bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 550: Differential Regulation of CD39 Expression by Il-1ß in Leukocytes and Endothelial Cells
Authors: Sutton Nadia R , Petrovic-Djergovic Danica , Baek Amy , Kanthi Yogen , Liao Hui , Visovatti Scott H , Pinsky David J
Published: July 2021

Background

CD39 (ENTPD-1) is a membrane-spanning ectonucleotidase that is responsible for the phosphohydrolysis of ATP to ADP and ADP to AMP. CD39 levels are modulated in multiple conditions, including pulmonary hypertension, cardiac, liver and renal injury, cancer, and autoimmune diseases. However, little is known about the upstream regulation of this molecule, or whether modulation of CD39 represents a response to inflammatory cytokines or is integral to the pathological process. Elevated levels of cytokine IL-1β are a hallmark of many inflammatory states.

Objectives

Objectives were to measure CD39 expression after stimulation with the IL-1β. CD39 is expressed on leukocytes and endothelial cells; therefore, CD39 expression was evaluated in these cell types.

Methods

Human umbilical vein endothelial cells (HUVECs) were treated with either 10 pg/ml or 1 ng/ml IL-1β for 12 hours. Cells were harvested and CD39 mRNA expression was measured using qPCR. CD39 protein expression was measured using Western Blot. Mononuclear leukocytes were isolated from whole blood of healthy humans via density gradient cell separation (n=3). Leukocytes were cultured for 12 hours with or without the addition of IL-1β (1ng/ml). Leukocytes were then assessed for expression of CD39, CD73, CD14, CD19, CD4, CD8, and CD25 by flow cytometry.

Findings

IL-1β (1 ng/ml) treatment resulted in decreased CD39 mRNA expression in HUVECs (1.1 vs. 3.9, p<0.01). IL-1β treatment also resulted in decreased CD39 protein expression in HUVECs (untreated, 1.56, 10pg/ml, 0.96, 1ng/ml, 0.48, untreated vs. 10pg/ml, p<0.01). IL-1β increased the percentage of T-reg CD4+CD25+ expressing CD39+ cells (treated vs untreated, 3.94 vs. 2.73%, p=0.013). There was a trend toward an increased percentage of CD4+ T cells expressing CD39+ with IL-1β treatment (13.85% vs.11.83%, p=0.25).

Conclusions

This is the first report of modulation of CD39 expression in response to the inflammatory cytokine IL-1β. Interestingly, IL-1β downregulated CD39 expression in HUVECs, but resulted in increased CD39 expression on a T-reg population positive for CD4 and CD25. CD39 could be differentially regulated depending on the inflammatory stimulus and target cell type.
Abstract 554: Rabbit Arteriovenous Fistula Model and Identification of a Therapeutic Agent to Increase Fistula Patency
Authors: MacAskill Mark , Wadsworth Roger M , Kingsmore David , Coats Paul
Published: July 2021

An arteriovenous fistula (AVF) is a vein graft which is created to permit vascular access allowing haemodialysis to be performed in renal failure patients. AVF using native tissues is the preferred surgical option; however AVF are associated with failure rates as high as 50% at 6 months. Failure is principally due to vascular cell proliferation, leading to the development of neointima causing stenosis and impaired blood flow. The aims of this study were to 1) develop a rabbit model of AVF remodelling, 2) investigate the contribution of cannulation injury in AVF remodelling and 3) identify a potential therapeutic which could be delivered topically to reduce AVF stenosis.

The proposed model was an AVF created between the femoral artery and vein of a New Zealand white rabbit. Briefly, the fistula was fashioned by creating a side to side anastomosis, followed by occlusion of the distal vein. Over phase I of the study (days 1-28) maturation of the vein was evident by the presence of a pulsatile flow in the venous branch of the AVF, as well as a significant increase in venous blood velocity from 55.2± 6.0cm/s at day 10 to 68.4± 3.1cm/s by day 28 (p<0.05). A degree of medial/adventitial thickening was also evident at this time point. Rabbits were assigned to 3 groups for phase II of the study (days 29-56). The control group received no intervention, the injury group received cannulation to the AVF every 48-72 hrs, and the injury plus diclofenac group received the same frequency of injury and topical diclofenac (1.16% of
diclofenac
diethylammonium) twice a day, 5 days a week. In the control group, vein wall width increased significantly from 10.5± 0.9μM in the counter lateral vein to 16.6± 1.6μM in the AVF (p<0.05). Cannulation injury caused vein wall thickness to significantly increase to 46.8± 5.7μM (p<0.05). With diclofenac treatment, vein wall width decreased significantly to 15.8± 1.8μM (p<0.05).

These results show for the first time that cannulation injury significantly contributes to AVF remodelling
in vivo
. Topical treatment with diclofenac, which has anti-inflammatory as well as anti-proliferative properties, appears to reduce injury driven remodelling within AVF. Therefore, topical diclofenac could be used as a prophylactic treatment to reduce vascular stenosis in AVF.
Abstract 557: Human S100/calgranulin Accelerates Ectopic Cardiac Calcification and Promotes Cardiac Hypertrophy in Transgenic Mice with Chronic Kidney Disease in a RAGE Dependent Manner
Authors: Hofmann Bowman Marion , Yan Ling , Mathew Liby , Chellan Bijoy , Gardner Brandon , Earley Judy , Puri Tipu S.
Published: July 2021

Purpose

Chronic kidney disease (CKD) is associated with accelerated cardiovascular disease. Elevated serum S100A12 predicts cardiovascular mortality in patients with end stage renal disease. We test the hypothesis that human S100/calgranulin transgenic mice exposed to the metabolic changes of CKD would develop accelerated cardiovascular disease.

