bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 370: Supernates from Stored Red Blood Cells Inhibit Collagen-Mediated Aggregation of Platelets Exposed to Arterial Shear Rates
Authors: McFaul Steve J , Garrastazu Hiram , Rodriguez Armando , Cap Andrew P , Campbell James E
Published: March 2022

Background:
Uncontrolled bleeding is a leading cause of death on the battlefield. We demonstrated previously that supernatant fluid from stored non-leukoreduced (NLR) red blood cells (RBC) inhibited aggregation of platelets in whole blood from healthy donors as measured by impedance aggregometry and decreased GPIIb/IIIa expression. In the current study, we examine stored NLR RBC supernatant fluid inhibition of platelet aggregation in a physiological model of arterial flow.

Methods:
Fresh citrated blood (500 ul) was mixed with supernate from stored NLR RBC (median age 44 days, 250 ul) and tyrodes buffer (250 ul) for impedance aggregometry. Flow model experiments utilized blood prepared with calcein-labeled platelet rich plasma, RBCs (40% hematocrit), and supernate (25% total volume). Arterial simulated flow (shear rate 1000/sec) over collagen I was carried out in a 75μm x 350μm x 5mm microchamber (Bioflux). GPIIb/IIIa expression was assessed by flow cytometry.

Results:
Supernates from nine stored NLR RBC units decreased platelet aggregation amplitude in fresh blood from eleven donors by an average of 40±12% vs Tyrode’s buffer controls while one supernate did not exhibit significant inhibition. In concordance with previously reported results, supernates decreased GPIIb/IIIa expression on platelets in whole blood by 53±1.2%. Linear regression analysis of a single flowed experiment showed a 60.5% decrease in aggregation rate (NLR RBC supernate vs HEPES buffered saline control). Inhibition of the rate of aggregation observed in the flow model vs aggregation amplitude by impedance correlated well (sup1: 85.1% vs. 52%; sup 2: 46.6% vs. 41%).

Conclusions:
Stored NLR RBC units significantly inhibit collagen-stimulated aggregation of platelets in the presence of high (arterial) and low (stirring) shear rates, and reduce expression of the fibrinogen-binding glycoprotein, IIb/IIIa, on the platelet surface. The substantial decrease in platelet aggregation in a physiologically relevant model of whole blood flow suggests massive transfusion of NLR RBC units may decrease the hemostatic capacity of platelets in combat casualties and contribute to uncontrolled hemorrhage. [apc1]unclear
Abstract 377: Soluble CD40 Ligand Reverses the Inhibitory Effect of Early Endothelial Progenitor Cells on Platelet Aggregation
Authors: Bou Khzam Lara , Hachem Ahmed , Zaid Younes , Mourad Walid , Merhi Yahye
Published: March 2022

Introduction and hypothesis:
Circulating Endothelial progenitor cells (EPCs) interact with other blood cells and platelets and may contribute to limit the thrombo-inflammatory reactions at the sites of vascular injury. Indeed, we have previously shown that EPCs inhibit platelet activation and aggregation. The CD40/CD40L axis is a thrombo-inflammatory mediator that affects platelet and endothelial functions. EPCs express CD40 while platelets represent the major source of soluble CD40 ligand (sCD40L) in the circulation. We aimed to demonstrate the existence of a sCD40L/CD40 signaling pathway in EPCs and to test the hypothesis that sCD40L affects the anti-platelet function of EPCs.

Methods and results:
Human peripheral blood mononuclear cell-derived early EPCs in culture express CD40 and its adaptor proteins, the tumor necrosis factor associated factors: TRAF1, TRAF2 and TRAF3. sCD40L stimulation of early EPCs had no effect on TRAF2 expression, but increased its binding to CD40. In contrast, sCD40L increased the expression of TRAF1 and affected its binding to CD40; whereas TRAF3 binding was not affected. In functional studies, pretreatment of early EPCs with sCD40L had no effect on their viability, but reduced their inhibitory effect on platelet aggregation. However, the release of the anti-aggregating agents prostacyclin and nitric oxide by early EPCs was not affected by sCD40L treatment.

Conclusion:
We have revealed the existence of the CD40L/CD40/TRAF axis in early EPCs, which reduced their anti-platelet properties. This was not related to an effect of sCD40L on early EPC survival nor on their release of prostacyclin or nitric oxide.
Abstract 39: Protein Disulfide Isomerase Plays an Important Role in αMβ2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation
Authors: Hahm Eunsil , Li Jing , Huh Sungjin , Wang Zhenjia , Rogelj Snezna , Cho Jaehyung
Published: March 2022

Introduction:
Protein disulfide isomerase (PDI), a cell-surface localized oxidoreductase, plays a crucial role in regulating integrin-mediated platelet functions. Although PDI is detected on the neutrophil surface, its role in neutrophil recruitment during vascular inflammation remains unknown.

Objectives:
To determine the physiological role of neutrophil surface PDI in integrin-mediated neutrophil recruitment during vascular inflammation.

Methods and Results:
Using real-time fluorescence intravital microscopy in myeloid-specific PDI knockout mice, we have demonstrated that neutrophil PDI is required for neutrophil recruitment into the site of TNF-alpha-induced cremaster venular inflammation. Infusion of wild-type PDI but not functionally-inactive PDI rapidly rescued the defect of neutrophil recruitment in the PDI knockout mice. Cell-impermeable PDI inhibitors also perturbed neutrophil recruitment to the site of TNF-alpha-inflamed venular inflammation in wild-type mice. Neutrophil recruitment was further inhibited when blocking anti-PDI antibodies were infused into alphaLbeta2 but not alphaMbeta2 knockout mice, suggesting that extracellular PDI regulates alphaMbeta2-mediated neutrophil recruitment. To further investigate the role of neutrophil surface PDI in integrin-mediated neutrophil adhesion during vascular inflammation, adhesion assays were performed using human neutrophils treated with cell-impermeable PDI inhibitors. PDI inhibition impaired human neutrophil adhesion to TNF-alpha-activated endothelial cells under venous shear and to ICAM-1-coated surfaces under static conditions. Neutrophil adhesion was further inhibited when anti-PDI was combined with anti-alphaL but not anti-alphaM antibodies. PDI inhibitors also perturbed fibrinogen binding to activated alphaMbeta2 integrin. Using PDI shRNA in neutrophil-like HL60 cells, we found that knockdown of PDI gene diminished alphaMbeta2-mediated cell adhesion to activated endothelial cells under shear. Immunoprecipitation assays and confocal microscopy revealed that surface PDI-alphaMbeta2 interaction is enhanced upon neutrophil activation and regulated by the isomerase activity.

