bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1126: Thiamine Derivative Enhances Mitochondrial Function To Reduce Vascular Calcification
Authors: Choi Eun Jung , Chanda Dipanjan , Sinam Ibotombi Singh , Kim Min-Ji , Jeon Jae-Han , Lee In-Kyu
Published: July 2024

Introduction/Background:
Vascular calcification significantly impacts cardiovascular disease and mortality. It's exacerbated by mitochondrial dysfunction in vascular smooth muscle cells (VSMCs), influenced by factors such as calcium and phosphate. This study examines the role of thiamine derivatives in improving mitochondrial function, potentially reducing vascular calcification.

Research Question/Hypothesis:
We hypothesize that thiamine derivatives enhance mitochondrial function, thereby attenuating vascular calcification.

Goals/Aims:
The aim is to assess the efficacy of thiamine derivatives in enhancing mitochondrial integrity and function to mitigate vascular calcification.

Methods/Approach:
VSMCs were treated in vitro with phosphate and calcium chloride to induce calcification, along with TTFD (Thiamine tetrahydrofurfuryl disulfide) treatment. Similarly, C57BL/6J mice were administered thiamine derivatives in vivo before inducing aortic calcification. Analysis included calcium deposition, apoptosis, and mitochondrial function assessment.

Results/Data:
TTFD significantly reduced vascular calcium deposition in VSMCs, both in vitro and in vivo. Mitochondrial function improvement was evidenced by increased oxygen consumption rates and JC-1 aggregate levels. Additionally, TTFD mitigated mitochondrial fission by inhibiting Drp1 phosphorylation at serine 616, leading to reduced apoptosis and osteogenic gene expression.

Conclusion:
Thiamine derivatives, particularly TTFD, demonstrate potential in mitigating vascular calcification through enhanced mitochondrial function and inhibition of mitochondrial fission. These findings highlight the therapeutic potential of thiamine derivatives in treating vascular calcification-related conditions.
Abstract 1144: Comparison Of Ahora And Rapido Mnemonics In Effectively Communicating Stroke Signs In Spanish
Authors: Bhati Maya , Singh Neeraj
Published: July 2024

Introduction:
Strokes affect 15 million people worldwide annually, making them a leading cause of death and disability. Mnemonics have been developed in multiple languages to inform the public how to recognize stroke signs, including the English “BEFAST.” For Spanish-speaking populations, the mnemonics “AHORA” and “RAPIDO” are both circulated with pictorial brochures. This study compares the effectiveness of both mnemonics in helping people recognize stroke signs, which can guide us on how to develop mnemonics to communicate similar information in other languages.

Hypothesis:
The brochures with AHORA and RAPIDO mnemonics are equally effective in communicating the strokes signs to recognize to Spanish-speaking populations.

Methods:
Between January and December 2023, 50 Spanish-speaking patients and relatives were interviewed in the Northwell Health Neurosciences clinic in New York. Each participant was shown the AHORA and RAPIDO brochures and asked to identify for each handout the purpose, favorable features, and ranking of quality on a scale of 0-5. To mitigate presentation bias, half of the participants viewed the AHORA brochure first and the other half viewed the RAPIDO brochure first. A paired t-test was conducted to determine the statistical significance of the difference in average ranking of each brochure.

Results:
The AHORA brochure received an average ranking of 3.66/5.00, significantly higher than the average ranking of 3.36/5.00 for the RAPIDO brochure (p=0.0096). Positive comments about the AHORA brochure included the brochure appearing concise, the mnemonic being shorter, and the emphasis on calling 911 when stroke signs are recognized. Comments about the RAPIDO brochure included the brochure including excessive information and the mnemonic being longer and more difficult to understand.

Conclusions:
Participants preferred the AHORA brochure over the RAPIDO brochure due to its more concise mnemonic, clearer descriptions of stroke signs, and emphasis on calling 911 when stroke signs are recognized. These findings provide valuable guidance for physicians in selecting brochures for Spanish-speaking patients and offer insights into the elements necessary to develop effective mnemonics in other languages.
Abstract 1150: Effect Of Diabetes On Clinical Outcomes In Patients With Peripheral Arterial Disease Undergoing Lower Extremity Revascularization: A Systematic Review And Meta-analysis
Authors: EDJIMBI JOHANN ALEXANDRE CHAFA , Aziri Buena , Begic Edin , Cleman Jacob , Smolderen Kim G , Mena-Hurtado Carlos
Published: July 2024

Background:
Peripheral arterial disease (PAD) is highly prevalent and has a well-known association with diabetes. It is still unknown if diabetes worsens clinical outcomes after lower extremity revascularization (LER).

Research question:
What is the impact of diabetes on clinical outcomes in patients with PAD undergoing LER.

Goals:
In this meta-analysis, we assessed the effect of diabetes on mortality, major limb amputation, and major adverse cardiovascular events (MACE) in patients with PAD following endovascular or open LER during the perioperative period and within 30 days of follow-up.

Methods:
We performed a systematic search of PubMed, Embase, and Cochrane databases to December 2023, including studies that compared the clinical outcomes of patients with diabetes and without diabetes following LER procedure. Review Manager 5.4 was used for statistical analysis. I
2
statistics were used to examine heterogeneity. A random-effects model was applied to all analyses.

Results:
Of the 3,810 articles screened, five observational studies with 55,444 patients were included. A total of 51.13 % had diabetes. There was no significant association between diabetes and mortality (RR 0.96; 95 % confidence interval (CI) 0.74 - 1.26; I
2
= 69%; P = 0.79; Figure 1A). However, diabetes was associated with a significantly increased risk of major limb amputation by 50% (RR 1.50; 95 % CI 1.03 - 2.21; I
2
= 94%; P = 0.04; Figure 1B), and an 18% significantly higher risk of experiencing MACE (RR 1.18; 95 % CI 1.08 - 1.29; I
2
= 0%; P = 0.0005; Figure 1C).

