bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 131: Identification Of A Molecule To Modulate A Platelet Olfactory Receptor And Limit Thrombosis
Authors: Aggarwal Anu , Wang Nancy , Prasad Josyula V , Jennings Courtney , Godwin Matthew , Bhandari Rohan , Ali Mariya , Stauffer Shaun , Cameron Scott J
Published: October 2023

Background and objective:
Olfactory Receptors (ORs) are G protein-coupled receptors (GPCR) we recently discovered in human platelets. OR2L13 activation by an olfactory ligand Levo-Carvone (L-Carvone) limits platelet activation. Our goal was to better characterize the pharmacology of OR2L13 ligands through structural modifications of L-Carvone and by using an 8K bioactive non-olfactory ligand library and deciphering their role as anti-thrombotic.

Methods and Results:
With an IC
50
of 58 μM, 2-tert-Butyl-5-Methylphenol was the best odorant for inhibiting ADP-mediated platelet activation (Fig. 1). 169 molecular hits >25% threshold in an OR2L13-expressiong reporter cell line for cyclic AMP (cAMP) production were found. By counter screen (cell line without OR2L13 receptor), 12 ligands were identified for OR2L13 and 6 (CCF0054500, CCF0054432, CCF0053070, CCF0052249, CCF0051970, CCF0058399) made endogenous cAMP. Healthy platelet rich plasma (PRP) (n=7) was incubated with OR2L13 ligand for 30 minutes, then subjected to Light Transmission Aggregometry (LTA). 3/6 ligands (CCF0054500, CCF0052249 and CCF0058399) inhibited platelet aggregation through the P2Y
12
receptor and so were further validated by FACS using washed healthy platelets (n=8, attenuation of agonist-activated platelets surface P-selectin expression) for the following receptors: ADP, TRAP-6, U46619, and CRP. CCF0054500 suppressed platelet activation and aggregation through P2Y
12,
PAR-1, Thromboxane and GPVI receptors. Whole blood (n=5) traversing collagen-coated capillaries reduced thrombosis when compared to vehicle under arterial sheer stress with CCF0054500.

Conclusions:
Like neurons, platelets have a fully functional OR signal transduction cascade. Platelet OR2L13 operates as a negative regulator of platelet reactivity and thrombosis in response to odorant and non-odorant surface agonists (biochemical activation) and under shear stress (biomechanical activation).
Abstract 132: Mechanism By Which Coagulation Factor XI Regulates Endothelial Cell Permeability And Barrier Function
In Vitro
And
In Vivo
Authors: Puy Cristina , Lorentz Christina U , Tucker Erik , Lupu Florea , Gailani David , McCarty Owen J
Published: October 2023

Introduction:
Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE (sVE)-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI) plays a regulatory role in inflammation, and while a relationship between FXI activity and VE-cadherin expression levels has been observed, the mechanism by which this process is mediated is unknown.

Hypothesis:
Activated FXI (FXIa) induces VE-cadherin proteolysis.

Methods:
Human umbilical vein ECs were stimulated with FXIa (30 and 5 nM) for six hours. Western blot analysis was used to study VE-cadherin proteolysis, disintegrin and metalloproteinase 10 (ADAM10) expression, FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions, and cell signaling events. SVE-cadherin in baboons challenged with
Staphylococcus aureus
was detected by ELISA.

Results:
FXIa (5 nM) generated a VE-cadherin fragment and increased ECs permeability; the serine protease inhibitor, PPACK, and an inhibitor of ADAM10 reversed these processes. FXIa induced the expression of active ADAM10 on ECs. The receptor-associated protein (RAP) blocked FXIa-induced VE-cadherin proteolysis by preventing FXIa interaction with PAI-1 and VLDLR on ECs. FXIa-VLDLR interaction induced Dab1 and Src kinases phosphorylation. FXIa activated the VEGFR2-PLCgamma1-ERK1/2 signaling pathway. The ERK1/2 inhibitor, LY3214996, and the Src kinases inhibitor, PP2, inhibited VE-cadherin proteolysis by FXIa. PLCgamma1 phosphorylation was also prevented with PP2. The VEGFR2 kinase inhibitor, SU1498, prevented the cleavage of VE-cadherin by FXIa; in contrast, a VEGF blocking antibody had no effect on FXIa-induced VE-cadherin cleavage. Finally, the inhibition of FXI activation in a baboon model of sepsis significantly decreased the levels of sVE-cadherin.

Conclusion:
The complex formation of FXIa with PAI-1 and VLDLR promoted Src kinase phosphorylation. Src activation triggered the VEGFR2-PLCgamma1-ERK1/2 signaling pathway, which induced the trafficking of active ADAM10 to the EC surfaces inducing the cleavage of VE-cadherin. FXI may be a druggable target to protect EC barrier function in inflammatory diseases.
Abstract 133: Engineering Plasminogen Activator Inhibitor-1 Variants With Novel Protease Inhibitor Activity
Authors: Haynes Laura M , Holding Matthew L , Trost Elise , DiGiovanni Hannah , Siemieniak David , Ginsburg David
Published: October 2023

