bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1232: Regulation of Mitochondria membrane Potential by Shear Stress: Implication of NO Signaling
Authors: Rouhanizadeh Mahsa , Hamilton Ryan , Cadenas Enrique , Hsiai Tzung
Published: July 2021

Introduction
: Mitochondria are the most important source of intracellular reactive oxygen species (ROS) in vascular endothelial cells (EC). Mitochondrial membrane potential (ΔΨm) is an indicator of mitochondrial energy state and mtROS production. Shear stress is known to induce eNOS and produce nitric oxide (NO).

Hypothesis:
We assessed whether NO is implicated in shear stress-induced ΔΨm and mtROS generation.

Methods
: Confluent HAEC were subjected to three conditions:

static control (τ
ave
= 0 dyn.cm
−2
at ∂τ/∂t = 0);

pulsatile shear stress (PSS) at a mean shear stress (τ
ave
) of 23 dyn.cm
−2
with a temporal variation (∂τ/∂t) at 71 dyn.cm
−2
.s
−1
; and

oscillatory shear stress (OSS) at τ
ave
= 0.02 with ∂τ/∂t at ±3 dyn.cm
−
2.s
−1
.

Tetramethyl rhodamine methyl ester (TMRM) and Mitosox red fluorescent dyes were used to assess mitochondrial ΔΨm and O
2
·−
production respectively. Intracellular H
2
O
2
production was assessed by carboxy-H
2
DCFDA. HAEC were treated with DETA-NO (NO donor) and L-NAME (eNOS inhibitor) to assess how shear stress-induced NO production regulates ΔΨm.

Results:
Delta;Ψm was increased by 12±5.3 mV in response to OSS, and by 33.5±8.9 mV in response to PSS (n=3, P <0.05) relative to static control. DETA-NO at 0.21mM and 0.94mM was used to simulate PSS-induced NO production during 1 hour and 4 hours of flow and it increased Delta;Ψm by 9.6±4.9 mV and 18.9 ±3.2 mV, respectively (n=3, P<0.05). L-NAME reduced Delta;Ψm (n=3, P<0.05) suggesting that NO is involved in Delta;Ψm regulation. PSS-induced Delta;Ψm was accompanied by a 1.4 fold increase in mtROS production (n=3, P<0.05) and a concomitant 10 fold upregulation in mitochondrial manganese superoxide dismutase (MnSOD) mRNA expression (n=6, P<0.05).

Conclusion:
Nitric oxide modulates Delta;Ψm and mtROS production. PSS, which upregulates eNOS, is more potent than OSS at increasing Delta;Ψm and mtROS, implicating NO as a signaling molecule in mitochondrial and endothelial function.
Abstract 1236: A Molecular and Functional Basis for Focal Arrhythmogenesis in Heart Failure-associated Atrial Fibrillation
Authors: Yeh Yung-Hsin , Wakili Reza , Qi Xiao Yan , Chartier Denis , Kääb Stefan , Ravens Ursula , Nattel Stanley , Dobrev Dobromir
Published: July 2021

Introduction:
Heart failure (HF) frequently causes atrial fibrillation (AF) and focal sources of unknown mechanism have been implicated. Here, we studied the potential role and molecular mechanisms of Ca
2+
handling abnormalities.

Methods:
Ca
2+
handling (microfluorescence, Indo-1 AM) and related protein expression (Western blot) were assessed in left atria of 20 dogs with ventricular tachypacing (240 bpm × 2 wks)-induced HF and 20 controls (CTLs). Whole-cell perforated-patch was used to record action potentials (APs), delayed afterdepolarizations (DADs) and triggered activity.

Results
: HF increased [Ca
2+
]
i
transient amplitude from 239±24 to 444±43* nM (*P<0.05), and [Ca
2+
]
i
release by 10 mM local caffeine puffs (an index of SR Ca
2+
content) from 849±71 (CTL) to 1574±169* nM (HF). Spontaneous Ca
2+
release events increased from 1.8±0.5 (CTL) to 10.7±2.1* events/run (HF). HF significantly increased APD (by ~40% at 1 Hz). DADs and triggered activity were more common in HF (15.2±2.6 triggered APs/run, vs CTL 0.4±0.2*), and were abolished by ryanodine (10 μM), but not by the I
f
-blocker Cs
+
(2 mM). HF caused profound changes in protein expression of key Ca
2+
handling and regulatory proteins (Table

). Calsequestrin, the major SR Ca
2+
-binding protein, was reduced by 32%*. Fractional RYR2 PKA (Ser2809) phosphorylation decreased by 63%*, whereas CaMKII (Ser2815) RYR2 phosphorylation increased by 221%*. The catalytic and regulatory (RII) PKA subunits were downregulated by 15%* and 73%*, whereas expression and autophosphorylation (Thr287) of CaMKIIδ were increased by 45%* and 81%* respectively. NCX1, SERCA and total, PKA and CaMKII phosphorylated SERCA-regulatory phospholamban were unchanged by HF.

Conclusions:
HF causes profound changes in regulation and expression of atrial Ca
2+
handling proteins, producing increased SR Ca
2+
load and release, along with DADs and triggered activity that may account for focal mechanisms that initiate and/or sustain HF-related AF.
Abstract 1283: Endothelial Nitric Oxide Synthase Promotes Mesenchymal Stem Cell Migration Towards the Ischemic Myocardium
Authors: Li Na , Xiang Fuli , Lu Xiangru , Karmazyn Morris , Feng Qingping
Published: July 2021

Background:
Bone marrow mesenchymal stem cells (MSCs) migrate from bone marrow towards the heart and contribute to cardiac repair post myocardial infarction. However, mechanisms by which MSCs migrate to the ischemic heart remain unclear. The present study investigated the role of endothelial nitric oxide synthase (eNOS) on MSC migration towards the ischemic myocardium and whether stromal cell derived factor-1 (SDF-1) contributes to the eNOS-mediated MSC migration.

