bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1152: Hydrodissection Facilitates Endoscopic Vein Harvesting Of The Great Saphenous Vein For Coronary Artery Bypass Grafting
Authors: Gallagher John F , Gallagher Pamela M , Escaravage Steven , Masciello Kaitlin , Gallagher Paul J , Garcia Victor , Fusco Dan
Published: July 2024

Coronary Artery Disease is the primary cause of mortality in the United States (US), ranking as the third leading cause of death globally. Coronary Artery Bypass Grafting (CABG) with left internal mammary artery to the left anterior descending artery is the gold standard for CABG with a patency rate of 90% at 15 years. The great saphenous vein (GSV) harvested by endoscopic vein harvesting (EVH) is the second most common vascular conduit used for CABG in the US. This is despite the poor long-term patency of this conduit due to vein graft failure (VGF). VGF is the number one technical problem associated with CABG, with 15-year patency rates of only 30 to 50%. While the "no touch" harvesting technique avoids VGF, with 15-year patency rates of 86%, it is technically demanding and results in increased leg wound healing, and not widely used. With this in mind, we developed the Minimally Invasive No Touch (MINT) technique that incorporates the best features of EVH and no touch. This procedure employs hydro dissection, a process that utilizes fluid pressure to gently harvest the GSV with minimal trauma. This approach effectively addresses two significant issues associated with using the GSV in CABG, namely VGF resulting from impaired endothelial function and excessive trauma to the GSV during the extended learning curve needed to master EVH. To evaluate this, harvesting was conducted by a PA, with over 2,000 standard EVH procedures. Our preliminary studies looked to evaluate the quality of GSVs harvested from patients using 1) standard EVH, 2) “no touch” harvesting techniques or 3) the MINT harvesting procedure, focused on evaluating the quality of the vessels by measuring the endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) bioavailability. eNOS activity assays across harvested GSVs revealed a 3.4-fold increase in NO generation across MINT harvested samples compared to EVH and “no touch” samples, respectively. This data suggests that the GSVs harvested by the MINT procedure result in a structurally intact vein with elevated eNOS activity and NO bioavailability consistent with a functionally intact endothelium. Additional studies are necessary to uncover the array of vascular benefits associated with MINT GSV harvesting for CABG procedures.
Abstract 125: The X Chromosome Gene
Kdm6a
Confers Sex Differences In Mature Adipocyte Function Impacting Cardiometabolic Health
Authors: Wiese Carrie B , Agle Zoey , Vergnes Laurent , Reue Karen
Published: July 2024

Biological sex impacts cardiovascular disease and other metabolic conditions that increase disease risk, including obesity. Physiological sex differences are determined by two components of sex: gonadal hormones and sex chromosome complement. Using mouse models to dissociate gonadal sex from chromosomal sex, we have identified sex chromosome-dependent regulation of circulating lipoproteins, atherosclerosis, and adiposity. Specifically for adiposity, mice with two X chromosomes had higher body weight gain compared to mice with XY sex chromosomes in the presence of ovaries or testes. In cells with XX chromosome complement, specific X chromosome genes escape X inactivation, which leads to higher expression in XX (female) compared to XY (male) tissues. We identified an X escape gene,
Kdm6a
, that encodes a histone demethylase enzyme and impacts adiposity by altering the function of mature adipocytes. Mice with mature adipocyte-specific
Kdm6a
reduction (Ad-
Kdm6a
-/-
) had lower body weight and adiposity compared to wildtype females. The white adipose triglyceride pool was altered with higher unsaturated and long chain fatty acids in Ad-
Kdm6a
-/-
mice compared to wildtype mice. Furthermore, siRNA knockdown of
Kdm6a
in cultured adipocytes altered lipid metabolism with reduced lipid droplet size. Given that KDM6A is a histone demethylase, we assessed the
Kdm6a
dosage effect on adipocyte gene expression and histone modifications across the genome. Differential gene expression associated with lipid homeostasis was identified in Ad-
Kdm6a
-/-
mice and in cultured adipocytes with
Kdm6a
knockdown compared to controls. KDM6A genomic targets and resultant histone modifications were identified in cultured adipocytes using CUT&RUN. We conclude that differential gene dosage of
Kdm6a
between female and male tissues is a determinant of adipose sex differences through modulation of histone modifications and gene expression. This leads to effects on adipocyte lipid composition and tissue mass, which impacts susceptibility to obesity and metabolic disease.
Abstract 127: Pushing The Limits Of Macrophage Efferocytosis In Atherosclerosis
Authors: Wu Xun , Li Fang , Shern Tyler P , Tall Alan , Tabas Ira , Zhang Hanrui
Published: July 2024

Introduction:
Enhancing macrophage efferocytosis represents a potential therapeutic strategy for residual risk reduction in atherosclerotic cardiovascular disease. Our genome-wide CRISPR screening discovered
Pdcd6ip
as a top-ranked negative regulator of efferocytosis. PDCD6IP, also known as ALIX, is an accessory protein of ESCRT machinery, regulating multivesicular body formation and cytokinesis. Yet, how PDCD6IP may act as a molecular break on macrophage efferocytosis has not been characterized.

Goals:
Determine if knockout of
Pdcd6ip
enhances macrophage efferocytosis and protects against atherosclerosis pathogenesis.

Methods:
In vitro
efferocytosis assays were performed in bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (HMDM).
In vivo
studies involved

Ldlr
-/-

recipient mice with bone marrow transplantation (BMT) of WT and

Pdcd6ip
-/-

BMs.

Results:

Pdcd6ip
-/-

induces cytokinesis arrest in proliferating BMDMs, resulting in an increased formation of binuclear polyploid BMDMs. Binuclear BMDMs have a remarkable capacity to engulf multiple apoptotic cells (ACs), termed continuing efferocytosis. While both mononuclear and binuclear

Pdcd6ip
-/-

BMDMs demonstrate enhanced efferocytosis in primary efferocytosis, binuclear

Pdcd6ip
-/-

BMDMs show enhanced continuing efferocytosis compared to binuclear WT BMDMs.

