bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2140: The Tgfb-Whsc1 Axis Drives Macrophage To Myofibroblast Transition During Wound Repair
Authors: Mangum Kevin D , Bauer Tyler , Joshi Amrita , Melvin William , Barrett Emily , Wolf Sonya , Moon Jadie , Shadiow James , Obi Andrea T , Gudjonsson Johann , Davis Frank , Gallagher Katherine A
Published: July 2024

During the proliferative phase of tissue repair macrophages can transition to myofibroblasts, which promote wound contraction and closure. The mechanisms that control this transition remain unknown and are not well studied. Here, using a combination of murine transgenic models of disease, loss of function approaches, pharmacologic inhibition in vivo, and single cell RNA sequencing (scRNA-seq) in human wounds, we identified that a histone methyltransferase, Whsc1, regulates the fibrotic gene program in macrophages during wound repair. Specifically, we identified that Whsc1 was upregulated in wound macrophages (CD3
-
CD19
-
NK1.1
-
Ly6G
-
CD11b
+
) in response to TGFβ. Genetic loss of TGFβ-mediated signaling as well as siRNA knockdown of Whsc1 in bone marrow derived macrophages (BMDMs) decreased TGFβ-dependent expression of
Acta2, Col1a1, Col3a1
(p<0.05). Further, TGFβ stimulation resulted in increased Whsc1 and H3K36 dimethylation at fibrotic gene promoters in wound macrophages (p<0.05). Importantly, local pharmacologic inhibition of Whsc1
in vivo
significantly impaired wound healing, suggesting that Whsc1 is critical for normal wound repair. We next examined the role of Whsc1 in pathologic healing conditions (i.e. diabetes) where the macrophage-myofibroblast transition is impaired. Whsc1 expression was decreased in human macrophages analyzed by scRNA-seq of diabetic wounds and in murine wound macrophages from a diabetic model (DIO) (p<0.05). Interestingly, in DIO BMDMs, Whsc1 did not traffic to fibrotic promoters after TGFβ treatment, but rather remained at NFkB-regulated inflammatory gene promoters (
Il1b, Il6, Tnf
). On further examination of this, using co-immunoprecipitation and proximity ligation assay in BMDMs, we demonstrated increased physical interaction between RelA and Whsc1 in diabetic macrophages. Knockdown of RelA in DIO macrophages increased Whsc1 enrichment at fibrotic promoters, suggesting RelA sequestered Whsc1 at inflammatory gene promoters (p<0.05). The identification of TGFβ-Whsc1 as a driver of macrophage-myofibroblast transition in normal and pathological tissues allows for development of novel therapies to improve tissue repair, and targeting RelA in macrophages may improve diabetic wound healing.
Abstract 3011: Parkin Mediated Mitophagy Deficiency In VSMCs Exacerbates Abdominal Aortic Aneurysm
Authors: Wang Hui , Blin Muriel , Luna Adrian , Wragg Kathleen , Gioscia-Ryan Rachel , Gao Xiaohua , Van den Bergh Fran , Wood Sherri , Hefter Charles , Beard Daniel , Ailawadi Gorav , Goldstein Daniel , Salmon Morgan
Published: July 2024

Background:
Parkin-mediated mitophagy eliminates dysfunctional mitochondria to reduce inflammatory responses caused by mtDAMPs. Given that reduced mitophagy is a hallmark of aging and AAA is an aging-associated disease, we sought to investigate the role of Parkin-mediated mitophagy in the development of AAAs.

Methods:
Groups of young (8-10 wks, n= 6/group) and aged (18-20 months, n=6/group) male Mito-QC mice either without intervention or underwent AAV.mPCSK9
D377Y
infection and western diet feeding followed by Angiotensin II (AngII) or saline pump infusion for 28 days. Separately, young male Myh11-creER
T2
Parkin
fl/fl
mice underwent tamoxifen (Parkin SMCKO) or vehicle injections (n=35-39/group), AAV.mPCSK9
D377Y
infection, western diet feeding and AngII infusion.
In vitro
, MOVAs were treated with AngII and the USP30 inhibitor, a mitophagy enhancer, or vehicle followed by assessment of Parkin and mtROS.

Results:
VSMCs in aged Mito-QC mice, identified by a CD45
-
CD90
-
α-actin
+
profile, exhibit diminished mitophagy activity (p=0.017) and decreased Parkin expression (p=0.029). In young AAAs, VSMCs characterized as CD45
-
CD90
-
α-actin
high
and α-actin
low
also demonstrated decreased mitophagy (p=0.0017, p=0.0020), decreased Parkin(p=0.029), and a decreased trend in SDHB in Complex II (p=0.103) and NDUFB8 in Complex I (p=0.040) of the electron transport chain. Parkin SMCKO mice demonstrated increased AAA rupture (p=0.038), greater aneurysm size (p=0.044), reduced mtDNA copy numbers (p=0.026) and elevated mtROS compared to their controls. Parkin SMCKO mice demonstrated trends of diminished expression of respiratory complexes (p=0.070) and an exacerbated decrease in the maximal OCR involving both coupled and uncoupled activities of Complex I plus II (p=0.045, 0.028).
In vitro
experiments demonstrated treatment with the USP30 inhibitor reversed the reduction in mitophagy activity and mtROS increasement induced by AngII in MOVAS.

Conclusions:
In conclusion, mitophagy activity in aortic VSMCs and Parkin decreased with aging and separately, in AAAs and Parkin SMCKO mice, associated with exacerbation of AAA progression. These studies suggest stabilization of Parkin-dependent mitophagy could represent a novel AAA therapeutic target.
Abstract 3013: Indoleamine 2,3-dioxygenase-mediated Tryptophan Catabolism Limits Experimental Abdominal Aortic Aneurysms
Authors: Xu Baohui , Li Gang , Deng Hongping , Li Yankui , Shen Fanru , Samura Makoto , Tsao Philip S , Dalman Ronald L
Published: July 2024

Background:
Indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression. Genetic IDO deficiency limits angiotensin II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic mice. We investigated the influence of IDO inhibition or IDO-catabolized tryptophan catabolite on elastase-induced AAAs in normolipidemic mice.