Conclusion:
Our findings suggest that diabetes is associated with an increased risk of major amputation and MACE but not with mortality in patients following LER.
Abstract 1153: Adipose-derived Mesenchymal Stem-cell Therapy Improves Arteriogenesis, Hemodynamics, And Walking Performance In A Porcine Model Of Peripheral Artery Disease
Authors: Hakim Ali H , Tian Yuqian , Al-Gahmi Al-Murtadha , Brunette Katya , Kim Julian K , zhu zhen , Park Song-young , Casale George , CARLSON MARK A , Pipinos Iraklis I
Published: July 2024

Background:
There are few effective alternatives to revascularization for peripheral artery disease (PAD). We used our porcine model of PAD to test the hypothesis that adipose derived mesenchymal stem cells (ADMSC) injected around the aortic trifurcation in the pelvis improve ischemic limb hemodynamics and walking performance (WP).

Methods:
In Ossabaw miniswine (N=13, Age=6 mo) with metabolic syndrome, right (R) hindlimb ischemia was induced with R External Iliac and Superficial Femoral artery coil occlusion (T0) via carotid artery access. All swine had weekly testing of WP on a treadmill. At weeks 0 (T0), 4 (T4), and 8 (T8), ankle brachial indices (ABI) and muscle tissue oximetry (STO
2
) were measured. At T4 and T8 all swine underwent angiography. At T4, in treatment (Tx) swine (N=4) ADMSC were isolated using stromal vascular fractionation of autologous abdominal wall fat. Using jugular vein access, a catheter with a retractable side-deploying needle was inserted in the R iliac vein and stabilized by a juxtaposed angioplasty balloon. The needle was deployed to access the retroperitoneum and ADMSC were infused around the aortic trifurcation.

Results:
Ischemic controls (IC) had no differences between T4 and T8 in ABIs, and mean STO
2
differences between R and L in calves and thighs. Tx improved both ABI and mean STO
2
measurements (table 1). IC had no changes in WP between T4 and T8, while Tx showed improvements (table1). On angiography, IC had growth of R Internal iliac artery (RIIA, the main inflow to the ischemic R hindlimb) and shorter appearance time (AT) of RIIA compared to L with no difference between T4 and T8. Tx showed increased growth of RIIA and shorter AT at T8 compared to T4 (table 1).

Conclusion:
In a clinically-relevant (similar to humans in size, anatomy, physiology and comorbidities) porcine model of PAD we found that ADMSC injection around the aortic trifurcation in the pelvis induces arteriogenesis and improves ischemic limb hemodynamics and walking performance.
Abstract 1156: Akkermansia (Strain BAA835) And The Microbial ADP-heptose Pathway Are Associated With Reduced Neointimal Hyperplasia After Arterial Injury
Authors: Ho Karen J , Niraula Sarbjeet , Pamulapati Vivek , Jung Jonathan , Jang Jae Woong , Plunkett Catherine , Xiong Liqun , Du James , Valdivia Raphael , Green Stefan , Seed Patrick
Published: July 2024

Introduction:
Despite advances in surgical therapy for atherosclerosis, up to 50% of procedures such as balloon angioplasty, stenting, and bypass fail due to restenosis from neointimal hyperplasia, a cell proliferative process potentiated by inflammation. We hypothesized that neointimal hyperplasia is associated with a distinct gut microbiome profile and can be modulated by fecal transplantation.

Methods:
We performed fecal transplantation (FT) of C57BL6 mice using donors with differential microbiome profiles and susceptibility to neointimal hyperplasia (Sprague-Dawley [SD] and Lewis [LE] rats, high and low susceptibility, respectively). In separate experiments, germ-free mice underwent FT using C57BL6 donors with and without
Akkermansia muciniphila
BAA835 (
Amuc
) prior to arterial injury. Microbial communities were characterized using 16S rRNA gene amplicon and shotgun metagenome sequencing. Arterial and immune effects were assessed using frozen arterial sections, multi-parameter flow cytometry of mesenteric lymph nodes, or multiplex cytokine assays after ex vivo stimulation of whole blood with lipopolysaccharide.

Results:
Paradoxically, neointimal hyperplasia after injury was 67.7% greater in FT recipients of low- compared to high-susceptibility stools (P=0.01). In fecal communities of both FT recipient groups, relative abundance of
A. muciniphila
(r=-0.66, P<0.001) and of the ADP-heptose biosynthesis pathway (r=-0.7, P<0.001) were negatively associated with neointimal hyperplasia.
A. muciniphila
was differentially elevated (P<0.001) in SD compared to LE FT recipients and was the main contributing taxa for the enrichment (P<0.001) of the ADP-heptose biosynthesis pathway in the SD recipient group. In mice colonized with communities that differed only by the presence of
Amuc
, neointimal hyperplasia was 57.8% greater in the
Amuc
- group compared to
Amuc+
(P=0.01).
Amuc+
recipients had lower levels of IL-12, MIP-1beta, and MCP-1 in unstimulated blood and higher IP-10 and MCP-1 levels in stimulated blood.

Conclusion:
A. muciniphila
and microbial ADP-heptose biosynthesis pathway are inversely associated with neointimal hyperplasia and are potential targets for novel microbe-based strategies to mitigate restenosis risk.
Abstract 116: Apolipoprotein E Registry In Discoidal Lipoproteins Influences Uptake
Authors: Strickland Michael R , Summers Brock , Basore Katherine , Wulf John , Jiang Hong , Sorci Thomas Mary G , Davis Albert A , Cashikar Anil G , Holtzman David M , Randolph Gwendalyn J
Published: July 2024

Introduction:
Apolipoprotein E (apoE) is of great interest due to its role as a risk factor for Alzheimer's disease and atherosclerosis. ApoE is secreted by astrocytes in the central nervous system as HDL-like lipoparticles. Currently there is no high-resolution structural model of lipidated apoE, resulting in a limited mechanistic understanding of how apoE functions in health and disease.

