bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 689: Notch Signaling Stimulates Expression Of Wnts And Contributes To Cardiac Markers Gene Expression In Human Progenitor Cell
Authors: Koyanagi Masamichi , Bushoven Philipp , Iwasaki Masayoshi , Urbich Carmen , Zeiher Andreas M , Dimmeler Stefanie
Published: July 2021

It has been demonstrated that adult human circulating endothelial progenitor cells (EPC) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a prominent role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPC in a co-culture system with neonatal rat cardiomyocyte (CM). EPC expressed the receptors Notch-1 and Notch-2, whereas CM expressed high level of Notch ligand Jagged-1. Therefore, we hypothesized that CM may activate Notch signaling within EPC. Indeed, after co-culture, Notch activation was detected in EPC by immunohistochemical detection of the intracellular cleavage fragment of Notch-1 (NICD), whereas NICD was only rarely detected in EPC before co-culture. Western blot analysis confirmed Notch cleavage after co-culture. RT-PCR directed against the human specific sequences of the Notch target genes Hey2 and Hes1 demonstrated a transient activation of the transcriptional activity of Notch in human EPC after co-culture with CM. Inhibition of γ-secretase significantly blocked Notch cleavages and NICD translocations. Furthermore, the expression of the cardiac marker protein γ-sarcomeric actinin and troponin T was significantly suppressed by γ-secretase inhibition (55.8 ± 8.1% and 54.0 ± 10.5% of control, respectively) or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because non-canonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and. Wnt5a and Wnt11 expressions in the human cells was induced by the co-culture and was blocked by γ-secretase inhibitor. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted NICD cleavage and Wnt5a expression in EPC. These data suggested that Notch is transiently activated upon co-culture of EPC with neonatal rat CM. γ-Secretase-dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of non-canonical Wnt proteins, which may act in an paracrine manner to further amplify cardiac differentiation.
Abstract 692: Class II HDACs Are Required During Early Stage Mouse Embryonic Stem Cell Differentiation in Response to Nitric Oxide
Authors: Illi Barbara , Spallotta Francesco , Rosati Jessica , Mattiussi Stefania , Nanni Simona , Rotili Dante , Valente Sergio , Mai Antonello , Farsetti Antonella , Capogrossi Maurizio C , Gaetano Carlo
Published: July 2021

INTRODUCTION & BACKGROUND: We have recently described that in mouse embryonic stem cells (ES) laminar shear stress provides chromatin programming signals. This effect is similar to that of nitric oxide (NO) which directly promotes cardiomyogenesis underlying the possible presence of a common mechanism of action the characterization of which has been the object of the present study.

METHODS & RESULTS: ES were treated with the NO donor DETANO from 1 to 24 hours (h) in the presence of absence of leukaemia inhibitory factor (LIF). In LIF-deprived cells the NO donor determined a marked global reduction in histone H3 acetylation paralleled by the expression of a number of cardiovascular markers. In this condition the stemness-associated gene product Oct4 decreased rapidly at mRNA and protein level underlying the potential opening of a NO-dependent differentiation program. Further class-II histone deacetylase (HDAC) 4, 5, and 7, rapidly shuttled to nucleus upon LIF deprivation. This phenomenon was transient as HDAC molecules returned to the cytoplasm in about 6 h. Intriguingly, the exposure of ES to the NO donor allowed the nuclear retention of these enzymes still detectable at the 6 h time-point. Further, we found that NO treatment stimulated the formation of nuclear complexes including the class II HDACs 4, 5 and the protein phosphatase 2A. Remarkably, ES treatment with the class II selective inhibitor MC 1568 prevented ES differentiation in the presence of NO.

CONCLUSIONS: These results suggest a potential role for class II HDACs in the NO-dependent early stage of ES commitment upon LIF removal.
Abstract 694: Ankyrin Repeat and SOCS Box Protein 4 (ASB4) is a Hydroxylation Substrate of Factor Inhibiting HIF1a (FIH) and Promotes Vascular Differentiation via an Oxygen-Dependent Mechanism
Authors: Ferguson James E , Wu Yaxu , Smith Kevin , Patterson Cam
Published: July 2021

The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development we employed a microarray screen using differentiating embryonic stem (ES) cells that identified the previously uncharacterized Ankyrin Repeat and SOCS Box Protein 4 (ASB4) as the most highly differentially expressed gene in the vascular lineage during early differentiation. Like other SOCS box-containing proteins, ASB4 acts as the substrate recognition molecule of an Elongin-B/Elongin-C/Cullin/Roc ubiquitin ligase complex that mediates the ubiquitination and degradation of substrate protein(s). We show here that high levels of ASB4 expression in the embryonic vasculature coincide with drastic increases in oxygen tension as placental blood flow is initiated. However, as vessels mature and oxygen levels stabilize, ASB4 expression is quickly downregulated, suggesting that ASB4 may function to modulate an endothelial-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 directly interacts with Factor Inhibiting HIF1-alpha (FIH), and is a substrate for FIH-mediated hydroxylation via an oxygen-dependent mechanism. Additionally, overexpression of ASB4 in ES cells promotes differentiation into the endothelial lineage in an oxygen-dependent manner. We propose that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.
Abstract 742: Inducibility of Atrial Fibrillation in the Myocardial Sleeve of the Superior Vena Cava is Mediated by the Ganglionated Plexi on the Right Pulmonary Artery
Authors: Niu Guo-Dong , Scherlag Benjamin J , Lu Zhibing , Ghias Muhammad , Lazzara Ralph , Jackman Warren M , Po Sunny S
Published: July 2021

