bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 396: A Unique Case of Artefactual and Real HDL Disappearance Due to Isolevuglandins Adducts
Authors: Tavori Hagai , Stringer Jonathan , Agarwal Monica , Lilley Jessica S , Zhang Linda , Davies Sean S , Linton MacRae F , Shapiro Michael D , Fazio Serg , Ormseth Michelle J
Published: March 2019

We report a case of acquired, progressive, and severe HDL cholesterol (HDL-C) deficiency that emerged in an individual with multiple myeloma. An 81 year-old white male presented with extremely low HDL-c (≤5 mg/dL). Between the ages of 69 - 73 years old, his annual lipid panels demonstrated HDL-c >70 mg/dL. At that point, his HDL-C levels progressively decreased until their virtual disappearance over a period of 12 years. During this time, he developed anemia, an IgG-kappa M-spike, and increased plasma cells on bone marrow biopsy, compatible with a diagnosis of multiple myeloma. Review of his medications, medical history, and metabolic status failed to identify a cause for his progressive decine of HDL-C. Parallel analyses of plasma samples using detergent to solubilize and release HDL-associated cholesterol (SYNCHRO LX, Beckman) and separation of non-HDL precipitated by dextran sulfate yielded HDL-C values of 5 mg/dL and 47 mg/dL, respectively. Furthermore, HDL-C levels of 52 mg/dL and 54 mg/dL were confirmed using ultracentrifugation and NMR-based methods, respectively. Serum apoAI concentration was 153 mg/dL, suggesting normal levels of HDL, in contrast with the ultra-low HDL-C reported in one method. The patient’s alpha migrating band on lipoprotein gel electrophoresis also appeared normal, suggesting the presence of roughly normal amounts of HDL. Methodological artifact alone, however, did not explain the significant “real” decrease in HDL-C (from the upper 70’s mg/dL to the 40’s mg/dL) recorded during 12 years of monitoring. Isolevuglandins (IsoLG) are highly reactive ketoaldehydes that interact with lysine residues of proteins and head groups of phosphatidylethanolamine. It was recently proposed that IsoLG adducts on HDL can result in deleterious structural and functional changes in the particle. Interestingly, the patient’s serum showed high titers of autoantibodies to IsoLG-apoAI, but low titers against native apoAI. We further studied the turnover of IsoLG-apoAI and native apoAI in mice. This case of artefactual and real HDL-C disappearance in a patient with multiple myeloma is likely due to paraprotein-related interference of HDL-C assays and increased plasma clearance of modified apoA1.
Abstract 412: Dietary Lectins Cause Coronary Artery Disease via an Autoimmune Endothelial Attack Mediated by Interleukin 16
Authors: Gundry Steven R
Published: March 2019

Interleukin 16 (IL-16) is a chemoattractant released by endothelial and other cells to attract activated T cells. It has previously been shown to have expression in many autoimmune diseases (AI). Based on our previous reports using elevated adiponectin levels of 16 ug/mL to predict lectin sensitivity, and showing an association between lectins and AI causation, we have treated a large pt population with known autoimmune disease (as confirmed by markers). We recently began using the PULS Cardiac Test (GD Biosciences Lab, Irvine, CA) a validated blood test for predicting 5 year risk of acute coronary syndrome (ACS) to examine which measured biomarkers predominate in these patients.

325 consecutive pts aged 25-89 had PULS tested as part of a comprehensive blood panel, which also screens for 14 autoimmune disease markers. The PULS tests ranks 9 biomarkers, including IL-16, MCP-3, Eotaxin, CTACK, sFas, Fas Ligand, HGF for ACS risk, ranking each biomarker from lowest to highest level contributing to risk.

241 pts (74%) were positive for elevated adiponectin, hx of AI, and/or markers for AI. Of these pts, 223 (93%) were positive for IL-16 as the highest PULS risk factor. In contrast, of the other 84 pts without AI or adiponectin elevation, only 5 (6%) had IL-16 recorded as a risk.

We conclude that based on IL-16 testing via PULS, that this provides further evidence to support an autoimmune cause of coronary artery disease at the endothelial level via T cell activation and attraction in patients with gluten and lectin sensitivity or known autoimmune disease. It also explains the known high association of autoimmune disease and ACS.
Abstract 416: Are Good and Evil Polarized? - How Murine Rgs5 knock-out Influences Macrophage Polarization and Aortic Atherosclerosis and Dissection
Authors: Schellinger Isabel N , Loghmani Houra , Hu Niandan , Ginski Greta , Wendt Franziska , Wibbe Nicolina , Raaz Uwe , Theilmeier Gregor
Published: March 2019

Background:
Aortic atherosclerosis and dissection cause significant morbidity and mortality. Although atherosclerosis is associated with aortic dissection, points of interaction are unclear. Regulator-of-G-protein signaling 5 (RGS5) is still insufficiently characterized regarding its role in aortic atherosclerosis and dissection.

Objectives:
We investigated Rgs5-deficiencie’s effects on aortic atherosclerosis and dissection.

Methods:
ApoE
-/-
Rgs5
GFP/GFP
double knockout mice (rraa, n=15) and WT littermates (RRaa, n=17) were fed a cholesterol-rich diet for 14weeks. Plaque size, plaque morphology (composition and a validated vulnerability score based on Stary criteria), macrophage phenotype and aortic dissection were evaluated.

