bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 451: Bone Marrow-derived Matricellular Protein CCN3 is Protective Against Atherosclerosis
Authors: Zhang Chao , Hu Xingjian , Shi Hong , Wu Wenconghui , Qing Yulan , Perbal Bernard , Dong Nianguo , Lin Zhiyong
Published: June 2021

Background:
Atherosclerosis and its complications (myocardial infarction, stroke, peripheral vascular disease) are the major cause of morbidity and mortality in developed countries. Despite considerable efforts, the underlying pathomechanisms remain incompletely understood. In this study, we examined the role of a matricellular protein termed CCN3 in the pathogenesis of atherosclerosis.

Methods and Results:
To investigate whether CCN3 deficiency affects the development of atherosclerosis, control (ApoE-/-) and CCN3/ApoE double knockout mice were subjected to high fat diet feeding. In response to 16-week high fat diet feeding, the aortas of CCN3/ApoE double knockout (DKO) mice demonstrated exquisite susceptibility to atherosclerosis formation as evidenced by significantly increased size of aortic lipid-rich plaques in aortic roots, arch, thoracic and abdominal aorta. Concomitant with this, the atherosclerosis phenotype of DKO mice was manifested as follows: (1) a profoundly enhanced immune cell infiltration; (2) significantly increased expression of inflammatory markers; (3) heightened reactive oxygen species generation. Next, to address the cellular contributor(s) within or outside of the vessel wall responsible for the atherosclerosis phenotype, we performed reciprocal bone marrow transplantation (BMT) experiments. Transplantation of DKO bone marrow to ApoE-/- mice resulted in an increase of atherosclerosis formation, while transplantation of ApoE-/- marrow to DKO mice caused a reduction of atherosclerosis. These results indicate CCN3 deficiency in the bone marrow plays a major role in the development of atherosclerosis. Mechanistically, our cell-based studies in isolated macrophages demonstrated that CCN3 deficiency leads to an increase of lipid uptake and foam cell formation, an effect attributed to the modulation of key factors (e.g., increase of CD36, decrease of ABCG1) involved in lipoprotein transport.

Conclusion:
These results demonstrate bone marrow-derived CCN3 as an essential regulator of atherosclerosis and suggest the potential for future therapeutic strategies by manipulating CCN3 levels.
Abstract 453: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function
Authors: Jarzebska Natalia , Rodionov Roman N , Burdin Dmitri , Brilloff Silke , Todorov Vladimir T , Martens-Lobenhoffer Jens , Hofmann Anja , Morawietz Henning , Demyanov Anton , Hilgers Karl , Cordasic Nada , Jacobi Johannes , Maas Renke , Chen YingJie , Bode-Böger Stefanie M , Hugo Christian P , Hohenstein Bernd , Weiss Norbert
Published: June 2021

Background:
Elevation of the endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been shown to be associated with increased risk of cardiovascular diseases. There are two major pathways of ADMA catabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is poorly characterized. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of systemic levels of ADMA and improvement of vasomotor function.

Methods and Results:
We generated transgenic mice (TG) with ubiquitous overexpression of
AGXT2
under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. There were no developmental or phenotypic changes in the TG animals. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels were decreased by 15% (p<0.05) in the TG mice, whereas ADGV plasma levels were 6 times higher in comparison with wild-types littermates (p<0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p<0.05). TG mice demonstrated improved endothelium-dependent vasodilation (in response to acetylcholine) in aortic rings. The endothelium-independent relaxation (in response to sodium nitroprusside) was unchanged. There was no difference in mean arterial blood pressure measured by telemetry between the wild type and AGXT2 TG mice. In further experiments, we crossed the AGXT2 TG mice with DDAH1 KO mice and showed that upregulation of AGXT2 protects DDAH1 KO mice from elevation of plasma ADMA levels.

Conclusion:
In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation
in vivo.
AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.
Abstract 455: Fimasartan Reduced Carotid Atherosclerosis Progression After Mechanical Injury in ApoE Knockout Mouse
Authors: Kim Jong-Ho , Lim I-Rang , Joo Hyung Joon , Choi Seung-Cheol , Hong Soon Jun
Published: June 2021

Aim:
The beneficial effects of angiotensin receptor blockers (ARBs) in atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan in reducing atherosclerosis progression and systemic inflammation after carotid artery injury in ApoE knockout mouse.

Methods:
Male ApoE knockout mice were randomly allocated to Group 1 (without carotid artery injury,
n
=20), Group 2 (without carotid artery injury + fimasartan,
n
=21), Group 3 (carotid artery injury,
n
=25) and Group 4 (carotid artery injury + fimasartan,
n
=24). Fimasartan (3mg/kg in distilled water) was orally injected on daily basis, and left carotid artery injury was induced with 0.014 inch guidewire. At 28 days, hematoxylin & eosin and elastic stains were used to measure cross-sectional atherosclerotic plaque from carotid artery. Moreover, inflammatory markers such as MMP9, IL-6, TNFα and ICAM were analyzed by ELISA, and anti-inflammatory cytokines such as IL-10 was measured in peripheral blood.