Research Questions:
The goal of this research was to utilize cryoEM to characterize the structure of apoE in discoidal lipoproteins.

Methods:
Astrocyte secreted apoE was immunopurified from culture media and characterized by TEM and cryoEM. Recombinant apoE (rapoE) was lipidated with DMPC to model astrocyte secreted lipoproteins.

Results:
TEM and cryoEM imaging of astrocyte apoE and rapoE found that apoE forms a dimer on discoidal lipoproteins. These results provide support for the “double belt” model of two apoE in an anti-parallel conformation. This conformation was independent of apoE isoform, suggesting it is a general feature of apoE in discoidal lipoproteins. ApoE isoforms differ by the addition or loss of a Cys residue. ApoE2 has 2 Cys residues (Cys112, Cys158), apoE3 has 1 Cys residue (Cys112), and apoE4 has no Cys residues. Accordingly, apoE4 cannot form disulfide bonds, apoE3 can form intermolecular disulfide bonds, and apoE2 can form both intramolecular and intermolecular disulfide bonds. Due to the presence of two apoE on discoidal lipoproteins, apoE3 can form a disulfide bridge between the two apoE protein. Disulfide bridge formation and experimental reduction of these disulfide bridges revealed that disulfide-linked apoE3 was uptaken significantly more by Neuro2a cells (P ≤ 0.0001) than nondisulfide-linked apoE3.

Conclusion:
All isoforms of apoE adopt an antiparallel dimer in discoidal lipoprotein. Different registries, regulated through the formation of disulfide bonds between the anti-parallel apoE polypeptides, leads to differential uptake of apoE by Neuro2a cells. Since the different isoforms of apoE have inherently different capacity to generate disulfide bridges, we conclude that this property affecting receptor uptake may strongly impact differential biological properties of the different isoforms.
Abstract 118:
Dennd5b
Affects Dietary Lipid Absorption And Metabolism In Male And Female Mice
Authors: Neupane Khaga , Karakashian Alexander , Mobilia Maura , Hage Olivia , Gordon Scott M
Published: July 2024

Background:
Dietary lipid absorption by the intestine is essential for systemic lipid homeostasis. We previously demonstrated a role for
Dennd5b
in intestinal lipid absorption. In humans,
DENND5B
gene variants are correlated with BMI in females, but not males. Female

Dennd5b
-/-

mice have reduced appearance of dietary triacylglyceride (TAG) in plasma and are resistant to diet-induced obesity, however, the metabolic impact of

Dennd5b
-/-

in male mice has not been reported. In this study, we test the hypothesis that the

Dennd5b
-/-

mouse model recapitulates the sex disparity observed in humans with
DENND5B
polymorphisms. We also investigate the fate of unsecreted intestinal TAG in this model of impaired dietary lipid absorption.

Methods and Results:
A non-absorbable fatty acid (FA) tracer was used to quantify the impact of
Dennd5b
-deficiency on lipid absorption in male and female mice (n=7-9 mice/genotype/sex). We observed significant, but relatively modest, reductions in lipid absorption efficiency in

Dennd5b
-/-

mice in both sexes, despite a complete absence of plasma TAG after oil gavage. We hypothesized that this was due to metabolic utilization of TAG by enterocytes. Metabolic cage studies showed that both wildtype and

Dennd5b
-/-

mice shift toward utilization of FAs when fed high-fat diet and Seahorse analysis revealed altered metabolic activity in

Dennd5b
-/-

intestinal tissue. Electron microscopy (EM) revealed large electron dense structures that resemble autophagosomes in

Dennd5b
-/-

enterocytes. Western blots detected altered Lc3 levels in

Dennd5b
-/-

intestine, implicating autophagy. RNA sequencing of intestinal tissue detected changes in expression of genes involved in beta oxidation and EM imaging revealed altered mitochondrial morphology in

Dennd5b
-/-

enterocytes.

Conclusions:
We conclude that autophagy contributes to degradation of unsecreted TAG in the

Dennd5b
-/-

enterocytes, liberating free FAs which are utilized in mitochondrial oxidation. The sexually dimorphic effect of
Dennd5b
does not appear to be as prominent in mice as it is in humans. Overall, our findings demonstrate that
Dennd5b
plays a critical role in TAG secretion by intestinal tissue and that disruption of this process can impact systemic metabolism in both sexes.
Abstract 126: Reduction Of Abdominal Aortic Aneurysm Rupture By Modulating Smooth Muscle Cell Phenotype Switching
Authors: Bontekoe Jack , Zhou Ting , Yang Huan , Conte Michelle , Silva-Pedraza Zulmari , Morgan Courtney , Aufhauser David , Dimusto Paul D , Liu Bo
Published: July 2024

Background:
Vascular smooth muscle cell (SMC) loss and phenotype switch promote abdominal aortic aneurysm (AAA) development, yet whether strategically enhancing positive aortic wall remodeling can reduce AAA rupture is unknown. We hypothesize inhibiting necroptosis signaling in SMCs may drive differentiation into resilient and regenerative phenotypes, thus reinforcing AAA wall integrity and rupture resistance.

Methods:
We developed Cre-loxP transgenic mice with SMC-specific loss-of-function mutation of the necroptosis regulatory gene,
Ripk3
(
Ripk3
Δ). AAAs were induced using the topical elastase plus β-aminopropionitrile murine model.

