bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 311: Cardiac Hypertrophy and Epicardial Fat Inflammation in Vitamin D-Deficient and Hypercholesterolemic Swine
Authors: Gupta Gaurav K , Agrawal Tanupriya , DelCore Michael G , Agrawal Devendra K
Published: March 2022

Introduction:
Vitamin D is a sectosteroid that functions through Vitamin D receptor (VDR), a transcription factor, which controls the transcription of many targets genes. Vitamin D deficiency has been linked with cardiovascular diseases, including heart failure and coronary artery disease. Suppressor of cytokine signaling (SOCS)-3 regulates different biological processes such as inflammation and cellular differentiation and is an endogenous negative regulator of cardiac hypertrophy.

Objective:
The purpose of this study was to test the hypothesis that vitamin D deficiency causes decreased expression of VDR and SOCS-3 in cardiomyocytes, and this correlates with cardiac hypertrophy and increased pro-inflammatory profile in epicardial adipose tissue.

Methods:
Eight female Yucatan miniswine were fed vitamin D-sufficient (900 IU/d) or vitamin D-deficient hypercholesterolemic diet. Complete lipid profile, comprehensive metabolic panel, and serum 25(OH)D levels were regularly measured. After 12 months animals were euthanized and histological and immunohistochemical studies were performed on myocardium and epicardial fat. The mRNA transcripts of VDR and SOCS-3 in the myocardium and epicardial fat tissue were quantified by qPCR.

Results:
Histological studies showed cardiac hypertrophy, as judged by cardiac myocyte cross sectional area, in vitamin D-deficient group. Immunohistochemical and real-time PCR analyses showed significantly decreased protein and mRNA expression of VDR and SOCS-3 in cardiomyocytes of vitamin D-deficient animals. Epicardial adipose tissue from vitamin D-deficient group had significantly higher expression of TNF-α, IL-6, MCP-1, and adiponectin in association with increased inflammatory cellular infiltrate. Interestingly, epicardial fat from vitamin D-deficient group had significantly decreased expression of SOCS-3.

Conclusion:
These data suggest that vitamin D deficiency induces hypertrophy in cardiomyocytes which is associated with decreased expression of VDR and SOCS-3. Vitamin D deficiency is also associated with increased inflammatory markers in epicardial fat. Activity of VDR in the body is controlled through regulation of vitamin D metabolites. Therefore, restoration of VDR function by supplementation of VDR ligands in vitamin D- deficient population might be helpful in reducing inflammation and cardiovascular risk.
Abstract 315: Potential Regulation of Intimal Hyperplasia by ADAM12 and Polo-Like Kinase-1 in Human Saphenous Vein Grafts in Coronary Artery Bypass Patients
Authors: Sur Swastika , Rao Velidi , Kansal Vikash , Agrawal Devendra K
Published: March 2022

Proliferation and migration of smooth muscle cells (SMCs) are the key events in the development of intimal hyperplasia, causing coronary artery bypass graft (CABG) failure. Although internal mammary artery (IMA) is comparatively immune to restenosis, saphenous veins (SV) are most commonly used bypass conduits. The underlying mechanisms that makes SV graft more prone to restenosis are not known as yet. The matrix metalloproteinases (MMPs) and kinases may be potential contributing factors in uncontrolled proliferation, migration and invasion of the graft conduit. In this study, we compared SMCs cultured from SV with IMA of the same patients undergoing CABG. The mRNA expression of polo-like kinases (PLKs), HER-2, ADAM 12L (long-form) and ADAM12S (short-form) were quantified by real-time PCR. The mRNA expression of cell proliferation related protein Ki-67 was measured in both conduits. Migration rate was determined using scratch assay. The mRNA expression of PLK-1, PLK-3, PLK-4, HER-2, ADAM 12L and ADAM12S were significantly higher in human SV SMCs compared to IMA SMCs. Also, SV SMCs showed higher proliferation and migration than IMA SMCs, which may be associated with increased expression of PLKs, ADAM 12 and HER-2. The protease domain of ADAM12 can cleave ECM substrates and cause migration. PLK1 is overexpressed in a variety of pathophysiological conditions and its expression correlates with cellular progression. In contrast, PLK-2 mRNA expression was higher in IMA SMCs compared to SV SMCs. The kinase activity of PLK-2 inhibits oncogenic transformation. Thus, overexpression of PLK 1 and ADAM12 in human SV bypass conduit may cause uncontrolled proliferation, migration and invasion leading to its increased incidence of intimal hyperplasia and vein graft failure in CABG patients.
Abstract 323: Dietary Saturated Fat Increases the Susceptibility of Low-Density Lipoprotein to Aggregation by Sphingomyelinase
Authors: öörni Katariina , Lähteenmäki Hanna , Aittokallio Tero , Taskinen Marja-Riitta , Jauhiainen Matti , Kovanen Petri T
Published: March 2022

Early steps in the development of atherosclerosis are characterized by accumulation of extracellular aggregated low density lipoprotein (LDL) particles in the arterial intima. Sphingomyelinase (SMase), an intimal enzyme that hydrolyses sphingomyelin on the surface monolayer of LDL particles, is particularly effective in inducing LDL aggregation. In this study, we analyzed individual differences in SMase-induced LDL aggregation with the aim of identifying specific lipid components of LDL that can be linked with their susceptibility for aggregation. For this purpose we isolated plasma LDL from 100 randomly selected subjects participating in the Finnish Health 2000 Health Examination Survey. LDL aggregation was analyzed by measuring the size of LDL aggregates with dynamic light scattering during their modification by human recombinant sphingomyelinase. Large variations were observed in the aggregation lag phase between the different LDL donors. In particular, LDL from some donors expressed rapid aggregation potential, whereas LDL from other donors was very resistant to aggregation. Large aggregates (>1000 nm) were observed in 13 of the LDL samples after incubation for 2 h in the presence of SMase and the size of the aggregates plateaued at about 4500 nm, the first samples reaching this size after incubation for 3 h. In four out of the 100 samples only very small aggregation was observed and LDL particles had an average size <350 nm after incubation for 6 h. The intake of saturated fat was found to correlate positively with increased susceptibility for LDL aggregation (
p
≤0.005
)
. Thus, LDL isolated from butter-using donors aggregated faster than LDL derived from vegetable oil -using donors. Lipidomic analysis demonstrated that LDL particles with the highest susceptibility for aggregation had a larger number of saturated fatty acids in their surface phospholipids when compared with that of the aggregation-resistant LDL particles. This study provides novel insights of the influence of dietary fats on LDL atherogenicity.
Abstract 328: High-Density Lipoproteins Increase miR-223 Levels in Human Endothelial Cells, Which Regulate Scavenger Receptor Class B Member 1
Authors: Tabet Fatiha , Vickers Kasey C , Catherinet Claire , Cuesta Torres Luisa F , Shoucri Bassem M , Barter Philip J , Remaley Alan T , Rye Kerry-Anne
Published: March 2022

