bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1045: Using Multiplex Imaging For Cell Phenotyping Of Early Human Atherosclerosis
Authors: Elishaev Maria , Li Boaz , Zhou Annie , Leeper Nicholas J , Francis Gordon A , Lai Chi , Wang Ying
Published: July 2024

Background:
In the past few years, animal models and cultured cells have shown that smooth muscle cells (SMCs) and inflammatory cytokines are important new players in atherosclerotic disease. Validating the roles of these new players in human atherogenesis is crucial for the development of therapeutics targeting them. Multiplex imaging is a powerful tool for mapping cell phenotypes and microenvironment in tissue sections, enabling proof-of-concept studies using biobanked human specimens. However, this technology has seldom been applied to human atherosclerotic lesions. This study is a trial to benchmark the application of the multiplex imaging platform PhenoCycler to biobanked human coronary lesions. We hypothesize that this technology is a powerful tool to prove theories raised by previous studies: SMCs initiate foam cell formation and inflammation is associated with phenotypic switching in SMCs.

Methods:
We created an 8-plex imaging panel to map lesion cells previously identified as key players in atherogenesis, including foam cells from SMC and macrophage origins, CD68
+
SMCs, apoptotic cells, and cells expressing inflammatory cytokines interleukin-1β and tumor necrosis factor α. They were imaged simultaneously on each section of early human lesions (n=9) to test theories related to foam cell formation, phenotypic transition, and inflammatory cytokines.

Results:
Foam cells of SMC origin greatly outnumbered those of macrophage origin and were enriched in the deep intima, where lipids accumulate in early atherogenesis, distinct from macrophage foam cells in the superficial intima. A higher presence of CD68
+
SMCs was observed among lesion SMCs highly expressing interleukin-1β, but not TNF-α. Highly inflamed SMCs exhibited a trend of increased apoptosis compared to macrophages with similar cytokine levels.

Conclusions:
Our multiplex imaging results confirmed that SMCs form the majority of foam cells in early atherogenesis, and their phenotypic transition to macrophage-like is related to inflammation. Applying multiplex imaging to biobanked human lesions unlocks opportunities to prove that theories based on animal models and cultured cells apply to human atherogenesis.
Abstract 1067: Floating Through The Aorta; Aortic Thrombus Takes It Course
Authors: Ehinmisan Ayara O , Najeeb Shehryar , Fomeche John , Koulova Anna , Homayounrooz Forugh
Published: July 2024

Background:
Aortic thrombus is an uncommon condition that is usually discovered as an incidental finding but in rare cases can be the cause of fatal conditions. The reported incidence of its intramural, pedunculated and floating types. The most common location is reported to be the descending aorta followed by the aortic arch. The pathophysiology remains unclear but possible predisposing factors include atherosclerosis, hypercoagulability disorders, malignancy, dissections, aneurysm, chronic inflammatory states, substance use and chronic steroid use. Treatment can vary from anticoagulation to thrombolysis and surgical removal based on the clinical presentation. Clinical presentation: 48-year-old male with a past medical history of hyperparathyroidism presented with three days of nausea, vomiting and lower abdominal pain. CT abdomen showed pancreatic inflammation and he was admitted for pancreatitis. MRCP was completed to evaluate for organic causes of pancreatitis which revealed left renal infarction. CTA C/A/P was done to evaluate infarction and was found to have large 1.5 x 1.1 x 2.1 cm mural thrombus, partially free-floating in distal thoracic aortic arch/proximal descending segment and focal narrowing at the origin left renal artery presumed to be of embolic origin. He was started on heparin drip for the rest of his hospital course and continued to improve. Hypercoagulable work up was unrevealing for any specific etiologies of the thrombus. Pancreatitis was determined as the presumed cause of thrombus as thromboembolism is a known possible complication of pancreatitis and splenic vein thrombosis may be seen in up to 12% of patients .Surgical intervention was initially considered, however repeat CT angiogram of chest/abdomen prior to discharge showed near resolution of thrombus with stable left renal infarct. He was discharged with apixaban and was recommended to follow-up outpatient.

Conclusion:
Aortic thrombus is rare due to the constant flow of pressure within the aorta. Early detection and management are crucial to avoid embolization which can lead to end organ dysfunction, limb ischemia, mesenteric ischemia, and stroke. DOAC anticoagulation is a viable and efficient therapy option if surgical intervention is not feasible
Abstract 1079: Pre-treatment Circulating Vascular Biomarker Signatures Predict Cancer-therapy Related Cardiac Dysfunction During Breast Cancer Treatment
Authors: Gustafson Dakota , Ching Crizza , Mistry Priya , McIntosh Chris , Thavendiranathan Paaladinesh , Fish Jason
Published: July 2024

Aim:
Breast cancer treatment with anthracycline and trastuzumab can result in cancer therapy-related cardiac dysfunction (CTRCD). It is not currently possible to accurately identify patients at risk of CTRCD from clinical and demographic information alone. We sought to identify circulating biomarkers that can be used to identify individuals at risk of CTRCD before or early during cancer treatment.

Methods:
Multi-omic analysis of circulating biomarkers of cardiac damage, inflammation, and endothelial activation, together with profiling of plasma microRNAs was performed before and early during cancer treatment in 136 women ≥ 18 years of age with HER2+ breast cancer (Stages I-III) undergoing treatment with sequential anthracycline and trastuzumab therapy.