Results:
Rupture-free survival at 56-days post-AAA induction was drastically improved with both homozygous and heterozygous loss of SMC RIPK3 function (
Ripk3
Δ

HO

: 100%,
Ripk3
Δ

Het

: 92.3% versus
Ripk3
Δ

WT

: 47.1%).
Ripk3
mutant aortas demonstrated rupture-resistance despite substantial aneurysmal dilations ranging 127-527%, and morphologically displayed significant medial thickening with SMC expansion. Bulk RNA-sequencing revealed stark transcriptional changes between 14-day
Ripk3
Δ

HO

and
Ripk3
Δ

WT

aortas. Both genotypes showed typical aneurysmal processes, however,
Ripk3
depletion greatly reduced aneurysm-induced inflammatory responses. We identified 54 uniquely altered transcripts in mutant tissues, coding for cell cycle regulators and extracellular matrix proteins. Ongoing tissue analysis will determine whether regenerative, matrix-producing SMCs vary among human AAAs and healthy aortas.

Conclusions:
AAA rupture is significantly reduced by inhibiting RIPK3 in SMCs, likely by promoting positive structural adaptations and down-regulating classic pathways of aneurysmal degeneration. We postulate pharmacologically inhibiting RIPK3-mediated SMC modulation could be a novel approach in AAA patient management, to reduce rupture risk from diagnosis to optimal timing of surgical repair.
Abstract 141: Lesion Site-specific Mass Spectrometry Analysis Of Human Carotid Plaques Reveals Strongest Association Of The Necrotic Core And Fibrous Cap Proteome With Plaque Stability
Authors: Sachs Nadja , Sinha Ankit , Kratz Elena , Pauli Jessica , Engl Marie-Luise , Winter Hanna , Eckstein Hans-Henning , Mann Matthias , Maegdefessel Lars
Published: July 2024

The spatial impact of the plaque proteome on atherosclerotic plaque stability remains largely unknown. In this study, we performed mass spectrometry-based proteomics of various carotid plaque subregions (media, necrotic core, fibrous cap), using guided laser capture micro-dissections (LCM) of FFPE tissue in a matched cohort of 112 patients undergoing carotid endarterectomy (CEA) to study the impact of the plaque proteome, not only on lesion stability, but also on subregional changes of expression. Further, we performed a histological analysis according to the AHA classification and combined the data with clinical information on risk factors, comorbidities, and patient symptoms. Our LCM mass spectrometry analysis yielded over 3000 proteins for each plaque subregion. Gene set enrichment analysis revealed a high abundance of extracellular proteins and
vice versa
for cellular proteins. Subregion analysis identified the necrotic core and the fibrous cap to display the most significant changes in proteins associated with the stability status, whereas the media proteome remained largely unaffected. Primary and secondary enrichment analysis revealed synthetic vascular smooth muscle cells as the major source of these proteins. In addition, we analyzed the serum proteome of more than 500 patients (including non-athero control patients) with overlap from the tissue cohort. We combined these with histological evaluation and a complete set of clinical data. We used these two datasets for developing tissue and serological markers associated with plaque stability. In summary, our approach utilized LCM mass spectrometry, histological evaluation, and clinical data to analyze the association of different lesion subregions on plaque stability. We are able to demonstrate a high through-put method to identify potential protein markers relevant to atherosclerotic plaque stability.
Abstract 151: Single Cell Transcriptional And Epigenetic Map Of Human Coronary Artery Disease In A Multi-ethnic Population
Authors: Amrute Junedh M , Lee Paul , Eres Ittai , Bredemeyer Andrea , Yamawaki Tracy , Kang Chul Joo , Lee Mick J , Gurung Rijan , Li Daniel , Zhao Quanyi , Sheth Maya , Tan Anthony , Gschwind Andreas , Cheng Paul , Jackson Simon , Hsu Yi-Hsiang , Foo Roger , Hall Ira , Gupta Rajat M , Engreitz Jesse , Li Chi-Ming , Quertermous Thomas , Ason Brandon , Lavine Kory J , Stitziel Nathan O
Published: July 2024

Identifying target genes that are causally linked to coronary artery disease (CAD) remains an elusive problem despite the plethora of CAD genome-wide association study (GWAS) risk loci. Here, we performed single nucleus multiomic (RNA + ATAC) sequencing in 44 human coronary arteries and bulk Hi-C in 16 coronaries from multi-ethnic participants. From sequencing 126,804 nuclei, we identified cell type specific transcriptional signatures, chromatin peaks, and gene regulatory networks through peak to gene linkage analysis. Single-cell quantitative trait loci (QTL) analysis identified cell-specific caQTL’s including cell-type specific caQTLs within CAD GWAS loci. The overlap of CAD GWAS loci with ATAC peaks demonstrated that vascular smooth muscle and endothelial cells harbor the greatest genetic risk. To identify proximal and distal putative target genes for CAD loci in a cell type specific manner, we built a enhancer-gene map by leveraging coronary artery ChiP-seq, cell specific RNA/ATAC-seq, and Hi-C based chromatin contact loops. Using this map, we identified distal target genes linked to CAD GWAS loci. Finally, given the effect of ancestry on CAD GWAS risk interpretation, we leveraged Hi-C data to compare the chromatin architecture in coronary tissue from 8 European Americans vs 8 African Americans. A meta-map, sample level, and local ancestry analysis identified ancestry-specific differences in chromatin looping and topologically associated domains. Collectively, our results provide a cell-type specific transcriptomic and epigenetic map which along with bulk chromatin contacts can be used to interpret and prioritize CAD GWAS candidates and nominate cell specific causal target genes.
Abstract 157: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease
Authors: Pan Huize , Ho Sebastian E , Xue Chenyi , Cui Jian , Johanson Quinian , Sachs Nadja , Ross Leila , Li Fang , Solomon Robert A , Connolly Edward , Patel Virendra , Maegdefessel Lars , Zhang Hanrui , Reilly Muredach P
Published: July 2024

Background:
Atherosclerosis, a leading cause of cardiovascular disease, involves the pathological activation of various cell types, including immunocytes (e.g., macrophages, T cells), smooth muscle cells (SMCs), and endothelial cells. Accumulating evidence suggests that transition of SMCs to other cell types, known as “phenotypic switching,” plays a central role in atherosclerosis development and complications. However, the characteristics of SMC-derived cells and the underlying mechanisms of SMC transition in disease pathogenesis remain poorly understood. Our objective is to characterize tumor cell-like behaviors of SMC-derived cells in atherosclerosis, with the ultimate goal of developing interventions targeting SMC transition for the prevention and treatment of atherosclerosis.

