bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1032: High Clopidogrel Maintenance Dosing in Patients with Inadequate Platelet Inhibition: Functional Insights on Thienopyridine Usage According to Updated ACC/AHA/SCAI 2005 Guidelines for Percutaneous Coronary Interventions
Authors: Angiolillo Dominick J , Costa Marco A , Shoemaker Steven B , Desai Bhaloo , Hang Yuan , Charlton Ronald , Suzuki Yoshie , Bernardo Esther , Zenni Martin Z , Guzman Luis A , Bass Theodore A
Published: July 2021

Background:
Clopidogrel under-dosing is a cause of inadequate platelet inhibition. Inadequate clopidogrel-induced antiplatelet effects have been associated with an increased risk of stent thrombosis. Updated guidelines for percutaneous coronary interventions (PCI) recommend to increase the dose of clopidogrel to 150mg in high risk patients if <50% platelet inhibition is demonstrated. However, to date there are no studies which have evaluated the functional impact of this recommendation. The aim of this study was determine the functional impact of a 150mg maintenance dose of clopidogrel in patients with 50% platelet inhibition while in their steady state phase of dual antiplatelet therapy.

Methods:
Platelet inhibition was screened by means of the VerifyNow P2Y
12
assay in patients in a steady state phase of dual antiplatelet therapy. Patients with <50% platelet inhibition were treated with 150mg of clopidogrel for one-month. Adenosine diphosphate-induced aggregation using light transmittance aggregometry and P2Y
12
reactivity ratio determined by vasodilator-stimulated phosphoprotein-phosphorylation analysis were also performed.

Results:
A total of 32 patients were screened to identify 20 with <50% platelet inhibition. Platelet inhibition increased from 28.3±12% to 43.2±18% in patients treated with 150mg of clopidogrel (p=0.004; primary endpoint). All other functional measures also showed that a high maintenance dose of clopidogrel reduces platelet function (Table

) . The degree of platelet inhibition achieved following one-month treatment with high dose clopidogrel broadly varied and only eight (40%) patients yielded a degree of platelet inhibition ≥50%.

Conclusions:
The use of a 150mg maintenance dose of clopidogrel in high risk patients with <50% platelet inhibition increases platelet inhibition. However, the antiplatelet effects achieved are non-uniform and a considerable number of patients persist with elevated platelet function.

Platelet function analysis pre and post high clopidogrel maintenance dose therapy
Abstract 1041: Endovascular Injury Affects Adipocytokine Profile Of The Periadventitial Adipose Tissue
Authors: Takaoka Minoru , Sata Masataka , Nagai Ryozo
Published: July 2021

Background:
Adipose tissue secretes not only proinflammatory cytokines but also anti-inflammatory cytokines such as adiponectin. Arteries are surrounded by abundant periadventitial fat tissues, which have been suggested to play a role in maintenance of vascular homeostasis. Here, we investigated whether endovascular injury affects the profile of adipocytokines expressed by perivascular adipose tissue (PAT).

Methods and Results:
First, the femoral artery of C57BL/6 mice was overstretched by insertion of a large wire. Real-time PCR analysis revealed that wire injury markedly down-regulated adiponectin expression (0.64±0.09 fold, p<0.01) and up-regulated expression of proinflammatory cytokines, TNF alpha, IL-6, PAI-1, and MCP-1 (p<0.01) 1 day after overstretch within PAT. Neutrophils and macrophages accumulated in the adventitia surrounding the injury site at 1 day after the injury. Inflammation was associated temporally with the expression of mRNAs encoding inflammatory adipocytokines. Second, the iliac artery of Wistar rats was dilated percutaneously with a balloon for 1 minute. There were up-regulation of proinflammatory cytokines and down-regulation of adiponectin 1 and 3days after endovascular injury within PAT (p<0.05). Third, abdominal aortas removed from Wistar rat with PAT were dilated with a balloon PTCA catheter for 1 minute ex vivo. After stretching, aortas with PAT were cultured for 6 hours, and we observed increased synthesis of proinflammatory cytokines and decreased synthesis of adiponectin within PAT (p<0.05). Fourth, the stimulation of TNF alpha, which markedly increased in injured arteries, caused significant down-regulation of adiponectin expression and up-regulation of IL-6, PAI-1, and MCP-1 in 3T3L1-derived adipocytes (p<0.01).

Conclusions:
Angioplasty may induce the changes of perivascular cytokines expression. The accumulation of neutrophils and macrophages at PAT in injured arteries and/or increased proinflammatory expression of injured vascular arteries may reflect these proinflammatory activities of PAT. Increased proinflammatory cytokines and decreased anti-inflammatory cytokine adiponectin in PAT possibly may have a role in vascular remodeling following PTCA-mediated vascular injury.
Abstract 1052: 26S Proteasome: Intracellular Localization and Regulation by Nitric Oxide
Authors: Kapadia Muneera R , Eng Jason W , Murar Jozef , Jiang Qun , Kibbe Melina R
Published: July 2021

Introduction:
Nitric oxide (NO) is known to inhibit vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome tightly regulates the degradation of most cell cycle proteins. Therefore, we previously studied the relationship between NO and the 26S proteasome, and found that NO directly inhibits 26S proteasome catalytic activity. The purpose of this study was two-fold:

to determine the effect of NO on 26S proteasome subunit expression, and

to determine the intracellular localization of these subunits.

Methods:
Protein expression was examined using western blot analysis conducted on rat aortic VSMC exposed to varying concentrations of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 125–1000 μM) for 24 hours. Intracellular localization was conducted with immunofluorescent staining of VSMC ± exposure to SNAP (500 μM). VSMC were also fractionated into their nuclear and cytoplasmic components and western blot analysis was conducted to confirm localization.