Background:
MicroRNAs (miRNAs) are recently discovered small non-encoding RNAs that control and regulate gene expression. We and others have recently demonstrated that miRNAs play key roles in lipoprotein metabolism. Here we aim to determine if miR-223 regulates scavenger receptor class B member 1 (SR-B1) in human endothelial cells.

Methods and results:
In silico target prediction studies suggest that SR-BI harbors a miR-223 target site within its 3’ untranslated region (3’UTR). This possibility was investigated by determining the miRNA profile of human coronary artery endothelial cells (HCAECs) using TaqMan arrays. Mature miR-223 levels were found in HCAECs and confirmed by real-time PCR. Here we demonstrate that incubation of HCAECs with native HDL (1 mg protein/ml) for 16 h increased miR-223 levels by 4-fold increase (p<0.007) as assessed by TaqMan Assay and decreased SR-B1 mRNA levels by 29±6% (p<0.05) as measured by real-time PCR. To determine if endogenous levels of miR-223 in HCAECs are sufficient to repress SR-BI mRNA, we used hairpin inhibitors (100 nM) to block miR-223 activity. Inhibition of miR-223 resulted in an increase in SR-B1 mRNA levels by 33±11% (p<0.05). Furthermore, transfection of HCAECs with miR-223 mimic (100 nM) decreased SR-B1 mRNA levels by 27±4% (p<0.01). Transient transfection of HCAECs with miR-223 mimics also reduced SR-BI protein levels by 30±9%. Using gene reporter (luciferase) assays, we determined that miR-223 directly targets the 3’UTR of SR-B1.

Conclusion:
miR-223 directly targets and represses endothelial cell SR-B1 levels, and may regulate HDL-induced cholesterol efflux and lipoprotein metabolism.
Abstract 337: Extensive Coronary Artery Atherosclerosis and Myocardial Infarction in SR-BI/LDLR Double Knockout Mice Fed 4 Different Atherogenic Diets
Authors: Fuller Mark T , Trigatti Bernardo L
Published: March 2022

Mice lacking the HDL receptor, SR-BI, are susceptible to spontaneous or diet-inducible occlusive coronary artery (CA) atherosclerosis and myocardial infarction (MI) in the context of an absence, or mutated form of apoE, respectively. We tested the effects of SR-BI deficiency on aortic sinus and CA atherosclerosis in LDL receptor deficient mice fed four different atherogenic diets; the HFCC and HFC diets are composed of 15% fat, 1.25% cholesterol and either contain or lack 0.5% sodium cholate, respectively, the HC diet is a normal chow diet supplemented with 2% cholesterol, and the western diet contains 21% butter fat and 0.15% cholesterol.

SR-BI/LDLR double knockout (dKO) mice fed the HFCC and HFC diets exhibited reduced survival; average survival was 3.5 and 9.5 weeks, respectively. Reduced survival was not observed in LDLR single knockout (sKO) control mice. Plasma lipoprotein cholesterol levels and triglyercide secretion rates were lower in dKO mice compared to sKO mice. Conversely, dKO mice developed significantly larger atherosclerotic plaques in their aortic sinuses compared to sKO mice, and severe occlusive CA atherosclerosis which was not observed in sKO mice. DKO mice fed the HC diet or western diet did not exhibit reduced survival up to 12 weeks of feeding, however CA atherosclerosis was still observed. CA atherosclerosis was accompanied by platelet positive staining and myocardial fibrosis, indicating MI. CA plaques in dKO mice were characterized by the presence of collagen and both CD68 and smooth muscle actin positive staining, indicating complex fibrous plaques consisting of both macrophages and smooth muscle cells. Plaque free CAs and CAs containing small, non-occlusive plaques stained positive for markers of activated endothelium. Importantly, dKO mice fed the HC diet had largest burden of atherosclerosis in their aortic sinuses whereas the extent of atherosclerosis in their CAs was the mild in comparison to that observed dKO mice fed the HFCC and HFC diets. This observation suggests that the development of atherosclerosis in the CAs and the aortic sinus are influenced by different factors.
Abstract 345: Associations Between Kidney Dysfunction and the Severity of Coronary and Aortic Atherosclerosis
Authors: Momiyama Yukihiko , Ohmori Reiko , Fayad Zahi A , Kato Ryuichi , Taniguchi Hiroaki , Ohsuzu Fumitaka
Published: March 2022

