bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1716: Calcium Channel Blockers Reduce the Antiplatelet Effect of Clopidogrel
Authors: Siller-Matula Jolanta , Lang Irene , Christ Guenter , Jilma Bernd
Published: July 2021

Due to the known CYP3A4 inhibition by calcium channel blockers, we hypothesized that there might be a pharmacodynamic interaction between clopidogrel and dihydropyridines in patients with coronary artery disease (CAD). Clopidogrel is activated by CYP3A4 which also metabolizes calcium channel blockers of the dihydropyridine class.

Methods: Responsiveness to clopidogrel was assessed by the vasodilator stimulated phosphoprotein (VASP) phosphorylation assay and aggregometry in 200 patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). The platelet reactivity index (PRI in the VASP assay, normal range 69–100%) was higher in patients on both clopidogrel and calcium channel blockers (61%) as compared to patients on clopidogrel without calcium channel blockers (48%). The absolute difference was 13% (95%CI: 6 –20%; p = 0.001) and the relative difference approached 21%. A high post-treatment platelet reactivity (PRI > 69%) was seen in 40% of patients with concomitant calcium channel blocker treatment compared to 20% without (X2-test: p = 0.008). Intake of calcium channel blocker remained an independent predictor of high platelet reactivity after adjustment for cardiovascular risk factors. This corresponded to an increased platelet aggregation of similar magnitude (p < 0.05). In vitro incubation with calcium channel blockers (nimodipine, verapamil, amlodipine and diltiazem) did not alter the PRI or the ADP-induced platelet aggregation of patients on clopidogrel, indicating that the interaction occurs in vivo, conceivably at the level of the CYP3A4 cytochrome. Co-administration of calcium channel blockers is associated with a decreased platelet inhibition by clopidogrel.
Abstract 1719: A Potential Mechanism for the Reduced Efficacy of Aspirin in Individuals with Diabetes
Authors: Ajjan Ramzi , Standeven Kristina F , Mughal Salihah , Phoenix Fladia A , Ariens Robert A , Grant Peter J
Published: July 2021

The protective effects of aspirin are reduced in individuals with diabetes by mechanisms that remain unclear. Diabetes is associated with changes in clot structure making it more resistant to lysis, which may be due to increased glycation of the fibrinogen molecule. In contrast, we have recently found that acetylation of fibrinogen enhances fibrinolysis of the fibrin clot. The aim of this study was to assess whether the reduced efficacy of aspirin in diabetes is due to an interaction between acetylation and glycation in fibrinogen and clotting factors.Type 1 diabetes subjects with poor diabetes control (n = 18) were recruited and insulin treatment was intensified. Blood samples were obtained before and after improving glycaemic control over a period of 7.3 months (CI 5.5, 9.2). Plasma samples were incubated with 0, 1, 10 and 100 mg L-1 aspirin for 5 h at 37°C, subsequently treated with thrombin and tissue plasminogen activator, and time to 50% lysis was assessed using turbidity measurements. Electron microscopy was used to study the structure of clots made from plasma-purified fibrinoge.HbA1c was reduced significantly post-intervention [10.52% (9.56, 11.48)
vs
. 9.23% (8.31, 10.15)]. In the absence of aspirin, improvement in glycaemic control significantly reduced lysis time [357 (312, 402) and 323 (278, 368) seconds at baseline and after intervention respectively; p < 0.05]. Low dose aspirin (1 mg L
−1
and 10 mg L
−1
) had an adverse effect on lysis time in the presence of poor diabetes control [367 (323, 413) and 385 (332, 437) seconds respectively, p < 0.05], whereas high dose aspirin (100 mg L
−1
) had a beneficial effect on lysis time only in individuals with improved glycaemic control [299 (264, 333) seconds, p < 0.05]. Experiments on plasma-purified fibrinogen showed the formation of thicker fibres and bigger pores with improved glycaemic control and after treatment with high dose aspirin. This study has shown that improvement in glycaemic control has a beneficial effect on fibrinolysis. Our work has also demonstrated that aspirin treatment can adversely affects lysis time in the presence of poor glycaemic control, which may explain the reduced efficacy of this agent in individuals with diabetes.
Abstract 1728: The Defect in HDL Function and Reverse Cholesterol Transport Found in Diabetic Mice with the Haptoglobin 2–2 Genotype Can Be Corrected with High Dose Antioxidant Therapy
Authors: Asleh Rabea O , Miller-Lotan Rachel , Abassi Zaid , Levy Andrew P
Published: July 2021

Background.
The primary function of the haptoglobin (Hp) protein is to clear free hemoglobin (Hb). Two common alleles exist at the Hp locus (1 and 2). We recently demonstrated that reverse cholesterol transport is impaired in individuals with Diabetes Mellitus (DM) and the Hp 2–2 genotype which may explain the increased incidence of cardiovascular disease in this population. We sought to

test the hypothesis that clearance of the Hp 2-Hb complex is slower in DM allowing more complex to bind to HDL thereby resulting in increased oxidative modification of HDL and inhibition of reverse cholesterol transport and

determine if antioxidant therapy could restore normal HDL function in Hp 2–2 DM mice.

Methods and Results.
Injection of
125
I-labeled Hp 1 or Hp 2-Hb complexes into non-DM mice demonstrated that the half-life of the Hp 2-Hb complex was 2–3 fold longer than the Hp 1-Hb complex (57.8 ± 2.8 vs. 20.4 ± 1.7 min). Moreover, in DM the half-life of the Hp 2-Hb complex was doubled while the half-life of the Hp 1-Hb complex was unchanged (103 ± 3.9 vs. 18.6 ± 1.8 min). Coimmunoprecipitation studies demonstrated that over 25% of the injected Hp 2-Hb complex was associated with HDL in DM mice representing a greater than 10 fold increase compared to Hp 1-Hb complex in non-DM mice. Coimmunoprecipitation studies in Hp 0 (knockout) mice demonstrated that the Hp protein was absolutely necessary for the interaction of Hb with HDL. Reverse cholesterol transport was impaired by DM in Hp 2 mice but this impairment was prevented by high dose antioxidant supplementation to these mice.