Conclusion:
Our results indicate that neutrophil surface PDI regulates the alphaMbeta2-ligand interaction, thereby playing an important role in neutrophil recruitment to the activated endothelium during vascular inflammation.
Abstract 402: Calpain-1 Deficiency Does Not Affect Angiotensin II-Induced Atherosclerosis or Abdominal Aortic Aneurysms in Male LDL Receptor--Deficient Mice
Authors: Subramanian Venkateswaran , Moorleghen Jessica J , Howatt Deborah A , Balakrishnan Anju
Published: March 2022

Background and Objective:
Chronic infusion of angiotensin II (AngII) augments the development of atherosclerosis and induces abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Recently, we demonstrated that pharmacological inhibition of calpains, a class of calcium-activated, neutral cysteine proteases, attenuated AngII-induced atherosclerosis and AAAs in mice. The purpose of this study was to determine the contribution of calpain-1 in the development of AngII-induced atherosclerosis and AAA using genetically modified mice.

Methods and Results:
Male LDL receptor
-/-
mice (10-12 weeks old) that were either calpain-1 +/+ or
-/-
(n=20 per group) were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for 5 weeks. After 1 week of feeding a saturated fat enriched diet, mice were infused subcutaneously with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. AngII increased systolic blood pressure similarly in both groups (pre-infusion- calpain-1 +/+: 122 ± 4; calpain-1 -/-: 123 ± 6 mmHg; post-infusion calpain-1 +/+: 156 ± 5; calpain-1 -/-: 153 ± 6 mmHg). Deficiency of calpain-1 had no effect on plasma cholesterol concentrations (calpain-1 +/+: 1589 ± 39; calpain-1 -/-: 1531 ± 70 mg/dL) or lipoprotein-cholesterol distributions during AngII infusion. AngII infusion led to development of AAA formation in 76% of calpain-1 +/+ mice and 65% in calpain-1
-/-
mice. This difference was not statistically significant. AngII infusion greatly augmented the development of aortic arch atherosclerosis that was not attenuated in calpain-1
-/-
mice, (calpain-1 +/+: 6.9 ± 1.0 %; calpain-1
-/-
: 5.1 ± 0.8 % of arch area intimal lesions; P = not significant).

Conclusion:
These findings suggest that calpain-1 does not contribute to the development of angiotensin II-induced atherosclerosis or AAAs.
Abstract 424: Novel Computational Algorithm to Quantify Blood Flow and Vascular Resistance from Contrast Angiography with High Accuracy
Authors: Mix Doran S , Schwarz Karl , Varble Nicole , Day Steven , Phillips Dan , Gillespie David , Chandra Ankur
Published: March 2022

OBJECTIVES:
Contrast angiography can diagnose arterial occlusive disease but cannot provide hemodynamic data. Past work has attempted to calculate angiographic blood flow but clinical use has been limited by measurement error of >10%. Our hypothesis was that blood flow could be calculated from a contrast angiogram with <10% error through the application of a novel computational algorithm.

METHODS:
A pulsatile,
in vitro
hemodynamic simulator with a light-based angiographic imaging system (InfiMed, Inc.) was used as the testing platform. Flow rates were varied through increases in outflow resistance and were directly measured with a Transonic flow meter (+/-4% error). An algorithm was designed to determine instantaneous flow from DICOM images using a combination of automatic vessel detection, segmentation, and time of flight bolus tracking. These calculated flow rates were compared to those directly measured.

RESULTS:
The calculated flow rates (cc/min) were highly accurate when compared to those directly measured (4.1+/-3% error). Furthermore, time-density curves were accurate enough to detect relative changes in flow of 1.7 cc/sec reflecting changes in distal vascular resistance (Figure 1).

CONCLUSION:
We conclude that using this approach, blood flow can be angiographically measured with increased accuracy relative to prior work. This may provide clinically reliable hemodynamic data to guide diagnostic and therapeutic interventions.
Abstract 440: Regional Differences in Fat Loss Following Weight Gain in Normal Adults
Authors: Singh Prachi , Somers Virend K , Romero-Corral Abel , Sert-Kuniyoshi Fatima H , Pusalavidyasagar Snigdha , Davison Diane E , Jensen Michael D
Published: March 2022

Background:
Normal weight adults gain leg fat gain via adipocyte hyperplasia and upper body subcutaneous (UBSQ) fat gain via adipocyte hypertrophy.

Objective:
To understand whether regional fat loss mirrors fat gain and whether leg fat loss results in decreased adipocyte number vs. size.

Design:
We assessed UBSQ, leg and visceral fat gain and loss in response to over- and underfeeding in 23 young, healthy normal weight adults (15 men). Participants gained ∼5% weight over 8 weeks and lost ∼80% of gained fat over the following 8 weeks. We measured abdominal subcutaneous and femoral adipocyte size and number after weight gain and loss.

Results:
Volunteers gained 3.1 ± 2.1 kg of body fat with overfeeding and lost 2.4 ± 1.7 kg with calorie restriction. Although upper body subcutaneous (UBSQ) fat and visceral fat gains were completely reversed with weight loss, leg fat had not yet returned to baseline values by 8 weeks. Abdominal and femoral adipocyte size decreased significantly with weight loss, but the number of adipocytes did not decrease. Abdominal adipocyte size decreases correlated (rho= 0.76, p = 0.001) with UBSQ fat mass decreases and femoral adipocyte size decreases correlated (rho= 0.49, p=0.05) with leg fat mass decreases.

