bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2013: Smooth Muscle Cell-specific Deficiency Of Fibrillin-1 Induces Aortic Aneurysms But Does Not Disrupt Elastic Fiber Formation In Mice
Authors: Li Bowen , Ito Sohei , Moorleghen Jessica J , Sawada Hisashi , Daugherty Alan , Lu Hong S
Published: July 2024

Background and Aim:
Fibrillin-1 has a complex biology that can influence elastic fiber formation and stability. Smooth muscle cells (SMCs), the most abundant cell type of the aortic wall, synthesize and secrete fibrillin-1. The purpose of this study was to determine the effects of fibrillin-1 (FBN1) synthesis in SMCs on elastic fiber formation and aortic pathologies.

Methods and Results:
We generated
Fbn1
floxed mice that included the insertion of tdTomato as a reporter for gene deletion. Female
Fbn1
floxed mice were bred with male
Fbn1
floxed mice expressing
Cre
transgene driven by a
Tagln
promotor that led to the deletion of FBN1 in SMCs (SMC-FBN1-/-). Gene recombination was confirmed by the presence of tdTomato in the aorta of SMC-FBN1-/- mice. To determine the impact of SMC-specific FBN1 deletion on aortic integrity, SMC-FBN1 +/+ and -/- littermates were either terminated at 4 weeks of age with capture of
in situ
and ex vivo aortic images or aortas of live animals at 8 weeks of age were monitored using ultrasound. For mice terminated at 4 weeks of age, SMC-specific FBN1 deletion resulted in increased ascending aortic length (+/+ vs -/-: 2.9 vs 3.9 mm in males, p=0.004; +/+ vs -/-: 2.9 vs 3.5 mm in females, p=0.004) and width (+/+ vs -/-: 1.5 vs 1.9 mm in males, p=0.03; +/+ vs -/-: 1.4 vs 1.7 mm in females, p=0.02) of the ascending aorta and the aortic arch (+/+ vs -/-: 1.4 vs 1.9 mm in males, p<0.001; +/+ vs -/-: 1.4 vs 1.7 mm in females, p=0.01), whereas diameters of the descending thoracic aorta and suprarenal aorta were not different between SMC-FBN1 +/+ and SMC-FBN1-/- mice in either sex. Surprisingly, elastic fibers, as determined by Verhoff staining, were present in the media of each aortic region in SMC-FBN1-/- mice at 4 weeks of age, with comparable numbers of elastic lamellae as SMC-FBN1+/+ littermates in each sex. At 8 weeks of age, ultrasound detected profound luminal dilatations of the ascending aortic region and the suprarenal abdominal region in SMC-FBN1-/- mice, compared to their wild-type littermates.

Conclusion:
SMC-specific deficiency of FBN1 induced aortic aneurysms progressively, increasing with age, but did not overtly affect elastic fiber formation and the number of elastic lamina layers.
Abstract 2015: Pyruvate Carboxylase-mediated Vascular Smooth Muscle Cell Mitochondrial Dysfunction Activates Cgas-sting Signaling In Aortic Dissection.
Authors: Liu Deshen , Zhao Li , Zhao Lingna , Huang Fangze , Zhu Zhengqiang , Zheng Shaoyi , Liu Xiu
Published: July 2024

Background:
Aortic Dissection (AD), characterized by the loss of vascular smooth muscle cells (VSMC) and degradation of the extracellular matrix, carries an exceptionally high mortality rate, and as of now, there are no effective drugs to impede its occurrence and progression. Deciphering the mechanisms governing the onset and progression of aortic dissection is paramount for the development of innovative therapeutic modalities. Recent evidence suggests a pivotal role of mitochondrial metabolism in vascular inflammation, with pyruvate carboxylase (PC) emerging as a crucial component of the tricarboxylic acid cycle. In this study, we investigated the influence of PC on the occurrence and development of AD, elucidating the underlying mechanisms.

Methods:
Investigating alterations in PC expression levels in aortic tissues of both humans and C57BL/6 mice, and assessing the impact of PC on the occurrence and AD in PC

SMKO

mice. The study further explores the role of PC through both
in vivo
and
in vitro
investigations to delineate the associated pathways.

Results:
We observed a significant upregulation in the expression of PC in AD tissues. In the AD model of mice, the occurrence rate of AD and the mortality resulting from aortic dissection rupture were significantly reduced in PC

SMKO

mice. Transcriptomic results revealed that overexpression of PC in VSMCs could activate the STING pathway. Inhibition of the STING pathway significantly reduced both the occurrence and mortality rates of AD in mice. Mechanistically, overexpression of PC induced mitochondrial metabolic dysregulation, leading to nuclear and mitochondrial DNA damage. The released DNA into the cytoplasm activated the transduction of the STING signaling pathway, consequently causing necrosis of VSMC.

Conclusions:
Our results indicated that PC could facilitate the occurrence and progression of AD by activating the STING pathway. Targeting PC has the potential to prevent the onset of AD.
Abstract 2016: Prior Diagnosis Of An Abdominal Aortic Aneurysm Is Associated With An Elevated Risk Of Subsequent Cancer Diagnosis
Authors: Luo Lingfeng , Haas Allen , Bell Caitlin , Baylis Richard , Adkar Shaunak , Fu Changhao , Angelov Ivan , Giordano Sharon , Klarin Derek , Leeper Nick J , Nead Kevin
Published: July 2024

Background:
Epidemiological and mechanistic evidence suggests a potential causal link between cardiovascular disease (CVD) and cancer. Abdominal aortic aneurysms (AAA), a prevalent form of CVD with distinct genetic and biological etiologies, lack clarity regarding cancer risk compared to other CVD types. We conducted a retrospective cohort study using the IBM MarketScan Research Database to investigate whether AAA individuals face an increased cancer risk independent of traditional shared risk factors.