Methods:
AAAs were induced in male C57B/6J mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with IDO inhibitor 1-methyl-DL-tryptophan (MT, 2 mg/ml in drinking water), tryptophan metabolite 3-hydroxyanthranilic acid (HAA, 200 mg/kg, oral gavage), or vehicle, starting 3 days prior to PPE infusion for 17 days. AAAs were assessed via ultrasonography and histopathology at sacrifice.

Results:
The Figure summarizes aortic diameters and histopathology in differentially treated PPE-infused mice. Rapid aortic enlargement was noted in MT-, as compared to vehicle-treated mice following PPE infusion. AAA rupture, unusual for this model, was noted in MT-treated AAA mice. No difference was noted between groups in medial elastin degradation, smooth muscle cell depletion, or macrophage accumulation. However, mural neoangiogenesis and lymphocyte accumulation were remarkably augmented in MT-treated mice. HAA Treatment attenuated aortic enlargement in conjunction with improved medial elastin and smooth muscle cell retention and reduced aortic leukocyte accumulation and neovessels.

Conclusion:
IDO activity and its tryptophan metabolite HAA limit experimental AAA progression in the PPE model. These results provide further support for investigating tryptophan metabolites in clinical AAA disease suppression.
Abstract 3035: Associations Of Genetic And Lifestyle Risk Factors With Incident Peripheral Artery Disease: A Prospective Cohort Study
Authors: Yuan Shuai , Sun Yuhao , Chen Jie , Sharma Pranav , Levin Michael G. , Larsson Susanna , Damrauer Scott M
Published: July 2024

Background:
The joint associations between genetic and lifestyle factors, and peripheral artery disease (PAD) remain unclear, yet understanding the connections is crucial for precise PAD prevention.

Methods:
Genetic susceptibility to PAD was estimated by a polygenic risk score of 50 genetic variants selected by
P
< 5х10
-8
and

r
2

<0.01 from a GWAS including 31,307 cases. Tertiles categorized genetic risk into low, intermediate, and high. A healthy lifestyle score was constructed based on 4 lifestyle factors (never smoking, non-excessive alcohol consumption, high cardiometabolic healthy diet adherence, and regular physical activity) and categorized into favorable (3-4 healthy factors), intermediate (2 healthy factors), and unfavorable (0-1 healthy factors) groups. Cox regression estimated hazard ratios (HR) and 95% confidence interval (CI) with adjustment for age (time scale), sex, BMI, education, baseline cardiovascular risk factors, 20 principal components.

Results:
Over 11.9 years, 4299 incident PAD cases were diagnosed in 379,228 UK Biobank participants free of baseline PAD. Genetic risk and unhealthy lifestyle categories were monotonically associated with PAD. Compared with low genetic risk, the HR was 1.69 (95% CI 1.56-1.89) for high genetic risk. Compared with favorable lifestyle, the HR for unfavorable lifestyle was 2.70 (95% CI 2.50-2.94). There was no multiplicative interaction between genetic and lifestyle factors (
P
= 0.461). The association between unfavorable lifestyle and higher PAD risk was consistent in genetic risk groups. Joint categorization analysis detected an HR for high genetic risk and favorable lifestyle was (1.85, 95% CI 1.54-2.22) reducing by > 50%, compared with high genetic risk and unfavorable lifestyle (HR 4.76, 95% CI 4.00-5.56).

Conclusions:
Genetic and lifestyle factors independently associated with incident PAD. Adhering to a favorable lifestyle substantially reduced PAD risk, even in individuals at high genetic risk.
Abstract 3038: Platelet-specific Deletion Of Transforming Growth Factor-β1 Augments Angiotensin Ii-induced Abdominal Aortic Aneurysm In Mice
Authors: Spuzzillo Anthony R , Stone Bailey , Benson Tyler W , Brunner Seth I , Wadding-Lee Caris A , Owens A. Phillip
Published: July 2024

Background:
Transforming growth factor beta (TGFβ) blockade via systemic antibody targeting has been shown to augment murine AAA progression in multiple independent studies. Despite these findings, the primary source of protective TGFβ within AAA remains unclear. Previous research conducted in our laboratory demonstrates deletion of platelet-specific TGFβ1 exacerbates aneurysms in the elastase mouse model of AAA. Our present study extends these results by showing a comparable phenotype in the angiotensin II (Ang II) murine model of AAA.

Methods and Results:
Low density lipoprotein receptor deficient (

Ldlr
-/-

) mice with platelet-specific deletion of
Tgfβ1
were created by breeding male mice expressing Cre recombinase under the control of a
Pf4
promoter to female
Tgfβ1
floxed mice (Jackson Labs). Male mice were fed high fat diet 1 week prior to and throughout subcutaneous infusion of AngII (1,000 ng/kg/min). After day 28 of infusion,
ex vivo
aortic diameter was significantly increased in Cre+ (n = 17) vs Cre- (n = 18) mice (
Cre-
: 1.75 ± 0.17 mm;
Cre+
: 2.43 ± 0.21 mm; P < 0.05). AAA incidence, defined as aortic diameter ≥ 1.2mm, was 56% for Cre- mice vs. 93% in the Cre+ group. Histological analysis of picrosirius red staining revealed a marked reduction in type 1 abdominal collagen deposition as a proportion of total cellular area in aortas from Cre+ mice versus Cre- controls. OCT embedded sections of aneurysms from both cohorts were also probed for macrophage marker CD68 and analyzed using immunofluorescent microscopy. We found Cre+ aortas had significantly increased macrophage infiltration compared to Cre- aortas.

