bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 290: The Transcription Factor Tead1 is Critical for Vascular Development in Mouse by Promoting Differentiation of Vascular Smooth Muscle Cells
Authors: Zhou Jiliang , Wen Tong , Dong Kunzhe , Hu Guoqing , Liu Jinhua , Wang Menghong , Zhang Wei
Published: March 2019

Background:
Our previous study has shown that the Hippo signaling effector YAP plays a crucial role in the phenotypic modulation of vascular smooth muscle cells (VSMCs) in response to arterial injury and cardiovascular development in mice. However, the role of TEAD1, a co-factor of YAP in VSMC development is unknown.

Objective:
The goal of this study to investigate the functional role of TEAD1 in cardiovascular development in mice.

Methods and Results:
TEAD1 was specifically ablated in cardiomyocytes and VSMCs by crossing TEAD1 flox mice with SM22alpha-Cre transgenic mice. Cardiac/VSMC-specific deletion of TEAD1 led to embryonic lethality by E14.5 in mice due to cardiovascular defects including severe hypoplastic cardiovascular wall, resulting from impaired cardiac/VSMC proliferation. Whole transcriptomic analysis revealed deletion of TEAD1 in mouse cardiomyocytes and VSMCs significantly down-regulated expression of cardiac/VSMC differentiation genes and key transcription factors including myocardin and pitx2c that are critical for cardiovascular development.
In vitro
studies demonstrated that myocardin and pitx2c rescued TEAD1-defiency induced defects of VSMC differentiation. Furthermore, we identified pitx2c was a direct transcriptional target of TEAD1 and pitx2c synergistically promoted VSMC differentiation with myocardin.

Conclusions:
This study revealed a critical role of TEAD1 in cardiovascular development by promoting VSMC proliferation and differentiation and provided novel insights into the regulatory mechanisms for smooth muscle development.
Abstract 302: TRAIL Protects Against Endothelial Dysfunction in vivo and Inhibits Angiotensin-II Induced Oxidative Stress in Vascular Endothelial Cells
in vitro
Authors: Manuneedhi Cholan Pradeep , Cartland Sian , Rayner Benjamin , Genner Scott , Dang Lei , Thomas Shane , Kavurma Mary
Published: March 2019

The vascular endothelium is critical for maintenance of cardiovascular homeostasis and vascular oxidative stress is a primary cause of endothelial dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) is increasingly recognised to play a protective role in atherosclerosis, however the molecular mechanisms by which it exerts its beneficial effects are unclear. Our aim was to identify whether TRAIL could protect against atherosclerosis by reducing vascular oxidative stress and improve endothelial cell (EC) function.
In vivo
, 12 w high fat diet (HFD)-fed

Trail
-/-
Apoe
-/-

mice had more severe atherosclerosis, with significant impairment in vasorelaxation in response to acetylcholine, but not sodium nitroprusside. HFD

Trail
-/-
Apoe
-/-

vessels also had increased vascular reactive oxygen species (ROS) as evident by dihydroethidium (DHE) staining compared to HFD

Apoe
-/-

mice. Following injection of Evan’s blue dye, blood vessels from highly vascularised organs of

Trail
-/-

mice demonstrated increased vascular permeability compared to

Trail
+/+

organs, and altered expression of adhesion molecule and tight junction proteins.
In vitro
, AngII (50 ng/ml) increased DHE and mitosox staining in ECs within 2 h, which was inhibited by apocynin, rotenone and L-Name, suggesting that NADPH oxidases (NOXs), the mitochondria and eNOS are major producers of ROS. siRNA targeting NOX-4, but not NOX-1, -2, or -5 reduced AngII-induced DHE, whereas siRNA targeting eNOS significantly augmented DHE staining. Importantly, pre-treatment of TRAIL at 1 ng/ml inhibited AngII-inducible DHE and mitosox staining. Functionally, TRAIL reduced eNOS monomer expression and monocyte recruitment in response to AngII. Furthermore, AngII reduced VCAM-1 and PECAM-1 mRNA was no rescued with TRAIL treatment. These findings demonstrate for the first time that TRAIL protects against endothelial dysfunction via its ability to modulate oxidative stress. Understanding the role TRAIL plays in normal physiology and disease, may lead to potential new therapies to improve endothelial function and atherosclerosis.
Abstract 305: Genetic Deficiency in Nox2-containing NADPH Oxidase Decreases Susceptibility to Venous Thrombosis in Mice
Authors: Sonkar Vijay K , Kumar Rahul , Gu Sean , Doddapattar Prakash , Lentz Steven R , Dayal Sanjana
Published: March 2019

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P<0.05). Markers for NETs were measured in plasma (nucleosome levels using ELISA) and venous thrombi (staining for citrullinated histones, Cit-H3). Levels of nucleosomes in the mice that did not develop thrombi were similar to those in baseline samples collected before IVC ligation. All of the mice that developed thrombi had significant elevation of plasma nucleosome levels compared with mice that did not develop thrombi (P<0.05), but nucleosome levels did not differ between WT and Nox2-KO mice. Likewise, the heterogeneous pattern of NET formation within thrombi detected by Cit-H3 staining was similar in WT and Nox2-KO mice. These findings suggest that Nox2 is an important determinant of thrombus size during IVC ligation. Interestingly, our study also provides evidence for Nox2-independent pathways for NET release during venous thrombosis.
Abstract 311: The Role of ADAMTS-5 in Aortic Dilatation and Extracellular Matrix Remodeling
Authors: Fava Marika , Barallobre-Barreiro Javier , Mayr Ursula , Lu Ruifang , Didangelos Athanasios , Baig Ferheen , Lynch Marc , Catibog Norman , Joshi Abhishek , Barwari Temo , Yin Xiaoke , Jahangiri Marjan , Mayr Manuel
Published: March 2019

Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic extracellular matrix (ECM). Evidence is emerging that ECM processing by members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family could play an important role in the progression of aortic dilatation. In particular, ADAMTS-1 and -4 have been implicated in TAA. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. A model of aortic dilatation by angiotensin II (AngII) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5
Δcat
). Adamts5
Δcat
mice showed an attenuated rise in blood pressure whilst displaying increased dilatation of the ascending aorta. Interestingly, a proteomics comparison of the aortic ECM from AngII-treated wildtype and Adamts5
Δcat
mice revealed versican as the most up-regulated ECM protein in Adamts5
Δcat
mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine), an increase in TGFβ and a decrease of low-density lipoprotein-related protein 1 (LRP1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of
ADAMTS5
, attenuated the generation of versikine but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5
Δcat
mice, but was not sufficient to maintain versican processing and prevent aortic dilatation. Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. A compensatory rise in ADAMTS-1 could not prevent aortic dilatation in Adamts5
Δcat
mice. LRP1, which has been involved in aneurysm pathology in both human and mice, appears to be linked to ADAMTS-5 and ADAMTS-5-mediated versican cleavage. Further studies are needed to explore the ADAMTS protease family as target for therapeutic approaches aimed to reduce or halt dilatation of the aorta resulting in aneurysm.
Abstract 338: Statin Therapy is Associated with Decreased Plaque Instability in Symptomatic Carotid Atherosclerotic Plaque: a Clinicopathological Analysis
Authors: Konishi Takao , Tanaka Shinya
Published: March 2019

Background:
Statin therapy has been shown to deplete atherosclerotic plaque instability in human carotid arteries by various imaging modalities or in animal models by pathological analysis. However, whether statin therapy attenuates plaque instability in human carotid plaque has not been fully described in a pathological analysis. The aim of this study was to analyze pathologically the possible relationships between statin therapy and plaque stability in patients undergoing carotid endarterectomy (CEA).

Methods:
Among consecutive 79 patients with advanced carotid artery stenosis (>70%) between May 2015 and February 2017, 66 patients without statin therapy (group 1) and 13 patients with statin therapy (group 2) were analyzed. The specimens were stained with hematoxylin/eosin and elastica-Masson. Immunohistochemistry was performed, using an endothelial specific antibody to CD31, CD34 and PDGFRβ.

Results:
Plaques from group 2 had significantly less plaque rupture (P=0.009), lumen thrombus (P=0.009), inflammatory cells (P=0.008), intraplaque hemorrhage (P=0.030) and intraplaque microvessels (P<0.001), compared with those from group 1. Among stroke patients with infarct size >1.0 cm
3
in MRI, the mean infarct volume in group 2 (4.2 ± 2.5 cm
3
) was significantly smaller than that in group 1 (8.2 ± 7.1 cm
3
, p = 0.031). There was no significant difference in the mean concentration of low-density lipoprotein cholesterol between group 1 and group 2 (121 ± 32 mg/dl vs. 105 ± 37 mg/dl, P=0.118).

Conclusions:
This clinicopathological analysis of carotid atherosclerotic plaque suggests that statin therapy is associated with decreased plaque instability, which might result in smaller infarct volume in stroke patients undergoing CEA. This plaque stability might be derived from pleiotropic effect beyond lipid lowering of statin therapy.
Abstract 349: The Association of Comorbid Depression with Inpatient Outcomes in Patients with Critical Limb Ischemia
Authors: Zahner Greg J , Cortez Abigail , Duralde Erin , Ramirez Joel L , Wang Sue , Hiramoto Jade , Cohen Beth E , Wolkowitz Owen M , Arya Shipra , Hills Nancy K , Grenon Marlene
Published: March 2019

Background:
Critical limb ischemia (CLI) is the end-stage of peripheral artery disease (PAD) and mounting evidence suggests an association between depression and PAD. The objective of this study was to determine whether outcomes, primarily major amputation, differed between CLI patients with and without comorbid depression in a large nationally-representative sample of patients in the United States.

Methods:
Patients hospitalized for CLI during 2012 and 2013 were identified from the National Inpatient Sample (NIS) using ICD-9 diagnostic and procedure codes. Depression, revascularization procedures, and relevant comorbidities were also identified. The primary outcome was major amputation and secondary outcomes were length of stay and other complications. Multivariable logistic models were run adjusting for sociodemographic factors, prior amputation, tobacco use, and several comorbidities.

Results:
The sample included 116,008 patients hospitalized for CLI of whom 10,512 (9.1%) had comorbid depression. Patients with depression were younger (64±14 yrs vs. 67±14, p<.0001) and more likely to be female (55% vs. 41%, p <.001), white (73% vs. 66%, p<.001), and tobacco users (46% vs. 41%, p<.001). They were also more likely to have prior amputations (9.8% vs. 7.9%, p<.001) and diabetes with chronic complications (25% vs. 20%, p<.001). During the hospitalization, the rate of major amputation was higher in patients with comorbid depression (11.5% vs. 9.1%, p<.001). In multivariable analysis, excluding patients who died prior to receiving an amputation (n=2,621), comorbid depression was associated with a 39% increased odds of major amputation (OR 1.39, 95%CI 1.30-1.49, p<.001). Across the entire sample, comorbid depression was also independently associated with a longer length of stay (β=0.199, 95%CI 0.155, 0.244, p<.001).

