bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 199: A 3-D Prevascularized Cardiac Muscle Construct for Analyzing hMSCs Engraftment and Differentiation Potential
in vitro
Authors: Valarmathi Mani T
Published: March 2022

Introduction:
The most fundamental problem facing cardiac therapy, unlike vascular grafts and heart valves, is to repair and/or regenerate the damaged myocardium. Restricted myocardial regeneration after tissue damage and shortage of donor organs for cardiac transplantation are the major constraints of conventional therapies. The most daunting task in the field of cardiovascular tissue engineering is the creation and/or regeneration of an
in vitro
engineered cardiac muscle; tissue engineering is associated with two common underlying concerns for clinical applicability, viz., contractility and thickness. However, both the thickness and the contractility of the derived cardiac tissue are dependent on the vascularity of the construct.

Hypothesis:
Whether functioning vascularized cardiac tissue can be generated by the simultaneous interaction of cardiac myocytes, endothelial cells, and somatic stem cells, as would expect to occur during myocardial reparative/regenerative processes; by utilizing, viz., the embryo-derived embryonic cardiac myocytes (eCMs) and the human adipose-derived multipotent mesenchymal stem cells (hMSCs) on a three-dimensional (3-D) prevascularized collagen cell carrier (CCC) scaffold.

Methods and Results:
First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D CCC for 7 days under vasculogenic culture conditions, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Lastly, expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, not only were hMSCs able to couple electromechanically with developing eCMs, but also able to contribute to the developing vasculature as mural cells, respectively.

Conclusions:
Hence, our unique 3-D co-culture system provides us a reproducible and quintessential
in vitro
3-D model of cardiomyogenesis, and a functional cardiac graft that can be utilized for personalized medicine.
Abstract 210: Soluble Tumor Necrosis Factor Receptors, Marker of Arterial Stiffness in Patients With Coronary Atherosclerosis
Authors: Kim Sang-Hyun , Kim Hack-Lyoung , Seo Jae-Bin , Zo Joo-Hee , Kim Myung-A
Published: March 2022

Background:
Soluble forms of tumor necrosis factor receptors (sTNFRs) are emerging target molecules of inflammatory disease. However, their role in vascular biology is not well known. This study was performed to investigate the association between serum concentrations of sTNFRs and arterial stiffness.

Methods and Results:
A total of 117 consecutive patients with suspected coronary artery disease (CAD) (63.6 ± 11.0 years; men, 65%) who were referred for invasive coronary angiography (ICA) were prospectively enrolled. Arterial blood sTNFR1 and sTNFR2 were measured using commercially available ELISA kits. Brachial-ankle pulse wave velocity (baPWV) measurements were made within 24 hours of blood sampling for sTNFRs measurement. Most of the patients (86.3%) had significant CAD (stenosis ≥ 50%) in ICA. In simple linear regression analyses, there were significant positive correlations of baPWV with sTNFR1 (
β
= 0.483,
P
< 0.001) and sTNFR2 (
β
= 0.366,
P
< 0.001). The baPWV values increased proportionally with increasing sTNFR1 (
P
= 0.002) and sTNFR2 (
P
= 0.076) tertiles. In multiple linear regression analyses, sTNFR1 (
β
= 0.300,
P
< 0.001) and sTNFR2 (
β
= 0.206,
P
= 0.013) had independent association with baPWV even after controlling for potential confounders including age, gender, systolic blood pressure, diabetes mellitus and hypertension.

Conclusions:
Taken together, sTNFR1 and sTNFR2 were independently associated with baPWV in patients undergoing ICA. This study suggests that sTNFR1 and sTNFR2 can be considered therapeutic targets as well as new makers of arterial stiffness.
Abstract 264: p38 Regulates Programmed Myofibroblast Differentiation and Fibrosis
Authors: Bugg Darrian , Ghearing Natasha , Dorn Lisa E , Sargent Michelle A , Gunajea Ambika , Kim Peter , Otsu Kinya , Molkentin Jeffery D , Davis Jennifer
Published: March 2022

Fibrosis is the excess collagen deposition that occurs with nearly every form of heart disease. A myriad of different stimuli including mechanical strain, cytokines and neuroendocrine effectors like TGFβ and Angiotensin II induce fibrotic matrix deposition. Current paradigms suggest that the differentiation of resident fibroblasts into myofibroblasts underlies the heart’s fibrotic response through yet unknown molecular mechanisms. Recently the p38 mitogen activated protein kinase (MAPK) signaling axis was implicated as a nodal pathway regulating myofibroblast differentiation
in vitro
, but never examined
in vivo
. To tactically address the role of p38 in the heart’s fibrotic response tamoxifen-inducible Cre recombinase knock-in mice (Tcf21
MCM
) were used to excise p38α from cardiac fibroblasts of conditional p38α knockout mice (p38
F/F
) subjected to ischemic injury. Deletion of p38 in fibroblasts reduced scar area by more than 50%, which we ascribe to the significantly reduced number of myofibroblasts in these hearts. Moreover, p38
F/F
- Tcf21
MCM
mice had markedly improved diastolic function relative to control littermates with intact p38 activity. Conversely, mice with fibroblast-specific expression of a constitutively active MAPK kinase 6 transgene (MKK6 Tg), which directly initiates p38α activity, developed interstitial and perivascular fibrosis at baseline. These mice had severe diastolic dysfunction in comparison to non-transgenic littermates with myofibroblasts present throughout heart. To investigate whether p38 MAPK regulates the fibrotic response in other tissues, p38α was deleted from activated dermal fibroblasts in mice using a tamoxifen-inducible periostin Cre recombinase (Postn
MCM
) that were subjected to subcutaneous circular wounds. Mice lacking p38 signaling in activated fibroblasts had significantly delayed wound closure relative to p38 replete littermates, whereas mice expressing the activated MKK6 transgene in activated fibroblasts had accelerated wound closure and scarring suggesting that p38α activity in fibroblasts is universally vital to the fibrotic phase of wound healing. Collectively, these data suggest that p38 inhibition is a viable candidate for anti-fibrotic therapies.
Abstract 27: Loss of GATA4 in Kit Lineage-derived Endothelium Leads to Vascular Permeability and Increased Heterotypic Fusion
Authors: Maliken Bryan D , Kanisicak Onur , Karch Jason , Molkentin Jeffery D
Published: March 2022