Methods and Results:
MSCs were isolated from wild-type (WT) bone marrow and cultured in vitro for 3 generations. Coronary microvascular endothelial cells were isolated from adult mouse hearts and seeded on inserts for transendothelial migration assay. Neonatal cardiomyocytes were isolated from WT, eNOS
−/−
and eNOS transgenic (Tg) mice, cultured to subconfluence and subjected to 30 min of anoxia followed by 6 hours of reoxygenation (A/R). The conditioned medium was collected and served as a chemoattractant. MSC migration was significantly decreased in eNOS
−/−
compared to WT conditioned medium (9.8± 1.8% vs. 14.7±2.3%), but increased in eNOS-Tg conditioned medium (38.0±4.5%,
P
<0.05). SDF-1 protein secretion was significantly decreased in eNOS
−/−
but increased in eNOS-Tg conditioned medium. To examine MSC migration in vivo, WT, eNOS
−/−
or eNOS-Tg mice were subjected to myocardial ischemia for 45 min followed by 24 hrs of reperfusion (I/R). Immediately after reperfusion, GFP
+
MSCs were administered via a tail vein. GFP
+
cells in the ischemic region were significantly decreased in eNOS
−/−
compared to WT hearts (3.4±0.3 vs. 5.6±0.4 cell per mm
2
, P<0.05) but significantly increased in eNOS-Tg compared to WT (10.2±1.6 vs. 5.6±0.4 cell per mm
2
, P<0.05). Furthermore, SDF-1 mRNA and protein expression was increased in eNOS-Tg as compared to WT and eNOS
−/−
after myocardial I/R.

Conclusions:
eNOS promotes MSC migration towards the ischemic myocardium. This is likely due to an upregulation of SDF-1.
Abstract 1289: Activation of the Transient Receptor Potential V4 Channel Causes Cardiovascular Collapse Mediated by Endothelial Failure
Authors: Willette Robert N , Bao Weike , Nerurkar Sandhya , Yue Tian-li , Doe Chris P , Stankus Gerald , Ju Haisong , Bentley Ross , Sulpizio Anthony , Behm David , Sandra Hoffman , Maniscalco Kristeen , Lin Zuojun , Linda Casillas N , Lin Min , Trout Robert E , Votta Bartholomew J , Marquis Robert W , Xiaoping Xu
Published: July 2021

Transient receptor potential V4 (TRPV4), is an osmo/mechano-sensitive ligand-gated non-selective cationic channel that contributes to intracellular Ca
2+
homeostasis and regulation of cell volume. TRPV4 is highly expressed in endothelial and various epithelial cells and to a lesser extent in vascular smooth muscle. The purpose of the present study was to evaluate the cardiovascular effects of a potent novel small molecule TRPV4 activator (TRPV4A; EC
50
= 19, 8 or 1 nM in mouse, rat or dog, respectively) and to examine its mechanism of action. In the three species (mouse, rat and dog) examined, the intravenous administration of TRPV4A in anesthetized preparations induced a dose-dependent reduction in blood pressure, followed by cardiovascular collapse at doses ≤300 ug/kg. TRPV4A had no acute cardiovascular effects in the TRPV4
−/−
null mouse (>5 mg/kg, iv). Hemodynamic analysis in the dog suggested that the terminal event was related to a profound reduction in cardiac output (decreased SV followed by decreased HR). However, TRPV4A had no effect on rate or contractility in the isolated, buffer-perfused rat heart. In contrast, TRPV4A produced potent endothelial-dependent relaxation of rodent isolated vascular ring segments that was abolished by NOS inhibition (L-NAME) and eNOS gene deletion. Surprisingly, the
in vivo
cardiovascular collapse induced by TRPV4A was not altered by NOS inhibition (L-NAME), eNOS gene deletion or by treatment with anticoagulant or antiplatelet agents. Cardiovascular collapse induced by TRPV4A was correlated (concentration and time appropriate) with profound fluid extravasation (Evans Blue) and tissue hemorrhage in the lung, intestine and kidney. Consistent with these results, TRPV4A induced a ruthenium-red sensitive non-selective cation current, reduction in cell volume and subsequent cell death in cultured endothelial cells. These results provide the first characterization of the cardiovascular effects of a potent and selective TRPV4 channel activator and suggest that inappropriate activation of TRPV4 produces acute cardiovascular collapse associated with endothelial activation/injury and failure of the microvascular permeability barrier.
Abstract 1301: NFATc- And SRF-induced Early Growth Response (Egr)-3 Is Essential For VEGF-mediated Endothelial Cell Migration And Tube Formation
Authors: Suehiro Jun-ichi , Miura Mai , Kodama Tatsuhiko , Minami Takashi
Published: July 2021

Endothelium is a dynamic cell layer constantly responding to changes in the various extracellular mediators. Responses are usually beneficial to host, but oversustained or dysregulated responses can result in vascular dysfunction, leading to the initiation of atherosclerosis, tumor growth, and inflammation. Such endothelial cell activation and dysfunction are mediated in large part by alterations in gene expression. Here we show, using comprehensive transcriptome analyses, that VEGF, thrombin and TNF-α each induces a dramatic and rapid up-regulation of early growth response (Egr)-3 in human umbilical vein endothelial cells (HUVEC). The effect of VEGF on Egr-3 was similar in human coronary artery, pulmonary artery, and dermal microvascular endothelial cells. In chemical inhibitor studies, VEGF-mediated induction of Egr-3 (mRNA peak 300-fold at 45 min) depended on MEK1/2, JNK, PI3K, PKA, and Ca-calcineurin. Egr-3 promoter luciferase and electrophoretic mobility shift analysis revealed that the 5′-flanking region at −57 to −130 was necessary and sufficient for transducing of VEGF-mediated Egr-3 upregulation and that region could bind NFATc1, c2, and SRF. Co-transfection assays with Egr-3 reporter and either NFATc or SRF expression plasmids resulted in 4 and 2 -fold Egr-3 promoter activation, respectively. In DNA microarray studies, HUVEC treated with VEGF for 1 and 4 hours in the presence of two independent siRNAs against Egr-3, 25 and 70 VEGF-inducible genes were strikingly downregulated, respectively. The pro-angiogenesis factors Ets-1, CXCL1, and tissue factor were among those genes downregu-lated. SiRNA knockdown of Egr-3 markedly impaired VEGF-mediated cell migration and tube formation, as determined by in vitro wound healing, boyden chamber, and collagen gel assays. Moreover in aortic ring assays, VEGF-stimulated neo-angiogenesis from the extracted mice aorta was completely abolished by administration of adenoviral-transferred miRNA against Egr-3. Collectively, these findings suggest that NFATc and SRF cooperatively upregulate Egr-3. Egr-3 might have an important function as a signal transducer in VEGF-mediated angiogenesis in activated endothelium.
Abstract 1313: Pro-atherogenic Effects Of Lipid Peroxidation Products: Role Of The Unfolded Protein Response
Authors: Vladykoskaya Elena , Haberzettl Petra , Ahmed Yonis , Hill Bradford G , Sithu Srinivas D , D’Souza Stanley E , Bhatnagar Aruni , Srivastava Sanjay
Published: July 2021