Chronic kidney disease (CKD) is considered to be associated with increased risk of coronary artery disease (CAD). To elucidate the associations between kidney dysfunction (KD) and the severity of coronary and aortic atherosclerosis, we performed aortic black-blood MRI in 149 patients (pts) (age 64±9 yrs) undergoing coronary angiography. Regarding MRI, 9 slices of thoracic and 9 slices of abdominal aorta were obtained at 12-mm intervals. Plaque extent in each slice was scored. Severity of aortic atherosclerosis was represented as the sum of scores. On coronary angiograms, severity of coronary atherosclerosis was represented as the numbers of stenotic vessels and segments. KD was evaluated by eGFR.
RESULTS:
The 149 pts were divided into tertiles by eGFR: 51±10 (T1), 60±3 (T2) and 86±12 ml/min/1.73
2
(T3). CKD (eGFR<60) was present in 41 (28%) pts. CAD (>50% stenosis) and thoracic and abdominal aortic plaques were found in 105 (70%), 95 (64%) and 136 (91%) pts, respectively. Regarding coronary atherosclerosis, the number of >25% stenotic coronary segments correlated with eGFR (r=-0.25, P<0.005). Stepwise decrease in the number of >50% stenotic vessels was found depending on eGFR tertiles: 1.5±1.0 (T1), 1.3±1.2 (T2) and 0.9±0.8 (T3) (P<0.025). In multivariate analysis, reduced eGFR was associated with the severity of coronary atherosclerosis independent of risk factors. Regarding aortic atherosclerosis, the severity of thoracic aortic atherosclerosis correlated with eGFR (r=-0.22, P<0.01), but abdominal aortic atherosclerosis did not (P=NS). The severity of thoracic aortic atherosclerosis decreased stepwise with eGFR tertiles: 3.0 (median) (T1), 2.0 (T2) and 1.0 (T3) (P<0.02), whereas the severity of abdominal atherosclerosis tended to decrease on eGFR tertiles: 8.0 (T1), 7.0 (T2) and 6.0 (T3) (P=NS). However, in multivariate analysis, reduced eGFR was not an independent factor for thoracic or abdominal aortic atherosclerosis.
CONCLUSION:
The severity of coronary and thoracic, but not abdominal, aortic atherosclerosis correlated with the degree of KD. However, KD was an independent factor for coronary atherosclerosis but not for aortic atherosclerosis. KD is more likely to be associated with coronary atherosclerosis than aortic atherosclerosis.
Abstract 349: Hypoxia Induces the Expression of the Neuroimmune Guidance Cue Netrin-1 in Atherosclerotic Plaques: A Mechanism for Macrophage Retention
Authors: Ramkhelawon Bhama , Van Gills Janine M , Rayner Katey J , Paranath Sajesh , Feig Jessica L , Fisher Edward A , Moore Kathryn J
Published: March 2022

Hypoxia is intimately linked to atherosclerosis and promotes its progression by increasing lipid accumulation, inflammation and angiogenesis. We recently demonstrated that the neuroimmune guidance cue, netrin-1 (Ntn1) is expressed in atherosclerotic lesions where it inactivates macrophage (Mφ) migration to chemokines implicated in the egress of these inflammatory cells from plaques, including MCP-1 and CCL19. However, the mechanisms governing Ntn1 expression in atherosclerosis are not known. Immunostaining of mouse atherosclerotic plaques revealed that Ntn1 colocalized with a hypoxia probe in Mφ-rich regions of the plaque. We hypothesized that oxidative stress in the plaque provokes Ntn1 expression in Mφ, leading to local secretion of this negative regulator of Mφ migration. In vitro, stimulation of Mφ with oxidized LDL induced mRNA levels of Ntn1 and its receptor Unc5b by 3- and 6-fold respectively (p<0.01), as well as the HIF-1α target gene, Vegf (3-fold). Similarly, hypoxia mimetics such as CoCL2 (0.1 mM) and DMOG (1 mM), or exposure of Mφ to 4% O2 also upregulated Mφ expression of Ntn1 and Unc5b. Notably, these responses were abrograted by inhibiting HIF1α suggesting a causative role for this transcription factor in regulating Ntn1 and Unc5b expression in Mφ. Indeed, oxLDL-induced hypoxia increased Ntn1 and Unc5b promoter-luciferase activity by 2-5-fold, which was lost upon treatment with a HIF1α inhibitor. Furthermore, gene expression profiling of J774 macrophages overexpressing a stable form of Hif-1α (J774Hif) showed that both Ntn1 and Unc5b mRNAs were upregulated compared to control J744 cells (J774Con). Moreover, J774Hif Mφ showed reduced migration to MCP-1 compared to J774Con, consistent with the secretion of Netrin1 by these cells, and this was reversed by pretreating J774Hif with recombinant Unc5b-Fc to block the effects of Netrin1. Finally, using a bone marrow transplant model we show that targeted deletion of Ntn1 in Mφ dramatically diminishes atherosclerosis progression in Ldlr
-/-
mice, and promotes Mφ emigration from plaques. Together, these data suggest a causative role for hypoxia in Mφ retention and chronic inflammation in atherosclerotic lesions though its induction of this negative regulator of Mφ migration.
Abstract 350: Inhibition of IGF-1 Signaling Increases Serum Cholesterol and Plaque Burden in ApoE Knockout Mice but Promotes a Stable Plaque Phenotype
Authors: Roy Joy , Joneberg Jeanna , Axelson Magnus , Larson Olle , Hedin Ulf , Razuvaev Anton
Published: March 2022

Insulin like growth factor-1 (IGF-1) has been shown to have a prominent effect on smooth muscle cell proliferation, migration and apoptosis. We and others have previously shown that inhibition of IGF-1 related signaling can attenuate intimal hyperplasia by attenuating smooth muscle cell proliferation and promoting apoptosis after vascular injury. We hypothesized with support from preliminary work in our laboratory that IGF-1 inhibition will decrease atherosclerosis burden, but may negatively affect plaque stability by increasing SMC apoptosis. Picropodophyllin(PPP) is a specific inhibitor of the IGF-1 receptor phosphorylation and is currently under development as an anti-cancer drug. The aim of this study was to study the short and long term effects of IGF-1 inhibition using PPP in ApoE deficient mice

Methods and Results
12 week old Apo E deficient mice (n=64) were treated with an oral preparation of PPP or vehicle (controls) for a period of 10 and 18 weeks. Blood samples were collected before sacrifice and aortas, brachiocephalic trunks and liver samples harvested. Semi-quantitative analysis of the aortic and brachiocephalic trunks using Sudan IV enface staining showed an increase in plaque burden and was significant after 18 weeks. There were no significant differences in weight, serum creatinine, albumin or glucose levels between the two groups. However, we noted 18% lower levels of IGF-1, doubled levels of growth hormone, 30% increase in serum cholesterol levels in the PPP treated group (p<0.05). RT-PCR analysis showed a 38% decrease in LDL receptor expression in the liver in the PPP group. Further immunohistochemical analysis of the plaques showed more fibrosis and lower cholesterol in the sections of the brachiocephalic trunks of the PPP group.