Results:
We identified three endothelial-centric biomarkers (i.e., Angiopoeitin-2, Endothelin-1 and Endoglin) that were elevated prior to and during cancer treatment, and one marker (i.e., E-Selectin) that was elevated during treatment in patients that went on to develop CTRCD. Additionally, there were significant elevations in inflammatory biomarkers (including Myeloperoxidase, Interferon gamma-induced protein-10 and Interferon-α2) before treatment in patients that developed CTRCD. No appreciable differences were observed in cardiac damage biomarkers (Troponin I, BNP, GDF-15) between groups. Assessment of plasma microRNAs prior to treatment revealed distinct microRNA signatures and predicted disease-relevant pathways in patients who went on to develop CTRCD. A Random Forest machine learning approach using all available baseline clinical, cardiac imaging and biomarker data revealed that pre-treatment Angiopoietin-2, Myeloperoxidase and Endoglin levels were the best predictors of CTRCD risk, and the model was further validated in an independent cohort.

Conclusion:
A combination of endothelial-centric and inflammatory biomarkers measured before treatment can accurately predict CTRCD during breast cancer therapy. Our findings suggest that sub-clinical vascular activation may predispose to cardiac dysfunction during cancer treatment, and that the vasculature may serve as a potential therapeutic target.
Abstract 1116: IL-33 Promotes Cardiovascular Lesion Development In A Murine Model Of Kawasaki Disease By Boosting IL-1β Production
Authors: Carvalho Thacyana T , Jena Prasant K , Aubuchon Emily A , Atici Asli E , Gomez Angela C , Fishbein Michael C , Crother Timothy R , Chen Shuang , Shimada Kenichi , Arditi Moshe , Noval Rivas Magali
Published: July 2024

Kawasaki Disease (KD), a pediatric acute febrile systemic vasculitis, is the leading cause of acquired heart diseases in children. Coronary artery aneurysms (CAAs) occur in up to 25% of untreated children, which is reduced to 5% with intravenous immunoglobulin (IVIG) treatment. However, up to 20% of KD patients are refractory to IVIG and at higher risk of developing CAAs. This highlights the need to characterize the immune mechanisms mediating KD to develop more efficient therapies. IL-1β plays a key role in KD pathogenesis. IL-33, a member of the IL-1 cytokine family, is released upon inflammation and tissue damage and exerts its effects by binding to its receptor ST2 (
Il1rl1
). Circulating levels of IL-33 are elevated in KD patients during the acute phase of the disease. However, if and how IL-33 contributes to cardiovascular lesion development in KD remains unknown. Using the
Lactobacillus casei
cell wall extract (LCWE) murine model of KD vasculitis, we observed increased
Il33
mRNA expression in hearts and abdominal aorta aneurysms of LCWE-injected mice. While ST2 was detected by immunofluorescence in the hearts of both control and LCWE-injected mice, its expression was increased in the abdominal aortas of LCWE-injected mice. In contrast, IL-33 expression was only increased in inflamed cardiovascular tissues of LCWE-injected mice. Single-cell RNA-sequencing and flow cytometric analysis of abdominal aortas, as well as spatial transcriptomics of heart tissues from LCWE-injected mice, indicated that
Il33
transcripts were expressed primarily by stromal cells, such as fibroblasts, endothelial cells, and vascular smooth muscle cells. On the other hand,
Il1rl1
transcripts were highly expressed by tissue infiltrating immune cells, such as macrophages, eosinophils, and neutrophils. Blocking IL-33, using either

Il33
-/-

mice or an anti-IL-33 antibody, significantly attenuated LCWE-induced KD vasculitis. In vitro, IL-33 treatment of LCWE-stimulated bone marrow-derived macrophages boosted their IL-1β production. Overall, our results indicate that IL-33, produced by stromal cells, may promote LCWE-induced KD vasculitis by increasing the release of IL-1β by tissue infiltrating immune cells, and this axis could therapeutically be targeted in KD.
Abstract 1119: Ultrafine Particle Inhalation Initiates Vascular Inflammation Via Hepatic-derived Xanthine Oxidoreductase-mediated Epigenetic Alterations
Authors: Kelley Eric E , McCallen Hailey , Nurkiewicz Timothy , Pilkerton Josilyn , Hussain Salik , Devallance Evan
Published: July 2024

Introduction:
Poor air quality has become a growing health concern. Ultrafine particles (UFP), a component of air pollution, has been associated with cardiovascular disease/ events, establishing a critical need to understand the cellular mechanisms involved. Using a model UFP we have shown elevated vascular oxidant production, and inflammation. However, the underlying mechanisms were unexplored. Xanthine oxidoreductase (XOR), increases in circulation following various insults, which we recently established is actively derived from hepatocytes. The binding of endothelial cells (EC) and the mechanisms by which EC bound XOR mediates impairment are not fully understood. Bound XOR produces almost exclusively H
2
O
2
, which can across the membrane and influence cellular signaling and gene expression. We
hypothesize
that UFP inhalation elevates hepatic XOR release into circulation and XOR binds to EC initiating redox signaling pathways leading to epigenetic-mediated inflammatory gene upregulation.

Methods & Results:
Male (M) and female (F) rats and mice (8 wk) were exposed to sham air or UFP for 3 days. Plasma XOR activity increased 5-fold M and 3-fold F exposed to UFP (both p<0.05). We then determined that UFP activation of PKC (rat liver 2.4-fold, mouse liver 2.3-fold, AML12 cells 1.7-fold, all p<0.05) leads to G9a-dependent upregulation and release of XOR in hepatocytes (Control 2± 0.3 vs PMA 8± 1 vs PMA G9a siRNA 2± 2 μU/ml, p<0.05). In arterioles, UFP arterioles showed greater XOR binding when perfused with Alexaflur tagged XOR (69±6 vs 51±4 AFU, p<0.05) and impaired dilation, which was prevented in hepatocyte XOR knockouts mice (KO) (max dilation sham 62± 3 vs UFP 35± 4 vs UFP KO 50± 4, p<0.05). In culture EC were treated with XOR for 1h (for binding) then washed with PBS and cultured for 24 h (XOR-EC). XOR-EC increased demethylase KDM4a expression (1.7-fold, p<0.05) and inflammatory markers ICAM, VCAM, TNF, and TLR4 (p<0.05), leading to enhanced leukocyte adhesion (~2-fold, p<0.05). KDM4a loss of function (iKDM4a) prevented ICAM expression (XOR-EC 8-fold vs iKDM4a 2-fold, p<0.05) and diminished leukocyte adhesion (p<0.05).