Methods

A novel humanized mouse with transgenic expression of human S100/calgranulin (hBAC-S100) was generated by expression of a bacterial artificial chromosome of the human S100/calgranulin gene cluster containing genes and regulatory elements for S1008/9 and S100A12 (60kb) in C57BL6/J mice. The hBAC-S100 mice were crossed with the RAGE KO mice (same C57BL6/J strain, gift from Ann Marie Schmidt, NYU) and F4 animals were used for the experiment. CKD was induced in hBAC-S100 and wild type (WT) littermate mice with intact or lacking RAGE signaling by surgical ligation of the ureters. The heart and aorta were analyzed after 10 weeks of elevated blood urea nitrogen.

Results

hBAC-S100 mice express human S100A12 in circulating myeloid cells and S100A12 was present in the serum of hBAC-S100 mice (25 ng/ml serum), but was not detected in WT mice. Importantly, serum hS100A12 was increased in hBAC-S100 mice with CKD compared to sham operated hBAC-S100 mice (42±17 ng/ml and 25±7 ng/ml, p=0.04). Moreover, hBAC-S100 mice with CKD exhibited ectopic cardiac calcification in fibroblast-rich annulus of the mitral and aortic valves upon Alizarin Red staining. In vivo ECHO showed abnormal mitral valve doppler flow with reduced ratio of early (E) to atrial (A) filling in hBAC-S100 mice with CKD compared to WT-CKD mice (1.17 ±0.1 vs 1.35 ±0.1, p=0.003), indicating diastolic dysfunction. Notably, hBAC-S100 mice with CKD develop cardiac hypertrophy as evidenced by increased heart weight/ body weight ratio and elevated gene expression of hypertrophic and fibrotic markers, such as ANP, β-MHC, TGF-β, CTGF, and Col 1a1. This phenotype was not observed in hBAC-S100 mice lacking RAGE, despite exposure to the same degree of CKD.

Conclusion

Circulating myeloid derived human S100/calgranulin is associated with the development of ectopic cardiac calcification and cardiac hypertrophy in chronic kidney disease in a RAGE dependent manner.
Abstract 562: Plgf is Crucial for the Hypertensive Response and T Cells Infiltration in Target Organs Induced by Angii
Authors: Carnevale Daniela , Pallante Fabio , Fardella Valentina , De Lucia Massimiliano , Fardella Stefania , Lembo Giuseppe
Published: July 2021

Although several therapeutic strategies have been developed against the main components involved in blood pressure (BP) regulation, an optimum control in some hypertensive patients is still flawed, suggesting that mechanisms so far unidentified, sustain BP increase. Interestingly, an emerging area of investigation revealed that adaptive immunity is central in hypertension, since mice devoid of lymphocytes are protected from hypertension, and adoptive transfer of T cells, but not B cells, restores the typical hypertensive response. How hypertensive stimuli can afford this, still represents a fascinating enigma, challenging the search for molecular players, capable to bridge vascular responses to immunity and probably orchestrating the still unveiled role of immune system in hypertension. Among the manifold cytokines and inflammatory mediators, Placental Growth Factor (PlGF), belonging to a family of VEGF related angiogenic factors, caught our attention, given its role of “angiogenic cytokine” and its expression by cells of both the cardiovascular and the immune system. Moreover, it has also been recently shown that PlGF is secreted in vascular cells in response to AngII. Thus, we hypothesized that PlGF might recruit a selected type of immune cell, expressing its receptor VEGFR1, influencing the process of T cells activation during hypertensive challenges. We found that the typical hypertensive response induced by chronic AngII infusion, was completely prevented in mice with genetic deletion of PlGF, as well as the typical end organ damage induced by hypertension, i.e. cardiac hypertrophy, renal damage, microvascular rarefaction and immune cells infiltration in target organs. To determine whether the involvement of PlGF in immune system activation and T cells infiltration during AngII-induced hypertension had a causal role or was merely a consequence of the failure in BP raise, we analyzed both vessels and kidneys, early after AngII infusion, i.e. before BP increase. Strikingly, PlGF absence protected from early infiltration of both CD4+ and CD8+ T cells. Overall our data indicate that PlGF is a pivotal player in the molecular mechanisms activating immune system recruitment during an hypertensive challenge, a central moment in the increase in BP.
Abstract 570: A Functional Mutation in WNT16a Up-regulates Pancreatic Expression of TCF7L2 Variants Through Wnt Signaling Pathway
Authors: Saleem Asma , Howard Eric W , Been Latonya F , Lerner Megan , Brackett Daniel , Lightfoot Stan , Bullen Elizabeth C , Sanghera Dharambir K
Published: July 2021

Type 2 (non-insulin-dependent) diabetes mellitus (T2DM) is associated with a marked increase in the risk of coronary heart disease. The discovery of TCF7L2 as a global T2DM gene has triggered investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates β-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon like protein-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2DM susceptibility in conjunction with gene variants in TCF7L2. Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay that would normally occur in the wild-type message, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2DM) from the Sikh Diabetes Study were used in this investigation, of which 32% were WNT16a insertion carriers. There was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p<0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003). Our results suggest synergistic effects of WNT16a insertion mutants and the at-risk ‘T ‘allele of TCF7L2 (rs7903146) for compounding the risk of T2DM, perhaps by impacting genes related to β-cell function in the Wnt/β-catenin/TCF7L2 signaling pathway.
Abstract 73: Atrial Fibrillation-induced Changes in Left Atrial Hemodynamics Directly Impact the Thrombotic Potential of Human Primary Endothelial Cell Cultures
Authors: Wamhoff Brian R , Simmers Michael B , Chen Zhu , Xu Yiming , Kong Ling-jie , Cole Banumathi , Pryor Andrew W , Berry Diana J , Day Nathan , Blackman Brett R , Ogletree Martin L
Published: July 2021

