bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 242: Functional Characterization of a Cis-eQTL Locus for Plasma Cholesterol Using CRISPR/Cas Genome Editing in Human Pluripotent Stem Cells
Authors: Raghavan Avanthi , Peters Derek , Kuperwasser Nicolas , Melnikov Alexandre , Rogov Peter , Wang Li , Zhang Xiaolan , Mikkelsen Tarjei , Musunuru Kiran 
Published: July 2021

Genome-wide association studies (GWASs) have discovered many novel genetic loci linked to serum lipid levels, yet pinpointing the causal variants remains a major challenge. Expression quantitative trait locus (eQTL) analysis of liver and adipose indicates that many lipid-associated variants influence gene expression in a cis-regulatory manner. To identify causal variants at 57 lipid-associated eQTL loci, we performed a massively parallel reporter assay (MPRA) in which the genomic region surrounding each candidate SNP was coupled to a reporter gene with a unique barcode identifier. This construct pool was transfected into 3T3-L1 adipocytes, and the copy number of each expressed barcode determined by RNAseq. Variants were prioritized according to allele-specific regulatory activity.
The top-ranked variant, rs2277862, is associated with total cholesterol and is flanked by the ERGIC3, CPNE1, and CEP250 genes, none of which have previous connections to lipid metabolism. We hypothesized that rs2277862 is causal for the eQTL at the 20q11 locus, and that it lies within a transcriptional regulatory site to influence local gene expression. We identified human pluripotent stem cell (hPSC) lines with different genotypes at rs2277862 and, using CRISPR/Cas genome-editing technology, either deleted the putative regulatory site encompassing rs2277862 or knocked in the alternate SNP allele. Analysis of undifferentiated rs2277862 homozygous major deletion mutants revealed diminished expression of ERGIC3, CPNE1, and CEP250; conversely, heterozygous deletion mutants had increased expression of the same three genes. Our results suggest that in hPSCs the major and minor alleles of rs2277862 have opposing effects on gene expression.
In ongoing work, we are validating these findings in differentiated adipocytes and seek to identify the factor(s) that binds the putative regulatory site to mediate allele-specific gene expression. We anticipate that this work will offer fresh insight into the mechanisms by which GWAS-implicated SNPs influence expression of causal genes for lipid metabolism and, more broadly, establish a novel methodology for finding causal variants for any phenotype of interest.
Abstract 258: A Pharmacological Approach to Promote Shear Stress-Dependent Anti-inflammatory Mechanisms in Arteries
Authors: Luong Le , Duckles Hayley , Schenkel Torsten , Arnold Nadine , Gsell Willy , Lungu Angela , Spencer Tim , Hose Rodney , Halliday Ian , Evans Paul 
Published: July 2021

