bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 150: Decoding The Variant-to-function Relationship For
LIPA
, A Risk Locus For Cad
Authors: Li Fang , Wu Xun , Ha Philip , Zebak Mazal , Xue Chenyi , Flynn Elise , Cheng Haoxiang , Hao Ke , Lappalainen Tuuli , Zhang Hanrui
Published: July 2024

Background:
Genome-wide association studies (GWASs) link genetic variation in the
LIPA
(lysosomal acid lipase) gene to coronary artery disease (CAD). Quantitative Trait Loci (QTL) studies indicate higher
LIPA
mRNA and enzyme activity in the monocytes of risk alleles carriers. Yet, the mechanism whereby this locus confers disease etiology has not been fully established. Herein, we aim to establish the variant-to-function relationship and elucidate how increased
LIPA
impacts atherosclerosis.

Methods:
Employing post-GWAS pipelines (eQTL, enzyme activity-QTL, Tri-HiC, luciferase assay, allele-specific binding, motif analysis, and mobility shift assay), we connect functional variants to target genes in causal myeloid cells. Myeloid-specific Lipa overexpression mice (

Lipa
Tg

) on

Ldlr
-/-

background were generated and characterized.

Results:
Post-GWAS pipelines support
LIPA
as the candidate causal gene at this locus. In human monocyte-derived macrophages, risk allele carriers of GWAS CAD variants exhibit higher
LIPA
mRNA, protein, and enzyme activity. Tri-HiC and luciferase assay identified an intronic region within
LIPA
, housing GWAS CAD noncoding variants and exhibiting monocyte-specific enhancer activity. Within the enhancer region, 1412445 and rs1320496 were identified as the two potential causal variants, with the risk alleles having increased enhancer activity in luciferase assay and increased binding to PU.1 in mobility shift assay. Collectively, the above data suggest that the risk alleles of CAD variants lead to increased
LIPA
in monocytes/macrophages. Consistently,

Lipa
Tg
, Ldlr
-/-

mice fed a Western diet showed significantly increased lesion size and lesion macrophage area compared to
Ctl, Ldlr

-/-

mice. scRNA-seq analysis followed by functional validation demonstrated that

Lipa
Tg

macrophages had higher free cholesterol yet lower neutral lipid accumulation, accompanied by a transcriptomic signature of increased interferon signaling. Lipa overexpression also increased monocyte infiltration into the plaque.

Conclusions:
We established that the risk alleles of common noncoding variants at the
LIPA
locus drive increased myeloid
LIPA
expression and aggravate atherosclerosis.
Abstract 152: Osteochondrogenic Transcription Factor SOX9 Regulates The Epigenetic Mechanisms Of Atherosclerosis And Intimal Calcification
Authors: Worssam Matthew D , Weldy Chad S , Monteiro Joao P , Palmisano Brian T , Kundu Ramendra K , Nguyen Trieu , Quertermous Thomas
Published: July 2024

Our lab has recently shown that several coronary artery disease (CAD)-protective genes inhibit the transition of smooth muscle cells from plaque-stabilising fibromyocytes (FMCs) to chondrocyte-like chondromyocytes (CMCs) that localise to areas associated with plaque vulnerability, namely acellular and calcified regions. To further investigate the molecular mechanisms underlying the transition from FMCs to CMCs, and to determine the CMC impact on lesional stability, we induced SMC-specific deletion of endochondral master regulator
Sox9
and performed single-cell genomics and histological assays in a murine model of atherosclerosis. Whilst deletion of
Sox9
resulted in only modest gene expression differences in the contractile SMC and FMC clusters, near total CMC ablation was observed, accompanied by marked downregulation of CMC marker genes, including
Col2a1
and
Ibsp
. Importantly, a substantial decrease in neointimal calcium deposition was also observed. Although
Sox9
deletion resulted in closure of chromatin at many CMC genes, overall, a net opening was observed in CMC cluster cells, particularly at genes implicated in FMC-associated processes including inflammation and cytoskeletal dynamics. Peaks of
de novo
open chromatin were enriched for the motifs of athero-protective, anti-chondrogenic transcription factors, including SMAD3 and AHR, implicating SOX9 as an epigenetic antagonist of anti-chondrogenic programs. In addition,
Sox9
ablation substantially reduced total plaque burden in non-aortic root arteries, which was not due to altered serum lipid levels, but which
in vitro
evidence suggested may in part be due to altered SMC proliferation. Taken together, these data implicate SOX9 as a major regulator of CMC formation and suggest pathological roles for SOX9 beyond intimal calcification. The elucidation of genes and pathways implicated in the FMC to CMC transition could identify novel targets to bias SMC towards plaque-stabilising and away from plaque-destabilising fates.
Abstract 160: Macrophage-specific Ablation Of Bmal1 Regulates Cholesterol Transport And Enhances Atherosclerosis
Authors: Pan Xiaoyue , O'Hare John , Mowdawalla Cyrus , Mota Samantha , Wang Nan , Hussain M.Mahmood
Published: July 2024

