bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 637: Genome-wide Crispr Screen in Primary Macrophages for Identification of a Regulatory Network for Macrophage Efferocytosis
Authors: Shi Jianting , Pan Huize , Tabas Ira , Zhang Hanrui
Published: March 2019

Defective macrophage efferocytosis drives important diseases including atherosclerosis, whereas enhancing efferocytosis has potential therapeutic benefits. A systematic approach to identify regulators of efferocytosis in an unbiased manner holds promise for novel molecular mechanisms and targets. This study establishes an experimental framework using a novel CRISPR knockout screen to systematically map the regulatory networks of macrophage efferocytosis. We infected bone marrow-derived macrophages (BMDM) isolated from Cas9 transgenic mice with a pooled, genome-wide lentiviral library with 78,637 gRNAs, and induced efferocytosis by fluorescently-labeled apoptotic Jurkat cells. We used FACS to isolate BMDM that engulfed multiple apoptotic cells (enriched in gRNAs targeting negative regulators) or did not engulf apoptotic cells (enriched in gRNAs targeting positive regulators), and determined gRNA abundance by deep sequencing. The top-ranked 1,000 genes analyzed by two published methods MAGeCK and PBNPA showed substantial overlap (53% for positive regulators, 36% for negative regulators). We recovered many of the known regulators. e.g.,
Actr2
,
Actr3
and
Arpc3
, key components of the Arp2/3 complex regulating actin polymerization, were ranked 2, 8 and 4;
Atp6v0b
and
Atp6v0c
, subunits of vacuolar ATPase responsible for acidification of intracellular organelles, were ranked 22 and 27.
Cd36
, a well-known efferocytosis receptor, was ranked 5. The top ranked positive regulators were enriched in GO terms “positive regulation of response to stimulus”, “Fcγ receptor signaling pathway” and “phagocytosis” (FDR-adjusted
P
= 2.19E-14, 1.10E-12 and 4.08E-11, respectively). Individual gRNAs verified a number of top hits from the pooled screen. In summary, we have developed a functional screen that has defined number of genes essential for macrophage efferocytosis. By focusing on a functional phenotype beyond cell viability and growth, we illustrate the versatility of CRISPR screens and provide a general strategy for systematically identifying gene of interest and uncovering novel regulators of complex macrophage functions.
Abstract 638: Delivery of Rapamycin-enriched Nanoparticles to Atherosclerotic Plaques Provides Targeted Regulation of the Macrophage Mtorc1-autophagy Signaling Axis
Authors: Zhang Xiangyu
Published: March 2019

Atherosclerotic vascular disease remains the leading cause of death in the United States with the majority of mortality due to coronary artery disease and myocardial infarction. Confirming several prior animal studies, we find the classic mTOR inhibitor, rapamycin, has potent atheroprotective effects in ApoE-null mice. However, an unfavorable system side-effect profile and a poor mechanistic understanding has significantly limited further evaluation of this promising therapy. Our previous work has indicated that macrophage mTORC1 signaling plays a critical role in plaque progression by inhibiting autophagy. We desired to develop a nanoparticle system to enable targeted delivery of rapamycin to plaque macrophages. Utilizing a chemokine receptor type 2 (CCR2)-binding peptide conjugated to tetraazacyclododecane tetraacetic acid (DOTA) and labeled with the fluorochrome Texas Red, we first prepared a nanoparticle system targeting macrophages and suitable for tracing in vivo. We show the specificity of these nanoparticles to macrophages in cell culture and plaque macrophages in vivo. We next loaded these nanoparticles with Rapamycin and show efficient inactivation of macrophage mTORC1 signaling and corresponding rescue of autophagy inhibition. Although the effects of this nanoparticle system in animal models of atherosclerosis is ongoing, our data support the utility of such targeted therapies in ameliorating the deleterious effects of macrophages in atherosclerosis.
Abstract 651: Hedgehog Responsive S100β
+
/Stem Cell Antigen-1
+
Vascular Stem Cells Contribute to Neointimal Formation
Authors: Redmond Eileen M , Fitzpatrick Emma , Liu Weimin , Helt Jay-Christian , Alshamrani Maryam , Hakimjavadi Roya , Harman Susan , Olayinka Abidemi , Burtenshaw Denise , Corcoran Eoin , Lally Catriona , Cahill Paul A
Published: March 2019

Intimal medial thickening (IMT) and vascular remodeling are hallmarks of arteriosclerotic disease. However, the origin of neointimal cells and the signaling molecules that dictate their fate and function remains controversial. Herein, we examined whether Hedgehog (Hh) responsive S100β
+
/Sca1
+
stem cells contribute to IMT within carotid arteries of transgenic mice following ligation-induced injury
in vivo
and myogenic differentiation of undifferentiated multipotent S100β
+
/Sca1
+
stem cells
in vitro
. Using Sca1-eGFP and S100β-eGFP transgenic mice, we demonstrated a significant accumulation in the number of eGFP
+
cells within the intima and medial layers of injured arteries following ligation concomitant with enhanced expression of Hh signaling components (ptch1 and Gli). Genetic lineage tracing analysis using S100β-eGFP/Cre/ERT2–dTomato transgenic mice to mark S100β
+
resident vascular stem cells before injury confirmed that S100β
+
progeny that are Sca1
+
significantly contribute to IMT, an effect significantly attenuated following treatment with the Hh smoothened inhibitor, cylopamine. In vitro, recombinant SHh (rSHh) treatment of multipotent S100β
+
/Sca1
+
resident stem cells increased Hh target gene Gli expression, decreased telomerase activity and promoted myogenic differentiation and cell growth; effects significantly attenuated following Hh inhibition. In human arteriosclerotic lesions, Hh components were upregulated concomitant with enhanced expression of S100β. Together, these findings suggest that S100β
+
/Sca1
+
stem cells are a major source of neointimal cells contributing to IMT and suggest that this cohort may be a relevant therapeutic target to prevent arteriosclerosis.
Abstract 662: Long Intergenic Non-coding RNA Rp11-10j5.1 is a Novel Regulator of Macrophage Activation
Authors: Li Daniel Y , Wang Ying , Zhang Hanrui , Xue Chenyi , Zhang Xuan , Liu Wen , Reilly Muredach P
Published: March 2019

Background:
Genetic discoveries for complex traits suggest roles for recently evolved regulatory elements, such as long intergenic non-coding RNAs (lincRNAs). The goal of the current study was to identify and functionally assess the contribution of a novel non-conserved, macrophage-enriched lincRNA, RP11-10J5.1, in macrophage inflammatory activation.

