bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 81: Pharmacological Inhibition of Calpain Attenuates Adipose Tissue Apoptosis, Macrophage Accumulation and Inflammation in Diet-induced Obese Mice
Authors: Subramanian Venkateswaran , Balakrishnan Anju , Howatt Deborah A , Moorleghen Jessica J , Katz Wendy S 
Published: July 2021

Background and Objective
Obesity is associated with low-grade chronic inflammation and apoptosis that contributes to development of insulin resistance and other metabolic complications. Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not defined. Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions, such as cytoskeletal remodeling and apoptotic cell death. Recent studies have demonstrated that activated calpain promotes adipocyte differentiation in vitro, and enhances macrophage recruitment during nephropathy. However, the functional role of calpain activation in adipose tissue macrophage accumulation and obesity remains to be elucidated. The purpose of this study was to define whether pharmacological inhibition of calpain influences diet-induced obesity and adipose tissue macrophage accumulation in mice.

Methods and Results
Male C57BL/6 mice (8 weeks old; n=10 per group) were fed either low (10% kcal) or high (60% kcal) fat diet for 12 weeks. Calpeptin, a calpain inhibitor (2.5 mg/kg/day) or vehicle (DMSO) was administered daily by osmotic mini-pumps for 12 weeks. Calpeptin administration did not influence high fat diet induced body weight and fat mass gain throughout the study. Calpain inhibition had no effect on glucose and insulin tolerance in obese mice. However, calpain inhibition highly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as evident by TUNEL, Picro Sirius and CD68 immunostaining. Real-time PCR analysis showed that calpain inhibition significantly suppressed inflammatory cytokines (TNFα, IL-6, MCP-1, F4/80) expression in adipose tissue (P<0.05 vs vehicle). In addition, Oil Red O staining revealed that calpain inhibition also suppressed accumulation of hepatic fat.

Conclusion
Pharmacological inhibition of calpain attenuated macrophage accumulation, adipocyte apoptosis, fibrosis and inflammation in diet-induced obese mice without influencing body weight gain and insulin tolerance.
Abstract 171: Role of α-Actinin-4-Dependent Endothelial Cell Stiffness in Neutrophil Transmigration
Authors: Hordijk Peter , Schaefer Antje , te Riet Joost , Ritz Katje , Hoogenboezem Mark , Anthony Eloise , Mul Erik , de Vries Carlie , Daemen Mat , Figdor Carl , van Buul Jaap 
Published: July 2021

Inflammation is causally linked to many chronic human disorders and constitutes a growing problem in the ageing population. The inflammatory process is driven by interactions of activated leukocytes with the endothelial lining of blood vessels. This requires binding of leukocyte β2-integrins to endothelial ICAM-1 (InterCellular Adhesion Molecule-1), which allows leukocyte adhesion, spreading, crawling and transendothelial migration (TEM). Integrin binding induces ICAM-1 clustering and its consequent association to F-actin which enforces leukocyte adhesion. Here, we analyzed the molecular basis of this positive feedback loop.
We show that ICAM-1 clustering promotes its binding to F-actin through distinct complexes with FilaminB, Cortactin and α-Actinin-4. We found that α-Actinin-4 regulates endothelial cell peripheral stiffness, which is sensed by adherent neutrophils and promotes adhesion, spreading, crawling and TEM. Conversely, increasing endothelial cell stiffness stimulates the ICAM-1-α-Actinin-4 interaction. Finally, we found that the endothelial lining of atherosclerotic plaques, which is characterized by increased stiffness and leukocyte infiltration, shows increased expression of α-Actinin-4. These results identify α-Actinin-4-regulated endothelial cell stiffness as a novel pro-inflammatory event that promotes ICAM-1-mediated leukocyte adhesion and TEM.
Abstract 378: Macrophage ß3 Integrin is an Important Player in Inflammation and Cellular Plasticity
Authors: Wolf Cristina A , Bobak Izabela , Su Xinming , Damm Ellen , Weilbaecher Katherine N , Schneider Jochen G 
Published: July 2021