Conclusions:
Comorbid depression was associated with 39% increased odds of major amputation among a large, nationally representative sample of CLI inpatients. These results provide further evidence that depression is a variable of interest in PAD and surgical quality databases should include mental health variables to enable further study of depression’s impact on surgical patients.
Abstract 368: Severe Obesity and Bariatric Surgery Alter the Platelet mRNA Profile
Authors: Heffron Sean P , Marier Christian , Parikh Manish , Fisher Edward A , Berger Jeffrey S
Published: March 2019

Mechanisms explaining the relationship between obesity and CVD are needed. Platelets are increasingly recognized as immune cells; the platelet transcriptome and sheddome can modulate the function of disparate cells and tissues. Despite growing recognition of the importance of the platelet transcriptome, low levels of RNA in platelets makes assessment difficult. Unbiased platelet RNA profiling specifically in obesity has not previously been performed.

We performed a prospective study investigating the association between severe obesity and weight loss via bariatric surgery and platelet function and RNA profile in 25 pre-menopausal, non-diabetic women (31.9 ± 7.6 years; 43.0 ± 6.5 kg/m
2
) who underwent sleeve gastrectomy. Ten women of similar age and racial background with normal BMI (22.8 ± 2.3kg/m
2
) served as control subjects.

Platelet function via light transmission aggregometry and surface expression via flow cytometry was assessed before and after surgery. RNAseq was performed on platelet RNA isolates of 8 obese women before and 5 after surgery and 5 control subjects.

Platelet count, size, age (reticulated platelets) and aggregation measures did not differ between control and obese women. However, surface markers (eg P-selectin and CD40) of platelet activation were significantly higher in obesity. mRNA sequencing demonstrated >600 RNA transcripts differentially abundant (P<0.05 and base mean counts ≥5) in obesity. Notably, platelet
S100A9
and
AGER
, established markers of CV risk, were 2 of the most highly upregulated transcripts in obesity. Pathway analyses identified eIF2 and mTOR signaling and protein translation broadly to be upregulated. At 6 months after surgery, subjects lost 26.1 ± 5.8% body weight, but only a small fraction of transcripts (46/630; which included
AGER)
exhibited significantly altered expression.

Platelet activation (not aggregation) was increased and the platelet transcriptome was altered in obesity. Given platelets’ ability to act as immune cells and transfer their RNA cargo to other cells important in atherothrombosis, the altered platelet RNA profile in obesity may contribute to increased risk of CVD. More substantial degrees or duration of weight loss may be necessary to rectify the altered platelet RNA profile.
Abstract 389: The Impact of Phospholipid Composition of Synthetic High-Density Lipoprotein on Its Pharmacological Activity
Authors: Kim Sang Yeop , Li Dan , Fawaz Maria V , Morin Emily E , Olsen Karl , Li Xiang-An , Schwendeman Anna
Published: March 2019

Objective:
Synthetic high-density lipoprotein (sHDL) is a nanoparticle that can mimic biological activities of endogenous HDL such as reverse cholesterol transport (RCT) and anti-inflammatory properties. We hypothesize that differences in the fluidity of sHDL phospholipids at body temperature effect plasma stability of sHDL, particle ability to efflux cholesterol and inhibit inflammation.

Methods:
sHDL particles with different membrane fluidities were prepared complexing phospholipids with different fatty acid chain length and saturation (POPC, DMPC, DPPC, and DSPC) with the apoA-I mimetic peptide, PVLDLFRELLNELLEALKQKLK (22A). The ability of various sHDL compositions to efflux cholesterol, inhibit NF-kB activation and cytokine release, and cause lipid raft disruption was examined in RAW264.7 macrophages. Various sHDL were administered to mice challenged by injection of 0.05 mg/kg LPS at 10 mg/kg dose and the levels of cytokine release were measured at 2 hours post-dose. Various sHDL were dosed to normal rats at 50 mg/kg and cholesterol mobilization and pharmacokinetics were examined.

Results:
22A-POPC and 22A-DMPC sHDL have relatively fluid phospholipid layer at body temperature compared to 22A-DPPC and 22A-DSPC sHDL due to lower phospholipid transition temperature. 22A-POPC and 22A-DMPC sHDL inhibited NF-κB activation and cytokine release in a concentration-dependent manner. From murine endotoxin infusion studies, 22A-DMPC displayed the significant inhibition of cytokine release. Cholesterol efflux studies demonstrated that 22A-POPC and 22A-DMPC displayed highest cholesterol efflux. Interestingly,
in vivo
RCT study showed that 22A-DSPC had longest plasma residence time and resulted in greatest cholesterol mobilization.

Conclusions:
Phospholipid composition of sHDL particles has a significant effect on its cholesterol efflux and anti-inflammatory properties, yet the effect appears to be different
in vitro
and
in vivo
.
in vitro
effect is driven by the ability of fluid phospholipid bilayer to bind LPS, cholesterol, and LCAT, while
in vivo
effect is defined by particle stability in plasma and residence time in the body.
Abstract 392: Nanoparticle-based Biosensors for Measuring Apolipoprotein A-I and HDL Function
Authors: Palekar Rohun U , Rink Jonathan S , McMahon Kaylin M , Thaxton C S
Published: March 2019

Background:
High-density lipoproteins (HDL) are dynamic circulating nanoparticles that carry cholesterol and have numerous functions. Rapid measurement of HDL, the HDL-associated protein apolipoprotein A-I (apoAI), or HDL function(s) is not currently possible. Such technology may provide new opportunities for HDL monitoring. We utilized nanoparticles to develop a method of sequestering apoAI from solution, including human serum, and for measuring the activity of the HDL-maturing and apoAI-dependent enzyme, lecithin:cholesterol acyl transferase (LCAT).

