bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2189: Role Of Innate Immune Response And Mitochondrial Ros In The Cardiac And Pulmonary Sequelae Of Covid-19
Authors: Liu Ting , Ashok Deepthi , Craney Morgan , Wong Amanda , Foster D B , Pekosz Andrew , Villano Jason , O'Rourke Brian
Published: October 2023

Intro:
Cardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome, but the mechanisms behind COVID-19-linked arrhythmias are unknown. Here, we test the hypothesis that innate immune activation and mitochondrial ROS contribute to cardiac conduction abnormalities and pulmonary dysfunction in a COVID-19 hamster model.

Results:
ECGs and subpleural pressures were recorded by radiotelemetry over a 4-week timespan after SARS-CoV-2 infection. Multiple cardiac arrhythmias were observed, including bradycardia, sinus pauses, and atrioventricular block (AVB). RR intervals and sinus arrhythmia (RR> mean+100ms, RR100) increased transiently 3-5 days after SARS-CoV-2 infection (baseline/peak: RR, 167±3ms/241±7ms; RR100, 5.5±0.4min
-1
/22.7±3.0min
-1
) and returned to levels ≤ baseline by 7 dpi. After the acute phase, these measures increased above baseline over 3 weeks (at 28dpi: RR, 190±2ms, p<0.05; RR100, 13.4±1.0 min
-1
, p<0.01). AVB count peaked at 3.1±0.1 min
-1
at 1 dpi and gradually decreased to 0.4±0.2 min
-1
by 28 dpi (vs. 0.004±0.002 at 0 dpi, p<0.05). These parameters did not change in Mock-infected controls. RT-PCR showed that RIG-I, CXCL-10, OAS1, IL-1b and CCL-2 expression increased 18, 7, 2, 3, and 2-fold, respectively, over Mock controls at 4 dpi. IF analysis of the His-bundle region showed increased abundance of inflammatory IBA-1
+
macrophages and decreased anti-inflammatory CD163
+
macrophages. Ruxolitinib (Jak/Stat inhibitor) or mitochondrial antioxidant (mitoTEMPO) treatments were administered via implanted osmotic pumps. Subpleural pressure recordings indicated that infection induced tachypnea peaking at 7 dpi (272±11 versus 81±17 breaths/min at 0 dpi, p<0.0001), which was attenuated by either treatment (peak: mitoTEMPO, 170±10; Ruxo,184±14). MitoTEMPO, but not Ruxolitinib, suppressed the increases in RR, RR100, and AVB during the acute phase and prevented the secondary changes in these measures in the post-acute phase (up to 28 dpi).

Conclusions:
The hamster model recapitulates a subset of arrhythmias observed in COVID-19 patients. Jak/Stat inhibition or mitoTEMPO treatment both mitigated acute respiratory distress, but only the latter suppressed arrhythmias during the acute and late phases of COVID-19.
Abstract P3001: Regulation Of Perivascular Adipose Tissue By Follistatin In Hypertension
Authors: Kuganathan Ann , Leal Marcos , Lu Vincent , MacDonald Melissa , Gao Bo , Dickhout Jeffrey , Krepinsky Joan
Published: October 2023

Follistatin lowers BP and improves resistance artery health in spontaneously hypertensive rats (SHR), a model of essential HTN. Functionally, follistatin improves smooth muscle contraction and endothelium-dependent relaxation. Structurally, increased medial collagen was inhibited. Vascular ROS were also reduced. Perivascular adipose tissue (PVAT) regulates vascular tone via release of vasoactive substances. Oxidative stress in PVAT, as in HTN, induces release of contractile agents. Transition from white to brown PVAT is associated with decreased risk of CVD. We analyze effects of follistatin on PVAT and its regulation of vessel function in SHR and normotensive Wistar Kyoto (WKY) rats.

White and brown PVAT were dissected from treated rats; arteries were isolated from untreated donors. PVAT was introduced near strain-matched donor arteries and assessed for ROS

levels and contractility using DHE and wire myography, respectively. In some vessels, endothelium was denuded to assess its contribution to contraction.

Follistatin reduced ROS, hypercontractility, and improved relaxation in a PVAT-dependent manner. Brown PVAT induced significant anti-contractile and antioxidant effects. Elevated expression of adipose tissue browning markers and decreased adipocyte area with follistatin, support PVAT browning effects. Endothelium removal results in augmented vessel constriction, effects reduced by follistatin.

Follistatin improves SHR smooth muscle tone in a PVAT-dependent manner, likely through the inhibition of vascular ROS and browning of PVAT. Future work will profile PVAT effects of follistatin through unbiased proteomic analysis. Although most vessels are surrounded by PVAT, studies of PVAT-vessel interaction in HTN are few. By characterizing PVAT dysfunction, we aim to outline specific PVAT-mediated pathways involved in lowering BP and improving vascular function and structure. Results will provide new insights into PVAT-targeted therapy for essential HTN, allowing potential translation of benefits to obesity and other diseases exhibiting PVAT dysfunction.
Abstract P3022: Smyd2 Deficiency Protects Against Atherosclerotic Plaque Formation Through Modulation Of Oxidative Stress
Authors: Sharma Shaligram , Guan Xiaoqing , Hou Yuning , Harvey Jada , Lamba Pranav , Li Jacob , Dorsey Arin , Diktonaite Kotryna , Li Chunying
Published: October 2023

Introduction:
Oxidative stress induced vascular remodeling promotes atherosclerosis. By scavenging excessive ROS levels, NRF2 protects vascular cells against oxidative stress and exerts anti-inflammatory activity through modulation of HO-1. This study aimed to explore smooth muscle cell (SMC)-specific role of SMYD2 in modulating atherosclerosis associated oxidative stress
in vivo
and
in vitro
.