Communication between cells and the surrounding environment is a crucial mechanism for survival. Integrins are membrane-bound molecules that are involved in signaling between the cells and the extracellular matrix, thereby influencing cytoskeletal stability and intracellular signaling. β3 integrin and its binding partner αv form the αvβ3 heterodimer that is expressed in various cells. We and others have described the consequences of its absence in inflammation, atherosclerosis and cancer in vivo. However, the distinct role of this integrin as a signaling molecule and the consequences of its absence for macrophage structure remain mostly elusive.
Our aim is to further characterize the phenotype of β3-deficient (β3-/-) bone marrow-derived macrophages (BMDM) under stimulatory conditions (LPS and LDLs) compared to control cells in vitro.
qPCR, WB, ELISA, migration, proliferation assays were used to investigate β3-/- BMDM and controls (wt BMDM and Raw 264.7).
LPS was described to be not only pro- but also anti-inflammatory in a time-dependent manner. We show that LPS stimulation leads to high expression of pro-inflammatory cytokines (IL-1β and TNFα) shortly after treatment, while expression of anti-inflammatory cytokine (IL-10) arises at a later stage (12h post stimulation). Interestingly, β3-/- BMDM express more IL-1β than controls. IL-10 expression appears much earlier in β3-/- BMDM (6h post stimulation) but is reduced after 12h, indicating a faster and higher cellular response in the absence of the β3 integrin.
OxLDL, the leading cause to foam cell formation, stimulates the expression of IL-1β in controls and β3-/- BMDM with the latter expressing significantly less of this cytokine indicating that lack of β3 causes differential cellular responses after LPS and oxLDL stimulation. Other LDL forms tested (nLDL, acLDL, cLDL) did not have any effect on IL-1β expression.
In addition, we identified a higher proliferation rate in the β3-/- BMDM when cultured with M-CSF and a migration deficit in response to LPS, M-CSF and VEGF.
Taken together, our results show that macrophage β3 deficiency causes differential cellular plasticity depending on the stimulus, with functional consequences that could be essential in inflammation and atherosclerosis.
Abstract 499: Development of a Novel Antithrombotic Therapy Targeting Platelet GPIbα: Assessment of Anfibatide In Vitro and in Phase I Clinical Trial
Authors: Hou Yan , Reheman Adili , Li Benjamin X , Dai Xiangrong , Yang Zhongqiang , Qian Fang , Zhang Guohui , Xu Zhengyu , Liu Jing , Liang Chaofan , Zhao Yanan , Shen Jie , Zhou Hui , Wang Yiming , Marshall Alexandra H , Lei Xi , Zarpellon Alessandro , Vanhoorelbeke Karen , Ruggeri Zaverio , Ni Heyu 
Published: July 2021

Platelet adhesion and aggregation are critical for hemostasis and atherothrombosis. Interaction between the GPIb complex and VWF initiates platelet adhesion and contributes to complete vessel occlusion, particularly at sites of stenosis. However, no therapeutic targeting this pathway has been developed. Previously, we demonstrated in murine thrombosis models that Anfibatide, a snake venom-derived GPIbα antagonist, inhibited botrocetin-induced VWF-GPIbα binding and inhibited thrombus formation in vitro and in vivo, without significantly activating platelets or changing bleeding. We subsequently hypothesized that Anfibatide would be a safe and potent anti-thrombotic agent in humans.
We tested Anfibatide with human platelets in vitro and conducted the first anti-GPIbα phase I clinical trial with 94 healthy volunteers. In vitro, Anfibatide inhibited ristocetin-induced and low dose thrombin-induced human platelet aggregation. Surface plasmon resonance studies showed that Anfibatide binds to GPIbα and blocks binding of both VWF A1 domain and thrombin. In ex vivo perfusion chambers, Anfibatide strongly inhibited human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface, especially at high shear flow conditions. Importantly, Anfibatide effectively dissolved the preformed thrombi in perfusion chambers. Anfibatide incubation with human whole blood did not significantly change coagulation parameters measured by thromboelastography. After treating volunteers with Anfibatide, no serious adverse events, premature discontinuations due to adverse events, or deaths occurred during the study. Anfibatide bound to 95% of GPIb and inhibited 90% of platelet aggregation without prolonging activated partial thromboplastin time, prothrombin time, thrombin time, or bleeding time. There was no bleeding tendency, no alteration of platelet count, and no detectable anti-Anfibatide antibodies.
In conclusion, Anfibatide provides anti-thrombotic effects through inhibition of both GPIbα-VWF and GPIbα-thrombin interactions. These results demonstrate that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.
Abstract 594: Macrophage Proteomics Identifies Mechanistic Links Between Type 2 Diabetes and Atherosclerosis
Authors: Coats Brittney , Schoenfelt Kelly , Becker Lev 
Published: July 2021