Results:
Of the 26S proteasome subunits examined, the α5, α6, β1, and inducible β1 subunits demonstrated increased protein expression with increasing concentrations of NO. However, β2, inducible β2, β5, and inducible β5 protein expression did not change with NO exposure. Immunofluorescent staining of VSMC for these subunits confirmed these results. Additionally, each of these subunits showed a distinct pattern of staining: the α5 subunit was mostly perinuclear, the α6 subunit appeared mostly nuclear, and the β1 and inducible β1 subunits were mostly cytoplasmic. Western blot analysis following nuclear and cytoplasmic separation confirmed that α5, β1, and inducible β1 subunits were mostly in the cytoplasmic fraction. Interestingly, while the α6 subunit localizes to the nucleus by immunofluorescent staining, western blotting showed it to be mostly in the cytoplasmic fraction, indicating that its cellular distribution is perinuclear.

Conclusion:
Our data indicate that the 26S proteasome subunits have distinct patterns of intracellular localization, and are differentially regulated by NO. Understanding the relationship between NO and the 26S proteasome provides insight into the mechanism by which NO inhibits VSMC proliferation.
Abstract 1057: Specific Targeting of Gi Signaling in Normal and Dysfunctional Myocardium
Authors: DeGeorge Brent R , Gao Erhe , Boucher Matthieu , Vinge Leif E , Chuprun J. K , Raake Philip , Martini Jeffrey S , Harris David M , Soltys Stephen , Eckhart Andrea D , Koch Walter J
Published: July 2021

Evidence has accumulated to suggest that cardiomyocyte apoptosis contributes significantly to the progression of heart failure. A salient characteristic of the failing heart is an upregulation of the adenylyl cyclase inhibitory G protein alpha subunit, Galpha(i2); however this upregulation has yet to be established as beneficial or deleterious. Of note, G protein-coupled receptor (GPCR) signaling through Gi has been implicated in the cardioprotective properties of adenosine, opioids, and bradykinin; however to date there has been no
in vivo
evidence that increased Gi in ischemic myocardium is cardioprotective. We have created a novel molecular tool to specifically address this question and other Gi-related mechanisms in the heart. We have developed a Gi inhibitor, GiCT - the carboxyl-terminal 63 amino acids of Galphai(i2), which represents the region of Gi that interacts specifically with GPCRs. Gi selectivity of GiCT has been shown
in vitro
as Gi signals are inhibited while Gs and Gq signals are not. Thus, a class-specific Gi inhibitor has been developed. We next created a transgenic mouse line with inducible, cardiac-specific expression of GiCT (Tg-GiCT mice). At baseline, Tg-GiCT mice display a structural and functional phenotype consistent with non-transgenic (nTg) mice. However, when subjected to stress in the form of myocardial ischemia/reperfusion (I/R) injury, Tg-GiCT mice demonstrate a significant increase in infarct size as compared to nTg mice. Furthermore, Tg-GiCT mice demonstrate increased myocardial apoptosis in response to I/R, with a resultant decrease in contractile performance. These results indicate first that ischemic damage induces apoptotic signals that can be countered by GPCR signaling specifically through Gi. Moreover, the beneficial survival effects of Gi signaling on the prevention of myocardial apoptosis in ischemia appears to outweigh any potential detrimental effects that Gi upregulation may have on contractility through inhibition of cAMP. Overall, our results demonstrate the
in vivo
utility of GiCT - to elucidate the novel finding that upregulation of Gi seen in dysfunctional myocardium induces survival signals and appears to be an adaptive mechanism post-ischemia to protect myocytes from apoptosis.
Abstract 1058: Novel Regulation of Wnt-Inducible Secreted Protein 1 by TNF-alpha in Primary Human Cardiac Fibroblasts
Authors: Colston James T , de la Rosa Sam D , Bailey Steven R , Chandrasekar Bysani
Published: July 2021

Wnt1-induced secreted protein-1 (WISP-1) is a member of the CCN family of growth factors known to play a role in cellular growth, transformation, and survival. We previously demonstrated that WISP-1 is upregulated in the heart post-infarct, stimulates fibroblast proliferation and collagen expression, and is induced by the proinflammatory cytokine TNF-a. The present study was designed to investigate the molecular mechanisms involved in TNF-a-mediated WISP-1 induction in primary human cardiac fibroblasts (hCF). hCF were treated with rhTNF-a. WISP-1 expression was analyzed by Northern, Western and promoter-reporter assays. ERK1/2 and JNK activations by Western blotting using activation-specific antibodies and imunecomplex kinase assays. CREB activation by Western blotting, EMSA and reporter assay. Results demonstrate that TNF-a potently induces WISP-1 mRNA and protein expression in a time- and dose-dependent manner, effects that were blocked by anti-TNFR2, but not anti-TNFR1, neutralizing antibodies. Further investigations revealed that TNF-a stimulates WISP-1 promoter-reporter activity, an effect that was blunted when the core CREB DNA binding sequence was mutated or following the overexpression of dnCREB. TNF-a treatment induced CREB phosphorylation, in vitro and in vivo DNA binding, and reporter gene activities. TNF-a also induced ERK1/2 phosphorylation and activity, effects that were blocked by the MEK1 inhibitor U0126 and the ERK1/2 inhibitor PD98059. Furthermore, inhibition of ERK1/2 blunted CREB activation while inhibition of ERK1/2 and overexpression of dnCREB attenuated TNF-a-mediated WISP-1 promoter-reporter activity and mRNA expression. However, inhibition of JNK by SP600125 failed to modulate TNF-a-mediated WISP-1 induction. Most importantly, siRNA-mediated WISP-1 knockdown attenuated TNF-a-mediated hCF proliferation. Thus, our results demonstrate for the first time that TNF-a is a potent inducer of WISP1 expression in hCF, and TNF-a induces WISP-1 expression via TNFR2, MEK1, ERK, and CREB. Since TNF-a and WISP-1 are upregulated post-infarct, our results indicate that TNF-a/WISP-1 signaling may play a critical role in post-infarct myocardial remodeling.
Abstract 1063: Intracardiac Injection of Epoetin-α Upregulates Stem Cell Chemoattractant Gene Expression In a Rat Myocardial Infarction Model
Authors: Klopsch Christian , Furlani Dario , Gäbel Ralf , Wagner Klaus , Wang Weiwei , Ong Lee-lee , Li Wenzhong , Nizze Horst , Titze Ulf , Lendlein Andreas , Lützow Karola , Li Ren-Ke , Ma Nan , steinhoff gustav
Published: July 2021