Methods:
SMC-specific SMYD2 knockout mice in ApoE
-/-
background (SMYD2

ΔSMC

ApoE
-/-
) and their littermate controls (SMYD2

fl/fl

ApoE
-/-
) were subjected to Western diet feeding for 18-26 weeks. Atherosclerotic plaque distribution was examined by
en-face
Oil Red O (ORO) staining of aortas from both groups. Hematoxylin & Eosin (H&E) staining on brachiocephalic artery tissues were performed to determine atherosclerotic plaque burden by measurements of total plaque size, lipid pool area, and necrotic core area. Lipid peroxidation was analyzed in aortic tissues and in blood serum. Primary VSMCs isolated from control and SMYD2 depleted mice and cultured C3H/
10T1
/
2
cells were used for
in vitro
studies.

Results:
SMC-specific SMYD2 depletion attenuated atherosclerotic plaque establishment and progression.
En
-face ORO analysis revealed significant suppression of ORO positive areas in SMYD2

ΔSMC

ApoE

-/-

mice compared to control mice. H&E analysis of lesions showed significant reduction of total plaque size, despite accumulation of lipids, in SMYD2

ΔSMC

ApoE
-/-
mice as compared to control mice. Immunofluorescence staining of aortic tissues showed increased expression of NRF2, HO-1 and GPX4 in SMYD2

ΔSMC

ApoE
-/-
mice compared to controls, as observed in serum by MDA assay. Mechanistic studies using C3H/
10T1
/
2
cells treated with PAz-PC (oxLDL), GPX4 inhibitor, with or without SMYD2 knockout, demonstrated that SMYD2 modulates HO-1 and GPX4 expression through NRF2. Specifically, SMYD2 binds NRF2 and reduces its translocation into the nucleus, thereby repressing HO-1 expression and inducing oxidative stress.

Conclusion:
SMYD2 depletion induces atheroprotective effect via reducing plaque burden and promoting antioxidant activity of NRF2/HO-1 axis, suggesting SMYD2 could be a therapeutic target against atherosclerosis.
Abstract P3024: Endothelial To Mesenchymal Transition Frequency Increases In Williams Syndrome Pulmonary Artery Endothelial Cells In Novel 3D Bioprinted
In Vitro
Arterial Model
Authors: Saadeh Maher R , Tomov Martin L , Dasi Lakshmi P , Avazmohammadi Reza , Serpooshan Vahid , Bauser Heaton Holly
Published: October 2023

Introduction:
Pulmonary artery stenosis (PAS) affects 1 in 2000 live births worldwide, with several causes, most commonly by genetic mutations that lead to or are associated with other congenital heart diseases (CHD). Despite its prevalence, long-term treatment strategies for patients with PAS remains ineffective due to its heterogenous etiology. In the last few decades, researchers have found hemodynamic forces to be a great contributor to stenosis development because it induces endothelial cell (EC) dysfunction through endothelial-to-mesenchymal transition (EndMT). Current pulmonary artery models,
in vivo
and
in vitro
, are ineffective in uncovering this mechanism in PAS because they either are unable to recapitulate the phenotype or the micromechanical environment.

Methods:
To address these limitations, we developed and optimized a high-throughput 3D bioprinted
in vitro
vascular model that recapitulates patient-specific disease states to determine how EndMT plays a role in stenosis development. This bioprinted model will be a cellularized bilayer comprised of EC and smooth muscle cells (SMC) sourced from either human primary cells or our established Williams Syndrome (WS) induced-pluripotent stem cell (iPSC) lines. WS was chosen due to its high predisposition in developing PAS, around 50%. Our hypothesis is that utilizing our 3D bioprinted model, we will demonstrate how alterations in hemodynamic forces, specifically flow regiments, will further promote EndMT in the WS group.

Results:
Encapsulated SMC functionality and differentiated phenotype was confirmed by calcium measurement and immunohistochemistry, respectively. Single-cell RNA sequencing (scRNA-seq) was used to examine EC, SMC, EC-SMC crosstalk, and EndMT gene expression profiles.

Conclusion:
This work represents a new and substantive departure from conventional
in vitro
vascular modeling, for it will allow us to identify initial vascular cellular responses in acute non-homeostatic conditions and uncover unique signaling pathways found in multiple CHD related PAS. This fundamental knowledge from our vascular mimics can then be applied to facilitate translational advancements in treatment of PAS via surgical and transcatheter intervention methods.
Abstract P3025: CXCL10 Impairs Endothelial Barrier Integrity In Endothelial Pparδ-deficient Mice Identified By Single-cell RNA Sequencing
Authors: Hong Huiling , Yang Kevin Y , Lui Kathy O , Tian Xiao-Yu
Published: October 2023

Rationale and Background:
CXCL10 is a pro-inflammatory chemokine produced by monocytes and endothelial cells (ECs) in response to IFNγ, acting as a chemoattractant for monocytes and T cells etc., through the CXCR3 receptor. The previous study from our group showed that selective deletion of PPARδ in the endothelium worsens ischemic vascular injury in mouse model of hindlimb ischemia. Single-cell RNA sequencing of muscle endothelial cells showed that PPARδ regulates genes involved in endothelial barrier function and vascular inflammatory responses.