Methods:
Enrolling individuals aged ≥18 with ≥36 months of continuous coverage between 2008 and 2020, we excluded those with potential Mendelian AAA etiologies, aortic aneurysms with nonspecific anatomic location, or pre-follow-up cancer diagnoses. A subgroup analysis was performed including individuals with Health Risk Assessment records that included smoking status and BMI. We compared three groups: 1) AAA, 2) non-AAA CVD, and 3) no-CVD.

Results:
The propensity score-matched cohort comprised 68,269 AAA, 136,538 non-AAA CVD, and 136,538 no-CVD individuals. The 5-year cumulative incidence of any cancer was 10.0% (9.7 - 10.3%) in AAA, 7.4% (7.2 - 7.6%) in non-AAA CVD, and 7.4% (7.2 - 7.5%) in no CVD. Time-dependent multivariable-adjusted Cox proportional hazards regression models revealed AAA patients had a higher cancer risk than non-AAA CVD (HR: 1.32; 95% CI: 1.28-1.37; p-value: <0.001) or no CVD (HR: 1.37; 95% CI: 1.32-1.44; p-value: <0.001). Results were consistent after excluding common smoking-related cancers and when adjusting for smoking status and BMI in a subgroup analysis.

Conclusions:
AAA patients may face a uniquely elevated cancer risk, warranting further mechanistic study and enhanced cancer screening investigations.
Abstract 2018: The Role Of Interleukin 27 Receptor Signaling In Regulation Of Intestinal Barrier, Gut Microbiota And Abdominal Aortic Aneurysm.
Authors: Marquez Sanchez Ana C , Titerina Elizaveta , Mazitova Aleksandra , Carnes Julianne M , Kossenkov Andrew , Rodrigues Richard , Trinchieri Giorgio , Koltsova Ekaterina
Published: July 2024

Abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by accumulation of immune cells, inflammation, and degradation of medial layer of the abdominal aorta. Angiotensin II (Ang II) plays a key role in controlling blood pressure but is also able to regulate various immune and non-immune cells. While some studies suggested the role of Ang II in dysbiosis, the link between alteration of intestinal microbiota and AAA is largely unexplored. Cytokines are established regulators of inflammatory milieu in the aortic wall in AAA, but their distant contribution, for example via control of intestinal microbial homeostasis and cooperation with Ang II, has never been explored. Interleukin (IL)27 is a member of the IL6/IL12 superfamily that regulates various immune and non-immune cells. We previously showed that IL27R signaling cooperates with Ang II in the regulation of myelopoiesis thereby promoting AAA. Myeloid progenitor expansion and myeloid cell production driven by AngII are blunted in

Apoe
-/-
Il27ra
-/-

mice. Here we found that

Apoe
-/-
Il27ra
-/-

mice were also protected from gut dysbiosis and contain less pro-inflammatory bacterial genera in their colons. Unexpectedly, microbiota depletion accelerated AAA development in

Apoe
-/-
Il27ra
-/-

mice, while

Apoe
-/-

controls were protected from the disease. RNA seq analysis revealed upregulation of inflammatory pathways and downregulation in tight junctions and collagen in colons of

Apoe
-/-
Il27ra
-/-

mice with ablated microbiota. Transcriptomic analysis of

Apoe
-/-

colonic organoids stimulated with recIL27 shows upregulation of pathways linked to cell proliferation, cell cycle, and junction genes, indicating direct role of IL27. The analysis of the bone marrow revealed elevation of early myeloid progenitors which correlated with heightened circulating monocytes and neutrophils in

Apoe
-/-
Il27ra
-/-

mice with ablated microbiota. Non-targeted metabolomics revealed microbiota and IL27R signaling-dependent alteration of serum metabolites likely mediating the effect on myeloid cells and AAA. Overall, our work uncovers novel role of IL27R signaling in the regulation of AAA, mechanistically connecting intestinal barrier and microbiota to the pathogenesis of this CVD.
Abstract 2029: Augmenting Mitochondrial Respiration In Immature Smooth Muscle Cells Is A Therapeutic Target For Moyamoya-like Cerebrovascular Disease
Authors: Kwartler Callie , Kaw Anita , Majumder Suravi , Wu Ting , Starosolski Zbigniew , Pinard Amelie C , Kernell Caroline , Zhou Zhen , Ghaghada Ketan , Marrelli Sean P , Milewicz Dianna M
Published: July 2024

Introduction:
Moyamoya disease (MMD), characterized by occlusive lesions in the distal internal carotid arteries, is a malignant cause of pediatric stroke. Surgical revascularization is the mainstay treatment, but peri- and postoperative ischemic complications are common. Multiple identified genes account for only a small percentage of cases. Identifying a common pathway of disease pathogenesis would enhance care for MMD patients.

Goal and Approach:
We used cellular and animal models for a rare cause of MMD-like cerebrovascular disease, heterozygous
ACTA2
p.R179 pathogenic variants, to define molecular mechanisms and assess therapeutic strategies. Our prior work showed that smooth muscle cells (SMCs) with
ACTA2
p.R179 variants are incompletely differentiated. We hypothesized that these immature phenotypes could be rescued by manipulation of cellular metabolism. We validated our results with cellular models of other pediatric MMD-associated variants: loss-of-function variants in
Pcnt
(explanted from a mouse model) and a pathogenic variant in
RNF213
(generated via Crispr/Cas9 in human iPSCs).

Results:
Acta2
SMC-R179C/+
SMCs have increased glycolysis and reduced oxidative respiration on Seahorse analyses. Molecular characterization showed that
Acta2
SMC-R179C/+
SMCs have reduced mitochondrial mass and decreased complex I activity, which is rescued by nicotinamide riboside (NR) treatment. NR surprisingly also increases SMC differentiation and decreases migration.
Acta2
SMC-R179C/+
mice and littermate controls were subjected to carotid artery ligation, and 33% of
Acta2
SMC-R179C/+
mice died due to strokes while all control mice survived (Kaplan-Meier p<0.01). Surviving
Acta2
SMC-R179C/+
mice have significant cerebrovascular remodeling and persistent occlusive carotid artery lesions. Treatment with NR reduces death (Kaplan-Meier p=0.08) and resolves persistent lesions (p<0.05) in
Acta2
SMC-R179C/+
mice after carotid ligation.
Pcnt
and
RNF213
mutant SMCs have a similar phenotype of incomplete differentiation and decreased mitochondrial respiration as
ACTA2
p.R179 and are responsive to NR treatment.