Methods:
Gold nanoparticles were surface-functionalized with phospholipids. The synthesized particles were incubated with various amounts of apoAI or with purchased, LDL-depleted, human serum samples where the apoAI amount was measured using an ELISA. After incubation, the particles were easily isolated and apoAI sequestration by the nanoparticles was measured using ELISA. As apoAI is a cofactor that activates LCAT, nanoparticles were incubated with various amounts of apoA1, LCAT, and free cholesterol or with the LDL-depleted human serum samples (diluted) for 1 hour at 37°C to enable binding of apoAI, cholesterol, and LCAT activity. The nanoparticles were easily isolated and the particle-associated cholesteryl ester was measured using an Amplex Red Cholesterol Assay kit.

Results:
The nanoparticles sequester apoAI according to the concentration of apoAI in solution (R = 0.668, p = 0.035, N = 10). Also, data reveal that the LCAT binds the nanoparticle-apoAI structures and catalyzes the conversion of cholesterol to cholesteryl ester. The amount of cholesteryl ester bound to the nanoparticles is directly dependent on the amount of apoAI (R = 0.729, p = 0.016, N=10).

Conclusion:
These results demonstrate that specific, functionalized nanoparticles can be added to serum to sequester apoAI in dose-response. Also, the formed conjugate structures activate LCAT whereby the amount of cholesteryl ester formed on the nanoparticle directly correlates with apoAI. Overall, this technology provides a framework and new approach for developing next generation HDL- and HDL-function specific biosensors.
Abstract 410: Statin Use May Alleviate Coagulation- and Inflammation-associated Gene Expression in Circulating Classical Monocytes in Women With Chronic HIV-1 Infection
Authors: Ehinger Erik , Akula Bala Pramod , Lin Juan , Hanna David B , Mueller Karin , Baas Livia , Qi Qibin , Wang Tao , Buscher Konrad , Liu Yongmei , Anastos Kathryn , Lazar Jason M , Mack Wendy J , Tien Phyllis C , Cohen Mardge H , Ofotokun Igho , Gange Stephen , Heath Sonya L , Hodis Howard N , Tracy Russell P , Landay Alan L , Kaplan Robert C , Ley Klaus
Published: March 2019

During virally-suppressed chronic human immunodeficiency virus (HIV) infection, persistent inflammation contributes to the development of cardiovascular disease, a major comorbidity in people living with HIV. Monocytes play a key role in atherosclerotic plaque development, inflammation, and stability, but their contribution to the CVD under viral suppression in people living with HIV remains unknown. Here, we investigated the transcriptomes of classical (CD14
++
CD16
-
) blood monocytes from 92 women with and without chronic HIV infection and subclinical cardiovascular disease (sCVD), defined as the presence of focal carotid artery plaque, from the Women's Interagency HIV Study (WIHS). Differential gene expression, based on four two-way comparisons among participant groups (HIV-sCVD-, HIV+sCVD-, HIV-sCVD+, and HIV+sCVD+, 23 subjects each), identified large pro-inflammatory gene signatures for both sCVD and virally-suppressed HIV. These findings were further corroborated by Ingenuity Pathway Analysis. We found that classical monocytes persistently express common CVD-related markers of inflammation, including IL6, IL1β, and IL12B; overlapping with many transcripts identified in sCVD+ participants. In comorbid disease (HIV+sCVD+), those reporting statin use showed dramatically reduced pro-coagulant tissue factor (F3) and tissue factor pathway inhibitor (TFPI and TFPI2) gene expression to a level comparable with healthy participants. Cytokine expression profiles associated with the tissue factor pathway were also modified in participants on statins, suggesting that statins may benefit women with chronic HIV infection by limiting pro-coagulation and inflammation pathways in classical monocytes.
Abstract 414: Local Artery Wall Inflammation Overrides Systemic Inflammation in Diabetes-Accelerated Atherosclerosis
Authors: Kanter Jenny E , Kramer Farah , Barnhart Shelley , Rahman Karishma , Amengual Jaume , Wei Xiaochao , Bergsbaken Tessa , Chait Alan , Fink Pamela J , Winer Daniel A , Fisher Edward A , Goldberg Ira J , Semenkovich Clay F , Bornfeldt Karin E
Published: March 2019

Human genomic studies have highlighted the importance of arterial wall-specific inflammatory processes in cardiovascular disease (CVD) risk. Diabetes increases systemic inflammation, local arterial inflammation, and CVD risk. To clarify the relative contributions of systemic inflammation versus artery wall inflammatory processes in atherosclerosis, we studied LDL receptor-deficient mice with streptozotocin-induced diabetes. The damage-associated molecular pattern protein S100A9 and toll-like receptor 4 (TLR4) have both been implicated in diabetes-induced inflammation. S100A9-deficient bone marrow chimeras were used to inhibit systemic inflammation, 5-aminosalicylic acid (5-ASA) was used to inhibit intestinal inflammation, and TLR4-deficient bone marrow chimeras were used to inhibit artery wall inflammation. No model affected the severity of diabetes, plasma cholesterol or blood leukocyte numbers. Hematopoietic S100A9-deficiency, but not TLR4-deficiency, reduced diabetes-associated systemic inflammation to levels observed in non-diabetic mice. 5-ASA differentially altered measures of systemic inflammation. Thus, diabetes induced a 2-fold increase in circulating leukocyte
Il1b
mRNA, which was normalized by S100A9-deficiency (p<0.01, n=7-10) and 5-ASA, but was not reduced by hematopoietic TLR4-deficiency (n=11-14). Similarly, diabetes increased plasma levels of the acute-phase protein serum amyloid-A (SAA), which were normalized by S100A9-deficiency (p<0.01, n=5-12), but not by TLR4-deficiency (n=5-10) or 5-ASA. Conversely, hematopoietic TLR4-deficiency (p<0.05), but not hematopoietic S100A9-deficiency or 5-ASA, reduced diabetes-accelerated myeloid cell accumulation in the artery wall determined by aortic
en face
Sudan IV staining (n=16-21). Finally, laser capture microdissection of CD68-positive lesional macrophages revealed that hematopoietic TLR4-deficiency prevents diabetes-induced inflammatory processes in the artery wall including expression of
Il1b, Ccr2
, and
S100a9
mRNA. Together, our data strongly suggest that although systemic inflammation is increased in diabetes, inhibition of inflammatory processes in the artery wall is required to prevent lesional macrophage accumulation.
Abstract 434: Nogo-B Regulates Hyperglycemia Induced Endothelial Dysfunction by Modulating Mitochondria Function
Authors: Zhang Cheng , Yu Jun
Published: March 2019

