bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3095: Correlating Spatial Wall Shear Stress With Transcriptomics In Pre-calcific Stages Of Bicuspid Aortic Valve Disease
Authors: Kazik Hail B , Kandail Harkamaljot S , Kessler Julie , Ibrahim El-sayed , Goot Ben , LaDisa John F , Lincoln Joy
Published: November 2022

Bicuspid aortic valve (BAV) is the most common congenital cardiovascular defect and characterized by formation of two, rather than three cusps. Approximately 50% of young adults with BAV prematurely develop calcification and significant stenosis, which is typically more severe in right/non-coronary (R/NC) fusion. Mechanisms underlying this process are unknown. However, based on BAV-induced narrowing of the valve orifice and obstruction of blood flow, it has been speculated that abnormal biomechanical stimuli play a major role. Despite this, validation studies correlating these stimuli to molecular changes that promote calcification in BAV are lacking. Our objective is to define biomechanical stimuli in young patients and mice with R/NC BAV prior to calcification onset, and further use the murine model to correlate biomechanical indices with transcriptomic changes indicative of mechanosensitive regulators of calcification.Fluid-structure interaction (FSI) models and quantified biomechanical indices (wall shear stress (WSS), von Mises stress) are being generated from echocardiography and MRI from young adults and

NfatC1
cre(+)
;Exoc5
fl/+

mice with R/NC BAV, and age-matched tricuspid aortic valve controls. In parallel, tissue sections collected from diseased mice are being subject to spatial transcriptomics (10x Visium) to determine the localization of differentially expressed genes relative to abnormal biomechanical stimuli. FSI results from a 23 y/o BAV patient indicate that cusp fusion leads to higher WSS on the fibrosa surface compared to the control, and high von Mises stress at the fusion site, both of which are regions prone to calcification in humans. To date, we have identified aortic valve stenosis in 25%

NfatC1
cre(+)
;Exoc5
fl/+

mice, and spatial transcriptomics in these mice is currently underway. To our knowledge these are the first FSI models to characterize biomechanical stimuli for a young adult with R/NC BAV prior to calcification. Our FSI studies in mice will validate the use of a mouse model to study human disease by comparison of biomechanical indices. Furthermore, parallel transcriptomic studies in BAV mice will allow us to correlate biomechanical stimuli with transcriptomic changes underlying premature calcification.
Abstract P3096: Cardiac-specific Hadc3 Loss Induces Reorganization Of Peripheral Heterochromatin And Promotes Diet-induced Heart Failure
Authors: Kiseleva Anna , Smith Cheryl L , Epstein Jonathan A
Published: November 2022

The nuclear periphery, including the nuclear lamina, serves as an important three-dimensional organizer of the genome. Together, with a subset of tethering proteins, nuclear lamina serves as a platform to anchor transcriptionally silent heterochromatin to the nuclear periphery. Peripheral heterochromatin, marked by the H3K9me2 histone modification, interacts with lamina proteins and is organized into lamina-associated domains (LADs). Our previous studies demonstrated that histone deacetylase 3 (HDAC3) is a scaffold for a nuclear lamina tethering complex and plays an important, non-enzymatic role in establishing cell identity in murine cardiac progenitor cells by reorganizing the LADs and releasing cardiac lineage-specific genes from the nuclear periphery during differentiation. This reorganization is critical as it makes specific genomic regions that are released from the nuclear periphery more accessible for transcriptional activation. Surprisingly,
Hdac3
deficiency in murine cardiomyocytes did not lead to any observable phenotype in adult animals. However, when the mice were maintained on a high-fat diet, this resulted in a severe disruption of fat metabolism and lead to heart failure within months. In the present study, we seek to determine if Hdac3 deletion is sufficient to re-organize LADs, release the genes normally found there, and make them more accessible for transcriptional activation as the mechanism leading to cardiac dysfunction. Since H3K9me2 marks peripherally localized genomic regions, we performed H3K9me2 CUT&RUN assay on cardiomyocytes derived from WT and Hdac3 KO mice, fed with normal and high-fat diet, to compare the positioning of chromatin genome-wide. From these data, we identified a list of genes and enhancers found in those regions that vary significantly in H3K9me2 enrichment depending on genotype and condition. We then performed Gene Ontology (GO) Analysis. A significant finding is that numerous enhancers with relatively reduced H3K9me2 in Hdac3 KO cardiomyocytes are regulators of neuronal genes. Loss of H3K9me2 is generally associated with increased gene transcription. We predict that disruption of the peripheral heterochromatin organization upon Hdac3 deletion may result in loss of identity in cardiomyocytes by activating non-cardiomyocyte program-specific genes, such as those of neuronal cell fate, which are normally found at the nuclear periphery and silent in cardiac cells. The results of this work will help to gain insight into the function of nuclear lamina scaffolding proteins, and open new avenues to design therapies to replace lost scaffolding proteins in patients predisposed to heart disease.
Abstract P3097: Post-transcriptional Mechanisms Of FTO-regulated Cardiac Contractility
Authors: Parikh Mihir , Phan Anh , Mathiyalagan Prabhu , Sahoo Susmita
Published: November 2022