The treatment of cardiac ischemic injury using heart-derived c-Kit stem cells showed promising results in clinical trials with scar reduction and improved ejection fraction following coronary infusion. We previously developed a c-Kit lineage tracing mouse model showing that these cells very rarely convert into
de novo
cardiomyocytes and most lineage-derived cardiomyocytes are due to fusion. To potentially reduce this
de novo
rate to zero, and unequivocally evaluate the cellular fusion process
in vivo
, we deleted the essential cardiomyogenic transcription factor Gata4 in c-Kit cells using a Cre-loxP approach (Kit-Gata4 KO). Mice with the tamoxifen inducible Kit-MerCreMer allele crossed into a Gata4 homozygous LoxP targeted background with the Rosa26-eGFP reporter were fed tamoxifen at weaning to delete Gata4 in all c-Kit expressing cells and show them and their progeny as eGFP
+
. Unexpectedly, we observed a greater than 10-fold increase in Kit lineage-traced cardiomyocytes in Kit-Gata4 KO mice compared to Kit only controls with up to 4 months of tamoxifen treatment (90% v. 81% fusion in Kit-Gata4 KO v. controls). A 6-fold increase in leukocyte (CD45
+
) infiltration was observed suggesting the possibility for greater rates of fusion in the heart. However, bone marrow transplant studies revealed that Kit-Gata4 KO bone marrow does not exhibit increased cardiac infiltration or heterotypic fusion with cardiomyocytes. The reciprocal experiment, donating control bone marrow to recipient mice with Kit-Gata4 KO hearts, showed a 4-fold increase in fusion of control bone marrow showing that increases in fusion was intrinsic to the cells within the heart. Analysis of cardiac endothelial cells in Kit-Gata4 KO mice revealed that total CD31
+
cells were increased by 30% with 2 months of tamoxifen treatment with a parallel increase in endothelial progenitor cells (CD133
+
) in the bone marrow. This endothelial phenotype was corroborated using endothelial-specific deletion of Gata4 in a Tie2-CreER mouse which showed a 5-fold increase in both CD31
+
cells and vascular permeability after 1 month of tamoxifen. Currently, studies are underway to assess the mechanism of altered endothelial integrity and expansion in the absence of Gata4 in c-kit hemoangioblast progenitors.
Abstract 270: Peroxisome Proliferator Activated Receptor- α is a Connecting Link Between Cardiac Fibrosis and Oxidative Stress in Atrial Fibrillation
Authors: Rizvi Farhan , Emelyanova Larisa , Edwards Stacie , Aldred Breanna , Homer Peter , Kress David , Ross Gracious , Sra Jasbir , Tajik A. Jamil , Jahangir Arshad
Published: March 2022

Background:
AF, a common sustained arrhythmia that predisposes heart failure and stroke, is associated with cardiac fibrosis and oxidative stress. Peroxisome proliferator-activated receptor (PPAR)-α, important for ROS and lipid degradation, is reported to negatively correlate with kidney, liver, and lung fibrosis, but its role in cardiac fibrosis is less clear. We hypothesize that reduced expression of PPARα in AF contributes to oxidative stress and interferes with TGFβ signaling, a cytokine implicated in cardiac fibrosis.

Methods:
Myocardial fibrosis was detected (Masson trichrome) in right atrial appendages of AF and non-AF patients. Levels of 4-hydroxynonenal (4-HNE) (ELISA) and superoxide production (MitoSOX) were assayed in patients’ atrial tissue homogenates and cardiac myofibers respectively. Using atrial fibroblasts (hAF) from AF and non-AF patients, α-SMA, COL1A1 and c-Jun/c-Fos expressions were determined (RT-PCR and/or Western Blot) in the absence and presence of TGF-β (5 ng) with or without ciprofibrate (200 μM), a PPARα agonist.

Results:
Increased atrial fibrosis, levels of 4-HNE (101.7 vs 7.4 ng/mg protein; p=0.02(n=6)) and oxidative stress (8.7 fold; p=0.04) correlated with reduced PPAR-α mRNA (2.1 fold; p<0.01) in AF patients. Activation of PPARα by ciprofibrate reduced TGFβ1-induced increased expression of α-SMA and COL1A1 by repressing AP-1 signaling in hAF.