Methods and Results:
We recently showed from RNA-seq that dozens of lincRNAs were upregulated during primary human macrophage inflammatory activation (LPS/IFN-γ) associated with cardiometabolic diseases including atherosclerosis. Among these differentially expressed (DE) lincRNAs, RP11-10J5.1 is one of the most substantially up-regulated, specifically expressed in human and enriched in macrophages. qRT-PCR confirmed upregulation of RP11-10J5.1 in THP1-derived macrophages (THP-1Φ) and human induced pluripotent stem cell-derived macrophages (IPSDM). Cellular fractionation revealed that RP11-10J5.1 localized almost exclusively in the nuclear fraction. We performed RACE (rapid amplification of cDNA ends) to accurately map the transcription start site and full-length sequence of the three isoforms of RP11-10J5.1. Knock-down (KD) of RP11-10J5.1 isoforms using site-specific antisense oligonucleotides (LNA-ASO) targeting the common exon in LPS/IFN-γ-stimulated HMDM (n=3 subjects) followed by RNA-seq has identified 43 DE genes (21 up, 22 down, fold change > 1.5, FDR < 0.05) that are potential targets of RP11-10J5.1. Validation in four additional subjects confirmed that KD of RP11-10J5.1 attenuated inflammatory-induction of
NTN1
and
IRF4
, key regulators of macrophage apoptosis and inflammatory activation. Conversely, overexpression of RP11-10J5.1 in HMDM enhanced inflammatory upregulation of
NTN1
and
IRF4
, supporting the potential role of RP11-10J5.1 in inducing
NTN1
and
IRF4
expression. Mechanistically,
in vitro
RNA pull-down followed by mass spectrometry analysis has identified interaction of RP11-10J5.1 with a number of candidate proteins that regulate transcription and mRNA splicing.

Conclusion:
These data suggest that lincRNA RP11-10J5.1 is a novel modulator of inflammatory macrophage activation.
Abstract 663: Nuclear RSK1 Induces Pro-inflammatory Activation of Macrophages through STAT1 Phosphorylation at Ser727
Authors: Nihira Keishi , Halu Arda , Lang Ho Lee , Mojcher Alexander C , Higashi Hideyuki , Yamada Iwao , Qiao Jiao , Wang Jianguo , Decano Julius L , Aikawa Elena , Singh Sasha A , Aikawa Masanori
Published: March 2019

Objective:
Maladaptive inflammatory responses involve macrophage activation by pro-inflammatory cytokines such as IFN-γ. Proteins that undergo nuclear translocation may regulate these processes.

Approach and Results:
To explore novel key regulators of macrophage activation, we performed quantitative proteomics to monitor protein translocation to the nuclei of human primary macrophages elicited with IFN-γ for 0, 10, 20, 30, or 60 min. Bioinformatics identified RSK1, a ribosomal protein kinase, as one of the candidates. We found that IFN-γ stimulation promotes RSK1 phosphorylation at Ser380. STAT1 is a key mediator of IFN-γ-triggered cellular responses. Mass spectrometry identified RSK1-mediated phosphorylation sites within STAT1, including Ser727. siRNA silencing of RSK1 attenuated STAT1 phosphorylation in IFN-γ-stimulated macrophages. In concert with these results, RSK1 silencing hindered IFN-γ-induced secretion of pro-inflammatory chemokines in human primary macrophages, such as CCL2/MCP-1, CCL7/MCP-3, and CCL8/MCP-2.

Conclusion:
We discovered that RSK1 nuclear translocation triggers STAT1 phosphorylation, resulting in pro-inflammatory activation of macrophages (Figure), a novel role for a ribosomal protein-associated kinase in nuclear signaling and inflammation.
Abstract 667: Endothelial Cell “Memory”: Rapid Recall of Chemokines After Prior Exposure to Inflammatory Stimuli
Authors: Valenzuela Nicole M
Published: March 2019

Background:
Surprisingly little has been studied on the capacity of cytokine-primed endothelial cells (EC) to respond upon secondary challenge. The principal hypothesis is that EC store and rapidly recall chemokines after upon secondary challenge.

Methods:
Confluent primary human aortic (AEC), dermal microvascular (DMVEC), umbilical vein (UVEC), and renal glomerular (RGEC) endothelial cells were pre-exposed to TNFα or IL-1β (10ng/mL, 18hr), allowed to rest (4-24hr), and re-challenged with the secretagogues PMA or histamine. The repertoire of rapidly recalled soluble mediators was determined by ELISA, and cell surface P-selectin by cell-based ELISA. Total P-selectin content was measured by Western Blot.

Results:
Priming with TNFα or IL-1β overnight increased IL-8 secretion by all EC. 4hr after removal of TNFα or IL-1β, IL-8 was still in all supernatants. After 7hr rest, IL-8 was detectable from RGEC but undetectable in other EC. After 24hr, IL-8 was undetectable in the supernatants from all EC. Secondary challenge with secretagogues 24hr after removal of TNFα or IL-1β stimulated rapid secretion of IL-8, but not from unprimed endothelium. P-selectin was rapidly externalized at the cell surface of unprimed cells after 20min exposure to PMA or 10min Histamine. TNFα-primed EC exhibited significantly lower P-selectin induction, mirrored by profoundly reduced total intracellular P-selectin expression. mRNA expression of LAMP3 (CD63, a tetraspanin critical for P-selectin sorting to Weibel-Palade bodies) was highest in microvascular EC and lowest in larger vessel EC. LAMP3 might be important for controlling WPb content as well as durability of exocytosis upon type 1 activation and sorting of newly synthesized type II mediators to WPb for rapid recall.