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor, or SERPIN, protein superfamily that canonically inhibits the plasminogen activators, tissue plasminogen activator (tPA) and urokinase (uPA). PAI-1, however, also exhibits less potent inhibitory activity against other proteases, including thrombin, coagulation factor XIIa (FXIIa), and transmembrane serine protease-2 (TMPRSS2). To identify amino acid substitutions in PAI-1 that lead to enhanced inhibitory activity toward non-canonical protease targets, we constructed a phage-display PAI-1 mutational library using error-prone PCR that contained 89% of all possible single amino acid substitutions. The library was screened for PAI-1 variants that efficiently inhibit FXIIa, thrombin, or TMPRSS2 by incubating the phage-displayed PAI-1 with the target protease, immunoprecipitating the covalently linked protease-PAI-1 complexes, and isolating the phage DNA that encodes for the mutant PAI-1 fusion protein. The input and selected libraries were subjected to high-throughput DNA sequencing at a depth of >400 million PAI-1 sequencing reads. Missense mutations that both enhance and diminish PAI-1’s inhibitory activity toward a given non-canonical serine protease are identified throughout the PAI-1 structure—highlighting the importance of exosite-driven interactions and allosteric networks in the determination of SERPIN specificity. Detailed examination of the unique set of amino acid substitutions associated with improved PAI-1 reactivity toward a specific protease compared to uPA demonstrated a clear correlation with the evolutionary relationship between the given target protease and uPA. As FXIIa is the most closely related of the coagulation/fibrinolytic serine proteases to uPA, it was selected as a target to further engineer a potent and specific inhibitor on the PAI-1 scaffold. For this purpose, we are utilizing unbiased directed evolution coupled with functional characterization of the PAI-1 variants identified with either the most robust FXIIa inhibitory response or those that exhibit high FXIIa selectivity. The development of a novel FXIIa inhibitor may be of potential value as a novel therapeutic for thrombotic disorders.
Abstract 134: A Single-cell Atlas Of Human Skeletal Muscle Tissue In Chronic Limb-threatening Ischemia
Authors: Peters Derek T , Xu Yueyuan , Otto James , Dai Qunsheng , Fei Kaileen , Kontos Christopher D , Southerland Kevin , Diao Yarui
Published: October 2023

Introduction:
Chronic limb-threatening ischemia (CLTI) represents end-stage peripheral arterial disease (PAD) and is a significant cause of limb amputation and mortality. Evidence supports the importance of skeletal muscle responses in the pathobiology of CLTI. Skeletal muscle regeneration depends on the coordinated activity of muscle stem cells (MuSCs) and an array of non-myogenic cells, including immune cells and fibroadipogenic progenitors (FAPs). The mechanisms underlying failed muscle regeneration in the CLTI limb are unknown. Single-cell RNA sequencing (scRNA-seq) facilitates the deconvolution of multicellular tissues in homeostatic and pathologic conditions. Herein, we profiled the transcriptomes of 135,203 cells from lower extremity skeletal muscle of PAD patients to construct a cellular atlas of CLTI.

Methods:
We recruited 12 PAD patients undergoing major limb amputation for CLTI. We obtained proximal (non-ischemic) and distal (ischemic) muscle samples from each patient and used scRNA-seq to profile the transcriptomes of 1,700 – 20,000 cells per sample.

Results:
We observed 10 distinct cellular populations in the CLTI limb. The predominant cell types were MuSCs, FAPs, and immune cells. There was significant transcriptional heterogeneity in each population; we defined a range of MuSC, FAP, and macrophage sub-populations and identified specific FAPs and macrophages associated with tissue ischemia. Using CellChat analysis, we found differential cell-cell signaling probabilities in ischemic vs. non-ischemic muscle. In ischemic muscle, FAPs are the primary source of outgoing signals, while macrophages are the primary receiver.

Conclusions:
Our single-cell atlas highlights the transcriptional heterogeneity of cellular responses in the CLTI limb. This atlas will serve as a resource for future studies of the molecular underpinnings of CLTI, advance our understanding of its pathogenesis, and support the development of novel therapies.
Abstract 145: CRISPRi-Perturb-seq In Endothelial Cells Links Atherosclerosis Risk Loci To Novel
Klf2/4
Regulatory Genes
Authors: Gupta Rajat M , Schnitzler Gavin R , Kang Helen , Angom Ramcharan Singh , Fang Shi , Lee-Kim Vivian , Zheng Tony , Ma Rosa , Barry Aurelie , Zhou Ronghao , Mukhopadhyay Debabrata , Engreitz Jesse
Published: October 2023

Introduction:
Genome-wide association studies (GWAS) have discovered >300 associations for CAD. Few loci are functionally characterized and may represent new mechanisms of disease.

Hypothesis:
Can an unbiased transcriptional screen for the >2000 candidate GWAS genes in endothelial cells (ECs) identify causal genes and disease-relevant pathways at CAD GWAS loci.

Methods:
We applied CRISPRi-Perturb-seq to knock down expression of genes within 500 Kb of coronary artery disease GWAS loci (2,300 genes in total) and measure their transcriptomic effects using single-cell RNA-seq. Validation of candidate causal genes was conducted in primary human ECs and zebrafish.

Results:
We identified 50 programs of co-expressed genes, which represent core cellular pathways (i.e. ribosome biogenesis) and 13 EC-specific pathways such as flow response and angiogenesis. The EC-specific programs show the greatest contribution to CAD heritability. 5 EC-specific programs have the greatest number of GWAS candidate genes. We find that 43 of 306 CAD GWAS signals converge onto these 5 gene programs, and all are regulated by genes in the cerebral cavernous malformations (CCM) pathway—which coordinates KLF2/4 transcriptional responses in endothelial cells, but has not been linked to CAD risk. The strongest regulator of these programs is
TLNRD1
, which we show is a new CAD gene and novel regulator of the CCM pathway.
TLNRD1
loss-of-function alters actin organization and barrier function in endothelial cells
in vitro
, and heart development in zebrafish
in vivo
.

