bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2109: Nox1-mediated Endothelial Ros Propagates Hypoxia-induced Pulmonary Hypertension Via The Unfolded Protein Response.
Authors: Goossen Christian J , Kracun Damir , Pagano Patrick J , Straub Adam C , Gomez Delphine A , Al Ghouleh Imad , Dustin Christopher , Cifuentes-Pagano Eugenia , Yuan Shuai
Published: July 2024

Pulmonary arterial hypertension (PAH) is a devastating disease in which reactive oxygen species (ROS) play an important role. The disease is characterized by lung arterial cell proliferation (inward remodeling) leading to right ventricle (RV) failure. We postulated that NADPH oxidase 1 (NOX1)-derived ROS mediate endothelial cell (EC) proliferation and hemodynamic changes in PAH. Indeed, we found increased NOX1 (1.6±0.10-fold; n=7-9, p<0.01) in human pulmonary artery ECs (HPAECs) exposed to 24 hr hypoxia (Hx) vs. normoxia (Nx) (1% v. 21% O
2
, in vitro PAH phenotype). To test an in vivo NOX1 role, C57BL/6J (WT), mice were implanted with pumps delivering a NOX1-selective peptidic inhibitor (NoxA1ds) or scrambled control (Scr) and exposed to Nx (21% O
2
) or Hx (10% O
2
) for 3 wks plus vehicle or VEGF receptor antagonist Sugen 5416. Sugen/Hx (Su/Hx) increased RV max pressure (RVMP) 2-fold ± 0.20-fold (n=7-9, p<0.001) along with elevated mean pulmonary artery pressure (mPAP, 2-fold 0.25-fold, n=7-9, p<0.001), pulmonary vascular resistance (PVR, 2-fold±0.2-fold, n=7-9, p<0.001), and right ventricular max dP/dt (RVM dP/dt, 2-fold±0.2-fold, n=7-9, p<0.001) vs. Nx. NoxA1ds infusion significantly attenuated these rises. In parallel, RNA-Seq of HPAECs treated with NOX1 siRNA and exposed to Nx or Hx revealed the protein kinase R-like ER kinase (PERK) branch of the unfolded protein response (UPR) as NOX1-mediated. UPR promotes tumor growth in cancer due to positive effects on cell survival and proliferation. Indeed, phospho-PERK expression measured via WB increased by 1.5-fold ± 0.3-fold (n=9, p<0.01) in HPAECs exposed to Hx, which was completely reversed by siNOX1. Further, downstream members of the PERK-UPR pathway followed similar trends including phospho-eukaryotic translation initiation factor 2A (eIF2α, 3-fold ± 0.6-fold, n=9, p<0.05), activating transcription factor 4 (ATF4, 2.1-fold ± 0.4-fold, n=9, p<0.01), and protein disulfide-isomerase (PDI, 2.3-fold ± 0.65-fold, n=9, p<0.01). We corroborated these findings in cross-sections of Su/Hx mouse lungs and in autopsied iPAH patient lungs. The findings are consistent with NOX1 mediating activation of the UPR through PERK, which, in turn, induces EC hyperproliferation in PAH.
Abstract 2118: Inhibition of Poldip2 Protects Against SARS-CoV-2 Inflammation
Authors: Hu Ruinan , White Taylor , Paiardini Mirko , Derdeyn Cynthia , Bosinger Steven , Lassegue Bernard P , Griendling Kathy K , Hernandes Marina
Published: July 2024

Introduction:
Polymerase delta-interacting protein-2 (Poldip2) mediates inflammation and endothelial permeability. Infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) results in lung inflammation and edema. We therefore hypothesized that Poldip2 inhibition may preserve endothelial barrier function and reduce inflammation in response to SARS-CoV-2 infection.

Methods:
Humanized ACE2 C57/Bl6 male and female mice were intranasally inoculated with SARS-CoV-2 or PBS vehicle. Poldip2 inhibition was achieved using BC-1215, an anti-inflammatory agent that targets Poldip2 based on molecular docking modeling. Mice received daily i.p. injections of 100 μg BC-1215 or saline for 7 days. Bronchoalveolar lavage (BAL) fluid was collected at 7 days for cell counts and ELISA determination of cytokine production. Lungs were harvested for qPCR.

Results:
Initial dosing experiments established a SARS-CoV-2 infection model and indicated that 4x10
5
infectious units of SARS-CoV-2 increased viral N2 copy number to 1.1x10
6
in the lung, but not heart and liver. The SARS-CoV-2 increase in CXCL1 mRNA in the lung was reduced by BC-1215 (5.9x10
-10
vs 7.6x10
-11
, P<0.05). TNF-α and MCP1 mRNA levels also trended down after BC-1215 treatment (5.9x10
-10
vs 2.1x10
-10
and 1.3x10
-09
vs 6.6x10
-10
, respectively). In BAL fluid, SARS-CoV-2-induced TNF-α (16.7pg/mL vs 6.0pg/mL, P<0.05) and IFN- γ (619.3pg/mL vs 1703.7pg/mL, P<0.05) levels were reduced by BC-1215. Protein accumulation and cell counts in BAL also trended down after BC-1215 treatment (protein 1325 mg/mL vs 805 mg/mL, cell number 202,833 vs 117,860).

