bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3560: Use of Hyperpolarized Magnetic Resonance for Non-Invasive Observation of Metabolic Regulation
Authors: Schroeder Marie A , Heather Lisa C , Atherton Helen J , Clarke Kieran , Radda George K , Tyler Damian J
Published: July 2021

Hyperpolarized magnetic resonance (HP MR) has enabled real time visualization of in vivo metabolism. In this study, we postulated that HP MR could also non-invasively provide a measure of metabolic regulation. We focused on regulation of pyruvate dehydrogenase (PDH), a highly controlled enzyme that catalyzes the oxidation of pyruvate to acetyl CoA and CO2/HCO3-. We compared PDH flux in conditions of normal and attenuated enzyme activity, and in the presence of normal and augmented Krebs cycle flux, to determine the contributions of PDH activity and end product inhibition to enzyme regulation. Six rats were examined in the fed and fasted states (to modulate PDH activity), with 40 μmol HP 13C1-pyruvate alone and 40 μmol HP pyruvate co-infused with 40 μmol malate (to manipulate Krebs cycle flux/acetyl CoA uptake). HP tracer was infused into the rats in an MR scanner and cardiac spectra were acquired every second for 1 min. Conversion of pyruvate to 13HCO3-was monitored and the 13HCO3-/pyruvate ratio was used as a marker of PDH flux. Infusion of malate increased PDH flux by 31% compared with pyruvate alone, indicating that removal of acetyl CoA by incorporation into the Krebs cycle increased PDH flux. PDH flux was 57% lower in fasted rats injected with pyruvate alone compared with fed rats, and did not change with malate co-infusion. Here, low PDH activity prevented additional enzyme flux. These results suggest that end product inhibition limits fed state PDH flux, whereas PDH activity regulates pyruvate oxidation in the fasted state. In conclusion, this study has provided evidence that HP MR may be useful to obtain details of metabolic regulation, rather than just reflecting metabolic state.

Figure 1

Bicarbonate/pyruvate ratio in fed and fasted rats, following an injection of pyruate or pyruate plus malate. In fed rats, co-infusion of malate increased PDH flux by 31% compared with injection of pyruvate alone (*p=0.02). Fasting reduced PDH flux by 57% (**p=0.002) following injection of pyruvate alone. Co-infusion with malate did not affect PDH flux in fasted rats.
Abstract 3612: Apelin Prevents Aortic Aneurysm Formation by Inhibiting Macrophage Inflammation and Infiltration
Authors: Leeper Nicholas J , Tedesco Maureen M , Kojima Yoko , Schultz Geoff , Ho Michael Y , Kundu Ramendra K , Anderson Joshua P , Tsao Philip S , Dalman Ronald L , Quertermous Thomas
Published: July 2021

To investigate the hypothesis that Apelin treatment would attenuate aortic injury in a murine model of elastase-induced abdominal aortic aneurysm (AAA) disease, by limiting disease-related vascular wall inflammatory. Male C57BL/6 mice were implanted with osmotic mini-pumps filled with Apelin (2 mg/kg/day) or saline (Control), and subsequently treated with intra-aortic porcine pancreatic elastase to create infrarenal AAA’s. Mice were sacrificed at day 3 for aortic PCR analysis or followed ultrasonographically until day 7 and then sacrificed for histological analysis (Elastic Masson, VCAM, ICAM-1, E-Selectin, and Mac-2 semi-quantitative analysis). The cellular expression of inflammatory cytokines and chemokines in response to Apelin was assessed by TaqMan analysis of both macrophages (J774, RAW, and harvested intraperitoneal cells) and rat aortic smooth muscle cells (A7R5) and compared to other stimuli. Apelin treatment resulted in diminished AAA formation, with a 30% reduction in in vivo aortic diameter by ultrasound (1.04 mm vs 1.48 mm, p < 0.05), and a 59% reduction in maximal aortic cross-sectional area by histology (0.59 mm2 vs 1.43 mm2, p < 0.05) relative to the saline-treated group. The macrophage infiltrate also was significantly reduced in the Apelin-treated mice compared to the Control group (68.6 cells/hpf vs 285.4 cells/hpf, p < 0.001). Apelin treatment was associated with reduced mean aortic MCP-1, MIP 1-alpha, IL-6 and TNF-alpha mRNA levels, although these did not reach statistical significance. Apelin stimulation of cultured macrophage models significantly reduced MCP-1 and TNF-alpha mRNA levels compared to basal conditions (−1.12 and −1.49 fold expression change, p < 0.05, respectively). No difference in endothelial cell adhesion molecule expression or smooth muscle cell cytokine production was found in response to Apelin. Apelin significantly reduces AAA formation in this accepted model of human AAA disease. The mechanism appears to be decreased macrophage infiltration, perhaps related to an apelin-mediated decrease in pro-inflammatory cytokine and chemokine activation.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 3633: Flow-induced Dilation is Mediated by Akt-dependent Activation of eNOS-derived H2O2 in Mouse Cerebral Arteries
Authors: Drouin Annick , Thorin Eric
Published: July 2021

Introduction
. Endothelial nitric oxide synthase (eNOS) produces superoxide leading to H
2
O
2
-dependent dilations to acetylcholine (ACh) in isolated mouse cerebral and human coronary arteries. Akt increases eNOS activity responsible for flow-mediated dilation (FMD). It is unknown, however, if endogenous H
2
O
2
mediates FMD.

Hypothesis
. Akt-dependent activation of eNOS leads to the production of functionally relevant levels of H
2
O
2
responsible for FMD.

