bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 51: Parasitic Soluble Egg Antigens Fight Atherosclerosis from 2 Different Angles
Authors: Wolfs Ine M , Goossens P , Stöger J L , Pöttgens C , Gijbels M , Wijnands E , van Die I , Kraal G , J de Winther M P
Published: March 2022

With intra-plaque inflammation being a major hallmark of atherosclerotic lesion growth and instability, modulation of the immune system may yield therapeutic potential in combating atherosclerosis. Helminths and their derivatives have immune modulating properties and are known for their anti-inflammatory effects. We investigated if immune modulation by Soluble Egg Antigens (SEA) derived from the helminth
Schistosoma Mansoni
can affect macrophage function and protect against atherosclerosis.

In vitro
, primary macrophages skewed by SEA showed an anti-inflammatory M2 phenotype. SEA induced the production of IL-10, an anti-inflammatory cytokine with known anti-atherogenic capacities, while inhibiting LPS-induced production of pro-inflammatory mediators. Likewise, peritoneal macrophages isolated from SEA-treated mice showed increased IL-10 production, thereby confirming the M2 macrophage-inducing potential of SEA
in vivo
. Additionally, T-lymphocytes showed a profound T
H
2 polarization after SEA treatment
in vivo
.

To study the effect of SEA treatment on atherosclerosis, LDLR
-/-
mice (n=20) were weekly injected with SEA or vehicle before and during 9 weeks of high fat feeding. Examination of the aortic root showed a 44% decrease in plaque size by SEA treatment (386.2x10
3
μm
2
± 159.3 vs. 214.3x10
3
μm
2
± 58.7, p<0.0001) along with less advanced lesions compared to controls. Furthermore, pathological assessment revealed smaller necrotic cores and less monocyte adhesion in lesions from SEA-treated mice. Besides an additional lowering of plasma cholesterol (45.17 mM ± 6.1 vs. 36.99 mM ± 4.28, p<0.0001), SEA reduced high fat diet-induced leukocytosis (9900.6 cells/μl μm
2
± 2503.6 vs. 7535.11x10
3
μm
2
± 1892.1, p<0.05), in particular at the myeloid level. Interestingly, both blood monocytes and isolated peritoneal macrophages pointed towards an anti-inflammatory phenotype by SEA, characterized by less Ly6C
high
cells and increased IL-10 production respectively. Finally, peritoneal macrophages from SEA-treated mice showed less lipid accumulation
.

In conclusion, SEA have the potential to reduce atherosclerosis from 2 perspectives: lowering plasma cholesterol levels and dampening the inflammatory (myeloid) immune status.
Abstract 511: Thioredoxin Interacting Protein Ablation Protects Vascular Smooth Muscle Cells from Oxidative Stress
Authors: Byon Chang Hyun , Hui Simon T
Published: March 2022

Vascular smooth muscle cells (SMC) play an important role in atherosclerosis. In response to increased oxidative stress, SMC secrete cytokines and express cell adhesion molecules which in turns, regulate monocyte/macrophage adhesion and other processes during atherosclerosis. Thioredoxin interacting protein (Txnip), an endogenous inhibitor of thioredoxin, is a key regulator of cellular sulfhydryl redox status. Recent studies also showed that Txnip is a key modulator of cellular glucose metabolism and insulin resistance. In addition, Txnip serves as a critical sensor linking inflammasome to inflammation associated oxidative stress. We hypothesized that Txnip ablation would lead to decreased inflammatory responses in SMC by limiting cellular oxidative stress. To examine this hypothesis, we isolated primary SMC from the thoracic aorta of Txnip knockout (TKO) mice. Comparing to SMC from wild type (WT) control mice, SMC from TKO mice have reduced levels of reactive oxygen species (ROS), as assessed by dihydroethidium (DHE) staining. Furthermore, Txnip ablation protected cells from oxidative stress when challenged with OxPAPC and hydrogen peroxide. Inflammatory markers (ICAM-1 and MCP-1) were down-regulated in Txnip-null SMC whereas oxidative stress-induced expression of inflammatory markers was inhibited by Txnip-deficiency. Interestingly, we found that macrophage adhesion to SMC was markedly reduced in Txnip-null cells. This is associated with a reduction in adhesion molecule and inflammatory marker expression in both SMC and macrophages from TKO mice. SMC from TKO mice exhibited an increased expression of KLF2 and decreased NF-κB (p65) activity, comparing to WT SMC. Taken together, our data demonstrate a critical role of Txnip-deficiency in inflammatory responses in SMC and suggest Txnip inhibition in SMC may represent a potential target for intervention of atherosclerosis.
Abstract 514: Endothelial Cell-Specific Molecule 1 or Endocan Is Required for Postnatal Cardiac Function
Authors: Menon Prashanthi , Dharaneeswaran Harita , Janes Lauren , Spokes Katherine , Beeler David , Aird William
Published: March 2022

Introduction:
Endothelial specific molecule 1 (ESM1) or Endocan is an endothelial secreted dermatan sulphate proteoglycan whose function remains elusive. ESM-1 is detected at a level of approximately 1 ng/ml in the plasma of healthy individuals and is significantly elevated in diseased states like cancer and sepsis. ESM1 expression in vivo is limited while its expression is up regulated in tumor neovessels. However there have been no in vivo studies of ESM1 function to date.

Objective:
The aim of this study was to understand the function of ESM1 in vivo using a global ESM1 knockout (KO) mouse model.

Methods and results:
ESM1 KO mice were generated by deleting the exon 1 containing the coding region of the mouse gene. Mice were backcrossed in C57BL/6 background for more than 10 generations. Targeted deletion of ESM1 was confirmed by real time quantitative RT-PCR (qRT-PCR). Analysis of ESM1 KO embryos and yolk sac at different time points (E12.5, E15.5) showed normal vasculature compared to WT controls. ESM1 KO mice were born at the normal mendelian but they showed 50% perinatal mortality by postnatal day 3 (P3). Pathological analysis of mice that died at P2 revealed dilated atria engorged with blood and thrombi indicative of possible defect in cardiac contractility. Pulmonary congestion was also observed indicative of possible cardiac failure. Fetal genes associated with cardiac stress response including atrial natriuretic peptide (Nppa), brain natriuretic pepetide (Nppb) and beta-myosin heavy chain (Myh7) were significantly up regulated in ESM1 KO postnatal (P2) hearts compared to controls.

