bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1021: Bulky Hydrophobic Side Chains In The β1-sandwich Of Microsomal Triglyceride Transfer Protein Are Critical For The Transfer Of Both Triglycerides And Phospholipids
Authors: Anaganti Narasimha , Valmiki Swati , Recacha Rosario , Islam Shahidul , Farber Steven , Ruddock Lloyd , Hussain M M
Published: July 2024

Hyperlipidemia predispose individuals to cardio-metabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apoB-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intestine. Previous studies suggested two lipid transfer domains in MTP and that specific inhibition of triglycerides (TG) and not phospholipids (PL) transfer can lower plasma lipids without significant tissue lipid accumulation. However, how MTP transfers different lipids and the domains involved in these activities are unknown. Here, we tested a hypothesis that two different β-sandwich domains in MTP transfer TG and PL. Mutagenesis of charged amino acids in β2-sandwich had no effect on PL transfer activity indicating that they are not critical. In contrast, amino acids with bulky hydrophobic side chains in β1-sandwich were critical for both TG and PL transfer activities. Substitutions of these residues with smaller hydrophobic side chains or positive charges reduced, while negatively charged side chains severely attenuated MTP lipid transfer activities. These studies point to a common lipid transfer domain for TG and PL in MTP that is enriched with bulky hydrophobic amino acids. Furthermore, we observed a strong correlation in different MTP mutants with respect to loss of both the lipid transfer activities, again implicating a common binding site for TG and PL in MTP. We propose that targeting of areas other than the identified common lipid transfer domain might reduce plasma lipids without causing cellular lipid retention.
Abstract 1024: A Genome-wide CRISPR Screen Identifies The LDL Receptor As The Primary Mediator Of Lp(a) Uptake By HuH7 Cells
Authors: Bragazzi Cunha Juliana , Gani Khan Taslima , Leix Kyle , Burroughs Michael , Smrcka Alan , Emmer Brian T
Published: July 2024

Elevated Lp(a) blood levels have been causally linked to the development of atherosclerotic and valvular heart disease, yet the identity of the specific receptor(s) that mediate hepatic clearance of Lp(a) remains controversial. Here, we report the development, execution, and validation of a genome-wide CRISPR screen for functional modifiers of Lp(a) uptake by HuH7 cells. We first optimized a method for fluorescently labeling purified human Lp(a). Sensitive and specific detection of fluorescent Lp(a) uptake by HuH7 cells was performed by flow cytometry. We then used this approach to isolate subpopulations of individual cells with aberrant Lp(a) uptake from a pool edited with a library of 123,411 gRNAs targeting 19,050 genes across the human genome. Strikingly, the top positive regulator of Lp(a) uptake in our screen was
LDLR
, which encodes the low-density lipoprotein receptor.
MYLIP
, which encodes a ubiquitin ligase that degrades LDLR, was the top negative regulator. We also observed a significant influence for several other established LDLR regulators, including those with a canonical role in SREBP signaling and those discovered in our analogous prior screen of LDL uptake. No other previously proposed Lp(a) receptor had a significant contribution to Lp(a) uptake in our screen. We subsequently validated our screen results by engineering HuH7 cell lines with targeted deletion or overexpression of
LDLR
and confirmed their corresponding impact on Lp(a) uptake. Lastly, we used biolayer interferometry to detect in vitro binding between purified Lp(a) and recombinant LDLR ectodomain. Together, our findings are consistent with a model in which hepatic clearance of Lp(a) is primarily mediated by the LDL receptor.
Abstract 1025: Structural Basis Of Apolipoprotein A1 Activation Of Lecithin:Cholesterol Acyl Transferase In High Density Lipoproteins
Authors: Coleman Bethany , Bedi Shimpi , Morris Jamie C , Hart Rachel , Jerome W. Gray , Manthei Kelly A , Vaisar Tomas , Tesmer John , Aller Stephen , Davidson William
Published: July 2024

Lecithin:cholesterol acyl transferase (LCAT) is responsible for the formation of extracellular cholesteryl ester, which is critical for high density lipoprotein (HDL) maturation. It also modulates numerous HDL physiological functions by impacting particle subspeciation patterns. LCAT is activated by apolipoprotein A1 (APOA1) which forms antiparallel dimers around the edge of nascent HDL. Previous work has suggested that the interface of APOA1 helices 4 and 6, when properly aligned, creates two symmetrical sites for productive LCAT binding. Size exclusion chromatography was used to isolate reconstituted HDL discs containing two molecules of APOA1 chemically crosslinked to 2 LCAT molecules. Mass spectrometry using multiple crosslinkers, including a novel application of formaldehyde, indicated that LCAT interacts with both APOA1 helices 4 and 6. Cryo-electron microscopy (cryoEM) was used to determine a 10 Å structure of the complex. Using the HADDOCK suite of molecular docking tools, we developed a structural model of LCAT bound to APOA1 discs that is consistent with the preponderance of data accumulated so far. The model shows that LCAT may bind at the two symmetrical sites in different orientations, possibly reflecting different steps of the LCAT reaction. Work to improve the resolution of the cryoEM datasets is ongoing. Further work, using strategically designed cysteine mutations in APOA1 to lock helices 4 and 6 together, suggests that the helices do not move apart during the LCAT reaction process. Determination of the structural interactions between LCAT and its activator, APOA1, is crucial to understanding HDL maturation, which has significant clinical implications. This work provides the highest resolution structural model available for this important interaction.
Abstract 1029: Angiotensin II-induced Hypertension Promotes Innate Immune Memory And Predisposes To Atherosclerosis Progression
Authors: Dungan Matthew M , Bracken Ronan C , Wassenaar Jean W , Fehrenbach Daniel J , Gonzalez Azuah L , Fuller Kristin K , Rahman Jamshed , Madhur Meena S , Brown Jonathan D , Doran Amanda C
Published: July 2024

Atherosclerotic cardiovascular disease is the leading cause of death globally. Hypertension is the most significant risk factor for atherosclerosis. Despite aggressive lowering of cholesterol and blood pressure, up to 50% of patients suffer recurrent cardiac events. This “residual risk” is thought to be due to untreated, chronic inflammation. Recently, a phenomenon that facilitates long-lasting innate immune activation called ‘Trained Immunity’ has been identified. Trained Immunity is driven by epigenetic and metabolic reprogramming of innate immune progenitors in response to specific stimuli. This ‘priming’ of progenitors results in innate immune cell progeny that are epigenetically and transcriptionally rewired to mount an augmented inflammatory response. We hypothesize that hypertension induces trained immunity in myeloid progenitor cells, leading to persistent monocyte activation and increased atherosclerosis progression.

