bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2110: Fasting-refeeding Dynamics Induce Distinct Vascular Responses In Lean And Obese Mice
Authors: Graham Elliot L , Risk Briana , Zhang Mingyue , Weir Tiffany , Gentile Christopher
Published: July 2024

Background:
Intermittent fasting (IF) can improve arterial stiffness, a predictor of cardiovascular disease. However, it is still unclear how many bouts of IF are needed to improve arterial stiffness, and once improvements occur, how long after the fasting period are they maintained. Thus, the primary purpose of this study was to determine how long changes in arterial stiffness are maintained after IF and how many IF cycles are required to alter arterial stiffness in lean and obese animals.

Methods:
Wild-type (WT) and genetically obese (Ob) mice were randomized to an 11-week protocol to receive either ad libitum control diet (Ad-Lib) or a once weekly 24 hr fast (IF), yielding 4 groups: WT+Ad-Lib; WT+IF; Ob+Ad-Lib; and Ob+IF. Body weight was measured weekly while arterial pulse wave velocity (aPWV) was measured at baseline and at least 24 hrs after fasting at mid- (weeks 5-6), and endpoints (weeks 9-10). To test the persistence of de-stiffening after one week of IF, IF groups were further split only during week 1 of the protocol such that body weight and aPWV were measured immediately after the first 24 hr fast, 1 day after refeeding, or 7 days after refeeding.

Results:
In WT mice, body weight was reduced by the 24 hr fast (p<0.01) but rebounded after a day of refeeding (p=0.22) when compared to baseline. Interestingly, aPWV was increased after the 24 hr fast (p=0.02) but was restored back to baseline levels within 24 hrs and did not change thereafter. In Ob animals, no significant differences in body weight were found between baseline and any timepoint during the first week. However, compared to baseline, Ob+IF mice had reduced body weight (p<0.01) and aPWV (p<0.01) at the midpoint, and modestly reduced aPWV at the endpoint (p=0.06).

Conclusions:
A single 24 hr fast increased aPWV in WT mice yet these acute changes returned to baseline within 24 hours of refeeding and did not change thereafter. A single 24 hr fast did not affect aPWV in Ob mice, whereas 5 weeks of IF ameliorated body weight and aPWV, with these vascular improvements being maintained with continued IF for at least another 5 weeks. These data suggest that once weekly 24 hr IF could be a viable dietary strategy to attenuate obesity-induced arterial stiffness.
Abstract 2113: Transcatheter Aortic Valve Replacement (TAVR) Complication: Intraoperative Mitral Regurgitation.
Authors: Singh Priyanka , Marwat Assad , Aurshina Afsha , Jajja Anaba , Khan Farid , Yarkoni Alon , Aurshiya Rasha
Published: July 2024

Introduction:
Transcatheter aortic valve replacement (TAVR) is the preferred treatment for aortic stenosis (AS) in patients with high surgical risk. This case report delves into a rare, significant intra-procedural mitral regurgitation (MR) of TAVR.

Case Presentation:
A 94-year-old female with severe AS and a STS score of 3.9% underwent TAVR. A recent 2D ECHO revealed severe AS. CT angiogram pre-TAVR exhibited scattered coronary artery calcifications, severe aortic valve calcification, and calcified atherosclerotic plaque. During the procedure, a 26mm CoreValve was deployed appropriately, with no perivalvular leak observed on angiography and transesophageal echocardiography (TEE). However, post-deployment, the patient experienced a sudden drop in blood pressure, and intra-procedural TEE revealed severe MR, not detected in previous studies. Treatment included pressors, non-invasive positive pressure ventilation, and diuretics, resulting in a favorable response. Subsequent TTE on the 4th day showed improvement in MR. A one-year follow-up TTE revealed only trace MR, confirming the normal functioning of the prosthetic valve.

Discussion:
Acute mitral regurgitation is an uncommon TAVR complication with potential morbidity and mortality. Various studies propose multiple mechanisms, and in this case, we attribute it to intraoperative global hypoperfusion causing transient MR. Hypoperfusion of the papillary muscle disrupted leaflet coaptation, leading to MR, as supported by its resolution with improved blood pressure. Studies have reported different mechanisms for acute MR, including papillary muscle rupture, leaflet perforation, guidewire impingement, and global hypoperfusion, with some indicating a higher incidence in CoreValve compared to other valve types.

Conclusion:
Given the rarity of acute MR during TAVR and the unclear pathophysiology, there are no established society guidelines for its management. With TAVR becoming routine, further clinical trials are crucial for optimizing treatment, including head-to-head comparisons of valves and procedural techniques, and developing comprehensive management strategies based on the specific mechanisms of this acute complication.
Abstract 2129: Gender Based Disparities In Outcomes, Resource Utilization & Readmission Rates In St-Elevation Myocardial Infarction
Authors: Kumar Manoj , Ali Shafaqat , Farooq Faryal , Kumar Sanjay
Published: July 2024

Background:
Cardiovascular disease is the leading cause of death among women; however acute coronary events tend to be under-managed in females due to higher atypical presentation & delay in diagnosis. We aim to investigate gender disparities in ST-elevation myocardial infarction (STEMI) outcomes.

Methods:
The national readmission database (2016-2020) was queried to identify hospitalizations for STEMI. Cohorts were stratified based on males and females admitted with STEMI. A Propensity Score Matching (PSM) model matched males and females with STEMI. Pearson’s x 2 test was applied to PSM-2 matched cohorts to compare outcomes.