Pdcd6ip
-/-

BMDMs have increased phagolysosomal acidification of the engulfed ACs, implicating proper degradation.

Pdcd6ip
-/-

did not alter the engulfment of live cells, beads, or zymosan, supporting specific regulation of AC clearance. We hypothesize that

Pdcd6ip
-/-

will be protective in atherosclerosis. Indeed, BMT of

Pdcd6ip
-/-

BMs into

Ldlr
-/-

mice led to increased atherosclerotic plaque stability after 22 weeks of Western diet feeding, characterized by decreased necrotic core area and increased fibrous cap thickness, without affecting the overall lesion area. Knockdown of PDCD6IP in HMDMs also led to an increased formation of binuclear HMDMs and enhanced continuing efferocytosis.

Conclusion:
We discovered a new role of PDCD6IP as a molecular break of macrophage efferocytosis. Inhibiting macrophage PDCD6IP may represent a therapeutic opportunity in atherosclerosis.
Abstract 133: Trem2 Agonist Promotes Atherosclerotic Plaque Stability
Authors: Patterson Michael , Xu Yingzheng , Hillman Hannah , Osinski Victoria , Schrank Patricia , Kennedy Ainsley , Barrow Fanta , Tollison Samuel , Shekhar Sia , Revelo Xavier , Binstadt Bryce , Williams Jesse W
Published: July 2024

Atherosclerosis is an inflammatory disease of the arteries that is a major cause of cardiovascular disease (CVD). Atherosclerotic plaque progression is driven by the deposition of low-density lipoprotein (LDL) within the intima, which is internalized by macrophages, forming lipid-engorged foamy cells. Importantly, the accumulation of excess LDL within foamy macrophages is cytotoxic, driving apoptosis, which ultimately promotes necrotic core formation and increased potential for plaque rupture. Recently, our lab identified the lipid receptor Trem2 to be highly expressed by foamy macrophages and regulate cell survival, proliferation, and cholesterol efflux. Thus, we tested whether agonizing Trem2 could drive macrophage survival to promote plaque stability. Using a Trem2 agonist antibody (AL002a, Alector Inc.), we treated Ldlr
-/-
mice that had preexisting atherosclerotic plaque with either isotype control or AL002a antibody for 8 weeks. AL002a treated mice showed an increase in plaque size in the aortic arch and aortic sinus. Plaque analysis determined that this was primarily due to an expansion of macrophage cellularity, through promotion of cell survival and proliferation. Furthermore, we found that plaques from AL002a treated mice had decreased necrotic core formation, increased fibrous cap size, and increased collagen deposition, consistent with improved stability. Single cell RNA-sequencing of atherosclerotic aortae from isotype and AL002a treated mice found that Trem2-agonism dramatically reshaped transcriptomes of both foamy macrophages and smooth muscle cells, including upregulation of oxidative phosphorylation (OXPHOS), cholesterol efflux, collagen, and pro-survival genes while downregulating inflammatory pathways. In vitro studies using bone marrow derived macrophages confirmed AL002a’s ability to drive cell survival, proliferation, OXPHOS, and cholesterol efflux in culture. Finally, we determined that AL002a’s ability to reshape aspects of foamy macrophage function was dependent on its ability to activate the kinase SYK downstream of Trem2. Overall, this data identifies Trem2 as a viable therapeutic target for promoting atherosclerotic plaque stability and improving outcomes in CVD.
Abstract 134: Lesion-specific Suppression Of YAP/TAZ By Biomimetic Nanoparticles Ameliorates Atherosclerosis
Authors: Wang Ting Yun , HUANG HUI-CHUN , Rousseau Joshua , Wang Mark Kuei-Chun K
Published: July 2024

Introduction:
Recent evidence highlights the potential therapeutic benefit of targeting YAP/TAZ in mitigating atherosclerosis. However, challenges associated with systemic delivery hinder the translation of the mechanistic discovery into an effective treatment. This study aimed to address the challenges by developing a lesion-specific nanodrug to achieve site-specific inhibition of YAP/TAZ for atherosclerosis therapy.

Hypothesis:
We have formulated a novel monocyte membrane-coated nanoparticle (MoNP) platform designed to facilitate precise delivery of payloads to atherosclerotic arteries. Our hypothesis is that leveraging MoNP for the targeted delivery of verteporfin (VP), a potent YAP/TAZ inhibitor, will significantly enhance the efficacy in mitigating atherosclerosis.

Methods:
Polymeric nanoparticles containing VP were prepared using an emulsion approach, followed by enclosing them with plasma membranes isolated from monocytes, yielding MoNP-VP. The pharmacological effects of MoNP-VP were assessed on cultured vascular and immune cells, as well as in the atherosclerotic mouse model.

Results:
Our
in vivo
findings demonstrated that the administration of MoNP-VP, as opposed to free VP, led to a substantial reduction in arterial inflammation and plaque development, suggesting that VP delivered by MoNP is a more effective route than its free drug counterpart. To dissect the mechanisms of actions, we analyzed the arterial tissues post-treatment using scRNA-seq analysis. Our results identified 9 distinct cell clusters within the affected arteries. Specifically, MoNP-VP treatment led to a decrease in macrophage (MΦ) clusters and an increase in the cluster of smooth muscle cells (SMC). Furthermore, our
in vitro
assays confirmed the efficacies of MoNP-VP in reducing the atheroprone phenotypes. These include diminished endothelial cell activation, less proliferation and migration of SMC, and a reduction in foam cell formation from MΦ.