Conclusion:
UBSQ and visceral fat depots increase and decrease proportionately with short term weight gain and loss, whereas gain and loss of leg fat are less well linked. Loss of lower body fat is due to reduced fat cell size, not number, which may result in long-term increases in leg fat cell number. This may have implications for long-term weight control.
Abstract 441: Cigarette Smoke Regulation of Cholesterol Efflux and Protease Production in Atherosclerosis Decreases Plaque Collagen Content and Increases Plaque Necrosis
Authors: Lemaitre Vincent , Sklepkiewicz Piotr L , D’Armiento Jeanine M
Published: March 2022

There is strong epidemiological data linking smoking to an increase in the incidence and severity of cardiovascular diseases. However, the molecular mechanisms responsible for this susceptibility are unknown. Recent studies report progression of atherosclerosis with smoke exposed individuals developing a higher incidence of acute coronary events. We investigated the influence of cigarette smoke exposure on the development of atherosclerotic lesions in apolipoprotein E (Apoe) knockout mice and demonstrated an increase in lesion size along with cholesterol levels in Apoe knockout mice exposed to cigarette smoke for 20 weeks. In addition, there was a dramatic increase in plaque necrosis with a decrease in the collagen content of lesions. The accumulation of lipids in macrophages induces an inflammatory response, with the secretion of cytokines and proteases, and leads to apoptosis and necrosis within the lesion. Our hypothesis is that cigarette smoke components impair cholesterol efflux and increase MMP expression in macrophages, leading to increased apoptosis and necrosis. Whole cigarette smoke extract (5% CSE) significantly impaired cholesterol efflux to ApoAI in elicited peritoneal macrophages correlating with the down regulation of ABCA1 and ABCG1 transporters. In addition, 5% CSE induced expression of several crucial matrix metalloproteinases in human THP-1 macrophages (MMP-1, 8, 13, 14) and mouse peritoneal macrophages (MMP-9, 13). After increasing ABCA1 and G1 expression with treatment with an LXR agonist the CSE-induced up regulation of MMPs (MMP-9 and 13) was inhibited suggesting a correlation between the loss of ABC transporters and MMP expression. Ex vivo experiments utilizing peritoneal macrophages elicited from mice exposed to cigarette smoke for 4 days demonstrated a decrease in ABCA1 and ABCG1 expression with the up regulation of MMPs (MMP-9, 12, 13 and 14) confirming that parallel to impairing cholesterol efflux through the down regulation of ABC transporters, cigarette smoke components induce MMPs ex vivo. The combined effect of cigarette smoke on protease expression and cholesterol efflux are likely synergistic in generating a more vulnerable plaque through an increase in necrosis and a lower collagen content whereby the mechanism is secondary to a direct effect of cigarette smoke on the expression of ABC transporters.
Abstract 473: Relationship of White Blood Cell Count with Coronary Atherosclerosis by Calcium Scores and Epicardial Fat Burden in Patients with Acute Chest Pain
Authors: Kassi Mahwash , Trevino Alejandro , Anwar Naveed , Forouzandeh Farshad , Nabi Faisal , Chang Su Min
Published: March 2022

Background:
Elevated white count ( WBC) as inflammatory marker has been associated with more severe CAD and worse outcome in patients with acute myocardial infarction (MI). However, the relationship between total white count and calcium score (CACS) and epicardial fat (EAT) in patients with acute chest pain but no MI is not known.

Methods:
:
CACS was evaluated by non contrast cardiac computed tomography in 974 consecutive patients with no history of CAD but admitted with acute chest pain and no MI by ECG and enzymes. Epicardial fat volume was measured in 713 pts . We divided the WBC count by the median and then at 10 K ( close to top quartile) . Pearson’s correlation was used to test association between log (CAC +1) and epicardial fat volume .

Results:
The mean age for this population was 57.4 with 60 % female. The predominant race was Caucasians (60%). The mean Framingham score was 8.16 ± 8.15. The mean WBC was 7.8± 4.38 ( x 10 K) . The median WBC was 7.3 x 10k. The mean CACS and EAT volume did not differ between the groups when different cut off of WBC was used ( see table)

There is no difference in the prevalence of CACS >0 or moderate CACS ( > 100) in different WBC group ( for WBC > 10 K 17 % has CACS > 100 vs 18 % in WBC <=10K p = NS) ) Similar result was found on the EAT volume analysis. The only predictor of CACS was age, FRS and EAT volume (log (CAC +1) vs epicardial volume: correlation coefficient 0.266 p <0.001).

Conclusion:
WBC does not predict calcium scores or epicaridal fat volume in patients with acute chest pain .
Abstract 478: Osteoprotogerin and Osteopontin Serum Levels Are Associated with Endothelial Function and Arterial Stiffness in Coronary Artery Disease
Authors: Maniatis Konstantinos , Siasos Gerasimos , Tousoulis Dimitrios , Oikonomou Evangelos , Zaromitidou Marina , Kioufis Stamatios , Paraskevopoulos Theodoros , Zisimos Konstantinos , Kollia Christina , Miliou Antigoni , Kokkou Eleni , Papavasiliou Athanasios G , Stefanadis Christodoulos
Published: March 2022

Background:
Osteoprotogerin (OPG) and osteopontin (OPN) have recently emerged as key factor in both vascular remodeling and development of atherosclerosis. Endothelial function and arterial stiffness are significant factors of clinical evaluation in patients with coronary artery disease (CAD). The study was designed to evaluate the relation between serum concentration of OPG, OPN and arterial function in CAD.

Methods:
We enrolled 280 patients with CAD (mean aged 61±11years), and 129 control subjects (mean aged 60±12 years). Serum OPG and OPN levels were measured, using ELISA. Endothelial function was evaluated by flow mediated dilation (FMD) in the brachial artery and carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness.