We have previously shown that global and liver-specific Bmal1 deficient mice develop enhanced atherosclerosis. Macrophages play a critical role in atherosclerosis, yet the role of macrophage Bmal1 in cholesterol uptake, intracellular transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific Bmal1 deficient mice to clarify the role of macrophage Bmal1 in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Mechanistically, Bmal1 deficient macrophages exhibited: (1) increased expression of Lox-1 and Cd36 and uptake of oxLDL; (2) decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux, and reverse cholesterol transport; and (3) diminished export of free cholesterol from lysosomes due to reduced expression of Npc1 and Npc2. Molecular studies showed that Bmal1 directly regulates Npc1 and Npc2 by binding to E-boxes in their promoters, whereas it indirectly regulates Abca1 and Abcg1 by regulating ZNF202. Furthermore, we provide evidence that Bmal1 interacts with both Clock and Npas2 to regulate ZNF202, Npc1 and Npc2. Our data demonstrate that macrophage Bmal1 is a key regulator of oxLDL uptake, lysosomal cholesterol export, and cholesterol efflux, and, therefore, plays a crucial role in the prevention of atherosclerosis.
Abstract 162: Post-transcriptional Regulation Of FGF21 In Obesity And Metabolism
Authors: Schmidt Heidi M , Jarrett Kelsey E , Rubert Gabriella , Steel Michelle , Cheng Angela , Tarling Elizabeth J , Vallim Thomas A
Published: July 2024

Imbalances in energy intake and expenditure are the leading cause of the dramatic surge in obesity and cardiovascular disease affecting >60% of Americans. Increasing fat content in the average diet alters metabolism and contributes to disease. FGF21, a secreted hepatokine, modulates feeding behavior and energy expenditure (EE) primarily through signaling in brain and adipose tissue. FGF21 analogues are actively in clinical trials for several metabolic diseases, and it is therefore important to understand the various pathways that control FGF21. FGF21 is regulated by well-defined transcriptional programs, but its post-transcriptional regulation is less understood. Post-transcriptional regulation is mediated by RNA binding proteins (RBPs) that control mRNA stability. One family of such RBPs is the ZFP36 family proteins (ZFP36, ZFP36L1, ZFP36L2) that bind to AU-rich regions in the 3’UTR, leading to mRNA degradation. All 3 ZFP36 family proteins are redundant, therefore we knocked out
Zfp36
,
Zfp36l1
and
Zfp36l2
in livers of adult mice.
Fgf21
was the most upregulated gene following loss of ZFP36 family proteins in the liver. Consistent with elevated FGF21, these mice rapidly lost weight and fat mass while remaining metabolically healthy. Using metabolic cages, we observed that mice lacking hepatic ZFP36 family proteins had increased food intake and EE and decreased respiratory exchange ratio, indicating preferential fat burning. We then generated mice lacking the ZFP36 family proteins and
Fgf21
which partially restored the metabolic phenotypes observed, leading to the hypothesis that ZFP36 family proteins post-transcriptionally regulate
Fgf21
in controlling food intake and energy homeostasis. To determine if loss of ZFP36 family proteins protects against diet-induced obesity, we fed mice lacking the ZFP36 family proteins a high fat diet, however, they were entirely protected from diet induced obesity. The protection was partially reversed by additional deletion of
Fgf21
. These findings demonstrate that FGF21 is regulated post-transcriptionally by the ZFP36 family proteins and loss of this mode of regulation has significant metabolic implications in diet-induced obesity and could lead to new therapeutic approaches to treat metabolic disease.
Abstract 2003: Epigenetic And Phenotypic Modulation Of Adventitial Fibroblasts In Atherosclerosis By Coronary Artery Risk Gene Tcf21
Authors: Gu Wenduo , Jackson William , Dalal Alex , Li Daniel , Zhao Quanyi , Nguyen Trieu , Worssam Matthew , Ramste Markus O , Monteiro Joao P , Weldy Chad S , Palmisano Brian , Fischbein Michael P , Quertermous Thomas , Cheng Paul
Published: July 2024

The vascular adventitia has long been postulated to play a key role in atherosclerosis, but the relevant adventitial cell populations and the mechanisms by which they influence atherosclerosis have remained elusive. To characterize relevant adventitial cells and their regulators, we have combined single cell multi-omic murine and human data with human genetics. Single cell transcriptomic and epigenetic analysis of both murine and human arterial tissue revealed a unique population of adventitial fibroblasts (AdvFib). Transcriptome and epigenome analysis revealed disproportionate enrichment for coronary artery disease (CAD) GWAS risk loci and genes in AdvFib, suggesting that AdvFib actively contributes to disease. Leveraging this rich information, we created a novel AdvFib specific CreERT2 murine model to enable AdvFib specific genetic manipulation. Murine lineage tracing using this novel AdvFib-specific model of atherosclerosis revealed early expansion and transcriptomic alteration of AdvFib during plaque development, akin to phenotypic modulation of SMC. Cell-cell communication analysis demonstrated an increase in AdvFib-driven crosstalk with other cell types during early phases of atherosclerosis, including activation of pro-inflammatory (Il6, Ccl2/7, Cxcl12) genes. Computational enrichment analysis of regulatory region of genes associated with AdvFib modulation points to a prominent role for
TCF21
, a CAD GWAS risk gene. Single cell time course data of atherosclerosis demonstrates that
Tcf21
is expressed primarily in AdvFib with dynamic alteration of its expression during atherosclerosis. AdvFib-specific
Tcf21
loss in murine model of atherosclerosis resulted in altered expression of ECM and inflammatory genes, with corresponding changes in recruitment of inflammatory cells. Mechanistically, through histone modification cut-and-tag in human coronary AdvFib, we showed that TCF21 regulates AdvFib activation through epigenetic reprograming of key cytokine and cell fate regulators by binding and altering H3K27Acetylation. Collectively, our results begin to establish the role of AdvFib in atherosclerosis and highlight the importance of TCF21 in this process through a precise epigenetic mechanism.
Abstract 2009: Computational Modeling Of Long-term Aortic Root Dilatation Following TEVAR Of Type B Aortic Dissection
Authors: Khabaz Kameel , Varsanik Alyssa , Kim Junsung , Nguyen Nhung , Milner Ross , Pocivavsek Luka
Published: July 2024

Objectives:
The long-term sequalae of thoracic endovascular aortic repair (TEVAR) are poorly understood. While several failure modes have been identified, the hemodynamic impact of TEVAR in the proximal native ascending aorta has yet to be fully appreciated. This study applies computational modeling tools to investigate a case of long-term aortic root dilatation following TEVAR for acute type B aortic dissection (TBAD).