Results:
rraa mice displayed more aortic dissections (5/15) compared to WT (0/17; p<0.05 in Chi
2
-test). Surprisingly, rraa showed significantly smaller (rraa plaque size 190 657 μm
2
± 93184 vs. control 398 279 μm
2
± 153339) and less complicated plaques (5 of 15 mice with >2 vulnerability features) compared to RRaa (14 of 15 with >2 vulnerability features, p<0.01). While relative areas of aSMA and lipids did not differ between genotypes, relative plaque areas positive for collagen and CD45 were increased in rraa (n=15/17, p<0.01) compared to RRaa. Due to smaller plaque sizes were anti-inflammatory macrophages reduced and pro-inflammatory macrophages increased in absolute numbers (p<0.05). Phenotyping of CD45
+
-myeloid cells revealed increased proportions of macrophages staining positive for pro-inflammatory markers (Cd86/Cd45: 24.71 area% ± 3.75 for RRaa; 60.95 area% ± 5.909, for rraa and iNOS/Cd45: 37.22 area% ± 6.37, for RRaa; 73.99 area% ± 3.997 for rraa, p<0.01), while anti-inflammatory markers (Cd163/Cd45 and Dectin/Cd45 double stainings) remained unchanged.

Conclusions:
rraa mice are more prone to aortic dissection despite smaller and less complex plaques with reduced anti-inflammatory but increased numbers of pro-inflammatory macrophages. We hypothesize that pro-inflammatory macrophage polarization along with evasion of anti-inflammatory macrophages in rraa mice could be causative for higher dissection numbers. Our data warrant further studies into the diverging pathomechanistic roles of Rgs5 in aortic dissection and atherosclerosis.
Abstract 417: CXCR4 Distinguishes and Maintains Atheroprotective IgM-producing B-1 cells
Authors: Upadhye Aditi , Srikakulapu Prasad , Perry Heather , Rosean Claire , Nguyen Anh , McSkimming Chantel , Gonen Ayelet , Hendrikx Sabrina , Taylor Angela , Tsimikas Sotirios , Witztum Joseph , McNamara Coleen
Published: March 2019

B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory IgM antibodies recognizing oxidation-specific epitopes, such as MDA-LDL, present in diseased arteries. However, factors mediating B1 IgM production are currently unclear. We evaluated MDA-LDL binding and chemokine receptor expression on human B1 cells in a cohort of subjects undergoing intravascular ultrasound (IVUS) for coronary artery assessment. Results demonstrate that a subset of human B1 cells (~35%) is able to bind MDA-LDL. Moreover, expression of the chemokine receptor CXCR4 on circulating B1 cells associates with increased plasma levels of anti-MDA-LDL IgM antibodies (p=0.0009), and decreased plaque burden in coronary arteries (p=0.0002). Mice with B cell-specific loss of CXCR4 on the atherogenic ApoE
-/-
background (CXCR4
BKO
) demonstrate fewer B1a cells (n=6-8,p<0.0001) and IgM antibody-secreting cells (n=6,p<0.01) in the bone marrow, and reduced plasma IgM levels (n=6-8,p<0.05), relative to littermate controls (CXCR4
WT
). Furthermore, retroviral-mediated overexpression of CXCR4 on B1a cells
in vivo
is associated with increased B1a localization to the bone marrow (p<0.01) and increased circulating levels of anti-MDA-LDL IgM antibodies (p<0.05). To determine the atheroprotective role of CXCR4 on the B1a cell subset, we adoptively transferred CXCR4
WT
or CXCR4
BKO
B1a cells into lymphocyte-deficient Rag1
-/-
ApoE
-/-
mice. After 16 weeks of Western diet feeding, recipients given CXCR4
BKO
B1a cells demonstrate reduced plasma IgM levels (n=7,p<0.001), and fewer donor B1a cells in the bone marrow and spleen (n=7,p<0.05) compared to recipients given CXCR4
WT
B1a cells. Intriguingly, B1a transfer reduces plasma cholesterol levels in mice regardless of CXCR4 expression (n=7,p<0.05). However, CXCR4 further strengthens the atheroprotective ability of B1a cells, as recipients given CXCR4
WT
B1a cells have reduced aortic lesion area compared to PBS controls (n=7,p<0.01) while recipients given CXCR4
BKO
B1a cells did not attain the same level of protection. Overall, these data suggest that CXCR4 is an important regulator of IgM production and B1a-mediated atheroprotection.
Abstract 430: Development of a Gut-microbe Targeted Non-lethal Therapeutic to Inhibit Thrombosis Potential Without Enhanced Bleeding
Authors: Roberts Adam , Gu Xiaodong , Buffa Jennifer A , Zhu Weifei , Hazen Stanley L
Published: March 2019

Recent clinical and animal studies show trimethylamine-N-oxide (TMAO), a microbiota-dependent metabolite derived from trimethylamine (TMA)-containing nutrients abundant in a Western diet, both promotes platelet hyperresponsiveness and thrombosis potential and is predictive of incident atherothrombotic event risks in large-scale clinical investigations. Utilizing a mechanism-based inhibitor approach targeting a major gut microbial enzyme (CutC/D) in the TMAO meta-organismal pathway, we developed choline analogues with cryptic reactive moieties that upon interaction with the microbial TMA generating enzyme, display potent, time-dependent and irreversible inhibition without affecting commensal microbial viability. In animal models, a single oral dose of mechanism-based microbial choline TMA lyase inhibitor significantly reduced (> 95% inhibition, p < 0.0001) plasma TMAO levels over a sustained period of time (up to 3 days), while limiting systemic exposure in the host. This was achieved by the selective accumulation of inhibitor at the site of action, within intestinal microbes, to millimolar levels, a concentration over a million-fold higher than needed for therapeutic effect. In animal models, inhibition of microbial TMA generation was shown to rescue diet-induced platelet hyperresponsiveness and enhanced thrombosis potential, both without increasing enhanced bleeding risk and observable toxicity. The present studies reveal mechanism-based inhibition of microbial TMA lyase activity as a therapeutic target for reducing thrombosis potential, a critical adverse complication in heart disease. They also suggest mechanism-based inhibitors designed to concomitantly accumulate within the gut microbe may serve as a generalizable approach for the selective targeting of microbial catabolic enzymes linked to host diseases, while limiting systemic exposure in the host.
Abstract 440: Targeting LOX-1 Scavenger Receptor and oxLDL Uptake With Affimers
Authors: Cuthbert Gary A , DeSiqueira Jonathan , Abdul-Zani Izma , Tomlinson Darren C , Ponnambalam Sreenivasan , Homer-Vanniasinkam Shervanthi
Published: March 2019