Conclusion
In contrast to our hypothesis, PPP treatment increased plaque burden but led to plaques with a more stable phenotype. The proposed mechanism is via an increase in serum cholesterol, however further work has to be performed to confirm the pathways involved. In summary, our results suggest that the effects of inhibition of IGF-1R phosphorylation in atherosclerotic animals cannot be extrapolated from results in healthy subjects and has to be considered when designing drugs in the future.
Abstract 357: Aldosterone Inactivates the Endothelin-B Receptor via a Thiol Redox Switch to Modulate Pulmonary Arterial Hypertension
Authors: Maron Bradley A , Zhang Ying-Yi , Loscalzo Joseph , Leopold Jane A
Published: March 2022

Hyperaldosteronism (ALDO) induces a vasculopathy in systemic blood vessels by increasing reactive oxygen species (ROS) that decrease nitric oxide (NO) levels. In pulmonary artery endothelial cells (PAECs), the endothelin-B receptor (ET
B
) activates endothelial NO synthase (eNOS) to generate NO. The effect of ALDO-induced ROS on ET
B
-NO signaling in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that in PAH, ALDO increases ROS levels in PAECs, which oxidatively modify the regulatory ET
B
cysteinyl thiol Cys405 to impair ET
B
activation of eNOS and decrease NO levels. To test this hypothesis, human PAECs were treated with vehicle (V) or ALDO (10
-7
mol/l) for 24 h and hydrogen peroxide (H
2
O
2
) levels were assessed by Amplex Red assay. Compared to V-treated cells, ALDO increased significantly H
2
O
2
levels (65.4 ± 1.6 vs. 100.6 ± 3.5 μM/μg protein, p<0.05). In cells exposed to endothelin-1 (ET-1)(10 nM) to stimulate ET
B
signaling, ALDO decreased eNOS activity by 59% (p<0.05) and nitrite (NO
2
-
) formation by 60% (p<0.05) without affecting eNOS protein levels. ALDO-induced ROS led to the oxidative formation of two disulfide bonds in ET
B
as detected by PEG-maleimide-labeled immunoblotting. To confirm that oxidative modification of Cys405 influenced ET
B
-dependent NO generation, COS7 cells were transfected with wild type (WT)-eNOS and WT-ET
B
or mutant ET
B
DNA containing an alanine substitution at position 405 (C405A-ET
B
), and cells were exposed to V or H
2
O
2
(200 μM) for 60 min. We observed that H
2
O
2
decreased ET
B
-mediated NO
2
-
formation by 78% (p<0.02) in WT-ET
B
-transfected cells; in C405A-ET
B
-transfected cells, H
2
O
2
decreased NO
2
-
levels by only 45%. Next, we assessed the effect of ALDO on NO signaling in PAH in vivo. In monocrotaline (MCT)-treated rats with PAH, plasma ALDO was increased by 406% compared to V-treated rats (p<0.02). ALDO antagonism with spironolactone (25 mg/kg/d) increased NO
2
-
levels in lung homogenates of MCT-treated rats by 50% (p<0.05) and decreased significantly pulmonary artery systolic pressure by cardiac catheterization (89 ± 5 vs. 69 ± 5 mm Hg, p<0.04). These data suggest that ALDO/the regulatory redox switch of ET
B
, Cys405, may represent novel therapeutic target(s) to restore ET
B
-NO signaling in patients with PAH.
Abstract 36: Fatty Acid Amide Hydrolase Deficiency Is Associated with a Vulnerable Plaque Phenotype in Atherosclerotic Mice
Authors: Steffens Sabine , Lenglet Sébastien , Thomas Aurélien , Montecucco Fabrizio , Burger Fabienne , Pelli Graziano , Galan Katia , Cravatt Benjamin , Staub Christian
Published: March 2022

Elevated endocannabinoid levels are linked with the development of atherosclerotic vascular disease and coronary circulatory dysfunction in obese individuals, a precursor of coronary artery disease. However, it remains unclear whether endocannabinoid levels represent a risk factor or diagnostic biomarker for acute atherosclerotic vascular events. So far, a causal role of increased endocannabinoid levels in atherosclerotic plaque vulnerability and occurrence of acute clinical events has not been investigated. Here, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability.

We interbred apolipoprotein E-deficient (ApoE-/-) mice with FAAH
-/-
mice to generate ApoE-/-FAAH
-/-
mice and measured serum levels of anandamide and related FAAH metabolites palmitoyl- and oleoylethanolamide. We assessed atherosclerosis in ApoE
-/-
and ApoE-/-FAAH
-/-
mice after 5, 10 and 15 weeks on high cholesterol diet (HCD; 1.25% cholesterol).

Levels of FAAH metabolites anandamide, palmitoyl- and oleoylethanolamide were 1.4 to 2-fold higher in FAAH-/-ApoE-/-mice. FAAH deficiency attenuated atherosclerotic plaque size increase (by ∼50% in thoraco-abdominal aortas after 15 weeks HCD; n=7-10; P=0.007), but plaques had significantly lower content of smooth muscle cells (36% less after 10 weeks HCD in aortic sinuses; n=10-15; P=0.01) and increased matrix metalloproteinase MMP-9 expression (by 73%; P=0.049). There was no difference in macrophage content, but a 65% increase in neutrophil infiltrates (P=0.0007) in aortic sinus plaques from ApoE-/-FAAH
-/-
mice compared to ApoE
-/-
controls. This was accompanied by 1.9-fold increased chemokine CXCL1 mRNA levels (P =0.004) in mouse aortas. CXCL1 expression was confirmed by immunostaining which revealed colocalization with lesional macrophages. MMP-9 mainly colocalized with neutrophils rather than macrophages (correlation coefficient r: 0.6529; P=0.006).