Conclusions:
These data provide evidence for a common and therapeutically targetable pathway in pediatric MMD patients.
Abstract 2030: Endothelial Deletion Of Bag3 Does Not Impair Ischemic Limb Recovery In A Murine Model Of Peripheral Artery Disease
Authors: Li Feifei , Terwilliger Zoe , Kolasa Makenzie , Green Thomas , karnekar Reema , Spangenburg Espen , Fisher-Wellman Kelsey , Yamaguchi Dean , McClung Joseph
Published: July 2024

Background:
Peripheral artery disease (PAD) is a highly prevalent vascular occlusive disease which mostly affects the lower limb. Emerging studies proved the critical functions of endothelial cells in arteriogenesis and ischemic limb blood flow recovery. However, very few targets with endothelial cell exclusivity have been identified. Here, we explored the role of endothelial BCL-2 associated athanogene 3 (Bag3) in ischemic limb recovery.

Hypothesis:
We hypothesized that the loss of endothelial specific BAG3 would attenuate blood flow and muscle recovery following HLI.

Methods:
Hind limb ischemia (HLI) was induced in Bag3 Cdh5(PAC)-CreERT2 male and female mice after tamoxifen (2mg/day x 5 days) or vehicle treatment (
n
≥12/group). Limb blood flow was measured by laser Doppler perfusion imaging (LDPI) and isometric muscle force production, vascular density and muscle morphology were assessed after HLI.

Results:
LDPI analysis revealed no differences in the blood flow between groups at HLI d7 (
n=9
; day 7, 21.86% ischemic/non-ischemic ± 3.80% vs 18.05% ± 1.96% SEM; p=0.05). There was no deficit in isometric force recovery of the EDL (
n
≥12; 57.76 ± 15.92 vs 83.05 ± 27.12 SEM;
p
=0.05) in KO animals at HLI d7. Similar rates of CD31+ vessel disorganization was observed in both groups at HLI d7, (
n
=7; 4626.0 um
2
CD31+ area ± 869 vs 4870.9 ± 1056 SEM) consistent with recovering ischemic tissue. There were also no differences in blood flow recovery assessed by LDPI at HLI d28 (
n
=7; 50.74% ischemic/non-ischemic ± 7.4% vs 59.17 ± 8.21; n=9).

Conclusions:
Endothelial cell specific Bag3 knockout did not impair vascular morphology or perfusion recovery at baseline or following HLI in a murine model of peripheral artery disease. No differences in muscle morphology or contractile function at baseline or after HLI at either timepoint were observed. In summary, we reveal an unexpectedly minor role of endothelial cell BAG3 in tissue recovery following ischemic injury.
Abstract 2034: Gut Microbial Markers Associated With Clinical Features Of Peripheral Artery Disease
Authors: Niraula Sarbjeet , Stirewalt Spencer L , Jung Jonathan , Alagna Megan E , Jang Jae , Wu Erik , Du James , Xiong Liqun , Green Stefan J , Seed Patrick C , Ho Karen J
Published: July 2024

Introduction:
The human gut microbiome and microbe-derived metabolites are known to be associated with cardiovascular disease. However, their associations with lower extremity peripheral artery disease (PAD) are not well understood. The goal of this study is to identify specific gut microbial markers associated with clinical features of PAD.

Methods:
Study participants aged ≥ 18 years with documented PAD and non-PAD controls were recruited from a single center and performed home fecal sample collection. Sample aliquots were stored at -80°C until DNA extraction and shotgun metagenome sequencing. Untargeted metabolite quantification was performed using nuclear magnetic resonance. Samples from participants on antibiotics were excluded. Raw paired end sequences were quality filtered using the KneadData pipeline followed by taxonomic annotation using Kraken2 against the UHGG database (v2.0.2) and species abundance estimation using Bracken. A species network was constructed with centered log-ratio transformed abundance data and statistical analyses were performed in R.

Results:
Among 115 study participants, 80 had PAD and 35 were non-PAD controls. Of those with PAD, 40 (50.0%) had chronic limb threatening ischemia (CLTI) and 39 (48.8%) had symptoms of claudication. There were significant differences in alpha and beta diversity between the claudication and CLTI subgroups but none between the non-PAD and PAD groups overall. There were multiple significantly differentially abundant microbial features in the PAD group compared to non-PAD controls, including species within the genera
Blautia
and
Clostridium
. Metabolite profiles were also significantly different between the PAD and non-PAD groups, including higher abundance of the short chain fatty acid butyrate in the non-PAD controls (p=0.02). Network analyses identified three clusters of species with higher relative abundance in the claudication group compared to the CLTI group, and members of these clusters included putative butyrate producers.

Conclusions:
We identified specific microbial and metabolomic patterns associated with the clinical presentation of PAD and highlight a potential role for microbially-mediated butyrate in disease state.
Abstract 2051: Monocyte Recruitment And Fate Specification Within Atherosclerotic Lesions
Authors: Hillman Hannah , Patterson Michael T , Xu Yingzheng , Schrank Patricia R , Kennedy Ainsley E , Williams Jesse W
Published: July 2024