Background:
N6-methyladenosine (m6A), the most abundant and functionally relevant mRNA modification, is reversibly demethylated by FTO, an m6A eraser and a primary regulator of m6A in the heart. In failing hearts, decreased FTO increases m6A, critically reducing cardiomyocyte contraction. FTO overexpression improved cardiac contraction and attenuated remodeling. Here, we investigate post-transcriptional and translational mechanisms of FTO-regulation of cardiomyocyte contraction in vitro and in vivo. We hypothesize that FTO directly binds to selective cardiac contractile mRNAs at their m6A site and affects their nuclear export, polysome binding, cytoplasmic availability, and protein expression.

Methods and Results:
We performed immunostaining to examine the intracellular location and expression of FTO under different pathological stimuli in human ventricular cardiomyocyte cell line (AC16 cells), rat primary cardiomyocytes and mouse and human hearts. FTO was detected in both cytoplasmic and nuclear compartments, and was significantly reduced in pathological hearts compared to healthy controls. MeRIP (immunoprecipitation of m6A-RNA) sequencing analysis uncovered that FTO targets different sets of cardiac mRNAs in the cytoplasm and nucleus of cardiomyocytes. Knocking down FTO increased nuclear retention of cardiac mRNAs, which was reversed with FTO overexpression. Immunoprecipitation experiments demonstrated that FTO directly binds to cardiac mRNAs. To determine the effect of FTO on translation of select cardiac mRNAs, we isolated the polysome fraction from the cytoplasm of AC16 cells. Interestingly, our stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry analyses suggested increased expression of cardiac contractile proteins with FTO overexpression.

Conclusion:
We demonstrate that FTO positively regulates post-transcriptional nuclear export, polysome binding of cardiac contractile mRNAs, and regulates their translation under pathological stimuli. By combining experimental and predictive models in human and mice, we have characterized the transcriptome-proteome molecular interplay regulating cardiac function.
Abstract P3099: Identification Of Hub Genes And Construction Of Protein-protein Interaction (PPI) Network Clusters Define Racially Distinct Signatures Of Ischemic Heart Failure
Authors: Bakshi Sayan , Potter Luke , Pepin Mark , Ha Chae-myeong , Wende Adam R
Published: November 2022

Cardiovascular diseases, including ischemic heart failure (IHF), are the leading cause of mortality worldwide; furthermore, African Americans (AA) have 30% higher mortality than Caucasian Americans (CA). Our recent study correlates racial differences in socioeconomic status with DNA methylation (DNAm), altered gene expression, and mortality in HF independent of etiology.

The current analysis focused on etiology-specific differences in DNAm with a specific focus on IHF, the related changes in gene expression, within self-reported race. RNA-seq expression (
P
< 0.05, ±1.5-fold) and array-based methylation (
P
< 0.05, ±5%) profiles were examined using cardiac biopsies from non-IHF (NIHF) and IHF AA (n = 9/5) and CA (n = 10/5) male patients. Network clusters were identified with MCODE (v2.0) and top hub genes identified via Cytohubba (v0.1). Both AA and CA patients showed similar numbers of IHF-specific differentially expressed genes (DEG) (591/365 AA; 439/486 CA; up/down). However, CA-specific methylation changes (29703/22703; up/down) were more abundant compared to AA (2904/2897; up/down). PPI network construction of these changes identified clusters unique to each group. The AA-specific upregulated cluster included AGPAT2, PPARG, and 10 other DEG associated with the fatty acid metabolism, while BMP2, SMAD6, and 4 other DEG that regulate BMP signaling and SMAD phosphorylation were downregulated. The CA-specific upregulated cluster included EZH2, CCNA1, and 18 other DEG that regulate mitotic sister chromatic separation, while EGFR, TNF, and 12 other DEG important for chronic inflammatory response formed the top downregulated cluster. Consistent with greater changes in DNAm the majority of DEGs in CA-specific clusters were also differentially methylated (58%) while fewer were in AA-specific clusters (17%). Within AA-specific PPI networks, PPARG/BMP2 (up/down) were the top ranked hub genes and KIF20A/KIT (up/down) in CA-specific networks. These racially distinct hub genes suggest altered regulatory mechanisms. Our analysis identified racially distinct clusters associated with specific pathways, which may connect the regulators of DNAm to gene expression and IHF, supporting the potential to use this information as prognostic biomarkers.
Abstract P3100: Integrating Single-Cell Transcriptomics And Light-Sheet Imaging To Elucidate The Mechanisms Of Cardiac Outflow Development
Authors: Wang Jing , Wang Zhaoqiang (Aaron) , O'donnell Ryan , Lien Ching-Ling , Hsiai Tzung K
Published: November 2022

Introduction:
Transcription factors Snai1 are recognized to regulate endo/epithelial-to-mesenchymal transitionduring cardiac morphogenesis. However, the relative importance of myocardial contractile vs. intracardiac fluidshear force-mediated Snai1 during outflow track (OFT) development remains undefined.