Methods:
We used SMC lineage tracing mice and human tissues and applied a range of methods, including molecular, cellular, histological, computational, human genetics, and pharmacological approaches to investigate the features of SMC-derived cells in atherosclerosis.

Results:
SMC-derived cells in mouse and human atherosclerosis exhibit multiple tumor cell-like characteristics, including genomic instability, evasion of senescence, hyperproliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. Specific expression of oncogenic mutant,

Kras
G12D

, in SMCs accelerates phenotypic switching and exacerbates atherosclerosis. Furthermore, we provide a proof of concept that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models.

Conclusions:
Our findings demonstrate that atherosclerosis is a SMC-driven tumor-like disease, advancing our understanding of its pathogenesis and opening prospects for innovative precision molecular strategies aimed at preventing and treating atherosclerotic cardiovascular disease.
Abstract 2021:
18
F-Sodium Fluoride Positron Emission Tomography To Assess Thoracic Aortopathy In Bicuspid Aortic Valve Disease
Authors: Nash Jennifer , Debono Samuel , Loganath Krithika , Whittington Beth , Tzolos Evangelos , Syed Maaz , Mitic Tijana , Tavares Adriana , Macaskill Mark , Clark Tim , Semple Scott , Van Beek Edwin , Dey Damini , Slomka Piotr J , Forsythe Rachael , Williams Michelle , Dweck Marc , Walker Niki , Newby David E , Fletcher Alexander J
Published: July 2024

Background:
Bicuspid aortic valve is associated with thoracic aortopathy with many patients suffering aortic dissection below the aortic diameter threshold for prophylactic aortic replacement surgery. In aortopathy, the aortic wall media initially undergoes increasing microcalcification due to elastin fibre fragmentation but with disease progression, elastin fibre degradation leads to loss of aortic microcalcification.
18
F-Sodium fluoride positron emission tomography (PET) is a non-invasive method of detecting arterial microcalcification that has been successfully used in cardiovascular disease risk stratification.

Aims:
To determine if aortic
18
F-sodium fluoride imaging can identify aortopathy and predict subsequent disease progression in patients with bicuspid aortic valve disease.

Methods:
In this prospective cohort study, 77 patients with bicuspid aortic valve from clinical sites across Scotland and 18 healthy volunteers, underwent
18
F-sodium fluoride PET-computed tomography (PET-CT) followed immediately by PET-magnetic resonance imaging (PET-MRI). Maximum standardised uptake values (SUVmax) were determined in the ascending aorta. Aortic diameters were measured in aortic axial CINE MRI sequences. Patients were followed-up at 24 months with a further PET-MRI. Annualised change in aortic size index (mm/m
2
/year) and annualised change in SUVmax stratified in tertiles were calculated.

Results:
Patients with bicuspid aortic valve disease had higher SUVmax than control subjects, especially in those with aortic dilatation. Baseline SUVmax was associated with annualised aortic expansion rate independent of baseline aortic size (β=0.45, p=0.001). Patients in tertile 1 (loss of SUVmax over time) exhibited greater aortic growth (0.60±0.50 mm/m
2
/year) than those with stable SUVmax (tertile 2, 0.26±0.36 mm/m
2
/year) or increasing SUVmax (tertile 3, 0.24±0.36 mm/m
2
/year; p=0.01).

Conclusion:
Aortic microcalcification activity is increased in aortopathy of patients with bicuspid aortic valve. Loss of aortic microcalcification is associated with more rapid disease progression and represents a promising new approach to the identification of patients at higher risk of aortic complications.
Abstract 2054: Adoptive Transfer Of Human Cd34+ Cells Promote Atherosclerosis In Rag2-ko/il2rg-ko/cd47-ko Immunocompromised Mice Independently Of Hypercholesterolaemia
Authors: Norata Giuseppe Danilo D , Moregola Annalisa , Bonacina Fabrizia
Published: July 2024

Background and aim:
Adaptive immune cells are involved in the development of atherosclerosis. Here we provide the immunemetabolic profile of an atheroprone immunodeficient mouse humanized with hCD34+ cells.

Methods:
LDLR-KO mice were crossed with the immunodeficient Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) mouse model to generate an immunocompromised dyslipidemic mouse (TKO-LDLR KO mice). These mice present an intact innate immune system but lack B and T lymphocytes as well as NK cells. New born mice (within three days) were sub lethally irradiated (200 cGy) and received 2 x 10^5 hCD34+ cells.

Results:
when fed a cholesterol rich diet, non humanized TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet. Moreover, they develop marked dyslipidemia (plasma chol levels >1200mg/dL), liver steatosis and moderate atherosclerosis at the aortic arch. The engraftment of human leukocytes (hCD45+) in the humanized group was evaluated after two months by flow cytometry analysis. Within the totality of circulating CD45+ cells, 10 to 30% were hCD45+, mainly B and T cells. Of note the presence of human lymphocytes was associated with a dramatic worsening of atherosclerosis as compared to non-humanized TKO-LDLR KO mice which was independent of plasma cholesterol levels which remained ex.

