bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 120: Neutrophilic Mice Are Protected Against
Staphylococcus Aureus
Sepsis And Immunothrombosis Through Peptidylarginine Deiminase 4-mediated Neutrophil Extracellular Trap Formation
Authors: Martens Caroline P , Stroobants Mathias , Zalghout Sara , Lox Marleen , Peetermans Marijke , Verhamme Peter , Vanassche Thomas , Martinod Kimberly
Published: July 2024

Background:
Neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) have been proposed as key drivers of immunothrombosis and thromboinflammation in
Staphylococcus
aureus
sepsis. Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and citrullinates coagulation proteins. NETs interact with VWF and platelets, thus providing a scaffold for the formation of microthrombi that cause microvascular perfusion deficits.

Aims:
To study the relationship between PAD4, NETs, and VWF in murine
S. aureus
sepsis.

Methods:
Murine sepsis was induced by injecting methicillin-resistant
S.
aureus
(MRSA, USA300 strain) intravenously in C57BL/6J, MRP8Cre
+
PAD4
flfl
(NET-defective) or PAD4
fl/fl
littermates. In separate experiments, granulocyte colony stimulating factor (G-CSF) was used to induce neutrophilia prior to administration of USA300. Mice were followed for up to 8 days for clinical scoring and reaching humane endpoints as a surrogate readout for survival. Cell counts and plasma levels of citrullinated histone H3-DNA (H3Cit-DNA) complexes, VWF antigen, and PAD4 were measured at days 1, 4, and 8.

Results:
WT mice injected with USA300 developed thrombocytopenia and had higher plasma levels of NETs, VWF, and PAD4 as compared to vehicle (saline)-injected mice. MRP8Cre
+
PAD4
flfl
mice had significantly reduced H3Cit-DNA and lower levels of VWF antigen 1d after USA300 injection as compared to littermate controls, with comparable VWF levels in the later disease phase. There was no difference in levels of cell-free PAD4 in plasma indicating a non-neutrophil source of PAD4, but the substantial reduction of plasma NET levels supporting that neutrophil PAD4 was essential for NET release. Neutrophilic mice (prior to sepsis induction) had lower sepsis disease scores and significantly improved survival rates as compared to mice receiving saline control. This protection was absent in the NET-defective mice, who surprisingly retained higher VWF levels.

Conclusion:
Individually eliminating NETs or VWF had little impact in MRSA sepsis in mice, and circulating PAD4 likely does not originate from neutrophils. Inducing neutrophilia protected mice against
S.
aureus
sepsis, an effect which was abrogated in the absence of NET formation.
Abstract 137: Identification Of A Noncoding Genetic Variant In The Tissue Factor Locus That Reverses Lethal Thrombosis In Mice
Authors: Rodionova Arina , Brake Marisa , Siebert-Mckenzie Amy E , Westrick Randal
Published: July 2024

Background:
Thrombosis is initiated by tissue factor (TF, gene name
F3
) binding to coagulationFVII, with tissue factor pathway inhibitor (TFPI) inhibiting this complex. Alterations in TF orTFPI expression significantly affect thrombosis. Reducing TFPI expression by 50% (
Tfpi
+/-) inmice results in a perinatal lethal phenotype on the Factor V Leiden homozygous(
F5

L/L

)prothrombotic background. We used the
F5

L/L

Tfpi
+/- lethal phenotype to conduct a dominantsensitized whole genome ENU mutagenesis screen to suppress the
F5

L/L

Tfpi
+/- lethality. Weidentified a Modifier of Factor 5 Leiden 6 (MF5L6) line with 72% penetrance and 85
F5

L/L

Tfpi
+/- offspring. A significant linkage peak (LOD=4.35),explaining half the suppressing effect andcontaining
F3
(Chromosome 3) was identified.

Goals/Hypothesis:
To identify the genomic variant controlling F3 expression in the MF5L6 line.

Methods:
To quantify
F3
expression in the surviving mice from MF5L6, quantitative PCR onliver, lung, and heart tissues from MF5L6 was performed. We used Sanger DNA and highthroughput sequencing to identify candidate TF regulatory variants in the F3 locus. Theprothrombin time assay was used to test the effects of reduced TF expression on in vitro bloodcoagulation.

Results:
Two distinct expression profiles in the lung and liver of the MF5L6 mice wereobserved, those that had a 50% reduction in
F3
mRNA and those that did not. Heart tissuesexhibited one expression profile, suggesting that the variant regulates F3 expressiontissue-specifically. Sanger sequencing of the
F3
coding region revealed no coding mutations inMF5L6 mice. Whole genome sequencing identified two novel candidate variants (in unknownF3 regulatory elements) in the 200 kilobase upstream region of
F3
. The 50% reduction in
F3
resulted in significant changes in coagulation by the prothrombin assay (n=18,p<0.0009).

Conclusion:
We identified novel candidate variants for regulating
F3
gene expression and aredetermining their mechanism of action. Investigation of these variants will provide new insightsinto the regulation of
F3
and enable us to identify the variant(s) responsible for the remainder ofthe thrombosis suppressing effect in MF5L6. Our findings provide new insights into the geneticregulation of thrombosis.
Abstract 145: Chronic Electronic Cigarette Exposure Accelerates SMC Phenotypic Modulation And Activates The NMDA-type Glutamate Receptor (NMDAR) Pathway In Mediating Vascular Calcification
Authors: Damiani Isabella , Weldy Chad , Easwaran Meena , Qin Guyu , Basu Sugandha , Direnzo Elizabeth , Quertermous Thomas , Kim Juyong B
Published: July 2024

Introduction:
Electronic cigarettes pose a serious emerging threat to cardiovascular health. The use of electronic nicotine delivery systems (ENDS) such as electronic cigarettes (e-cigs) have increased exponentially, especially among adolescents. Emerging data suggest e-cig use has been implicated in promoting atherosclerosis. This work explores the mechanism of e-cig exposure accelerated the atherosclerotic lesion development and vascular remodeling.