Methods and Results:
To further understand the role of PPARδ in endothelial cells from different vascular beds, we induced acute lung inflammation by lipopolysaccharide (LPS) in EC-selective PPARδ knockout mice (Ppard
EC-KO
) and their wild-type littermates (Ppard
EC-WT
) and analyzed the lung ECs by scRNAseq. After LPS injection, lung ECs were collected as CD45
-
CD31
+
CD144
+
cells by FACSorting, and scRNAseq was performed using 10X genomics method. General capillary ECs and aerocyte capillary ECs were identified by distinctive marker genes as the two most abundant lung ECs. The capillary ECs from LPS-challenged Ppard
EC-KO
mice showed high enrichment of inflammatory signaling including IFN-IRF, IL6-STAT3, and TNFα-NFκB. Further sub-clustering analysis revealed a capillary EC subset with upregulation of IFN signaling and identified CXCL10 as the top marker-gene in LPS-challenged mice. CXCL10 expression was indeed upregulated in the lung tissue of LPS-treated Ppard
EC-KO
mice and in TNFα-treated human lung microvascular endothelial cells (HLMVECs). CXCL10 decreased the expression of EC tight junctional proteins ZO-1 and Claudin-5 through the ubiquitin-proteosome system-mediated degradation in HLMVECs. Blocking the CXCR3 by AMG487 suppressed vascular leakage and EC activation in response to LPS.

Conclusion:
These results suggested the possibility of targeting CXCL10 signaling for restoring vascular homeostasis in acute lung inflammation. (Supported by Hong Kong Research Grant Council GRF 14107822 and 14105321)
Abstract P3033: Thrombospondin-1 Promotes Vascular Smooth Muscle Cell De-differentiation In Diabetes
Authors: Gupta Shreya , Mathias Amy , Lallo Jason , Bhavnani Neha , Raman Priya
Published: October 2023

Diabetes is a major risk factor for atherosclerosis. Diabetic patients are prone to increased vascular smooth muscle cell (VSMC) migration and proliferation, hallmarks of VSMC de-differentiation, critical for the evolution of atherosclerosis. Multiple studies, including our earlier work, support the role of thrombospondin-1 (TSP1), a proatherogenic matricellular protein, as a putative link between diabetes and vascular disease. However, molecular mechanism(s) by which TSP1 promotes diabetic vasculopathy are incompletely understood. The goal of this work was to explore whether smooth muscle TSP1 (smTSP1) regulates VSMC phenotypic switch in diabetes. We generated conditional SMC-specific TSP1 knockout (smTSP1
KO
) mice by crossing female TSP1
fl/fl
mice with tamoxifen (tmx)-inducible

Myh11-CreER
T2

male mice. For Cre recombination, 5-wks-old male TSP1
fl/fl
Cre
tg
and age-matched TSP1
+/+
Cre
tg
littermate mice were treated with 60 mg/Kg/day tmx i.p. once daily for 5 consecutive days. To induce type 2 diabetes, mice cohorts were placed on western diet at 7-wks-age followed by streptozotocin (50 mg/Kg/day, i.p. x 5 days) at 16-wks-age. Mice were harvested at 20-wks-age for plasma and tissue collection. Immunoblotting of aortic lysates revealed significant decrease in TSP1 expression in tmx-treated TSP1
fl/fl
Cre
tg
, validating smTSP1
KO
mice. Lack of smTSP1 increased ACTA2 (SM contractile marker) expression by 2-fold accompanied with 50% reduction in CDK4 (cell cycle regulator) expression in diabetic smTSP1
KO
vs. smTSP1
WT
aortic vessels. Consistently, in murine aortic SMC (MASMC) primary cultures derived from smTSP1
KO
mice, loss of smTSP1 augmented (>65%) LMOD1 (SM contractile marker) and SRF (transcriptional activator of SM contractile genes) expression coupled with diminished (45%) vimentin (SM synthetic marker) expression in 25mM glucose-treated smTSP1
KO
vs. smTSP1
WT
cells. Further, propidium iodide staining via flow cytometry revealed reduced S-phase cell counts concomitant to increased G
0
-G
1
cell distribution in glucose-treated smTSP1
KO
vs. smTSP1
WT
MASMC. Together, our data demonstrates a direct role of TSP1 in VSMC phenotypic transition and opens novel avenues for cell-specific TSP1-based therapies for treatment of diabetes.
Abstract P3037: MicroRNA-205 Is Required For Normal Cell Cycle And Cardiac Growth In The Neonatal Mouse Heart.
Authors: Burgon Patrick G , Weldrick Jonathon , Yi Rui , Megeney Lynn
Published: October 2023

After birth, most newborn mouse cardiomyocytes (CM) undergo a final cycle of cell division in the absence of cytokinesis, which leads to binucleation. The majority of subsequent increases in cardiac mass are attributable to cardiomyocyte hypertrophy, as a negligible number of new CM are created after birth. Mice with a heart-specific deletion of Dicer, a crucial enzyme necessary for microRNA maturation, die by the fifth postnatal day in spite of having normal cardiac morphology and function at birth. Despite the significance of this phenomenon, little is known about the molecular/genetic foundation for the change from hyperplastic to hypertrophic-based myocardial expansion that occurs in the neonatal mouse heart. We hypothesize that a microRNA is required for the normal transition from fetal to adult heart during the neonatal period. Heart microRNA profiles (3 arrays/time point) were measured from RNA was isolated from E19, 1, 3, 5, 7, 10, and 35-day-old mouse hearts (9 hearts/time point pooled). Bioinformatic analysis identified microRNA-205 as a possible participant in the cardiac transitional program. We observed a transient 20-fold increase in miR205 expression between postnatal days 1 and 5, with levels returning to baseline by day 10.
In-situ
hybridization revealed miR-205 expression is restricted to the heart's epicardium.We next generated mice with a CM-specific deletion of miR205 using MHCcre targeting of a floxed-miR205 allele. Mice lacking miR-205 in their CM appear normal at birth. Elevated cell cycle markers (Ki67 & phospho-pH3) were detected at postnatal day-14 with 20% larger hearts at postnatal day 14, and adult hearts measuring up to 50% larger than controls. In contrast, a CM-specific miR205 over-expression mouse model resulted in more CM being present at both 5-day and 14-day post-birth. Interestingly, no difference in cell-cycle markers or heart size was observed. miR205 directly targets YAP within the evolutionarily conserved hippo pathway, which regulates organ size. YAP protein expression is significantly increased in hearts lacking miR205. Both of the miR205-heart models present with altered Hippo signaling kinetics after birth. We conclude that miR205 regulates postnatal heart size by directly modulating the Hippo pathway.
Abstract P3039: The Biophysical Action Of Microrna-1 Is Critical To Maintaining The Homeostasis Of The Heart
Authors: Yang Dandan , Wan Xiaoping , Kacira Ege , xu Xianyao , Tang Feng , Hund Thomas J , Mohler Peter J , Deschenes Isabelle , Fu Jidong A
Published: October 2023