Conclusion:
Adoptive transfer of human CD34+ cells in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO immunocompromised mice promotes atherosclerosis independently of hypercholesterolemia, by affecting the maturation and distribution of the different B and T lymphocyte subsets.
Abstract 2056: Impaired Cellular Metabolic Adaptations In Autosomal-Dominant Hyper-IgE Syndrome: Possible Implications To Deficient Angiogenesis And Wound Healing
Authors: Joseph Benjamin T , Dmitrieva Natalia I , Nguyen Dai , Boehm Manfred
Published: July 2024

Background:
Autosomal dominant Hyper-IgE syndrome (AD-HIES; Job’s syndrome) is caused by dominant negative mutations in signal transducer and activator of transcription 3 (STAT3). Patients present with immunodeficiency accompanied by severe non-immunological features including poor post infection lung healing, subsequent pulmonary failure, and skeletal, connective tissue, and vascular abnormalities including arterial tortuosity and aneurisms which can lead to myocardial infarction and subarachnoid hemorrhage. HIF1a-dependent deficient angiogenesis has been implicated in the abnormal wound healing response of AD-HIES. Both STAT3 and HIF1a are major regulators of cellular metabolism. In this study we are testing the hypothesis that dysregulation of cellular metabolic pathways may contribute to AD-HIES pathophysiology.

Methods:
We used skin fibroblasts (SF) from 4 AD-HIES patients and 4 normal volunteers. To investigate ability of the cells to adapt and function in nutrient-deficient conditions (to model tissue damages disrupting normal tissue perfusion) we exposed them to a culture media lacking glucose, pyruvate and glutamine (deficient media). We used a live imaging approach (IncucyteS3 System, Sartorius) to analyze cell proliferation, reactive oxygen species (ROS), cell death, and wound closing ability (scratch-wound assay).

Results:
In deficient media, both AD-HIES and control SF decreased their proliferation rate. While control SF adapted, AD-HIES SF showed decreased survival after prolong nutrient deprivation. Level of ROS, measured at 12, 24, 48h in deficient media was 25% higher in AD-HIES SF (n=8, P=0.003). We used a scratch-wound assay to assess the ability of fibroblasts to migrate and close the wound after 48h of adaptation to deficient media. In full media, both control and AD-HIES fibroblast completely closed the wound within 36h. In deficient media, wound closure was delayed to much higher degree by AD-HIES fibroblasts (60±14% closure by control SF vs 18±9% by AD-HIES SF, n=4, P=0.016).

Conclusions:
Adaptive remodeling of cellular metabolism is impaired in AD-HIES SF that may contribute to deficient angiogenesis and poor wound healing, suggesting possible therapeutic targets within cellular metabolic pathways.
Abstract 2075: Vascular Smooth Muscle Cell Derived Pdgfd Promotes Vascular Remodeling And Plaque Progression In Atherosclerosis
Authors: Monteiro João P , Weldy Chad S , Worssam Matthew , Palmisano Brian , Li Daniel , Gu Wenduo , Ramste Markus O , Sharma Disha , Zhao Quanyi , Kundu Ramendra K , Nguyen Trieu , Cheng Paul , Quertermous Thomas
Published: July 2024

We have recently identified rs2019090 and
PDGFD
as the functional variant and gene mediating CAD risk at the 11q22.3 locus, with our initial analysis using a global knockout (KO) model showing that this gene may promote phenotypic changes in smooth muscle cells (SMCs) in the plaque and contribute to neointimal vascular calcification. Nonetheless, the specific cell-type and phenotypic states through which PDGFD may confer disease risk remains unexplored.To delineate the impact of SMC-derived PDGFD signalling on cell state transitions and plaque progression in atherosclerosis, we have developed a novel SMC-specific lineage tracing and
Pdgfd
KO mouse (

Pdgfd
ΔSMC/ΔSMC
, Myh11
CreERT2
, ROSA
tdT/+
, ApoE
-/-

) allowing us to confidently define the impact of SMC-derived Pdgfd in the vascular SMC lineage as well as in neighbouring populations. Leveraging our lineage tracing KO mutants on a hypercholesterolemic diet, we have employed single cell RNA sequencing (scRNAseq) and histological analyses to characterize the cellular and molecular effect of Pdgfd in vascular disease. SMC-specific Pdgfd deletion resulted in alterations in the distribution of transitioning SMC populations, as well as previously unexplored gene expression differences within these cell types. This was accompanied by a significant reduction in atherosclerotic burden and plaque size across the aorta, including aortic root as well as descending abdominal aorta. Histological analysis revealed decreased monocyte recruitment, show by quantifying CD68+ cells within the plaque. To explore this further we interrogated the scRNAseq dataset to identify major pathways of cell-cell communication and the impact of altered Pdgfd signalling across different cell types. Overall, these data reveal that SMC-derived PDGFD substantially alters lesional SMC cell phenotype transitions, as well as inflammatory cell recruitment, implicating it as a major regulator of atherosclerotic disease progression.
Abstract 2119: Transcriptomic Profiling Reveals Therapeutic Potential Of Docosahexaenoic Acid For SARS Coronaviruses Infection Management
Authors: Huang Shiqi , TAYLOR Carla G , Zahradka Peter
Published: July 2024