Methods
. Cerebral arteries (100 to 150 μm in diameter) were isolated from 12±2 week-old C57Bl/6 male mice. FMD (0 to 10 μl/min, 2-mu;l step-increase at constant internal pressure of 60 mm Hg; shear stress = 0 to 50 dyn/cm
2
) were induced in vessels pre-constricted with phenylephrine (30 μM) at a pO
2
value of 150 mm Hg using a gas mixture of 12% O
2
, 5% CO
2
, 83% N
2
. Simultaneously to diameter acquisition, H
2
O
2
or NO production was detected by the fluorescent dyes CMH
2
CFDA or DAF-2, respectively. Results are expressed as mean±SEM of n = 6 to 8 mice per experiment.

Results
. FMD (at 10 μl/min, 25±3% of maximal diameter) were reduced (P<0.05) by endothelial removal (6±1%) or eNOS inhibition with N-nitro-L-arginine (L-NNA; 11±1%), but not by the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO; 24±3%). Likewise, FMD was not associated with a rise in NO-associated fluorescence, while addition of PEG-catalase and silver diethyldithio-carbamate (DETC; superoxide dismutase inhibitor) reduced (P<0.05) FMD to 10±2% and 15±1%, respectively, suggestive of the implication of H
2
O
2
in FMD. Accordingly, a rise in H
2
O
2
-associated fluorescence (+133±19 a.u.) was observed simultaneously to FMD, and was reduced by L-NNA, PEG-catalase and DETC (+55±10, +64±4 and +50±10 a.u., respectively; P<0.05). Furthermore, specific inhibition of Akt by triciribine (1 μM) prevented FMD together with H
2
O
2
-associated rise in fluorescence (3±1% and +23±4 a.u., respectively, P<0.05). Triciribine, however, neither limited ACh-induced dilation nor the rise in H
2
O
2
-associated fluorescence.

Conclusion
. In C57Bl/6 mouse cerebral arteries, Akt-dependent activation of eNOS-derived H
2
O
2
accounts for flow-mediated dilation. This suggests a physiological role for eNOS-derived free radicals.
Abstract 3666: Major Role of Ly6chi/7/4hi/ccr2+ Monocytes in Post-ischemic Neovascularization
Authors: Cochain Clément , Rodéro Mathieu , Vilar José , Loinard Céline , Récalde Alice , Zouggari Yasmine , Duriez Micheline , Lévy Bernard , Mallat Ziad , Combadière Christophe , Silvestre Jean-Sébastien
Published: July 2021

Circulating mouse monocytes consist of two main subsets, Ly6CHi7/ 4HiCCR2+CX3CR1loCCR5+ and Ly6Clo7/4loCCR2-CX3CR1HiCCR5+ monocytes.We sought to investigate their respective role in postischemic neovascularization and unravel the mechanisms involved in their recruitment to ischemic tissues. Postischemic neovascularization was impaired in MCP-1−/− or CCR2−/− but not in CCR5−/− or CX3CR1−/− mice with operatively-induced hindlimb ischemia (n=10 per groups). Overexpression of MCP-1 in the ischemic muscle by plasmid electrotransfer improved foot perfusion, angiographic score and capillary density by 1.4-, 1.8- and 1.4-fold, respectively (p<0 .01 versus control). In contrast, overexpression of Fractalkine or Rantes, the ligands of CX3CR1 and CCR5 respectively, had no significant effects (n=10 per groups) suggesting that recruitment of 7/4HiCCR2+ monocytes play a significant role in this setting. In this line, flow cytometry analysis revealed that monocytes infiltration in ischemic muscle peaked at day 3 and dropped to levels comparable to those in non ischemic muscle on day 7. 7/4Hi monocytes predominated from day 1 to day 3 (52% of infiltrated Cd11b+ cells) whereas 7/4lo prevailed from day 7 onward. MCP-1 or CCR2 deficiency fully abrogated infiltration of both monocytes subsets, while overexpression of MCP-1 in the ischemic muscle selectively increased 7/4Hi monocytes infiltration (287% of wild-type, p<0.05). In contrast, monocytes subsets infiltration was slightly reduced in CX3CR1−/− mice (p<0.05 versus MCP-1−/−) and unchanged in CCR5−/− mice. Interestingly, MCP-1 deficiency or upregulation also led to changes in monocyte subtypes levels in both bone marrow and blood. MCP-1/CCR2 signaling plays a major role in monocytes mobilization from the bone marrow and recruitment to the ischemic tissue. We also suggest that inflammatory 7/4Hi/CCR2 monocytes represent a major component of the inflammatory response in post-ischemic neovascularization.
Abstract 3672: Loss of Perivascular Adipocyte Paracrine Function Leads to Increased Vascular Tone and Glucose Intolerance in Hypertension
Authors: Khavandi Kaivan , Greenstein Adam , Withers Sarah , Sonoyama Kazuhiko , Lewis Sarah , Jeziorska Maria , Laing Ian , Yates Allen , Pemberton Philip , Malik Rayaz A , Heagerty Anthony M
Published: July 2021