Introduction:
Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1, SREC-1, OLR1) is a class E scavenger receptor which binds oxidized low density lipoprotein (oxLDL) particles implicated in promoting atherosclerosis. Affimers are non-antibody synthetic protein-based scaffolds that enable recognition of a wide range of molecular targets.

Aims:
To investigate the ability of Affimers to bind LOX-1 and modulate oxLDL accumulation and effects on cellular responses.

Methods:
Recombinant soluble human LOX-1 was screened with an Affimer library and LOX-1-specific Affimers were isolated and characterized. Human embryonic kidney (HEK293T) cells inducibly expressing human LOX-1 were incubated with labeled oxLDL and Affimers to modulate LOX-1 and oxLDL uptake and trafficking. Furthermore, Affimer-mediated effects on oxLDL-regulated metabolism and apoptosis was evaluated.

Results:
Five Affimers specific for human LOX-1 were identified. Fluorescent labelled Affimers demonstrate specific binding to LOX-1 expressing HEK293T cells and modulate LOX-1 and oxLDL trafficking and accumulation in endosomes and lysosomes. LOX-1-specific Affimers also show effects in modulating oxLDL-mediated effects in specific cellular responses.

Conclusion:
LOX-1-specific Affimers are a new class of synthetic molecules that have therapeutic potential in targeting LOX-1, a key mediator of inflammation and arterial dysfunction in cardiovascular disease.

Membrane specific binding is demonstrated only in induced LOX-1 expressing cells (LOX+).

Increasing concentration of affimer prior to incubation with DiI labelled oxLDL.
Abstract 449: Dimerization of Sortilin Regulates its Trafficking to Extracellular Vesicles and Leads to Secretion of Soluble Sortilin Homodimers, a Potential Novel Biomarker
Authors: Itoh Shinsuke , Mizuno Ken , Aikawa Masanori , Aikawa Elena
Published: March 2019

Objective:
Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretary or endocytic compartments of cells, is found in both EVs and cells. In many diseases, including cardiovascular calcification and cancer, sortilin expression levels are atypically high. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Therefore, suppressing trafficking of sortilin to EVs may act as a novel therapy against EV-associated diseases. However, the precise mechanisms of sortilin trafficking remain to be determined. In this study, we hypothesized that dimerization of sortilin regulates its trafficking to EVs.

Approach and Results:
We found that sortilin forms a homodimer with an intermolecular disulfide bond (IDB) at Cysteine783 (Cys783) residue. Palmitoylation and the IDB formation compete for Cys783. Formation of the IDB led to trafficking of sortilin to EVs by preventing palmitoylation, which further promoted sortilin trafficking to the Golgi apparatus. Moreover, addition of sortilin-derived propeptide decreased sortilin homodimers within EVs, suggesting that the propeptide binding suppresses sortilin trafficking to EVs through prevention of its dimerization. We also found that soluble sortilin secreted by cells overexpressing full-length sortilin forms homodimers with IDBs using various commercially available antibodies against sortilin, thus providing an opportunity for diagnosis of EV-associated diseases by detection of sortilin homodimers in patients.

Conclusions:
Sortilin is transported to EVs via the formation of homodimers with an IDB, which is endogenously regulated by its own propeptide. Therefore, inhibiting sortilin dimerization may provide a novel therapy against EV-associated diseases, including cardiovascular calcification and cancer. In addition, soluble sortilin homodimer with IDBs could be detected and used as a novel diagnostic biomarker for EV-associated diseases.
Abstract 462: Low Carbohydrate High Protein (LCHP) Diets Are Atherogenic by Modulating Macrophage Mtorc1-mitophagy Signaling to Accelerate Apoptosis and Plaque Complexity
Authors: Zhang Xiangyu
Published: March 2019

Low carbohydrate high protein (LCHP) diets which are commonly utilized for weight loss have been reported to raise cardiovascular risk. However, the mechanisms underlying this risk are unknown. We first confirm that LCHP diets increase atherosclerotic lesion area with a particular rise in plaque complexity. Mass spectrometry analysis reveals protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques with concomitant stimulation of macrophage mTORC1 signaling. This activation is causal as LCHP diet-induced plaque progression is abrogated in macrophage-specific Raptor-null mice. A prominent effect of certain amino acids, such as leucine, on macrophages is the synergistic exacerbation of macrophage apoptosis induced by atherogenic lipids. More specifically, leucine stimulates mTORC1-dependent inhibition of autophagy which hinders the mitophagic removal of dysfunctional and pro-apoptotic mitochondria. This amino acid-mTORC1-autophagy signaling axis is supported in vivo by 1) the absence of reduced atherosclerosis in mice dually deficient in macrophage mTORC1 and autophagy (Raptor/ATG5-/-) and 2) the absence of increased atherosclerosis in macrophage autophagy-deficient (ATG5-/-) mice fed a LCHP diet. Our data provide the first mechanistic details of the deleterious effects of high protein diets on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of dietary protein.
Abstract 467: Lipoproteins and Cardiovacular Disease Risk
Authors: Ikezaki Hiroaki , Fisher Virginia A , Liu Ching-ti , Cupples L. Adrienne , Schaefer Ernst J
Published: March 2019