Conclusions:
This study of platelet-specific deletion of TGFβ1 in a secondary model of murine AAA supports the conclusion that TGFβ1 sourced form platelets plays a critically important role in the protection of AAA.
Abstract 3039:
TOMM40
Knockdown In Macrophages Inhibits Oxidized LDL-induced NLRP3 Activation And Promotes Reverse Cholesterol Transport
Authors: Chelluri Sai S , Yang Neil V , Theusch Elizabeth V , Krauss Ronald M
Published: July 2024

Atherogenesis has been shown to be promoted by activation of the NLRP3 inflammasome, a cytosolic innate immune sensor activated by a broad range of factors, including oxidized LDL (oxLDL). Our group has shown that hepatocyte knockdown of Translocase of Outer Mitochondrial Membrane 40 (
TOMM40),
which encodes a key component of the mitochondrial importer TOM complex, results in increased levels of oxysterols that activate the transcription factors LXRα and β. As LXR agonists have been shown to inhibit NLRP3 activation, we tested whether
TOMM40
knockdown reduces NLRP3 activation in THP-1 monocyte-derived macrophages. On stimulating these cells with both the NLRP3 activator Nigericin and oxLDL following
TOMM40
knockdown by siRNA, we found that pro-inflammatory cytokine release and NLRP3 activation were significantly inhibited (Two-way ANOVA, p<0.0001, n=6). We further showed that
TOMM40
knockdown resulted in significant
LXRβ
upregulation (unpaired t-test, p=0.007, n=3), and reduced oxLDL-induced upregulation of both
NLRP3
and
CASP1
, which encodes a protein recruited in NLRP3 activation (One-way ANOVA, p=0.0007, n=3). Moreover, pre-treatment with an LXR antagonist, GSK2033, abrogated
TOMM40
knockdown-induced NLRP3 inhibition, supporting our hypothesis that this inhibition is likely LXR dependent. Since LXRs are known to promote cholesterol efflux from macrophages, we also examined the effect of
TOMM40
knockdown on expression of the cholesterol transporter genes,
ABCA1, ABCG1,
and
SR-B1
, and found that
ABCG1
and
SR-B1
were significantly upregulated (unpaired t-test, p=0.02 and p=0.003 respectively, n=3). Taken together, these findings identify mechanisms by which
TOMM40
knockdown exerts anti-inflammatory effects and promotes reverse cholesterol transport. While
TOMM40
has been studied largely in relation to
APOE
and neurodegenerative disease due to its genomic location, we identify a novel potential impact of TOMM40 on atherogenesis.
Abstract 3046: Macrophage-smooth Muscle Cell Interactions In Atherosclerotic Foam Cell Formation
Authors: Xiang Pinhao , Allahverdian Sima , Chan Teddy , Ortega Carleena , Francis Gordon A , Sahin Basak
Published: July 2024

Background:
A hallmark of atherosclerosis is uncontrolled uptake of atherogenic lipoproteins by artery wall cells that accumulate large amounts of cholesteryl esters (“foam cells”). While smooth muscle cells (SMCs) and macrophages are in close proximity in lesions and contribute to cholesterol accumulation, whether these cells influence each other’s tendency to form foam cells is not clearly established.

Hypothesis:
Direct and indirect interactions between SMCs and macrophages promote foam cell formation by both cell types.

Method:
Human vascular SMCs were seeded onto macrophages to mimic direct macrophage-SMC interactions (n=5). Alternatively, conditioned media generated from human macrophages or SMCs with or without lipoprotein exposure was incubated with the alternate cell type (n=3). Aggregated lipoproteins were added in both models to induce cellular cholesterol accumulation. Intracellular cholesterol was quantified to compare lipoprotein uptake in cocultures relative to monocultures. Crosstalk based on CellChat analysis using single-cell RNA sequencing data from human coronary samples was performed to identify potential factors responsible for foam cell development (n=3). One-way ANOVA was used for statistical analysis.

Result:
SMCs showed a 4-fold (+/-0.3, p=0.002) increase in cell cholesterol accumulation when in direct contact with macrophages and a 3-fold (+/- 0.3, p<0.0001) increase when exposed to the conditioned media from macrophage foam cells. In contrast, macrophages showed a 3-fold reduction (+/- 0.3, p=0.001) in intracellular cholesterol accumulation when in direct contact with SMCs and a 2-fold reduction (+/-0.2, p=0.0005) when exposed to SMC foam cell-conditioned media. CellChat analysis suggested macrophage foam cells but not macrophage non-foam cells promote SMC foam cell formation by secretion of inflammatory cytokines, and that SMCs promote macrophage foam cell formation by secretion of extracellular matrix components.

Conclusion:
Our study provides novel understanding of how macrophage-SMC interactions influence macrophage and SMC foam cell formation. Further studies will be presented examining the role of SMC extracellular matrix proteins and macrophage-secreted cytokines on foam cell formation.
Abstract 3054: Proinflammatory Phenotypes And Role Of Ly-6c
Low
Monocytes In Atherosclerosis With Hypertriglyceridemia
Authors: Xiao Jing , Peng Xueying , OBrien Veronica , Litchfield Benjamin , Gao Feng , Lian Zeqin , Perrard Xiaoyuan , Pownall Henry J , Ballantyne Christie M , Wu Huaizhu
Published: July 2024

Background:
Monocytes play pivotal roles in atherosclerosis. Ly-6C
low
monocytes, also known as patrolling monocytes, become foamy in the circulation of mice with hyperlipidemia. However, the role of Ly-6C
low
monocytes in atherosclerosis with hypertriglyceridemia (HTG) remains to be determined.

Aim:
To examine the phenotypes and role of Ly-6C
low
monocytes in atherosclerosis in HTG.

Method:
Ldlr
-/-
ApoC3Tg (transgenic expression of human ApoC3) mice fed a western high-fat diet (WD) were used to examine the effects of HTG on monocyte lipid accumulation (foamy monocyte formation), phenotypes, and contributions to atherosclerosis.