In order to investigate the contribution of perivascular adipose tissue (PVAT) to arterial function, a total of 55 small arteries harvested from 35 skin biopsies of patients with Metabolic Syndrome and matched controls were mounted as ring preparations in a wire myograph. Contractility to cumulative doses of Norepinephrine in the presence or absence of PVAT showed an anticontractile effect in arteries from healthy volunteers (p=0.009), which was lost in patients with Metabolic Syndrome. Bioassay studies confirmed that PVAT releases a hydrophilic anticontractile factor in health, which is absent in obesity. Using a soluble fragment of the human Type 1 receptor, we identified that the anticontractile factor was adiponectin, which is the sole mediator of vasodilation, acting by increasing endothelial bioavailability of nitric oxide. Significant endothelial dysfunction was observed in patients with Metabolic Syndrome (p<0.001). Quantitative image analysis of adipose tissue revealed significantly increased adipocyte cell size in patients with Metabolic Syndrome, compared with healthy controls (p<0.006). There was immunohistochemical evidence of inflammation with upregulation of TNF-alpha receptor 1 in these patients (p<0.001). Application of exogenous TNF-alpha abolished the anticontractile effect of PVAT by reducing adiponectin bioavailability. Oxidative stress also induced by cytokines TNF-alpha and IL-6 but not IL-1, reduced adiponectin production from PVAT and increased basal tone. When the obese microenvironment was replicated
in vitro
by inflicting hypoxia on PVAT, adiponectin activity was lost but then rescued by incubation with cytokine antagonists. Further application of the adiponectin receptor fragment abolished PVAT relaxation. We conclude that in healthy arteries, PVAT releases adiponectin which reduces vascular tone. In obesity, this is lost by a cascade of adipocyte hypertrophy, hypoxia, inflammation and oxidative stress. The resulting vasoconstriction contributes to hypertension, hypertriglyceridaemia and insulin resistance. Direct targeting of adiponectin release from PVAT therefore provides a novel therapeutic opportunity in the Metabolic Syndrome.
Abstract 3691: Deletion of Suppressor Of Cytokine Signaling-1 in a Murine Model of Atherosclerosis Results in a Lethal Systemic Inflammation
Authors: Grothusen Christina , Schuett Harald , Lumpe Stefan , Bleich Andre , Glage Silke , Ihle James N , Schieffer Bernhard
Published: July 2021

Introduction: Atherosclerosis is a chronic inflammatory disease of the cardiovascular system which may result in myocardial infarction and sudden cardiac death. While the role of pro-inflammatory signaling pathways in atherogenesis has been well characterized, the impact of their negative regulators, e.g. suppressor of cytokine signaling (SOCS)-1 remains to be elucidated. Deficiency of SOCS-1 leads to death 3 weeks post-partum due to an overwhelming inflammation caused by an uncontrolled signalling of interferon-gamma (IFNγ). This phenotype can be rescued by generating recombination activating gene (rag)-2, SOCS-1 double knock out (KO) mice lacking mature lymphocytes, the major source of IFNγ. Since the role of SOCS-1 during atherogenesis is unknown, we investigated the impact of a systemic SOCS-1 deficiency in the low-density lipoprotein receptor (ldlr) KO model of atherosclerosis.

Material and Methods:
socs-1
−/−
/rag-2
−/−
deficient mice were crossed with ldlr-KO animals. Mice were kept under sterile conditions on a normal chow diet. For in-vitro analyses, murine
socs-1
−/−
macrophages were stimulated with native low density lipoprotein (nLDL) or oxidized (ox)LDL. SOCS-1 expression was determined by quantitative PCR and western blot. Foam cell formation was determined by Oil red O staining.

Results:
socs-1
−/−
/rag-2
−/−
/ldlr
−/−
mice were born according to mendelian law. Tripel-KO mice showed a reduced weight and size, were more sensitive to bacterial infections and died within 120 days (N=17). Histological analyses revealed a systemic, necrotic, inflammation in Tripel-KO mice. All other genotypes developed no phenotype. In-vitro observations revealed that SOCS-1 mRNA and protein is upregulated in response to stimulation with oxLDL but not with nLDL. Foam cell formation of
socs-1
−/−
macrophages was increased compared to controls.

Conclusion: SOCS-1 seemingly controls critical steps of atherogenesis by modulating foam cell formation in response to stimulation with oxLDL. SOCS-1 deficiency in the ldlr-KO mouse leads to a lethal inflammation. These observations suggest a critical role for SOCS-1 in the regulation of early inflammatory responses in atherogenesis.
Abstract 3717: Receptor for Advanced Glycation End Products (RAGE) Plays an Integral Role in Non-Diabetic Atherosclerosis and Oxidized LDL Stimulation
Authors: Uekita Hironori , Ishibashi Toshiyuki , Shiomi Masashi , Sugimoto Koichi , Ohkawara Hiroshi , Kamioka Masashi , Yamagishi Shoichi , Takeishi Yasuchika
Published: July 2021

Background: RAGE is a multifunctional receptor which identifies AGE, S100/calgranulins and HMGB-1 as the ligands. An AGE/RAGE axis plays a central role in the pathogenesis in diabetic vascular remodeling. However, the issue that hyperlipidemia associated with oxidized LDL mediates vascular responses via RAGE remains to be elucidated. In this study, we attempted to clarify the role of RAGE in non-diabetic atherosclerosis and oxidized LDL (ox-LDL) stimulation.

Methods and Results: We used the aortic and coronary atherosclerotic lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits at ages of 1 to 14 months, which had high concentrations of serum ox-LDL independent of aging. Immunohistochemistry demonstrated the significant expression of RAGE as early as at age of 1 month with the stronger expression at 3 and 7 months, which was diminished at ages of 14 months. The major original sources of RAGE expression were macrophages and endothelial cells, as well as smooth muscle cells. The concentrations of serum AGE did not alter significantly with aging, while there was no difference in serum AGE level between WHHL rabbits and control New Zealand White rabbits. These data suggest the integral role of RAGE in the initiation and progression of non-diabetic atherosclerosis associated with hyperlipidemia. We further investigated the relevance of RAGE expression in a monocyte/macrophage lineage. Fluorescent immunohistochemistry and Western blotting showed the expression of RAGE on plasma membrane in isolated human peripheral monocytes, with the stronger expression in cultured monocyte-derived macrophages. Inhibition of RAGE by siRNA suppressed AGE-induced MCP-1, MMP-9 and PAI-1 expression in cultured human macrophages. Inhibition of RAGE did not significantly suppress the incorporation of DiI-labeled oxidized LDL in monocyte-derived macrophage by fluorescent study. However, oxidized LDL- triggered MCP-1, MMP-9 and PAI-1 expression was attenuated by inhibition of RAGE.