MicroRNAs (miRs) are evolutionarily-conserved small noncoding RNAs (~22 nucleotides) and are involved in most biological events through the classical mechanism of RNA interference (RNAi). Recently, we discovered a novel action of miR to biophysically modulate the function of bound proteins. miR1, the most predominant miR in the heart, physically binds to the inward rectifier potassium channel Kir2.1, directly represses the I
K1
current, and biophysically modulates cardiac electrophysiology. An arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts biophysical modulation while retaining the RNAi function. However, the physiological significance of miR’s biophysical action on the heart remains unknown. To study this, we created hSNP14A/G single nucleotide mutation on miR1-1 and miR1-2 by using CRISPR/Cas9 technology and successfully generated 14G-mutated transgenic homozygous mice (14G-Homo, miR1-1
14G/G
:miR1-2
14G/G
), in which the mutated miR1 maintains the RNAi while with a specific deletion of the biophysical action. We found that 14G-Homo mice had high mortality with impaired heart pumping function and some mice developed heart failure at 6 months of age. The whole transcriptome RNA-sequencing assays revealed cardiac electrical remodeling in 14G-Homo hearts. Consistently, 14G-Homo hearts had prolonged QRS and QT intervals with slower electrical conduction through the ventricle, and 14G-Homo mice are susceptible to arrhythmia. Our patch clamping showed that 14G-Homo cardiomyocytes had significantly prolonged action potential with bigger I
K1
and L-type Ca
2+
current (I
Ca,L
) than WT cells. We also performed a CLIP RNA pulldown-mass spectrometry assay and identified more ion channel proteins that interact with miR1. From the validation, miR1 biophysically suppresses I
Ca,L
via directly binding to Cavβ2, the β subunit of the voltage-gated Ca
2+
channel. We conclude that miR1 biophysically modulates the function of the heart, which is critical to maintaining heart homeostasis. Our discovery expands the biological significance of miR biology and broadens its implication of RNA medicine development for cardiovascular diseases.
Abstract P3042: Cardiomyocyte MicroRNA-125a-5p Plays A Vital Protective Role In Myocardial Infarction And Directly Represses Proapoptotic GTP-Cyclohydrolase I Feedback Regulator
Authors: Hayasaka Taiki , Sepulveda Marisa , Kim Il-man
Published: October 2023

Background:
We reported that microRNA-125a-5p (miR-125a) is activated by β-blocker carvedilol acting through β-arrestin1-mediated β1-adrenergic receptor (receptor found mainly in cardiomyocytes [CMs]) signaling (Figure A). GTP cyclohydrolase I feedback regulator (GCHFR) inhibits tetrahydrobiopterin which is protective in myocardial infarction (MI). Despite these findings, the roles of miR-125a and GCHFR in MI and their mechanisms of actions remain elusive. Here, we hypothesize that miR-125a in CMs plays a protective role in MI in part by repressing GCHFR, a novel target of miR-125a.

Methods and Results:
Fractionation of cardiac cell types isolated from mouse hearts reveals that miR-125a expression is higher in CMs than other cells. Using cultured CM and
in vivo
approaches, we show that miR-125a is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125a exhibit an increased sensitivity to apoptosis, whereas CMs overexpressing miR-125a have increased phospho-AKT prosurvival signaling. MiR-125a is downregulated in failing hearts, and miR-125a overexpression protects mouse hearts against MI. Systemic deletion of miR-125a in mice worsens maladaptive post-MI remodeling. Genome-wide profiling in miR-125a KO mouse hearts identifies GCHFR as a novel target of miR-125a. Mechanistically, protective roles of miR-125a in CMs are in part attributed to the direct and functional repression of proapoptotic GCHFR. Last, novel conditional CM-specific miR-125a KO in mice exacerbates maladaptive cardiac remodeling after MI (Figure B).

Conclusions:
Our findings suggest a crucial role for the miR-125a/GCHFR axis in regulating CM survival post-MI.
Abstract P3044: MicroRNA-127-3p Regulates Inflammatory Response In Injury-induced Vascular Remodeling And Cellular Senescence In Vascular Smooth Muscle Cells
Authors: Daniel Fiene , Kalies Katrin , Hehl Laura , Guerlach Simon , Köster Jan Michael , Boettcher Solveig , Knoepp Kai , Dutzmann Jochen , SEDDING Daniel
Published: October 2023

The accumulation of senescent cells in the vasculature over the lifespan contributes to vascular remodeling due to the severely impaired functional properties of the cells. MicroRNAs (miRs) are promising therapeutical targets as they exhibit diverse and cell-specific functions and can be effectively targeted
in vivo
. Here, we propose that targeting miR-127-3p might be an attractive approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications.

