bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 508: Pyruvate Induction of Glyoxalase 1 in Porcine Brain Post-cardiac Arrest: Enhanced Methylglyoxal and Glutamate Detoxification
Authors: Scott Gary F
Published: July 2021

Background:
After cardiac arrest (CA) and cardio-cerebral resuscitation (CCR), enhanced glycolysis elevates toxic methylglyoxal (MG) that contributes to carbonyl-exacerbated oxidative stress, enzyme inactivation and death of neurons and astroglia. Post cardiac arrest cognitive deficits may be prevented by pyruvate infusions that boost glyoxalase 1 (GLO1) detoxification of MG and brain glutamate efflux via Nrf2-regulated Phase II gene expression.

Purpose:
To define pyruvate-induced cytoprotective mechanisms that curtail brain injury and cognitive deficit after cardiac arrest and resuscitation in pigs.

Methods:
Pigs were subjected to CA/CCR, infused with 4 mM pyruvate or NaCl control, then carotid artery and frontal cortical lysates were assayed for Nrf2/ARE binding activity and GLO1 activity, while plasma glutamate concentrations were measured.

Results:
Compared with saline controls, pyruvate infused pigs demonstrated increased Nrf2/ARE binding activity and 3-fold higher GLO1 activity supporting MG detoxification. Plasma glutamate concentrations were substantially reduced by pyruvate, which would support greater brain efflux of excitotoxic glutamate.

Conclusions:
Intravenous pyruvate induces Nrf2-regulated gene activation for augmented GLO1 biological activity via mechanisms that may lower glutamate-inflicted excitotoxicity and the heretofore untreatable cognitive deficits.
Abstract 509: Accurate Measurement of Carotid Lumen Diameter and Narrowing Utilizing Ultrasound
Authors: Sharma Aditya M , Araki Tadashi , Kumar Krishna , Ikeda Nobutaka , Lavra Francesco , Rajan Jeny , Saba Luca , Nicolaides Andrew , Laird John , Shafique Shoaib , Suri Jasjit S
Published: July 2021

Introduction:
Ultrasound is often used for monitoring of carotid disease. In current clinical practice, degree of stenosis is an important predictor to assess stroke risk. Lumen narrowing from plaque is currently measured via techniques such as computed tomographic scan, magnetic resonance angiogram or conventional angiogram. Duplex ultrasound measures degree of stenosis based on peak systolic velocities and other parameters and only provides a wide range of level of stenosis. Pursuing lumen size measurement in ultrasound via manual quantification of lumen diameter is tedious. Furthermore, non-uniformity in plaque growth makes it more challenging and time-consuming. There has been an increasing interest in the automatic and robust delineation of the lumen boundaries of the carotids and to measure the lumen diameter via ultrasound given its non-invasive and safe approach.

Methods:
Deidentified carotid ultrasound images were obtained on patients retrospectively who underwent carotid ultrasound at Toho University Ohashi Medical Center, Tokyo, Japan. A higher order derivative Gaussian filter is applied on these images to highlight the edges. Using pixel classification, lumen region is detected and lumen boundaries are estimated.

Results:
Of the 202 patients with common carotid artery images, 155 were males and 47 were females.Mean age 69 ± 15.9 years. Mean HbA1c, LDL, HDL and Cholesterol of patients were 6.28±1.1 mg/dl, 101.27±31.6 mg/dl, 50.26±14.8 mg/dl and 175.04±38 mg/dl, respectively. Specialist trained in carotid ultrasound manually traced lumen diameter. Automated tracing and lumen measurements were obtained. The coefficient of correlation between automated diameter and manual diameter was: 0.88, 0.91 and 0.93. The mean diameter error between automated and manual tracing were: 0.50±0.37 mm, 0.36±0.34 mm and 0.30±0.28 mm. Precision of merit between automated diameter and manual diameter was: 93.28%, 95.33% and 96.32% corresponding to manual tracers.

Conclusions:
The automated lumen diameter measurement is near real time, quick, accurate, fully automated and reliable to assess carotid lumen diameter and narrowing.
Abstract 533: Salicylate Restores Macrophage Cholesterol Homeostasis via Activation of AMP-Activated Protein Kinase
Authors: Fullerton Morgan D , McGregor Chelsea P , LeBlond Nicholas D , Snider Shayne A , Ford Rebecca J , Austin Rick C , Steinberg Gregory R , Kemp Bruce E
Published: July 2021

Objectives:
Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage foam cell cholesterol homeostasis remains unclear.

Methods:
We sought to address this question by testing the effects of AMPK-specific activators in primary bone marrow-derived macrophages from AMPK β1-deficient (β1-/-) mice.

Results:
Macrophages from AMPK β1-/- mice had enhanced lipogenic potential and diminished cholesterol efflux, although cholesterol uptake was unaffected. Specific activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of both fatty acids and sterols in WT but not AMPK β1-/- macrophages. In lipid-laden macrophage foam cells, salicylate and A-769662 decreased cholesterol uptake and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an AMPK β1-dependent manner. Increased cholesterol efflux was also associated with increased gene and protein expression of the ATP binding cassette transporters, ABCG1 and ABCA1. Moreover, in vivo reverse cholesterol transport was significantly suppressed in mice that received AMPK β1-/- macrophages compared to WT control.