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are associated with atherosclerosis. Expression of UPR target genes such as activating transcription factor 3 (ATF3) and ATF4 is markedly increased in human atherosclerotic lesions. Staining for these proteins co-localizes with the staining with antibodies that recognize the aldehydic epitopes of oxidized LDL, suggesting that lipid-derived aldehydes could be involved in mediating ER stress and UPR. We examined the role of phospholipid aldehyde, 1-palmitoyl-2-(5-oxovaleroyl)-
sn
-glycero-3-phosphocholine (POVPC), unsaturated lipid-derived aldehydes- 4-hydroxy,
trans
-2-nonenal (HNE) and acrolein in the induction of ER-stress and UPR in human aortic endothelial cells (HAEC) and human umbical vein endothelial cells (HUVEC). POVPC, HNE and acrolein (10 –25 μM) increased the phosphorylation of eIF2α (eukaryotic initiation factor-2α) by 1.5–5 fold (P<0.001) and induced its downstream effector proteins - ATF4 (1.5–3.5 fold; P<0.001) and ATF3 (4–10 fold; P<0.0001). Incubation of HAEC with these aldehydes also increased the adhesion of THP-1 cells (monocyte) to HAEC by 1.4–1.6 fold (P<0.01). Moreover, incubation of endothelial cells with POVPC increased the mRNA level of the pro-inflammatory cytokine IL-8 by >25 fold (P<0.0001). Chemical chaperone, phenyl butyric acid (PBA), diminished aldehydes-induced expression of ATF3 and ATF4 proteins, endothelial cell-monocyte adhesion and IL-8 formation by 80–95% (P<0.001). POVPC (10–25 μM) also activated JNK by (3–6 fold) in HAEC. Reduction of POVPC to its corresponding alcohol, 1-palmitoyl-2-(5-hydroxyvaleroyl)-
sn
-glycero-3-phosphocholine (PHVPC) inhibited JNK activation by 74 ± 14 % (P<0.001). Pharmacological inhibition of JNK, inhibited the aldehyde-induced induction of ATF3 and ATF4 proteins by 70–90 % (P<0.001) but not the phosphorylation of eIF2α, and PBA inhibited the POVPC-induced JNK activation by 85 ± 11 % (P<0.001). These data suggest that lipoprotein oxidation products activate endothelial cells in part by inducing ER-stress and their inflammatory signaling could be attenuated by chemical chaperones of protein folding.
Abstract 240: Inhibition of Glycogen Synthase Kinase 3 Beta Improves EPC Yield, Function and Ameliorates Arterial Repair Post-injury and Stenting
Authors: Hibbert Benjamin , Ma Xiaoli , Holm Erik , Pourdjabbar Ali , Rayner Katey , Chen Yong-Xiang , Sun Jianfeng , O’Brien Edward
Published: July 2021

Endothelial progenitor cells (EPCs) are circulating undifferentiated pluripotent cells capable of reconstituting endothelium
in vivo
and diminishing lesion formation following arterial injury. We tested the hypothesis that treatment with a specific soluble GSK 3β-inhibitor (GSK3βi) would improve the
in vitro
yield and function of EPCs from patients with coronary artery disease (CAD) and augment their
in vivo
potential to improve arterial repair after injury and stent implantation.
In vitro
treatment with GSK3βi at 1× (IC50) and 2× (IC50) increased the number of cells that dual labelled for acLDL and UEA-1 lectin on day 7 (13.4±3.2 vs. 39.9±5.3 vs. 43.8±10.1; p < 0.05). Furthermore, GSK3βi significantly augmented CAD-EPC survival to levels equal to those of healthy controls, increased VEGF secretion (7.1±1.6 vs 30.0±8.9, p<0.05) and resulted in a 4× increase in adhesion (p<0.05). Using Q-PCR, mRNA copy numbers for VLA-1,2,3,4 and 5 were assayed; however, only VLA-4 levels increased (4×, p<0.01). Use of a VLA-4 specific blocking antibody confirmed that the improved EPC adhesion was specifically mediated by VLA-4. Finally, the effects of GSK 3β inhibition were tested in two
in vivo
models of arterial injury. CD-1 athymic nude mice underwent femoral artery wire injury and injection of CAD-EPCs that were either GSK3βi treated, untreated, or injection with vehicle alone. GSK3βi treated EPCs improved the ability of xenotransplanted cells to inhibit lesion formation when compared to untreated and vehicle arteries respectively (intima/media ratios: 0.3±0.1 vs. 5.3±1.2 vs 11.2±2.7 μm, p<0.01). GSK3βi coated stents were inserted into rabbit carotid arteries. Fourteen days after stent deployment, GSK3βi stents resulted in a 44% increase in re-endothelialization (assessed by scanning electron microscopy and compared to vehicle coated stents; p<0.01) and a 16% trend towards a reduction in neointima formation (p<0.09). In summary, our data demonstrates that GSK3βi improves EPC function
in vitro
and ameliorates arterial repair following injury and stent insertion
in vivo
.
Abstract 1318: Aging Inhibits The Apoptotic Resolution Of Inflammation Leading To An Exaggerated Neointimal Development In Response To Vascular Injury.
Authors: Vazquez-Padron Roberto I , Wei Yuntao , Mateo Dania , Li Sen , Salgar Sashi , Pham Si M
Published: July 2021

Objective
: This study aims to determine the mechanisms by which aging prolongs inflammation in response to vascular injury, and exaggerate neointimal formation.