Conclusion:
Together, these findings support the notion that targeting YAP/TAZ dysregulation is a feasible strategy for reducing plaque development and progression and demonstrate that MoNP-VP is a highly effective nanodrug for treating atherosclerosis.
Abstract 138: Platelet RIPK1 Promotes Platelet Activation And Aggregation In Arteriolar Thrombosis And Ischemic Stroke
Authors: Kim Jae-Sung , Cho Jaehyung , Brown Gavriel
Published: July 2024

Receptor-interacting protein Ser/Thr kinase 1 (RIPK1) is a molecular switch for inflammation and cell death in nucleated cells, and its function is controlled by phosphorylation at the Ser166 residue. However, it is unknown whether RIPK1 regulates platelet function in arterial thrombosis. Intravital microscopy using megakaryocyte-specific Ripk1 conditional knockout (CKO) mice (
Ripk1
fl/fl;Pf4-cre
) revealed that platelet Ripk1 promoted platelet thrombus formation at sites of laser-induced cremaster arteriolar thrombosis. Deletion of platelet Ripk1 prolonged the time to occlusion in FeCl
3
-induced carotid arterial thrombosis without affecting tail bleeding times. Further, compared to wild-type (WT) mice, Ripk1 CKO mice exhibited improved neurological deficits and reduced infarct volume in ischemic stroke induced by transient middle cerebral artery occlusion. Flow cytometry and aggregation assays showed that deletion of platelet Ripk1 reduced P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by thrombin, collagen-related peptides (CRP), and U46619, but not ADP. Pretreatment of Ripk1-null platelets with ADP rescued the aggregation defect. Treatment of platelets with Nec-1s, a RIPK1 inhibitor that blocks RIPK1-Ser166 phosphorylation, did not affect platelet activation and aggregation. Instead, RIPK1-Ser320/321 was rapidly phosphorylated in thrombin- or CRP-stimulated human and mouse platelets. Treatment of platelets with an inhibitor of phospholipase C, p38, or MAPK-activated protein kinase 2 (MK2), but not a protein kinase C inhibitor, decreased RIPK1-Ser320/321 phosphorylation after thrombin or CRP stimulation, implying that the p38-MK2 axis phosphorylates RIPK1-Ser320/321. Using a genetic approach in human megakaryoblastic MEG-01 cells, we found that RIPK1 knockdown reduced granule secretion from stimulated MEG-01 cells, which was rescued by overexpressing WT RIPK1, but not mutant RIPK1-Ser320Ala, in the RIPK1 knockdown MEG-01 cells. These results suggest that platelet RIPK1 enhances granule secretion during platelet activation through p38-MK2-mediated phosphorylation of RIPK1-Ser320/321, thereby contributing to the pathology of thrombotic disease.
Abstract 140: Exploring Protein Kinase C Theta (PKCθ) Signaling In The Generation Of Procoagulant Platelet: Insights From Phosphoproteomics And Systems Analysis
Authors: Zheng Yiheng , Reitsma Stephanie , Jordan Sandler , Babur Ozgun , Melrose Alexander , pang jiaqing , Hodapp Stefanie , Newton Alexandra , Wilmarth Phillip , Reddy Ashok , McCarty Owen J , Aslan Joseph E
Published: July 2024

Background:
Platelet activation plays a pivotal role in physiological hemostasis and thrombosis. Protein Kinase C theta (PKCθ) isoforms exert significant regulatory influence on platelet activation. While specific PKC family members have well-defined roles in platelet function, the roles of PKCθ remain less explored.

Objectives:
To determine mechanisms of PKCθ signaling that orchestrate procoagulant platelet generation.

Methods:
Platelets were prepared from healthy human donors for
ex vivo
experiments. Platelet signaling and function were studied following stimulation with GPVI agonist crosslinked collagen-related peptide (CRP-XL). To investigate PKCθ inhibition mechanisms in platelets, small molecule inhibitors targeting the PKCθ (CC-90005 and C20) were employed.

Results:
From phosphoproteomics, we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKCα/β, PKCθ, and PKCΔ activities determine PKCθ Ser685 phosphorylation. From the PKC Inhibitors Activity Curves, we noted a robust and selective inhibitory impact of CC-90005 on PKCθ, with specificity surpassing that of C20. Parallel platelet function analyses revealed that inhibiting PKCθ had minimal impact on platelet spreading and aggregation. However, PKCθ inhibition in the presence of Ca
2+
upregulated platelet phosphatidylserine (PS) exposure, as measured by flow cytometry. This was accompanied by an increase in platelet procoagulant activity, where PKCθ inhibition reduced the lag time for fibrin formation. Furthermore, fluorescence microscopy also found that PKCθ inhibition significantly augmented the proportion of procoagulant platelets and microparticles. Additionally, compared to the control group, the use of cyclosporin A (CsA) reduced the production of procoagulant platelets, but the addition of CC-90005 or C20 rescued this phenomenon, with signal intensity surpassing that of the control group.

Conclusions:
Our study proposes a model wherein PKCΔ and PKCα/β activities induce the phosphorylation of PKCθ at S685, influencing mitochondrial calcium signaling and supporting procoagulant platelet generation.
Abstract 2004: Sex Differences In Bile Acid Metabolism Impact Cholesterol Homeostasis And Cardiovascular Disease
Authors: Jarrett Kelsey E , Schmidt Heidi , Chan Alvin , Sholto Madelaine C , Cheng Angela , Steel Michelle , Taveras Maria , Tarling Elizabeth J , Vallim Thomas A
Published: July 2024

Cardiovascular disease (CVD) remains the leading cause of death in the United States for both men and women, despite cholesterol-lowering drugs such as statins. Diets rich in cholesterol are well-known to contribute to disease progression. The absorption of dietary cholesterol is facilitated by liver-synthesized detergents called bile acids. We hypothesized that manipulating bile acid metabolism to reduce cholesterol absorption would result in decreased plasma cholesterol levels and protect from atherosclerosis.

