bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 551: A Novel Retrievable Drug Delivery Stentgraft To Enhance Paclitaxel Delivery For Restenosis
Authors: Stafford Jordan F , Kenawy Dahlia , Amari Foued , Yazdani Saami K , Tillman Bryan
Published: October 2023

Introduction:
Half of vascular interventions will fail in under 5 years due to restenosis. While drug coated balloons (DCB) are used to minimize restenosis, there are several disadvantages: 1) Limitations of the amount of drug that can be adhered to a balloon, 2) Significant drug is washed off the balloon by the circulation and 3) There are limits on the time of balloon inflation due to ischemia concerns. We developed a dumbbell shaped, retrievable drug delivery stentgraft (RDDS) that isolates a segment of artery with an outer chamber (
OC
) (Fig 1A). The center lumen of the stent maintains distal perfusion to allow longer drug exposure and this stent is removed after use.

Methods:
A RDDS stentgraft was fashioned from laser cut nitinol scaffold, covered in polymer and an infusion cannula was installed (Fig B). In a porcine model, bilateral common iliac arteries were dilated by plain balloon angioplasty to simulate endovascular intervention. Next a commercial DCB with paclitaxel (PXL) was used to treat one iliac, while an RDDS infused a similar dose of PXL to treat the contralateral iliac. Vessels and serum had PXL quantitation by liquid chromatography-mass spectrophotometry.

Results:
Angiographically, the RDDS maintained distal flow while the OC isolated the arterial wall for PXL delivery (Fig C, D). Blood samples distal to the treated areas demonstrated lower concentrations of PXL during treatment with the RDDS compared to DCB (10.50 ± 0.77 vs. 2310.07 ± 471.69 ng/mL, p < 0.0001). There was equivalent drug delivery to target vessel wall between the two approaches (33.85 ± 8.78 vs. 41.45 ± 12.51 ng/mg, p = 0.251, RDDS vs. DCB).

Conclusions:
The RDDS angiographically isolated a segment of artery, while maintaining distal perfusion. Moreover, compared to a DCB, the RDDS was able to prevent loss of drug to the circulation and effectively deliver paclitaxel to the vessel wall. This this study concludes that chambered retrievable stentgrafts may have a role in locoregional drug delivery.
Abstract 554: Monocyte Toll-like Receptor 4 (tlr4) Signaling Is Essential For Post Thrombotic Upa Expression
Authors: Sharma Sriganesh , Kumar Kiran , Bamezai Sharika , Moreno Rocha Oscar Y , Rocco Sabrina , Mangum Kevin D , Luke Catherine , Gallagher Katherine A , Moore Bethany , Henke Peter , Obi Andrea T
Published: October 2023

Background:
Leukocyte expression of Toll-like receptor 4 (TLR4) increases with transformation to chronic deep venous thrombosis (DVT) & in non-resolving thrombi, but TLR4 function during thrombus clearance is unclear. We hypothesized TLR4 signaling to regulate expression of fibrinolytic factors in monocytes & macrophages (MO/MFs) for VT resolution.

Methods:
Small interfering RNA (siRNA) knockdown (KD) of TLR4 & disruption of Myd88-dependent/independent pathways occurred in immortalized MFs (RAW264.7), with quantitative real-time PCR (qRT-PCR) for a panel of fibrinolytic factors & immunoblotting for urokinase (uPA). Tlr4-/- mice & wild-type controls underwent IVC stasis/ligation, yielding VT at 2h, 2d, & 8d. Murine/chronic human thrombi underwent immunofluorescent staining for indicated targets & murine thrombi weight-length ratios were found. Splenic & Bone marrow-derived MFs from Tlr4 Cre mice were immunoblotted for uPA.

Results:
TLR4 silencing in RAW264.7 cells yielded suppression of uPA mRNA (Fig 1B) & protein levels (not shown). TRIF KD yielded decreased uPA mRNA (Fig 1C) & protein levels (not shown), implicating Myd88-independent signaling in MO/MF uPA production. Human (not shown) & murine chronic thrombi staining saw TLR4 co-localization with MF marker CCR2 (Fig 1D). Global knockout (KO) of murine TLR4 did not affect early thrombogenesis (Fig 1E left), with delayed VT resolution seen from 2d (Fig 1E right). Decreased uPA protein levels were observed in Tlr4 KO mice (Fig 1F).

Conclusions:
TLR4 signaling stimulates in vivo VT resolution. Expression of fibrinolytic factor uPA is enforced in MO/MFs by TLR4-Myd88-independent signaling, a potential target for VT resolution.
Abstract 562: Ischemic Heart Disease Mortality And Systemic Lupus Erythematosus
Authors: Terrani Kristina , Ibrahim Ramzi , Razzaq Faryal , Habib Adam , Salih Mohammed , Lee Justin Z
Published: October 2023

Introduction:
Systematic lupus erythematosus (SLE) is associated with significant morbidity and mortality, contributing to accelerated atherosclerotic disease. We sought to quantify ischemic heart disease (IHD) mortality trends in individuals with SLE in the United States (U.S.).

Hypothesis:
Disparities related to IHD mortality in individuals with SLE exist.

Methods:
Cross-sectional analyses were conducted using the CDC Wide-ranging Online Data for Epidemiologic Research database to capture all mortality data in the U.S. using death certificate information from 1999 to 2020. We identified individuals with IHD (ICD10: I20-I25) as the underlying cause of death and SLE (ICD10: M32) as the multiple causes of death. Underlying cause of death was defined as the diagnosis that led to mortality and multiple causes of death were defined as the diagnoses that contributed to mortality. Quantified measures included age-adjusted mortality rate (AAMR) per 100,000 population and corresponding 95% confidence intervals. AAMR was adjusted to the U.S. population in the year 2000.