The initial screenings revealed miR-127-3p as a highly regulated miR in the process of restenosis 7 and 21 days (p<0.001) after wire induced-injury and in aged (20 months) C57BL/6 mice (p<0.05)
in vivo
. Additionally, replicative senescent vascular smooth muscle cells (SMC) and human coronary artery endothelial cells (EC) showed an enhanced expression of miR-127-3p (p<0.05) compared to non-senescent cells. Functional effects of miR-127-3p on SMCs and ECs were assessed via transfection with either pre-miR or antagomir. Overexpression of miR-127-3p
in vitro
reduced SMC proliferation (p<0.05) and altered migrational capacity. Downregulation of miR-127-3p resulted in a reverse effect. In contrast, cellular functions were not altered in ECs. We further identified potential targets with effects on inflammation and cellular aging by
in silico
analysis and verified them on mRNA and protein levels by qRT-PCR and western blot analysis. As a direct target of miR-127-3p, the senescence-associated gene CD44 was identified by TargetScan. Expression of CD44 was altered on mRNA and protein levels after overexpression respectively downregulation of miR-127-3p in SMCs. Further, p14arf and laminB1 were downregulated after overexpression of miR-127-3p in SMCs, contrary to the effect after downregulation of miR-127-3p with increased mRNA levels of p14arf and laminB1.

In this study, we identified miR-127-3p as a contributing factor in SMC function during the development of cellular senescence and show that it might therewith also be a modulating factor in the process of restenosis. However, further studies need to be carried out to validate the potential of miR-127-3p as a therapeutic target in vascular aging and remodeling
in vitro
and
in vivo
.
Abstract P3046: The Effect Of MicroRNA-367-3p And MicroRNA-148a-3p On Vascular Endothelial Cells In Context Of Senescence And Infarct Healing
Authors: Hehl Laura , Kalies Katrin , Daniel Fiene , Knoepp Kai , Dutzmann Jochen , SEDDING Daniel , Köster Jan Michael , Guerlach Simon , Boettcher Solveig
Published: October 2023

To improve the healing after myocardial infarction (MI), a well-coordinated angiogenic response is necessary. MicroRNAs (miRNAs) were identified as regulators of angiogenesis, however, strategies to improve angiogenesis by targeting miRNAs are challenging. Here, we evaluate miRNA-367-3p and 148a-3p as novel miRNA candidates regarding their therapeutic potential toward neovascularization after MI.

Following expression analysis of the respective miRNAs in human vascular endothelial cells (EC) and mice tissue, their contribution to EC function after either pre- or anti-miR transfection was assessed. Further, potential targets were identified and studied on mRNA and protein level.

Initial screenings show that miRNA-148a-3p and miRNA-367-3p are differently expressed in both a restenosis model via wire-induced injury and aged mice tissue (p<0.05). Under growth conditions, miRNA-148a-3p expression was upregulated in ECs. In senescent ECs, miRNA-367-3p shows a significant 50-fold upregulation of its expression (p<0.01); miRNA-148a-3p is also overexpressed in senescent ECs (p<0.01). Hypoxia increases the effect observed in senescence (p<0.05). Downregulation of either miRNA-367-3p or miRNA 148a-3p leads to an improvement in migration capacity (p<0.0001) and proliferation of ECs (p<0.0001). Systematic target research
in silico
identified DKK3 as potential target of miRNA-367-3p and DDX6 as potential target of miRNA-148a-3p. Downregulation of either miRNA-148a-3p or miRNA-367-3p verified these predictions by significantly upregulating DKK3 respectively DDX6. In further experiments, we study the dysfunctional effect of miRNA-148a-3p on vascular endothelial cells by targeting DDX6 as well as the anti-angiogenic impact of miRNA-367-3p by targeting DKK3.

Summarizing different expression levels of miRNA-367-3p and miRNA-148a-3p exerted distinct effects on EC function, especially in senescent cells and under hypoxic conditions. Our experimental data regarding cellular function show that these miRNAs are involved in the angiogenetic response and hold the potential to serve as therapeutic targets. In future experiments, we will evaluate the miRNA potential on MI healing
in vivo.
Abstract P3048:
Ppp1r1b-lncRNA
Regulates Cardiomyocytes Differentiation Via Epigenetic Modulation Of
Tbx5
Promoter
Authors: Kang Xuedong , Hwang John , Touma Marlin
Published: October 2023

Introduction:
Myogenic differentiation is essential to cardiac development as well as regeneration and repair in response to genetic lesions or injury in the context of cardiomyopathy and congenital heart defects (CHDs). We have previously shown that mouse
Ppp1r1b-lncRNA
modulates epigenetic features of myogenic master regulators through targeted interaction with histone-modifying complex and other co-factors. However, the molecular mechanisms that underlie human
PPP1R1B-lncRNA
-mediated regulation of cardiomyocyte differentiation remain unknown.

Objectives:
To elucidate the mechanisms by which human
PPP1R1B-lncRNA
regulates cardiomyocyte differentiation.

Methods/Results:
PPP1R1B-lncRNA
possessed conserved function in promoting myogenic differentiation in human cardiac and skeletal myocytes and its expression was dysregulated in CHDs. Mechanistically, by silencing
PPP1R1B-lncRNA
, myogenic differentiation was impaired in
PPP1R1B-lncRNA
-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The expressions of myogenic transcription factors, including
GATA4
and
TBX5
, as well as sarcomere proteins, including
TNNT2
and
MYOZ2,
were significantly suppressed in
PPP1R1B-lncRNA
deficient hiPSC-CMs. Epigenetic analysis revealed increased H3K27 tri-methylation at
TBX5
promoter in the
PPP1R1B-lncRNA
knockdown cardiac progenitors.
PPP1R1B-lncRNA
was found to bind to EZH2, the enzymatic core of the polycomb repressive complex (PRC2). Furthermore, chromatin isolation by RNA purification (ChIRP)-seq revealed enriched interaction of
PPP1R1B-lncRNA
with
TBX5
,
MEF2C,
and
GATA4
promoters. Meanwhile, chromatin immunoprecipitation (ChIP)-PCR demonstrated increased enrichments of EZH2 at
TBX5
promoter in the
PPP1R1B-lncRNA-depleted
cells, resembling what was observed in the pre-differentiation state.