To alter the bile acid pool, we used liver-directed AAV-CRISPR to disrupt
Cyp7a1,
which catalyzes the rate-limiting step of bile acid synthesis. Consequently, we show that loss of CYP7A1 in adult male mice reduces the size of the bile acid pool. To study atherosclerosis, we co-disrupted
Cyp7a1
concurrently with
Low Density Lipoprotein Receptor
(
Ldlr
) to induce hypercholesterolemia on a Western diet for 20 weeks. Loss of hepatic CYP7A1 in males reduced atherosclerosis and reduced cholesterol absorption.

In stark contrast to what we observed in male mice, the absence of this rate limiting enzyme in female mice resulted in only a modest reduction in the bile acid pool. This blunted reduction in bile acids in female
Cyp7a1
CRISPR mice was not sufficient to decrease cholesterol absorption, resulting in accumulation of cholesterol, and consequently increasing cardiovascular risk. We hypothesize that there is a female-specific mechanism to preserve bile acid production that operates in the absence of CYP7A1. We are currently in the process of testing candidate female-specific enzymes for bile acid production.

These studies indicate new avenues by which female bile acid and cholesterol metabolism regulation differs from than that of males. The resulting differences in cardiovascular risk highlight the essential need for male- and female-specific personalized medicine approaches for cardiovascular disease prevention.
Abstract 2017: Interleukin-10 Hydrogel-Based Therapy For Abdominal Aortic Aneurysm In Rodent Model
Authors: Madison Mackenzie K , Doiron Theresa S , Stashevsky Jennifer , Chen Stone , Jimenez Olivia , Aridi Hanaa , Basu Rohan , Gil Chang-Hyun , Miller Steven J , Murphy Michael P
Published: July 2024

Abdominal aortic aneurysms (AAA) are diagnosed in over 200,000 patients per year in the United States. Endovascular AAA repair offers a strategy for those unable to undergo open operation; however there still exists a treatment gap for those whose anatomy is not suitable for current stent graft options. In response to this need for novel therapeutics to prevent aneurysm proliferation, the objective of this work was to expand AAA treatment options via anti-inflammatory hydrogel application with a handheld bioprinting device. Induction of AAA in 8-week-old, male, Sprague-Dawley rats was achieved via intraluminal infusion of porcine elastase and topical application of calcium chloride to the infrarenal aorta. Following a 4-week incubation period, rats were treated with IL-10 loaded hydrogel or blank control hydrogel. The IL-10 loaded hydrogel was placed concentrically around the aorta from the lowest renal artery to the aortic bifurcation. Rats were sacrificed 4 weeks post-treatment. Histologic analysis included hematoxylin and eosin, picrosirius red, and Verhoeff-Van Gieson for inflammatory cell infiltration, collagen and elastin content, respectively. Three-dimensional aneurysm reconstruction and analysis was performed using SkyScan1076 micro-CT at baseline, post-induction/pre-treatment and sacrifice. Immunohistochemistry (IHC) was used to examine the presence of macrophage infiltration (ED1/CD68) as a marker of inflammation. Three-dimensional reconstruction demonstrated appropriate aneurysm formation in both groups and successful aneurysm mitigation in the treatment group only. Post-mortem histologic analysis showed irregularity of the aortic intima, fibrosis, abnormal collagen organization and dramatically decreased elastin concentration in control rodents while treated rats demonstrated preservation of aortic architecture. IHC demonstrated a significant increase in macrophage infiltration in the control group when compared to treated. Taken together, the results demonstrate that peri-aortic application of an IL-10 loaded hydrogel mitigates AAA expansion in a rat model. This novel approach may be a potential strategy for treating patients with AAA whose anatomy is unsuitable for endovascular intervention.
Abstract 2024: Elucidating The Role Of Shape In Aortic Disease State Pathology With Machine Learning
Authors: Pugar Joseph , Khabaz Kameel , Jiang David , Pocivavsek Luka
Published: July 2024

Maximum aortic diameter has been the mainstay metric for aortic disease management and surgical intervention for decades, specifically acute aortic dissection and aneurysm. While this size metric has been effective at separating patient populations into groupings associated with required intervention and monitoring for eventual intervention respectively, size alone ignores the role of complex geometric shape and how it may be further indicative of disease state. Analyses of aortic shape in addition to size have been discussed at length in literature, however the representations and methods to convey shape are siloed and often highly subjective. Machine learning methodologies offer a means to explore high dimensional feature spaces and can identify a sparse set of metrics which result in a division of patient responses parametrized by physical and interpretable variables. Additionally, by introducing a modeling workflow which contains engineered parameters designed to describe aortic shape, in addition to traditional size metrics, the exploration of shape dependence on intervention outcomes becomes more comprehensive across size-shape parameter spaces. The engineered parameters presented primarily incorporate estimations of integrated Gaussian surface curvature into features that contain both the magnitude and spatial variation in shape of the aortas. The models discussed accurately classify over two hundred aortic disease patients spanning aneurysm, dissection, and other modalities with accuracies above 90% and performance across multiple modeling methodologies is discussed. The classification success of the geometrically informed feature space suggests the underlying dependence on pathology shape on the success of the intervention outcome. The most clinically effective models will preserve the wealth of information in modern medical diagnostics and imaging into descriptive, and physically interpretable, parameters to aid in clinical decision making.
Abstract 2036: Prognostic Impact Of Pfkfb3 Expression In Endothelial Cells Insights From Peripheral Arterial Disease Patients And The Effects Of Supervised Exercise
Authors: Nofal Mohamed , Alhusban Suhib , Green Thomas D , Terwilliger Zoe , Li Feifei , karnekar Reema , Yamaguchi Dean , McClung Joseph , Annex Brian H
Published: July 2024

Background:
Glycolysis constitutes 85% of ATP production in endothelial cells (ECs). Although upregulating PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3), the rate-controlling enzyme for EC glycolysis, has been considered a mean to induce angiogenesis, our group found this approach ineffective or even harmful in pre-clinical peripheral arterial disease (PAD) models. However, no EC specific PFKFB3 data in human PAD patients exists.