Recruited monocytes that subsequently differentiate into macrophages within atherosclerotic lesions are a primary driver of plaque progression and a risk factor for rupture. Advances in single cell RNA sequencing have expanded our understanding of macrophage diversity within plaques. However, limited analysis has explored regulators of monocyte lineage commitment and spatial localization in atherosclerotic plaque. Examining scRNAseq gene expression data of plaque-associated monocyte and macrophage lineages, pseudotime trajectory analysis predicted a binary fate decision by monocytes differentiating toward either an inflammatory or lipid-loaded “foamy” differentiation state. Foamy state was associated with lipid processing genes (
Fabp5, Lgals3, Trem2
), whereas inflammatory state was associated with inflammasome genes (
Il1b, Nlrp3
) and chemokines (
Ccl3, Ccl4
). To test the in silico analysis, we established a monocyte fate mapping approach using a tamoxifen-inducible reporter mouse (CCR2
creERT2
Rosa26
lsl-tdTomato
) and performed kinetic analysis of tdTomato+ cells within plaque. tdTomato-labeled cells could be found shortly after tamoxifen treatment with small, rounded morphology, consistent with newly recruited monocytes. Within days, small, rounded cells were absent and labeled cells appeared to be lipid loaded with bloated morphology. Immunostaining revealed inflammasome activation in a large subset of monocytes immediately following recruitment to lesions. However, IL-1b
hi
cells were no longer detectable days after recruitment. Together, these data support a model where recruited monocytes commit toward either inflammatory or foamy lineage immediately upon plaque entry. Further, data support that inflammatory cells are short lived in atherosclerotic plaques, whereas foamy cells persist for longer periods of time. Ongoing work in our lab will address the role of known foamy macrophage regulators, including Trem2, in modulating fate commitment of monocytes entering early and late stage atherosclerotic lesions. Understanding the spatial-temporal dynamics of monocyte recruitment and differentiation will illuminate new mechanisms of atherosclerosis progression and potential therapeutic targets.
Abstract 2061: Single-cell Analysis Reveals Insights Into Visceral Adiposity In Mice With Diabetes-associated Atherosclerosis
Authors: HU YI , Chen Jingshu , Jamaiyar Anurag , Feinberg Mark W
Published: July 2024

Introduction:
Visceral white adipose tissue (vWAT), previously seen as passive and uniform, plays a dynamic role in metabolism and immune response. Dietary shifts affect its structure and function, impacting conditions like insulin resistance, dyslipidemia, and diabetes. vWAT is also linked to vascular diseases, but its role in atherosclerosis in mouse models is not fully understood.

Methods:
vWAT from male
Ldlr
-/-
mice fed either a chow diet or high fat, high sucrose containing (HFSC) diet for 24 weeks were harvested. scRNA-seq, histology, and immunofluorescence were used to analyze the cellular and transcriptomic profiles in Chow and HFSC groups.

Results:
Distinct subpopulations of endothelial cells (ECs), immune cells, and adipose stem and progenitor cells (ASPCs) were identified, highlighting differences between healthy and diseased states. Notably, HFSC-fed mice exhibited an increase in immune cell composition, coupled with a decrease in ECs and progenitor cells. Cell subtype distribution in macrophages shifted notably, with more Lipid-associated (LAM), but fewer Perivascular-like macrophages (PVM). Novel markers Lyz2 and Trem2, linked to atherosclerosis in
Ldlr
-/-
mice, were identified specific to LAM. Pathway analysis showed Pdgfa, S100a8, and Mmp13 upregulation in LAM, crucial for atherosclerosis development. The probability model of cell-cell communication was established and characterized as 3 distinct cell patterns that enriched in pathways (Chemokine, Wnt & BMP signaling) implicated in atherosclerosis progression.

Conclusion:
scRNA-seq atlas reveals interactions among adipose cell types in lean and obese state. Inflammation triggers cellular distribution changes, with overlapping markers and pathways in vWAT and atherosclerosis emerging in disease progression. This dataset serves as a valuable resource for exploring genes and cell types involved in vWAT function in diabetes-associated atherosclerosis.
Abstract 2063: Endothelial Cell Scavenger Receptor Class B, Type I Expression Is Transcriptionally Controlled By HIF-1α
Authors: Huang Yibin , Peng Jun , Chambliss Ken , Chen Kenian , Xu Lin , Huang Linzhang , Mineo Chieko , Shaul Philip W
Published: July 2024

Background:
Scavenger Receptor Class B, Type I (SR-BI), encoded by Scarb1, mediates LDL cholesterol transcytosis in endothelial cells (EC) to deliver circulating LDL into the subendothelial space to drive atherogenesis.

Research Questions:
It is unknown whether EC SR-BI expression is altered in atherosclerosis, and how EC SR-BI expression is regulated.

Approach:
Single cell RNAseq was performed on coronary arteries from 5 ischemic heart disease patients, in segments both with and without a grossly apparent atheroma. Transcription factor (TF) network analysis was done to reveal possible regulatory relationships between differentially-expressed genes (DEGs). The modulation of EC SR-BI was studied in primary human aortic EC (HAEC) and in vivo in mouse aortic EC. ChIP-seq, ChIP-qPCR and CRISPR-based mutagenesis were used to interrogate TF binding to the human Scarb1 gene. EC SR-BI expression was determined by immunoblotting and qPCR, and EC LDL transcytosis was studied in transwells using DiI-labeled LDL.

Results:
SR-BI expression was greater in EC in coronary segments with atheroma versus those without atheroma (p=0.003). TF networks for DEGs in atheroma versus non-atheroma EC, and for DEGs in EC with low versus high SR-BI revealed multiple TF with possible regulatory relationships with Scarb1. One TF in common was HIF-1α. In HAEC, an increase in HIF-1α with the HIF prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) caused upregulation of SR-BI mRNA and protein, and a parallel increase in LDL transcytosis that was entirely SR-BI-dependent. Expression of a constitutively-active HIF-1α variant also increased SR-BI and LDL transcytosis. ChIP-seq revealed a HIF-1α binding site in Scarb1 intron 1, and HIF-1α binding there increased 4-fold with DMOG. CRISPR deletion of the binding site fully prevented SR-BI upregulation by DMOG without affecting known HIF-1α target genes. In mice, 3d treatment with DMOG increased SR-BI in aortic EC.