Results:
Among 1 million hospitalizations for STEMI, 332,165 (31.86%) were females. After propensity matching (N=176,688), females had higher inpatient mortality (14.7% vs 14.5%, aOR: 1.13), CVA (3.3% s 2.7%; aOR 1.25), vascular complications (0.9% vs 0.3%; aOR 1.58%) and transfusion requirement (5.8% vs 5.2%, aOR:1.19) [p<0.05]. Females were less likely to receive a PCI (63.1% vs 65.8%, aOR: 0.83) or undergo an emergent CABG (0.8% vs 1.1%, aOR: 0.72) [p<0.001] for acute STEMI. From 2016-2020; STEMI related mortality has decreased from 12.7% to 12.4% in females (p-trend:0.047) while utilization of PCI has increased from 58.2 to 66.2 (p-trend<0.05). Furthermore, female patients had higher readmission rates on 30-day (10.8% vs. 10.2%), 90-day & 180-day intervals (p<0.001).

Conclusion:
In STEMI patients, female patients had higher mortality, cardiovascular complications & readmission rates. They less frequently underwent cardiac interventions than males. However, utilization of PCI is increasing, and associated mortality is decreasing.
Abstract 2137: Ligands Of The Receptor For Advanced Glycation End Products Induce Trained Immunity In Mouse Bone Marrow-Derived Macrophages
Authors: Kumar Vikas
Published: July 2024

Background:
The bone marrow undergoes a process of "training," and glucose-related mechanisms play a role in the accelerated development of atherosclerosis in diabetic mouse models. In diabetes, the accumulation of advanced glycation end-products (AGEs), resulting from the nonenzymatic glycation of proteins, is observed. We hypothesized that the receptor for AGEs (RAGE) ligand carboxymethyllysine (CML) induces trained immunity in macrophages, contributing to the pathogenesis of diabetic complications.

Methods:
Bone marrow-derived macrophages (BMDMs) from Wild Type (WT) C57BL/6J mice were exposed to CML-AGE for 24 hrs, followed by a 48-hr washout period without CML-AGE. Subsequently, BMDMs were stimulated with LPS or CML-AGE vs. vehicle for 24 hrs. The roles of RAGE and Diaphanous 1 (DIAPH1) were explored using Agerfl/fl Lysm Cre+/+, control Lysm Cre+/+, Diaph1fl/fl Lysm Cre+/- and control Lysm Cre+/- mice.

Results:
In CML-AGE-trained BMDMs from WT mice, significant upregulation of pro-inflammatory IL1b, IL6, and TNFα, and downregulation of anti-inflammatory IL10, was observed, but not in Ager-deleted BMDMs. Seahorse Analyzer studies revealed an increased reliance on glycolysis in CML-AGE-trained Lysm Cre+/+ vs. Agerfl/fl Lysm Cre+/+ and Lysm Cre+/- vs. Diaph1fl/fl Lysm Cre+/- macrophages

In vivo, CML-AGE induced trained immunity in BMDMs and splenic monocytes isolated from Ager- and -Diaph1-expressing mice but not those devoid of Ager or Diaph1. Western blotting indicated that CML-AGE training induced upregulation of H3K27ac (histone acetylation marks) and H3K4me3 (histone methylation marks). The non-selective methylation inhibitor MTA blocked the development of CML-AGE-induced trained immunity, with reduced Il6 and Tnfa expression. CML-AGE stimulation or training in control but not Ager- or Diaph1-devoid BMDMs led to a decrease in Mito-ER distance; cristae number, volume density, and area; while increasing the interaction of DIAPH1 with Mitofusin 2 (MFN2), which was recently identified as a mediator of mitochondrial dysfunction.

Conclusions:
CML-AGE induces trained immunity through modulation of cellular metabolism, epigenetic marks, and mitochondrial dysfunction, ultimately activating proinflammatory gene expression
Abstract 2150: PTEN Protects Against Vascular Smooth Muscle Cell Pro-inflammatory Dedifferentiation Through Metabolic Re-wiring
Authors: Lu Sizhao , Dubner Allison M , Jolly Austin J , Noble Tysen D , Hinthorn Tyler , Nemenoff Raphael A , Moulton Karen S , Weiser-Evans Mary C
Published: July 2024

Vascular smooth muscle cells (SMCs) play a critical role in intimal hyperplasia in atherosclerosis and restenosis. SMCs in diseased arteries exhibit a modulated phenotype with reduced contractile gene expression, elevated proliferation, and a proinflammatory/profibrotic phenotype. Metabolic reprogramming accompanies and is essential for SMC phenotypic modulation. Our group previously demonstrated that the tumor suppressor gene, PTEN, contributes to the maintenance of the SMC contractile phenotype. Analysis of human GTEx project data established a negative correlation between expression of PTEN and inflammatory genes in human arterial tissues. As a metabolic regulator, the function of PTEN in SMC metabolism has not been examined. We subjected SMC lineage tracing mice with systemic (sPTEN) and inducible, SMC-specific (iPTEN) PTEN overexpression to disease models of atherosclerosis and carotid artery (CA) injury. Compared to control mice, sPTEN and iPTEN mice are protected from atherosclerotic lesion formation and ligation-induced neointima formation. The observed vascular protection effect of PTEN was associated with reduced inflammation and reduced expression of key regulators of glycolysis, including GLUT1, HIF1a and PKM2, which were up-regulated in dedifferentiated SMCs. Consistently, unbiased metabolomics analysis confirmed that CA injury induced a SMC metabolic phenotype, including accumulation of pyruvate, reduction of OXPHOS, and increased fatty acid synthesis, which was rescued in iPTEN mice. Through high throughput screening, we previously identified and validated 5-azacytidine (5-aza) as potent transcriptional activator of PTEN expression. 5-aza reversed injury-induced SMC dedifferentiation in WT, but not PTEN inducible KO (PTEN iKO) mice. Our new scRNA-seq data further suggest that 5-aza inhibited injury-induced SMC metabolic reprogramming and dedifferentiation in a PTEN-dependent manner. Altogether, the data support that PTEN antagonizes the metabolic changes in SMC dedifferentiation and thereby maintains a contractile SMC phenotype. Pharmacological induction of PTEN by 5-aza can serve as a viable approach to rescue SMC metabolic reprogramming and phenotypic modulation in vascular disease.
Abstract 3004: Intermittent Hypoxia Decreases Aortic Stiffness And Facilitates Abdominal Aortic Aneurysm
Authors: Sharma Neekun , Khalyfa Abdelnaby , Ramirez-Perez Francisco I , Morales Quinones Mariana , Gozal David , Padilla Jaume , Manrique Acevedo Camila , Martinez-Lemus Luis A
Published: July 2024