Results:
There was no difference between control subjects and CAD patients according to age (p=0.48) and sex (p=0.07). CAD patients had significantly impaired FMD (4.75 ± 2.22 % vs.6.50± 3.13 %, p<0.001) and increased PWV (8.94 ± 2.21 m/sec vs. 8.28 ± 1.91 m/sec, p=0.006) compared to control subjects. CAD patients had also significantly higher levels of OPG (3.91±1.87 pmol/l vs. 2.88±2.17 pmol/l, p<0.001) and logOPN (1.81±0.18 ng/ml vs. 1.71±0.24 ng/ml, p<0.001) compared to control subjects. More importantly, PWV were positively associated with serum OPG levels (r=0.19, p<0.01) and with serum logOPN levels (r=0.10, p=0.049) and FMD was negatively associated with OPG levels (r=-0.126, p=0.048).

Conclusion:
Our findings indicate that CAD patients had increased OPG and logOPN levels. Moreover, there is a consistent association between endothelial dysfunction and impaired arterial stiffness with serum levels of OPG and OPN in CAD patients.
Abstract 488: Acute Fluvastatin Pleiotropy Is Mevalonate Independent
Authors: Stein Jeffrey J , Maier Kristopher G , Gahtan Vivian
Published: March 2022

Objectives
Thrombospondin 1 (TSP1) is a matricellular glycoprotein present in injured arteries, and acts as a chemoattractant for vascular smooth muscle cells (VSMC). Migration of VSMCs to an area of injury plays a role in the development of intimal hyperplasia. We have recently shown that acute treatment of VSMCs with statins leads to a decrease in TSP1-induced VSMC chemotaxis. The purpose of this study was to determine whether the acute statin effect is dependent on the mevalonate pathway or is a pleiotropic effect.

Methods
Quiescent VSMCs were treated for 20 hours (standard time) or 20 minutes (acute treatment) with 100μM mevalonolactone, 0.5 μM fluvastatin, or mevalonolactone and fluvastatin combined. Migration of VSMCs towards 20 μg/ml TSP1 was assessed using a modified Boyden microchemotaxis chamber. Data was recorded as cells migrated per 5 high power fields and converted to percent positive control. Extracellular signal-regulated kinase 1/2 (ERK 1/2) activation was determined by Western blot. Densitometry is presented as percent positive control.

Results
TSP1 induced VSMC migration compared to SFM, 100% + 7.6 vs. 35.3% + 4.5. Treatment with fluvastatin for 20 hours (34.3% + 6.9) or 20 minutes (38.4% + 9) inhibited TSP1-induced VSMC migration. 20 hour mevalonate treatment together with fluvastatin restored the migratory effect of TSP1, 81% +9.9, p=0.11. Addition of mevalonate to acute fluvastatin treatment, did not reverse the anti-migratory effect of fluvastatin compared to TSP1 alone, 35.6% + 9.9, p<0.0001. TSP1 significantly activated ERK 1/2 compared to basal medium, p42: 100% + 0 vs. 62.3% + 12.6; p44: 100% + 0 vs. 56.9% + 8.7. Acute fluvastatin did not inhibit ERK1/2 activation compared to TSP1, p42: 86.7% + 12.7; p44: 81.7% + 9.9.

Conclusion
These data indicate long-term fluvastatin inhibition of TSP1 stimulated migration is at least partially dependent on the mevalonate pathway. In contrast, acute fluvastatin inhibition of TSP1-induced chemotaxis involves an alternate pathway resulting in pleiotropic statin effects. Previous studies found that standard treatment time with statins inhibited ERK 1/2 activation. Our current study found that acute fluvastatin inhibition of TSP1-induced chemotaxis does not appear to be ERK1/2 dependent.
Abstract 494: Platelet Aggregation in a Model of Simulated Arterial Flow: Red Blood Cells Are Integral Players in Hemostatic Control Resuscitation
Authors: Campbell James E , Cap Andrew P , Blackbourne Lorne H
Published: March 2022

Background:
Approximately one in four trauma patients exhibits an early bleeding diathesis, termed the acute coagulopathy of trauma and defined by an initial INR >1.2, which is associated with a 3-fold increase in mortality. In recent years, the concept of damage control resuscitation has been developed to address this problem. This approach emphasizes hemorrhage control and early resuscitation with blood products in a 1:1:1 ratio of plasma:platelets:RBCs in an attempt to replace whole blood lost in hemorrhage. The optimal ratio of blood components has not been established and few pre-clinical studies have explored this problem under arterial shear conditions.

Objective:
Our objective is to establish stepped ranges of hematocrit values and platelet counts to identify the component and concentration of that component most responsible for the largest loss of platelet-collagen interaction.

Methods:
Donor citrated whole blood containing calcein tagged platelets was flowed at an arterial shear rate of 1000/second through a 70μm x 350μm x 5mm collagen-coated chamber at 37°C for 10 min, n=6. Images were captured every 15 seconds and analyzed for fluorescence intensity.

Results:
Linear regression analysis generated slopes (fluorescent intensity units/second) that were most different as hematocrit dropped below physiologic levels (HCT40control=100%, HCT25=39.5%, HCT20=21.4%, HCT18=17.4%, HCT16=11.6%). Non-linear fitting of slopes vs. HCT yielded the equation y=1.27e4 x
2
-3.51e4 x +4.43e5 describing an exponential decrease in platelet-collagen interaction with linear decreases in HCT.