Conclusion:
Our findings demonstrate for the first time that ESM1 is required for postnatal cardiac function. Future studies are aimed at understanding the mechanisms leading to impaired cardiac function in ESM1 KO mice.
Abstract 516: Strain-Specific Differences in Transcriptome Regulation by Cholesterol Loading in Macrophages from AKR and DBA/2 ApoE-Deficient Mice
Authors: Berisha Stela Z , Hsu Jeffrey , Robinet Peggy , Smith Jonathan D
Published: March 2022

Background:
Macrophages are a key cell type in atherogenesis as upon cholesterol loading they become foam cells in the arterial intima that provide the earliest histological evidence of atherosclerosis. Thus, atherosclerosis can be viewed as a disease caused by a combination of elevated cholesterol and inflammation.

Methods and Results:
We studied the cholesterol loading response of apoE
-/-
macrophages from atherosclerosis resistant (AKR) and atherosclerosis susceptible (DBA/2) strains. Bone marrow macrophages (BMM) from male and female mice were treated ex vivo with or without AcLDL and whole genome transcriptome data was obtained using Illumina expression microarrays. Transcriptome data were analyzed in gender-specific and combined manners using the Limma R package. Probe data were fitted to a linear model with strain, gender, and cholesterol loading as additive variables to delineate the strain:loading interaction effect. We identified 790 transcripts with a significant strain:loading interaction effect, suggesting that the response to AcLDL was altered in BMM between the two strains. For example the Cd274 gene was up regulated by cholesterol loading in AKR, while down regulated in DBA/2 (p=1.4E-09). Gene enrichment analysis was performed using the Romer R package. The strain:loading differentially regulated transcripts were found to be significantly enriched in sterol regulatory element binding protein (SREBP)-motif-containing genes, which contain binding sites for the SREBP-1a and SREBP-1c transcription factors within 1000 bp of their transcription start site. Nuclear SREBP was found abundantly only in unloaded cells from both strains. The SREBP-motif-containing geneset was significantly enriched in transcripts that were down-regulated in loaded DBA/2 BMM relative to the other samples. We also identified many SREBP-motif associated genes that were up regulated upon cholesterol loading, suggesting a novel function for SREBP as a transcriptional repressor in unloaded macrophages.

Conclusions:
Transcriptome profiling can yield insights into strain differences in cholesterol loading and atherosclerosis susceptibility. The SREBP pathway may be alternatively regulated in atherosclerosis resistant and susceptible strains.
Abstract 518: Antithrombotic Effects of Apelin: Implication During Metabolic Disorders
Authors: Adam Frederic , Lopez José , Vatier Camille , Turpin Sabrina , Muscat Adeline , Lalou Claude , Valet Phillipe , Khatib Abdel-Majid , Bobe Regis , Feve Bruno , Siegfried Geraldine
Published: March 2022

Thrombotic risk, that is partly related to a disturbed adipokine/cytokine profile, is strongly increased in obese and type 2 diabetic patients and. Apelin is an adipokine with pleiotropic function and cardiovascular fluid, and energetic homeostasis, and acts through the APJ transmembrane receptor.

In this project, we show for the first time that apelin displays a potent antithrombotic effect The antithrombotic effect is APJ- mediated and requires the proteolytic maturation of apelin. Apelin strongly inhibits thrombin- or collagen- induced platelet aggregation, independently of ADP secretion. It prevents platelet activation and thrombin-activated intracellular signaling pathways.

As a consequence, in mice models, intravenous apelin injection increases tail-bleeding time and delays chemically- induced vessel occlusion and thrombus stabilization.

We also determined that functional APJ is strongly over-expressed in platelet from obese subjects or mice (DIO mice, db/db), but apelin preserved its antithrombotic function during these metabolic disorder conditions.

In conclusion, in this original study, we enlighten a new biological function of apelin that could be considered as a new strategy to reduce the increased risk of thrombosis in patients with metabolic disorders.
Abstract 519: Calmodulin Is a Marker of a Distinct Smooth Muscle Cell Population Recruited by Plaque-Derived Macrophages from the Human Carotid Artery Media
Authors: Coen Matteo , Marchetti Giovanna , Zerbinati Carlotta , Guastella Giuseppe , Gagliano Teresa , Bernardi Francesco , Palagi Patricia M , Mascoli Francesco , Bochaton-Piallat Marie-Luce
Published: March 2022

Background:
Different experimental observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset during atheromatous plaque establishment and restenosis.

Objectives:
We investigated the presence and features of such SMC subset in cell cultures derived from human carotid endarterectomy (CEA) specimens; moreover, we aimed at finding markers that could play a role in SMC behavior.

Methods and Results:
CEA specimens comprised a grossly undiseased (UP) and a diseased portion (DP). The UP contained a thin intimal thickening with the underlying media and the DP an atherosclerotic plaque with the underlying media. Cell cultures were initiated by tissue explantation. From plaque tissues only macrophage-derived foam cells were retrieved. From medial tissues 2 distinct SMC phenotypes were identified: 1) large SMCs, flat with a monolayered growth pattern, isolated from the UP media; 2) small SMCs, fusiform and growing in multilayers, isolated from both the UP and DP media only when cocultured with plaque-derived macrophages. Small SMCs displayed higher proliferative and migratory activities and a poor level of differentiation compared with large SMCs. Two-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry showed that calmodulin (CaM), a calcium-binding protein involved in cell-cycle regulation, was mainly expressed in small SMCs. This result was confirmed using a specific CaM antibody. Coculture of large SMCs with plaque-derived macrophages induced a switch to the small phenotype and was associated with increased CaM expression. Consistently, the specific CaM inhibitor W-7 hampered the large-to-small switch and predominantly inhibited the proliferation of small SMCs. In vivo, CaM was markedly expressed in atherosclerotic plaques whereas it was barely detectable in the media.