Conclusions:
High-throughput functional analysis of 2,300 genes proximal to CAD GWAS loci prioritized pathways—such as angiogenesis and EC migration—that are regulated by multiple risk SNPs. Our study identifies new genes that likely influence risk for CAD, identifies convergence of CAD genes into certain pathways in endothelial cells, and prioritizes study of genes in the CCM-signaling pathway for contribution to CAD risk.
Abstract 153: Involvement of Peptidylarginine Deiminase 4 in Eosinophil Extracellular Trap Formation and Contribution to Citrullinated Histone Signal in Thrombi
Authors: Martinod Kimberly , Meyers Severien , Crescente Marilena , Van Bruggen Stijn , Stroobants Mathias , Siegel Patrick M , Glatz Katharina , Witsch Thilo
Published: October 2023

Background:
Extracellular traps (ETs) formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4 and the contribution of PAD4 to EET formation is not known.

Methods:
We performed immunohistological analysis of a large, intact and eosinophil-containing coronary thrombus retrieved from the right coronary artery using an aspiration catheter. Moreover, we studied isolated human eosinophils regarding their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria.

Results:
Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and intracellular and extracellular citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase exclusively, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4-dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live
Staphylococci
. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis.

Conclusions:
As in NETosis, PAD4 can catalyze the formation of EETs. In turn, inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents for the coronaries.
Abstract 154: Crossroads Of Immunity And Coagulation: Protease Activity Of Complement Component C1s Inhibits Protein C Activation In Covid-19
Authors: Eustes Alicia S , Nguyen Kathy H , ahmed azaj , Lentz Steven R , Dayal Sanjana
Published: October 2023

Introduction:
There is an abundance of crosstalk between innate immunity and coagulation in thrombo-inflammatory diseases such as COVID-19. Several potential procoagulant effects of complement activation have been described, but whether complement proteases affect antithrombotic pathways in is not known.
Aims:
We tested the hypothesis that complement component 1 (C1) impairs the protein C antithrombotic pathway.

Methods:
Plasma levels of the C1 complex were measured in plasma collected from healthy individuals and COVID-19 patients hospitalized early in the COVID-19 pandemic (NCT04360824). Activation of protein C by recombinant thrombomodulin was measured in a two-stage assay. In the first stage, protein C, thrombin, and thrombomodulin were incubated to generate activated protein C. The reaction mix was incubated with either healthy or COVID-19 plasma in the presence or absence of C1 inhibitor (which binds to C1 to inhibit its activity) or sutimlimab (a specific C1s inhibitor). During the second stage, protein C activation was measured using spectrophotometry with the chromogenic substrate S-2366.

Results:
In the inflammatory condition COVID-19, plasma levels of the C1 complex were increased compared to healthy controls (
p
= 0.004). Generation of activated protein C by recombinant thrombomodulin was decreased in the presence of COVID-19 plasma (
p
= 0.001), whereas the addition of healthy plasma had no significant effect (
p
= 0.469). Addition of C1 inhibitor or sutimlimab normalized protein C activation in the presence of COVID-19 plasma (
p
= 0.022 and 0.039, respectively). Addition of recombinant C1s protease, but not C1r protease or C1q, impaired the generation of activated protein C in a dose dependent fashion.

Conclusions:
We have uncovered a novel role of the protease C1s in regulating the generation of anticoagulant activated protein C. These findings suggest that the classical pathway of complement activation may impair anticoagulant responses in COVID-19.
Abstract 158: The Histone Demethylase JMJD3 Regulates Smooth Muscle Differentiation And Is Associated With Blood Pressure
Authors: Mangum Kevin , Joshi Amrita , Wolf Sonya , Barrett Emily , Moon Jadie , Moore Bethany , Obi Andrea T , Davis Frank , Gallagher Katherine A
Published: October 2023

Hypertension is associated with significant morbidity and mortality in the United States, and despite its prevalence an unmet need exists for more personalized targeted therapies. Blood pressure is regulated by environmental and genetic factors, and genome wide association studies (GWAS) have identified numerous genes in the human population associated with hypertension. Recently, one GWAS identified the single nucleotide polymorphism rs62059712 that is associated with systolic blood pressure in humans. rs62059712 is located upstream of the gene JMJD3, a histone demethylase that our lab has studied in a variety of disease contexts, and the rs62059712 major C allele (MAF 0.92) is associated with increased blood pressure in humans. Based on our analysis of data from the UCSC genome browser, we found that rs62059712 is located in a DNase Hypersensitive Site marked by H3K27Ac, suggesting it may regulate JMJD3 transcription. In luciferase assays, the fragment containing rs62059712 displayed transcription activity in smooth muscle cells (SMC), and the major C allele exhibited increased luciferase activity compared to the minor T allele in mouse aortic SMC (p<0.05). Notably, rs62059712 is in linkage disequilibrium with rs74480102, which did not display allele-specific transcription activity in SMC. siRNA knockdown of JMJD3 in mouse aortic SMC significantly decreased SMC-specific gene expression (p<0.05), and SMC marker gene expression was also reduced by treatment of SMC with the JMJD3 selective inhibitor GSK4J (p<0.05). Likewise, SMC contractile gene expression was reduced in aortas from SM22Cre;JMJD3
flox/flox
mice harboring SMC-specific deletion of JMJD3. Further, angiotensin II increased JMJD3 expression in human aortic SMC. Finally, in chromatin immunoprecipitation assays in mouse aortic SMC, JMJD3 was significantly enriched at smooth muscle gene promoters (p<0.05). Our results identify the blood pressure-associated gene JMJD3 as a key epigenetic regulator of smooth muscle differentiation. Additionally, we show that the rs62059712 major C allele, which is associated with increased blood pressure, increases JMJD3 transcription activity, and likely expression, to drive increased downstream contractile smooth muscle gene expression.
Abstract 160: Atherosclerosis Regression At The Single-Cell Level
Authors: Barcia Durán José Gabriel , Gourvest Morgane , van Solingen Coen , Fisher Edward , Moore Kathryn J
Published: October 2023

