bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 156: Uric acid is an Independent Predictor of Plaque Stability and Future Cerebrovascular Events in Patients Undergoing Carotid Endarterectomy
Authors: Prasad Megha , Nardi Valentina , Lerman Lilach , Lerman amir
Published: March 2019

Background:
Uric acid is a known risk factor for atherosclerosis. We aimed to determine whether baseline uric acid levels predict plaque stability on presentation and future cerebrovascular events (CVA) in patients undergoing carotid endarterectomy (CEA).

Methods:
We analyzed 317 patients who underwent CEA between 2/2002 and 6/2017. Patients were stratified according to plaque stability as unstable or stable --those who presented with a history of
>
1 neurologic event were “unstable” and those with no history of CVA were classified as stable. All patients underwent comprehensive baseline laboratory testing at baseline prior to CEA and then were followed by a nurse coordinator and through the medical record for the development of cerebrovascular events including stroke and transient ischemic attack for a median of 10.9 years (IQR 7.3, 13.4).

Results:
Baseline uric acid was significantly higher in patients presenting with unstable plaque vs. stable plaque(6.5 (IQR 5.6, 7.9) vs. 5.8(IQR 4.8,6.6); p<0.001), and was an independent predictor of plaque stability when adjusting for potential confounders including glucose, BMI, hsCRP, cholesterol, and systolic blood pressure (L-R 6.9, p=0.0096). There were a total of 58 CVA during follow-up, and baseline uric acid was higher among patients who developed CVA vs those without CVA [(6.9
+
2.1) mg/dL vs. (6.4
+
1.7) mg/dL; p=0.037]. After adjusting for diabetes, cholesterol, systolic blood pressure, smoking , age, aspirin, and creatinine, uric acid and cholesterol were both independent predictors of stroke in this population [uric acid: (LR 4.84, p=0.03); cholesterol: (L-R 11.8, p=0.0006)].

Discussion:
While cholesterol has been associated with poor outcomes in patients with vascular disease, our data suggest that uric acid may be a novel risk factor for stroke in patients undergoing CEA. Further investigation is necessary to understand the therapeutic potential of reducing uric acid levels in these patients and the potential benefit of reducing overall cerebrovascular risk.
Abstract 158: Indoleamine 2,3-dioxygenase 1 is Expressed in Macrophages in Human Coronary Atherosclerotic Plaque and is Associated With Tissue Factor Expression in Activated THP-1 Macrophages
Authors: Watanabe Yuki , Koyama Shohei , Matsuura Yunosuke , Nishihira Kensaku , Yamashita Atsushi , Kitamura Kazuo , Asada Yujiro
Published: March 2019

Background:
Recently, several clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme of the Kyn pathway and is induced by cytokines, particularly IFNγ. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown.

Objective:
The present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP) and define its role in plaque thrombogenicity.

Methods:
Immunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with SAP and UAP. We examined the role of IDO1 in tissue factor (TF) expression in THP-1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α.

Results:
We localized IDO1 mainly in CD68-positive macrophages in atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3-positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. THP-1 macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF-κB (p65) binding activity in activated THP-1 macrophages. Inhibition of the aryl hydrocarbon receptor (AHR) that binds to Kyn, also reduced Kyn-induced TF expression in activated THP-1 macrophages. Kynurenine dose-dependently increased TF expression under IDO1 inhibition whereas hydroxyanthranilic and quinolinic acids did not.

Conclusion:
The IDO1 is expressed in coronary atherosclerotic plaques and might contribute to thrombus formation through TF upregulation via NF-κB (p65) and AHR in activated macrophages.
Abstract 166: Effects of Dietary Unsaturated Fat and Carbohydrate on the HDL Proteome and Metabolism of 9 HDL Proteins Across 6 HDL Size Fractions in Humans
Authors: Andraski Allison B , Singh Sasha A , Lee Lang , Higashi Hideyuki , Smith Nathaniel , Aikawa Masanori , Sacks Frank M
Published: March 2019

Introduction:
When dietary carbohydrate replaces fat, HDL-C and apoA1 decrease. The proportion in plasma of large HDL2 decreases while small HDL3 increases. These findings suggest that diet affects metabolically important attributes of HDL that produce the size changes. We studied in humans the effect of dietary carbohydrate and unsaturated fat on the HDL proteome and metabolism of 9 HDL proteins across 6 HDL sizes.

Methods and Results:
Twelve participants were placed on a controlled high unsaturated fat (HF) or high carbohydrate (HC) diet in a randomized crossover design. At the end of each 4-week diet period, subjects were infused with D3-Leu tracer, and blood was collected for 70 hrs. ApoA1-HDL was prepared by immunoaffinity purification, separated into 6 sizes α0, α1, α2, α3, preβ, and <preβ by ND-PAGE, and in-gel trypsinized for mass spectrometry. We used label free quantification to characterize the HDL proteome. The proteome composition and distribution across the 6 HDL sizes were remarkably conserved in all subjects on both diets. Diet altered the abundance of several proteins on the major HDL sizes α2 and α3. The HC diet increased proteins involved in lipid metabolism (apoC3, apoC1, apoC2-C4) and acute phase response (SAA1, 4), and decreased antioxidant (PON1, 3) and protease inhibitor (SERPINA3, G1) proteins. We also monitored the metabolism of 9 proteins that likely affect HDL metabolism – apoA1, apoA2, apoA4, apoC3, apoE, apoM, apoJ, apoL1, and LCAT. We used high resolution parallel reaction monitoring and XPI software to measure tracer enrichment in these 9 proteins across the 6 HDL sizes. We found that the HC diet increased apoA2 and decreased apoA1 and apoA4 pool sizes on large HDL. The HC diet increased apoA1 and apoE turnover on large α1 and α2 and 3, respectively.