Conclusions:
Human
PPP1R1B-lncRNA
promotes cardiomyocyte differentiation via interactions with promoters of key cardiac transcription factors.
PPP1R1B-lncRNA
enhances chromatin accessibility at
TBX5
promoter by interfering with PRC2 binding. Understanding
PPP1R1B-lncRNA
function in cardiomyocyte differentiation may lead to novel therapies for cardiopathies and CHDs.
Abstract P3050:
Cpne5
Is Necessary For Normal Murine Sinoatrial Node Function
In Vivo
Authors: Ren Lu , Thai Phung , Mantri Sruthi , Duan Lauren , Atzert Felix , Beyersdorf Benjamin , Chiamvimonvat Nipavan , Wu Sean M , Goodyer William R
Published: October 2023

Cpne5
(
Copine 5
) is a Ca
2+
-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing of the developing mouse heart, we previously found
Cpne5
gene expression to be highly enriched in the cardiac conduction system (CCS). Further, variants in
CPNE5
were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesized that
Cpne5
is necessary for normal CCS function through Ca
2+
-dependent membrane trafficking of ion channels.

Whole mount immunostaining with 3D volumetric imaging (iDISCO+) of intact WT hearts demonstrated robust and specific protein expression of Cpne5 throughout all CCS components, including the sinoatrial node (SAN) and SAN-transitional cells. Next, to assess the necessity of
Cpne5
within the CCS, systemic knockout mice (Cpne5KO) were generated using CRISPR/Cas9. Successful deletion of
Cpne5
in CRISPR-generated KO mice was confirmed by PCR and, at the protein level, immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed a slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, with notable sinus pauses, sinus bradycardia, and increased heart rate variability as compared to WT littermate controls on long term telemetry.

Systemic loss-of-function of Cpne5 in mice resulted in marked sinus node dysfunction, consistent with human GWAS data, implicating a novel role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies, including CPNE5 deletion in human induced-pluripotent cell-derived SAN-like cells, are ongoing to further elucidate its underlying mechanism.
Abstract P3059: Recombinant Adeno-associated Viral Vector 2/8 Model Of Interleukin 6 Mediated Chronic Inflammation
Authors: Jacobs Johanna E , Maes Wannes , Caluwe Ellen , Lox Marleen , Gillijns Hilde , Pokreisz Peter , Roskams Tania , Van Den Haute Chris , Gijsbers Rik , Janssens Stefan P , Van Aelst Lucas
Published: October 2023

Background:
Interleukin 6 (IL6) is a pleiotropic cytokine mostly known as a proinflammatory cytokine. There is a need for a reliable in vivo model to study IL6 mediated chronic inflammation.

Hypothesis:
The aim of our study was to create a mouse model mimicking the chronic inflammatory state as observed in cancer and inflammatory diseases. We opted for a recombinant adeno-associated viral vector (rAAV) to mimic the acute initiation of chronic inflammation later in life.

Methods:
We opted for a rAAV2/8 subtype, human EF1a short (EFS) promoter and firefly luciferase led by an internal ribosome entry site (IL6-IRES-fLuc) DNA cassette to create a stable low-grade expression of IL6 in liver and serum, with minimal expression in other organs, particularly the heart. rAAV2/8_EFS-IL6-IRES-fLuc and the control vector rAAV2/8_EFS-MCS-IRES-fLuc were generated and different dosages were tested.

Results:
All mice treated with high dose AAV2/8-IL6 vector (8.8* 10
10
genome copies (GC)/animal) showed severe weight loss and hepatosplenomegaly, starting between week 4-6 post injection. Histological examination of the liver showed features of chronic hepatitis. Infiltrating cells were found in both the liver and the heart. In the spleen, the histological structure of red and white pulp was disturbed and more giant cells could be detected. Cardiac function remained normal at 6 weeks post injection. Assuming that the previous results were due to an overexpression of IL6 due to too high amounts of rAAV2/8_EFS-IL6-IRES-fLuc, we subsequently challenged mice with a rAAV2/8_EFS-IL6-IRES-fLuc dilution series to test which vector dose could be used for reliable long-term low grade IL6 overexpression. After extensive testing, the ideal dose for a 12 weeks IL6 overexpression model was between 2.2 and 2.93* 10
10
GC. Using this dose range, we saw stable overexpression of IL6 without weight loss. Hepatosplenomegaly, disturbed anatomy of the spleen and infiltrating cells in the liver were still present yet less pronounced.

Conclusion:
We successfully generated and characterized a mouse model of IL6 overexpression to mimic chronic IL6 mediated inflammation. To the best of our knowledge this is the first model employing IL6 overexpression using a rAAV viral vector.
Abstract P3069: ScRNA-seq And Flow Cytometry Based Analysis Reveals That Chronic Heart Failure Associates With Enhanced Activation And Clonal Expansion Of T Cells With Predictive Autoreactive Capacity
Authors: Abplanalp Wesley T , Merten Maximilian , Cremer Sebastian , Rasper Tina , Holz Kathrin , Schuhmacher Bianca , Macinkovic Igor , Tombor Lukas , John David , Zeiher Andreas M , Dimmeler Stefanie
Published: October 2023

Background:
Heart failure (HF) is marked by adverse remodelling and chronic inflammation causing impaired function of the heart and poor prognosis. While much is known about the immune response immediately after MI, little is known of the role of the immune system in chronic HF. Our aim is to elucidate the response of T cells to HF.