Methods and Results
: Using balloon injury model in rat iliac arteries, we found that aging rats (22 month-old) developed thicker neointima at 4 weeks after injury than their younger counter parts (4 month-old) (I/M: 0.8 ± 0.2 vs. 0.54 ± 0.15, p<0.008). We also found that injured arteries in aging animals accumulated more alpha-actin positive VSMC in the neointima than those from the young ones (500 ± 100 vs. 320 ± 120 cell/mm
2
). Although similar number of macrophages (CD68+; detected by immunohistochemistry; IHC) appeared in the adventitia 24 h after injury in both groups, these cells disappeared in the young arteries after 3 days, while remained in the media and neointima of aging arteries until 30 days after injury. Macrophages in aging arteries were CD163 negative and IL-6 and IL-18 positive, indicating they are of pro-inflammatory phenotype. There were no differences in the number of vascular T cells and dendritic cells between groups. Vascular apoptotic cells were determined by ISOL combined with IHC either for VSMC or macrophages. Aging arteries had fewer apoptotic VSMC or macrophages (0.2% ± 0.05) at any time after vascular injury when compared with the younger ones which had abundant vascular apoptotic cells in the media and neointima at 7 days after surgery (2.7% ± 0.45). Finally, the vascular cytokines production was assessed after balloon injury using the LincoPlex system. At three days after injury aging arteries contained three folds or higher levels of IL18, IL-6, Gro KC, and Leptin than the young ones. These pro-inflammatory cytokines stayed elevated in the aging vessels for most of the arterial remodeling process. In contrast, when compared with aging arteries, young arteries produced three times or higher levels of anti-inflammatory cytokines IL-10, IL-17, IL13 and IL-9 at seven days after injury.

Conclusions
: Our data suggests that aging inhibits the apoptotic resolution of the vascular inflammatory response to injury, leading to exaggerated neointimal formation.
Abstract 1330: Divergent Control of the Mitochondrial Death Gene BNIP3 by Opposing Actions of the Cellular Factors Nuclear Factor kappaB and E2F-1 in Ventricular Myocytes.
Authors: Shaw James , Yurkova Natalia , Regula Kelly , Zhang Tong , Aguilar Floribeth , Kirshenbaum Lorrie A
Published: July 2021

The hypoxia-inducible death factor Bnip3 is known to provoke mitochondrial perturbations and cell death of ventricular myocytes. The transcriptional control processes that govern Bnip3 gene expression under basal and inducible conditions remain cryptic. Sequence analysis of the Bnip3 promoter revealed the presence of distinct but overlapping DNA binding elements for the cell cycle factor E2F-1 and cellular factor NF-κB. Previously, we reported a survival role for NF-κB in ventricular myocytes. As a step toward elucidating the regulation of Bnip3 gene expression in ventricular myocytes, we tested the impact of E2F-1 and NF-κB on basal and inducible expression of Bnip3. A 2.0 fold increase in Bnip3 gene transcription was observed in cells expression wild type E2F-1 but not in cells expressing an E2F-1 mutant defective for DNA binding. Interestingly, basal Bnip3 gene transcription was increased by 2.5 fold in myocytes rendered defective for NF-κB activation with a non-phosphorylatable form of IκBα. Importantly, genetic ablation of E2F-1 inhibited basal and inducible Bnip3 transcription in NF-κB defective cells. Expression of the p65 subunit of NF-κB in NF-κB defective cells inhibited E2F-1 mediated Bnip3 transcription. Western blot analysis of cardiac cell lysate revealed that p65 NF-κB immunoprecipitated with E2F-1. ChIP analysis of the Bnip3 promoter indicated that the p65 NF-κB bound DNA under normoxic conditions. During hypoxia E2F-1 activity increased where as p65 NF-κB protein levels were decreased. ChIP analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia which coincided with a 3.5 fold increase in Bnip3 gene transcription. IKKβ mediated activation of NF-κB activation abrogated hypoxia-induced E2F-1 binding to the Bnip3 promoter and Bnip3 gene transcription. To our knowledge our data provide the first direct evidence that a novel relationship exists between p65 NF-κB and E2F-1 for basal and hypoxia-inducible regulation of the Bnip3 promoter. Furthermore, our data highlight a novel survival pathway by which NF-κB averts hypoxia - induced cell death by antagonizing the E2F-1 dependent transcription of Bnip3 in ventricular myocytes.
Abstract 1338: The Role of the Estrogen Receptor 46 Hydrophobic Domain on Endothelial Membrane-initiated Signaling
Authors: Kim Kyung Hee , Li Lei , Hisamoto Koji , Stepensky David , Cresswell Peter , Bender Jeffrey R
Published: July 2021

The expression of Estrogen Receptor 46 (ER46, 174 –595 aa), an N-terminus truncated isoform of the Estrogen Receptor α, in human endothelial cells (EC), and its critical role in membrane-initiated, rapid responses to 17β-estradiol (E2) have been reported. ER46 is targeted to human EC caveolar membranes, where it is a component of a complex containing c-Src, PI3-Kinase (PI3K), Akt and eNOS. E2 rapidly activates eNOS via c-Src/PI3K/Akt in EC. A highly conserved hydrophobic region of ER46, adjacent to a critical palmitoylation site (Cys447), may be required for its membrane localization and function. We generated a hydrophobic region mutant, “4Gly” (I451G/I452G/L453G/L454G), to evaluate the importance of this domain on ER46 membrane localization, eNOS activation, ER46 dimerization, and protein-protein (ER46-c-Src) interactions. Peripheral plasma membrane staining with cell impermeant E2-horse radish peroxidase (E2-HRP) was observed in COS-7 cells transiently transfected with ER46, but not with 4Gly. E2-BSA (30 nM, 10 min) induced eNOS phosphorylation (Ser1177) in plasma membrane fractions of ER46- but not 4Gly- transfected COS-7 cells. Using Fluorescence Resonance Energy Transfer (FRET), we demonstrated that ER46 and the 4Gly mutant both undergo homodimerization, although 4Gly less efficiently. The protein-protein interaction between ER46 and c-Src was also assessed by FRET. The FRET efficiency between mCerulean-tagged ER46 and EYFP-tagged c-Src was significantly increased after E2-BSA (40 nM, 10 min) stimulation in transiently transfected HeLa cells, indicating an E2-induced ER46-c-Src interaction within 75 Å. The FRET efficiency of c-Src-4Gly was 50% compared to that seen with the wild type ER46 in these reconstitution experiments. These results demonstrate that this conserved hydrophobic region of ER46 is critical for the subcellular localization, for required protein-protein interactions and for consequent membrane-initiated rapid signaling responses to E2.
Abstract 1340: Disruption of a Hepatocyte Growth Factor/c-Met Receptor Autocrine Loop Severely Impairs the Potency of Pluripotent Adipose Stromal Cells
Authors: Cai Liying , Johnstone Brian H , Liang Zhong , Traktuev Dmitry , Cook Todd G , Li Jingling , Feng Dongni , Cornetta Kenneth , Rosen Elliot D , March Keith L
Published: July 2021