To study hypertension’s effects on hematopoietic stem and progenitor cells (HSPCs) in atherosclerosis, we induced angiotensin II (Ang II)-mediated hypertension in donor mice for 2 weeks, followed by bone marrow transplant from the hypertensive mice into irradiated atherosclerosis-prone

Ldlr
-/-

mice. After 10 weeks on a high-fat diet, we observed that Ang II-induced hypertensive marrow recipients had accelerated atherosclerosis compared to normotensive marrow recipients. Further, RNA-seq and ATAC-seq analysis of monocyte progeny from transplanted Ang II-trained HSPCs demonstrated durable transcriptional and epigenetic signatures indicative of Trained Immunity. In vitro treatment of HSPCs with Ang II before differentiation into macrophages led to an augmented pro-inflammatory cytokine response to LPS stimulation. We found that Ang II training activated Dectin-1, a receptor involved in other forms of immune training, and blocking of Dectin-1 inhibits the augmented cytokine response. Our results suggest that Ang II induces trained immunity of immune progenitors via a Dectin-1-mediated pathway. These findings represent a novel mechanism by which hypertension contributes to the long-term increased risk of atherosclerosis in hypertensive patients.
Abstract 1038: Map Kinase Phosphatase-5 Deficiency Impairs Macrophage Efferocytosis And Exacerbates Late-stage Atherosclerosis
Authors: Baldini Margaret , Palos-Jasso Andrea , Azuma Masahi , Yu Jun , Shan Dan , Roy Rajika , Koch Walter , Gao Erhe , Zhong Chao
Published: July 2024

Objective:
Atherosclerosis is a vascular inflammatory disease that is a major cause of death in Westernized society. Late-stage atherosclerosis is characterized by a vulnerable plaque, which consists of a thin fibrous cap and a large necrotic core that is prone to rupture. Efferocytosis is a repair mechanism that clears apoptotic cells within the plaque, contributing to plaque stability. In late-stage atherosclerotic conditions efferocytosis is impaired and promotes rupture. MAPK phosphatase-5 (MKP5) regulates various processes such as immune diseases and cardiac fibrosis. The goal of this project is to identify the role of MKP5 in late-stage atherosclerotic plaque stability.

Approach/Results:
To induce late-stage atherosclerosis, male and female LDLR
-/-
and LDLR
-/-
MKP5
-/-
mice were subjected to transverse aortic constriction (TAC) surgery and fed on a highfat diet (western diet with 1.25% cholesterol) for 12 weeks. Our data show that MKP5 deficiency exacerbates plaque instability, through decreased cap thickness, decreased collagen content, increased lesion size and necrosis in the plaque, and severe coronary lesions. Furthermore, there are higher instances of intraplaque hemorrhage in the right carotids of the LDLR
-/-
MKP5
-/-
mice. Mechanistically, TUNEL staining of the aortic roots was performed to determine efferocytotic capabilities in vivo. Results showed fewer macrophage-associated TUNEL-positive cells in LDLR
-/-
MKP5
-/-
aortic roots. To further investigate this mechanism, bone-marrow-derived macrophages (BMDMs) isolated from WT and MKP5
-/-
mice were incubated with apoptotic Jurkat cells and then analyzed to determine the engulfment of apoptotic cells by the macrophages via confocal microscopy and flow cytometry. Results showed that efferocytosis was significantly inhibited in MKP5
-/-
BMDMs. Bulk-RNA sequencing analysis revealed that the expression of efferocytotic genes in MKP5
-/-
BMDMs was decreased at basal and oxLDL stimulated conditions.

Conclusion:
Results suggest that MKP5 deficiency impairs efferocytosis, exacerbates atherosclerosis, and causes late-stage plaque to move toward unstable phenotypes.
Abstract 1040: Investigating The Role Of Lipoproteins In Macrophage Er Stress
Authors: Bobek Jordan M , Sahoo Daisy
Published: July 2024

Atherosclerosis, a major cause of cardiovascular disease (CVD), is an inflammatory disease defined by the progressive buildup of lipid-laden macrophages and cholesterol-filled plaques. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) play critical roles in the pathogenesis of atherosclerosis due to their roles in transporting cholesterol between tissues and cell types, including macrophages. Epidemiological studies have shown that high levels of HDL-cholesterol (HDL-C) are correlated with decreased CVD. However, HDL-C raising therapeutics failed to show clinical benefits, suggesting that HDL function may be a better indicator of CVD risk. Recent studies from our lab demonstrated that HDL modified by reactive aldehydes has impaired cardioprotective functions in macrophages. Other studies have also shown that oxidized LDL triggers ER stress in vascular cells and macrophages, a process that can be blunted by the presence of HDL. Therefore, we tested the hypothesis that HDL modified by reactive aldehydes triggers ER stress in macrophages, with the goal of identifying mechanisms by which dysfunctional HDL contributes to atherosclerosis progression. Primary murine macrophages were incubated with native or modified forms of lipoproteins and ER stress markers (XBP1, BiP, p-eIF2a, ATF6) were assessed by immunoblot analysis or semi-quantitative PCR. Through a comprehensive and extensive analysis, our findings led us to discover three novel findings in primary murine macrophages: (i) oxLDL alone was unable to induce ER stress; (ii) co-incubation with oxLDL or HDL in the presence of thapsigargin (an inducer of ER stress) resulted in increased and prolonged expression of ER stress markers as compared to cells treated with thapsigargin alone; and (iii) neither HDL nor HDL modified by reactive aldehydes was able blunt the ER stress induced by thapsigargin in the presence or absence of oxLDL. In summary, our highly systematic approach to assess the role of lipoproteins in mediating ER stress in primary macrophages generated convincing data that refutes previously-published findings, thus resulting in an inability to make definitive conclusions on the role of HDL modification in ER stress.
Abstract 1054: Toll-like Receptor 4 Is A New Regulator Of Smooth Muscle Cell Phenotypic Transitions
Authors: Hong Junyoung , Cherepanova Olga A
Published: July 2024