Conclusion:
These findings suggest that vascular inflammation caused by UFP inhalation is mediated in part by an EC bound XOR- KDM4a pathway.
Abstract 1129: Ddx24 In Vascular Smooth Muscle Cells Is Essential For Embryonic Vascular Development
Authors: Gong Yujiao , He Huanhuan
Published: July 2024

Background:
The DEAD-box family plays a crucial role in tumorigenesis and immune responses. Our previous research has identified an association between the dysfunction of DEAD-box RNA helicase 24 (DDX24) and a specific type of vascular malformation. However, the role and mechanism of DDX24 in vascular development remains unclear.Research questions: What is the role of DDX24 in regulating vascular smooth muscle cell (VSMC) function and embryonic vascular development? Aims: Our aim is to investigate the function and regulatory mechanism of DDX24 in embryonic vascular development.

Methods:
Cardiomyocyte/VSMC-specific
Ddx24
knockout mice were generated by crossing
Tagln
-Cre mice with

Ddx24
flox/flox

transgenic mice. Stereomicroscopic photography and immunofluorescence staining were used to examine the development of blood vessels. Flow cytometry and cell proliferation assays were employed to assess the effect of DDX24 on VSMC functions. RNA sequencing and RNA immunoprecipitation were performed to uncover the downstream target of DDX24. Additionally, RNA pull-down and RNA stability experiments were conducted to elucidate the regulatory mechanism.

Results:
Cardiomyocyte/VSMC-specific
Ddx24
knockout mice died before embryonic day 13.5 and exhibited defects in embryonic vascular formation and vascular remodeling in extraembryonic tissues. Ddx24 knockdown led to the suppression of VSMC proliferation through cell cycle arrest, likely due to increased DNA damage. DDX24 protein was found to bind to and stabilize the mRNA of FA complementation group A (
FANCA
), a gene that responds to DNA damage. Depletion of FANCA also affected cell cycle and DNA repair in VSMCs. Overexpression of FANCA successfully rescued the alterations induced by DDX24 deficiency.

Conclusion:
Our study illuminates the role of DDX24 in VSMC-mediated vascular development, suggesting a potential therapeutic target for diseases associated with VSMC dysfunction.
Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment
Authors: Coleman Bethany , Cady Tabitha , Erramilli Satchal , Kossiakoff Anthony , Davidson William
Published: July 2024

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.
Abstract 1154: Length And Proximal Extent Of Occlusion Dictates Severity Of Disease In A Mini-swine Model Of PAD
Authors: Hakim Ali H , Tian Yuqian , Al-Gahmi Al-Murtadha , Cartwright Sara , Salaymeh Jamal K , Kim Julian K , zhu zhen , Casale George , Pipinos Iraklis I , CARLSON MARK A
Published: July 2024

Background:
There is ongoing need for large animal models of peripheral artery disease (PAD) that replicate human disease. We present mild (MI), moderate (MD) and Severe (S) phenotypes of PAD in an Ossabaw mini-swine platform.

Methods:
Metabolic syndrome was induced in swine (N=16; age= 4 mo) with a high fat/fructose/sodium diet for 8 weeks. Ischemia induction (T0) consisted of: (1) open ligation and resection of R Superficial Femoral Artery (SFA) for MI(N=4); (2) endovascular coil occlusion of R External Iliac Artery (REIA), RSFA and popliteal artery for MD (N=9); and (3) coil occlusion of REIA, RSFA, and right internal iliac artery (RIIA) for S (N=4). At 4 weeks (T4) and 8 (T8), all swine underwent angiography via carotid access, and bilateral gastrocnemius biopsy. All swine had measurements of ankle brachial indices (ABIs), and calf muscle tissue oximetry (STO
2
) measurements at T0, T4 and T8, along with weekly measurement of walking performance on a treadmill (WP).

Results:
At T4 and T8 R ABI were increasingly decreased from MI to MD to S (table 1). Mean STO
2
difference between R and L calf at T4 and T8 had no changes in MI, while the differences were larger in S compared to MD (table 1). WP was increasingly decreased from MI to MD to S (table 1). The artery that grew the most on angiography at T4 and T8 were R profunda artery (RPFA) in MI; RIIA in MD; and LIIA in S (table 1). In MI, R PFA had faster flow at T4 and T8 compared to L PFA. At T4, MD and S had similar delays of RPFA reconstitution, this improved at T8 in MD but did not in S (table 1). On histology, MI had no obvious myopathy, MD and S had moderate and severe myopathy respectively. All S subjects developed poorly-healing ischemic ulcers.