Conclusions:
Plaque-derived macrophages promote the selective migration of a distinct SMC subpopulation exhibiting features of an “atheroma-prone” phenotype. CaM is a marker of small SMCs in vitro and of plaque SMCs in vivo, and plays a role in SMC phenotypic modulation. Further studies on these SMC populations can be instrumental in understanding and influencing the evolution of atherosclerosis.
Abstract 520: Chronic Overexpression of Heat Shock Protein 27 Facilitates Favorable Plaque Remodeling?
Authors: Cuerrier Charles M , Tremblay Dominique , Chen Yong-Xiang , Huot-Marchand Julie-émilie , deBlois Denis , Pelling Andrew E , O’Brien Edward R
Published: March 2022

Background:
Previously, we discovered that over-expression of Heat Shock Protein 27 (HSP27) in atherosclerosis-prone apoE
-/-
mice (apoE
-/-
HSP27
o/e
) maintained on a high fat diet (HFD) for 4 weeks reduces aortic lesion area - however, only in females.

Purpose/Objective:
To determine if chronic HSP27 over-expression modulates plaque lesion characteristics including aortic mechanical properties.

Methods/Results:
Male and female apoE
-/-
and apoE
-/-
HSP27
o/e
mice fed a high fat diet for 12 weeks were studied. Relative to apoE
-/-
mice, female apoE
-/-
HSP27
o/e
mice had aortic lesion area reductions of 35% for
en face
and 30% for cross-sectional sinus tissue sections. Interestingly, the same parameters were also reduced by 21% and 24%, respectively, in male cohorts. Aortic plaques from apoE
-/-
HSP27
o/e
mice showed almost 50% reductions in the area occupied by cholesterol clefts and free cholesterol, with fewer macrophages and reduced macrophage apoptosis. The mechanical properties of the aorta were studied
ex vivo
using 2-mm vessel segments (n=14-20/group). We developed an apparatus to determine vessel stiffness during radial stretching at a constant rate, recording tension via a force transducer. There was an increase in vessel stiffness in apoE
-/-
HSP27
o/e
mice compare to apoE
-/-
(female: 628±30Pa
vs
444±38Pa, p<0.001; male: 679±19Pa
vs
505±22Pa; p<0.001). The vasoactive response of aortic segments (n=12/group) to phenylephrine (PE), carbachol (CCh) and sodium nitroprusside (SNP) were also determined. While the vasorelaxation response induced by CCh and SNP were similar between the two animal groups, PE (10μM) produced a greater increase in vasoconstriction in apoE
-/-
HSP27
o/e
mice compared to apoE
-/-
(female: 205±9%
vs
162±10%, p<0.001; male: 190±9%
vs
139±12%, p<0.01).

Conclusion:
Chronic over-expression of HSP27 provides a persistent atheroprotective effect that benefits both sexes and coincides with remarkable reductions in lesion cholesterol accumulation as well as macrophage apoptosis. In addition, it increases the aortic stiffness and the vasoactive response to PE in apoE
-/-
mice model. These data provide new clues as to how HSP27 may improve atherosclerotic lesion stability.
Abstract 526: Macrophage Clearance from Resolving Inflammation Depends on Apoptosis Rather than Migration to Lymph Nodes
Authors: Gautier Emmanuel L , Randolph Gwendalyn J
Published: March 2022

Resolution of acute inflammation is a key step in the return to homeostasis after initiation of tissue injury, and its failure ultimately leads to a state of chronic inflammation known to favor the initiation and/or the progression of numerous diseases such as atherosclerosis. However, the cellular mechanisms governing inflammatory macrophages (iM
f
) clearance during the process of resolution are not completely understood. To better investigate iM
f
clearance in acute inflammation, we for the first time compared the importance of emigration through lymphatics to that of local death using a classical model of acute resolving peritonitis induced by thioglycollate. Of note, Ly-6C
hi
monocyte-derived iM
f
that accumulated in the peritoneum became CD11c+ cells, but gene expression profiling revealed these cells as macrophages, not dendritic cells. Although these iM
f
migrate to draining lymph nodes, this appeared in a kinetic and a quantity that doesn’t fit with the onset and extend of macrophage contraction during resolution. We found that migration out of the peritoneal cavity surprisingly did not rely on CCR7 but was partially dependent on CCR2. Moreover, blocking migration using a Gαi signaling inhibitors (that block all chemokine-dependent migrations and others) minimally impact on iM
f
clearance. By contrast, competition assays between WT and CD68-Bcl2 transgenic or Bim
-/-
iM
f
revealed that macrophage protected from apoptosis were much less susceptible to be cleared from the cavity during resolution. Thus, we conclude that in a model previously claimed to remove macrophages via emigration, local death of macrophages is quantitatively the main mode of clearance. As we recently reported that iM
f
removal from regressing atherosclerotic plaques is an emigration-independent process, we are now completing experiments to study the role of apoptosis in this phenomenon. Overall, removal by local death and control of new recruitment may be universally dominant mechanisms for macrophage clearance.
Abstract 528: Bone Marrow Angiotensin AT2 Receptor Deficiency Aggravates Atherosclerosis by Eliminating Macrophage Liver X Receptor-Mediated Antiatherogenic Actions
Authors: Kato Taku , Yamada Hiroyuki , Kawahito Hiroyuki , Irie Daisuke , Ikeda Kouji , Okigaki Mitsuhiko , Mogi Masaki , Iwai Masaru , Horiuchi Masatsugu , Matsubara Hiroaki
Published: March 2022

[BACKGROUND]
The angiotensin II (Ang II) type 2 (AT2) receptor is crucially involved in atherogenesis; however, bone marrow (BM) AT2-mediated anti-atherogenic action remains undefined.