Atherosclerosis is influenced by blood flow whichS exert wall shear stress (WSS) on endothelial cells (EC). Low oscillatory WSS promotes atherosclerosis by inducing inflammatory molecules (e.g. VCAM-1), whereas high uniform WSS induces anti-inflammatory genes (e.g. eNOS). Ivabradine decreases heart rate by inhibiting the If current in the sinus node, without affecting blood pressure or cardiac contractility. Besides cardio-protection, ivabradine protects arteries from atherosclerosis via unknown mechanisms. We hypothesised that ivabradine reduces arterial inflammation by increasing blood flow-induced WSS.
Mice (C57BL/6, male, aged 10 weeks) were treated with ivabradine for 1 week (162 mg/L in drinking water) or remained untreated. WSS was mapped at atherosusceptible (inner curvature) and protected (outer curvature) sites of the aorta by defining the geometry (CT angiography; Siemens) and flow characteristics (ultrasound; Visualsonics). Segmentation and smoothing were performed to reconstruct the volume of interest (Amira, VMTK) and a tetrahedral mesh with prismatic boundary layer was generated (IcemCFD). Fluid behavior was modeled over the cardiac cycle using Fluent 14.5 (ANSYS). Computational fluid dynamics revealed that WSS at the inner curvature of the aorta had lower magnitude and higher oscillatory index compared to the outer curvature. Ivabradine treatment reduced heart rate by 20% (p<0.05; n=5 per group) and altered WSS at the inner curvature by increasing magnitude and reducing oscillatory index. En face immunostaining and confocal microscopy demonstrated that treatment with ivabradine led to reduced VCAM-1 expression (mean fluorescence intensities (MFIs): 710.6 (untreated) vs 4.4 (treated); p<0.01; n=5 per group) and enhanced eNOS expression (MFIs: 3.48 (untreated) and 6.25 (treated); p=0.05; n=5 per group) at the inner curvature.
We conclude that ivabradine treatment altered hemodynamics in the murine aorta by increasing the magnitude and uniformity of shear stress. These changes were accompanied by induction of a mechanosensitive protective gene (eNOS) and suppression of inflammatory VCAM-1. Thus ivabradine may protect arteries by altering local mechanical conditions to trigger an anti-inflammatory response.
Abstract 342: The Antiapoptotic Function of APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 in Endothelial Cells Is Linked to Changes in Transcription Factor Activities and Independent of Its DNA Repair Domain
Authors: Dyballa-Rukes Nadine , Rabanter Lothar , Goy Christine , Altschmied Joachim , Haendeler Judith 
Published: July 2021

APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has both DNA repair and redox regulatory activity. Interestingly, APEX1 is also able to modulate transcription factor reduction, a prerequisite for DNA binding, independent of its intrinsic redox activity, probably by recruiting other reducing molecules like Thioredoxin-1 (TXN). APEX1 has been shown to exert anti-apoptotic properties in different cell lines and is localized in the cytosol and in the nucleus. So far it has not been studied whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and whether this is dependent on its nuclear or cytosolic localization.
After cloning human APEX1 and overexpression in EC we demonstrated that it inhibits not only basal but also H2O2-induced apoptosis. To further characterize the anti-apoptotic properties of APEX1 we generated mutants that lacked the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) (APEX1ΔN 21-318). We first analyzed the localization of the mutants by immunostaining. APEX1wt and APEX1ΔC 1-127 showed a predominant nuclear staining whereas APEX1ΔN 21-318 was mainly localized in the cytosol. With respect to the anti-apoptotic properties, only the mutant with intact NLS and redox domain APEX1ΔC 1-127 prevented apoptosis both under basal conditions as well as after H2O2 treatment similar to APEX1wt.
The association between nuclear localization of APEX1wt and its anti-apoptotic properties in EC suggests that this function is linked to changes in transcription factor activities. It is known that the p65 subunit of the transcription factor complex NF-kB is activated by pro-apoptotic stimuli in EC. Using a specific DNA binding assay we could show that overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant had no effect.
In conclusion, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity, is independent of its DNA repair domain and is at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyze the impact of APEX1 on other transcription factors in concert with TXN.
Abstract 485: Effect of Bone Marrow Transplantation on Aortic Inflammatory Disease and Accelerated Atherosclerosis
Authors: Douglas Gillian , Patel Jyoti , Hale Ashley , Channon Keith M 
Published: July 2021