Obesity and Type 2 Diabetes (T2Ds) are associated with increased risk of atherosclerosis and CHD. However, the mechanism(s) linking these diseases are incompletely understood. Macrophages play essential roles in the pathogenesis of both disorders, suggesting that changes in macrophage biology link obesity/T2D and atherosclerosis. We previously showed that the atherogenic actions of cholesterol-loaded macrophages (foam cells) are an emergent property of a dysregulated sterol responsive protein network secreted by macrophages (MSRN). Thus, we hypothesized that obesity/T2Ds may impact atherogenesis by similarly disrupting the MSRN.
To test this hypothesis, we isolated peritoneal macrophages (PMΦ) from C57BL/6 mice fed a chow or obesogenic diet and interrogated their secreted proteomes using mass spectrometry. Based on stringent statistical analysis (FDR<5%), we identified 36 proteins that were differentially expressed in media conditioned by macrophages from obese relative to lean mice. Unsupervised gene set enrichment analysis demonstrated that obesity-induced changes to the PMΦ proteome significantly targeted the MSRN. Importantly, this phenomenon could not be explained by cholesterol accumulation or inflammation, since PMΦ’s from obese mice did not accumulate cholesterol and pro-inflammatory cytokine expression was not induced in PMΦ’s from obese or atherogenic mice.

Our previous studies showed that treating hypercholesterolemic
Ldlr
-/- mice with TZD (insulin sensitizer) could normalize MSRN protein expression
in vivo
, suggesting that insulin resistance is a key regulator of the network. To test this hypothesis, we placed C57BL/6 mice on an obesogenic diet for 7 weeks and treated the mice with TZD for 2 weeks. TZD therapy had no effect on body weight, but normalized fasting glucose levels. Moreover, PMΦ’s isolated from TZD-treated mice, demonstrated normalized protein expression patterns. Collectively, our studies suggest that insulin resistance in T2Ds reproduces macrophage protein dysfunction observed in atherogenic models, and provides a plausible mechanism by which T2Ds may act to increase risk of atherosclerosis.
Abstract 131: Restoration of ER Stress and Induction of FGF15/19 Independently Rescue ABCG5 ABCG8 Sterol Transporter
Authors: Wang Yuhuan , Su Kai , Sabeva Nadezhda , Ji Ailing , van der Westhuyzen Deneys , Foufelle Fabienne , Gao Xia , Graf Gregory 
Published: July 2021

Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic ABCG5 ABCG8 (G5G8). Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanism responsible for G5G8 regulation remains unclear.
In the current study, we conducted a series of experiments to address potential mechanisms. To determine if leptin signaling directly regulates hepatic G5G8 abundance, we acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8.
Alternatively, the G5G8 protein complex may be decreased due to compromised ER stress. It has been previously reported that tauroursodeoxycholate (TUDCA) alleviates ER stress. It also increases G5G8 and biliary cholesterol in both lean and db/db mice. The ER chaperone protein, glucose regulated protein 78-kDa (GRP78) can restore ER function and reduce unfolded protein response (UPR) signaling. Therefore, we tested the hypothesis that expression of GRP78 could rescue G5G8 in db/db mice. Adenovirus encoding GRP78 was administered to db/db mice and the effect on hepatic G5G8 was determined. G5 and G8 proteins and biliary cholesterol were increased in the absence of changes in mRNAs encoding either protein.
However, TUDCA has also been shown to induce FGF15. In several models of bile acid feeding, FGF15/19 is stimulated in ileum and activates its receptor in liver to repress bile acid synthesis. Simultaneously, G5G8 and biliary cholesterol secretion are elevated. To determine if FGF15/19 had a direct effect on hepatic G5G8, we injected C57BL/6 mice with recombinant FGF19. CYP7A1 and CYP8B1 mRNA expression were both strongly suppressed, whereas G5G8 increased at both mRNA and protein levels.
In conclusion, G5G8 can be rescued in ob/ob and db/db mice through multiple mechanisms that include restoration of ER functions and FGF15/19 signaling. Counter regulation of CYP7A1, CYP8B1, and G5G8 by FGF15/19 allows for the maintenance of hepatic sterol elimination in the face of expanded bile acid pool.
Abstract 238: Distinct Vascular Effects of Celecoxib and Rofecoxib in vivo
Authors: Ricciotti Emanuela , Grosser Tilo , Grant Gregory R , Liu Shu-Lin , Lawson John A , Assoian Richard , FitzGerald Garret A 
Published: July 2021