Conclusions.
These data may explain why the Hp 2 genotype promotes less efficient reverse cholesterol transport in DM and suggests that strategies targeted to decrease oxidation of HDL by the Hp 2-Hb complex may improve HDL function.
Abstract 1735: Targeted Application of Interleukin-2 Activates T-Lymphocytes and Reduces Plaque Formation
Authors: Graf Kristof , Dietrich Thore , Schneemann Christiane , Atrott Kirstin , Stawowy Philipp , Menssen Dietrich , Willuda Jörg , Fleck Eckart
Published: July 2021

A newly developed fusion protein (L19-IL2) consisting of an anti-ED-B fibronectin single chain antibody and the cytokine interleukin-2 (IL2), is a potent antitumor agent for renal cell cancer. ED-B fibronectin (ED-B) is expressed in inflammatory plaque lesions. Preliminary studies suggested that delivering IL2 locally to atherosclerotic tissue seem to reduce plaque progression. Therefore we investigated the application of L19-Il2 on lesion formation in apoE-deficient mice (apoE
−/−
). 6-month old apoE
−/−
, fed with high fat diet (n = 5/group) were injected intravenously L19-IL2 (4290IU/g bodyweight), with L19 (antibody alone), with the control fusion protein D1-IL2 (4290IU/g bodyweight) or NaCL (control) at day 1, 3 and 5 and were sacrificed at day 7. Plaque lesion formation was analyzed by histology, immunohisto-chemistry (Mac3 = macrophages, CD31; actin, human IL2, ED-B, CD25, CD4), morphometry of the aortic root and by Sudan stain of the thoracic aorta followed by densitometry. Macrophage, ED-B, CD4, CD25 (IL-2 receptor subunit) content was analysed after immunohistochemistry.

Results:
Treatment with L19-IL2, L19, and D1-IL2 did not affect bodyweight, survival of mice compared to control (NaCL). Serum levels for blood glucose, cholesterol, liver and cardiac enzymes, and troponin were not different between L19-IL2, L19, and NaCL. Cardiac histology was also unaffected. Human IL2 was only detectable in plaque lesions after L19-IL2, but not after L19 or D1-IL2 treatment. L19-IL2 application significantly reduced plaque lesion size in the aortic root (L19-Il2: 23.2 ± 1.7%, L19: 35.2 ± 1.2%, D1-IL2: 31.6 ± 3.0%, NaCL: 31.0 ± 3.2%, p < 0.01), plaque extension in the thoracic aorta (L19-Il2: 5.3 ± 0.7%, L19: 8.2 ± 0.4%, D1-IL2: 9.8 ± 1.0%, NaCL: 8.9 ± 1.2%, p < 0.01). Mac3 immunoreactivity was reduced under L19-IL2, whereas CD4 and CD25 were strongly increased compared to L19, D1-IL2 and NaCL treated groups.

Conclusion:
A fusion protein, which delivers human IL-2 into plaque lesions, induces T-lymphocyte activation, reduction of macrophages and a reduction in plaque growth. The present study suggests that local application of IL2 activates cellular mechanisms involving T-lymphocytes leading to a reduction of atherosclerotic disease.
Abstract 1741: Bone Marrow Mononuclear Cell Therapy upon Reperfusion Reduces Myocardial Infarct Size by Activating the PI3K/Akt Survival Pathway
Authors: Yasin Mohammed , Shintani Yasunori , Collino Massimo , Takahashi Kunihiko , Salem Husein , Kapoor Amar , Rucklidge Garry , Suzuki Ken , Thiemermann Christoph
Published: July 2021

Cardioprotective molecular mechanisms underlying adult bone marrow progenitor cell therapy have not been fully elucidated. We hypothesized that intravenous bone marrow mononuclear cells (BMMNC) might be:

protective when administered upon reperfusion

protective by paracrine mechanisms.

Freshly isolated 10 million BMMNC,
c-Kit
+
(7 ± 1%, n =10),
CD34
+
(7 ± 1%, n =10),
CD45
+
(54 ± 6 %, n = 10), and
CD133
+
(15 ± 1%, n = 10), were intravenously administered upon reperfusion in a rat model of 25 minutes left anterior descending coronary artery occlusion followed by reperfusion lasting either 2 hours (I/R2h) or 7 days (I/R7D). At the end of I/R2h, BMMNC caused significant reductions in infarct size (BMMNC 33 ± 3 %; control 57 ± 2 %, n = 10, P < 0.01). Following I/R7D, BMMNC caused significant improvements in systolic function (LVEF: BMMNC 71 ± 3 %; control 48 ± 4 %, n = 11, P < 0.0001) and diastolic function (LVEDP: BMMNC 5 ± 2 mmHg; control 15 ± 2 mmHg, n = 9, P < 0.01). Comparable cardioprotection was achieved when BMMNC supernatant was intravenously administered instead of BMMNC. Cardioprotection by BMMNC was associated with Akt phosphorylation, which could be abolished by inhibition of PI3K with LY294002. When compared with controls, proteomics of hearts subjected to I/R2h and treated with BMMNC demonstrated higher expression of

the anti-oxidants catalase (3.3 fold), and peroxiredoxin 6 (2.0 fold);

heat shock proteins HSP20 (2.9 fold), alpha B crystallin (1.7 fold), HSP72 (2.8 fold), ischemia responsive protein-94 (1.6 fold), and TNF receptor-associated protein 1 (2.3 fold);

glycolytic enzymes alpha-enolase (1.6 fold), glyceraldehyde-3-phosphate dehydrogenase (2 fold), and pyruvate kinase (2 fold); and

mitochondrial respiratory proteins, aconitase (4.8 fold), ATPase synthase (3.5 fold), citrate synthase (2.5 fold), and voltage-dependent anion-selective channel protein 1 (3.4 fold).