Methods:
We investigate a patient who presented with heart failure 4.5 years after initially successful TEVAR. Cross-sectional aortic root diameter was measured in a pre-operative scan and 57 months post-TEVAR. Three-dimensional localized surface dilatation was calculated using pre-operative and post-operative segmentations, followed by a non-rigid surface mapping method of calculating area change in corresponding regions. The hemodynamic impact of TEVAR on the proximal aorta was analyzed with a computational fluid dynamics (CFD) simulation of the post-operative patient geometry.

Results:
Aortic root diameter increased from 39.2 mm pre-op to 47.1 mm (+ 20.1%) postop and 34.9 mm preop to 40.8 mm (+ 16.9%) postop on axial and coronal images respectively. The three-dimensional area mapping between pre and post-op scans demonstrates a maximum 14% area increase near the aortic root. The CFD simulations show increased velocity and static pressure in the ascending aorta near the aortic root region.

Conclusions:
Three different computational approaches demonstrate long-term changes in ascending aortic morphology following TEVAR. Computed tomography measurements demonstrate significant aortic root dilatation, and three-dimensional surface mapping shows increasing area in the proximal ascending aorta. The CFD results emphasize localization of increased velocity and stress in the same region. These observations point to potential avenues for further mechanistic investigation.
Abstract 2010: Atherosclerotic Plaque Macrophages Drive Abdominal Aortic Aneurysm Formation
Authors: Khan Aniqa , Thayaparan Danya , Emoto Takuo , Besla Rickvinder , Hamidzada Homaira , Vohra Shabana , Lindquist Moritz , Botts Steven R , El-Maklizi Mahmoud , Schroer Stephanie , Althagafi Marwan , Zhang Hangjun , Libby Peter , Howe Kathryn L , Heximer Scott P , Rubin Barry B , Cybulsky Myron I , Roy Joy , Epelman Slava , Ken-ichi Hirata , Stampfli Martin , Robbins Clint S
Published: July 2024

Introduction:
Cigarette smoking (CS) contributes to the most deaths in abdominal aortic aneurysms (AAA) and tobacco use associates with early damage to the abdominal aorta. Associated risk factors, such as hyperlipidemia, link clinical AAA to the atherosclerotic process, suggesting common pathogenetic features with plaque development. We hypothesized that CS exposure exacerbates atherosclerotic disease in the abdominal aorta by increasing the recruitment of inflammatory macrophages that mediate arterial degradation and AAA formation.

Methods:
Adult male
Apoe
deficient mice commenced a high cholesterol diet and were concomitantly exposed to CS or room air (RA) for 4, 8, 12, and 16 weeks. As plaque macrophages are highly prevalent in advanced AAA, additional CS exposed mice received a selective CSF1R inhibitor, PLX3397, for up to 16 weeks to deplete macrophages. AAA incidence, atherosclerotic plaque burden and lesion composition were assessed in the aorta by immunofluorescence, Movat, EVG, and Oil Red O staining.

Results:
CS induced AAA at all timepoints with the highest incidence of 37% at 16 weeks of exposure (n=151 CS, n=75 RA p< 0.05). Aneurysms always coincided with atherosclerosis (n=7 CS, n=7 CS/AAA p<0.05), and severe elastin fragmentation was consistently overlaid by plaque (n=20 CS p<0.05, Fig 1A and B). In some cases, lesions also associated with aortic rupture, causing death in ~11% of animals (n = 35 RA, n = 46 CS p<0.04). CD68+ macrophages associated highly with severe elastin damage but less at intact regions (2.6-fold p<0.0001, Fig 1C). PLX3397-mediated macrophage depletion attenuated plaque development and prevented AAA (n=20 treated, n=27 control p<0.05, Fig 1D).

Conclusion:
CS exacerbates atherosclerosis and increases the accumulation of macrophages at sites of arterial injury. Depletion of macrophages results in a lack of aneurysm formation, highlighting their capacity to directly injure the aortic wall and necessity to mediate AAA.
Abstract 2022: AlphaV Integrins Inhibition Downregulates Pro-fibrotic Events In Human Aortic Smooth Muscle Cells And Limits Thoracic Aortic Aneurysm Progression In Marfan Mice
Authors: Rega Sara , Fanotti Nadia , Casaburo Manuel , Poggio Paolo , Vescio Martina , Pattini Linda , Lambroia Luca , Climent Montserrat , Elia Leonardo , Bonalumi Giorgia , Giambuzzi Ilaria , Udugampolage Nathasha , Pini Alessandro , Pompilio Giulio , Perrucci Gianluca Lorenzo
Published: July 2024

Aortic dilation can often lead to lethal events, such as aortic dissection and/or vessel rupture. The aneurysms occurring in the thoracic aortic trait (TAA) may depend on comorbidities, such as hypertension, or on genetically determined conditions, such as Marfan syndrome (MFS). To date, the surgical replacement of aorta represents the most effective therapeutic approach in all TAA patients, since an etiologic pharmacological treatment is still lacking. Integrin receptors are key players in cellular mechanotransduction responses and the role of integrins containing αV subunit have been described as responsible for pro-fibrotic molecular events. Recently, it has been reported the effectiveness of a
pan
αV integrin inhibitor (
i.e.
, GLPG0187) in limiting the TAA progression in MFS
in vivo
model. However, the specific αV integrin subgroup directly involved in this detrimental process is yet not known.