Objectives:
There is growing evidence that smooth muscle cell (
SMC
) phenotypic transitions have a critical role in cardiovascular pathologies, including atherosclerosis. However, molecular mechanisms regulating different aspects of these transitions are still not fully elucidated. We previously found that the pluripotency factor OCT4 plays a protective role in SMC in atherogenesis and neointioma formation after vascular injury. Using a bioinformatics approach, we identified Toll-like receptor 4 (
TLR4
), an integral component of the innate immune system, pivotal in eliciting inflammatory responses, as an indirect target of OCT4 in SMC. However, it is not yet elucidated whether OCT4/TLR4 axis has a role in SMC transitions.

Methods and Results:
First, we used blocking siRNA against
Oct4
and different downstream targets of the TLR4 signaling (
Trif
,
Irf3
), as well as a TLR4 inhibitor TAK242 (Resatorvid) in cultured mouse aortic SMC treated with the oxidized phospholipid, POVPC. These experiments demonstrated that blocking TLR4 signaling in wild-type SMC resulted in suppression of SMC contractile genes and up-regulation of LGALS3 (SMC transition marker). In contrast, TAK242 treatment decreased LGALS3 levels in OCT4 deficient SMC, indicating that TLR4, at least in part, is responsible for the SMC phenotype in OCT4 deficient SMC. Next, using the bioinformatics approach and Chromatin Immunoprecipitation assays, we identified the EST Proto-oncogene 1 (
ETS1
) transcriptional factor as a target of OCT4 that directly regulates TLR4. Interestingly, on the other hand, we found that overexpression of TLR4 led to OCT4 activation in SMC, and, oppositely, the inhibition of TLR4 signaling by TAK242 resulted in significant decreases in the POVPC-induced OCT4 activation, indicating that TLR4 signaling is critical for OCT4 activation in SMC. Using an SMC-lineage tracing mouse model combined with a TLR4-IRES-YFP reporter, we demonstrated that TLR4 is highly expressed in the healthy arteries of young mice but not in the phenotypically modulated SMC within the atherosclerotic plaques.

Conclusions:
These results suggest a negative feedback loop between TLR4 and OCT4 regulating SMC phenotypic transitions.
Abstract 1057: Association Between Remnant Cholesterol And All-cause Mortality And Cardiovascular Mortality In Young And Middle-aged American Adults: A Retrospective Cohort Study
Authors: Du Dongjie
Published: July 2024

Background:
The association of remnant cholesterol with all-cause mortality and cardiovascular mortality in young, middle-aged American adults remains unclear. The purpose of the study is to explore this relationship.

Methods:
This retrospective cohort study included 16428 patients aged from 18 to 65 years old from the National Health and Nutrition Examination Survey (1999–2018) with follow-up mortality outcomes records linked to the National Death Index (NDI) until December 31, 2019. Participants were divided into four groups based on the RC quartile: a Q1 group (≤0.359 mmol/L), a Q2 group (0.359–0.517 mmol/L), a Q3 group (0.518–0.768 mmol/L), and a Q4 group (≥0.768 mmol/L). Multivariable Cox proportional hazards models were used to estimate the risk of mortality.

Results:
During a 123-month follow-up, a total of 931 (5.02%) all-cause deaths and 239 (1.22%) CVD-related deaths were recorded. After adjusting for multiple potential confounding factors and comparing them with the Q1 RC group of the participants, the adjusted hazard ratios for all-cause mortality for the Q2–Q4 groups were 0.98 (0.69, 1.38), 1.02 (0.74, 1.39), and 1.45 (1.09, 1.92) (
P
for trend = 0.001), and those for cardiovascular mortality were 1.41 (0.68, 2.94), 1.77 (0.86, 3.62), and 2.98 (1.38, 6.43), respectively (
P
for trend =0.001). The restricted cubic splines revealed a non-linear association between the baseline RC and cardiovascular mortality. Interestingly, an interaction effect was found between gender and RC in all-cause mortality.

Conclusion:
RC was associated with an increased risk of all-cause mortality and cardiovascular mortality in young and middle-aged American populations.
Abstract 1060: Association Of Inflammation And Cholesterol With Long-term Clinical Outcomes After In-stent Restenosis Revascularization
Authors: Han Zhang
Published: July 2024

Background:
Among patients with coronary artery disease, residual inflammatory risk assessed by high-sensitivity C-reactive protein (hsCRP) is at least as strong a predictor of future cardiovascular events as is residual risk assessed by low-density lipoprotein cholesterol (LDL-C).

Objectives:
To evaluate the relationships between relative importance of hsCRP and LDL-C as determinants of clinical outcomes among patients who underwent percutaneous coronary intervention (PCI) for in-stent restenosis (ISR) lesions.

Methods:
Between January 2017 and December 2018, a total of 2079 patients who underwent PCI for ISR were consecutively enrolled. The primary outcome was the rate of major adverse cardiac events (MACE), defined as a composite endpoint of all-cause death, spontaneous myocardial infarction (MI), or repeat revascularization. Quartiles of increasing hsCRP and LDL-C were assessed as predictors of clinical outcomes after adjustment for covariates. Patients were then categorized as four predefined residual risk groups according to hsCRP (≥2 mg/L vs. <2mg/L) and LDL-C levels (≥70mg/dL vs. <70mg/dL).