Results:
Histomorphometric staining showed significantly reduced neointimal hyperplasia in Group 2 (2.30±0.66%) compared to Group 1 (12.31±2.97%,
p
<0.05). In addition, Group 4 (32.03±4.62%) showed significantly reduced neointimal hyperplasia compared to Group 3 (50.06±7.50%,
p
<0.05). All fimasartan treated groups revealed decreased smooth muscle cell proliferation and CD68+ macrophages in carotid artery. Furthermore, inflammatory cytokines such as MMP9 and IL-6 were significantly lower in Group 4 (0.32±0.02ng/mL and 11.68±2.13pg/mL) compared to Group 3 (0.54±0.13ng/mL and 16.68±3.03pg/mL,
p
<0.05, respectively). In particular, TNFα and ICAM at were significantly decreased in Group 2 (5.83±2.28pg/mL and 3.76±0.84ng/mL) and Group 4 (7.32±0.95pg/mL and 5.04±1.47ng/mL) compared to Group 1 (6.64±1.34pg/mL and 4.42±0.88ng/mL) and Group 3 (9.28±1.57pg/mL and 6.31±1.60ng/mL,
p
<0.05, respectively). IL-10 increased significantly in Group 4 (29.20±0.52pg/mL) compared to Group 3 (26.18±1.14pg/mL,
p
<0.05).

Conclusions:
Fimasartan reduced neointimal hyperplasia with decreases in macrophages in carotid atherosclerotic plaque and with reductions in systemic inflammation in ApoE knockout mice.
Abstract 462: Transgenic Overexpression of Tissue-nonspecific Alkaline Phosphatase in Monocytes/Macrophages Induces Calcification of Atherosclerotic Plaques
Authors: Corbo Anthony Marco , Romanelli Filippo , Huang Yuan , Dedkov Eduard I , Millan Jose Luis , Savinov Alexei Y , Savinova Olga V
Published: June 2021

Introduction:
Calcification of atherosclerotic plaques is associated with different histopathological and clinical manifestations. Microcalcification promotes inflammation and increases plaque instability, whereas macrocalcification is associated with stable fibroatheroma. The mechanisms of initiation and progression of calcification in atherosclerosis are not completely understood.

Hypothesis:
Upregulation of tissue-nonspecific alkaline phosphatase (TNAP) in monocytes/macrophages can lead to calcification of atherosclerotic plaques in a mouse model.

Methods:
TNAP overexpression was induced from a targeted transgene in
Hprt
locus utilizing Cre recombinase driven by monocyte-specific
LyzM
gene promoter. Resultant mice overexpressing TNAP in cells of monocyte/macrophage lineage (TNAP-Tg) were bred with animals bearing low density lipoprotein receptor mutation. Hypercholesterolemia was induced by feeding Paigen diet for ten weeks. Average plaque area and calcification (area %) were determined from histological sections of the aortic root (5-10 sections/sample). Blood flow velocity in the ascending aorta and left ventricular function were assessed by echocardiography.

Results:
In the absence of atherosclerosis, no appreciable soft tissue calcification was observed in TNAP-Tg animals (n=2). During progression of atherosclerosis, calcification was detected in the aortic root lesions of TNAP-Tg mice (4.85% of total plaque area) in significant excess compared to controls (0.02%, p<0.001, n=8 per group). Total plaque area in the aortic root was not affected by TNAP overexpression (0.40 mm^2 in TNAP-Tg; 0.38 in controls; p=0.75). Blood flow velocity in the ascending aorta and heart function were not significantly different between groups.

Conclusions:
We developed an animal model in which the interaction between early calcium deposition inside atherosclerotic lesions and the stability of such lesions can be studied experimentally.
Abstract 464: Chemokine Receptor CCR6 Expression on B Cells Augments Local IgM Production and Atheroprotection
Authors: Srikakulapu Prasad , McSkimming Chantel , McNamara Coleen
Published: June 2021

Background:
CCR6 mediates immune cell recruitment to inflammatory sites and has cell type-specific effects on diet-induced atherosclerosis in mice. Recent studies implicate the local immune responses in the adventitia/perivascular adipose tissue (PVAT) in atherosclerosis development. We have previously demonstrated that adoptive transfer of CD43
-
splenocytes (B cells) into B cell deficient

μMT
-/-

ApoE
-/-

mice results in reduced diet-induced atherosclerosis in a CCR6-dependent manner. Notably, there were significantly greater numbers of B cells in the aorta including PVAT of

μMT
-/-
ApoE
-/-

mice which received splenic B cells from

CCR6
+/+

mice compared to

CCR6
-/-

mice, despite no difference in B cell numbers in blood, spleen and peritoneal cavity, suggesting that CCR6 expression on B cells is important in B cell aortic homing. Production of IgM antibodies is thought to be a major mechanism whereby B cells limit atherosclerosis development. Yet whether B cells produce IgM locally in the PVAT and whether this is regulated by chemokine receptors such as CCR6 is unknown.

Methods and Results:
FACS experiments demonstrated high numbers of B cells available in the PVAT than aorta of young

ApoE
-/-

(49121±11190 and 80±11; p<0.001, n=7) mice. ELISPOT experiments demonstrated significantly fewer IgM secreting cells were in the PVAT of

ApoE
-/-
CCR6
-/-

mice compared to

ApoE
-/-
CCR6
+/+

mice (100±25 vs 850±150, p<0.05, n=5), despite no differences in IgM secreting cell numbers in spleen and bone marrow. Adoptive transfer of CD43
-
splenic B cells from

ApoE
-/-
CCR6
-/-

and

ApoE
-/-
CCR6
+/+

mice into secretory IgM deficient

ApoE
-/-

sIgM
-/-

mice demonstrated significantly reduced atherosclerosis in mice that received B cells from

ApoE
-/-
CCR6
+/+

mice compared to those that received B cells from

ApoE
-/-
CCR6
-/-

mice. Moreover, the B cells from

ApoE
-/-
CCR6
+/+

mice attenuated atherosclerosis only when they were capable of secreting IgM. FACS data from human blood demonstrated that circulating B and T cells but not monocytes express CCR6, suggesting potential human relevance to our murine findings.