We evaluated the effectiveness of the specific αVβ3 and αVβ5 integrin inhibitor cilengitide in downregulating pro-fibrotic
in vitro
events on MFS patients’ vascular smooth muscle cells (MFS-VSMC), and in limiting
in vivo
thoracic aortic dilation on a MFS mice model.

By comparison with healthy control (HC)-VSMC, MFS-VSMC showed higher levels of αVβ3 and αVβ5 integrins as well as pro-fibrotic collagen I. When compared with GLPG0187, the cilengitide-mediated
in vitro
inhibition of these integrins determined a more effective downregulation of TGF-β downstream pathways (SMAD2/3 and ERK1/2) as well as a statistical tempered expression of pro-fibrotic mechanotransduction mediators, such as RhoA GTPase. The 2D-echocardiographic results on MFS mice (Fbn1
C1039G/+
) treated with cilengitide confirmed the greater effectiveness of this drug in limiting the TAA progression, not only in comparison with vehicle but also with the broad
pan
αV integrin inhibitor GLPG0187.

Altogether, our results improve the know-how on αV integrin involvement in MFS-TAA scenario, unveiling the specific detrimental role of αVβ3 and αVβ5 in this pathological context. The selective inhibition of these integrins exerted by cilengitide highlights the role of this compound as a potential and more targeted tool for future therapeutic approaches to limit TAA progression in MFS patients.
Abstract 2025: Evolving Burden Of Peripheral Artery Disease Attributable To Smoking In 38 Oecd Countries From 1990-2019: Global, Regional, National Variations, Age Dynamics, And Implications For Global Health
Authors: Ahmed Danish , Islam Hamza , Reyaz Ibrahim , Goel Shrey , Vemparala Priyatha , Patel Dhruval , Vavaliya Nihar , MADHU KRUPA , Islam Rabia , DESAI HARDIK D , PATEL JUHI , AMIN VISHRANT P
Published: July 2024

Introduction and Background:
Peripheral artery disease (PAD) ranks as the 11th leading cause of mortality and disability in countries within the Organization for Economic Cooperation and Development (OECD). Smoking is recognized as a leading risk factor for PAD, and its impact on the prevalence and outcomes of PAD has significant public health implications.

Method:
Using Global Burden of Disease framework, Deaths, disability adjusted life years (DALYs), years lived with disability (YLDs) due to stroke attributable to Smoking were analyzed by age, sex, year and location across the 38 OECD Countries from the last 3 decades.

Results:
From 1990 to 2019, the total number of deaths increased from 7,226 to 10,738, and Disability-Adjusted Life Years (DALYs) rose from 191,915 to 238,577. In 2019, the United States had the highest number of deaths (3,596) and DALYs (83,073). Hungary had the highest age-standardized mortality rate in 2019 (1.7 deaths per 100,000 person-years). The 80-84 age group had the most deaths (2,233), while the 70-74 age group had the highest DALYs (45,947) in 2019. Over the past three decades, males consistently had a higher burden, with an annual percentage change in deaths of 50% compared to 45% for females, and a 32% change in DALYs for males compared to 12% for females.

Conclusion:
The findings highlight the urgent need for public health efforts to address the increasing burden of peripheral artery disease (PAD), particularly focusing on smoking cessation and tailored interventions for high-risk groups. Clinicians should prioritize early detection and patient counseling to mitigate PAD risk factors, especially among males with a consistently higher burden.
Abstract 2026: Assessing The Global Burden And Trends Of Stroke Attributable To Smoking In 38 Oecd Countries From 1990 To 2019: A Benchmarking Analysis
Authors: Vemparala Priyatha , Islam Rabia , Vanodia Mihir , Savaliya Prashantkumar , Vashi Mandeepsinh , Lakkimsetti Mohit , Patel Dhruval , Goel Shrey , Islam Hamza , PATEL JUHI , AMIN VISHRANT P , DESAI HARDIK D
Published: July 2024

Introduction:
Stroke has remained a 2
nd
leading cause of both mortality and disability among cardiovascular diseases (CVD) on a global scale. Smoking is responsible for 12.27% of CVD-related deaths in countries belonging to the Organization for Economic Cooperation and Development (OECD).

Method:
Using Global burden of disease tool, we analyzed deaths, disability adjusted life years (DALYs), years lived with disability (YLDs) due to Stroke attributable to Smoking by age, sex, year and location across the 38 OECD countries.

Results:
The total number of deaths attributed to smoking-related strokes saw a notable reduction of 45% (with a 95% uncertainty interval of 42-48%), while DALYs decreased by 41% (with a range of 38-44%). However, YLDs showed a marginal 1% decrease (with a range of 2-5%). Regarding age-standardized rates, Latvia recorded the highest death rate in 2019 at 12.42 deaths per 100,000 person-years, followed by Hungary with 9.72 deaths. Conversely, Australia had the lowest rate at 1.97 deaths, while the United States reported 4.45 deaths per 100,000 person-years. In terms of specific age groups, the 80-84 age group witnessed the highest number of deaths, with 16,069 fatalities, while the 60-64 age group had the highest DALYs, totaling 392,824 in 2019. Over the past three decades, males consistently bore a greater burden compared to females.