Introduction:
Intermittent hypoxia (IH) is a phenomenon observed with obstructive sleep apnea (OSA), which is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, it remains unknown if IH is a risk factor for the development of AAA. The goal of this study was to determine the effects of IH exposure on arterial stiffness, and to assess whether IH increases the susceptibility to angiotensin (Ang) II-induced AAA in mice.

Methods:
Male C57BL/6 mice (8-week-old) were subjected to IH (6.5%-21% O
2
, 90 s/cycle, 12 h/day) or room air (RA, 21% O
2
, 24 h/day) for short-term (6 weeks) or long-term (6 months) (n = 10/group). Short-term mice were examined for arterial stiffness by pulse wave velocity (PWV) and atomic force microscopy, while long-term mice were used for AAA studies. To induce AAA, mice were infused with Ang II (1 μg/kg/min) using osmotic minipumps during final 14 days of IH or RA exposure. Sham surgery was performed in control mice. Outcomes were assessed via transabdominal ultrasound imaging and histology.

Results:
Short-term IH exposure decreased the stiffness of the abdominal (0.88 vs. 2.32 m/s in RA;
p=0.0006
) and thoracic (2.0 vs. 3.5 kPa in RA;
p=0.01
) aorta. Ang II increased abdominal aortic diameter in RA mice compared to sham and long-term IH with Ang II further augmented the aortic diameter (1.56 vs. 1.34 mm in RA Ang II;
p=0.03
) and incidence of AAA (50 vs. 10% in RA Ang II;
p=0.05
). Histologically, long-term IH plus Ang II augmented medial elastin fragmentation (7.7 vs. 5.3 in RA Ang II;
p=0.04
) and aortic collagen content (3.1 vs. 1.3 in RA Ang II;
p=0.06
). Ang II tended to non-significantly increase PWV in both groups (RA sham 1.9 vs. RA Ang II 2.5 and IH Ang II 2.3 m/s). Notably, a negative correlation between maximum aortic diameter and PWV was observed in AAA-induced mice (R
2
=0.48,
p=0.0009
).

Conclusions:
IH favors AAA development in association with changes in aortic stiffness. These findings support a potential causal link between OSA and AAA pathogenesis. Clarification of the molecular mechanisms may reveal novel therapeutic targets for the management of AAA.
Abstract 3005: Transgenerational Impact Of E-cigarette Nicotine Vaping On Abdominal Aortic Aneurysm
Authors: Spin Joshua M , Shen Wen-Jun , Chavan Sohil , Amirahmadi Maryam , Rhee Yae Hyun , Deng Alicia , Tsao Philip S
Published: July 2024

Background:
Smoking and family history are major risk factors for abdominal aortic aneurysm (AAA). Smoking is a powerful modulator of DNA methylation, and nicotine can cause heritable epigenetic alterations in animals and humans. We found that nicotine infusion and e-cigarette (e-cig) vaping augment murine AAA, and that parental nicotine infusion augments model AAA in offspring, altering tissue DNA methylation patterns.

Hypothesis:
We investigated the effects of maternal e-cig vaping in mice on their offspring’s experimental AAA risk, and transgenerational DNA methylation.

Methods:
Two treatment arms were employed: 1) pre-fertilization or 2) peri-natal. Female ApoE-/- or C57BL6 mice (F0) were exposed to e-cig nicotine (24 mg/ml) puffs, 9 sec/min, 1 hour/day (or room air). Arm 1: treated 28 days, then mated to untreated controls. Arm 2: mated after 1 week of e-cig, then maintained on treatment until gestation was complete. Aortic tissue of 10-week-old F1 offspring was evaluated using RRBS-Seq DNA methylation analysis. Parallel offspring underwent angiotensin-II infusion (1μg/kg/min; ApoE-/-) or elastase infusion (2U/ml for 5 min at 120 mmHg; C57) to induce AAA. Aneurysm growth was ultrasound tracked over 28 days.

Results:
Maternal vape exposure altered aortic DNA methylation in offspring, with numerous differentially methylated regions (DMRs; q<0.1). Hundreds of these DMRs were located in the same genes previously identified in offspring of nicotine-infused mice (see Background). Vaping altered offspring aortic methylation patterns in AAA-related genes, including miRNAs. DMR pathway analysis was enriched in transcription factors. Further, many DMRs were located in genes harboring risk loci for human AAA from our recent collaborative publication. Maternal e-cig exposure augmented AAA size in both models, arms, and genders (p<0.05). AAA incidence and mortality were increased in ApoE-/- in both genders and arms. Effects were most significant in males.