Background
Paracrine stimulation of endogenous repair, rather than direct tissue regeneration, is increasingly accepted as a major mode of therapeutic stem and progenitor cell action; yet, this principle has not been fully established
in vivo
. Adipose-derived stem cells (ASCs) secrete many factors and promote reperfusion and tissue repair in ischemia models. RNA interference was used to silence the expression of the abundant protein, hepatocyte growth factor (HGF), to determine its contribution to ASC potency
in vivo
.

Methods and Results
Dual-cassette lentiviral vectors, expressing GFP and either a small hairpin RNA (shRNA) specific for HGF mRNA (shHGF) or a control sequence (shCtrl), were used to stably transduce ASCs (ASC-shHGF or ASC-shCtrl). ASC-shHGF secreted 5-fold less HGF, which resulted in a reduced ability of these cells to promote survival, proliferation and migration of mature and progenitor endothelial cells
in vitro
(
p
<0.01). HGF knockdown also severely impaired the ability of ASCs to promote reperfusion in a mouse hindlimb ischemia model. Perfusion of the ischemic leg at 15 d in mice treated with ASC-Ctrl was 84±4%, compared to only 69±5% for ASC-shHGF (
p
<0.05). Even so, ASC-shHGF retained residual activity as indicated by greater reperfusion (
p
<0.05) than with saline treatment (58±6%). Capillary densities in ischemic tissues from each group followed a similar rank order (ASC-Ctrl>ASC-shHGF>saline) (
p
<0.05 between each group). While there was no difference in total GFP
+
cells in ischemic limbs at 5 d after infusion, indicating similar homing potentials, 3-fold fewer ASC-shHGF were present in ischemic tissues at 15 d compared to ASC-shCtrl (
p
<0.01). This was accompanied by an increase in TUNEL-positive ASC-shHGF cells (61 ± 0.1%) compared to ASC-Ctrl (41% ± 3.2%) in ischemic tissues at 5 d (
p
<0.01); suggesting that attenuated potency of ASC-shHGF was related to reduced survival in ischemic tissues.

Conclusions
These results indicate that secretion of HGF is critically important for ASC potency. In addition to promoting endogenous repair, the data suggest that an important effect of HGF is autocrine promotion of ASC survival in ischemic tissue. Enhanced donor cell survival is an important goal for increasing the efficacy of cell therapy.
Abstract 1357: Mobility of Epicardial Rotors During Human Ventricular Fibrillation
Authors: Nash Martyn P , Mourad Ayman , Bradley Chris P , Paterson David J , Sutton Peter M , Hayward Martin , Taggart Peter
Published: July 2021

Introduction:
Stability of reentry during VF may depend on the dynamics of rotor cores. We determined the mobility of rotors during human VF using global epicardial mapping and phase singularity analysis.

Methods:
In 10 patients undergoing cardiac surgery (6 with coronary artery disease; 4 with aortic valve disease), VF was induced by burst pacing prior to (n=7) or immediately following (n=3) the onset of cardiopulmonary bypass. For each subject, a 20 – 40 s episode of fibrillatory activity was sampled at 1 kHz using an epicardial sock containing 256 unipolar contact electrodes connected to a UnEmap system. Trajectories of persistent epicardial rotors (singularities of phase maps, based on de-trended voltage versus its Hilbert transform, lasting for > 1000 ms) were tracked. The mean core location was determined across the lifetime of each persistent rotor. The rotor was classified as stationary if its core remained within 15 mm of the mean location for more than 90% of its duration.

Results:
Using the above criteria, the numbers of mobile and stationary rotors varied from patient-to-patient (see figure

). In all but one patient, there were more mobile than stationary rotors. Over all patients, the mean ± SD number of mobile rotors (32 ± 21) was significantly greater than stationary rotors (7 ± 6, P<0.01).

Conclusions:
At least one stationary rotor was always present during human VF, but it is predominantly characterised by a small number of persistent mobile rotors.
Abstract 1367: Altered Transmural Expression Of I
Kr
In Cardiac Memory Is Regulated By The Transcription Factor, Activator Protein-1
Authors: Ozgen Nazira , Plotnikov Alexei N , Shlapakova Iryna N , Danilo Peter , Rosen Michael R
Published: July 2021

Introduction:
We previously reported that the transient outward current (I
to
) and mRNA and protein levels of its accessory subunit KChIP2 decrease in ventricular pacing-induced cardiac memory (CM). These changes appear dependent on a decrease of the cAMP response element binding protein (CREB). We have also reported that the transmural gradients of the delayed rectifier potassium current component, I
kr
and the mRNA and protein of its subunit ERG (all showing epicardium (EPI) > endocardium (ENDO)), are diminished in CM. However, there is no CREB binding site in the ERG promoter region to explain the change in ERG. We now tested the hypothesis that 1) the normal transmural gradient of ERG expression in canine left ventricle (LV) is paralleled by a transmural gradient of the Activator protein-1 (AP-1) components c-Jun and c-Fos, and 2) altered transmural expression of c-Jun and c-Fos parallels the altered transmural ERG in CM.

Methods:
We screened 2000bp upstream from the first exon of ERG for transcription factor binding sites. Then 10 dogs were chronically instrumented with LV posterior wall and LA pacing electrodes. Five underwent 3w AV-sequential pacing (CM), and 5, 3w of atrial pacing (Sham). CM was quantified as change in T wave vector displacement (TVD). At 3w biopsies were taken 1–2 cm from the LV pacing site. Real time PCR was used to determine c-Jun and c-Fos.