Methods and Results:
Using single-cell RNA analysis, we identified a population of epicardial Snai1b
+
cells inthe zebrafish hearts at 5-days post-fertilization that carry both smooth muscle cells and fibroblast markers. sc-RNA data from Weinberger
et al.
(2020) was analyzed via the
scater
package in R. Snai1b
+
cells in the
Tg(Snai1b:EGFP; Flk:mCherry)
embryos were localized by confocal microscopy and the real-time valvular dynamics by our custom-built 4-D (space + time) light-sheet fluorescence microscope (LSFM) at 200 frames/second. Consistent with Weinberger
et al.
(2020), Snai1b was highly expressed in the “Epi2” epicardial cluster (Elnb
+
/Mylka
+
/ Sema3fb
+
) (
Figure 1A, E-G
). In addition, this cluster expressed alpha-smooth muscle actin (a-SMA orActa2), vimentin (Vim), and fibronectin 1a (Fn1a). We also observed the activation of BMP-Snai-Pitx2 pathway, where Nodal and Pitx2 were suppressed due to the expression of BMPs and Snai1/2. Next, confocal imaging localized the Snai1b+ cells at the bulbus arteriosus (BA) base, separating the ventricle from the passiveBA (
Figure 1B
). LSFM further captured the moving boundary of endocardium in BA for intra-tissue strain and stress simulation (
Figure 1C
).

Conclusions:
Snai1b may contribute to OFT development in a myocardial contractile force- rather than intra-cardiac shear stress-dependent manner. This finding paves the way to elucidatethe 4-D genomic interaction between Snai1b
+
cells and developmental transcriptomics.
Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells
Authors: Liu Juli , Liu Sheng , Wan Jun , Yang Lei
Published: November 2022

Background:
Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of
in vivo
studies, the molecular mechanisms that govern both early human heart and brain development remain elusive.

Results:
Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A
–/–
) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A
–/–
hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF.

Conclusions:
We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.
Abstract P3102: Matrine Inhibits Advanced Glycation End Products- Inducedmatrine Inhibits Advanced Glycation End Products- Induced Macrophage M1 Polarization By Reducing Dnmt3a/b- Mediated Dna Methylation Of Gpx1 Promoter
Authors: Liu Zhongwei , Xing Yujie
Published: November 2022

Background and Objective:
Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes- exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms.

Methods:
Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation.

Results:
Matrine inhibited AGEs- induced macrophage M1 polarization which was impaired by RAGE and TLR4 over-expressions. Matrine reduced AGEs exposure elevated expressions of DNMT3a and DNMT3b in macrophages, which further decreased DNA methylation of GPX1 promoter. Increased GPX1 by matrine suppressed oxidative stress in AGEs- exposed macrophages which was impaired by RAGE over-expression.

Conclusions:
Matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b- induced GPX1 promoter DNA methylation.
Abstract P3109: Identification Of Drug Transporter Genomic Variants And Inhibitors That Protect Against Doxorubicin Induced Cardiotoxicity
Authors: Magdy Tarek , weddle Carly J , Lyra Leite Davi M , Fonoudi Hananeh , Burridge Paul W
Published: November 2022

Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. To answer these questions, six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human-induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. We demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. The role of SLC28A3 in DIC was then confirmed using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes. To identify the potential causal variant, Nanopore-based fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing. An extended in silico haplotype and functional analysis followed by base editing, identified a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus. Next, using high-throughput drug screening of 1200 drugs in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. Finally, using eight cancer cell lines, we demonstrate that desipramine does not impede doxorubicin cytotoxicity.
Abstract P3113: RAF 'Paradox Breaker' Inhibitor, PLX8394, Enhances Inflammation And Apoptosis In Murine Hypertension
In Vivo
Authors: Cooper Susanna T , Malone Giles O , Haines Zoe H , Meijles Daniel N
Published: November 2022

Introduction:
The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension.

Hypothesis:
We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling.

Methods & Results:
To assess the role of RAF inhibition in cardiac adaptation to hypertension
in vivo
, C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic
Nppa
(p=0.77) or
Nppb
(p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate
Col1a1
(p=0.22) and
Col3a1
(p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines (
Il1b
, p=0.02;
Il6
, p=0.008) and the inflammatory cell marker
CD45
(p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use.