Atherosclerosis is a chronic inflammatory disease, but lipid-lowering therapies continue to be the standard of care, and substantial regression of human atheromata remains an elusive goal. To address this gap, our lab has devised multiple models to achieve plaque regression in atherosclerotic mice using genetic or RNA-based approaches. Here, we set out to learn how the immune cell composition of murine plaques changes with the induction of a regression phenotype irrespective of model. To achieve this, we performed single cell RNA-seq on CD45
+
leukocytes from the aortic arch plaques of

Ldlr
-/-

mice fed an atherogenic diet (baseline progression) and from two additional cohorts that were switched to control chow following atherogenesis and either treated with antisense oligonucleotides targeting
ApoB
(regression) or not (halted progression). The resultant transcriptomic data were integrated with those from our previous regression studies in a batch-corrected manner to distill the common features of atherosclerosis regression. This approach yielded a dataset of 35,996 cells, a degree of granularity one order of magnitude higher than that of the average comparable study, which allowed us to draw a comprehensive picture of the lymphoid and myeloid transcriptional dynamics that give rise to pro-resolving phenotypes. Specifically, we found two novel macrophage populations with distinct transcriptional profiles and unique distribution across conditions that nonetheless shared the same differentiation path. The first was characteristic of the halted progression groups and showed upregulation of genes involved in oxidative phosphorylation; the other was enriched in regression and displayed a transcriptional program consistent with plaque remodeling by means of efferocytosis and cholesterol efflux. Our findings suggest that pro-resolving macrophage fate acquisition in regression occurs at the expense of a separate, potentially pro-inflammatory lineage. A deeper understanding of the molecular and cellular purview of these pro-resolving macrophages as well as the ontogenic path of bone marrow monocytes toward that fate would contribute to the development of anti-inflammatory therapies to treat and prevent atherosclerosis.
Abstract 167: Immunomodulatory Properties of HDL are Associated with Lipidomic and Proteomic Composition and are Modifiable by Diet
Authors: Zhai Fangyi , Huang Lindsey , Garcia Chelsea , Woodruff Rachael , Jang Hyungryun , Corvino Olivia , Woo Hayoung , Blesso Christopher N , Lee Ji-Young , Palancia Esposito Christa , Zhang Ruijie , Andersen Catherine J
Published: October 2023

HDL is proposed to be an important immune regulator; however, the direct effects of HDL lipidomic and proteomic composition on immune activity, and the capacity to modulate immunomodulatory properties of HDL through dietary intervention, requires further study. We hypothesized that daily intake of different egg-based diets, which we have reported to induce HDL lipidome and proteome remodeling, would correspond to changes in the capacity of HDL to modulate macrophage and T cell inflammation. Healthy men and women (n = 26) participated in a 16-week randomized, crossover intervention trial in which they followed a 4-week egg-free diet, followed by random assignment to a 4-week diet period in which they consumed 3 whole eggs/day or 3 egg whites/day. Subjects then switched to the alternative egg treatment following a second 4-week egg-free washout period. Fasted blood samples were collected after each diet period to isolate HDL for compositional analysis and perform immune activation assays. We observed that subject-derived HDL increased lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) secretion from RAW 264.7 macrophages following the whole egg diet period, whereas there was a trend toward reduced LPS-induced interleukin 6 (IL-6) secretion and no effect on IL-10. Further, HDL dose-dependently increased Jurkat T cell counts and interleukin-2 (IL-2) secretion following activation, and these changes were inversely associated with lipid raft formation. HDL following the whole egg period increased IL-2 secretion from activated T cells, with greater increases in cell numbers observed in female subjects. Interestingly, changes in HDL-phosphatidylcholine, -phosphatidylethanolamine, and -ceramide species correlated with changes in macrophage TNFα between diet periods, whereas changes in macrophage IL-1β and T cell IL-2 were associated with changes in HDL-triacylglycerol and sphingomyelin species. Changes in macrophage and T cell cytokine production correlated with changes in 25 and 34 HDL proteins, respectively, but not HDL-cholesterol or cholesterol efflux capacity. HDL lipidomic and proteomic composition are determinants of its immunomodulatory properties, which can be modified through dietary intervention.
Abstract 210: The Interleukin-1 Signaling In Endothelial Reprogramming By Disturbed Flow
Authors: Kidder Evan , Pea Meleah , Cheng Siyuan , Koppada Satya-Priya , Visvanathan Suren , Hendrson Quartina , Yu Xiuping , Alfaidi Mabruka
Published: October 2023

Background and Objective:
Accumulative evidence has demonstrated the phenotypic transition of endothelium to endothelial-to-mesenchymal transition (EndMT) in human atheroma. Disturbed flow (d-flow) is one of the main triggers to EndMT formation, however, there is little evidence on the signaling intermediaries that link d-flow to EndMT formation. Herein, we described a novel role for endothelial interleukin-1 receptor (IL-1R1) in d-flow-induced EndMT
in vitro
and
in vivo
.