[METHOD AND RESULT]
We generated BM chimera apoE-deficient (apoE-/-) mice whose BM cells were repopulated with AT2-deficient (Agtr2-/-) or wild type (Agtr2+/+) cells. Eight weeks after BM transplantation, all mice were fed a western diet for two months. Atherosclerotic lesion area was significantly increased in apoE-/-/BM-Agtr2
-/-
mice compared with apoE-/-/BM-Agtr2+/+ mice (51%, P<0.05), accompanied by the enhanced accumulation of MOMA-2 positive cells (P<0.05). Hemodynamic data and lipid profile did not differ between the two groups. We first examined the expression levels of macrophage liver X receptors (LXRα and β), which have been reported to exert anti-atherogenic actions by reducing pro-inflammatory response and promoting cholesterol efflux and macrophage emigration. The mRNA expression of LXRβ, but not LXRα, was significantly decreased by 21% in Agtr2
-/-
thioglycollate-induced peritoneal macrophages (TGPM) compared with Agtr2+/+ TGPM (P<0.01). Inversely, LXRβ mRNA expression was markedly increased by co-treatment of angiotensin II (100nM) with AT1 receptor blocker, Olmesartan (10μM) (47%, p<0.01). Treatment with LXR agonist (GW3965) significantly attenuated LPS-induced mRNA expressions of TNF-α and IL-1β in Agtr2+/+ TGPM (35%, 46%, respectively, P<0.01), however, anti-inflammatory effect of LXR agonist was markedly reduced in Agtr2
-/-
TGPM (p<0.05). Furthermore, the expression levels of ATP-binding cassette transporter ABCA1 and CCR7, essentially implicated in cholesterol efflux and macrophage emigration, were much lower in Agtr2
-/-
TGPM than Agtr2+/+ TGPM (34%, 53%, p<0.01). Flow cytometric analysis also showed a significantly lower expression of CCR7 in Agtr2
-/-
TGPM (24%, p<0.05).

[CONCLUSION]
Our findings demonstrate that BM-AT2 deficiency aggravates atherosclerosis, at least in part, by eliminating anti-atherogenic properties elicited by macrophage LXR activation, suggesting that AT2-mediated regulation of macrophage LXR activity could be a novel therapeutic target for the prevention of atherosclerosis.
Abstract 529: Increased 18F-FDG Uptake in the Aortic Wall of β-aminopropionitrile Exposed Rats May be Predictive of AAA Rupture
Authors: English Sean J , Piert Morand R , Diaz Jose A , Ghosh Abhijit , Sherman Phillip S , Quesada Carole A , Pechota Angela , Eliason Jonathan L , Henke Peter K , Upchurch Gilbert R
Published: March 2022

Objectives:
Ruptured abdominal aortic aneurysm (AAA) is associated with a uniformly high mortality rate, and a noninvasive method is necessary to detect vascular wall inflammation that may assist in predicting AAA rupture. The ability of 18F-FDG positron emission tomography to identify increased inflammation in the porcine pancreatic elastase (PPE) perfusion model in Sprague-Dawley rats exposed to β-aminopropionitrile (BAPN) to stimulate AAA rupture was evaluated.

Methods:
BAPN (300mg/kg subcutaneous) was administered daily starting 3d prior to infrarenal abdominal aortic exposure to PPE and daily until AAA harvest or rupture (BAPN, n=10). Control rats only underwent PPE exposure (n=8). Aortic diameter was evaluated at 3, 7, and 14d post PPE exposure utilizing ultrasound (US, 12MHz). Both groups were imaged with 18F-FDG microPET. AAA maximum to mean muscle 18F-FDG uptake ratios (AMR) were determined. AAA rupture sites were identified at post rupture harvest. Matrix metalloproteinase (MMP) 2 and 9 zymogramatic activities, in terms of integrated optical density (IOD), were assessed at 7d (control n=3, BAPN n=4) and 14d post PPE exposure (control n=3, rupture n=3).

Results:
Mean 7d aortic diameter increases of 251 ± 29.4% and 340 ± 56.0% (p=0.20) were observed in control and rupture groups, respectively. Four BAPN exposed rats ruptured within 24h after 18F-FDG microPET. Considering the entire AAA, mean AMRs for control and ruptured groups were not significantly different at 15.3 ± 1.6 and 15.5 ± 1.9, respectively. However, considering the 18F-FDG uptake at known rupture sites, the mean AMR for ruptured AAAs was significantly greater than that of control AAAs (AMR 13.2 ± 1.1 vs. 8.4 ± 1.3, p=0.02). IOD values of 8373 ± 827 and 4655 ± 1851 (p=0.14) were observed for MMP9 activity in 14d control and ruptured AAA tissue, representing an 80% increase in ruptured AAA tissue. IOD values of 4760 ± 647 and 1190 ± 87 (p=0.03) were observed for MMP2 activity in 14d control and ruptured AAA tissue, respectively, representing a 75% decrease in ruptured AAA tissue. No differences in MMP2 or 9 activities for control and BAPN exposed rats were appreciated at 7d post PPE exposure.