Results:
We identified 2,733 deaths from 1999 to 2020. AAMR decreased from 0.055 (95% CI, 0.046 - 0.064) in 1999 to 0.027 (95% CI, 0.023 - 0.032) in 2020. Females were disproportionately affected by higher AAMR compared to males (0.054 [95% CI, 0.051 - 0.056] and 0.013 [95% CI, 0.011 - 0.014], respectively). Black populations had the highest AAMR (0.084 [95% CI, 0.077 - 0.090]), with no significant differences among other racial groups such as White (0.029 [95% CI, 0.028 - 0.030], American Indian/Alaska Native (0.041 [95% CI, 0.025 - 0.063]), and Asian/Pacific Islander (0.023 [95% CI, 0.017 - 0.030]) populations. No differences in AAMR were observed between metropolitan and non-metropolitan regions (0.027 [95% CI, 0.026 - 0.029] and 0.029 [95% CI, 0.026 - 0.032], respectively). The West had the highest AAMR (0.036 [95% CI, 0.033 - 0.039]), with no significant differences among the Northeastern (0.027 [95% CI, 0.024 - 0.030]), Midwestern (0.027 [95% CI, 0.025 - 0.030]), and Southern regions (0.029 [95% CI, 0.027 - 0.031]).

Conclusion:
IHD mortality in individuals with SLE remains disproportionately concentrated in females, Black populations, and Western regions.
Abstract 564: Genetic Basis Of Thoracic Aortic Aneurysms In A Large Unselected Clinical Population
Authors: Mirshahi Hannah S , Haley Jeremy S , Smelser Diane T , Ryer Evan J , Elmore James R , Carey David J
Published: October 2023

Objectives:
Thoracic aortic aneurysms (TAAs) are associated with protein coding variants in genes associated with Loeys-Dietz Syndrome (LDS), vascular Ehlers-Danlos Syndrome (VEDS) and
ACTA2
. The purpose of this study was to estimate the prevalence of these variants, along with potential novel variants, in a large unselected clinical population.

Methods:
We analyzed exome sequence data from 174,452 participants. Rare variants (frequency <0.001) were annotated by functional consequence and classification as pathogenic or likely pathogenic (P/LP) in the ClinVar database. TAA diagnoses were determined by ICD-10 codes.

Results:
2,670 (1.5%) individuals had a TAA diagnosis; mean age was 66.1 years, compared to 56.7 for the study population (p=0.014). 69% of TAA cases were male and 71% were smokers. There were 20 carriers of ClinVar P/LP variants in LDS genes (8 variants in 10 carriers for
SMAD3
, 3 variants in 3 carriers for
TGFB2
, 2 variants in 2 carriers for
TGFBR1
, 3 variants in 5 carriers for
TGFBR2);
5 had a diagnosis of TAA (2 for
SMAD
3, 1 each for the others; ORs =16-65); the age of diagnosis was significantly lower than for non-carriers (p<0.0001). 11 P/LP carriers without a TAA diagnosis were <55 years old and could be at risk for TAA. There was 1 TAA case in 21
COL3A1
P/LP carriers and 0 cases in 10
COL1A1
P/LP carriers. We also identified LDS and VEDS variants that are classified as uncertain significance that could be reclassified as P/LP based on their association with early TAA (Table 1).
ACTA2
P/LP variants were also strongly associated with TAA (OR=26; p<0.001). The prevalence of P/LP variants in these genes is 1/3,300 (1/1,350 if potential novel pathogenic variants are included).

Conclusions:
Our results provide estimates of the prevalence and penetrance of variants associated with TAA in a large unselected clinical population, reveal potential novel pathogenic variants, and support the use of genetic data to identify individuals with high disease risk.
Abstract 580: Temporal Profile Of Catheter-directed Thrombolysis On Post-thrombotic Syndrome Outcomes In Iliofemoral DVT Patients: Post-hoc Analysis Of ATTRACT Trial
Authors: Li Wenzhu , Weinberg Ido , Lee Hang , Rosovsky Rachel , Reed Guy L , Libby Peter , Tawakol Ahmed A , Henke Peter , Jaffer Farouc A
Published: October 2023

Introduction:
Patients with iliofemoral deep vein thrombosis (IFDVT) may preferentially benefit from catheter-directed thrombolysis/thrombectomy (CDT) to reduce the post-thrombotic syndrome (PTS). Our prior analysis of the full NIH ATTRACT trial cohort demonstrated that the maximal benefit of CDT occurs at the day (D) 4-8 symptom-onset-to-randomization (SOR) timeframe (Circulation 2021; 143:1224-1238). However, the optimal time window for CDT benefit specifically for IFDVT patients has not been determined.

Hypothesis:
IFDVT patients may have a broader time window and greater magnitude for CDT benefit compared to all-comer DVT patients.

Methods:
We analyzed the IFDVT subgroup (N=391) of the NIH ATTRACT trial to assess optimal SOR timeframes for improved PTS outcomes (Villalta and VEINES-QoL scores). In a series of sensitivity analyses, IFDVT patients were non-prespecified divided into two (early, late) or three (early, intermediate, late) SOR groups. Mean scores were estimated by a piecewise linear-regression growth-curve model, with adjustment for strata including clinical center and baseline covariates (age, sex, BMI) by intention-to-treat analysis.

Results:
In the IFDVT patients, sensitivity analyses revealed that CDT significantly and maximally improved PTS outcomes in the intermediate D4-9 SOR [CDT +anticoagulation (AC) vs. AC alone; between-group difference= -2.54 (Villalta), +13.13 (VEINES-QoL), p<0.0001 for each]. However, CDT did not provide a benefit at early SOR ≤D3 and later SOR ≥D10 (p>0.05). Moreover, CDT effects differed across SOR 0-3, 4-9 and 10+ days (Villalta: -0.77, -2.54, -1.98; p=0.027), (VEINES-QoL: -1.89, +13.13, +1.92; p=0.022).