Results:
Screening revealed an AP-1 response element upstream to the ERG promoter region, supporting the hypothesis that c-Jun and c-Fos might be reasonable candidate transcription factors here. TVD increased in CM compared to Sham. As previously, mRNA and protein transmural gradients of ERG decreased in CM. C-jun and c-Fos exhibited transmural gradients, EPI>ENDO, in Sham (Table

). This gradient was lost in CM.

Conclusion AP-1 is a transcription factor for ERG and the altered ERG transmural gradient in CM may be regulated by the altered c-Jun and c-Fos gradient.

T vector displacement (TVD) and mRNA of c-Jun and c-Fos
Abstract 1385: The ATP-binding Cassette A1 (ABCA1)-mediated Apolipoprotein A-I Lipidation Occurs at the Plasma Membrane and not in the Endocytic Compartments
Authors: Denis Maxime , Landry Yves D , Zha Xiaohui
Published: July 2021

The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of HDL through the lipidation of lipid-poor apolipoprotein A-I (apoA-I). ApoA-I has been suggested to internalize with ABCA1, presumably to acquire lipids from the endosomal compartments, an important hub for cholesterol trafficking. The aim of our work is to determine how apoA-I get endocytosed and whether this internalization contributes to the biogenesis of HDL. When examined by confocal microscopy, we found that Cy3.5-apoA-I endocytosed rather slowly (t
1/2
= 15 min, in baby hamster kidney cells expressing ABCA1) and poorly co-localized with transferrin, consistent with a pathway independent of clathrin-coated pits. ApoA-I was instead found perfectly co-localized with FITC-dextran, a bulk phase uptake marker and, at later time points, with LysoTracker. This strongly indicates that majority of internalized apoA-I was delivered to the lysosomes. ABCA1 was not found to co-localize with endocytosed apoA-I. Similar observations were obtained in mouse macrophages and normal human fibroblasts. Next, in order to determine the functional significance of endocytosis to cholesterol efflux, we used sucrose or latrunculin A to inhibit apoA-I endocytosis. We show that apoA-I, transferrin and dextran uptake were abolished, but cholesterol efflux was not decreased. To specifically determine whether internalized apoA-I contributes to the biogenesis of HDL, we developed a method to strip off apoA-I from the cell surface. Our results show that internalized apoA-I do not significantly contribute to the biogenesis of HDL (< 20%). Together, our results suggest that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs. Internalized apoA-I is mostly targeted for lysosomal degradation and therefore does not significantly contribute to the biogenesis of HDL.
Abstract 1392: Increased High Density Lipoprotein Cholesterol induced by Human Apolipoprotein A-I Gene Transfer increases Adiponectin Expression in abdominal Adipose Tissue
Authors: Van Linthout Sophie , Spillmann Frank , Foryst-Ludwig Anna , Bücker-Gärtner Carola , Meloni Marco , Feng Yingmei , Kintscher Ulrich , De Geest Bart , Schultheiss Heinz-Peter , Tschoepe Carsten
Published: July 2021

Introduction: High density lipoprotein (HDL) cholesterol (C) levels positively correlate with plasma adiponectin levels. However, the role of HDL on adiponectin expression is unkown. To investigate the effect of increased HDL levels on adiponectin expression, 1) gene transfer with human apolipoprotein (apo) A-I, the main apo of HDL, was performed in control mice and in lipopolysaccharide (LPS)-injected mice, associated with decreased adiponectin levels and 2) HDL was supplemented in vitro on (pre)-adipocytes.

Methods: Eight weeks old male C57BL/6 mice were i.v. injected with 5 × 10e10 total particles of the E1E3E4-deleted adenoviral vector Ad.hapoA-I, expressing human apo A-I or with the same dose of Ad.Null, containing no expression cassette. Fourteen days hereafter, mice were i.p. injected with LPS from Escherichia coli at a dose of 80 mg/kg or with saline. Mice were sacrificed 12 hours after LPS or saline injection. Human apo A-I and mouse adiponectin plasma concentrations were determined by ELISA. Abdominal fat phospho (p) and total (tot.) Akt protein levels were determined by Western Blot; adiponectin mRNA expression of (pre)-adipocytes by real-time PCR.

Results: Ad.hapoA-I GT resulted in human apo A-I expression levels of 83Â27>4.6 mg/dl at day 14, which was associated with 1.8-fold (p<0.05) and 1.5-fold (p<0.05) higher HDL-C and adiponectin levels compared to Ad.Null mice, respectively. After LPS-injection, human apo A-I levels decreased by 1.7-fold (p<0.001), leading to 1.7-fold lower adiponectin levels compared to Ad.hapoA-I control mice, but still 1.5-fold (p<0.01) higher compared to LPS-Ad.Null mice. The increased adiponectin levels in Ad.hapoA-I versus Ad.Null LPS-injected mice were associated with a 1.7-fold (p<0.05) increase in p-Akt/tot.Akt ratio. In vitro, LPS administration decreased adiponectin expression by 2.1-fold (p<0.01), which was normalized to control levels by HDL supplementation in a phosphatidylinositol-3-kinase (PI3K)-dependent manner, since Ly 294002 reversed the HDL-mediated increase in adiponectin expression.

Conclusion: HDL increases adiponectin expression via the PI3K-Akt pathway, which may contribute to some of the pleiotropic actions of HDL such as its well-known anti-inflammatory effects.
Abstract 1400: Reduced Cx43 Expression Reduces Scar Formation After Coronary Ligation
Authors: Zhang Yan , Kanter Evelyn M , Weinheimer Carla J , Gribben Erin M , Kovacs Attila , Yamada Kathryn A
Published: July 2021

Gap junctions mediate cell-to-cell electrical coupling required for synchronized cardiac contraction. They also serve as conduits for exchange of small signaling molecules. Reduction in connexin43 (Cx43), the major gap junction protein in ventricular tissue, likely underlies malignant arrhythmias in infarcted hearts. However, little is known about the role of reduced Cx43 expression in infarct healing. This study was performed to test the hypothesis that reduced expression of Cx43 influences cardiac fibroblast function and wound healing after myocardial infarction (MI).