Conclusion:
Our data highlight the therapeutic potential of targeting macrophage AMPK with new or existing drugs for the restoration of cholesterol homeostasis during the early stages of atherosclerosis.
Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance
Authors: Robinet Peggy , Smith Jonathan D
Published: July 2021

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaired in DBA/2 foam cells. To assess lysosome function and number, cells were labeled with Lysotracker red DND-99 (LyT) and analyzed by flow cytometry. We found that AcLDL loading did not affect LyT intensity. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). In addition, upon loading, the mature form of cathepsin L was increased in AKR (43%, p<0.05) but not DBA/2 cells. Together these data suggest an impairment of lysosomal degradation capacity in DBA/2 foam cells. Finally, we investigated the role of TPC2, a lysosomal membrane protein which over expression has been previously linked to a defect in autolysosome formation. We found that upon AcLDL loading TPC2 protein levels were increased by 35% in DBA/2 cells, which are defective in autolysosome formation, while they were unchanged in AKR cells. In conclusion, we found that DBA/2 vs. AKR foam cells express more TPC2 and have fewer and/or dysfunctional lysosomes that may explain the autolysosome formation defect in these cells.
Abstract 542: The Effects of Soluble Epoxide Hydrolase Inhibition in Atherogenesis and Atherosclerotic Progression
Authors: Li Dan , Zhang Xu , Zhu Yi
Published: July 2021

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA), are known to have anti-inflammatory effects and hydrolyzed by soluble epoxide hydrolase (sEH). We aimed to investigate the effect of sEH inhibition on atherosclerotic progression.

LDLR-/- mice with or without sEH inhibitor and LDLR and sEH double knockout (DK) mice were fed with western diet for 6 weeks to induce arteriosclerosis, and then half of the mice switched to chow diet supplemented with sEH inhibitor for another 6 weeks. To further assess the role of sEH in atherosgenesis, we transplanted LDLR-/- or DK bone marrow into LDLR-/- recipients and also fed with 6 weeks western diet. The progression of atherosclerosis was evaluated and AA metabolites were analyzed by LC-MS/MS.

The results revealed that sEH inhibition and gene knockout decreased western diet-induced high plasma LDL cholesterol level and the size of the atherosclerotic lesion in the aorta of the mice. Furthermore, mice with DK bone marrow transplantation developed less aortic lesion by less ly6chi monocyte infiltiation without diminished the cholesterol level in plasma. After diet switch, the cholesterol level dropped back to control level and the size of arotic lesion became smaller compared to the western diet group. When sEH inhibitor decreased sEH activity in the liver and increased EETs/DHETs ratio in the plasma, the plaque size seemed not being further reduced after diet switch. However, M2 type macrophage marker at protein and mRNA level was increased in the aortic lesions. In cultured macrophage, sEH inhibition and EETs supplements could increase the markers of M2 macrophage at mRNA level.

Thus, sEH inhibition and depletion in macrophage attenuated atherosclerosis when fed with western diet. Although the role of the sEH inhibitor on atherosclerosis progression and plaque stability need to be further investigated. EETs levels and sEH activity may play a pivotal role in atherogenesis and monocyte/macrophage in filtration and differentiation.
Abstract 553: Siglec-1 (CD169) on Monocytes/Macrophages: A New Receptor For Extracellular Self-RNA in Triggering Inflammatory Responses via the Sheddase TACE/ADAM17
Authors: Cabrera-Fuentes Hector A , Preissner Klaus T , Boisvert William A
Published: July 2021

As an important component of atherosclerosis, monocytes/macrophages respond to external stimuli with rapid changes in their expression of many inflammation-related genes to undergo polarization towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. Although sialoadhesin (Sn), also known as SIGLEC-1 or CD169, is a transmembrane protein receptor expressed on monocytes and macrophages whether it has a role in macrophage polarization and ultimately, macrophage-driven atherogenesis, has not been investigated. We have previously shown that, independently of Toll-like receptor signaling, extracellular RNA (eRNA) could exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system by inducing cytokine mobilization. In the current study, recombinant mouse macrophage CSF[[Unable to Display Character: –]]driven bone marrow-derived macrophage (BMDM) differentiation was found to be skewed towards the M1 phenotype by exposure of cells to eRNA. This resulted in up-regulation of inflammatory markers, whereas anti-inflammatory genes were significantly down-regulated by eRNA. Interestingly, eRNA was released from BMDM under hypoxia and induced TNF-α liberation by activating TNF-α converting enzyme (TACE) to provoke inflammation. Conversely, TNF-α promoted eRNA release, especially under hypoxia, feeding a vicious cycle of cell damage. Administration of RNase1 or TAPI (a TACE-inhibitor) prevented the production of inflammatory mediators. Murine BMDM isolated from mice deficient in sialoadhesin had the opposite reaction to eRNA treatment with a prominent down-regulation of pro-inflammatory cytokines/M1 phenotype markers, while anti-inflammatory cytokines/M2 phenotype markers were significantly raised. In keeping with the proposed role of eRNA as a pro-inflammatory “alarm signal”, these data further shed light on the role of eRNA in macrophage function in the context of chronic inflammatory diseases such as atherosclerosis. The identification of sialoadhesin as putative eRNA recognition site on macrophages may allow further investigation of the underlying mechanisms of eRNA-macrophage interaction and related signal transduction pathways. Siglec-1 thereby may provides a new target to treat eRNA-mediated vascular diseases.
Abstract 565: Ambient Fine Particulate Matter Decreases Endothelial Progenitor Cells Through Reactive Oxygen Species-mediated Apoptosis
Authors: Cui Yuqi , Xie Xiaoyun , Liu Jason Z , Jia Fengpeng , He Jianfeng , Hao Hong , Liu Ying , Cowan Peter J , Zhu Hua , Sun Qinghua , Liu Zhenguo
Published: July 2021