Conclusions:
The data confirm that prior exposure of EC to inflammatory stimuli evokes storage of IL-8 that can be rapidly secreted upon exposure to a second stimulus. Moreover, constitutive expression of P-selectin is suppressed by TNFα exposure, but not IL-1β. Thus, EC exhibit a chronic inflammatory phenotype after cytokines have been encountered and removed, due to storage of chemokines within Weibel-Palade bodies or other granules.
Abstract 671: Looking with the Poiseuille’s Glasses - a Proposal of a Novel Hypothetical Method to Increase the Dimensions of the Coronary Arteries when Required
Authors: Arokiaraj Mark C
Published: March 2019

Background:
The purpose was to develop a novel hypothetical method to increase the dimensions of coronary arteries.

Methods:
In the long-term observation, the coronary sizes were dilated in three unexpected scenarios. The coronary artery sizes were observed in patients with mitral stenosis patients (n=12) by angiogram. The coronaries of patients with patent ductus arteriosus who underwent surgical/coil closure in the past (n=7) were examined by echocardiogram. These patients also had normal aortic and left ventricular dimensions, and all had surgeries/coil closure more than four years prior to this observation. Patients with renal failure on long-term dialysis through peripheral arterio-venous fistula without left ventricular hypertrophy (n=13) were studied by echocardiography for coronary dimensions. Normal age and sex-matched coronary sizes served as controls in the study. All these observations were made over a period of 10 years.

Results:
The sizes of coronaries in patients with mitral stenosis were greater than 22% (± 4) of the normal. The coronary sizes were 32% (±4) higher than normal in patients with patent ductus arteriosus without left ventricular or aortic dilatation. Patients with renal failure on hemodialysis without left ventricular hypertrophy had coronary sizes 28% (±5) higher than similarly sized ventricles. Reducing the diastolic blood pressures, the increase in coronary angulation by sine (θ) function, and increase in the coronary length and diameters can be achieved by observing the differential equations of Poiseuille’s. A hypothetical model to increase the coronary sizes could be developed based on the analysis of the differential equations. The proposed method is creating a peripheral arterio-venous fistula, which can be closed later by a percutaneous method/surgery. The closure time needs to be determined by experimental studies. The other methods could be a continuous exercise program or usage of beta-blockers.

Conclusion:
A novel hypothetical method of peripheral arterialvenous fistula formation could potentially increase the size of the coronaries. Further experiments need to be performed to observe the results.
Abstract 672: SUMOylation of Vps34 by SUMO1 Promotes Phenotypic Switching of Vascular Smooth Muscle Cells by Activating Autophagy in Pulmonary Arterial Hypertension
Authors: Yao Yufeng , Li Hui , Li Yong , Hu Changqing , Tang Bo , Hu Zhenkun , Da Xingwen , Song Qixue , Yu Yubing , Chen Qiuyun , Wang Qing K
Published: March 2019

Pulmonary arterial hypertension (PAH) is a life-threatening disease without effective therapies. PAH is associated with a progressive increase in pulmonary vascular resistance and irreversible pulmonary vascular remodeling. SUMO1 (small ubiquitin-related modifier 1) can bind to target proteins and lead to protein SUMOylation, an important post-translational modification mechanism with a key role in many diseases. However, the contribution of SUMO1 to PAH remains to be fully characterized. In this study, we explored the role of SUMO1 in the dedifferentiation
of vascular smooth muscle cells (VSMCs) involved
in hypoxia-induced pulmonary vascular remodeling and PAH. In a mouse model of hypoxic PAH, SUMO1 expression was significantly increased, which was associated with activation of autophagy (increased LC3b and decreased p62), dedifferentiation
of VSMCs (reduced α-SMA, SM22 and SM-MHC),
and pulmonary vascular remodeling. Overexpression of SUMO1 significantly increased VSMC proliferation, migration, hypoxia-induced VSMC dedifferentiation, and autophagy, but these effects were abolished by inhibition of autophagy by 3-MA. Mechanistically, SUMO1 promotes Vps34 SUMOylation and the assembly of the Beclin-1-Vps34-Atg14 complex, thereby inducing autophagy, whereas Vps34 mutation K840R reduces Vps34 SUMOylation and inhibits VSMC dedifferentiation. In conclusion, our data uncovers an important role of SUMO1 in VSMCs proliferation, migration, activation of autophagy, and phenotypic switching (dedifferentiation) involved in pulmonary vascular remodeling and PAH. Targeting of the SUMO1-Vps34-autophagy signaling axis may be exploited to develop therapeutic strategies to treat PAH.
Abstract 676: Hypoxia Upregulates NADPH Oxidase 4-Mediated Hydrogen Peroxide Release by a HIF-Independent Mechanism in Human Endothelial Cells
Authors: Brunssen Coy , Arsov Alexander , Poitz David M , Eickholt Claudia , Hofmann Anja , Langbein Heike , Brux Melanie , Engelmann Felix , Goettsch Claudia , Goettsch Winfried , Augstein Antje , Bornstein Stefan R , Strasser Ruth H , Breier Georg , Morawietz Henning
Published: March 2019