Thrombus formation in the left atrial appendage (LAA) of the heart is a source of stroke risk in patients with atrial fibrillation (Afib). Strategies to prevent thromboembolism from the LAA via restoration of normal rhythm or prevention of clot formation are limited by lack of adequate human surrogate systems that recapitulate the biology ex vivo. Endothelial cell phenotype and function in blood vessels and chambers of the heart are regulated in part by regional hemodynamics. Blood flow shear forces in the LAA are substantially altered during Afib. Thus, we hypothesized that Afib-induced alterations in hemodynamics directly impacts the thrombotic potential of the endothelium in the LAA. To test this, human primary aortic endothelial cell cultures were primed under hemodynamic shear forces simulating the human LAA during normal sinus rhythm for 24 hrs and continued under sinus rhythm or switched to simulate Afib for an additional 24 hrs. Compared to sustained sinus rhythm, Afib hemodynamics suppressed genes (qRTPCR) that define a healthy/quiescent endothelial cell phenotype (e.g. KLF2 and NOS3/eNOS) while inducing the pro-thrombotic gene F3 (tissue factor) and suppressing ADAMSTS13, MMP14 and TIMP2. To determine whether the observed molecular response produced a functional prothrombotic phenotype, hemodynamic primed aortic endothelial cells were exposed to dilute (~27% final concentration) human platelet-free plasma supplemented with Alexa488-labeled human fibrinogen, corn trypsin inhibitor and calcium. Cleavage of fibrinogen to fibrin and concomitant deposition on the endothelium was measured by quantitative confocal microscopy. When exposed to Afib hemodynamics, endothelial cells supported significant fibrin deposition that extended to greater depth (i.e., thicker) on the endothelium compared to sinus rhythm hemodynamics. In conclusion, we show for the first time a direct mechanistic link between changes in LAA hemodynamics associated with development of Afib and functional alterations in the thrombotic potential of the endothelium. This study produced a novel insight enabled by studying potential triggers of thrombosis in a context that more closely mimics the normal and pathological human in vivo environment.
Abstract 76: MicroRNA-144 Regulates Hepatic ABCA1 and Plasma HDL Following Activation of the Nuclear Receptor FXR
Authors: Vallim Thomas , Tarling Elizabeth , Kim Tammy , Civelek Mete , Baldan Angel , Esau Christine , Edwards Peter A
Published: July 2021

Rationale

The bile acid receptor Farnesoid-X-Receptor (FXR) regulates many aspects of lipid metabolism by various complex and not fully understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism.

Objective

To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism.

Methods and Results

ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma High Density Lipoprotein (HDL)-cholesterol levels. Here we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lower hepatic ABCA1 and plasma HDL levels. We identified two complementary sequences to miR-144 in the 3’ untranslated region (UTR) of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I (ApoA-I) protein, whilst overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL- cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL- cholesterol. In addition, we utilized tissue-specific FXR deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal FXR. Finally, we identified functional FXR response elements (FXREs) upstream of the miR-144 locus, consistent with direct FXR regulation.

Conclusion

In conclusion, we have identified a pathway involving FXR, miR-144 and ABCA1 that together regulate plasma HDL cholesterol. This pathway may be therapeutically targeted in the future in order to increase HDL levels.
Abstract 83: Bone Marrow Derived Cells Do Not Mediate Reductions in Cholesterol, Atherosclerosis and Adiposity in Microsomal Prostaglindin E Synthase 1 Deficient Mice Fed High Fat Diets Enriched in Cholesterol
Authors: Srodulski Sarah , King Victoria L
Published: July 2021

Microsomal prostaglandin E
2
synthase-1 (mPGES-1) catalyzes the conversion of COX-2 generated PGH
2
to PGE
2
and is the predominate source of PGE
2
during and inflammatory response. We and others have demonstrated that mPGES-1 deficiency attenuates atherosclerosis in mice on a mixed background. The present study investigated the effect of mPGES-1 deficiency on atherosclerosis in C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice. mPGES-1 deficiency attenuated atherosclerosis in LDLr-/- mice fed either a low fat (LF) (P = 0.02) or high fat (HF) (P = 0.0026) diet enriched with cholesterol, or a western diet (P = 0.02) for 17 weeks. mPGES-1 deficiency attenuated weight gain and cholesterol concentrations in mice fed a western (P = 0.004 and P < 0.05; respectively) or HF diet (P = 0.01 and P = 0.012, respectively). However, body weight and cholesterol concentrations were not different in mice fed the LF diet. These data suggest that different mechanisms mediate the reduction in atherosclerosis in mPGES-1 deficient mice fed LF and HF diets. To determine if mPGES-1 deficiency in macrophages contributed to the reduction in atherosclerosis in mice fed HF diets, 4 groups of chimeric mice were generated. Four weeks post bone marrow cell transplant (BMT) mice were fed a western diet. BMT attenuated weight gain in all groups of chimeric mice; however, weight gain was not different between any of the groups. BMT decreased atherosclerotic lesion formation 10 fold in all groups of mice. Neither bone marrow cell specific deficiency of mPGES-1 (KO>WT) or mPGES-1 specific expression in bone marrow derived cells (WT>KO) had an effect on lesion formation compared to WT>WT or KO>KO mice. Cholesterol concentrations were decreased in KO>KO and WT>KO mice compared to WT>WT (P < 0.01) and KO>WT (P< 0.05) mice. These data suggest that mPGES-1 expression in bone marrow derived cells does not contribute to the development of atherosclerosis. Moreover, these data suggest that prostanoids may play a role in hepatic cholesterol homeostasis in mice fed HF diets enriched in cholesterol thereby contributing to atherosclerotic lesion formation. Moreover, these data provide further evidence that prostanoids play a role in regulating the accumulation of diet-induced adiposity.
Abstract 94: Ca
2
+/calmodulin-dependent Protein Kinase II ? Gene Deletion Enhances Vascular Remodeling in Response to Injury
Authors: Saddouk Fatima Zahra , Jiang Miao , Sun LiYan , Schwarz John J , Singer Harold A
Published: July 2021