Conclusions:
EC expression of SR-BI is upregulated with increasing atherosclerosis severity in human coronary artery. HIF-1α is upregulated in parallel, and HIF-1α causes the direct transcriptional upregulation of EC SR-BI leading to enhanced EC LDL transport. HIF-1α control of SR-BI in EC may be critical to atherogenesis.
Abstract 2065: Activation Of Cgas-sting Axis By DNA Double-strand Breaks In Atherosclerosis: A Possible Link From DNA Damage To Inflammation And Cell Senescence
Authors: Ishida Mari , Sakai Chiemi , Kobayashi Yusuke , Ishida Takafumi
Published: July 2024

Aim:
Premature atherosclerosis in some progeroid syndromes suggests the involvement of DNA damage in the progression of atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be determined. The aim of this study was to elucidate the role of DSBs in atherosclerosis using mice and vascular smooth muscle cells (VSMCs) deficient in Ku80, a DSB repair protein.

Methods and Results:
The plaque size and DSBs were increased in the aorta of Ku80-deficient apolipoprotein E knockout mice (Ku80
+/-
ApoE
-/-
) compared to those of ApoE
-/-
control after 4 weeks of a high-fat diet. At the early stages of atherosclerosis (two-week high-fat diet), although the plaque size of Ku80
+/-
ApoE
-/-
was similar to that of ApoE
-/-
control, the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80
+/-
ApoE
-/-
aortas. We further investigated molecular links between DSBs and inflammatory responses using VSMCs isolated from Ku80
+/-
mice. In VSMCs from Ku80
+/-
mice, IL-6 mRNA level was elevated along with increased cellular senescence markers, such as p16 mRNA and senescence-associated (SA) β-gal activity. Additionally, Ku80
+/-
VSMCs showed accumulated cytosolic free DNA, an increased level of cGAMP, and activation of TBK1, interferon regulatory factor 3 (IRF3) and NF-κB, suggesting the activation of cGAS-STING axis, a cytosolic DNA sensing machinery. Activation of cGAS-STING axis was also shown in the aortae from Ku80
+/-
ApoE
-/-
in comparison with those from ApoE
-/-
control. Silencing IRF3, but not NF-κB attenuated the IL-6 mRNA level in Ku80
+/-
VSMCs, so did cGAS-silencing. Of interest, cGAS-silencing also attenuated DNA damage-induced SA β-gal activity in wild-type VSMCs. Furthermore, depletion of either p16 or p53 suppressed IRF3 activation by DNA damage.

Conclusions:
These results suggest that accumulation of cytosolic DNA induced by DNA damage accelerates atherosclerosis via the activation of cGAS-STING-IRF3 axis. cGAS-STING-IRF3 axis may play a pivotal role as a common signaling pathway that serve as a link from DNA damage to inflammatory and cell senescence response in arterial wall.
Abstract 2067: Myeloid Cell Lgr4 Contributes To Atherosclerotic Lesion Formation
Authors: Kathuria Ishita , Aithabathula Ravi V , Pervaiz Naveed , Le Britney , Kim Ki-Suk , Wang Jiqiu , Singh Udai P , Singla Bhupesh
Published: July 2024

Background:
R-Spondin 2 (RSPO2), a matricellular protein, primarily functions via binding to cell surface receptors including leucine-rich repeat-containing G protein-coupled receptor (LGR) 4, 5, and 6. We previously identified elevated RSPO2 levels in both human and mouse atherosclerotic arteries. Our preliminary experiments demonstrated increased expression of LGR4 in human atherosclerotic aortic segments, identified
Lgr4
as the major RSPO2’s receptor in primary murine macrophages, and atherogenic molecule oxLDL stimulated LGR4 levels in macrophages. However, the precise role of macrophage LGR4 in atherosclerosis development is unknown.

Methods and Results:
We generated novel myeloid cell-specific
Lgr4
knockout mice (
Lgr4
fl/fl
LysM
Cre
+/-
,
Lgr4
ΔM
) by breeding
Lgr4
fl/fl
mice with
LysM
Cre
+/-
mice. AAV8-h
PCSK9
-injected
Lgr4
ΔM
and littermate control
Lgr4
fl/fl
(
Lgr4
WT
) were fed a Western diet (16 weeks) to investigate for atherosclerosis. Oil red O (ORO) staining of whole aortas (en face) and aortic root sections showed reduced atherosclerosis in male
Lgr4
ΔM
mice compared with sex-matched
Lgr4
WT
mice. Further histochemical staining performed on aortic root sections revealed smaller necrotic cores in
Lgr4
ΔM
mice. However, there were no significant differences in plasma total cholesterol and body weight. Interestingly, male
Lgr4
ΔM
mice had decreased fat mass percentage, blood glucose, and epididymal adipose tissue weight. Initial studies with female mice (
n
= 4/group) demonstrated no differences in atherosclerosis and metabolic parameters. Additional
in vitro
studies exhibited that RSPO2 treatment induces oxLDL uptake, reduces efferocytic capacity, and promotes activation of VASP protein (inducer of actin aggregates) in macrophages. Further, the deletion of
Lgr4
reduces lipid accumulation in macrophages.

Conclusions:
Taken together, these findings suggest that deletion of myeloid cell
Lgr4
suppresses atherosclerotic lesion formation and identify Lgr4 as a novel therapeutic target for atherosclerosis.
Abstract 2069: Impact Of Endothelial Stiffening And CD36-mediated Lipid Uptake On Atherosclerotic Lesion Formation
Authors: Le Master Elizabeth , Aguilar Victor , Lee James , Levitan Irena
Published: July 2024

Objective:
To determine the impact of endothelial stiffening induced by CD36 (cluster of differentiation 36) lipid uptake in the development of atherosclerosis.