Conclusions:
The largest drop in platelet adherence to collagen in a physiological model of arterial flow occurs as hematocrit falls from 40%. The commonly suggested 30HCT target for minimal RBC transfusion during resuscitation may not adequately support hemostasis and should be reconsidered.
Abstract 499: Rac1 Plays a Stimulatory Role in Glycoprotein Ib-IX-Dependent Platelet Activation
Authors: Delaney Michael K , Zheng Yi , Du Xiaoping
Published: March 2022

The platelet receptor for von Willebrand factor (VWF), the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. Ligand occupancy of GPIb-IX stimulates the sequential activation of the Src family kinase (SFK) Lyn, phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin aIIbβ3, synthesis of thromboxane, granule secretion, and integrin-dependent stable platelet adhesion and aggregation. It remains unclear, however, as to how the PI3K/Akt pathway is activated following ligand binding to GPIb-IX. Using platelet-specific conditional Rac1
-/-
mice and the Rac1 selective inhibitor
NSC23766
, we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1-deficient platelets are defective in VWF/GPIb-induced aggregation, thromboxane synthesis and granule secretion, and stable adhesion to VWF under shear stress. Importantly, GPIb-IX-induced activation of integrin aIIbβ3 was also significantly impaired in Rac1
-/-
or Rac1-inhibitor treated platelets, indicating that Rac1 is critical for GPIb-mediated integrin activation. However, Rac1 deficiency does not alter the VWF binding function of GPIb-IX, demonstrating that the functional defects observed in Rac1-deficient platelets derives from a defect in VWF-induced GPIb-IX signaling. To determine why Rac1 is important in GPIb-IX signaling, we further show that VWF/GPIb-induced activation of Rac1 was abolished in Lyn
-/-
mouse platelets or SFK-inhibitor treated human platelets but remained unaffected by the PI3K inhibitor LY-294002, suggesting that Rac1 is activated downstream of Lyn independent of PI3K. Moreover, VWF/GPIb-induced PI3K-dependent activation of Akt and P38 MAPK was abolished in Rac1-deficient platelets. Thus, our study reveals that Rac1 is an important new link in the GPIb-IX signaling pathway that mediates Lyn-dependent activation of the PI3K/Akt pathway leading to integrin activation.
Abstract 509: Identification of Macrophage Phenotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome
Authors: Everaert Bert R , Van Laere Steven J , Hoymans Vicky Y , Timmermans Jean-Pierre , Vrints Christiaan J
Published: March 2022

Objective:
Circulating angiogenic cells (CACs) have been identified as important regulators of vascular biology. However, there is still considerable debate about the phenotype and function of CACs.

Methods and results:
DNA microarray transcriptome analysis was used to study the CAC phenotype and investigate the key biological pathways in CAC biology. We determined a CAC-specific gene signature, which revealed the close relationship between CACs and members of the macrophage lineage, such as tumor-associated macrophages (TAMs), dendritic cells (DCs), osteoclasts and atherosclerotic plaque-remodeling macrophages. We identified CCL2, CCL17, CCL18 and CCL22 as a chemokinetic fingerprint for CACs and indicated cholesterol transporter pathways (LXR/RXR, FXR/RXR) and riboflavin metabolism as highly important biological pathways in the CAC transcriptome. These findings, together with upregulation of the chemokine receptor CCR7, suggest the involvement of CACs in reverse plaque remodeling.

Conclusions:
CACs are phenotypically similar to regulatory M2 macrophages and lack signs of endothelial differentiation. However, the expression profile of CACs prompted us to suggest new mechanisms by which these cells could engage in cardiovascular pathophysiology, such as immunomodulation, tissue remodeling, enhancement of cholesterol efflux and vasculoprotection. These findings expand the current knowledge on CAC biology and suggest new indications for CAC therapy in future clinical trials.
Abstract 510: Gene Set Enrichment Analysis of the Dual PPAR-α/γ Agonists Aleglitazar and Muraglitazar Reveals Distinct Metabolic Signatures in Human Cardiomyocytes
Authors: Wright Matthew B , Ebeling Martin , Steiner Guido , März-Weiss Pia
Published: March 2022

Dual-acting agonists of the peroxisome proliferator-activated receptors α and γ (PPAR-α/γ) have been pursued to reduce cardiovascular risk in type 2 diabetes. Although core PPAR signaling appears conserved, compound-specific on- and off-target effects contribute to profiles of specific molecules. Here, we used gene set enrichment analysis (GSEA) to compare the effect of dual PPAR-α/γ agonists aleglitazar (Ale) and muraglitazar (Mura).

Primary human cardiomyocytes were incubated with non-cytotoxic concentrations of Ale or Mura (both 1 nM to 25 μM). Whole-genome expression studies were carried out using Illumina microarrays. Principal Component Analysis, hierarchical clustering and GSEA using the publicly available Pathway Commons database identified multiple signaling and metabolic networks significantly regulated by both compounds. Effects of Mura at the higher concentrations substantially diverged from both Ale and low concentrations of Mura, suggesting potential off-target effects. Within the Pathways Commons collection analyzed, four networks were significantly differentially affected by 25 μM Mura versus the other treatments.

Nuclear Factor Y, which recognizes CCAAT elements, was identified as a common signature of Mura-specific effects on the cholesterol biosynthesis pathway (seven genes,
p
= 0.003 for enrichment).

The divergent effects of Ale and Mura on metabolic, energy regulation and interferon signaling pathways may contribute to their unique biological profile.
Abstract 515: Increased Adventitial Inflammation Occurs in Regions of Low Endothelial Shear Stress in a Swine Model of Coronary Atherosclerosis
Authors: Antoniadis Antonios P , Papafaklis Michail I , Chatzizisis Yiannis S , Sukhova Galina K , Takahashi Saeko , Tsuda Masaya , Coskun Ahmet U , Stone Peter H , Feldman Charles L
Published: March 2022

Introduction:
Plaque inflammation is a critical step in the initiation and progression of atherosclerosis. Such inflammation is thought to originate from the luminal surface of plaque and infiltrate the intima-media in advanced lesions. Low endothelial shear stress (ESS) is known to induce intima-media inflammation and plaque growth. In this study we investigated in-vivo the hypothesis that low ESS induces also adventitial inflammation.