Conclusions:
Dietary carbohydrate when it replaces unsaturated fat alters the HDL proteome and metabolism of several HDL proteins on specific HDL sizes. Carbohydrate increases the abundance of proteins involved in lipid metabolism and the acute phase response, decreases antioxidant and protease inhibitor proteins, and enhances turnover of apoE and apoA1. This study suggests that diet modulates HDL function by affecting HDL composition and the metabolism of major HDL proteins.
Abstract 187: Host Age Enhances Atherosclerotic Plaque Necrosis and Macrophage Infiltration in Mice Independently of Metabolic Alterations
Authors: Tyrrell Daniel J , Goldstein Daniel R
Published: March 2019

Traditional murine models of atherosclerosis age with mild hypercholesterolemia and metabolic derangement, which confounds determination of the intrinsic effects of aging on atherogenesis. To examine if host age enhances atherogenesis independently of metabolic alterations, we induced hypercholesterolemia in wild-type (WT) aging mice using a PCSK9 adeno-associated viral approach. Intraperitoneal PCSK9 transfection and high-fat (42% fat) feeding led to similar elevated blood cholesterol levels between young (3-months) and aged (18-months) WT mice, which were also similar to young low-density lipoprotein receptor knockout (LDLR
-/-
) mice fed the same high-fat diet within 2 weeks. Elevated cholesterol levels persisted during the 10-week feeding period. Although aged mice exhibited an elevated body weight at baseline, both groups gained the same amount of weight during the high-fat feeding. Glucose and insulin resistance measured via tolerance tests were dysregulated but similar between young and aged mice. We next measured atherosclerotic plaque size and composition using H and E, Masson's trichrome, and immunohistochemical staining along with flow cytometry. There was no difference in total plaque size in the aortic sinus between the groups; however, the atherosclerotic necrotic core size and macrophage staining was significantly greater in aged mice. Flow cytometry revealed greater numbers of inflammatory monocytes in the blood along with increased macrophage infiltration in the aorta of aged hyperlipidemic mice compared to young mice. We conclude that aging induces inflammatory monocytosis and macrophage infiltration during atherosclerosis, resulting in increased plaque necrosis and progression of atherosclerosis, independently of confounding metabolic alterations. Our results indicate that aging directly impacts atherogenesis by increasing plaque instability.
Abstract 191: Plan-Based Diet and Lifestyle Changes Improve Stress Test Induced ST Depression in 18 Days
Authors: Ramirez Francisco E , Nedley Neil
Published: March 2019

Background:
This study documents the effects of an intensive 18-day lifestyle program on EKG stress test readings of the ST segment. Improving EKG readings could be a positive indicator of cardiovascular improvement.

Methods:
The medical residential program takes place in Weimar California. The intervention could be summarized in the word NEWSTART which means Nutrition, Exercise, Water, proper Sunlight exposure, fresh Air, Rest and Trust (in relational, spiritual, and psychological aspects). Physicians, nutritionist, exercise physiologist, physical therapist, psychological and spiritual counselors are part of the team. Every patient does a treadmill test (using the Bruce protocol) before and after the 18 day program and a before and after blood tests. Board certified physicians monitored the progress of each patient. Data from 11 years of retrospective recordings were used. From n=2091 patients in the data base, n=145 were used in the study because they had a stress test induced ST depression of 2 mm on two EKG leads or more at baseline. No angiography was available at the institution.

Results:
Average age of patients was 64 (SD 10.5), n=81 (56%) female, and n=131 (90%) were Americans. At baseline the group with ST depression in more than two leads had a mean ST depression in mm of 3.9, SD 2.3, max 13, median 3, mode 2 in the most affected lead. At the end of the 18 days the same group had a mean ST depression in mm was 2.7, SD 2.7, max 12, median 0, mode 0 on the most affected lead. After the 18 days the stress test reported that n=70 (48.2%) had improvement as seen in an improvement of the ST depression during the stress test. While n=64 (44.1%) had the same level of ST depression during the last stress test and n=11 (7.5%) worsen the ST depression during the end stress test. The changes in the ST segment were significant according to the paired t-test t(144)=5.421 with a p<.001. The n=145 lost 4 pounds of weight on average (SD 4) at the end of the 18 days, No heart infarctions occurred during the intervention.

Conclusion:
The intensive lifestyle interventions seem to be effective in improving ST depression. Improvement of ST depression could improve long term prognosis. Further follow up is advised.
Abstract 210: Beta-carotene Conversion to Vitamin a Delays Atherosclerosis Progression and Accelerates Atherosclerosis Regression
Authors: Amengual Jaume , Zhou Felix , Barrett Tessa J , Nikain Cyrus A , von Lintig Johannes , Fisher Edward A
Published: March 2019

β-carotene (BC) from plants is strong antioxidant and is the precursor of vitamin A in animals. Human studies show that elevated BC blood levels correlate with lower incidence of heart disease, but the molecular mechanisms are unclear. While humans accumulate high amounts of BC in blood, rodents completely metabolize BC to vitamin A, precluding the possibility of studying if BC influences heart disease using mouse models. To overcome this, we used a mouse model lacking the enzyme that converts BC to vitamin A (BC oxygenase 1, BCO1) and studied if BC plays a role in atherosclerosis, the main cause of heart disease. We first crossed

Bco1
-/-

mice with

Ldlr
-/-

mice to generate

Bco1
-/-

/

Ldlr
-/-

mice. These mice were fed Western diet (WD) containing BC (WD-BC) for 12 wks, accumulated levels of BC in blood comparable to those observed in humans (12±2 μM), as well as in atherosclerotic lesions, while

Ldlr
-/-

mice fed WD-BC only accumulated trace amounts of BC in blood (0.3±0.1 μM).