Conclusion:
Reduced expression of PPARα is associated with impaired cardiac mitochondrial metabolism and promotes TGFβ-induced profibrotic pathway. Preliminary data suggest PPARα agonist might confer therapeutic benefits for patients with cardiac fibrosis and oxidative stress in AF.
Abstract 275: Activating Transcription Factor 3 Protects Against Obesity and Metabolic Dysfunction Through Promoting Lipolysis and Adipocytes Browning
Authors: Cheng Ching-Feng , Ku Hui-Chen , Lai Pei-Fang , Lin Heng
Published: March 2022

Recent studies implying that promoting lipolysis or enhancing brown remodeling of white adipose tissue is a potential strategy to treat obesity and metabolic syndrome. Activating transcription factor 3 (ATF3) is a member of the ATF/cAMP-response element binding protein family of transcriptional factors. It is upregulated under stress condition in a variety of tissues, including the adipocytes. However, the mechanism of ATF3 in adipocytes and obesity regulation are not clear. We gave high-fat-diet (HFD) to both ATF3-/- (KO) and their wild type littermates (WT) for 16 weeks. KO showed obvious obesity as compared to WT. The obese KO mice then received AAV8-mediated gene transfer of ATF3 (AAV8-ATF3) at 16 weeks, in which decrease body weights were observed as compared to KO receiving AAV8-GFP. Histology demonstrated increased adipocyte cell diameter in KO, in which such enlargement of adipocytes can be reversed by AAV8-ATF3 therapy. We then used ATF3-overexpressing 3T3-L1 preadipocytes for in vitro studies. These cells demonstrated decreased lipid accumulation with diminished adipogenic markers (C/EBPα, PPARγ2, FABP4, and perilipin 1) and lipogenic markers (ACC1, ACC2, FAS, ChREBP, and SREBP1) as compared to the control cells. To our surprise, ATF3 overexpressed 3T3-L1 cells also demonstrated increased lipolysis (ATGL, HSL, and MGL), upregulated mitochondrial protein UCP1 and PCG1α expression, and enhanced brown/beige genes expression (Prdm16, Dio2, CIDEA, Tbx1, and Elovl3) as compared to control cells. Mechanistically, our ChIP assay found that ATF3 can directly repress ChREBP activity and ATF3 enhanced adipocyte browning via inhibition of SCD1. Lastly, we established a screening platform for ATF3 inducer, and found compound 5 could induce ATF3 expression. Our results demonstrated that compound 5 could induce UCP1 and promote lipolysis and white adipocyte browning in vitro and inhibit HFD induced obesity and increase insulin sensitivity in vivo. In summary, our findings confirmed ATF3 and its inducer, can promote lipolysis, increase UCP1 expression, and enhance adipocytes white-to-brown trans-differentiation through ChREBP and SCD1 pathways, thus represent an attractive drug candidate to protect against obesity and metabolic dysfunction.
Abstract 281: Hepatic Liver Receptor Homolog-1, a Key Regulator of Lipid Storage and Phospholipid Diversity
Authors: Miranda Diego A , Krause William C , Cazenave Amaury , Escusa Hazel A , Shihadih Diyala S , Stahl Andreas , Wenk Markus R , Silver David L , Ingraham Holly A
Published: March 2022

Cardiovascular disease and malignancy are the most common cause of death in Non-alcoholic Steatohepatitis (NASH) patients. Aside from lifestyle modification, there is currently no treatment for NASH. Activation of Liver Receptor Homolog-1 (Lrh-1), known to bind phospholipid ligands, has been shown to effectively reduce liver triglyceride (TG) in DIO mice, raising Lrh-1 as a possible target for treating NASH. Despite this finding, hepatic TGs are equivalent in controls and liver-specific Lrh-1 knockout (LKO or

Lrh1
AlbCre

) mice, regardless of diet. Given this discrepancy, we sought to characterize Lrh-1’s role in hepatic lipid metabolism by acutely deleting Lrh-1 in the adult liver, thus eliminating potential compensatory developmental effects associated with LKO. To acutely eliminate Lrh-1 in hepatocytes, 6-week old

Lrh-1
fl/f

l
male mice were infected with AVV8-TBG-eGFP (Control) or AAV8-TBG-Cre (LKO

AAVCre

) via retro-orbital injection and fed chow or high fat diet. LKO

AAVCre

mice developed hepatic steatosis after six weeks on standard chow or high fat diet. Furthermore, LKO

AAVCre

hepatocytes exhibited large lipid droplets, which were visible as early as 2 wks post-infection, thus suggesting that lipid handling is significantly altered in LKO

AAVCre

hepatocytes, independent of fatty acid transport or oxidation. LKO

AAVCre

exhibited lower
Pcsk9
expression, which correlated with decreased fasting plasma LDL-C. Consistent with other studies showing that perturbations in phospholipid pools affect lipid storage, lipidomic analyses revealed a significant reduction in phospholipid species containing arachidonic acid (AA), thus reducing the overall diversity of key membrane phospholipids. RNA-Seq analyses from LKO

AAVCre

livers confirmed that factors promoting lipid droplet size (
Cidec
,
Plin4
) were greatly increased while key enzymes in biosynthesis of unsaturated fatty acids were reduced (
Fads1, Fads2
and
Elovl5
). In addition, expression of human LRH-1 in LKO

AAVCre

decreased hepatic TG and improved glucose tolerance in DIO mice, in a ligand dependent manner. Collectively our data establish a novel role for Lrh-1 as a key regulator of lipid storage, thereby providing the first in vivo evidence as to why phospholipid serve as Lrh-1 ligands.
Abstract 290: Concurrent Gout is Associated with Reduced In-hospital Mortality Among Postmenopausal Women with Acute Myocardial Infarction: Insights from a Nationwide Sample
Authors: Akinjero Akintunde M , Adegbala Oluwole , Edo-Osagie Eseosa , Akinjero Nike E , Akinyemiju Tomi
Published: March 2022

Background:
Gout has been shown to increase the risk of cardiovascular diseases among postmenopausal women. The mechanisms remain unclear. We sought to evaluate in-hospital outcomes among postmenopausal women hospitalized with coexisting gout and acute myocardial infarction (AMI).