Prior work from our laboratory not only suggested that docosahexaenoic acid (DHA) may upregulate immune response pathways in human monocytes, it also revealed that DHA reduces angiotensin converting enzyme 2 (ACE2), the cellular receptor for SARS coronaviruses (SARS-CoV), in various rat tissues and human cultured cells. Also, DHA was found to inhibit cellular entry of SARS-CoV-2 pseudovirus. Thus, it was hypothesized that DHA has the potential to help manage SARS-CoV infection. RNA-seq was performed on DHA-treated (20 or 125 μM for 8 h) and control human EA.hy926 cells in both the growing and quiescent states. The data were processed by the RSEM-STAR-DESeq2 pipeline, and then subjected to gene set enrichment analysis (GSEA) with clusterProfiler. GSEA revealed that only in quiescent cells, 20 μM DHA downregulated pathways related to SARS-CoV-1/2-host interactions, specifically the processes by which the virus disrupts host protein translation and global mRNA splicing to suppress host defenses. The Reactome term “potential therapeutics for SARS” was positively enriched by both 20 and 125 μM DHA in quiescent cells only, including genes related to nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, interleukin-6 pathway, heat shock proteins, and
TBK1
. However, in growing cells, terms related to SARS-CoV-1/2-host interactions were upregulated by DHA. Beyond derailing SARS-CoV via translation machinery and ACE2, DHA was also previously found to concomitantly reduce ACE1 protein levels, thus preserving the ACE1/ACE2 balance. This is significant because the balance of ACE1/ACE2 is important for maintaining renin-angiotensin system homeostasis, a factor critical in the pathogenesis of long COVID. Overall, our findings advance a novel perspective on the therapeutic potential of DHA for managing SARS-CoV infection, via its ability to hinder virus-host interactions. Moreover, the beneficial effects of DHA only occurred in quiescent (healthy) endothelial cells but not growing (dysfunctional) endothelial cells, implying that COVID-19 patients without CVD may be more responsive to DHA treatment compared to patients with underlying CVD. Further
in vitro
,
in vivo
, and even clinical studies are required to validate these effects of DHA on SARS-CoV.
Abstract 2136: The Effects Of Dual Antiplatelet Therapy On Neutrophil Function
Authors: Hummel Sonja , Larsen Peter , Hally Kathryn
Published: July 2024

Introduction:
Dual anti-platelet therapy (DAPT) is routinely administered to inhibit platelet aggregation in acute myocardial infarction (AMI) and, in doing so, reduces the risk of recurrent adverse cardiovascular events. Alongside platelets, neutrophils are also key mediators of inflammation during AMI, both secreting cytokines and chemokines and producing Neutrophil Extracellular Traps (NETs). While DAPT is known to mediate off-target anti-inflammatory effects, the extent to which DAPT affects neutrophil function is not well understood.

Objective:
To investigate the effects of DAPT on neutrophil-mediated inflammation.

Methods:
Neutrophils were isolated from whole blood of ten healthy subjects pre- and post-DAPT (ticagrelor and aspirin) treatment and NET formation in response to classical NET-inducing agonists (PMA and ionomycin) was measured by SYTOX Green fluorescence and MPO activity. Cytokine and chemokine production following stimulation with an activating agonist (FSL-1) was measured by cytometric bead array and reported as median fluorescence intensity (MFI). DAPT was effective in inhibiting platelet aggregation in response to AA and ADP.

Results:
NET formation in response to ionomycin, but not PMA, was greater in DAPT-treated neutrophils compared to treatment-naïve neutrophils (69.9 % ± 30.66 SEM). Levels of inflammatory mediators were also markedly different pre- and post- DAPT treatment. For example, MFI of IL-8 was decreased by 18% ± 9.97 SEM post-DAPT compared with pre-DAPT MFI, while MFI of IL-12p and IFN-α were significantly increased (32.54% ± 12.12 SEM, 58.68% ± 18.72 SEM respectively) compared to pre-DAPT.

Conclusion:
DAPT primes neutrophils for inducing some, but not all, NETotic pathways and releasing some, but not all, inflammatory mediators. This suggests that our primary strategy for inhibiting platelet aggregation in AMI does not provide broad off-target anti-inflammatory effects on neutrophils. In fact, DAPT may encourage a pro-inflammatory neutrophil phenotype.
Abstract 2142: IL-17 Signaling In Intestinal Epithelial Cells Regulates Microbiota, Metabolism And Neuroinflammation In Atherosclerosis.
Authors: Mazitova Aleksandra , Marquez Sanchez Ana C , Terrell Meghan , Rodrigues Richard , Trinchieri Giorgio , Dzutsev Amiran , Kossenkov Andrew , Grivennikov Sergei , Koltsova Ekaterina
Published: July 2024

Microbiota plays a crucial role in regulating immunity and has been suggested to contribute to atherosclerosis, the precise role of cytokine signaling in intestinal epithelial cells (IEC) in controlling microbiota, inflammation, and vascular disease is poorly understood. Here we investigate the role of IL-17RC signaling in intestinal epithelial cells (IEC) using newly generated

Ldlr
-/-
Il17rc

Δ

IEC

mice. We found that IEC-specific ablation of IL-17RC heightened intestinal inflammation, altered microbiota composition, and led to enhanced atherosclerosis development accompanied by accumulation of activated myeloid and T cells in aortas along with upregulation of genes reminiscent of neuroinflammatory signature. IL-17RC ablation in IEC reduced tight junction proteins contributing to enhanced gut permeability. RNAseq analysis of intestines revealed upregulation of multiple inflammatory pathways, including interferon and TLR signaling, while several metabolic pathways were downregulated in

Ldlr
-/-
Il17rc

Δ

IEC

mice; implying an important role of IL-17R signaling in the control of IEC metabolism, microbes and metabolites at the host-microbiota interface. Metagenomic sequencing revealed alteration of microbiota composition in

Ldlr
-/-
Il17rc

Δ

IEC

. Atherosclerosis development was microbiota dependent as co-housing enhanced the disease in control mice, while microbiota depletion significantly suppressed the disease.Our work uncovers novel protective role of IL-17 signaling in intestine which regulates the barrier, microbiota and metabolism leading to reduced neuroinflammation in atherosclerotic aorta.
Abstract 3010: Inhibition Of Endothelial Cell-Mediated Efferocytosis Promotes Abdominal Aortic Aneurysm Formation
Authors: Valisno Jeff Arni C , Su Gang , Bellotti Paolo , Krebs Jonathan , Cai Guoshuai , Lu Guanyi , Sharma Ashish K , Upchurch Gilbert R
Published: July 2024

Introduction:
Aortic wall inflammation and vascular smooth muscle (VSMC) apoptosis can lead to accumulation of apoptotic cells and debris during abdominal aortic aneurysm (AAA) formation. We hypothesize that dysregulation of MerTK receptor-mediated efferocytosis (clearance of apoptotic cell debris) by endothelial cells (ECs) can significantly contribute to aortic inflammation and vascular remodeling in AAA pathogenesis.