Ldlr
-/-

mice fed WD-BC showed delayed atherosclerosis progression (40% reduction in plaque size, n=10-12 mice/group) when compared to those fed WD without BC (WD-noBC). These changes were accompanied by decreased plasma cholesterol (35%) and triglyceride (~55%) levels (n=10-12 mice/group). People typically present for treatment of atherosclerotic disease after plaques have advanced. Thus, we next tested whether BC plays a role in atherosclerosis regression after lipid lowering. For this purpose, we utilized a LDLR antisense oligonucleotide (LDLR-ASO) strategy to block expression of the LDLR to induce hypercholesterolemia. Wild-type and

Bco1
-/-

mice were fed WD-noBC and simultaneously treated with LDLR-ASO for 16 wks. After LDLR-ASO treatment was stopped, plasma cholesterol levels fell from 700mg/mL to 70mg/mL. Mice were also switched to WD-BC or continued with WD-noBC for 3 wks. Wild-type mice, but not

Bco1
-/-

mice, fed WD-BC showed a reduced content of plaque macrophages (30%) and newly recruited monocytes (25%) (n=7 to 12 mice/group) when compared to those fed WD-noBC. We are currently in the process of elucidating the molecular mechanisms responsible of these beneficial effects of BC on atherosclerosis progression and regression.
Abstract 215: Glycosphingolipids Contribute to Pro-atherogenic Pathways in the Pathogenesis of Hyperglycemia-induced Atherosclerosis
Authors: Dang Vi
Published: March 2019

Three out of every four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which diabetes promotes atherosclerosis are not clear. In this study, comprehensive metabolomic techniques were used to investigate the molecular mechanisms by which hyperglycemia promotes accelerated atherogenesis in three distinct disease mouse models. Normoglycemic apolipoprotein E-deficient mice served as the atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections or by introducing a point mutation in one copy of the insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as the hyperglycemia-induced models, without alterations in the levels of glucose or insulin, were also included in the analysis. Mice with accelerated atherosclerosis showed distinct metabolomic profiles compared to the controls. We detected 6369 metabolite features in the plasma of each mouse. Second-order analysis of pair comparisons between each disease model and the control resulted in 62 commonly altered features (p<0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolisms, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated glycosphingolipid metabolism is the most significantly altered pathway. Glycosphingolipid metabolites induced oxidative stress and inflammation in cultured human vascular cells including macrophages, endothelial and smooth muscle cells. Treatment with a known antioxidant, α-tocopherol, reduced oxidative stress and inflammation induced by glycosphingolipids. Our findings suggest that the glycosphingolipid pathway contributes to pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced atherosclerosis, making it a potential therapeutic target to block or slow atherogenesis in diabetic patients.
Abstract 220: Targeting Cholesterol Efflux, Autophagy, and Inflammation to Prevent Atherosclerosis
Authors: Iacano Amanda , Lewis Harvey , Andro Heather , Hazen Jennie , Brubaker Greg , Raheem Bani , Ritchey Brian , Lorkowski Shuhui , Smith Jonathan D , Gulshan Kailash
Published: March 2019

Introduction:
The NLRP3 inflammasome and Toll-like receptor signaling is activated in advanced human atherosclerotic plaques, while autophagy and reverse cholesterol transport (RCT) become dysfunctional. Simultaneous targeting of these pathways can prevent CVD.

Objectives:
To determine if Miltefosine can prevent atherosclerosis by inducing RCT/autophagy and dampening inflammation.

Methods and results:
Here, we report that Miltefosine, an anti-leishmanial drug, acts to decrease atherosclerosis in vivo, and that it induced RCT/autophagy while dampening TLR signaling and NLRP3 inflammasome activity. Miltefosine treatment of macrophages disrupted lipid rafts; ~26 % decrease vs. control via alexa647-CTB binding by flow-cytometry, (p<0.005, n=4), and increased ABCA1 mediated cholesterol efflux to apoA1; ~20% increase in treated vs. control (p<0.001, n=3). Macrophages treated with Miltefosine vs. control exhibited a marked increase in autophagosomes, indicated by p62 and LC3 staining puncta. Autophagic degradative flux was not inhibited by Miltefosine. Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester:free cholesterol) ratio (p<0.005, n=3). Miltefosine treated vs. control macrophages showed ~75% reduction in pro-IL1β mRNA levels upon LPS induction (p<0.05, n=3). Miltefosine potently inhibited NLRP3 inflammasome assembly upon LPS/ATP treatment leading to ~70% reduction in ASC1 speck positive cells. Gasdermin D mediated release of mature IL1β was reduced by ~80% in Miltefosine treated vs. controls (p<0.01, n=3), while only ~20% reduction was observed by cyclodextrin treatment, indicating that inflammasome inhibition by Miltefosine was not only due to cholesterol depletion. In vivo pilot studies showed that Miltefosine added to a chow diet increased RCT to plasma (~70% increase in 24h vs. control), and feces (~60% increase in 24h vs. control, p< 0.005, n=5) in C57BL6 mice. Atherosclerotic lesions were =50% smaller in apoE
-/-
mice fed chow diet + Miltefosine vs. controls (p<0.02, n=5).