Methods and Results:
We found that IL-1R1 is predominantly expressed in endothelium of human coronary stable atherosclerotic plaques. The endothelial expression of IL-1R1 is increased in unstable lesions and that is associated with the EndMT transcriptional factor Snail1. In our endothelial cell-specific lineage tracing mice, we observed the predominant expression of IL-1R1 and Snail1 in areas exposed to d-flow. Inhibition of IL-1R1 resulted in downregulation of EndMT markers after d-flow
in vitro
. Importantly, in our endothelial cell-specific IL-1R1 KO mice we confirmed the direct effect of IL-1R1 inhibition on d-flow-induced EndMT in the partial carotid ligation model of d-flow.

Conclusions:
Global inhibition of IL-1 in patients with underlying atherosclerosis has already been tested in the completed CANTOS study with some promising effects. However, the data on interleukin-1 receptor IL-1R1 in mice are still conflicting. Our study illustrates a crosstalk between d-flow and IL-1R1 in endothelium, and represents a new direction for potential precision therapy by targeting IL-1R1 within the endothelium to maintain plaque stability in patients.
Abstract 212: Transcriptional Profiling Of Vascular Smooth Muscle Cell-foam Cells In Atherosclerotic Lesions
Authors: Ibikunle Chinyere O , Bashore Alexander C , Pan Huize , Xue Chenyi , Kim Eunyoung , Ross Leila S , Reilly Muredach P
Published: October 2023

Background:
One of the hallmarks of atherosclerosis progression is the uptake of oxidized lipids by cells to become Foam/Foamy cells - the most prominent being vascular smooth muscle cells (VSMC)-foam cells. Although studies have identified the ability of VSMC to become VSMC-foam cells, their transcriptomic profile and the phenotypic modulations which distinguish them from contractile VSMC remain poorly defined and understood.

Hypothesis:
We hypothesize that VSMC-foamy cells differ from contractile VSMC not only by modulation of genes shared by both cell types, but also by the expression of a unique set of genes which contribute to their formation and maintenance within atherosclerotic lesions.

Method:
To address this, we performed bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing mice models on high fat diet. VSMC and VSMC-foam cells were isolated and sequenced at specific time points reflective of late and advanced atherosclerosis disease progression. Computational analysis enabled us identify differentially expressed genes, biological pathways and molecular functions which distinguished VSMC from VSMC-foam cells.

Result and Conclusion:
Our computational analysis identified differential genes and pathways unique to VSMC-foam cells which might be associated with disease progression. From our analysis, VSMC showed a lower expression of contractile VSMC marker genes and a higher expression of genes in synthetic/intermediary multipotent state, we term “SEM” state. Our study also showed that VSMC-foam cells also expressed unique genes which distinguished them from contractile VSMC. These genes not only serve as potential genetic markers, but might further explain the nuclear composition, as well as the inflammatory and metabolic profile of VSMC-foam cells. Defining and understanding the molecular mechanisms and drivers which promote VSMC-foam cell formation and maintenance in atherosclerosis, would aid in our understanding of the disease with potentials for more effective therapeutic solutions.
Abstract 218: Characterization Of Mineral Maturity In Vascular Calcification Using Phosphorus-31 Solid State Magnetic Resonance Spectroscopy
Authors: Yao Jingting , Farrar Christian , Aikawa Elena , Sosnovik David E , Kulkarni Aditi , Ackerman Jerome
Published: October 2023

Introduction:
Cardiovascular calcification involving abnormal mineral depositions within the vessel walls and aortic valves underlies pathophysiologic mechanisms of cardiovascular diseases such as stroke and heart attack. To date, no effective therapeutic strategies are available to prevent or treat cardiovascular calcification, except for surgical interventions performed after formation of advanced calcification. Developing medical options requires a thorough understanding of mechanisms of the mineralization process, which are still insufficiently investigated.

Goal and Method:
The goal of the present study is to use a specialized solid state magnetic resonance spectroscopy (MRS) to characterize cardiovascular calcification. This MRS technique has been previously demonstrated to be effective in quantifying bone mineral and solid organic matrix, determining compositional features of minerals in solids, as well as characterizing the degree of bone mineral maturity, which is unattainable by existing non-destructive methods. The novelty of this study is the application of solid state MRS in combination with Herzfeld-Berger analysis to arterial calcification. A common model of cardiovascular calcification is apolipoprotein E-deficient (ApoE-/-) mice fed with a high cholesterol diet that develop substantial calcification in the aortic valves and carotid arteries.

Hypothesis:
We hypothesize that the specialized solid state MRS technique is capable of capturing differences in the mineralization of calcified human aortic valves and vascular calcification in the ApoE-/- knockout mouse.

Results and Conclusions:
Here we show the pattern of calcification in the human aortic valves resembles a dense, well-organized matrix characteristic of mature human trabecular bone and human calcified arterial plaque. In contrast, calcification in the mice resembles that of immature or embryonic bone, exhibiting a more disorganized pattern of mineralization that likely models early preclinical calcification of the aortic valve.
Abstract 228: Lipoprotein(a), Apolipoprotein(a) Size Polymorphism, And Cardiovascular Risk In Chronic Kidney Disease: Findings In The Sugar Study
Authors: Khan Muhammad A , Law Hayley G , Zhang Wei , Bang Heejung , Roshanravan Baback , De Boer Ian H , Berglund Lars , Byambaa Enkhmaa
Published: October 2023