Background/Aims:
Bone marrow (BM)-derived endothelial progenitor cells (EPCs) play a critical role in angiogenesis and vascular repair. Some environmental insults, like fine particulate matter (PM) exposure, significantly impair cardiovascular functions. However, the mechanisms for PM-induced adverse effects on cardiovascular system remain largely unknown. The present research was to study the detrimental effects of PM on EPCs and explore the potential mechanisms.

Methods:
PM was intranasal-distilled into male C57BL/6 mice for one month. Flow cytometry was used to measure the number of EPCs, apoptosis level of circulating EPCs and intracellular reactive oxygen species (ROS) formation. Serum TNF- α and IL-1β were measured using ELISA. To determine the role of PM-induced ROS in EPC apoptosis, PM was co-administrated with the antioxidant N-acetylcysteine (NAC) in wild type mice or used in a triple transgenic mouse line (TG) with overexpression of antioxidant enzyme network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase (Gpx-1) with decreased in vivo ROS production.

Results:
PM treatment significantly decreased circulating EPC population, promoted apoptosis of EPCs in association with increased ROS production and serum TNF-α and IL-1β levels, which could be effectively reversed by either NAC treatment or overexpression of AON.

Conclusion:
PM exposure significantly decreased circulating EPCs population due to increased apoptosis via ROS formation in mice.
Abstract 566: Experimental Investigation of Blood Flow in the Femoral Bifurcated Artery
Authors: Lotfi Azadeh , Lawler Zachary , Jan Omar , Barber Tracie , Simmons Anne
Published: July 2021

Stent implantation is one of the most widely used interventional treatments for arterial stenosis which occurs predominantly due to atherosclerosis. Although stent placement can ensure very good patency of the lumen, stent-induced hemodynamic disturbance, which can lead to further stenosis, still remains a common clinical complication.

This study investigates the degree of hemodynamic disturbance induced by stenting an idealized bifurcated popliteal artery, which branches into the anterior and posterior tibial arteries, and is known as a site prone to atherosclerosis. Both stent-free and stented bifurcated arteries were examined, and the local flow patterns analysed for the comparative disturbance through the use of Micro Particle Image Velocity (micro-PIV) system. A life-size model of the artery was reconstructed using dimensions obtained from a patient specific MRI scan. The experiments were conducted under steady flow conditions, and the flow rates across the bifurcation were visualized and measured using the micro-PIV system.

It was shown that hemodynamic disturbances induced by the blood flow over the stent can further disrupt the arterial wall downstream of the stent causing further downstream vascular damage in addition to the in-stent restenosis. This downstream vascular disruption may require additional treatment depending on the type and severity of the damage. The results also support the hypothesis that links certain flow dynamic behaviour with the development of early intimal thickening, as the near wall low fluid momentum regions are found at locations where thickening was localized in bifurcated arteries in clinical studies.
Abstract 57: Platelet Extracellular-signal-regulated Kinase 5 is a Redox Switch which Regulates Myocardial Infarct Expansion via Matrix Metalloproteinases
Authors: Cameron Scott J , Ture Sara K , Mickelsen Deanne , Chakrabarti Enakshi , Modjeski Kristina L , Seaberry Michael , Field David , Abe Jun-ichi , Morrell Craig
Published: July 2021

Background:
Dysregulated platelet activation in an ischemic microvascular environment may play a role in myocardial infarction (MI). Platelet receptor signaling is well-characterized, but mechanisms of receptor-independent activation, such as by reactive oxygen species (ROS) generated in ischemic conditions, are less well understood. We discovered that ERK5, a nuclear protein which is ROS-activated in others cells, is abundantly present in platelets. We investigated whether ERK5 could regulate platelet activation and thrombosis in healthy and diseased states.

Methods:
Human and mouse platelets were stimulated with agonists including ADP, U46619, TRAP, convulxin, or ROS (H
2
O
2
or 5% O
2
). ERK5 activity was assessed by immunoblotting. Platelet activation was assessed via fluorescent-activated cell sorting (FACS) for P-selectin or activated GPIIb/IIIa. Intravascular thrombus (pulmonary embolus) or mesenteric thrombus (oxidative injury) formation was assessed by
ex vivo
fluorescent imaging and
in vivo
intravital microscopy, respectively. MI was performed in wild-type (WT) and in platelet specific ERK5 deficient (ERK5
-/-
) mice by LAD coronary artery ligation. Left ventricular (LV) function was determined by echocardiography. Matrix metalloproteinase (MMP) activity was determined by in-gel zymography.