Methods:
SMC-lineage tracing mice (n=16) on a ApoE null hyperlipidemic background were put on high fat diet for 12 weeks and exposed to pod-based e-cigs (Juul) three times a week for 12 weeks in a whole-body chamber (inExpose, Scireq) and compared to WT mice exposed to air (n=16). The aortic sinus was dissected and digested, FACS was performed to sort for live single cells, then single-cell RNA-Seq (sc-RNAseq) and ATAC-seq were performed on the 10X Genomics platform. Analyses were completed with Seurat and Signac tools. The osteogenic load was assessed by histology with alkaline phosphatase (AP) enzymatic assay using the Ferangi Blue Chromogen kit.

Results:
scRNA-Seq analysis revealed that e-cig accelerated the SMC transition to a chondrogenic phenotype (CMC). We found a significantly increased proportion of chondromyocytes (CMC) expressing specific markers such as
Col2a1
and
Acan
in the e-cig exposed mice compared to control. The scATAC-seq profiles showed that e-cig exposed mice develop a cell cluster that enriched for markers related to nicotine, glutamatergic and dopaminergic pathways. The genes highly enriched for chromatin accessibility in this cluster included
Grin2a
, a gene that encodes a N-methyl-D-aspartate receptor (NMDAR) GluN2A, which was found to be expressed in the fibromyocyte and CMC population of the scRNA-seq. Consistent with our finding from the scRNA-Seq data showing a greater CMC proportion in the e-cig exposed mice, we found the relative AP-stained area to be larger in the exposed group than in the WT group.

Conclusion:
Overall, these data indicate that e-cig exposure can promote atherosclerosis through adverse SMC phenotypic modulation. Moreover, e-cig exposure induces NMDAR activation that may be implicated in SMC modulation to a chondrogenic phenotype.
Abstract 148: Loss Of Tlr4 Signaling Inhibits Thrombus Resolution In A Non-monocyte Dependent Manner
Authors: Kumar Kiran , Moreno Oscar Y , Parchment Nathaniel , Sharma Sriganesh , Rocco Sabrina , Luke Catherine , Lambert Sylviane , Holinstat Michael A , Davis Frank , Gallagher Katherine A , Moore Bethany , Henke Peter , Obi Andrea T
Published: July 2024

Background:
Loss of TLR4 signaling reduces Venous Thrombosis (VT) resolution. Leukocyte expression of TLR4 is elevated in humans undergoing acute to chronic DVT transformation. We hypothesized that TLR4 signaling in monocytes directs thrombus clearance.

Methods:
Wild type,

Tlr4
-/-

,

LysM
cre+/-
Tlr4
fl/fl

mice underwent VT via IVC stasis at 4/7/14d timepoints. Thrombi subtypes were identified by flow cytometry. Thrombus size was assessed, and fibrinolytic mediators were assayed in splenic/bone marrow Mφs. Whole blood from mice was stimulated with TLR2/4 and assayed for thrombolytic potential via HALO assay. Immunohistochemistry was performed for cellular infiltrate.

Results:
We observed elevated TLR4 cell surface expression on CD11b+/Ly6C- Mo/Mφ from wild type thrombi. Thrombus weight in

Tlr4
-/

mice was elevated at 4d stasis, confirming impaired thrombus clearance. In basal conditions, circulating Mo/Mφ saw decreased Urokinase & MMP9 mRNA levels in

LysM
cre+/-

mice. In post thrombotic conditions, we saw decreased protein levels of Urokinase and MMP9 in Cre+ bone marrow-derived Mφs. Whole blood thrombolysis efficiency increased with TLR4 agonism in Cre- mice compared to Cre+. Efficiency was restored with LPS-mediated dual TLR2/4 agonism. Total thrombus weight was unchanged. No difference was identified in monocyte infiltrate into the thrombus (Fig. 1)

Conclusions:
Genetic deficiency of Mo/Mφ TLR4 signaling alters induction of fibrinolytic/matrix remodeling gene expression. Ex vivo TLR4 agonism increased whole blood thrombolysis in Cre- but not Cre+ mice. Additional TLR2 agonism of Cre+ mice restored thrombolytic activity, suggesting TLR2 as a compensatory pathway that may account for the unchanged
in vivo
phenotype in LysMcreTlr4fl/fl mice. Further clarifying these cellular pathways may provide an alternate pathway to target VT resolution.
Abstract 149: The Protective Effect Of Vamp8 Deficiency In Aortopathies: Potential Impact Of Impaired Platelet Cargo Release
Authors: Mohammadmoradi Shayan , Driehaus Elizabeth , Heier Kory , Alfar Hammodah , Lykins Joshua , Levitan Bryana , Zhang Ming , Joshi Smita , McQuerry Kristen , Whiteheart Sidney W
Published: July 2024

Introduction and Objective:
Platelet activation and subsequent cargo secretion play a critical role in thrombus formation and aortic remodeling, factors potentially influencing the growth and rupture of abdominal aortic aneurysms (AAA). Despite the compelling data that platelet activation is associated with AAA and rupture, the underlying mechanisms remain poorly understood. This study hypothesizes that VAMP8, a key element of platelets’ secretory machinery, significantly contributes to AAA formation.

Approach and Results:
A retrospective, single-center multiple comparisons study of adult aneurysmal patients admitted to UKHealthCare between 2004 and 2023 revealed a significantly lower platelet count in AAA patients (n=3131, 213.76 ± 3.46) vs. healthy individuals (n= 6309, 243.18 ± 4.09) suggesting an increase in platelets consumption, potentially in the aneurysmal sack. Thoracic aortic aneurysm and aortic dissection patients also indicated lower platelet count.
In vivo
, platelets were shown to accumulate at elastin break sites and false lumens of abdominal aortas from 28-day AngII-infused hypercholesteremic mice. To assess the role of platelets, specifically platelet secretion, we utilized VAMP8 null mice. We first validated the platelet secretion defect in these mice and then used RNASeq and proteomics array to define the role of VAMP8 in platelet cargo packaging. Next, VAMP8
-/-
or WT hypercholesterolemic male mice fed a Western diet were infused with AngII (1,000 ng/kg/min) for 4 weeks. Our results revealed that VAMP8
-/-
mice are protected against AngII-driven aortic rupture. Additionally, aortic
ex vivo
analysis indicated that the VAMP8 deficiency (n=15, aortic diameter: 0.9 mm ± 0.03) profoundly attenuated AngII-induced AAA compared to control (n=9, aortic diameter: 2.0 mm ± 0.2). Further analysis also indicated a marked reduction in thoracic aortic aneurysm and atherosclerosis development in VAMP8
-\-
vs. Control group.