In ApoE-/- mice Ang II infusion leads to vascular inflammation resulting in the formation of aortic aneurysms and accelerated atherosclerosis. In order to elucidate the role of hemopoetic cells in these pathologies, bone marrow transplant (BMT) has been utilized. However, if BMT is used, it is critical that we fully understand the effect of BMT on aortic inflammatory disease and atherosclerosis. In this study we used Ang II to investigate the effects of bone marrow transplant on aortic inflammatory disease and accelerated atherosclerosis.
ApoE-/- mice (12wk age) were either lethally irradiated (2x 5Gy) and reconstituted with ApoE-/- bone marrow (1x10-6 cells) or non-irradiated. . BMT mice and non-irradiated littermates were treated with Ang II (0.8mg/kg/day) to induce aortic inflammation and accelerate atherosclerosis. After 14 days of treatment mice were culled and the aorta and aortic root harvested. AngII infusion in non-irradiate mice resulted in a 33% incidence in aortic aneurysm, however, in BMT mice no aneurysms were observed. Ang II infusion lead to an increase in atherosclerosis with a significant increase in inflammatory cell infiltration observed. Irradiation resulted in a significant decrease in Ang II-induced accelerated atherosclerosis (0.049±0.003 vs. 0.037±0.016; P<0.05; non-irradiated vs. irradiated) and a significant reduction in macrophage infiltration (0.018±0.003 vs. 0.007±0.002; P<0.05; non-irradiated vs. irradiated). However, in order to achieve a similar incidence of aortic aneurysms in BMT mice as observed in non-BMT mice treated with 0.8 mg/kg/day Ang II, the dose of Ang II had to be increased to 3mg/kg/day. A similar result was observed in atherosclerosis studies, with a dose of 3mg/kg/day required to induce comparable levels of macrophage infiltration and plaque size to those in non-BMT mice treated with a dose of 0.8mg/kg/day.
In conclusion, BMT resulted in a significant decrease in AngII-induced accelerated atherosclerosis and a reduction in Ang II-mediated aortic inflammation. Interestingly, BMT mice had a significant reduction in inflammatory cell infiltration compared with non-irradiated littermates indicating that the reduction maybe due to an alteration in inflammatory cell recruitment after BMT.
Abstract 513: Endothelial Barrier Function is Maintained by the RNA-Binding Protein Quaking
Authors: de Bruin Ruben G , Dane Martijn J , Lee DaeHyun , van der Veer Eric P , Roeten Marko K , Schmidt Iris , Sundararajah Janani , Bijkerk Roel , de Boer Hetty C , Rabelink Ton J , van Zonneveld Anton-Jan , van Gils Janine M 
Published: July 2021

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is maintained largely by adherens junctions. Remarkably, little is known about their regulation at the post–transcriptional level. We found that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar, atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression.
Subsequently, we aimed to investigate the role of QKI in EC vascular integrity. Interestingly, the mRNA of VE-cadherin, the prime adhesion protein in EC adherens junctions, contains a conserved QKI-binding site. We identified that the targeted reduction of QKI results in a reduction of VE-cadherin expression and organization at the cell periphery. These studies revealed a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA-immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and induce transcript translation (4 fold ± 0.4; p<0.01), respectively. This effect was perturbed when the QKI-binding site was mutated. These results suggest that QKI acts to enhance barrier function. Overexpression of QKI markedly increased the capacity to form a high resistance endothelial monolayer (1.3 fold ± 0.96), while silencing of QKI markedly impaired EC barrier function (0.65 fold ± 0.13; p<0.05). To validate in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p<0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to WT littermates.
In conclusion, we show that QKI functions as a critical regulator of VE-cadherin, and the modulation of QKI expression affects endothelial monolayer integrity. These studies provide novel insight into the importance of post-transcriptional regulation on endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.
Abstract 518: Differential Effects of Alcohol and Its Primary Metabolite Acetaldehyde on Vascular Smooth Muscle Cell Notch Signaling and Growth
Authors: Hatch Ekaterina , Morrow David , Hamm Katie , Cahill Paul A , Redmond Eileen M 
Published: July 2021