In conclusion, intravenous administration of BMMNC upon reperfusion reduced infarct size by activation of the PI3K/Akt survival pathway. Further survival benefits were also demonstrated by up-regulated expression of recognised cardioprotective anti-oxidants, heat shock proteins, and augmented glycolytic mitochondrial bioenergetics.
Abstract 1746: Insulin Like Growth Factor Binding Protein-3 (IGFBP-3) Mediates Vascular Repair By HDL Receptor Activation And Sphingosine Kinase (SK-1)/Sphingosine 1-Phosphate (S1P) Dependent Nitric Oxide (NO) Generation
Authors: Calzi Sergio Li , Kielczewski Jennifer L , McFarland Evan , Chang Kyung Hee , Afzal Aqeela , Shaw Lynn C , Jarajapu Yagna , Chan-Ling Tailoi , Grant Maria B
Published: July 2021

Purpose:
We demonstrated that IGFBP-3 stimulates hematopoietic stem cells (HSC) to differentiate into endothelial cells, form capillaries, and stabilize the vasculature (Chang, et al, PNAS 2007). Local IGFBP- 3 production is increased by hypoxia and facilitates the homing of HSC to areas of injury. In the circulation, IGFBP-3 is bound to HDL. In this study, we investigated the signaling pathways responsible for the robust migratory effects of IGFBP-3.

Methods:
The effects of IGFBP-3 on NO generation in human vascular precursors (CD 34
+
, CD14
−
), human lung microvascular endothelial cells, and human umbilical vein endothelial cells were examined using DAF-FM fluorescence. Western analysis was use for detection of eNOS and vasodilator-stimulated phosphoprotein (VASP), which redistributes to lamellipodia forming an active motor complex that supports motility and is phosphorylated in response to NO. Localization of VASP was performed by immunohistochemistry. SK-1 was assessed following IGFBP-3 stimulation.

Results:
In CD34
+
cells and endothelial cells, IGFBP-3 stimulated eNOS phosphorylation at Ser1177 (102 ± 1.8%, P = 0.0002) and increased NO generation (275 ± 50%, P = 0.05) by increasing SK-1 and S1P generation. IGFBP-3 was bound and internalized by the HDL receptor, scavenger receptor 1B (SR1B). NO generation following IGFBP-3 exposure was reduced by SK inhibitors or SR-1B blocking antibody pretreatment (35 ± 5%, P < 0.02). IGFBP-3 generated NO increased phosphorylation of VASP at Ser239 and promoted the redistribution of VASP to lamellipodia.

Conclusions:
IGFBP-3 effects on cell migration are NO dependent and mediated in part by activation of the HDL receptor SR1B suggesting that some of the beneficial effects of HDL are mediated by the association of IGFBP-3.
Abstract 1770: Proteasome Switching is Associated with Adverse Cardiac Remodeling in a Transgenic Mouse Model of Sustained Inflammatory Signaling
Authors: Wang Wei , Szalai Gabor , Sivasubramanian Natarajan , Mann Douglas L
Published: July 2021

The 26S proteasome possess proteolytic activity and deubiquitinating (DUB) activity of ubiquitin tagged proteins. Whereas the proteolytic activity of the 26S proteasome facilitates protein degradation, proteasome DUB activity spares proteins from degradation by shortening the length of the ubiquitin chains, thereby preventing proteins from being degraded by the 26S proteasome. In yeast, increased DUB activity is beneficial by preventing depletion of ubiquitin pools critical for cell signaling. However, the role of DUB activity in mammalian systems is not known. We have shown that mice with cardiac restricted overexpression of tumor necrosis factor (sTNF mice) develop a heart failure phenotype characterized by progressive left ventricular (LV) remodeling and accumulation of pro-apoptotic proteins, including Smac/Diablo. To determine whether the adverse LV remodeling in sTNF mice was related to alterations in DUB activity we measured the cleavage of ubiquitin-AMC, an in vitro fluorescent substrate for DUBs, in purified preparations of the 26S proteasome obtained from hearts of 4 week old sTNF and littermate (LM) control mice. Compared to LM controls we observed a significant (p < 0.001) 60.8% decrease in activity of the 26S proteasome and a significant (p < 0.01) 24.2% increase in DUB activity in sTNF mouse hearts. There was also a significant (p < 0.01) 11-fold increase myocardial protein levels of USP14, a critical DUB associated with the 26S proteasome in sTNF mouse hearts. The decrease in 26S proteasome activity and increased DUB activity in sTNF mouse hearts was accompanied by an increase in myocardial levels of ubiquitinated SMAC/Diablo. Taken together these results show for the first time that sustained myocardial inflammation leads to switch in the function of the proteasome from a proteolytic function to a protein sparing function. Although this “proteasome switching” may provide a short-term adaptive benefit by preventing depletion of critical ubiquitin pools, it may lead to long-term maladaptive consequences by allowing the progressive accumulation of potentially harmful pro-apoptotic proteins in the cytosol, which may in turn promote programmed cell death and adverse cardiac remodeling.
Abstract 1805: Chromosome 9p21.3 Genetic Variation is Associated with Angiographic CAD but not with CAD Disease Burden
Authors: Mower Chrissa P , Anderson Jeffrey L , Horne Benjamin D , Park James J , Coleman Jesse L , May Heidi T , Jahromi Mona S , Carlquist John F
Published: July 2021

Genetic variation at the 9p21.3 locus rs2383206 is associated with coronary heart disease (CHD) phenotypes. In a comparison of patients with and without angiographically confirmed CHD, the G allele of rs2383206 was present more frequently in diseased vs controls (normal angiograms). However, the pathophysiologic impact, whether it affects initiation, severity, or triggers an event, of the 9p21.3 locus remains unknown. We sought to determine whether 9p21.3 variation affects disease severity (promotion) by assessing its association with CAD burden.

Methods: Genotyping for rs2383206 using 5′exonuclease chemistry (Taqman) was performed on 1759 subjects. Subjects were grouped as homozygous wild-type (low risk), heterozygous (intermediate risk) or homozygous risk-associated genotype (high risk). Disease burden was assessed by 1, 2, or 3 vessels; ≥70% stenosis and the validated Duke CAD Index (DCI). Comparison used a chi-square test (single vs multivessel disease) and analysis of variance (ANOVA) for the DCI comparison.