Conclusion:
Results provide evidence that CCR6 expression on B cells mediates B cell recruitment into aorta and/or PVAT to provide atheroprotection via IgM secretion.
Abstract 468: Induction of Autophagy with Beclin-1 is not Sufficient to Reduce Atherosclerosis
Authors: Zhang Xiangyu , Bhattacharya Soumashubhra , Sergin Ismail , Evans Trent , Razani Babak
Published: June 2021

Autophagy is a highly conserved cellular process designed to degrade long-lived and dysfunctional protein aggregates and organelles. Recent work has established an important role for macrophage autophagy in the control of inflammatory signaling, maintenance of efficient cholesterol homeostasis, and degradation of deleterious protein aggregates. The absence of macrophage autophagy renders many of these processes dysfunctional and leads to significant increases in atherosclerotic plaque formation. Thus, there has been intense interest in the cardiovascular community to devise strategies to induce autophagy as an atheroprotective measure. We approached this by pharmacologically manipulating a critical upstream activator of autophagy, Beclin-1, a protein involved in the formation of the autophagy initiation complex and biogenesis of the autophagosome. A cell-permeable Beclin-1 peptide (known to induce autophagy both in vitro and in vivo) was administered to pro-atherogenic ApoE-null mice fed a Western diet for a period of 8 weeks. Despite its autophagy-inducing properties in vivo, Beclin-1 peptide failed to reduce the development of atherosclerosis. Moreover, the peptide’s autophagy-inducing properties in vitro were also insufficient to blunt atherogenic lipid-induced hyperactivation of the inflammasome/IL-1β or stimulate the clearance of cytotoxic protein aggregates. These data lead us to conclude that the induction of autophagy is not sufficient to curb atherosclerotic progression. It is increasingly appreciated that macrophages of the atherosclerotic plaque also progressively develop a profound lysosomal dysfunction which likely precedes and exacerbates autophagy dysfunction. This raises that notion that combined stimulation of both autophagy and lysosomes would be necessary to ameliorate atherosclerosis and that efforts to stimulate autophagy in isolation are unlikely to be successful.
Abstract 469: Residential Lifestyle Interventions Reduce Blood Pressure in 18 Days
Authors: Ramirez Francisco E , Siebold Jill , Ivy Linda , Antuna Kathelyn , Sanchez Albert , Nedley Neil
Published: June 2021

Background:
Tight control of blood pressure can have long term positive effects, non-pharmacological interventions to achieve it are documented in this study.

Hypothesis:
Vegan diets together with other lifestyle changes can safely decrease blood pressure.

Methods:
Twelve years of data was used, 1196 patients participated in a lifestyle program in Weimar California. The intervention consisted in whole food plant based diet (WFPD), exercise, sleep hygiene, medical, psychological and spiritual therapies. Treatments of massage and hydrotherapy were also given. A physician monitored the progress of each patient during the 18 days. Blood pressure was measured at baseline, at the end of the first week (time 1) and at the end of the second week (time 2) and at the end of 18 days. Some patients need it to decrease their blood pressure medication during the 18 days. Data was process with SPSS statistics package.

Results:
From 1196 patients, 764 of them were females. Mean age at baseline was 60.7 SD 15. Both systolic and diastolic blood pressure were reduced throughout treatment. For systolic on average (in mm HG) at baseline: 132.21, at time 1: 125.3, at rime 2: 121.96, at the end : 121.4. For diastolic on average (in mm HG) at baseline: 76.1, time 1: 72.2, time 2: 70.86, at the end : 69.9. A repeated measures ANOVA determined that mean systolic blood pressure differed significantly between time points (F(3,3585) = 69.008, P < .001). Post hoc polynomial contrasts revealed a significant linear trend in the data from start to end (F(1,1195 = 206.474. p<.001, partial η
2
= .14)). A similar pattern was found for diastolic blood pressure. The overall ANOVA found a significant difference in time points (F(3,3585) = 34.738, P < .001). Post hoc polynomial contrasts also showed a significant linear trend (F(1,1195 = 109.031. p<.001, partial η
2
= .08). Participants lost 4.8 pounds on average by the end of the program.

Conclusion:
WFPD together with lifestyle interventions are associated with a significant reduction of systolic and diastolic blood pressures. Many patients reduced their blood pressure medication. Lifestyle interventions options should be offered to patients with hypertension.
Abstract 47: Quaking Post-Transcriptionally Promotes Differentiation of Monocytes Into Pro-Atherogenic Macrophages by Controling Pre-mRNA Splicing and Gene Expression
Authors: de Bruin Ruben G , Shiue Lily , Prins Jurrien , de Boer Hetty C , Djaramshi Anjana , Fagg W. Samuel , van Gils Janine M , Katzman Sol , Donahue John P , van Esch Hilde , Rabelink Ton J , Kazan Hilal , Biessen Erik A , Ares Manuel , van Zonneveld Anton Jan , van der Veer Eric P
Published: June 2021

Aim:
Atherosclerosis is accelerated by excessive monocyte recruitment, influx and differentiation into pro-inflammatory macrophages. While the genome-wide mRNA expression profiles of human monocytes and pro-inflammatory macrophages are well-established, their transcriptomes are ultimately defined by factors, such as RNA-binding proteins, that modulate pre-mRNA splicing patterns and mRNA transcript abundance. This prompted us to investigate the role of the RNA-binding protein Quaking in regulating global changes in pre-mRNA splicing and mature mRNA expression as human monocytes acquire the pro-inflammatory macrophage identity.

Methods:
We employed RNA-sequencing and splicing-sensitive microarrays to determine genome-wide changes in pre-mRNA splicing and mRNA expression upon conversion of human monocytes into pro-inflammatory macrophages, including those derived from a unique Quaking haploinsufficient patient.