Conclusions:
E-cig nicotine exposure augments experimental AAA growth in multiple models across generations. These effects are accompanied by broad aortic epigenetic changes, including in key AAA modulator genes, with enrichment in transcription factors that target known AAA-related genes.
Abstract 3018: Expression Of The Aim2 Inflammasome In Skeletal Is Modulated By Surgical Bypass And Is A Target To Reduce Ischemic Myopathy In Pad
Authors: Xie Bowen , Assaf Edwyn , Ferrari Ricardo , Casale George , Pipinos Iraklis I , Sachdev Ulka
Published: July 2024

Introduction:
Peripheral arterial disease (PAD) causes skeletal muscle myopathy and impairment, yet relevant pathways are understudied. We reported a new role for the absent in melanoma 2 (AIM2) inflammasome in PAD myocyte function. We hypothesize that AIM2 is an important mediator of cell death pathways in PAD muscle and is reduced by revascularization.

Methods:
Myocytes were obtained commercially or from ischemic and perfused PAD muscle using cell sorting. mRNA libraries underwent next generation sequencing (NGS) and differential gene expression (DGE) mapped to the GRCh368 human genome. Ingenuity Pathway Analysis (IPA) identified relevant biological pathways filtered by both fold change (fc) >2 and false discovery rate of p< 0.05. Sections were procured from human gastrocnemius before and after vascular bypass and analyzed for AIM2 with immunofluorescence (IF) and western blot (WB). Inflammatory caspase-1 activity was measured +/- siRNA against AIM2. Statistical analysis included t- test and ANOVA + post-hoc analysis. P<0.05 was considered significant.

Results:
DGE and IPA on isolated myocytes highlighted Neutrophil Degranulation and Pathogen Induced Cytokine Storm Signaling Pathways to be significantly altered in ischemic muscle cells (Fig1, p < 1xE-30 each, N=4) emphasizing AIM2 inflammasome activation (CASP1, fc 30.73; NLRP3/ASC/CASP1, fc 27.23; PYCARD fc 40.63; IL1B fc 63.49). AIM2 protein was high in PAD by IF and WB. Caspase-1 activity was attenuated in the presence of AIM2 inhibition (N=3; ****p<0.0001 in PAD). AIM2 expression was significantly reduced after surgery for PAD (p<0.05, N=6/group, t-test).

Conclusion:
AIM2 inflammasome expression in PAD induces pyroptotic caspase activity and is attenuated by surgical bypass, directly demonstrating a unique biologic benefit of muscle revascularization in PAD. Targeting AIM2 pathways either pharmacologically or by surgery may help to reduce myopathic damage in ischemic muscle from PAD.
Abstract 3024: Mitochondrial Volume Abnormalities In Gastrocnemius Muscle Of Patients With Peripheral Artery Disease
Authors: Sullivan Alexander , Sugahara Sho , Brooks Craig R , Beckman Joshua A , Gamboa Jorge L
Published: July 2024

Background:
The mechanisms of claudication in patients with peripheral artery disease (PAD) are poorly understood. Mitochondria are responsible for ATP production during aerobic conditions but may become structurally impaired in patients with chronic ischemia and PAD.

Research Question:
Are mitochondrial volumes different in patients with PAD compared with age-matched controls?

Aims:
To better understand mitochondrial abnormalities and ultrastructure in patients with PAD.

Methods/Approach:
Patients with PAD (defined as ABI <0.85) and age-matched controls (ABI 1.00-1.40) were recruited and underwent gastrocnemius muscle biopsy. Muscle samples were collected in optimal cutting temperature compound. Mitochondria were labeled with cytochrome oxidase IV antibody and Cy3 fluorescence secondary antibody and images were rendered in three dimensions using structured illumination microscopy to quantify mitochondrial size, content, and morphology. Comparisons were done with a Mann-Whitney U test.

Results/Data:
Forty-eight subjects (median [IQR] age 66.5 [59.0, 74.0] years, 32 (66.7%) male) were recruited and underwent gastrocnemius muscle biopsy, including 16 controls and 32 with PAD. The largest singular mitochondrial volume was greater in those with PAD (22.6 vs. 4.2μm
3
, p=0.01, Figure 1A) but the median mitochondrial volume was smaller (0.07 vs. 0.01μm
3
, p=0.03, Figure 1B). While no control mitochondria were larger than 7.2μm
3
, 1% of mitochondria in patients with PAD were larger than 10μm
3
(Figure 1C). There was no difference in the total mitochondrial volume or content between PAD and controls.

Conclusion:
Patients with PAD have larger maximum but smaller median mitochondria sizes in the gastrocnemius muscle, suggesting greater heterogeneity of mitochondrial size in those with PAD. The changes in mitochondrial morphology may result in mitochondrial inefficiency and an impaired compensatory response in the setting of chronic ischemia from PAD.
Abstract 3027: Home-based, High-pressure Intermittent Limb Compression Following Lower Extremity Revascularization In Patients With Severe Peripheral Artery Disease: A Pilot Trial
Authors: Tesini Roseguini Bruno , Klein Janet , Perkins Susan , Han Yan , Westin Gregory , Motaganahalli Raghunandan
Published: July 2024

Objective:
Many patients who undergo lower extremity revascularization (LER) for peripheral artery disease (PAD) continue to experience mobility impairment despite adequate revascularization. High-pressure intermittent limb compression (HPILC) has been shown to augment leg blood flow and improve walking endurance in symptomatic PAD patients. This pilot trial aimed to examine the feasibility of home-based HPILC for 2 hours daily over 3 consecutive months in patients who underwent LER for PAD. The primary outcome was the change in 6-minute walk distance from baseline to 3-month follow-up.