Hypothesis:
EC PFKFB3 upregulation is associated with poor prognosis in PAD patients.

Goal:
Assess PFKFB3 in ECs and correlate its’ expression with PAD clinical presentation and outcome.

Methods:
CD31 (EC marker) and PFKFB3 immunostaining was performed on age-matched gastrocnemius (GA) muscle sections from healthy adult control (HA), PAD patients with intermittent claudication (IC; patients experience pain during activity), and patients with chronic limb threatening ischemia (CLTI; the most severe presentation of PAD -patients experience pain at rest with increased risk of ulcers, gangrene, and amputation). A similar approach was used on GA muscle sections from PAD patients before and after 3 weeks of supervised exercise. qPCR was done on flow sorted live CD56-CD31+ EC population from HA and CLTI GA muscle biopsies, for CD31 and PFKFB3 expression.

Results:
The IC presentation did not increase the %PFKFB3
+
ECs (17.08 ± 1.19 vs. 14.40 ± 2.32 SEM, n=6-7/group, p= 0.69) from HA. However, in CLTI tissues, the %PFKFB3
+
ECs increased vs both HA (42.01 ± 4.32 SEM vs. 14.40 ± 2.327 SEM, n=7/group, p= 0.0025) and IC (42.01 ± 4.32 SEM vs. 17.08 ± 1.19 SEM, n=6-7/group, p= 0.01). Furthermore, qPCR analysis of PFKFB3 expression in flow sorted CD56-CD31+ EC fraction from CLTI vs. HA (1.855 ± 0.084 SEM vs. 1 ± 0.1768 SEM, n=4/group, P= 0.0052). Three weeks of supervised exercise in PAD patients, with tolerance for activity, decreased the %PFKFB3
+
ECs compared vs. pre-exercise values (7.02 ± 1.74 SEM vs. 18.24 ± 2.15 SEM, n= 4 /group, p= 0.0077) respectively.

Conclusion:
In humans with PAD, higher EC expression of PFKFB3
+
uniquely associated with CLTI, but not the IC presentation. Supervised exercise, the only non-surgical approved method to improve functional performance and angiogenesis in PAD patients, reduced EC PFKFB3 expression.
Abstract 2062: Engineering Extracellular Matrix Cues To Study The Progression Of Endothelial To Mesenchymal Transition
Authors: Jain Ishita , Huang Ngan F
Published: July 2024

Endothelial-mesenchymal transition (EndoMT) is a biological process in which vascular endothelial cells (ECs) acquire a mesenchymal identity that give rise to lineages like fibroblasts and smooth muscle-like cells. Pathologically, EndoMT is involved in the progression of cardiovascular diseases such as atherosclerosis, in which ECs give rise to smooth muscle-like cells within the plaque. However, there is essentially a dearth of knowledge of how the biochemical and biomechanical cues from the extracellular matrix (ECM) milieu directly influence the progression of EndoMT. To address the limited knowledge of ECM effects on EndoMT, we aim to develop a 3D tissue engineered model with independently tunable biochemical and biomechanical cues, including stiffness, stress relaxation rate and ECM composition to study EndoMT progression in 3D. Alginate hydrogels were prepared at 2% w/v final concentration and crosslinked using calcium sulfate and different concentrations. Human primary coronary artery endothelial cells (HCAECs, Lonza) were formed into spheroids and then co-encapuslated with fibroblasts in the alginate hydrogels for 7 days. EndoMT was induced by using media with no supplements such as VEGF and bFGF (that inhibit EndoMT) and using TGFb treatment. We successfully fabricated a family of alginate hydrogels with different mechanical and viscoelastic properties. HCAEC spheroids survived well in the 3D alginate hydrogel system and actively interacted with the fibroblasts in the hydrogels. Additionally, we observed viscoelasticity dependent migration of ECs from the spheroid into the alginate hydrogel and vessel like tube formation in the 3D model. Lastly, we quantified higher expression of the mesenchymal marker, SM22, in ECs in slow relaxing hydrogels, pointing towards viscoelasticity dependent progression of EndoMT. These results underscore the importance of mechanical factors such viscoelasticity in addition to stiffness in progression of diseases like atherosclerosis. Future work involves performing transcriptomics analysis to uncover the mechanism behind viscoelasticity dependent EndoMT progression.
Abstract 2078: Integrin α5β1-mediated Interactions With Fibronectin Enhance Tim3 Expression To Drive Efferocytosis By Macrophages
Authors: Pandit Rajan , Wilhite Peyton , Kumar Dhananjay , Governale Theresea-Anne , Pearson Brenna , Cockerham Elizabeth , Orr Wayne A , Yurdagul Arif
Published: July 2024