Differentiated vascular smooth muscle (VSM) cells undergo phenotypic switching following injury or primary cell culture. This process includes changes in expression of ion channels, proteins involved in intracellular Ca
2+
homeostasis, as well as changes in Ca
2+
signal effectors. Our previous studies showed that the delta2 isoform of the multifunctional serine/threonine protein kinase Ca
2+
/calmodulin-dependent protein kinase II (CaMKIIδ
2
or CaMKIIδc), was up-regulated in rat carotid artery VSM following balloon injury and contributed positively to cell proliferation, migration and neointima formation. Conversely, CaMKII γ gene isoforms are down-regulated following vascular injury in rats, with as yet unknown functional consequences.
We hypothesized that CaMKIIγ expression opposes the vasculoproliferative response promoted by CaMKIIδ.
To test this, we generated CaMKIIγ conditional knockout mice by crossing CaMKIIγ
loxp/loxp
mice with a SM22α (transgelin)-Cre line. Efficient deletion of CaMKIIγ was confirmed in VSM and heart with no compensatory increases in CaMKIIδ expression or observable effects on mouse development, expression of VSMC differentiation marker genes or vascular morphology. Carotid artery ligation-induced neointima formation was markedly enhanced in CaMKIIγ conditional knockout mutant mice compared to littermate controls (Neointima/media area ratios: 0.52 ± 0.20, n=8 vs. 0.03±0.03, n=7) 3 weeks post-ligation with a nearly 2-fold increase in vascular wall proliferation index (0.09±0.006, n=6 vs. 0.05±0.009, n=6) and no change in apoptosis index. CaMKIIγ protein expression is down-regulated following primary culture of aortic VSM. Ectopic re-expression of CaMKIIγ in cultured VSMC from control or global CaMKIIδ-/- knockout mice resulted in a significant reduction in proliferation rates as measured by cell counting and BrdU incorporation and cell migration as assessed by scratch wounding. We conclude from these studies that CaMKIIγ and δ-isoforms have non-equivalent functions in VSM, and specifically, that CaMKIIγ isoforms inhibit the vasculoproliferative response.
Abstract 102: Granular Media Calcinosis in the Aortic Wall of Patients With Bicuspid and Tricuspid Aortic Valves
Authors: von Salisch Sandy , Haunschild Josephina , Misfeld Martin , Borger Michael A , Dhein Stefan , Mohr Friedrich-Wilhelm , Etz Christian D
Published: July 2021

Background:
Bicuspid aortic valve is the most frequent congenital cardiac abnormality and associated with proximal aortic disease (i.e. aneurysm, dissection or rupture).
Granular media calcinosis(GMC)--suggested to increase stiffness and play a pathogenetic role in dissecting aneurysm--has not yet been quantified in BAV.

Methods:
Specimen of the proximal aortic wall from 76 patients--32 with tricuspid (TAV) and 44 with bicuspid aortic valve (BAV)--were obtained during surgery to quantify media thickness and
GMC
by von Kossa staining (panel C), comparing the convexity (Cvx) and concavity (Ccv) in BAV vs. TAV.

Results:
Interlamellar GMC affected the most central layers of the media and those adjacent to the outer adventitia with a doubling within both--the Cvx and Ccv--of pts with BAV compared to patients with TAV (13.3±9.6
vs.
6.6±7.4 and 12.8±10.8
vs.
6.4±7.1; p<0.05, panel A) was seen, but neither a difference in calcification between the Ccx and the Ccv side within the BAV nor the TAV group.
No association between age and calcification grade
, neither in the Cvx nor the Ccv (r=0.132, p=0.218 and 0.103, p=0.341) was seen. There was a significant difference in the total media thickness between BAV and TAV at the Cvx (867±162μm
vs
. 993±158μm; p<0.05) and the Ccv (1005 ± 236
vs
1223 ± 217μm; p<0.05, panel B). Independent of aortic valve morphology, the Cvx was thinner than the Ccv side (TAV: 993 ± 158 vs.1223 ± 217μm; p<0.001; BAV: 869 ± 162 vs.1005 ± 236μm; p<0.05, panel B).