Conclusion:
Inhibition of Poldip2 by BC-1215 treatment reduced TNF-α, CXCL1, IFN-γ in SARS-CoV-2 infected mice, suggesting that BC-1215 is a promising agent for treatment of COVID-19.
Abstract 2122: A Multiomic Timecourse Atlas Of Murine Vascular Disease Smooth Muscle Cell Transition Phenotypes And Their Perturbation With
Tcf21
Knockout
Authors: Li Daniel Y , Kundu Soumya , Gu Wenduo , Cheng Paul , Jackson William R , Zhao Quanyi , Nguyen Trieu , Ramste Markus O , Monteiro João P , Li Kevin , Kundaje Anshul , Wirka Robert , Quertermous Thomas
Published: July 2024

Background:
Vascular smooth muscle cells (SMC) contribute to heritable CAD risk and undergo cell changes to fibroblast-like (FMC) and osteochondrogenic (CMC) phenotypes. We perform molecular characterization of this transition process over the course of atherosclerosis development to better understand how causal SMC CAD genes such as
TCF21
drive the phenotypic transition process to alter CAD risk.

Methods/Results:
Deep multiomic profiling of aortic roots from control atherosclerosis model (

Myh11Cre
ERT2
, ROSA
tdT/+
, ApoE
-/-

) mice with SMC specific lineage tracing on high fat diet were harvested for 7 scRNA and 6 scATAC time points across 16 weeks. Similarly, aortic roots from
Tcf21
knockout (

Myh11Cre
ERT2
, Tcf21
ΔSMC/ΔSMC
, ROSA
tdT/+
, ApoE
-/-

) were collected at 3 timepoints for paired scRNA/scATAC. Waddington-Optimal Transport (WOT) mapped transcriptional trajectories across SMC lineage clusters which terminate in FMC and CMC phenotype clusters. PANDO/CellOracle frameworks were used to create gene regulatory networks with the ability to model SMC transition perturbations
in silico
. These analyses generated a multiomic timecourse atlas demonstrating the lineage traced phenotypic progression of SMC to FMC and CMC. Upon knockout of
Tcf21
, we discern notable shifts in the SMC to FMC transition characterized by differential scRNA transcriptional pathways and scATAC shifts in transcription factor (TF) motif accessibility which also overlap with WOT predicted TF enrichment for each cell state. These integrative analyses revealed
Tcf21
regulation of
Cebpb
and the
Tead1
-Hippo pathways as novel mechanisms for mediating its role in modulation of disease related processes such as inflammation, response to
TGF-
β
signaling and cell proliferation. Further mechanistic validation is performed through a combination of TF binding site analysis with
TEAD1
and
CEBPB
ChIP-Seq in human coronary artery SMCs, dual luciferase reporter assays and proximity ligation assays.

Conclusion:
We construct a single cell atherosclerosis timecourse to model mouse SMC cell states and perturb CAD gene
Tcf21
to elucidate the interplay between epigenetic and transcriptional mechanisms which affect key SMC transition elements such as
CEBPB
and the
TEAD1
-Hippo axis.
Abstract 2124: Genetic Variation Associated With Progression And Severity Of Peripheral Artery Disease
Authors: Levin Michael G , Adkar Shaunak , Ghouse Jonas , Depaolo John , Pedersen Ole B , Ostrowski Sisse , lynch julie , Bundgaard Henning , Damrauer Scott M , Klarin Derek
Published: July 2024

Background:
Peripheral Artery Disease (PAD) is a major source of cardiovascular morbidity and mortality, affecting >230 million individuals worldwide. While PAD may initially cause exertional limb pain (claudication), progression leads to chronic limb threatening ischemia (CLTI; rest pain and/or tissue loss) and amputation, which are associated with high rates of hospitalization and death. Genetic variants associated with PAD susceptibility have been identified using genome-wide association studies (GWAS). However, the role of genetic variation in the progression of PAD and disease severity remains poorly understood.

Methods:
GWAS of progression to CLTI were performed among individuals with PAD in the VA Million Veteran Program (MVP) and Copenhagen Hospital Biobank (CHB), accounting for age, sex, and genetic principal components. GWAS were first performed among genetically inferred ancestry subpopulations (EUR: European; AFR: African; AMR: admixed American), and combined using fixed-effects meta-analysis. SlopeHunter was used to account for index-event bias using a GWAS of prevalent PAD. Genetic associations with PAD severity (claudication, CLTI, amputation) were evaluated using multinomial logistic regression.

Results:
Up to 21.1 million genetic variants were tested for association with CLTI (17,641 individuals) in comparison to PAD (75,133 individuals). A total of 3 genetic loci were associated with progression to CLTI (p < 5x10
-8
). The strongest association with CLTI was rs6025 (OR 1.25, 95% CI 1.18 to 1.34, p = 4.62x10
-12
), a common missense variant (p.Arg534Gln) encoding Factor V Leiden, with consistent effects accounting for index-event bias. Compared to 489,538 individuals without PAD in MVP, rs6025 was associated with increased risk of non-specific PAD (OR 1.15, 95% CI 1.1 to 1.2, p = 5x10
-8
), claudication (OR 1.14, 95% CI 1.1 to 1.2, p = 1x10
-4
), CLTI (OR 1.42, 95% CI 1.3 to 1.6, p = 7x10
-23
), and amputation (OR 1.54, 95% CI 1.4 to 1.8, p = 2x10
-10
).