Methods:
Data was retrieved from the Nationwide Inpatient Sample (NIS), using ICD-9-CM codes. We excluded women less than 55yrs old. Patients with acute myocardial infarction (AMI) and co-occurring gout were compared with those without gout. We then tested for associations with stroke, in-hospital mortality and length of stay using multivariate analysis to adjust for confounders. All analyses were performed using SAS.

Results:
There was a total of 335,403 hospital admissions for AMI. Out of these, 484 had co-occurring gout. Women with both co-occurring conditions were more likely to be white (64.1%). In adjusted models, co-occurring gout and AMI was significantly associated with a reduced likelihood of in-hospital mortality (aOR=0.50, 95% CI: 0.35-0.72, Table 1). However, co-occurring gout and AMI was not significantly associated with stroke rates (aOR=0.95, 95% CI: 0.59-1.55) or length of stay (aOR=1.46, 95% CI: 0.77-2.16).

Conclusion:
Results from this nationally representative sample suggests that co-occurring gout is associated with reduced in-hospital mortality among postmenopausal women admitted for AMI, while no significant associations were found for length of stay and stroke rates. Further prospective studies are needed to explore these associations.
Abstract 304: Molecular Mechanisms of Electrically-induced Cardioprotection
Authors: Roessler Anne E , Luther Kristin , Ren Xiaoping , Wang Yang , Shen Zilong , Miao Qing , Jones W. Keith
Published: March 2022

Ischemic preconditioning (IPC) is a well-characterized cardioprotective phenomenon involving multiple brief, non-lethal ischemic episodes to the heart. Remote ischemic preconditioning (RIPC) elicits cardioprotection via repetitive ischemia and reperfusion to a distant vascular bed, organ, or limb. A non-ischemic, nociceptive stimulus in distant tissues can result in the most powerful cardioprotection reported - an 80% decrease in infarct size - called remote nociceptor-induced cardioprotection (NIC). Electrically induced cardioprotection via electrical stimulation with electroacupuncture needles (EA) or skin patches (ES) on the abdomen may be a clinically feasible way to induce NIC. A 5 volt, 15 minute electrical stimulus applied as either preconditioning or postconditioning reduces infarct size in a mouse surgical model of myocardial infarction. This study’s objective is to determine the molecular mechanism of electrically-induced NIC. Our previous work shows that IPC requires upregulation and post-transcriptional regulation of heat shock proteins (HSPs), which act in unity to assist in protein folding, repair, and degradation following myocardial injury. However, Western blotting and PCR revealed non-significant changes in HSP mRNA and protein relative to sham, which were inconsistent with the large, significant increases in HSPs observed after IPC. Studies using mice with a genetic deletion of HSP70.1 revealed that this heat shock protein is not required for protection. The transcription factor NF-κB regulates many cardioprotective genes in the heart after IPC, including, but not limited to, heat shock proteins. Based on our work in IPC, we hypothesize that electrical stimulation of the skin is cardioprotective via a unique induction of NF-κB-dependent genes. Pharmacological and genetic inhibition of NF-κB were utilized to evaluate the role of this transcription factor in electrically-induced cardioprotection. Deep sequencing studies are in progress to delineate the presence and quantity of additional transcripts that may be involved in electrically-induced cardioprotection. Future investigations will address the neurobiology of electrically-induced cardioprotection via skin stimulation.
Abstract 306: Endothelial Arnt Mediates Cardiac Remodeling After Ischemia Reperfusion Injury Through an MMP Pathway
Authors: Zheng Mei , Knapp Maura , Sladojevic Nikola , Chen Anna , Andrade Jorge , Zhao Qiong , Liao James K , Wu Rongxue
Published: March 2022

Background:
Prevention of myocardial injury following heart attack is a challenge due to lack of effective treatments and the potential to develop chronic heart failure (HF). Impaired endothelial function is thought to contribute to the progression of HF after cardiac ischemia-reperfusion injury (IR). However, the underlying mechanisms are unknown. Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), also known as hypoxia-inducible factor 1-beta (HIF1-β), plays a major role in vascular endothelial function and is implicated in ischemic diseases. We hypothesize that endothelial ARNT mediates cardiac remodeling following IR.

Methods and Results:
We generated inducible endothelial specific ARNT knockout mice (ecARNT
-/-
) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT
-/-
mice exhibit severe cardiac blood vessel leakage and impaired cardiac function after IR. Following 1 month of IR, ecARNT
-/-
mice had a 50% mortality rate, while no mortality was observed in the control group (ARNTf/f mice treated with tamoxifen). To determine the underlying mechanisms by which ARNT regulates endothelial function, we performed DNA sequencing on cardiac microvascular endothelial cells (CMECs) isolated from control and ecARNT
-/-
hearts. Data suggest a significant increase in the matrix metalloproteinases (MMP) family of proteins following ARNT knockdown. An increase in MMP activity in the serum of ecARNT
-/-
CMECs after IR was also detected. Moreover, use of an MMP inhibitor in vivo rescues heart failure induced by IR in ecARNT
-/-
mice. Taken together, these results suggest that ecARNT may mediate heart failure during IR through an MMP pathway.

Conclusion:
Endothelial ARNT is a critical regulator of endothelial function and could potentially serve as a therapeutic target for HF in response to IR injury.
Abstract 31: Nifedipine Increases Vascular Formations in Skin Wounds
Authors: Oliveira Dinaldo C , Brasileiro Augusto , Dantas Lais , Oliveira Danielle , Nunes Vitor , Gadelha Maria Isabel , Amorim Jessica
Published: March 2022

Introduction:
Loss of skin integrity can result in a wound. Wounds are associated with high cost and disability for patients. Peripheral arterial disease may be the cause of wounds that infect and lead to amputations. Our objective was to evaluate whether the use of nifedipine in wounds is associated with greater amounts of new vascular formations and collagen when compared to placebo.