Objective:
As an important part of the vasculature, the endothelium plays multiple functions in maintenance of vascular homeostasis. Endothelial dysfunction is associated with several pathologies, including but not limited to diabetes, hypertension and atherosclerosis. Emerging evidence suggest that mitochondrial have a profound impact on endothelial cell function. Hyperglycemia induced mitochondrial defects are associated with endothelial dysfunction and pathogenesis of diabetes related microvascular and macrovascular diseases. However, the mechanism by which hyperglycemia causes mitochondrial dysfunction are not fully elaborate.

Approach and Results:
Here we examined the role of Nogo-B, a regulator of mitochondria-endoplasmic reticulum unit, in mitochondrial dysfunction that triggered by hyperglycemia. Our data show that compared to control, depletion of Nogo-B enhanced high glucose induced mitochondrial fission; suppressed mitochondrial membrane potential and respiration rate in endothelial cells. Furthermore, Nogo-B deficiency resulted in elevated JNK phosphorylation, which then led to MFN2 degradation and cell apoptosis.

Conclusion:
Taken together, our findings provide novel insights into an unexpected role of Nogo-B. Nogo-B is a regulator of mitochondrial morphology and function under high glucose treatment in endothelial cells. The results from this study deepen our understanding on the mechanism of diabetic related endothelium dysfunction.
Abstract 447: Lipid Droplet Associated Hydrolase (LDAH) Impacts Oxysterol Metabolism and Prevents Atherosclerosis
Authors: Goo Young-Hwa , Lee Inhwa , Lydic Todd A , Bangru Sushant , Saha Pradip , Kalsotra Auinash , Chan Lawrence C , Paul Antoni
Published: March 2019

Macrophage/foam cells in the arterial wall engulf apolipoprotein B-containing lipoproteins and store the surplus of lipids derived from these particles in lipid droplets (LDs). Even though cholesterol is abundant in the LDs of atheroma, the lipidome of LDs displays a variety of lipid species, including other sterols that have a deep impact on foam cell and plaque biology. While the LD is increasingly seen as a reservoir of signaling precursors, how the bioactive lipids are mobilized from the LDs remains elusive. Previously, we identified LDAH as a novel LD protein that contains a lipase/esterase sequence and reported it as a CE hydrolase. Our in vivo atherosclerosis studies on LDAH knock-out (KO) and transgenic (Tg) mice in apoE KO background revealed an athero-protective role of LDAH driven by its ability to reduce lipid accumulation in the lesional foam cells. LDAH increases expression of ATP-binding cassettes (ABC) A1 and G1, two cholesterol transporters that are regulated by liver X receptor (LXR). Interestingly, we also found that LDAH promotes favorable tissue remodeling, evidenced by a remarkable increase in lesional collagen content, which is known to contribute to plaque stability. To determine molecular mechanism behind these phenotypes we performed combinatory “omics” studies of LDAH WT, KO, and Tg foam cells: lipidomics (targeted and untargeted) and transcriptomics. Lipidomics analyses confirmed that LDAH inversely regulates total cholesterol levels, and revealed that several other lipid species were also altered by LDAH. Interestingly, among sterol lipids, LDAH preferentially targets esterified oxysterols whose free forms are LXR ligands. RNA-seq analysis identified several collagen synthesis genes regulated by LDAH, supporting the phenotype seen in the atheroma. Overall, our studies suggest that LDAH might play a central role in the production of messengers from ester precursors stored in LDs of foam cells. Therefore, unraveling the mechanisms of trafficking through LDs may be key to exploit the atheroprotective potential of endogenous lipid mediators.
Abstract 456: LncRNA
CHROME
is Increased in Cardiovascular Disease and Regulates Inflammatory Gene Expression
Authors: Scacalossi Kaitlyn R , van Solingen Coen , Hennessy Elizabeth J , Rizzacasa Barbara , Berger Jeffrey S , Kazan Hilal , Moore Kathryn J
Published: March 2019