Results:
Plasma triglycerides in Ldlr
-/-
ApoC3Tg mice were 5.1 times higher than in Ldlr
-/-
controls at 6 weeks on WD, correlating with more than twice the increase in aortic atherosclerosis (P<0.01). Consistently, Ly-6C
low
compared to Ly-6C
high
monocytes in Ldlr
-/-
mice had increased lipid accumulation and exhibited elevated levels of TNFα, IL-6, IL-1β, Plin2, and PPAR-γ. In Ldlr
-/-
ApoC3Tg mice, Ly-6C
low
(also CD11c
+
) monocytes demonstrated even greater lipid accumulation and adhesion to VCAM-1 and had further elevations in levels of TNFα, IL-6, IL-1β, Plin2, and PPAR-γ than CD11c
+
(Ly-6C
low
) monocytes in Ldlr
-/-
controls, indicating an enhanced inflammatory response and lipid accumulation under HTG. Furthermore, compared to Ldlr
-/-
control mice, Ldlr
-/-
ApoC3Tg mice had an increase in the level of SIRPα (a ligand of the “don’t-eat me” signal CD47) on Ly-6C
low
monocytes, indicative of impairment in efferocytosis and clearance of apoptotic cells. Additionally, nuclear c-Rel mRNA and protein levels were upregulated in Ly-6C
low
monocytes of Ldlr
-/-
ApoC3Tg mice compared to Ldlr
-/-
controls, suggesting activation of the NF-κB pathway in Ly-6C
low
monocytes by HTG. Finally, depletion of Ly-6C
low
monocytes reduced atherosclerosis by 45% (P<0.05) in Ldlr
-/-
ApoC3Tg mice, indicating a role of Ly-6C
low
monocytes in atherosclerosis in HTG.

Conclusions:
HTG accelerates atherogenesis in Ldlr
-/-
mice, possibly by increasing lipid accumulation and inflammatory phenotypic changes in monocytes, primarily CD11c
+
Ly-6C
low
monocytes.
Abstract 3056: IL-19 Is A VEGFC-inducible Lymphokine That Regulates Prox1 Through RNA Stability
Authors: Peluzzo Amanda , Kelemen Sheri E , Autieri Michael
Published: July 2024

Introduction:
During inflammatory diseases, such as atherosclerosis, malfunctional lymphangiogenesis occurs. Studies suggest that inflammation induces a zipper morphology in lymphatic junctions which limits lymphatic function, but this phenomenon has never been evaluated under atherogenic conditions. We previously reported that IL-19 is anti-inflammatory, anti-atherosclerotic, and pro-angiogenic. The purpose of this study is to test the hypothesis that IL-19 induces
functional
lymphangiogenesis under atherosclerotic conditions.

Methods & Results:
Both IL-19 and VEGFC, a vital pro-lymphangiogenic factor, induce IL-19 protein expression in lymphatic endothelial cells (LECs). Interestingly, IL-19 does not induce VEGFC protein expression. IL-19 treatment does induce expression of Prox1, the master regulator of lymphatic development. Proliferation, migration, and tube formation assays confirmed IL-19 induces lymphangiogenesis. IL-19 also promotes short- and long-term regulation of lymphatic permeability by rapidly inducing VE-cadherin phosphorylation and slowly increasing Angpt2 expression. In the presence of oxLDL, LECs have decreased permeability via electric cell-substrate impedance sensing and increased zippers via immunocytochemistry, while co-treatment with IL-19 maintains LEC barrier function.

Ldlr
-/-
Il19
-/-

double knockout (dKO) mice, known to have exacerbated atherosclerosis, have significantly worse lymphatic function compared to

Ldlr
-/-

on a high fat diet. However,
dKO
and

Ldlr
-/-

mice on chow diet have similar lymphatic function, again suggesting a role for IL-19 by maintaining lymphatic flux under pro-inflammatory conditions. Although IL-19 canonically phosphorylates STAT3 in LECs, STAT3 knockdown does not affect Prox1 expression. However, Prox1 knockdown prevents IL-19-associated lymphangiogenesis and Angpt2 upregulation. Since IL-19 is known to alter mRNA stability, we performed Actinomycin D assays and determined IL-19 increases Prox1 mRNA stability, possibly contributing to Prox1 upregulation independent of STAT3 activity.

Conclusion:
IL-19 is a previously un-recognized regulator of lymphangiogenesis and could potentially promote lymphatic function during inflammation.
Abstract 3057: Vascular Smooth Muscle Cell
Cd47
Contributes To Atherosclerosis Development
Authors: Pervaiz Naveed , Aithabathula Ravi V , Kathuria Ishita , Le Britany , Kim Ki-Suk , Ahn Wonmo , Csanyi Gabor , Singla Bhupesh
Published: July 2024

Aims:
Thrombospondin-1 (TSP1), a secretory protein, has been associated with various cardiovascular diseases. TSP1 mainly functions via its cognate receptors CD47 and CD36. Recently, we observed elevated TSP1 expression in human and murine atherosclerotic arteries. However, the role of vascular smooth muscle cell (VSMC) TSP1-CD47 signaling in regulating VSMC phenotype and atherogenesis is unclear.

Methods and Results:
Human arterial SMCs were treated with human TSP1 and analyzed for SMC markers and proliferation. The data revealed decreased expression of SMC markers, ACTA, CNN1, and SM22α in TSP1-exposed VSMCs compared with vehicle-treated control cells. Further, TSP1 treatment increased VSMC proliferation
in vitro
, suggesting TSP1 induces VSMC hyperproliferative phenotype. Additional experiments showed CD47 as the major TSP1’s receptor in VSMCs. To investigate the
in vivo
role of VSMC
Cd47
in atherosclerosis, we generated tamoxifen-inducible SMC-specific
Cd47
knockout mice (

Cd47
f/f
Myh11 Cre
+/–

,

Cd47
ΔVSMC

) by crossing female

Cd47
f/f

mice with male

Myh11 Cre
+/-

mice. Male tamoxifen-injected

Cd47
ΔVSMC

and corn oil-administered

Cd47
WT

mice were given a single injection of AAV8-
hPCSK9
(1х10
11
viral genomes/mouse, IP) and fed a Western diet for 12 weeks. En face oil red O (ORO) staining of whole aortas exhibited reduced atherosclerosis in

Cd47
ΔVSMC

mice compared with control

Cd47
WT

mice. Further histochemical staining performed on aortic root sections revealed attenuated neointimal lesion area, lipid accumulation and necrotic area in

Cd47
ΔVSMC

mice. However, there were no significant differences in weight gain, body composition (fat and lean mass), plasma total cholesterol, and fasting blood glucose between

Cd47
ΔVSMC

and

Cd47
WT

mice.