Emerging evidence suggests that Erythropoietin (EPO) protects the myocardium from ischemic injury and promotes beneficial remodelling. However, the role of EPO and its receptor (EPO-R) in mediating cardiac regeneration remains unclear. We hypothese that stem cell homing and proliferation modulated by EPO could contribute to its cardio-protective effects. Epoetin-α (3000 U/kg) was injected along the infarction border after the induction of a rat myocardial infarction (MI) by permanent ligation of the left descending coronary artery. At six weeks after MI, cardiac function was measured by pressure-volume loops in left and right ventricles. Infarction size, angiogenesis and pathologic effects were evaluated. Gene expressions of EPO-R, SDF-1α, CXCR-4, c-kit, eNOS, TNF-α, IL-8, Integrin-β and CdK4 were analyzed by RT-PCR at different time points of the first week (24h, 48h, 96h and 7 days). We found EPO treatment improved left ventricular function both at baseline levels and under Dobutamine stress (tau, cardiac output, stroke work, ejection fraction, dp/dt maximum and minimum, n=11–14, p<0.05) and decreased right ventricular wall stress (maximum and endsystolic pressure, n=5– 8, p<0.05). Infarction size was reduced from 27.8±3.4% to 20.1±2.8% (n=6 – 8, p<0.01). Capillary density was increased from 257.7±24.5 to 338.5±35.9 (vessels per square mm, n=6 – 8, p<0.05). Mortality was decreased from 29.0% to 22.2% (n=53– 69). EPO-R was down regulated in infarcted, peri-infarcted and non infarcted areas at all time points (n=7, p<0.05). Cardiac SDF-1α, CXCR-4 and eNOS gene expressions were increased at 24 hours. C-kit was up regulated significantly at 48 hours compared to 24 hours in the EPO treated hearts. In untreated hearts, c-kit expression remained constant. Proinflammatory cytokines (TNF-β, IL-8 and Integrin-γ) were down regulated. Cell cycle re-entry marker (CdK4) was increased at 24 hours in non infarcted zones. In conclusion, we demonstrate intramyocardium Epoetin-α injection induces an earlier up regulation of stem cell chemoattractants, reduces inflammation, enhances angiogenesis and restores heart function after MI.
Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K
+
Channels
Authors: Underkofler Heather A , Balijepalli Sadguna , Moungey Brooke M , Slind Jessica K , Best Jabe M , Kamp Timothy J , January Craig T , Delisle Brian P
Published: July 2021

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the
human Ether-a-go-go Related Gene
(
hERG
). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I
hERG
) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I
hERG
. This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I
hERG
by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I
hERG
by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.
Abstract 1073: Cardiac Voltage-gated Nav channel Na
v
1.5 Requires An AnkyrinG-dependent Pathway For Targeting in Cardiomyocytes
Authors: Lowe John S , Palygin Oleg , Wright Patrick , Shibata Erwin , Mohler Peter
Published: July 2021

Membrane localization of ion channels is very important for normal function in excitable cells. In heart, voltage-gated Na
+
channels are necessary for the rapid upstroke of the cardiomyocyte action potential, and variants in
SCN5A
(encodes Na
v
1.5) are associated with fatal arrhythmias. We have identified ankyrin family proteins as critical components for normal ion channel and transporter targeting in cardiomyocytes. Humans with
ANK2
(encodes ankyrin-B) loss of function variants display abnormal cardiac phenotypes and risk for sudden cardiac death. Mice that lack ankyrin-B expression display a similar phenotype. Our most recent results demonstrate that a second ankyrin gene product, ankyrin-G (encoded by
ANK
3) is critical for targeting Na
v
1.5 to specific cardiomyocyte membrane domains. We assessed the hypothesis that Na
v
1.5 membrane expression and localization is controlled by an ankyrin-G-dependent pathway and disruption of ankyrin-G/Na
v
1.5 interactions lead to human cardiac disease in this study. We used a combination of techniques including biochemistry, confocal microscopy, lentiviral expression, and electrophysiology to evaluate the functional relationship between ankyrin-G and Na
v
1.5. We defined the structural elements on ankyrin-G and Na
v
1.5 for their interaction using site-directed mutagenesis and
in vitro
binding assays. Lentiviral expression of shRNA targeted to rat 190 kD ankyrin-G effectively reduced the expression of ankyrin-G with a concomitant reduction of Na
v
1.5 in immunofluorescence and immunoblot assays. Further-more, primary cardiomyocytes with reduced ankyrin-G expression have a significant reduction in Na
+
current density with no evident biophysical effects on Ca
2+
current or inactivation-gating of Na
v
1.5 These results confirm the importance of ankyrin polypeptides for normal cardiac function and shed new light on the importance of intracellular trafficking pathways for the delivery and stability of critical ion channels and transporters in excitable cells.
Abstract 1080: Calmodulin Plays an Important Role in Electrical Storm: Insights from a New Experimental Model
Authors: Tsuji Yukiomi , Harada Masahide , Hojo Mayumi , Shimano Masayuki , Uchikawa Tomohiro , Inden Yasuya , Murohara Toyoaki , Nattel Stanley , Kodama Itsuo , Kamiya Kaichiro
Published: July 2021

Background:
Electrical storm (ES), characterized by recurrent episodes of ventricular fibrillation (VF), is a serious clinical condition, importantly complicating implantable cardioverter-defibrillator (ICD) therapy. The pathophysiology and molecular basis of ES are poorly understood because of a lack of suitable animal models. Here, we report a new experimental model that features repetitive episodes of ICD firing for recurrent VF, in which we assessed the potential involvement of calmodulin in ES.