NADPH oxidases are important sources of reactive oxygen species in the vascular wall. Recent evidence supports a vasoprotective role of H
2
O
2
produced by the main endothelial isoform Nox4. The impact of hypoxia on NOX4 expression in human endothelial cells and the underlying mechanism remains to be elucidated. In this study, we show that NOX4 mRNA and protein expression was upregulated by hypoxia (1 % O
2
) in human umbilical vein endothelial cells (HUVEC). Correspondingly, H
2
O
2
production was 2-fold elevated in HUVEC after hypoxia. In contrast to rotenone and oxypurinol, lentiviral downregulation via shNOX4 abolished the elevated hypoxic hydrogen peroxide levels to normoxic values. Hypoxia stabilized the hypoxia-inducible factor (HIF)-1α protein in endothelial cells. Furthermore, VEGF promoter activity and a control promoter containing 3 hypoxia-responsive elements (HRE) were induced by hypoxia. NOX4 promoter deletions up to -119/+239 had an increased basal activity compared to control vector. A full-length and a terminally deleted NOX4 promoter construct missing a putative HRE showed a comparable activity under hypoxic and normoxic conditions, suggesting that NOX4 is not induced on the transcriptional level by hypoxia in endothelial cells. In addition, stabilization of HIF-1α protein under normoxic conditions using DMOG did not change NOX4 mRNA expression in HUVEC. Furthermore, overexpression of HIF-1α did not alter NOX4 promoter activity. Blockade of active transcription by actinomycin D revealed an increased stability of the NOX4 mRNA under hypoxic conditions. In conclusion, this study demonstrates an HIF-independent upregulation of NOX4 as major source of endothelial hydrogen peroxide generation in response to hypoxia. Our data support as a novel mechanism an increased NOX4 mRNA stability under hypoxic conditions in human endothelial cells.
Abstract 678: microRNA 146a Reduces Activity of Matrix-Metalloproteinases in the Context of Arterial Stiffness
Authors: Dannert Angelika , Schellinger Isabel N , Jakubiczka-Smorag Joanna , Mattern Karin , Petzold Anne , Hasenfuss Gerd , Raaz Uwe
Published: March 2019

Background:
Arterial stiffness is part of arterial aging and strongly associated with increased risk for cardiovascular events. Unfortunately, until today there is no treatment available effectively counteracting arterial stiffening. One key feature of arterial stiffening is elastin fragmentation. Breakdown of elastin is mediated by matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9 play a pivotal role in this process. Non-coding micro RNAs (miRs) were found to effectively regulate gene expression, consequentially representing potential therapeutic targets. We hypothesize that miR146a inhibits MMP-2 and MMP-9, thus leading to decreased elastin fragmentation in the vascular extracellular matrix.

Methods and Results:
RNA Sequencing found miR146a to be upregulated in a stiffness-dependent manner
in vitro
and in an age dependent manner
in vivo
. Since both MMP-2 and MMP-9 are predicted targets of miR146a, we transfected aortic smooth muscle cells with constructs either increasing (mimicking construct) or decreasing (inhibitory anti-sense construct) effective miR146a levels and evaluated the effect on MMP-2 and MMP-9 expression and activity
in vitro
. qRT-PCR analysis demonstrated significantly de-repressed MMP-2 and MMP-9 levels upon transfection with inhibitor constructs, while mimic constructs further repressed the expression of MMP-2 and MMP-9. Overall MMP activity was significantly altered accordingly after transfection with the respective constructs as determined by a fluorimetric MMP Activity Assay. MMP-2 activity could be specifically assessed using Zymography. After transfection with miR146a inhibitor, both active MMP-2 as well as pro-MMP-2 activity were found to be significantly increased.

Conclusion:
Our study provides first evidence that miR146a reduces the levels and activity of MMP-2 and MMP-9 in vascular smooth muscle cells. We conclude that miR146a might have protective function on the integrity of the elastin network in the arterial media. We therefore consider miR146a a potential drug target in the context of elastin fragmentation and arterial stiffness, thus potentially alleviating the development of aortic aneurysm.
Abstract 679: Pdgf/snail-mediated Endothelial Plasticity Drives Non-productive Neovascularization and Impedes Tissue Repair After Myocardial Infarction
Authors: Fan Yi , Huang Menggui , Yang Fan , Duan Hao
Published: March 2019

Ischemic heart disease is the most common cause of death in the Western world, largely due to myocardial infarction (MI). After MI, formation of new blood vessels,
i.e.
, neovascularization, is crucial for ischemic tissue reperfusion, repair and regeneration. However, the newly formed vasculatures in infarcted tissue are characterized by functional and structural abnormalities, which compromise vascular delivery function and impede cardiac recovery after MI. Likewise, aberrant non-productive neovascularization, albeit previously under-appreciated, represents a promising therapeutic target for post-MI treatment. Here we reveal that mesenchymal transformation-mediated endothelial cell (EC) plasticity induces aberrant post-ischemic neovascularization. In contrast to the old concept implicating that ECs undergo endothelial mesenchymal transitions to generate fibroblasts
de novo
in infarcted cardiac tissue, we suggest that ECs acquire mesenchymal phenotypes including high proliferation and motility to generate excessive abnormal vasculatures after MI. By utilizing genetic EC lineage tracing and single-cell RNAseq technologies with a mouse MI model induced by ligation of left anterior descending coronary artery, our transcriptome analysis uncovers that ECs undergo mesenchymal transformation in infarcted tissues. Moreover, exposure of cardiac ECs to ischemic microenvironment
in vitro
induces EC expression of mesenchymal genes including S100A4, ACTA2, and CDH2, and interestingly, EC functions including tube formation and uptake of ac-LDL are retained, suggesting EC mesenchymal transformation without lineage transition. Furthermore, we identify a PDGF/Snail-mediated axis that controls EC transformation under hypoxia. Notably, genetic ablation of PDGF receptor-β in ECs promotes blood perfusion and tissue repair after hindlimb ischemia and MI in mice. These findings identify a novel cellular mechanism controlling non-productive neovascularization after ischemia, and suggest that targeting EC plasticity may offer promising therapeutic opportunities for normalization of aberrant neovasculature and improvement of tissue repair in ischemic heart diseases.
Abstract 686: The Impact of Tobacco Smoke on Level of Endothelial Dysfunction Markers in Patients with Essential Hypertension
Authors: Podzolkov Valery , Bragina Anna , Druzhina Natalia , Mohammadi Leila , Murashko Natalia
Published: March 2019

Objectives:
to study the markers of endothelial dysfunction (ED): metabolites of Nitric Oxide (NO), endothelin1 (E1), homocysteine (HC), Von Willebrand factor (vWF), tissue plasminogen activator (tPA) in plasma of tobacco smokers and nonsmokers with essential hypertension (EH).