Conclusion:
BAVs had significantly thinner media and twice as much GMC than their tricuspid peers possibly associated with the loosening of the bond between the elastic lamellae causing a
decrease in elasticity
possibly explaining a higher risk for dissection and rupture.
Abstract 109: IL-1ß-Induced Ceramide Synthesis Accentuates NETosis and Inflammation in Abdominal Aortic Aneurysms
Authors: Meher Akshaya K , Pope Nicolas , Su Gang , Davis John P , Serbulea Vlad , Laubach Victor E , Leitinger Norbert , Ailawadi Gorav , Upchurch Gilbert R
Published: July 2021

Objective:
We have previously shown that circulating neutrophils are required for experimental aortic aneurysm (AA) formation via a non-MMP2/9-mediated mechanism. In other experiments we documented that IL-1β signaling is required for AA formation. Therefore, we hypothesized that increased levels of IL-1β in AA triggers inflammatory ceramide synthesis and thus extracellular trap (NET) formation (NETosis) by neutrophils, thereby augmenting inflammation.

Methods:
Immunohistochemical techniques were used to identify neutrophils and NETs. Ceramides from human AA and control aortas from transplant donors were quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Neutrophils isolated from human blood were treated with IL-1β to determine if IL-1β induces ceramide synthesis and NETosis in vitro. Experimental AAs were induced by elastase perfusion in C57BL/6 wild-type (WT) and IL-1β knock-out (KO) mice and cellular content of AA was analyzed using flow cytometry. Groups were compared using two-tailed unpaired t-test.

Results:
Immunohistochemical staining showed that neutrophils were scattered or grouped near lymphoid-like structures in the adventitia of human AA. Three-dimensional confocal imaging showed NETs in the adventitia of human AA. Importantly, the levels of long chain ceramides C16 and C24:1 were higher (2.6 and 1.8-fold, respectively), whereas, the levels of short chain ceramides C4, C6 and C8 were lower (4.3, 3.9 and 3.5-fold, respectively) in human AA compared to controls. In isolated neutrophils, IL-1β treatment induced synthesis of long chain ceramides and discharge of NETs. Neutrophils and NETs were also detected in aortic media at day 7 after induction of AA in WT mice. On day 7 following elastase perfusion, despite the increase in number of circulating immune cells, AA size was smaller (91.08±11.7% vs 53.8±5.4%, p=0.03), and, infiltration of neutrophils (12179±2697 vs 3046±628, p=0.01) and B cells was lower in the aorta of IL-1β KO mice compared to WT mice.

Conclusions:
These data document for the first time the presence of NETs in human AAs. Furthermore, the data suggests that neutrophils augment inflammation in AA by increasing pro-inflammatory long chain ceramide synthesis and NETosis in response to IL-1β.
Abstract 125: Inflammatory Markers Influence Microembolization in Patients Undergoing Carotid Interventions
Authors: Hitchner Elizabeth , White Sharla P , Lahey Lauren , Robinson William , Zhou Wei
Published: July 2021

INTRODUCTION:
Despite the use of embolic protection and improvement in surgical technique for carotid interventions, perioperative cerebral microembolization is still relatively common. Susceptibility to microembolization is largely uncontrolled and the effects remain poorly understood. We hypothesized that inflammation may play a role in this process and sought to identify plasma markers predicting embolization in patients undergoing carotid interventions.

METHODS:
Patients scheduled for carotid interventions at an academic center were prospectively recruited to the study. MRI with diffusion-weighted imaging (DWI) was performed prior to and within 48 hours following the procedure and used to identify new cerebral microemboli. Blood samples were collected at the time of intervention prior to carotid interrogation (Pre) and at 24 hours following intervention (Post). Plasma was isolated and analyzed by a 27-plex Luminex assay. The assay was performed in triplicate and Significance Analysis of Microarrays (SAM) identified significant inflammatory markers. Mann-Whitney U tests were performed to further evaluate these findings.

RESULTS:
71 male patients were included with an average age of 70 years; 38 underwent CAS and 33 underwent CEA. 59% of the cohort exhibited postoperative DWI lesions. Post plasma samples were collected for all patients, and Pre samples were collected for a cohort of 19 patients. We observed significantly lower levels of several cytokines in patients with procedure-related DWI lesions, particularly platelet-derived growth factor (PDGF) (150.8 vs 389.6pg/ml, p<.001). Patients with more complex plaques were also observed to have lower levels of PDGF. For the cohort with both Pre and Post samples, there was a significantly greater decrease in PDGF for patients with DWI lesions compared to those who did not. (U=382, Z=-2.6, p=.008).

CONCLUSIONS:
Lower plasma levels of cytokines were observed in patients with procedure-related DWI lesions, suggesting that patients who illicit an inappropriate immune response may be more susceptible to procedure-related microembolization. Impaired PDGF response may indicate poor repair mechanism to injury. Further investigation is warranted.
Abstract 126: Are Carotid Plaques With Intraplaque Hemorrhage Different in Plaque Size, Distribution and Shear Stress?
Authors: Kassar Zaid A , Speelman Lambert , van Dijk Anouk , Selwaness Mariana , Cibis Merih , Gijsen Frank J , Van der steen Anton F , van der Lugt Aad , Wentzel Jolanda J
Published: July 2021

Introduction:
Intraplaque hemorrhage (IPH) is associated with risk at ischemic stroke. In addition, plaques that contain IPH show accelerated plaque growth, which might affect the local shear stress (SS) distribution over the plaque. Blood flow induced SS is known to be involved in the development and destabilization of atherosclerotic plaques. It is unknown whether the SS distribution over plaques with IPH differs from plaques without IPH and whether SS is involved in plaque progression/destabilization of plaques with IPH. We compared the plaque size, distribution and SS over plaques with and without IPH in symptomatic patients.