Objectives:
Elevated serum levels of low density lipoprotein cholesterol (LDL-C), small dense LDL-C (sdLDL-C), remnant lipoprotein cholesterol (RLP-C), and lipoprotein (a) or Lp(a) and low high density lipoprotein cholesterol (HDL-C) have all been associated with increased cardiovascular disease (CVD) risk. Our goal was to assess direct measurements of these lipoproteins as compared to standard risk factors (age, gender, hypertension, hypertension treatment, diabetes, smoking, total cholesterol, and HDL-C) in the Framingham Offspring Study (FOS).

Methods:
Stored frozen plasma samples (-80 degrees C) obtained after an overnight fast from male and female participants free of CVD at cycle 6 of FOS were used (n=3,147, mean age 58 years, with 677 (21.5%) developing CVD over 16 years. Total cholesterol, HDL-C, direct LDL-C, sdLDL-C, remnant lipoprotein cholesterol (RLP-C), Lp(a), and high sensitivity C reactive protein (hsCRP) were measured by standardized automated analysis. Large buoyant LDL-C (lbLDL-C) (direct LDL-C - sdLDL-C) and calculated LDL-C (total cholesterol-HDL-C - TG/5) were also calculated.

Results:
For all CVD risk on univariate analysis significant factors with p values in parentheses in order of significance were: age (8.1x10
-41
), hypertension (3.2x10
-23
), HDL-C (4.2x10
-16
), sdLDL-C (4.2x10
-14
), hypertension treatment (1.5x10
-14
), gender (1.7x10
-10
), diabetes (5.1x10
-9
), direct LDL-C (8.2x10
-9
), body mass index (9.2x10
-7
), calculated LDL-C (6.2X10
-6
), RLP-C (8.0 X 10
-4
) cholesterol medication (1.8 X 10
-4
), total cholesterol (0.00081) smoking (0.0024), hsCRP (0.005), and Lp(a) (0.024). On multivariate analysis sdLDL-C, direct LDL-C, hsCRP, and Lp(a) were still significant using the model including all standard risk factors. All four parameters significantly improved the model C statistic and net risk reclassification.

Conclusions:
Our data indicate that for CVD risk prediction in FOS direct LDL-C is significantly better than calculated LDL-C, that HDL-C, sdLDL-C, direct LDL-C, RLP-C, and Lp(a) are all atherogenic, and that after standard risk factors, sdLDL-C, direct LDL-C, Lp(a), and hsCRP all add significant information.
Abstract 480: Injury-Activated Vascular Cells Share a Common Photonic Fingerprint with Stem Cell-Derived Myogenic Progeny Following Interrogation Using a Lab-on-a-Disc (Load) Platform
Authors: Molony Claire , King Damien , Julius Lourdes A , Fitzpatrick Emma , Di Luca Mariana , Casey Gillian , Hakimjavadi Roya , Burtenshaw Denise , Healy Killian , Canning Daniel , Kernan David , Llobera Andreu , Liu Weimin , Morrow David , Redmond Eileen M , Ducree Jens , Cahill Paul A
Published: March 2019

The accumulation of vascular smooth muscle (SMC)-like cells within the intima contributes significantly to intimal medial thickening (IMT) and vascular remodeling typical of arteriosclerotic disease. Light has emerged as a powerful tool to interrogate cells label-free and facilitates discriminant observations both
in vitro
and
in vivo
. The auto-fluorescence (AF) profile of individual cells isolated from arteriosclerotic vessels, captured on V-cup array and interrogated across five wavelengths using a novel Lab-on-a-Disc platform, was significantly increased at the 565 ± 20nm wavelength concomitant with a reduction in Myh11 expression, when compared to differentiated vascular smooth muscle (SMC) cells from control vessels. In vitro, TGF-β1 promoted myogenic differentiation of murine bone-marrow derived Sca1
+
/CD44
+
mesenchymal stem cells (MSC) and murine Sca1
+
C3H 10T1/2 cells concomitant with enrichment of the specific SMC epigenetic histone mark, H3K4me2 at the Myh11 promoter, Myh11 promoter transactivation and increased SMC differentiation marker mRNA and protein expression. Myogenic differentiation resulted in a significant increase in the AF intensity across 565 ± 20nm wavelength, an effect not observed for TGF-β1 treated RAMOS human B lymphocytes but mimicked by Notch activation of resident Sca1
+
multipotent vascular stem cells (MVSCs) with Jagged1 and inhibited following elastin and collagen III depletion, respectively. Moreover, the temporal increase in the AF intensity at 565 ± 20nm wavelength during myogenic differentiation was similar to the AF profile of dissociated cells from arteriosclerotic vessels at this same wavelength. These data suggest that an AF photonic fingerprint of stem cell-derived myogenic progeny
in vitro
mimics that of vascular cells
ex vivo
following injury.
Abstract 487: Myocardial Infarction is Associated with Changes in Innate Immunity that Drive Breast Cancer Progression
Authors: Koelwyn Graeme J
Published: March 2019