Conclusion:
Our results implicate F5 p.Arg534Gln as a risk factor for PAD progression and severe PAD manifestations including CLTI and amputation. This result provides genetic support for a potential role of anticoagulation as a treatment to prevent PAD progression and severe outcomes.
Abstract 2125: Aneurysmal Disease Among Hospitalized Patients With Fibromuscular Dysplasia - Types And Prevalence: Findings From The National Inpatient Sample
Authors: Nriagu Bede N , Ozbay Mustafa , Sherpally Deepak , Quintanilla Bryan , Abdelmottaleb Wael , Evbayekha Endurance O , Nriagu Ugonwa N , Munguia Galaz Cesar
Published: July 2024

Introduction:
Fibromuscular dysplasia (FMD) is a non-atherosclerotic, non-inflammatory disease that can lead to various arterial abnormalities, including dissection, occlusion, and aneurysm. Only a few studies have examined the common types of aneurysmal disease among patients with FMD. We examine the types and prevalence of aneurysmal disease among hospitalized patients with FMD.

Methods:
Using the International Classification of Diseases, 10th edition, Clinical Modification (ICD-10-CM) diagnosis codes, we queried the National Inpatient Sample 2016 to 2020 for hospitalizations among patients ≥18 years old with a diagnosis of Fibromuscular dysplasia. Aneurysmal disease of the iliac, carotid, upper extremity, lower extremity, renal, vertebral artery, thoracic aorta, and abdominal aorta was further identified in this cohort of patients using ICD-10 codes. We examined prevalence of FMD and further compared the prevalence of aneurysmal disease among hospitalized patients with and without FMD.

Results:
Our study included 9,315 weighted hospitalizations among patients with fibromuscular dysplasia (mean [SD] age 61.2 [15.5] years). FMD was five times more common in females than males (84.1% vs. 15.9%). Compared to hospitalized patients without FMD, aneurysmal disease was more common among hospitalized patients with FMD. The most common aneurysmal site among patients with and without FMD was the renal artery (1.4%) and abdominal aorta (0.71%) respectively. The prevalence of aneurysmal disease among hospitalized patients with FMD was renal artery aneurysm (1.40%), abdominal aortic aneurysm (1.29%), carotid artery aneurysm (1.23%), thoracic artery aneurysm (1.02%), vertebral artery aneurysm (0.86%), lower extremity artery aneurysm (0.38%), iliac artery aneurysm (0.27%), and upper extremity artery aneurysm (0.16%).

Conclusion:
Aneurysmal disease is prevalent among hospitalized patients with Fibromuscular dysplasia, with the renal artery identified as the most common site of aneurysm. Further research is needed to understand better the relationship between FMD and other vascular abnormalities.
Abstract 2126: Impact Of Pulmonary Hypertension On In-Hospital Outcomes Among Patients With Atrial Fibrillation Undergoing Catheter Ablation: Insights From The National Inpatient Sample Database
Authors: Nriagu Bede N , Ozbay Mustafa , Nriagu Ugonwa N , Sherpally Deepak
Published: July 2024

Introduction:
Catheter Ablation (CA) is a treatment option for patients with Atrial Fibrillation (AF) when medications are intolerable or are unable to control symptoms. Pulmonary hypertension is characterized by elevated pulmonary artery pressure and affects all ages, genders and races. In this study, we examine the impact of pulmonary hypertension (PH) on hospitalized patients with AF undergoing catheter ablation.

Methods:
Using the International Classification of Diseases, 10th edition, Clinical Modification (ICD-10-CM) diagnosis and procedure codes, we queried the National Inpatient Sample 2016 to 2020 for hospitalizations among patients ≥18 years old diagnosed with PH, and AF who underwent CA procedure. Among this cohort, we identified the percentage of all hospitalizations for AF undergoing CA and stratified this cohort into those with and without PH. Mortality and length of hospital stay were compared between those with and without PH.

Results:
Our study included 232,150 weighted hospitalizations among patients with atrial fibrillation who underwent catheter ablation. Of these, 28,980 (12.5%) had pulmonary hypertension, and the majority (51.1%) were males. Among this cohort, those with pulmonary hypertension were older compared to those without pulmonary hypertension (mean [SD] age: 70.0 [11.2] vs. 68.2 [11.4] years). Patients with pulmonary hypertension had a higher length of hospital stay (10.2 vs. 6.2 days, p<0.0001) and higher total hospital charges ($241,212 vs. $187,987; p<0.0001) compared to those without pulmonary hypertension. Higher in-hospital mortality was recorded among patients with pulmonary hypertension compared to those without pulmonary hypertension (3.4% vs. 1.2%,
p<0.0001
).

Conclusion:
Among patients with atrial fibrillation undergoing catheter ablation, pulmonary hypertension was associated with worse outcomes, including higher length of stay and mortality. Further research is needed to better understand the impact of pulmonary hypertension among patients with atrial fibrillation undergoing catheter ablation.
Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation
Authors: Mangum Kevin D , Bauer Tyler , Joshi Amrita , Barrett Emily , Wolf Sonya , Moon Jadie , Shadiow James , Obi Andrea T , Gudjonsson Johann , Davis Frank , Gallagher Katherine A
Published: July 2024

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers,
Acta2
and
Tagln
(p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased
Setd4
expression and inhibited smooth muscle gene expression (
Acta2, Tagln, Cnn1, Myh11
), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased
Setd4
expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.
Abstract 2144: Smooth Muscle Cell Specific Beclin-1 Deficiency Promotes Aging-accelerated Aortic Stiffness In Mice
Authors: Kannan Arun , Ramesh Nithya , Frony Ana Clara S , Downey Edward , Subramanian Venkateswaran
Published: July 2024

Background and Objective:
Aortic stiffness, a hallmark of aging, is a significant riskfactor for cardiovascular diseases like hypertension and atherosclerosis. Vascularsmooth muscle cells (SMCs) play a crucial role in maintaining aortic structural integrity,and increased SMC stiffness contributes to age-accelerated aortic stiffness.Autophagy, a self-regulatory cellular process, declines with aging, predisposingindividuals to age-related diseases. Reduced levels of Beclin-1, a key autophagyinduction gene, have been observed in aging-associated diseases. However, thecontribution of SMC-derived Beclin-1 in aortic stiffness progression remains unknown.This study examined the contribution of SMC-derived Beclin-1 to aortic stiffnessdevelopment in mice.