Conclusion:
Our results elucidate that a deficiency in VAMP8 results in the profound attenuation of aortic aneurysms and atherosclerosis development, introducing a novel paradigm for understanding the impact of platelet cargo secretion in the development of aortopathies.
Abstract 2008: Characterizing Changes In Abdominal Aortic Aneurysm Principal Wall Strain Using Ultrasound Elastography
Authors: Kedwai Baqir J , Zottola Zachary , Geiger Joshua , Lehane Daniel , Stoner Michael , Richards Michael , Mix Doran S
Published: July 2024

Pressure-normalized aortic principal wall strain (ep+/pp, %/mmHg) is a biomechanical parameter for abdominal aortic aneurysm (AAA) stability correlated with aneurysm growth rate. We hypothesized that ep+/pp decreases as AAAs degenerate. This study aimed to characterize ep+/pp change over time using ultrasound elastography (USE). Axial ultrasound images of patient AAAs were collected at two consecutive clinic visits. The USE algorithm applied a finite element mesh over the aneurysm wall at the point of maximum diameter. A custom image-registration protocol tracked frame-to-frame displacement fields of the images over one cardiac cycle to calculate maximum ep+/pp (Fig 1). Clinical data were collected from patient records. The cohort was separated by index ep+/pp terciles. Thirty-one patients with a mean age of 71.7 ± 10.1 at index visit were included, with follow-up imaging at an interval of 7.1 ± 3.4 months. For the cohort, maximum ep+/pp decreased from 2.3 ± 1.0 %/mmHg to 2.0 ± 0.9 %/mmHg (p=0.08), and average AAA diameter increased from 4.4 ± 0.6 cm to 4.5 ± 0.7 cm (p=0.04). The “high-strain” tercile was associated with a strain reduction of -1.7 ± 0.8 %/mmHg, compared to “low” (0.1 ± 1.0 %/mmHg, p<0.01) and “intermediate” (-0.4 ± 0.5 %/mmHg, p=0.04) terciles (Fig 2). There was no difference in AAA growth or rupture between terciles. The present findings indicate that AAA ep+/pp at baseline predicts the degree of ep+/pp decrease over time. These data suggest that biomechanical changes in the aortic wall related to aneurysmal degeneration can be quantized by a novel ultrasound technique. Research correlating these results with histopathologic AAA remodeling may inform monitoring.
Abstract 2014: Long Non-coding RNA LINC-PINT Regulates Smooth Muscle Cell Plasticity In Developing Abdominal Aortic Aneurysms
Authors: Li Zhaolong , Fasolo Francesca , Wu Zhiyuan , Pauli Jessica , Glukha Nadiya , Jin Hong , Ritzer Julia , Sachs Nadja , Paloschi Valentina , Maegdefessel Lars
Published: July 2024

Background:
Long noncoding RNAs (lncRNAs) play pivotal roles as molecular regulators in diverse vascular biological processes and diseases. This study utilized multi-transcriptomic profiling to investigate the contribution of lncRNAs in Abdominal Aortic Aneurysm (AAA) disease.

Materials & Methods:
Utilizing bulk and single-cell RNA sequencing, we identified lncRNA
long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT)
as a significantly deregulated transcript in AAA compared to non-dilated aortas. Deconvolution of single-cell and bulk RNA-sequencing
via
the BayesPrism tool identified plasticity of smooth muscle cells (SMCs) as the predominant shifted condition between dilated
vs.
non-dilated aortas. Hybridization-based RNA
in situ
sequencing (HybRISS) confirmed the presence of
LINC-PINT
in de-differentiating SMCs.

Results:
Experimental knock-down of
LINC-PINT
, using site-specific antisense oligonucleotides (LNA-GapmeRs) markedly decreased proliferation rates of cultured human aortic smooth muscle cells (AoSMCs), but also increased their apoptosis, whereas overexpression of
LINC-PINT
had the opposite effect. Transcriptomic profiling of
LINC-PINT
-silenced AoSMCs indicated down-regulation of contractility markers, accompanied by an increase of inflammation and extracellular matrix (ECM) degradation. In line with this,
LINC-PINT
knock-down abolished the transforming growth factor beta (TGF-β)- induced AoSMCs polarization towards a contractile phenotype. Notably, RNA immunoprecipitation (RIP) demonstrated an interaction between
LINC-PINT
and p65, inhibiting its translocation to the promoter region of inflammatory genes.
LINC-PINT
knock-down increased p65 binding to its coactivator p50, which was confirmed by Co-immunoprecipitation (CO-IP). Combined
in silico
prediction and luciferase reporter assays further revealed that
LINC-PINT
expression was controlled by the ZNF93 and ZNF263 transcription factors.

Conclusion:
We provide evidence of
LINC-PINT
being a novel regulator influencing SMC dynamics and fate decisions of central importance to AAA pathophysiology. Currently ongoing in vivo studies in
Lncpint
-deficient mice explore its relevance in experimental AAA development.
Abstract 2023: Plasminogen Activator Inhibitor-1 Deficiency Augments Angiotensin II-induced Cardiac Fibrosis, But Not Aortic Aneurysm Formation In Mice
Authors: Pettey Alex , Ito Sohei , Howatt Deborah , Franklin Michael , Zhang Nancy , Graf David , Sawada Hisashi , Lu Hong S , Daugherty Alan
Published: July 2024

Background:
Plasminogen activator inhibitor-1 (PAI-1) is highly abundant in human thoracic aortic aneurysms (TAA). This high abundance is mimicked in TAAs created by chronic infusion of angiotensin II (AngII) into mice. The purpose of this study was to determine whether deletion of PAI-1 influenced the development of thoracic aortic aneurysms during AngII infusion.