Conclusion:
Miltefosine induced RCT and autophagy while dampening TLR signaling and NLRP3 inflammasome activity, and it decreased atherosclerosis lesion burden in mice.
Abstract 221: Crif1 deficiency-induced Mitochondrial Dysfunction Stimulates Endothelial Inflammation
Authors: Kim Cuk Seong , Choi Su-jung , Kim Seonhee , Song Hee Jung
Published: March 2019

CR6 interacting factor 1 (CRIF1) deficiency impairs mitochondrial oxidative phosphorylation complexes, contributing to increased mitochondrial and cellular reactive oxygen species (ROS) production. CRIF1 downregulation has also been revealed to decrease sirtuin 1 (SIRT1) expression and impair vascular function. Inhibition of SIRT1 disturbs oxidative energy metabolism and stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B)-induced inflammation. Therefore, we hypothesized that both CRIF1 deficiency-induced mitochondrial ROS production and SIRT1 reduction play stimulatory roles in vascular inflammation. Plasma levels and mRNA expression of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) were markedly elevated in endothelium-specific CRIF1-knockout mice and CRIF1-silenced endothelial cells, respectively. Moreover, CRIF1 deficiency-induced vascular adhesion molecule-1 (VCAM-1) expression was consistently attenuated by the antioxidant N-acetyl-cysteine and NF-κB inhibitor (BAY11). We next showed that siRNA-mediated CRIF1 downregulation markedly activated NF-κB. SIRT1 overexpression not only rescued CRIF1 deficiency-induced NF-κB activation but also decreased inflammatory cytokines (TNF-α, IL-1β, and IL-6) and VCAM-1 expression levels in endothelial cells.

These results strongly suggest that CRIF1 deficiency promotes endothelial cell inflammation by increasing VCAM-1 expression, elevating inflammatory cytokines levels, and activating the transcription factor NF-κB, all of which were inhibited by SIRT1 overexpression.
Abstract 231: The Role of Epsin in Regulating SR-B1 Function and Lipid Metabolism in Atherosclerosis
Authors: Song Kai , Brophy Megan L , Dong Yunzhou , Wu Hao , Rahman Ashiqur , Chen Hong
Published: March 2019

Atherosclerosis is both a metabolic and inflammatory disease. Vulnerable atherosclerotic plaque forms in response to hyperlipidemia and arterial wall inflammation-associated stimuli, where macrophages represent the "Trojan horse". Owing to extreme paucity of appropriate targets that effectively block dyslipidemia and concomitantly inhibit macrophage-mediated inflammation, the battle fighting against atherosclerosis remains daunting. The discovery of new molecules and pathways that hit two birds with one stone would therefore significantly advance the development of new and more effective therapeutics to treat heart disease. In our latest study, we show that epsins 1 and 2 are upregulated in atherosclerotic plaques in apolipoprotein E deficient ApoE-/- mice fed western diet. Consequently, we show that macrophage-specific and hepatocyte-specific epsin deficiency inhibits atherosclerosis in ApoE-/- mice fed western diet. We have found that epsin interacts with and reduces the levels of anti-atherogenic SR-B1. Furthermore, given a paramount role for epsin in promoting atherosclerosis, whether targeting epsin systemically is a viable strategy to impede atheroma progression is an extremely important and medically relevant question to be addressed. Thus, our central hypothesis is that in macrophage foam cells, epsin reduces levels of SR-B1, impeding atheroma resolution; and, in liver, epsin-mediated downregulation of SR-B1 elevates cholesterol level thus facilitating atheroma progression.
Abstract 232: Development and Validation of a Mathematical Model Representing Complex Cellular Interactions Within the Artery Wall: in Silico Approach to Test Targeted Interventions
Authors: Torres Marcella , Wang Jing , Yannie Paul , Segal Rebecca , Reynolds Angela , Ghosh Shobha
Published: March 2019

Atherosclerotic cardiovascular diseases remain the number one cause of morbidity and mortality despite significant advances in lipid management. Complex cellular interactions occur within the artery wall requiring timely infiltration/egress of immune cells and lipoproteins within a changing inflammatory milieu that determine the progression of an atherosclerotic plaque. Lack of detailed understanding of the multiple processes involved and their potential interactions has hindered development of targeted therapies. The objective of this study was to develop a computational model of the sequential influx of immune cells in response to a trigger to permit a system-level analyses of the processes involved using ordinary differential equations (ODEs). Thioglycollate induced peritonitis was used as a model to examine the infiltration of immune cells and phenotypic polarization of macrophages in response to a stimulus. Peritoneal exudates obtained at 10 different time-points over 7 days were analyzed by FACS to determine the distribution of neutrophils, macrophages and Ly6C
Hi
(M1) or Ly6C
Lo
(M2) polarization. Weighted least squares of the different parameters were used to calibrate the model developed in our laboratory (Panel A) to simulate inflammation/repair during sepsis using the Levenberg-Marquardt algorithm. Panels B-D show the “match” of the experimental data to the calibrated model demonstrating the validity of this model. Since the ODEs are derived from a combination of known and hypothesized kinetics of the biological system, model the changes in physiological variables over time and are based on biological interactions, our calibrated mathematical model will permit the evaluation of changes in one or more targeted parameters
in silico
on immune cell distribution/phenotype. Efficacy of various macrophage-specific interventions (e.g., reduction in cholesterol content or inflammation) can be predicted prior to preclinical experimentation.
Abstract 233: CHI3L1 and Its Role in Vascular Smooth Muscle Cell Proliferation and Migration in Advanced Atherosclerosis
Authors: Tsantilas Pavlos , Lao Shen , Vaerst Monika , Li Yuhuang , Nanda Vivek , Maegdefessel Lars , Leeper Nicholas
Published: March 2019

Objectives:
Stroke is one of the leading causes for death and disability worldwide. The underlying pathomechanism is most likely a thrombotic event caused by acute rupture or erosion of an unstable plaque in the carotid artery. Vascular smooth muscle cells (VSMCs) play an important role in both, development and rupture of atherosclerotic plaques. Thus, efforts have been devoted determining the characteristics of VSMCs in atherosclerotic plaques to develop novel diagnostic and therapeutic strategies to prevent unstable plaque rupture.