Conclusion:
Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.
Abstract P3117: Validating Genetic Risk Markers Causing Doxorubicin-induced Cardiotoxicity In African American And European American Pediatric Patients Using A Hipsc Model
Authors: Shah Disheet A , Sapkota Yadav , Burridge Paul W
Published: November 2022

The W.H.O. estimates that globally 400,000 children under 19 are diagnosed with cancer annually. 60% of pediatric cancers are treated with chemotherapy using anthracyclines, mainly doxorubicin. Survivors of childhood cancer are approximately 15 times more likely to be diagnosed with congestive heart failure. African American (AA) survivors had a 2.5-fold increased risk of severe or life-threatening cardiomyopathy vs European Americans (EA). In recent WGS studies, 2 novel loci in chromosome 1 & 6 were identified to be correlated with an increased risk of doxorubicin-induced cardiotoxicity (DIC) in AA and EA population groups respectively and were reduced or absent in the other group. Our study aims to validate these loci using patient specific hiPSC-derived cardiomyocytes (hiPSC-CM), and for that we recruited and generated 20 hiPSC lines from the blood of AA and EA pediatric patients. Using a novel bioreactor system, a large-scale production of hiPSC-CM for this patient specific samples is underway, and the cells will be utilized for phenotypic and genotypic characterization. Our aim is to assess the effect of doxorubicin on cardiomyocyte viability, apoptosis, mitochondrial function, and calcium dynamics. Differential cardiac gene regulation after exposure to doxorubicin will be studied using RNA sequencing. In conclusion, our findings will validate the genetic variants predisposing AA and EA populations to DIC. This study will clinically impact therapeutic approaches on both AA and EA populations and at individual levels while using doxorubicin. The validated hiPSC-CMs could help in identifying personalized cardioprotectants to reduce DIC without modifying chemotherapy. This study could also potentially provide data for genomically-informed drug discovery for novel cardioprotectants.
Abstract P3121: Serological Mechanisms Driving Cardiotoxicity In Covid-19
Authors: Colzani Maria T , Bargehr Johannes , Mescia Federica , Knight-Schrijver Vincent , Summers Charlotte , Smith Kenneth G , Lyons Paul , Sinha Sanjay
Published: November 2022

Cardiac involvement is common in patients severely ill with COVID-19 and is associated with increased morbidity and mortality. The mechanism for myocardial injury in COVID-19 has been proposed to be at least in part due to a direct viral cytotoxic effect on cardiomyocytes. Preclinical data corroborate the notion that SARSCoV-2 can enter and replicate within hiPSC-derived cardiomyocytes. However, clinical evidence demonstrating direct viral effects on the heart remains limited. Alternatively, cardiac injury may result from excessive inflammatory cytokine production which is the main driver for the pathogenesis in COVID-19. Despite this, cardiotoxicity secondary to hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. To investigate serological drivers of cardiotoxicity in COVID-19 we have established a bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell-derived cardiomyocytes (hESC-CMs). We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability and contractility independent of viral transduction. Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-β, IL-10, CRP and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partially reversed the phenotype and preserved cardiomyocyte viability and function. Interestingly, treatment with serum from Systemic Inflammatory Response Syndrome (SIRS) patients recapitulates the effect observed with COVID-19 patients’ serum indicating that the phenotype observed is most likely not unique to COVID-19 disease. In conclusion, our results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and indicate possible targets for therapeutic intervention.
Abstract P3122: Latent Mouse Model Of HIV-1
Authors: Kondrachuk Olena , Rappaport Jay , Gupta Manish K
Published: November 2022

Background:
Antiretroviral therapy (ART) significantly improves the life span of people living with HIV/AIDS (PLWHA). However, HIV- associated cardiovascular diseases (CVDs) remain one of the leading causes of heart failure and comorbidity in PLWHA. Modern ART regimens reduce viral loads to undetectable levels in HIV+ patients while being less toxic. Nevertheless, HIV proteins are present in the plasma of PLWHA. The HIV protein, Nef, has been found circulating in the plasma of ART-treated patients within extracellular vesicles and in association with immune cells. We hypothesized that circulating HIV Nef proteins may cause an adverse effect on various organ functions. To further understand the effect of Nef on differing organ functions, we have generated a transgenic mouse model which expresses the Nef protein in the presence of Cre recombinase.

Methods and Results:
Nef transgenic mouse model was generated by cloning HIV-1 Nef ORF in CAG-Lox-CAT vector. Transgenic mice lines were crossed with the alpha myosin heavy chain promoter Cre mice to express Nef protein in the heart. Western blot data validated Nef protein expression in the adult mouse heart. Histological evaluation of the effect of Nef protein expression on heart morphology revealed cardiac hypertrophy. Furthermore, heart function analysis shows that Nef expressing mouse lines have depressed heart function.