Methods and Results:
The incidence of ES was assessed in 8 rabbits that had been in chronic complete atrioventricular block (CAVB) for 90±12 days (M±SEM; range 50 –129 days), all of whom had undergone extracardiac ICD system implantation. The maximal number of intervals to detect VF (NID: 105/140) was programmed to avoid inappropriate shocks for incessant ventricular tachyarrhythmias (VTs). Shock intensities were set >twice the defibrillation threshold (1.5±0.2 J). All rabbits had ICD-detected self-terminating VTs and corrected QT-interval (QTc) prolongation (226±12 versus 185±11 ms at baseline, P<0.05) before the first VF episode, and 7/8 rabbits subsequently developed ES, defined as ≥3 VF episodes per 24-hour. The ES appeared on day 30±7 and occurred for 19±5 days follow-up. The QTc was remarkably increased on the day of ES compared with the value on the day without ES (271±12 versus 219±10 ms, P<0.01). Analysis of 1006 VF episodes obtained from 8 rabbits showed no circadian variation of VF incidence. One-week infusion of W-7, a calmodulin antagonist, to 3 rabbits by osmotic pump caused dose-dependent reductions in days of VF (from 5.3±1.2 to 3.0±1.1 and 0.6±0.3*), VF episodes (from 20±3 to 19±7 and 1.7±1.2*) and self-terminating VT episodes (from 2572±854 to 997±197* and 178±24* for 1 week; all results are shown as before infusion to data during infusion of W-7 at doses of 297 and 744 μg/hr respectively; *P<0.05 versus before W-7) without shortening the QTc.

Conclusions:
This model shows a high incidence of ES, allowing for study of mechanisms and treatment of ES, as well as consequences of multiple high-energy shocks. The positive response of ES to W-7 suggests that the calmodulin/calmodulin kinase system may be crucial to the generation of ES in this model.
Abstract 1084: Myosin Light Chain Kinase Signaling Regulates Ectopic Atrial Activity
Authors: Wolkowicz Paul , Grenett Hernan E , Huang Jian , Ku David D , Urthaler Ferdinand
Published: July 2021

Objective
: Determine whether calmodulin (CaM) acts through novel signaling pathways to elicit left atrial ectopic activity.

Methods
: Rat left atria (n=6 –9 per group) were isolated and superfused at 30°C. Mechanical function was recorded using a Grass recorder. Left atrial proteins were analyzed using Western analysis.

Findings
: Our rat left atria are quiescent in the absence of external pacing. However, in the presence of 2-aminoethoxydiphenyl borate (20μM; 2APB), a novel sarcoplasmic reticulum (SR) calcium leak inducer, left atria contract spontaneously at 67±12 contractions (min)
− 1
in the
absence
of pacing. Pre-treating left atria with two irreversible CaM inhibitors, phenoxybenzamine and fluphenazine chloroethane (90μM each), decreases spontaneous contraction frequency to 5±3 and 2±1 (min)
− 1
, respectively, indicating CaM signaling is activated and required for this ectopic activity. Pre-treating left atria with 20μM KN62 did not decrease the frequency of 2APB-induced ectopic activity (72±8 contractions (min)
− 1
) indicating calmodulin-dependent protein kinase II (CAMKII) is not a downstream target here. In contrast, 20μM ML7, a specific inhibitor of myosin light chain kinase (MLCK), a second cardiac CaM-regulated kinase, decreases 2APB-induced spontaneous contractions to 7±3 min
−1
. Western analysis shows left atria contain smooth muscle MLCK. This kinase preferentially phosphorylates
non-muscle
myosin light chain 2 (nmMLC2). Western analyses also show left atria contain nmMLC2 which is not phosphorylated in untreated muscles. However, nmMLC2 phosphorylation increases significantly in left atria exposed to 2APB.

Conclusions
: CaM and MLCK inhibitors prevent the ectopic atrial activity induced by 2APB. This suggests a unique SR calcium leak occurs under this condition which activates CaM signaling whose specific downstream target is MLCK, a major myocardial CaM-dependent kinase. MLCK signaling via its sole substrate, nmMLC2, is required for this novel ectopic atrial activity. Hyperphosphorylation of SR ryanodine receptors by CAMKII is known to induce an SR calcium leak that underlies triggered ectopic activity. Our data suggest
multiple
calcium leaks and CaM signaling pathways regulate the stability of intact atrial muscle.
Abstract 1105: Gadofluorine M-Cy 3, a Novel Paramegnetic Contrast Agent for the in vivo and Histolgical Identification of Transplanted Cells.
Authors: Adler Eric , Bystrup Anne , Saebo Karen , Mani Venketesh , Giovannone Steve , Young Wilson , Frank Joseph , Weinmann Joachim , Keller Gordon , Fayad Zahi
Published: July 2021

PURPOSE: Gadofluorine M with a fluorescent dye (GdFMCy3) is a lipophilic paramegnetic contrast agent that is readily absorbed by cultured cells. We hypothesized that this agent would be superior to iron oxide based techniques for cell tracking post cell transplantation.