Conclusions:
CDT maximally improves PTS outcomes for IFDVT patients in the D4-9 timeframe, which is a more selective time window and of greater magnitude of PTS benefit than the full ATTRACT trial cohort. However, CDT does not improve outcomes beyond AC at early D0-3 and late D10+ timeframes. These findings may have practice implications to consider symptom duration to optimize AC and CDT therapies in IFDVT patients. Mechanisms underlying the benefit of AC and CDT on PTS outcomes need further study.
Abstract 588: Androgen Promotes Aortic Aneurysms Via Suppressing PD-1 In Male Mice
Authors: Mu Xufang , Liu Shu , Wang Zhuoran , Jiang Kai , McClintock Timothy , Stromberg Arnold , Tezanos Alejandro , Gong Ming C , Guo Zhenheng
Published: October 2023

Objective:
Male sex is a well-established risk factor for abdominal aortic aneurysms but the underlying mechanisms remain to be fully understood. Our lab reported aldosterone and high salt (aldo-salt) induced aortic aneurysms in an age-dependent manner in male mice but whether it shows sex dimorphisms as in human is unknown. The current study tested the hypothesis that male mice are more susceptible to aldo-salt induced aortic aneurysms through androgen mediation and investigated the potential mechanisms.

Results:
Ten-month-old male and female mice were used in all experiments. We first demonstrated the sex differences in the aldo-salt model with 70% incidence in male mice (7/10) compared to complete protection in female mice (0/9). Next, we showed androgen and androgen receptor (AR) mediated the process by several independent experiments: 1) orchiectomy provided strong protection (13.3%, 2/15) compared to sham control (69.2%, 9/13); 2) Exogenous DHT administration to orchiectomized mice restored the aneurysms formation (55%, 6/11); 3) inhibition of AR function by drug decreased total incidence compared to control (2/11 vs. 7/11). To dissect the mechanism, we conducted bulk RNA sequencing in aortas from 3 groups of mice given aldo-salt 1) intact male mice; 2) orchiectomized mice; and 3) orchiectomized mice with DHT. Through a series of bioinformatic analyses, we found PD-1 signaling pathway was significantly enriched in orchiectomized mice compared to control and reversed by DHT addition. Next, we found a significantly higher expression of PD-1 in spleen from orchiectomized mice than sham mice with aldo-salt presence, at both protein and mRNA levels. Mechanistically we showed AR could bind to PD-1 promoter region and suppressed PD-1 expression. Finally, we injected αPD-1 antibody or control IgG antibody to orchiectomized mice during the aldo-salt administration to confirm phenotypes. Results showed that neutralization of PD-1 partially abolished the orchiectomy protection (0/8 vs. 5/12).

Conclusions:
Our results demonstrated that male mice are more susceptible to aldosterone and high salt-induced aortic aneurysms. Androgen and its receptor mediate the high susceptibility in male mice partially through PD-1 signaling pathway.
Abstract 589: A Fluid Structure Interaction Workflow For Studying Aortic Dissections
Authors: Nnate Blessing , Khabaz Kameel , Dhara Sanjeev , Nguyen Nhung , Li Willa , Pocivavsek Luka
Published: October 2023

Introduction:
After diagnosis and treatment of aortic dissections, there is the potential for future complications such as the growth or rupture of pre-existing aneurysms or further dissection of the aorta. This susceptibility to future complications is what we refer to as “aortic fragility”. Current computational models of aortic dissection rely upon computational fluid dynamics (CFD) or finite element analysis (FEA); however, there are limitations to using either method alone. Using a healthy patient aorta as our control case, we developed a novel method of coupling CFD with FEA in a fluid surface interaction (FSI) model for studying aortic dissection.

Methods:
We developed this FSI model by first segmenting aortas from CT scans to generate patient-specific models. With these meshes, we used a cardiac cycle fluid velocity profile to perform CFD and extracted static pressure values on the aortic wall at systole and diastole. We applied these pressures to our FEA model as the mechanical load driving wall deformation.

Results:
We successfully completed the CFD portion of our FSI simulation with results consistent with our expectations. Velocity data at the ascending and descending aorta, branches and arch correctly reflect our input cardiac cycle velocity profile. Histograms of static pressure values on the aortic wall at systole and diastole show that the mean systolic pressure is greater than the mean diastolic pressure, thus reflecting our expectations. Heat maps of this data reveal the aortic arch and branches to be areas of highest pressure, with pressure gradually decreasing in the distal aorta.

Conclusion:
With our healthy aorta control, we have successfully validated the CFD component of our FSI model. Our methods improve upon previous models by coupling CFD with FEA, allowing the use of realistic pressure loads derived from fluid analysis. We plan to further improve by validating the FEA component of our model and applying our methodology to dissection geometries. With this FSI workflow, we hope to more accurately model and predict the deformation patterns of aortic dissection in fragile aortas.
Abstract 593: Efficacy Of Induced Pluripotent Stem Cell-derived Endothelial Cell Therapy Is Impaired By Chronic Nicotine Exposure In A Murine Model Of Peripheral Artery Disease
Authors: Chan Alex H , Hu Caroline , Chiang Gladys , Huang Ngan F
Published: October 2023