Results:
Using laser-capture micro-dissection and immunohistochemistry, we discovered that expression levels of Quaking mRNA and protein are low in monocytes of early human atherosclerotic lesions, but abundant in macrophages of advanced plaques. Depletion of Quaking protein using both siRNA and GapmeR technology significantly impaired monocyte adhesion and migration; delayed differentiation into pro-inflammatory macrophages while maintaining the capacity to adopt the anti-inflammatory phenotype; and diminished foam cell formation in vitro and in vivo. RNA-sequencing and microarray analysis of human monocyte and macrophage transcriptomes revealed striking changes in Quaking-dependent pre-mRNA splicing and mRNA transcript levels, with gene ontology analyses identifying an enrichment in transcripts involved in cellular migration and lipid metabolism. Furthermore, these studies uncovered common as well as novel alternatively spliced transcripts with unknown biological functions in monocytes and macrophages.

Conclusions:
Our studies illustrate a central role for Quaking in post-transcriptionally guiding pro-inflammatory macrophage identity and function.
Abstract 472: PPARγ-agonist Rosiglitazone Modulates Macrophage Proliferation, Polarization, and Inflammatory Function
Authors: Hyatt Danielle , Alenghat Francis J
Published: June 2021

Background:
Macrophages are central to the initiation, progression, and repair of inflammation. Rosiglitazone (Rosi), an antidiabetic thiazolidinedione (TZD), is a PPAR-γ agonist. Although TZDs improve certain cardiovascular risk factors and surrogate endpoints, they do not appear to reduce major cardiovascular events, and cardiovascular risk of Rosi has been a particular concern. Previously undetected effects on macrophages could provide a more nuanced understanding of how Rosi and other TZDs impact inflammation.

Results:
To this end, we have characterized the effects of Rosi on bone marrow-derived macrophages (BMMs)
in vitro
. M2-polarized BMMs express canonical mRNA markers, IL10 and Ym1. In this system, compared to polarized untreated cells and M2s treated with 1μM of the TZD Pioglitazone (Pio), M2s treated with 1μM Rosi express lower levels of anti-inflammatory M2 markers (IL10: 0.07 ± 0.01 for Rosi vs. 0.73 ± 0.04 for untreated vs. 1.00 ± 0.06 a.u. for Pio; Ym1: 0.06 ± 0.00 for Rosi vs. 0.81 ± 0.06 for untreated vs. 1 ± 0.04 a.u. for Pio). M2s polarized in the presence of Rosi express diminished protein levels of the M2 marker CD206 when compared to untreated M2s (Rosi 0.44 vs. untreated 1.00 a.u.). Culture of marrow with BMM-differentiating media in the presence of Rosi increased proliferation compared to untreated marrow and to marrow treated with Pio or the PPARγ antagonist GW9662, as measured by cell counts (Rosi 189 ± 19% relative to untreated BMM counts; Pio 115 ± 4%; GW9662 103 ± 4%; n=4, p < 0.001). There was no significant difference in monocyte-to-macrophage differentiation. Mature BMMs showed greater increase in cell counts with Rosi when compared to untreated BMMs or those treated with Pio or GW9662 (Rosi +70 ± 7%, untreated +2 ± 1%, Pio +24 ± 10%, GW9662 +8 ± 5%, n=3, p < 3 x 10
-3
). In an
in vitro
system of efferocytosis, macrophages differentiated in the presence of Rosi show impaired uptake of apoptotic foam cells in comparison to untreated BMMs (Rosi 0.77 vs. untreated 1.00 a.u.).

Conclusion:
Overall, we find that rosiglitazone, more than pioglitazone, increases macrophage proliferation and reduces anti-inflammatory markers and effector functions. Future work will seek to delineate the mechanism of this apparent inflammatory effect on macrophages.
Abstract 49: Elevating Apolipoprotein A-I Levels Promotes Atherosclerosis Regression in Diabetic Mice by Inhibiting Proliferation of Bone Marrow Monocyte Precursors
Authors: Barrett Tessa J , Distel Emilie , Ogando Yoscar , Astudillo Yaritzy M , Liu Jianhua , Goldberg Ira J , Murphy Andrew J , Fisher Edward A
Published: June 2021

Diabetes is a primary risk factor for cardiovascular diseases (CVD) and in clinical imaging studies is shown to impair the resolution of CVD, a process termed regression. We have also reported this failure of lesion regression in mouse models of diabetes, despite effective lipid lowering. This, in part, can be attributed to diabetes-driven monocytosis promoting continued monocyte infiltration into plaques. In non-diabetic settings apolipoprotein (apo) A-I and high-density lipoprotein (HDL) suppress leukocytosis and promote lesion regression. As low apoA-I/HDL is a typical feature of diabetic dyslipidemia this study aimed to establish whether raising apoA-I/HDL levels
in vivo
is an effective approach to reduce diabetes-driven leukocytosis and promote lesion regression.

Aortic arches from hyperlipidemic Ldlr
-/-
mice were transplanted into WT, diabetic WT, and diabetic human apoA-I-overexpressing transgenic mice (transgenic mice have a 3-fold increase in HDL-cholesterol), and lesion composition assessed 2 weeks post-surgery. Following aortic transplantation into WT mice (i.e. normal lipid levels) we found regression, as assessed by change in plaque macrophage (mΦ) content relative to baseline control mice was achieved (68% mΦ reduction, P<0.001). Regression was impaired when aortas were transplanted into diabetic WT recipients (50% mΦ reduction, P<0.01). However, raising apoA-I/HDL levels in the setting of diabetes restored regression in diabetic mice (62% mΦ reduction, P<0.001).
In vivo
monocyte/mΦ trafficking analyses revealed that elevating apoA-I/HDL levels in diabetes improves atherosclerosis regression by reducing monocyte entry by 60% (P<0.01), and promoting mΦ egress from lesions (30% increase). We also found that greater apoA-I/HDL reduced blood monocytes by decreasing the proliferation of monocyte progenitors in the bone marrow (15-20% reduction, P<0.05), explaining, in part, how apoA-I/HDL promotes regression.