Material and Methods:
124 hypertensive were recruited: 45 men and 79 women, with the mean age of 51,4±6,5 yrs and average duration of EH for 8,5±7,6 yrs. The control group included 25 healthy subjects (10 men and 15 women) with mean age of 48,2±7,8 yrs. Plasma NOx levels were determined by spectrometry, E1, HC, Wf, tPA levels by enzyme immunoassay. The results of study were analyzed by using Statistica 10.0.

Results:
to determine the effects of smoking on level of ED markers, we divided our subjects to subgroups: hypertensive smoker (35%), hypertensive nonsmokers (65%), smokers without hypertension (38%) and healthy nonsmokers (62%). In hypertensive smokers, in comparison to nonsmokers, we found a significant induction in NOx level (48,2±18,8 umol/l and 40,3±21,2 umol/l respectively, p<0,05), E1 (1,2±0,16 and 0,6±0,2 fmol/l respectively, p<0,05), HC (25,7±6,04 and 16,2±6,5 umol/l, respectively, p<0,05), vWF (1,3±0,8 and 1,1±0,6 mg/dl, respectively, p<0,05) and tPA (13,05±6,2 and 8,5±6,2 umol/l respectively, p<0,05). The NOx level correlated with smoking (r=0,46, p<0,05), HC level with smoking (r=0,4, p<0,05), and vWF level with the risk level of SCORE (r=0,43, p<0,05). In a controled group, the smokers, turned out to have significantly higher level of HC (20,7±5,3 and 17,2±4,7 umol.l, respectively, p<0,05), vWF (1,3±0,8 and 0,8±0,6 mg/dl, respectively, p<0,05) and tPA (11,1±6,5 and 6,6±5,2 umol/l, respectively, p<0,05). The changes in NOx and E-1 level were not significant.

Conclusion:
we observed an increase in level of ED markers in smokers with EH and of the control group. That confirms an important influence of smoking not only on development, but also progression of ED.
Abstract 690: Endothelial-Specific Krüppel-Like Factor 2 Inactivation Promotes Endothelial Dysfunction in the Setting of Uremia
Authors: Saum Keith L , Campos-Naciff Begoña , Celdran-Bonafonte Diego , Roy-Chaudhury Prabir , Owens A. Phillip
Published: March 2019

Background:
Uremic solutes that accumulate in end-stage renal disease (ESRD) contribute to endothelial dysfunction and subsequent cardiovascular disease in ESRD patients. However, the specific mechanisms which mediate uremia-induced endothelial dysfunction in ESRD are not understood. Kruppel-like factors (KLFs) are important regulators of endothelial homeostasis which may be affected by the uremic milieu. In this study, we examined the role and regulation of endothelial KLF2 in mediating endothelial dysfunction in the setting of uremia.

Methods and Results:
First, we assessed the impact of the uremic milieu on endothelial gene expression utilizing serum from uremic and non-uremic pigs with chronic renal insufficiency. We demonstrate that KLF2 expression was dose-dependently decreased with uremic serum in human umbilical vein endothelial cells (HUVECS) versus normal serum which was reversed by prolonged serum dialysis or the application of laminar shear stress. Carboxymethyl-lysine (CML) modified albumin, a uremic advanced glycation end-product (AGE), also inhibited KLF2 expression. This effect was completely abrogated by receptor for AGE (RAGE) siRNA, as well as by a constitutively active form of IκBα, implicating a role for RAGE activation and the NF-κB signaling pathway. KLF2 suppression also promoted endothelial dysfunction in vitro, as adenoviral overexpression of KLF2 inhibited reactive oxygen species production and leukocyte adhesion in HUVECs treated with uremic serum or CML-AGE. For in vivo translation, we utilized high-frequency ultrasound and quantified flow-mediated dilation of the femoral artery in endothelial-specific KLF2 conditional knockout (cKO) mice after 5 minutes of hindlimb ischemia and demonstrated a 50% reduction in vasodilation in KLF2 cKO mice compared to controls. The lack of femoral artery vasodilation in KLF2 cKO mice was also accompanied by attenuated return of wall shear stress to baseline independent of blood velocity.

Conclusions:
Collectively, these observations implicate loss of endothelial KLF2 as mediator of endothelial dysfunction in the setting of uremia and suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of cardiovascular disease in ESRD.
Abstract 693: RNA Stability Protein ILF3 Mediates IL-19 Induced Angiogenesis
Authors: Vrakas Christine N , Herman Allison B , Ray Mitali , Landesberg Gavin , Kelemen Sheri E , Scalia Rosario , Autieri Michael V
Published: March 2019