Results:
During a median follow-up of 36 months, 436 MACEs occurred. Baseline hsCRP was significantly associated with MACE (highest versus lowest quartile; adjusted hazard ratio [aHR], 1.90 [95% CI, 1.39-2.59]; P<0.001). By contrast, the baseline LDL-C quartile was not associated with MACE (highest versus lowest quartile; aHR, 0.93 [95% CI, 0.71- 1.22]; P=0.59). Compared with patients without residual risk (hsCRP <2 mg/L and LDL-C <70 mg/dL), participants with both residual inflammatory and LDL-C risk (hsCRP ≥2 mg/L and LDL-C ≥70 mg/dL) (aHR, 1.39 [95% CI, 1.06-1.83]; P=0.02) and those with residual inflammatory risk only (hsCRP ≥2 mg/L and LDL-C <70 mg/dL) (aHR, 1.34 [95% CI, 1.01-1.72]; P=0.04) had significantly higher risks of MACE.

Conclusions:
In the current cohort of patients after ISR PCI, inflammation assessed by hsCRP predicted higher risk of adverse clinical outcomes, whereas the level of LDL-C was not associated with prognosis.
Abstract 1073: Longitudinal Imaging Of NF-kappaB In Murine Pulmonary Embolism
Authors: Negron Steven , McCarthy Jason , Kessinger Chase W
Published: July 2024

Objective:
Pulmonary embolism (PE) is a common condition that urgently needs novel diagnostic and therapeutic strategies. Here, we investigated PE-induced inflammation via longitudinal imaging of transcription factor NF-kappaB (NFkB) activation signals in murine PE. Understanding the temporal activation of inflammatory mediators, such as NFkB, in PE may lead to future medical interventions or better patient stratification.

Methods:
NFkB activation was assessed using male, 12-16w old, NFkB-GFP-luciferase (NGL) reporter mice (n=20 PE animals). Stasis venous thrombosis (VT) was induced in the femoral veins of donor mice on day 0. On day 1, the thrombus was resected and intravenously injected into recipient mice to create the PE model. PE animals underwent bioluminescence imaging on days -1, 1, 4, and 7. Regions-of-interest (ROI) areas were kept constant and placed over areas of PE as verified in other modalities. Lung and PE morphometrics were imaged by fluorescence-mediated tomography and computed tomography using a fibrin-specific molecular imaging agent, FTP11 (i.v. 150nmol/kg). Cardiac function pre- and post-PE was assessed by ultrasound and pulmonary arterial pressure measurements. Mouse cohorts were sacrificed 1, 4, and 7 days following stasis-VT embolization and underwent histological analysis of the PE.

Results:
The mean volume of the femoral stasis-VT was 1.21±0.25 mm
3
. The bioluminescence signal plateau was found at 4-8 minutes post-intravenous injection of D-luciferin in PE animals. The six-minute post-injection image was utilized in comparing longitudinal images. Bioluminescence from the activation of NFkB in the PE ROI showed a maximal signal in the day 1 PE with a 55.0% (334 to 151RU) decrease in signal from day 1 to day 7(p<0.05). Histological and protein assays validated the activation and localization of NFkB. In PE animals, immunohistochemistry of infiltrating CD45-positive cells showed increased cell numbers from day 1 to day 7 (p<0.05). Since PE is confided spatially to a lung lobe, contralateral PE-negative lobes also showed increased infiltrating cells compared to sham animals.

Conclusions:
Quantitative serial imaging pre- and post-PE demonstrates an inflammatory timeline in PE-induced NFkB activation.
Abstract 1078: A Cross-species Platform And Phosphoproteomics Analysis To Mitigate Vegfri-induced Hypertension And Endothelial Dysfunction
Authors: Camarda Nicholas D , Lu Qing , Man Joshua , Martin Gregory , Travers Richard J , Lopez Katherine E , Meola Dawn M , Powers Sarah N , Song Zeyuan , Papanastasiou Malvina , DeRuff Katherine , Mullahoo James , Egri Shawn , Davison Desiree , Eblen Scott , Jaffe Jacob , Huggins Gordon , London Cheryl , Upshaw Jenica , Yang Vicky , Jaffe Iris Z
Published: July 2024

Introduction:
Vascular endothelial growth factor receptor inhibitors (VEGFRis) are a class of tyrosine kinase inhibitors (TKIs) used to treat human and canine cancers, but these drugs also cause hypertension (HTN) and endothelial cell (EC) dysfunction.

Purpose:
We combined phosphoproteomic analysis in ECs with a cross-species in-vitro and in-vivo approach to identify mitigating therapies for VEGFRi-induced EC dysfunction and HTN.

Methods and Results:
We developed a sorafenib-induced HTN mouse model showing increased blood pressure, mesenteric resistance vessel EC dysfunction (decreased phosphorylation of Ser1177 on endothelial nitric oxide synthase [p-eNOS], increased endothelin-1 [ET-1]), and renal glomerular endotheliosis. Human umbilical vein ECs were treated with VEGFR TKIs, non-VEGFR TKIs, and cardiovascular drugs. Phosphoproteomic mass spectrometry and a marker selection algorithm identified a 14-phosphopeptide signature differentiating VEGFR TKIs from non-VEGFR TKIs. The signature peptide RBM17 pS222 increased with VEGFRi treatment in human, canine, and mouse aortic ECs, correlating with reduced p-eNOS. By screening for cardiovascular drugs that reverse the VEGFRi phosphoproteomic signature, we found alpha-adrenergic antagonists, particularly doxazosin, had the most anti-correlated signatures. Doxazosin prevented VEGFRi-induced EC dysfunction in primary aortic ECs across species. In mice, doxazosin reversed sorafenib-induced EC dysfunction in resistance vessels without affecting blood pressure or renal glomerular endotheliosis. Conversely, lisinopril reduced HTN and glomerular endotheliosis but not EC dysfunction. Preliminary results from our clinical trial in canine cancer patients confirmed that toceranib induced HTN and showed that both doxazosin and lisinopril significantly lowered blood pressure in dogs with cancer and VEGFRi-induced HTN.