Results:
Human and platelet ERK5 was activated by ROS and via the thrombin and thromboxane receptors, but not via the purinergic or collagen receptors. Murine
in vivo
thrombosis was regulated by platelet ERK5 only if the injury involved oxidative stress. MI in mice promoted sustained platelet activation over one week in an ERK5-dependent manner. Following MI, platelet ERK5
-/-
mice had less reactive platelets, less platelet MMP activity and thromboxane production, attenuated MMP activity in the LV, less remodeling with smaller infarcts, and enhanced myocardial systolic performance.

Conclusions:
ERK5 is an ischemic sensor in platelets which regulates ongoing platelet activation after MI as well as remodeling via myocardial microvasculature. These observations may explain ischemic microvascular aberrations like the no-reflow phenomenon following percutaneous coronary intervention, suggesting a novel pharmacologic target.
Abstract 573: Endothelial α5 Integrins Regulate OxLDL-Induced Fibronectin Deposition, Proinflammatory Gene Expression, and Early Atherosclerosis
Authors: Yurdagul Arif , Green Jonette , Orr Wayne
Published: July 2021

Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to a fibronectin-rich matrix preceding lesion development. Published data from our lab and others demonstrate that endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli. However, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. Work from our group previously demonstrated that oxidized LDL (oxLDL) promotes endothelial proinflammatory gene expression by activating the integrin α5β1, a classic mediator of fibronectin fibrillogenesis. We now show that treating endothelial cells with oxLDL induces fibronectin deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced fibronectin deposition. While endothelial fibronectin expression remains unchanged, oxLDL robustly stimulates the deposition of endothelial cell-derived fibronectin associated with a significant reduction in intracellular fibronectin. Interestingly, loss of endothelial cell-derived fibronectin, but not plasma fibronectin, prevents integrin α5 localization to focal adhesions, reduces fibronectin fibril length, and inhibits oxLDL-induced VCAM-1 expression. In addition, inducible endothelial-specific deletion of α5 integrins significantly blunts atherosclerotic plaque formation in ApoE knockout mice, suggesting an important role for this integrin in early endothelial activation. Taken together, our data demonstrate that oxLDL stimulates α5 integrin-dependent subendothelial matrix remodeling and endothelial proinflammatory gene expression through the deposition of fibronectin.
Abstract 574: Whole Body and Hepatocyte-specific Deletion of Bmal1 Induces Hyperlipidemia and Enhances Atherosclerosis
Authors: Pan Xiaoyue , Hussain Mahmood M
Published: July 2021

The aberrant accumulation of lipids in the plasma is associated with high incidence of atherosclerosis. Plasma lipids exhibit diurnal variations. Diurnal rhythms are controlled by two major transcription factors, Clock and Bmal1. Previously, we have shown that expression of a Clock mutant protein predisposes mice to hyperlipidemia and atherosclerosis. Here we show that the complete and liver-specific ablation of Bmal1 gene expression in

Apoe
-/-

mice promotes hyperlipidemia and atherosclerosis. We observed that Bmal1 deficiency increases of microsomal triglyceride transfer protein (MTP) expression. Molecular studies indicated that Bmal1 deficiency decreases the expression of small heterodimer partner (SHP), a repressor MTP, to increase MTP expression and hepatic lipoprotein production. The effect of Bmal1 deficiency on MTP can be circumvented by the hepatic over expression of SHP. Over expression of SHP in the liver-specific Bmal1 deficient

Apoe
-/-

mice reduced atherosclerosis and plasma lipids. These studies show that Bmal1 regulates hepatic lipoprotein production by regulating SHP. Deregulation of these circadian regulatory mechanisms and physiologic pathways predisposes mice to hyperlipidemia and atherosclerosis.
Abstract 581: Lipid Peroxide Derived Dicarboxylic Acids Induce Atherosclerotic Calcification
Authors: Riad Aladdin , Narasimhulu Chandrakala A , Litvinov Dmitry , Jiang Xueting , Ruiz Irene F , Parthasarathy Sampath
Published: July 2021

Background:
Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, which contribute to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor its relationship to lipid peroxidation is known.

Hypothesis:
Lipid peroxide derived AZA induces calcification of smooth muscle cells thereby providing the link between calcification and overall plaque burden, and association of calcification with the lipophilic region of the lesion.

Methods:
In this study, the potential of lipid peroxide-derived lipophilic dicarboxylic acid (DCA, e.g. azelaic acid) to promote calcification upon exposure to vascular smooth muscle cells was tested. Using 13-Hydroperoxyoctadecadienoic acid (13-hydroperoxylinoleic acid, 13-HPODE) and thin-layer and gas chromatography[[Unable to Display Character: –]]mass spectrometry we characterized the conditions where HPODE is decomposed to aldehyde product 9-oxo-nonanoic acid and its corresponding DCA azelaic acid (AZA).

Results:
Both free AZA and intracellular delivery of AZA via lysophosphatidylcholine (lysoPtdCho) micelles induced calcification of aortic smooth muscle cells, as determined by Von Kossa and alizarin red staining. HPODE treatment resulted in the cellular conversion to ONA and AZA as determined by GC-MS.