Long non-coding RNAs (lncRNAs) are a class of regulatory RNAs capable of binding DNA, RNA, and/or protein to regulate transcriptional and epigenetic networks. Although thousands of lncRNAs have been identified, relatively few have been functionally characterized. Here we identify a primate-specific lncRNA,
CHROME
, encoded in a locus associated with cardiovascular disease. We found that
CHROME
expression is increased in the plasma of patients with inflammatory conditions, including coronary artery disease and lupus, compared to control subjects. Using FANTOM, a database of transcriptome analyses, we found the
CHROME
locus is transcriptionally activated in human monocytes and macrophages stimulated with microbial ligands and inflammatory cytokines. To investigate CHROME’s molecular mechanisms, we used RNA immunoprecipitation (RIP) and chromatin isolation by RNA purification (ChIRP) to map
CHROME
lncRNA -protein and -DNA interactions, respectively. We found that
CHROME
has a strong histone binding affinity and its DNA binding pattern was consistent with interaction of
CHROME
with numerous inflammatory transcription factor motifs, including sites for CEBPβ, SPI1, NFKB, and RELA. To investigate the impact of CHROME’s interaction with DNA in macrophages, we used gain and loss of function studies combined with RNA-sequencing. Ingenuity Pathway Analysis of genes most significantly altered with both CHROME knockdown and overexpression identified the inflammatory response as the pathway most significantly altered by
CHROME
. In particular, repression of
CHROME
led to a decrease in the expression of interferon stimulated genes and receptors involved in macrophage motility. Together, these data suggest that
CHROME
contributes to the transcriptional regulation of inflammatory gene expression and its dysregulation in the setting of atherosclerotic cardiovascular disease may contribute to the maintenance of chronic inflammation.
Abstract 472: Identifying and Characterizing Causal Genes in GWAS-identified Loci for Triglyceride Levels Using High-throughput, Image-based Screens in Zebrafish Larvae
Authors: von der Heyde Benedikt , Masiero Mauro , Emmanouilidou Anastasia , Klingström Tiffany , den Hoed Marcel
Published: March 2019

Background:
High triglyceride levels are an established risk factor for coronary artery disease (CAD). Unpublished results from our proof-of-principle studies show that triglyceride-levels are also associated with atherosclerosis in zebrafish larvae. Identifying and characterizing causal genes for triglyceride levels may yield novel drug targets. In 2013, a meta-analysis of genome-wide association studies identified 37 previously unanticipated loci that are associated with triglyceride levels. We aim to characterize positional candidate genes in these loci using a zebrafish model system.

Methods:
We prioritized 37 candidate genes for functional-follow up. These genes together have 41 zebrafish orthologues, which were targeted in five lines of ~8 genes each using a multiplex CRISPR-Cas9 approach. Founder mutants have been raised and 384 offspring for two of the five lines have so far been screened for body size as well as fluorescently labeled lipids, macrophages and neutrophils in the vessel wall, using a high-throughput imaging set-up. Image quantification was performed using automated, custom-written pipelines in CellProfiler and ImageJ. Additionally, whole body lipid fractions and glucose levels were measured in each larva using enzymatic assays. CRISPR-induced mutations were quantified using paired-end sequencing and data were analyzed using hierarchical mixed models and (zero-inflated) negative binomial regression.

Results:
Each additional disrupted allele in
pepd
resulted in higher triglyceride levels (beta±SE 0.21±0.09 SD). Mutants for
met
have less vascular infiltration by macrophages (-0.31±0.13), and less co-localization of macrophages with lipids (-0.56±0.21) and neutrophils (-0.49±0.18). Mutants for
lpar2
have lower LDLc (-0.54±0.27) and HDLc levels (-0.57±0.27), while
gmip
mutants have less vascular lipid deposition (-1.05±0.43), and less co-localizing lipids and neutrophils (-1.26±0.56).

Conclusion:
By characterizing candidate genes for triglyceride levels using a high-throughput, largely image-based approach in zebrafish larvae, we identified previously unanticipated genes that influence a range of cardiometabolic risk factors. This approach is anticipated to identify novel drug targets.
Abstract 486: Type 2 Cytokines Are Required for the Resolution of Injury in Neonatal Hearts Following Ischemic Injury
Authors: Chen Francis M , Leung Fung Ping , Tse Gary , Wong Jack Wing Tak
Published: March 2019

Background:
Type 2 signals such as interleukin-4 (IL-4) and interleukin-13 (IL-13) have been canonically defined as skewing naïve T-cells to Th2 cells and upregulating anti-inflammatory immune programs following injury. As opposed to adults, the immature immune system of the neonate basally prefers a Th2 versus a Th1 profile to maintain fetomaternal tolerance during development.

Hypothesis:
Due to the required activation of type 2 immunity for proper resolution of injury coupled with the inherent Th2 immune profile of the neonate, we
hypothesized
that IL-4 and IL-13 play an indispensable role in the proper resolution of injury in the neonatal heart niche by fostering an anti-inflammatory response conducive for niche remodeling and growth factor release.

Methods and Results:
Neonatal Balb/C and IL-4
-/-
/IL-13
-/-
(DKO) immune populations and overall heart regenerative capacity after left anterior descending (LAD) coronary artery ligation were profiled using flow cytometry and transthoracic echocardiography. DKO mice had significantly reduced CD4/CD8 ratios and suppression of CD206
+
alternatively activated macrophages whilst increasing the population of pro-inflammatory Ly6c
Hi
monocytes. WT Balb/C mice had high CD4/CD8 ratios and actively reduced Ly6c
Hi
populations, preferring Ly6c
Mid
populations and anti-inflammatory CD206
+
alternatively-activated macrophages. Furthermore, echocardiography demonstrated a reduction in both EF and FS in DKO mice following LAD ligation compared to control mice, which had complete restoration of EF and FS.