Methods:
We studied 11 swine at 23 (baseline) and 30 (followup) weeks after the induction of diabetes and hyperlipidemia. Using angiography and intravascular ultrasound data, we performed 3D coronary reconstruction of coronary arteries and calculated the ESS with computational fluid dynamics. In 56 segments, we assessed the adventitial inflammatory (CD45) and antigen-presenting (MHC-II) cell content at followup with immunohistochemistry. Segments were classified as low (≤ 1 Pa) or higher (>1 Pa) ESS.

Results:
MHC-II content in the adventitia (1.3±0.3%) was higher than in the media (0.3±0.1%, p<0.001) but lower than in the intima (9.3±2.3%, p<0.001). Low-ESS regions had increased adventitial MHC-II content compared to high-ESS regions (1.6±0.4 vs. 0.9±0.3%, p<0.05). Adventitial MHC-II content was not influenced by plaque size (large plaques 1.7±0.5 vs. intermediate plaques 0.8±0.3%, p=0.24), internal elastic lamina fragmentation (IEL) grade (minimal/absent 0.6±0.3 vs. moderate/large 1.4±0.3, p=0.9), or remodeling pattern (inadequate 1.7±0.6 vs. compensatory 1.2±0.4 vs. expansive 0.6±0.2%, p=0.6). Adventitial CD45 content was not significantly different between low- and high-ESS segments, and also not different in large vs. intermediate plaques, in segments with various IEL fragmentation grades or remodeling patterns.

Conclusion:
Although total inflammation is not dependent on ESS, low ESS induces higher adventitial activated inflammatory cell content, as assessed by MHC-II immunostaining. This, in conjunction with the higher MHC-II content in the adventitia than in the media and the presence of an intact IEL suggests an additional source of inflammation in low-ESS plaque regions, originating from the vessel outer wall. The induction of neovascularization possibly accounts for this phenomenon.
Abstract 524: The Interaction of Agt Gene Polymorphism with Cardiovascular Risk Traits in Atherosclerosis
Authors: Dzimiri Nduna , Al-Najai Mohammed , Elhawari Samar , Gueco Daisy , Vigilla Mary Grace , Andres Editha , Muiya Paul , Mazher Nejat , Alshahid Maie , Meyer Brian F
Published: March 2022

Angiotensinogen (AGT) is an important component of the renin-angiotensin system which is involved in the regulation of systemic blood pressure and many other cardiovascular functions. In the present study, we evaluated possible interaction of the
AGT
gene polymorphism with cardiovascular risk traits in triggering atherosclerosis in 4525 angiographed candidates, including 3232 coronary artery disease (CAD) and 2292 non-CAD individuals. Association studies were performed for 8 SNPs by the Applied Biosystems real-time PCR system. Four of the studied variants, i.e. recessive TT of rs699C>T [Odds ratio(95% Confidence Interval) = 1.29(1.16-1.43); p<0.00001], combined CT+TT of rs2148582C>T [1.28(1.08-1.52); p=0.004], AG+GG of rs5051A>G [1.30(1.09-1.55); p=0.003] and AG+GG [1.44(1.16-1.77); p=0.001] of rs2067853C>T conferred risk for primary hypertension (HTN; 3521 cases versus 1094) as well as CAD. Interestingly, the rs699 was also causative for hypercholesterolaemia [1.15(1.05-1.28); p<0.0002] and hypertriglyceridaemia 1.22(1.11-1.35); p<0.00001]. In contrast, the rs2148582 [0.90(0.82-0.980; p=0.012] and rs5051 [0.89(0.82-0.98); 0.011] showed some protective properties against acquiring type 2 diabetes mellitus (T2DM; 2544 cases versus 2070 controls), while the rs3789679 appeared be protective against HTN (0.0001) but causative for T2DM. Several haplotypes constructed from the 8 studied SNPs were either independently associated, or shared by some of the traits among themselves or with CAD. Important ones include two 8-mers GTGGGTGG (χ
2
=6.97; p=0.0083) and GTAGGCGG (χ
2
=5.69; p=0.017) associated with HTN, ACGAGTAT (χ
2
=6.76; p=0.0093) and ACGGATAT (χ
2
=9.83; p=0.0017) associated with T2DM, GTAGGCGG (χ
2
=9.43; p=0.0021) associated with obesity, and ACGGATAT (χ
2
=6.56; p=0.0104) associated with hyperlipidaemia. Put together, the data shows that
AGT
causative variants are partly shared among cardiovascular risk traits and CAD, pointing to the likelihood of gene-disease interaction as important contributing factor in atherosclerosis disease pathways.
Abstract 534: GPR30 Stimulates Capillary Formation by Human Endothelial Progenitor Cells
Authors: Dubey Raghvendra K , Baruscotti Isabella , Hauser Christine , Imthurn Bruno , Barchiesi Federica , Rosselli Marinella
Published: March 2022