Methods:
Laser capture microdissections of the fibrous cap of ruptured plaques in symptomatic patients (n=20) and stable plaques in asymptomatic patients (n=20) were used to perform a GeneChip 2.0 Human Transcriptome Array. CHI3L1 could be detected as the most significantly upregulated transcript in ruptured plaques. Following, immunohistochemistry was performed to compare distribution of CHI3L1-positive cells, VSMCs and macrophages in ruptured and stable plaques. In-vitro MTT-assay, Boyden Chamber assay and TUNEL assay were performed to investigate the role of this gene in human VSMC proliferation, migration and apoptosis.

Results:
The MTT-assay revealed that silencing of CHI3L1 lead to a decreased proliferation of human VSMCs. Further, migration was reduced in absence of CHI3L1. Immunohistochemistry of human carotid plaques indicated that in stable plaques mainly macrophages were CHI3L1-postive, whereas in unstable plaques both macrophages and VSMCs were CHI3L1-positive. In-vitro knockdown of CHI3L1 in cultured human VSMCs significantly increased CD68 expression, suggesting a potential role in transformation of VSMCs to macrophages. CHI3L1/CD68-positive cells (putative macrophages) in unstable plaques appeared hyperproliferative (Ki-67 positive).

Conclusion:
In summary, CHI3L1 plays a role in VSMC-regulation in atherosclerotic plaques. Expression levels in unstable plaques are increased. Further, it seems to induce proliferation as well as migration in VSMCs. Results of CHI3L1-induced CD68 expression indicate a potential role in VSMC-macrophage transformation. 
Abstract 237: The Inhibitory Effect on Vascular Smooth Muscle Cell Hyperplasia in Artificial Atherosclerotic Mouse Model via miR-155 Induced NoxA1 Down Regulation
Authors: Yan Wenwen , Yao Yian , Tang Yu , Chen Zhisong , Wang Lemin , Liu Xuebo
Published: March 2019

Objective:
To investigate the protective efficacy of miR-155 on down regulating NoxA1 gene expression, resulting in an inhibitory effect of vascular smooth muscle cell (VSMC) over proliferation and thus alleviating the progression of coronary artery atherosclerosis in mouse disease model.

Methods:
Carotid artery of C57BL6 mouse was used for VSMC primary culture. The isolated VSMC was transfected with recombinant Pad2YFG adenovirus fluorescent vector with miR-155 fragment (mimic or mutant). Fluorescence microscope was applied to observe the transfection rate of miR-155 into VSMC. Artificial artery-injured mouse model fed with high-fat diet was established as host to accept VSMC transplantation via tail vein injection for 3 consecutive days. Aortic and carotid arteries were extracted at week 6. Distribution of miR-155 was quantified; protein and RNA were extracted to detect NoxA1 and NADPH expression in vehicle control, miR-155 mimic group, and the inhibitor group. Immunohistochemistry was performed in arteries section to compare the thickness of neointima and assess the severity of AS of each group.

Results:
The miR-155 distribution was observed varied at week 6 in control, miR-155 mimic and inhibitor groups. The NoxA1 protein expression in VSMC was decreased in mimic group at week 6 vs control and inhibitor groups (p<0.05); no significant difference of NADPH expression was observed in all groups. The NoxA1 and NADPH gene expression in VSMC were both found reduced compared with those of control group at week 6 (p<0.05). Immunohistochemistry of artery frozen sections figured out that the thickness of neointima of carotid artery in miR-155 mimic group was significantly lower vs control and inhibitor groups (p<0.01) at week 6.

Conclusion:
miR-155 transfection into VSMC seems to have reversed regulatory effect on NoxA1 expression resulting in amelioration of atherosclerotic lesion in artificial AS mouse model. A therapeutic strategy based on the functional role of miR-155 in VSMC over proliferation-based atherosclerosis would need to complementarily-synthesize miRNAs, which is a promising therapeutic tool due to the enhanced stability and sustained effects after intravenous injection in the arterial wall.
Abstract 242: Sexual Dimorphic Effects of Serum Amyloid A3 in Atherosclerosis in Mice
Authors: den Hartigh Laura J , Goodspeed Leela , Gomes Kjerulf Diego , Wang Shari , Turk Katherine E , Houston Barbara , Kramer Farah , Kanter Jenny E , Bornfeldt Karin E , Chait Alan
Published: March 2019

Introduction:
The acute-phase protein serum amyloid A (SAA) exists as 4 different subtypes in mice. Levels of the SAA3 subtype increase in response to acute inflammatory stimuli, and its expression is modestly and chronically elevated in adipose tissue and macrophages in obese mice. Previously we showed that SAA3 deficiency protected C57Bl/6 female mice, but not males, from obesity, adipose tissue inflammation, and hyperlipidemia in response to a high fat high sucrose diet (HFHS: 36% calories from fat, 36% calories from sucrose with 0.15% added cholesterol). We therefore investigated whether SAA3 deficiency modulates atherosclerosis in mice deficient in the low-density lipoprotein receptor (LDLR).