Conclusions: Our in vivo and in vitro study shows that RAGE plays an integral role in the initiation and development of atherosclerosis independent of diabetes and that ox-LDL mediates vascular responses via RAGE.
Abstract 3731: Beta2-Integrin Activation on T-cell Subsets is an Independent Prognostic Factor in Patients with Unstable Angina Pectoris
Authors: Konstandin Mathias H , Acksoy Hülya , Wabnitz Guido H , Erbel Christian , Giannitsis Evangelos , Samstag Yvonne , Katus Hugo A , Dengler Thomas J
Published: July 2021

Cardiac troponins provide excellent risk stratification in unstable angina (UA), but no reliable markers are available in troponin-negative patients. Beta2-integrin mediated T cell recruitment plays a pivotal role in coronary atherosclerotic plaque rupture. The present study investigates beta2-integrin activation on T cell subsets as a risk marker in UA. Functional activation (affinity/avidity) of beta2-integrins on T cells was measured using a flow cytometry-based whole blood assay in 87 patients with UA. Beta2-integrin activation was significantly higher in patients with severe coronary artery disease (sC) and myocardial infarction (MI) compared to patients with no/minimal coronary atherosclerosis (no/mC), irrespective of troponin status. Adjusted for cardiovascular risk factors, medication, left ventricular function, MI at enrollment and high sensitivity C-reactive protein (hsCRP), beta2-integrin activation was independently associated with incidence of revascularization, hospitalization and all major cardiovascular events during 9 months of follow-up after index investigation. The highest prognostic value of beta2-integrin activation was seen in troponin-and hsCRP-negative patients. Quantitative assessment of T cell beta2-integrin activation allows to identify high risk patients with UA and sC without established MI; furthermore, it is associated with incidence of future cardiovascular events independent of conventional risk factors (troponin, hsCRP).
Abstract 3741: Stress Promotes Arterial Thrombosis through Activation of the Sympathetic Nervous System
Authors: Stampfli Simon F , Camici Giovanni G , Garcia Irene , Luscher Thomas F , Tanner Felix C
Published: July 2021

Background:
Several lines of evidence demonstrate a correlation between stress and cardiovascular mortality and morbidity. Myocardial infarction can be triggered by life threatening events such as earthquakes or missile attacks, and even every day stressful situations such as anger were shown to correlate with adverse cardiovascular events, primarily caused by arterial thrombus formation. The sympathetic nervous system is known to mediate various stress-related effects; however, a mechanism linking stress and thrombosis has not been described.

Methods:
To investigate the influence of acute stress on arterial thrombosis, mice were subjected to a restraint stress protocol for 20 hours. Subsequently, arterial thrombosis was induced by photochemical injury
in vivo
. Furthermore, coagulation times (PT, aPTT) and tissue factor activity were assayed in plasma and carotid artery, respectively. In addition, tail bleeding time was assessed. To investigate the role of the sympathetic nervous system, chemical sympathectomy was performed by treating mice with 6-Hydroxydopamine (OHDA) or control vehicle, respectively.

Results:
Time to thrombotic occlusion was significantly decreased in stressed mice as compared to controls (−26.7 ± 4.0 minutes, p<0.001, n=5). In sympathectomized mice stress failed to decrease time to occlusion (−8.1 ± 5.6 minutes, n.s., n=5); Sympathectomy alone had no effect on time to occlusion as compared to vehicle treatment. Tissue factor activity, tail bleeding time, and coagulation times remained unchanged under all conditions, indicating an unaltered state of vessel wall tissue factor, systemic coagulation and platelet function, respectively.

Conclusions:
Stress enhances arterial thrombosis via the sympathetic nervous system. The link between increased sympathetic activity and thrombus formation is still under investigation. These findings may open novel perspectives for the understanding of stress-induced cardiovascular events.
Abstract 3747: Endothelial Specific Inactivation of CXCR4 Receptor in Adult Mice Reveals the Pivotal Role of SDF-1/CXCR4 Axis in the Development of Endothelial Progenitor Cells (EPCs) for Myocardiac Infarction
Authors: Yokoyama Ayumi , Kwon Sangmo , Ii Massaki , Asahara Takayuki
Published: July 2021

The development of endothelial progenitor cells (EPCs) is markedly modulated by vascular niche-derived factors for maintaining EPC pool in adult bone marrow. Although SDF-1a/CXCR4 axis have shown to play a critical role in maintaining hematopoietic stem cell (HSC) pool, the precise regulatory mechanism of EPC development remain unclear. In the analysis of tie-2 specific inactivation of CXCR4 in adult mice, we have found that the number of CD34(−/low)/c-kit(+)/Sca-1(+)/Lin(−) or side population (SP) stem cells is profoundly increased. In addition, EPC colony forming capacity of CD34(−)/c-kit(+)/Sca-1(+)/Lin(−) stem cells in endothelial specific CXCR4 null mice are drastically enhanced. Notably, the development of definitive EPC, a late stage of EPC development, is specifically impaired in Tie2cre-CXCR4 null mice, although the colony number of primitive EPCs, an early stage of EPC development, is profoundly increased. EPCs derived from endothelial specific CXCR4 null mice showed showed immatured EPC function, such as migration capacity, proliferation, cell survival. When cultured with SDF-1, niche-derived stem cells gave rise to promoted EPC colony forming capacity. In myocardiac infarction model, we observed the significantly reduced cardiac function,when measured by echocardiography. Furthermore, capillary density was also significantly decreased, as well as induced fibrosis in endothelial specific CXCR4 null mice. In conclusion, the present report clearly showed for the first time that the SDF-1a/CXCR4 axis plays a pivotal role in EPC maturation in special niche, suggesting that the full understanding of EPC development via regulating SDF-1-CXCR4 axis provide a key source of therapeutic tissue regeneration.
Abstract 3783: An Insulin-independent AMPK-dependent Survival Pathway Is Activated In Glucose-starved Cardiac Myocytes through IRS-1/PI3-kinase and Rictor-mediated Activation of PDK1 and PDK2
Authors: Chopra Ines , Li Huifang , Bishopric Nanette H , Webster Keith A
Published: July 2021

INTRODUCTION: Akt (PKB) is the downstream effector of insulin and IGF-1 and regulates multiple targets that control cell survival and growth. Akt is phosphorylated at Thr-308 (activating) and Ser-473 (regulatory) sites by PI3-kinase dependent kinase-1 (PDK1) and TOR2-rictor/PDK2 respectively. When energy is abundant Akt supports cell growth by stimulating TORC1 and inhibiting GSK3β. When energy and insulin are low such as during ischemia, AMPK is activated and depresses TORC1 by activating the repressive TSC1/2 complex. It is not known how AMPK affects TORC2 in this setting.