Conclusion:
These results demonstrate that DCAs could contribute to atherosclerotic calcification thus accounting for the latter’s relationship to plaque burden and association with lipids. This study also challenges the dogma that arterial calcification represents the deposition of calcium phosphate. Our future work aims to delineate the association of calcium with lipid rich plaques and lipid oxidation with calcification in animal and human atherosclerosis.
Abstract 598: A Platelet Glycoprotein Ib-IX-specific 14-3-3/Rac1/LIMK1 Signaling Pathway Promotes Thrombin-Induced Platelet Activation
Authors: Estevez Brian , Delaney Keegan , Stojanovic-Terpo Aleksandra , Du Xiaoping
Published: July 2021

Rationale:
Thrombin-induced platelet activation requires protease-activated receptors. However, the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds to thrombin and is important for low dose thrombin-induced platelet activation. It is unclear how GPIb-IX promotes thrombin-induced platelet activation.

Objective:
To clearly elucidate the mechanism by which GPIb-IX promotes thrombin-induced platelet activation.

Methods and Results:
We reconstituted GPIb-IX (GPIb) /Protease-activated receptors (PARs) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression. This effect of GPIb appears to require GPIb signaling, as mutation of a cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-IX-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown by pretreating human platelets with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of GPIbα, which inhibited thrombin-induced platelet activation, but did not affect thrombin binding to platelets. Furthermore, 14-3-3 binding site deletion in GPIbα or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1. To determine the role of the Rac1/LIMK1 signaling pathway in mediating thrombin-induced GPIb signaling and platelet activation, we examined the effects of Rac1 knockout, LIMK1 knockout and Rac1-inhibitor on low dose thrombin-induced calcium response and platelet activation. Rac1 inhibitor, NSC23766, abolished the GPIb-dependent cell response in a reconstituted CHO cell model. Rac1 knockout platelets showed diminished platelet response to thrombin and were not different from wild type platelets in the presence of MPαC. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation.

Conclusions:
The stimulatory role of GPIb in thrombin-induced platelet activation requires a thrombin-induced GPIb-specific 14-3-3/Rac1/LIMK1 signaling pathway.
Abstract 607: Kininogen Regulates Thrombin Generation in a Mouse Model of Sickle Cell Disease
Authors: Sparkenbaugh Erica M , Chandarajoti Kasemsiri , Key Nigel S , Gruber Andras , Mackman Nigel , McCrae Keith , Pawlinski Rafal
Published: July 2021

Sickle cell disease (SCD) is associated with activation of coagulation and vascular inflammation. We previously demonstrated that, in mouse models of SCD, tissue factor (TF) expressed on leukocytes activates coagulation and contributes to inflammation via microvascular thrombosis, whereas non-coagulant form of TF expressed by endothelial cells mediates factor Xa (FXa)-PAR2 signalling and contributes to inflammation via IL-6 expression. We also showed that both rivaroxaban and dabigatran, oral inhibitors of FXa and thrombin, attenuated the hypercoagulable state in sickle mice.

It has been proposed that anticoagulants that target the intrinsic coagulation pathway may be associated with a lower risk of bleeding compared to targeting the extrinsic or common coagulation pathways. Therefore, we tested if the intrinsic pathway contributes to the activation of coagulation in sickle cell mice. Bone marrow from sickle (SS) and non-sickle (AA) Townes mice was transplanted into wild-type (WT) (n=18 AA, 15 SS), FXII-/- (n=14 AA, 22 SS), or FXI-/- (n=21 AA, 23 SS) recipients (all on C57Bl/6 genetic background). In addition, bone marrow from AA or SS mice was transplanted into high molecular weight kininogen (HK)+/+ (n=17 AA, 16 SS) or HK-/- (n=10 AA, 19 SS) mice. All mice were used for experiments 5 months after BMT.

Thrombin generation, measured by plasma thrombin anti-thrombin (TAT) levels, was increased in WT mice injected with SS bone marrow (WT/BMSS) compared to WT/BMAA mice (4.4±0.7 vs 2.6±0.3 ng/mL, p<0.05, mean ± S.E.M), however neither FXII nor FXI deficiency affected this parameter. Consistent with these data, inhibition of FXIIa-dependent activation of FXI with 14E11 antibody also did not reduce plasma TAT levels in SS Townes mice. Interestingly, elevated plasma TAT levels observed in HK+/+/BMSS mice was significantly reduced in HK-/-/BMSS mice (4.8±0.5 versus 3.48±0.2 ng/mL, p<0.05). These data indicate that HK, but not FXII and FXI, contributes to thrombin generation in SCD at steady state of disease. In vitro, HK fragments induce TF expression on monocytes via activation of CD11b/18. We are now investigating if inhibition of HK fragments-induced TF expression on monocytes may attenuate the hypercoagulabIe state in SCD mice without impacting hemostasis.
Abstract 617: Phenotype as a Basis for Selection of BM-MSC-Derived SMCs for Regenerative Repair of Abdominal Aortic Aneurysms
Authors: Swaminathan Ganesh , Stoilov Ivan , Bratz Ian , Mecham Robert P , Ramamurthi Anand
Published: July 2021