Angiogenesis is a physiological process vital for growth, development and wound healing but is also a promising therapeutic target for the treatment of ischemic cardiovascular disease. Presently, there is nothing known regarding the role of, the RNA binding protein, Interleukin Enhancer-binding Factor 3 (ILF3) in angiogenesis. RNA binding proteins play crucial roles in cellular processes, specifically, post-transcriptional control of RNAs through mRNA stabilization. We hypothesized that ILF3 plays an essential role in angiogenesis through the stabilization of pro-angiogenic mRNA transcripts. Using immunohistochemistry we identified abundant ILF3 expression in CD31
+
vessels of hypoxic porcine cardiac tissue exposed to ischemic myocardial infarction. Using both western blotting and qRT-PCR, we found that pro-angiogenic stimuli, IL-19 and VEGF, induce ILF3 expression in cultured human coronary artery endothelial cells (hEC). Angiogenic proliferation, migration and tube formation are all significantly reduced in hEC when ILF3 is knocked down using siRNA. Importantly, these angiogenic assays are significantly increased when ILF3 is overexpressed in hEC using adenovirus. Using ILF3 siRNA in addition to VEGF stimulation in hEC, several angiogenic factors including CXCL1, IL-8, and HGF are decreased at the transcript and protein level. However, these angiogenic factors are increased when hEC are infected with AdILF3 and stimulated with VEGF. Through immunohistochemistry we found that ILF3 translocates from the nucleus to the cytoplasm of hEC stimulated with VEGF or IL-19 at various time points, suggesting a role in mRNA stability. Using the transcription inhibitor actinomycin D, we found that ILF3 stabilizes pro-angiogenic transcripts including VEGF, CXCL1, and IL-8 in hEC. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. We believe that ILF3 is impacting functional angiogenesis through cytokine-inducible mRNA stabilization of pro-angiogenic transcripts.
Abstract 695: Rage-mDia1 Mediates Vascular Remodeling in Angiotensin Ii Induced Thoracic Aortic Aneurysm
Authors: Kreibich Maximilian , Awosanya Samiat , Bavaria Joseph E , Branchetti Emanuela
Published: March 2019

Background:
The Receptor for Advanced Glycation End Products (RAGE), when activated, induces irreversible vascular tissue injury by promoting pro-oxidative and pro-inflammatory signaling pathways. RAGE signal transduction requires the engagement of RAGE cytoplasmic domain with the formin Diaphanous 1 (mDia1) and results in the release of soluble RAGE in the circulation. We have previously demonstrated that the RAGE/sRAGE axis is involved in the development and progression of thoracic aortic aneurysm (TAA) in humans and mice. Elevated levels of sRAGE are found in aneurysmal patients and in mouse models of TAA. In addition, RAGE inhibition counteracts aortic dilatation and the release of sRAGE in the circulation. We hypothesize that RAGE induced vascular remodeling in TAA is mediated by mDia1.

Methods:
AngII infusion (1000 ng/Kg/min) was performed in C57BL6 male mice, fed with hypercolesterolaemic diet. Mice were treated for 4 weeks with losartan or with a RAGE antagonist (RAP). Ascending aortas of treated animals were harvested at day 28 post-infusion. Aortic dilatation and degeneration were assessed by echocardiography and histology. Immunofluorescence and Real Time PCR were performed to evaluate the expression of RAGE, mDia1, extracellular matrix proteins (ECM) and pro-inflammatory genes.

Results:
AngII infusion induces ascending aorta dilatation and medial thickening characterized by a substantial ECM deposition. RAGE and mDia1 expression is significantly increased in the aneurysmal aorta of mice chronically infused with AngII together with up-regulation of pro-inflammatory molecules (IL-6, MCP-1, IL1beta, TLR4, CCR2) and markers of extracellular matrix remodeling (Col1, Col3, MMP-2 and MMP12). Inhibition of RAGE dampens mDia1 expression and significantly reduces medial pro-inflammatory and pro-fibrotic signals while counteracting aneurysm formation.

Conclusion:
Formin mDia1 is involved in RAGE mediated TAA formation. Strategies that block RAGE-mDia1 interaction may unveil novel therapeutic approaches for the treatment of TAA.
Abstract 707: Vascular Smooth Muscle Cell Tfeb Deletion Promotes Abdominal Aortic Aneurysms
Authors: Lu Haocheng , Fan Yanbo , Liang Wenying , Hu Wenting , Xiong Wenhao , Chang Ziyi , Garcia-Barrio Minerva , Zhang Jifeng , Chen Yuqing
Published: March 2019

Rationale:
Abdominal aortic aneurysm (AAA) is a vascular disease with a very high mortality rate in the case of rupture. Vascular smooth muscle cells (VSMCs) are crucial to maintaining vascular integrity and function. Transcription Factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis in a variety of cell types via inducing the transcription of a coordinated lysosomal regulatory gene network. TFEB shows anti-inflammatory and anti-atherosclerotic effects in vascular endothelial cells and macrophages. However, the role of TFEB in vascular disease remains to be further explored.

Objectives:
To investigate the role of VSMC TFEB in AAA.

Methods and Results:
We found that TFEB was down-regulated in human aortic aneurysmal lesion compared with the non-lesion area by QRT-PCR and immunostaining. In human aortic smooth muscle cells (HASMCs), TFEB mRNA and protein abundance were decreased upon treatment with pro-inflammatory factors while adenovirus-mediated TFEB overexpression potently inhibits inflammation, apoptosis (caspase 3 cleavage and Annexin V staining) and matrix metalloproteinase activity (zymography). A consistent phenotype was observed in the TFEB knockdown HASMCs. Mechanistically, TFEB activates the PI3K-Akt pathway, and PI3K inhibitors (wortmannin and LY294002) abolished the anti-apoptotic effect of TFEB in HASMCs. Utilizing VSMC-specific TFEB deficiency mice (floxed-TFEB/myh11-ERT2 cre+), we determined the effect of TFEB on AAA formation in vivo. In the mouse aneurysm model induced by the combination of angiotensin II and 3-aminopropionitrile infusion, TFEB VSMC-deletion significantly increases aneurysm formation, rupture, and mortality (n =13-14 for each group, p< 0.01).

Conclusions:
Our data reveal a critical protective role of TFEB in VSMC homeostasis, suggesting TFEB to be a potential target to treat aortic aneurysm.
Abstract 709: Computational Seal Zone Mechanics - Predicting EVAR Failures
Authors: Pocivavsek Luka , Skelly Christopher , Milner Ross
Published: March 2019

Introduction:
Endovascular aortic stent-graft technology (EVAR) radically altered aortic aneurysm repair from a maximally invasive procedure to a minimally invasive approach. While the overall principle of the repair remained the same, the surgeon ceded control of graft/aorta coupling with EVAR from the time-honored surgical approach of suturing, which creates a mechanical kinematic coupling constraint at the graft/aorta interface. In EVAR, the coupling condition is replaced by contact mechanics between the outer graft surface and the aorta. We hypothesize that proximal endograft failure is linked to the complex contact mechanics occurring at the graft/aorta interface.