Conclusion:
Our study suggests that Nef causes cardiac dysfunction that mimics cardiomyopathy secondary to latent HIV in PLWHA. Further, this mouse model could be useful in studying the effect of Nef protein in different organs or cells using cell-specific Cre recombinase. Moreover, Nef transgenic mice could be useful in generating new therapeutic tools to control comorbidity in PLWHA.
Abstract P3125: Cldn11 Is A Candidate For Vascular Smooth Muscle Cell Plasticity Regulator In Atherosclerosis Pathology
Authors: Kim Hyung-Seok , Lee Su-Jin , Kim Soojin , Lee Jungho , Park Jong-Tae
Published: November 2022

Claudin-11(CLDN11) is a component of tight junction, and important for normal CNS functions. The function of CLDN11 is well elucidated in oligodendrocytes and CNS system, but its expression pattern according to human vascular bed such as intracranial and extracranial, role in the vascular smooth muscles (vSMCs) plasticity and atherosclerosis is still unknown. Human left anterior descending coronary artery (LAD) and middle cerebral artery (MCA) vascular smooth muscle layer was dissected from the dead body. CLDN11 expression is identified in LAD and MCA vascular smooth muscle cell layer, and the CLDN11 expression is higher in MCA than in LAD (p<0.01). Interestingly, the CLDN11 expression is decreased in vSMCs around the atherosclerotic lesions compare to non-atheromatous lesions by immunohistochemistry. To determine the functional role of CLDN11 in vSMCs, microarray analysis was performed following siCLDN11 knockdown. Compared to normal HASMC (human aortic vascular smooth muscle cells), siCLDN11-HASMC was showed increased gene expression such as RSPO3, CXCL8, EGR3, CYP1B1, TNFAIP2, PTGS2, HEY1, BMPER and these set was related to cell migration, proliferation and angiogenesis. CLDN11 knockdown vSMCs showed increased the cell migration of HASMC measured by wound healing, tube formation, and cell cycle assay, and reduced cas-3 cleavage. Finally, CLDN11 expression was inversely correlated with the progression of coronary atherosclerosis and the sudden cardiac death with complicated coronary atherosclerosis. This study shows that CLDN11 play a role in the initiation and propagation of atherosclerosis by regulating the plasticity of vSMCs, suggesting the possibility of biological marker for atherosclerosis in clinical diagnosis (This work was supported by NRF-2019R1A2C1089108, NRF-2019M3E5D1A02068082, COMPA-2022C100, and BK21 Program, Center for Glocal Future Biomedical Scientists at Chonnam National University).
Abstract P3127: Wnt1-inducible Signalling Pathway Protein 1 Contributes To Vascular Fibrosis By Promoting Fibroblasts Phenotypic Switch And Extracellular Matrix Deposition
Authors: Li Ze , Williams Helen , Johnson Jason , George Sarah
Published: November 2022

Background:
Vascular fibrosis is linked with many diseases, including hypertension. Upon pathophysiological stimulation, fibroblasts undergo phenotypic switch and increase extracellular matrix (ECM) synthesis and deposition. Wnt1-inducible signalling pathway protein 1 (WISP-1) is a growth factor implicated in fibrotic remodelling. We tested the hypothesis that WISP-1 induces cardiac fibroblast activation and increases ECM deposition, thereby contributing to vascular fibrosis in hypertension.

Methods and Results:
Primary human cardiac fibroblasts (HCFs) were treated with recombinant WISP-1. Western blotting analysis showed that WISP-1 treatment for 30 minutes activated Akt pathway (phospho-Akt:total-Akt, fold change: 1.33±0.14 vs. 1.00±0, n=9,
P
<0.05). WISP-1 induced Akt phosphorylation was inhibited by an integrin β1 blocking antibody (phospho-Akt:total-Akt, fold change, 0.52±0.12 vs. 1.00±0, n=5,
P
<0.05). WISP-1 treatment for 24 hours induced collagen 1 maturation in conditioned media and coincided with decreased meprin α metalloproteinase protein levels (fold change: 0.44±0.14 vs. 1.00±0, n=5,
P
<0.05). The collagen maturation was inhibited by a zinc-mediated proteinase inhibitor (GM6001) or an Akt inhibitor. Additionally, immunocytochemistry analysis illustrated that 24-hour WISP-1 treatment increased the percentage of α-smooth muscle actin-positive HCFs compared to the control (1.89±0.37 vs. 1.00±0, n=8,
P
<0.05).
In vivo
, WISP-1 knockout mice exhibited reduced collagen 1 content (0.39±0.09 vs. 1.19±0.25, n=5-7,
P
<0.05) and proteoglycan content (2.20±0.37 vs. 6.22±1.10, n=5-7,
P
<0.05,) in the perivascular area compared to their wild-type control after 28 days angiotensin II (1000ng/kg/min) infusion.