METHODS: Embryonic Stem Cell derived cardiac progenitor cells (ES-CPCs) were generated using previously established methods and incubated for 12 hours with 5 mM GdFMCy3, or transfected with iron oxide using published protocols. Cell survival was >95% for cells incubated with both GdFMCy3 and iron. 500,000 cells labelled with GdFMCy3, iron oxide or control (no contrast agent) were directly injected into the myocardium of mice (n=5/group). Mice were scanned over a two week interval post injection at 9.4T using gated T1-weighted sequences (GdFMCy3), T2* weighted GRE sequences (iron oxide) or the positive contrast sequence GRASP (iron oxide). Mice were sacrificed and the hearts sectioned for microscopy. Perl staining and fluorescence microscopy were used to identify iron oxide and GdFMCy3 within the myocardium, respectively.

RESULTS: GdFMCy3 labelled cells were successfully identified in vivo at 9.4T (figure

panel B). Good correlation between MRI and histology was observed for both cell labels (figure

). Contrast to noise ratios were significantly higher in the GdFMCy3 group relative to the iron oxide group (figure

).

CONCLUSIONS: GdFM-Cy3 is readily taken up by stem cells and easily identified by both MRI and fluorescence microscopy. Given its superior contrast to noise ratio GdFM-Cy3 may be an excellent alternative to iron oxide for in vivo detection and tracking of transplanted cells.
Abstract 1111: Embryoinc Stem Cell Derived Cardiovascular Progenitor Cells Improve Function More Than Hemangioblasts in a Mouse Model of Myocardial Infarction
Authors: Adler Eric , Chen Vincient , Bystrup Anne , Young Wilson , Giovannone Steve , Kattman Steve , Fayad Zahi , Keller Gordon
Published: July 2021

BACKGROUND: Intramyocardial transplantation of stem cells improves left ventricular ejection fraction (EF) in animal studies and preliminary clinical trials. The mechanism may involve either replacement of myocytes or improved vascular supply to existing myocytes. We recently identified an Embyronic Stem cell derived cardiovascular progenitor cell (ES-CPC) that is the common precursor of cardiomyocyte and vascular cell lineages. To determine whether myocyte transplantation improves myocardial function more than angiogenesis alone does, we compared the effect of ES-CPCs to hemangioblasts (vascular/hematopoetic progenitor cells) on EF in a mouse model of myocardial infarction.

METHODS: ES-CPC and hemangioblasts were isolated from a doxycycline-responsive, Notch-inducible ES cell line containing Notch 4 cDNA under the control of a tetracycline-inducible promoter. Notch induction of mesoderm-derived ES cells resulted in a CPC phenotype, whereas non-induced cells developed into hemangioblasts. Mice underwent transplantation of 500,000 ES-CPC (n=20), hemangioblasts (n=16), or an equal volume of serum-free media (n=12) 30 minutes after surgically-induced myocardial infarction. All cell lines constitutively expressed green fluorescent protein (GFP). EF was assessed two weeks post-transplantation using 9.4 Tesla MRI. Mice were then euthanized and frozen heart sections were examined using fluorescent microscopy.

RESULTS: The mean EF was 59Â ± 15, 46Â ± 17, and 39Â ± 13% in the ES-CPC, hemangioblast, and control groups, respectively (p<0.05 for the differences among all 3 groups; ANOVA). GFP + cells were detected in frozen sections of both the ES-CPC and hemangioblast groups. GFP + cells in ES-CPC treated hearts expressed markers associated with both cardiomyocyte and vascular phenotypes, whereas the GFP + cells in the hemangioblast group expressed markers associated with vascular phenotypes.

CONCLUSIONS: Both hemangioblast and ES-CPC transplantation improves EF in a mouse model of myocardial infarction, but ES-CPC transplantation was more effective. This suggests that enhancement of myocardial function by transplantation of both cardiomyocyte and vascular phenotypes exceeds that with vascular phenotypes alone.
Abstract 1117: Genetic Modification with Sonic Hedgehog Mitigates Aging-induced EPC Dysfunction and Augments Therapeutic Angiogenesis of Endogenous EPCs following Acute Myocardial Infarction
Authors: Ii Masaaki , Horii Miki , Akimaru Hiroshi , Kusano Kengo F , Thorne Tina , Asahara Takayuki , Losordo Douglas
Published: July 2021

Background: Clinical trials of autologous stem/progenitor cell transplantation in ischemic heart diseases are now on-going. Simultaneously, mounting evidence suggests that age related decreases in EPC number or function may limit this therapeutic approach. We tested the hypothesis that gene modification of aged EPCs, using the embryonic signaling molecule, sonic hedgehog (Shh), may ameliorate aging induced dysfunction of EPCs and thereby enhance the reparative effect of autologous EPCs.

Methods and Results: EPCs from aged mice (24 m.o.), previously shown to exhibit impaired potency, were used and compared to EPCs from young (3 m.o.) mice. Shh gene transfer significantly improved proliferation and anti-apoptosis activities vs. empty vector transfected (EV) aged EPCs, up to a similar level of EV young EPCs. Next, we evaluated the therapeutic effect of Shh-gene-modified aged EPCs (Shh-EPC) vs. EV aged EPCs (EV-EPC) vs. PBS (Ctrl) injection in a mouse AMI model. Shh-EPCs or EV-EPCs (1000 cells) or PBS was injected into the ischemic myocardium immediately following MI. Scar length in the infarcted LV 14 days after surgery was significantly reduced in the Shh-EPC group compared to the EV-EPC group and the PBS group associated with increased capillary density in the peri-infarct area. In addition, Ki67 positive cardiomyocytes were observed with increased frequency in the Shh-EPC group. In Tie2-LacZ BMT mice, beta-gal positive-endogenous BM-derived EPC recruitment was significantly greater in ischemic myocardium in the Shh-EPC group than that in the EV-EPC group. Differentiation of recruited EPCs into endothelial cells or cardiomyocytes was also observed more frequently in the Shh-EPC group compared to the EV-EPC group. These data suggest that cardiac repair was induced by a small number of transplanted Shh gene modified aged EPCs with the increased paracrine effects following MI.