Randomized controlled trials of cyclooxygenase (COX)-2 selective NSAIDs revealed that these drugs confer a cardiovascular hazard, resulting mainly from suppression of COX-2 derived prostacyclin (PGI2).
Disruption of rodent COX-2-derived PGI2 in vascular cells and cardiomyocytes has recapitulated all elements of the cardiovascular hazard attributable to NSAIDs in humans.
To investigate further the impact of COX-2 inhibition in the vasculature, we examined the effects of therapeutic concentrations of two selective COX-2 inhibitors, celecoxib and rofecoxib, on thrombosis and vascular injury. Furthermore, we used RNA sequencing (RNA-Seq) to assess whether celecoxib and rofecoxib might elicit distinct genomic effects in mouse aorta.
Celecoxib (100 mg/Kg) and rofecoxib (50 mg/Kg) caused a selective and equipotent COX-2 inhibition as indicated by measuring thromboxane (TXB-M, an in vivo index of COX-1 activity) and prostacyclin (PGI-M, an in vivo index of COX-2 activity) urinary metabolites by mass spectrometry.
Both celecoxib and rofecoxib augmented platelet deposition and prolonged platelet disaggregation after laser-induced injury in the cremaster arterioles.
A similar effect was observed in mice lacking PGI2 receptor IP. There was no additional pro-thrombotic effect of the drugs on an IPKO background, consistent with suppression of COX-2 dependent PGI2 mediating the predisposition to thrombosis induced by celecoxib and rofecoxib.
Celecoxib, but not rofecoxib, caused a reduction of neointimal hyperplasia and the intima/media ratio of injured femoral artery at 3 weeks after wire injury.
RNA-seq analysis revealed 903 transcripts differentially expressed between celecoxib and control group and 3,127 transcripts between rofecoxib and control group (at a false discovery rate of 10%) in mouse aorta.
Thus, both celecoxib and rofecoxib cause a similar predisposition to thrombosis in mice.
Celecoxib, but not rofecoxib, reduces neointimal hyperplasia after vascular injury and COX-2 independent genetic actions might modulate this effect.
The predisposition to thrombosis and attenuation of the response to vascular injury by celecoxib recapitulates what has been found in humans.
Abstract 260: Identification of Micrornas Specifically Expressed in Isolated Cells of Human Abdominal Aortic Aneurysm
Authors: Pinet Florence , Spear Rafaelle , Hot David , Staels Bart , Chwastyniak Maggy , Amouyel Philippe , Haulon Stephan 
Published: July 2021

Abdominal aortic aneurysm (AAA) is a vascular asymptomatic disease responsible for 4% of mortality in the elderly population. MicroRNAs (miRNA) have recently been shown to be potential biomarkers due to their stability in plasma.
The aim of this study was to investigate the potentiality of miRNAS as biomarkers of AAA by identifying miRNAs specifically expressed in the key cells present in the human AAA tissue.
The distribution of inflammatory cells such as smooth muscle cells (SMC), M1 and M2 macrophages, neutrophils, B and T lymphocytes and mast cells were located by immunohistochemistry in 20 human AAA biopsies, showing a specific distribution towards the aneurysmal aortic wall and the presence of adventitial tertiary lymphoid organs (ATLOs) in 10 samples. We isolated by laser microdissection (LMD) area enriched in aneurysmal SMC, M1 and M2 macrophages, and ATLOs and SMC from control aorta. RNA extracted from 2 samples of LMD-isolated cells was screened on human miRNAs microarray.
Out of the 850 human miRNAs, more than 200 miRNAs were detected in LMD-isolated aneurysmal cells, with 164 detected in ATLOs, 49 in SMC, and 87 miRNAs in macrophages. We selected the 3 miRs with the highest expression for ATLOs and 10 miRs (based on their miR-29b levels) for SMC and macrophages for validation by qRT-PCR in LMD-isolated samples (n=4).
We found that miR-15a-3p (0.1 fold) and miR-30a-5p (0.2 fold) were down-regulated and miR-489-3p up-regulated (2 fold) in ATLOs which was inversed for miR-489-3p in the whole aneurysmal aorta. Of the 10 miRNAs selected, six (miR-199a-3p and miR-451 upregulated and miR-24, miR-29a, miR-29b, and miR-29c downregulated) were modulated similarly in SMC and macrophages and whole aneurysmal aorta, except for miR-199-3p. Let-7f and miR-34a were similarly upregulated ed in both subtypes of macrophages and aneurysmal aorta.
Expression in the plasma of AAA (n=24) compared to PAD (n=18) patients was significantly down-regulated for miR-15a-3p (p = 0.03) and miR-30a-5p (p = 0.04) and upregulated for let-7f (p=0.048) and miR-29b (p=0.035).
In conclusion, this non-hypothesis driven screening of miRNAs expressed in isolated aneurysmal cells allowed to identify four miRNAs as potential AAA biomarkers.
Abstract 420: Exploring the Role of TNFα in TLR4-/NLPR3 Inflammasome-driven Inflammation in Humans
Authors: Moerland Matthijs , Malone Karen , Dillingh Marlous , Grievink Wieke , Reijers Joannes , Burggraaf Koos 
Published: July 2021