Abdominal aortic aneurysms (AAA) are a leading cause of death in the elderly with over 150,000 new diagnoses annually in the US. They involve bulging of the abdominal aorta due to chronic proteolytic degradation of the wall structural extracellular matrix (ECM), mainly elastin and collagen. There are currently no established medical management approaches for treating small AAAs and early surgical intervention provides no benefit. AAA growth arrest and regression are also prevented by inherently poor auto regenerative repair of elastic matrix by adult vascular smooth muscle cells (SMCs). We have identified bone marrow mesenchymal stem cell-derived SMCs (BM-SMCs) as superior cell types for regenerative cell therapy due to their high elastogenicity and pro-elastogenic and anti-proteolytic effects on AAA SMCs. In this study we investigated if and how phenotypic coordinates of BM-SMCs on a contractile to synthetic phenotypic continuum impact their above functional benefits. BM-SMC subpopulations were generated under 2% & 10% v/v FBS conditions in presence of transforming growth factor beta-1 (TGFβ), with or without platelet derived growth factor (PDGF-BB) under low & high glucose conditions. Phenotypic coordinates of the BM-SMCs were established by analysis of SMC marker (alpha SM actin (αSMA), SM22, caldesmon, smoothelin, MHC) expression with RT-PCR, western blotting, and immunofluorescence (IF) staining. Elastogenicities of the cell subpopulations were compared by analysis of tropoelastin and matrix elastin synthesis (Fastin assay), crosslinking (ELISA for desmosine), IF, and TEM; contractile properties were compared via a carbachol assay and whole cell patch-clamp for intracellular Ca
2+
/K
+
activity. Overall, our results indicate that 10% FBS + TGF with/without PDGF express higher contractile markers and higher elastic matrix yield when compared to controls (aortic SMCs and MSCs) and other test cases. Together with ongoing studies that are evaluating pro-elastogenic and anti-proteolytic effects of our generated BM-SMCs on AAA SMCs in non-contact coculture, we believe that the results are promising towards identifying superior elastogenic cell sources for a regenerative cell therapy for AAAs, which we are validating in a rat AAA model.
Abstract 62: Molecular Basis of Regulatory Variation at Coronary Heart Disease-Associated Loci
Authors: Miller Clint L , Pjanic Milos , Lee Jonathan D , Liu Boxiang , Greenleaf William J , Montgomery Stephen B , Quertermous Thomas
Published: July 2021

Genome-wide association studies have identified 46 replicated genetic loci for coronary heart disease (CHD), and 104 loci associated at a 5% false discovery rate. However, the regulatory mechanisms of these associations largely remain elusive. Given that the majority of these CHD-associated loci reside in non-coding regions, they are predicted to function via context-specific gene regulation. Recent high-throughput assays of regulatory function include the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq). ATAC-seq utilizes a Tn5 transposase to fragment and tag accessible DNA sequences, which are often coupled to transcription factor occupancy identified by ChIP-seq. Importantly, this assay may reveal the spatio-temporal regulatory profiles in limited numbers of primary cells. Using ATAC-seq in human coronary artery smooth muscle cells (HCASMC) we identified 147,173 accessible chromatin peaks in control versus 198,976 peaks in TGF-beta-stimulated cells (136,446 shared peaks). Using
de novo
motif enrichment analysis we identified significant enrichment of specific AP-1 family members (29.2% vs. 5.1% background), chromatin remodeling, and SMC differentiation transcription factors. Using functional enrichment analysis of ChIP-seq and CHD-overlapping regions we observed enrichment of the hypoxia inducible factor 1 (HIF-1) and TGF-beta signaling pathways (1.5x10
-22
and 5.6x10
-18
, respectively) and relevant phenotypes, including cell migration and blood vessel morphology. Finally, we utilized these regulatory maps to explore the causal mechanisms underlying CHD-associated variants at four loci using haplotype-specific chromatin immunoprecipitation (haploChIP) and luciferase reporter assays. Taken together, these results suggest that genome-wide approaches such as ATAC-seq can be leveraged to map context-specific regulatory mechanisms of non-coding variants associated with complex diseases such as CHD, and reveal new biological and molecular insights into targeting heritable disease risk.
Abstract 629: Sensing Nutrient Overload: The Role of the Endothelium
Authors: Preston Kyle , Scalia Rosario
Published: July 2021

Recent data suggest that leukocyte accumulation into visceral fat depots occurs within 72 following high fat feeding. At this early stage, neutrophils have been found to be the predominant leukocyte population to infiltrate the visceral fat. Other studies have shown that deletion of the high affinity counter-receptor for P-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), attenuate immune cell infiltration into fat depots of chronically high fat fed mice. Accordingly, we tested the hypothesis that endothelial, expressed P-selectin serves as a molecular sensor for initiating inflammatory responses in the microcirculation of the visceral fat following nutrient overload. Wild-type (WT) and P-sel-/- C57BL/6 mice were given either a low fat (10% fat) or a high fat (60% fat) meal by gavage. All mice were studied 1, 2, 3, 4 and 24 hours post gavage by intravital microscopy (IVM) to measure postprandial kinetics of leukocyte rolling (LR) and leukocyte adhesion (LA) in the microcirculation of the mesenteric fat pads. In WT mice, administration of high fat meals acutely and transiently increased LR and LA in the visceral fat microcirculation (p<0.001 vs low fat meal). In contrast, high fat meals failed to increase LR and LA in P-sel-/- mice (NS vs low fat meal WT). Immunofluorescence studies demonstrated that fat meals upregulate cell surface expression of P-selectin in the visceral fat microcirculation, but not in the subcutaneous one. Flow cytometry studies demonstrated that neutrophils are the main leukocyte population to acutely infiltrate visceral fat depots after lipid overload. Mechanistic studies were also undertaken using palmitate, a postprandially elevated, unsaturated free fatty acid. Superfusion of (dose) palmitate during IVM acutely increased LR and LA in the microcirculation of the mesenteric fat pads of WT mice (p<0.01 vs control), a response that was prevented in vivo by P-selectin antibody blockade. Taken together, these data demonstrate that endothelial P-selectin plays a key role in the early inflammatory responses to fat overload by promoting neutrophil trafficking in the microcirculation of the visceral fat. These data first uncover a novel nutrient-sensory role of the vascular endothelium, which warrants further investigation.
Abstract 630: Broad-spectrum Inhibition of the CC-Chemokine Class Accelerates Wound Healing.
Authors: Ridiandries Anisyah , Tan Joanne T , Bursill Christina A
Published: July 2021