Cardiovascular (CV) disease and cancer-specific mortality compete as the leading causes of mortality in women with early-stage breast cancer. Following treatment for early-stage disease, breast cancer patients are 5 times more likely to develop CV complications such as myocardial infarction (MI) compared to age-matched controls, due to the ‘cardiotoxic’ effects of anti-cancer therapies on CV tissues (e.g. chemotherapy, radiation). As CV disease and cancer progression share many common risk factors and etiologies, we hypothesized that CV disease may also influence cancer progression. To investigate this potential cross-disease communication, we developed a mouse model using the left anterior descending (LAD) coronary artery permanent ligation technique of MI in the E0771 orthotopic breast cancer model. LAD ligation significantly increased tumor growth over 20 days compared to sham surgery controls (SHAM) (p<0.001). This was associated with an altered tumor microenvironment and tumor immune cell landscape, most notably increased proportions of CD11b+Ly6C+Ly6G- myeloid cells in LAD vs SHAM (p<0.01). RNA and ATAC sequencing of these cells from the bone marrow, circulation, and tumor showed distinct functional and chromatin-based changes following LAD ligation, including increased expression of genes associated with cell proliferation, mitochondria and lipid transport, and enrichment of transcription factor binding motifs within differentially accessible regions, including PU.1, Ets1, Runx1 and Cebp. Using monocyte adoptive transfer experiments, we also show that recruitment of bone marrow monocytes to the tumor microenvironment was increased in LAD vs SHAM mice. Collectively, these findings suggest that MI can accelerate breast cancer progression and provide insight into molecular mechanisms driving innate immune adaptations that promote cross-disease communication.
Abstract 491: Production of Fatty Acid Binding Protein-4 by Macrophages in Atherosclerotic Human Coronary Arteries
Authors: Prathipati Priyanka , Walton Brian
Published: March 2019

Background:
Fatty acid binding protein-4 (FABP4) is a lipid chaperon that carries fatty acids and other lipophilic substances to specific compartments in the cell. It is mostly expressed in macrophages and adipocytes and is associated with insulin resistance, obesity and atherosclerosis. Previous studies have shown the role of FABP4 in high risk phenotypes of atherosclerotic plaques featuring inflammation and vulnerability of the plaques in cardiovascular diseases. Hence we hypothesis that FABP4 is produced by macrophages in the human coronary atherosclerosis and might be involved in aggravation of the disease.

Materials and methods:
Human coronary arteries which were formalin fixed and paraffin embedded were used with IRB review. Immunohistochemistry was performed on these samples to analyze the expression of FABP4 and co-localization of CD68 (macrophage marker) in the atherosclerotic plaques of these human coronary arteries.

Results:
Atherosclerotic plaques in the human coronary arteries expressed both FABP4 and CD68.

Conclusion:
Macrophages in the plaques of human plaques secrete FABP4 which is evident from the co-existence of both molecules in the atherosclerotic coronary arteries.
Abstract 510: Extracellular cAMP as a Novel Therapeutic Strategy in Pulmonary Arterial Hypertension
Authors: Sassi Yassine , Bueno-Beti Carlos , Jones Carly , Bonnet Guillaume , Neves Susana , Hadri Lahouaria , Hajjar Roger J
Published: March 2019

Cyclic adenosine monophosphate (cAMP) is a cellular second messenger that mediates physiological effects of G protein-coupled receptors. Yet, the last two decades of research have shown that there is more to the role of cAMP than ever expected from this small molecule. Stimulated cells transport cAMP outside the cells, a process that is mediated by the multidrug resistance-associated proteins (MRPs). The present study investigated the role of extracellular cAMP in the lung and asked whether it may act on pulmonary vasculature remodeling. By employing a fluorescence resonance energy transfer (FRET)-based sensor, we found that extracellular cAMP activates intracellular cAMP formation in human pulmonary artery smooth muscle cells (hPASMC) and endothelial cells (hPAEC). Extracellular cAMP, via binding of its metabolite adenosine to the type A2 receptor, reduced hPASMC and hPAEC growth by controlling the PKA/CREB pathway. To test for a role of extracellular cAMP in the pulmonary vasculature, we chose rat-monocrotaline and mouse-hypoxia as
in vivo
models for pulmonary arterial hypertension (PAH). Rats treated with monocrotaline developed PAH with increased pulmonary artery pressures (PAP) and right ventricular (RV) hypertrophy. Extracellular cAMP infusion significantly prevented and reversed these structural changes. Extracellular cAMP-treated rats displayed lower RV systolic pressure and Fulton index, as well as decreased PAP. In line with these observations, we found infused cAMP to potently repress RV hypertrophy, RV systolic pressure, perivascular lung fibrosis and pulmonary arteries remodeling in mice exposed to Sugen5416 and chronic hypoxia. Together, our results assign extracellular cAMP a potent regulatory role in pulmonary vascular cells, and suggest targeting the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PAH.
Abstract 511: Mef2 Transcription Factors are Essential Regulators of Endothelial Morphology and Functions
Authors: Schwarz John , Lu Yao Wei
Published: March 2019

Background:
Laminar shear stress on the endothelium promotes atheroprotecive gene expression. In vitro experiments support a model of laminar shear stress activating the Mef2 transcription factors that in turn induce transcription of Klf2 and Klf4, which regulate many anti-inflammatory and anti-thrombotic genes. However, this model has not been tested in vivo. Three Mef2 transcription factors (Mef2a, -c, and -d) are expressed in the endothelium. To understand their functions and test this model, we generated mice with inducible, endothelial-specific deletions of Mef2c, Mef2a/c, and Mef2a/c/d.