Summary:
Our results confirmed that both IL-4 and IL-13 are required for mediating a Th2/M2 immune response following LAD ligation in the neonate, thereby permitting a niche that is conducive for regeneration. Future studies are needed to determine whether application of IL-4 and IL-13 in adult mice with myocardial infarction can similarly promote cardiac regeneration.
Abstract 500: Endothelial Specific
Meis1
Knockout Protects Cells from Doxorubicin-Induced Apoptosis
Authors: Chen Miao , Ledford Benjamin , Barron Catherine M , He Jia-Qiang
Published: March 2019

Introduction:
Meis1
belongs to the TALE (three amino-acid loop extension) subclass of the homeobox gene families. It is a highly conserved transcription factor in all eukaryotes and plays a crucial role in anatomical development during embryogenesis and maintaining homeostasis after birth. However, little is known about the role of
Meis1
gene on regulating functions of endothelial cells (ECs). Our preliminary study found that EC-specific knockout (KO) of
Meis1
showed protective benefits in response to hindlimb ischemia, such as increased blood flow and decreased toe apoptosis. We hypothesize that deletion of
Meis1
may enhance EC viability or resistance to harsh environment. The present study aims to explore the potential underlying mechanisms using an
in vitro
apoptotic condition induced by doxorubicin (Dox).

Methods:
EC-specific deletion of
Meis1
gene was generated by crossbreeding

Meis1
flox/flox

mice with
Tie2-Cre
mice. Following confirmation of genotyping, ECs were isolated from the lungs of 3-5 WT and KO newborn pups using CD31 microbeads. Cells were then plated and treated with 0, 0.5, and 1 μM Dox for 24 hours prior to various assays.

Results:
Our qPCR data indicated that
KDR
expression was ~100-times more in CD31
+
fraction than in CD31
-
fraction both in WT and KO groups (n=4); while in KO group,
Meis1
expression was significantly decreased to 20% of WT control, suggesting the successful isolation of
Meis1-KO
ECs. Further analysis with immunocytochemistry indicated that 83.1±2.3% CD31
+
cells were also KDR
+
, compared to 8.3±1.2% KDR
+
cells in CD31
-
fraction (n=7-8). Using EC-specific dye uptake assay, we found that 84.6±2.4% CD31
+
cells were positive for Dil-Ac-LDL, compared to 7.9±3.2% in CD31
-
cells (n=6), which appears to correspond to more tube formation observed in KO than WT groups. As we expected,
Meis1-KO
ECs demonstrated greater cell viability (81.2±6.1%) than WT cells (58.0±1.6%) under 0.5 μM Dox treatment (n=5-6). The increased viability in KO ECs may be due to the decreased expression of
Bax
, a pro-apoptotic factor.

Conclusions:
We conclude that activation of
Meis1
gene may facilitate EC death under ischemic condition while deletion of
Mei1
gene promote protective effects on ECs.
Abstract 504: MicroRNA Signatures in Murine Aorta and Carotid Arteries
Authors: Faffe Debora , Curty-Costa Ernesto , Hoffmann Luisa , Silva Rosane , Urmenyi Turan P
Published: March 2019

Atherosclerosis, a major cause of mortality, affects arteries diffusely. However, some vascular beds are preferably involved. In the last decade microRNAs (miRs) have emerged as key regulators of gene expression in physiological or pathophysiological processes. Here, we investigated whether different arteries, commonly affected by atherosclerosis, present specific miR expression signatures. For this purpose, aorta (Ao) and carotid (Ca) arteries of four healthy male Wistar rats were dissected, total RNA was extracted with Trizol, and enriched for small RNA fraction. MicroRNAs were then sequenced using massive parallel sequencing (RNA-Seq) on Ion Torrent PGM platform. Data were analyzed using CLC Genomics Workbench software. We identified 266 mature miRs in Ao and 421 in Ca, 260 were common between arteries. Differential expression analysis (EDGE) showed increased expression (IE) of 16 miRs in Ao and reduced expression (RE) of 54 miRs in Ao, relative to Ca. The lists of differently expressed miRs were subjected to
in silico
functional analyzes: (1) computational target prediction, using miRWalk tool, identified 1,094 target genes for IE list and 3,574 for RE list; (2) gene-set enrichment analysis in the lists of putative target genes, using EnrichR tool, showed differences in enriched biological processes, such as response to shear stress, regulation of endothelial proliferation, smooth muscle metabolism, and response to FGF signaling (which were enriched in RE list); (3) construction of regulatory networks for differently expressed miRs, using miRnet, identified metabolic pathway components differently regulated between arteries, such as inhibition of SIRT1 and increased expression of HIF1-alpha in Ao. Taken together, our results show differences in miR signature between arteries, suggesting that specific expression profiles of miR may play a role in the vascular behavior as well as in atherosclerosis pathophysiology.
Abstract 529: Digoxin and Platelet Activation in Atrial fibrillation Patients
Authors: Pastori Daniele , carnevale Roberto , Bartimoccia Simona , Nocella Cristina , Novo Marta , Violi Francesco , Pignatelli Pasquale
Published: March 2019

Background:
digoxin use was shown to be associated with an increased risk of cardiovascular events In atrial fibrillation (AF) . We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation.

Methods:
This is a post-hoc analysis from an observational prospective study of AF patients treated with vitamin K antagonists. Patients were divided into two groups balanced for age, sex and cardiovascular risk factors: digoxin users (n=132) and non-users (n=388). Urinary excretion of 11-dehydro-thromboxane B
2
(TxB
2
), a marker of platelet activation and serum digoxin concentration (SDC) were measured. In vitro experiments were performed on platelets from healthy subjects treated with scalar doses of digoxin (0.6 - 2.4 ng/ml).