Methods:
We used two models of LDLR-deficiency to promote atherosclerosis in
Saa3
+/+
and
Saa3
-/-
male and female mice: (1) 16 weekly injections of an LDLR antisense oligonucleotide (LDLR-ASO), or (2) genetic deletion of LDLR (
Ldlr
-/-
). All mice consumed the HFHS diet for 16 weeks.

Results:
While both models of LDLR deficiency promoted hypercholesterolemia,
Ldlr
-/-
mice had nearly 1.5- and 2-fold higher cholesterol levels than LDLR-ASO mice (males: 753±109 vs. 560±62 mg/dL, n=8, p<0.05; females: 519±20 vs. 302±22 mg/dL, n=10, p<0.0001). There was no effect of SAA3 deficiency on plasma cholesterol in male mice from either model. However, SAA3 deficiency unexpectedly enhanced body weight gain by 15% and body fat gain by 40%, and exacerbated cholesterol levels in female
Ldlr
-/-
mice (
Saa3
-/-
Ldlr
-/-
: 623±26 vs.
Saa3
+/+
Ldlr
-/-
: 519±20 mg/dL, n=10, p<0.01). Consistent with worsened plasma cholesterol levels,
en face
aortic atherosclerotic lesion area was 2-fold greater in female
Saa3
-/-
Ldlr
-/-
mice than in their wild type counterparts (n=10, p<0.05). Conversely, atherosclerosis was improved by 40% in male
Saa3
-/-
mice in both models of hypercholesterolemia (n=8).

Summary:
While both LDLR-ASO and
Ldlr
-/-
promote hypercholesterolemia in male and female mice, SAA3 deficiency worsens hypercholesterolemia and atherosclerosis in female mice, and reduces atherosclerosis in male mice.

Conclusion:
In the setting of hypercholesterolemia, SAA3 may be pro-atherogenic in male and atheroprotective in female mice, solidifying the sexual dimorphic nature of SAA3.
Abstract 263: Inhibition of Angiotensin II Signaling on LysM+ Myelomonocytic Cells and Their Depletion Improves Vascular Endothelial Dysfunction in Mice With Heart Failure After Myocardial Infarction
Authors: Rudi Wolf-Stephan
Published: March 2019

Background:
Myocardial infarction (MI) induced heart failure (HF) leads to impaired left ventricular function and endothelial dysfunction accompanied by a systemic inflammatory reaction. So far the effects of inhibition of angiotensin II-signaling in heart failure after MI regarding the endothelial function are not fully understood.

Methods and Results:
8-12 weeks old male C57BL/6 mice were subjected to permanent ligation of the left anterior descending artery (LAD) to induce ischemic heart failure. We measured a reduced vascular endothelial and smooth muscle function in isolated aortic segments 7d and 28d post MI in isometric tension studies. Vascular superoxide formation and vascular mRNA expression of monocyte chemoattractant protein-1 (
ccl2
), vascular cellular adhesion molecule-1 (
vcam-1
), inducible NO synthase (
nos2
), and angiotensin II receptor type 1 (
agtr1
) were increased in HF mice compared to sham. Flow cytometry analysis of aortic tissue of HF mice revealed an increased accumulation of CD45+ immune cells in the aortic wall, including CD11b+Gr-1lowF4/80+ macrophages and Ly6C+ monocytes. Diphteria toxin mediated depletion of lysozyme M positive cells starting 28d after MI using LysM-Cre-iDTR mice improved the endothelial function in HF mice. To test specific effects of ATII-signaling on endothelial function in heart failure we administered ramipril (10mg/kg bodyweight) or telmisartan (40mg/kg bodyweight), respectively, via drinking water, starting 1d after MI. After 28d, we assessed an improved vascular endothelial and smooth muscle function, less vascular superoxide production, less accumulation of CD45+ immune cells, reduced aortic expression of
ccl2, vcam-1, nos2
and
agtr1
mRNA of HF mice treated with ramipril as well as with telmisartan in comparison to non-treated HF controls. Importantly, left ventricular dimensions and ejection fraction was not different in treated and non-treated mice, implying a direct anti-inflammatory effect on the vasculature.

Conclusion:
Our results suggest that vascular dysfunction post MI is at least in part mediated by angiotensin II, driving vascular accumulation of inflammatory monocytes and direct effects on vascular oxidative stress levels.
Abstract 265: Oral Nitrite Supplementation Improves Rates of Wound Healing in Diabetic Mice
Authors: Salem Karim M , Nath Nandan , Aggarwal Ankur , Tzeng Edith
Published: March 2019

Introduction:
Nitric oxide (NO) is required for cutaneous wound healing. Impaired diabetic wound healing has been linked to a deficiency in local NO production and can be enhanced with NO delivery. NO is a short-lived, highly reactive molecule and local delivery is complicated by these properties. An alternate source of NO can be achieved through the ability of nitrite reductases to convert the stable NO end-product, nitrite, back to NO. We have previously demonstrated that skin and wound edge express high levels of xanthine oxidoreductase (XOR). XOR is a strong nitrite reductase. We hypothesize that dietary nitrite supplementation will improve wound healing in diabetic mice.

Methods:
Db/db mice (N>8/group) were pretreated for 1 week with sodium nitrite supplemented drinking water (50 mg/L), nitrite-free chow, or standard chow. Additionally, nitrite supplemented mice were gavaged with nitrite supplemented water (0.2 ml) every other day for the duration of the experiment. A 1 cm
2
excisional wound was created on the back of each mouse. Wounds were photographed every other day until closure and wound areas calculated with ImageJ and compared with ANOVA and Kaplan Meier.