Hypothesis:
The new vascular formation in wounds is greater in those treated with nifedipine than in those treated with placebo.

Methods:
We performed 32 wounds in pigs and locally applied placebo or nifedipine (1%, 10% or 20%). Wounds were evaluated macro and microscopically at 6 different moments. A logistic longitudinal model of mixed effects was applied. For this purpose, the response to moderate or marked polymorphonuclear cells was considered as a dependend variable, with the comparison groups (placebo, nifedipine 1%, 10% and 20%) as the explanatory variable. Based on the placebo group, the OR was estimated with its respective confidence interval for the nifedipine groups at the different doses. Value of p <0.05 was considered significant. This study was approved by the animal research ethics committee.

Results:
The figure below shows the comparison of new vascular formation and collagen between groups at six different times.

Conclusions:
The use of nifedipine in wounds was associated with greater vascular formation when compared to placebo. There were differences in the production of collagen between the groups evaluated (in the groups of nifedipine 10% and 20% less amount of collagen). Nifedipine may have a positive impact on the healing of skin wounds.
Abstract 315: Mechanical Stretch Activates Biased AT1R Signaling and a Distinct β-arrestin Conformation
Authors: Gareri Clarice , Wang Jialu , Hanada Kenji , Kashai Alem W , Xiong Xinyu , Rajagopal Sudarshan , Rockman Howard A
Published: March 2022

Introduction:
Angiotensin II Type 1 receptor (AT1R) is a member of the G protein-coupled receptors (GPCRs) family, playing an important role in several cardiovascular diseases. Depending on the stimulus, AT1R activates a cascade of signaling pathways including those mediated by G proteins and the multifunctional proteins β-arrestins. AT1R is also mechanosensor and respond to membrane stretch by activating ligand-independent β-arrestin-biased signaling.

Objective:
The aim of this study is to investigate the precise molecular mechanism for mechanoactivation of the AT1R.

Methods and Results:
Our previous work demonstrated that mechanical stretch induced by hypotonicity (osmotic-stretch, OSM) allosterically activates the AT1R to mediate β-arrestin signaling. Using Proximity Ligation Assays (PLA) and co-IP we now demonstrate that OSM uniquely promotes Gαi recruitment to AT1R to initiate β-arrestin signaling. In sharp contrast to the β-arrestin-biased ligand TRV120023, the Gαi inhibitor Pertussis Toxin (PTX) blocked the OSM-induced β-arrestin recruitment, EGFR transactivation and ERK signaling. To determine whether the two biased stimuli (OSM and TRV120023) can activate different β-arrestin conformations, we used β-arrestin FLAsH constructs and monitored BRET following AT1R activation by either ligand or stretch stimulation. A FlAsH constructs located in the N-domain domain and two located in the C-domain domain showed a distinct BRET pattern indicating that in response to OSM, β-arrestin assumes a distinct conformation from either the balanced ligand Angiontensin II and the TRV120023 (N=7; p<0.05). Current studies are underway using site-specific stable-isotope labeling strategies coupled with mass spectrometry-based quantitative analysis (CDSiL-MS) to obtain biophysical information regarding the precise AT1R conformations induced by these balanced and β-arrestin-biased stimuli.

Conclusion:
The principal driver for differential signaling is thought to be different receptor conformations stabilized by each ligand. Our data suggest that in response to two β-arrestin-biased stimuli, AT1R likely adopt distinct conformations indicating remarkable conformational heterogeneity in activating intracellular responses.
Abstract 318: Jnk3 Regulates Angiogenesis via a Sirtuin1, Creb1 Axis in Peripheral Tissue
Authors: Kant Shashi , Craige Siobhan M , Chen Kai , Reif Michaella M , Ramo Kasmir , Davis Roger J , Keaney John F
Published: March 2022

Peripheral artery disease (PAD) affects nearly 10 million people in the United States alone, yet patients with clinical manifestations of PAD (e.g. claudication and limb ischemia) have limited and ineffective treatment options. In ischemic tissues, stress kinases, c-Jun N-terminal kinase (JNK), are activated. Here we show that inhibition of the JNK3 isoform, which is most highly expressed in the peripheral nerves, strikingly potentiates angiogenesis and blood flow recovery from mouse hind limb ischemia. JNK3 deficiency leads to increased pro-angiogenetic growth factors such as
Vegfa
,
Pdgfb
,
Pgf
,
Hbegf
and
Tgfb3
in ischemic muscle and cells by repression of the transcription factor Creb1. JNK3 acts through sirtuin 1 (SirT1) to suppress the activity of Creb1. With Sirt1 suppression, as in JNK3 -deficient mice, Creb1 is more active and upregulates pro-angiogenic factors. Together these data suggest that the JNK3/Sirtuin1/Creb1 axis coordinate the vascular remodeling response in peripheral ischemia.
Abstract 324: Identification of Regulators of Cardiac DNA Methylation Using a Systems Genetics Approach in Mice
Authors: Rau Christoph D , Romay Milagros , Wong Eleanor , Tan Wilson , Lusis Aldons J , Foo Roger , Wang Yibin
Published: March 2022

Systems biology approaches to studying heart failure in humans are hampered by numerous environmental factors and inciting incidents which combine to complicate the application of standard population-based tools and algorithms to the syndrome. We recently reported a study of heart failure in a large cohort of inbred mouse strains (the Hybrid Mouse Diversity Panel; HMDP) treated with the beta-adrenergic agonist isoproterenol. We demonstrated that it was possible to combine systems-level tools with the benefits of an animal model to capture and identify numerous genetic variations which drive heart failure.