Methods and Results:
To determine the role of PAI-1 in acute and chronic phases of AngII-induced pathology, whole-body PAI-1 deficient mice (PAI-1 -/-) and wild type littermates (PAI-1 +/+) were infused with AngII (1,000 ng/kg/min) for 7 or 28 days, respectively. Aortic diameters were measured by ultrasonography and
in situ
measurement. PAI-1 deficiency did not alter maximal luminal or external aortic diameters at 7 or 28 days of AngII. At 7 days of AngII, ventricular hemorrhage was visibly evident in PAI-1 -/- mice. Mid-ventricular heart sections were next examined by hematoxylin and eosin staining, and red blood cell accumulation was found primarily within the epicardium. Masson’s trichrome staining revealed interstitial fibrosis in the epicardium, myocardium, and posterior septum of PAI-1 -/- mice. At 28 days of AngII, PAI-1 -/- mice displayed primarily epicardial fibrosis evident by trichrome staining and gross appearance. The posterior septum presented less fibrosis after 28 days of AngII relative to 7 days of AngII.

Conclusions:
PAI-1 deficiency did not alter aortic dilatation, indicating that PAI-1 is not a key contributor to AngII-induced TAAs. Paradoxically, PAI-1 deficiency augmented AngII-induced cardiac fibrosis with distinct distributions between acute and chronic phases.
Abstract 2037: Investigating Changes In Satellite Cell Proliferation And Angiogenic Potential In Response To Exercise
Authors: Oh Dong Gun
Published: July 2024

Background:
Peripheral Artery Disease (PAD) is an atherosclerotic process leading to vessel obstruction. Supervised walking exercise is a first-line treatment to improve walking ability and quality of life scores of patients. Despite its known benefits, the underlying mechanism is not fully understood. Thus, this study focuses on satellite cells (SCs), known for their role in muscular regeneration but also found to have an under investigated role as mediators of vascular growth through the secretion of cytokines and growth factors. Previous in vivo models have demonstrated increased migration of endothelial cells and smooth muscle cells, as well as blood vessel growth in response to SC activity. Aims: The project aims to enhance the efficacy of exercise therapy through a better understanding satellite cell biology and their pro-vasculogenic ability.

Hypothesis:
Exercise therapy will enhance SCs proliferation and secretion of paracrine growth factors inducing increased cell migration and vascular growth.

Methods:
Aortic Ring Assay: Murine thoracic aortic sections were embedded in a Type I collagen matrix in 96-wells. The wells were seeded with Opti-MEM (control), Opti-MEM+0.3% Vascular Endothelial Growth Factors (VEGF)(+ control), SC Conditioned Media (CM) (24hr incubation, 2M cells/plate), or denatured CM (56°C, 1 hr). The total length and number of branches were measured via ImageJ. Flow Cytometry: 18+ months old C57BL/6 were housed with voluntary running wheels with activity counter until day 28. Satellite cells were mechanically and chemically digested and were labeled as: CD11b-, CD45-, CD31-, Sca1-, and α7-integrin+ at 7-day intervals.

Results:
Aortic Ring Assay: SC CM showed significantly higher total branch length (13381.89±2470.44μm, n=6) (****p<0.0001) and numbers (19.24±3.87, n=6) (**p<0.01) compared to control (2158.18±382.31μm, n=9), (5.76±0.81, n=9). Flow Cytometry: SC proportion on day 21 (2.55±0.14,n=2) was significantly higher than control (1.54±0.298,n=8)(*p<0.05).

Conclusion(s):
These findings indicate that SC CM induces angiogenesis by secreting pro-angiogenic factors. The angiogenic activities could potentially be heightened through the proliferation of SCs under exercise therapy.
Abstract 2041: Enhanced Platelet Reactivity In The Presence Of Abdominal Aortic Aneurysm: Unveiling Vascular Dynamics
Authors: Benson Tyler W , Spuzzillo Anthony R , Wadding-Lee Caris A , Stage Hayley , Cameron Scott J , Owens A. Phillip
Published: July 2024

Introduction:
Abdominal aortic aneurysms (AAA) are a pro-inflammatory disease that frequently feature a nonocclusive intraluminal thrombus (ILT) at the expansion site. Platelet abundance is notable at the luminal interface of an ILT, and recent investigations conducted by our lab and others elucidate the pivotal involvement of platelets activation and adhesion in the pathogenesis of AAA. While inflammation is recognized to affect platelet physiology, the impact of inflammation associated with AAA on the platelet phenotype remains unclear. Therefore, our objective was to elucidate the impact of AAA on platelet phenotype.

Methods and Results:
Male
C57BL/6J
mice underwent laparotomy and either a sterile solution of 10 mg/mL elastase solution (n =6) or heat inactivated (HI) elastase solution (n = 4), used as control, was applied to the infrarenal aorta. Weekly ultrasounds demonstrated that mice treated with elastase had significantly elevated aortic diameter and AAA incidence as compared to HI treated controls. Platelet counts and volumes were assessed weekly via a veterinary hematology analyzer. While there was no significant difference in mean platelet volume, platelet counts in AAA mice were on the lower end of the reference range, comparable to human AAA patients. At 4 weeks following laparotomy, platelets were isolated via terminal IVC blood collection, washed, and subjected to a dose response curve of both thrombin (protease-activated receptor agonist; 0.1 U/mL - 1 U/mL) and convulxin (glycoprotein VI agonist; 10 ng/mL - 100 ng/mL). Surface expression of P-selectin was the measured by flow cytometry. Platelets isolated from mice with aneurysms had greater surface P-selectin after agonist exposure compared to HI control platelets. Additionally, we reanalyzed published RNA sequencing data from control and AAA patient isolated platelets and found several upregulated platelet surface markers in AAA patients as compared to controls.