Introduction:
Previous studies have shown that electrical stimulation of the ganglionated plexi (GP) on the right pulmonary artery (RPA), the so-called “3rd fat pad (FP)” causes slowing of the sinus rate and AV conduction. Ablation of this GP prevents induction of atrial fibrillation (AF) caused by vagosympathetic nerve (VNS) stimulation and atrial premature beats (APBs).

Methods:
Eleven dogs anesthetized with Na-pentobarbital were subjected to a right thoracotomy at the 4
th
intercostal space. The RPA at the upper lobe of the right lung was dissected and the distal end tied in order to insert and stabilize an 8 Fr. Sheath into the RPA. A small basket electrode catheter, consisting of 5 splines, each spline containing 3 pairs of bipolar electrodes, was inserted into the RPA underneath the superior vena cava (SVC). A Lasso catheter, inserted through a sheath in the right jugular vein was positioned in the SVC contacting the sleeve of myocardium at the SVC-right atrial (RA) junction. Octapolar electrode catheters were sutured against the right superior, inferior pulmonary veins (PVs), RA and RA appendage. Through a left sided thoracotomy, similar placements of recording electrode catheters were made at the left superior, inferior PVs and left atrium (LA) body and appendage. Right and left vagosympathetic nerve stimulation (VNS, frequency, 20 Hz; stimulus duration, 0.01 ms; voltage 1.5– 4.5 Volts) slowed the heart rate (HR) by 50% or induced 2:1 AV block. The RPA GP was also stimulated to achieve similar effects on HR and AV block.

Results:
RPA GP stimulation consistently and significantly reduced the threshold for AF inducibility (control 8±3; RPA GP stimulation 3.2±1.5 volts, p<0.01) whereas after RPA GP ablation, the averaged voltage to induce AF was increased to 11.5±1.5 although 7 of 11 showed non-inducibility at the maximum voltage used (12 volts). The inducibility threshold at the other atrial and PV sites were unchanged by RPA GP stimulation before or after RPA GP ablation (p=NS). Moreover, there was a loss of HR slowing and AV block with VNS stimulation.

Conclusion:
RPA GP stimulation which markedly decreased HR or AV conduction selectively suppressed AF inducibility at the myocardial sleeve of the SVC but did not affect AF inducibility at other atrial sites.
Abstract 759: Inhibition Of Pro-inflammatory Cytokine Il-17 Reduces Atherosclerotic Lesion Development In Apo E −/ − Mice
Authors: Erbel Christian , Klingenberg Roland , Celik Sultan , Wambsganss Nadine , Funke Benjamin , Boeckler Dittmar , Dengler Thomas J
Published: July 2021

Atherosclerotic plaque typically contains infiltrates of activated macrophages and T cells. In human carotid artery plaques, we previously showed the presence of IL-17 producing T cells (Th17) and IL-23. Effects of inhibition of the pro-inflammatory Th1 cytokine IL-17 on atherosclerosis development was studied in a rodent model. 25 female ApoE −/ − mice fed a normal diet were treated with a specific blocking antibody against IL-17 for 12 weeks (100 μg intraperitoneally once per week). For analysis, aortic root was embedded in OCT and serially cryosectioned in 5μm intervals; distal aorta was snap frozen for mRNA analysis. Sections every 75μm were stained with Oil RedO, the lesion area was quantified using PC-based image analysis. In IL-17 treated mice no changes in LDL cholesterol and anti-ox LDL antibodies were seen. Subsets of immune cells, surface marker expression and fraction of regulatory T cells in peripheral blood / spleen were unchanged. Inhibition of IL-17 markedly reduced atherosclerotic plaque volume by 65% (p=0.004) and fractional stenosis by 68% (p=0.01). Immunohistochemistry revealed significant reduction of cellular infiltration by T cells and macrophages in atherosclerotic lesions. By quantitative RT-PCR, significantly reduced expression was shown (among others) for CD3ϵ, egr-1, Akt and RANKL (all p< 0.01). Functional blockade of Th1 cytokine IL-17 reduces atherosclerotic lesion development in an established animal model without major derangement of peripheral cellular immunity. Our findings suggest a relevant role for IL17-producing T cells in atherosclerosis warranting further study of pathogenic mechanisms and therapeutic potential.
Abstract 762: Atherosclerosis Regression Promoted By An LXR Agonist Is Dependent On The Chemokine Receptor CCR7
Authors: Feig Jonathan E , Pineda-Torra Ines , Shamir Raanan , Joaquin Victor A , Grauer Lisa S , Garabedian Michael J , Fisher Edward A
Published: July 2021