Conclusion:
Our study demonstrates the utility of phosphoproteomics and cross-species analysis in identifying EC-protective effects of doxazosin in VEGFRi-induced HTN. Our mouse data suggest distinct mechanisms for VEGFRi-induced EC dysfunction and HTN, potentially involving renal glomerular damage.
Abstract 1084: Differential Expression Of MicroRNAs In Patients With End Stage Renal Disease Target The Klotho/FGF23 Signaling Axis And May Attenuate Left Ventricular Hypertrophy
Authors: Alberga Carmine A , Hauck Ludger , Grothe Daniela , Premsingh Benny , Chan Christopher T , Billia Filio
Published: July 2024

Introduction:
Conventional hemodialysis (CHD) is the standard method of dialysis with concomitant changes in the cardiovascular system that may result in left ventricular hypertrophy (LVH). Importantly, increased frequency of dialysis reduces cardiac complications by regulating fluid status. Klotho and Fibroblast Growth Factor 23 (FGF23) has been shown to be expressed in a reciprocal fashion in patients with end-stage renal disease with LVH.

Research Questions/Hypothesis:
The Klotho/FGF23 axis may be regulated by circulating microRNAs (miRs) in CHD patients that transition to nocturnal hemodialysis (NHD) thereby regulating the development of LV mass.

Goals/ Aims:
To define alterations in expression of circulating miRs in patients transitioning from CHD to NHD and correlate the miR signatures with alterations in LV mass.

Methods/Approach:
Total RNA was isolated from serum of patients undergoing CHD and NHD and used to define the differential expression of miRs via microarray analysis. In parallel, circulating levels of Klotho and FGF23 were measured via ELISA analysis.

Results/Data:
We identified miRs with differential expression patterns in CHD vs NHD that targeted the Klotho/FGF23 axis. In patients with ≥10% increase or decrease of LV mass, a subset of miRs were identified that regulated the Klotho/FGF23 axis, namely let-7 members, miR-200c, miR-1 and miR-30c. There was no significant correlation between Klotho levels and LV mass in ESRD patients (p >0.05). However, we observed an association between FGF23 levels and LV mass in patients who were treated with NHD such that there were increased circulating FGF23 levels with increased LV mass, and vice versa (p=0.008, two-way ANOVA).

Conclusion:
Our novel findings have identified the differential expression of miRs in patients that transition from CHD to NHD with concomitant alterations in LV mass. Therapeutic delivery of miR antagomirs or mimics may mitigate cardiac hypertrophy in patients with renal disease by regulating the Klotho/FGF23 axis.
Abstract 1089: Noninvasive Assessment Of Aortic Biomechanics Using 4-D Flow And Cardiovascular Magnetic Resonance In A Porcine Model Of Aortic Coarctation
Authors: Gober Leah M , Stellon Michael , Lamers Luke , Frenkel Amy , Irrer Dana , Roldan Alzate Alejandro
Published: July 2024

Objectives:
Aortic coarctation (COA) is a congenital heart disease that results from narrowing of the aorta, contributing to increased cardiovascular morbidity. Current animal models are limited by stenotic mechanism and study longevity. We introduce a porcine model of COA, treated with a novel growth stent and imaged with advanced MRI techniques, to obtain hemodynamic metrics.

Methods:
A total of 14 piglets, 4 sham, 4 coarcted controls (CC) and 6 coarcted stents (CS), underwent surgical creation of a COA. The timeline is detailed in Figure 1. Serial imaging was obtained on a 3.0T MRI scanner. Hemodynamic metrics were acquired in predefined vascular planes (Ensight, CEI) to assess net flow, velocities and helical or vortical flow. CMR image analysis using cvi42 (Circle, Canada) included T1 maps and cardiac function.

Results:
Analyses are shown at 20 weeks. For CMR, there were no differences in cardiac function metrics, left ventricular mass or T1 mapping. In the 4D Flow analysis, shown in Figure 2, ascending aortic flow averages 62.5 mL/cycle in the CS group, higher than the sham and CC groups (p= <0.05). Both CS and CC animals have increased post-stenotic velocities (>1.5m/s), compared to the sham aorta (p= 0.01). All coarcted aortas, both CS and CC, exhibited helical flow and post-stenotic dilatation.

Conclusions:
No hemodynamic differences were seen with CMR, reflecting minimal adverse myocardial remodeling. 4D MRI data revealed locational differences in flow, velocity and collateralization. In conclusion, this work illustrates a successful longitudinal large animal model of COA and validates advanced imaging methods in the study of vascular hemodynamics.
Abstract 1103: The Relationship Between PAI-1, Corticosterone, And Splenic Activity: Data From The Washington, D.C. Cardiovascular Health And Needs Assessment
Authors: Aquino Peterson Elizabeth M , Baumer Yvonne , Ortiz-Whittingham Lola R , Baez Andrew S , Gutierrez-Huerta Cristhian A , Anyanwu Ezinne , Mitchell Valerie , Collins Billy S , Powell-Wiley Tiffany M
Published: July 2024

Background:
Chronic psychosocial stress (CS) has been associated with cardiovascular disease (CVD) which disproportionally affects African Americans (AA) at least partially via activation of the neuro-hematopoietic axis. Prior studies suggest plasminogen activator inhibitor 1 (PAI-1) is associated with hypertension, obesity, atherosclerosis, and major adverse cardiac events. Yet, little is known about the connection between CS and PAI-1 and its potential association with the neuro-hematopoietic axis in AA.

Research Question:
We examined associations between measures of CS and PAI-1 in a community-based cohort of AA residing in resource-limited Washington, D.C. neighborhoods, at risk for CVD.