Methods & Results:
We previously reported that endothelial Mef2c inhibits the formation of endothelial actin stress fibers and migration of smooth muscle cells across the internal elastic lamina into the intima. Combined deletion of endothelial Mef2a/c produced a similar phenotype. Neither of these deletions affected survival or altered the levels of Klf2 or Klf4. However, combined deletions of Mef2a/c/d led to death 10-14 days after induction. Pulmonary hemorrhage was consistently observed as was variable amounts in other organs. En face imaging of the aorta and vena cava revealed a substantial increase in endothelial cell density and proliferation. The aortic endothelium displayed extensive actin stress fibers but the overall organization was not substantially changed. However, the vena cava was disorganized with endothelial cell aggregation. Transcriptome analysis showed ≥ 2-fold alterations in expression of 894 genes, with many important for endothelial function. Notably, Klf2 and Klf4 were both decreased in Mef2a/c/d deficient aortic endothelium to a similar extent as the Mef2s. This is consistent with the phenotypic similarity of Klf2/4 and Mef2a/c/d deletions. Comparison of genes altered by Klf2/4 and Mef2a/c/d deletions revealed that 37% of Mef2-dependent genes are also Klf2/4-dependent.

Conclusions:
Together, these data support a model in which Mef2 transcription factors redundantly regulate endothelial expression of Klf2 and Klf4 in response to shear stress to promote atheroprotective gene expression. They further regulate expression of many genes important for endothelial function independently of Klf2/4.
Abstract 525: Contraction of Blood Clots Influences Their Susceptibility to Fibrinolysis
Authors: Tutwiler Valerie , Peshkova Alina D , Le Minh Giang , Zaitsev Sergei , Litvinov Rustem I , Weisel John W
Published: March 2019

The success of endogenous and therapeutic fibrinolysis is governed in part by the structure of the fibrin clot. An understudied feature that potentially affects fibrinolysis is the degree of platelet-driven shrinkage, i.e. contraction, of blood clots and thrombi. The aim of this work was to examine the effect of clot contraction on the rate of naturally occurring fibrinolysis from within and external fibrinolysis to simulate therapeutic thrombolysis. Clot contraction, which was initatied with 2mM CaCl
2
and 1U/ml thrombin, was impaired by ~75% by inhibiting platelet non-muscle myosin IIa activity (blebbistatin), actin polymerization (latrunculin A), and platelet-fibin(ogen) binding (abciximab). Internal fibrinolysis measured using dynamic optical tracking of clot size as a function of time occurred 2X faster in contracted clots compared to uncontracted clots in the presence of 75ng/ml tPA. In direct contrast, the dynamic release of radioactive fibrin degradation products as a measure of external fibrinolysis was 4X faster in uncontracted clots when 75 ng/ml tPA was added to the surface of preformed clots. This difference in the susceptibility of contracted and uncontracted clots to internal versus external lysis suggests that the lysis rate is dominated by the interplay of clot permeability to fibrinolytic enzymes as well as the spatial proximity of the fibrin fibers themselves. These results have implications for understanding clot stability in patients with thrombotic disorders and improving clot lysis by impairing platelet-mediated clot contraction.
Abstract 538: Procedural Characteristics and Outcomes in Women with Hyperuricemia after Percutaneous Coronary Intervention
Authors: Prasad Megha , Rihal Charanjit , Lerman Lilach , Lennon Ryan , Lerman Amir
Published: March 2019

Background:
Serum uric acid may serve as a marker of cardiovascular events and outcomes in men, but further studies are required to determine its role in women. We aimed to determine differences in procedural characteristics and outcomes in women undergoing percutaneous coronary intervention(PCI).

Methods:
We retrospectively analyzed the Mayo Clinic Cardiovascular Catheterization Laboratory database, and included patients undergoing PCI from 1/1/2000 to 12/31/2007. There were 495 women who underwent PCI in this time period. Patients were divided into normal uric acid (n=240) and high uric acid (n=255) groups, and high uric acid was defined as >6.0 mg/dL. Patients were followed for adverse outcomes for a median of 47.1 (IQR 23.8, 62.8) months.

Results:
Mean age was 66.6
+
12.7 years in the normal group vs. 70.5
+
11.2 years in the elevated uric acid group(p=0.01). Hypertension and diabetes were more prevalent in the elevated uric acid group (p<0.01). BMI was significantly higher in the elevated uric acid group (p=0.001). There was a higher prevalence of calcified lesions among women with elevated uric acid when compared to those with normal uric acid levels (47% vs. 30% p<0.001). There was significantly lower TIMI=3 flow postprocedurally in those with elevated uric acid levels when compared to those with normal uric acid levels (91 % vs. 97%, p=0.003). After adjusting for potential confounders including age, hyperlipidemia, hypertension, BMI, diabetes, and chronic kidney disease, elevated uric acid was associated with increased likelihood of death (HR 1.8, p=0.01) and the composite endpoint of death or subsequent myocardial infarction (HR 1.5, p=0.03).

Discussion:
Uric acid may be an independent determinant of procedural characteristics and a predictor of outcomes after percutaneous intervention. Further studies are required to better understand the effect of uric acid on cardiovascular events in women.
Abstract 547: High-density Lipoprotein Particle Diversity From Selective Combinatorial Assembly of Particle Constituents Result in a Dynamic Particle Population
Authors: Fried Steven G , Altmann Scott W
Published: March 2019

Introduction:
Mass spectrometry is starting to elucidate the extent of lipoprotein diversity. It is believed that HDL is comprised of proteins encoded by >100 genes and ~200 distinct lipid species in an undetermined number of combinations. Multiple proteoforms derived from a single gene contribute to particle complexity, confound proteome identification and makes differentiation of distinct subpopulations a challenge. Subtle proteoform variants can account for alterations in particle physicochemical properties and critical functions. Refining high-definition constituent identification within a structured atlas may reveal mechanism-based pathophysiology of a variety of diseases.