An elevated level of lipoprotein(a) [Lp(a)] is a genetically regulated, casual, and prevalent risk factor for cardiovascular disease (CVD). A few non-genetic factors including chronic kidney disease (CKD) influence Lp(a) levels. In this cross-sectional study, we evaluated associations of CKD, defined by estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m
2
, with Lp(a) level and its major genetic regulator—apolipoprotein(a) [apo(a)] Kringle (K) repeats—in non-diabetic, non-dialysis patients with CKD (n=54) and healthy controls (n=39) enrolled in the Study of Glucose and Insulin in Renal Disease. The mean age of participants was 63 years (SD=14), 48% were women and 77% were of European descent. The mean eGFR was 37 and 89 mL/min/1.73 m
2
in the CKD and the control groups, respectively. Lp(a) concentration was higher in participants with CKD vs controls [median (IQR): 14 (5; 44) vs 11 (4; 30) mg/dL]. Notably, this increase in Lp(a) level was observed for both smaller and larger apo(a) sizes (i.e., independent of number of K repeats). Across the eGFR groups using common cut-offs of ≥90, 60-89, 30-59, <30 mL/min/1.73 m
2
, the median (IQR) Lp(a) concentration was 12 (3; 29), 11 (6; 31), 8 (4; 44), and 20 (8; 71) mg/dL, respectively, with a similar distribution of apo(a) K repeats across the eGFR groups. Among CKD patients, Lp(a) concentration was higher in those with CVD (n=17) vs without CVD (n=37) [median (IQR): 26 (4; 73) vs 11 (4; 43) mg/dL). In conclusion, among persons without diabetes, severe kidney impairment is characterized by markedly higher Lp(a) levels compared to those without it. Among persons with CKD, prevalent CVD is associated with higher Lp(a) levels. Mechanisms underlying CKD-induced increases in Lp(a) levels unrelated to apo(a) size polymorphism and their relevance in CVD risk warrant further investigations.
Abstract 229: Endothelial Cell Handling Of Atherogenic ApoB-carrying Lipoproteins
Authors: Cabodevilla Ainara G , Nasias Dimitrios , Tilp Anna , Kore Rufina , Anaganti Narashima , Davidson Nicholas , Madl Tobias , Hussain M M , Goldberg Ira J
Published: October 2023

Atherosclerosis is triggered by the subendothelial retention of cholesterol-rich,
apoB
-containing LDL (which carries apoB100) and chylomicron remnants (which carry apoB48, the 48% N terminal sequence of apoB). In addition, multiple studies have established elevated postprandial TRLs as an independent risk factor for CVD. However, the mechanisms whereby postprandial TRLs (which circulate mostly within chylomicrons) exert their atherogenic effects remain unknown. Two receptors -
SR-BI
and
ALK1
- have been shown to bind apoB and mediate transcytosis of LDL across the endothelium. We have shown that SR-BI also mediates EC uptake of nascent chylomicrons. RNAseq analysis revealed that ECs exposed to chylomicrons express a host of inflammatory and immune response genes, which does not occur with knockdown of SR-BI. In addition, following chylomicron uptake ECs release small extracellular vesicles that induce lipid accumulation in macrophages and lead to reduced eNOS phosphorylation in naïve ECs. Conversely, although binding of ALK1 to apoB is believed to occur through the N-terminus of apoB, the receptor is not involved in chylomicron uptake. Using a series of N terminal apoB peptides (apoB8-18, with sequences comprising the 8-18% N terminus of apoB), we found that chylomicron uptake was inhibited by apoB15 and apoB18, and LDL uptake was additionally inhibited by apoB12. These observations suggest that a) the binding sequence to SR-BI is between residues 547-684 of apoB, and b) an additional sequence between residues 365-547 participates in the binding to ALK1. Molecular modeling of apoB interactions identified an additional sequence in the C terminus of apoB potentially involved in the binding to ALK1. This sequence is absent in chylomicrons, which may explain why ALK1 inhibition and competition with apoB12 fail to significantly inhibit chylomicron uptake. Consistently, uptake of apoB100-carrying chylomicrons from Apobec
-/-
mice was inhibited by both ALK1 siRNA and competition with apoB12. Our observations suggest that a) the N terminus of apoB harbors distinct binding sequences to SR-BI and ALK1, and b) chylomicron uptake by ECs contributes to atherogenesis by inducing endothelial dysfunction, inflammation, and macrophage foam cell formation.
Abstract 230: A Novel Crosstalk Between Ehbp1-mediated Cholesterol Metabolism And Inflammation In Nash
Authors: Cai Bishuang
Published: October 2023

Background:
Emerging evidence shows that excessive cholesterol accumulation in hepatocytes promotes liver fibrosis in non-alcoholic steatohepatitis (NASH), but how disrupted hepatic cholesterol homeostasis contributes to NASH is not well understood. Human genome-wide association studies (GWAS) implicate the EH domain binding protein 1 (EHBP1) in correlating with low-density lipoprotein (LDL) cholesterol levels. Moreover, EHBP1 expression is dramatically reduced in hepatocytes from cirrhotic livers with advanced fibrosis based on single-cell RNA sequencing (scRNA-seq) of human livers, indicating that EHBP1 may play a role in liver fibrosis by modulating cholesterol metabolism in hepatocytes. As dysregulation of cholesterol metabolism is frequently associated with inflammation, our
aim
was to dissect the crosstalk between EHBP1-mediated cholesterol metabolism and inflammation in NASH.

Methods:
An established murine NASH model, the FAT-NASH mouse was used to study the role of EHBP1 in NASH. Primary murine and human hepatocytes were isolated for mechanistic studies. Human NASH livers obtained from the Liver Tissue Cell Distribution System (LTCDS) were used to establish the clinical relevance of EHBP1-mediated cholesterol metabolism. In addition, we have leveraged publicly available human genetic studies, and unbiased scRNA- and ChIP-seq datasets to support our study.