HYPOTHESIS: Under low energy states AMPK promotes survival by differentially regulating TORC1 and 2 thereby maintaining Akt phosphorylation and survival in the absence of insulin.

METHODS: Cardiac myocytes (CM) were subjected to glucose and insulin-free incubation for 4h and the activities of insulin signaling components were measured by western blot and co-IP. Survival was measured ± siRNAs by Hoechst/PI staining.

RESULTS: After 4h of glucose/insulin deprivation there was increased phosphorylation of Akt-Thr-308 (11.2±2.4; n=6, p<0.001) and Akt-Ser-473 (9.7± 2; n=6, p<0.001). Phosphorylation of GSK-3β (Ser-9) also increased but there was decreased phosphorylation of p70S6-kinase (Thr389) and 4EBP (Ser65) indicating global down-regulation of TORC1. We identified 2 separate pathways for the insulin-independent phosphorylation of Akt at both sites in glucose-depleted CM. Activated AMPK promoted phosphorylation of insulin receptor substrate-1 (IRS-1) on Ser-789. AraA or a Ser-789 decoy peptide blocked this, and IRS-1-P-Ser-789 co-IP’d with PI3-kinase suggesting positive regulation of PDK1. Glucose depletion did not change the levels of rictor or raptor or their relative binding to TOR. However TSC1 and rictor were quantitatively associated with PI3-kinase selectively under glucose/insulin depletion and this correlated with Akt phosphorylation. CM survival under glucose/insulin depletion was impaired by siRNA-mediated knockdown of IRS-1 or rictor (n=3; P<0.05).

CONCLUSIONS: AMPK maintains phosphorylation of Akt under low energy states in the absence of insulin. The mechanism involves IRS-1 phosphorylation by AMPK and enhanced binding of rictor and TSC1 to PI3K.

This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
Abstract 3889: Persistent Cardioprotection by Regular Ethanol Consumption After Abstention Depends on eNOS but not iNOS Activity After Reperfusion
Authors: Kaneda Kazuhiro , Miyamae Masami , Sugioka Shingo , Okusa Chika , Inamura Yoshitaka , Onishi Anna , Domae Naochika , Figueredo Vincent M , Kotani Junichiro
Published: July 2021

We previously found regular ethanol consumption induces cardioprotection against ischemia-reperfusion (I-R) injury with persistent protection after abstention. The mechanism of this continued protection remains unclear. We now determine how long ethanol cardioprotection persists after abstention and whether inducible and/or endothelial nitric oxide synthase (iNOS and eNOS) play a role in this continued cardioprotection. Isolated perfused hearts from control (CTL) and ethanol-treated (ETOH) guinea pigs were subjected to 30 min global ischemia and 120 min reperfusion. ETOH received 5% ethanol in their drinking water for 8 weeks. I-R was performed on ETOH hearts at 0, 4, 7 and 14 days after abstention. To determine whether iNOS and eNOS play a role in this persistent cardioprotection, a NOS inhibitor, L-NAME (100μM) was given before ischemia or during reperfusion. Contractile recovery was monitored by left ventricular developed (LVDP) and end-diastolic (LVEDP) pressures. Infarct size (IS) was determined by triphenyltetrazolium chloride staining. Myocardial iNOS and eNOS were determined by Western blot and immunohistochemically. After I-R, ETOH at 0, 4 and 7, but not 14 days abstention, had significantly higher LVDP (62±5, 64±5, 54±4, 34±3
vs
. 26±3 mmHg, respectively), and lower LVEDP (17±6, 21±3, 30±6, 43±1
vs.
59±5 mmHg, respectively) compared to CTL. IS was significantly reduced in ETOH at 0, 4 and 7, but not 14 days abstention, compared to CTL (24±3, 24±2, 30±3, 37±2
vs.
47±3%, respectively). L-NAME given during reperfusion, but not before ischemia, abolished cardioprotection at 0 and 7 days abstention ( IS: 42±3, 45±3%, N.S.
vs.
CTL, respectively). Western blot analysis demonstrated upregulation of eNOS expression up to 7 days, but not 14 days abstention. Expression of iNOS was reduced at 0 day. eNOS immunoreactivity was greater at 0, 4 and 7 days and iNOS at 14 days, compared to CTL. Chronic cardioprotection against I-R by regular ethanol consumption persists for at least 7 days after abstention. Increased eNOS activity after reperfusion plays a role in this persistent cardioprotection.
Abstract 3952: Late Outgrowth Endothelial Progenitor Cells From Patients With AMI Improve Left Ventricular Ejection Fraction In Experimental Myocardial Infarction
Authors: Knoedler Martina , Weidl Eliane , Gawehn Sandra , Schurmann Maren , Stein Andreas , Seidl Stefan , Peschel Christian , Oostendorp Robert , Ott Ilka
Published: July 2021

Background: Recent studies suggest that endothelial progenitor cells (EPC) from bone marrow or peripheral blood improve myocardial function in experimental myocardial infarction (MI). Since applications for cell therapy are limited by the number of available cells, expansion of EPC may facilitate its therapeutic use in ischemic disease. The aim of this study was to expand late outgrowth EPC from peripheral blood from patients with acute myocardial infarction, characterize them and investigate their therapeutic effect in experimental MI.