Methods:
Participants were enrolled in the Washington, D.C. CV Health and Needs Assessment (DC-CHNA). CS was evaluated using the Cohen Stress Scale questionnaire. Fasting blood samples were taken to measure circulating levels of PAI-1 as well as cortisol and corticosterone, known biomarkers of stress. Participants underwent
18
FDG PET/CT to measure metabolic activity of the amygdala, bone marrow, and spleen, indicators of the neuro-hematopoietic axis. Multivariable linear regression analyses were adjusted for BMI and ASCVD 10-year risk score.

Results:
The DC-CHNA consisted of AA, the majority of whom were women, (N=60, mean age: 61±10.52, mean BMI: 33±7.85) at intermediate risk for CVD. Higher CS scores associated with higher PAI-1 levels (β=0.32, p=0.02). Higher PAI-1 associated with higher corticosterone (β=0.35, p=0.01) but not cortisol. Additionally, higher corticosterone associated with splenic activity (β=0.46, p=0.03) but not amygdala or bone marrow activity. Subsequently, higher PAI-1 also associated with higher splenic activity (β=0.22, p=0.01), while bone marrow activity was shown to be trending (β=0.14, p=0.06).

Conclusions:
We found that chronic psychosocial stress is associated with higher PAI-1 levels, which are associated with corticosterone and splenic activity, thus providing a potential link to how CS contributes to disparate CVD risk and health outcomes. Larger longitudinal studies are needed to investigate the predictive and potential therapeutic value of PAI-1 in individuals at risk for CVD.
Abstract 1105: Genome-first Approach To Rare And Common Variant Risk Of Thoracic Aortic Aneurysm And Dissection
Authors: Depaolo John , Biagetti Gina M , Judy Renae , Abramowitz Sarah , Desai Nimesh , Szeto Wilson Y , Bavaria Joseph E , Levin Michael , Guo Dongchuan , Milewicz Dianna M , Damrauer Scott M
Published: July 2024

Background:
Thoracic aortic aneurysm and dissection (TAAD) can have catastrophic health consequences. Eleven genes have strong or definitive evidence for causing hereditary TAAD (HTAAD). However, patients are seldom tested for rare pathogenic (P) or likely pathogenic (LP) variants in these HTAAD genes, and little is known about their prevalence in the general population. The degree to which common genetic variation associated with TAAD modifies rare variant pathogenicity is unknown.

Methods:
Penn Medicine Biobank (PMBB) participants volunteered to have electronic health records linked to biospecimen data including DNA that has undergone genome-wide genotyping and whole exome sequencing. P/LP HTAAD gene variants were curated according to American College of Medical Genetics and Genomics standards and analyzed to determine the associated risk of TAAD. A TAAD polygenic risk score (PRS) was derived from a multi-trait genome-wide association study (GWAS) of TAAD and aortic diameter and analyzed to determine the modifying effect this PRS had on rare variant penetrance.

Results:
Of the 43,731 participants in the analytic cohort, 309 individuals carried a P/LP HTAAD gene variant, 79 (26%) of whom had a diagnosis of TAAD. Among non-carriers, 2,106 (4.9%) had a diagnosis of TAAD. Compared to individuals without a P/LP variant, those with a P/LP HTAAD variant had a 5.4-fold increased risk of a TAAD diagnosis (95% confidence interval [CI] 1.8 to 16.8,
P
=0.003). Similar results were seen among carriers stratified by individual HTAAD gene. Using a multi-trait GWAS to isolate the diameter-independent common genetic changes linked to TAAD, we constructed a PRS that strongly associated with TAAD in the PMBB (OR 1.70 per 1 s.d. increase in PRS, 95% CI 1.62 to 1.78,
P
<0.001). Finally, TAAD prevalence was increased among individuals carrying a P/LP HTAAD gene variant in the highest PRS quintile (38.2%, 95% CI 25.4% to 52.3%) compared to carriers in the lowest PRS quintile (8.9%, 95% CI 3.0% to 19.6%) suggesting that common variant risk modifies rare variant TAAD risk.

Conclusions:
Our results indicate that P/LP HTAAD gene variants confer a 5.4-fold increased risk of TAAD in the population at-large, and that common variant risk may be an important modifier of rare variant TAAD risk.
Abstract 111: COL8A1 Modulates Endothelial Phenotype During Inflammatory Endothelial-to-Mesenchymal Transition
Authors: Li Qian , Talapaneni Sriharsha , Meng Yonghong , Demer Linda L , Tintut Yin , Hsu Jeffrey J
Published: July 2024

Background:
Endothelial-to-mesenchymal transition (EndMT), the dynamic process whereby endothelial cells (ECs) gradually lose endothelial identity while acquiring mesenchymal characteristics, plays significant roles in inflammatory cardiovascular pathologies such as atherosclerosis. The mechanisms of EC phenotypic modulation during EndMT remain widely unexplored. Understanding the factors driving the endothelial changes during EndMT may allow for the development of novel therapeutic targets to modulate this transition.

Methods/Results:
Inflammatory EndMT was induced in human aortic ECs (HAECs) by treatment with tumor necrosis factor-α (TNF-α), which showed a partial EndMT as evidenced by morphological changes, downregulation of endothelial markers, decreased angiogenic capacity, upregulation of mesenchymal markers, and increased cellular invasion. Results indicated that TNF-α treatment resulted in the loss of endothelial markers after short-term (24h) treatment. Interestingly, the expression of
COL8A1
, which encodes for one of the two alpha chains of the non-fibrillar type VIII collagen, was selectively downregulated among collagens by TNF-a treatment at early time-points. In HAECs, overexpression of
COL8A1
, as well as exposure to exogenous recombinant COL8A1, led to increased endothelial marker expression.
COL8A1
knockdown potentiates TNF-α induced endothelial phenotype loss (reduced endothelial markers and decreased angiogenic capacity), while
COL8A1
overexpression partially rescues TNF-α induced endothelial phenotype loss. RNA sequencing revealed that
COL8A1
knockdown resulted in the decreased expression of genes related to endothelial function (e.g., focal adhesion, cell-cell connections). Gene Set Enrichment Analysis (GSEA) showed significant enrichment of the TNF-α/NFKB signaling geneset and cardiac epithelial-mesenchymal transition geneset following
COL8A1
knockdown.