Conclusion: Shh gene transfer to aged EPCs ameliorates aging-induced EPC dysfunction. Intramyocardial transplantation of Shh-gene-modified aged EPCs augments therapeutic efficacy for myocardial repair following MI by promoting endogenous EPC recruitment. Shh gene modified EPCs may represent a second-generation EPC-based therapeutic strategy in ischemic heart disease.
Abstract 1126: Adiponectin Prevents Neointimal Hyperplasia After Vascular Injury In Adiponectin Transgenic Mice
Authors: omori yasutoshi
Published: July 2021

Background: Adiponectin has been reported to accumulate in the human injured artery and regulate the development of atherosclerosis, decreasing the expression of adhesion molecules in vascular endothelial cells, and inhibiting the proliferation of vascular smooth muscle cells. However, the role of adiponection after vascular injury is not fully elucidated. Therefore, we investigate whether adiponectin prevents neointimal hyperplasia after vascular injury in adiponectin transgenic mice (TG).

Methods: C57/BL6 mice (WT) and TG of 6 –7 week age were used. We inserted a large wire (0.38 mm in diameter) into femoral artery from distal side to proximal side to make the vascular endothelium damaged model. Mice were sacrificed at 1, 2 and 4 weeks, and non injured mice were used as control. Tissue concentration of fat and liver and serum concentration of adiponectin was measured using ELISA. Intima/ media ratio was measured in morphometrical analysis. Immunohistochemical staining of anti-adiponectin, anti-α-smooth muscle embryo (α-SMemb), anti-α-smooth muscle cell actin, von Willebrand Factor (vWF) and intercellular adhesion molecule-1 (ICAM-1) was performed using paraffin embedded sections.

Result: In immunohistochemical analysis at 1 week, α-SMemb positive cells, which were also positive for ICAM-1 were observed in WT, but were not seen in TG. Adiponectin positive cells, which were also positive for vWF were observed in the endothelial cells in TG, but were not detected in WT at 1 week, but those cells were observed in th endothelial cells in TG and WT at 2 and 4 weeks. In α-SMA staining at 4 weeks most neointimal area was stained with this anti-body in TG. On the other hand, in neointima of WT, only some area was stained with this anti-body. In morphometrical analysis, intima/media ratio was significantly smaller in TG than in WT at 1, 2 and 4 weeks. Adiponectin concentration of fat, liver and serum was significantly higher in TG than in WT in non-injured model and at 4 weeks. At 1 week liver and fat adiponectin level is significantly higher in TG than in WT, and serum adiponectin level in TG tended to be high as compared to that of WT.

Conclusions: Adiponectin prevents neointimal hyperplasia after vascular injury, probably through the inhibition of inflammatory reaction.
Abstract 1182: Inflammation Impairs Reverse Cholesterol Transport
in vivo
Authors: McGillicuddy Fiona C , Hinkle Christine C , Joshi Michelle R , Chiquoine Elise H , Billheimer Jeffrey T , Rothblat George H , Rader Daniel J , Reilly Muredach P
Published: July 2021

Introduction
: Activation of innate immune responses have been postulated to impair reverse cholesterol transport (RCT). In this proof of concept study we provide the first
in vivo
functional evidence to support this hypothesis by tracking macrophage
3
H-cholesterol into plasma, liver, bile and feces in C57BL/6 mice during endotoxemia.

Methods:
C57BL/6 mice were injected subcutaneously with lipopolysaccharide (LPS) (10mg/kg daily for 2 days) or saline prior to intraperitoneal (IP) administration of
3
H-cholesterol-loaded macrophages.
3
H-cholesterol levels in plasma, liver, spleen, bile and feces were measured over 48 h. Lipid profiles were analyzed, enzymatically, using a Cobas FARA analyzer. Plasma (5 %), isolated from control or LPS treated mice (without macrophage injection), was used as an acceptor in
ex vivo
cholesterol efflux studies from
3
H-cholesterol-loaded J774 macrophages.

Results:
In a pilot non-RCT study (n = 4), as previously reported, LPS significantly increased total and HDL cholesterol, phospholipid and triglyceride levels (2.05 ± 0.09, 2.41 ± 0.28, 1.98 ± 0.08 and 2.57 ± 0.33 fold increase respectively, p < 0.01). In RCT studies, despite increased HDL cholesterol, LPS significantly decreased
3
H-cholesterol plasma counts at 4 h (−20.4 ± 2.0 %, p < 0.001) and 24 h (−27.1 ± 3.4 %, p < 0.001), as well as
3
H-cholesterol in liver, bile and feces (22.9 ± 3.2, 41.9 ± 10.7, and 75.3 ± 4.1 % decrease, p < 0.05, p = 0.05 and p < 0.01 respectively) (n = 8 –12 per group). LPS decreased hepatic SRB1, ABCG1, ABCG5 and HL mRNA expression.
Ex vivo
efflux to plasma isolated from LPS treated mice was significantly impaired relative to control (77.5 ± 7.4 % of control, p < 0.05, n = 5).