Introduction:
Wound healing is a multistep process involving inflammation, proliferation and remodelling at the wound site. Macrophages are important in the inflammatory stage to remove debris, however excessive macrophage accumulation may prolong the inflammatory response leading to prolonged wound healing, excessive scar formation and loss of function at the injury site. In the wound healing process, the CC-chemokine class is involved in the recruitment of macrophages to the wound site. Inhibition of the CC-chemokine class to reduce macrophage infiltration may present as strategy to increase wound closure and reduce scar formation.

Aim:
To determine if broad spectrum CC-chemokine inhibitor, 35K, can improve wound healing by reducing the inflammatory response and preventing scar formation.

Results:
Two full-thickness wounds were created on the dorsum, one each side of the midline, of C57BL6 mice. Wounds were treated topically with PBS or 35K protein daily for 10 days. Wounds treated with 35K closed significantly faster than PBS treated wounds between days 4 - 6 (p<0.05) post-wounding. Laser Doppler measurements revealed 35K significantly increased blood flow at the early (day 3-4) and late stages (day 10) of wound repair. Ten days after wounding, collagen was significantly lower in the 35K treated wounds (25.3% p<0.05). There were also reductions in neovessels (CD31, 39%, p<0.001), arterioles (alpha actin, 48% p<0.01) and macrophages (CD68, 25%), as a percentage of wound area, in 35K treated wounds. In wound tissue collected on day 4 (early stage) post-wounding, 35K treatment had an inhibitory effect on CCL2 and CCL5 protein by 41% (p<0.05) and 36% (p<0.05) respectively, and were reduced by 22% and 66% (p<0.05) respectively at day 10 (late stage) wound healing. Consistent with this, mRNA levels of p65 also decreased in both day 4 (34%, p<0.05) and day 10 (62%, p<0.05) wounds treated with 35K.

Conclusion:
The CC-chemokine class appears to play a key role in wound repair as topical application of CC-chemokine inhibitor 35K promoted wound healing. This was likely to be via inhibition of inflammation and promotion of blood flow recovery in the early stages of wound repair, thereby resulting in less scar formation.
Abstract 637: Hyperhomocysteinemia-mediated sCD40L induction and CD16
+
CD40
+
Monocyte Differentiation in Chronic Kidney Disease
Authors: Yang Jiyeon , Choi Eric T , Kunapuli Satya P , Yang Xiaofeng , Wang Hong
Published: July 2021

Chronic kidney disease (CKD) with uremia is associated with high mortality of cardiovascular disease (CVD) and Hyperhomocysteinemia (HHcy) is highly prevalent in uremic CKD patients. Elevated inflammatory monocyte (MC) is a cellular hallmark of chronic inflammation and CVD. Here we investigated mechanism in uremia-associated HHcy on MC differentiation in CKD-associated CVD. Data base mining revealed that CD40 is induced in MC from CKD subjects and associated with CVD, inflammatory disease and MC activation. Blood samples were obtained from 28 vascular disease patients with or without CKD.

By flow cytometric analysis using CD14+ as a MC marker, we observed inflammatory CD16+ MC is increased in CVD, whereas CD40+ MC and CD16+CD40+ MC are increased in CKD-associated CVD. CD40+ MC expresses T cell activation markers CD86, HLA-DR, adhesion receptor CD62L, and chemokine receptor Ccr2. Plasma CD40L levels are increased in CVD, positively correlated with CD16+ MC. Interestingly, plasma Hcy levels are increased in CKD-associated CVD, positively correlated with cellular Hcy, plasma creatinine, CD16+CD40+ MC, and negatively correlated with S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH), an indicator of methylation. In addition, MC and T cell inflammatory cytokines TNFα, IL-6, and IFN[[Unable to Display Character: ɤ]] are induced in CKD-associated CVD subjects.

Next, we examined mechanism of CD16+CD40+ MC differentiation using cultured human peripheral blood mononuclear cells (hPBMC). CKD serum, Hcy, and CD40L induced CD16+CD40+ MC differentiation, which were prevented by folic acid and CD40L antibody. IFN[[Unable to Display Character: ϫ]], TNFα, and IL-6 synergistically induced CD16+CD40+ MC differentiation, which was blocked by neutralizing antibodies to TNFα and IL-6.