Despite significant advances in diagnosing and treating cardiovascular disease, atherosclerosis remains the leading cause of death worldwide. While most atherosclerotic plaques remain asymptomatic, a subset can lead to myocardial infarction, stroke, or sudden death. A plethora of evidence has demonstrated that rupture-prone atheromas contains a large necrotic core owed to defective efferocytosis, the process by which apoptotic cells (ACs) are engulfed and cleared by macrophages. The efficient clearance of apoptotic cells blocks post-apoptotic necrosis and prevents the release of tissue-degrading enzymes, immunogenic epitopes, and proinflammatory mediators. As atherosclerosis advances, substantial remodeling of the extracellular matrix (ECM) occurs whereby transitional ECM proteins, notably fibronectin (FN), deposit into the subendothelial matrix normally dominated by collagen IV and laminins. However, whether macrophage interactions with the ECM alters efferocytosis, inflammation resolution, and atherosclerosis remains unknown. Surprisingly, we discovered that macrophage interactions with FN enhance efferocytosis compared to their interactions with basement membrane proteins. Furthermore, we demonstrate that the primary FN receptor, integrin α5β1, is required for FN-mediated efferocytosis and TIM3 expression, (encoded by
Havcr2
), a known phosphatidylserine receptor that binds to ACs. Moreover, treating mice with established atherosclerosis with the integrin α5β1 antagonistic peptide ATN-161 reduced TIM3 expression in macrophages and expanded necrotic core formation. We also found lesional macrophages from human unstable atheromas displayed significantly lower TIM3 expression compared to macrophages within stable plaques. In a mouse model of atherosclerosis regression, where atherogenic stimuli dissipate, efferocytosis is restored, and FN remains, lesional macrophages showed a substantial increase in TIM3 expression. Thus, while FN is traditionally considered atherogenic in endothelial and smooth muscle cells in the initial stages of atherosclerosis, our data uncovers that macrophage adhesion to FN upregulates TIM3 expression, mediating efferocytosis and promoting features of plaque stability.
Abstract 2151: VascularRegulationbySuper Enhancer-DerivedLINC00607
Authors: Luo Yingjun
Published: July 2024

Vascular endothelial cells (ECs) play a pivotal role in whole-body homeostasis. Recent advances have revealed enhancer-associated long non-coding RNAs (lncRNAs) as essential regulators in EC function. We investigated LINC00607, a super enhancer-derived lncRNA (SE-lncRNA) in human arteries with an emphasis on ECs. Based on public databases and our single-cell RNA-sequencing (scRNA-seq) data from human arteries collected from healthy and diabetic donors, we found that LINC00607 is abundantly expressed in the arteries and its level is increased in diabetic patients. Furthermore, through transcriptomic and promoter analysis, we identified c-Myc as an upstream transcription factor of LINC00607 in diabetic conditions. Interestingly, we found that N6-methyladenosine modification also contributes to the induced abundance in diabetic patients. Using RNA sequencing, we characterized the transcriptomes regulated by LINC00607 in ECs and vascular smooth muscle cells (VSMCs) and in basal and diabetic conditions in ECs. Finally, using scRNA-seq, we demonstrated that the modified antisense oligonucleotide inhibitor of LINC00607 can reverse dysfunctional changes induced by high glucose and TNF alpha in ECs. Collectively, our study demonstrates a multi-pronged approach to characterize LINC00607 in vascular cells and its gene regulatory networks in ECs and VSMCs. Our findings provide new insights into the regulation and function of SE-derived lncRNAs in both vascular homeostasis and dysfunction in a cell-type and context-dependent manner, which could have a significant impact on our understanding of epigenetic regulation implicated in cardiovascular health and diseases like diabetes.
Abstract 3007: The Role Of Myeloperoxidase In The Development Of Abdominal Aortic Aneurysm
Authors: Ku Jacqueline , Bello Idris , Glaros Elias , Hung Tzong-Tyng , Kavurma Mary M , Rye Kerry Anne , Witting Paul , Thomas Shane R
Published: July 2024

Background:
Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disorder characterized by inflammation in the aortic media, extracellular matrix degradation, aortic dilation and rupture. Increasing evidence identifies a role for oxidative stress, although the oxidative reactions involved requires clarification. Myeloperoxidase (MPO) is a pro-inflammatory neutrophil-derived enzyme that promotes cardiovascular disease by catalyzing tissue damaging oxidative reactions. Here we investigated the impact of MPO pharmacological inhibition and gene deletion on AAA. For pharmacological inhibition, the clinically trialed 2-thioxanthine class of irreversible MPO inhibitors was employed.

Methods:
Apolipoprotein E (ApoE
-/-
) or ApoE
-/-
/MPO
-/-
gene-knockout mice were infused with angiotensin II (AngII) by osmotic mini-pumps for 28 days and received 2-thioxanthine or vehicle via daily oral gavage. AAA development was monitored by micro-ultrasound measurement of aortic diameter and post-mortem measurement of maximal aortic width at the supra-renal aorta. Atherosclerosis was measured at the aortic sinus by morphometry and aortic arch by
en face
analysis of oil-red-O-stained lipids. Cardiac fibrosis was measured by picrosirius red staining.

Results:
AngII infusion afforded an AAA incidence rate of 60% in ApoE
-/-
mice that was reduced to 25% by 2-thioxanthine treatment. 2-Thioxanthines also inhibited the AngII-mediated increase in aortic diameter and cardiac fibrosis, but not the AngII-induced accelerated development of atherosclerosis at the aortic sinus and arch. Paradoxically, studies in ApoE
-/-
/MPO
-/-
mice established that MPO gene-deletion did not affect AngII-induced AAA but significantly attenuated the increase in aortic arch atherosclerosis. Notably, 2-thioxanthines significantly inhibited AAA incidence and severity in AngII-infused ApoE
-/-
/MPO
-/-
mice.

Conclusion:
These studies highlight the complexity surrounding the role of MPO in a murine model of AAA and atherosclerosis. Importantly, this work establishes that 2-thioxanthines can protect against AAA in the absence of MPO, supporting that this clinically trialed MPO inhibitor drug class can target other heme peroxidases
in vivo
.
Abstract 3030: Age-sex Differences In The Global Burden Of Stroke Attributable To Particulate Matter Air Pollution In G20 Countries From 1990-2019: A Benchmarking Analysis
Authors: Vemparala Priyatha , Patel Dhruval , Vashi Mandeepsinh , Savaliya Prashantkumar , Singh Kushagrita , Reyaz Ibrahim , MADHU KRUPA , Lakkimsetti Mohit , PATEL JUHI , AMIN VISHRANT P , DESAI HARDIK D
Published: July 2024

Introduction:
Stroke stands as the primary contributor to both fatalities and disability worldwide, accounting for 12.68% of deaths within G20 nations. Among the various modifiable risk factors for stroke, air pollution emerges as a prominent culprit, particularly due to the escalating levels of industrial and automotive emissions on a global scale.