Conclusions:
Sub-acute endotoxemia impaired RCT in mice, despite increased plasma HDL cholesterol levels. This coincided with reduced hepatic expression of the HDL receptor, SRB1, and the transporters responsible for cholesterol transport to bile, ABCG5/8. In addition,
ex vivo
studies suggest impaired HDL particle efflux function during endotoxemia. In summary, we demonstrate for the first time
in vivo
that inflammation impairs several components of the reverse cholesterol transport pathway.
Abstract 1187: PDZ Domain Protein Arrays Define a Network of ABCA1 and SPLTC1 Protein-Protein Interactions that Regulate ABCA1 Trafficking and Efflux Activity
Authors: Fitzgerald Michael L , Tamehiro Norimasa , Zhou Suiping , Okuhira Keiichiro
Published: July 2021

Protein microarrays are an emerging high throughput proteomic tool that can be used to study protein-protein interactions. Here we use protein arrays that have 123 PDZ domains spotted in duplicate from 77 PDZ proteins testing over 700 potential interactions and mapping a network of protein interactions that encompasses the ABCA1 transporter and SPLTC1. SPTLC1, through its associated serine palmitoyltransferase activity, catalyzes the initial step in the synthesis of sphingomyelin whose plasma level has been associated with an increased risk of cardiovascular disease. Conversely, ABCA1 effluxes cellular cholesterol to maintain plasma HDL levels which prevents cardiovascular disease. Using hierarchical clustering and networking algorithms analysis of our data set shows PDZ proteins are able to discriminate between closely related PDZ binding motifs contained in the C-termini of ABCA1 and SPTLC1. Mutation of the SPTLC1 PDZ motif disrupted SPTLC1’s ability to bind PDZ proteins and destabilized SPTLC1 protein expression demonstrating the functional importance of the motif. Co-expression of SPTLC1 with ABCA1 in 293 cells inhibited cholesterol efflux activity of the transporter by up to 80% (p < 0.01) as we previously reported and here we show this is through a mechanism that blocks the exit of the transporter from the endoplasmic reticulum. In contrast, treatment of primary human fibroblasts or mouse macrophages with myriocin, a specific serine palmitoyltransferase inhibitor, increased efflux by nearly 60% (p < 0.05). Our results demonstrate the utility using protein arrays to map protein-protein interactions that we hypothesis form a regulatory network that integrates the cholesterol efflux and sphingolipid synthesis pathways. That SPTLC1 activity negatively regulates ABCA1 efflux further suggests inhibition of SPTLC1 activity may represent a new therapeutic target to prevent cardiovascular disease.
Abstract 1193: Mast Cell Activation Promotes Leukocyte Recruitment And Adhesion To The Atherosclerotic Plaque
Authors: Bot Ilze , de Jager Saskia C , Zernecke Alma , Weber Christian , van Berkel Theo J , Biessen Erik A
Published: July 2021

Activated mast cells have been identified in the perivascular tissue of human coronary artery plaques. As mast cells have been described to release a whole array of chemokines including interleukin 8 (IL-8) and MIP1 α, we propose that activated mast cells play a pivotal role in leukocyte recruitment at advanced stages of atherosclerotic plaque development. Peritoneal mast cells of either C57Bl/6 or mast cell deficient Kit(W
−sh
/W
−sh
) mice were activated by injection of compound 48/80 (1.2 mg/kg). Interestingly, mast cell activation led to a massive neutrophil influx into the peritoneal cavity at 3 hours after activation (controls: 1 ± 0.7*10
4
Gr1
+
-neutrophils/ml up to 8 ± 0.2*10
4
Gr1
+
neutrophils/ml at 3 hours after activation, *P<0.05), while neutrophil numbers in Kit(W
−sh
/W
−sh
) mice were not affected by compound 48/80 administration. Moreover, increased levels of CXCR2
+
Gr1
+
neutrophils (t=0: 0.55 ± 0.07% versus t=3 hours: 1.00 ± 0.12%, *P<0.05) were observed after mast cell activation. Next, we investigated whether mast cell activation also translated in induced leukocyte adhesion to advanced atherosclerotic plaques. Adventitial mast cells of advanced collar aided carotid artery plaques were activated by local application of a dinitrophenyl-BSA (DNP) challenge in ApoE
−/−
mice. Three days later, the carotid artery segments carrying the plaques were isolated and perfused ex vivo with rhodamine labeled leukocytes, showing a dramatically increased number of adherent leukocytes after mast cell activation (49 ± 6 versus 19 ± 4 leukocytes/microscopic field for DNP versus control plaques, respectively, **P<0.001). Strikingly, antibody blockade of either the CXCR2 or VCAM-1 receptor VLA-4 on labeled leukocytes completely inhibited leukocyte adhesion to the atherosclerotic plaque (*P<0.05), while blockade of CCR1, -3 and -5 with Met-RANTES had no effect. In conclusion, our data suggest that chemokines such as IL-8 released from activated perivascular mast cells induce leukocyte recruitment and adhesion to the atherosclerotic plaque, aggravating the ongoing inflammatory response and thus effecting plaque destabilization. We propose that mast cell stabilization could be a new therapeutic approach in the prevention of acute coronary syndromes.
Abstract 1205: Role for Nox1 NADPH Oxidase in Atherosclerosis
Authors: Sheehan Andrea L , Takapoo Maysam , Banfi Botond , Miller Francis J
Published: July 2021