Methods:
Three months following LER, 9 participants were randomly assigned to either HPILC (n=5) or a sham (n=4) group. Participants were provided with a portable pneumatic air pump and inflatable cuffs that were wrapped around the feet, ankles, and calves (ArtAssist, ACI Medical). The inflation pressures were 120 mmHg and 30 mmHg in the HPILC and sham devices, respectively. A built-in timer, inaccessible to participants, provided one estimate of treatment compliance. Outcomes were assessed before and after the completion of the 3-month interventions.

Results:
Among the 9 randomized participants, one individual required reintervention and was withdrawn from the study. Adherence to treatment was 74% for HPILC and 92% for sham. As shown in Figure 1, the change in 6-minute walk distance from baseline tended to be higher in the HPILC group compared to the sham-treated group (HPILC: 35.2±38.7 m vs. Sham: -14.7±25 m, p=0.07). There were no group differences in the secondary outcomes, including the changes in the ankle-brachial index, the toe-brachial index, and the Vascular Quality of Life questionnaire scores.

Conclusions:
Home-based HPILC following LER for PAD is feasible, well-tolerated, and appears to improve walking performance. These preliminary findings indicate that HPILC may be a useful adjunct therapy to enhance lower extremity functioning in patients recovering from LER.
Abstract 3028: Diabetic Complication Ischemia Triggers Muscle Mass And Muscle Weight Loss By Reducing Mesenchymal Progenitor Cells Population Proliferation
Authors: Uribe Echevarria Zubizarreta Veronica , Lopez-Diez Raquel , Vakil Raveena , Nimma Ramesh , Vasquez Carolina , Kumar Vikas , Ramasamy Ravichandran , Schmidt Ann Marie M
Published: July 2024

Introduction:
Mesenchymal progenitor cells (MPCs), such as myoblasts and fibroadipogenic progenitors (FAPs), and their communications with each other and with immune and vascular cells are responsible for muscle maintenance and regeneration. We hypothesize that in diabetes, loss of muscle mass after induction of hind limb ischemia is mediated by reduction in myoblast proliferation, thereby impairing muscle regeneration.

Methods:
Ischemic injury was induced in male diabetic (Akita) and littermate Wild Type (WT) mice via unilateral femoral artery ligation (FAL). Gastrocnemius muscle was collected at baseline, 3 and 7 days after surgery for single-cell RNA sequencing (scRNA seq). We identified the different cell populations in clusters, determined differentially-expressed genes, utilized Reactome Pathway Analysis and analyzed the cell cycle progression status and cell-cell interactions.

Results:
scRNA seq analysis of diabetic mice muscle, but not WT muscle, showed a reduction in MPCs, myoblasts and fibroadipogenic progenitors (FAPs) at 3 days post-FAL vs. baseline. FAPs were identified as two distinct subclusters; FAPs/Lum+ (associated with cell cycle regulation) and FAPs/Prg4+ (associated with extracellular matrix remodeling). We observed a lack of pathways involved in cell cycle progression in myoblasts at 3 days post- FAL in diabetic vs. WT muscle. At day 7 post-FAL, we observed an increased number of cell cycle pathways vs. day 3 post-FAL in diabetic muscle, implying a delayed response in the myoblasts. Cell cycle progression analysis revealed fewer diabetic myoblasts in S/G2M phase, with lower expression of proliferation markers (Mki67, Top2a, Mcm6 and Pcna), but no significant differences were observed in FAP/Lum+ cells. Cell-cell interaction analysis at day 3 post-FAL revealed fewer FAP-myoblast interactions in diabetic muscle, while interactions between either FAPs/myoblasts and myeloid cells were higher.

Conclusion:
These findings suggest that understanding the network of inter-cellular communications among MPCs, immune and vascular cells, and their roles in reduced myoblast proliferation after ischemia, is critical to identify new therapies to stimulate regeneration in diabetes and peripheral vascular disease.
Abstract 3040: Quantitative Proteomic Analysis Of HDL Shows Apolipoprotein M (ApoM) As A Possible Marker For COVID-19 Mortality
Authors: Souza Junior Douglas R , Silva Amanda R , Fernandes Lívia R , Palmisano Giuseppe , Ronsein Graziella E
Published: July 2024

Introduction:
High-density lipoprotein (HDL) comprises a heterogeneous group of particles that differ in many aspects, such as density, size, composition and function. The use of mass spectrometry-based proteomics to investigate HDL protein composition has brought some new insights into the field. The qualitative and quantitative HDL proteome is remodeled in different disease states, such as diabetes mellitus, hypercholesterolemia and kidney dysfunction. Beyond its established role in inflammatory and cardiovascular diseases, HDL's involvement in infectious diseases, such as COVID-19, has recently come to light. Here, we investigate the remodeling of HDL proteome in hospitalized and non-hospitalized COVID-19 patients.

Hypothesis:
We hypothesize HDL proteome is remodeled in infectious diseases and may be a marker and perhaps a predictor of a poor prognosis.

Methods:
We implemented a robust, global label-free quantification proteomic approach to unravel relative changes in HDL protein composition. We analyzed the proteome of COVID-19 subjects from Brazil categorized in two groups, hospitalized (n=30) and non-hospitalized (n=11).

Results:
Comparative analysis between hospitalized and non-hospitalized patients revealed a more than 50% increase in relative levels of certain proteins in hospitalized patients, including the inflammatory proteins serum amyloid A1 and A2, as well as pulmonary surfactant-associated protein B. We also observed reductions in the lipid-metabolism related apolipoprotein A2 and phospholipid transfer protein. These findings, independent of sex, triglyceride and HDL-cholesterol, suggest an inflammatory remodeling of the HDL particles. Further analysis of surviving (n=35) and deceased patients (n=6) indicated a negative association between ApoM levels and odds of death due to COVID-19 complications (odds ratio per 1-SD increase in ApoM was 0.27 [0.07 to 0.72], P=0.007).