Recent research on the epigenome has identified changes to DNA methylation which affect the progression of cardiovascular diseases and suggests that DNA methylation may serve as an important additional marker or even driver of genes involved in heart failure-associated phenotypes. We have supplemented our prior study by completing reduced representational bisulfite sequencing on 88 strains from our panel both with and without catecholamine challenge.

We have identified 75,000 CpGs which vary among the strains of the HMDP across the genome. Each CpG was mapped to the genome using GWAS to identify methylation quantitative trait loci (mQTLs). These mQTLs were then grouped together to identify mQTL hotspots, regions on the genome which regulate a sizable fractions of all observed CpGs. We then overlapped these hotspots with hotspots which regulate the expression of gene transcripts.

Three genomic regions were identified as multi-omic master regulators which affect both DNA methylation and gene expression for over 5% of the expressed transcripts and varying CpGs. Closer examination of these loci identified the serine peptidase inhibitor
Serpina3n
as a likely candidate gene for the regulation of DNA methylation, gene expression and cardiac phenotypes. Subsequent
in vitro
and
in vivo
work demonstrate that
Serpina3n
knockdown results in reduced cellular hypertrophy and changes to hypertrophy-related gene expression. Further analysis of the other master regulatory regions will likely reveal additional genes which will improve our understanding of how DNA methylation acts to regulate pathways underlying heart failure.
Abstract 330: MicroRNA Dysregulation in Patients with Hereditary Hemorrhagic Telangiectasia
Authors: Cannavicci Anthony
Published: March 2022

Hereditary hemorrhagic telangiectasia (HHT) is a rare, understudied, autosomal dominant disorder that is characterized by angiogenic abnormalities. Gene mutations in HHT involve key angiogenic factors. Clinical manifestations of HHT include benign telangiectasias and potentially life threatening arteriovenous malformations (AVMs) that can occur in the pulmonary and cerebral circulation. Currently, there is no effective treatment for HHT. PBMCs have been implicated in the pathogenesis of HHT and the goal of this study was to profile and characterize PBMC microRNAs, to determine whether they had a role in the observed angiogenic abnormalities. A total of 28 HHT patients with confirmed mutations in either
ENG
,
ACVRL1
or
SMAD4
, and 25 controls were recruited. PBMCs were isolated from 16 ml of peripheral blood and total RNA was obtained. PBMC expression profiling was conducted with a human miR array analysis. Dysregulated miRs identified from the analysis were then validated with RT-qPCR. Significant differences were determined using a two-tailed t- test. Of the 800 miRs screened, 167 dysregulated miRs were identified in PBMCs. Selected PBMC miRs (MiRs-28-5p, -30b-5p, -361-3p and -374a-5p) were validated with RT-qPCR. PBMC miR-361-3p (
p
=0.025) known to target IGF1, was found to be significantly decreased compared to controls. IGF1 messenger RNA (mRNA) levels in PBMCS of HHT patients was significantly elevated (p=0.005) compared to controls. Our results show dysregulation of select miRs in PBMCs from HHT patients. We also show an increase in IGF1 mRNA, an important angiogenic factor, in PBMCs that may be related to the downregulation of miR-361-3p in HHT patients. This may represent an important pathogenic mechanism that is involved in the angiogenic dysfunction observed in HHT and may provide for a unique miR therapeutic target.
Abstract 332: A Data Mining Strategy Identifies microRNA-15b-5p as a Potencial Bio-Marker in Non-Ischemic Heart Failure
Authors: Mancilla Georthan , Oyarzun Ingrid , Artigas Rocio , Quiroga Clara , Wichmann Ignacio , Verdejo Hugo , Castro Pablo
Published: March 2022

Advances in bioinformatics has provided us with large databases that characterize the complex genetic and epigenetic changes associated with human diseases. The use of data mining strategies on public access databases to identify previously unknown disease markers is an innovative approach to identify potential biomarkers or even new therapeutic targets in complex diseases such as heart failure (HF). We assessed the hypothesis that a bioinformatic strategy of meta-analysis using existing public databases may predict novel differentially expressed microRNA (miR) in non-ischemic HF patients.We systematically reviewed the literature for miR profiling and HF (2006-2014). Four studies fulfilled quality criteria for analysis. Raw data from the largest study (discovery dataset, n=35) was normalized and analyzed by unsupervised hierarchical clustering. Significance analysis selected miRs with a fold change of 2 and a false discovery rate of less that 1%. The miRs reported as significant in the other three papers were incorporated in a meta-analysis using a robust rank aggregator approach and then contrasted with the results of the unsupervised analysis of raw data. To validate the in-silico analysis, we evaluated the miRs with higher predicted fold change in plasma samples from 23 patients with HF and five healthy controls using RT-qPCR. Internal controls included miR-39 for standardization of the extraction procedures and miR23a, miR451 as hemolysis markers.