Results:
Time to complete healing was significantly different between nitrite supplemented (NS), nitrite depleted (ND), and control mice (18.9±0.7, 21.6±3.3, and 23±3.5 days, respectively, P = 0.035). NS mice reached 75% and 100% healed faster than ND or control mice (P = .009 and P = .042, respectively) [Figure]. Initial wound expansion on day 2 was decreased in NS mice compared to ND mice. XOR activity was increased in wounds compared to skin but similar between all treatment groups.

Conclusion:
Nitric oxide is a critical component of wound healing. Oral systemic nitrite supplementation improves diabetic wound healing in mice. Dietary nitrite may be an inexpensive and safe method of augmenting NO production through wound XOR expression to improve wound repair.
Abstract 275: Identification of Smooth Muscle Derived Macrophages as a Novel Potential Source of Tissue Resident Macrophages
Authors: Bulut Gamze B , Nguyen Anh T
Published: March 2019

Smooth muscle cells (SMC) are not terminally differentiated but display remarkable plasticity to differentiate into other cell types in response to injury or chronic disease states such as atherosclerosis. Of major significance, we previously demonstrated that nearly a third of the macrophage marker positive cells within advanced mouse and human atherosclerotic plaques are of SMC not myeloid origin. These transitions in SMC phenotype are dependent on the stem cell pluripotency gene Klf4 and play a critical role in overall lesion pathogenesis. Since atherosclerosis happens well beyond our reproductive ages, we hypothesized that transitions of SMC to a macrophage-like state also occur in normal mice and play some protective role that is likely to be evolutionarily conserved. To test this hypothesis, we performed flow cytometric evaluations of transitions of SMC to a macrophage-like state within various microvascular tissue beds of our Myh11ERT2Cre eYFP SMC-specific lineage tracing mice. Remarkably, we found that 15-20% of eYFP+ cells within the stromovascular fraction of epidydimal and mesenteric fat depots were CD45+ of which >90% were also positive for the macrophage markers CD11b, F4/80, and Lgals3. Similar SMC derived macrophage-like cells were also observed in skeletal muscle and subcutaneous fat, but at much reduced levels. The percentage of SMC-derived macrophages in pathological fat depots increased about 2-fold upon four-weeks of diet-induced obesity (DIO). Interestingly, these macrophages appear to exhibit an M2-like polarization in that they were CD206+ but CD86-. We are currently ascertaining if the frequency or function of these SMC derived macrophage like cells within pathological fat depots is altered by SMC specific conditional knockout of Klf4, and/or if this is associated with alterations in insulin sensitivity or glycemia. In summary, we have discovered a novel source of tissue resident macrophages which we postulate play a protective role in regulating the inflammatory and metabolic properties of pathological adipose tissue depots.
Abstract 281: miR-155: a Negative Modulator of Acute Oscillatory Shear Stress (OSS)-induced Vascular Inflammation and Dysfunction
Authors: Mohamed Islam , Thomas Sheena , Searles Charles
Published: March 2019

Introduction:
Shear-sensitive micro-RNAs play an integral role in dictating vascular wall pro-inflammatory response and development of atherosclerosis. Previously, our group and others have identified an inverse relationship between micro-RNA-155 (miR-155) expression and inflammation in atheroprone areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and in-vitro.

Hypothesis:
we hypothesized that miR-155 negatively regulates acute OSS-induced vascular inflammation and dysfunction, via modulation of the MAPK-ETS-1 pathway.

Methods:
12-week old C57B/6J wild type (WT) and miR-155 knockout mice (KO) were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of OSS, for 3-7 days. Downstream acute OSS segments were compared to upstream unidirectional shear stress (USS) segments of thoracic aorta using RT-PCR, western blot and two-way ANOVA followed by Tukey’s multiple comparison analyses.

Results:
In WT mice, acute OSS induced vascular inflammation evidenced by upregulation of MCP-1 and VCAM-1 expression in OSS segments compared with USS. This was associated with loss of vascular barrier function as evaluated by extravasation of Evans-blue dye assay along with increased MMP-9 and MMP-3 expression. However, vascular miR-155 levels were also higher in OSS segments compared with USS (n=6-12, P<0.05). Nevertheless, miR-155 KO mice showed enhanced expression and activation of ERK and p-38 MAPKs and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus WT controls (n=3-4, P<0.05). Tail vein injections of miR-155 overexpressing lentivirus particles in WT mice after AAC resulted in further upregulation of miR-155 and abolished OSS-induced upregulation of p-38 and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus scramble controls (n=5-6, P<0.05).

Conclusions:
Despite the early upregulation of shear-sensitive miR-155, our data suggest that miR-155 serves as a negative feedback regulator to acute OSS-induced vascular inflammation via inhibition of p-38 and ETS-1. Further studies are in progress to evaluate the effect of exogenous miR-155 on OSS-induced oxidative stress and vascular function, which can serve as basis for developing novel miRNA-based therapeutic modalities.
Abstract 282: Differential Effects of Thrombospondins on Angiogenesis Gene Expression: Contribution of Statins
Authors: Muqri Furqan , Helkin Alex , Maier Kristopher G , Gahtan Vivian
Published: March 2019

Objectives:
Angiogenesis is important for vascular repair and of interest for treating peripheral arterial disease (PAD). Thrombospondins (TSP) are matricellular proteins involved in PAD. TSP-1 is the first endogenous angiogenesis inhibitor found. TSP-2, which is structurally similar, is also anti-angiogenic. TSP-5 (Cartilage Oligomeric Matrix Protein) differs structurally and may not be anti-angiogenic. Statins have pleiotropic vascular effects that impact TSPs. We studied the effects of TSPs and statin on endothelial cells (ECs).

