SHF) and cardiac neural crest (CNC). The purpose of this study was to determine whether LRP1 depletion in these two SMC origins has differential effects on AngII-induced ascending aortic aneurysm.

Methods and Results:
Mef2c-Cre was used to delete LRP1 in SMCs of SHF origin; while Wnt-1-Cre was utilized to delete LRP1 in SMCs of CNC origin in mice. Saline or AngII (1,000 ng/kg/min) was infused for 28 days into 12 - 14 week-old male mice with LRP1 depletion in either SHF or CNC origin as well as their wild type littermates. No mice died in saline-infused groups of either genotype. In the AngII-infused mouse group, LRP1 depletion in SMCs of SHF origin led to 38% death due to ascending aortic rupture, compared to a 4% rupture rate in wild type littermates (p = 0.002). In the survivors of AngII-infused group, LRP1 deletion in SMCs of SHF origin resulted in larger ascending aortic diameter compared to wild type littermates (1.6 ± 0.1 vs 2.0 ± 0.1 mm, p < 0.05), as measured by ultrasonography. In contrast to the increases of aortic rupture and luminal dilation in mice with LRP1 depletion in SMCs of SHF origin, LRP1 deletion in SMCs of CNC origin did not affect AngII-induced aortic rupture rate or luminal dilation, compared to wild type littermates. To explore potential signaling mechanisms on how LRP1 depletion in SMCs of SHF origin augments AngII-induced ascending aortic aneurysm, mice were infused with either saline or AngII for 24 hours, and ascending aortic tissues were harvested for Western blot analyses. Aortic LRP1 protein abundance was decreased in mice with LRP1 depletion in SMCs of SHF origin regardless of infusion. Although SMAD2 and ERK signaling contribute to aortic wall integrity, this short interval of AngII infusion did not change these activities in the aorta of SHF-SMC specific LRP1 deleted mice.

Conclusion:
LRP1 expression in SHF, but not in CNC, -derived SMCs exerts a critical role in the augmentation of AngII-induced ascending aortic aneurysm.
Abstract 130: Exosomal Cellular Communication in Peripheral Arterial Disease
Authors: Lewis Sarah , Konig Julia , Lawson Charlotte , Collinson Lucy , Shiwen Xu , Hamilton George , Baker Daryll , Tsui Janice
Published: March 2019

Background:
Exosomes are nano-meter sized vesicles that play a role in cellular communication. Their effects on target cells are influenced by their cellular source and associated niche. We hypothesize that exosomal communication is involved in the local and systemic pathology of peripheral arterial disease (PAD), where exosomes derived from ischaemic muscle contribute to impaired muscle regeneration and endothelial dysfunction. This study aims to investigate the effect of ischaemia on skeletal muscle exosomes and their potential role in PAD.

Methods:
Exosomes were isolated from C2C12 myoblasts (MB), MTs and ischaemic MTs (IMTs) through ultracentrifugation. Comparative analysis of MT and IMT exosomes was performed using Nanosight, western blotting (WB) and electron microscopy (EM). PKH26 labelled MB, MT and IMT exosomes were co-cultured with myoblasts or human umbilical vein endothelial cells (HUVECs) for 6, 12 and 24hrs. Exosome uptake was assessed by fluorescent microscopy and FACs. The effect of IMT exosomes on myoblast differentiation was assessed by WB for myogenin and myosin heavy chain.

Results:
Exosomes were secreted by MTs and IMTs at similar frequencies and were structurally similar. Both were positive for exosomal markers CD81 and Alix. Following 24hr co-culture of PKH26 labelled exosomes with myoblasts or HUVECs, over 80% (over 4000 of 5000) of cells fluoresced positively, signifying internalisation of the exosomes by myoblasts and HUVECs. IMT but not MT derived exosomes impaired myoblast differentiation.

Conclusion:
MT derived exosomes are taken up by both myoblasts and endothelial cells. Whilst ischaemia does not significantly alter MT derived exosomes quantitatively or structurally, internalisation of IMT exosomes impairs differentiation of myoblasts to myotubes. This suggests that in PAD, altered exosomal message secondary to ischaemia, contributes to muscle dysfunction. Our data also indicates that there is muscle-endothelial cell cross-talk via exosomes, which may also contribute to systemic cardiovascular morbidity. Clarification of the functional effects and delineation of the mechanisms involved may lead to new targets that can be manipulated to improve outcomes of patients with PAD.
Abstract 147: The Role of Thrombin Generation in Cardiovascular Disease and Mortality - Results from the Population-based Gutenberg Health Study
Authors: van Paridon Pauline C , Panova-Noeva Marina , van Oerle Rene , Schulz Andreas , Hermanns Iris M , Prochaska Jürgen H , Arnold Natalie , Binder Harald , Schmidtmann Irene , Beutel Manfred E , Pfeiffer Norbert , Münzel Thomas , Lackner Karl J , ten Cate Hugo , Wild Philipp S , Spronk Henri M
Published: March 2019

Background:
Thrombin formation is one of the key enzymatic processes that direct the activity of the hemostatic system. Thrombin generation (TG), a method addressing the overall potential of a given plasma sample to form thrombin, may be a potential tool to improve risk stratification for cardiovascular diseases (CVD). This study aims to explore the relation between TG and cardiovascular risk factors (CVRFs), CVD, and total mortality.