Raising apoA-I/HDL levels promotes atherosclerotic lesion regression in diabetic mice. This may serve as a therapeutic strategy for patients with diabetes, who unlike WT mice, have reduced HDL levels and remain at an elevated risk for CVD despite effective plasma cholesterol lowering.
Abstract 5: Lipid Droplet Associated Hydrolase: A New Player in Cholesterol Mobilization From Foam Cells
Authors: Goo Younghwa , Saha Pradip , Chan Larry , Paul Antoni
Published: June 2021

Lipid laden macrophages/foam cells are a hallmark of atherosclerotic lesions from early to late stages of development. Macrophages take-up modified low-density lipoprotein (mLDL) particles and store surplus mLDL-derived cholesterol as cholesterol ester (CE) in cytoplasmic lipid droplets (LDs). Accelerating CE hydrolysis from the LDs is a plausible strategy to promote reverse cholesterol transport from the atheroma. However, the identity of the CE hydrolases that function on LDs remains unknown. Previously we identified lipid droplet-associated hydrolase (LDAH) in LDs purified from macrophages and reported that
in vitro
LDAH regulates CE levels by increasing CE hydrolysis. To determine the relevance of LDAH in atherogenesis, we have generated LDAH knockout (LDAH-/-) mice. Mouse peritoneal macrophages (MPM) isolated from LDAH-/- mice had increased cytoplasmic LDs, increased net CE content, and decreased cholesterol efflux. In atherosclerosis studies, both male and female LDAH-/- mice crossed with apolipoprotein E knockout (apoE-/-) mice fed a Western diet developed larger lesions. Lesions of LDAH-/-/ apoE-/- mice were characterized by increased areas of macrophages containing enlarged cytoplasms with large LDs. Supporting a direct atheroprotective role of LDAH in macrophages, lesions of apoE-/- mice that received bone marrows from LDAH-/-/apoE-/- mice progressed faster than those that received bone marrow cells from LDAH+/+/apoE-/- mice. In qPCR analyses of genes involved in cholesterol homeostasis in macrophages, we found that ABC binding cassette transporters ABCA1 and ABCG1, which mediate cholesterol efflux through the plasma membrane, were consistently decreased in LDAH-/- MPM. Further
in vivo
gene expression studies on macrophages selectively obtained from lesions using laser capture microdissection are underway. In conclusion, our study suggests that LDAH promotes LD CE hydrolysis and cholesterol efflux from foam cells within the atheroma, and uncovers a potential target to promote reverse cholesterol from arteries as a means of ameliorating atherosclerosis development.
Abstract 504: Telomere-based Assessment of Biological Age in Patients with Advanced Vascular Disease
Authors: Yin Hao , Akawi Oula , Fox Stephanie A , Li Fuyan , O'Neil Caroline , Wong Jorge , Arpino John-Michael , Balint Brittany , Watson Alanna , Guo L. Ray , Quantz MacKenzie A , Nagpal A. Dave , Kiaii Bob , Chu Michael W , Pickering J. Geoffrey
Published: June 2021

Introduction:
Ascertaining the biological age of patients with advanced vascular disease could advance risk assessment and management. The extent to which telomeres shorten in leukocytes could be a marker of biological age because it reflects the accumulation of replication stresses imposed on leukocyte progenitors. However, because of wide, genetic variability in leukocyte telomere length (TL), a single leukocyte TL measurement does not reliably indicate telomere shortening.

Hypothesis:
We hypothesized that the difference in length of telomeres in “non-replicating” muscle-rich tissue and that of circulating leukocytes provides a patient-specific index of telomere shortening in patients with advanced vascular disease.

Methods:
TL in leukocytes, skeletal muscle, and right atrial cardiac muscle were measured from 134 patients undergoing coronary or thoracic aortic surgery, using quantitative polymerase chain reaction. Relationships between leukocyte TL or the muscle-leukocyte TL difference (ΔTL) and early post-operative outcomes were tested using Cox proportional hazard and binary logistic regression analyses.

Results:
Telomeres in cardiac muscle and skeletal muscle were significantly longer than those in leukocytes (p<0.001) but with synchrony among these measures in an individual (p<0.001). Leukocyte TL and skeletal muscle TL inversely correlated with chronological age (p<0.001) however cardiac muscle TL did not (p=0.283). There was no relationship between leukocyte TL and either the post-operative length of stay in the intensive care unit (ICU) or major complications. However, cardiac-leukocyte ΔTL was associated with length of ICU stay (hazard ratio 1.26, p=0.043) and post-operative complications (odds ratio 3.03, p=0.029).

Conclusions:
Right atrium-leukocyte ΔTL provides an index of telomere shortening and may inform outcomes in patients with advanced vascular disease. This two-component telomere measurement may reflect the biological age of individuals with chronic vascular disease.
Abstract 505: Circulating Monocyte-Platelet Aggregates are Different Across Phenotypes of Vascular Disease
Authors: Allen Nicole , Guo Yu , Perez Adriana , Bissoon Edna , Cambria Matthew , Rubin Maya , Lerner Gabriel , Adelman Mark , Rockman Caron B , Berger Jeffrey S
Published: June 2021

Background:
Platelets are a major culprit in the pathogenesis of cardiovascular disease (CVD). Although vascular disease in different arterial beds share common risk factors, the role of platelet activity may differ by vascular bed. Clinical trials suggest that more potent antiplatelet therapy is needed in lower extremity peripheral artery disease (PAD) than coronary artery disease (CAD). We investigated circulating monocyte platelet aggregates (MPA), a reproducible
ex vivo
measurement of platelet activity, in patients with CVD (PAD, CAD, carotid artery stenosis [CAS], and abdominal aortic aneurysm [AAA]) and disease controls.