Results: Average age 51.1± 7.4 years, 64.0% male. Frequency of the CAD risk allele did not differ among groups with 1, 2, or 3 vessel disease. There was no difference among groups with respect to the DCI. After adjustment for standard cardiac risk factors, the rs2383206 genotype was associated with CAD compared to controls (OR (CI)=1.73(1.26–1.85), p=0.001).

Conclusion: The rs2383206 polymorphism was not associated with CAD disease burden. Findings suggest the rs2383206 polymorphism, although associated with disease onset is not likely involved in its progression. These findings will aid in refining the application of 9p21.3 for risk assessment and developing novel preventive and therapeutic strategies.
Abstract 1828: Dyxin/Lmcd1 Mediates Cardiac Hypertrophy Both
In Vitro
And
In Vivo
Authors: Frank Derk , Frauen Robert , Hanselmann Christiane , Kuhn Christian , Will Rainer , Katus Hugo A , Frey Norbert
Published: July 2021

In order to identify new molecular mediators of cardiomyocyte hypertrophy, we performed a genome wide mRNA microarray screen of biomechanically stretched neonatal rat cardiomyocytes (NRCM). We found the novel sarcomeric LIM protein Dyxin/Lmcd1 being significantly upregulated (5.6x, p<0.001). Moreover, Dyxin was also significantly induced in several mouse models of myocardial hypertrophy including aortic banding, calcineurin overexpression and angiotensin stimulation, suggesting a potential role as a mediator of cardiac hypertrophy. To further test this hypothesis, we adenovirally overexpressed Dyxin in NRCM which potently induced cellular hypertrophy (150%, p<0.001) and the hypertrophic gene program (ANF, BNP). Consistent with an induction of calcineurin signalling, the calcineurin-responsive gene Rcan1– 4 (MCIP1.4) was found significantly upregulated (3.2x, p<0.001). Conversely, knockdown of Dyxin (−75% on protein level) via miRNA completely blunted the hypertrophic response to hypertrophic stimuli, including stretch and PE (both p<0.001). Furthermore, PE-mediated activation of calcineurin signaling (Upregulation of Rcan1– 4 by 7.3x, p<0.001) was completely blocked by knockdown of Dyxin. To confirm these results in vivo, we next generated transgenic mice with cardiac-restricted overexpression of Dyxin using the
α
-MHC promoter. Despite normal cardiac function as assessed by echocardiography, adult transgenic mice displayed significant cardiac hypertrophy in morphometrical analyses (3.9 vs. 3.5 mg/g LV/heart weight, n=8–11, p<0.05). This finding was supplemented by a robust induction of the hypertrophic gene program including ANF (3.7-fold, n=6, p=0.01) and
α
-skeletal actin (2.8-fold, n=6, p<0.05). Likewise, Rcan1– 4 was found upregulated (+112%, n=5, p<0.05), Taken together, we show that the novel sarcomeric z-disc protein Dyxin/Lmcd1 is significantly upregulated in several models of cardiac hypertrophy and potently induces cardiomyocyte hypertrophy both in vitro and in vivo. Mechanistically, Lmcd1/Dyxin appears to signal through the calcineurin pathway.
Abstract 1845: Adult Cardiac Progenitor Cells Promote Angiogenesis and Cardioprotection through Their Secreted Soluble Vcam-1
Authors: Matsuura Katsuhisa , Honda Atsushi , Nagai Toshio , Fukushima Noritoshi , Shimizu Tatsuya , Okano Teruo , Komuro Issei , Hagiwara Nobuhisa
Published: July 2021

Although cardiac progenitor cells have been thought to be the promising source of cell therapy, the precise mechanisms of their paracrine action have not been fully elucidated. Since we observed that the transplantation of clonal expanded Sca-1 positive cardiac progenitor cells (cSca-1 cells) derived from adult murine heart by using cell sheet technique improved cardiac function of infarcted heart compared to adipose tissue derived mesenchymal cells (ATMC), we explored the secreted factors highly expressed in cSca-1 cells and identified that soluble VCAM-1 (sVCAM-1) was much abundant in cSca-1 cells compared to ATMC by using cytokine antibody array. cSca-1 cells-derived conditioned medium (CM) significantly enhanced endothelial migration and matrigel tube formation and these effects were abolished by knock down of VCAM-1 (Fold increase: control, 1.0; CM, 2.97 ± 0.21; siVCAM-1, 1.98 ± 0.09; siControl, 2.76 ± 0.05, p<0.01), suggesting that cSca-1 cells promote angiogenesis via their secreted sVCAM-1. We next examined whether sVCAM-1 conferred direct protective effects on cardiomyocytes. We exposed cardiomyocytes to 0.2 mM H
2
O
2
in the absence or presence of sVCAM-1 or CM and examined cardiomyocyte viability by MTT assay. The exposure of cardiomyocytes to H
2
O
2
significantly induced the cell injury. Interestingly when pretreated with sVCAM-1 or CM, the cell damages of cardiomyocytes by H
2
O
2
were significantly reduced. However when pretreated with anti-VLA4 antibody, a principal coreceptor of sVCAM-1, CM mediated cell protected effect was completely inhibited (Fold increase: control, 1.0; anti-VLA4, 0.89 ± 0.33; sVCAM-1, 1.69 ± 0.27; CM, 2.08 ± 0.28; CM+anti-VLA4, 1.07 ± 0.07, p<0.01), suggesting that a crucial role of the VLA4 in inducing survival of cardiomyocytes by CM. sVCAM-1 and CM induced phosphorylation of FAK, Akt, Erk and p38 MAPK in neonatal rat cardiomyocytes. When pretreated with wortmannin, SB203580 and PD98059, the cardioprotective effects of sVCAM-1 and CM significantly inhibited, suggesting that sVCAM-1 might protect cardiomyocytes from oxidative stress via integrated upregulation of Akt, Erk and p38MAPK. These findings suggest cardiac progenitor cells promote angiogenesis and cardioprotection through their secreted sVCAM-1.
Abstract 1900: Preconditioning in Cynomolgus Monkey Heart Protects Against Both Ischemic and Reperfusion Injury
Authors: Yang Xi-Ming , Liu Yanping , Liu Yongge , Tandon Narendra , Kambayashi Junichi , Downey James M , Cohen Michael V
Published: July 2021