Methods and Results:
In a pilot study assessing healthy controls (HC) and HF patients (n=16), scRNA-seq showed an enhanced T cell activation profile in HF patients. A confirmation cohort of n=180 HC and HF patients using flow cytometry of immune cells confirmed an increase of T cell memory/effector cells (HF=65%, HC=53% activated T cells). Correspondingly, circulating antigen presenting cells showed augmented inflammatory profiles (ICAM-1, TREM-1). Given that T cells showed strong activation profiles in HF patients, we investigated whether T cells are preferentially clonally expanded in the same patients. Bulk T cell receptor (TCR) sequencing revealed that TCR diversity was significantly reduced (p=0.03) along with clonal expansion of T cells in HF patients. To assess what T cells might be targeting, we utilized epitope prediction tools to identify 22 unique human-derived epitopes found only in HF patients. ScRNA-seq of CD45+ sorted cardiac-derived and circulating cells of the same HF patient (N=1) versus healthy controls (n=4) showed clonal expansion of TCRs in the cardiac tissue and blood in HF and epitope prediction confirmed epitopes found in bulk TCR sequencing. When modelling this in mice, a scRNA-seq post-MI time course also found an accumulation of specifically pro-inflammatory (Th17) T cells in the heart at days 14, 28 and 48 post-MI. To assess the impact of immune dysregulation in HF on cardiac cells, we cultured the secretome of HF or HC immune cells with endothelial cells and found decreased viability of endothelial cells and increased monocyte adhesion to stimulated endothelial cells (p=0.005).

Conclusion:
This study suggests that chronic HF may drive persistent T cell activation and clonal expansion, with potential cardiac-relevant autoimmune implications causing or promoting adverse remodelling. These data also provide additional insights for therapeutic cardio-immunological interventions.
Abstract P3076: Immune Checkpoint Regulator Impairs Left Ventricular Remodeling In A Hypertension-Fibrosis Model Of Cardiac Injury.
Authors: Jimenez Jesus , Ma Pan , Lavine Kory J
Published: October 2023

Cancer treatments directly result in cardiovascular toxicity and may exacerbate typical age-related diseases such as hypertension and diabetes. Among cancer treatments contributing to survivorship, immunotherapies including immune checkpoint inhibitors (ICIs) are now widely utilized due to their groundbreaking efficacy. CD40 agonist (CD40ag) antibodies are immune checkpoint regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse left ventricular remodeling in a hypertensive-fibrosis model of cardiac injury, C57BL/6J mice were pretreated with isotype or CD40ag antibodies 7 days prior to myocardial injury, then continued for a total of 35 days. Myocardial injury was induced by continuous infusion of angiotensin II and phenylephrine (AngII/PE) for 4 weeks. Masson’s trichrome stain revealed increased fibrosis and immunohistochemistry demonstrated increased immune cell abundance in heart sections. There was impaired cardiac function assessed by 2-D echocardiography in minimally sedated mice showing reduced ejection fraction and radial strain. Finally, CD40 agonism resulted in gene expression upregulation of multiple inflammatory cytokines and chemokines post-cardiac injury. These results reveal that pre-treatment with CD40 agonist prior to injury with AngII/PE infusion increased myocardial inflammation and impaired left ventricular remodeling.
Abstract P3077: Withdrawal Of High Fat Diet And L-NAME Dampens T Cell Activation And Ameliorates Pathology In Experimental Heart Failure With Preserved Ejection Fraction (HFPEF) Through Modulation Of The T Cell IRE1a-XBP1 Axis
Authors: Smolgovsky Sasha , Bayer Abraham , kaur Kuljeet , Aronovitz Mark , Sanders Erin , Blanton Robert M , Thorp Edward B , Schiattarella Gabriele G , Hill Joseph A , Cubillos-Ruiz Juan R , Alcaide Pilar
Published: October 2023

Introduction:
Cardiometabolic HFpEF is characterized by T cell systemic inflammation and can be modeled preclinically with high fat diet (HFD) and L-NAME. Metabolic perturbations can alter T cell function through modulation of the unfolded protein response (UPR). How T cells are activated in HFpEF and whether T cell inflammation can be tuned by removing cardiometabolic stress remains unknown.

Hypothesis:
HFD and L-NAME induce reversible alterations in T cell UPR signaling that modulate T cell dependent cardiac inflammation and pathology in experimental HFpEF.

Methods:
Male C57/BL6J mice (wild-type, WT), OT-II antigen restricted mice, and CD4
Cre
Xbp1

fl/fl

mice were fed a high-fat diet (HFD) and L-NAME (H/L) for 5 weeks. WT mice were switched to standard diet (STD) for two additional weeks. WT mice receiving HFD or L-NAME only, or TAC surgery (a model of HF with reduced EF) were used as controls. Left ventricular (LV) end diastolic pressure volume relationship (EDPVR) was assessed using invasive hemodynamic analyses and LV EF by echocardiography. Cardiac and splenic T cells were characterized by flow cytometry and T cell UPR was evaluated by qPCR and Western blot.

Results:
5 weeks of H/L was sufficient to induce cardiac T cell infiltration and splenic T cell activation. OTII mice fed H/L showed increased cardiac CD4
+
T cell infiltration compared to STD controls, indicating T cell activation is not cardiac antigen restricted. Furthermore, splenic T cell characterization revealed H/L resulted in decreased phosphorylation of the UPR protein inositol-requiring enzyme 1α (IRE1α) and reduced expression of X box-binding protein 1 (
Xbp1s/u
) compared to T cells from mice exposed to HFD or L-NAME alone, STD, or WT TAC controls. CD4
Cre+
Xbp1

fl/fl

mice (T cell Xbp1
-/-
) fed with H/L had increased cardiac T cell infiltration compared to Cre
-
controls. Lastly,
Xbp1s
expression in splenic CD4
+
T cells was recovered after withdrawal of H/L and resulted in partial reduction of cardiac hypertrophy, decreased EDPVR, as well as less cardiac T cell infiltration compared to mice kept on H/L.