Increased levels of endocannabinoid anandamide and related FAAH metabolite levels are associated with the development of smaller atherosclerotic plaques with more vulnerable phenotype.
Abstract 360: Molecular and Cellular Dynamics of Merlin in Vascular Cells via Mechanotransduction
Authors: Shan Chun X , McDonnell Brian , McGinn Ciaran , Dowling Paul , Cummins Philip , Murphy Ronan
Published: March 2022

Endothelial integrity plays a pivotal role in normal blood vessel function and disruption of which will lead to the initiation and progression of atherosclerosis. The actin cytoskeleton which provides endothelial structural integrity can be greatly disrupted by many factors, such as inflammation and haemodynamic forces. Altered actin dynamics underpins many (patho-) physiological processes in endothelial cells such as migration, differentiation, proliferation and adhesion.

The ERM (ezrin/radixin/moesin) proteins which belong to erythrocyte 4.1 superfamily are actin adaptor proteins and play an important role in controlling cytoskeleton dynamics. This study focuses on one member of the ERM protein family- Merlin. Merlin (moesin, ezrin and radixin-like protein), has been well characterised as a tumour suppressor, contributing to brain tumours such as schwannoma and meningiomas. However, the role of merlin within the vasculature is poorly understood as is its function in various cellular processes such as migration, proliferation and cell adhesion.

In this current study we exposed Human Aortic Endothelial Cells (HAECs), in an in vitro model, to haemodynamic force (i.e. shear stress and cyclic strain) to exam its influence on the temporal and spatial expression of Merlin. We also investigate if Merlin mediates the regulation of Cell-ECM adhesion, migration and proliferation. Results demonstrate that the expression of Merlin is decreased by exposed to haemodynamic force. Furthermore, we have concluded that Merlin knock-down, via siRNA, decreases HAECs adhesion, migration and proliferation as studied in real-time by an xCELLigence system.
Abstract 364: ROCK2 Regulates Endothelial Hyperpermeability Responses Through Regulation of Basal Junctional Forces
Authors: Van Nieuw Amerongen Geerten , Aman Jurjan , van Bezu Jan , Krishnan Rama , Rajendran Kanitha , van Hinsbergh Victor
Published: March 2022

Rationale
Our previous studies suggested distinct roles for the Rho kinase isoforms ROCK1 and ROCK2 in regulating vascular permeability in vivo. Although being the dominant isoform, it remained unclear how ROCK2 regulates endothelial permeability.

Objective
To investigate the mechanism of ROCK2-mediated endothelial hyperpermeability responses through selective targeting of either Rho kinase isoform.

Methods
and
Results
Endothelial hyperpermeability was induced
in vitro
by thrombin and was assessed by the passage of a tracer or by Electrical Cell-substrate Impedance Sensing (ECIS). Endothelial contractile forces were mapped by traction force microscopy. Silencing of ROCK2, but not ROCK1 attenuated thrombin-induced hyperpermeability of culured human endothelial monolayers. Simultaneous downregulation of both Rho kinase isoforms further reduced the hyperpermeability. Surprisingly, ROCK2 inhibition did affect neither the thrombin-induced formation of contractile F-actin stress fibers nor the thrombin-induced contractile force enhancements, but rather protected the endothelium from barrier disruption by lowering basal tension in endothelial junctions.

Conclusions
These data identify a novel mechanism for regulating endothelial hyperpermeability: ROCK2 activity is essential for the development of a critical basal isometric cellular tone (the so-called prestress), which presence is necessary for opening of the endothelial barrier in response to inflammatory mediators. GPvNA is supported by the Dutch Heart Foundation (grant 2011T072)
Abstract 365: The Impact of CYP2C19*2 Polymorphism on Endothelial Function and Arterial Stiffness in Patients with Coronary Artery Disease Receiving Clopidogrel
Authors: Zaromitidou Marina , Siasos Gerasimos , Tousoulis Dimitrios , Kioufis Stamatios , Oikonomou Evangelos , Maniatis Konstantinos , Miliou Antigoni , Kolia Christina , Michalea Stavroula , Kokkou Eleni , Papavasiliou Athanasios G , Stefanadis Christodoulos
Published: March 2022

Background:
The role of CYP2C19*2 polymorphism in clopidogrel response and cardiovascular prognosis has come under question in recent studies. Measurement of endothelial function and arterial stiffness are well validated in large population studies as strong predictors of adverse cardiovascular outcomes. We examined the impact of CYP2C19*2 polymorphism on endothelial function and arterial stiffness in coronary artery disease (CAD) patients.

Methods:
The study included 245 patients with stable CAD, receiving clopidogrel regimen (75mg/d). CYP2C19*2 genotyping was performed by real-time polymerase chain reaction. CAD patients were divided in two groups. Group A consisted of patients homozygous for the wild type and group B consisted of patients carrying at least one CYP2C19*2 loss-of-function allele. Endothelial function was evaluated by flow-mediated dilatation (FMD). Carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness.

Results:
Group A included 153 patients (62.4%) and group B 92 patients (37.6%). There was no statistically significant difference between the two groups in age (62±10 vs. 60±12 years, p=0.12), male sex (90% vs. 91%, p=0.65) and the presence of dyslipidemia (74% vs. 70%, p=0.57), diabetes mellitus (27% vs. 27%, p=0.97) and arterial hypertension (80% vs. 81%, p=0.74). Importantly, there was no difference in FMD (4.82±2.17% vs. 4.92±2.47%, p=0.74) and PWV values (9.08±2.02m/sec vs. 8.89±2.49m/sec, p=0.55) between the two groups.