We have developed a transplantation-based mouse model of atherosclerosis regression by allowing plaques to form in apoE−/ − mice, then changing the plaque’s plasma environment from hyperlipidemia to normolipidemia. In this model, we reported emigration of plaque foam cells to regional and systemic lymph nodes after 3 days in the regression environment (Llodrá et al. PNAS 2004). This emigration required the expression of the chemokine receptor CCR7 on the foam cells and was associated with up-regulation of their LXR mRNA (Trogan, Feig et al. PNAS 2006). Because the human and murine promoters have LXREs, we hypothesized that LXR was a regulator of CCR7. Using a murine model of immature DCs, D2.4 cells, we found that CCR7 expression is increased 8 –9X upon LXR activation by the agonist T0901317 (T09). Importantly, this increase is dependent on CCR7 gene transcription, because pre-treatment with actinomycin D abolished the observed response. When siRNA to LXR alpha and LXR beta was used in conjunction with T09, CCR7 gene expression was significantly blunted to only 2X that of the control (no T09). To extend the studies in vivo, we treated atherosclerotic apoE−/ − mice (n=9/group) with the LXR agonist (50 mg/kg/day) or saline for 2 weeks. Using laser capture microdissection, we isolated lesional macrophages and found expression of downstream targets of LXR such as ABCA1, ABCG1, and ABCG4 to be up-regulated by 3.7, 4.4, and 3.1X, respectively, as assessed by quantitative real-time PCR. Interestingly, CCR7 expression was found to be increased by 3.7X. This result was confirmed at the protein level using immunohistochemistry, which showed that CCR7 expression was increased in foam cells from the LXR-agonist treated but not the saline-treated group. Morphometric analyses revealed that foam cell content and lesion area decreased by 21% and 24%, respectively. The dependence of these relatively rapid (2 wks) effects on CCR7 was shown by treating apoE−/ − CCR7−/ − atherosclerotic mice (n=8) with T09. After 2 wks, foam cell content only decreased by approximately 9%. In conclusion, CCR7 is a required factor for atherosclerosis regression in a mouse model and LXR regulates its expression in vitro and in vivo.
Abstract 770: Coordinated Upregulation of Cardiomyocyte Urotensin II (U-II) and its Receptor (UT) Exhibits Enhanced Inhibition of L-Type Calcium Current in Heart Failure
Authors: Cheng Heng-Jie , Han Qing-Hua , Zhou Peng , Morimoto Atsushi , Masutani Satoshi , Little William C , Cheng Che-Ping
Published: July 2021

Background
. Heart failure has been linked to urotensin II (U-II), the most potent mammalian vasoconstrictor. However, the alteration and functional significance of U-II in HF are uncertain.

Methods
. We compared LV myocyte U-II and its receptor (UT) protein levels and I
Ca,L
response to U-II stimulation in 10 normal (N) adult rats and 10 age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg, sq, for 2 days). I
Ca,L
was measured using whole-cell voltage clamp technique. In 3 subgroups, U-II-mediated actions were determined after pretreatment of myocytes with U-II receptor antagonist, Urantide (10
−5
M); pertussis toxin (PTX, 2μg/ml, 36°C, 6h); and dibutyryl-cAMP (Db-cAMP, 10
−4
M).

Results
. Compared with normal myocytes, in HF myocytes, both U-II (93%, 0.081 vs 0.042) and UT (52%, 0.038 vs 0.025) protein levels were significantly increased. Importantly, these changes were associated with enhanced U-II -mediated negative modulation on L
Ca,L
. Superfusion of U-II (10
−8
-10
−5
M) caused a dose-dependent decrease in I
Ca,L
in both normal and HF myocytes with maximal inhibition at 10
−5
M. In normal myocytes, U-II (10
−5
M) decreased I
Ca,L
by 18% (5.0±0.29 vs 6.1±0.5 pA/pF, p<0.01). In HF myocytes, the baseline I
Ca,L
was significantly lower (3.3±0.1 vs 5.9±0.3 pA/pF, p<0.01) and was further reduced by 27% with U-II 10
−5
M (2.4±0.1 vs 3.3±0.1 pA/pF), that was significantly greater than U-II-induced changes in normal myocytes. U-II-induced reductions in I
Ca,L
were significantly attenuated by U-II-antagonist. In both groups of rats, U-II-induced decreases of I
Ca,L
were prevented with pretreatment myocytes with PTX (normal: 5.6±0.5 vs 5.5±0.4; HF: 3.4±0.4 vs 3.5±0.2 pA/pF) or Db-cAMP (normal: 5.6±0.5 vs 5.6±0.7; HF: 3.7±0.3 vs 3.8±0.4 pA/pF).

Conclusions
. In HF rat cardiomyocytes, U-II and UT-coupled, G
i
-mediated signaling pathway is upregulated, which amplifies U-II stimulation-induced inhibition of I
Ca,L
. This may lead to exacerbation of the dysfunctional [Ca
2+
]
i
homeostasis and enhance cardiac depression of the failing myocardium.
Abstract 774: Decreased Number And Functional Activities Of Endothelial Progenitor Cells In CuZnSOD-deficient Mice.
Authors: Groleau Jessika , Dussault Sylvie , Haddad Paola , Turgeon Julie , Ménard Catherine , Rivard Alain
Published: July 2021

Background:
Several risk factors for cardiovascular diseases are associated with reduced neovascularization in response to ischemia. A common feature of all cardiovascular risk factors is increased production of reactive oxygen species (ROS) such as superoxide. We have recently demonstrated that mice that are deficient for CuZnSOD present impaired neovascularization in response to ischemia. Here we tested the hypothesis that endothelial progenitor cell (EPC) dysfunction might be involved in this neovascularization defect.