Methods:
38 patients with a recent (<3 months) TIA or minor stroke of the PARISK (Plaque At Risk) study and with carotid artery stenosis 2 mm were selected for analysis. To calculate local SS, the lumen contours were used in FIDAP, a CFD software package. The mean and maximum shear stress at the plaque was normalized to the average shear stress in the common carotid artery (NSSmean, NSSmax). The NSSmean and NSSmax were compared for plaques with and without IPH.

Results:
In 75 carotid arteries plaque was observed (99%). The 29 plaques (39%) that contained IPH were longer (17±5 vs 11±6 mm, p<0.05) and located more proximal in the carotid bulb than plaques without IPH. Symptomatic vessels contained more often IPH (76%) than plaques from asymptomatic arteries (23%). Plaques that contained IPH in symptomatic arteries were not different in length (17±4 mm) compared to plaques with IPH in asymptomatic arteries (16±5 mm). In 18 symptomatic vessels the SS could be calculated. The NSSmean did not differ for plaques with or without IPH (0.98±0.35 vs 0.75±0.40), whereas the NSSmax for plaques with IPH was 2x higher than for plaques without IPH (1.32 ±0.35 vs 3.06±1.45, p<0.05).

Conclusion:
Carotid plaques that contain IPH are longer, located more proximal in the carotid bulb and exposed to higher SS than plaques without IPH. IPH determines the plaque length, which was not influenced by the presence or absence of symptoms. Since IPH is associated with stroke risk, the higher SS and larger plaque size potentially contribute to this increased stroke risk.
Abstract 127: Pharmacological Regression of Pulse Pressure Prevents Further Cerebrovascular Dilatation in Rats with Elastase-Induced Intracranial Aneurysm
Authors: Ng Keith , Li Jonathan , Gupta Vivek , Qian Yi
Published: July 2021

Objective:
Highly pulsatile blood pressure (BP) transmits into small vessels in the brain, potentially damages cerebrovasculature, causing transient ischaemic attack or stroke. We examined the effects of pulse pressure (PP) regression using an angiotensin converting enzyme inhibitor (perindopril, ACEI) or a thiazide diuretic (hydrochlorothiazide, HCTZ) on the incidence of intracranial aneurysms.

Materials and methods:
12 weeks old pre-hypertensive Spontaneously Hypertensive Rat (SHR) received ACEI (3mg/kg/day) or HCTZ 20mg/kg/day in drinking water a week before aneurysm induction. Aneurysms were induced with a single stereotaxic microinjection of elastase (30milli-units) into the right basal cistern using a fine glass pipette (tip size <0.05mm) through a burr hole at 0.9mm posterior, 1.4mm lateral to the bregma, and 3.3mm deep. Control SHR received inactivated elastase heated to 100
0
C and were untreated. Rats were recovered with ACEI or HCTZ continued for 8 weeks. Rats were then anaesthetised (urethane1.3 g/kg, i.p.), their BP measured at common carotid artery before euthanised and fixed with a bromophenol blue dye and gelatin mixture for examination of the cerebrovasculature. Incidence was counted positive when the rat showed at least one localised outward bulging of the vascular wall with diameter >50% of the basilar artery diameter.

Results:
13/30 rats developed intracranial aneurysms 8 weeks post injection. Table shows both ACEI and HCTZ reduced the incidence of intracranial aneurysms with ACEI more efficacious compared with HCTZ. Both ACEI and HCTZ effectively reduced MAP, SBP, and DBP in SHR. However, only ACEI significantly reduced PP.

Conclusion:
ACEI was more efficacious in preventing further dilatation of elastase-induced intracranial aneurysms. This effect was associated with marked regression of PP. This study suggests that pharmacological interventions to delay aneurysm growth should target at regressing PP rather than MAP per se.
Abstract 14: Caskin2 is a Novel Regulator of Endothelial Cell Quiescence
Authors: Mueller Sarah B , Arora Shubhangi , Mattocks Natalie , Gurley Susan B , Kontos Christopher D
Published: July 2021

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. Morpholino knockdown of Caskin2 in zebrafish results in abnormal vascular development characterized by overly branched intersegmental vessels and failure to form the dorsal longitudinal vessels. Interestingly, Caskin2 knockout mice are viable and fertile. However, compared to wild-type mice, adult Caskin2 knockout mice have significantly more abdominal adipose and higher fasting blood glucose levels even when fed a standard chow diet. In vitro, Caskin2 overexpression inhibits serum-induced EC proliferation and DNA synthesis but promotes EC survival during serum starvation. Caskin2 localizes to the nucleus as well as the cytoplasm and promotes the downregulation of genes associated with EC activation (e.g., IL-8, VEGFR2, ANG2) and the upregulation of genes associated with EC quiescence (e.g., Notch1, KLF2/4, ANG1). More broadly, pathway analysis of microarray data demonstrates that Caskin2 primarily regulates cell cycle and metabolic genes. Additional data suggest that these effects result from the interaction of Caskin2 with the Ser/Thr phosphatase PP1alpha, which is mediated by a consensus PP1 binding motif at the Caskin2 N-terminus. Taken together, our data demonstrate that Caskin2 is sufficient to suppress EC proliferation in vitro and necessary to prevent dysregulated angiogenesis in at least one in vivo model. These findings indicate that Caskin2 is a novel regulator of EC quiescence and suggest a role for Caskin2 in the prevention of EC dysfunction in vivo. Understanding Caskin2’s function on the molecular level may lead to the development of novel pharmacological approaches to prevent inappropriate angiogenesis, normalize dysfunctional vessels, and improve vascular function in a variety of cardiovascular diseases.
Abstract 143: Role of Chronic Kidney Disease Promoted Caspase-1 Activation in Neointimal Hyperplasia
Authors: Ferrer Lucas M , Monroy Alexandra , Lopez Jahaira , Yang Xiao-Feng , Wang Hong , Choi Eric
Published: July 2021