Methods and Results: Venous blood samples were obtained from patients with acute MI (n=51), stable angina (sAP, n=57) and healthy controls (H,n=47). CD34+ cells were isolated using immunomagnetic beads (Miltenyi Biotec). CD34+ cells cultured on fibronectin in endothelial cell medium formed colonies after 1–2 weeks and were further expanded for up to 3 months to generate late outgrowth EPC (eEPC). Expansion was observed up to 2.9x10′9 (MI), 11x10′9 (sAP) and 7x10′9 cells (H) with a mean culture duration of 61 days. Expanded cells showed an endothelial morphology and expressed endothelial surface markers (CD31, VEGF-R2, CD105). Intramyocardial transplantion of 1x10′6 eEPC in experimental myocardial infarction in athymic nude rats revealed improvement in echocardiographic ejection fraction after 2 weeks. This was associated with enhanced vessel density after 1 week and increased mRNA expression of HGF. No differences in infarct size were observed. Similarly in a chronic model of myocardial infarction (eEPC transplantation 1 week after MI) myocardial function significantly improved after 5 weeks in comparison to the control group.

Conclusion: Expansion of eEPC from circulating CD34+ cells in patients with coronary artery disease is feasible and improves myocardial function after local transplantation in acute and chronic myocardial infarction. The large number of generated eEPC may prove benefical for therapeutic use and this advantage may prevail time-consuming expansion procedures.
Abstract 3964: Nanoparticle-Mediated Monocyte-Selective Transfection of Dominant-Negative Monocyte Chemoattractant Protein-1 (MCP-1) Gene Inhibits Plaque Rupture in ApoE-Deficient Mice
Authors: Koga Jun-ichiro , Egashira Kensuke , Matoba Tetsuya , Hara Kaori , Tsujimoto Hiroyuki , Sunagawa Kenji
Published: July 2021

Preventing rupture of unstable plaque is the most effective strategy for acute coronary syndrome (ACS). Monocytes/macrophages play a key role in plaque destabilization and rupture. Monocyte chemoattractant protein-1 (MCP-1) and its receptor (CCR2) play an essential role in regulating monocyte recruitment and activation. Blockade of the MCP-1/CCR2 pathway by nanoparticle (NP)-mediated monocyte-selective transfection of dominant-negative MCP-1 (7ND) gene inhibits plaque rupture. We prepared bioabsorbable PLGA NP (mean diameter = 200 nm). Intravenous administration of NP incorporated with 7ND gene (5 mcg plasmid/1 mg PLGA) resulted in incorporation of NP via phagocytosis and gene transfer into CD11b
+
circulating monocytes, and suppressed monocyte function. We then examined the effect of 7ND-NP on plaque rupture of brachiocephalic arteries in apoE-deficient mice fed with high-fat diet and infused with angiotensin II (Figure A

). At 20 weeks, animals were divided into no treatment group and those treated weekly with intravenously 7ND-NP or FITC-NP (Figure B

). FITC-NP was detected in macrophages of destabilized brachiocephalic arterial plaques, but not in those of stable aortic plaques. 7ND-NP did not affect the size of atheroma, but did reduce the occurrence of plaque rupture (Figure C

), macrophage infiltration, and tissue factor expression. Blockade of MCP-1/CCR2 pathway by NP-mediated monocyte-targeting gene transfer stabilized atheroma and prevented plaque rupture. This nanotechnology platform may be a less invasive therapeutic strategy for stabilizing rupture-prone plaques and thus preventing ACS.
Abstract 5255: Differential Effect Of A Novel Adenosine-Agonist On Stimulation-Induced Norepinephrine Release From Rat Hearts
Authors: Bott-Flugel Lorenz , Bernshausen Alexandra , Laugwitz Karl-Ludwig , Ehmke Heimo , Seyfarth Melchior
Published: July 2021

Presynaptic modulation of norepinephrine (NE) release plays a major role in the regulation of cardiac sympathetic activity. We tested the effect of the novel, highly selective adenosine A1 subtype agonist BAY 68–4986 on stimulation-induced NE release. Isolated rat hearts (Wistar or SHR) were perfused with a modified Krebs-Henseleit solution (Langendorff technique). Exocytotic NE release was induced by electrical field stimulation with 5 V, 6 Hz for 1 minute. NE was detected by HPLC. Two stimulations (S1, S2) were performed to compare intraindividually NE release under control conditions (S1) and with BAY 68–4986 30ug/ml, 300 ug/ml or CCPA 10–6M (S2). Neuronal uptake was inhibited by desipramine (100 nM) in order to prevent amine elimination and nonexocytotic release of NE. Stimulation of adenosine A1 receptors with BAY 68–4986 led to a concentration dependent decrease of the stimulation-induced NE release only in SHR rats, whereas no effect could be seen in Wistar rats (SHR: S2/S1 = 0.90 +/− 0.08 with 30 ug/l, S2/S1 = 0.54 +/− 0.02 with 300 ug/l, Wistar: S2/S1 = 1.05 +/− 0.12 with 30 ug/l, S2/S1 = 1.03 +/− 0.09 with 300 ug/l, p=0.003). The effect of BAY 68–4986 on stimulation-induced NE release in SHR was similar to the effect of 10–6 M CCPA (S2/S1 = 0.59 ± 0.05). This differential effect in genetically different rats was also observed in vivo, with a blunted increase of the heart rate during stress reaction only in SHR rats, when treated with BAY 68–4986. We could demonstrate that the novel, highly selective A1-adenosine agonist BAY 68–4986 has a differential effect on stimulation-induced NE release and stress responde in SHR rats compared to Wistar rats.
Abstract 5261: Platelet-Depletion Ameliorates Cardiac Function and Disease Severity in Experimental Autoimmune Myocarditis
Authors: Russ Martin , Seliger Barbara , Hauptmann Steffen , Marty Rene , Bukur Jürgen , Eriksson Urs , Schubert Sebastian , Werdan Karl , Buerke Michael
Published: July 2021

Introduction: Experimental Autoimmune Myocarditis (EAM) is a CD4+ T cell mediated model of inflammatory cardiomyopathy. Activated platelets express CD154, a molecule critical to adaptive immune responses, which has been implicated in platelet-mediated modulation of inflammation.