Conclusions:
Increased MMP9 activity in ruptured rat AAA tissue parallels what has been observed in ruptured human AAA tissue. Decreased MMP2 activity in ruptured AAA tissue may represent decreased smooth muscle cell presence. Increased 18F-FDG uptake by rat AAA walls, in regions of known rupture, suggests that FDG-PET may be used to predict AAA rupture.
Abstract 54: Overexpression of Glutathione Peroxidase-1 Protects Mice from Aging-Related Enhancement of Experimental Thrombosis and Platelet Activation
Authors: Dayal Sanjana , Wilson Katina M , Lentz Steven R
Published: March 2022

Despite a well-established clinical association between aging and thrombosis, little is known about the mechanisms by which aging increases susceptibility to thrombotic events such as stroke, myocardial infarction, and deep venous thrombosis. Since aging is associated with increased levels of reactive oxygen species and decreased levels of the antioxidant enzyme glutathione peroxidase (Gpx), we tested the hypothesis that overexpression of a cellular form of Gpx (Gpx1) protects aged mice from increased thrombotic susceptibility. Susceptibility to arterial thrombosis was first examined in C57BL/6J mice at 4, 12, or 18 months of age. The time to stable occlusion after photochemical injury of the carotid artery was significantly shortened in both 12 and 18 month old mice compared to 4 month old mice (P<0.01). To examine the protective role of Gpx1, transgenic mice overexpressing Gpx1 (Gpx1 Tg mice) were studied. Unlike wild type littermates, no shortening of the time to occlusion of the carotid artery was seen in 12 or 18 month old Gpx1 Tg mice. In addition, we also found that aged wild type mice exhibited increased susceptibility to venous thrombosis, and that overexpression of Gpx1 was protective in an inferior vena cava stasis model. An age-dependent increase in platelet hydrogen peroxide generation was observed in thrombin-activated platelets from wild type mice but not Gpx1 Tg mice (P<0.05). Markers of platelet activation, including fibrinogen binding and activation of αIIbβ3, were also increased in thrombin-activated platelets from aged mice compared to young mice (P<0.05). Overexpression of Gpx1 or treatment with catalase prevented these enhanced platelet activation responses in platelets from aged mice. Our findings demonstrate that aged mice exhibit increased susceptibility to both arterial and venous thrombosis and that enhanced thrombosis and platelet activation in aged mice are mediated by peroxide. Therapeutic strategies to prevent the accumulation of hydrogen peroxide in platelets may reduce the incidence of aging-associated thrombosis.
Abstract 541: Nogo-B Receptor Is Essential for Embryonic Stem Cell Differentiation to Endothelial Cell Lineage and Primitive Blood Vessel Formation
Authors: Liu Zhong , Zhao Baofeng , Ramchandran Ramani , Miao Qing (Robert)
Published: March 2022

Nogo isoforms-A, -B, and -C, are members of the reticulon family of proteins. Nogo-B was previously identified as a protein that is highly expressed in caveolin-1 enriched microdomains and/or lipid rafts of endothelial cells (EC) and vascular smooth muscle cells. Mice deficient in Nogo-A/B show exaggerated neointimal proliferation, abnormal remodeling and a deficit in ischemia induced arteriogenesis and angiogenesis. Nogo-B receptor (NgBR) is a type I receptor with a single transmembrane domain, and was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation in zebrafish via Akt pathway. Here, we further demonstrated the role of NgBR in regulating the differentiation of murine embryonic stem cells (ESC) into endothelial cell lineage and primitive blood vessel formation in 2D or 3D embryoid body culture systems. Murine NgBR gene-targeting ESC was generated by either gene-trap or homologous recombination approaches, respectively. Gene-targeting of NgBR was confirmed by Southern blot and qPCR. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but decreases the endothelial cell lineage commitment as well as reduces the formation and branching of primitive blood vessels in 2D or 3D embryoid body culture systems. However, there is no effect on the differentiation of ESC to vascular smooth muscle cell lineage. Mechanistically, NgBR has two potential regulatory roles during embryonic vasculature development. NgBR knockdown not only impairs both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases endothelial cell lineage commitment by delaying BMP4 production during the period of mesoderm formation. These results suggest that NgBR may be one of important genes coordinating the vasculature development.
Abstract 548: Variations in Venous Wall Strain Can Be Modeled and Measured Using IVUS-Derived Elastography
Authors: Rubenstein Lindsay A , Richards Michael S , Mix Doran , Chandra Ankur , Doyley Marvin M , Gillespie David L
Published: March 2022

Objectives:
Iliofemoral venous outflow stenting has been used to treat both acute and chronic venous thrombosis. It may be beneficial to understand the properties of the normal venous wall and the flow through collapsible tubes to develop new stent technology and improve long-term patency. We hypothesize that radial and circumferential strain can be modeled and measured using intravascular ultrasound (IVUS) techniques to describe venous geometry and asymmetric vessel wall characteristics.

Methods:
A hydrogel phantom was used to mimic the mechanical properties of a homogeneous vein, including geometry and wall compliance. Radio-frequency (RF) echo frames were acquired using a modified IVUS system with an 8.5 Mhz catheter. All RF signals were digitized to 14 bits at 50 MHz. Radial strain was estimated by applying an intensity matching, image registration based algorithm to the digitized frames.

Results:
The radial strain images (Figure 1) show increasing positive strain (radial wall thickening) on the top and bottom of the phantom with decreasing negative values (radial wall thinning) at either side. Results suggest an asymmetric compression pattern typical of a vessel collapsing under negative pressure.

Conclusions:
In conclusion, our data suggest that novel strain analysis can be performed on a phantom vessel consistent with the mechanical properties of the venous system. Further work with venous elastography will help define the mechanical properties of the venous wall and facilitate the development of new stent technology to treat venous disease.
Abstract 550: Nuclear Targeting of Apelin in Vascular Smooth Muscle Cells: Implications in Cell Proliferation and Atherosclerotic Plaque Formation?
Authors: Chaabane Chiraz , Brun Cécile , Roatti Angela , Audigier Yves , Baertschi Alex J , Bochaton-Piallat Marie-Luce
Published: March 2022

Background:
The main circulatory form of Apelin is a highly conserved 13 amino acid hypotensive peptide derived from a 77 amino acid prepropeptide. The very low plasma levels suggest autocrine, paracrine and possibly intracrine functions of Apelin. It is synthesized in smooth muscle cells (SMCs), and we found a nuclear expression in rhomboid type (R), poorly differentiated SMCs. The biological features of R-SMCs (i.e. enhanced proliferative and migratory activities) explain their capacity to accumulate in the intima.

Objectives:
Investigate the role of intracrine Apelin in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation.