Methods and Results:
Ultrasound measurements revealed increased aortic stiffnessin 60-week-old aged mice, characterized by elevated pulse-wave velocity (PWV) andreduced aortic distensibility and radial strain compared to 8-week-old young mice.Western blot and immunohistochemistry analyses showed a decline of aortic Beclin-1in the SMC-rich aortic medial layer of aged mice. To investigate the role of SMC-Beclin-1 in aortic stiffness, mice with inducible SMC-Beclin-1 deletion were generated bybreeding Acta2-CreERT2 hemizygous mice with Beclin-1 floxed mice. Baselinemeasurements at 8 weeks showed no PWV difference between Beclin-1 x Acta2-CreERT2 (Cre+) and non-Cre littermates (Cre-). However, following tamoxifen-induced Cre recombination, SMC-Beclin-1 deficiency significantly accelerated PWVby 18 weeks (Cre-: 2.4 ± 0.1 vs Cre+: 4.1 ± 0.2 m/s; P<0.001), accompanied byreduced aortic distensibility (Cre-:115.4 ± 3.2 vs Cre+: 93.1 ± 3.4; P<0.001) and radialstrain(Cre-:20.3 ± 0.7 vs Cre+:15.8 ± 0.7; P<0.001). Histological analyses indicatedthat Beclin-1 deficiency increased collagen deposition in aortic media and adventitia.RNA Sequencing and Western blot analyses identified extracellular matrix regulatingGalectin-3 and S100A4 as novel targets of Beclin-1, selectively upregulated in SMC-Beclin-1 deficient aorta.

Conclusion:
These findings suggest that SMC-derived Beclin-1 plays a critical rolein suppressing aging-accelerated aortic stiffness in mice.
Abstract 2148: Lung Capillary Endothelium To Arterial Endothelium Transition In Pulmonary Arterial Hypertension
Authors: Liu Bin
Published: July 2024

Introduction:
Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear.

Method and Results:
Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggests that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for distal arterial ECs in PH. General capillary EC lineage tracing approach using Plvap-DreERT2; Tdtomato mice demonstrated that general capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or Notch4 neutralized antibodies normalized the arterial programming in PH.

Conclusion:
In conclusion, our study demonstrates that general capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients.

Acknowledgment:
This work was supported in part by NIH grants R00HL138278, R01HL158596, R01HL62794, R01HL169509, R01HL170096, AHA Career Development Award 20CDA35310084, The Cardiovascular Research and Education Foundation, Arizona Biomedical Research Centre funding (RFGA2022-01-06), and University of Arizona institution funding to Z.D.
Abstract 3003: Ketogenic Diet Limits Experimental Abdominal Aortic Aneurysms
Authors: Samura Makoto , Xu Baohui , Hojong Park , Youngjin Han , Tsao Philip S , Dalman Ronald L
Published: July 2024

Background:
Intermittent fasting and ketogenic diet may improve cardiovascular health. We studied the impact of ketogenic dietary intervention on experimental abdominal aortic aneurysms (AAAs).

Methods:
AAAs were induced in male mice via topical abluminal porcine pancreatic elastase (PPE) application at laparotomy in C57BL/6J, and 28 day administration of exogenous angiotensin (Ang) II in apolipoprotein E deficient strains, respectively. As feeding regimens, mice were fed standard or ketogenic diets 3 days prior to or 3 days following, AAA induction. Influence on AAA development was assessed by serial transabdominal ultrasonography and histopathologic analysis at sacrifice.

Results:
Ketogenic diet promotes cardiovascular health in part by increasing ketone body production. The Figure summarizes the influence of ketogenic diet on experimental AAAs in two distinct models. Following PPE application, subsequent aortic diameter enlargement was significantly reduced in mice receiving ketogenic vs. standard diets at 7 and 14 days regardless of specific feeding regimen. On histologic analysis, ketogenic diet feeding resulted in improved aortic medial elastin and smooth muscle cell retention, reduced mural macrophage, CD4
+
and CD8
+
T and B cell infiltration, and attenuated mural angiogenesis. In the Ang II infusion model, ketogenic diet limited Ang II-induced aortic diameter expansion, lowered AAA incidence and reduced AAA severity.

Conclusion:
Ketogenic dietary intervention suppressed experimental AAA progression in complementary murine modeling systems. Already popular clinically, this dietary modification may prove effective in limiting clinical AAA disease progression as well as other cardiovascular disease endpoints.
Abstract 3015: Matrix Metalloproteinase Inhibition In Rat Abdominal Aortic Aneurysm
Authors: Zaghloul Mohamed , Elizondo-Benedetto Santiago , Arif Batool , Heo Gyu Seong , Luehmann Hannah , Liu Yongjian , Gropler Robert J , Sadeghi Mehran M , Zayed Mohamed A
Published: July 2024

Background:
Matrix metalloproteinases (MMPs) have a robust proteolytic activity and play a fundamental role in degradation of extracellular matrix proteins. This degradation is a hallmark of the initiation, progression, and rupture of abdominal aortic aneurysms (AAAs).