Treatment strategies for peripheral artery disease (PAD) remain limited due to the many challenges of surgical intervention. Stem cell therapy offer a promising option, but barriers exist to their clinical translatability. Among these barriers include lifestyle choices like nicotine use that may influence the efficacy of stem cell therapy. The objective of this study aims to examine the efficacy of iPSC-EC therapy for treatment of PAD in mice with prior chronic exposure to nicotine. We hypothesize that chronic nicotine exposure negatively affects the therapeutic potential of implanted iPSC-ECs in a murine model of PAD. Immunocompromised (NSG) mice were implanted with an osmotic pump for controlled exposed to nicotine or saline (control) for 28 days. Cotinine levels confirmed plasma nicotine presence. After 28 days of nicotine or saline exposure, the mice underwent induction of hindlimb ischemia and before receiving an injection of 10
6
iPSC-ECs. A second dose of cells were injected to the ischemic limb on day 7 post-surgery. All mice were monitored for blood perfusion recovery using laser Doppler spectroscopy imaging. At day 14 post-surgery, mice were euthanized, and the gastrocnemius muscle was harvested for histological analysis. The results show that the average cotinine concentration in sera was ~200 ng/mL, which is within physiological range for a tobacco user. Blood perfusion recovery at day 14 showed that nicotine treated groups have impaired perfusion recovery, compared to control mice treated with saline. This finding was supported by histological analysis of capillary density showing decreased angiogenesis in the nicotine group after iPSC-EC therapy. In conclusion, nicotine exposure was associated with poorer blood perfusion recovery after iPSC-EC therapy, compared to control saline exposure, suggesting that chronic nicotine exposure may have adverse effects on the efficacy of iPSC-EC therapy for treatment of PAD.
Abstract 594: E-selectin-Overexpressing Mesenchymal Stem Cell-based Therapy Augments Cutaneous Wound Healing In Ischemic Limbs
Authors: Huerta Carlos T , Ortiz Yulexi , Li Yan , Ribieras Antoine , Voza Francesca , Le Nga , Liu Zhao-Jun , Velazquez Omaida C
Published: October 2023

Objective:
Tissue loss considerably worsens the risk of extremity amputation in patients with chronic limb-threatening ischemia (CLTI), and unmodified mesenchymal stem cell (MSC) therapies have failed to demonstrate more than minor clinical benefits for inducing therapeutic angiogenesis and wound healing. Here we describe the effects of MSCs engineered to overexpress E-selectin, a cell-adhesion molecule capable of stimulating neovascularization, using a translational murine model recapitulating hindlimb ischemia with cutaneous tissue loss.

Methods:
MSCs were harvested from bone marrow of 8-10-week-old FVB/Rosa26Sor
mTmG
donor mice and verified as CD44
+
/CD73+/CD105
+
/CD29
+
/Sca-1
+
by flow cytometry. MSCs underwent viral transduction with E-selectin-GFP/AAV or GFP/AAV. Femoral artery ligation was performed in 12-14-week-old recipient FVB mice followed by a 4 mm cutaneous wound in the ipsilateral limb and local injection of PBS or 1x10^6 donor GFP
+
/MSCs or E-selectin
+
/MSCs (E-sel
+
/MSC). Wounds were monitored daily for 7 days and then harvested.

Results:
Unmodified MSCs do not express E-selectin, and E-sel
+
/MSCs retained MSC phenotype. Mice receiving E-sel
+
/MSC treatment (n=10) demonstrated accelerated wound closure compared to both GFP
+
/MSCs (n=10) and PBS (n=10) treated mice at each post-operative day (POD) with the highest degree of difference observed at POD 5 (94 ± 3%
vs.
79 ± 10% GFP
vs.
72 ± 9% PBS, p<0.001) and 7 (98 ± 2%
vs.
86 ± 8% GFP
vs.
87 ± 6% PBS; p<0.010). Collagen deposition was greater in wounds treated with E-sel
+
/MSCs (21 ± 4%
vs.
5 ± 1% GFP
vs.
9 ± 1% PBS). DiI perfusion at POD 10 demonstrated greater vessel density in wounds treated with E-sel
+
/MSCs (76 ± 15%
vs.
29 ± 7% GFP
vs.
9 ± 5% PBS relative vascular density). E-sel
+
/MSCs demonstrated improved viability with more mTmG
+
-E-sel
+
/MSCs (13 ± 3 cells/high powered field (HPF)
vs.
GFP
+
/MSCs 3 ± 2 cells/HPF
vs.
PBS 0 ± 0 cells/HPF, p<0.010) at POD 7 on immunofluorescent microscopy.

Conclusion:
Cell therapy using E-selectin-overexpressing MSCs overcomes impairments in wound healing of ischemic limbs. These data exhibit the potential role for E-selectin-modified MSCs as a novel cell therapy in future clinical applications for delayed and non-healing wounds associated with CLTI.
Abstract 600: Percutaneous Transhepatic Approach To Patent Foramen Ovale Closure In A Patient With Congenital Inferior Vena Cava Interruption
Authors: Nwaedozie Somto , Soodi Deepa , Sharma Param , Girotra Sudhakar , Shah Milind
Published: October 2023

Percutaneous closure of Patent foramen ovale (PFO) is indicated in patients with cryptogenic stroke to reduce the risk of recurrent embolic events. Transfemoral access has been the mainstay approach to PFO closure, but in patients with congenital IVC interruption, trans-hepatic approach can be successfully performed. A 59 year old man with medical history of Type II DM presented with sudden left sided weakness of seven hours duration. On brain imaging he was found to have right hemispheric cortical infarcts without significant cerebrovascular stenosis. Hypercoagulable work up was unremarkable and underlying Atrial fibrillation was ruled out by continuous cardiac telemetry. Transthoracic echocardiography with bubble study demonstrated a PFO with interatrial shunt. As a result, the etiology of his stroke was attributed to paradoxical embolism and he was referred for a PFO closure. Intra-procedure, IVC angiogram demonstrated an interrupted IVC at the intrahepatic segment with continuation of the IVC by prominent hemiazygos and azygos veins which re-directed the IVC catheter into the thorco-abdominal hemi-azygos system (Fig 1A). An MRI done demonstrated similar findings and a supra-hepatic continuation of the IVC into the right atrium (Fig 1B). His Internal-jugular veins were also considered unsuitable access, as a result an alternative trans-hepatic venous approach was adopted in collaboration with interventional radiology which resulted in successful closure without any complications. Congenital anomalies of the IVC are uncommon but can preclude conventional trans-femoral venous interventions. Recognition of these anomalies can aid pre-procedural planning and multidisciplinary collaboration essential for optimal outcome. Percutaneous PFO closure in patients with interrupted IVC anomalies can be successfully performed through a trans-hepatic approach in collaboration with interventional radiologists.
Abstract 605: Mechanistic And Therapeutic Implications Of A Non-canonical Amino Acid Starvation Response In Restenosis Post Angioplasty
Authors: Yin Li , Shirasu Takuro , Islam Zain , Kent Eric W , Hoyt Nicholas , Le Quang , Huang Yitao , Zhang Mengxue , Patel Paranjay , Ahn JungEun , Lian-Wang Guo , Wang Bowen
Published: October 2023