Hypothesis:
The ongoing identification of HDL constituents necessitates an effort to catalogue, categorize, map and relate entities in a framework that organizes this knowledge in a form of testable observations, explanations and predictions. Defining constituent interactions will advance knowledge and guide inquiry that enables HDL research, clinical diagnostics and subsequent therapeutic strategies.

Methods:
Informatic analysis of the literature produced an HDL proteome reference set. Proteome specific analysis of gene isoforms, proteolytic products, amino acid modifications and cSNPs were used to prepare a proteoform index. Theoretical tryptic peptide maps were generated and referenced to the PeptideAtlas and available published mass spec peptide lists.

Results:
Over 380 non-immunoglobulin proteins were identified in high-density lipoprotein fractions from human samples. A consensus-based scoring produced an “unofficial” list of 122 genes. UniProtKB was used to expand an index of potential proteoforms and derive a theoretical peptide mass library used to map empirically defined peptide spectral lists.

Conclusions:
Utilizing a reductionism model to HDL measurement has proven insufficient and undermines the application of Precision Medicine for CAD. Understanding the health benefits of HDL requires clinical diagnostics that captures particle diversity and population heterogeneity. Constructing a conceptual framework that unifies a constituent map is the initial step to resolving their relational context and functional consequences.
Abstract 554: FoxO Transcription Factors are Required for Hepatic HDL-Cholesterol Clearance
Authors: Lee Samuel X
Published: March 2019

Insulin resistance and type 2 diabetes are associated with low levels of high-density lipoprotein-cholesterol (HDL-C). The insulin-repressible FoxO transcription factors are potential mediators of insulin’s effect on HDL-C. FoxOs mediate a substantial portion of insulin-regulated transcription, and poor FoxO repression is thought to contribute to the excessive glucose production in diabetes. In this work, we show that mice with liver-specific triple FoxO knockout (L-FoxO1,3,4), which are known to have reduced hepatic glucose production, also have increased HDL-C. This was associated with decreased expression of HDL-C clearance factors, scavenger receptor class B type I (SR-BI) and hepatic lipase, and defective selective uptake of HDL-cholesteryl ester by the liver. The phenotype could be rescued by re-expression of SR-BI. These findings demonstrate that hepatic FoxOs are required for cholesterol homeostasis and HDL-mediated reverse cholesterol transport to the liver.
Abstract 576: LDL-Trafficked Small RNAs Promote Atherosclerosis through TLR Signaling in Macrophages
Authors: Allen Ryan M , Zhao Shilin , Ramirez-Solano Marisol A , Zhu Wanying , Richmond Bradley W , Blackwell Timothy , Sheng Quanhu , Vickers Kasey C
Published: March 2019

Atherosclerosis is a chronic inflammatory disease, and despite resounding success in lipid management to reduce cardiovascular events, most heart attacks in the US occur in patients with clinically normal LDL-C levels. The next generation of therapies for cardiovascular disease (CVD) will include inflammation-based targets; however, systemic immune suppression has limitations. Thus, the critical barrier for successful targeting of inflammation in CVD is the identification of vascular ligands that drive immune cell activation. Previously, we demonstrated that low-density lipoproteins (LDL) transport microRNAs. Here, we report that LDL traffic a wide-diversity of small non-coding RNAs (sRNA) in circulation, many of which are derived from exogenous species, e.g. bacteria and fungi. Moreover, using high-throughput sRNA sequencing, we found that LDL-sRNA signatures are influenced primarily by environmental microbiota, as opposed to commensal bacteria in the gut microbiome. Using a mouse model of compromised mucosal immunity, we identified a process by which LDL accumulates bacterial sRNAs. Next, we demonstrated that native LDL potently stimulated cytokine release from macrophages through activation of single-stranded RNA (ssRNA)-sensing toll-like receptor (TLR) signaling, which was blunted by partial silencing of ssRNA-sensing TLRs in macrophages. Most importantly, preliminary results in mice suggest that targeting ssRNA-sensing TLRs is a potential strategy to suppress atherosclerosis. Taken together, we put forth a novel paradigm in which, LDL scavenge microbial sRNAs to promote clearance during health, yet serve as TLR-ligands that propagate macrophage inflammation in hypercholesterolemia, which likely contributes to the underlying inflammation in the pathogenesis of CVD.
Abstract 587: Inflammatory Status and Macrophage Infiltration in Relation to Insulin Resistance and Dyslipidemia among Morbid Obese Subjects
Authors: Chu Nain-Feng , Chang Tian-Jong
Published: March 2019