Hypothesis:
TSP-1 and -2 induce anti-angiogenic genes, and inhibit EC tube formation while TSP-5 or statin induce pro-angiogenic genes and stimulate EC tube formation.

Methods:
ECs were exposed to serum free medium (SFM), TSP-1, -2, or -5 (20μg/mL) for 6 hours with or without 24 hour statin (0.5μM) pre-treatment. qrtPCR was done on 94 angiogenesis related genes. Analysis was by t-test and ANOVA, p < 0.05 was significant. Canonical pathways were examined with IPA software. EC tube formation and disruption were assessed with Matrigel and SFM, TSP-1, -2, or -5 (20μg/mL) treatment with or without statin.

Results:
TSP-1 and -2 downregulated pro-angiogenic genes while TSP-5 both up-regulated and downregulated pro-angiogenic genes (Fig. 1). All TSPs were affected by statin. TSP-1, and -2 decreased EC tube formation (47% and 28% respectively, p<0.05) and disrupted EC tubes (36% and 26% respectively, p<0.05). Addition of statin trends towards attentuation of TSP tube disruption. TSP-5 had no effect on EC tubes formation.

Conclusions:
TSP-1, -2, and -5 impact EC angiogenesis pathways. TSP-1 and -2 downregulated pro-angiogenic genes in similar pathways. TSP-5 upregulated pro-angiogenic genes, and statin augmented this effect. TSP-1 and -2 are anti-angiogenic while TSP-5 is not
in vitro
. The role of statin in EC tube formation is unclear but statin does impact TSP regulation of angiogenesis gene expression and thus further studies are needed.
Abstract 288: Nitric Oxide Mediates Active Downregulation of Tissue Factor Expression in Human Pericytes
Authors: Sommerville Laura , Monroe Dougald , Hoffman Maureane
Published: March 2019

Tissue Factor (TF) is a transmembrane protein that not only expresses pro-coagulant enzymatic activity, but also mediates signal transduction by several second messenger pathways. It plays an obvious role in hemostasis and thrombosis, as well as modulating angiogenesis, inflammation, immune responses and progression of malignancy and metastasis. TF is normally expressed by a limited subset of cell types. It can also be induced on other cells by a plethora of mediators, mostly linked to inflammation and immune responses. By contrast, TF has only been shown to be actively downregulated in a single circumstance: we have reported that constitutive expression of TF by pericytes around dermal vessels disappears during angiogenesis associated with wound healing. This suggests that downregulation of pericyte TF may play a functional role in the process of angiogenesis. We previously reported that TF protein in cultured human pericytes is significantly reduced 8 hours after treatment with phorbol 12-myristate 13-acetate (PMA) (94.71% reduction
+
2.18%, p<0.001). This loss of TF protein is facilitated by reduction of TF mRNA. Downregulation of TF protein and mRNA depends on Protein Kinase C. The goal of the current study was to identify a physiologic mediator of pericyte TF downregulation. Treatment of human pericytes with growth factors and cytokines involved in wound healing did not change TF expression. Co-culture of human pericytes with human microvascular endothelial cells in transwells in the presence of basic fibroblast growth factor (bFGF) resulted in downregulation of pericyte TF. However, conditioned media from endothelial cells grown in the presence of bFGF did not trigger a decrease of TF in pericytes. bFGF has been shown to stimulate NO production, and both bFGF and NO have been linked to angiogenesis. This led us to consider nitric oxide (NO) as a potential labile mediator of TF downregulation. TF protein was reduced 8 hours after treatment with DETA/NO (44% reduction
+
6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Our working hypothesis is that TF downregulation in pericytes is partially mediated by endothelial cell-derived NO, but is also mediated by additional PKC-dependent pathways.
Abstract 289: Sirtuin-1 Inhibition by Peroxynitrite Contributes to Nicotine-induced Arterial Stiffness in Mice
Authors: Ding Ye , Lu Qiulun , Zhu Huaiping , Xie Zhonglin , Zou Ming-Hui , Song Ping
Published: March 2019

Cigarette smoking is a key contributor to arterial stiffness, which is a major and independent risk factor for serious cardiovascular events. Sirtuin-1 (SIRT1), a nicotinamide adenine dinucleotide-dependent protein deacetylase, plays an essential role in maintaining cardiovascular homeostasis, and
Sirt1
inhibition is linked to smoking-induced arterial damage and aortic stiffness. The mechanism behind key component of cigarette smoking, nicotine-induced SIRT1 inactivation remains unknown; therefore, we aimed to characterize the relationship between nicotine, SIRT1 inhibition, and arterial stiffness. Arterial stiffness and SIRT1 expression and activity in wild-type and
Sirt1-
overexpressing mice treated with nicotine or vehicle were monitored. Nicotine significantly increased arterial stiffness in wild type, but not
Sirt1
-overexpressing mice. Additionally, SIRT1 protein levels and activity were decreased in nicotine-treated wild-type mice, but not in
Sirt1-
overexpressing mice, and these results were reproducible in human aortic smooth muscle cells. We further determined that SIRT1 inhibition was caused by zinc release from and subsequent inhibition of deacetylation activity of SIRT1, leading to Yes-associated protein activation, and, ultimately, extracellular matrix remodeling associated with arterial stiffness. Zinc release from SIRT1 was mediated by peroxynitrite resulting from nicotine-induced increases in inducible nitric oxide synthase. Taken together, nicotine triggers arterial stiffness via extracellular matrix remodeling mediated by SIRT1 inhibition due to peroxynitrite-induced zinc release from SIRT1.