Methods:
We performed a detailed geometric/finite element analysis (FEA) of an abdominal aortic aneurysm in a patient who presented with a ruptured juxta-renal AAA. We used the pre and post-operative CTA imaging to perform FEA and defined the stent graft (SG)/aortic wall interactions using a fully integrated cohesive zone model (CZM) (see figure 1).

Results:
Three degrees of adhesion strength were studied, corresponding to F~0.1, 10, and 1000 Newton. In the case of the two weaker adhesion strengths, the SG and neck separated at local points along the interface. These areas of separation (interfacial failure) corresponded to zones of high stress (~ 10MPa) within the aortic wall. This points to a new physical model of aortic/SG stability, where local elastic deformation energy in the neck (G) competes with aortic/SG adhesion energy (gamma~Gc) giving rise to the criterion for seal failure: G>Gc .

Conclusions:
The energy balance of deformation of the aortic neck with stent graft adhesion strength provides a key new parameter in evaluating future EVAR repairs. Non-linear neck geometries give rise to complex non-linear stress distributions that can greatly impact the stability of a repair. We provide a computational methodology in which this can be systematically studied for any given patient anatomy.
Abstract 715: Peripheral Artery Disease in Patients with End Stage Renal Disease: Nationwide Trends in Management and Outcomes
Authors: Al-Ogaili Ahmed , Ayoub Ali , Paz Luis , Torres Christian , Fuentes Harry , Tafur Alfonso
Published: March 2019

Background:
Patients with end stage renal disease (ESRD) are at high risk for peripheral artery disease (PAD). We sought to determine the national prevalence of PAD, trends in management and outcomes among this high risk population.

Methods:
We queried the 2003-2014 National Inpatient Sample databases to identify PAD related admissions (claudication and critical limb ischemia) among patients with ESRD using ICD-9 codes. Temporal trends in treatment options, and in-hospital mortality were analyzed. Multivariate logistic regression was used to assess predictors of major amputation.

Results:
Among 9 million ESRD patient admissions, 357,949 (3.9%) had PAD related diagnosis. Patients were mainly white (39%), men (58%), with mean age of 65.6 years. The prevalence of PAD slightly decreased from 4.3% in 2003 to 3.9% in 2014 (P trend < 0.001). Utilization of percutaneous transluminal angioplasty (PTA) increased by 142% (10.6% to 25.7%), whereas, open bypass use decreased by 51% (14.1% to 6.9%) during the study period (P trend < 0.001). Moreover, the total number of major amputations was 65,669. There was a notable decline in its usage from 25.9% to 15.3% (adjusted odds ratio [OR] per year: 0.96; 95% confidence interval: 0.95-0.98, P<0.001). Revascularization appeared to reduce the risk of amputation (OR 0.49, P<0.001). On the contrary, hypertension (OR 2.5, P<0.001), heart failure (OR 1.5, P<0.001), and black race (OR 1.5, P<0.001) were independent predictors of amputation. Overall in-hospital mortality rate was 6.3%, with no statistically significant trend throughout the years.

Conclusion:
PAD is a common disease in ESRD patients. Among therapeutic options, a significant increase in less invasive endovascular revascularization methods and a decline in open bypass surgeries were noted. Despite the significant drop in amputation rates, racial disparities persist, with black patients having higher odds of major amputation.
Abstract 716: Generation of γ-secretase Inhibitor-loaded PLGA-Fe
3
O
4
- Magnetic Nanoparticles
Authors: Bashmail Roa , Mckenna Niamh , O'Shea Ciaran , Hakimjavadi Roya , Molony Claire , Kozlowska Dorota , Cahill Paul A
Published: March 2019

Cardiovascular disease (CVD) is the number one killer in Ireland and the wider EU. A hallmark of the disease is the obstruction to blood flow due to the build-up of vascular smooth muscle (SMCs)-like cells within the vessel wall. While polymer-coated DES have significantly reduced the incident of in-stent restenosis, current DESs lack the fundamental capacity for (i) adjustment of the drug dose and release kinetics and the (ii) ability to replenish the stent with a new drug on depletion. This limitation can be overcome by a strategy combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of specific drugs that target the resident vascular stem cell source of the SMC. Magnetic nanoparticles (MNP’s) containing magnetite (Fe3O4) were fabricated, polymer coated with poly (DL-lactide-co-glycolide) polyvinyl alcohol [PLGA-PVA] and loaded with a γ-secretase inhibitor (GSI) of Notch signalling, DAPT using an oil in water emulsification technique. The free GSI’s and GSI-loaded MNP’s were assessed for drug release, the efficacy at controlling mesenchymal stem cell (MSC) growth (proliferation and apoptosis) and inhibiting myogenic differentiation under magnetic and non-magnetic conditions. The DAPT-loaded MNPs had an average hydrodynamic diameter of 351 d.nm Up to 40% of drug was released from MNPs within 48 h rising to 65% after 1 week under magnetic conditions. The Notch ligand, Jagged1 increased Hey1 mRNA levels and promoted myogenic differentiation of MSCs in vitro by increasing SMC differentiation markers, myosin heavy chain 11 (Myh11) and calponin1 (CNN1) expression, respectively. This effect was significantly attenuated following treatment of cells with MNP’s loaded with DAPT when compared to unloaded MNP’s. Notch GSI -loaded magnetic nanoparticles are functional at targeting vascular stem cells in vitro.
Abstract 719: Radiotracer Imaging of Lower Extremity Skeletal Muscle Perfusion is Significantly Correlated With Exercise Tolerance
Authors: Chou Ting-Heng , Stacy Mitchel R
Published: March 2019

Introduction:
Exercise therapy is a proven treatment for peripheral artery disease (PAD) that is now reimbursable by the US Centers for Medicare and Medicaid Services. Traditional methods for detecting responses to exercise in PAD have been peak walking time and the distance walked until onset of claudication, as well as the ankle-brachial index and quality of life questionnaires. However, a quantitative approach for assessing regional perfusion responses to exercise programs does not exist, thus hindering the ability to monitor physiological responses to exercise therapy. Therefore, we investigated if radiotracer-based perfusion imaging of the lower extremities could be utilized as a non-invasive correlate to exercise tolerance for future application in evaluating PAD patients undergoing exercise therapy.