Conclusion:
Length and proximal extent of occlusion dictated the characteristics and severity of phenotypes in our Ossabaw swine model of PAD. This mirrors PAD in humans and can be used to develop therapies for different disease severities.
Abstract 153: Multi-parametric Thrombus Profiling Microfluidics Uncovers Mechanobiology Of Hypertension And Aging-related Thrombosis
Authors: Din Misbahud , Paul Souvik , Ullah Sana , Yang Haoyi , Xu Rongguang , Abidin Nurul A , Chowdhury Bari , Zhou Fangyuan , Rogers Stephenie , Miller Mariel , Biswas Atreyee , Hu Liang , Fan Zhichao , Zahner Christopher , Berman Megan , Xue Lingzhou , Ju Lining , Chen Yunfeng
Published: July 2024

Arterial thrombosis, which represents a critical complication of cardiovascular diseases, is a leading cause of death and disability worldwide. A symbolic feature of arterial thrombosis is large-scale platelet aggregation due to high shear stress generated by stenosis. However, current hematological tests cannot evaluate arterial thrombosis in such physiological settings.

To bridge this gap, we developed a microfluidics-based thrombus profiling assay (Fig. 1A) that combines multi-fluorescence imaging with a stenotic channel design to comprehensively characterize thrombi formed in high shear blood flow (Fig. 1B, C). Thrombi are characterized by a 7-dimensional profile indicating the thrombus size (Plt) and levels of fibrinogen (Fg), VWF, P-selectin, phosphatidylserine (PS) and extended (E
+
) and activated α
IIb
β
3
in the thrombus (Fig. 1D).

Using this assay, we discovered intensified thrombus formation in subjects with hypertension and/or aging (Fig. 1D). We also found extended integrin α
IIb
β
3
as a superior biomarker for platelet hyperreactivity than P-selectin and activated integrin α
IIb
β
3
and a potential predictor for arterial thrombosis (Fig. 1E). By studying the effects of anti-thrombotic agents NMC4 and 7E3 on hypertension patients’ blood, our work reveals a ‘treatment mismatch’ phenomenon that can seriously affect the efficacy and safety of anti-thrombotic drugs (Fig. 1F).

Our findings provide mechanistic insights into the high risk of arterial thrombosis and antiplatelet resistance in populations with hypertension and aging. The discovered ‘treatment mismatch’ heralds a new era in thrombosis management where patients’ thrombus profiles dictate personalized therapeutic decisions to optimize treatment efficacy and reduce adverse outcomes. The outstanding performance of our assay underscores its translational potential for anti-thrombotic drug screening, thrombosis diagnosis, and personalized anti-thrombotic regimen selection.
Abstract 2006: Estrogen Therapy Is Associated With A Greater Risk Of Cardiovascular-kidney-metabolic Syndrome And Thrombosis In Cisgender Women, But Spares Transgender Women: A Global Retrospective Study
Authors: Manchkanti Srija , Ramakrishna Akhila , Streeter Jennifer , Dayal Sanjana
Published: July 2024

Despite the use of estrogen-based therapies in cisgender- and transgender-women, and the presumed cardioprotective effect of endogenous estrogen, there are no comprehensive studies investigating the risk of cardiovascular-kidney-metabolic (CKM) syndrome in patients taking exogenous estrogen. CKM syndrome is a health disorder attributable to connections among obesity, type 2 diabetes mellitus (T2DM), chronic kidney disease (CKD), and cardiovascular disease, including heart failure (HF), ischemic heart disease (IHD), and stroke. We hypothesized that cis- and trans-women would have decreased risk of CKM syndrome. We conducted a retrospective analysis using the TriNetX database, which contains patient information from 83 healthcare organizations worldwide. We identified 9,271,702 cis-women +/- estrogen and 17,729 trans-women on estrogen matched 1:1 with cis-men and separated patients into age deciles from 20-69 years. Cis-women taking estrogen showed a significantly increased risk of CKM comorbidities including dyslipidemia, HTN, T2DM, overweight/obesity (OB), HF, IHD, and CKD (p<0.0001 for all), and an elevated risk of thrombotic outcomes, such as myocardial infarction (MI), stroke, pulmonary embolism (PE), and deep vein thrombosis (DVT) compared to cis-women not taking estrogen (p<0.0001 for all). In contrast, the use of estrogen in trans-women compared to cis-men was not associated with a greater risk of MI, stroke, PE, or DVT, (except in the 60-69 year-aged cohort for DVT, p=0.0056). Trans-women taking estrogen also had a lower risk of HTN, T2DM, HF, IHD, and CKD, despite a higher risk of dyslipidemia, and OB compared to cis-men (p=0.02-<0.0001). This is the first comprehensive study revealing a significant association between exogenous estrogen use and CKM syndrome in cis-women. Additionally, our novel findings indicate that estrogen use in trans-women is associated with dyslipidemia, and OB, but is not associated with a greater overall risk for CKM syndrome or thrombosis. Given these findings, additional prospective studies are needed to confirm the risks or protections provided by estrogen therapy in cisgender and transgender women.
Abstract 2011: Development Of Machine Learning Model To Combine Clinical And Imaging Data For Prediction Of Endoleaks: Proof-of-concept Study
Authors: Kiang Sharon C , Roh Daniel , Dardik Alan , Tomihama Roger T
Published: July 2024

Objective:
To develop a foundational ensemble machine learning (ML) classifier to train on computed tomography angiography (CTA) scans and clinical data to predict the occurrence of endoleaks after endovascular aneurysm repair (EVAR) of the abdominal aorta.

Methods:
An ensemble supervised ML model was developed by using the Convolutional Neural Network (CNN) classifier to transform imaging data into a vector that can be analyzed by a supervised ML model. To test this model, we applied CTA scans from 14 patients with an endoleak after infra-renal EVAR and 14 propensity-matched patients without an endoleak. Sixty DICOM images in batches of 3 were selected from each patient for a total of 20 batches per patient. These batches were assessed by the CNN model to output a singular representative vector for the respective patient (n=28). A supervised ML model was then trained on these cases using Leave-One-Out Cross Validation. To account for variability based on the random state, a for-loop was used to iterate through random states 0 to 49 and the average accuracies and AUC were taken from these 50 iterations. The clinical data was then standardized using a MinMaxScaler and appended to the representative vectors so that both the clinical and imaging data could be used as input for the supervised ML model. The ensemble model was assessed by testing accuracy and the area under the receiver operating characteristic curve (AUROC) and compared to the best ML model alone and imaging alone predictions for validation.