Conclusions:
Our data indicates that AAA influences platelet reactivity and modifies surface marker expression. While more work is needed to fully elucidate the underlying mechanisms, our data has significant implications for understanding how systemic inflammatory diseases like AAA affects platelets.
Abstract 2044: Disruption Of
DENND5B
Attenuates Triglyceride-rich Lipoprotein Secretion From Human Enterocytes And Hepatocytes
Authors: Hage Olivia , Neupane Khaga , Karakashian Alexander , Gordon Scott M
Published: July 2024

Background and Hypothesis:
Elevated plasma triacylglycerides (TAGs) are an established risk factor for atherosclerotic cardiovascular disease, and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory formerly reported that
Dennd5b
-deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of the intestinal epithelium of
Dennd5b
-/-
mice reveals significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Given that
Dennd5b
is expressed by the two major triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine), we hypothesized that disruption of
DENND5B
attenuates TRL secretion in both of these cell types in humans.

Methods and Results:
To determine if
DENND5B
disruption impairs TRL secretion in human cells, we generated
DENND5B

-/-

intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR gene editing. Western blots verified DENND5B protein reduction in
DENND5B

-/-

cells. The impact of
DENND5B
deficiency on TRL secretion was quantified by measuring the cellular and secreted TAG content of
DENND5B

+/+

and
DENND5B

-/-

cells under standard growth conditions and after oleic acid loading. Caco-2 cells were differentiated on membrane supports for 21 days prior to experiments.
DENND5B

-/-

Caco-2 cells secreted significantly less TAGs compared to wild-type Caco-2 cells (-67.8%, p<0.0001).
DENND5B

-/-

HepG2 cells also had significantly reduced TAG secretion (-91.0%, p<0.0001). Disruption of
DENND5B
in both cell lines reduced TAG secretion without affecting cellular TAG content. Immunofluorescence microscopy showed intracellular DENND5B protein localization consistent with secretory pathway involvement with detection on a perinuclear structure (likely Golgi), cytosolic vesicles, and plasma membrane.

Conclusions:
These studies suggest that DENND5B plays a role in TRL secretion from human enterocytes and hepatocytes and provide new model systems for investigating the molecular function of DENND5B in TRL secretion from human cells.
Abstract 2046: Overexpression Of Endothelial Lipase Enhances The Cellular Uptake Of Low-density Lipoproteins In Cultured Hepatocytes In The Absence Of Low-density Lipoprotein Receptor.
Authors: Lucero Diego , Aligabi Zahra , Burks Kendall H , Stitziel Nathan O , Goldberg Ira J , Remaley Alan T
Published: July 2024

Background:
Inhibitors of angiopoietin-like3 (ANGPTL3) reduce low-density lipoprotein (LDL) cholesterol, even in Familial Hypercholesterolemia (FH) patients with a dysfunctional LDL receptor (LDLR). Although the exact mechanism remains elusive, it may depend upon endothelial lipase (EL). Early studies showed that EL mediates the cellular uptake of high-density lipoproteins in hepatocytes; however, whether EL similarly mediates LDL uptake is unknown.

Aim:
Investigate the role of EL in mediating the cellular uptake of LDL in an LDLR-deficient model.

Methods:
Cas9-expressing HepG2 cells were transfected with a scrambled or an
LDLR
-targeting guide RNA to produce control and
LDLR
-knockout (KO) cells. Control and KO cells were transfected by lipofection with an empty plasmid or a plasmid containing the human
LIPG
gene (EL). Cellular uptake of fluorescent human LDL was measured by FACS. In addition, we measured the uptake of LDL in cells with and without the pre-incubation with 5 U/ml heparin or a cocktail of heparinases I, II, and II to test the contribution of heparan sulfate proteoglycans (HSPG).

Results:
LDLR
-KO HepG2 cells showed a 20-25% residual uptake of LDL compared to controls (p<0.001) and 10-fold higher levels of EL mRNA. Remarkably,
LIPG
-transfected
LDLR
-KO hepatocytes showed a 2-fold increase in LDL uptake compared to LDLR-KO cells non-overexpressing EL (p<0.001). The pre-incubation with either heparinases or heparin completely blocked the uptake of LDL not only in
LIPG
-transfected
LDLR
-KO cells but also in those
LDLR
-KO cells transfected with the empty plasmid, suggesting a crucial role of HSPG in the LDL uptake. Conversely, overexpression of EL decreased LDL uptake in control cells (-11%, p=0.02), while unchanged or slightly increased LDL uptake was observed after incubating with heparinases or heparin.

Conclusions:
Our findings demonstrate that EL facilitates the uptake of LDL through an LDLR-independent, HSPG-dependent pathway. This pathway may be responsible for the residual uptake of LDL observed in
LDLR
-KO hepatocytes and may represent a potential druggable target to treat FH. Our data provides an alternative mechanism to explain the reduction of LDL cholesterol induced by ANGPTL3 inhibitors.
Abstract 2047: Pioglitazone Improves Apolipoprotein A1-associated Proteomic Composition More Than Weight Loss In Insulin Resistance
Authors: Parsa Shyon , Collier Timothy S , McPhaul Michael , Mellander Olle , Knowles Josh , Rohatgi Anand , Abbasi Fahim
Published: July 2024