Conclusion:
CYP2C19*2 polymorphism is not associated with endothelial function and arterial stiffness in CAD patients receiving clopidogrel. These findings are consisted with the absence of an effect of the CYP2C19*2 loss-of-function alleles on cardiovascular risk among CAD patients treated with clopidogrel, as it is documented in recent large trials.
Abstract 366: Sirtuin 6 Reduces Osteogenic Signaling in Aortic Valve Interstitial Cells and Vascular Smooth Muscle Cells
Authors: Zhang Heyu , Greiten Lawrence E , Zhang Bin , Miller Jordan D
Published: March 2022

Cardiovascular calcification is often an active process involving re-differentiation of cells to an osteoblast-like phenotype, and is strongly associated with aging. Sirtuins are histone and protein deacetylases that play a critical role in a number of age-related diseases, and SIRT6-deficient mice have the most profound progeroid phenotype. The role of SIRT6 in the regulation of cardiovascular calcification, however, is poorly understood. We hypothesized that age-related reductions in sirtuins promote osteogenic differentiation in cells from aortic valve and aorta. We used aortic valve interstitial cells isolated from SIRT6-deficient mice, pigs, or humans, and vascular smooth muscle cells from mouse aorta to determine whether osteogenic differentiation of cardiovascular cells is regulated by SIRT6 levels. In line with our hypothesis, we found increased p-SMAD1/5/8 levels in cells from SIRT6-deficient aortic valve and aorta. Second, global SIRT inhibition with NAD+-depletion increased histone acetylation and protein levels of the osteogenic proteins Sp7 and Runx2. Third, inhibition of SIRTs enhanced the transcriptional responses of Sp7 and Runx2 when cells were treated with BMP2. Fourth, knockdown of SIRT6 in mouse and human aorta and aortic valve cells by RNA interference increased Sp7 expression. Finally, chronic pharmacological inhibition of SIRTs promoted calcified nodule formation and calcium deposition in cultured mouse/swine aortic valve cells. Collectively, our data suggest that reductions in SIRT6 levels favor amplification of pro-osteogenic signaling and osteogenic differentiation of cardiovascular cells and promote the formation of calcified nodules
in vitro
. These data also suggest that sirtuin re-activation may be a useful therapy to slow initiation and/or progression of cardiovascular calcification
in vivo.
Abstract 368: Increased Platelet Activity in HIV-Infected Subjects on Antiretroviral Therapy Is Inhibited with Low-Dose Aspirin
Authors: Berger Jeffrey S , Nardi Michael A , Montenont Emilie , Valdes Vanessa , Hu Liang , Merolla Michael , Aberg Judith , O’Brien Meagan
Published: March 2022

Introduction:
HIV-infected adults are at increased risk for cardiovascular events and increased platelet activity may contribute to this risk.

Hypothesis:
Platelet activity measurements are increased in subjects with HIV on fully suppressive antiretroviral therapy (ART) and low-dose aspirin attenuates this increase in platelet activity.

Methods:
We studied platelet aggregation in 25 HIV infected subjects on ART with undetectable plasma HIV-1 RNA and median CD4 521 cells/mm
3
and 29 healthy HIV seronegative controls in response to submaximal adenosine diphosphate (ADP, 0.4uM), collagen (0.05ug/ml), epinephrine (0.1uM ), arachidonic acid (AA, 0.15mM) and without agonist (spontaneous platelet aggregation [SPA]). The effects of a 1-week course of aspirin 81mg daily on platelet activity were also investigated. Results are presented as median (interquartile range) and analyzed using the Mann-Whitney U test.

Results:
Compared to controls, subjects with HIV had increased platelet aggregation in response to ADP (13.5% [6.8, 81.6] vs 6.4% [2.8, 10.7], p<0.01), collagen (13.5% [6.8, 81.6] vs 6.4% [2.8, 10.7], p=0.01), AA (81.9% [9.4, 90.2] vs 9.6% [2.8, 77], p<0.01), and SPA, 8.2% [4.7, 13.2] vs 4.5% [2.2, 8.9], p=0.04). There was no difference in baseline aggregation in response to epinephrine. Following aspirin therapy, percent aggregation in response to AA decreased more in subjects with HIV (-43% vs -20%, P=0.04), yet remained significantly higher in subjects with HIV versus controls (9.8% [5.8, 16.6] vs 7.4 [4.4, 9.8], P=0.02). In response to ADP, collagen, epinephrine, and SPA, subjects with HIV experienced numerically greater platelet inhibition than controls and residual platelet aggregation was not significantly different between groups.

Conclusions:
Baseline platelet activity is increased in subjects with HIV infection virologically suppressed on ART which may contribute to the excess cardiovascular risk commonly observed in this population. Subjects with HIV experienced greater decreases in platelet activity in response to aspirin, retaining modestly more platelet activity compared to controls. Cardiovascular prevention studies of aspirin in subjects with HIV are warranted.
Abstract 376: Erythrocyte Microparticles Can Induce Kidney Vaso-Occlusions in Sickle Cell Disease
Authors: Camus Stéphane M , Gausseres Blandine , Bonnin Philippe , Loufrani Laurent , Grimaud Linda , de Moraes Joao A , Renard Jean-Marie , Tedgui Alain , Boulanger Chantal , Tharaux Pierre-Louis , Blanc-Brude Olivier
Published: March 2022

Background—
Patients with sickle cell disease suffer from painful vaso-occlusive crises. These crises are associated with disseminated vaso-occlusions, raised levels of circulating erythrocyte microparticles as well as thrombospondin-1 (TSP1). Microparticules (MP) are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates the shedding of erythrocyte MP and participates in vaso-occlusions.

Methods and Results—
We injected TSP1 to transgenic SAD mice with sickle cell disease, sensitive to renal vaso-occlusions. We characterized circulating MP by FACS after phosphatidylserine labeling. TSP1 injection strongly stimulated MP levels (+260%) and initiated renal vaso-occlusions within 5 minutes, without impacting systemic parameters (heart rate, cardiac output). This was consistent with increased local vascular resistance. In vitro, TSP1 triggered a rapid conversion of purified erythrocytes into echinocytes carrying long and apparently fragile spicules. This was followed by MP shedding. These modifications of erythrocyte phenotype and MP shedding were recapitulated by peptides derived from TSP1 carboxyterminus. We purified the MP shed by erythrocytes in vitro, and administered them back into SAD mice. Erythrocyte MP triggered immediate renal vaso-occlusions, similar to those induced by TSP1. In vitro, purified erythrocyte MP triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion to them and induced endothelial apoptosis. Purified erythrocyte MP also compromised acetylcholine-dependent vasodilation in perfused microvessels. These effects of MP were inhibited by saturating surface phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production.