Results:
Using the clinical data alone, ML accuracy and AUROC performed with and accuracy of 57% and AUROC curve of 0.57. Using the imaging data alone, the final RFC model reported an accuracy of 59% and an AUROC curve of 0.59. Combining the imaging and clinical data, the final RFC model reported an accuracy of 60% and an area under the receiver operating curve of 0.60.

Conclusions:
Preliminary data from these models have shown proof-of-concept that a deep learning model such as a CNN can be combined with a supervised machine learning model to combine CTA imaging and clinical data to predict complications from an endovascular intervention. These models can serve as a proof of concept for future studies analyzing endovascular cases with larger datasets.
Abstract 2020: Vascular Glucose Transporter 1 Is Dispensable For Abdominal Aortic Aneurysm Induced By Angiotensin II
Authors: Nakayama Yuki , Torimoto Keiichi , Vari Hymavathi R , Eguchi Satoru
Published: July 2024

Background:
Pharmacological inhibition of glycolysis attenuated abdominal aortic aneurysm (AAA) development in mice. Glucose transporters (GLUTs) incorporate glucose as a first step of glycolysis. GLUT1 over-expression in vascular smooth muscle cells (VSMC) leads to enhancement of atherosclerosis. However, the role of VSMC GLUT1 in AAA remains unclear.

Hypothesis:
VSMC GLUT1 deletion attenuates AAA development and rupture in mice.

Goals/Aims:
Our goal was to establish contribution of VSMC GLUT1 in AAA.

Methods:
The glucose consumption of cultured VSMCs was evaluated in the presence or absence of AngII stimulation and GLUT1 inhibitor BAY-876. GLUT1 floxed mice were crossed with tamoxifen-inducible SMMHC-Cre mice. At 6 weeks of age, mice were injected with tamoxifen. As a control, tamoxifen-treated SMMHC-Cre male mice were utilized. At 8 weeks, angiotensin II (AngII, 1 μg/kg/min) was delivered for 4 weeks. In addition, mice received 1 mg/mL BAPN during the first 2 weeks of AngII infusion. After 4weeks, all animals were euthanized and maximal external diameter of the abdominal aorta was measured.

Results:
In cultured VSMC, the glucose consumption increased by AngII was attenuated by pretreatment with GLUT1 inhibitor (media glucose 257.9±4.5 vs 238.9±6.2 mg/dL, p<0.05). AngII plus BAPN significantly developed AAA in control mice (diameter 1.05±0.07 vs 2.37±0.31mm, p<0.05). However, silencing of VSMC GLUT1 did not affect AAA development by AngII plus BAPN (2.41±0.46mm, p=0.66). The survival rate due to aortic rupture in VSMC GLUT1 silenced mice and control mice treated with BAPN and AngII were 60.0% and 57.1%, respectively (p=0.820). AngII induced hypertension in control mice (102±3.8 vs 133±7.1mmHg, p<0.05) and VSMC GLUT1 silenced mice (149±11.4mmHg, p=0.99). Discussion Although GLUT1 may be a primary VSMC GLUT to enhance glucose utilization in response to AngII stimulation, VSMC GLUT1 silencing did not alter AngII-dependent AAA development or rupture in a mouse model. Since pharmacological inhibition of GLUT attenuated AAA and associated macrophage infiltration, it is possible that GLUT1 expressed in macrophages may contribute to development of AAA.

Conclusion:
VSMC GLUT1 silencing did not alter AngII-dependent AAA development in a mouse model.
Abstract 2031: Supramolecularly Fabricated Non-thrombogenic Small Diameter Vascular Graft
Authors: Mehrotra Nitisha , Adumbumkulath Aparna , Kim Paul , Somawardana Suran , Mhatre Kirti , Mattar Aladdein , Elgalad Abdelmotagaly , Salas Nilson , Gilani Ramyar , Shin Crystal , Acharya Ghanashyam , Ajayan Pulickel M.
Published: July 2024

Background:
Small diameter (<6mm) arterial reconstruction often requires the use of autologous and synthetic vascular substitutes. While autologous conduits are the gold standard, because of their limited availability synthetic conduits are often used. However, synthetic conduits can lead to poor outcomes due to thrombosis and lumen occlusion occluding the lumen and result in graft failure. By modulating the biopolymer properties of the synthetic conduits, graft failure can be prevented. In this study, we present a thromboresistant synthetic vascular graft (zetagraft) fabricated with tunable biopolymers for small diameter arterial reconstructions.

Methods:
Zetagrafts were fabricated by the solvent extraction driven self-assembly using a concentric tubular assembly device using polyvinyl alcohol dissolved in dimethyl sulfoxide (15% w/v). We characterized the physicochemical and mechanical properties of zetagraft by fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), optical and scanning electron microscopy, and tensiometry. In-vitro studies were performed on the zetagraft using fibroblast, HUVEC and MVEC cell lines. Ex vivo hemocompatibility analysis was performed with fresh porcine blood. The zetagrafts were implanted in a rat aorta replacement model to evaluate its thromboresistance.