Moderate consumption of alcohol (ethanol/EtOH) is a negative risk factor for atherosclerosis while binge drinking and chronic alcohol abuse is associated with increased cardiovascular disease and mortality. As the balance between the levels of EtOH itself and its primary metabolite, acetaldehyde, is likely different in these two drinking groups, we assessed the hypothesis that differential effects of EtOH and acetaldehyde on vascular smooth muscle cell (SMC) Notch signaling and growth may be key in mediating the effects of various drinking patterns on cardiovascular disease.
Human coronary artery smooth muscle cells (HCASMC) were treated (24 h) with either EtOH (25 mM) or acetaldehyde (10 or 25 μM). We confirmed our previously reported inhibitory effects of EtOH on Notch signaling and subsequently on the growth of HCASMC. In contrast, acetaldehyde treatment stimulated HCASMC proliferation (determined by cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling as determined by increased Notch target gene (HRT 1-3) mRNA levels. Interaction of a ligand (e.g., Dll4) with the Notch receptor initiates proteolytic cleavage by γ-secretase, resulting in the release of the active Notch-intracellular domain (NICD). A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Dll4-stimulated γ-secretase activity in a solubilized membrane fraction from HCASMC (similar to the effect of the control inhibitor, DAPT), but no effect of acetaldehyde treatment. Similarly, EtOH, but not acetaldehyde, affected the association and distribution of the γ-secretase catalytic subunit, presenilin-1, with lipid rafts as determined by dual fluorescent labeling and confocal microscopic visualization (Alexa 488-conjugated cholera toxin B subunit for lipid rafts, and Alexa 594-conjugated anti-presenilin-1 antibody).
In conclusion, EtOH and acetaldehyde have opposite effects on SMC Notch signaling and growth. These differential effects of alcohol and its metabolite may be relevant in mediating the opposing effects of different drinking patterns (e.g., daily moderate vs weekend binge) on cardiovascular disease.
Abstract 529: Cbfa1 (Runx2) is a Mediator of Aortic Stiffness and Hypertension in a Murine Model of Diabetes Mellitus Type 2
Authors: Raaz Uwe , Schellinger Isabel N , Adam Matti , Nakagami Futoshi , Deng Alicia , Emrich Fabian C , Jagger Ann , Kayama Yosuke , Findley Clarence , Spin Joshua M , Tsao Philip S 
Published: July 2021

Accelerated arterial stiffening is a complication of diabetes mellitus and associated with the development of hypertension. Arterial stiffening results from extensive extracellular matrix remodeling (elastin breakdown, collagen accumulation). The osteogenic transcription factor Cbfa1 (Runx2) has been identified as a mediator of aortic calcification and regulator of matrix protein expression. However, its impact on aortic stiffness is unclear.
This study was designed to elucidate the temporal relation between aortic stiffening and the development of arterial hypertension in a murine model of diabetes mellitus type 2. Moreover we aimed to investigate the role of Cbfa1 as potential mediator diabetic aortic stiffening.
Serial ex vivo mechanical testing of the thoracic aorta and volume-pressure recording (VPR) based tail-cuff blood pressure measurements revealed that aortic stiffening precedes blood (pulse) pressure elevations in diabetic db/db mice. Vascular stiffening was accompanied by increased medial collagen deposition Picrosirius Red staining). qRT-PCR and immunohistochemistry revealed enhanced expression of Cbfa1 and target genes (Col1a1, Col1a2, FN1, Spp1) in db/db mice compared to controls. Moreover, overexpression of Cbfa1 in vascular smooth muscle (Cbfa1-smTg mice) results in increased medial collagen deposition, aortic stiffness and augmented pulse pressure. Interestingly, Cbfa1-smTg mice did not exhibit enhanced vascular calcification (by von Kossa and Alizarin Red staining).
In conclusion we demonstrated that aortic stiffening precedes the onset of hypertension in db/db mice and identified Cbfa1 as mediator of aortic stiffening - presumably via pro-fibrotic mechanisms.
Abstract 568: Biomarkers of Hemostasis and Thrombosis in Hemophilia A and B Patients
Authors: Chen Zhu , Wu Weizhen , Ge Lan , Ichetovkin Marina , Desai Kunal , Wang Weixun , Xu Yiming , Ogletree Martin L , Seiffert Dietmar A 
Published: July 2021