Methods and results:
Seven days after surgically induced hindlimb ischemia, EPCs were isolated from CuZnSOD deficient mice (CuZnSOD
−/ −
, n=8 –10) or control mice (CuZnSOD
+/+
, n=8 –10). We found that the number of EPCs was significantly reduced in the bone marrow (40.6±3.0 vs 70.5±5.0, p<0.05) and the spleen (0.29±0.06 vs 0.54±0.11, p<0.001) of CuZnSOD
−/ −
mice compared to controls. Moreover, EPC functional activities were significantly impaired in CuZnSOD
−/ −
mice. More specifically, we found that EPC adhesion was reduced by 41%, migration by 88%, proliferation by 46% and integration into tubules by 77% in CuZnSOD
−/ −
mice. To confirm the role of EPCs in vivo, we isolated CD117+ cells (also acLDL and lectin positive when cultured for 4 days in vitro) from the bone marrow of CuZnSOD
+/+
mice and performed an intravenous injection (5X10
5
cells) into CuZnSOD
−/ −
mice at 24 hours after surgically-induced hindlimb ischemia. Interestingly, EPC supplementation in CuZnSOD
−/ −
mice led to a complete rescue of ischemia-induced neovascularization, as assessed by blood flow recuperation in the ischemic hindlimb (Laser-Doppler analysis). Moreover, EPC therapy in CuZnSOD
−/ −
mice was also associated with a significant reduction of oxidative stress in ischemic tissues (nitrotyrosine and DHE staining).

Conclusions:
CuZnSOD deficiency is associated with a significant reduction in the number and the functional activities of EPCs. Cellular therapy with EPC can rescue the impaired neovascularization in CuZnSOD
−/ −
mice. Our results suggest that increased oxidative stress could contribute to explain EPC dysfunction and impaired neovascularization associated with different cardiovascular risk factors.
Abstract 782: Fra2 Mediates Oxygen-induced TGFbeta-1 mRNA Expression In Adult Cardiac Fibroblasts: Significance In Myocardial Fibrosis Following Ischemia-reperfusion Injury
Authors: Roy Sashwati , Khanna Savita , Sen Chandan K
Published: July 2021

Background
. Transforming growth factor beta-1 (TGFbeta-1) is a key cytokine implicated in the development of cardiac fibrosis following ischemia-reperfusion (IR) injury. The profibrotic effects of TGFbeta-1 are primarily attributable to the differentiation of cardiac fibroblasts (CF) to myofibroblasts. Previously, we have reported perceived hyperoxia (Circ Res 92:264 –71), sub-lethal reoxygenation shock during IR, induces differentiation of CF to myofibroblasts at the infarct site. The mechanisms underlying oxygen-sensitive induction of TGFbeta-1 mRNA remain to be characterized.

Hypothesis
. Fra2 mediates oxygen-induced TGFbeta-1 mRNA expression in adult cardiac fibroblasts.

Methods.
TGFbeta-1 mRNA expression in infarct tissue was investigated in an IR injury model. The left anterior descending coronary artery of mice was transiently occluded for 60 minutes followed by reperfusion to induce IR injury. Spatially resolved infarct and non-infarct tissues were collected at 0, 1, 3, 5, and 7 days post-IR using laser capture microdissection. TGFbeta-1 mRNA levels were measured using real-time PCR. To investigate the role of oxygen in the regulation of TGFbeta-1, we used our previously reported model of perceived hyperoxia where CF (from 5wks old mice) after isolation were cultured at 5%O
2
(physiological pO
2
) followed by transferring them to 20%O
2
to induce hyperoxic insult.

Results & Conclusions.
In vivo, a significant increase (p<0.01; n=5) in TGFbeta-1 mRNA was observed at the infarct site already at day 1 post-IR. The levels continued to increase until day 7 post-IR. In vitro, exposure of CF to 20%O
2
hyperoxic insult induced TGFbeta-1 mRNA (p<0.001; n=4) and protein (p<0.01; n=4) expression. Using a TGFbeta-1 promoter-luciferase reporter and DNA binding assays, we collected first evidence that AP-1 and its component Fra2 as major mediators of oxygen-induced TGFbeta-1 expression. Exposure to 20%O
2
resulted in increased localization of Fra2 in nucleus. siRNA-dependent Fra-2 knock-down completely abrogated oxygen-induced TGFbeta1 expression. In conclusion, this study presents first evidence that Fra-2 is involved in inducible TGFbeta1 expression in CF. Fra2 was noted as being central in regulating oxygen-induced TGFbeta-1 expression.s
Abstract 791: A Critical Role for Bmper (BMP-binding Endothelial cell precursor-derived Regulator) in Cardiac Muscle Mass Regulation during Development and Pressure Overload Induced Hypertrophy
Authors: Willis Monte , Ren Rongqin , Patterson Cam
Published: July 2021