The major cause of vascular access failure is venous stenosis due to neointimal hyperplasia (NH) [62]. Vascular smooth muscle cells (VSMC) are critical for the development of NH lesions, as they have the ability to modulate their phenotype from “contractile” to a “synthetic” in the presence of uremia, through the regulation of sensor genes for uremia danger signals and VSMC-specific differentiation genes. Recent research indicates that Caspase-1 (casp-1) activation plays an essential role in sensing metabolic danger signal-associated molecular patterns[4] and initiating vascular inflammation. Therefore, the goal of this project is to examine the role of CKD-driven casp-1 activation in VSMC and CKD-related NH.

We have established a CKD mouse model and published CKD-associated vascular remodeling. We exposed wild type and caspase-1 knockout mice to our CKD model, analyzed and quantified the NH lesion formed. We also examined in vitro and ex-vivo changes in VSMC-specific differentiation genes when exposed to uremic serum and cLDL, in the presence or absence of casp-1 inhibitor.

We found that CKD serum induces with casp-1 activation and phenotypic changes in VSMCs from a “contractile” to a “synthetic” phenotype, which are reversed with casp-1 inhibition. In an ex-vivo model using relative quantification we found that VSMC contractile markers α -Actin, Calponin, SM-22, and Smoothelin gene expression of CKD mouse carotid VSMC were higher in casp-1 knockout mice when compared to wild-type (1.40, 1.28, 1.22, 1.41 respectively). Also using an in-vivo model, relative quantification of α-actin decreased from 1.0 to 0.329 when VSMCs were exposed to uremic serum and increased back to 0.588 when casp-1 inhibitor is added. The relative quantification of Calponin also decreased from 1.0 to 0.394 when exposed to uremic serum and increased back to 0.601 with caspa-1 inhibitor. We also found that casp-1 deficiency significantly reversed CKD-related vascular remodeling by reducing NH volume from 1,440,023.70 in wild-type mice to 71,069.97 μm2 in casp-1 knockouts (p-value 0.002). Our results provide a novel insight over the therapeutic potential of casp-1 inhibitors for CKD induced NH.
Abstract 147: Hepatic Scavenger Receptor BI Protects Against Polymicrobial-Induced Sepsis Through Promoting LPS Clearance in Mice
Authors: Guo Ling , Zheng Zhong , Ai Junting , Huang Bin , Li Xiang-An
Published: July 2021

Recent studies revealed that scavenger receptor BI (SR-BI or Scarb1) plays a critical protective role in sepsis. However, the mechanisms underlying this protection remain largely unknown. In this study, using Scarb1
I179N
mice, a mouse model specifically deficient in hepatic SR-BI, we report that hepatic SR-BI protects against cecal ligation and puncture (CLP)-induced sepsis as shown by 75% fatality in Scarb1
I179N
mice, but only 21% fatality in C57BL/6J control mice. The increase in fatality in Scarb1
I179N
mice was associated with an exacerbated inflammatory cytokine production. Further study demonstrated that hepatic SR-BI exerts its protection against sepsis through its role in promoting LPS clearance without affecting the inflammatory response in macrophages, the glucocorticoid production in adrenal glands, the leukocyte recruitment to peritoneum or the bacterial clearance in liver. Our findings reveal hepatic SR-BI as a critical protective factor in sepsis and point out that promoting hepatic SR-BI-mediated LPS clearance may provide a therapeutic approach for sepsis.
Abstract 15: Novel Functions of Small GTPase Rap1 in Regulating Endothelial Homeostasis: Control of Nitric Oxide Release, Vascular Function and Blood Pressure
Authors: Lakshmikanthan Sribalaji , Zheng Xiaodong , Nishijima Yoshinori , Vasquez-Vivar Jeannette , Zhang David X , Chrzanowska-Wodnicka Magdalena
Published: July 2021

Endothelial dysfunction, resulting from decreased nitric oxide (NO) bioavailability is a pathology linked to endothelial vasomotor dysfunction and hypertension, inflammation and atherosclerosis, perturbed endothelial barrier and progression of diabetes. In blood vessels, NO is produced by the endothelial NO synthase (eNOS), the activity of which is regulated by Ca2+/calmodulin, binding of regulatory cofactors, and posttranslational modifications, including phosphorylation events on Ser1177, which stimulate NO production. Rap1 is a ubiquitously expressed small GTPase implicated in promoting vascular barrier. We have shown that endothelial cell (EC)-specific Rap1 deletion leads to defective angiogenesis in vivo due to faulty VEGFR2 activation and signaling. Importantly, EC-specific Rap1 knockout mice developed hypertension and pathological left ventricular hypertrophy.

The objective of the study was to determine the role of small G protein Rap1 in regulating endothelial NO production and endothelial-dependent vasorelaxation in vivo and ex vivo.