Method:
Using Global Burden of Disease tool, we analyzed deaths, disability-adjusted life years (DALYs), and years lived with disability (YLDs), attributable to particulate matter pollution. This pollution encompasses ambient particulate matter and household air pollution arising from solid fuels. Our analysis was stratified by age, gender, year, and location across G20 Countries, spanning the period from 1990 to 2019.

Result:
From 1990 to 2019, the total number of deaths saw an average annual increase of 17% (95%UI: 1-36%), and DALYs rose by 11% (3-26%). China experienced the highest increase in deaths (353%), followed by India (349%) and Turkey (208%), while Estonia saw a decrease. In 2019, Saudi Arabia had the highest mortality rate at 31.3 per 100,000, with Bulgaria at 24.33. The 70-74 age group had the highest deaths from household air pollution (44,241), and the most deaths from ambient air pollution (132,188). The 65-69 age group saw the highest DALYs due to household (1,077,344) and ambient particulate matter pollution (3,083,347). In 2019, strokes linked to household air pollution were higher in females, while males were more impacted by ambient particulate matter pollution.

Conclusion:
This study underscores the pervasive threat posed by stroke on a global scale and emphasizes the significant role of air pollution, in exacerbating this crisis. The observed upward trend in stroke-related deaths and DALYs over the past three decades calls for immediate action in implementing pollution control measures and public health interventions to mitigate the growing burden of stroke.
Abstract 3031: Novel Therapeutic Approach In Diabetic Wound Healing Using Inducible Mesenchymal Stem Cell-derived Fibroblasts In A Unique Murine Model
Authors: Voza Francesca A , Shao Hongwei , Ortiz Yuli , Le Nga , Li Yan , Ozafo Lucy , Liu Zhao-Jun , Velazquez Omaida C
Published: July 2024

Introduction:
It is estimated that about 40 to 60 million people in the United States suffer from diabetic foot ulcers (DFUs), one of the most severe DM complications. A significant proportion of affected individuals undergo amputation and are exposed to high mortality risks. Stem cell-based therapies offer a promising therapeutic avenue for these patients. Fibroblasts hold a crucial role in wound healing and angiogenesis, and we further highlighted that activated Notch signaling in fibroblasts derived from diabetic wounds leads to dysregulated fibroblast function. In this study, the potential therapeutic outcomes from genetically modified fibroblasts derived from mesenchymal stem cells (MSC-DF) in treating DFU is assessed, utilizing a unique genetic mouse model.

Methods:
A fibroblast-specific and inducible diabetic murine model with triple-gene engineering was created via inducible intracellular Notch1 pathway activation (RosaN1IC/Col1a2-Cre/ERT/Leprdb). MSC-DFs harvested from 10-12 week old
B6db/
ROSAN1I/
/
Cola2-Cre/ERT (LLRRCc)
donor mice were cultured
in vitro
with activation versus silencing of Notch 1 signaling. Recipient
LLRRCc
mice (8-12-week-old) underwent a 6mm excisional dorsal skin wound and subsequent local injection of 1x10^6 donor syngeneic activated or silenced GOF
Notch1
-MSC-DFs (n= 5/group). Wound closure was monitored daily for 9 consecutive days post-injury, and tissue was obtained for histological sectioning.

Results:
GOF
Notch1
-MSC-DFs were characterized by immunostaining based on expression of markers specific to fibroblasts and a panel of Notch signaling genes. LLRRCc murine subjects receiving silenced GOF
Notch1
-MSC-DFs treatment (n=5) demonstrated faster rates of wound closure over activated GOF
Notch1
-MSC-DFs (n=5) treated mice. Collagen deposition was more abundant in wounds treated with silenced GOF
Notch1
-MSC-DFs.

Conclusion:
MSC-DF deactivated Notch1 signaling therapy improves the rate of wound healing in GOF Notch Diabetic mice. These promising outcomes demonstrate a potential role for this innovative stem cell-based therapy targeting Notch-1-modified MSC-DF in future clinical applications for DFU patients through restoration of impaired fibroblast function.
Abstract 3058: Cofilin 1 Mediates Fatty Acid-induced Foam Cell Formation And Inflammation In Mouse Bone Marrow-derived Macrophages
Authors: Poles Jillian , Arivazhagan Lakshmi , Kumar Vikas , Schmidt Ann Marie M
Published: July 2024

Background:
Macrophages contribute to the development of atherosclerosis, in part through the accumulation of lipid droplets and subsequent transformation into highly inflammatory, lipid laden foam cells. Our previous work suggested that the RAGE/DIAPH1 axis plays a key role in regulating lipid metabolism, as deletion of
Diaph1
in hepatocytes reduced nuclear translocation of SREBP1 through the actin cytoskeleton. Cofilin 1 (CFL1) is an actin polymerization molecule which has also been linked to the nuclear translocation of SRBP1, as well as triglyceride synthesis through activation of phospholipase D1. In the aforementioned study by our group,
Diaph1
deletion was found to increase the Serine 3 phosphorylation (i.e., inactivation) of
Cfl1
.

Hypothesis:
Accordingly, we hypothesized that CFL1 plays a role in lipid droplet accumulation in macrophages, potentially increasing the risk of atherosclerotic development.

Methods:
To investigate its role in macrophage lipid handling, we silenced
Cfl1
in bone marrow derived macrophages (BMDMs) extracted from male C57BL/6J mice, using silencing RNAs and scramble controls. BMDMs were treated with a mixture of fatty acids palmitate and oleate (250 uM). The effects of
Cfl1
knockdown on lipid accumulation were assessed using immunohistochemical staining (Bodipy). Quantitative (q)PCR was used to interrogate the mechanisms underlying these effects, and their impact on inflammation.