Bone morphogenetic proteins (BMPs) of the TGF-beta superfamily, have been implicated in multiple processes during cardiac development. Our laboratory recently described an unprecedented role for Bmper in antagonizing BMP-2, BMP-4, and BMP-6. To determine the role of Bmper on cardiac development in vivo, we created Bmper null (Bmper −/−) mice by replacing exons 1 and 2 with GFP. Since Bmper −/− mice are perinatally lethal, we determined pre-natal cardiac function of Bmper −/− mice in utero just before birth. By echocardiography, E18.5 Bmper −/− embryos had decreased cardiac function (24.2 +/− 8.1% fractional shortening) compared to Bmper +/− and Bmper +/+ siblings (52.2 +/− 1.6% fractional shortening) (N=4/group). To further characterize the role of Bmper on cardiac function in adult mice, we performed echocardiography on 8-week old male and female Bmper +/− and littermate control Bmper +/+. Bmper +/− mice had an approximately 15% decrease in anterior and posterior wall thickness compared to sibling Bmper +/+ mice at baseline (n=10/group). Cross-sectional areas of Bmper +/− cardiomyocytes were approximately 20% less than wild type controls, indicating cardiomyocyte hypoplasia in adult Bmper +/− mice at baseline. Histologically, no significant differences were identified in representative H&E and trichrome stained adult Bmper +/− and Bmper +/+ cardiac sections at baseline. To determine the effects of Bmper expression on the development of cardiac hypertrophy, both Bmper +/− and Bmper +/+ sibling controls underwent transaortic constriction (TAC), followed by weekly echocardiography. While a deficit was identified in Bmper +/− mice at baseline, both anterior and posterior wall thicknesses increased after TAC, such that identical wall thicknesses were identified in Bmper +/− and Bmper +/+ mice 1–4 weeks after TAC. Notably, cardiac function (fractional shortening %) and histological evaluation revealed no differences between Bmper +/− and Bmper +/+ any time after TAC. These studies identify for the first time that Bmper expression plays a critical role in regulating cardiac muscle mass during development, and that Bmper regulates the development of hypertrophy in response to pressure overload in vivo.
Abstract 803: Diesel Exhaust Inhalation Enhances Thrombus Formation In Man
Authors: Lucking Andrew J , Lundback Magnus , Mills Nicholas L , Faratian Dana , Cassee Fleming , Donaldson Ken , Boon Nicholas , Badimon Juan J , Sandstrom Thomas , Blomberg Anders , Newby David E
Published: July 2021

Background: Transient exposure to traffic-derived air pollution may be a trigger for acute myocardial infarction although the mechanism is unclear. The aim of this study was to investigate the effect of diesel exhaust inhalation on thrombus formation in man using an ex vivo model of thrombosis.

Methods and Results: In a double-blind randomized cross-over study, 20 healthy volunteers were exposed to diluted diesel exhaust (300 μg/m3) or filtered air during intermittent exercise for 1 or 2 hours. Thrombus formation, coagulation, platelet activation and inflammatory markers were measured at 2 and 6 hours after exposure. Thrombus formation was measured using the Badimon ex vivo perfusion chamber at low (212 /s) and high (1,690 /s) shear rates with porcine aortic tunica media as the thrombogenic substrate. Specimens were fixed, stained and thrombus area measured using computerized planimetry. Compared to filtered air, diesel exhaust increased thrombus formation in the low and high shear chambers by 24.2% (p<0.001) and 19.1% (p<0.001) respectively. This increased thrombogenicity was seen at two and six hours, and using two different types of diesel exposure. Although there were no effects on coagulation variables, diesel exhaust inhalation increased platelet-neutrophil (6.5% to 9.2%; P<0.05) and platelet-monocyte (21.0% to 25.0%; P<0.05) aggregates 2 hours following exposure.

Conclusions: Inhalation of diesel exhaust increases ex vivo thrombus formation and causes platelet activation in man. These findings provide a potential mechanism that links exposure to traffic-derived air pollution with acute atherothrombotic events including acute myocardial infarction.
Abstract 813: Tnf-α Alters Superoxide And Nitric Oxide In The Brain Stem And Hypothalamus Contributes To Sympathoexcitation In Heart Failure Mice
Authors: Guggilam Anuradha , Yu Li , Ebenezer Philip J , Zucker Irving H , Francis Joseph
Published: July 2021

Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine that plays an important role in the pathophysiology of cardiovascular disease. Recent evidence suggests that TNF-αinduced oxidative stress contributes to the development of cardiovascular disease. The present study examined the effect of TNF-αon the imbalance between nitric oxide (NO) and superoxide (O
2
. −
) production in the brain stem and hypothalamus and its contribution to enhanced sympathetic drive in mice with heart failure.