Methods:
Subjects with CVD (n=351) and disease controls (n=73) had MPA measured using strict quality control, all of whom were on aspirin monotherapy. MPA were identified by CD14/CD61 positivity. Data is represented as median (IQR, interquartile range). Wilcoxon rank sum, Wilcoxon signed rank, and multivariable linear regression were used to analyze data.

Results:
Platelets aggregated with monocytes had a higher platelet surface expression of PAC-1, P-selectin, and CD40 versus platelets not aggregated with monocytes (P<0.05 for each comparison). Compared with controls, MPA was significantly higher in CVD (14.5 [10.3, 27.8] vs. 9.4 [8.2, 11.5], P<0.001) which remained significant after multivariable adjustment for demographics and risk factors (β=9.1 (SER=3.9), P=0.02). For each vascular disease phenotype, MPA was higher than controls (
Figure
). In a multivariable linear regression model including demographics, risk factors and each vascular bed, PAD was the only vascular disease associated with a higher value of MPA (β=10.2 (SER=2.4), P<0.001).

Conclusions:
Platelets aggregated with monocytes have increased platelet activity and are significantly elevated in subjects with PAD. Future studies understanding the platelet profile in PAD and its clinical relevance are needed.
Abstract 514: Development and Validation of Risk Model for Predicting Prevalence of Peripheral Artery Disease in a Low to Intermediate Risk Population
Authors: Malick Waqas A , Rockman Caron B , Guo Yu , Xu Jinfeng , Adelman Mark A , Berger Jeffrey S
Published: June 2021

Background:
Peripheral artery disease (PAD) is associated with impaired quality of life and significant cardiovascular morbidity and mortality, yet remains under recognized and under diagnosed.

Objectives:
This study sought to develop and validate a risk model for the identification of individuals at risk for PAD that could be useful in the clinical setting.

Methods:
Twenty three variables assessed in ≈3.2 million self-referred participants without established cardiovascular disease from 2003 to 2008 who completed a medical and lifestyle questionnaire in the United States were evaluated by screening ankle brachial indices <0.90 for PAD. Subjects were divided into a derivation cohort (1.57 million) and a validation cohort (1.57 million). Lasso variable selection was used in the derivation cohort to develop the best-fitting parsimonious prediction models. Discrimination and calibrations was evaluated using the C statistic and the Hosmer-Lemeshow calibration statistic.

Results:
The overall prevalence of PAD was 3.96%. Using lasso variable selection, 11 variables were included in complex best-fitting model: age, sex, race, marital status, BMI group, smoking status, hypertension, diabetes, family history of PAD, physical activity, and inter-arm systolic blood pressure difference. In the validation cohort, the C-statistics for this model was .746 and the calibration was excellent (P=0.38; no significant deviation between predicted and observed outcomes). The current risk score has improved discrimination and calibration compared with other existing risk scores for PAD.

Conclusion:
This robust PAD risk calculator derived from a diverse population across the US provides a good risk estimate of PAD and is anticipated to assist in identifying subjects at risk for PAD.
Abstract 515: Comparison of Aging and Disturbed Flow Effects on Arterial Wall Biomechanics
Authors: Pokutta-Paskaleva Anastassia , Gleason Rudolph L , Brewster Luke P
Published: June 2021

Introduction:
Arterial stiffness is an age related disease that doubles ones cardiovascular mortality and functions as an intermediary step in the formation of atherosclerosis. We have recently demonstrated that low and oscillatory wall shear stress, termed disturbed flow (d-flow), leads to arterial stiffness in otherwise normal arteries. Since d-flow has been linked to atherosclerotic plaque formation, our objective is to compare the carotid artery mechanics of 12 week arteries exposed to 4 weeks of d-flow to that of untreated 80 week old s129 mice. We hypothesize that low and oscillatory shear governs the remodeling of the arteries in a way that mimics the effect of arterial stiffening associated with aging.

Materials and Methods:
We used a partial carotid ligation model in 12-week-old s129 male (n=3) and female (n=3) mice. 4 weeks post surgery we euthanized the animals and performed biaxial extension-inflation biomechanical testing, confocal imaging, histological studies and opening angle studies on the left (partially ligated) carotid artery (LCA) and right (non-ligated) carotid artery (RCA) in order to characterize their biomechanical behavior. The same testing was performed on s129 80-week-old male animals (n=4). Pressure-diameter (P-d) data were collected from cyclic pressurization ramps from 0 to 160 mmHg at constant axial extension levels ranging from 1.3 to 1.9. Compliance was calculated at the in-vivo axial stretch level of 1.7 for both age groups. Intima media thickness was analyzed from H&E stained histological slides. Results were compared with the unpaired, two-tailed t-test and significance was taken at p<0.05.