Ischemic preconditioning (IPC) and postconditioning (IPoC) are equally cardioprotective in some species tested but IPoC has been less protective in others. Although IPoC decreases infarct size in humans, no comparable data exist for IPC making a comparison impossible. We therefore compared IPoC and IPC in sub-human primate hearts which would be expected to most closely mimic that of humans. Cynomolgus monkeys underwent a 90-min occlusion of a coronary artery and 4h of reperfusion. In IPC 2 cycles of 10-min coronary occlusion/10-min reperfusion preceded the 90-min occlusion, and in IPoC 6 cycles of 30-sec reperfusion/30-sec coronary reocclusion were performed immediately after the 90-min occlusion. The regression line for the infarct-risk zone plot was very linear (r=0.99) and intersected the risk zone axis at 0.82 cm
3
. Collateral flow averaged only 6.6% of preocclusion flow and did not influence infarction. In control monkeys 44% of the risk zone infarcted indicating cynomolgus myocardium is much more resistant to infarction than that of rabbits or rats. While IPC reduced infarct size to near zero (2.4±0.9% infarction of risk zone), IPoC was much less protective (27.5±4.8% infarction of risk zone). Increasing the number of IPoC cycles to 10 did not increase salvage. When wortmannin was administered at the end of the ischemic period to block any protection that IPC might elicit during reperfusion, infarct size increased to only 28%. These findings suggest that only half of IPC’s protection in monkey heart derives from prevention of cell death during reperfusion. The other half of IPC’s salvage would have derived from suppression of cell death during the prolonged ischemic period. If human heart is similar to cynomolgus heart, then interventions applied at reperfusion such as IPoC may be much less effective than would be predicted from the non-primate animal studies. Effective clinical salvage may require cardioprotective interventions that target both the ischemic phase as well as reperfusion injury.
Abstract 1915: Simvastatin Regulates Insulin Signaling in Endothelial Cells via Activation of AMP-Activated Protein Kinase
Authors: Levine Yehoshua C , Michel Thomas
Published: July 2021

Statins exert pleiotropic effects upon the cardiovascular system, but the role of statins in modulation of insulin signaling remains incompletely defined. In these studies, we explore the effects of simvastatin on insulin pathways in bovine aortic endothelial cells (BAEC), and propose a novel mechanism linking these pathways that involves AMP-activated protein kinase (AMPK). Treatment of BAEC with 100 nM insulin promotes the rapid phosphorylation of Akt (3.8±0.2-fold increase; n=4; p<0.01), glycogen synthase kinase-3β (GSK3β, 2.7±0.5-fold increase; n=4; p<0.01), as well as that of critical substrates in the mammalian target of rapamycin (mTOR) axis including mTOR (2.8±0.2-fold increase; n=4; p<0.02) and p70S6K (3.2±0.5-fold increase; n=4; p<0.02). However, in cells pretreated with simvastatin (10 μM for 24 hr), these insulin-dependent phosphorylations are significantly attenuated (62±10%, 48±12%, 53±9%, and 58±13% reduction in the phosphorylation of Akt, GSK3β, mTOR, and p70S6K, respectively; p<0.02; n=4). Given our findings on the modulation of these key metabolic enzymes by simvastatin, we explored the effects of insulin and simvastatin on AMPK. We found that insulin promotes a rapid (within minutes) and robust increase in phosphorylation of both AMPK and its substrate acetyl CoA carboxylase (2.5±0.4-fold increase; n=4; p<0.01). Importantly, transfection of BAEC with siRNA targeting AMPK results in enhanced insulin-dependent phosphorylation of Akt (57±9% enhancement compared to cells transfected with control siRNA; n=4; p<0.015), as well as significant increases in phosphorylation of GSK3β, mTOR, and p70S6K (55±12% enhancement; n=4; p<0.02), indicating a key regulatory role for AMPK in insulin-dependent metabolic signaling. Importantly, in cells transfected with AMPK siRNA, simvastatin treatment no longer attenuates insulin signaling to these substrates, suggesting that AMPK is required for the regulation of insulin pathways by statins. Taken together, these results establish that simvastatin modulates critical insulin responses in endothelial cells, and provide new insights into the roles of AMPK in linking insulin- and statin-regulated metabolic signaling pathways in the vascular wall.
Abstract 1918 P122 Protein, a Positive Regulator for Phospholipase C-σ1, Is Upregulated and Involved in Enhanced Calcium Signaling in Human Coronary Spasm
Authors: Murakami Reiichi , Osanai Tomohiro , Tomita Hirofumi , Okumura Ken
Published: July 2021