Methods and Results:
Myocardial infarction (MI) was induced by ligation of the left coronary artery in wild type (WT) and TNF knockout (TNF KO) mice. After 5 weeks, WT + MI mice exhibited left ventricular (LV) dilatation and a decrease in fractional shortening (%FS). Real time RT-PCR exhibited an increase in the mRNA expression for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the LV of WT + MI mice. The mRNA expression for nNOS was significantly reduced in the brain stem, while that of iNOS and Nox2 were elevated in the brain stem as well as hypothalamus of WT + MI mice. Plasma norepinephrine (NE) levels in WT + MI mice were greater in WT + MI mice than that in sham, or TNF KO + MI mice indicating that sympathetic drive was enhanced by TNF-αin this model.

Conclusions:

The present study shows that TNF-α contributes to left ventricular hypertrophy leading to ventricular dysfunction.

TNF-αinduces the production of O
2
. −
and modulates the production of nitric oxide in the brain stem of HF mice.

TNF-αcontributes to sympatho-excitation in HF.
Abstract 827: Total Body ABCG1-Deficiency Increases Atherosclerotic Lesion Development In LDL-Receptor Knockout Mice
Authors: Meurs Illiana , Out Ruud , Hoekstra Menno , Hildebrand Reeni B , Van Berkel Theo J , Van Eck Miranda
Published: July 2021

ATP-binding cassette transporter G1 (ABCG1) facilitates the efflux of cholesterol from macrophages to HDL. As ABCG1 plays a role in preventing cellular lipid accumulation, macrophage ABCG1 expression has been suggested to protect against atherosclerosis. In bone marrow transplantation studies, however, we and others obtained different effects of specific macrophage ABCG1-deficiency on atherosclerotic lesion development under different experimental settings. The objective of this study was to further define the effect of ABCG1-deficiency on lipid metabolism and atherosclerotic lesion development. ABCG1 knockout (KO) mice upon atherogenic diet feeding develop only modest atherosclerotic lesions, therefore, we recently cross-bred ABCG1 KO mice with LDL-receptor KO (LDLr KO) mice. LDLr KO and LDLr/ABCG1 double KO (dKO) littermates were fed a high-fat, high-cholesterol Western-type diet (WTD) containing 0.25% cholesterol and 15% fat for 10 weeks to induce atherosclerotic lesion formation. Both on chow diet and on WTD, serum lipid levels and lipoprotein profiles showed no significant differences between the LDLr KO and LDLr/ABCG1 dKO mice. However, after 10 weeks of diet feeding, a significant 1.5-fold increase in atherosclerotic lesion size was observed in the aortic root of LDLr/ABCG1 dKO mice (130 ± 12x10
3
μm
2
[n = 8] compared to 88 ± 11x10
3
μm
2
[n=7] for LDLr KO mice;
p
=0.026). In addition, macrophage-rich areas of the lungs and spleens of LDLr/ABCG1 dKO mice exhibited excessive lipid accumulation compared with LDLr KO mice. Furthermore,
in vitro
studies using bone marrow derived macrophages of LDLr KO and LDLr/ABCG1 dKO mice showed that disruption of ABCG1 results in a 15% decrease in cholesterol efflux to HDL (
p<
0.001), whereas the cholesterol efflux to ApoAI was unaffected. These studies reveal that total-body ABCG1 deficiency leads to increased atherosclerotic lesion development and massive lipid accumulation in macrophages of the lung and spleen, possibly due to impaired efflux of cholesterol from macrophages deficient in ABCG1. In conclusion, the expression of ABCG1 protects against atherosclerotic lesion development.
Abstract 832: The Farnesoid X Receptor (FXR) Antagonizes Oxidized LDL Receptor, LOX-1, Activation
Authors: Harnish Douglas , Hahn Ashleigh , Hartman Helen , Huard Christine , Martinez Robert , Mahaney Paige , Evans Mark , Zhang Songwen
Published: July 2021

The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of enterohepatic circulation and lipid homeostasis and may also positively impact vascular inflammation and endothelial function through the antagonism of oxidized LDL receptor, LOX-1, activation. LOX-1 is a endothelial membrane bound scavenger receptor that upon ligand binding stimulates NF-κB and MAPK pathways leading to adhesion molecule and inflammatory gene expression with subsequent nitric oxide inhibition. Since LOX-1 is activated by oxLDL, LOX-1 expression was monitored in LDLR −/− mice fed a western diet. Hepatic LOX-1 mRNA was significantly induced by 7 days on the western diet compared to chow fed controls. Moreover, this LOX-1 induction was dose dependently inhibited with increasing concentrations of a potent synthetic FXR ligand WAY-362450 (FXR-450; 1–30 mg/kg po) as were downstream LOX-1 dependent genes, VCAM-1 and ICAM-1. The regulation of LOX-1 was FXR-specific since another oxLDL hepatic scavenger receptor, CD36, expression was not affected by FXR-450. To determine whether this signaling pathway was active in other settings, the FXR mediated regulation of LOX-1 was studied in the diabetic mouse strain, KKAy. Both hepatic and renal LOX-1 expression was inhibited by 30 mg/kg FXR-450 after 7 days of dosing on a chow diet and resulted in the reduction of serum soluble LOX-1 levels as well. Renal gene profiling studies indicated that FXR-450 stimulated genes involved in nitric oxide production, including DDAH-1, ASS-1 and GTPCH that were all confirmed to be induced in the KKAy mouse model. Since the nitric oxide synthase antagonist ADMA stimulates LOX-1 expression, we postulated that the positive impact of FXR-450 was due to the decrease in local ADMA concentrations via DDAH-1, ASS-1 and GTPCH inductions. In support of this, ADMA mediated induction of LOX-1 was also inhibited by FXR-450 treatment. Therefore, these results identify a novel cross-talk between FXR and LOX-1 signaling pathways through their co-ordinate modulation of the nitric oxide pathway and suggest that FXR agonists may have a beneficial impact on vascular inflammation and endothelial function.
Abstract 833: MicroRNA-206 Controls Cardiac Myosin Heavy Chain Gene Expression By Targeting RXR-α
Authors: Nishi Hitoo , Ono Koh , Iwanaga Yoshitaka , Horie Takahiro , Kinoshita Minako , Nagao Kazuya , Mori Rieko , Hasegawa Koji , Kita Toru
Published: July 2021