Methods:
The left anterior coronary artery was ligated in wild-type (WTs) and Cx43-deficient (Cx43
+/−
) mice studied 6 d later. We quantified infarct size by Masson’s trichrome staining; cell proliferation by bromodeoxyuridine staining; matrix metallo-proteinase (MMP) activity in the noninfarcted, border and infarct regions (IZ) by zymography; myofibroblasts by α-smooth muscle actin (αSMA) immunostaining; and collagen deposition by Picrosirius red staining.

Results:
6 d post-MI mean infarct size by trichrome staining was not different in Cx43
+/−
(46±4%) vs WTs (46±5%). However, significant differences were observed in the Cx43
+/−
infarcts. The area of unresorbed necrotic myocardium in the IZ was larger (p<0.01) in Cx43
+/−
(40±11%, n=12) vs WTs (17±8%, n=6). Fibroblast proliferation in the IZ was increased (p<0.05) in Cx43
+/−
(6.1±0.5%, n=4) vs WTs (4.1±0.7%, n=5), and activated MMP-2 was increased even more (p< 0.05) in the IZ of Cx43
+/−
(40±6 relative density units, n=5) vs WTs (29±4 units, n=3) consistent with enhanced fibroblast proliferation. On the other hand, transformation to myofibroblasts in the IZ was diminished as reflected by reduced (p<0.01) αSMA-positive immunostaining in Cx43
+/−
(32±16%, n=6) vs WTs (57±5%, n=12), and collagen deposition in the IZ was less (p<0.01) in Cx43
+/−
(36±6%, n=6) vs WTs (50±13%, n=8) consistent with fewer myofibroblasts.

Conclusion:
Reduced Cx43 expression increases fibroblast proliferation and MMP activity, and reduces myofibro-blasts and collagen deposition indicating that remodeling of Cx43 can influence fibroblast function and delay scar formation and cardiac wound healing after MI.
Abstract 1405: The Alpha-1A is the Predominant Alpha-1-Adrenergic Receptor in the Human Heart at the mRNA but not the Protein Level
Authors: Jensen Brian C , Swigart Philip M , Myagmar Bat-Erdene , Shah Sanjay , DeMarco Teresa , Hoopes Charles , Simpson Paul C
Published: July 2021

Background:
Alpha-1-adrenergic receptors (ARs) play an adaptive role in the heart and protect against the development of HF. There are three alpha-1-AR subtypes: A, B, and D. Limited prior studies find that the human heart contains primarily the A subtype mRNA. Alpha-1-AR subtype proteins have never been measured in human heart.

Hypothesis:
This study tests the hypothesis that all three alpha-1-AR subtypes are present in both the failing (F) and non-failing (NF) human heart.

Methods:
F human hearts [n =6, mean EF 26%, mean age 47, 3 males (M), 3 females (Fe)] were obtained after cardioplegia during heart transplant. NF controls were unused donor hearts (n =6, mean EF 58%, mean age 41, 4 M, 2 Fe). Left (LV) and right ventricular (RV) tissue from the mid free wall was flash frozen in liquid nitrogen. mRNA was purified and treated with DNase. Quantitative real time reverse transcription PCR results were normalized to beta-actin and TATA binding protein. Saturation binding used tritiated prazosin and competition binding used 5-methylurapidil, an alpha-1A-specific antagonist.

Results:
All three alpha-1-AR subtype mRNAs were identified, and the A subtype was the most abundant in both LV and RV (p <0.05). Subtype mRNA levels did not change significantly in F versus NF, in either LV or RV [see table

. Arbitrary Units ±SEM (% of total alpha-1-AR mRNA )]. Mean alpha-1-AR protein density was unchanged in HF by saturation binding (NF: 4.6 ±0.7 fmol/mg vs. F: 3.9 ±0.8 fmol/mg). Competition binding in NF LV tissue (n =6) showed a classic two-site binding curve with 38% high affinity (alpha-1A) binding sites and 62% low affinity (alpha-1B/D) sites.

Conclusions:
These results are the first to measure alpha-1-AR subtype proteins in the human heart. At the protein level, alpha-1A is less abundant than the sum of alpha-1B and D, even though alpha-1A mRNA is most abundant. Given that alpha-1-ARs protect against HF in animal models, specific activation of cardiac alpha-1-AR subtypes might be feasible.
Abstract 250: Over-expression of Human Arginase (ARG1) in Macrophages Reduces Atherosclerosis in LDL-receptor Deficient Mice
Authors: Teupser Daniel , Haffner Ivonne , Wilde Johannes , Burkhardt Ralph , Thiery Joachim
Published: July 2021

Background:
In previous work we have identified arginase I (ARG1) as a novel candidate gene of atherosclerosis resistance in macrophages (Teupser et al ATVB 2006). In macrophages, L-arginine can be metabolized by NO synthase and arginase to form NO and urea, respectively. Thus, high expression of ARG1 in macrophages (as observed in atherosclerosis resistant our rabbits) may be an effective mechanism of reducing macrophage NO-production and thus vascular inflammation.

Methods and Results:
To test the hypothesis that ARG1 exerts anti-atherogenic properties, we generated ARG1-transgenic mice, expressing human ARG1 under the control of a CMV promoter. In these animals, ARG1 was over-expressed in various tissues including bone-marrow-derived macrophages. Under basal conditions, expression of human ARG1 in macrophages of transgenic mice was significantly higher compared to endogenous murine ARG1 (27062±13652 vs. 176±81 copies ARG1/10
6
copies cyclophilin, respectively). To test the functionality of the human transgene, macrophages were incubated with lipopolysaccharide (1 μg/mL). After 6 hours of incubation, NO-release was significantly diminished in macrophages of transgenic mice, compared to non-transgenic controls (94±9 vs. 140±16 μmol/ng cell protein, respectively p = 0.03), indicating that hARG1 transgene expression influences NO synthesis by competing with iNOS for the common substrate L-arginine. To test the effect of the transgene on atherosclerosis susceptibility, bone marrow from ARG1-transgenic and non-transgenic animals were transplanted onto LDL-receptor deficient C57BL/6 recipients at 8 weeks of age. Animals were sacrified at 20 weeks of age and no significant differences in lipid and lipoprotein levels were noted. However, atherosclerosis of animals transplanted with transgenic bone marrow (n=16) was significantly reduced compared to controls (n=15) (75160±9914 vs. 121500±18410 μm
2
, p=0.03).