Conclusion:
Our findings suggest that COL8A1 mediates the EC transition during inflammatory EndMT by serving to maintain normal endothelial function, whereby TNF-α decreases
COL8A1
expression to facilitate the loss of the EC phenotype.
Abstract 1118: The Effect Of Type 2 Diabetes On Macrophage Differentiation In Non-healing Diabetic Foot Ulcers
Authors: Cirka Haley , Samant Amruta , Khair Lyne , Nguyen Tammy
Published: July 2024

Background:
Diabetic (DM) patients have a 30-fold increased risk of developing diabetic foot ulcers (DFUs), with 43% of non-healing DFUs requiring a lower extremity amputation (LEA). Non-healing DFUs is associated with impaired macrophage differentiation. Bone marrow (BM) hematopoietic stem cells (HSCs) differentiate into monocytes, which enter the circulation to become pro-inflammatory macrophages (M1) or anti-inflammatory macrophages (M2). In murine studies, DM HSCs transplantation was sufficient for impaired wound healing and skewed differentiation towards M1 when compared to non-DM control mice. This study suggests a causal relationship between DM BM-derived HSCs and impaired wound healing. However, it is unclear if these results translate to humans. We hypothesize that human DM BM-derived HSCs would be skewed towards M1 and away from M2 when compared to non-DM BM-derived HSCs.

Methods:
BM was collected from patients undergoing LEAs. Comparative analysis was made between DM patients with non-healing DFUs and from non-DM patients. Organoid BM samples were cultured for CD34+ HSC expansion.
In vitro
differentiation was performed using CD34+ HSCs induced to differentiated into monocytes (M-CSF) and subsequently to M1 (IFNy) or M2 (IL-4 and IL-13). Differentiated cells were quantified using flow cytometry and compared using student t-test statistical analysis.

Results:
HSCs derived from DM and non-DM BM yielded comparable percentages of monocytes, M1, and M2. We found no difference in the efficiency of monocyte or M1 differentiation between groups. Interestingly, M2 differentiated at a lower efficiency from DM HSCs than from non-DM HSCs (0.685 versus 1.15, respectively; p = 0.047).

Conclusions:
HSCs derived from DM BM have a comparable efficiency to differentiate towards monocytes and M1, however a decreased efficiency to differentiate towards M2 when compared to HSCs derived from non-DM BM
in vitro
. This finding suggests that the mechanism of non-healing DFUs involves dysfunction of BM-derived HSC/monocytes. In DFUs, monocytes may be skewed towards M1 differentiation because of reduced M2 differentiation capacity, thus favoring a pro-inflammatory state and preventing healing.
Abstract 1121: Neutrophil Proteobacteria Sequences Increases After Open Aortic Surgery
Authors: Geiger Joshua , Mahamed Zahra , Gill Ann , Kedwai Baqir , Lehane Daniel , Kruger Joel , Stoner Michael , Gill Steven , Kim Minsoo , Mix Doran S
Published: July 2024

Introduction:
Bacterial extracellular vesicles (bEV) transport diverse cargoes, including nucleic acids, and play a crucial role during host-microbe interactions, including modifying host immune response. Neutrophil activation is prevalent in aortic disease pathogenesis, but the cause is unclear. The baseline neutrophilic bEV content and its dynamics are unknown and may provide insight into neutrophil activation in aortic disease.

Research Question:
Does open aortic surgery change neutrophil bEV content?

Methods:
Blood was collected pre and post-operatively from 6 patients undergoing open aortic surgery. Neutrophils were isolated using standard centrifugation techniques, and nucleic acids were purified. The V1-V3 16s rRNA segments were amplified with universal primers and sequenced using an Illumina MiSeq platform. Reads were processed in QIIME2 and aligned to reference banks of known bacterial sequences. Sequence abundance was analyzed with Kruskal-Wallis and Wilcoxon-rank tests.

Results:
The average patient age was 60.2 years, with 4 male patients and 4 current smokers. The most common phylum and genus represented in the neutrophil bacterial reads were Proteobacteria (Figure 1A) and
Delftia
, respectively. The relative abundance of Proteobacteria sequences increased overtime (p=0.042) from a median of 45.43% (IQR:35.3-52.0) to 70.8% (IQR:66.1-81.5, p=0.026) post-operatively, to 83.5% (IQR:60.7-89.7, p=0.004) on POD1 (Figure 1B). POD3 Proteobacteria sequence abundance was stable from POD1 at 81.4% (IQR:64.2-89.6).

Conclusions:
Neutrophils of patients with aortic disease rapidly change their bacterial sequence content in response to open aortic surgery. Bacteria in the phylum Proteobacteria change most dramatically and equilibrate between POD 1 and 3. Further studies will elucidate the impact changing bacterial sequence abundance and bEV content has on neutrophil activity and its relationship to disease pathogenesis.
Abstract 1125: Percutaneous Optofluidic Catheter-guided Photo-triggered Nanogoldsomes Reduce Infarct Size And Mitigate Myocardial Dysfunction In A Swine Model Of Acute Myocardial Infarction
Authors: Chang Wei-Tien , Meng Shih-Wei , Tseng HsinYu , Lin Mao-Shin , Wang Liu-Chun , Min-Hsuan Hsu , Chang Kally , Huang Hung Ji , Liau Ian , Yeh Cheng-Shan
Published: July 2024

Introduction:
S-nitrosoglutathione (GSNO) is an endogenous S-nitrosothiol that plays important roles in nitric oxide (NO) generation and signaling. We previously designed an Au-polymersomes/GSNO nanoparticle (nanogoldsome) that can be triggered by laser or ultrasound to accelerate the release of NO, which effectively dilates the arteries of the target organ and induces anti-apoptotic protection against ischemia-reperfusion injury. In this study we integrated an optofluidic coronary catheter with the photo-triggered nanogoldsomes, and investigate their roles in mitigating the infarct size in a swine model of acute myocardial infarction (AMI).