Conclusion:
From a public health policy perspective, the significant decline in smoking-related stroke deaths and DALYs signals the success of anti-smoking efforts, yet the varying rates among countries underscore the need for tailored strategies. Clinicians should take heed of the enduring higher burden in males, emphasizing early intervention and smoking cessation counseling, especially for vulnerable age groups. Integrating prevention and smoking cessation programs into stroke care is pivotal to further mitigating smoking's impact on stroke outcomes.
Abstract 2035: In-Depth Analysis Of Tibial Artery Calcification Through Microct And Histology
Authors: Niu Hong , Kim Kevin , Kamada Keisuke , Liu Hui , Tang Gale L
Published: July 2024

Background:
Peripheral artery disease (PAD) impacts over 200 million people worldwide. It is traditionally linked to atherosclerotic calcification, or intima arterial calcification (IAC). Recent findings emphasize the role of medial arterial calcification (MAC). Current studies have identified some factors in MAC and IAC, however, a comprehensive understanding of their distinct features and pathogenesis is lacking. This study addresses this gap by examining tibial arteries from PAD patients post-amputation. We characterized the calcification using microCT and histology.

Methods:
Human tibial arteries were obtained from PAD patients post amputation, and segmented into posterior, anterior, and peroneal arteries. The arteries were dissected, fixed in 4% paraformaldehyde, and stained with iodine. High-resolution microCT images were captured using a Scanco μCT40, and 3D models were reconstructed by ImageJ and Slicer3D. The samples were then processed for histological analysis, including Hematoxylin and eosin (H&E) stain and Alizarin red staining.

Results:
The study design is shown in Fig. A, featuring the serial dissection of a posterior artery as an example. The microCT scan protocol is designed for imaging continuous sections under a specific orientation. The 3D models enable a comprehensive assessment of calcification and quantification for distinct categorization, and the peroneal artery showed the least calcified lesions (*P<0.05, ***P<0.001; Fig. B & C). MAC/IAC or co-existing lesions were characterized by cross-sectional images and H&E staining. The results indicated a predominance of MAC in the highly calcified group, comprising over 98% of the entire tissue (Fig. D & E).

Conclusions:
We characterized calcified lesions in tibial arteries by microCT and histology. Calcification categories were determined through 3D models. Histology and cross-section images enabled the assessment and quantification of MAC/IAC or co-existing lesions.
Abstract 2049: Regulation Of MTP-ATGL Interactions In Adipocytes And Their Impact On Hepatic Lipid Metabolism
Authors: Rajan Sujith , Verano Michael , Aleman Jose O , Hussain M M
Published: July 2024

Introduction:
We have shown that microsomal triglyceride transfer protein (MTP) in adipocytes regulates lipolysis by inhibiting adipose triglyceride lipase (ATGL). Adipose MTP knockout mice (A-

Mttp
-/-

) exhibit smaller adipocytes, more thermogenesis, and less weight gain on an obesogenic diet.

Aim:
To investigate the MTP-ATGL interaction in adipocytes and its impact on hepatic lipid metabolism.

Methods:
We studied MTP-ATGL interactions using
in silico
modeling, ELISA, and co-IP. Lipoprotein profiles were analyzed using FPLC. Adipose-liver cross-talk was assessed through co-culture experiments, adipokine profiling and lipidomics. Hepatic triglyceride (TG) production and fatty acid (FA) oxidation were assessed using poloxamer-407 and
14
C-palmitate. Statistical analyses were done using GraphPad Prism (P <.05 significant).

Results:
Our study suggests MTP-ATGL interactions are hydrophobic involving C-terminal of ATGL and N-terminal β-barrel of MTP. These interactions vary in different metabolic states like fasting and adipocyte differentiation. A-

Mttp
-/-

mice had higher plasma TG content in VLDL and LDL fractions. Despite increased movement of FAs from adipose to the liver as indicated by lipidomics of eWAT, plasma and liver, A-

Mttp
-/-

mice liver showed less steatosis. Adipokine profiling showed significantly higher amounts of adiponectin in the plasma of A-

Mttp
-/-

mice. Reduced ceramide levels and significantly increased AMPK phosphorylation suggest higher adiponectin activity in A-

Mttp
-/-

livers. A-

Mttp
-/-

livers showed higher expression of FA uptake and utilization related genes, but no change in lipid synthesis. Hepatocytes treated with

Mttp
-/-

adipocytes conditioned media (CM) showed higher FA oxidation than those treated with

Mttp
f/f

adipocytes CM.

Mttp
-/-

adipocytes CM showed significantly higher PPARα reporter activity than

Mttp
f/f

adipocytes CM.

Conclusion:
Our study reveals dynamic hydrophobic interactions between MTP and ATGL that vary with metabolic states. Enhanced movement of PPARα ligands (FAs) and adiponectin from A-

Mttp
-/-

adipocytes boosts VLDL production and FA oxidation, to protect livers from hepatic steatosis. This highlights a novel role of adipose MTP in the regulation of hepatic lipid homeostasis.
Abstract 2055: Pyruvic Carboxylase In Macrophages Aggravates Atherosclerosis By Regulating Metabolism Reprogramming To Promote Inflammatory Responses Through Hif-1 Signal Pathway
Authors: Huang Fangze , Zhao Lingna , Liu Deshen , Zhao Li , Liu Xiu , Zheng Shaoyi
Published: July 2024

Background:
Atherosclerotic patients exhibit abnormal glucose metabolism. Pyruvic carboxylase (PC) is a key enzyme in glucose metabolism which converts pyruvate into oxaloacetate, an intermediate reactant in both gluconeogenesis and tricarboxylic acid cycle. However, the role of PC in atherosclerosis remains unclear.
Aims:
We aimed to explore a potential target in macrophage metabolism for atherosclerosis treatment.