Approach and Results:
Endothelial stiffening of intact
en face
monolayers, as assessed by atomic force microscopy, reveal the hyperlipidemic mouse model of atherosclerosis, LDLR
-/-
(low density lipoprotein receptor knockout) is significantly stiffer, especially in the atheroprone aortic arch, as compared to wild type controls (n=4 male mice with 8-15 tissue measurement sites per condition per mouse, p<0.05). Furthermore, a 4-5 month high fat diet (HFD) feeding in LDLR
-/-
mice resulted in significantly enhanced stiffening compared to regular chow feeding, an effect that was observed in males but not females (n=6-8 mice, p<0.05). There was a significantly greater plaque development, as analyzed using Oil O Red staining for lipids, in male HFD-fed LDLR
-/-
mice, as compared to age-matched females (n=5-6, p<0.05). No significant difference between males and females was found in lipid panel parameters (n=4-6) or weight gain (n=10-14). Specific downregulation of CD36 in male hypercholesterolemic Cdh5.CreERT2CD36fl/fl LDLR
-/-
mice led to both a significant decrease in endothelial stiffening (n=5 mice, p<0.05) and in the areas of atherosclerotic lesion formation (n=5-6 mice, p<0.05). Mechanistically, we show that the saturated fatty acid, palmitic acid, increased endothelial stiffening in the
en face
monolayers of wild type mice, an effect which is prevented by the pre-treatment of the CD36 irreversible inhibitor, SSO (sulfo-N-succinimidyl oleate, n=5-6 male mice, p<0.05).

Conclusions:
These results highlight that endothelial stiffness from intact aortas and atherosclerotic plaque formation following HFD feeding is strongly dependent on endothelial CD36.
Abstract 2071: Identification Of Genetic Loci Influencing Lethal Atherothrombosis In Mice
Authors: Lichtenberg Katherine
Published: July 2024

Introduction:
Atherothrombosis leading to myocardial infarction and stroke is a major cause of mortality. Ruptured atherosclerotic plaques release prothrombotic molecules such as tissue factor, which initiates thrombosis. Both genetic and environmental factors contribute to atherothrombosis. Combined complete deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes (dKO) on a mixed C57BL/6J (B6) and 129S1/SvImJ (129S1) background leads to an atherothrombotic phenotype and early death before 8 weeks of age. However, the dKO phenotype is dramatically different if the Scarb1 and ApoE deficient mice are backcrossed extensively to the B6 mouse strain, with no dKOs surviving to the weaning age.

Hypothesis/Goals:
We hypothesized that 129S1 genetic variants reduce atherothrombosis severity in dKO mice and used mouse genetics to identify these loci.

Methods:
To identify potential genetic factors, we produced and analyzed Scarb1 and ApoE dKO mice from 6 backcross/intercross generations with progressively more B6 genomic contribution. Weights were taken for 38 dKO mice and littermates from 3 weeks old until death of the dKO mice at 8 weeks of age. dKO mice from F1-F3 generations were divided into 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse DNA was subjected to genome wide marker genotyping using the GigaMUGA platform. Quantitative trait loci analysis (QTL) was performed to identify loci responsible for the phenotypic weight differences.

Results:
dKO weights fell into 3 categories: indistinguishable from littermates, 14%-39% less than littermates, and greater than 39% less than littermates. QTL analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. Niemann Pick type C2 (Npc2), an intracellular cholesterol transporter, is within the interaction region on Chr 12 and is a prime candidate dKO modifier.

Conclusion:
We used weight data as a representative of overall health to categorize dKO phenotypes relative to non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate modifier gene for the Chromosome 12 locus. Identification of atherothrombosis modifier genes will provide novel therapeutic targets for this complex disease.
Abstract 2080: Chip E3 Ligase Mediates Vascular Smooth Muscle Cell Proliferation And Migration By Regulating Mitochondrial Fission
Authors: Heo Kyung-Sun
Published: July 2024

Objective:
Excessive mitochondrial fission promotes the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to subsequent vascular disorders such as atherosclerosis and abdominal aortic aneurysms (AAAs). Therefore, targeting the signaling pathways linked to mitochondrial fission-driven VSMC dysfunction could be a promising therapy for vascular diseases. This study aims to explore novel target molecules and mechanisms underlying mitochondrial fission-induced VSMC phenotypic switching.

Methods:
To investigate the role of CHIP in VSMC function, VSMCs were isolated from the aorta of wild-type (WT) and CHIP-knock-out (KO) mice, and their proliferation and migration rates were compared. Western blotting, qRT-PCR analysis, MitoTracker staining, JC-1 staining, MitoSOX staining, and a luciferase assay were employed to elucidate the molecular mechanisms.

Results:
We found that angiotensin II (Ang II)-induced VSMC migration and proliferation were reduced by CHIP-KO. Consistently, CHIP deficiency rescued the effects of Ang II on MMP2, MMP9, PCNA, and cyclin D1, as observed in both protein and mRNA levels. Notably, the localization of DRP1 phosphorylation (Ser616) in mitochondria and the mitochondrial fragmentation induced by Ang II were inhibited in CHIP-KO cells. The inactivation of DRP1 using mitochondrial division inhibitor 1 (Mdivi-1) inhibited Ang II-induced KLF4 protein expression and promoter activity. Furthermore, DRP1 inhibition significantly suppressed Ang II-induced VSMC migration and proliferation. Interestingly, Ang II-induced transcriptional activity and protein expression of KLF4 were prevented by CHIP inhibition. Concomitantly, the inhibition of DRP1 or CHIP prevented Ang II from increasing intracellular and mitochondrial ROS production. Consistent with the in vitro data, the CHIP KO and Mdivi-1 treatment group did not show abdominal aortic aneurysm (AAA) formation when challenged with Ang II infusion (1000 ng/kg/min) and β-aminopropionitrile (1 mg/ml in drinking water).