Insulin resistance (IR) is linked to an atherogenic dyslipidemia and an increased risk of ASCVD. Apolipoprotein A1 (ApoA1)-associated lipoproteins exert varying anti-atherogenic functions and have a large diverse proteome. It is, however, unknown whether IR contributes to atherogenic perturbations in these ApoA1-associated proteins. We thus tested the hypothesis that IR is associated with an atherogenic ApoA1 proteome and that insulin-sensitizing interventions improve the proteome to a more anti-atherogenic composition. The study included 861 participants without diabetes (age 48 ± 12 years, female = 65.5 %). We measured IR directly using the steady-state plasma glucose (SSPG) concentration during the insulin suppression test, where a higher SSPG indicates greater IR. The ApoA1-associated proteome lipoproteins were measured by mass spectrometry. A subgroup of participants underwent pioglitazone treatment (PIO; n = 38) or weight loss (WL; n = 70) for 3 months. Paired t-testing was used to quantify within-subject changes in outcomes of interest pre- to post-treatment. At baseline, several components of ApoA1-associated proteome significantly correlated with SSPG concentration (
Figure
). Both PIO and WL significantly improved IR (p<0.01). PIO led to significant increases in several ApoA1-associated proteins: ApoC1; ApoC2; ApoC3; ApoC4; ApoA2; ApoA4; ApoD; ApoE; ApoM; LCAT; LpPLA2; PLTP; PON1; and PON3. WL also led to significant increases in ApoA1-associated proteins: ANGT; ApoA4; ApoD; ApoM; LpPLA2; PLTP; PON1; and PON3. In conclusion, IR correlates with an atherogenic ApoA1-associated proteome. Both PIO and WL improve IR and the composition of the ApoA1-associated proteome, but PIO has a more pronounced effect on the proteome compared to WL. These findings support further exploration into the ApoA1-associated proteome for both understanding residual lipid-related ASCVD risk and targeted therapeutics for ASCVD reduction.
Abstract 2052: Macrophage-specific Ripk1 Knockdown Reduces Macrophage Inflammation And Atherosclerosis Progression In Ldlr
-/-
Mice.
Authors: Xu Han , Tapia Caceres Felipe , Eng Vik , Wang Yizhuo , Bisht Kavita , Cochran Blake , Rye Kerry Anne , Karunakaran Denuja
Published: July 2024

Background:
Chronic activation of the innate immune system drives inflammation and contributes to atherosclerosis. Pro-inflammatory agents (oxidised LDL, IL-1β) drive lesional inflammation and development, whereas anti-inflammatory agents (IL-10, HDL, Apolipoprotein-AI), promote an anti-inflammatory milieu and resolution. RIPK1 (Receptor interacting protein kinase 1) is a master regulatory gene that decides whether a cell undergoes inflammation, apoptosis and/or necroptosis, depending on its phosphorylation or ubiquitination status. We have previously observed that RIPK1 gene expression is increased in both endothelial cells and macrophages in early atherosclerotic lesions (Karunakaran et al, Circulation 2022). Here, we seek to determine whether macrophage-specific RIPK1 contributes to atherosclerosis.

Methods:
Given that Ripk1
-/-
mice are lethal, we performed bone marrow (BM) transplants of Ripk1
+/-
and wild-type (wt) littermate BM into female and male Ldlr
-/-
mice (n=8-12/grp) and mice were fed a western diet for 12-16 wks. In vitro experiments were performed in BM derived macrophages from wt and Ripk1
+/-
mice.

Results:
Ldlr
-/-
mice recipient of Ripk1
+/-
BM and western diet feeding for 12 or 16 wk had a marked reduction in aortic sinus lesion area (68.9% and 49.7% respectively, p<0.05) compared to their wt controls. Preliminary analysis show that there is a reduction in lesional macrophage area in Ripk1
+/-
BM recipient Ldlr
-/-
mice relative to control (0.02±0.01 vs 0.01±0.006mm, p<0.01), suggesting that macrophage infiltration was reduced. Interestingly, anti-inflammatory agents, including recombinant IL10, IL13 (both 100ng/ml), Apolipoprotein-AI and discoidal HDL (both 500ug/mL) markedly reduced macrophage Ripk1 expression in vitro (by ~50%, p<0.05), likely reducing Ripk1-dependent inflammation and promoting an anti-inflammatory milieu. Current mechanistic studies are investigating the transcriptional regulator, STAT3, as a key regulator of Ripk1 expression.

Conclusion:
We identified macrophage-RIPK1 as a central driver of atherosclerosis. These timely studies highlight the therapeutic potential of dampening Ripk1 overexpression, as opposed to the detrimental abolishment of Ripk1, in treating atherosclerosis.
Abstract 2059: Therapeutic Delivery Of MICRORNA-369-3p Promotes Atherosclerosis Regression In Diabetic Mice By Regulating Bone Marrow Myeloid Progenitors
Authors: Rawal Shruti , Wara Akm Khyrul , Sachan Madhur , de Oliveira Vaz Camila , Randhawa Vinay , Feinberg Mark W
Published: July 2024

Introduction:
Hyperglycemia-induced immunometabolic alterations promote myelopoiesis, increase proinflammatory monocytes, and impair atherosclerosis regression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet medical need. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages and regulate inflammation. Using miR-seq, we identified miR-369-3p as a novel miR expressed higher in regressing mouse plaques.

Hypothesis:
Therapeutic administration of miR-369-3p promotes plaque regression of diabetes-associated atherosclerosis in mice.

Aim:
To identify the therapeutic benefits of miR-369-3p overexpression, and the mechanism by which it promotes plaque regression in diabetic mice.

Methods:
Ldlr-/
-
mice were fed HFSC-diet for 12-weeks to develop atherosclerotic plaques and were switched to chow-diet for 6-weeks to induce regression. During regression, mice received either non-specific control mimic (NS-m) or miR-369-3p mimic (369-m) intravenously twice every week. Immunohistological, morphometric, and flow cytometric analyses were performed to define the changes in plaque, circulation, and bone marrow following 369-m delivery.

Results:
Therapeutic delivery of 369-m promoted plaque resolution in diabetic mice, evident by reduced plaque area and macrophage content when compared to NS-m injected mice. Circulating proinflammatory cytokines (IL1β, IL6 and TNF-α), total circulating monocytes, and M1 macrophages were decreased in lesions while M2 macrophages were increased in 369-m group. 369-m also increased plaque stability and increased FOXP3+ Tregs in plaques when compared to NS-m group. Bioinformatic analyses showed GPR91 and CEBPB as conserved miR targets. Mechanistically, we found that 369-m decreased bone marrow granulocyte-monocyte progenitor population (GMPs), resulting in less inflammatory monocytes, attributed to reduced CEBPB and GPR91 target expression in GMPs.