Hypothesis: Platelets are critical for the generation of heart-specific, autoreactive T-cell responses in a model of experimental Autoimmune Myocarditis.

Methods: BALB/c mice were immunized twice at day 0 and day 7 with 100μg alpha-MyHC-peptide (614–29) together with Complete Freund‘s adjuvant. Platelets were markedly depleted by i.v. injection of a GP1alpha antibody every 5th day (n=8). Control mice were injected with a non-depleting isotype antibody (n=8). Mice were assessed at day 28 for heart dimensions and cardiac function using echocardiography. After lethal anesthesia, hearts were removed and analyzed for heart weight/body weight ratio and histological severity scores. CD4+ T-cells were isolated from spleens, and analyzed for CD154 and IL-17 expression using FACS after in vitro re-stimulation on alpha-MyHC pulsed, irradiated antigen presenting cells.

Results: Modest platelet depletion protected from left ventricular dilatation, and preserved left ventricular ejection fraction in immunized mice. Myocarditis prevalence and severity scores were significantly reduced in depleted animals. Relative numbers of spleen-derived CD4+ T-cells expressing CD154 or IL-17, were significantly reduced in the treatment group (table

).

Conclusion: Our findings suggest that platelets might play a critical role in the development of heart-specific autoimmunity and cardiomyopathy. Further studies are needed to confirm these findings, which suggest a novel treatment approach for inflammatory cardiomyopathy.

Echocardiography - Histology - FACS
Abstract 5276: Sumoylation Of Klf5 Is A Molecular Switch Regulating Ppar-δ-containing Transcriptional Programs Of Lipid Metabolism
Authors: Oishi Yumiko , Manabe Ichiro , Tobe Kazuyuki , Kadowaki Takashi , Nagai Ryozo
Published: July 2021

Metabolic syndrome is increasingly recognized as a major risk factor for cardiovascular disease. We have previously shown that a zinc finger transcription factor, Krüppel-like factor 5 (KLF5), plays an important role in cardiovascular diseases, such as atherosclerosis and cardiac hypertrophy. Interestingly, KLF5 is also expressed in metabolic tissues, such as adipose tissue, skeletal muscle and pancreatic β-cells. Moreover, we found that KLF5 is crucial for adipocyte differentiation. Therefore, it is very likely that KLF5 plays multiple roles in development and progression of metabolic syndrome and its cardiovascular and metabolic consequences including atherosclerotic cardiovascular disease. Indeed,
KLF5
heterozygous knockout (
KLF5
+/−
) mice were resistant to high-fat-induced obesity and metabolic syndrome, despite consuming more food than wild-type mice. This appears to in part reflect their enhanced energy expenditure. Expression of the genes involved in lipid oxidation and energy uncoupling, including
uncoupling protein (UCP)
and
carnitine-palmitoyl transferase 1b (CPT1b)
was upregulated in the soleus muscles of
KLF5
+/−
mice. KLF5 could be reversibly modified by small ubiquitin-like modifier 1 (SUMO1), after which SUMOylated KLF5 strongly inhibited
CPT1b
,
UCP3 and UCP2
promoter activity. Results of chromatin immunoprecipitation, two-hybrid, and reporter assays showed that under basal conditions SUMOylated KLF5 associated with transcriptionally repressive regulatory complexes containing unliganded PPARδ and corepressors. However, upon agonist stimulation of PPARδ, the deSUMOylating enzyme was recruited and KLF5 was deSUMOylated. The unSUMOylated KLF5 now formed transactivating complexes with liganded PPARδ and CBP. Thus, SUMOylation appears to be a molecular switch affecting function of KLF5 and the transcriptional regulatory programs governing lipid metabolism. Moreover, KLF5 is essential for the PPARδ agonist-dependent transcriptional control. Results of the present study have established KLF5 as a novel key molecule in lipid metabolism and suggest that the posttranscriptional modification of KLF5 is an attractive novel therapeutic target for both metabolic and cardiovascular diseases.
Abstract 5281: Bioluminescence Imaging of X-Ray Visible Microencapsulated Mesenchymal Stem Cells for Cell Based Therapy of Peripheral Arterial Disease
Authors: Kedziorek Dorota A , Walczak Piotr , Fu Yingli , Azene Nicole , Arepally Aravind , Bulte Jeff W , Kraitchman Dara L
Published: July 2021

Introduction:
Therapeutic angiogenesis in Peripheral Arterial Disease (PAD) using stem cell therapy is potentially complicated by immunorejection. To overcome this problem, microen-capsulation using the alginate-poly-L-lysine (PLL)-alginate (APA) method was developed to provide a protective porous bubble to block antibodies but allow exchange of small molecules. Recently, we have developed a method to enable X-ray detection of these capsules. However, cell survival within the capsules could not be determined. Plus PLL can be mildly cytotoxic. In the present study, we combined reporter gene methods to verify cell survival with X-ray detection of the microcapsules in a rabbit PAD model and studied the PLL impact on cell viability.

Methods:
Rabbit mesenchymal stem cells (MSCs) were transfected with triple fusion (TF) reporter gene for bioluminescence (firefly luciferase), fluorescence (red fluorescent protein) and PET (truncated thymidine kinase). TF-MSCs were encapsulated in the perfluorooctyl bromide (PFOB) capsules to enable computed tomographic detection. Capsule crosslinking was performed with three PLL concentrations, i.e., 0.005%, 0.025% and 0.05%. Bioluminescent imaging (BLI) was used to monitor cells survival for one week
in vitro
and after intramuscular injection
in vivo
.