Conclusions:
Our results point out HDL remodeling is a marker of COVID-19 severity and ApoM-carried sphingosine-1-phosphate signaling pathway may play a pivotal role in the disease outcome.
Abstract 3041:
APOB
Gene Deletion Increases The Expression Of Vitamin D Binding Protein In Human Hepatoma Cells
Authors: SWATI FNU , Rosario Sara , Mooring Ashley , Prakashmurthy Chandana , Ansari Abulaish , Aleman Jose , Pamir Nathalie , Hussain M M , Chattopadhyay Atrayee , Schlamp Maria Florencia
Published: July 2024

High plasma apolipoprotein B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many metabolic diseases. Inhibition of APOB mRNA decreases plasma lipid levels but at a risk of lipid accumulation in tissues. To understand the global consequences of reduced apoB synthesis, we generated a human hepatoma (Huh-7) cell culture model system deficient in apoB. ApoB deficient Huh-7 cells (Ako) were generated using the CRISPR/Cas9 system. Total RNA for transcriptomics and overnight conditioned media for proteomics and lipidomics was obtained from the Ako and Huh-7 cells (n=3). Statistical analysis was done using Student’s t-test while comparing two groups, p < 0.05 was considered significant.
APOB
gene deletion significantly reduced APOB mRNA and protein levels in the Ako cells compared to control cells. Ako cells supported apoB48 secretion when transfected with apoB48 expression plasmids. These cells did not accumulate significant amounts of triglyceride. KEGG pathway analysis of RNA-Seq data showed a significant increase in DNA replication/repair pathway and complement/coagulation cascade in the Ako cells. The most downregulated pathways included the PI3k-Akt signaling and MAPK signaling pathways. Differential gene expression analysis identified vitamin D binding protein (VDB, gene
GC
) mRNA as the most highly upregulated transcript in Ako cells. The Ako media secretome contained significantly higher amounts of VDB compared to control Huh-7. Furthermore, targeted lipidomics of the secretome showed significant increases in media ergocalciferol. Our qPCR and western blot analysis confirmed increased expression of VDB mRNA and protein secretion in the Ako cells as compared to the Huh-7 cells. We established a cell culture model deficient in secreting apoB-containing lipoproteins. These cells, however, support the assembly and secretion of apoB48 when transfected with apoB48 expression plasmid. A major consequence of apoB deficiency was upregulation of the VDB expression. We interpret these data to suggest that human hepatoma cells have two differentially regulated pathways for the transport of vitamin D. In the absence of apoB-lipoprotein assembly, these cells upregulate VDB to augment secretion of vitamin D.
Abstract 3045: Dietary Whole-grain Rice Differentially Regulates HDL-associated MicroRNAs And Hepatic Lipase In Healthy Japanese Volunteers.
Authors: Tsujita Maki , Nakasuka Kosuke , Massick Clark , Miura Yutaka , Miida Takashi , Vickers Kasey C
Published: July 2024

Background:
The Japanese population consumes a diet high in rice. Dietary whole-grain (brown) rice has advantageous health benefits over white rice, largely predicted to be conferred by brown rice bran, germ, and fiber. We have previously reported that high-density lipoproteins (HDL) transport microRNAs (miRNA) and deliver functional RNA to recipient hepatocytes. Here, we hypothesize that dietary brown rice reorganizes functional small RNA (sRNA) cargo on circulating HDL and regulates systemic lipid metabolism through hepatic HDL-miRNA uptake.

Methods:
Healthy subjects (n=13, 20-65 y.o., 38.5% male) consumed 150 g/serving for all 3 meals of (1) white rice, (2) brown rice, and (3) white rice for 3 days with intervals of 4 to 7 days. At day 4, plasma HDL-cholesterol (HDL-C) and total plasma lipids were measured by direct and enzymatic methods. Total RNA was isolated HDL purified by size-exclusion chromatography from human plasma. HDL-sRNA sequencing was completed using NovaSeq6000 (Illumina). HTGL protein levels were measured by ELISA (IBL).

Results:
Plasma cholesterol, HDL-C, and non-HDL-C levels were not immediately affected by dietary brown rice; however, HDL-C levels were increased after a delay of 10 days. Nevertheless, HDL-associated miR-203-3p and miR-205-5p were found to be significantly decreased in subjects with brown rice consumption compared to white rice. miR-203-3p is predicted to target and suppress hepatic lipase (
LIPC
, HTGL). Plasma levels of HTGL were not altered by brown rice, but HTGL protein levels were found to be significantly decreased when the brown rice diet was switched back to white rice, and miR-203-3p levels returned to HDL particles.

Conclusions:
Dietary brown rice reduces HDL-associated miR-203-3p and miR-205-5p after 3 days and differentially regulates the systemic level of a putative miR-203-3p target gene. These results support that HDL-miR-203-3p functionality may be responsive to brown rice diets and post-transcriptionally regulate
LIPC
gene expression in recipient hepatocytes. These observations can potentially be used to design dietary supplements to regulate HDL-miRNA function in the liver to control systemic lipid metabolism.
Abstract 3072: GDF15 Represses The Interferon Response Early In Vascular Senescence
Authors: Tsitsipatis Dimitrios , Rodriguez Rivera Tatiana , Mazan-Mamczarz Krystyna , Blew Cassandra , Duggan Michael , Walker Keenan , Gorospe Myriam , Herman Ali
Published: July 2024