Results:
Mean age of digoxin users was 75.2±7.2 years and 47.7% were women. Median overall 11-dehydro-TxB
2
was 105.0 (IQR 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 (IQR 0.40-1.00) ng/ml. Urinary 11-dehydro-TxB
2
and SDC were significantly correlated (rS=0.350, p<0.001). Patients in the upper tertile of SDC showed significantly higher urinary11-dehydro-TxB
2
compared to non-digoxin users (p=0.019). In vitro study showed that digoxin at concentration ≥1.2 ng/ml induced calcium mobilization, arachidonic acid (AA) release, TxB
2
biosynthesis and platelet aggregation, that were inhibited by the calcium chelator Ethylenediaminetetraacetic acid (EDTA) or by the specific phospholipase A
2
(PLA
2
) inhibitor Arachidonyl trifluoromethyl ketone (ACOOCF3).

Conclusion:
We found a significant in vivo correlation between SDC and platelet activation. In vitro study showed that supra-therapeutic digoxin concentrations increased platelet aggregation via calcium-related PLA
2
phosphorylation. This finding provides new insights into the association between digoxin use and cardiovascular complications in AF patients.
Abstract 545: Evolocumab Alters LDL-cholesterol Levels but Not Glycemic Profile and Liver Lipids in Chow Fed and High fat/Cholesterol/Fructose Fed Golden Syrian Hamsters
Authors: Briand Francois , Burr Noémie , Brousseau Emmanuel , Sulpice Thierry
Published: March 2019

Background:
Beside its effect on LDL-cholesterol levels, proprotein convertase subtilisin/kexin9 (PCSK9) could also be linked to glucose metabolism and non-alcoholic fatty liver diseases. Here we evaluated whether Evolocumab (EVO), a monoclonal antibody targeting PCSK9, may alter glycemic profile and liver lipids in hamsters.

Methods:
Hamsters were maintained on either a control chow (CC) or high fat/cholesterol/fructose (HFCF) diet for 5 weeks. After 2 weeks of diet, hamsters were randomized into 2 groups, control or EVO at 30mg/kg s.c. once weekly for 3 weeks, to evaluate the effects on lipoprotein/glycemic profiles, and liver lipids.

Results:
When compared with CC diet, HFCF diet significantly increased total cholesterol and LDL-cholesterol levels by 87% and 102%. Hamsters fed the HFCF diet also showed higher HOMA-IR index of insulin resistance, higher blood glucose area under the curve (AUC) during an oral glucose tolerance test (OGTT), and higher liver lipids (triglycerides: +197%, total cholesterol: +421%, both p<0.001 vs. CC). However, plasma PCSK9 levels were markedly reduced by 47% in HFCF fed hamsters (p<0.001 vs. CC). As expected, treatment with EVO significantly increased hepatic LDL-receptor protein levels by 1.74-fold and reduced plasma LDL-cholesterol by up to 28% in CC fed hamsters (p<0.01 vs. CC control). The LDL-cholesterol reduction was also observed in HFCF fed hamsters treated with EVO, but to a lesser extent (up to 16%). In both CC and HFCF fed hamsters, EVO did not alter fasting glycemia and plasma insulin levels. IN CC fed hamsters, EVO transiently reduced blood glucose levels by 15% at time 15 minutes (p<0.01 vs. CC control) after an oral glucose load. However, EVO did not change blood glucose levels AUC or plasma insulin levels during the OGTT, in neither CC nor HFCF fed hamsters. As well, no change in liver lipids levels was observed in CC or HFCF fed hamsters treated with EVO.

Conclusion:
Our data indicate that diet-induced insulin resistance and liver steatosis in hamsters are not associated with elevated plasma PCSK9 levels, and are not altered by Evolocumab. Whether these results are specific to our hamster model and can translate to humans remains to be further investigated.
Abstract 556: Structural Characterization of Distinct Subparticles of Human High-density Lipoproteins Isolated from Plasma
Authors: Melchior John T , Street Scott E , Shah Amy S , Jerome W. Jay , Hart Rachel , Heinecke Jay W , He Yi , Andraski Allison B , Furtado Jeremy D , Sacks Frank M , Davidson W. Sean
Published: March 2019

High-density lipoproteins (HDL) play a critical role in lipid transport and the maintenance of vascular homeostasis. HDL consists of a heterogeneous population of particles that vary in size, composition, and functionality. HDL is proteomically diverse; it can host at least 95 different proteins and structure-function studies suggest that specific protein complements on the surface of the particles are responsible for the broad spectrum of HDL function.

The goal of this study was to isolate and fully characterize specific subspecies of HDL based on their protein complement and particle size. We built upon our previous studies where we segregated lipoproteins using immunoaffinity chromatography using antibodies specific to the two major scaffold proteins of HDL; apolipoprotein (apo)A-I and apoA-II. Two populations were obtained directly from plasma that either had both apoA-I and apoA-II (LpA-I/A-II) or just apoA-I with no apoA-II (LpA-I). These subpopulations were further fractionated using size exclusion chromatography using four superdex columns in series.

We observed at least three distinctly sized particles within the LpA-I/A-II subfraction and two distinctly sized particles in the LpA-I subfraction which all elute in the size range of HDL. We successfully isolated the “large” and “small” subpopulations observed in the LpA-I subfraction and compositional analysis revealed profound proteomic differences between the two populations. Particles in the larger fraction were enriched in more common apolipoprotiens important for lipid metabolic processes such as apoL-I, apoD, apoM and apoE. Conversely, the smaller LpA-I particles were enriched in proteins important for immune response and complement activation. Using multiple cross-linking reagents, we have mapped the protein-protein interactions on each of these subpopulations to begin generating detailed structural models of these particles
in silico
.