Conclusions:
Taken together, these findings suggest that TSP1 promotes VSMC phenotype switch and VSMC-specific
Cd47
deletion in hypercholesterolemic mice suppresses atherosclerosis progression.
Abstract 3062: Activation Of Adam17-dlk1 Axis Induces Aortic Valve Calcification.
Authors: Djellal Merouane , Rehman Safur , Bouchareb Rihab
Published: July 2024

Aortic valve stenosis (AS) is characterized by valve architecture disruption through mineralization and valve cusps fibrosis. Currently, there are no proven pharmacotherapies to prevent or decelerate AS progression. Understanding the molecular mechanisms leading to aortic valve mineralization is of inestimable importance. A balance between calcium deposition and regression may be involved in the pathogenesis of calcified aortic valve disease. Delta-like 1 homolog (Dlk1) is an imprinted gene that can be proteolytically cleaved within the juxta-membrane region by TACE, resulting in the release of an extracellular part of Dlk1 as a soluble fragment known as fetal antigen 1 (FA1). DLK1 is associated with increased adiposity and fibrosis.

Objective:
extracellular matrix (ECM) remodeling disorder in response to TACE-Dlk1 defect might affect valve structure through ECM-lipid interaction in patients with abdominal obesity. We investigated the potential role of TACE/DLK1 in activating osteoblastic differentiation in mouse valve interstitial cells (VICs) in vitro.

Method:
We first isolated VICs from WT and DLK1-/- mice and treated them with a calcifying medium for seven days. Mass spectrometry examined extracellular protein expression (ECM) and increased lipid retention.

Results:
The loss of function of Dlk1 was associated with disrupted ECM expression and the increase of osteogenic genes (RUNX2, SPP1, BMP2, MSX2) expression.

Conclusion:
Dlk1 affects ECM expression and lipid retention, enhancing aortic valve degeneration.
Abstract 3079: Overexpression Of Abca1 In Carotid Endothelium Of Hyperlipidemic Rabbits Decreases Vascular Inflammation
Authors: Wacker Bradley K , Bi Lianxiang , Saenz De Pipaon Echa Goren , Sanford Nicole , Regan Abigail , Lim Natalie S , Kim Francis , Dichek David A
Published: July 2024

Introduction:
Vascular gene therapy that reduces inflammation or increases cholesterol efflux might reduce atherosclerosis. ATP-binding cassette subfamily A member 1 (ABCA1) has both anti-inflammatory and cholesterol efflux activities. We tested whether vessel wall overexpression of ABCA1 would reduce inflammation, vessel wall lipid accumulation, and atherosclerotic lesion size.

Methods:
We used a helper-dependent adenoviral vector (HDAdABCA1) to overexpress ABCA1 in carotid endothelium of rabbits fed a normal or high-fat diet (HFD). Control arteries received a “Null” vector. We measured ABCA1 expression in transduced carotids of rabbits fed both diets. We used HFD-fed rabbits (n=30) to test whether HDAdABCA1 infusion reduced atherosclerosis. Measurements were made on whole carotid extracts, on extracts of laser-microdissected endothelium, and on histologic sections.

Results:
At 3 days after infusion, HDAdABCA1 increased whole vessel ABCA1 mRNA by ~2-fold in rabbits fed a normal diet (P=0.07) or a HFD (P=0.04). Endothelial ABCA1 protein was ~1.6-fold higher in HFD-fed rabbits (P=0.06). 24 wks after vector infusion in HFD-fed rabbits, endothelial ABCA1 mRNA was increased (46%; P=0.07), but whole vessel ABCA1 mRNA was slightly decreased (10%; P=0.004). Whole vessel CD68 expression was decreased, as were mRNA encoding several markers of M1-like macrophages: IL-1β: –44%; IL-6: –40%; MCP1: –25%; (P=0.02 for all) and M2-like macrophages: TGF-β1: –13% (P<0.001); ARG-1: –27% (P=0.03); IL-10: –23% (P=0.09). mRNA encoding IL-6 (100%; P<0.001) and TNF-α (P<0.05) was also reduced in endothelium of HDAdABCA1 arteries. Intimal/medial area ratio was slightly less in HDAdABCA1 carotids (–11%; P=0.06). Intimal lipid as well as macrophage and smooth muscle cell content were unchanged (P≥0.5 for all). Mass spectrometry showed no difference in unesterified or esterified cholesterol (P≥0.6 for both).

Conclusion:
ABCA1 overexpression in carotid endothelium reduced CD68 expression along with levels of several M1- and M2-like macrophage markers and inflammatory cytokines. ABCA1 overexpression had a small effect on intimal mass and did not reduce intimal lipid accumulation. ABCA1 overexpression in endothelium has mild atheroprotective effects.
Abstract 3089: Do Arteries And Veins Have Different Remodeling Programs?
Authors: Rojas Miguel G , Pereira-Simon Simone , Varona Santos Javier , Zigmond Zachary , Yang Xiaofeng , Tabbara Marwan , Martinez Laisel , Vazquez-Padron Roberto I
Published: July 2024

Aims:
Arteries and veins respond differently to injury, with significantly higher rates of postsurgical failure in the latter. The biological reasons for this discrepancy are not well understood, which is a limiting factor for the development of vein-targeted therapies. This study contrasts peripheral arteries and veins at the single-cell level, with a focus on cell populations with remodeling potential.