Hypothesis:
We hypothesized that broad inhibition of MMPs with a 3
rd
generation MMP inhibitor (GM6001) can reduce AAA inflammation and rupture using an established rat model.

Aims:
Our study aimed to investigate the potential of GM6001 in reducing AAA-related inflammation using a rat model, filling a critical gap in AAA research.

Methods:
Male Sprague-Dawley rats underwent intraluminal infrarenal perfusion of abdominal aorta using porcine pancreatic elastase (PPE), and daily β-aminopropionitrile (BAPN) to promote AAA rupture. Starting on postoperative day 3 (POD3) rats were treated daily either with or without GM6001 via intraperitoneal injections. On POD6, treated and untreated groups, underwent PET/CT imaging using a pan-MMP radiotracer 64Cu-RYM2 and
in vivo
aortic diameter measurement using ultrasound. Rate of rupture of treated and untreated groups was observed until POD14 (Fig 1A).

Results:
Treated rats demonstrated no evidence of toxicity or weight loss. Compared to untreated rats, treated rats had lower AAA rupture rates (P<0.01; Fig 1B), smaller AAA diameters (P<0.05; Fig 1C), and lower AAA MMP radiotracer uptake (P<0.05; Fig 1D,1G). Total and active MMP-9 were significantly lower in treated rats (Fig 1E-F). In addition, IL-1B, IL-17A, IL-18, IFN-y, and RANTAS were significantly lower in treated rats, but TNF-a was higher (Fig 1H-M). VVG staining demonstrated a higher content of preserved elastin in treated rats (Fig 1N).

Conclusion:
Inhibition of MMPs with a 3
rd
generation MMP inhibitor appears to be a promising therapeutic approach to impede the progression, inflammation, and rupture of AAAs in rats. This may represent a renewed pathway for MMP inhibition in human subjects with AAAs.
Abstract 3016: Cytoplasmic And Nuclear Nfatc3 Cooperatively Contributes To Vascular Smooth Muscle Cell Dysfunction And Drives Aortic Aneurysm And Dissection
Authors: Zhao Li , Liu Deshen , Zhao Lingna , Huang Fangze , Peng Qin-Bao , Liu Xiu , Zheng Shaoyi
Published: July 2024

Background:
Aortic aneurysm and aortic dissection (AAD) are fatal diseases resulting in catastrophic aortic rupture and sudden death. The degradation of the extracellular matrix (ECM) and the phenotypic transformation of vascular smooth muscle cells (VSMCs) are crucial pathological mechanisms in AAD. We previously found that macrophage NFATc3 inhibits atherosclerosis and foam cell formation. However, the precise role of VSMC-NFATc3 in AAD remains unknown.

Aims:
This study investigates the role of NFATc3 in VSMCs in AAD progression and the molecular mechanisms.

Methods:
To investigate the role of NFATc3 in VSMCs in AAD, we used VSMC-NFATc3-specific knockout and knock-in mice. By establishing mouse models for thoracic AAD (TAAD) and abdominal aortic aneurysm (AAA), we employed techniques such as RNA sequencing, mass spectrometry, and chromatin immunoprecipitation to study the mechanistic role of VSMC-NFATc3 in AAD.

Results:
Cytoplasmic and nuclear NFATc3 levels are elevated in human and mouse AAD. VSMC-NFATc3 deletion reduces TAAD and AAA progression in mice, contrary to VSMC-NFATc3 overexpression. VSMC-NFATc3 deletion reduces ECM degradation and maintains the VSMC contractile phenotype. Nuclear NFATc3 targets and transcriptionally upregulates MMP9 and MMP2, promoting ECM degradation and AAD development. NFATc3 promotes VSMC phenotypic switching by binding to eukaryotic elongation factor 2 (eEF2) and inhibiting its phosphorylation in the VSMC cytoplasm. Restoring eEF2 reverses the beneficial effects in VSMC-specific NFATc3-knockout mice. Cabamiquine—targets eEF2 and inhibits protein synthesis—inhibited AAD development and progression in VSMC-NFATc3-overexpressing mice.

Conclusion:
VSMC-NFATc3 promotes VSMC switch and ECM degradation while exacerbating AAD development, making it a novel potential therapeutic target for preventing and treating AAD.
Abstract 3017: Fluorine-18 Sodium Fluoride Positron Emission Tomography/ Computed Tomography Imaging Of Calcium Progression In Patients With Peripheral Artery Disease
Authors: Rimmerman Eleanor T , Chou Ting-Heng , Musini Kumudha N , Patel Surina , Wynveen Molly , Trainer Jenna , Mevis Katy , Atway Said , Go Michael , Stacy Mitchel R
Published: July 2024

Introduction:
Peripheral artery disease (PAD) is an atherosclerotic disease that promotes lower extremity ischemia and is associated with necrosis, limb loss, and premature death. Fluorine-18 sodium fluoride (
18
F-NaF) PET/CT imaging has been utilized for early detection of calcific disease progression in the coronary arteries; however, this technique has not been assessed in PAD to predict developing arterial calcification.

Hypothesis:
We hypothesized that
18
F-NaF PET/CT imaging could identify sites undergoing active microcalcification and predict the formation or expansion of vascular calcium.