Senescent cells have been identified as major contributors of the progression of vascular diseases including atherosclerosis by increasing the senescence-associated secretory phenotype (SASP) and dysregulating vascular cells. Notably, senescence and the SASP can contribute to plaque rupture and atherosclerotic progression by promoting inflammation and plaque vulnerability. A time course microarray analysis of vascular smooth muscle cell (VSMC) senescence revealed p53-regulated
GDF15
mRNA was increased early and remained elevated throughout senescence. Notably, GDF15 has been implicated as a biomarker for cardiovascular disease mortality and is part of the conserved SASP across cell types and tissues. Interestingly, intracellular or secreted GDF15 protein levels were increased in monkeys fed a high-fat diet or in mice fed a high-fat diet and in individuals with high Framingham risk scores, respectively. We sought to identify the role of GDF15 in senescent VSMCs. Using CRISPRi, we silenced GDF15 during early senescence and performed RNA-sequencing. Pathway analysis revealed a enrichment in interferon signaling with the loss of GDF15. Coinciding with the increase in interferon signaling, phosphorylation of STAT1 and CREB were increased by loss of GDF15 in early senescence, aiding in the elevation of interferon expression. Given the role of interferons in communication and the prominence of macrophages in atherosclerosis, we tested the effects of conditioned media, derived from either proliferating or senescent VSMCs, on the activation and the expression of scavenger receptors on human macrophages. Senescent conditioned media increased expression of
ABCG1
,
CD36
,
LOX-1
,
SCARF1
, and
GDF15
mRNA as well as TNFα and CCL2 secretion in macrophages. We propose that elevated GDF15 levels in early senescence suppress interferon signaling, thus preventing macrophage chemotaxis and in turn leading to the accumulation of senescent cells in the vasculature. Thus, disrupting the senescent VSMC-macrophage communication through secreted proteins may offer promising avenues of therapeutic development to prevent or improve inflammatory vascular diseases.
Abstract 3073: Gene Regulation By RNP Granule Formation In Atherosclerosis
Authors: Wang Kristin M , De Jong Annika , Cyr Yannick , Newman Alexandra , Bozal Fazli , van Coen , Moore Kathryn J
Published: July 2024

Background:
Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages in the arterial intima. Macrophages become lipid laden foam cells that persist in the arterial vasculature, inciting chronic inflammation and plaque formation. A better understanding of the mechanisms underlying this maladaptive inflammatory response may reveal novel therapeutic strategies for intervention.

Hypothesis:
We hypothesize that the transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SG). SGs are cytoplasmic membrane-less organelles that sequester translation initiation factors, mRNAs, and noncoding RNAs due to stress. We previously reported that SG formation is increased in plaque macrophages of atherosclerotic mice and can be induced
in vitro
during cellular cholesterol loading. However, the consequences of SG formation and the transcripts sequestered by these organelles is unknown.

Methods and Results:
To investigate whether SGs influence macrophage phenotype, we used siRNA to knockdown
G3bp1
and treated with Dil-oxLDL. We found that
G3bp1
knockdown reduced macrophage lipid loading and induced transcriptional changes associated with canonical M2 anti-inflammatory macrophage polarization. Furthermore, RNA-seq of macrophages treated with si
G3bp1
and oxLDL showed downregulation of pathways involved in phagosome formation and S100 family signaling. RNA immunoprecipitation of G3BP1 from macrophages treated with an ER stressor revealed preferential binding of G3BP1 to transcripts implicated in cholesterol metabolism. Altogether, these data support a role for SGs in maintaining a pro-inflammatory state and regulating cholesterol pathways in plaque macrophages.

Conclusion:
Our data support a role for SGs in repressing the translation of RNA transcripts in lipid-laden foam cells that accumulate in atherosclerotic plaque. Identification of the specific proteins and RNAs sequestered in SGs will provide insight into how they regulate gene expression and cellular phenotypic switching that prevents the resolution of inflammation.
Abstract 3074: RNA Editing Enzyme
ADAR1
In Vascular Smooth Muscle Controls Vascular Integrity And Progression Of Atherosclerosis
Authors: Weldy Chad S , Li Qin , Monteiro Joao P , Guo Hongchao , Galls Drew , Gu Wenduo , Cheng Paul P , Ramste Markus O , Li Daniel , Palmisano Brian , Sharma Disha , Worssam Matthew , Zhao Quanyi , Kundu Ramendra K , Nguyen Trieu , Li Jin Billy , Quertermous Thomas
Published: July 2024

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) to prevent the dsRNA sensor, MDA5 (
IFIH1
), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism for coronary artery disease (CAD) risk. Here, we show that human atherosclerotic smooth muscle cells (SMCs) express immunogenic RNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC) and loss of RNA editing causes activation of MDA5, characterized by upregulation of ISG genes (i.e.
ISG15
) and others with known roles in SMC phenotypic transition and CAD risk (i.e.
KLF4
). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification
in vitro
. In a high fat diet atherosclerosis model, scRNAseq analysis revealed SMC to have increasing ISG expression with phenotypic modulation. To evaluate SMC RNA editing
in vivo
, we generated a conditional SMC specific
Adar1
KD mouse (
Adar1
flox/flox
,

Myh11
CreERT2
, ROSA
tdT/+

,

ApoE
-/-

). At 2 weeks following SMC-
Adar1
KD, we observed 50% mortality with a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. scRNAseq of aortic tissue showed that SMC-
Adar1
KD causes ISG activation and coordinates a complex transcriptional response throughout the vessel wall with robust macrophage cell infiltration. Through evaluation of ligand-receptor interaction, we show that
Ccl5:Ccr5
is the principal regulator of cell infiltration following SMC-
Adar1
KD. Through study of SMC-
Adar1
haploinsufficiency in the atherosclerosis mouse model, we further reveal that MDA5 activation occurs in SMC phenotypic modulation and accelerates formation of chondromyocytes — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA mediates disease progression, promoting our understanding of disease causality and CAD genetic risk.
Abstract 3083: Macrophage Klf15 Prevents Foam Cell Formation And Atherosclerosis Via Transcriptional Suppression Of Olr-1
Authors: Zhao Ling-Na , Song Zheng-Kun , Zhao Li , Liu De-Shen , Huang Fang-Ze , Zheng Shao-Yi , Liu Xiu
Published: July 2024

Background:
Macrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis.