Conclusion:
miR-369-3p overexpression reduced plaque-inflammatory macrophages, bone marrow GMPs, and proinflammatory monocytes by regulating macrophage immunometabolism in diabetes-accelerated atherosclerosis in part by targeting CEBPB and GPR91.
Abstract 2097:

Jak2
V617f

Clonal Hematopoiesis Promotes Arterial Thrombosis Via Platelet Activation And Cross-talk
Authors: Liu Wenli , Pircher Joachim , Schulz Christian , Wang Nan
Published: July 2024

JAK2
V617F

(

JAK2
VF

) clonal hematopoiesis (CH) is associated with a marked increase in atherothrombotic cardiovascular disease (CVD). The role of

JAK2
VF

CH in arterial thrombosis has not been assessed. Using mice simulating

JAK2
VF

CH, we showed accelerated carotid artery thrombosis, with increased platelet counts and activation and increased leukocytes and red blood cells. There was increased platelet activation in both

Jak2
VF

and wild type (WT) platelets in

Jak2
VF

CH suggesting cross-talk between mutant and WT platelets. To assess platelet-specific effects, we used Gp1ba-Cre (VFGp1ba) to selectively express

Jak2
VF

in platelets. VFGp1ba increased platelet counts to a similar level as in 20%

Jak2
VF

CH mice but had no effect on leukocyte counts. Like

Jak2
VF

CH, VFGp1ba increased platelet activation and accelerated arterial thrombosis. Mechanistically,

Jak2
VF

platelets showed 2.8-fold up-regulation of COX-1, increased activation of cytosolic phospholipase A2, an enzyme important for arachidonic acid generation, and increased thromboxane A2 production. Conditioned media from activated

Jak2
VF

platelets induced WT platelet activation that was reversed by a thromboxane receptor antagonist. Low dose aspirin ameliorated carotid thrombosis in VFGp1ba and

Jak2
VF

CH but not in WT control mice. This study shows increased arterial thrombosis in

Jak2
VF

CH related to

Jak2
VF

platelet activation and cross-talk with WT platelets via thromboxane, suggesting a potential beneficial role of aspirin treatment in

JAK2
VF

CH.
Abstract 2114: Differences In The Circulating Transcriptome Between Peripheral Artery Disease And Coronary Artery Disease
Authors: Ni Richard , Muller Matthew , Ruggles Kelly , Barrett Tessa , Berger Jeffrey
Published: July 2024

Introduction:
Despite common risk factors, peripheral artery disease (PAD) and coronary artery disease (CAD) exhibit distinct pathological presentations. Previous gene expression profiling of blood from CAD and PAD patients shows alterations in inflammatory and immune related transcripts relative to controls. Studies have not directly compared gene expression in PAD versus CAD. Here, we aimed to use whole-blood RNA sequencing to investigate transcriptomic differences between PAD and CAD.

Methods:
Whole blood from subjects age 40-65 years with symptomatic PAD (n=20, mean age 59, 75% male, 35% white) and CAD (n=51, mean age 52, 82% male, 78% white) was collected into PAXgene tubes. Patients with concomitant CAD and PAD were excluded. RNA was isolated, sequenced and analyzed to investigate differential circulating gene expression between PAD and CAD subjects adjusting for age, sex, and race/ethnicity. Cell type deconvolution method dtangle was applied to characterize cell type proportions in the whole blood transcriptome.

Results:
Between PAD and CAD subjects, 106 transcripts were differentially expressed (adjP < 0.1), of which 38 were enriched in PAD and 68 were enriched in CAD. Gene set enrichment analysis (GSEA) revealed 29 pathways differentially expressed (FDR 0.05) between PAD and CAD, of which 8 were enriched in PAD and 21 enriched in CAD. The top enriched pathways in PAD were “specific granule” (Normalized enrichment score [NES] = 2.1, q value = 2.5 x 10
-5
) and “secretory granule membrane” (NES = 1.9, q = 1.0 x 10
-4
). Top enriched pathways in CAD were “T cell receptor complex” (NES = 3.1, q = 2.2 x 10
-7
) and “response to interferon-alpha” (NES = 3.1, q = 2.2 x 10
-9
). Cellular deconvolution using dtangle showed increased CD8 T-cells (p=0.01) and CD4 Naïve T-cells (p=0.007) in subjects with CAD versus PAD.

Conclusion:
Whole blood transcriptomics revealed significant differences in the circulating transcriptome between patients presenting with PAD and CAD. Our data suggest upregulation of pathways related to secretory granules in PAD, and enrichment of CD8 and CD4 Naïve T-cells and pathways related to T-cell signaling in CAD. Differences in the circulating transcriptome may help explain pathological and clinical differences between PAD and CAD.
Abstract 2128: Hepcidin Loss Demonstrates Sex Dependent Differences In Aortic Aneurysm Size
Authors: Kazaleh Matthew , Gioscia-Ryan Rachel , Gao Xiaohua , Golla Ashna , Scott Erik , Swaminathan Sundaraman , Hawkins Robert , Ailawadi Gorav , Salmon Morgan
Published: July 2024

Background:
Studies suggest circulating iron levels play a role in the pathophysiology of abdominal aortic aneurysms (AAAs). Low iron levels lead to medial degeneration and VSMC dysfunction while elevated iron levels promote oxidative stress. Hepcidin is a major iron regulatory hormone that binds ferroportin to reduce serum iron levels, and its removal in VSMCs has been shown to exacerbate AAAs; however, the role of sex in this mechanism remains understudied. The goal of the current study is to investigate the potential role of sex in iron metabolism in AAAs.