Conclusions:
Reversible downregulation of the T cell IRE1α-XBP1 axis is a unique T cell signature of HFpEF that contributes to T cell inflammation and adverse cardiac remodeling.
Abstract P3085: The Macrophage Landscape Across The Lifespan Of A Human Cardiac Allograft
Authors: Li Xiao , Turaga Diwakar , Tsai Chang-Ru , Li Rich Gang , Spinner Joseph , Hickey Edward , Adachi Iki , Martin James F
Published: October 2023

Much of our knowledge of organ rejection after transplantation is derived from rodent studies. We used single-nucleus RNA sequencing (snRNA-seq) to investigate the inflammatory milieu in pediatric cardiac allografts at different stages post transplantation. We distinguished donor from recipient-derived cardiac cells using naturally occurring genetic variants embedded in snRNA-seq data. Donor-derived cardiac resident macrophages dMPs, which accompany the cardiac allograft into the recipient, are rapidly lost post transplantation. On the other hand, monocyte-derived macrophages from the recipient (rMPs) quickly populate the heart post transplantation and form two macrophage populations, rMP1 and rMP2. While rMP2s have cell signatures similar to dMPs, they lack pro-reparative phagocytic activity and instead express profibrotic genes. In contrast, rMP1 express genes consistent with hallmarks of cellular rejection. Our data further support the conclusion that rMP1s activate a subset of natural killer (NK) cells, turning them into a cytotoxic cell population. Self-amplifying reciprocal signaling occurs between rMP1s and NKs, which is consistent with a key mechanism for clinical rejection. Our data reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft after heart transplantation. Moreover, we observed a clear functional dichotomy of recipient-derived macrophages. At different stages after heart transplantation, rMPs contribute to acute adverse damage to the myocardium and chronic organ failure.
Abstract P3092: Histone Deacetylase 6 Inhibitor Suppresses The Inflammation Through MIR17HG In LPS-Induced Macrophages
Authors: Perales Selene , Sigamani Vinoth , Rajasingh Sheeja , Johnson Rajasingh
Published: October 2023

Background:
Long non-coding RNAs (lncRNAs) have been linked to a wide range of pathologic diseases despite having a limited capacity for protein coding. MIR17HG is the host gene for the miR17-92a-1 cluster, a group of six microRNAs involved in several cellular functions. Dysregulation of MIR17HG has been associated with multiple diseases such as Feingold Syndrome, osteoarthritis, Parkinson’s disease, and cancer. Treatment with histone deacetylase-6 inhibitor (Tubastatin A -TBA) reduces inflammatory response on macrophages after LPS challenge. Here, we analyze the role of MIR17HG as an epigenetic regulator of inflammation in LPS-induced THP1 cells.

Hypothesis:
We hypothesize that TBA suppresses the effect of MIR17HG and promotes protection in macrophages under LPS-induced inflammation.

Methods and Results:
To elucidate potential lncRNAs of interest during macrophage inflammatory response, we treated macrophages with LPS (1 μg/mL) and TBA (2.5 μM) for 1hr and performed RNA sequencing. We selected MIR17HG for further investigation based on the sequencing data
(Fig. A)
and bioinformatic analysis. The expression of MIR17HG in LPS and TBA treatment conditions was validated by qRT-PCR. MIR17HG expression was significantly higher in LPS and treatment with TBA suppressed the LPS-stimulated MIR17HG expression
(Fig. B, C)
. We also observed the IL1β behaved in a similar manner
(Fig. D)
. Further overexpression and knockdown experiments will be performed to identify the link between MIR17HG and ILβ.

Conclusion:
We report for the first time that MIR17HG is an important mediator of the pro-inflammatory response of macrophages.
Abstract P3095: Il-1 Signaling On Vascular Smooth Muscle Cells Accelerates Atherosclerosis After Murine Kawasaki Disease Vasculitis
Authors: Chen Shuang , Lee Youngho , Lane Malcolm , Moreira Debbie , Aubuchon Emily , Shimada Kenichi , Crother Timothy R , Noval Rivas magali , Arditi Moshe
Published: October 2023

Objective:
We have previously shown that
Lactobacillus casei
cell wall extract (LCWE)-induced Kawasaki disease (KD) vasculitis significantly accelerates atherosclerosis in hypercholesterolemic mice. IL-1 signaling is known to play a key role in both KD vasculitis and in the development of atherosclerosis. Here, we investigated the contribution of IL-1 signaling on vascular smooth muscle cells (VSMCs) during KD vasculitis-induced acceleration of atherosclerosis.

Methods:
Tamoxifen-inducible VSMC-specific IL-1 receptor (
Il1r1)
knockout (

Myh11
Cre

-ERT2

Il1r1
Δ/Δ

)and

Il1r1
fl/fl

littermate control mice, all on ApoE
-/-
background, were injected with either PBS or LCWE. Following induction of KD vasculitis, mice were fed a tamoxifen diet for 2 weeks to induce
Il1r1
deletion on VSMCs, before being exposed to 8 weeks of Western diet (WD) to promote atherosclerosis.

Results:
KD vasculitis was associated with a significant acceleration of atherosclerosis, as expected. Specific deletion of
Il1r1
on VSMCs reduced this effect, as characterized by significantly decreased plaque size, lipid composition, macrophage infiltration and necrotic core formation in aortic root, as well as diminished lipid accumulation in aorta en face measurements.

Conclusions:
Our results suggest an important pathophysiologic link between IL-1 signaling, specifically on VSMCs, and subsequent acceleration of atherosclerosis in hypercholesterolemic mice following KD vasculitis. Thus, further studies are warranted on the role of IL-1 signaling not only in acute KD, but also in the subsequent vascular remodeling and complications following acute KD vasculitis.