Introduction:
Branched-chain amino acid-limited diet, such as leucine restriction (LR), has demonstrated pleiotropic cardiometabolic benefits. Prior studies exclusively focused on the GCN2-mediated, canonical amino acid starvation response (AASR) as the primary mechanism. Recent experimental evidence points to the existence of a non-canonical AASR, driven by GCN1. However, neither the therapeutic potential of dietary LR nor the implication of non-canonical AASR has been studied in restenosis following endovascular repairs from smooth muscle cells (SMC) hyperplasia. In this study, we aimed to probe the outcome of LR and dissect the roles of GCN1 vs GCN2 in restenosis.

Methods and Results:
Male Sprague Dawley rats were subjected to dietary LR (20% leucine level) or iso-caloric control two weeks before carotid artery angioplasty. Two weeks post-procedure, animals in the LR group showed reduced restenosis, as evidenced by a 53.6% reduction in intima-to-media (I/M) ratio (0.61±0.23 vs 1.32±0.15, p<0.01). To dissect the contribution from canonical vs non-canonical AASRs, we conducted intraluminal infusion of specific shRNA-expressing lentivirus and scrambled control in angioplastied arteries of rats subjected to LR. While GCN2 local knockdown showed minimal impact on I/M ratio compared to control (0.59±0.12 vs 0.74±0.20, p=0.62), GCN1 silencing effectively nullified LR-mediated protection against restenosis (1.21±0.39 vs 0.74±0.20, P<0.05). Moreover, GCN2 over-expression (via adenovirus) or GCN1 silencing (via siRNA) aggravated PDGF-BB-induced hyperproliferation measured by BrdU assay in primary culture of human aortic SMC, further indicating the divergent roles of GCN1 vs GCN2.

Conclusions:
Our study provides experimental evidence supporting the role of dietary LR in restenosis management. Additionally, the GCN1-mediated, non-canonical AASR is demonstrated as the primary mediator of the anti-restenotic benefits of LR, while the GCN2-mediated canonical AASR displays a paradoxical, disease-promoting role in this context.
Abstract 612: Biochemical And Structural Regulation Of The Enzymatic Degradation Of Blood Clots
Authors: Risman Rebcca , Percoco Victoria , Shroff Mitali , Bannish Brittany , Tutwiler Valerie
Published: October 2023

Blood clots perform a critical biological function in cessation of bleeding following injury. However, their function is transient and after performing their physiological function they must be resolved via fibrinolysis. Fibrinolysis is the enzymatic cleavage of the blood clot component fibrin(ogen). Excessive, early fibrinolysis can lead to bleeding whereas impaired fibrinolysis can result in heart attacks and strokes (thrombosis). The balance of resolution is a critical step in preventing life threatening complications, but the influence of changes in the fibrin structure on the biochemical regulation of fibrinolysis is poorly understood. We used turbidimetric experiments to assess the biochemical regulation of fibrinolysis and microscopy studies to characterize the microstructure of human blood plasma with varying network structure and its effect on fibrinolysis. Experiments are combined with a 3D stochastic multiscale computational model to probe the individual influence of changes in network density, pore size, and fiber diameter. Experimental studies show that increasing fibrinogen concentration leads to changes in fibrin density, pore size, and fiber diameter and ultimately results in impaired fibrinolysis. Analysis of simulations and experiments indicate that the pore size of the fibrin network makes the most significant contribution to the rate of fibrinolysis. We show that this effect is strongly influenced by the ratio of protein:enzyme when compared to absolute enzyme concentration. Our findings suggest that fibrin structure, which is altered in pathological conditions, is an important factor for the development of enzymatic treatments with increased efficacy in treating bleeding and/or thrombosis.
Abstract 623: A Novel Strategy For Reducing Future Events In Myocardial Infarction Survivors
Authors: Zhang Yunjia , Glaros Elias , Hung Tzong , Zhang Richard , Rayner Benjamin S , Wu Ben , Ku Jacqueline , Cochran Blake J , Devalaraja Matt , Thomas Shane , Rye Kerry Anne
Published: October 2023

Objective:
Myocardial infarction (MI) is the primary cause of ischaemic heart disease. ApoA-I, the main apolipoprotein constituent of high-density lipoproteins (HDLs) has anti-inflammatory properties and protects against post-MI ischemia/reperfusion (I/R) injury. However, HDLs from MI survivors do not protect against I/R injury. This study asks whether a newly developed peptide that has anti-inflammatory properties can mimic the ability of apoA-I to reduce I/R injury in a rat model of MI, and protect myocardial fibroblasts from hypoxic damage.

Methods:
The left anterior descending (LAD) coronary artery of Wistar rats (n≥6/group) was temporarily ligated for 1 h to induce I/R injury. Rats that underwent the procedure were randomly allocated to no treatment or treatment with PBS or peptide. The procedure was also performed on rats without LAD ligation (sham control). The rats were treated immediately after the procedure with peptide (10 mg/kg, ip) or PBS, then re-treated every second day for 7 days. H9C2 myocardial fibroblasts were incubated for 24 h under hypoxic (0.4 % oxygen) conditions ± peptide (0.05-1 mg/ml).

Results:
Peptide treatment improved the ejection fraction of rats that underwent LAD ligation by 16.4±7% (p<0.05) compared to control. It also reduced fibrosis in the left ventricle by 12.6±2.6% (p<0.0001). These results were recapitulated
in vitro
in myocardial fibroblasts incubated under hypoxic conditions, with peptide treatment (1 mg/ml) decreasing cell death by 19.8±4.0% (p<0.05).