Methods:
Male and female C57BL/6 wild-type (WT/WT), heterozygous hepcidin knock-out (WT/KO) and hepcidin KO (KO/KO) underwent peri-adventitial application of porcine pancreatic elastase on the infrarenal aorta (n=10-19/group). At day 14, the aortas were measured using video micrometry and harvested for immunohistochemistry. In separate studies, male and female hepcidin WT/WT and KO/KO underwent elastase application and systemic b-aminopropionitrile (BAPN, 0.02%) administration and were harvested at 28 days. Finally, male and female LysM Cre Ferroportin flx/flx mice and their controls underwent elastase application and harvested at 14 days (n=5-10/group).

Results:
Hepcidin WT/KO and KO/KO male mice had dose dependent protection of aneurysm formation compared with controls (WT/WT: 145.5±33.8% vs WT/KO: 110.7±34.8% vs KO/KO: 53.7±13.7%, p=0.0001 by ANOVA). Female Hepcidin WT/KO and KO/KO mice exhibited increased AAA size compared with their controls (KO/KO: 119.0±23.5% vs WT/KO: 101.6 ±28.6% vs WT/WT: 71.2±22.0%, p =.0011 by ANOVA). In the elastase + BAPN model, Hepcidin KO/KO male mice had less aortic dilation compared to controls (KO/KO: 196.0 ±31.9% vs WT/WT: 476.8±82.2%, p =.0002 by Mann-Whitney) while female KO/KO mice exhibited greater aortic dilation (WT/WT: 186.7±26.0% vs. KO/KO: 406.9±62.2%, p=0.006). Male LysM Cre Ferroportin flx/flx mice had decreased aortic dilation (WT/WT: 132.5±19.3% vs flx/flx: 53.5±7.8%, p=0.001).

Conclusion:
Elimination of hepcidin in AAA formation exhibited protective effects in male and deleterious effects in female mice. This data suggests targeting of hepcidin using drug-based therapies could have gender-specific clinical implications.
Abstract 2130: Sex-based Differences In Patients With Thromboangiitis Obliterans (Buerger’s Disease): Insights From Nationwide Inpatient Sample
Authors: Li Wenzhu , Wu Fangqin , Hu Zhizhi , Ai Lisha , Su Guanhua
Published: July 2024

Introduction:
Thromboangiitis obliterans (TAO) has been characterized as an inflammatory disease predominantly affecting young male smokers. The epidemiologic spectrum of TAO has evolved, with increased proportion of women and non-smokers. However, sex-based differences among patients with TAO in real-world datasets remain unclear.

Research Question:
How do the TAO prevalence and limb amputation rate differ between women and men in a real world dataset?

Methods:
We assess sex-based differences in a large, administrative Nationwide Inpatients Sample (NIS) 2016-2019. The diagnosis of TAO and variables were identified by ICD-10-CM codes. Univariable comparisons between female and male patients were assessed by Mann-Whitney. Multivariate logistic models were used to analysis associations between sex and TAO prevalence, and cumulative limb amputation, adjusted with demographics characteristics and comorbidities.

Results:
In this cohort study of patients with TAO (N=836), 301 (36.0%) of them were women and 535 (64.0%) of them were men. TAO prevalence rate showed 96.4% higher in men compared to women (
Fig. 1A
, female:7.28%ooo, male: 14.30%ooo, p<0.001), with adjusted odd ratio (OR) at 1.51 (
Fig. 1C
, 95% CI, 1.30-1.76; p<0.001) for male. The cumulative limb amputation rate was up to 32.9% in male patients and 20.9% in female patients (
Fig.1B
, p<0.001), with adjusted OR at 2.22 (
Fig. 1C
, 95% CI; 1.51-3.26; p<0.001) for male patients.

Conclusions:
Male patients showed significant higher TAO prevalence and cumulative limb amputation rate. Notably, although TAO is a rare vascular disease, the cumulative limb amputation rate was up to 32.9% in male and 20.9% in female patients. The sex-based disparities spur further studies on subgroup analysis, mechanism, health equity and multidisciplinary management for patients with TAO.
Abstract 2139: Galectin-3 Plays A Critical Intracellular Role In Macrophages By Promoting The Repair, Removal, And Replacement Of Damaged Lysosomes And Protecting Against Atherosclerosis
Authors: Liu Ziyang , Khan Saifur Rahman , Rodriguez-Velez Astrid , Zhang Xiangyu , Razani Babak
Published: July 2024

Galectin-3 (Gal3) is a β-galactoside-binding lectin predominantly known as a secreted inflammatory biomarker in cardiovascular disease, with significantly elevated circulating levels in patients with atherosclerosis and myocardial infarction. However, the molecular mechanisms of Galectin-3 action and whether its intracellular role contributes to atherogenesis remain unclear. Using several databases of bulk and single cell RNAseq, we first show that Gal3 transcripts are upregulated during plaque progression with particular abundance in the myeloid/macrophage lineage and foamy macrophages. Furthermore, Gal3 transcripts correlate significantly with increases in a network of lysosomal genes, suggesting a possible link between macrophage Gal3 and lysosomal function. Indeed, instigation of lysosome membrane damage in primary macrophages via cholesterol crystals and LLOMe triggers robust recruitment of Gal3 to lysosomes by sensing exposed carbohydrate moieties of proteins including Lamp1. Gal3 recruitment initiates a two-pronged lysosomal recovery program composed of either lysosomal repair involving the ESCRT complex or lysosomal removal and replacement involving autophagy/lysophagy and TFEB-mediated lysosomal biogenesis. Gal3-KO macrophages corroborate these findings displaying blunted ESCRT recruitment, diminished autophagy and TFEB activation, and resultant accumulation of damaged lysosomes. Gal3-deficiency also results in enhanced apoptosis and inflammasome/IL-1β activation upon triggering of lysosomal stress in macrophages. These findings are recapitulated in vivo, where Gal3-null bone marrow transplanted in atherogenic LDLR-null mice yields increased lesion size as well as altered plaque composition with macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of the advanced plaque. Taken together, our data implicate unique and previously unrecognized protective function for intracellular Galectin-3 in macrophages and atherosclerosis which are distinct from its traditional role as an inflammatory biomarker in cardiovascular disease.