Results:
The in-silico strategy identified 54 miR that fulfilled our criteria. The meta-analysis confirmed nine differentially expressed miRs, six of them previously reported (let-7b, miR-100, miR-103, miR-199a, miR-23a) and three with no known relation with HF (miR-125b, miR-140, miR-15b). RT-qPCR of HF plasma samples revealed that miR-15b-5p was significantly reduced in HF subjects (p=0.004). Conversely, the previously described miR-23a (p = 0.0021) and let-7b (p = 0.0195) were largely increased in HF subjects. In conclusion, bioinformatics analysis allows the identification of previously unreported miR associated with HF. This novel approach using publicly available data for the identification of new potential biomarkers may accelerated the pre-analytic phase of biomarker research.
Abstract 336: Reactive Ion Surface Modification of Vascular Graft Materials Enhances Endothelialization Without Promoting Thrombosis
Authors: Anderson Deirdre E , Jurney Patrick L , Cutiongco Marie F , Pohan Grace , Chevallier Pascale , Mantovani Diego , Yim Evelyn K , Hinds Monica T
Published: March 2022

Previous work demonstrated that poly(vinyl alcohol) (PVA) holds great potential as a cardiovascular biomaterial. PVA prevents thrombosis at least as well as expanded polytetrafluoroethylene (ePTFE) clinical vascular grafts in whole blood studies. However, long term
in vivo
success of this material will likely depend on its ability to support an endothelial cell (EC) monolayer. To promote EC attachment and growth we are treating the PVA surface with high energy reactive ions. We hypothesize that increasing the concentration of reactive groups on the surface of the PVA will increase endothelial outgrowth cell (EOC, a type of endothelial progenitor cell isolated from whole blood) attachment without increasing thrombosis. To test this hypothesis we exposed PVA films to reactive ion modifications using O
2
, N
2
, and Ar gases at a variety of powers and durations. Treated samples were then characterized with X-ray photoelectron spectroscopy to quantify surface chemistries. Samples were seeded with EOCs to quantify attachment and proliferation using immunohistochemistry. Platelet and fibrin accumulation was measured dynamically in PVA tubes using a baboon arteriovenous shunt with radiolabeled platelets for 1hr. Flow rate was controlled and no anticoagulants were given. O
2
and N
2
treatments encouraged cell adhesion on the PVA films in an energy dependent manner, which correlated to an increase in surface nitrogen. Initial shunt data (n=3) indicate that treated samples did not have increased platelet attachment compared to untreated PVA or ePTFE. In conclusion, reactive ion modified-PVA demonstrates a promising biomaterial by supporting EC growth without increasing thrombosis.
Abstract 346: Glucose Inhibits Cardiomyocyte Maturation Through Nucleotide Biosynthesis
Authors: Nakano Haruko , Minami Itsunari , Braas Daniel , Pappoe Herman , Wu Xiuju , Sagadevan Addelynn , Vergnes Laurent , Fu Kai , Morselli Marco , Ding Xueqin , Stieg Adam Z , Gimzewski James K , Pellegrini Matteo , Clark Peter M , Reue Karen , Lusis Aldons J , Ribalet Bernard , Kurdistani Siavash K , Cristofk Heather , Nakatsuji Norio , Nakatsuji Norio , Nakano Atsushi
Published: March 2022

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is known to be associated with congenital heart disease. However, little is known about the mechanism how blood glucose impacts heart formation. Using a chemically-defined human pluripotent stem cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic and electrophysiological levels via nucleotide biosynthesis through the pentose phosphate pathway. Even though blood glucose level stays stable in utero during normal pregnancy, glucose uptake by fetal cardiac tissue was found drastically reduced at late gestational stages in the mouse. Interestingly, perturbation of glucose dynamics during gestation in a murine model of diabetic pregnancy promoted mitosis and inhibited maturation of fetal cardiomyocytes. Therefore, this observation suggests that the metabolic switch is not only to meet the energy demand but also to induce a genetic program to facilitate cardiac maturation, providing a possible mechanistic basis for the congenital heart disease in diabetic pregnancy.
Abstract 347: The Success of Humanizing Miniature Hearts is the Right Cannulation
Authors: Nguyen Duong T , O’Hara Matthew , Graneli Cecilia , Peng Bo , Gunther Navina , Hammer David , Gan Li-Ming , Althage Magnus , Hagvall Sepideh
Published: March 2022

The development of humanized miniature organs through decellularization of rodent organs and recellularization with human cells will provide higher quality translational evidence for in vitro models from animal to human within basic research and pharmacological applications. Here, we have focused on the preservation of the circulation network within rat hearts by cannulating the superior vena cava (SVC), ascending aorta (A), pulmonary vein (PV), and pulmonary artery (PA) (deemed 4-Flow). Compared to the conventional Langendorff, the 4-Flow cannulation is superior by allowing control of both atria and ventricles for non-invasive mechanical stimulation and most advantageous is access to both the coronary arteries and cardiac veins. Hence, the objectives are to thoroughly recellularize the heart and to conduct ventricular mechanical stimulation. Controlled perfusion was conducted with inflow via the SVC and A, and outflow via PV and PA. The resin cast of a decellularized heart in Figure 1A demonstrates that the retrograde flow in addition to antegrade flow through both coronary vascular networks permit efficient whole heart perfusion of solutions, cells, and factors. Multiphoton microscopy (1B) reveals that cells are perfused to capillaries and tissues but more so with enzymatic treatment (1C-D). All of the valves’ functionality are maintained, hence, mechanical stimulation of the left ventricle is possible via perfusion through the PV to expand the left chamber until the aortic valve opens to relieve the pressure (1E-F). Consequently, a 4-Flow humanized miniature heart is the complex in vitro model that will revolutionize the translation into patient studies.
Abstract 35: Transendocardial Mesenchymal Stem Cell Injection Demonstrates Reverse Remodeling Effects of Global LV Volumes and Enhanced Lateral Papillary Muscle Shortening
Authors: Vincent Louis , Natsumeda Makoto , Rieger Angela C , Florea Victoria , Tompkins Bryon A , Balkan Wayne , Leroux Lionel , Hare Joshua M
Published: March 2022

Rationale:
Secondary mitral regurgitation (MR) carries a poor prognosis despite improvements in surgical and transcatheter interventions. Mesenchymal stem cell (MSC) therapy for heart disease reduces infarct size and left ventricle (LV) dilatation, reverses remodeling, and improves regional contractility and functional capacity. However, it is unknown if the benefits of MSC therapy on LV structure and function apply to lateral papillary muscle shortening, an important predictor of secondary MR severity.