Methods:
Single-cell RNA sequencing of human AAA tissue from a published dataset (PMID: 33779682) was evaluated for cells of the aortic wall (VSMC/EC) identified via SingleR and cell-specific marker s. Differentially expressed genes (DEGs) were identified via edgeR using the QL F-test and efferocytosis-related genes (ERGs) were recognized using a list of 141 ERGs curated from GeneCards. MerTK expression was assessed in human aortic tissue and ECs via western blot and immunofluorescence. PKH67-labelled apoptotic VSMCs were overlayed on ECs to assess efferocytosis activity and visualized via confocal microscopy. In a murine elastase AAA model, endothelial-specific MerTK knockout (
Cdh5
Cre-ERT2
/
MerTK
fl/fl
) and littermate male mice were analyzed on postoperative day 14 for aortic diameter. Statistical analyses were performed by Welch’s T-test and p<0.05 was considered statistically significant.

Results:
ERGs (ANXA2, PHACTR1, PPARG, ADAM9, CD14, IL6) were differentially expressed (p<0.05, log
2
FC > |1|) between aortic wall cells of human AAAs and control. Human AAA tissue displayed a significant increase in MerTK protein compared to controls (1±0.1 vs. 1.51±0.4, n=6/group, p=0.01). The expression of MerTK in aortic ECs was confirmed by western blot and immunofluorescence(n=3). Pharmacologic inhibition of MerTK using UNC5293, a specific MerTK inhibitor, attenuated EC-mediated efferocytosis of apoptotic VSMCs (qualitative data; 10 images/group n=4). Importantly, EC-specific
MerTK
-/-
mice had a significant increase in aortic diameter compared to littermate controls (155±40.4 vs. 103±30.9%, n=5-6/group, p=0.04) on day 14.

Conclusion:
EC-mediated efferocytosis can be regulated by MerTK to mediate AAA formation. Ongoing studies will delineate mechanisms of EC-MerTK signaling in AAA formation.
Abstract 3014: Bulk And Single-cell Characterization Of The Immune Microenvironment Of Abdominal Aortic Aneurysm
Authors: Yuan Lushun , Chen Song
Published: July 2024

Background:
Abdominal aortic aneurysm (AAA), characterized by localized dilation of the abdominal aorta, involves an immune microenvironment that plays a crucial role in disease development. This study aimed to investigate immune cell heterogeneity and functional dynamics in AAA using single-cell RNA sequencing (scRNA-seq) analysis of AAA content and healthy aorta samples.

Methods:
By analyzing scRNA-seq datasets from AAA content and healthy aorta samples, along with bulk datasets comprising AAA and healthy aortic tissues, we comprehensively profiled the immune microenvironment.

Results:
The analysis revealed five major cell types in both AAA contents and healthy aortic tissues. Further integrative analysis identified 18 distinct subpopulations, unraveling specific immune cell subtypes within AAA. These subtypes were distinguished based on tissue infiltration, functional variations, immune dynamics, metabolic changes, and communication patterns. Notably, B cells and plasma cells were found to contribute to AAA and thrombus formation. Additionally, cytotoxic CD8+ T cells showed enrichment in cytokine and chemokine receptor interactions, indicating their involvement in AAA development. Two pro-inflammatory macrophage subtypes (LY6E+ and CCL5+) and two pro-inflammatory neutrophil subtypes specific to AAA emerged as significant findings. Particularly, MME+ neutrophils exhibited heightened immune receptor activity and infiltrated AAA tissues, as supported by bulk dataset analysis. Metabolically active, these neutrophils played a vital role in regulating CXCL signaling, enabling effective cell-to-cell communication among immune cells.

Conclusions:
This study provides a comprehensive understanding of the diverse immune cell populations within AAA and healthy aorta tissues. The findings shed light on their functional characteristics, developmental trajectories, metabolic differences, and intercellular communication patterns, significantly advancing our knowledge of AAA formation and progression.
Abstract 3019: Piezo-1 Expression Decreases Under Disturbed Flow Model Of Femoral Atherosclerosis.
Authors: Sanchez Gloriani , Zhao Fujie , Wood Levi , Brewster Luke P
Published: July 2024

Background:
Peripheral arterial disease (PAD) is often manifest by focal atherosclerotic plaque formation in the infra-inguinal arteries. However, murine models of atherosclerosis in the infra-inguinal arteries are lacking. It is well known that low and oscillatory wall shear stress, which occurs around blockages in PAD, promotes focal atherosclerotic plaque, but this has not been tested in vivo. Piezo1 is a mechanosensitive ion channel associated with hemodynamic changes in the arterial wall, but its role in flow-mediated atherosclerotic plaque deposition is not known.

Objective:
To develop a flow-mediated murine model of focal atherosclerotic plaque in the femoral arteries.

Methods:
8 male and female S129 mice underwent partial femoral artery ligation (Figure A/B). Wall shear stress was calculated from duplex ultrasound imaging (Figure C/D). Atherogenic conditions were imposed with PCSK9 infection and high fat diet; this was initiated 1 week prior to partial femoral ligation. Histologic comparison of the left (partial femoral ligation) and right (normal stable flow) was performed.

Results:
Low wall shear stress was induced in this model (Figure C/D). Piezo1 expression was decreased in both male and female arteries under these conditions in this model (Figure E). Atherosclerotic plaque was induced by the partial femoral artery ligation but not on the normal stable flow side (Figure F).

Conclusions:
We have successfully developed a flow-mediated model of focal atherosclerotic plaque in murine femoral arteries. This is associated with downregulation of Piezo1 gene expression. Ongoing work is examining the causative role of Piezo1 on endothelial response to disturbed flow.