Methods and Results:
Upper arm arteries (brachial) and veins (basilic/cephalic) from 27 organ donors were compared using a combination of bulk and single-cell RNA sequencing, proteomics, flow cytometry, and histology. The cellular atlases of six arteries and veins demonstrated a significantly higher proportion of smooth muscle cells (SMCs) in arteries vs. higher counts of fibroblasts in veins. In both types of vessels, clustering analyses identified an intermediate myofibroblast population with repair characteristics. Both arterial and venous myofibroblasts were characterized by upregulation of
F2R
,
FN1, POSTN, COL8A1,
and
ITGA10
, and were confirmed by histology and F2R/CD90 double staining by flow cytometry. However, bioinformatic analyses indicate that arterial myofibroblasts are phenotypically related to both SMCs and fibroblasts, whereas in veins they are predominantly fibroblast derived. Differences in wall structure further explain the distinct contributions of SMC and fibroblasts to these reparative populations. PDGFRA
+
fibroblasts are almost exclusively located outside the external elastic lamina in arteries, while widely distributed throughout the venous wall. In line with a higher abundance of fibroblasts and fibroblast-derived reparative cells, ECM-targeted proteomics confirmed a higher abundance of fibrillar collagens in veins vs. more basement ECM components in arteries. These differences in ECM composition are also associated with vessel-specific modulation of myofibroblast differentiation.

Conclusions:
The distinct cellular origins and transcriptional programs of myofibroblast populations in arteries and veins may explain differences in acute and chronic wall remodeling between vessels. This information is relevant for the development of anti-stenotic therapies.
Abstract 3104: Adenine Base Editing To Correct Pathogenic Variants Causing Pseudoxanthoma Elasticum
Authors: Testa Lauren , Volpp Daphne , Wang Xiao , Musunuru Kiran
Published: July 2024

Pseudoxanthoma elasticum (PXE) is a rare, autosomal recessive disorder caused by pathogenic variants in
ABCC6
, resulting in systemic depletion of the anti-mineralization compound pyrophosphate. Pyrophosphate depletion drives ectopic calcification of elastic fibers within the skin, retina, and vasculature. Arterial calcification can lead to intermittent claudication of peripheral arteries and coronary artery disease. There are currently no targeted therapies for PXE. Genome editing may be a promising therapeutic option because
ABCC6
is predominantly expressed in the liver, a site that is amenable to current genome editing delivery modalities. We sought to assess whether adenine base editing could efficiently correct one of the most frequent
ABCC6
variants in PXE patients, c.3490C>T (p.R1164X) in human hepatocytes and a humanized mouse model. We used prime editing to generate a homozygous R1164X human hepatoma cell line. Treatment of R1164X cells with mRNA encoding the adenine base editor ABE8.8 and a synthetic guide RNA (gRNA) resulted in efficient correction of the variant. OligoNucleotide Enrichment and sequencing (ONE-seq) was used to nominate candidate off-target sites, which were evaluated by targeted amplicon sequencing of edited R1164X cells. To mitigate off-target editing, we generated 21 hybrid gRNAs in which various RNA nucleotides in the protospacer were replaced by DNA nucleotides. All hybrid gRNAs maintained on-target editing, and interestingly, several hybrid gRNAs improved on-target editing compared to the standard gRNA. All hybrid gRNAs improved off-target editing, with multiple hybrid gRNAs eliminating off-target editing at multiple off-target sites. To assess
in vivo
correction of the R1164X variant, we generated a homozygous R1164X humanized mouse model which displays depletion of plasma pyrophosphate. Studies to correct the variant in the humanized mouse model by lipid nanoparticle delivery of the lead candidate hybrid gRNA are underway. Efficient correction of the R1164X variant in the liver by adenine base editing and restoration of pyrophosphate would represent one of the first examples of using genome editing to improve the multisystemic manifestations of a heritable connective tissue disorder.
Abstract 3106: Targeted Mrna Delivery To The Inflamed Lung For The Treatment Of Ards
Authors: Zhou Zhengjie , Fang Yun
Published: July 2024

Acute respiratory distress syndrome (ARDS), caused by widespread endothelial barrier disruption and uncontrolled cytokine storm, is the major feared complication of pandemic influenza and COVID-19. Currently, there are few pharmacological treatments that directly target ARDS. We have previously discovered that reduction of Krüppel-like Factor 2 (KLF2, a transcription factor) in inflamed endothelium leads to ARDS induced by LPS, influenza, and high-tidal volume ventilation. Recently, we detected a significant reduction of KLF2 in lung autopsies from COVID-19 patients and demonstrated the anti-viral function of endothelial KLF2. Here, we devised a VCAM1-targeting nanomedicine strategy to restore KLF2 in inflamed endothelial cells. We engineered a rationally designed liposome which possesses potent mRNA delivery and transfection capacity. This VCAM1-targeting liposome selectively delivered functional KLF2 mRNA to inflamed vascular endothelium and significantly reduced mouse ARDS induced by influenza virus H1N1 and SARS-CoV-2, by calming local cytokine storm and promoting endothelium health. Moreover, we demonstrated that this targeted liposome serves as a broad platform to effectively deliver various mRNA molecules to targeted tissues of interest by displaying different targeting motifs. Our results demonstrate that this KLF2 mRNA encapsulated, VCAM1-targeting liposome is a promising mRNA therapeutic strategy to treat ARDS and vascular diseases.
Abstract 3111: A Systematic Review Of Vaccination-induced Takotsubo Cardiomyopathy: A 2024 Update
Authors: Taj Saher , Haseeb Muhammad , Ahmed Zahoor , Ong Kenneth
Published: July 2024

Background:
Vaccine has potential to cause severe cardiac complications. Takotsubo cardiomyopathy (TCM) induced by vaccine is a life-threatening complication, albeit rarely reported. Our study provides a comprehensive insight into this patient cohort on a large scale.

Methods:
We conducted a systemic literature search in PubMed and EMBASE up to January 5, 2024, using MeSH terms for "vaccine" and "takotsubo cardiomyopathy" to identify cases of vaccine-induced TCM (Figure 1).

Results:
Our study included 25 cases of TCM with a median age of 63 ± 18.28 years. Of those, 80% were female. TCM was reported in patients after the coronavirus virus 2019 (COVID-19) vaccine (92%) and influenza vaccine (8%). Of COVID-19 vaccines, 88% were mRNA (Moderna, Pfizer) types. Most TCM cases were reported after 2nd dose of vaccine (44%). Chest pain (56%) and dyspnea (44%) were the most common symptoms, with a median time of 3 ± 2.02 days from vaccination to the first symptom onset. Cardiac troponins were elevated in 92% of patients, and ST-segment change (76%) was the most common ECG finding. All patients had left ventricular ejection fraction (LVEF) of 50%, with severe cardiomyopathy (EF 30%) in 24%. LV dysfunction was the most common finding (88%) on echocardiogram. A total of 92% of patients were discharged with improved symptoms after a median length of hospital stay of 7 ± 3.42 days (Figure 2).