Methods:
For this study, N=5000 subjects from the population-based Gutenberg Health Study were analyzed in a highly standardized setting. TG was measured by the Calibrated Automated Thrombogram method at 1 and 5 pM tissue factor (TF) trigger in platelet poor plasma. Lag time, endogenous thrombin potential (ETP), and peak height were derived from the TG curve. Sex-specific multivariable linear regression analysis adjusted for age, CVRFs, CVD and therapy (vitamin K antagonists, oral contraceptives and hormonal replacement therapy), was used to analyze the determinants of TG. Cox regression models adjusted for age, sex, CVRFs and vitamin K antagonists investigated the association between TG parameters and total mortality.

Results:
Lag time (at 1 and 5 pM TF) was positively associated with obesity and dyslipidemia for both sexes (p<0.0001). Additionally, obesity was a positive determinant of ETP (at 1pM and 5 pM TF) in both sexes (p<0.0001) and peak height in males (1 pM TF, p=0.0048) and females (1 pM TF and 5 pM TF, p<0.0001). Cox regression models showed an increased mortality in individuals with a lag time (1 pM TF, HR=1.46, [95% CI: 1.07; 2.00], p=0.018) and ETP (5 pM TF, HR = 1.50, [1.06; 2.13], p=0.023) above the 95th percentile of the reference group, independent of the cardiovascular risk profile. Kaplan Meier survival curves showed a decreased survival in individuals with a lag time above the 90th percentile of the reference (1 and 5 pM TF, p<0.0001) and ETP above the 97.5th percentile of the reference (1 pM TF, p=0.00097).

Conclusion:
This large-scale study provides important insights in the effects of traditional CVRFs, particularly obesity, on TG in males and females. Lag time and ETP were found as potentially relevant predictors of increased mortality in the general population, which deserves further investigation.
Abstract 151: Plasminogen Activator Inhibitor-1 Stimulates Smooth Muscle Senescence and Atherosclerosis Formation
Authors: Braet Drew J , Ji Yan , Fay William P
Published: March 2019

Cell senescence, which is characterized by loss of replicative capacity and secretion of inflammatory mediators, was recently shown to play a causal role in atherosclerosis. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that regulates fibrinolysis, cell migration, and cell proliferation, promotes cell senescence. This study tested the hypotheses that 1) PAI-1 stimulates vascular smooth muscle cell (SMC) senescence and 2) pharmacologic PAI-1 inhibition attenuates senescence of SMCs and atherosclerotic plaques. Human coronary artery SMCs were grown in culture and treated with recombinant PAI-1 and PAI-039 (tiplaxtinin), a specific pharmacological inhibitor of PAI-1. SMCs were scored for the presence of senescence-associated β-galactosidase (SA-βgal), an enzyme whose expression is specifically upregulated in senescent cells. PAI-1 (10 μg/mL) significantly increased SMC senescence (28.3±1.2% senescent cells, n=6) compared to vehicle control (14.3±1.4%, n=7, p <0.05). PAI-039 (25 μM) significantly decreased PAI-1-induced SMC senescence (19.1±1.1%, n=5, p <0.05 vs PAI-1-treated cells). To examine the significance of our findings in vivo, we studied the effects of PAI-039 on cell senescence in aortas of

ldlr
-/-

mice fed a western diet (WD) with or without PAI-039 (5 mg/g of diet). After twelve weeks of WD aortas were harvested and SA-βgal activity was measured in intact specimens with FDG substrate. SA-βgal activity was significantly less in

ldrl
-/-

mice fed WD containing PAI-039 (366±64 FU/mg, n=7) compared to controls (591±82 FU/mg, n=7, p<0.05)
.
The decrease in senescence was associated with a statistically significant reduction in atherosclerosis formation, assessed by quantitative Oil Red O imaging. In summary, SMC senescence is stimulated by PAI-1 and down-regulated by PAI-039, a specific PAI-1 inhibitor. PAI-039 also significantly inhibited cell senescence in aortas of
ldlr
-/-
mice fed WD. These results suggest that PAI-1 is an important mediator of SMC senescence and that pharmacologic targeting of PAI-1 is an effective strategy to inhibit vascular senescence and atherogenesis in vivo.
Abstract 155: High sensitivity C-Reactive Protein Predicts Future Cerebrovascular Events in Patients Undergoing Carotid Endarterectomy
Authors: Prasad Megha , Nardi Valentina , Lerman Lilach , Lerman Amir
Published: March 2019

Background:
High sensitivity C-reactive protein is a risk factor atherosclerosis and plaque rupture. We aimed to determine the role of high-sensitivity C reactive protein in predicting future cerebrovascular events (CVA), including stroke and transient ischemic attack, in patients undergoing routine carotid endarterectomy (CEA).

Methods:
A total of 251 patients underwent routine CEA without any history of stroke. At baseline prior to CEA, patients underwent routine laboratory testing, including testing for novel biomarkers including hs-CRP. Patients were followed for a median of 10.9 years (IQR 7.3, 13.4) for the development of CVA with regular nursing follow-up and/or chart review.

Results:
Of 251 patients enrolled in the study, 19 patients without prior CVA subsequently developed CVA during follow-up. In univariate analysis, baseline hsCRP was significantly higher in patients who developed CVA during follow-up vs. those that did not (8.1 (IQR 2.2, 46.3) mg/L vs. 3.85(0.9, 11.75) mg/L; p=0.0054)(Figure 1). After adjusting for confounders including age. diabetes mellitus, cholesterol, systolic blood pressure, history of smoking, aspirin use and duration of follow-up, log hsCRP was an independent predictor of CVA during follow-up after CEA (L-R 9.9; p=0.0016).

Discussion:
Hs-CRP appears to be a risk factor for CVA during follow-up after CEA. Further investigation is necessary to determine the role of risk modification in these patients and the therapeutic potential of reducing inflammation in this population.