Methods and Results:
As shown in our previous work, a 3’-shift in translation can lead to N-truncated peptides with new address sequences (FASEB J. 20(6): 732-4, 2006). P-SORT software analysis of preproApelin sequence suggests that N-terminal truncated Apelin may target the nucleus, and we confirm this in many cell types by overexpression of mutated preproApelin-EGFP and preproApelin-His-tag. Transfection of mutated preproApelin-His-tag encoding plasmid in differentiated spindle-shaped (S) SMCs induces a transition towards a R-phenotype associated with increased proliferative activity, as well as downregulation of SMC differentiation markers (i.e. α-smooth muscle actin) and increased nuclear expression of S100A4 (a marker typical of R-SMCs). In contrast, transfection of S-SMCs with wild type preproApelin-His-tag encoding plasmid does not induce nuclear targeting of Apelin or S100A4, and does not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB also induces nuclear targeting of both Apelin and S100A4 and transition to the R-phenotype. In vivo, Apelin is expressed in SMC nuclei of stent-induced intimal thickening, while its expression in SMCs of the media is mainly cytoplasmic.

Conclusions:
Altogether, theses preliminary studies provide evidence for nuclear targeting of Apelin in SMCs and suggest actions on gene expression, proliferation, and differentiation. The pathophysiological consequences of this retargeting could be of importance in the understanding of artherosclerosis.
Abstract 57: Hyperglycemia-Induced miR-467 Regulates Angiogenesis and Tumor Growth in a Tissue-Specific Manner
Authors: Stenina Olga , Sul Kristina , Krukovets Irene
Published: March 2022

Recent emerging evidence suggests that diabetes is associated with increased incidence of several cancers (e.g., breast, bladder, pancreas, liver) but decreased incidence of others (e.g., prostate). We suggest that the tissue-specific association with cancers is caused by a tissue-specific regulation of angiogenesis in diabetic patients. A well-known aberrant angiogenesis of diabetes (increased neovascularization in some tissues, e.g., retina and kidney, and deficient angiogenesis causing ischemia in others, e.g., skin and myocardium) has been recognized for many years and is the cause of diabetic microvascular complications (retinopathy, neuropathy, cardiomyopathy, and nephropathy). However, the molecular mechanisms of the tissue-specific diabetic angiogenesis are unknown. We have discovered a novel tissue-specific mechanism that is activated by hyperglycemia: the levels of microRNA-467 (miR-467) are upregulated in response to hyperglycemia in mouse breast cancer but not in prostate cancer. miR-467 inhibits production of a potent anti-angiogenic protein thrombospondin-1 (TSP-1), and, as a result, angiogenesis is increased in a tissue-specific manner. Our preliminary data identified miR-467 as a regulator of TSP-1 production and angiogenesis in response to hyperglycemia, established a correlation between hyperglycemia-induced tumor growth and miR-467 and TSP-1
in vivo
, confirmed the effect of miR-467 on angiogenesis
in vivo
and the effect of miR-467 antagonist on hyperglycemia-induced tumor growth. In a mouse breast cancer model, hyperglycemia increased the tumor size 8 fold (0.24 g ± 0.012 vs 0.03 ± 0.002, p = 0.003), miR-467 levels 4 fold increase, and angiogenesis 7-fold (endothelial marker CD31), but decreased TSP-1 levels > 3-fold. When miR-467 antagonist was expressed in breast cancer cells, the changes detected in response to hyperglycemia (increase in angiogenesis, size of tumors and miR-467 levels and decrease in TSP-1 level) were completely prevented.

We described a tissue-specific mechanism of diabetes-induced cancer growth, which suggests a novel breakthrough approach to block hyperglycemia-induced cancer angiogenesis and growth without affecting physiological angiogenesis.
Abstract 59: Altering Levels of Invariant Natural Killer T Cells Does Not Improve Adipose Tissue Inflammation or Insulin Sensitivity in Diet-Induced Obesity
Authors: Subramanian Savitha , Ding Yilei , Goodspeed Leela , Wang Shari , Kim Jinkyu , Reardon Catherine , Getz Godfrey , O’Brien Kevin D , Chait Alan
Published: March 2022

Obesity is a chronic inflammatory state characterized by adipose tissue immune cell infiltration. While macrophages are a key player, recent evidence also implicates T lymphocytes in adipose inflammation. Natural killer T (NKT) cells are a specialized lymphocyte subset expressing natural killer cell properties and T cell receptors (TCR). The invariant NKT (iNKT) cell has a limited TCR repertoire (Vα14/Jα18) and recognizes lipid antigens. Existing data on the role of iNKT cells in obesity is inconclusive. We hypothesized that lipid antigen recognition in obese adipose tissue by iNKT cells contributes to inflammation and insulin resistance. To evaluate whether lack of iNKT cells protects from obesity and its complications, Jα18-/-Ldlr
-/-
mice lacking iNKT cells were placed on a high fat, high carbohydrate diabetogenic diet +0.15% cholesterol (DDC) for 16 weeks. Effects on body weight, dyslipidemia, insulin resistance, visceral adipose tissue macrophage accumulation and systemic inflammation were analyzed. Jα18-/-Ldlr
-/-
mice gained more weight than control Ldlr
-/-
mice. Hypertriglyceridemia developed in the Jα18
-/-
mice without differences in glucose homeostasis. Increased F4/80 and MCP1 gene expression and macrophage Mac2 immunostaining suggested worsened adipose inflammation. No differences in circulating serum amyloid A, an inflammatory marker, were observed. Overall, mice lacking iNKT cells were not protected from becoming obese but had worsened metabolic features. We also evaluated effects of overabundance of iNKT cells on obesity using Vα14 transgenic(Tg) mice. Vα14TgLdlr
-/-
mice gained 25% more weight on DDC, had increased fat mass on body composition analysis, hepatomegaly, hyperlipidemia and hyperinsulinemia compared to controls. Adipose tissue revealed increased macrophage, chemotactic and cytokine gene expression as well as Mac2 staining. Similar inflammatory changes were also observed in the liver. Thus the Vα14Tg mice also had significantly worsened metabolic features of obesity. These results suggest that the relationship of iNKT cells in metabolism is complex and that simple excess or depletion of these immunological cells is not sufficient to beneficially modify metabolic abnormalities associated with obesity.
Abstract 60: The ABCG5 ABCG8 Sterol Transporter Opposes Insulin Resistance and Fatty Liver Disease Independent of Phytosterol Accumulation
Authors: Su Kai , Sabeva Nadezhda S , Liu Jingjing , Wang Yuhuan , Bhatnagar Saloni , Westhuyzen Deneys R , Graf Gregory A
Published: March 2022