Conclusions:
Ang II-increased CHIP expression promotes DRP1-dependent mitochondrial fission, leading to activation of p90RSK/KLF4 signaling pathway to VSMC proliferation and migration.
Abstract 2085: Study Of The Relationship Between Brachial Artery Volumetric Elastic Modulus And Estimated Brachial Artery Lumenal Area And Prognosis In Hemodialysis Patients
Authors: Sakurabu Yoshimasa , Uchida Haruhito A , Okamoto Shugo , komoto Kiichi , Haraguchi Souichirou , Hashimoto Masami , Hayashi Ruiko , Wada Kentarou , Asakawa Tomohiko , Katayama Katsuyoshi , Onishi Yasuhiro , Uchiyama Natsumi , Tanaka Keiko , Takeuchi Hidemi , Umebayashi Ryoko , Takemoto Rika , Wada Jun
Published: July 2024

Objectives:
Patients with hemodialysis (HD) have a higher incidence of CVD and worse prognosis than non-dialysis patients. Recently, a new device can measure novel parameters of arterial stiffness such as brachial artery volume elastic modulus (VE) and estimated brachial artery lumen cross-sectional area (eA) in a minimally invasive and simplified manner. Using this device, we investigated the relationship between these parameters and prognosis in HD patients.

Methods and Results:
This was a multicenter, prospective, observational study. A total of 246 patients on maintenance dialysis at six maintenance dialysis centers were registered. They were 158 men (64%), mean age 69±13 years, mean years on dialysis 5.8±6.2 years, BP 143±23/78±13 mmHg, Hb 11.5±6.9 g/dL, Alb 3.5±0.4 g/dL, corrected-Ca 9.2±0.8 mg/dL, P 5.4±2.7 mg/dL, iPTH 164±180 pg/mL. 125 patients (51%) had diabetes mellitus and 122 patients (50%) were current and past smokers. 51 (20%) had a history of ischemic heart disease, 23 (9%) had a history of symptomatic stroke, and 45 (18%) had a history of peripheral arterial disease. VE and eA were measured in the non-shunt limb during dialysis. Outcomes were death and MACE (non-fatal ischemic heart disease, symptomatic stroke, and PAD requiring intervention). 44 patients died, 20 had ischemic heart disease, 6 had symptomatic stroke, and 6 had PAD during the 3-year observation period. Patients were divided into 2 groups based on the median VE value and analyzed using the Kaplan-Meier method for death plus MACE, death, and MACE each, but no significant differences were found. eA was also examined in the same way, but no significant differences were found. Further, patients were divided into 4 groups based on the VE and eA values (high&high, high&low, low&high, and low&low for VE&eA, respectively) and were studied in the same fashion, the high&low and low&high groups had significantly higher incidence of death plus MACE (log-rank p=0.0184) and MACE (log-rank p=0.0310) than the high&high and low&low groups.

Conclusion:
In HD patients, we found that the combined assessment of VE and eA was significantly associated with their prognosis, suggesting that these parameters may be clinically useful.
Abstract 2086: Pyruvate Dehydrogenase - Induced Metabolic Reprogramming In Macrophages Ameliorates Atherosclerosis
Authors: SINAM IBOTOMBI SINGH , Chanda Dipanjan , Kim Min-Ji , Jeon Jae-Han , Lee In-Kyu
Published: July 2024

Introduction/Backgrounds:
Atherosclerosis is a cardiovascular disease initiated by dysregulated lipid homeostasis. Excessive lipid uptake of macrophage leads to forming foam cells in the intima of the arterial wall. Several transcriptome analyses in macrophage have revealed that oxidized-LDL (ox-LDL)-induced glycolysis are correlated with the degree of foam cell formation. Pyruvate dehydrogenase kinases (PDKs) regulate the activity of pyruvate dehydrogenase (PDH), gate-keeping enzyme responsible for switching the pyruvate metabolism from glycolysis to oxidative phosphorylation (OXPHOS). Here, we investigated the role of the metabolic switch by PDH in macrophages during foam cell formation.

Research Question/Hypothesis:
Here we tested the hypothesis that metabolic switching from glycolysis to OXPHOS in macrophage by PDK inhibition ameliorates atherosclerosis by suppressing iNOS-induced foam cell formation.

Goals/Aims:
The aim to asses a potential therapeutic approach for the management of atherosclerosis by inhibition of PDK

Methods/Approach:
Bone marrow cells from wild-type or PDK X-deficient mice were transplanted into irradiated LDLR-/- mice, followed by western diet (WD) for 18 weeks. Mouse bone marrow-derived macrophages (BMDMs) were treated with PDK inhibitor, followed by activation with ox-LDL for 24hr.

Results/Data:
Oil red O staining of the whole aorta and heart valve revealed that PDK X-deficient bone marrow transplanted WD-fed LDLR-/- mice significantly reduced atherosclerotic plaques. PDK inhibitor hampered ox-LDL-induced M1 polarization, confirmed by Nos2 expression. Moreover, PDK inhibitor-treated BMDM showed a marked decrease in nitric oxide production.

Conclusion:
These findings suggest that PDH-mediated metabolic switch ameliorates atherosclerosis by suppressing iNOS-induced foam cell formation. Our data provide evidence that inhibition of PDK may be a promising therapeutic agent for atherosclerosis.
Abstract 2092: Ultrasound And Histology Protocol For Characterization Of Venous Stents Effect In Deep Veins In Vivo
Authors: Moreno Oscar Y , Clay Amber , Luke Catherine , Kumar Kiran , Rocco Sabrina , Parchment Nathaniel , Myers Daniel D , Wakefield Thomas , Henke Peter , Obi Andrea T
Published: July 2024

Background:
Deep venous stenting is a common strategy to treat deep vein thrombosis (DVT) decreasing future symptoms and complications. We propose a histopathological characterization of stents deployed in the inferior vena cava of rats and demonstrate how it correlates with ultrasound evaluation.

Methods:
Sprague Dawley rats (643±62g) were anesthetized (isoflurane). IVC diameter was measured by ultrasound on B mode. Through a midline laparotomy, infrarenal IVC side branches were ligated, posterior venous branches cauterized, micro-clips temporarily placed, and a U-stich is placed in the IVC. Using retrograde canulation of the IVC a sharpened guidewire with a stent sheath backloaded is inserted through the U-stich. The stent sheath is advanced with enough running room to deploy the venous self-expanding stent. After removal, U-stitch was tightened for hemostasis. No anticoagulation was used. Venous flow and patency were measured using a color/pulsed-wave Doppler 10-mHz probe post-procedure (PO) and 24 hours after (POD1). Vein wall thickness (VWT), in-stent-restenosis (ISR), and residual thrombosis percentage were quantified using trichrome staining at 7-, 14-, and 21-days.