Conclusions:
WISP-1 activates cardiac fibroblasts via integrin β1-Akt pathway and meprin α metalloproteinase, thereby inducing fibroblast phenotypic switch and promoting collagen deposition. Deletion of WISP-1 alleviated hypertension induced vascular fibrosis
in vivo
. Modulation of WISP-1 levels may be beneficial for attenuating the effects of hypertension on vascular fibrosis, and could provide potential therapeutic targets in clinical treatment.
Abstract P3128: Obesogenic Diet Promotes Endothelial-to-Mesenchymal Transition In Adipose Tissue
Authors: Chavkin Nicholas W , Hirschi Karen , GOKCE Noyan , Walsh Kenneth
Published: November 2022

Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive Endothelial-to-Mesenchymal Transition (EndoMT), where endothelial cells transition into mesenchymal-like interstitial cells that leave the vascular wall and promote tissue inflammation and fibrosis. In this study, we aimed to determine whether obesogenic diet can promote EndoMT in adipose tissue. EndoMT was assessed in 1) transgenic endothelial cell lineage-tracing mice subjected to High-Fat/High-Sucrose (HF/HS) diet, 2) available single cell RNA sequencing datasets from human obese adipose tissue, and 3) cell culture models using primary human adipose-derived endothelial cells. In lineage-tracing mice, obesogenic diet induced EndoMT in a time-dependent manner at 13, 26, and 52 weeks on HF/HS diet in both subcutaneous and visceral adipose tissue. Interestingly, rate of EndoMT was greater in visceral compared to subcutaneous adipose tissue. After 52-weeks on HF/HS diet, EndoMT cells in visceral adipose tissue upregulate inflammatory response and TGF-Beta signaling transcriptional pathways compared to endothelial cells in the same adipose tissue depot. Initiation of EndoMT was also found in single cell RNA sequencing datasets of stromal cells from human obese adipose tissue, indicated by endothelial cells in these datasets containing sub-clusters with reduced
Pecam1
and increased
Acta2
expression. Finally, the capacity for EndoMT in primary human adipose-derived endothelial cells was validated by bulk RNA sequencing after treatment with proinflammatory cytokines (TNF-Alpha, IFN-Gamma, TGF-Beta1), confirming decreased expression of endothelial cell genes and increased expression of genes associated with EndoMT, inflammatory response, and TGF-Beta signaling. Together, these clinical and experimental findings indicate that chronic obesity can promote EndoMT in adipose endothelial cells.
Abstract P3129: Functional Coupling Of TRPM2 And Extrasynaptic NMDARs Exacerbates Excitotoxicity In Ischemic Stroke
Authors: Zong Pengyu , feng jianlin , Yue Zhichao , Li Yunfeng , Wu Gongxiong , Sun Baonan , He Yanlin , Miller Barbara , Yu Albert S , Su Zhongping , Xie Jia , Mori Yasuo , Hao Bing , Yue Lixia
Published: November 2022

Introduction:
Ischemic stroke caused a heavy burden on public health. NMDA receptor (NMDAR) mediated excitotoxicity was thought to be the culprit for neuronal death during ischemic stroke. However, NMDAR antagonists all failed to show protective effects in human patients. The heat-sensitive ion channel TRPM2 is Ca
2+
-permeable and usually activated under oxidative stress conditions. TRPM2 is abundantly expressed in the brain and promotes neuronal death during ischemic stroke, whereas the underlying mechanisms remain elusive.

Hypothesis:
Oxidative stress is a hallmark of brain damage during ischemic stroke. We hypothesized that TRPM2 is important in magnifying NMDAR-mediated excitotoxicity.

Methods:
Neuron specific TRPM2 knockout was achieved by crossing
nestin-cre
mice with

Trpm2
fl/fl

mice. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) were performed to mimic ischemic stroke in vivo and in vitro, respectively. Co-immunoprecipitation and direct binding assay were used to examine protein-protein interaction. Subcloning and mutagenesis were used to identify interaction details. Interfering peptide specifically disrupting TRPM2-NMDAR interaction was designed and synthesized. Cortical neurons were isolated and cultured, Fura-2-AM and Rhodamine-123 imaging were used to examine Ca
2+
overload and mitochondrial dysfunction, respectively. Synaptic and extrasynaptic NMDAR mediated responses were separated, and synaptosome was isolated to examine the influence of TRPM2 on NMDAR and expression of TRPM2 in neurons at different sites, respectively.

Results:
Neuron specific TRPM2 knockout alleviates ischemic stroke in mice. TRPM2 physically and functionally interacts with extrasynaptic NMDAR, which enhances excitotoxity. The EE
3
motif in TRPM2 directly associates with the KKR motif in NMDAR. Uncoupling of TRPM2-NMDAR association using a disrupting peptide TAT-EE
3
prevents OGD-induced Ca
2+
overload and mitochondrial dysfunction in neurons, and protects mice against MCAO-induced brain injury.