Hypothesis:
Test the hypothesis that administration of MSCs promotes interpapillary muscle distance (IPMD) shortening.

Methods/Results:
This retrospective analysis draws on results from autologous or allogeneic MSC injection therapies in a Göttingen swine model of chronic ischemic cardiomyopathy (ICM). MRI was used to measure end-diastolic volume (EDV), end-systolic volume (ESV), diastolic/systolic IPMD, and IPMD shortening. NOGA mapping and angiographic tracings of left ventriculography allowed for assessment of the effect of injection localized to papillary muscles (defined as injection within cardiac segments 4, 6, 10 and 12 in the 16-segment model). Three months after stem cell injection, EDV increased in both placebo- (12.2±3.6 mL; p=0.002) and MSC- (10.2±2.6 mL; p=0.03) treated swine. ESV increased only in placebo- (7.1±2.2 mL; p=0.003) but not MSC- treated swine. Systolic IPMD was maintained with MSC therapy (1.20±0.74 mm; p=0.33) but increased in placebo (1.83±0.60 mm; p=0.01). Systolic IPMD was preserved whether MSC injection was localized to papillary muscle (0.53±0.49 mm; p=0.44) or not (0.22±0.40 mm; p=0.24). Notably, IPMD shortening was significantly greater in MSC- (8.1±5.6%; p=0.02) but not placebo-injected (4.7±5.0%; p=0.69) swine. There were no between group differences in IPMD shortening (p=0.08).

Conclusion:
This study is the first to show that transendocardial MSC injections significantly enhanced IPMD shortening and lateral interpapillary muscle contraction in a model of chronic ICM. This effect was independent of injection site.
Abstract 361: Regulation of Activity of Apoptosis Inducing Factor by SUMO Conjugation
Authors: KIim Eun Y , Zhang Yi , Grover Abha , Yu Wei , Wang Jun
Published: March 2022

Apoptosis inducing factor (AIF), a mitochondrial oxidoreductase, is primarily involved in maintaining mitochondrial function. However, in response to apoptotic insults, AIF is released from mitochondria via cleavage and relocates to the nucleus, where it binds chromatin, subsequently causing its condensation and cell death. Thus, AIF plays an important role in both cell survival and death. The regulatory pathways/signaling events that govern the key step of AIF translocation and activity in cell death are not completely deciphered. SUMO conjugation, a highly conserved posttranslational modification, plays an important role in cardiac homeostasis. Herein, we report AIF as a novel SUMO substrate, and SENP5, a SUMO deconjugation enzyme, increased cardiomyocyte death once overexpressed in the mouse hearts, which coincided with an increase level of truncated AIF in cytosol. In line with the above findings, adenoviral mediated transduction of SENP5 wild type (wt), but not the C713L mutant, the latter of which lacks de-sumoylation activity, triggered cell death and AIF nuclear occupancy. Also, the SENP5-wt, but not the catalytically inactive mutant, de-sumoylated AIF. We further identified lysine residue 89 (K89) on AIF as one of the major SUMO attachment sites, and mutation of K89 to arginine (K89R) promoted AIF nuclear translocation in AC16, a cardiac cell line, and necrosis in L929 cells induced by TNFα/Z-vad-fmk. Our findings provide a novel mechanism by which SUMO conjugation is involved in mediating the function of AIF, which is important for cardiomyocyte survival and death.
Abstract 37: Interleukin-10 Deficiency Can Alter Contents and Further Impair Functions of Exosomes Derived From Endothelial Progenitor Cells
Authors: Yue Yujia , Garikipati Venkata N , Verma Suresh K , Goukassian David , Benedict Cindy , Kishore Raj
Published: March 2022

Endothelial progenitor cell (EPC) based therapy has been shown to have an immense potential to promote cardiac neovascularization and to attenuate ischemic injury. Recent research revealed that the therapeutic performance of stem cells is largely due to paracrine effects, in which exosomes (Exo) play an essential role. However, the autologous transplantation of EPCs in patients with systemic inflammation, which is a common symptom in patients with ischemic heart diseases, yield modest results, suggesting the compromised cell function and altered Exo performance. So we hypothesized that EPCs under inflammatory stress might change Exo contents, which eventually compromises their repair ability in ischemic heart disease. Whether modulation of identified targets like specific microRNAs in Exo cargo can rescue and/or enhance their reparative properties of dysfunctional Exo is not known. We have previously shown in IL-10 KO mice (model mimicking systemic inflammation) that loss of Interleukin-10 (IL-10) impairs EPCs functions via miR-375. After cell expansion, we isolated Exo from these two groups and compared their functions in terms of cell survival, proliferation, migration and angiogenic capacity
in vitro
. Our studies revealed that WT-EPC-Exo treatment enhanced endothelial cells proliferation and tube formation and inhibited apoptosis; whereas IL-10 KO-Exo exhibited opposite effects, suggesting that reparative capacity of WT-EPC Exo is lost in Exo derived from IL-10-KO-EPCs. The deep sequencing and proteomic analysis between WT and IL-10KO-Exo revealed drastically altered Exosomal cargo. Importantly, IL-10KO-EPC-Exos were highly enriched in microRNAs and proteins related to pro-inflammation, cell apoptosis and anti-angiogenesis. Through modulation of specific target (here as miR-375), we partially rescued IL-10KO-EPC-Exo dysfunction. Taken together, our study revealed that Exo display impaired function under inflammatory stimulus via changing Exo contents, and the dysfunction can be rescued by modulation specific target packed in Exo.