We previously showed that the activity of phospholipase C (PLC)-σ1, a key enzyme for Ca
2+
signaling in the coronary artery smooth muscle, was enhanced threefold in patients with coronary spastic angina (CSA) compared with control subjects. Structural mutation of PLC-σ1 (864G-A) variant with the amino acid replacement of arginine 257 by histidine is one mechanism for the enhanced PLC-σ1 activity, but this was observed in only 10% of CSA patients. PLC-σ1 was shown to be positively regulated by p122 protein. We examined the possible role of p122 protein in the mechanism for enhanced PLC-σ1 activity. In 11 patients with CSA and 9 control subjects without CSA, skin fibroblasts were obtained at the coronary angiography and were cultured. Protein and gene expressions of p122 were determined by Western blot analysis and real-time quantitative RT-PCR, respectively. The protein expression of p122 was enhanced in CSA threefold compared with control subjects (237±17 vs 85±13 units, p<0.0001). The gene expression of p122 was also enhanced in CSA by 36.1±8.7% compared with control (p<0.01). We further examined whether the upregulated p122 protein is associated with the enhancement of intracellular Ca
2+
signaling. Human embryonic kidney cells (HEK293) were cultured and transfected by muscarine M1 receptor. In the cells expressing normal PLC-σ1, acethylcholine (ACh) at 10
−6
and 10
−5
mol/L caused a dose-dependent, rapid transient increase in [Ca
2+
]
i
followed by a lower but sustained phase of the increase. We further transfected the HEK293 cells by p122, which resulted in the increased expression of p122 protein two-to threefold. [Ca
2+
]
i
at baseline was 23±1 nmol/L in the cells without p122 transfection and 39±2 nmol/L in those with p122 (P<0.01). The peak increase in [Ca
2+
]
i
from the baseline after ACh was significantly greater in the cells transfected with p122 than in those without transfection (68±6 versus 33±4 nmol/L at 10
−6
mol/L Ach, and 128±11 versus 67±8 nmol/L at 10
−5
mol/L ACh, both P<0.01). The sustained phase of [Ca
2+
]
i
increase was prolonged in the cells with p122 transfection compared with those without transfection. In conclusion, the enhanced p122 protein expression is involved in the pathogenesis of CSA by enhancing intracellular Ca
2+
signaling.
Abstract 1921: Clinical Impact of Heterozygous Carrier of Autosomal Recessive Hypercholesterolemia on Asymptomatic Hyperlipidemic Patients: Evidence From Familial Gene Analysis
Authors: Tada Hayato , Kawashiri Masa-aki , Noguchi Tohru , Nakanishi Chiaki , Tsuchida Masayuki , Takata Mutsuko , Nohara Atsushi , Inazu Akihiro , Kobayashi Junji , Mabuchi Hiroshi , Yamagishi Masakazu
Published: July 2021

Autosomal recessive hypercholesterolemia (ARH) is an extremely rare genetic defect, and is recognized only around 50 cases in all over the world. The genetic cause of ARH is mutation in an adaptor protein involved in low-density lipoprotein (LDL) receptor internalization, which results in marked elevation of serum LDL cholesterol (LDL-C) and premature atherosclerosis. Therefore, few data exist regarding the clinical significance about the heterozygous carrier of ARH. We recently identified the second ARH patient (Ins C
599
) in Japan and his some relatives who have the same single mutation (heterozygous). We identified 11 heterozygous ARH carriers (male=5, mean age=48.2) and 7 non-carriers (male=3, mean age=53.3) in the same family. In addition, we screened the same mutations in unrelated consecutive 500 hyperlipidemic patients (male=32, mean age=49.4) with mean LDL-C of 225.2±6.1mg/dl using PCR to determine its frequency and examined their clinical features. We identified an unrelated heterozygous ARH carrier and a non-carrier in the same family among unrelated to the original family. Serum LDL-C levels of heterozygous ARH carriers (mean=153.8±35.8mg/dl) were significantly higher than those of non-carriers (mean=108.2±41.4mg/dl, p<0.05). Serum triglyceride (151.9±110.7mg/dl vs 140.4±48.8mg/dl) and high-density lipoprotein cholesterol (57.4±11.0mg/dl vs 54.0±11.9mg/dl) levels were not different between them. Interestingly, heterozygous ARH carriers didn’t show any xanthomas including Achilles tendon (6.4±1.1mm vs 5.7±1.1mm) in contrast to FH, which is frequently associated with typical xanthoma. These results demonstrate that heterozygous ARH (Ins C
599
) carriers show higher LDL-C levels compared with non-carrier family member without tendon xanthomas observed in FH. We suggest that heterozygous ARH (Ins C
599
) carrier may explain a part of primary hypercholesterolemia.
Abstract 1927: High-dose Simvastatin Alone Inhibits Rho Kinase and Improves Flow-mediated Vasodilatation to a Greater Extent than Combination of Low-dose Simvastatin and Ezetimibe, Despite Comparable Lipid-Lowering
Authors: Liu Ping-Yen , Liu Ping-Yen , Liu Yen-Wen , Lin Li-Jen , Chen Jyh-Hong , Liao James K
Published: July 2021

Background:
By inhibiting HMG-CoA reductase, statins not only inhibit cholesterol biosynthesis, but also decrease the formation of isoprenoids, which are important for mediating signaling through the Rho/Rho kinase (ROCK) pathway. In animal studies, inhibition of ROCK by statins improves endothelial function, decreases inflammation, and prevents atherosclerosis. These so-called cholesterol-independent effects of statins are dose-related and may contribute to some of their clinical benefits. We hypothesize that ezetimibe, which inhibits cholesterol absorption, does not exert these cholesterol-independent effects in humans.

Methods and Results:
We studied 60 dyslipidemia subjects (n=20 in each arm) in a prospective, randomized, observer-blinded study comparing treatment with simvastatin 40 mg/d or simvastatin/ezetimibe 10/10 mg/d to corresponding placebo tablets for 28 days. Prior statin usage was comparable between the groups and a washout period of 2 weeks was instituted before enrollment. Blood samples for fasting lipids, leukocyte ROCK activity and C-reactive protein (CRP) were collected at days 0 and 28. Baseline demographics, lipid levels, ROCK activity and CRP were not different between the 3 groups. Compared to placebo group, both treatment regimens decreased low-density lipoprotein cholesterol (LDL-C) by 38% and CRP by 32– 42% after 28 days (p<0.001 for both compared to placebo). Although LDL-C and CRP were reduced to comparable levels by either lipid-lowering regimen (p>0.05 between the groups), only simvastatin 40 mg reduced ROCK activity and improved forearm flow-mediated vasodilatation (FMD) (p<0.01 for both compared to baseline). The reduction of ROCK activity with simvastatin 40 mg remained significant even after controlling for changes in LDL-C (p=0.01) and correlated with improvement in FMD (R
2
=0.78, p<0.01). However, there was no correlation between changes in FMD with changes in LDL-C or CRP.