Methods:
We generated macrophage-specific PC knockout mice (
PC
M

KO

) by crossbreeding the
PC

fl/fl

mice with
L
yz
2
-cre mice expressing Cre-recombinase in macrophages. Bone marrow transplantation was conducted to generate
PC

fl/fl

/
Apoe

-/-

and
PC
M

KO

/
Apoe

-/-

chimaera. Mice were administered with AAV-
PCSK9

DY

followed by a 12-week high-fat diet (HFD) to induce atherosclerosis.

Results:
PC was upregulated in macrophages of human and mice with atherosclerosis. Macrophage-specific PC deficiency mitigated HFD-induced atherosclerotic lesions in both
Apoe

-/-

mice and mice injected with AAV-
PCSK9

DY

. PC deletion reduced reactive oxygen species overproduction and mitochondrial damage in macrophages. Transcriptomics revealed that PC activated the HIF-1 signal pathway by initiating metabolic reprogramming in macrophages. By preventing HIF-1α from proteasome degradation, PC induced nuclear translocation of HIF-1α in atherosclerotic aortic roots. HIF-1α stabilizer, dimethyloxalylglycine, reversed the anti-inflammatory effect of PC in macrophages exposed to hypoxia, whereas the inhibition of HIF-1α impeded the transformation of pro-inflammatory macrophages to anti-inflammatory macrophages induced by PC overexpression. Dimethyloxalylglycine reversed the beneficial effects of macrophage PC deficiency on atherosclerosis.

Conclusion:
Macrophage PC aggravates atherosclerosis by regulating metabolic reprogramming, thereby promoting inflammatory responses in macrophages through HIF-1 signal pathway.
Abstract 2068: Novel Mechanisms Underlying The Role Of Ornithine Decarboxylase In Inflammation Resolution And Atherosclerosis Regression
Authors: Kumar Dhananjay , Pandit Rajan , Yurdagul Arif
Published: July 2024

The effective removal of apoptotic cells (ACs) by macrophages, termed “efferocytosis”, is essential for resolving inflammation and promoting tissue repair. Accordingly, impaired efferocytosis underlies chronic diseases, such as atherosclerosis, and underscores the need to restore this process. Pro-resolving macrophages execute successive rounds of efferocytosis and produce the pro-resolving polyamine putrescine, which is critically regulated by its rate-limiting enzyme ornithine decarboxylase (ODC1). Despite valuable insights, critical gaps remain in our understanding of how putrescine biosynthesis is regulated during inflammation resolution. Therefore, we sought to explore the mechanisms underlying the signaling pathways governing ODC1 gene expression, putrescine biosynthesis, and their role in inflammation resolution and atherosclerosis regression. We found that ODC1 expression in lesional macrophages decreased during atherosclerosis progression (
Ldlr
-/-
mice fed a high-fat, Western-type diet (HFD) for 16 weeks), whereas ODC1 expression substantially increased during atherosclerosis regression (
Ldlr
-/-
mice fed an HFD for 16 weeks then treated with a virus that restores hepatic LDLR and switched to normal chow for 5 more weeks). Consistently, early-stage human atherosclerotic plaques (Type 1-2) showed high levels of ODC1 in CD68
+
macrophages, yet lesional macrophages in advanced-stage human atheromas (Type 3-5) showed low levels of ODC1.
In vitro
studies in humans and mice indicated that engulfment of ACs enhanced ODC1 expression, which was unaffected by AC binding or its degradation. However, blocking actin polymerization prevented ODC1 upregulation by apoptotic cells whereas stimulating its polymerization enhanced ODC1 expression. Furthermore, uptake of inert beads similarly increased ODC1 expression, relying on a novel pathway involving integrin αV-mediated Stat3 activation and Myc-dependent
Odc1
transcription. These newly identified mechanisms illuminate previously unknown mechanisms driving inflammation resolution and reveal new avenues for developing targeted therapies in atherosclerotic cardiovascular disease.
Abstract 2070: Myeloid Smyd2 Deficiency Exacerbates Atherosclerosis Via Promoting Plaque Necrosis And Calcification
Authors: Sharma Shaligram , Ilse Nadia , Harvey Jada , Li Jacob , Li Chunying
Published: July 2024

Introduction:
The accumulation of lipoprotein-laden macrophages within arterial walls marks the earliest phase of atherosclerotic cardiovascular disease, giving rise to foam cells. Here, we aim to define the previously unexplored role of myeloid specific SMYD2 (a histone lysine methyltransferase) in atherosclerosis and plaque formation.

Methods:
Myeloid-specific Smyd2 knockout mice in ApoE
-/-
background (Smyd2
mKO
ApoE
-/-
) and their littermate controls (Smyd2
fl/fl
ApoE
-/-
) were subjected to Western diet (WD) feeding for up to 26 weeks. Atherosclerotic plaque distribution was examined by
en-face
Oil Red O (ORO) staining of freshly collected aortas. Hematoxylin & Eosin (H&E) staining on brachiocephalic artery (BCA) tissues were performed to determine total plaque size, lipid pool area, and necrotic core area. Plaque calcification was determined by von Kossa staining and Alizarin Red S staining. Primary bone marrow derived macrophages (BMDMs) isolated from Smyd2
fl/fl
ApoE
-/-
and Smyd2
mKO
ApoE
-/-
mice and cultured RAW 246.7 cells (with Smyd2 knock-down) were used for in vitro studies including calcification assay, PR-PCR, and Western blot analysis.

Results:
Myeloid Smyd2 deficient mice demonstrated significantly increased aortic plaque burden on ORO staining as compared to control mice following WD feeding, though with slower WD-induced body weight gain relative to Smyd2
fl/fl
ApoE
-/-
control mice. H&E staining of BCA indicated that there was significant reduction in total lumen and lipid core area, while significantly increased total plaque and necrotic core in Smyd2
mKO
ApoE
-/-
mice. Myeloid Smyd2 deletion promotes plaque calcification in mice fed with WD, and in cultured BMDMs and RAW 246.7 cells treated with calcification promoting media. Gene expression analysis of key calcification factors of aortic tissues and cultured cells showed significant upregulation of Sox9 and Msx2 genes in aortas of WD-fed Smyd2
mKO
ApoE
-/-
mice and cultured Smyd2 deficient cells, as compared to controls.