Results:
The zetagraft was uniform, highly stretchable, and self-healing when punctured with a needle. Blood circulation followed by FT-IR and XRD analysis showed no protein adsorption on the luminal surface. Immuno fluorescence staining revealed the absence of platelet and blood cell adsorption on the zetagraft lumen, demonstrating its thromboresistance. In vivo implantation of the zetagraft in rat abdominal aorta replacement model revealed the absence of thrombosis and other blood component adsorption on the zetagraft lumen.

Conclusions:
Advances in material science allow for fabrication of more tunable biocompatible small vascular grafts. In this study we demonstrated the development of a thromboresistant zetagraft. Our early results are very promising and zetagraft will provide a novel small vascular graft and thus fulfil an important unmet clinical need.
Abstract 2050: Slc43a3 And Intracellular Fatty Acid Trafficking
Authors: shen wen-jun , Zhang YiQiang , Qiu Shuo , Deng Alicia , Hasbargen Kathrin B , Bernlohr David A , azhar salman , Tsao Phillip , Kraemer Fredric B
Published: July 2024

Background:
Fatty acids (FAs) have important roles in many cellular functions. Dysregulation of FA flux is both a characteristic and effector of many prevalent clinical conditions, including diabetes mellitus, obesity as well as insulin resistance and/or metabolic syndrome. Adipocytes take up long chain fatty acids through diffusion and protein mediated transport, whereas fatty acid efflux is considered to occur by diffusion. Our previous study suggests the possibility of a protein facilitated/regulated process of FA efflux by demonstrating that the organic anion transport inhibitor DIDS strongly inhibits FA efflux in lipolytically-stimulated adipocytes, leading to an accumulation of intracellular FAs, while not affecting glycerol efflux. We
hypothesize
that there is potential regulator(s) for FA efflux.

Methods and Results:
In a screen of subcutaneous adipose samples from obese patients for expression levels of transporters without known function, Slc43a3 (a member of the solute carrier transporter family) shown increased expression after bariatric surgery. In addition, Slc43a3 regulates fatty acid flux in adipocytes. To study the mechanism of Slc43a3 function, clonal lines of preadipocyte OP9 cells were generated, in which green fluorescent protein has been knocked-in at the C-terminus of endogenous Slc43a3 using CRISPR/Cas9. In parallel, Slc43a3 knockout (KO) line has also been generated. Examination of the cells showed that expression of Slc43a3 induced in the cytoplasm of the adipocytes during differentiation. Slc43a3KO adipocytes accumulate bigger lipid droplets and less small lipid droplets. To study the functional significance of Slc43a3 in vivo, adipose tissue specific KO of Slc43a3 were generated with floxed Slc43a3 and tamoxifen induced adiponectin-Cre recombinase. The Slc43a3 KO mice have less body weight and less % body fat on a normal chow diet; however, no differences are noted when the mice are switched to high fat diet, indicating the lack of lipid signaling molecules in the Slc43a3 KO mice with normal chow diet. Functional analysis shows a blunted stimulated lipolysis in the Slc43a3 KO mice.

Conclusions:
Both in vitro and in vivo data confirmed an unique role of Slc43a3 in regulating fatty acid flux in adipose tissue.
Abstract 2057: Serine Hydroxymethyltransferase 2 In Hepatocytes Regulates One-carbon Metabolism, Circulating VLDL And Atherosclerosis
Authors: McKinney Mary P , Ghrayeb Alia , Finney Alexandra , ANAND SUMIT K , Rohilla Sumati , Das Sandeep , Pearson-gallion Brenna , Tan Lin , Lorenzi Philip , Orr Wayne W , Yurdagul Arif , Mor Inbal , Gottlieb Eyal , Rom Oren
Published: July 2024

While the role of lipids in atherosclerotic cardiovascular disease (ASCVD) is well studied, the contribution of amino acids is becoming increasingly recognized. We and others reported that the ratio of glycine to serine is reduced in association with ASCVD severity. Primarily expressed in the liver, serine hydroxymethyltransferase 2 (SHMT2) is thought to be responsible for the bulk of glycine biosynthesis from serine. We recently reported that reversed SHMT2 activity in fatty livers drives glycine depletion (
Ghrayeb et al. Cell Metab 2024
). Nevertheless, whether the regulation of glycine/serine metabolism by hepatic SHMT2 affects atherosclerosis remains unknown. Utilizing novel hepatocyte-specific
Shmt2
knockout mice (
Shmt2
HKO
) crossed with apolipoprotein E-deficient (
Apoe
-/-
) mice, metabolomics, and transcriptomics, we studied the role of hepatocyte SHMT2 in atherosclerosis. We found a significant increase in glycine and the glycine:serine ratio in plasma from
Shmt2
HKO
/
Apoe
-/-
mice compered to
Shmt2
flox
/
Apoe
-/-
mice fed a Western diet. In livers from
Shmt2
HKO
/
Apoe
-/-
mice, decreased S-adenosylmethionine (SAM) and increased adenosylhomocysteine (SAH) resulted in reduced SAM:SAH ratio. While hepatic steatosis was unaltered,
Shmt2
HKO
/
Apoe
-/-
mice had lower phosphatidylcholine (PC) and PC:phosphatidylethanolamine (PE) ratio, key regulators of hepatic VLDL assembly and secretion. Accordingly, FPLC demonstrated reduced triglyceride and cholesterol contents in VLDL from
Shmt2
HKO
/
Apoe
-/-
mice. Moreover, RNA-seq and qPCR analyses revealed a downregulation of proinflammatory mediators (
Tnf, Ir1b, Ccr5
) and an upregulation of fatty acid β-oxidation regulators (
Acadm
,
Acadl
) in livers from
Shmt2
HKO
/
Apoe
-/-
mice. Finally, histological analysis of aortic sinuses revealed lower plaque size in female
Shmt2
HKO
/
Apoe
-/-
mice. These studies uncover hepatic SHMT2 as a regulator of one-carbon and VLDL metabolism in atherosclerosis.
Abstract 2060: Hv1 Contributes To Atherosclerosis
Authors: Zhang Lu , Wu Xin , Liu Jianhua , Cao Xinyu , Hong Liang
Published: July 2024