Conclusion:
Although very rare, TCM is a life-threatening complication of vaccine. TCM should be included in the differential diagnosis of patients presenting with post-vaccination chest pain or dyspnea, especially after mRNA vaccines, warranting continued surveillance and regulated reporting of vaccine-related cardiac complications.
Abstract 3116: Elucidating The Molecular Progression Of Hereditary Vascular Dementia In A Novel Mouse Model
Authors: Romay Milagros , Ma Feiyang , MOMPEON CAMPOS ANA , Salvador Jocelynda , Knutsen Russell H , Kozel Beth A , Iruela-Arispe M. Luisa
Published: July 2024

Aging is an immutable risk factor for the development of numerous cardiovascular diseases, including vascular dementia. Vascular dementia is the second most common cause of dementia with up to 20% of dementia cases in the elderly attributed to dysfunctional vasculature. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian vascular dementia characterized by progressive dysfunction of vascular smooth muscle cells (vSMCs) due to gene variants in
NOTCH3
. With over 250 known disease - causing variants in
NOTCH3
identified in the past 24 years, the allelic heterogeneity observed in CADASIL has long contributed to our impaired understanding of the molecular progression from NOTCH3 variation to clinical presentation. Recent work published by our group has shown that
Notch3
is lost from in the cerebral vasculature of mouse (

Notch3
-/-

) and humans during aging. With time, the progressive loss of
Notch3
in the aging mouse brain vasculature drives the formation of cerebral micro-aneurysms as well as impairs vascular reactivity and cerebral blood flow. These vascular consequences also hinder glymphatic flow which promotes an accelerated aggregation of glycosaminoglycans and the development of neuronal gene signature analogous to those observed in other neurodegenerative diseases such as Alzheimer’s disease. To assess if CADASIL represents a loss of NOTCH3 function and thus an accelerated cerebral aging or novel gain of function in NOTCH3, we used CRISPR-Cas9 genome editing to create a novel CADASIL mouse model: Notch3C175Y (N3Y). This alteration faithfully recapitulates the human variant within the same locus and shows the pathological hallmark of CADASIL: GOMs (granular osmiophilic material). Histopathological assessment of the N3Y mouse shows dysregulation of the vSMC organization as early as 8 weeks of age along with novel structural abnormalities as compared to the

Notch3
-/-

. Using single-cell RNA sequencing we also performed in-depth molecular characterization of the vascular changes in N3Y. Analysis at 2 months and at 24 months allowed us to identify temporal progression of the disease and provided novel molecular insights into the mechanisms of this mendelian form of vascular dementia.
Abstract 3119: Angiographic Burden Of Coronary Atherosclerosis Contributes To Adverse ASCVD Outcomes Independent Of Traditional Risk Factors
Authors: Tsao Noah L , Abramowitz Sarah , Shakt Gabrielle , Judy Renae , Damrauer Scott M , Levin Michael
Published: July 2024

Background:
Coronary artery disease (CAD) is a major contributor to cardiovascular morbidity (including myocardial infarction [MI] and heart failure [HF]), and mortality. Although burden of CAD (number and degree of coronary artery stenosis) has been observationally linked to these outcomes, the causal contribution (and independence from traditional cardiovascular risk factors) has been poorly defined.

Methods:
The primary exposure for this experiment was a genetic instrument for burden of CAD that was constructed from a GWAS of semi-quantitative CAD burden among VA Million Veteran Program participants undergoing coronary angiography. Two-sample Mendelian randomization (MR) was performed to test 1) bidirectional associations between traditional ASCVD risk factors (including blood lipids, blood pressure, diabetes, obesity, and smoking) and CAD burden and 2) between CAD burden and adverse cardiovascular outcomes (MI, HF, and parental longevity as a proxy of lifespan). Multivariable MR (MVMR) for mediation was then performed to determine whether traditional risk factors mediate the association between CAD burden and adverse outcomes.

Results:
As expected, traditional cardiovascular risk factors including lipid levels, diabetes and obesity were associated with increased burden of CAD, MI, HF, and lifespan. Angiographic CAD burden was associated with increased risk of MI, HF, and lifespan. MVMR demonstrated experiment wide independent contributions of CAD burden to HF (OR 1.13 [95% CI 1.08-1.19], p = 2.3E-6), lifespan (b = -0.06, se = 0.01, p = 9.7E-05) and MI (OR 1.74 [95% CI 1.55-1.95], p = 3.4E-21), after accounting for traditional ASCVD risk factors. The effects of lipids on HF and lifespan were partially mediated by angiographic burden of CAD.

Conclusion:
This study suggests that increased angiographic burden of coronary artery disease is associated with adverse cardiovascular outcomes, with an independent contribution beyond traditional cardiovascular risk factors. More aggressive primary prevention of traditional cardiovascular risk factors may be indicated to prevent development and progression/growth of coronary plaques and mitigate subsequent adverse cardiovascular outcomes.
Abstract 3138: Atheroprone Flow, Aging, And Oxysterols Impair Endothelial Cell Nuclear Health Via Disruption Of Autoregulatory Perinuclear Cytoskeletal Dynamics
Authors: Paul Amit , Lazarko Dana , Levitan Irena
Published: July 2024