Estradiol (E2) induces vascular repair by promoting endothelial progenitor cell (EPC) growth and capillary formation. Based on our previous findings that the capillary stimulating effects of E2 are mimicked by its non-permeable analog, BSA-tagged E2, we hypothesize that the stimulatory effects are potentially mediated via the newly discovered membrane bound estrogen receptor (ER) GPR30. To investigate this, we assessed capillary formation by EPC derived endothelial cells in response to E2, and GPR30 agonists (G1) and antagonist (G15) in a matrigel based assay. Capillary formation was assessed microscopically by quantifying junction/sprout formation and capillary length. E2 (10nM) increased EPC-induced capillary formation by 126 ± 5% and these effects were mimicked by G1 (stimulated by 133 ± 8.9% and 210 ± 7.2% by 1 and 10 nM, respectively) and propyl-pyrazole-triol [PPT; stimulated by 143 ± 9%; 100 nM; ER-α agonist], but not by diarylpropionitrile (ER-β agonist). The stimulatory effects of estradiol, G1 and PPT were significantly (p<.05) abrogated by G15 (20nM), a GPR30 antagonist, suggesting a role for GPR30 in mediating the capillary stimulatory effects of E2. Interestingly, the stimulatory of estradiol, PPT and G1 were also abrogated by methyl-piperidino-pyrazole (ER-α antagonist), implying that the stimulatory effects of E2 are mediated via cross-talk between GPR30 and membrane bound ER-α. Because, receptor tyrosine kinase (RTK), hemeoxygenase (HO), and G-protein coupled mechanisms are involved in capillary formation we investigated their role in GPR30 induced capillary formation. The stimulatory effects of G1 were blocked by SU5416 (RTK inhibitor), ZnPP (HO inhibitor), pertusis toxin (G protein pathway inhibitor), and LY294002 (Akt pathway inhibitor). Moreover, treatment of EPCs with G1 induced expression of phosphorylated-Akt and HO-1, but not MAPK. We conclude that E2 via GPR30 or GPR30-coupled ER-α promotes EPC-mediated capillary formation by a mechanism that involves non-genomic activation of RTKs and HO-1 activation. E2 in particular and GPR30 agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation following injury.
Abstract 537: S-Nitrosoglutathione Reductase (GSNOR) Deficiency Reduces Vascular Inflammation and Atherosclerosis.
Authors: Marchini Julio F , Manica Andre , Griffin Gabriel , Quillard Thibaut , Dutzmann Jochen , Travers Richard , Criado Veronica A , Tesmenitsky Yevgenia , Luscinskas Francis W , Stamler Jonathan S , Simon Daniel I , Croce Kevin
Published: March 2022

Introduction:
Diminished NO production and altered NO bioavailability promote vascular inflammation and atherosclerosis. S-nitrosoglutathione reductase (GSNOR) controls cellular NO balance by degrading S-nitrosoglutathione (GSNO), which is a major source of nitric oxide (NO) bioactivity. GSNOR inhibition increases protein S-nitrosylation and potentiates NO action. The role of GSNOR in vascular inflammation is undefined, and the purpose of our investigation was to determine if GSNOR targeting dampens inflammation and reduces atherosclerosis.

Methods and results:
We evaluated vascular inflammation and atherosclerosis in wild type (WT) and GSNOR-deficient (GSNOR
-/-
) mice. TNF-induced NFκB activation was reduced by 40% (p<0.05) in endothelial cells (ECs) isolated from GSNOR
-/-
compared to WT mice, which was accompanied by attenuated expression of P-selectin, E-selectin, and VCAM-1 (>34% reduction, p<0.05). Decreased EC adhesion molecule expression led to a 45% reduction in leukocyte rolling in the cremaster muscle microvasculature of GSNOR
-/-
mice (p<0.01). Deficiency of GSNOR also reduced macrophage production of MCP-1 and PDGF-stimulated aortic smooth muscle cell (SMC) proliferation (p<0.01). Furthermore, GSNOR
-/-
SMCs displayed a more mature phenotype compared to WT as evidenced by 105% increased mRNA expression of myosin heavy chain 11 (p<0.05), and 45% decreased expression of the SMC embryonic marker tropomyosin-4 (p<0.005). High fat-fed mice doubly deficient in ApoE and GSNOR (GSNOR
-/-
/ApoE
-/-
) had a 27% decrease in aortic atherosclerotic lesion area compared to ApoE deficient controls (p<0.03).

Conclusion:
GSNOR deficiency attenuates vascular inflammation and atherosclerosis. These data highlight a novel role for GSNOR in regulating NO balance and vascular inflammation
in vivo
, thereby suggesting that GSNOR is an attractive therapeutic target for atherosclerotic vascular disease.
Abstract 9: Two Distinct Roles of Plasminogen Activator Inhibitor-2 (PAI) and PAI-1 in Deep Vein Thrombosis
Authors: Siefert Suzanne A , Chabasse Christine , Netzel-Arnett Sarah , Hoofnagle Mark H , Sarkar Rajabrata , Antalis Toni M
Published: March 2022

Objectives:
Urokinase-plasminogen activator (uPA) has a vital role in deep vein thrombosis (DVT). While PAI-1 is the main inhibitor of uPA
in vivo
, PAI-2, found mostly intracellularly, has proven to have more anti-inflammatory and anti-apoptotic than anti-fibrinolytic activities. We explored the hypothesis that PAI-1 and PAI-2 play roles in DVT through either fibrinolytic or inflammatory pathways.

Methods:
Wild type C57Bl/6 (WT), PAI-2
-/-
, and PAI-1
-/-
mice underwent surgical caval ligation. Subsequent thrombus was harvested at various time points and measured for thrombus weight (TW) normalized to body weight. Tissue was analyzed by western blot, ELISAs, zymography and flow cytometry.

Results:
PAI-2 deficiency resulted in unchanged thrombus formation (TW day 4: WT = 1.037 ± .043 mg/g n=17; PAI-2
-/-
= 1.028 ± .067 mg/g n=17, p=.91), while it enhanced thrombus resolution (TW day 12: WT = .43 ± .03 mg/g n=13; PAI-2
-/-
= .319 ± .021 mg/g, n=16, p=.004). PAI-1 deficiency caused decreased formation (TW day 12: PAI-1
-/-
= .901 ± .043 mg/g n=11, p=.045) and enhanced resolution (TW day 12: PAI-1
-/-
= .248 ± .011 mg/g n=10, p=.00004). Though total uPA levels in thrombi were similar between groups, active uPA by ELISA was increased by 5 fold in PAI-1
-/-
mice and 40 fold in PAI-2
-/-
mice compared to WT mice at day 12, indicating increased uPA-mediated fibrinolysis. Exploring other mediators of resolution, we found that PAI-1 and PAI-2 deficiencies lead to contrasting results. By zymography, PAI-1 deficiency caused an early increase in matrix metalloproteinase (MMP) activity that decreased over time. Conversely, PAI-2 deficiency trended towards less early MMP activity that then increased. Using flow cytometry to assess clot cell populations, on day 4, PAI-2
-/-
mice had an increase in neutrophils with a decrease in macrophages, whereas PAI-1
-/-
mice had an increase in macrophages.