Results:
Serial
in vitro
BLI enabled the detection of viable encapsulated MSCs without detrimental signal degradation (~13% decrease of BLI signal intensity after PFOB encapsulation comparing to equal number of naked MSCs). PLL did not result in cell death; higher PLL concentrations were correlated with stronger BLI signal. BLI signal production was only slightly reduced by second layer of alginate (~80% for 0.05% PLL).
In vivo
BLI demonstrated the detection of naked, APA, and PFOB-encapsulated TF-MSCs. X-ray imaging enabled PFOB microcapsules detection relative to vasculature.

Conclusion:
BLI allows monitoring of encapsulated cells survival. PLL concentrations ≤ 0.05% appear safe for encapsulated cells with higher concentration being associated with enhanced crosslinking and capsule stability. MSCs expressing TF reporter in PFOB microcapsules enables dual monitoring of cell delivery/capsule tracking by X-ray imaging and cell viability with BLI.
Abstract 5289: Abnormally Tight Domain-Domain Interaction at Mutation Site Could be a Primary Cause of Catecholaminergic Polymorphic Ventricular Tachycardia: Insight from RyR2
S2246L/+
Knock-In Mouse Model
Authors: Suetomi Takeshi , Yano Masafumi , Xu Xiaojuan , Ono Makoto , Uchinoumi Hitoshi , Tateishi Hiroki , Okuda Shinichi , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Ohkusa Tomoko , Matsuzaki Masunori
Published: July 2021

Mutations in cardiac ryanodine receptor 2 (RyR2) have been shown to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of this lethal arrhythmia, we developed knock-in (KI:RyR2
S2246L/+
) mice model with the human CPVT-associated RyR2 mutation (S2246L). The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n=6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7). In isolated cardiomyocytes, line scan images were obtained to measure local Ca
2+
release events using a confocal microscopy with fluo-4 AM as a Ca
2+
indicator. In the KI cardiomyocytes, the frequency of Ca
2+
sparks (SpF: s
−1
·100μm
−1
) was much more increased in response to 100 nM isoproterenol than in WT cardiomyocytes (KI:4.7±0.5 vs WT:1.9±0.2, p<0.01). Using the canine cardiac SR, we fluorescently labeled RyR2 with methylcoumarin acetamido (MCA), using either DP
2232–2266
or DP
2232–2266
mut; Ser is mutated to Leu (S2246L), as a carrier; DP
2232–2266
harbors the same CPVT mutation site as KI mice (S2246L). The binding affinity of DP
2232–2266
mut to RyR2 (Kd=0.08μM) was higher than that of DP
2232–2266
(Kd=0.32μM), suggesting that abnormally tight interaction of the domain pair (between the domain2232–2266 and another putative domain) may be formed by the S2246L mutation. Interestingly, addition of DP
2232–2266
to the SR dose-dependently inhibited the cAMP (30μM)-induced Ca
2+
leak; IC50=0.1μM, although DP
2232–2266
had no effect on the cAMP-induced increase in 2808Ser phosphorylation. In conclusion, some type of RyR2 mutation in CPVT may causatively induce hyper-activated channel gating by forming abnormally tight domain-domain interaction, triggering diastolic Ca
2+
release and hence lethal arrhythmia. Interruption of such abnormal domain-domain interaction (by competing with native domain) may lead to a new therapeutic strategy against CPVT.
Abstract 5294: Sequestration of Prosurvival Akt Pathway by Reductive Stress Underlies hR120GCryAB Protein Aggregation Cardiomyopathy in Mice
Authors: Soorappan Rajasekaran Namakkal , Zhang Huali , Odelberg Shannon , Benjamni Ivor J
Published: July 2021

Recent genetic studies of protein aggregation R120GCryAB cardiomyopathy in mice have implicated a novel ‘toxic’ gain-of-function mechanism linked to increased G6PD activity and levels of reduced glutathione (GSH), termed ‘reductive stress.’ Because the intercrosses between hR120GCryAB High Tg and G6PD-deficient (20% normal activity) rescues the phenotype, we hypothesized that the pathogenesis of hR120GCryAB cardiomyopathy are causally linked to redox sensitive signaling transduction pathways controlling cardiac hypertrophy and failure. We examined the temporo-spatial effects on activation of the major stress-dependent survival pathways. Human R120GCryAB expression in mice induces modest activation of total and phosphospecific p38 and ERK1/2 kinases but significantly increases phospho-specific JNK1/2 in vivo. Both total and phosphospecific AKT1/2 expression were similarly increased in hR120GCryAB and hR120GCryAB/G6PDmut in vivo. In contrast, immunohistochemical analysis indicated cytosolic Akt expression co-localized with in large protein aggregates, whereas G6PD deficiency completely prevented protein aggregation, indicating hR120GCryAB-induced reductive stress governs conformational states of PI3-kinase/AKT signaling in compartment-specific manner. Consistent with this notion, the cytosolic abundance of forkhead FOXO3a, a phosphorylation-dependent downstream target of nuclear AKT1/2 signaling, was decreased significantly in R120GCryAB High Tg heart homogenates and reciprocally increased in hR120GCryAB/G6PDmut in vivo. To investigate the critical roles of AktCys60 and AktCys77 residues, which form disulfide bond pairing under oxidized conditions, we next performed site-directed mutagenesis and demonstrated that redox-insensitive Akt mutants (AktC60A and AktC77A, respectively) were exclusively localized in the nucleus when expressed transiently in H9c2 myocytes. Redox-dependent mechanisms play a key role in the cardiotoxicity associated with hR120GCryAB-induced reductive stress. Furthermore, G6PD inhibition is a novel therapeutic strategy for mitigating cardiac hypertrophy and heart failure in hR120GCryAB cardiomyopathy.