Results:
In primary human and murine hepatocytes, EHBP1 deficiency decreases PCSK9 secretion by destabilizing SORT1, leading to enhanced LDL receptor (LDLR) and the Hippo pathway effector TAZ, a novel cholesterol sensor that can induce liver fibrosis in NASH. Consistent with these
in vitro
findings, our
in vivo
studies showed that hepatocyte-specific silencing of EHBP1 reduces PCSK9 secretion and induces liver LDLR, cholesterol, TAZ, and liver fibrosis in NASH mice. Importantly, the NASH-associated inflammatory cytokine TNFα suppresses EHBP1 expression by reducing PPARα expression.

Conclusion:
Hepatocyte EHBP1 protects against liver fibrosis by promoting PCSK9 secretion and suppressing cholesterol-TAZ pathway and this protective process is antagonized by the inflammatory cytokine TNFα in NASH.
Abstract 240: Bioactive Lipids In Circulation: Biomarker Of Ageing?
Authors: Duchez Anne-Claire , Caroline SUT , Portier Mailys , Heestermans Marco , ARTHAUD Charles-Antoine , EYRAUD Marie-Ange , Prier Amelie , BERTRAND-MICHEL Justine , Hind Hamzeh-Cognasse , COGNASSE Fabrice
Published: October 2023

Blood transports cells and nutrients. However, it is less known that the major plasma compound is the lipid family in the broad sense. Among these lipids, cholesterol, triglyceride but also the family of polyunsaturated fatty acids (PUFAs) and their metabolites. These lipid mediators are involved in several chronic, inflammatory diseases such as rheumatoid arthritis. But, ageing is associated with chronic inflammation and affects almost 20% of the world’s population. Thus, it is by far the most important population before cardiovascular disease and cancer. Using mass spectrometry analyses, we evaluated the levels of different classes of lipid mediators from healthy young and elderly donors (single donor apheresis). Our data showed a significant decrease of lysophosphatidic acid species‘s expression, modulation of the 5-lipoxygenase product’s, as well as lipid mediators derived from PUFA oxidation. Their effect on blood vessel and circulating cells influenced their function. Indeed, lipid mediator intake is involved in transfusion reactions, and we observed an activation of blood cells, especially platelet, with significant increase of p-selectin. These data highlighted the importance of lipid mediator level in a context of transfusion and ageing of blood donor independently of the storage time of single donor apheresis. They can serve as biomarker of ageing.
Abstract 246: Glycosylation Engineered Platelet Membrane Coated Nanoparticle For Targeted Inhibition Of Vascular Restenosis
Authors: Li Fengshi , Rong Zhihua , Liu Changwei
Published: October 2023

Introduction:
IL10 nanoparticles coated with platelet membrane (IL10-PNP) has good targeting and local immune regulation function, and hence can effectively reduce restenosis after vascular injury. However, IL10-PNP still accumulated in the liver and spleen in a significant amount. Consequently, it is imperative that present PNP be further improved in terms of their immune-compatibility and targeting abilities. Glycosylation is one of the most important post-translational modifications of proteins, and platelet function is closely related to surface glycosylation.

Hypothesis:
We assessed the hypothesis that glycosylation engineered IL10-PNP (IL10-GE-PNP) with surface sialic acid removed and fucose conjunct has better biocompatibility and target ability.

Methods:
Glycosylation engineer of platelet membrane glycoprotein was performed by treating with neuraminidase and adding GDP-Fucose, fucosyltransferase I and fucosyltransferase VII. IL10 nanoparticles were prepared by recombinant IL-10 and PLGA nanoparticles and then coated by a glycosylation engineered platelet membrane. In vitro experiments were carried out to verify that glycosylation engineering of IL10-PNP enhanced the binding ability and regulation function of macrophages, smooth muscle cells (SMCs) and endothelial cells (ECs). In vivo experiments were carried out in a carotid artery injury model to verify the effect on reducing deposition in liver and spleen and ameliorating vascular restenosis.

Results:
The above methods could effectively remove sialic acids and add α (1,2)-fucose and α (1,3)-fucose. IL10-GE-PNP was successfully constructed and demonstrated its ability to target and regulate macrophage polarization in vitro and in vivo. The regulation of macrophages by IL10-GE-PNP can reduce the proliferation and migration of SMCs and promote the repair of ECs in an inflammatory environment both in vitro and in vivo, thereby reducing restenosis after vascular injury. And glycosylation engineer significantly reduces the deposition in liver and spleen of IL10-PNP.

Conclusions:
In conclusion, IL10-GE-PNP is an excellent candidate for targeting vascular injury and holds promise as an innovative drug delivery system for suppressing vascular restenosis.
Abstract 247:
LMNA
Mutation-induced Endothelial Dysfunction Contributes To The Pathogenesis Of Atrial Fibrillation
Authors: Owais Asia , Chen Hanna , Chen Yining , Arif Mahmud , Barney Miles , Sridhar Arvind , Ong Sang-Ging , Darbar Dawood
Published: October 2023

Introduction:
The genetic basis of atrial fibrillation (AF), a commonly encountered arrhythmia in clinical practice, is well established. Clinical and epidemiologic studies have implicated endothelial dysfunction in early-onset AF; however, the molecular mechanisms underlying this association remain unclear. Early-onset AF is a common occurrence in Lamin A/C (
LMNA
) cardiomyopathy and usually precedes DCM. As variants in
LMNA
can cause endothelial dysfunction, we have explored the mechanistic link between endothelial dysfunction and early onset AF using human induced pluripotent stem cell -derived atrial cardiomyocytes (iPSC-aCMs) and iPSC-endothelial cells (ECs) generated from the proband of an affected family harboring a pathogenic
LMNA
-S143P mutation (
Figure 1
).