The vascular hemodynamic environment plays a well-known role with progressive pathological conditions such as atherosclerosis, hypercholesterolemia, and aging. Our lab has previously shown that the exposure of oxysterols to endothelial cells (ECs) causes increased cell stiffening. This stiffening effect is compounded when the hemodynamic environment changes from atheroprotective laminar flow to atheroprone oscillatory flow. The increasing EC stiffening demonstrates that these disruptive effects significantly alter cellular biomechanics. The complexity of events associated with these altered biomechanical states leaves many questions unanswered about spatiotemporal changes in the biomechanical structures of endothelial cells exposed to a varying hemodynamic environment and oxysterols. To address this void, we observed the effects of 7-Ketocholesterol exposure (a major oxysterol) on the primary cytoskeletal/biomechanical components (F-actin, Microtubules, Intermediate Filaments, etc.) of human aortic endothelial cells during various time points of laminar/oscillatory flow conditions through a combination of sophisticated multiplexed 6-color labeling, ultra-high resolution 3D microscopy, 3D computer vision, and machine learning. Additionally, we applied these techniques to extracted aortic tissues from young and aged mice (on low fat diet vs high fat diet) and focused on obtaining information from both the laminar flow areas (descending aorta) and the oscillatory flow areas (aortic arch). Our findings demonstrate that oxysterol exposure and aging cause significant remodeling & alteration of crucial endothelial cytoskeletal/biomechanical components and that these alterations are unique to each combinatorial condition. Notably, we find that cytoskeletal remodeling associated with endothelial stiffening is accompanied by major nuclear distortion, a hallmark of aging and senescence which subsequently causes DNA damage and genomic instability. We also find that this nuclear distortion is driven by dynamic biomechanical changes and mediated by the hemodynamic environment, where atheroprotective flow promotes cytoskeletal configurations protecting the nuclei, and atheroprone flow promotes cytoskeletal configurations that leave the nuclei unprotected.
Abstract 3144: Mitochondrial Rnas Regulate Endothelial Function Through Nuclear Transcription
Authors: Sriram Kiran , Yuan Dongqiang , Malhi Naseeb , Tapia Alonso , Luo Yingjun , Liu Xuejing , Zhen Chen
Published: July 2024

Mitochondrial-nuclear communication is vital for cellular homeostasis and response to environmental stress. The known messengers for mitochondria to communicate their functional status to the nucleus include ATP, ROS, and Ca
2+
. It remains unclear whether the immediate output of mitochondrial transcription, i.e., the mitochondrial RNAs (mtRNAs), are a type of messenger to communicate to the nucleus. Herein, we show that
mt
RNAs are attached to the nuclear genome and thus constitute a subset of the
c
hromatin-
a
ssociated
RNA
(mt-caRNA). Mt-caRNAs preferentially attach to promoter regions and the attachment levels change in response to cellular stress, i.e. high glucose and TNFα (HT). We identify the mitochondrial non-coding RNA SncmtRNA as a mt-caRNA. In human endothelial cells (ECs), suppression of SncmtRNA attenuates HT stress induction of nascent RNA transcribed from the nuclear genome, including the cell adhesion molecules ICAM1 and VCAM1, and abolishes stress-induced monocyte-EC adhesion. In addition to SncmtRNA, we show nuclear localization of other mtRNAs such as mt-CYB and mt-ND5, which is increased in the diabetic human condition. Collectively, our findings suggest the potential involvement of many mtRNAs in mitochondrial-nuclear communications and that mt-caRNAs may regulate nuclear transcription and thus play a role in mediating cellular response in health and disease.
Cardiac Arrest and Cardiopulmonary Resuscitation Outcome Reports: 2024 Update of the Utstein Out-of-Hospital Cardiac Arrest Registry Template
Authors: Bray Janet E. , Grasner Jan-Thorsten , Nolan Jerry P. , Iwami Taku , Ong Marcus E.H. , Finn Judith , McNally Bryan , Nehme Ziad , Sasson Comilla , Tijssen Janice , Lim Shir Lynn , Tjelmeland Ingvild , Wnent Jan , Dicker Bridget , Nishiyama Chika , Doherty Zakary , Welsford Michelle , Perkins Gavin D. ,   on behalf of the International Liaison Committee on Resuscitation
Published: July 2024

The Utstein Out-of-Hospital Cardiac Arrest Resuscitation Registry Template, introduced in 1991 and updated in 2004 and 2015, standardizes data collection to enable research, evaluation, and comparisons of systems of care. The impetus for the current update stemmed from significant advances in the field and insights from registry development and regional comparisons. This 2024 update involved representatives of the International Liaison Committee on Resuscitation and used a modified Delphi process. Every 2015 Utstein data element was reviewed for relevance, priority (core or supplemental), and improvement. New variables were proposed and refined. All changes were voted on for inclusion. The 2015 domains—system, dispatch, patient, process, and outcomes—were retained. Further clarity is provided for the definitions of out-of-hospital cardiac arrest attended resuscitation and attempted resuscitation. Changes reflect advancements in dispatch, early response systems, and resuscitation care, as well as the importance of prehospital outcomes. Time intervals such as emergency medical service response time now emphasize precise reporting of the times used. New flowcharts aid the reporting of system effectiveness for patients with an attempted resuscitation and system efficacy for the Utstein comparator group. Recognizing the varying capacities of emergency systems globally, the writing group provided a minimal dataset for settings with developing emergency medical systems. Supplementary variables are considered useful for research purposes. These revisions aim to elevate data collection and reporting transparency by registries and researchers and to advance international comparisons and collaborations. The overarching objective remains the improvement of outcomes for patients with out-of-hospital cardiac arrest.
Model-Mediated Teleoperation: An Operator-Centric Perspective
Authors: van der Walt Christophe , Falcone Sara , van Erp Jan , Stramigioli Stefano , Dresscher Douwe
Published: July 2024

Haptic teleoperation is a means of dexterously manipulating environments wherein a human operator can be situated several hundreds of kilometers away from the site of manipulation. Time delays between the local and remote sites pose problems for the user experience. Model-mediated teleoperation addresses these problems, but assumes a sufficiently representative model of the operator and of the environment. When applied to complex environments, this model becomes difficult to work with. A solution to this difficulty lies in the fact that human perceptual capabilities are limited. So too, possibly, is the amount of feedback needed in order to accomplish tasks effectively. In this work, we review information on human perception of the environmental properties involved in environmental perception (inertia, friction, roughness, compliance, and geometry). Moreover, we detail how these properties can be modeled. Finally, we propose some open questions that are relevant in understanding the link between the environment presented to the operator and their effectiveness at accomplishing tasks.