Atherosclerosis is a complex process that involves the interaction between oxidized low-density lipoprotein (oxLDL) and macrophage. It is an inflammatory disease of the large artery driven by macrophage activation and infiltration. Through scavenger receptors, macrophages can uptake oxLDL and other lipids, leading to foam cell formation and the progression of atherosclerosis. Hv1 proton channel is highly expressed in phagocytes and promotes NADPH oxidase-derived ROS (reactive oxygen species) production. So far, most of the research on Hv1 has been focused on immune, reproductive, and neurological diseases. However, the role of Hv1 in cardiovascular diseases, such as arteriosclerosis, remains unclear. To explore the potential roles of Hv1 in atherosclerosis, we detected the expression of Hv1 in atherosclerotic lesions. It was shown that Hv1 is abundantly expressed in human and murine atherosclerotic lesions and highly expressed in macrophages in the aortic root lesions. In addition, Hv1 knockout mice fed with a high-fat western diet exhibit remarkably decreased atherosclerotic plaques compared with control mice. Moreover, deficiency of Hv1 in macrophages impedes macrophage phagocytosis and foam cell formation essential for atherosclerosis development, and Hv1 enhances macrophage ROS production associated with the progression of atherosclerosis. Our results suggested that Hv1 is a pro-atherosclerotic factor and contributes to the pathogenesis of atherosclerosis. The research identified a new function of the Hv1 channel in human diseases and may present a novel target against atherosclerosis and atherosclerosis-related diseases.
Abstract 2073: StarD4 Is Required For Efficient Clearance Of Extracellular Free Cholesterol Generated From Digestive Exophagy Of Aggregated Low-density Lipoprotein.
Authors: Ma Cheng-I J , Wang Weixiang A , Maxfield Frederick R
Published: July 2024

In the development of atherosclerotic plaques, macrophages infiltrate the intima of the aortic wall to digest modified lipoproteins. Over time, these macrophages become overloaded with lipids and become plaque-associated foam cells. Most modified lipoproteins are aggregated and cross-linked to the extracellular matrix of the vessel wall, which prevents them from being phagocytosed by macrophages. Our lab has described how macrophages catabolize aggregated LDL extracellularly in a process termed digestive exophagy. Macrophage forms a tightly sealed, acidified extracellular compartment around agLDL that is stabilized by F-actin. Lysosomes are secreted into this extracellular compartment, allowing lysosomal acid lipase to catabolize cholesteryl esters in the core of agLDL to free cholesterol. However, it remains unclear how the extracellular free cholesterol is transported into macrophages for lipid droplet formation. Here, we identify the cholesterol transport protein StarD4 as a major player in the intracellular movement of free cholesterol released by digestive exophagy. We show that StarD4 knockout bone marrow-derived macrophages have delayed onset in foam cell formation when encountering agLDL. However, no difference is observed when StarD4 knockout macrophages are treated with monomeric acetylated LDL, suggesting the effect of StarD4 is specific to cholesterol delivered to the plasma membrane. Moreover, loss of StarD4 does not alter the rate of digestive exophagy, and no difference in lysosome exocytosis is observed between StarD4 knockout and wildtype macrophages. Therefore, our results indicate that StarD4 mediated plasma membrane to ER cholesterol transport is a critical step in digestive exophagy of agLDL. Loss of StarD4 can delay the onset of foam cell formation, which may provide insight into novel therapeutic strategies for atherosclerosis.
Abstract 2077: The Epigenetic Regulator
PRDM16
Controls Smooth Muscle Phenotypic Modulation And Atherosclerosis Risk
Authors: Palmisano Brian T , Nguyen Trieu , Zhao Quanyi , Worssam Matthew , Monteiro Joao P , Li Daniel , Weldy Chad S , Kundu Ramendra K , Gu Wenduo , Cheng Paul , Knowles Josh , Quertermous Thomas
Published: July 2024

Despite decades of progress, coronary artery disease (CAD) remains the top cause of death worldwide. Additionally, trends in outcomes have worsened recently, highlighting the critical need for additional treatments. Human genetics has identified over 300 loci associated with CAD, but understanding the molecular mechanisms leading to disease remains a huge barrier to developing new therapies. These CAD-associated loci are enriched in smooth muscle cells (SMC) of the vascular wall, but no current therapies target these cells. Since insulin resistance is an important risk factor for CAD, understanding the molecular functions of genes associated with both insulin resistance and CAD will prioritize therapeutic candidates, especially if expressed in SMCs. One promising candidate is the gene
PRDM16
(PR domain containing 16), which is highly expressed in vascular tissue. PRDM16 regulates cell fate decisions and insulin resistance in adipose tissue, but its role in atherosclerosis and SMC function is unknown. Recent advances in lineage tracing and conditional SMC knockout mouse models in conjunction with single cell technologies have demonstrated the cellular trajectories of SMC into several cellular states, including fibromyocytes (FMC) and chondromyocytes (CMC). Here, we employ this innovative approach to understand the role of
Prdm16
on SMC phenotypic modulation in vivo and its role in the development of FMC and CMC. We also demonstrate the impact of Prdm16 in atherosclerosis and other important lesion characteristics relating to disease risk. We validate these effects in vitro and employ epigenetic analysis to identify the gene regulatory mechanisms whereby
PRDM16
mediates its effects on SMC phenotypic modulation. Collectively these data demonstrate that
PRDM16
is a causal factor that promotes risk of CAD.
Abstract 2079: Single-cell And Spatially Resolved Transcriptome Analysis Reveals A Novel Lncrna Regulator Of Human Atherosclerosis
Authors: Pauli Jessica , Schanda Sara , Wu Zhiyuan , Chernogubova Ekaterina - , Ritzer Julia , Meunier Sandro , Dueck Anne , Fasolo Francesca , Yokota Chika , Wirth Johannes , Sachs Nadja , Winter Hanna , Jin Hong , Steiger Katja , Engelhardt Stefan , Eckstein Hans-Henning , Maegdefessel Lars
Published: July 2024