Conclusion:
Therapeutic interfering peptide TAT-EE
3
attenuates ischemic stroke. Targeting the TRPM2-NMDAR coupling could be a promising strategy for screening more effective therapies for ischemic stroke.
Abstract P3131: Aquaglyceroporin Expression In Resistance-sized Arteries
Authors: Reinert Angela , Choi Young , Lay Saline , Nguyen Tiffany , Gevorgyan Nare , Bulley Simon
Published: November 2022

Due to its importance in maintaining homeostasis of many physiological processes in the body, including the cardiovascular system, it’s crucial that humans consume water, to replace any loss that occurs through urine, sweat, feces, and expelled air. Unfortunately, a significant proportion of the global population either does not have a safe drinking water source or consistent access to water throughout the year and with water scarcity projected to only impact more people in the future, it’s important to understand what is the effect of water deprivation on the human body. Once the body loses a greater amount of water than it’s able to intake it enters the process of dehydration, resulting in a body water deficit, known as hypohydration. Even mild dehydration can affect an individual's mental and physical well-being, with an association between chronic dehydration and adverse cardiovascular outcomes, including hypertension, stroke, and coronary heart disease (CHD). While the majority of research is focused on acute dehydration, chronic low water intake, also termed underhydration, is also associated with adverse health outcomes. However, the effect of acute and chronic reductions in water intake on the function of resistance-sized arteries, and particularly vascular smooth muscle cells (VSMCs), that regulate systemic blood pressure, is poorly understood. Aquaporins (AQPs) are membrane proteins primarily responsible for facilitating water movement across cells in tissues and organs,
,
with the aquaglyceroporin (AQP3, AQP7, AQP9-10), subfamily also exhibiting permeability to glycerol, and other small solutes. Previous studies have identified AQP7 expression in endothelial cells (ECs) and the heart, where it is proposed to enable the uptake of glycerol as a cardiac energy substrate, however, its function in VSMCs is unclear. Preliminary data demonstrate that in mice the expression of glycerol-permeable aquaglyceroporins, AQP7 and AQP9, are elevated in mesenteric arteries by 48 hours of water deprivation and 28 days of reduced water intake, respectively. Mesenteric artery glycerol content is higher in mice dehydrated for 48 hours compared to 0-hr controls. Male mice administered 10% glycerol
ad libitum
with water have elevated mesenteric artery AQP7 expression when compared to 0-hr controls. In female mice, ovariectomy (OVX) attenuates increased AQP7 expression in 48-hr dehydrated females, with, 17β-estradiol treatment restoring dehydrated-mediated elevations in expression. These findings suggest that mice adjust to alterations in water intake differently short- and long-term, and suggest a potential mechanism for increasing VSMC glycerol-uptake as an energy source during water duress.
Abstract P3139: A Pluripotent Stem Cell Model Of Nilotinib-induced Arterial Disease
Authors: Pinheiro Emily A , Burridge Paul W
Published: November 2022

Nilotinib is a highly effective tyrosine kinase inhibitor used to treat Chronic Myeloid Leukemia (CML). While nilotinib successfully treats CML, a common and serious side effect is development of nilotinib-induced arterial disease (NAD) even in patients without pre-existing risk factors. This side effect is not seen with imatinib, a CML drug in the same class that inhibits the same target as nilotinib. To date, the mechanisms of NAD are unknown, and the cell types involved in this adverse effect have yet to be identified. Furthermore, no tools currently exist to predict which patients will develop NAD. In this study, we used endothelial and vascular smooth cells derived from human-induced pluripotent stem cells (hiPSCs) as a model to identify which cell types are involved in NAD and which cellular functions are perturbed. We first developed a set of efficient, reproducible endothelial and vascular smooth muscle differentiation and purification protocols that consistently yield >95% pure cells. For both hiPSC-derived endothelial cells (hiPSC-ECs) and hiPSC-derived vascular smooth muscle cells (hiPSC-VSMCs) we next identified a panel of assays relevant to various functions of atherogenesis. In comparing the effects of nilotinib and imatinib on hiPSC-ECs, we see a nilotinib-specific decrease in cell capacity for proliferation and migration, in addition to disturbances in Ac-LDL uptake and NO homeostasis. Nilotinib is not differentially toxic to hiPSC-ECs compared to imatinib and has no effect on barrier function as measured by multielectrode array. The data collected to date demonstrate the feasibility of using hiPSCs to model NAD
in vitro
and recapitulate the clinical discrepancy in vascular effects between nilotinib and imatinib.
Abstract P3140: Targeting Of Kinin B1r/il-6 Signaling Prevents Microvascular Leakage And Mitochondrial Dysfunction In Sepsis Induced Heart Failure
Authors: Pan Kaichao , Zhang Qin , Ullah Karim , Solanki Ani , Sitikov Allbert , Liao James K , Wu Rongxue
Published: November 2022

Introduction:
Cardiovascular dysfunction is a major complication of severe sepsis. Microvascular leakage and mitochondrial dysfunction during sepsis are critically involved in heart failure. However, the underlying mechanisms between cardiac microvascular barrier function and sepsis-induced heart failure are not well studied. Kinin B1 receptor (B1R) regulates inflammation and induces capillary leakage. Previous findings from our lab also revealed an increase in B1R serum level in both septic patients and animal models within 24 hours.