Inflammatory response and macrophage infiltration in adipocyte is associated with the development of insulin resistance status and obesity-related co-morbidities. This study is to examine the inflammatory status and macrophage infiltration in adipose tissues and their relationship with insulin resistance (IR) and dyslipidemia among morbidly obese patients.We collected blood and omental visceral fat samples from 35 morbidly obese subjects (BMI ≧ 35 kg/m
2
) and used HOMA-IR (≧2.4) and prescription history to divide the subjects into normal and insulin resistant sub-groups. We used enzyme-linked immunosorbent assay (ELISA) to determine the advanced glycation end products (AGEs) and plasma growth arrest-specific protein 6 (GAS-6) expression levels. We used turbidimetric method to determine the concentration of high sensitivity C-reactive protein (hs-CRP) level. For adipocyte, the cluster of differentiation 68 (CD68) antibody, as macrophages infiltration of adipocyte, was determined using immunohistochemistry (IHC) methods.The mean BMI among these 35 morbidly obese subjects (20 male, 15 female) is 41.2 kg/m
2
. There are 15 subjects with normal HOMA and 20 subjects with insulin resistant HOMA. Among the IR group, the fasting glucose, insulin, triglyceride, GAS-6, CD68, levels are significantly higher than those of the normal HOMA group. CD68 level are positively correlated with fasting glucose, insulin, cholesterol and HOMA-IR level and negatively correlated with HDL-C level. After adjusting for age, gender and BMI, every increase of 1ng/mL AGEs, 1ng/mL GAS-6, 1mg/L hs-CRP, and 1μm
2
CD68 is associated with increasing of HOMA-IR by 1.323 units (p=0.030), 0.118 unit (p=0.681), 2.787 units (p=0.059), and 0.0002 unit (p=0.019), respectively.Inflammation response, especially CD68 level, is associated with the insulin resistance and dyslipidemia among the morbidly obese subjects. Additionally, it is imperative to examine the interactions between inflammation response, macrophage infiltration and inflammatory status on insulin resistance and obesity-related co-morbidities.
Abstract 589: Amygdala Activity Assessed by 18-FDG PET/CT Associates with Coronary Plaque Burden Quantified by Coronary Computed Tomography Angiography in Psoriasis
Authors: Goyal Aditya , Chaturvedi Abhishek , Elnabawi Youssef , Chung Jonathan , Dey Amit , Aberra Tsion , Belur Agastya , Rivers Joshua , Genovese Leonard , Groenendyk Jacob , Lerman Joseph , Joshi Aditya , Rodante Justin , Playford Martin , Bluemke David , Chen Marcus , Gelfand Joel , Mehta Nehal
Published: March 2019

Introduction:
Psoriasis (PSO), a chronic inflammatory disease associated with increased prevalence of both stress and coronary artery disease, provides a model to study the role of perceived stress in cardiovascular (CV) disease. While stress perception quantified as resting amygdala activity (RAA) is associated with CV events, its association psoriasis severity as well subclinical CV indices is not well known. We thus, sought to look into the relationship between RAA, psoriasis area severity index (PASI) score and Non-calcified burden (NCB) in PSO.

Methods:
150 consecutive PSO patients and 58 healthy volunteers (HV) underwent 18-FDG PET/CT and CCTA scans (Toshiba, 320-detector row) to quantify RAA and NCB respectively. Skin disease severity was assessed as PASI score, RAA as target-to-background ratio (Osirix) and NCB using prior published methods (QAngio). The relationship between them was analyzed using multivariable regressions (STATA 12).

Results:
Despite older age, both PSO & HV were at low CV risk by traditional risk score. RAA was higher in PSO compared to HV (Table 1). Moreover, PASI score correlated positively with RAA in treatment naïve psoriasis patients (β=0.22, p=0.03). Furthermore, RAA associated with NCB (β=0.27, p<0.001), which persisted independent of traditional risk factors (β=0.19, p<0.001). Similar relationship was not observed in HV.

Conclusion:
In conclusion, PSO skin disease severity relates to amygdalar activity by 18-FDG PET/CT suggesting the role of chronic inflammatory disease like PSO in triggering perceived psychological stress. Moreover, there is a direct association between amygdalar activity and coronary plaque burden suggesting a role for perceived stress in subclinical CV disease in these patients. Larger studies are needed to confirm these findings.
Abstract 596: Disruption of Cholesterol Metabolism and Substrate Stiffness Coordinate to Regulate Cellular Biomechanics and Induce Cytoskeletal Remodeling in Vascular Smooth Muscle Cells
Authors: Sanyour Hanna , Li Na , RIckel Alex , Hong Zhongkui
Published: March 2019

Cholesterol is the main culprit contributing to the development of atherosclerosis by inducing the endothelial dysfunction, triggering the inflammatory response in the blood vessel wall, and deposition in the foam cell at the atherosclerotic plaque. However, growing evidence suggests that the role cholesterol plays during the process of atherogenesis is not only triggering the inflammation, but also plays an important role as a regulator of cellular mechanics, cell spreading, and cell migration, which is the critical step in atherogenesis. In addition, the mechanical environment of the VSMCs in blood vessel undergoes progressive stiffening along with aging, which may potentially affect the development of atherosclerosis. In this study, our main hypothesis is that membrane cholesterol affects the VSMCs migration during the progression of atherosclerosis by regulating the integrin- and cadherin-mediated cell adhesion and this effect highly depends on the mechanical properties of extracellular matrix (ECM). VSMCs used were enzymatically isolated from the descending thoracic aorta of male Sprague-Dawley rats. Cell mechanics was measured using an atomic force microscope (AFM). F-actin cytoskeleton was visualized with confocal microscopy and AFM imaging. 1 μM fluvastatin was used to reduce the endogenous cellular cholesterol. Our results showed that fluvastatin treatment dramatically decreased cell E-modulus, but, significantly increased the average rupture force of collagen I-, fibronectin-, and N-cadherin-mediated cell adhesion. In addition, the confocal microscopy and AFM imaging studies showed that the fluvastatin treatment significantly disrupted the actin stress fiber organization. Interestingly, the effect of fluvastatin on the biomechanics and the architecture of cytoskeleton of VSMCs decreased on the soft substrate. These results indicate that cholesterol and ECM stiffness play the roles synergistically in biomechanics, cytoskeleton architecture, and thus in migration of VSMCs. The knowledge that we obtained in this project will lead to a novel therapeutic strategy to prevent the VSMCs migration and then formation of atherosclerotic plaque by regulating VSMCs biomechanics.