MicroRNA (miRNA) is a small noncoding RNA that modulates posttranscriptional translation. Growing evidence indicates that miRNA is involved in basic cell functions and oncogenesis. In order to find a possible functional role of miRNA in the heart, we first suppressed the expression of Dicer and Ago2, which are required for miRNA biogenesis and function, by lenti-virus-mediated siRNA in neonatal rat cardiomyocytes. Knockdown of Dicer and Ago2 expression significantly increased their beating rate; therefore, we hypothesized that miRNA may regulate the expression of cardiomyocyte contractile protein since cardiac contractility depends on the expression of two myosin heavy chain (MHC) genes, α and β, which are regulated in an antithetical manner by developmental, physiological, and pathological signals. As siRNA against Dicer, which results in the loss of all miRNA function, upregulated α-MHC and downregulated β-MHC mRNA expression, we tried to identify functional miRNAs which have an opposite effect against Dicer siRNA on α/β-MHC expression in the heart. We first performed miRNA microarray analysis in the heart of Dahl salt-sensitive rats in which systemic hypertension caused compensated concentric left ventricular hypertrophy (LVH) at the age of 11 weeks, followed by congestive heart failure (CHF) at the age of 17 weeks. In this model, decreased α/β-MHC ratio is observed at LVH and CHF stages. Next we overexpressed miRNAs which are upregulated in LVH and CHF stages in neonatal rat cardiomyocytes by lenti-virus. After screening these miRNAs, miR-206 was found to decrease the α/β-MHC mRNA expression ratio. The computational miRNA target prediction algorithm showed one of the targets of miR-206 is RXR-α, which regulates α/β-MHC transcription. Forced expression of miR-206 reduced the translation of RXR-α. Moreover, when the expression of RXR-α was downregulated by siRNA, the α/β-MHC mRNA expression ratio decreased in the same manner as miR-206. In conclusion, miR-206 controls the gene expression of MHC isoforms by targeting RXR-α, and these results suggest that miR-206 regulates cardiac contractility.
Abstract 848: Growth-differentiation Factor-15 Promotes Anti-inflammatory Effects And Enhances Survival After Experimental Myocardial Infarction
Authors: Kempf Tibor , Widera Christian , Bavendiek Udo , Willenbockel Christian , Korf-Klingebiel Mortimer , Kanwischer Anna , Wollert Kai C
Published: July 2021

Growth-differentiation factor (GDF)-15 is a stress-responsive TGF-ß related cytokine. GDF-15 plasma levels are increased and provide independent prognostic information in patients with acute coronary syndrome. After coronary ligation in mice, GDF-15 protein expression is rapidly induced and remains elevated within the infarcted myocardium for at least 7 days. We therefore postulated that GDF-15 is involved in wound healing post MI. The left descending coronary artery was ligated in male GDF-15 knock-out (KO, n=36), heterozygous (+/-, n=72) and wild-type mice (WT, n=26). Six-week mortality was significantly enhanced in KO (81%) and +/- (67%), as compared to WT mice (35%, P=0.004 by log-rank test). Four days after MI, no differences in infarct size (H.E. staining), cardiac dimensions, wall thickness, and systolic function (echocardiography) were observed (n=8 –12 per group). However, cardiac rupture was more frequent in KO (31%) as compared to WT mice (4%, P<0.01). Immunohistochemistry on day 4 demonstrated significantly elevated numbers of granulocytes (KO, 514±61/mm
2
vs. WT, 181±22/mm
2
; P=0.003) and macrophages (KO, 176±19/mm
2
vs. WT, 89±11/mm
2
; P=0.002) within the infarct border zone of KO mice (n=8–12 per group). This was paralleled by an increase in MMP-9 activity (in-gel zymography) within the infarct border zone (KO, 246±33% vs. WT, 100±14%; n=8–10; P=0.004); no differences in TIMP1 expression were noted between both genotypes. In vitro, pre-stimulation with GDF-15 dose-dependently inhibited the migration of human granulocytes towards the chemoattractant fMLP (10
−8
mol/L) in transwell migration chambers (3 μm pore size), control 100%; GDF-15 0.5ng/mL 78±5%, 5ng/mL 47±10%, 50ng/mL 34±10% (P<0.001). Our data identify GDF-15 as an endogenous anti-inflammatory cytokine that inhibits leukocyte infiltration, and promotes wound healing and survival after myocardial infarction.
Abstract 859: Hypoxia-activated CXCR4 Expression In Cardiac Progenitor Cells For Replenishing Stem Cell Pool In Acute Ischemic Myocardium
Authors: Tang Yao Liang , Shen Leping , Qian Keping , Phillips M. Ian
Published: July 2021