Methods:
Patients with PAD (n=26) underwent
18
F-NaF PET/CT imaging at baseline and CT imaging 18 months later. Regions of interest were manually drawn on axial PET and CT images for the arteries of the symptomatic limb (totaling 8,001 arterial segments). Arterial uptake of
18
F-NaF was quantified and reported on a segment-by-segment basis as maximum standard uptake values (SUV
max
). Baseline and 18-month CT images were co-registered to quantify serial changes in calcium density for each arterial segment using CT image cutoffs for calcium (≥130 Hounsfield Units). Differences in arterial uptake of
18
F-NaF were compared across 3 categories of segments: 1) no calcium at baseline or 18 months, 2) newly formed calcium and 3) existing calcified lesions with expansion of calcium.

Results:
Arterial segments containing newly formed calcium had significantly higher baseline uptake of
18
F-NaF than segments without calcium progression. Existing calcified arterial lesions with progression showed significantly higher
18
F-NaF uptake compared to segments with newly formed calcium and segments without calcium progression (
Figure 1
).

Conclusions:
In conclusion, calcium progression can be detected and potentially predicted using
18
F-NaF PET/CT imaging, thereby providing a molecular imaging tool for monitoring PAD pathophysiology and responses to medical therapy.
Abstract 3020: The Effects Of High Glucose On The Release Of Exosomes From Adipocytes
Authors: Sawant Harshal , Sun Bowen , Meyer Regan , Bihl Ji C
Published: July 2024

Type 2 Diabetes Mellitus is primarily characterized by hyperglycemia accompanied by adipose tissue dysfunction. More importantly, diabetes is a risk factor for stroke and other cerebrovascular diseases, which might be related to poorly explored adipose tissue dysfunction. Even though adipose tissue is comprised of various cells, it is found that predominantly adipocyte dysfunction contributes to diabetes and related complications. Exosomes are extracellular vesicles released from almost all cells with varying functions in different conditions. Here, we aim to determine the function of adipocyte-derived exosomes (Adp-EXs) in high glucose (HG) conditions. We hypothesized that Adp-EXs from the HG group would induce cytotoxicity and oxidative stress in primary human brain microvascular endothelial cells (HBMECs). First, the level of Adp-EXs was measured after HG treatment. Secondly, their effects were studied on HBMECs after co-incubation. Adipocytes were cultured in normal glucose (NG) or HG (25mM) media to isolate Adp-EXs by serial ultracentrifugation method. The size and concentration of Adp-EXs were determined via the Nanoparticle Tracking Analysis system. Adp-EXs at three concentrations (1x10
8
, 5x10
8
and 1x10
9
particles/mL) were co-incubated with HBMECs for 24 to 48 hours to evaluate optimal cellular communication. PKH26-labelling (2uL/mL) of Adp-EX was performed to track its incorporation into HBMECs. Dihydroethidium staining and flow cytometry quantified reactive oxygen species production and cytotoxicity was determined by lactate dehydrogenase assay. For all experiments, n=5-10 and mean SEM=8.600±6.194. Results showed: 1) HG increased Adp-EX levels without size differences between NG and HG groups. 2) Adp-EXs integrated with HBMECs in a time and dose-dependent manner, more prominently in the HG group. 3) Adp-EXs from the HG group increased Dihydroethidium-positive HBMECs. 4) Adp-EXs from the HG group increased fluorescence for lactate dehydrogenase. In conclusion, the data suggest that HG could promote Adp-EX release, and trigger oxidative stress and cytotoxicity in HBMECs.
Abstract 3022: Comparison Of Stroke Mnemonics
Authors: Singh Neeraj
Published: July 2024

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

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

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

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

Conclusions:
Participants preferred the AHORA brochure over the RAPIDO brochure due to its more concise mnemonic, clearer descriptions of stroke signs, and emphasis on calling 911 when stroke signs are recognized. These findings provide valuable guidance for physicians in selecting brochures for Spanish-speaking patients and offer insights into the elements necessary to develop effective mnemonics in other languages.
Abstract 3026: Genetic Deletion Of Skeletal Muscle Bag3 Exacerbates Ischemic Myopathy
Authors: Terwilliger Zoe S , Kolasa Makenzie , Li Feifei , Green Thomas , karnekar Reema , Spangenburg Espen , Fisher-Wellman Kelsey , Yamaguchi Dean , McClung Joseph
Published: July 2024

Background:
Bcl2-associated athanogene 3 (BAG3) is a multifunctional adaptor protein that regulates diverse cellular functions. Mutations in BAG3 are causally linked to myofibrillar myopathy and dilated cardiomyopathy in humans and now appear to be one of the most common to cause human disease. We previously identified a murine coding variant in
Bag3
that contributes to hindlimb ischemia (HLI) pathology, however, no cell specific data on Bag3’s role in PAD pathology exists.

Hypothesis:
We hypothesized that muscle specific BAG3 is required for both the restoration of blood flow and muscle recovery following HLI.

Methods:
We generated a genetic model of lifelong muscle cell (mature fiber and progenitor)
Bag3
deletion (Pax7-Cre; Bag3
-/-
) on a BL6 parental background. Male and Female mice (12-20 weeks; WT: n=45; KO: n=32) were evaluated at baseline and HLI d7 and d28 for blood flow via LDPI, isometric muscle force production, vascular density and muscle morphology.
Bag3
deletion was validated by recombination, qRT-PCR and Western Blotting in whole tissue, isolated myofibers, and FACS muscle progenitor cells.