Results:
Cfl1-
silenced (75% knockdown shown by qPCR) vs. scramble cells exhibited a significant decrease in the ratio of Bodipy to DAPI % area (p=0.0001). By qPCR, compared to scramble controls, BMDMs subjected to silencing of
Cfl1
and incubated with fatty acids (palmitate-oleate) displayed significantly lower mRNA expression of
Cd36
(p=0.0018)
; Ppargc1a
(p=0.004)
,
Il6
(p<0.0001), and
Tnfa
(p=0.013).

Conclusions:
Our results indicate that silencing of
Cfl1
leads to a decrease in lipid accumulation and markers of inflammation in BMDMs. Collectively, our data suggest that the effects of
Cfl1
silencing on lipid handling result from decreased fatty acid uptake, which limits fatty acid oxidation on account of fatty acid availability in the cell. Studies are underway to probe the implications of these findings in atherosclerosis.
Abstract 3067: Chronic Knockout Of The ETS Transcription Factor ERG Promotes Loss Of Endothelial Cell Identity And Endothelial Cell Dysfunction In An Aortic Endothelial Cell Model Relevant For Atherosclerosis
Authors: Schulz Kristen , Botts Steven R , Ellis Kai , Khyzha Nadiya , Kumaragurubaran Rathnakumar , Wilson Michael , Howe Kathryn L , Fish Jason E
Published: July 2024

Background:
The ETS transcription factor ERG is a key endothelial cell (EC) transcription factor. ERG represses vascular inflammation and is downregulated in chronic inflammatory diseases (e.g. liver fibrogenesis, atherosclerosis). Previously, transient ERG knock-down has been associated with endothelial-mesenchymal transition (EndMT) and EC dysfunction; however, the mechanisms underlying EndMT and the EC dysfunction associated with chronic ERG loss have not been elucidated in an atherosclerosis-relevant model.
We hypothesize that chronic ERG knockout (KO) in telomerase-immortalized human aortic ECs (teloHAECs) induces EndMT and contributes to EC dysfunction underlying vascular dysregulation in atherosclerosis.

Methods:
TeloHAECs with CRIPSR/Cas9-mediated deletion of
ERG
were used. Chromatin accessibility and gene expression in ERG KO and wildtype (WT) teloHAECs were assessed using ATAC-seq (n=2) and RNA-seq (n=5-6). TOBIAS and diffTF were used to identify enriched motifs by footprinting (P<0.05) and to profile TF activity (FDR<0.05), respectively. ERG-mediated EC dysfunction was investigated using immunofluorescence (IF; n=3), western blot (WB; n=3), and a timelapse migration assay (n=3).

Results:
There were ~ 21,000 differentially accessible chromatin peaks between
ERG
KO and WT ECs. Integrated analysis of chromatin accessibility and gene expression demonstrated increased activity of TGFβ-SMAD and IL-1β signaling constituents and classical EndMT transcription factors. Loss of ERG downregulated EC genes (e.g.,
CLDN5
,
TIE1
) and protein expression (e.g., CDH5, vWF) and upregulated mesenchymal genes (e.g.,
ACTA2
,
FSP1
) and protein distribution (e.g., FN1, COL1A1). Investigation of EC-dysfunction phenotypes revealed increased migration and proliferation in ERG KO teloHAEC - with the percentage of Ki67 positive ECs increasing from 14% to 49% (P<0.05, n=3).

Conclusion:
ERG KO in cultured aortic ECs markedly impacts chromatin accessibility and gene expression and induces EC identity loss, migration, and proliferation, providing mechanistic insight into the role of ERG in vascular dysfunction in atherosclerosis.
Abstract 3070: Investigating CD36 Oligomerization In Macrophages
Authors: Tillison Emma , Sahoo Daisy
Published: July 2024

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Atherosclerosis, the main cause of CVD, results from the buildup of cholesterol-filled plaques in artery walls. Accumulation of cholesterol from oxidized low-density lipoprotein (oxLDL) leads to the formation of macrophage foam cells, a process that is mediated by CD36, a scavenger receptor that serves as the primary receptor for oxLDL. The structure of full-length CD36 has not been resolved, nor have the details or role of quaternary structure been fully elucidated. Studies have shown that CD36 has the ability to form homodimers and multimers in some cell types. However, the role of CD36 oligomerization in oxLDL uptake remains unknown. The goal of this study is to investigate CD36 oligomerization in macrophages, a cell type important in the initiation of atherosclerosis. We hypothesize that CD36 oligomerizes in macrophages to facilitate oxLDL uptake. To begin testing this hypothesis, we harvested elicited peritoneal macrophages from wild-type (WT) or CD36 knockout (CD36-KO) mice. We performed crosslinking experiments in which macrophages were incubated with BS3, a membrane-impermeable crosslinker, to stabilize CD36 oligomers. When crosslinked samples were separated by SDS-PAGE electrophoresis, we observed a decrease in monomeric species with increasing crosslinker concentration, however, BS3 also induced aggregation of CD36 rather than oligomer formation. We next turned to Native-PAGE electrophoresis, which lacks detergent entirely, to identify oligomeric species within macrophages, as well as PFO-PAGE electrophoresis, which contains a milder detergent than SDS. Both methods provided evidence of higher-order complexes in WT, but not in CD36-KO macrophages. To further validate our studies, we purified human full-length CD36 using an Sf9 insect cell system and demonstrated that pure CD36 forms homo-oligomers by PFO-PAGE. Together, these experiments provide strong evidence for CD36 oligomerization. Future studies will focus on correlating CD36 oligomerization with its functions in oxLDL binding and uptake. Understanding the higher-order structure of CD36 and how it impacts function will allow us to target CD36 to prevent foam cell formation and atherosclerosis.