Conclusions:
These findings suggest that despite comparable decrease in LDL-C and CRP, high-dose statin monotherapy has greater effects on both ROCK activity and endothelial function than low-dose statin and ezetimibe. These findings provide additional evidence of potential statin benefits beyond cholesterol lowering.
Abstract 1935: A Magnetic Resonance Imaging Contrast Agent Targeted Towards Activated Platelets Allows In Vivo Detection of Thrombosis and Monitoring of Thrombolysis
Authors: von zur Muehlen Constantin , Elverfeldt Dominik , Moeller Julia , Choudhury Robin , Hagemeyer Christoph , Schwarz Meike , Bode Christoph , Peter Karlheinz
Published: July 2021

Platelets are the key to thrombus formation in atherothrombosis and play a major role in plaque rupture. Non-invasive imaging of activated platelets would be of great clinical interest. Here, we evaluate the ability of a magnetic resonance imaging (MRI) contrast agent consisting of microparticles of iron oxide (MPIO) and a single-chain antibody targeting ligand-induced binding sites (LIBS) on activated GPIIb/IIIa to image carotid artery thrombosis and. Anti-LIBS or control antibody were conjugated to 1μm-sized MPIOs (LIBS-MPIO/control-MPIO). Non-occlusive wall-adherent thrombi were induced in BL/6 mice using 6% ferric chloride. MRI (at 9.4 Tesla) of the carotid artery was performed once before (figure

, A) and repeatedly in 12min long sequences after LIBS-MPIO/control-MPIO injection. After 36min, a significant signal void, which is the typical effect of iron oxide-based contrast agents, was observed with LIBS-MPIO (figure

, B), but not control-MPIO (P<0.01) and corresponded to LIBS-MPIO binding as confirmed by histology. After thrombolysis, in LIBS-MPIO injected mice the signal void subsided, indicating successful thrombolysis (figure

, C). On histology, MPIO-content of thrombus, as well as thrombus size, correlated significantly with LIBS-MPIO-induced signal void (both P<0.01). LIBS-MPIO allows in vivo MRI of activated platelets with excellent contrast properties and monitoring of thrombolytic therapy. This approach represents a novel non-invasive technique allowing rapid detection and quantification of platelet-containing thrombi, such as found on the surface of ruptured atherosclerotic plaques.
Abstract 1941: The Anti Von Willebrand Factor Aptamer ARC1779 Effectively Inhibits Platelet Activation and Adhesion
Authors: Duerschmied Daniel , Wagner Patricia , Gilbert Madaline , Gilbert James , Wagner Denisa , Schaub Robert
Published: July 2021

Objectives Immediately after vessel wall damage, plasma von Willebrand factor (VWF) binds to exposed collagen and laminin, enabling the subsequent adhesion of platelets to immobilized VWF via glycoprotein (GP) Ibα. The GPIbα-VWF interaction is able to withstand high (arterial and intrastenotic) shear conditions, rendering this mechanism crucial for the initiation of hemostatic and thrombotic processes in the arterial circulation. In addition, released VWF promotes platelet activation. We have generated a nuclease-resistant anti-VWF A1 domain aptamer, ARC1779. Aptamers are nucleic acid molecules with high affinity and specificity due to the ability to fold into unique three dimensional structures. We examined the ability of ARC1779 to inhibit these platelet activation processes.

Methods and Results Botrocetin-induced aggregation of platelet-rich plasma (IC90 ~100–300 nM) and shear force-induced platelet aggregation in whole blood (IC95 ~100 nM) were inhibited by ARC1779. Platelet adhesion was assessed under high shear conditions (1500/s) in a parallel plate flow chamber using calcein orange labeled platelets. ARC1779 at a concentration range of 7.5 nM − 375 nM produced a concentration dependent inhibition of platelet adhesion with an IC50 of 7.5 nM and IC90 of 75 nM. Almost complete abolition of platelet adhesion was achieved with 400 nM of the aptamer. Firm adhesion of platelets was assessed by monitoring time of adhesion of randomly selected adherent platelets treated with 200 nM of ARC1779. The time that ARC1779 treated platelets remained attached was significantly reduced compared to untreated platelets (p<0.001). The occlusion time after electrical injury of carotid arteries in cynomolgus macaques was assessed and found to be significantly prolonged during ARC1779 treatment. At a concentration of 700 nM, ARC1779 inhibited occlusion of the injured carotid arteries as effectively as the GPIIb/IIIa antagonist abciximab at 1300 nM. The bleeding time was significantly shorter with ARC1779 than with abciximab.

Conclusions ARC1779 constitutes a promising novel anti-platelet agent for various thrombotic indications.
Abstract 1947: Chymase Inhibition Reduces the Incidence of Intraplaque Hemorrhage and Induces a Stable Atherosclerotic Plaque Phenotype in ApoE Deficient Mice
Authors: Bot Ilze , Westra Marijke M , van Heiningen Sandra H , Hilpert Hans , Lankhuizen Inge M , van Berkel Theo J , Fingerle Jürgen , Biessen Erik A
Published: July 2021