Hypothesis:
LMNA
-S143P mutation causes endothelial dysfunction which leads to ion channel and structural remodeling generating an arrhythmic substrate for AF.

Methods:
Human iPSC-aCMs and iPSC-ECs were generated from the wildtype (WT) and
LMNA
-S143P mutation proband. Electrophysiological assessment was performed in matured iPSC-aCMs by optical voltage mapping and measuring calcium transients. EC function was assessed by angiogenesis assay.

Results:
The
LMNA
-S143P-iPSC-ECs exhibited impaired angiogenesis compared to WT-iPSC-ECs. The action potential duration at 90% repolarization (APD90) was prolonged in
LMNA
-S143P-iPSC-aCMs compared to WT-iPSC-aCMs (
Figure 1
).
LMNA
-S143P-iPSC-aCMs had irregular calcium transients resembling triggered delayed afterdepolarizations.

Conclusions:
We showed ion channel remodeling and abnormal calcium homeostasis as a potential mechanistic link between endothelial dysfunction and triggered AF due to a pathogenic
LMNA
-S143P mutation. Our findings suggest that treatment of endothelial dysfunction with calcium channel blockers and/or statins may prevent the progression of early-onset AF associated with
LMNA
variants.
Abstract 250: Genome-wide Association Study Of Intracranial Aneurysms Reveals Shared Heritability With Aortic Aneurysms And Atherosclerosis
Authors: Adkar Shaunak , Lynch Julie , Bamezai Sharika , Sorondo Sabina , Choi Ryan , Levin Michael , Damrauer Scott M , Pyarajan Saiju , Tsao Philip S , Skirboll Stephen , Leeper Nick J , Klarin Derek
Published: October 2023

Introduction:
Genetic susceptibility to intracranial aneurysms (ICAs) has been ascribed largely to genetic predisposition to hypertension. The shared heritability with other vascular phenotypes, including atherosclerosis and aortic aneurysms, has not been described.

Hypothesis:
We hypothesized that a subset of genetic loci associated with ICA are shared with atherosclerosis and aortic aneurysmal disease independent of hypertension.

Methods:
We identified participants enrolled in the Million Veterans Program (MVP) with and without ICA. Thirty-two million genotyped and imputed variants were tested for association with ICA using mixed effect regression modeling, controlling for age, sex, and population structure prior to external replication and meta-analysis. Multi-trait colocalization was performed with summary statistics for systolic and diastolic blood pressure, peripheral and coronary atherosclerosis, and thoracic aortic size/aneurysm and abdominal aortic aneurysm. Single nucleus RNA sequencing data yielded 147 thousand nuclei to assess cell type specific expression of candidate causal genes.

Results:
We identified 3165 participants (2280 European, 627 African, and 258 Hispanic ancestry) with and 592927 without ICA in the MVP. After replication and meta-analysis with data from an additional 12273 individuals with and 591046 without ICA, we replicated 16 of 17 previously described loci and identified 5 novel loci associated with ICA (
NAV1
,
EXOC2
,
TWIST1
,
FGF5
, and
DLEU1
). These loci, excluding
EXOC2
, had previously been associated with thoracic aortic aneurysm/size, AAA, or atherosclerosis. Multi-trait colocalization of both novel and replicated loci revealed 11 ICA susceptibility loci that strongly colocalized with loci associated with atherosclerotic and aneurysmal traits (posterior probability > 0.7). Five of these did not colocalize with loci associated with hypertensive traits (
EDNRA, SOX17, RPPB8, NAV1,
and
CDKN2B
). Cell type specific expression of candidate causal genes revealed enrichment in endothelial cells and fibroblasts.

Conclusion:
Here, we identify novel loci associated with ICA and identified shared heritability with atherosclerotic and aneurysmal disease independent of hypertension.
Abstract 251: Regulation Of Endotheliopathy By Chromatin-associated Long Non-coding RNA
Authors: Chen Zhen B , Tang Xiaofang , Sriram Kiran , Luo Yingjun , Yuan Dongqiang , Malhi Naseeb K , Liu Xuejing , Natarajan Rama
Published: October 2023

Lining the critical interface between circulating blood and vascular wall, endothelial cells (ECs) play vital functions in health and disease. The optimal gene expression in ECs is essential to maintain endothelial homeostasis, and its dysregulation can lead to EC dysfunction or endotheliopathy, a common mechanism underlying many metabolic and cardiovascular diseases. Through chromatin-RNA contact profiling, we have identified several chromatin-associated long non-coding RNA (lncRNAs) that play essential roles in the regulation of endothelial function, including endothelial nitric oxide production, angiogenesis, endothelial-mesenchymal transition, and inflammatory activation. These include nuclear genome-transcribed lncRNAs, e.g. lncRNA-enhancing endothelial nitric oxide synthase (LEENE) and LINC00607 that promotes expression of fibronectin and plasminogen activator inhibitor-1. Our recent study has also revealed that mitochondria transcribed RNAs (mtRNA) are pervasively attached to nuclear chromatin and such mtRNA-association with chromatin is increased by pro-inflammatory, diabetes-mimicking stimuli. Suppression of a mitochondrial lncRNA in ECs attenuates nascent RNA transcription from the nuclear genome, including that of critical genes promoting pro-inflammatory activation. Finally, we show increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors. Together, these data highlight an emerging role of chromatin-associated lncRNAs in regulating endothelial biology.