Results and Conclusions:
Partial ligation for 4 weeks drastically decreased the compliance of the 12-week-old left carotid in a way that exactly follows the stiffening of the arteries in the 80-week-old mice. The compliance of the non-manipulated right carotid arteries reflected the age related difference in the stiffness of young vs old arteries with statistical difference in the 30 - 90 mmHg range. We have established that low shear stress and oscillatory flow promote the increase in arterial stiffening in the partially ligated young mice in a way that mimics the age-related decrease in arterial compliance in old mice.
Abstract 526: 4-hydroxy-2-nonenal (HNE), a Lipid Peroxidation Product, Exerts Both Pro- and Anti-thrombotic Effects on Vascular Cells
Authors: Kothari Hema , Vatsyayan Rit , Rao Vijaya Mohan
Published: June 2021

Background:
Oxidative stress and generation of lipid peroxidation (LPO) products are detrimental in the pathogenesis of atherosclerosis and associated acute thrombotic events. However, recent studies suggest that moderate oxidative stress and low levels of LPO products can induce adaptive immune responses and exert beneficial effects. Tissue factor (TF) is a critical initiator of coagulation and aberrant TF expression on vascular cells under inflammation triggers intravascular thrombosis. HNE, a highly reactive LPO product and TF have been shown to be associated with atherosclerosis. Recently, we demonstrated that HNE decrypts procoagulant activity of pre-existing TF on activated monocytes and endothelial cells and generates TF+ microparticles. Here, we investigated the effect of HNE on induction of TF and cell adhesion molecules in monocytes and endothelial cells that were not perturbed earlier.

Methods and results:
THP-1 monocytic cells and endothelial cells (HUVEC) were stimulated with LPS and TNF-α/IL1-β, respectively, in the presence of a control vehicle or varying concentrations of HNE that are pathophysiologically relevant. TF induction was measured at mRNA (by qRT-PCR), protein (by immunoblotting) and activity levels (in factor X activation assay). Pre-treating cells with HNE inhibited TNF-α/IL1-β- or LPS-stimulated TF procoagulant activity in a dose-dependent manner. THP-1 and HUVEC varied in their sensitivities to HNE (THP-1> HUVEC). HNE-mediated inhibition of TF activity correlated with lower TF mRNA and protein levels. Our results demonstrate that HNE prevents TNF-α/IL1-β- and LPS-induced IKKβ degradation and thereby inhibits NFκβ activation. In addition to inhibiting TF expression, HNE significantly reduced monocyte adhesion to endothelial cells through downregulating TNF-α/IL1-β-induced expression of endothelial adhesion molecules VCAM-1 and ICAM-1.

Conclusion:
HNE may play a dual role in regulating TF activity in atherosclerosis. HNE could act as a prothrombotic mediator by increasing coagulant activity of pre-existing TF through decryption process. HNE can also elicit anti-thrombotic and anti-inflammatory effect by inhibiting TF and adhesion molecules in response to stimulus by impairing the NF-ĸB pathway.
Abstract 53: Role of Serotonin through 5HT
2A
in alphaIIbBeta3 Outside-in Activation
Authors: Oliver Kendra H , Duvernay Matthew , Hamm Heidi E , Carneiro Ana M
Published: June 2021

Patients taking selective serotonin reuptake inhibitors (SSRIs) have an increased risk of bleeding. However it remains unclear how SSRIs, which inhibit the serotonin transporter (SERT), modulate hemostasis, particularly platelet activation. 5HT (serotonin, 5-hydroxytryptamine) is taken up by platelets via SERT and activates the 5HT
2A
receptor. αIIbβ3 is an integrin that binds fibrinogen and plays a critical role in platelet aggregation, which is central to hemostasis and thrombosis. Futhermore, it is known that αIIbβ3 and SERT interact genetically, physically, and functionally. To gain a clear understanding of how SERT function regulates platelet activity we examined two independent models of SERT inhibition - constitutive genetic deletion (SERT
-/-
) and 6-day water citalopram administration. Through SERT, platelets are the major storage site for 5HT in whole blood. Our works suggests that ADP-mediated αIIbβ3 activation is decreased as assessed by JON/a binding. Additionally while 5HT alone does not cause any platelet activation in SERT
+/+
, 5HT synergizes with submaximal ADP to potentiate αIIbβ3 activation. The synergy of 5HT-ADP synergy is lost in SERT
-/-
platelets as determined by the addition of exogenous 5HT. Furthermore, blocking 5HT
2A
receptor with an antagonist, ketanserin, inhibited ADP-mediated αIIbβ3 activation, suggesting that 5HT-ADP synergy could be mediated through activation of the 5HT
2A
receptor. Additionally, we found that 5HT
2A
receptor surface expression was reduced in SERT
-/-
platelets. SERT
-/-
platelet membranes incubated with a 5HT
2A
agonist, DOI, also exhibited reduced
35S
GTPys incorporation. These findings suggest that long-term inhibition of SERT leads to reduced 5HT
2A
receptor-dependent potentiation of αIIbβ3 activation, perhaps through altered hemostatic regulation of 5HT. Further experiments are being carried out to determine a possible novel regulation of αIIbβ3 via 5HT
2A
that could alter the functionality of αIIbβ3. However, these findings suggest a novel mechanism central to the increased bleeding observed in patients taking SSRIs and support the continued investigation of 5HT
2A
receptor inhibitor as anti-platelet drugs for treatment of cardiovascular diseases.
Abstract 530: Decreased NSF S-Nitrosylation in the Aorta and an Age-Dependent Endothelial Activation in Mice with Fabry Disease
Authors: Kang Justin J , Shu Liming , Desch Karl C , Bodary Peter F , Shayman James A
Published: June 2021

Fabry disease is an X-linked lysosomal storage disorder caused by loss of activity of the enzyme, α-galactosidase A (GLA). The loss of GLA function leads to an age-dependent accumulation of globotriaosylceramide (Gb3) in endothelial cells. Endothelial Gb3 accumulation is associated with endothelial nitric oxide synthase (eNOS) uncoupling and decreased nitric oxide (NO) bioavailability. We hypothesized that GLA deficiency promotes endothelial inflammation. von Willebrand factor (VWF) is the main component of Weibel Palade bodies (WPB) and is secreted upon endothelial inflammation. We observed significantly elevated plasma VWF in GLA null mice (FA) compared to age-matched Wild-Type mice (WT) (p=0.046 at 2 months and p<0.001 at 5 and 17 months), indicating increased endothelial inflammation in FA. Out of the many proteins that regulate WPB exocytosis, N-ethylmaleimide sensitive factor (NSF) is a critical mediator of the exocytic machinery and a major target of NO. NO can reversibly modify NSF cysteine residues via a process called S-nitrosylation (SNO), leading to decreased WPB exocytosis. To evaluate whether GLA deficiency promotes a decrease in SNO-NSF, nitrosylated cysteines of aortic homogenates from male WT and FA were first labeled with biotin through a biotin switch assay. Next, biotinylated proteins were isolated with streptavidin-agarose beads. We observed an approximately 60% decrease in the level of SNO-NSF in the aorta of FA compared to that of WT by Western blot (WT vs. Fabry: 1.0±0.03 vs. 0.4±0.11, p=0.002, n=4-5/group) whereas total protein expression of NSF and GAPDH were not different between groups. The level of thioredoxin-1 (TRX-1) was significantly elevated in FA compared to WT (WT vs. Fabry: 1.0±0.05 vs. 1.4±0.14, p=0.01, n=6-9/group), suggesting high levels of reactive oxygen species and protein denitrosylase activity in Fabry disease. In conclusion, these results provide evidence of endothelial activation in Fabry disease. GLA deficiency resulted in decreased SNO-NSF and increased TRX-1 in parallel with the robust elevation of VWF. Future study is required for further understanding of the mechanistic links between these observations, and to determine whether this is a reversible phenomenon in the setting of Fabry disease.
Abstract 531: In Patients with Uncontrolled Arterial Hypertension Platelets and Coagulation Factor XI are Responsible of Modifications of Thrombin Generation
Authors: Lagrange Jeremy , Weihert Yvonne , Münzel Thomas , Karbach Susanne , Wenzel Philip
Published: June 2021

Background:
Hypertensive crisis is an extreme phenotype of increased blood pressure that can lead to organ failure and thrombotic complications. We recently found in experimental hypertension an angiotensin II driven factor XI (FXI)-thrombin amplification loop leading to vascular injury.

Objective:
We wanted to evaluate, in patients with uncontrolled hypertension (HT), the role of platelet and factor XI in thrombin generation (TG).

Patients and methods:
Prospectively, 52 patients with uncontrolled HT (16 grade I and 36 grade II/III) were examined at the outpatient clinic and emergency room of the University Medical Center Mainz, and 16 controls. Calibrated automated thrombography (CAT) was used to measure TG in platelet rich plasma (PRP, adjusted to 150.109/L of platelets), platelet free plasma (PFP), with platelets from controls and uncontrolled HT patients resupsended in a pool of healthy controls and in the presence or absence of an antibody blocking FXI activation pathway.

Results:
We found an increased systolic blood pressure in HT patients, compared to control patients (179±1.9 mmHg for grade II/III patients, 139±1.2 mmHg for grade I patients and 123±2.5 mmHg for controls); age, BMI as well as weight were not different. TG in PRP showed a positive and significant correlation between blood pressure and the other TG parameters as Endogenous thrombin potential (ETP), peak TG and velocity of TG. ETP was significantly increased in grade II/III patients compared to the control group. TG parameters in HT patients and controls were completely blunted by the inhibition of FXI pathway by blocking its apple 3 domain. This result indicates an involvement of the FXI thrombin loop in thrombin generation in PRP of uncontrolled HT patients. Without platelets TG showed no difference between HT patients and controls. Platelets resuspended from HT or controls restored the differences in ETP, peak and velocity between the 2 groups indicating that increased TG in HT patient mostly depends on platelet overreactivity.

Conclusion:
These results point out the important role of platelet overreactivity as well as FXI-dependent thrombin activation pathway in hypertension. Monitoring the prothrombotic state of platelets might add to risk stratification of patients with HT.
Abstract 533: Lymph is a Vehicle for Extracellular Vesicles in Mice
Authors: Milasan Andreea , Tessandier Nicolas , Tan Sisareuth , Brisson Alain , Boilard Eric , Martel Catherine
Published: June 2021

Introduction:
Although for a long time considered as simple cellular debris, extracellular vesicles (EVs) are now known to be involved in many pathophysiological processes such as thrombosis, autoimmune diseases and inflammation. Due to their diversity and presence in different tissues, EVs are considered important biomarkers and thus, their precise detection in various biological fluids is important to better understand all their different functional activities. The lymphatic system works in close collaboration with the cardiovascular system to preserve fluid balance throughout the body. Lymphatic vessels are present in almost all vascularized tissues, including the brain and the artery wall, and their role in these organ-related pathologies are under intense investigations.

Hypothesis:
Since lymphatic vessels are often perceived as "sewers", due to their role in removing interstitial fluid and waste products from peripheral tissues such as the artery wall, we herein want to qualitatively and quantitatively assess the presence of EVs in circulating lymph.

Methods and Results:
Using several approaches such as a Zetasizer Nano S, electron microscopy and flow cytometry analysis, we have detected and characterized EVs in lymph of healthy animals, and found that these EVs are inclusively derived from red blood cells, platelets and lymphatic endothelial cells. Analysis of lymph from atherosclerotic mice (Ldlr
-/-
) confirmed the idea that EVs number and origin varies according to the pathological setting.

Conclusion:
Herein, we show for the first time that EVs are present in lymph and that their level and origin vary in atherosclerosis. Our work will be setting the stage to a better understanding of the mechanism underlying EV accumulation in peripheral tissues during inflammation, and to better control related diseases.