Results:
KO reduced muscle force production (n=18; 145.8mN Force ± 28.6 vs 56.9 ± 16.8 SEM; p=0.0208) and capillary perfusion (n=7; 0.17 CD31+/Lectin+ vessels ± 0.02 vs 0.09 ± 0.01 SEM p=0.0491) indicative of the multi-compartmental effect of BAG3 loss in skeletal muscle at HLI d7. Extended HLI (d28) revealed a KO phenotype characterized by reduced blood flow recovery (LDPI; n= 7; .68 ischemic/non limb ± 0.5 vs .38 ± 0.6 SEM p=0.0011) and sustained myopathy including reduced myofiber size (
n
=7; 1585um
2
± 161 vs 1093 ± 169 SEM;
p
=0.05) and increased tissue lesion size (
n
=7; 2.5% muscle area ± 2.0 vs 48.3% ± 21.6;
p
=0.03).

Conclusion:
Loss of skeletal muscle Bag3 exacerbates myopathy and reduces capillary perfusion and blood flow recovery following HLI and demonstrate a requirement for BAG3 in recovery from limb ischemia.
Abstract 3033: Registration And Segmentation Framework For 3d Instrument Visualization Relative To Preoperative Ct Imaging Via Spatially-tracked Ultrasound: A Phantom Study
Authors: Warner William , Fan Xiaoyao , Powell Richard J , Bellamkonda Kirthi , Paulsen Keith
Published: July 2024

Objectives:
Surgical navigation guides surgery without radiation by mapping instrument locations onto preoperative CT (pCT). Adoption to vascular surgery remains novel. Feasibility of spatially-tracked ultrasound (US) for surgical registration and wire localization relative to pCT is demonstrated.

Methods:
pCT was taken (0.6mm) of a carotid artery phantom and a CT artery model (CTM) segmented. An US probe (Philips L17-5), was rigidly attached to an optical tracker and swept along the artery (1000 images). A navigation system (Medtronic StealthStation) acquired the tracker and 2D US image's positions via a spatial calibration (1.6mm accuracy). The artery in each image was segmented and tracking data combined to reconstruct a 3D US artery model (USM) (Fig. 1). The CTM and USM were registered via an iterative closest point algorithm. A Rosen wire (0.035in) was inserted into the artery. Acquiring an US sweep of the wire (500 images), it was segmented in each image via thresholding. Segmentations and tracking were combined into a 3D US wire-model (USWM). Its position was overlaid onto pCT via the USM-CTM registration. Another CT was acquired, registered via a fiducial point-based registration (0.86 mm accuracy), and the ground truth wire position segmented (Fig. 2). A tip-to-tip distance was measured between the USWM and CT wire positions.

Results:
CTM-to-USM registration error was 0.93 mm (RMSE) between artery point clouds. The tip-to-tip distance between CT and USWM wire models was 0.2 (longitudinal) and 1.4 (radial) mm.

Conclusions:
A vascular navigation framework capable of patient registration and highly accurate instrument localization is provided using tracked iUS.
Abstract 3043: Apolipoprotein A5 Alters The VLDL Proteome To Promote Increased Particle Uptake By Hepatocytes
Authors: Tarapore Pheruza , Swertfeger Debi , Morris Jamie C , Sarkar Snigdha , He Yi , Melchior John T , Liu Min , Davidson W. Sean
Published: July 2024

The levels of circulating triglycerides (TG) carried in TG-rich lipoproteins (TGRL) and their remnants are causally associated with cardiovascular disease (CVD). Liberation of TG from lipoproteins to peripheral tissues is modulated by TGRL-associated proteins including apolipoproteln A5 (ApoA5), which has been reported to profoundly impact TG metabolism. Present at ~150 ng/ml in plasma, it has been thought to work via stimulation (or de-repression) of lipoprotein lipase (LPL), which hydrolyzes TG to deliver fatty acids to cells. To determine whether ApoA5 may alter the proteomic composition of VLDL, we examined the VLDL particle proteome from C57BL/6J WT and C57BL/6J apoA5 KO mice by mass spectrometry. ApoA5 presence was associated with increased VLDL apoE and apoM content and reduced levels of apoA1 and apoD, compared to VLDL from apoA5 KO mice. Presence of apoA5 also tracked with increased levels of the serine protease plasmin and decreased levels of its inhibitor alpha2-microglobulin on the VLDL. Immunoblotting analyses confirmed these results. Despite large differences in apoA5 content, VLDL from WT or KO mice behaved similarly as a substrate for recombinant human LPL
in vitro
. However, we observed that VLDL from apoA5 KO mice was internalized by cultured mouse hepatocytes less efficiently than WT VLDL (Kd 80±9.0 vs. 35±10 μg/ml). Notably, when recombinant mouse apoA5 was added back to apoA5 KO plasma, levels of VLDL apoE and apoM were rescued, and uptake efficiency was restored (Kd 29±8.0). Our current working model is that ApoA5 alters the proteome of TGRL in ways that affect remnant particle uptake by the liver, perhaps via apoE. ApoA5 may also affect plasmin levels/activity which was recently linked to LPL regulation.
Abstract 3047: Extracellular Vesicles Produced By IL4-polarized Human Macrophages Suppress Atherosclerosis By Reprograming Ly6c
hi
Monocytes
Authors: Raffai Robert L , Vu Ngan K , Phu Tuan Anh , Ng Martin , Gao Alex
Published: July 2024

Introduction:
Extracellular vesicles (EVs) produced by IL4-polarized macrophages (Mφ) suppress inflammation by communicating microRNA-controlled immunometabolic signaling to recipient immune cells and adipocytes. However, detailed cellular and molecular pathways responsible for these effects remain incompletely understood.