Conclusions:
The peptide protects myocardial cell viability and reduces myocardial death and is therefore of potential therapeutic benefit for MI survivors with I/R injury.
Abstract 630: Cardiac Myxoma Tumor Dynamics-a Mathematical Monte Carlo Simulation As A Basis For Tumor Vaccine
Authors: Khalid Yaser , Dasu Kirti , Lee Michael , Gabriel Nielsen , Umrani Rohan , Roy Trinava , Dasu Neethi , Avuthu Sindhupavani
Published: October 2023

Introduction:
Mathematical models analyzing tumor-immune interactions can be used to better understand tumors and specifically target tumor antigens. Cardiac myxomas produce vascular endothelial growth factor, which likely induces angiogenesis for tumor growth. We created a model using synthetic antigens which are tumor epitopes expressed by cardiac myxomas, to both test the strength of a model human immune system and the immune system’s response.

Methods:
We constructed a model utilizing MATLAB to numerically simulate all equations describing tumor-immune growth, antigen presentation, host immune response, and interaction rates. The immune system is modeled through thirteen coupled differential equations, in which each equation exhibits the rate of change of a cell population in terms of growth, death, cell-cell kill, cell recruitment, and cell inactivation. This model has been modified further to introduce the addition of cardiac myxoma “vaccines” using Monte-Carlo processes to simulate an antigen stimulation response to a variety of HLA epitopes.

Results:
We used 12 different tumor antigen epitopes from cardiac myxomas to test the immune system response: 5 from the ANXA3 gene, 1 from ACOX2, 1 from MIA, 1 from PLA2GA2, 1 from PRKAR1, 1 fromNKX2-5, 1 from MEF2, and 1 from GATA4. Our mathematical model and Monte Carlo simulation showed that a robust immune response can be generated if the immune system recognizes epitopes that are between 41 to 452 amino acids long.

Conclusion:
We showed that if a synthetic epitope is between 41-452 amino acids long, a host will produce an immune response to cardiac myxomas.
Abstract 651: The Chromatin Remodeler Brg1 Drives Adventitial Progenitor Cell Myofibroblast Differentiation And Vascular Fibrosis
Authors: Jolly Austin , Lu Sizhao , Dubner Allison , Noble Tysen , Weiser-Evans Mary C
Published: October 2023

Cardiovascular diseases lead to stiffening of blood vessels. We identified a population of smooth muscle-derived progenitor cells that reside in the adventitial layer (AdvSca1-SM cells) that drive vascular fibrosis after acute vascular injury. AdvSca1-SM cells can differentiate into myofibroblasts and secrete pro-remodeling factors including extracellular matrix proteins and pro-inflammatory cytokines. While the response of AdvSca1-SM cells to vascular injury has been studied, the epigenetic changes that influence their differentiation towards myofibroblasts is poorly understood. We observed Brahma-related gene 1 (Brg1), a highly conserved chromatin remodeling ATPase, is upregulated in AdvSca1-SM cells in response to vascular injury and may be a key factor in regulating gene expression to influence AdvSca1-SM differentiation. We hypothesize that Brg1 induces expression of gene sets associated with remodeling and fibrosis and ultimately drives AdvSca1-SM cell differentiation towards pathologic myofibroblasts.

Results:
Carotid arteries from mice subject to carotid ligation exhibit decreased perivascular collagen deposition, smaller neointima, decreased adventitial expansion, decreased numbers of alpha-actin positive AdvSca1-SM cells, and fewer infiltrating macrophages when treated with the pharmacological Brg1 bromodomain inhibitor PFI-3 as compared to mice that received vehicle control. In a cell culture system, TGF-β induces AdvSca1-SM cells to express myofibroblast-specific genes, including alpha actin and periostin, and demonstrate enhanced contractile function in collagen contraction assays. PFI-3 blocks TGF-β induced myofibroblast gene expression and dampens contractile function. Mechanistically, CUT&RUN assays reveal that TGF-β leads to recruitment of Brg1 to the promoters of fibrosis-related genes, and this recruitment is blocked by PFI-3. This suggests that TGF-β induces Brg1 to remodel chromatin and drive differentiation towards myofibroblasts. These findings suggest Brg1 is a major regulator of vascular remodeling and may be a targetable protein to help people who suffer from disease-related vascular stiffening.
Abstract 660: Injury Induced Connexin 43 Expression Plays A Role In Vascular Endothelial Wound Healing
Authors: Sedovy Meghan W , Leng Xinyan , Iqbal Farwah , Malek Arya , Roberts Kailynn , Johnstone Scott R
Published: October 2023

Vascular surgeries, such as stent placement and coronary artery bypass, damage the endothelial lining of blood vessels. Impaired healing of endothelial damage is a primary contributor to vascular stent and bypass failure, putting patients at risk for myocardial infarction and death. As such, it is necessary to identify regulators of normal endothelial healing and understand how these factors are dysregulated when healing is impaired. Retrospective analysis of RNAseq data from a previous study investigating non-injured vs. regenerating endothelial cells (EC) in mouse aortas revealed a significant increase in the expression of connexin 43 (Cx43) throughout the EC healing process. Although Cx43 functions are important for skin wound healing, the role of connexin 43 in large artery endothelial wound healing has never been investigated. Here, we test the hypothesis that injury-induced increases in Cx43 expression and gap junction functions promote vascular endothelial wound healing. To study Cx43 in the regenerating endothelium in vivo, we developed a novel model of endothelial cell (EC) injury in mouse carotid arteries. In this model, carotids are ligated for a period of one minute, resulting in an EC denudation injury that heals over 24-hours. Western blotting of EC isolated from damaged carotids demonstrated an increase in Cx43 protein in injured mouse EC while immunofluorescent imaging revealed increases in Cx43 staining at EC borders indicative of increased gap junction signaling. Human coronary artery EC scratch assays were also used to study the process of endothelial wound repair in vitro. As Cx43 phosphorylation is the primary regulator of gap junction channel signaling, we used pharmacological strategies (novel peptide treatments) to increase protein kinase C (PKC) phosphorylation at Cx43 serine 368 (Cx43-pS368), associated with gap junction closure and reduced intracellular communication. In cultured EC, increasing Cx43-pS368 resulted in slower wound healing times, suggesting that limiting Cx43 gap junction communication impairs EC wound healing. In conclusion, our data indicate that injury-induced EC upregulation of Cx43 and increases in gap junction intracellular communication may play a key role in EC wound healing responses.
Abstract 661: Wnt16 Promotes Smooth Muscle Mitochondrial Function And Contractile Phenotype Via Taz (Wwtr1) Relays, Limiting Aneurysmal Dilatation Of Ascending Aorta
Authors: Behrmann Abraham S , Zhong Dalian , Xie Shangkui , Li Li , Lemoff Andrew , Kozlitina Julia , Towler Dwight A
Published: October 2023