Methods:
In a swine AMI model of 90 min coronary artery (CA) occlusion, nanogoldsomes (0.145 mg Au and 3.66 μg GSNO/kg) were administered 5 min after balloon-off using a percutaneous coronary optofluidic catheter with 532 nm laser stimulation at the tip. The CA diameter was measured at baseline and at 0 and 15 min of reperfusion. Left ventricular (LV) function (max dp/dt) were measured at baseline, and 1 h and 7 days after reperfusion. The infarct area was determined at day 7 using triphenyltetrazolium chloride stain with the infarct fraction calculated in reference to the area at risk stained by Evan’s blue coronary infusion with balloon occlusion at the same site.

Results:
Compared to AMI group, the CA diameter showed significant dilatation 10 min after nanogoldsome/laser treatment (1.22 vs. 0.98 folds,
P
< 0.05), while systemic blood pressure showed no significant drop. The infarct fraction was significantly reduced (49% vs. 73%,
P
< 0.05) and LV function markedly better 7 days after AMI. By contrast, no significant differences were noted in the control groups of GSNO (3.66 μg/kg) and nanogoldsomes without laser stimulation.

Conclusion:
Percutaneous optofluidic catheter-guided infusion of nanogoldsomes at coronary orifice with simultaneous tip laser stimulation in the first minutes of AMI reperfusion magnified NO release in the most critical reperfusion period. This instantly dilated coronary arteries, and reduced infarct area and mitigated LV dysfunction 7 days after AMI. With large animal experiments showing pathophysiological benefits, further translation to pre-clinical studies is encouraged.
Abstract 113: Endothelial Argonaute 1: A Novel Regulator Of Hyperlipidemia And Atherosclerosis
Authors: Liu Xuejing , Yuan Dongqiang , Tapia Alonso , Luo Yingjun , Malhi Naseeb K , Chen Zhen
Published: July 2024

Background:
Dysfunctional endothelial cells (ECs) crucially contribute to atherosclerosis (AS) largely through vascular inflammation. Beyond this, understanding the role of EC dysfunction (ECD) in liver dysfunction and hyperlipidemia, another AS driver, is unclear. Argonaute 1 (AGO1), a core protein in the RNA-induced silencing complex, was found to regulate EC response to hypoxic and metabolic stresses in our previous studies. EC conditional AGO1 knockout (EC-AGO1-KO) mice are protected from diet-induced obesity. However, the role of AGO1 in EC response to pro-atherogenic stimuli and its importance in hyperlipemia and AS are unknown.

Methods:
We subjected EC-AGO1-KO and wild-type mice to an AS model induced by AAV9-PCSK9 with a high-fat-high-cholesterol diet. We measured plasma lipids, liver enzymes and analyzed liver and AS lesions. In human EC cultures, AGO1 knockdown and oxPAPC challenged were performed. RNA-sequencing evaluated gene expression, and monocyte adhesion assays assessed EC function were then performed. Furthermore, we performed CUT&Tag assay to probe a non-canonical role of AGO1 in the nucleus through transcriptional modulation.

Results:
EC-AGO1-KO mice showed remarkable atheroprotective effects, with attenuated hyperlipidemia, improved liver function, and reduced AS lesion. AGO1 inhibition in oxPAPC-treated ECs demonstrated a profound anti-inflammatory effect, decreased ICAM1, VCAM1, MCP1 mRNA levels and monocyte adhesion. Additionally, a potential novel AGO1-mediated gene regulatory mechanism was identified at the molecular level through transcriptional induction.

Conclusions:
Our study identifies AGO1 as a key regulator in ECD, hyperlipidemia, and vascular inflammation, contributing to AS. Understanding the molecular mechanisms and functional significance of AGO1-regulated ECD provides valuable insights for novel therapeutic strategies to intervene in metabolic and cardiovascular diseases involving ECD.
Abstract 1131: Delivery Of Allogeneic Mitochondria Reverses Signatures Of Endothelial Aging & Senescence
Authors: Headley Colwyn A , Hu Caroline , Chiang Gladys , Oropeza Beu , Tsao Philip S , Huang Ngan F
Published: July 2024

Mitochondrial dysfunction is a key factor in vascular aging, including endothelial cell (EC) dysfunction. This research investigates whether introducing mitochondria from allogeneic fibroblasts into human microvascular ECs can mitigate the cellular abnormalities associated with aging.

Mitochondria were transferred into ECs of different passages (p6-14) using centrifugation. Post mito-transfer, the metabolic activity, mitochondrial network (density), and the proliferative capacity of ECs were evaluated through extracellular flux analysis, confocal microscopy, and 5-ethynyl-2'-deoxyuridine incorporation (proliferation assay), respectively.

Results show that compared to age matched control ECs, mito-transferred ECs exhibited no change in their oxygen consumption rates linked to mitochondrial respiration. Additionally, while early passage ECs (p6-7) had no change in glycolytic activity (compared to controls), middle (p10-11) and late passage ECs (p10-14) had considerable increases in their glycolytic activity post mito-transfer. Mitochondrial network analysis additionally revealed that the mitochondrial density of early passage ECs were unaffected by mito-transfer, while late passage ECs had enhanced mitochondrial networks, including total volume, and branching post mito-transfer. Lastly, late passage ECs demonstrated a 25% increase in proliferation after mito-transfer.

These findings indicate that aging in ECs is linked to increased oxidative phosphorylation metabolism, and that mito-transfer can reduce senescence-associated oxidative glucose metabolism and enhance EC proliferation. This positions mito-transfer as a promising strategy for improve EC function in cardiovascular aging.