Using ex vivo myography and tamoxifen-inducible, endothelial-specific Rap1-knockout mice (Cadh5-CreERT2+/0;Rap1f/f), we demonstrate that Rap1 deficiency completely abrogates NO-dependent vasodilation and attenuates NO production. Mechanistically, we show that Rap1 is rapidly activated in response to receptor agonists that activate eNOS via Ca2+/calmodulin- dependent pathway and in response to shear flow, which modules eNOS activity by its phosphorylation. Rap1 deletion in human ECs, in vitro, leads to deficient NO release in response to both these stimuli, and interferes with PI3K/Akt pathway and eNOS Ser1177 phosphorylation. Further, we demonstrate Rap1 is required for transducing signals from the endothelial mechanosensing complex comprising PECAM-1, VE-cadherin and VEGFR2 in response to shear flow, leading to ligand-independent VEGFR2 activation and signaling to stimulate NO production.

We conclude that Rap1 in endothelium is critically required for endothelial homeostasis and NO production, thereby affecting vascular tone and regulation of blood pressure. Furthermore, this study establishes Rap1 as a novel regulator of mechanotransduction in response to shear flow.
Abstract 152: Oxidized Low-Density Lipoprotein Immune Complexes Generate an Inflammatory Immune Response by Signaling Through Multiple Receptors on Dendritic Cells
Authors: Rhoads Jillian P , Wilhelm Ashley J , Major Amy S
Published: July 2021

Antibodies to oxidized LDL (oxLDL) and resulting immune complexes (ICs) are a prominent feature of atherosclerosis and diseases associated with increased atherosclerosis including Type 2-diabetes and rheumatoid arthritis. Although levels of oxLDL-ICs correlate with disease severity and associate with pro-inflammatory activation of macrophages
in vitro
, it is currently unclear whether these ICs are simply biomarkers or play an active role in disease pathogenesis. One possible mechanism by which oxLDL-ICs may regulate inflammation in atherosclerosis is by interacting with Fc gamma receptors (FcgRs) expressed on the surface of antigen presenting cells, such as dendritic cells (DCs). Not surprisingly, signaling through FcgRs is tightly regulated and dependent on their relative cell surface density. In addition, FcgR signaling has been shown to be linked to Toll like receptor-4 (TLR-4), a pattern recognition receptor. We hypothesize that oxLDL-ICs exacerbate atherosclerosis via signaling through FcgRs and TLR4 on DCs resulting in a pro-inflammatory T cell response. To test this hypothesis, bone marrow derived DCs (BMDCs) were treated with
in vitro
generated oxLDL ICs. Interestingly, BMDCs treated with oxLDL-ICs had increased expression of the activation markers MHC-II and CD40 and produced greater levels of the Th17 polarizing cytokines IL-1beta and IL-23 compared to cells treated with oxLDL alone. Secretion of pro-inflammatory cytokines was significantly decreased by pre-treatment of BMDCs with a TLR4 inhibitor and by blocking FcgR signaling. This suggests that responses to oxLDL-ICs involve both TLR-4 and FcgRs. Furthermore, incubation of OT-II T cells with BMDCs treated with oxLDL-IC prior to incubation with ovalbumin peptide displayed increased pro-inflammatory cytokine secretion compared to incubation with oxLDL alone. In conclusion, our studies provide new evidence that oxLDL-ICs signal through multiple receptors on DCs resulting in increased inflammatory potential. Because of their critical function in shaping the T cell response, we believe that DC signaling via FcgRs and oxLDL-ICs represents an important link between innate and adaptive immunity in atherosclerosis.
Abstract 175: The Nck Adaptor Proteins Couple Oxidant Stress-Driven Tyrosine Phosphorylation to NF-κB Activation and Proinflammatory Gene Expression
Authors: Chen Jie , Leskov Igor , Albert Patrick , Kevil Christopher G , Stokes Karen , Orr A W
Published: July 2021

Reactive oxygen species production drives endothelial NF-kappaB activation and NF-kappaB-dependent proinflammatory gene expression in several pathological conditions, including ischemia-reperfusion injury. While multiple reports link oxidant stress-induced tyrosine kinase signaling to NF-kappaB, we previously showed that the serine/threonine kinase p21 activated kinase (PAK) critically mediates NF-kappaB activation by oxidant stress. The adaptor protein Nck classically couples PAK to tyrosine kinase signaling by recruiting PAK to the plasma membrane. However, the mechanisms linking oxidant stress to PAK signaling are currently unknown. We now show that H2O2 stimulates both Nck interactions with tyrosine phosphorylated proteins and membrane translocation, whereas inhibiting Nck (siRNA, blocking peptide) abrogates H2O2-induced PAK activation. Consistent with PAK’s role in NF-kappaB signaling, Nck knockdown completely blocks H2O2-induced NF-kappaB activation, proinflammatory gene expression, and monocyte adhesion, suggesting that Nck couples tyrosine kinase signaling to PAK-dependent NF-kappaB activation. In contrast to these proinflammatory responses, antioxidant gene expression following oxidant stress is not affected by Nck inhibition. In addition to exogenous H2O2, endogenously produced oxidant stress following hypoxia/reoxygenation injury shows a similar requirement for Nck for proinflammatory signaling (PAK, NF-kappaB), proinflammatory gene expression (ICAM-1, VCAM-1) and monocyte adhesion. Finally, leukocyte firm adhesion and emigration following ischemia-reperfusion injury in the cremaster muscle of C57Bl/6J mice is completely inhibited by treatment with the Nck blocking peptide. Taken together, these data identify Nck as an important mediator of oxidant stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury.