bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 303: Risk Factors for Venous Thromboembolism (VTE) among Patients with Neurologic Disease and Leg Paresis: A Population-based Case-control Study.
Authors: Cohoon Kevin P , Andrews Karen L , Ashrani Aneel A , Petterson Tanya M , Bailey Kent R , Heit John A
Published: March 2022

Background:
Whether VTE risk can be further stratified among patients with neurologic disease and leg paresis is unknown.

Objective:
To identify independent risk factors for deep vein thrombosis (DVT) and pulmonary embolism (PE) among patients with neurologic disease and leg paresis.

Methods:
Using the population-based resources of the Rochester Epidemiology Project, we identified all Olmsted County, MN residents with incident DVT or PE over the 45-year period, 1966 to 2010. We performed a case-control study nested within the Olmsted County population with incident VTE associated with neurologic disease and leg paresis over the 18-year period, 1988-2010 (n=192). For each VTE case, we identified 1 Olmsted County resident with neurologic disease, leg paresis and no VTE (control) matched to each case on sex, date of birth (+/- 5 years), calendar year (+/- 1 year) and duration of medical records. Using conditional logistic regression, we tested duration of paresis (index-3 months, 3-12 months, and > 12 months), paresis etiology, and degree of immobility as potential risk factors for VTE after adjusting for other known VTE risk factors, including hospitalization for major surgery or acute medical illness, trauma/fracture, and active cancer.

Results:
In univariate analyses of the 192 pairs, trauma/fracture, hospitalization for major surgery or for acute medical illness, active cancer, duration of paresis, and degree of immobility were associated with VTE. In the multivariable analysis, hospitalization for acute medical illness, active cancer, degree of immobility (ambulatory without/with assistance, function from a wheelchair base, bed bound; OR: 2.60; 95%Cl: 1.65-4.11); p=<0.0001) were independent predictors of VTE.

Conclusion:
Among patients with neurologic disease and leg paresis, VTE risk can be further stratified by hospitalization for acute medical illness, active cancer and degree of immobility; these patients should be considered for VTE prophylaxis.
Abstract 310: Oxidized LDL-binding Cationic Peptides and Proteins in Inflammation and Atherosclerosis: Novel Insights
Authors: Aluganti Narasimhulu Chandrakala , Selvarajan Krithika , Burge Kathryn , Parthasarathy Sampath
Published: March 2022

Background:
Apolipoprotein A1 and apolipoprotein E mimetic peptides have attracted attention due to their ability to reduce atherosclerosis and exhibit antioxidant, anti-inflammatory, and hypolipidemic properties. We previously reported that the non-lipoprotein-related Lys-Arg-rich cationic peptides have anti-inflammatory properties both in vitro and in vivo. In this study, we have determined whether ApoB100 of low density lipoprotein (N-LDL), which is Lys-Arg rich, has similar properties.

Methods:
5F-mimetic peptide of ApoA1, LL27 derived from the anti-microbial peptide CAMP, and a human glycodelin derived peptide, were commercially synthesized. N-LDL was prepared and used. The ability of these peptides and protein to neutralize charges of modified lipoproteins, as well as attenuate macrophage uptake and inflammation, were analyzed. Oxidized LDL (Ox-LDL) was pretreated with increasing concentrations of peptides and N-LDL to evaluate charge neutralizing properties of the peptides as well as that of the protein (ApoB100). RAW cells were incubated with LPS or Ox-LDL pretreated with and without peptides and N-LDL. RNA was isolated from treated cells and real time PCR was performed using mouse IL-1α and IL-6 primers.

Results:
Cationic peptides as well as ApoB100 protein of N-LDL decomposed the peroxide content of 13-HPODE. Incubation of Ox-LDL and Ac-LDL with the peptides as well as ApoB100 resulted in charge neutralization as noted by agarose gel electrophoresis. Pre-incubation of the peptides and N-LDL with modified lipoproteins reduced the uptake of the latter by macrophages and foam cell formation as detected by Oil-Red O staining. Reduced inflammation was observed in the presence of N-LDL as compared to LPS/Ox-LDL.

Conclusions:
Based on these studies, we postulate that cationic peptides and protein might have properties that would a) affect events that are unrelated to lipid lowering, b) might play an additional role in immune competent cells, including macrophages, and c) might interact with other biologically important anionic molecules, including lipids and proteins. We also predict that lysine-rich cationic peptides and proteins could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.
Abstract 313: Perinatal Growth Restriction Decreases Hepatic RNA Editing of Apolipoprotein B in Male Rat Offspring
Authors: Devarajan Asokan , Thamotharan Shanthie , Valburg Claire , Devaskar Sherin , Freije William A
Published: March 2022

Introduction:
Individuals born with intrauterine growth restriction develop cardiovascular disease. We employed hepatic microarray-based expression profiling of male rat offspring born to mothers subjected to combined intrauterine and postnatal calorie restriction (IPCR) as a discovery driven experiment to interrogate intrauterine growth restriction. These studies identified that the expression of Apobec1 and Apobec1 complementation factor (A1CF), genes known to edit apolipoprotein B (ApoB) mRNA through the epigenetic process of cytosine deamination, are decreased in IPCR. Editing of ApoB mRNA at position 666 changes the ribonucleotide base from a cytosine to a uracil (U) and controls the production of ApoB100 and ApoB48 lipoproteins.

Hypothesis:
IPCR decreases hepatic ApoB mRNA editing in male offspring through transcriptional regulation of Apobec1 and A1CF.

Methods:
Pyrosequencing designed to interrogate ApoB mRNA editing was used to quantify hepatic ApoB100 and ApoB48 transcripts in an experiment employing the male offspring (litters culled to 6 male offspring for each mother) from 4 control fed mothers and 4 mothers subjected to 50% calorie restriction from embryonic day 11 to postnatal day 21 (IPCR). RT-qPCR and western blotting were employed to quantify the hepatic expression of Apobec1 and A1CF. Serum ApoB100 and Apo48 were quantified by ELISA.

Results:
ApoB transcripts in the control fed offspring were edited, with a U base call of 55 ± 2% at position 666, whereas IPCR offspring possessed decreased editing with a U base call of 12 ± 1% (n=12 for each group; F=368; p<0.0001). The RT-qPCR relative quotient (RQ) in the IPCR group was 0.2 ± 0.1 (F=107;p<0.0001) and 0.4 ± 0.1 (F=25;p<0.0001) for Apobec1 and A1CF, respectively (RQ=1 in the control fed offspring for both Apobec1 and A1CF). Relative to control, IPCR treatment significantly decreased hepatic Apobec1 and A1CF proteins by 45% ± 10 and 27 % ± 9.2 respectively. IPCR treatment increased serum ApoB100 by 1.5 fold ± 0.43 (p= 0.051) and significantly decreased ApoB48 by 0.8 fold ± 0.1 (n=6) compared to control.

Conclusion:
These data define a novel association of IPCR and ApoB editing through a nutritionally controlled epigenetic process working through transcriptional control of Apobec1 and A1CF.
Abstract 315: Inflamed HDL Loses Its Anti-inflammatory Properties on Adipocytes: Role of Interaction of Serum Amyloid A with the Extracellular Matrix
Authors: Han Chang Yeop , Tang Chongren , Omer Mohamed , Wang Shari , Vaisar Tomas , Wight Thomas N , Osborne William R , Chait Alan
Published: March 2022

Obesity induces adipocyte hypertrophy and accumulation of macrophages in adipose tissue, which associates with insulin resistance and cardiovascular disease risk. HDL and apoA-I have anti-inflammatory properties in addition to facilitating reverse cholesterol transport. We recently showed that HDL from healthy humans and lean mice inhibits palmitate-induced monocyte chemotactic factor expression by decreasing lipid raft (LR) content in adipocytes. We also have shown that adipocytes produced extracellular matrix (ECM)
in vivo
and
in vitro
when they became hypertrophic. Moreover, inflamed HDL loses its anti-inflammatory effect and ability to stimulate cholesterol efflux from adipocytes. From gain of function and loss of function experiments, it appears that serum amyloid A (SAA) is partially responsible for these effects. However, the mechanism by which SAA impairs HDL’s anti-inflammatory function is unknown. To generate HDL under conditions of sterile inflammation, HDL was purified from plasma of AgNO
3
-injected C57BL/6 mice. Also, to generate SAA-enriched HDL, HDL was purified after HDL from healthy humans was exposed to 293 HEK cells with lentiviral overexpression of SAA2. Control, inflamed and SAA-enriched HDL were labelled using DiI dye and the ECM of adipocytes was stained by Alexa 488 conjugated wheat germ agglutinin. After exposure of control HDL to adipocytes, there was no co-localization of DiI and Alexa 488 staining. However, we found that inflamed and SAA-enriched HDL were co-localized with the ECM of adipocytes, suggesting that inflamed and SAA-enriched HDL are being trapped at the ECM and therefore are blocked from accessing the plasma membrane. Enzymatic digestion of the ECM restored inflamed and SAA-enriched HDL’s abilities to facilitate cholesterol efflux from adipocytes and to inhibit palmitate-induced chemotactic factor expression. Taken together, our results suggest that the presence of SAA on HDL makes the lipoprotein lose its anti-inflammatory effect on adipocytes by blocking accessibility of HDL to the plasma membrane. The interaction between SAA in HDL and the ECM of adipocytes should be considered as a new therapeutic target for improvement of HDL’s anti-inflammatory properties.
Abstract 326: Streptococcal Serum Opacity Factor Product, Cholesteryl Ester Rich Particles, Induce Cholesterol Ester Metabolism and Bile Acid Secretion after Uptake by Human Huh7 Hepatocytes
Authors: Gillard Baiba K , Rodriguez Perla J , Fields David W , Raya Joe L , Lagor William R , Courtney Harry S , Gotto Antonio M , Pownall Henry J
Published: March 2022

Plasma concentrations of HDL-cholesterol (C) negatively correlate with atherosclerotic cardiovascular disease. Nevertheless, current evidence suggests that this correlation is not axiomatic and that some forms of HDL are dysfunctional and atherogenic. Interventions that raise HDL-C have not been uniformly successful suggesting that mechanisms by which HDL-C is increased determine its anti atherogenic potency. Additionally, mice overexpressing the HDL receptor, SR-BI, have lower plasma HDL-C levels and
less
atherosclerosis. Thus, new strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. Serum opacity factor (SOF) disrupts HDL and forms three products, including a cholesteryl ester-rich microemulsion (CERM) containing apo E and the CE of ~400,000 HDL particles. Hepatic CE uptake in Huh7 cells is faster when delivered by CERM than by HDL, and cleared both
in vitro
and
in vivo,
in part, via the LDL-receptor (LDLR). We investigated the therapeutic potential of SOF in promoting RCT by comparing the final RCT steps, hepatic uptake, CE metabolism to cholesterol and bile salts, and secretion, of [
14
C]-CE-HDL, -CERM and [[Unable to Display Character: –]]LDL. Cells were pulse-labeled for 2 h with 20-50 ug/mL HDL protein (6-17 ug/mL [
14
C]-CE), or the CE-equivalent as CERM or LDL, and chased for 0, 2 or 6 h. Cells, pulse media and chase media were analyzed for sterols by beta-counting, TLC and solvent partitioning. [
14
C]CE uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [
14
C]CE hydrolysis were 3.0 ± 0.2, 4.4 ± 0.6 and 5.4 ± 0.7 h respectively for HDL, CERM and LDL-CE. Bile acids in cells after uptake from HDL or CERM were low, <0.2% of total sterol but higher in cells after uptake from LDL, 4.2 ± 2.2 % of total sterol (
p = 0.03
) at the 2 h chase time. The fraction of sterols secreted as bile acids was comparable for all three donor particles. After the 2 h chase, ~40% of the secreted sterols were bile acids, and after 6 h, ~ 50% were bile acids. These ratios were the same for cells treated with
14
C-CE-HDL, CERM or LDL. Thus, the rates of hepatic metabolism of CERM-CE to free cholesterol, to bile acids and secretion are intermediate between those for HDL- and LDL-CE, supporting the therapeutic potential of SOF as a promoter of RCT.
Abstract 332: Coronary Artery Disease-Protective Variant A43T in APOC3 Alters Circulating ApoC-III Levels In vivo
Authors: Khetarpal Sumeet A , Varshini Amritha , Larach Daniel B , Tabita-Martinez Jennifer , McParland James T , McCoy Mary G , Kiss Daniel , Zanoni Paolo , Mucksavage Megan , Millar John S , Cuchel Marina , Lund-Katz Sissel , Phillips Michael C , Kathiresan Sekar , Rader Daniel J
Published: March 2022

Elevated plasma triglycerides (TG) raise risk of coronary artery disease (CAD) independently of low-density lipoprotein cholesterol (LDL-C). Recent human genetics studies have shown that genetically lower TG through loss-of-function (LoF) of APOC3 lowers risk of developing CAD. APOC3 encodes apolipoprotein C-III (ApoC-III), an apolipoprotein on VLDLs and HDLs that inhibits the lipoprotein lipase (LPL) pathway of postprandial TG clearance. The specific molecular mechanisms underlying the reduction of TG and CAD risk by APOC3 LoF mutations are not known. Here, we study the mechanism of LoF for one of the 4 disease-protective APOC3 coding variants, A43T, in humans and rodent models. We recruited human subjects with this variant for deep phenotyping of TG metabolism and show that carriers of this variant have lower plasma ApoC-III in vivo, and possibly increased LPL activity. Using an adeno-associated virus (AAV) vector to express WT vs. mutant human APOC3 in mice, we show that the A43T variant also lowers circulating ApoC-III levels. Additional studies are ongoing to determine the mechanism of lower stability of ApoC-III A43T on TG-rich lipoprotein particles.
Abstract 338: Effects of RVX-208 a Selective Bromodomain Extra-Terminal Protein Inhibitor Beyond Raising ApoA-I/HDL.
Authors: Wong Norman C , Kulikowski Ewelina , Wasiak Sylwia , Attwell Sarah , Halliday Christopher , Gilham Dean , Tsujikawa Laura , Jahagirdar Ravi , Lebioda Kenneth , Johansson Jan , Sweeney Mike
Published: March 2022

The recently completed human trials SUSTAIN and ASSURE (n=499) showed a 55% reduction of major adverse cardiovascular events (MACE) in atherosclerotic patients treated with statins and oral RVX-208 (200 mg/day). RVX-208 increases ApoA-I, the dominant protein of HDL-c, by selectively inhibiting bromodomain extra-terminal proteins (BET) that are epigenetic readers which regulate chromatin function via binding to acetylated histones. Patients given RVX-208 had increased ApoA-I and HDL-c by 10.3% and 7.7%, respectively. Although significant, these increases may account, in part, for lower MACE. Thus, effects of RVX-208 on other biological processes that impacted atherosclerosis were examined. Specifically, biomarkers of vascular inflammation were measured and microarray techniques were used in studying human primary hepatocytes and whole blood. Results in human aortic endothelial cells (HAEC) exposed to RVX-208 showed suppressed VCAM-1 and MCP-1 with an IC50 of 1uM and 10uM, respectively. In U937 macrophages RVX-208 suppressed the pro-inflammatory cytokine IL-6 (IC50 1uM). Primary hepatocytes treated with RVX-208 lead to upregulation of gene sets within processes such as; transcription, translation and chromatin modeling, as previously seen with other BET inhibitors. More importantly RVX-208 down regulated gene sets within the; complement cascade, fibrin clotting, cholesterol and fatty acid synthesis, innate immune system and diabetes mellitus pathways. Any one of these changes, independently or cumulatively, may underlie the observed MACE reduction. Blood treated ex vivo with RVX-208 suppressed gene sets involved in pro-inflammatory signaling of monocytes and neutrophils, monocyte-endothelial cell interactions, extracellular matrix remodeling and Th1/Th2 cell responses. Moreover, RVX-208 affected genes with known roles in atherosclerosis and/or vascular inflammation yielding an overall anti-atherogenic profile. In summary, the orally active selective BET inhibitor RVX-208 significantly lowers MACE in patients with residual CVD risk receiving standard of care therapy including statins. The above results suggest that RVX-208 alters several biological pathways in a cardioprotective manner which may lead to lower MACE.
Abstract 339: Hyperhomocysteinemia Reduces Large HDL Particle and EL Expression via Hypomethylation Related Mechanism in Mice
Authors: Xi Hang , Choi Seungbum , Dipaul James M , Zhang Yuling , Yang Xiaofeng , Wang Hong
Published: March 2022

Hyper-homocysteinemia (HHcy) has been identified as an independent risk factors of cardiovascular diseases (CVD). We previous reported HHcy is associated with reduced HDL-cholesterol and decreased large HDL particle in mouse and human CVD. Here, we investigated underlying mechanism of homocysteine (Hcy)-related HDL biosynthesis and degradation.

We examined protein/gene expression, activities of four HDL-degradation related lipases including endothelial lipase (EL), lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL). Severe HHcy (98.4±22 μM) was established in cystathionine betasynthase-gene mutant mice (
Cbs
+/-
), moderate HHcy (23.5±5 μM) was also developed in
Cbs
+/+
mice, both fed a high methionine (HM) diet for 8-week. Moderate and severe HHcy increased protein levels of EL in aorta (204% and 319%), lung (201% and 264%), and decreased LPL in aorta (98% and 34%), lung (99% and 61%), spleen (98% and 51%). HL and PL protein levels were not changed by HHcy. Activities of overall lipases were increased in moderate and severe HHcy in aorta (19.8±5.2 and 27.5±7.1, arbitrary unit), plasma (64.3±5.5 and 126.5±26.2), adipose (147.0±8.9 and 210.9±38.9) and liver (160.5±41.3 and 276.2±51.3).

Next, we examined tissue levels of Hcy, and its metabolites (SAM, SAH) in heart, lung, brain, spleen, kidney and liver from wild type mice. Tissue expression profile of Hcy degradation and synthesis-related enzymes were established by database mining analysis. We found that mRNA levels of EL, HL, and PL were positively correlated with Hcy and negatively correlated with SAM/SAH ratio, which indicated of methylation status. In contrast, LPL mRNA levels were negatively correlated with Hcy and positively correlated with SAM/SAH ratio. These data suggest that EL, HL, PL gene expression may be induced by Hcy-related hypomethylation.

We identified a large CpG island with 113 CG dinucleotide pairs in human EL promoter region, where four HDL-related single nucleotide polymorphisms (SNPs) was overlapping with CpG containing transcription factor consensus element.

Our study suggests that HHcy inhibits HDL biosynthesis, promotes HDL degradation via EL induction and DNA hypomethylation related mechanisms.
Abstract 341: Role of Cell-to-Cell Communication in Subendothelial Cell Functions
Authors: Orekhov Alexander N , Andreeva Elena R
Published: March 2022

In subendothelial intima of adult human aorta cells form a common three-dimensional network contacting each other with their processes. This network consists mainly of pericyte-like stellate cells and smooth muscle cells. Cellular network disintegrated in early atherosclerotic lesions, in advanced atherosclerotic plaque cells lost all contacts. We believe that integration of smooth muscle cells in the form of a common cellular network is necessary for the normal physiology of the tissue while disintegration of this network play significant role in atherosclerosis. In present study, using immunocytochemistry we have found that connexin43 (Cx43), the major protein of gap junctions, drops in atherosclerotic lesions as compared with uninvolved intima. In atherosclerotic lesions, we observed that the number of Cx43 plaques is lower on lipid-laden cells than on cells free from lipid inclusions. In primary culture, subendothelial intimal cells tend to create multicellular structures in the form of clusters. Cluster creation is accompanied by the formation of gap junctions between cells; the degree of gap junctional communication depends on the density of cells in culture. We showed that such important atherosclerosis-related processes as proliferation (DNA synthesis), fibrosis (protein synthesis) and lipidosis (accumulation of intracellular cholesterol) depend on the degree of cell-to-cell communication. Dependence of proliferation and fibrosis was bell-shaped. We have incubated cells cultured from uninvolved subendothelial intima with various forms of modified LDL causing intracellular cholesterol accumulation. After incubated with modified LDL intercellular communication dropped considerably. This suggests that intracellular lipid accumulation is a reason for decrease of gap junctions. However, latex beads decrease of gap junctions, too. Thus, not cholesterol accumulation but stimulation of phagocytosis reduces intercellular contact communication. We believe that stimulation of phagocytosis accompanied by accumulation of modified LDL in intimal cells is the cause of reduction and disruption of cell-to-cell contacts. Supported by Russian Scientific Foundation (Grant # 14-15-00112).
Abstract 344: Association Between Serum Retinol Binding Protein -4 Level and Coronary Artery Disease: A Meta Analysis
Authors: Agasthi Pradyumna , Aloor Sivakanth , Chenna Avantika , Harris Rachel
Published: March 2022

Background:
Retinol binding protein 4 (RBP-4) is hepatokine/adipokine shown to be strongly associated with traditional and non-traditional (e.g. cytokines) cardiovascular risk factors via impairment of glucose and lipid metabolism and adipose tissue dysfunction. RBP-4 induce endothelial inflammation by stimulating expression of proinflammatory molecules whose effects are mediated via the activation of NADPH oxidase and NF-κB. The association of coronary artery disease (CAD) and RBP4 remains obscure. The objective of this study is to conduct a meta-analysis to evaluate the relationship between serum RBP-4 levels and CAD.

Methods:
We searched MEDLINE, CIINHAL and COCHRANE databases for studies reporting serum RBP-4 levels in the CAD and non CAD study population. We included case controls, cohort and cross-sectional studies. We calculated the weighted standardized mean difference (SMD) in serum RBP-4 levels between the CAD and control groups.

Results:
Our search strategy yielded 52 articles and we included 6 case control and 1 cohort study enrolling 4234 participants. The median age of the CAD group was 62yrs (56 - 65.4) compared to 59.4yrs (42.1 - 65.2) in the control group. The median percentage of female population in the CAD group was 63(IQR 53 -82.5) compared to 62(IQR 35.5 - 72) in the control group. The unweighted median serum RBP-4 levels in the CAD group was 33.4mcg/ml (26.59 - 65.7) compared to 29.8mcg/ml (16.5 - 62.5) in the control group. The SMD of RBP-4 level was 0.14 (95% CI 0.08 - 0.20) p<0.001 comparing those in the CAD group and control group.

Conclusion:
Elevated serum RBP-4 levels is significantly associated with CAD. Current findings warrant the need to further investigate the role of RBP-4 in the development of CAD and its potential to predict incident CAD events.
Abstract 345: A Proteomic Study of Human and Mouse Plaques Supports Involvement of Proteolysis, Extracellular Matrix, and Cytoskeleton in Atherosclerotic Plaque Rupture
Authors: Airhart Nathan , Hong Jie Hu , Vaisar Tomas , Fox Kate , Kohler Ted , Nicosia Roberto , Dichek David
Published: March 2022

Introduction:
Atherosclerotic plaque rupture is the most common trigger of myocardial infarctions and strokes. The molecular mechanisms of plaque rupture are uncertain; accordingly, there are no specific interventions aimed at preventing plaque rupture. We used unbiased “shotgun” proteomics to identify proteins and pathways that are associated with human plaque rupture. We performed parallel proteomic analyses in a mouse model that develops plaque rupture.

Methods:
We used tandem mass spectrometry, performed on tissue extracts, to compare the proteomes of: 1) stable and unstable areas of human carotid plaques (n = 6); and 2) aortic arches of ApoE-null mice with (n = 6) or without (n = 6) macrophage-specific overexpression of urokinase plasminogen activator (uPA). Protein abundance was compared using the PepC statistical program, with correction for multiple comparisons. Gene ontology and pathway enrichment analysis were performed with DAVID.

Results:
Mass spectrometry detected a total of 1161 proteins in human plaque tissue. There were 150 proteins with increased abundance in the ruptured versus stable regions, including proteins involved in coagulation, inflammation, and proteolysis. 339 proteins were significantly decreased in ruptured areas, including smooth muscle-specific and extracellular matrix proteins. We identified 775 total proteins in the mouse aortas. 61 proteins with increased abundance in aortas of uPA-overexpressing mice included cytoskeletal proteins and proteases. 39 proteins with decreased abundance in aortas of uPA-overexpressing mice were predominantly extracellular matrix proteins. Gene ontology and pathway analysis revealed significant alterations in cytoskeletal proteins, extracellular matrix, and cell-adhesion in both human and mouse samples.

Conclusion:
We used shotgun proteomics to define alterations in proteomes of ruptured human plaques and of arterial tissue from a mouse model that develops plaque rupture. Specific proteolytic, cell-adhesion, and cytoskeletal organization networks are altered in unstable human plaques. The mouse model demonstrated remarkable similarity to these findings, supporting its use in investigating the molecular pathogenesis of plaque rupture.
Abstract 346: 9p21.3 Coronary Artery Disease Risk Variants Disrupt Tead Transcription Factor Binding and Tead-dependent Tgfβ Induction of P16 Expression
Authors: Almontashiri Naif , Antoine Darlene , Vilmundarson Ragnar , Chen Hsiao-Huei , Stewart Alexandre
Published: March 2022

Background:
The mechanism by which 9p21.3 locus increases the risk of CAD is elusive. The 9p21.3 risk locus is associated with reduced expression of the cell cycle suppressor genes CDKN2A (p16) and CDKN2B (p15) and increased vascular smooth muscle cell proliferation. We asked whether risk alleles disrupt transcription factor binding that could account for this effect.

Methods and Results:
A bioinformatic screen predicted which of 59 single nucleotide polymorphisms (SNPs) disrupt or create transcription factor binding sites at the 9p21.3 risk locus. Electrophoretic mobility shift assay (EMSA) and luciferase reporter assays examined the binding and functionality of the predicted regulatory sequences. Primary human aortic smooth muscle cells (HAoSMCs) genotyped for the 9p21.3 risk allele were used in the functional studies. HAoSMCs homozygous for the risk allele showed reduced expression of p15 and p16 protein levels and increased cellular proliferation compared to the other genotypes. Although 14 out of the 59 SNPs were predicted to have an effect on transcription factor binding,. only rs10811656 and rs4977757 disrupted functional binding sites for TEAD transcription factors by EMSA and luciferase reporter assays. TEAD3 and TEAD4 over-expression induced p16 but not p15 in HAoSMCs homozygous for the non-risk allele, but not for the risk allele. TGFβ is known to activate p16 expression and also to functionally interact with TEAD3 and TEAD4. In HAoSMCs homozygous for the risk allele, TGFβ failed to induce p16 or to inhibit cell proliferation. Knockdown of TEAD3 blocked TGFβ-induced p16 expression and dual knockdown of TEAD3 and TEAD4 markedly reduced p16 expression in heterozygous HAoSMCs.

Conclusion:
We show for the first time a novel mechanism whereby p16 expression is perturbed by the 9p21.3 risk locus through the disruption of TEAD3 and TEAD4 binding. Furthermore, we show that TGFβ induction of p16 is TEAD3-dependent in HAoSMCs. This mechanism accounts, in part, for the effect of the 9p21.3 risk alleles on CAD. Emerging therapies that activate TEAD factors to inhibit cellular proliferation may also be useful to limit atherosclerosis progression in individuals heterozygous or homozygous for the non-risk allele of 9p21.3.
Abstract 352: Adventitial Stem Cell Progenitors Express Neural Stem Cell Markers
Authors: Fitzpatrick Emma , Kennedy Eimear , Hakimjavadi Roya , Cahill Paul A
Published: March 2022

Many forms of cardiovascular disease (CVD) are charaterised by intimal medial thickening (IMT) due to the formation of a neointimal layer as a result of the accumulation of vascular smooth muscle cells (vSMC) within the vessel wall. There is strong evidence, albeit controversial, that indicates a putative role for both adventitial (Sca1
+
, CD34
+
and Lin
-
) and medial multipotent vascular progenitor stem cell-derived [Sox10, Sox17 and S100β] vSMC in contributing to IMT.

The objective of this study was to characterise adventitial progenitor stem cells (APCs) from aortic explants and compare them to medial MVSCs. APC and MVSC phenotype was determined by immunocytochemistry using antisera specific for adventitial stem cell marker Sca1 and stem cell markers Sox10, Sox 17 and S100β respectively. Differentiated vSMC markers, α-actin, calponin 1 and smooth muscle myosin hevy chain II (Myh11) were used to detect SMCs. Discrete APC populations were positive for neural stem cell markers [Sox 10, Sox17 and S100β] as well as marker α-actin. Notably a significant number of intial cells migrating from the adventitial explant were Sox 10, Sox 17 and S100β positive respectively. Similarly, MVSC from medial explants were positive for neural stem cell markers [Sox 10, Sox17 and S100β] and Sca1 (when grown in maintenance media). There was a distinct absence of smooth muscle myosin heavy chain II filaments (differentiated vSMC markers) in both the APC and MVSC cell populations.

We conclude that APCs and MVSCs may be derived from a similar source.
Abstract 36: Concurrent Ultrasound Diagnosis and Treatment of Thrombosis Using Activated Platelet Targeted Theranostic Microbubbles
Authors: Wang Xiaowei , Gkanatsas Yannik , Palasubramaniam Jathushan , Hohmann Jan David , Hagemeyer Christoph E , Peter Karlheinz
Published: March 2022

Molecular ultrasound imaging is an attractive non-invasive technology widely available for rapid clinical diagnosis. We hypothesized that thrombolytic drugs loaded microbubbles (MBs), which are selectively targeted to activated platelets, will allow high-resolution, real-time imaging of thrombosis, and at the same time offer potent thrombolytic efficacy without bleeding complications, and enable the immediate monitoring of success or failure of thrombolysis.

Our therapeutic agents/imaging particles, targeted theranostic microbubbles (TT-MB), consist of a fusion construct that combines the fibrinolytic drug urokinase, echo-enhancing microbubbles for visualization by ultrasonography, and an activated-platelet-specific single-chain antibody for targeting specifically to thrombi. In the ferric-chloride induced carotid artery thrombosis mouse model, treatment with TT-MB significantly reduced thrombus size after 45 min, while no significant difference was observed in the MB that were targeted but without urokinase (37.09 ± 5.6 vs. 97.16 ± 4.3, mean % change ± SEM, normalized to baseline thrombus size, p<0.001). The same degree of efficient thrombolysis was only achievable using a high dose of urokinase (NS). We also show that the targeting and thus clot-enrichment effect of TT-MBs results in a highly potent fibrinolysis that could only be matched using high doses of non-targeted urokinase. However, the latter is associated with a highly prolonged bleeding time (79.25 ± 6.5 vs. 1079.25 ± 260.7, seconds ± SEM, p<0.001). In contrast, TT-MB does not prolong bleeding time (NS).

In conclusion, activated platelet targeted microbubbles conjugated with recombinant urokinase represent a novel and unique theranostic approach to simultaneously diagnose and treat thrombosis as well as to immediately monitor success or failure of thrombolysis. This unique technology holds promise for major progress towards rapid diagnosis and bleeding-free, potent therapy of the vast number of patients suffering from thrombotic diseases.
Abstract 365: Adiponectin has Greater Association with Left Ventricular Mass Index and Coronary Artery Calcification Score in Lean Compared to Overweight-Obese Subjects: The CARDIA Study
Authors: Ostovaneh Mohammad R , Ambale Venkatesh Bharath , Nwabuo Chike C , Turin Moreira Henrique , Liu Kiang J , Jacobs David R , Schreiner Pamela J , Steffes Michael W , Lima Joao A
Published: March 2022

Objective:
To compare the association of adiponectin with left ventricular mass index (LVMi) and coronary artery calcification (CAC) score in lean and overweight-obese subjects.

Methods:
The CARDIA (Coronary Artery Risk Development in Young Adults) study enrolled black and white adults aged 18 to 30 years in 1985 to 1986. Adiponectin was measured 15 years after baseline. At Years-20 and -25, subjects underwent CT-derived CAC score measurement and M-mode echocardiography, respectively. BMI at Year-15 was categorized into 3 groups (below <25, 25-30, ≥30). Adiponectin and CAC score were log-transformed. Effect modification by BMI for association of adiponectin with LVMi and CAC score was assessed using stratified multivariable linear regression analysis adjusted for demographics and traditional cardiovascular risk factors.

Results:
Mean age of the 2121 included participants was 50.2±3.6 years and 58.7% were females. Adiponectin level was positively associated with LVMi and CAC score (table). In stratified analysis, positive association of adiponectin with LVMi was observed in subjects with BMI <25 (β=3.1, p=0.005) but not in those with BMI of 25-30 (β=-1.5, p=0.14) or ≥30 (β=-0.23, p=0.8). Adiponectin also showed strong association with CAC score in subjects with BMI<25 (β=0.3, p=0.03) and not in those with BMI of 25-30 (β=0.03, p=0.8) or ≥30 (β=0.18, p=0.28).

Conclusion:
Adiponectin at Year 15 has pronounced association with LVMi measured 10-years later and CAC score measured 5 years later in lean subjects but not in overweight-obese subjects. Higher adiponectin levels in young-middle aged adults are associated with subclinical cardiovascular disease later in life.
Abstract 367: Pcsk6 Is a Key Protease Modulating Smooth Muscle Cell Activation in Vascular Remodeling and Plaque Vulnerability
Authors: Perisic Matic Ljubica , Razuvaev Anton , Lengquist Mariette , Sabater-Lleal Maria , Folkersen Lasse , Du Lei , Österholm Cecilia , Roy Joy , Kronqvist Malin , Frebelius Siw , Gonzalez Diez Maria , Hedin Erika , Paulsson-Berne Gabrielle , Hansson Göran K , Odeberg Jacob , Hamsten Anders , Eriksson Per , Hedin Ulf
Published: March 2022

Proprotein convertases (PCSKs) are conserved among species and involved in processing of MMPs and growth factors, but poorly characterised in atherosclerosis. Previously we demonstrated upregulation of PCSK6 in a large cohort of plaques from symptomatic vs. asymptomatic patients. This protease localized to smooth muscle cells (SMCs) and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines in plaques. Here we aimed at elucidating its role in vascular development and disease.

In zebrafish embryos PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. Increased expression of PCSK6 in vascular pathologies was validated by microarrays from carotid plaques vs. undiseased arteries (n=32 patients, p<0.0001), abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By immunohistochemistry, PCSK6 localized mainly to SMCs in the fibrous cap and neo-vessels in plaques, AAA and TAA tissues. Investigation of mouse carotid ligation, rat artery balloon injury and human restenosis tissues with pronounced intimal hyperplasia confirmed induction of PCSK6 in proliferating SMCs. By microarrays following progression of rat carotid injury, PCSK6 was downregulated in early phases defined by inflammatory pathways, while upregulated in later phases with SMCs activation and localized in SMCs. Expression of PCSK6 in this model was positively correlated to PDGFB and IGF1 (Spearman r>0.7, p<0.0001). PCSK6 overexpression in SMCs in vitro increased their migration in a wound-healing assay, especially upon PDGFBB stimulation. By eQTL analyses several polymorphisms in the PCSK6 gene were found to influence its expression in plaques and aneurysm tissue. Among these, rs6598465 showed association with maximum progression of arterial intima-media thickness in high-risk coronary artery disease subjects (n=3400, p=0.037).

We identified a functional link between elevated expression of PCSK6 and vascular remodeling characterized by SMC activation. The present study establishes PCSK6 as one of the key modulators of pathological processes in relation to plaque vulnerability.
Abstract 368: Inhibition of Ido-mediated Tryptophan Metabolism Aggravates Atherosclerosis in Hypercholesterolemic Mice
Authors: Polyzos Konstantinos A , Ovchinnikova Olga , Berg Martin , Baumgartner Roland , Agardh Hanna , Pirault John , Gisterå Anton , Assinger Alice , Laguna Fernandez Andres , Bäck Magnus , Hansson Göran K , Ketelhuth Daniel F
Published: March 2022

Introduction:
Indoleamine 2,3-dioxygenase (IDO), an enzyme catalyzing the first step of tryptophan (Trp) degradation along the kynurenine pathway, has been shown to control autoimmunity and inflammation. Pharmacological inhibition of IDO by 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO, aggravates disease in several chronic inflammation models. Yet, the role of IDO-mediated Trp metabolism in the pathogenesis of atherosclerosis is unknown.

Hypothesis:
We assessed the hypothesis that inhibition of tryptophan metabolism affects the development of atherosclerosis in hypercholesterolemic Apoe-/- mice.

Methods/Results:
Twelve-week old Apoe-/- mice were treated with 1-MT in the drinking water for 8 weeks. Systemic IDO inhibition led to significantly larger atherosclerotic lesions in the aortic root (0.211± 0.024 vs 0.118± 0.025 mm2; mean ± SEM for 1-MT and controls, respectively; n=9-10). IDO inhibition resulted in increased aortic mRNA levels of TNF, MCP-1, and VCAM-1. Immunohistochemical staining of plaques showed increased CD68+ macrophage infiltration and VCAM-1 expression in 1-MT-treated mice. Additionally, IDO inhibition increased VCAM-1 expression in the smooth muscle cells (SMCs) of the tunica media. Moreover, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis can be reversed by exogenous administration of the downstream Trp metabolite 3-hydroxyanthranilic acid (3-HAA). Importantly, 3-HAA supplementation reversed the 1-MT-induced VCAM-1 expression in the medial compartment.

Conclusion:
IDO-mediated Trp metabolism plays a major role in vascular inflammation and atherosclerosis in Apoe-/- mice.
Abstract 370: Dendritic Cells Express Triggering Receptor Expressed on Myeloid Cells-1 and Correlate with Plaque Stability in Symptomatic and Asymptomatic Patients with Carotid Stenosis
Authors: Rai Vikrant , Rao Velidi H , Agrawal Devendra K
Published: March 2022

Aim:
Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory infiltrates. Immune cells, including T-lymphocytes, dendritic cells (DCs) and macrophages recruited from blood stream into atherosclerotic lesions, are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. Upon activation, DCs, macrophages, and neutrophils produce several inflammatory cytokines and growth factors. In atherosclerotic tissue, the number of DCs increases with the progression of the lesion. In this study, we examined the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in plaque DCs and compared between the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis.

Methods and Results:
Surgical specimens of human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. The expression patterns of TREM-1, HLA-DR, and fascin (DC) were determined by immunofluorescence with α-actin as specific marker to identify smooth muscle cells (VSMCs). There was a marked decrease in VSMCs and an increased density of HLA-DR+ cells in the plaques of S than AS patients. Fascin expression to show DCs was undetectable in normal carotid artery specimens but significantly increased in the carotid plaques of S compared to AS. TREM-1 expression was increased in S and localized with HLA-DR and fascin. However, TREM-1 was increased in S plaques containing less number of VSMCs. The mRNA expression of MMP-9, MMP-1 was increased while Col 1 (α1) and Col III (α1) mRNA transcripts were decreased in S plaques.

Conclusions:
Increased expression of TREM-1 in plaque DCs of S compared to AS patients suggests a potential role of TREM-1 in atherosclerotic plaque vulnerability. Collectively, these data demonstrate that selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing atherosclerotic plaques.
Abstract 38: Cyclin-Dependent Kinase Inhibitor 2B Regulates Transforming Growth Factor Beta 1 Mediated Smooth Muscle Cell Recruitment to Ischemic Blood Vessels
Authors: Nanda Vivek , Downing Kelly , Kojima Yoko , Dalman Jessie , DiRenzo Daniel M , Connolly Andrew J , Maegdefessel Lars , Perisic Ljubica , Dandona Sonny , Guo Liang , Davis Harry R , Virmani Renu , Spin Joshua M , Leeper Nicholas J
Published: March 2022

Peripheral artery disease (PAD) is a highly morbid condition affecting nearly 8.5 million Americans. Genome wide association studies (GWAS) have identified genetic variation at the chromosome 9p21 cardiovascular risk locus as an important source of heritable PAD risk. However, it is unknown whether this association is secondary to an increase in atherosclerosis or is the result of a separate angiogenesis-related mechanism.

Quantitative ultrastructural evaluation of human plaque laden vascular samples revealed that carriers of the 9p21 risk allele displayed a significantly increased burden of immature intraplaque microvessels than carriers of the ancestral allele. To determine whether this process occurs under non-atherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking
Cdkn2b;
a candidate gene we previously identified to have reduced expression in human carriers of the 9p21 risk allele. These animals developed advanced hind-limb ischemia and digital auto-amputation, relative to wild-type controls. Interestingly,
in situ
and
in vitro
hypoxic assays identified this defect to be a consequence of pro-angiogenic behavior displayed by
CDKN2B
deficient endothelial cells (EC) and impaired smooth muscle cell (SMC) recruitment to the developing neovessel. Exploratory microarray studies performed to identify the mechanism involved, revealed that TGFβ1 signaling is significantly induced in cultured
CDKN2B
-deficient cells; a finding later confirmed in the vasculature of individuals carrying the 9p21 risk allele. Subsequent molecular signaling studies reveal this increase to be a result of impaired expression of the inhibitory factor,
SMAD-7.
Increased TGFβ1 signaling was ultimately found to manifest the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-‘rheostat’ known to have antagonistic effects on the EC and SMC. Dual knockdown and rescue studies confirmed the reversibility of the proposed mechanism, in vitro.

Taken together these findings suggest that loss of
CDKN2B
may not only promote cardiovascular disease through the development of atherosclerosis, but may also impair TGFβ1 signaling and hypoxic neovessel maturation consequently resulting in PAD.
Abstract 383: A Functional Polymorphism T29C in Transforming Growth Factor Beta 1 Gene is Associated with the Severity of Coronary Artery Disease
Authors: Yang Minjun , Jiang Jianjun , Zhu Min , Zhu Huanhuan , Lu Yifei , Lin Jiangbo , Chen Xiaofeng
Published: March 2022

Background and Objective:
Transforming growth factor beta 1 (TGF-β1) has been previously reported to be involved in pathogenesis of atherosclerosis. However, there are conflicting views about whether TGF-β1 is pro- or anti-atherogenic. The aim of the present study was to establish the contributions of TGF-β1 and its key receptor TGF-β receptor type II (TGFBR2) functional gene polymorphisms to the severity of atherosclerotic coronary artery disease (CAD).

Methods and Results:
Five hundred and three consecutive patients with angiographically documented CAD were prospectively enrolled in this study. Coronary stenosis severity was assessed by the number of coronary narrowed vessels and the Gensini-score. All patients were genotyped for two TGF-β1 polymorphisms [rs1800471 (+T29C), rs1800470 (+915G/C)] and two TGF BR2 polymorphisms [rs67855385 (-3779A/G), rs764522 (-1444C/G)] by the means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and then confirmed by direct sequencing. No association between TGF-β1 rs1800470 (+915G/C), TGF BR2 rs67855385 (-3779A/G) and rs764522 (-1444C/G) gene polymorphisms and the severity of CAD was observed (P>0.05). TGF-β1 rs1800471 (+T29C) polymorphism was also not significantly associated with severity of CAD in female patients. However, in male CAD patients, polymorphisms at rs1800471 (+T29C) were found to be associated with the severity of CAD. There was a significant difference of the T allele frequency in patients with different numbers of vessels involved (three or more vessels: 49.3%, two vessels: 44.1%, single vessel: 35.8%) (P=0.039). Gensini scores were 34.33 ± 2.23, 32.06 ± 4.7877, and 26.90 ± 3.83 in those with the TT, CT, and CC genotype respectively (P=0.045). By using multiple linear regression, adjusting for other risk factors such as gender, blood pressure, diabetes, history of smoking, and serum lipid levels, the T allele was still positively correlated with the Gensini score and number of vessels involved (β=0.034).

Conclusion:
Our findings suggest that TGF-β1 T29C gene polymorphism is associated with severity of CAD in Chinese populations.
Abstract 389: Coronary Endothelial Dysfunction Is Associated With Increased Risk of Bleeding Events
Authors: Prasad Megha , Reriani Martin , Lerman Lilach O , Lerman Amir
Published: March 2022

Background:
Coronary endothelial dysfunction(CED) may reflect loss of vascular integrity and permeability. We aimed to test the hypothesis that CED may be associated with increased risk of bleeding events.

Methods:
Coronary vascular reactivity was evaluated in 444 patients with non-obstructive coronary arteries by administration of intracoronary acetylcholine during angiography. CED was defined as a ≤ 50% increase in coronary blood flow (CBF) from baseline in response to maximal dose of acetylcholine. After a median follow-up of 7 years, patients were assessed for the occurrence of any significant bleeding events.

Results:
Of 444 patients, 70.7% (314) were female. Average age was 52.3 years. Of 252 patients with CED, 7.9% (20) developed bleeding events compared to 2.1% (4 of 192) without CED (p=0.004). Patients who developed bleeding events had a lower percent change in CBF vs. those who did not median: (IQR) 14.1 (-28.1, 98.7) vs. 40.0 (-11.0, 103.5); p=0.03 (Figure 1). CED was significantly associated with the development of bleeding in univariate analysis(p=0.004, relative risk: 3.81 CI, 1.32, 10.96). In multivariate analysis CED was an independent predictor of development of bleeding (p=0.01)

Conclusions:
CED is associated with increased risk of bleeding events. This can be explained by the altered vascular function that is inherent to CED. This study underscores the systemic involvement of the endothelium in vascular disease, and the concept of the ‘vulnerable patient’.