Conclusions:
This identifies PAI-2 as a novel regulator of venous thrombus resolution and suggests the first context
in vivo
in which PAI-2 has an anti-fibrinolytic role. Both PAI-1 and PAI-2 deficiencies enhance thrombus resolution, but through diverging mechanisms. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution.
Abstract 100: Initial Characterization of Oxidant-Resistant Human ApoAI Transgenic Mice
Authors: Berisha Stela Z , Hung Kim T , Brubaker Greg D , Smith Jonathan D
Published: March 2022

Introduction

HDL functions, such as its cholesterol acceptor and anti-inflammatory activities, are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes HDL and apoAI. We previously found that the 4WF apoAI isoform, in which the 4 Trp residues are substituted with Phe, is equally competent as wild type (WT) human apoAI to interact with lipids and accept cholesterol in an ABCA1-dependent manner, but is resistant to MPO mediated loss of function.

Hypothesis

The oxidant resistant 4WF apoAI isoform will be superior in mediating reverse cholesterol transport than WT human apoAI in vivo under conditions of elevated oxidative stress.

Methods and Results

We generated transgenic mice expressing the 4WF human apoAI (h-apoAI) isoform. A genomic fragment containing the h-apoAI gene was subjected to site directed mutagenesis to convert the Trp to Phe residues, and microinjected into C57BL/6 fertilized oocytes. We selected the line with the highest h-apoAI expression to compare with existing WT h-apoAI transgenics. We assessed plasma apoAI and HDL-C levels in male and female mice. Male mice have significantly higher h-apoAI levels than females for both isoforms (p<0.05). In general, the WT apoAI transgenics have higher plasma h-apoAI levels than the 4WF transgenics; however there is enough overlap that we were able to select male 4WF and WT h-apoAI transgenic mice having matched plasma h-apoAI levels of ~550 mg/dl, which are described below. HDL-C levels were slightly lower in 4WF vs. WT transgenics (157±4 vs 184±8 mg/dl, respectively, N=5 per group, p=0.03). We performed in vivo reverse cholesterol transport (RCT) assay by s.c. injection of cholesterol labeled and loaded bone-marrow macrophages. RCT to the plasma reflected the HDL-C levels and was lower in the 4WF vs. WT transgenic mice (4.5% vs 6.6%, respectively p=0.007). However, RCT to the liver and feces was equivalent in the 4WF and WT transgenics (NS). This suggests that the flux of HDL-C in the 4WF mice may be higher than in the WT apoAI transgenics, leading to equal RCT to the feces and liver despite lower HDL-C levels.

Conclusion

4WF transgenic mice are now breeding with MPO transgenic mice in order to test our hypothesis that this isoform retains more HDL function under oxidative stress conditions.
Abstract 15: The Helix-Loop-Helix Factor Id3 Links Natural Helper Cells to Proliferation of Atheroprotective B-1a Cells
Authors: Perry Heather , Oldham Stephanie , Fahl Shawn , Harmon Daniel , Que Xuchu , Tsimikas Sotirios , Witztum Joseph , Bender Timothy , McNamara Coleen
Published: March 2022

Natural immunity is emerging as important for atheroprotection. Natural IgM antibodies, such as E06, that recognize oxidative epitopes on LDL or phospholipids prevent atherosclerosis and are produced by the atheroprotective B cell subset, the B-1a B cell. IL5, an important B-1a B cell mitogen, can be produced by the innate lymphoid cell, the natural helper (NH) cell. We previously demonstrated that splenic B cells provide atheroprotection to B cell deficient mice, an effect dependent on the helix-loop-helix transcription factor, Id3. Taken together, these results raise the hypothesis that Id3 may be an important regulator of splenic B-1a B cells and natural immunity in atheroprotection.

Methods and Results

Id3
-/-
Apoe
-/-

mice at 8 weeks old had fewer B-1a cells in the spleen (200672 ±58019, n = 9) compared to

Id3
+/+
Apoe
-/-

mice (511840 ±88811, n = 9) as determined by flow cytometry. Consistent with fewer B-1a B cells, there were lower levels of serum E06 in

Id3
-/-
Apoe
-/-

mice compared to

Id3
+/+
Apoe
-/-

mice (1573 ±300 RLU vs. 2807 ±263 RLU respectively, n = 8 for each group) as assessed by ELISA.

Id3
-/-
Apoe
-/-

mice had decreased B-1a B cell homeostatic proliferation compared to

Id3
+/+
Apoe
-/-

mice (30.0 ±3.3% vs. 54.4 ± 1.2% respectively, n = 4) as measured by
in situ
CFSE. However, B-1a B cell proliferation and number and levels of E06 were unchanged in mice with B cell specific deletion of Id3 compared to controls. To determine if Id3 is required for IL33 induced levels of IL5, IL33 or vehicle control was i.p. injected into

Id3
+/+
Apoe
-/-

mice or

Id3
-/-
Apoe
-/-

mice every 3 days for 7 days.

Id3
-/-
Apoe
-/-

mice had less IL33 induced serum IL5 levels (5.1 ±3.1 pg mL
-1
, n = 5) compared to

Id3
+/+
Apoe
-/-

mice (67.4 ±22.9 pg mL
-1
, n = 4). Indeed, loss of Id3 significantly attenuated IL33 induced IL5 production in NH cells measured by flow cytometric intracellular cytokine staining (

Id3
-/-
Apoe
-/-

: 31.1 ±5.4%, n = 6 vs.

Id3
+/+
Apoe
-/-

: 50.3 ±4.3%, n = 6). NH cells are also present and can to produce IL5 in response to exogenous IL33 (vehicle: 4.0 ±1.4%, n = 4 vs. IL33: 14.7 ±2.4%, n = 4) in the aortic adventitia/perivascular adipose tissue of

Apoe
-/-

mice.

Conclusion

Results provide the first evidence implicating Id3 as a key regulator of NH cell IL5 production and B-1a B cell proliferation.