Conclusion:
Our data show for the first time that over-expression of human ARG1 in macrophages has direct anti-atherogenic properties by reducing NO-release in macrophages. We conclude that ARG1 might constitute an interesting novel target for the prevention of atherosclerosis.
Abstract 1415: Activation of Soluble Guanylate Cyclase Regulates Phosphodiesterase 5A Localization and Restores Anti-adrenergic Action of Sildenafil despite Nitric Oxide Synthase Inhibition
Authors: Nagayama Takahiro , Zhang Manling , Takimoto Eiki , Kass David A
Published: July 2021

Background:
We have shown that inhibition of cyclic GMP-phosphodiesterase 5A (PDE5A) by sildenafil (SIL) blunts cardiomyocyte β-adrenergic stimulation, but this effect depends on the activity of endothelial nitric oxide synthase (eNOS) to generate a specific pool of cyclic GMP. PDE5A normally localizes at Z-bands in myocytes, but localization is more diffuse in cells with eNOS chronically inhibited. Here, we tested whether the influence of eNOS on PDE5A localization and anti-adrenergic action depends upon cyclic GMP.

Methods and Results:
Mouse
in vivo
hemodynamics were assessed by pressure-volume analysis. Isoproterenol (ISO: 20 ng/kg/min,
iv
) stimulated contractility was inhibited by SIL (100 μg/kg/min,
iv
), however this did not occur in mice given N
w
-nitro-L-arginine methyl ester (L-NAME: 1 mg/mL in drinking water for 1 week) to inhibit NOS. Myocytes transfected with an adenoviral vector encoding a fusion protein (PDE5A-DSred)
in vivo
were subsequently isolated and examined for PDE5A/α-actinin localization. Normal cells showed strong co-localization, whereas L-NAME-treated cells had diffuse PDE5A distribution. If L-NAME was stopped for 1-wk washout, SIL regained anti-adrenergic activity, and PDE5A z-band localization was restored. If L-NAME was continued but combined with Bay 41– 8543 (BAY: 30 mg/kg/day,
po
), a soluble guanylate cyclase (sGC) activator, both PDE5A localization and SIL anti-adrenergic action were also restored. Chronic L-NAME suppressed phosphorylation of vasodilator-stimulated protein (VASP), a marker of protein kinase G (PKG) activity, in hearts acutely exposed to ISO+SIL. After L-NAME washout or L-NAME+BAY, VASP phosphorylation with ISO+SIL was restored.

Conclusion:
NOS-dependent modulation of both PDE5A sarcomere localization and anti-adrenergic activity depends upon sGC-derived cyclic GMP, and is linked to PKG activation. This suggests sGC activators may have synergistic effects with PDE5A inhibitors.
Abstract 1417: Combined Assessment of Left Ventricular Strain and Rotation - A Novel Approach for Assessment of Function in Infarcted Myocardium and its Border Zones
Authors: Helle-Valle Thomas , Remme Espen , Lyseggen Erik , Vartdal Trond , Pettersen Eirik , Smith Hans Jørgen , Osman Nael F , Lunde Ketil , Edvardsen Thor , Smiseth Otto A
Published: July 2021

Background
: LV circumferential strain and rotation are markers of myocardial function, but the two measures dissociate in the ischemic LV. This study investigates the mechanism and clinical significance of this dissociation.

Methods
: In healthy subjects (n=15) and patients with LAD-related myocardial infarction (n=15), LV apical rotation and strain were measured from apical short-axis recordings by STE and MRI tagging. Infarct extent was determined by contrast MRI. To define mechanisms of the dissociation between strain and rotation we used a finite element model.

Results
: Mean apical rotation and strain by STE were lower in the patients than in healthy subjects (13±4 vs. 10±5° and −24±2 vs. −12±10%, respectively,
P
<0.05). In patients, segmental strain was reduced in proportion to extent of infarction (r=0.84,
P
<0.0001). Maximum and minimum rotation, however, were found at the infarct borders; maximum rotation at the border in direction of rotation (counter-clockwise) and minimum rotation at the opposite infarct border (Figure

). The simulation study reproduced the clinical findings and indicated that the dissociation was mainly caused by mechanical interactions between infarcted and viable myocardium transmitted along circumferentially oriented LV fibers.

Conclusions
: Systolic strain reflects regional myocardial function and infarct extent, while systolic rotation defines infarct borders. Strain and rotation provides complementary information in assessment of myocardial dysfunction and infarct extent.
Abstract 1421: In Vivo Measurement of Flow-Mediated Vasodilation in Living Rats using High Resolution Ultrasound: Age-Dependent Endothelial Dysfunction
Authors: Heiss Christian , Sievers Richard E , Amabile Nicolas , Momma Tony Y , Natarajan Shobha , Yeghiazarians Yerem , Springer Matthew L
Published: July 2021

In humans, endothelial function serves as a surrogate marker for cardiovascular health and is measured as changes in arterial diameter after temporary ischemia (flow-mediated dilation; FMD). We developed an FMD-related approach to study conduit artery vasodilation in living rats, and demonstrate a reduction in FMD in older versus younger animals consistent with age-related endothelial dysfunction. Diameter and Doppler-flow measurements were obtained from the femoral artery using high-resolution ultrasound (35 MHz). We observed dose-dependent vasodilation using both endothelium-dependent and endothelium-independent pharmacologic vasodilators (acetylcholine and nitroglycerine). Flow-dependent vasodilation was observed in response to flow increase induced both by adenosine and local saline infusion. Transient hindlimb ischemia led to reactive hyperemia with sequential flow velocity increase and femoral artery dilation, the latter of which was completely abolished by NO-synthase (NOS) inhibition with L-NMMA. To demonstrate its applicability in a model of endothelial dysfunction, we show that FMD is significantly reduced in older versus younger animals. While FMD was completely NOS-dependent in younger animals, NOS-dependent mechanisms accounted for only half of the FMD in older animals, with the remainder being blocked by charybdotoxin (CTx) and apamin suggesting contribution of endothelium-derived-hyperpolarizing-factor. Using this new integrative physiologic model to reproducibly study FMD in living rats, we show that age-dependent endothelial dysfunction is accompanied by a shift in mechanisms underlying vasodilatory endothelial function.