Hypothesis:
We hypothesized that treatment with B1R antagonist protects against sepsis-induced microvascular leakage and mitochondrial dysfunction.

Methods and Results:
To address the hypothesis, we pre-treated WT male mice with either vehicle (control) or a specific B1R antagonist B6929 via subcutaneous injection, then subjected them to 40% cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Echocardiographic analysis revealed an early cardioprotective effect of B1R inhibition, as evidenced by the improvement of EF%, FS%, and cardiac output in mice pre-treated with B1R antagonist (n=6-8, p<0.01) at 4-6 hours post-CLP. Furthermore, mitochondrial H
2
O
2
measurement of the freshly isolated cardiac mitochondria from CLP-induced septic mice also showed that B1R inhibition partially abolished the production of H
2
O
2
. The opening of mPTP was also reduced with B1R antagonist treatment. In vivo cardiac vascular permeability assessed using the Miles assay further demonstrated that B1R inhibition prevented sepsis-induced cardiac microvascular leakage and edema. Similar improvements were also observed in the liver and kidney. Interestingly,B1R antagonist treatment abolished CLP-induced serum level of IL-6 by 70%. In
in-vitro
study, B1R antagonist treatment inhibited LPS-induced IL-6 expressions in human cardiac microvacuolar endothelial cells (HCMECs). Finally, ECIS based analysis demonstrated that IL-6 deletion using either siRNA or B1R antagonist treatment rescued LPS-induced endothelial barrier leakage.

Conclusions:
Our findings suggest that targeting B1R via IL-6 may be a novel therapeutic approach for the treatment of sepsis-associated cardiovascular disease.
Nonoptimal component placement of the human connectome supports variable brain dynamics
Authors: Hayward Christopher James , Huo Siyu , Chen Xue , Kaiser Marcus
Published: November 2022

Neural systems are shaped by multiple constraints, balancing region communication with the cost of establishing and maintaining physical connections. It has been suggested that the lengths of neural projections be minimized, reducing their spatial and metabolic impact on the organism. However, long-range connections are prevalent in the connectomes across various species, and thus, rather than rewiring connections to reduce length, an alternative theory proposes that the brain minimizes total wiring length through a suitable positioning of regions, termed component placement optimization. Previous studies in nonhuman primates have refuted this idea by identifying a nonoptimal component placement, where a spatial rearrangement of brain regions in silico leads to a reduced total wiring length. Here, for the first time in humans, we test for component placement optimization. We show a nonoptimal component placement for all subjects in our sample from the Human Connectome Project (N = 280; aged 22–30 years; 138 females), suggesting the presence of constraints—such as the reduction of processing steps between regions—that compete with the elevated spatial and metabolic costs. Additionally, by simulating communication between brain regions, we argue that this suboptimal component placement supports dynamics that benefit cognition.
Research on multi-moving target location algorithm based on improved TDOA/FDOA
Authors: Wu Hao , Wu Zhong-Hong , Shi Zhang-Song , Sun Shi-Yan
Published: November 2022

Wireless positioning and tracking technology are key technologies in applying wireless sensor networks and have vital research significance and application value. In this paper, in terms of positioning multiple non-coincident dynamic targets, aiming at the problem of low precision and large computational complexity when locating multiple dynamic targets by the two-step least squares algorithm and the constrained total least squares algorithm, an improved constrained total least squares algorithm is proposed. This algorithm fully considers the constraints, introduces the Lagrangian multiplier technology and the quasi-Newton BFGS iterative formula, avoids the calculation of the Hessian matrix, reduces the amount of calculation, and improves the positioning accuracy. Simulation experiments show that when the measurement error and sensor position error are moderate, the ICTLS positioning algorithm has a smaller RMSE than the TSWLS and CTLS positioning algorithms, showing higher positioning accuracy and stronger robustness. Secondly, aiming at the problem that the target is close to the reference node or any coordinate axis causes the positioning error of the traditional positioning algorithm to increase sharply, an optimized two-step least squares algorithm is proposed. This algorithm corrects the defects of the TSWLS algorithm by selecting the reference station and rotating the coordinate system again, and improves the positioning performance of the algorithm while reducing the amount of calculation.