ABCG5 and ABCG8 form a complex (G5G8) that opposes the absorption of plant sterols, but is also expressed in liver where it promotes the excretion of cholesterol into bile. Hepatic G5G8 is transcriptionally regulated by a number of factors implicated in the development of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Therefore, we hypothesized that G5G8 may influence the development of diet-induced obesity phenotypes independently of its role in opposing phytosterol absorption. G5G8 knockout (KO) mice and their wild type (WT) littermates were challenged with plant sterol free low fat (LF) or high fat (HF) diets. Weight gain and the rise in fasting glucose were accelerated in G5G8 KO mice following HF feeding. HF-fed G5G8 KO mice had increased liver weight, hepatic lipids and plasma ALT compared to WT controls. Consistent with the development of NAFLD, macrophage infiltration, the number of TUNEL positive cells and the expression of proinflammatory cytokines were also increased in G5G8 KO mice, but there was no evidence of fibrosis. Hepatic lipid accumulation was associated with increased mRNA levels for PPARγ and PPARγ target genes, but not genes involved in lipogenesis. Phosphorylation of eukaryotic translation initiation factor 2α (eiF2α) and expression activating transcription factor 4 (ATF4) and tribbles 3 (Trb3) were elevated in HF-fed G5G8 KO mice, a pathway that links the unfolded protein response (UPR) to the development if insulin resistance through inhibition of protein kinase B (Akt) phosphorylation. However, the remaining components of the UPR were unchanged.

Conclusion:
G5G8 plays a previously unappreciated role in the development of insulin resistance and fatty liver disease by opposing hepatic accumulation of cholesterol and phosphorylation of eiF2α.
Abstract 62: Azathioprine Is Protective in Aneurysm Formation and Progression
Authors: Marinkovic Goran , Hibender Stijntje , de Vries Carlie J , de Waard Vivian
Published: March 2022

Objective:
An aortic aneurysm is a dilatation of the arterial wall caused by destruction of its integrity, which results in often lethal vessel ruptures. Currently, only surgical treatment is available for patients with growing aortic aneurysms, therefore there is a clinical need to identify drugs to attenuate aneurysm growth. Chronic inflammation is fundamental in aneurysm pathology with excessive leukocyte infiltration. We studied the role of the immunosuppressive drug Azathioprine (AZA) in aneurysm formation.

Methods and Results:
To investigate AZA in aneurysm formation, we used the Angiotensin-II (AngII) aneurysm mouse model. AngII in ApoE
-/-
mice on a Western Type diet resulted in 11/14 (79%) mice with aneurysms, whereas only 3/13 (23%) of the AZA-treated mice developed aneurysms. In the aorta altered signalling downstream of the AngII receptor type 1 was observed on AZA treatment; phosphorylation of Jak2 and JNK was decreased and of p38 and p44/42 was increased. In addition, reduced levels of matrix metalloproteinases (MMP)2 and MMP9 and decreased leukocyte influx was observed in AZA-treated mice.

To study aneurysm progression, AZA treatment was started 10 days after AngII infusion; all mice developed aneurysms, however the aneurysm type was more severe in the control group demonstrating the inhibitory effect of AZA on aneurysm progression.

To delineate the mechanism of AZA protection, we performed experiments with macrophages and endothelial cells in the presence of 6-mercaptopurine (6MP), the active metabolite of AZA. Macrophages produce less IL-12 and IFNgamma and more IL-10 after 6MP incubation and endothelial cells express less CCL5 (Rantes), IL-12 and VCAM1. Adhesion of monocytes to activated endothelial cells is reduced in the presence of 6MP. These data underscore the anti-inflammatory effect of AZA on macrophages and endothelial cells, which may explain the inhibition of aneurysm formation.

Conclusion:
We report a protective effect of AZA in aortic aneurysm initiation and, most importantly, inhibition of progression of the disease. AZA modulates the inflammatory response of macrophages and endothelial cells, inhibits monocyte adhesion to endothelial cells, and modulates AngII signalling in the aortic vessel wall.
Abstract 69: The Increase in Dalcetrapib-Induced HDL Remodeling Is Dependent on Cysteine 13 of Cholesteryl Ester Transfer Protein and Does Not Involve Phospholipid Transfer Protein
Authors: Maugeais Cyrille , Perez Anne , von der Mark Elisabeth , Tetaz Tim , Schmid Georg , Niesor Eric J
Published: March 2022

Introduction:
Inhibition of the cholesteryl ester transfer between HDL and apoB-containing lipoproteins by dalcetrapib requires cysteine 13 (C13) of CETP. In contrast to the CETP inhibitors torcetrapib or anacetrapib, the CETP modulator dalcetrapib stimulates CETP-dependent generation of pre-β-HDL in human plasma or isolated HDL
in vitro
.

Hypothesis:
We hypothesized that the effects of dalcetrapib on CETP-dependent HDL remodeling also require C13 of CETP and are not the result of interaction with the other cysteine-containing phospholipid transfer protein (PLTP).

Results:
The CETP C13S mutant was as active as wild-type (wt) CETP both i) in transferring cholesteryl ester between HDL and apoB-containing lipoproteins and ii) in generating pre-β1-HDL. Dalcetrapib inhibited the transfer of cholesteryl ester between HDL and apoB-containing lipoproteins by wtCETP (IC
50
= 200 nM) but increased pre-β1-HDL formation from isolated HDL, and ABCA1-mediated efflux (3-fold and 2.5-fold increase at 10 μM, respectively, p<0.01). These properties of dalcetrapib were not observed with the CETP C13S mutant. Native HDL was remodeled by wtCETP as evidenced by the formation of large and small particles; a process further enhanced by dalcetrapib.