NADPH oxidase-derived reactive oxygen species (ROS) contribute to the pathobiology of vascular disease. However, studies investigating NADPH oxidases in atherosclerosis have been limited in their ability to distinguish between the role of vascular cell and inflammatory cell -derived ROS. In this study, we examined the contribution of Nox1-derived ROS in a mouse model of atherosclerosis. Nox1 is a primary catalytic subunit of vascular cell, but not inflammatory cell, NADPH oxidase. At weaning, male apolipoprotein E deficient mice (AS, n=12) and male mice deficient in both apolipoprotein E and Nox1 (AS/Nox, n=16) received an atherogenic diet for 18 weeks. Mean blood pressures (116±3 vs. 110 ±3 mmHg; AS vs. AS/Nox, n=6), weights, and serum cholesterol levels (1578±150 vs. 1631±80 mg/dl; AS vs. AS/Nox) were similar between the AS and AS/Nox mice. As measured by lucigenin-enhanced chemiluminescence, superoxide levels were increased in segments of thoracic aorta from AS mice as compared to aorta from control mice (22±2 vs. 14±2 RLU/sec/mm
2
; AS vs. C57BL/6; p<0.05, n=6). In contrast, superoxide levels in segments of thoracic aorta from AS/Nox mice were significantly lower (9±1 RLU/sec/mm
2
, n=6) than both AS and C57BL/6 mice. Dihydroethidium staining confirmed decreased superoxide levels in aorta of AS/Nox mice. Atherosclerotic lesion area was measured by staining the aorta
en face
with Oil Red O. Although atherosclerotic lesion area was reduced over the entire length of aorta in AS/Nox mice as compared to AS mice (10±1% vs. 14±1%, p<0.01), the reduction in lesion was primarily limited to the aortic arch (28±3% vs. 43±2%, p<0.001). In summary, Nox1 contributes to generation of ROS and lesion formation in atherosclerosis. These data confirm a role for vascular cell NADPH oxidases, and in particular Nox1, in vascular disease.
Abstract 1216: The Cardioactive Peptide Apelin Increases Adipocyte Glucose Uptake and is Necessary for the Maintenance of Systemic Insulin Sensitivity
Authors: Yue Patrick , Asagami Tomoko , Kundu Ramendra K , Yee Yin-Gail , Glassford Alexander J , Azuma Junya , Quertermous Thomas , Tsao Philip S
Published: July 2021

Background
: Apelin, a peptide hormone with unique cardioactive properties, is also an adipokine, secreted by adipocytes in response to insulin. However, the overall effect of apelin on insulin sensitivity remains largely uncharacterized.

Methods
: For
in vitro
experiments, 3T3L1 cells were differentiated into adipocytes over 8 days, with apelin (1 microM) added daily to the media. Cells were then treated with insulin (100 nM; n = 5) for 30 minutes and incubated with 2-[
3
H]-deoxyglucose. Glucose incorporation was then measured by scintillation counting. For
in vivo
experiments (n = 4 all studies), apelin-deficient (KO) mice were created by homologous recombination in embryonic stem cells. At age 7 weeks, insulin and glucose tolerance tests, as well as an enzyme immunosorbent assay for insulin, were performed after a 6-hour fast. The mice were then scanned by computed tomography using a GE eXplore RS MicroCT system, and visceral adipose content was determined with MicroView software. Upon sacrifice 1 week later, visceral adipocytes were isolated via collagenase digestion, exposed to insulin, and assessed for glucose uptake as above.

Results
: Because apelin is upregulated by insulin in adipocytes, we measured glucose uptake in differentiated 3T3L1 cells chronically dosed with apelin. Though no differences were observed in basal uptake, insulin-induced uptake was increased versus control (p < 0.05). To further investigate the role of apelin
in vivo
, we assessed for insulin resistance in apelin KO mice. At 8 weeks of age, apelin KOs were heavier than age-matched wild type controls (25 vs. 22 g; p < 0.05). Though fasting glucose levels were not significantly different between groups, insulin levels were increased in the KOs (895 vs. 477 pg/microL; p < 0.05). In addition, both insulin and glucose tolerance tests were significantly abnormal in the KOs compared to wild type. Moreover, visceral fat volume was greater in the KOs (274 vs. 248 mm
3
/g body weight; p < 0.05). Finally, insulin-stimulated uptake was reduced (p < 0.05).

Conclusions
: Apelin is necessary for the proper maintenance of glucose homeostasis. Furthermore, apelin potentiates insulin-induced glucose uptake in adipocytes, suggesting a possible mechanism for its insulin sensitizing effects.
Abstract 1226: Augmentation Of Cardiac Function In Response To Pressure Overload In Transgenic Mice With A Cardiac-Specific Expression Of Adenylyl Cyclase Type 2
Authors: Hu Che-Lin , Gao Shumin , Hong Chull , Liu Huasheng , Tian Yimin , Reinhardt Heather , Ishikawa Yoshihiro , Vatner Stephen F , Vatner Dorothy E
Published: July 2021

The results from studies examining the effects of transgenic (TG) mice with overexpression of the major adenylyl cyclase (AC) isoforms in the heart, AC5 and 6, has been controversial. Accordingly, we examined the effects of a non-calcium regulated AC isoform, AC2, by overexpressing it in the heart. This resulted in a 3 fold increase in overall cardiac AC activity. Baseline left ventricular ejection fraction (LVEF), measured by echocardiography, was higher (P<0.05) in TG mice when compared to WT (75±1% vs 72±1%) while heart rate was similar (P<0.05). The extent of increased LVEF stimulated by isoproterenol (ISO) was also higher in TG suggesting an increase in functional response of beta-AR signaling in these transgenic mice. Aortic banding for 1 wk induced significantly greater (P<0.05) LV hypertrophy in TG compared to WT as indicated by LV weight (LVW)/tibial length (TL) ratio (7.1±0.4 vs 6.0±0.3 mg/mm, P<0.05). Aortic banding resulted in no change of LVEF in the AC2TG (76±3%) or in WT (70±3%), i.e., LV function was maintained. Pathological examination of the 1wk banded hearts demonstrated fewer TUNEL positive myocytes in the AC2TG (0.16±0.08 cells/microm2) vs WT (1.00±0013 cell/microm2) (P<0.05). Cross-sectional myocyte area in AC2TG was also larger (316±16microm2) than WT (289±20microm2) which confirmed the increased in LV/TL. Thus, a moderate level of AC2 overexpression in the heart increases the amount of cardiac hypertrophy, in response to pressure overload, but exerts a beneficial effect on apoptosis and LV function.