Methods and Results:
Patients (n=24) underwent single-photon emission computed tomography (SPECT)/CT imaging of the calves after a standard Bruce protocol treadmill stress test, as well as under resting conditions (Fig. 1A). Average SPECT image intensity values were normalized to injected radiotracer dose and patient body weight to generate standardized uptake values (SUVs) within CT-defined muscle groups of the calf (gastrocnemius, soleus, anterior tibias; Fig. 1B). Perfusion reserve was calculated as the percent change of SUV from the rest to stress condition for each muscle group. SPECT/CT-derived measures of perfusion reserve in the soleus and anterior tibialis muscle groups were significantly correlated with peak METs during treadmill stress testing (Fig. 1C).

Conclusion:
Lower extremity SPECT/CT perfusion imaging is significantly correlated with exercise tolerance and therefore may offer a novel non-invasive tool for physiologically characterizing responders and non-responders to structured exercise therapy in the setting of PAD.
Abstract 721: The Exercise Pressor Response to Lower Extremity Dynamic Exercise is Accompanied by an Abnormal Change in Total Peripheral Resistance in Peripheral Arterial Disease Subjects
Authors: Kuroki Marcos T , Kim Danielle J , Cui Jian , Gao Zhaohui , Blaha Cheryl , Leuenberger Urs A , Sinoway Lawrence I
Published: March 2019

Patients with peripheral arterial disease (PAD) have an exaggerated pressor response to dynamic exercise of the affected limb. We tested the hypothesis that this response is mediated by an increase in total peripheral resistance (TPR). We measured heart rate, blood pressure, stroke volume, cardiac output (CO), and total peripheral resistance (TPR) during exercise in 9 subjects with PAD and 9 age matched controls using continuous finger arterial pressure monitoring (Finometer). Finometer derived values were adjusted to values obtained by echocardiography, and brachial pressure at baseline. The average age and ABI was 65 ± 7 and 1.06 ± 0.07 in controls, and 66 ± 7 and 0.64 ± 0.08 in PAD. Participants performed dynamic plantar flexion at progressive workloads of 2 to 12kg (increased by 1kg/min until onset of fatigue/pain) using their symptomatic leg or leg with lower ABI. Those who reached 12kg continued exercise at a constant load until onset of fatigue/pain or a maximum of 4 minutes. Participants were then asked to perform an isometric handgrip exercise at 30% of maximum voluntary contraction (MVC) until fatigue. Results are shown in Table 1. There were no differences in hemodynamic parameters at baseline. The average weight at end of plantar flexion was 12.0 ± 0.2 and 9.2 ± 2.5 kg (p<0.0001) for control and PAD. Perceived exertion (Borg scale) and pain (NPRS scale) at end of exercise was 15 ± 2 and 0, and 15 ± 2 and 6 ± 3 in control and PAD. Plantar flexion elicited a significantly higher rise in blood pressure in PAD compared to control. TPR decreased in controls, but slightly increased in PAD. In contrast, isometric handgrip elicited a similar pressor response, as well as changes in CO and TPR in both groups. We conclude that the exaggerated pressor response in PAD is mediated by an abnormal response in TPR, which counteracts the normal vasodilatory response to dynamic leg exercise seen in controls. This response appears to be specific to dynamic plantar flexion exercise in PAD.
Abstract 722: Evaluation of Peripheral Calcium Score as a Measure of Peripheral Artery Disease Severity
Authors: Lee Sujin , Kalra Kanika , Redpath Benjamin , Little Brent , Bernheim Adam , Brewster Luke , Shaw Leslee , Arya Shipra
Published: March 2019

The current gold standard for diagnosing PAD is Ankle Brachial Index (ABI). However, vascular calcification can falsely elevate ABI. No studies have compared the diagnostic value of peripheral calcium score (PCS) in lower extremity arteries with ABI.

Primary aim of this study was to describe the association of PCS with continuous ABI values and categories of ABI in a retrospective cohort design. We identified 50 patients who underwent CTA and ABI measurements [ABI categories for PAD severity: severe (<0.5), moderate (0.5-0.9), normal (0.9-1.4), noncompressible (>1.4)]. We evaluated CTAs imaged from abdominal aorta through lower extremities and determined total calcium volume of plaques with density >130 HU and area >1mm
2
from infrarenal abdominal aorta to the foot using TeraRecon by two independent readers (Intra class correlation 99%). We explored the association between ABI and PCS in SAS using multiple linear regression and analysis of covariance adjusting for age, race, smoking status, hypertension, hyperlipidemia, type II diabetes, and chronic kidney disease.

We found that ABI was inversely associated with PCS in linear regression (p<0.01, Figure 1A). Differences in mean PCS were statistically significant across ABI categories [F(3,29)= 5.03, p=0.01, Figure 1B]. Across subgroups, the mean PCS was significantly different for ABIs <0.5 and 0.5-0.9 (p=0.02), <0.5 and >1.4 (p<0.001), 0.5-0.9 and >1.4 (p=0.04), 0.9-1.4 and >1.4 (p=0.05). Proportion of tibial calcium to overall PCS was much lower in ABI<0.5: 0.0003 vs ABI>1.4: 0.357 (p=0.02).

Mean PCS may be a valid measure of PAD severity and percentage of tibial calcium may help quantify PAD burden in non-compressible vessels. Our study serves as proof of concept for a comprehensive PCS system to diagnose and evaluate PAD severity, particularly in high-risk subpopulations where non-invasive studies may be unreliable.