When reconstituted HDL (rHDL), containing only apoA-I and phospholipids, was substituted for isolated HDL, dalcetrapib did not increase wtCETP-induced pre-β1-HDL or ABCA1-dependent efflux (0.9-fold at 10 μM). PLTP induced pre-β1-HDL generation and ABCA1 efflux from native HDL and rHDL. PLTP-dependent HDL remodeling was not affected by dalcetrapib (0.9- and 1-fold at 10 μM for HDL and rHDL respectively).

Conclusions:
The enhanced generation of pre-β1-HDL by wtCETP and dalcetrapib was associated with an increase in ABCA1-specific efflux and HDL remodeling. As observed for cholesteryl ester transfer activity, dalcetrapib effects on CETP-dependent HDL remodeling required C13 of CETP and dalcetrapib had no effect on PLTP-dependent HDL remodeling.
Abstract 71: Assessment of Reverse Cholesterol Transport in Vivo in Humans: A Novel Method
Authors: Cuchel Marina , Billheimer Jeffrey T , Millar John S , Schwartz Charles C , Raper Anna C , Ochotny Rachel , Baer Amanda L , Su Ching , Carlsson Bjorn C , Rader Daniel J
Published: March 2022

Aim:
Reverse cholesterol transport (RCT) is one of the main atheroprotective functions of HDL; however no method exists to assess RCT in vivo in humans. We developed a macrophage-specific method using 3H-cholesterol/albumin complexes that has been validated in animal studies. We present the results of a feasibility study in humans.

Methods:
Thirty subjects received 3H-cholesterol/albumin complexes as i.v. bolus, followed by blood and stool sample collection up to 8 days. Tracer counts were assessed in plasma, non-HDL, HDL and fecal fractions. Data were analyzed using multi-compartmental modeling.

Results:
Figure 1 shows the tracer kinetics as free cholesterol (FC; panel A) and cholesteryl ester (CE; panel B) specific activity in plasma, HDL and non-HDL. 3H-cholesterol disappeared from plasma rapidly after injection (macrophage uptake of 3H-cholesterol/albumin complexes); nadir was reached by 60 min. Counts present as FC in the HDL fraction increase rapidly and linearly in the first 240 min after nadir (129.9±85.5 cpm/μmols per hour)(3H-cholesterol uptake from macrophages by HDL). Linearity is lost after ∼240 min, as FC is transformed to CE and exchanged with other fractions. In a subset of subjects (n=10), multi-compartmental analysis was used to calculate fractional transfer rates, including macrophage FC to HDL-FC (0.040 ± 0.014 fraction/hour), HDL-FC to HDL-CE (0.238 ± 0.083 fraction/hour), HDL-CE to nonHDL-CE (0.200 ± 0.113 fraction/hour) and fecal excretion (0.032 ± 0.006 fraction/hour).

Conclusions:
These preliminary data support the feasibility of this approach and suggest that it may be used to measure macrophage RCT in vivo in humans.
Abstract 72: Endothelial Cell Tissue Factor Enhances Inflammation but Not Coagulation in Mouse Model of Sickle Cell Disease
Authors: Chantrathammachart Pichika , Mackman Nigel , Sparkenbaugh Erica , Key Nigel S , Pawlinski Rafal
Published: March 2022

Sickle cell disease (SCD) is associated with increased tissue factor (TF) expression and activation of coagulation. Using a mouse model of SCD, we have previously demonstrated that inhibition of TF with a rat-anti mouse TF (1H1) antibody not only abolishes activation of coagulation (measured by plasma levels of thrombin anti-thrombin (TAT) complexes) but also reduces inflammation and endothelial cell (EC) injury, which was indicated by attenuation of plasma levels of IL-6 and sVCAM-1, respectively. SCD is one of the few diseases where TF has been shown to be expressed on ECs. Therefore, we investigated the effect of EC-specific deletion of TF gene on activation of coagulation, inflammation and EC injury in a mouse model of SCD (BERK mice). Sickle cell (SS) mice that expressed normal levels of TF or have EC-specific deletion of TF gene (TFfl/flEC) were generated by transplanting TFfl/fl (normal expression of TF) and TFfl/fl Tie2 Cre+ mice (lacking TF gene in both hematopoietic cells and ECs) with bone marrow from SS mice (bone marrow transplantation reconstituted expression of TF in all hematopoietic cells in TFfl/fl Tie2 Cre+). Interestingly, we found that activation of coagulation (TAT) and EC injury (sVCAM-1) were not affected by EC-specific deletion of TF gene. In contrast, plasma levels of IL-6 were significantly reduced in TFfl/flEC SS mice compared to SS mice with normal levels of TF expression (mean+/-SEM; 6.9+/-1.8 vs 14.9+/-3.0 pg/ml; p<0.05). Similar results were observed in SS mice lacking protease activated receptor-2 (PAR-2) expression in all non-hematopoietic cells compared to SS mice expressing normal levels of PAR-2 (no difference in plasma levels of TAT and sVCAM-1; reduction of IL-6, 9.4+/-0.9 vs 18.9+/-4.5 pg/ml; p<0.05). In contrast, SS mice with PAR-1 deficiency in all non-hematopoietic cells demonstrated reduced plasma levels of sVCAM-1 (835+/-41 vs 1003+/-52 ng/ml; p<0.05) but not IL-6 or TAT. Our data indicates that increased expression of EC TF contributes to the inflammation (possibly via PAR-2 activation on ECs) whereas TF expressed by other cellular sources (monocytes,perivascular cells) contributes to the activation of coagulation and EC injury (likely via thrombin-dependent mechanisms) in this mouse model of SCD.