Results:
Complete stent patency was observed in 50% of animals, partial patency in 15%, and obstruction in 35% after insertion. Table I. Recanalization at 1-day improved flow was observed in 41% of obstructed or partially patent stents. ISR was 6.2±3.8% of the luminal stent area (LSA), residual thrombosis in 29.9±23%, VWT mean 80±13.5 μm, no statistical differences found between timepoints.
Figure 1.

Conclusion:
Characterization and follow-up of deep vein stents can be achieved by ultrasound and histology. This viable animal model can identify human pathological stent processes as early thrombosis, resolution, recanalization, and ISR. This model can reduce costs for feasibility studies to test novel stents and simulate real-life patency outcomes at multiple time points.
Abstract 2096: Role Of Divalent Cations In The Enzymatic Activation Of Coagulation Factor XII By Lipopolysaccharides
Authors: Liu Ting , Lira da Sliva Andre , Lorentz Christina U , Tucker Erik , Lupu Florea , Gailani David , Puy Cristina , McCarty Owen J
Published: July 2024

Background:
Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. LPS is a polyanionic molecule that may act as a site of initiation of coagulation by activating factor (F)XII. While the physicochemical properties of LPS are known to be sensitive to interactions with divalent cations, it is unknown whether divalent cations modulate the activation of FXII by LPS.

Aims:
To determine whether divalent cations regulate the kinetics of FXII activation by select LPS chemotypes from
E. coli
.

Methods:
Aggregates of
E. Coli
LPS chemotypes O26:B6 and Rd2 were prepared in ultrapure water in the presence or absence of calcium (Ca
2+
). The biophysical characterization of LPS chemotypes were determined by dynamic light scattering (DLS) and measurement of zeta potential (ZP). Chromogenic substrate assays were used to study the effects of LPS on FXII activation.

Results:
The LPS O26:B6 and Rd2 in the absence of Ca
2+
produced aggregates in form of micelles with hydrodynamic diameters of 190±10 and 200±7 nm and ZP of -25±0.8 and -60±2 mV, respectively. The presence of Ca
2+
produced aggregates in the form of vesicles with hydrodynamic diameters of 400±15 and 1300±60 nm and ZP of –11±2 and –3±0.5 mV, respectively. FXII was incubated in HEPES buffer with the distinct LPS morphologies in varying salt concentrations. We found maximal autoactivation of FXII by O26:B6 and Rd2 chemotypes at lower NaCl concentrations. The presence of Ca
2+
, however, selectively reduced the ability of O26:B6 to activate FXII. Strikingly, the neutralization of the surface net charge of the LPS chemotype Rd2 by Ca
2+
ions abrogated the amidolytic activity of Rd2.

Conclusions:
The presence of calcium ions induces changes in the physicochemical properties of LPS that result in distinct effects on FXII activation
in vitro
. We are currently studying whether this phenomena is conserved for other divalent cations including Zn, Mg, Cu, and Mn.
Abstract 2098: A Novel Mechanism Of Focal Adhesion Kinase Activation By Integrins And Its Role In Outside-in Signaling In Platelets
Authors: Mack Andrew , Wang Can , Du Xiaoping
Published: July 2024

Ligand binding to integrin αIIbβ3 mediates platelet adhesion/aggregation and transmits outside-in signaling, leading to platelet spreading, granule secretion, and significant expansion of platelet thrombi resulting in thrombosis. Focal Adhesion Kinase (FAK) has been recognized in many cells to play an important role during integrin outside-in signaling. Whereas FAK activation downstream of integrins is characterized by autophosphorylation of FAK at Y
397
followed by Y
576/7
phosphorylation, it is not clear how FAK phosphorylation and activation is regulated and how FAK play roles in outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) plays a key role in integrin-dependent stimulation of FAK phosphorylation and activation and ROS-stimulated FAK activation is downstream of the Gα13-integrin interaction, NOX2 activation, and the Src signaling pathway. Furthermore, FAK, following ROS-dependent activation, is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We further show that FAK is important in amplification of platelet activation and aggregation as well as in vivo thrombosis. Thus, we have identified a novel mechanism of FAK activation and a key role of FAK in integrin outside-in signaling and integrin-dependent ITAM pathway activation, leading to amplification of platelet activation, aggregation, and thrombosis.
Abstract 2099: Coronary Artery Thrombosis Due To Intramural Hematoma
Authors: Pimentel Filho Pedro , Galvao Andre , Willes Jessica , Leivas Justo , Couto Guilherme , Azmus Alexandre
Published: July 2024

BJMC ,male 61 years old, presented with acute chest pain and non ST acute myocardial infarction. ECG showed ST segment depression on anterior and lateral precordial leads and ST elevation on aVR. Ultra-sensitive Troponin T rised . Coronary angiography showed calcified Left main(LM) with critical lesion extending to anterior descending artery. Percutaneous Coronary Intervention (PCI) was performed succesfully with stent implantation. Two hours later he started with epigastric discomfort , anxiety ,chest pain , diaphoresis , tachycardia and acute ECG changes , irresponsive to intravenous nitroglycerin . He evolved to ventricular tachycardia and cardiac arrest , with CPR. New PCI was followed with immediate clinical improvement . Clinical symptoms of ischemia and hemodynamic instability were due to significant (LM) caliber reduction .IMH is a pathological process were blood accumulate within the media space, suggesting new stenosis or spasm. IMH is unfrequent - may occur in initial hours and even at several days after apparently succesfull intervention. IMH causes acute lumen obstruction and thrombosis. The diagnosis is frequently missed and the clinical course can be dramatic and life threatening. Intravascular ultrasound (IVUS) is a very importante tool to identify IMH and should be available in complex cases interventions.