Cardiovascular diseases, such as atherosclerosis, are the major cause of death in Western societies. Still, molecular mechanisms of plaque destabilization remain unclear. Vascular Smooth Muscle Cells (VSMCs) are the most abundant cell type in atherosclerotic lesions. These cells keep their plasticity also with disease progression and are, therefore, an ideal target cell type for potential therapies. Long non-coding RNAs (lncRNAs) are one example of highly cell type- and disease-state-specific novel molecular modulators. We utilized combined total (bulk) RNA and single cell (sc) RNA sequencing of advanced human carotid artery lesions (patients who underwent carotid endarterectomy in our vascular surgery clinic) to discover novel regulators. Additionally, we performed hybridization-based RNA
in situ
sequencing (HybRISS, Xenium) to indicate where these genes are located within the plaques. We also performed scSeq in our inducible carotid artery plaque rupture mice (
ApoE-/-
).
In vivo
and
in vitro
modulation (KD; knock-down) of the newly identified lncRNA
DLX6-AS1
(
D
istal-
L
ess Homeobo
x
6
A
nti
s
ense
1
) using Antisense LNA (Locked nucleic acid) GapmeRs is performed in this mouse model as well as in primary human VSMC culture.
DLX6-AS1
shows a highly specific expression pattern in a distinct sub-cluster of VSMCs, as well as a striking location within human atherosclerotic lesions shown by spatial transcriptomics in advanced plaques.
DLX6-AS1
is mainly expressed in the nucleus of cells, and KD of this lncRNA leads to changes in VSMC behavior
in vitro
and
in vivo
. The KD also has only little effect on the other genes of this locus,
DLX6
and
DLX5
(
D
istal-
L
ess Homeobo
x
6
and
5
) We found
Dlx6os1
(
D
istal-
l
ess homeobo
x
6,
o
pposite
s
trand
1
) in our scRNA-seq dataset of the inducible carotid artery plaque rupture mice (
ApoE-/-
) where the KD leads to an increased plaque rupture rate. Taken together, our datasets, methods, and animal models demonstrate that utilizing bulk- and scRNA-seq data, together with spatially resolved sequencing methods, are powerful tools to identify and characterize novel lncRNA regulators that can facilitate fate decisions in VSMCs in atherosclerosis.
DLX6-AS1
is one of these novel regulators and will be further analyzed
in vivo
and
in vitro
.
Abstract 2094: Sex Differences In Venous Thrombus Resolution In Mice With Metabolic Syndrome
Authors: Nguyen Khanh P , Nguyen Kevin , Setthavongsack Naly , Ngu Daphne , Vo Anthony , Faik Sarah , Woltjer Randall , Hinds Monica T , Loftis Jennifer , Maloyan Alina
Published: July 2024

Introduction:
Metabolic syndrome (MS) is highly prevalent and have been shown to be associated with mitochondrial impairment and increased cardiovascular and thrombotic risk. It is less understood how MS impacts venous thrombosis and thrombus resolution and the effect of sex as a biological variable on these processes. We hypothesize that mice with MS will have impaired thrombus resolution, particularly males compared to females.

Methods:
We used a model of diet-independent MS where wildtype FVB mice that have been born to high fat diet fed mothers exhibit multiple organ manifestations of MS. 29 adult mice underwent surgical induction of deep vein thrombosis (DVT) via inferior vena cava ligation (stasis model) resulting in thrombi formation until day 3 and thrombus resolution until day 7. Immunohistochemistry was used to assess vascular tissue remodeling.

Results:
Comparing control mice to mice born to obese mothers after DVT, there were no significant differences in thrombus weights on day 3 (1.83±.62 vs 1.48±.53 g/kg,
p
=.186), suggesting a similar initial thrombus burden. However, mice born to obese mothers had significantly impaired thrombus resolution at day 7 (1.85±.53 vs 1.14±.42 g/kg, Student’s t-test
p
=.007) as compared to control mice born from mothers fed a regular diet. Importantly, thrombus weights were significantly larger in
male
offspring of obese mothers as compared to
females
(2.17±.67 vs 1.43±.26 g/kg, Student’s t test
p
=.013). Additionally, histology analyses showed associations with increased vein wall collagen deposition (Masson’s trichrome) (25±3 vs 13±4% area stained, Student’s t test,
p
=.0018) and neovascularization (smooth muscle cell actin) within the thrombi of mice with signs of MS as compared to control mice (70+/-10 vs 40+/-8% area stained, Student’s t test,
p
=.009).

Conclusions:
Wildtype mice with diet-independent MS demonstrate diminished thrombus resolution, with males having significantly larger thrombi than females. Impaired DVT resolution was associated with decreased tissue remodeling within the thrombi. These data suggest that MS may induce negative tissue remodeling decreasing thrombus involution and increasing vein wall fibrosis, factors that may increase the likelihood of post thrombotic syndrome.