Wnt16 is selectively expressed in arterial vascular smooth muscle (VSM) as well as in bone and skin. Non-synonymous
WNT16
cSNPs that increase risk for low bone mass also convey risk for composite cardiovascular events (stroke, MI, heart failure, CV death) in the Dallas Heart Study after adjustment for age, sex, ancestry, systolic blood pressure, and body mass index (OR = 1.25, p = 0.02) . To better understand Wnt16’s role in cardiovascular biology, we generated Wnt16-/-;LDLR-/- mice. Blood pressure and pulse wave velocity were reduced in Wnt16-/-;LDLR-/- mice vs. LDLR-/- controls. Ascending aorta (AscAo) dilatation following angiotensin-II (AngII) infusion was worsened with Wnt16 deficiency, while no differences existed at baseline. Contractile markers such as
Acta2
- known to convey AscAo aneurysm risk and reduce pressure with genetic deficiency - were down-regulated in Wnt16-/- VSM, with reduction in TGFbeta-induced contraction. De-differentiation markers (C3, Sca1) were upregulated, and mitochondrial respiration (Seahorse oximetry) was significantly impaired with Wnt16 deficiency. Acta2 protein was reduced with Wnt16 deficiency as was aortic Ankrd1, a target of Yap and Taz(Wwtr1) activation via nuclear TEAD-directed transcription. Wnt16-/- VSM exhibited reduced nuclear Yap+Taz. RNAi targeting
Wnt16
or
Taz
(not
Yap1
) phenocopied Wnt16 deficiency, inhibited Wnt16 up-regulation of contractile markers and reversed
C3
suppression. Wnt16 deficiency increased the Yap-Taz inhibitory protein Ccm2 in VSM. Moreover, RNAi targeting Yap-Taz inhibitory kinases
Lats1/2
or
Ccm2 --
but not
Ccm3
or
Stk3/Stk4 --
elicited responses reciprocal to Wnt16 deficiency. Biotin proximity labeling following expression of Taz-BioID2 fusion protein identified TEAD1, IQGAP3 and specific MAP4K components of the STRIPAK complex as functionally important Wnt16-Taz signaling mediators. TEAD cognates in
Acta2
and
C3
promoters convey responses to Wnt16 and Taz+Tead1, and Wnt16 treatment increased Taz association with the
Acta2
promoter in aortic VSM. Thus, Wnt16 promotes the contractile phenotype and mitochondrial function in VSM and limits AngII aneurysmal remodeling in AscAo. Strategies that augment the VSM Wnt16-Taz relay may mitigate aneurysmal remodeling.
Abstract 664: Essential Roles Of Prdm16 In Repression Of Venous Marker In The Artery
Authors: Thompson Michael , Sakabe Masahide , Xin Mei
Published: October 2023

Proper vascular formation is regulated by multiple signaling pathways. The vascular endothelial growth factor (VEGF) signaling promotes endothelial proliferation, and Notch and its downstream targets act to lead endothelial cells toward an arterial fate through regulation of arterial gene expression. However, the mechanisms of how endothelial cells (ECs) in the artery maintain their arterial characteristics remain unclear. Here, we show that PRDM16 (positive regulatory domain-containing protein 16), a zinc finger transcription factor, is only expressed in arterial ECs but not venous ECs in developing embryos and neonatal retinas. Endothelial-specific deletion of
Prdm16
induced ectopic activation of venous gene expression in the arterial ECs and reduced vascular smooth muscle cell (vSMC) recruitment around arteries. Whole-genome transcriptome analysis using isolated brain ECs showed that the expression of
Angpt2
(encoding ANGIOPOIETIN2 that inhibits vSMC recruitment) is upregulated in the
Prdm16
knockout ECs. Conversely, forced expression of
Prdm16
in venous ECs is sufficient to induce arterial gene expression and repress ANGPT2 level. Together, these results reveal an arterial cell-autonomous function for PRDM16 in suppressing venous characteristics in arterial ECs.
Abstract 676: Novel Human Variants Of SR-B1 Impact HDL-cholesterol Clearance
Authors: Powers Hayley , Sahoo Daisy
Published: October 2023