The defective hemostatic process in hemophiliacs overlaps with the underlying physiology contributing to bleeding risk in anticoagulated patients. Hemophiliacs may also develop compensatory changes in fibrinolysis, platelet reactivity, vascular inflammation, and remodeling secondarily. We therefore explored markers for these processes in hemophilia A and B as part of our larger effort to develop translational biomarkers to guide antithrombotic therapy and minimize bleeding risk.
Plasmas from hemophilia A patients (11 severe; 1 moderate) and hemophilia B patients (3 severe; 3 mild or moderate), sampled after a treatment washout period, and 7 normal subjects were procured. Markers/assays pursued include: coagulation (F1+2, FPA, TAT, D-dimer), fibrinolysis (PAP, PAI-1, tPA), platelet activation (soluble P-selectin (sP-sel)), vascular inflammation (MCP-1), angiogenesis (VEGF), PT, aPTT, and thrombin generation assay (TGA). Consistent with aPTT prolongation and changes in TGA, both hemophilia groups displayed numerically lower than normal levels of all 4 coagulation markers, with hemophilia A reaching statistical significance for F1+2 and TAT. Hemophilia A also exhibited a significant decrease in PAP and a numerical increase in PAI-1 compared to normal. VEGF and MCP-1 exhibited no appreciable difference. In the combined analysis for hemophilia A and B (“A+B”), PAP displayed a strong positive correlation with F1+2 and a strong negative correlation with PAI-1; A+B exhibited a statistically significant reduction in F1+2, FPA, and TAT, and a non-significant increase in sP-sel and non-significant decrease in PAP, compared to normal.
In conclusion, hemophilia A and B plasma samples had significantly reduced markers of in vivo thrombin generation, thrombin activity, and coagulation, and trends evidencing increased platelet activation and decreased fibrinolysis, possibly as compensation. Lack of signal in vascular inflammation or angiogenesis could be due to the (near optimal) hemostatic therapies. Our results illustrate the potential value of biomarkers of in vivo coagulation, fibrinolysis, and platelet activation in translational research on antithrombotic therapies for both their efficacy and bleeding risk.
Abstract 585: Endotoxin Contamination of Apolipoprotein A1: Effect on Macrophage Proliferation---a Cautionary Tale
Authors: Jin Xueting , Xu Qing , Champion Keith , Kruth Howard S 
Published: July 2021

Incubation of human macrophages with ApoA1 (purified from human plasma) for 1-2 days resulted in decreased cell density that was independent of cell toxicity. Although ApoA1 treatment caused macrophages to become more rounded and clustered, with some macrophages detaching, this did not account for the decrease in cell density. Thus, we considered that ApoA1 decreased macrophage cell proliferation. Using a BrdU incorporation cell proliferation assay, we demonstrated that ApoA1 (20 ug/ml) resulted in a >90% inhibition in macrophage proliferation. Using a recombinant version of ApoA1, we found that the results were dependent on the expression system: ApoA1 expressed in human cells did not inhibit proliferation but ApoA1 expressed in E. coli did. Human plasma-derived ApoA2 did not inhibit proliferation. All apolipoproteins were obtained commercially.
We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation >90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus protein-bound endotoxin, suggested endotoxin mediated inhibition of macrophage proliferation. While human cell-derived recombinant ApoA1 and human plasma-derived ApoA1 showed similar levels of total endotoxin contamination (levels that were sufficient to inhibit macrophage proliferation), the levels of free endotoxin in the human cell-derived recombinant ApoA1 were more than 1000-fold less than levels in the human plasma-derived ApoA1. This finding helps explain why the recombinant ApoA1 did not inhibit macrophage proliferation, but the plasma-derived ApoA1 did.

Conclusion:
Our findings show that apparent apolipoprotein-mediated cell effects (or effects of any other biological products) can be significantly influenced by endotoxin contamination, especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages. Lastly, endotoxin potently inhibits macrophage proliferation, which may be a significant pathogenic mechanism by which bacteria escape the immune system.