The role of TNFα in the pathogenesis of atherosclerosis is incompletely understood. TNFα blockade reduces the severity of various autoimmune diseases and the often related atherosclerosis. However, excessively released TNFα is only one component of the hyperactive innate immune system in such diseases. To provide more insight into the role of TNFα in the induction of inflammation, we explored the effects of TNFα blockade in human whole blood.
TLR4/NLPR3 inflammasome challenges were applied to induce an inflammatory response. For this purpose, whole blood was incubated 4 hours with LPS and aluminium hydroxide (Alhydrogel). TNFα blockade was evaluated in vitro (LPS/Alhydrogel challenge in whole blood of 4 healthy human subjects, +concentration range of adalimumab) and ex vivo (LPS/Alhydrogel challenge in whole blood of 13 healthy human subjects receiving a single subcutaneous (sc) dose of 40 mg adalimumab). Cytokine release was evaluated in culture supernatants.
In vitro, TNFα blockade strongly reduced TNFα levels detected; -97±1% at the lowest adalimumab concentration (0.3125 μg/mL). TNFα blockade did not affect LPS/Alhydrogel-induced IL-6, IL-1β and IL-18 release, but reduced IFNγ release; maximally -93±4% at 5 μg/mL adalimumab. A single sc adalimumab dose in healthy subjects reduced LPS/Alhydrogel-induced TNFα levels (maximally -98±1% on day 4, and still -58±59% on day 64; versus baseline). IL-6, IL-1β and IL-8 release were not reduced after anti-TNFα treatment. The effect of TNFα blockade on IFNγ release could not be reliably estimated due to highly variable IFNγ levels, especially between genders (baseline IFNγ levels 1248±1771 and 140±283 pg/mL, males vs females).
TNFα is a major inducer of NFκB-driven cytokine gene transcription, but TNFα blocking did not reduce LPS/Alhydrogel-induced release of IL-1β, IL-6, IL-8 or IL-18 by primary human cells. This suggests that primary TLR4- and inflammasome-mediated signalling is sufficient to drive secretion of these cytokines. However, in vitro TNFα blockade did impair IFNγ release. Since IFNγ is a key factor in atherogenesis, exerting both pro- and anti-atherogenic properties, our data warrant further mechanistic investigation of the role of TNFα and anti-TNFα therapies in atherosclerosis.
Abstract 491: Detection of Subclinical Coronary Artery Disease by Calcium Score in Patients With Atrial Fibrillation: Potential Clinical Implications
Authors: Chaikriangkrai Kongkiat , Valderrabano Miguel , Khaleel Bala Sayf , Alchalabi Sama , Graviss Edward , Nabi Faisal , Mahmarian John , Chang Su Min 
Published: July 2021

Background:
Clinical implications of detecting subclinical coronary artery disease (CAD) in patients with atrial fibrillation (AF) are unclear.

Methods:
A total of 430 AF patients (age 63 ± 10 y, 65% male, 62% hypertensive, 16% diabetic, 42% dyslipidemic) without known CAD undergoing pre-procedural CT for catheter ablation were included. We evaluated the change in:

1) numbers of patients with CACS-diagnosed CAD who could potentially be on statin.
2) CHA2DS2-VASc score after incorporating CACS>100 (related to increased risk of stroke) into the original definition of vascular diseases who could potentially be on anticoagulants.

Results:
1) Prevalence of subclinical CAD (CACS>0) was 74% (319/430) and 25% (106/430) had CACS>100. There were 62% (267/430) who were not on statin. Of these patients, 71% (190/267) had subclinical CAD while 21% (34/163) of statin users had CACS of 0.

2) The median original CHA2DS2-VASc score was 2. After incorporating CACS>100 into the original score, 24% (18/75) with the original score of 0 had the score changed to 1 (7/35 in persistent AF [PST-AF] and 11/40 in paroxysmal AF [PRX-AF]) (figure A) and 17% (22/131) with the original score of 1 had the score changed to ≥ 2 (10/83 in PST-AF and 12/48 in PRX-AF) (figure B). PRX-AF had more frequent increase in CHA2DS2-VASc score than PST-AF (p=0.035)(figure C).

Conclusion:
In AF patients without known history of CAD, detecting subclinical CAD by CACS potentially has important therapeutic implications for prevention forprogression of CAD and stroke.