Mast cells are abundantly present in perivascular tissue during atherosclerotic plaque progression and were previously demonstrated to contribute significantly to plaque destabilization. In this study, we aimed to investigate to what extent inhibition of chymase, one of the mast cell proteases, could enhance plaque stability. We demonstrated earlier that the specific chymase inhibitor RO501 (1 μM) was able to quench mast cell activation
in vitro
, as illustrated by a decreased β-hexosaminidase (−41% and −80%, respectively; P<0.05) as well as chymase activity in the releasate of activated MC/9 and peritoneal mast cells (−71% and −65%, respectively; P<0.05). Next, we assessed whether chymase inhibition was also effective
in vivo.
Atherosclerotic carotid artery lesions were induced in ApoE
−/−
mice by perivascular collar placement and mast cells were activated by DNP challenge systemically during lesion development. RO501 (50 mg/kg/day) was administered as diet supplement, leading to serum concentrations of ~2 μM. While plaque size after 6 weeks of treatment did not differ, collagen content of the lesions was 2-fold enhanced in mice treated with RO501 compared to controls (1.4 ± 0.5% and 0.7 ± 0.2%, respectively). This was accompanied by a significant decrease in necrotic core size of the plaques (controls: 52 ± 3% versus RO501: 41 ± 4%, P<0.05). To determine the effects of chymase inhibition after acute mast cell activation in advanced plaques, perivascular mast cells were focally activated in the adventitia of advanced lesions in ApoE
−/−
mice, which were treated with RO501 as described above. At three days after focal mast cell challenge, the incidence of intraplaque hemorrhage (IPH) was inhibited from 23% in control mice to 4.5% in RO501 treated mice, while also the plaque erythrocyte area was reduced by >90% from 1.2 ± 0.6*10
3
to 0.1 ± 0.08*10
3
μm
2
(P<0.05). Also, we observed a reduction in apoptotic cells (RO501: 0.68 ± 0.20% vs. 1.01 ± 0.36% for IPH negative controls and 1.23 ± 0.42% for IPH
+
plaques). In conclusion, our data suggest that chymase inhibition at least partly prevents the detrimental effects of perivascular mast cells on plaque stability, identifying chymase inhibition as a new therapeutic approach in the prevention of acute coronary syndromes.
Abstract 1948: Over-expression of HSP27 Provides Chronic Atheroprotection: Reductions in Foam Cell Content and Inflammatory Responses
Authors: Chen Yong-Xiang , Rayner Katey , Deb-Rinker Paromita , Simard Trevor , McNulty Melissa , Zhao Xiaoling , O’Brien Edward R
Published: July 2021

Introduction: Heat Shock Protein 27 (HSP27) was recently shown by our laboratory to be a biomarker of atherosclerosis and after 4 weeks of a high fat diet (HFD) apoE
−/−
mice that over-express HSP27 (or apoE
−/−
HSP27
o/e
mice) show a marked attenuation of aortic lesion size compared to apoE
−/−
mice - but only in females (ATVB 20005; Circ Res in press). Moreover, we demonstrated that

serum HSP27 levels were inversely related to atherosclerotic burden,

estrogens mediated the release of HSP27 from cells and

extracellular HSP27 bound to Scavenger Receptor-A where it could competitively inhibit acLDL uptake.

Purpose/Methods: The goal of this study was to determine if and how HSP27 over-expression persistently attenuates atherogenesis in apoE
−/−
mice fed a HFD for 12 weeks.

Results: Compared to female apoE
−/−
mice, female apoE
−/−
HSP27
o/e
mice showed persistent reductions in aortic (en face) lesion area and aortic sinus cross-sectional lesion area at 12 weeks (e.g., 38% and 39% respectively vs. 35% and 40% after 4 weeks of HFD). Although our original studies revealed that male apoE
−/−
HSP27
o/e
mice had no protection from atherogenesis after 4 weeks of a HFD, in the current study we noted that 12 weeks of a HFD resulted in a 17% reduction in aortic (en face) lesion area and a 24% attenuation in aortic sinus cross-sectional lesion area with HSP27 over-expression. While lesions observed after 4 weeks of HFD consisted mainly of lipid-laden foam macrophages, after 12 weeks of HFD the histopathology was different and included a distinct foam cell layer (or FCL) luminal to a deeper necrotic layer (e.g., with cholesterol crystals). The FCL area was reduced in size in apoE
−/−
HSP27
o/e
vs. apoE
−/−
mice (e.g., −63% in females, p=0.03; and −48% in males, p=0.02).
In vitro,
macrophages from apoE
−/−
HSP27
o/e
mice showed a reduction in TNF-α expression compared to apoE
−/−
mice (e.g., 40% at baseline, and 55% after LPS provocation).

Conclusions: Over-expression of HSP27 provides a persistent atheroprotective effect that benefits both sexes. Chronic lesions are not only smaller in size with HSP27 over-expression but show fewer foam cells and macrophages are less pro-inflammatory. These results highlight a potential clinical role for chronic HSP27 modulation for atheroprotection.
Abstract 3390: Impairment of Post-ischemic Neovascularization in Mice Lacking IQGAP1, an Actin-binding Scaffold Protein: Role in Macrophage Infiltration and ROS production
Authors: Urao Norifumi , McKinney Ronald , Fukai Tohru , Ushio-Fukai Masuko
Published: July 2021

Neovascularization is important repair mechanism in response to ischemic injury and its process is in part dependent on reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is key regulator for actin cytoskeleton and motility. We demonstrated that IQGAP1 is expressed in endothelial cells (ECs) and mediates VEGF-induced ROS production and migration of cultured ECs. However, its role in postnatal neovascularization is unknown. Here we show that neovascularization induced by hindlimb ischemia is significantly inhibited in IQGAP1-deficient mice as evaluated by laser Doppler blood flow (38%), capillary density (48%) and alpha-actin positive arterioles (68%). In wild-type (WT) mice, western and immunofluorescence analysis reveal that IQGAP1 protein expression in ischemic tissues is significantly increased at Mac3+ macrophages and lectin+ capillary-like ECs at 3 days and 7 days after ischemia, respectively, which is associated with increased superoxide production. Of note, macrophage infiltration is important for post-ischemic revascularization. Mice lacking IQGAP1 show significant decrease in the number of infiltrated Mac3-positive macrophage and superoxide production in ischemic muscles (30% and 52%, respectively) as compared to WT mice. Numbers of inflammatory cells and cKit+/Flk1+ endothelial progenitor cells in peripheral blood are not affected in these knockout mice. Moreover, thioglycollate-induced peritoneal leukocyte recruitment and accumulation are significantly inhibited in IQGAP1-deficient mice (53%), whereas white blood cell number is not changed. In vitro, peritonitis-elicited (activated) macrophages obtained from IQGAP1-deficient mice show impaired polarization of actin, as visualized by phalloidin staining as well as complete inhibition of migration towards stromal derived factor-1 and VEGF, as measured by modified Boyden-chambered method. In summary, IQGAP1 plays a key role in reparative neovascularization in response to tissue ischemia by regulating not only ECs but also macrophage migration/infiltration as well as ROS production in the vessel growth area. Thus, IQGAP1 is a potential therapeutic target to promote neovascularization in ischemic cardiovascular diseases.

This research has received full or partial funding support from the American Heart Association, AHA National Center.