Methods:
We used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and

ApoE
-/

-
mice on a HFD. We subsequently injected mice with AAV-
KLF15
and AAV-
LacZ
to assess the role of KLF15 in the development of atherosclerosis
in vivo
. Oil Red O, H&E, and Masson’s trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively.

Results:
We determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis.

Conclusion:
Macrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.
Abstract 3090: Oral Administration Of Akkermansia Muciniphila Ameliorates Venous Wall Fibrosis: Therapeutic Implication Of A Novel Probiotic Therapy Against Post-thrombotic Syndrome
Authors: Wang Bowen , Yin Li , Islam Zain H , Marcum William A , Zhang Kaijie , Hoyt Nicholas , Le Quang , Kent Eric W , Johnston Campbell , Kanchetty Rohan , Tetz Zoe , Stanisic Sophia , Huang Yitao , Guo Lian-Wang , Cowardin Carrie A
Published: July 2024

Introduction:
Post-thrombotic syndrome (PTS) is the most common sequelae of deep vein thrombosis (DVT), manifested as venous insufficiency in up to 50% of patients. Unfortunately, none of the existing therapies, including anticoagulants and thrombolytics, have shown definitive effects for PTS, presenting a vital unmet clinical need.
Akkermansia Muciniphila (AKK)
, a gut commensal anaerobic bacterium, has been recognized with pleiotropic benefits in a wide range of diseases including cardiometabolic disorders. However, its therapeutic and mechanistic implications in thromboembolic diseases and the subsequent PTS remain unknown. We hypothesize that probiotic therapy with live
AKK
after DVT formation could ameliorate venous wall fibrosis, indicative of PTS.

Methods:
In male specific-pathogen free c57b6/j mice (10-12 weeks), DVT was experimentally created via partial ligation of inferior vena cava (IVC) as previously established, leading to progressive venous fibrosis. For the probiotic treatment,
AKK
(strain BAA-835) was cultured using BHI broth in an anaerobic Coy chamber, followed by daily oral gavage to mice beginning from day 1 post-DVT induction. At day 14 post-procedure, IVC was collected for Masson Trichrome and immunofluorescent staining, followed by quantitation of venous collagen deposition/fibrosis and phosphorylation of endoplasmic reticulum (ER) stress PERK kinase. Endpoint plasma specimens were collected to quantify the circulating levels of trimethylamine N-oxide (TMAO).

Results:
Post-DVT probiotic therapy with live
AKK
effectively mitigated venous wall fibrosis in comparison to BHI culture media gavage alone (fibrotic area: 61.12±6.64 vs 53.17±5.99; p<0.05). TMAO, the only microbial metabolite associated with thromboembolic risk, was significantly reduced in plasma following
AKK
probiotic therapy. Furthermore, phosphorylation of ER stress PERK, which is directly activated by TMAO and critically contributes to myofibroblast activation and organ fibrosis, was inhibited in the
AKK
-treated IVC (435.09±26.49 vs 326.86±17.61; p<0.01).

Conclusion:
Our current study supports the potential use of Post-DVT
AKK
probiotic therapy for the treatment of PTS, possibly via modulating the TMAO-PERK axis.
Abstract 3091: Long-term Association Between Hip Or Knee Replacement Surgery And Venous Thromboembolism Risk: A Prospective Matched Cohort Study In The Uk Biobank
Authors: Wu Peng , Yang Zhirong , Sha Feng , Tang Jinling
Published: July 2024

Background:
Hip and knee replacement surgeries have been linked to a transient increase in venous thromboembolism (VTE) risk in the short term. However, there is limited evidence regarding the extended impact of these surgeries on VTE risk beyond the first year.

Objective:
This prospective matched cohort study aimed to investigate the associations between hip or knee replacement surgery and VTE risk over a 10-year period, examining how these associations evolved over time.

Methods:
Analyzing data from 496,862 adults recruited between 2006 and 2010 in the UK Biobank study, 21,155 hip replacement surgery patients and 21,899 knee replacement surgery patients were identified and matched with the general population using exposure density sampling. The piecewise Cox proportional hazards model was employed to estimate hazard ratios (HR) and 95% confidence intervals (CI) for VTE outcomes at different intervals within the 10 years post-surgery.

Results:
For the hip replacement surgery cohort (n=221,138), the mean age was 61.5 (standard deviation [SD] 6.8), 40.7% were men, and 407 (1.9%) patients with hip surgery developed VTE during follow-up. In the knee replacement surgery cohort (n=199,487), the mean age was 61.7 (SD 6.4), 44.5% were men, and 436 (2.5%) patients with knee surgery developed VTE. Adjusting for various factors, the elevated risk peaked within 30 days post-surgery, with HRs (95% CIs) of 21.4 (15.4-29.6) and 33.0 (23.4-46.4) for hip and knee replacement, respectively. The elevated risk persisted until the tenth year, with HRs (95% CIs) of 1.59 (1.23-2.04) and 1.68 (1.21-2.33) for hip and knee replacement, respectively. These associations were held across participant characteristics and in sensitivity analysis.

Conclusions:
In middle-aged and older British adults, hip or knee replacement surgery was associated with both short- and long-term VTE risk, with the heightened risk persisting for up to a decade.