The reverse cholesterol transport (RCT) pathway is the primary avenue by which our bodies lower plasma cholesterol levels. In RCT, peripheral cholesterol is transported by high density lipoproteins (HDL) to the liver for excretion. Cholesterol delivery is facilitated by a high-affinity interaction between HDL and its receptor, scavenger receptor class B member 1 (SR-B1). Recently, novel variants within the gene encoding SR-B1 were identified in a population of Icelandic people. Four missense mutations - V32M-, V111M-, V135I-, and G319V-SR-B1- were associated with elevations in plasma HDL-cholesterol. Based on these clinical phenotypes, we hypothesized that these conserved variants of SR-B1 would disrupt HDL-cholesterol transport. To assess the functions of the variants, we transiently transfected vector, wild-type, or mutant SR-B1 constructs into COS7 cells which express low endogenous SR-B1. All mutants displayed decreased DiI-HDL binding, yet only G319V-SR-B1 displayed a significant decrease in DiI lipid uptake, by 42% compared to wild-type. SR-B1 also mediates cholesterol efflux from cells into HDL. When assayed, only V32M- and G319V-SR-B1 decreased cholesterol efflux to HDL by 25 and 52%, respectively, compared to wild-type. Additionally, we demonstrated a decrease in total and cell surface expression of the G319V-SR-B1 variant compared to wild-type SR-B1, suggesting that impairments in function could be due to decreased expression. SR-B1 forms oligomers to facilitate its functions, and all variants maintained the ability to form higher order oligomers. To further characterize the SR-B1 variants, we utilized a recently-developed purification system to generate full-length SR-B1 protein and performed microscale thermophoresis to monitor changes in affinity for HDL. Paralleling our results from cellular assays, all SR-B1 variants displayed impaired HDL binding. When mapped to a homology model of full-length SR-B1, all four mutations are within the extracellular region, but the functional perturbations caused by each mutation warrants further studies. These mutants, combined with previously characterized SR-B1 variants, can be integrated as important pieces in the puzzle of cholesterol homeostasis and cardiovascular disease.
Abstract 677: Short Hydrocarbon-Stapled Apolipoprotein E Mimetic Peptides Enhance Lipoprotein Uptake
Authors: Reimund Mart , Graziano Giorgio T , Lucero Diego , Sviridov Denis O , Pryor Milton , Tang Jingrong , Remaley Alan T
Published: October 2023

ApoE facilitates the clearance of apoB-containing lipoproteins by binding to heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1). ApoE has a receptor binding and a lipid binding domain, which are both needed for its biological function. Several apoE-based peptides have been developed that lower plasma lipid levels in animal models and in early-stage clinical trials. However, these peptides are long (28-61 residues), costly to synthesize and are potentially immunogenic as they often contain many amino acid substitutions that vary from the native apoE sequence. We developed novel short (10-12 residues) apoE mimetic peptides based on the apoE receptor binding region (residues 141-150) containing only 2 amino acid substitutions. We used hydrocarbon stapling to stabilize the native alpha helical structure of this region and to increase the lipid binding affinity of the peptides. In addition, octanoic or myristic acid was attached to the N-terminus of the peptides to further increase lipid binding affinity. Helical conformation and lipid binding of the peptides were confirmed by circular dichroism spectroscopy and phospholipid vesicle solubilization assay. Our peptides increased the uptake of fluorescent tagged VLDL and LDL in a concentration dependent manner (0.625-20 μM peptides) in both wild-type (WT) and LDLR knockout (KO) HepG2 cells as measured by flow cytometry. In addition, an all D-amino acid version of one of the peptides had a similar effect on LDL uptake as its all L-amino acid version. Altogether, this suggests that peptide-mediated uptake is LDLR independent. Treatment of WT HepG2 cells with heparinases diminished the low-dose effect (up to 2.5 μM) of the peptides on LDL uptake, indicating that HSPGs plays a role in peptide-mediated LDL uptake. Finally, the peptides (4.6 mg/kg, IV) lowered plasma total cholesterol (37% at 3h; 29% at 6h) and triglycerides (33% at 1h) in apoE-KO mice on a standard chow diet. In summary, the novel apoE peptides are shorter than previously reported peptides, are more similar to the native sequence of apoE, and are likely resistant to proteolysis, because of hydrocarbon stapling and, therefore, could potentially be developed into an oral therapy.
Abstract 684: Hepatic Oxalate Overproduction As A Driver And Therapeutic Target In NASH And Atherosclerosis
Authors: Das Sandeep , Finney Alexandra C , kumar Dhananjay , Liu Yuhao , Rohilla Sumati , Anand Sumit Kumar , Nunez Kelly , liu zhipeng , Pearson-Gallion Brenna , Abdullah Chowdhury S. , Cockerham Elizabeth D , Bhuiyan Md Shenuarin , Orr Wayne W , Garcia-Barrio Minerva T , Zhang Jifeng , Liu Wanqing , Chen Y. Eugene , Thevenot Paul , Cohen Ari , Yurdagul Arif , Rom Oren
Published: October 2023

Affecting one third of the global population, with no pharmacotherapy available, nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease. Surprisingly, the major cause of death in patients with NAFLD, particularly in those with the more severe nonalcoholic steatohepatitis (NASH), is atherosclerotic cardiovascular disease (CVD). Thus, there is a critical need to identify targetable pathways for concurrent treatment of NASH and atherosclerotic CVD, which has been hampered by limited understanding of the pathophysiology and metabolic pathways linking these two diseases. Recently, we and others uncovered oxalate metabolism commonly dysregulated in NAFLD and CVD. Using unbiased transcriptomics, we identified suppression of genes that limit oxalate production in livers from humans and mice with NASH. Alanine-glyoxylate aminotransferase (AGXT), a liver-specific enzyme that detoxifies glyoxylate, the oxalate precursor, was reduced and oxalate was markedly increased in correlation with NASH severity. Remarkably, oxalate was also increased both in patients and mice with atherosclerosis. In our mouse models of hepatic oxalate overproduction (
Agxt
-/-
and
Agxt
-/-
/

Apoe
-/-

), both NASH and atherosclerosis were increased with suppressed hepatic fatty acid β-oxidation (FAO) and induction of proinflammatory pathways. In hepatocytes, oxalate induced mitochondrial dysfunction and lipid accumulation while downregulating peroxisome proliferator-activated receptor α (PPARα), inhibiting FAO and upregulating C-C motif chemokine ligand 5 (CCL5). Importantly, limiting hepatic oxalate production via hepatocyte-specific overexpression of AGXT (AAV8-TBG-AGXT) reduced NASH and atherosclerosis in mice by stimulating FAO and attenuating proinflammatory responses. Together, our findings uncover hepatic oxalate overproduction as a dysregulated metabolic pathway linking NASH and atherosclerotic CVD, highlighting the potential of oxalate reduction for concurrent treatment of these two prominent diseases.