Conclusions—
We conclude that TSP1 can trigger the production of microparticles by erythrocytes in sickle cell disease. Second, our data reveal that erythrocyte MP can induce endothelial injury, and this mechanism facilitates the occurrence of vaso-occlusive crises in mice. Therefore, our work supports a novel concept that toxic erythrocyte microparticles constitute a direct connection between sickle cell anemia and vascular disease.
Abstract 379: Protective Effect of Ceria Nanoparticle on Focal Cerebral Ischemia in Rats
Authors: Kim Jun Yup , Kim Chi K , Kim Dohoung , Jang Hyunduk , Lee Seung-Hoon
Published: March 2022

Background and Objectives
Ceria, one of the rare earth resources, is a potent catalytic agent using in many industrial fields. Interestingly, after converting ceria into biocompatible nanoparticle via chemical engineering, ceria nanoparticle may be used as anti-oxidant agent to treat diseases related to oxidative injuries. However, few studies have elaborated on the association between ceria nanoparticle and ischemia-reperfusion injury in experimental stroke model.

Methods
We used 3 nm ceria nanoparticle via the combining process using thermal decomposition and reverse micelle method. To make biocompatible, ceria nanoparticle was coated with phospholipids-PEG. We induced ischemia-reperfusion injury in SD rats via modified Longa method, and ceria nanoparticle or PBS was administered intravenously. Infarct volume and the number of apoptotic cells (TUNEL-positive cells) were measured at 24 hours. To identify the anti-oxidant role of ceria nanoparticle, hydroethidine, a marker of superoxide activity, was administered 15 minutes before inducing focal ischemia, and detect ethidine, the product of reaction between superoxide and hydroethidine, using fluorescent microscopy at 3 hours.

Results
We compared three doses of ceria nanoparticle and the infarct volumes were decreased dose-dependently (ρ = -0.39, p < 0.05). The infarct volumes were 130.8 mm3 in ceria-treated group (0.5 mg/kg) and 236.2 mm3 in control group (p < 0.05). The ceria-treated group exhibited a lower number of TUNEL-positive apoptotic cells (ceria-treated group: 32.5 ± 18.3 cells/mm3 ; control group: 63.4 ± 2.4 cell/mm3; p < 0.05). Ethidine (oxidezed hydroethidine) signals were weakly observed in the ischemic cortices of ceria-treated rats compared to control group.

Conclusion
Our study documented that ischemia-reperfusion injury was decreased by ceria nanoparticle via superoxide scavenging effects. This result may indicate the potentially protective effect of ceria nanoparticle on ischemic stroke.
Abstract 382: Mice with Endothelial-Targeted Inactivation of PPAP2B (Lipid Phosphate Phosphatase 3) Display Enhanced Vascular Inflammation and Permeability
Authors: Panchatcharam Manikandan , Miriyala Sumitra , Patil Pooja , Nguyen Adrienne , Sunkara Manjula , Morris Andrew , Escalante-Alcalde Diana , Smyth Susan S
Published: March 2022

Meta-analysis of data from a series of genome-wide association studies (GWAS) revealed a striking association between the
PPAP2B
loci and coronary artery disease. The
PPAP2B
gene encodes for the lipid phosphate phosphatase 3 (LPP3) integral membrane enzyme, that dephosphorylates lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and related bioactive lipids. While emerging evidence suggests a role for both LPA and S1P in experimental models of atherosclerosis, essentially nothing is understood about the pathophysiologic role of LPP3, largely due to the fact that genetic inactivation results embryonic lethality. We report that constitutive inactivation of LPP3 in vascular endothelial cells accomplished by breeding mice with floxed
Ppap2b
to mice expressing Cre recombinase under control of the Tie1 promoter results in embryonic lethality, indicating that dysregulation of LPP3-dependent vascular endothelial cell function likely underlies the developmental phenotype observed in
Ppap2b
-null embryos. Using an estrogen inducible Cre transgene under control of the Tie1 promoter, we have inactivated
Ppap2b
expression in endothelial cells in adult mice and confirmed the lack of vascular endothelial LPP3 by immunologic and biochemical approaches. The absence of vascular endothelial LPP3 results in a 2.2±0.5 fold increase in basal vascular permeability, as assessed by Evans blue dye extravasation in lung tissue. The permeability difference is exaggerated following an inflammatory challenge (LPS; 2 mg/kg), and LPS-induced expression of plasma inflammatory markers IL6 and KC were 3.3±0.5 and 1.9±0.6 fold higher in mice lacking endothelial LPP3. LPS-enhanced permeability in the absence of LPP3 was attenuated by administration of an LPA receptor antagonist. Mice lacking LPA receptors 1 and 2 (LPA1, LPA2) or LPA receptor 4 (LPA4) were protected from LPS-mediated permeability. These results demonstrate a fundamental role for LPP3, possibility via modulation of local LPA levels and signaling through LPA1, LPA2, and LPA4 receptors, in maintaining the integrity of the vascular endothelium.
Abstract 389: Mitochondrial p27 Is Absolutely Required for Endothelial Cell Migration and ATP Production
Authors: Jakob Sascha , Beylebens Marius , Altschmied Joachim , Haendeler Judith
Published: March 2022