Objectives:
Examine transcriptional responses sensitive to IL4-polarized Mφ EVs among circulating Ly6C
hi
monocytes derived from mice with hyperlipidemia.

Methods/Approach:
EVs were produced by culturing THP1 macrophages with human IL4, and purified using cushioned-density gradient ultracentrifugation. Twenty-week old male LDL-receptor deficient mice fed high-fat diet for 16 weeks were treated to a 6-week course of 3-weekly intraperitoneal infusions of 10e
10
THP1-IL4 EVs or sham. Circulating Ly6C
hi
monocytes were collected using a cell sorter and RNA was examined using nanoString nCounter inflammatory and metabolic pathway panels. Plasma cytokines were detected with a V-Plex MesoScale assay, while atherosclerosis was assessed by detecting myeloid cell subsets in enzymatically-digested aortae and oil red O positive lesions in the aortic root.

Results:
THP1-IL4 EVs caused profound transcriptional reprogramming among Ly6C
hi
monocytes, resulting in reduced signaling pathways controlled by MAPK, PI3K, TLR and inflammasomes. We identified reduced levels of Casp-1, Nlrp1a, as well as Aim2, all recognized to drive atherosclerosis including in JAK2
vf
clonal hematopoiesis. Furthermore, THP1-IL4 EVs upregulated metabolic pathways involved in arginine metabolism, IDH1/2 activity, redox stress control, nucleotide salvage, fatty acid oxidation and mitochondrial respiration. In contrast they reduced pathways involved in glucose transport, hypoxia, KEAP-NRF2 pathway and tryptophan metabolism. Control of these pathways in Ly6C
hi
monocytes contributed to reduced levels of IL-1β, IL-6, IFNγ and TNFα in plasma, as well as CD45
+
aortic myeloid cells and oil red O positive aortic lesions.

Conclusions:
IL4-polarized Mφ EVs suppress atherosclerosis by communicating profound transcriptional reprogramming to Ly6C
hi
monocytes. Ongoing studies examine their capacity to drive atherosclerosis regression.
Abstract 3050: The Multifaceted Roles Of Long Noncoding Rna
CHROMR
In Cardiovascular Disease
Authors: van Solingen Coen , Cyr Yannick , Barcia Duran Jose Gabriel , Eberhardt Natalia , Smyrnis Panagiotis , Giannarelli Chiara , Moore Kathryn J
Published: July 2024

The last decade revealed powerful roles for long noncoding RNA (lncRNA) in chromatin organization and orchestration of gene expression in healthy and pathological states, including atherosclerotic cardiovascular disease (ACVD). Yet, fewer than 10% of human lncRNAs have been characterized, representing unique opportunity for discoveries in the pathogenesis and treatment of ACVD. We previously showed that the primate-specific lncRNA
CHROMR
(CHolesterol-induced Regulator Of Metabolism RNA) is induced by cholesterol excess to restrict the availability of functionally related microRNAs that normally repress cholesterol efflux and HDL biogenesis. Using single-cell RNA-seq analysis of human carotid plaques from the NYU ATHERO-IN study, we now show that
CHROMR
is robustly expressed in myeloid cells within carotid plaques and strongly correlates with expression of interferon (IFN) stimulated genes (ISGs). Previously we established that
CHROMR
functions in the nucleus to license the expression of interferon (IFN) stimulated genes (ISGs).
CHROMR
acts through sequestering IFN regulatory factor-2 binding protein 2, part of the IRF2 repressor complex that antagonizes transcriptional activation of ISGs. While this coordinated regulation of lipid metabolism and IFN responses by
CHROMR
is beneficial in accelerating an inflammatory response upon viral infection, it has detrimental effects in the context of ACVD. As such, therapeutic inhibition of
CHROMR
holds potential to reduce plaque inflammation. Therefore, we treated human atherosclerotic 3D carotid vascular explants with GapmeRs to deplete
CHROMR
and subsequently measured the cellular response upon inflammatory stimulus. We found that Gap
CHROMR
reduced
CHROMR
levels and blunted pro-atherogenic cytokine/chemokine secretion, whereas lentiviral delivery of
CHROMR
increased cytokine/chemokine secretion when compared to controls. Collectively, our findings underscore the merit of investigating
CHROMR,
as well as other lncRNAs, to decipher novel regulatory mechanisms that govern lipid metabolism and inflammation in humans.
Abstract 3053: An Emerging Role Of The RNA Binding Protein Human Antigen R In Atherosclerotic Vascular Smooth Muscle Cell Heterogeneity
Authors: Wadding-Lee Caris , Coughlin Taylor , Jones Shannon , Jay Megan S , Lusis Aldons J , Camerer Eric , Civelek Mete , Tranter Michael , Owens A. Phillip
Published: July 2024

Objective:
Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis.

Methods and Results:
Downregulation of VSMC migration and cytokine release in

Par2
-/-

mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation,

Par2
+/+

and

Par2
-/-

VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While

Par2
+/+

VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers,

Par2
-/-

VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where

Par2
-/-

VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in

Par2
-/-

VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female

Ldlr
-/-

HuR
flox/flox

SM22
Cre+

mice have attenuated atherosclerosis compared to their littermate Cre
-
controls at both 12 and 24 weeks.

Conclusions:
These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.