Hcy inhibited DNA methyltransferase 1 activity in isolated human blood MC. Finally, by gene analysis and pyrosequencing, we identified that the core promoter of CD40 gene is located at sole CpG island and hypomethylated at p65 consensus element in WBC from CKD-associated CVD subjects with low SAM/SAH ratio.

In conclusion, we identified CD16+CD40+ MC as a novel inflammatory MC subset which is increased in uremic HHcy-associated CVD. CD16+CD40+ MC differentiation may be due to CD40 promoter DNA hypomethylation.
Abstract 638: Flow Modulates the Endothelial Expression of Neuronal Guidance Cues
Authors: Zhang Huayu , de Bruin Ruben G , Dane Martijn J , Sol Wendy M , van der Veer Eric P , van den Berg Bernard M , Rabelink Ton J , van Zonneveld Anton Jan , van Gils Janine M
Published: July 2021

Disturbed hemodynamic conditions lead to impaired homeostasis of the vascular endothelium and, subsequently, influx and retention of monocytes in the arterial wall. Emerging data suggest that, aside from a role in the development and maintenance of the nervous system, neuronal guidance cues (NGCs) are also required for physiological and pathological immune responses. We recently reported that NGCs are differentially expressed in atherosclerosis-prone and -resistant aortic sites in LDLR-/-mice during early atherogenesis. Here, we detail our investigation of the role of shear stress on the expression levels of NGCs (ephrins, slits, netrins and semaphorins) in human umbilical vein endothelial cells (ECs) under atherosclerosis-prone (static) and -protective (laminar flow) conditions.

To gain insight into the consequences of laminar flow on the mRNA expression levels of NGCs in ECs, the cells were exposed to 0, 1 or 7 days of laminar flow (shear stress 10 dynes/cm2, IBIDI-flow system). The mRNA levels of the leukocyte attractant NGCs EphrinA1, B1 and B2 were all reduced by laminar flow, in particular for EphrinB2 (52%[[Unable to Display Character: ]], p=0.02). In contrast, the potentially anti-inflammatory Slit2 displayed a striking early response to flow, with a marked increase in mRNA expression after 1day (2.2-fold, p=0.02), but was not sustained at Day 7. Interestingly, the expression levels of netrin-4, semaphorin 3F and semaphorin 4B were upregulated in ECs exposed to laminar flow for 1 and 7 days (2.1-fold, p=0.05; 3.0-fold, p=0.04; 2.1-fold, p=0.05; respectively), suggesting these NGCs as potentially athero-protective anti-inflammatory leukocyte repellent. Moreover, overexpression of the shear-induced transcription factor Krüppel-like factor 2 (KLF2) in static cultured ECs induced the expression of both semaphorin3F mRNA and protein (3.8 fold and 3.9 fold, respectively), while netrin-4 mRNA and protein upregulation appeared to be independent of KLF2.

Taken together, our study provides novel insight into regulation of the endothelial NGC expression profile by flow, which could implicate NGCs as mediators of the EC function by facilitating repulsion or attraction of monocytes under varying hemodynamic conditions.
Abstract 653: The Role of "Saposin-Like Domain" in Wnt Signaling
Authors: Krishnamoorthy Aparna
Published: July 2021

Hyperlipidemia is a major contributor to atherosclerosis that results in endothelial dysfunction and is driven by several cell types including macrophages and vascular smooth muscle cells. Among the various cytokines and signaling molecules that contribute to the disease, dysregulated Wnt signaling is known to result in uncontrolled cell division within bulging aortic plaques. Wnt proteins are morphogens that signal through interactions with two membrane bound co-receptors: Frizzled and Low-density lipoprotein receptor related protein 5/6. Wnt binding to these receptors transduces a signal, leading to increased cellular levels of ß-catenin that migrates to the nucleus and leads to gene activation. Normally ß-catenin is rapidly degraded, a process that attenuates Wnt signaling. However, in many diseases, including atherosclerosis, ß-catenin levels remain elevated leading to uncontrolled activation of the Wnt pathway genes. Interestingly, while the intracellular events of the Wnt signaling pathway are well studied, questions about how a hydrophobic molecule like Wnt engages with cell surface receptors remain unexplored in the field. A recent X-ray crystal structure revealed Wnt exists as a two-domain protein: a C-terminal “cytokine like domain” and an N-terminal “saposin-like domain” that contains a covalently bound fatty acid. Saposins are a family of proteins that possess membrane / lipid binding properties and undergo a conformational change when they contact membrane surfaces. My hypothesis is that the saposin-like domain is a membrane active component that plays a critical role in initiating Wnt signaling by undergoing a conformational change upon reaching cell membrane in order to promote Wnt-receptor engagement and signaling. I plan to isolate and express the saposin-like domain, which will exist as a water-soluble helical bundle in the absence of lipid. When presented with an appropriate lipid surface, the saposin-like domain will unfurl, thereby exposing a hydrophobic interior that contacts the membrane surface. The ability of the isolated domain to interfere with Wnt signaling will also be determined. Findings from my study reveal the molecular basis underlying initial events of Wnt receptor interaction in atherosclerosis.