Conclusion:
We delineated the consequences of Smyd2 deficiency in macrophages on atherosclerotic plaque development, particularly its impact on plaque necrosis and calcification.
Abstract 2072: The 9p21.3 Coronary Artery Disease Risk Locus Induces Cell State Transitions In Vascular Smooth Muscle Cells
Authors: Lo Sardo Valentina , Salido Elsa , de Medeiros Vieira Carolina
Published: July 2024

Background:
Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms - SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect.

Hypothesis:
The 9p21.3 risk haplotype alters VSMCs plasticity and induces dedifferentiation and alternative lineage acquisition.

Aims:
Identify transcriptomic signatures and trajectories of VSMCs induced by the 9p21.3 risk haplotype

Methods:
We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform.

Results:
Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators.

Conclusions:
Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of CAD genetic risk factors in preserving a healthy cellular state within the vasculature.
Abstract 2074:
De Novo
Nucleotide Synthesis Is Necessary For DNA Damage And Repair In Atheroprone Endothelial Cells
Authors: Ma Qian , Yang Qiuhua , Cai Yongfeng , Varadarajan Sudhahar , Zhang Zhidan , Fukai Tohru , Huo Yuqing
Published: July 2024

Aims:
Endothelial cell (EC) metabolism is tightly regulated by hemodynamic changes to protect them from atherogenic injury induced by disturbed flow (d-flow) and other atherogenic risk factors. Therefore, characterizing the role of flow-induced metabolic reprogramming is important to develop new treatments for atherosclerosis and similar diseases. Currently, the effect of proatherogenic d-flow on EC nucleotide metabolism, including
de novo
purine and pyrimidine synthesis, remains under explored. ATIC and CAD are critical enzymes for
de novo
purine synthesis and pyrimidine synthesis, respectively. We investigated in the role of ATIC and CAD-mediated
de novo
nucleotide metabolism in atheroprone ECs.

Methods and Results:
(i) scRNA-Seq analysis results showed that DNA damage response was increased in ECs exposed to d-flow, this was accompanied by increased ATIC-mediated
de novo
purine synthesis and CAD-mediated
de novo
pyrimidine synthesis; (ii) Knockdown of ATIC or CAD with siRNA aggravated EC DNA damage, apoptosis and EC permeability induced by d-flow or other atherogenic factors; (iii) Supplement of nucleobases can rescue
ATIC
KD-mediated DNA damage and apoptosis in ECs; (iv) MYC, predicted high stringency binding with ATIC promoter, was highly expressed in d-flow induced ECs and MYC knockdown reduced the increased ATIC expression in d-flow induced ECs; (v) The atherosclerotic lesion size was markedly increased in EC-specific
Atic
deficient mice in atherosclerosis.

Conclusion:
ATIC and CAD-mediated
de novo
nucleotide synthesis in ECs supply nucleotides to repair DNA damage and preserve EC barrier integrity in vulnerable atheroprone regions and ultimately protect against the development and progression of atherosclerosis.
Abstract 2076: Small Extracellular Vesicles From Chylomicron-treated Endothelial Cells Affect Macrophage Inflammatory Profile
Authors: Tilp Anna , Nasias Dimitris , Tirumalasetty Munichandra , Mooring Ashley , Abumrad Nada A , Miao Qing R , Jose Aleman Jose , Goldberg Ira J , Cabodevilla Ainara G
Published: July 2024

Multiple clinical studies have established elevated postprandial triglyceride-rich lipoproteins (TRLs) as an independent risk factor for cardiovascular disease. However, the mechanisms whereby postprandial TRLs, which circulate mostly within chylomicrons, exert their atherogenic effects remain unknown. We have previously reported that media from chylomicron-treated endothelial cells (ECs) leads to lipid droplet (LD) accumulation in cultured macrophages. To determine the reason for this observation, we assessed whether treated ECs produced small extracellular vesicles (sEVs) that were responsible for this effect. When we removed sEVs from the media, LD production diminished. The EVs contained very little free fatty acids and triglycerides, but abundant phospholipids, esp. phosphatidyl serine, and diacylglycerols. Compared to EVs not treated with chylomicrons, these EVs were larger, more abundant, and contained a specific repertoire of microRNAs. Treatment of macrophages with conditioned media increased expression of genes related to several inflammatory processes and genes that promote macrophage adhesion to the vascular wall. In this context, deletion of the endothelial SR-B1, which mediates chylomicrons uptake, mitigates the macrophage inflammatory response at the transcriptional level. Thus, EC chylomicron uptake leads to production of EVs that alter the phenotype of macrophages.
Abstract 2082: Neutrophil Programming In Atherosclerosis
Authors: Geng Shuo , Li Liwu
Published: July 2024