The cell cycle inhibitor p27 has been shown to exert non-nuclear functions. Moreover, migration of fibroblasts depends on p27 expression. However, the underlying mechanisms of this phenomenon are largely unknown. Therefore, we first investigated the role of p27 in endothelial cell (EC) migration and showed that its downregulation abrogated migration. Since we previously demonstrated that migration of EC requires intact mitochondria, we analyzed whether it depends on the localization of p27 within the cell. We generated p27 variants, which are exclusively localized either in the nucleus (nucp27) or in the mitochondria (mitop27). Overexpression of mitop27 enhanced EC migration, whereas nucp27 decreased it. Along the same line, mitop27 induced ATP production, which is required for migratory processes. As a potential mechanism for the increased ATP production, we found that mitochondrial p27 interacts with prohibitin, which forms multimeric complexes thereby enhancing respiration and ATP production. Next, we analyzed, which domains in p27 are involved in these processes. Therefore, we created p27 deletion mutants and found that both the N- and the C-terminus are necessary for cell migration and ATP-production. These regions contain several phosphorylation sites, two of which (Ser 10 and Thr 187) are phosphorylated by pro-migratory stimuli. Thus, we cloned corresponding nonphosphorylatable (p27 S10A/T187A) and phosphomimetic (p27 S10D/T187D) mutants of mitop27. Overexpression of the nonphosphorylatable mutant decreased EC migration, while the phosphomimetic mutant increased it. Finally, we investigated the mitochondrial import mechanism of p27. Since p27 lacks a typical mitochondrial targeting sequence, we determined whether it interacts with cytosolic heat shock protein 60 (HSP60), which is known to import proteins into the mitochondria. Indeed, cytosolic p27 is associated with HSP60 and this association is increased under promigratory stimuli.

Taken together, these data demonstrate that mitochondrial p27 and its phosphorylation at the N- and C-termini are required for migration and ATP production and that p27 is imported into the mitochondria in a HSP60 dependent manner.
Abstract 396: Serum Osteoprotegerin (OPG) Differs Significantly in Patients with Calcified Aortic Valve Stenosis in Relation to the Presence of Coronary Atherosclerosis
Authors: Motovska Zuzana , Odvodyova Denisa , Fojt Richard , Karpisek Michal , Vichova Teodora , Tousek Petr , Kocka Viktor , Widimsky Petr
Published: March 2022

Background.
We concentrated on OPG/RANKL in patients with symptomatic calcified AoS of the trileaflet valve, who were being considered for aortic valve replacement (AVR).

Methods.
Serum levels of cytokines were measured in: Group A (N = 101) patients with AoS and normal coronary arteries (assessed using coronary angiography), Group B (N = 105) patients with AoS and significant (stenosis > 50%) coronary atherosclerosis. Control groups consisted of patients without AoS and with normal coronary arteries (group C, N = 39) or with significant coronary atherosclerosis (Group D, N = 30).

Results.
Values of OPG were significantly higher in patients with AoS and normal coronary arteries compared to controls with normal coronary arteries and without AoS. No difference was observed between levels of OPG in patients with AoS and CAD and control group of patients with CAD and without AoS. Multivariable analyses underscored these results (table). Serum levels of RANKL did not differ significantly between groups A vs. C. Serum OPG did not change significantly in patients after AVR (Group A). The difference in serum concentration of OPG in patients after AVR and CABG (Group B) was significant (p<0.001).

Conclusion.
Serum OPG differs significantly in patients with calcified aortic stenosis in relation to the presence of significant coronary atherosclerosis. Our results suggest that OPG plays and important role in pathomechanism of calcified aortic valve disease in patients without coronary atherosclerosis
Abstract 399: Deficiency of Angiotensin Type 1a Receptors in Adipocytes Increases Fat Mass in Angiotensin II-Infused LDLR-Deficient Mice but Has No Effect on Vascular Pathologies
Authors: Putnam Kelly , Karounos Michael , Lewis Eboni , Yiannikouris Frederique , Daugherty Alan , Cassis Lisa A
Published: March 2022

Objectives:
Angiotensin II (AngII) promotes atherosclerosis and abdominal aortic aneurysms (AAA) through the angiotensin type 1a receptor (AT1aR). However, the cell type mediating these effects has not been defined. Adipose tissue surrounding the aorta may influence the inflammatory milieu within the vessel wall and thus contribute to disease progression. The purpose of this study was to determine the effect of adipocyte-AT1aR deficiency on development of AngII and diet-induced vascular pathologies in low density lipoprotein receptor deficient (LDLR-/-) mice.

Methods and results:
LDLR
-/-
mice with adipocyte-AT1aR deficiency (AT1aRaP2, n = 21) and littermates (AT1aRfl/fl, n=17) were fed a high-fat diet (HF, 42% kcal fat; 0.15 cholesterol) for 1 week prior to, and throughout, 28 days of AngII infusion (1,000 ng/kg/min). Adipocyte-AT1aR deficiency had no effect on AAA incidence (external diameter of aorta; AT1aRfl/fl 1.4±0.1 vs. AT1aRaP2 1.5±0.1 mm, p=0.41) and atherosclerotic lesion areas in the aortic arch (AT1aRfl/fl 7.1±0.8 vs. AT1aRaP2 8.3+0.8% lesion area, p=0.49). Serum cholesterol (AT1aRfl/fl 1981±109 vs. AT1aRaP2 2044±85 mg/dL, p=0.65) and triglyceride (AT1aRfl/fl 450±31 vs. AT1aRaP2 496±33 mg/dL, p=0.32) concentrations were also unaffected by adipocyte-AT1aR deficiency. Interestingly, body weight was significantly increased in AngII-infused AT1aRaP2 mice (AT1aRfl/fl 26.8±0.5 vs. AT1aRaP2 28.0±0.5 g, p=0.013 for interaction between genotype and time), due to increases in adipose mass (retroperitoneal fat; AT1aRfl/fl 0.26±0.03 vs. AT1aRaP2 0.36±0.02 g, p=0.017). Mice fed the HF diet for 3 months without AngII infusion did not exhibit any changes in atherosclerotic lesion area in the aortic arch (AT1aRfl/fl 14.3±1% vs. AT1aRaP2 11.7±1%, p=0.14).

Conclusions:
Adipocyte-AT1aR deficiency has no effect on development of diet- or AngII-induced atherosclerosis or AAAs in hypercholesterolemic mice. Interestingly, body weight and fat mass were increased in AngII-infused adipocyte AT1aR deficient mice. Future studies will elucidate the mechanisms through which AngII regulates body weight and fat mass under these conditions.