Emerging evidence suggest the involvement of neutrophils during the progression of human atherosclerosis through secreting inflammatory mediators and causing damages to endothelial vasculature. However, compared to other well-recognized immune cells such as monocytes/macrophages, mechanisms underlying the regulation of neutrophils during atherosclerosis are relatively less studied and understood. This presents a major impediment in our thorough understanding of the pathogenesis of atherosclerosis. In this study, we reported that both murine and human neutrophil population possesses a minor homeostatic resolving subset characterized by reduced expression of inflammatory mediators (LTB4, elastase), elevated expression of anti-inflammatory resolving mediators (RvD1, CD200R), correlated with reduced expression of inflammatory signaling adaptor TRAM. We observed that TRAM deficient neutrophils are effectively reprogrammed into the resolving state, and can potently improve vascular integrity and suppress atherosclerosis pathogenesis when adoptively transfused into recipient atherosclerotic animals. Mechanistically, TRAM deficiency reduces the expression of LOX5AP, dislodges nuclear localization of LOX5, and favors the generation of RvD1 instead of LTB4. We also observed that human TRAM-low neutrophils are less inflammatory, as reflected in reduced swarming and reduced induction of LTB4/elastase. TRAM serves as a stress sensor of oxLDL and/or free cholesterol, triggers inflammatory signaling processes that facilitate elastase release. Our data defines a novel resolving neutrophil subset due to reduced TRAM expression which may hold therapeutic potential for reducing inflammation and treating atherosclerosis.
Abstract 2083: Apolipoprotein A-II Increases Cholesterol Efflux Capacity Of HDL Via The Apolipoprotein A-I C-terminus
Authors: Morris Jamie C , Sexmith Hannah , Street Scott E , Melchior John T , Shah Amy S , Davidson William
Published: July 2024

The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux (CEC) is a more robust predictor of cardiovascular disease protection than its plasma quantity. Previously, we found that fully lipidated HDL containing apolipoprotein A-II (APOA2) promotes cholesterol efflux via the ATP binding cassette transporter (ABCA1). This was surprising given that ABCA1 is thought to primarily interact with lipid-poor apolipoproteins. Having previously focused on isolated lipoproteins, we moved into human plasma with the hypothesis APOA2 can enhance ABCA1-mediated CEC in this more complex environment. Human plasma was incubated with increasing amounts of lipid-free APOA2. The samples were assayed for CEC from macrophages +/- cAMP for ABCA1 stimulation. APOA2 dose dependently increased whole plasma CEC - due mostly to HDL as the effect persisted in apoB-depleted plasma. Next, plasma was incubated with lipid-free APOA1 or APOA2, immediately fractionated by size exclusion chromatography and each fraction assayed for CEC. In unmodified plasma, cholesterol effluxed to peaks corresponding to LDL, HDL and lipid-free apolipoproteins with the latter exclusively increased by ABCA1 expression, as expected. When lipid-free APOA1 was added, efflux to LDL and HDL remained largely unchanged. Strikingly, when APOA2 was added, APOA2 increased ABCA1-mediated cholesterol efflux to the fully lipidated HDL fractions. Proteomic analyses indicated that APOA2 increased in HDL though some APOA1 was retained. To explore the mechanism, we reconstituted HDL particles with APOA1 that either contained or lacked its C-terminal lipid binding helix. APOA2 lost the ability to stimulate ABCA1 efflux to HDL when the C-terminal domain of APOA1 was deleted. Our current hypothesis is that APOA2 displaces the C-terminal helix of APOA1 from the HDL surface which can then interact with ABCA1 - much like it does in lipid-poor APOA1. Reconstituted forms of APOA1 are under development as a therapy to rapidly clear cholesterol from coronary arteries in patients with acute coronary syndrome. Our work suggests APOA2 may be a novel target given its dual benefits for cholesterol efflux.
Abstract 2093: Quantitative Assessment Of Venous Thrombus Resolution And Macrophage Content In Mice Using Ferumoxytol-enhanced Magnetic Resonance Imaging
Authors: Morrison Logan , Nguyen Khanh P
Published: July 2024

Objective:
The objective of this study is to explore the feasibility of ferumoxytol-enhanced magnetic resonance imaging (Fe-MRI) for noninvasive assessment of deep venous thrombus (DVT) resolution and macrophage content in resolving thrombi in a surgical murine model of inferior vena cava (IVC) ligation.

Background:
Imaging evaluation of acute DVT or post-thrombotic syndrome (PTS) in animal or clinical models is limited to assessment of anatomy. We hypothesize that Fe-MRI, used to evaluate macrophage content in other inflammatory diseases, can be useful to evaluate thromboinflammatory features after DVT.

Methods:
Nineteen wild-type CD-1 mice underwent surgical IVC ligation to induce DVT. Mice received either saline or administered 5 mg/kg of 14E11, a Factor XI inhibitor, before the procedure. Fe-MRI was performed from days 6-7 after ligation to evaluate thrombus volume, perfusion and macrophage content via T2, and T2 *-weighted images. Mice were euthanized at days 3-15 after surgery. The thrombi and adjacent vein walls were excised, weighed, formalin fixed, and paraffin embedded for histological analysis. Specimens were stained with specific antibodies to evaluate macrophage activity, collagen deposition, neovascularization, and recanalization. Significance was determined using the Mann-Whitney U or Student’s t-test.

Results:
After IVC-ligation in control mice, thrombus weights decreased by 59% from day 3 to day 15. FXI inhibition led to reduced macrophage content in both thrombus (p = .008) and vein wall (p = .01), decreased thrombus volume (p = .03), decreased thrombus mass (p = .01) and impaired thrombus perfusion (p = .01) compared to control mice. Iba1 staining supported these findings, showing significantly reduced macrophage presence in thrombi (p = .02).

Conclusions:
Fe-MRI T2 and T2 * relaxation times can be used to characterize and quantify post-thrombotic changes of perfusion, macrophage activity, and volume over time in a surgical mouse model of venous thrombosis. This approach could lead to better quantification of in vivo inflammation correlating monocyte and macrophage activity within resolving thrombi and veins and may serve as a useful tool in research and clinically in the evaluation of DVT or PTS.