The resident cardiac progenitor cells (CPCs) have been identified in the adult myocardium, however, the CPC pool is rapidly depleted after acute myocardial infarction (AMI). The strategy to home ex vivo cultured CPCs to ischemic myocardium might be an important strategy to replenish resident CPC pool, and improve cardiac function. The CXC chemokine SDF-1α and its receptor CXCR4 have been identified as critical mediator for the ischemia-specific recruitment of progenitor cells, yet the level of CXCR4 expression in CPCs is quite low in normal conditions. Our previous studies showed that hypoxia can optimize CPCs via inducing functional CXCR4 expression in vitro, therefore, in this study, we want to investigate whether the transplantation of hypoxia treated CPCs (Hypo:CPCs) have the benefit of improving cardiac function resulted from increased cell homing in ischemic myocardium through mechanisms of hypoxia induced CXCR4 expression. To compare the homing capability between CPCs and Hypo:CPCs in response to endogenous SDF-1 signal in acute ischemic myocardium, we used retrovirus (pCL-MFG-β-gal) to label c-kit
+
Lin
−
CPCs with β-gal, and then injected labeled CPCs and Hypo:CPCs (1×10
6
) intravenously via intra-jugular vein to mice 10 min after induction of MI in the C56BL/6 mice. We harvested the hearts 1d after cell transplantation to quantify the cell retention by chemiluminescent β-galactosidase assay. We observed that hypoxia treatment resulted in about 2.5 fold increase in β-gal cell recruitment in ischemic hearts (p<0.001, n=7– 8/group), however, AMD3100 can partially reduce Hypo:CPCs homing, therefore, the beneficial effects of Hypo:CPCs transplantation were mediated primarily through increasing cell homing via SDF-1:CXCR4 binding within the ischemic hearts. In addtion, the infusion of Hypo:CPCs also led to improved left ventricular performance, as assessed by LV development pressure by Millar catheter, by 23.67% compared with CPCs at 1 month after cell therapy (p<0.001, n=6/group). These results indicate that replenish CPC pool after AMI using ex vivo cultured CPC transplantation has significant beneficial effects on injured heart function dependent of hypoxia induced CXCR4 upregulation.
Abstract 885: Arginase Promotes Endothelial Dysfunction and Hypertension in Obesity
Authors: Durante William , Johnson Fruzsina K , Johnson Robert A , Azam Mohammed A , Peyton Kelly J
Published: July 2021

Endothelial dysfunction (ED) resulting from impaired nitric oxide (NO) synthesis is a salient feature in obesity and contributes to the development of cardiovascular disease, including hypertension. Recently, we reported that arginase, which metabolizes L-arginine to L-ornithine and urea, competes with endothelial NO synthase (eNOS) for substrate, L-arginine, and triggers ED in salt-sensitive hypertension. This study tests the hypothesis that arginase contributes to ED and hypertension by limiting L-arginine availability in obese Zucker rats (ZR), which are a well-established genetic model of obesity. Male obese (516 ± 8g) ZR display a significant increase in plasma (5.2 ± 0.3-fold) and vascular (57.6 ± 6.1%) arginase activity that is associated with a selective 2.2 ± 0.1-fold rise in vascular arginase I protein expression compared to age-and sex-matched lean (344 ± 8g) ZR. In contrast, arginase II and eNOS protein levels are unchanged in obese ZR. In obese ZR, the vasodilation of isolated, first-order gracilis muscle arterioles to acetylcholine (Δ
max
lean: 62 ± 7 vs obese 32 ± 4 μm) and luminal flow (Δ
max
lean: 22 ± 2 vs obese −3 ± 2 μm) are significantly impaired. However, acute in vitro pretreatment of arterioles with the arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC; 100μM) or L-arginine (1mM) restores endothelium-dependent dilation in obese ZR, and abolishes the difference between the obese and lean animals. However, D-arginine (1mM), which is not a substrate for eNOS, fails to restore endothelium-dependent dilation in obese ZR. Finally, obese ZR develop hypertension (158 ± 5mmHg) that is reversed by the daily administration of L-arginine (10g/L) in the drinking water (118 ± 5 mmHg) or by the chronic intraperitoneal administration of BEC (55.6μg/hour) via an osmotic minipump (115 ± 6mmHg) for one week. In contrast, the blood pressure of awake lean ZR (116 ± 4mmHg) is unaffected by either the chronic administration of L-arginine (112 ± 3mmHg) or BEC (111 ± 5mmHg). In conclusion, the present study indicates that obesity promotes ED and hypertension by restricting L-arginine availability via the induction of arginase I, and identifies arginase I as a novel therapeutic target in treating vascular disease.
Abstract 889: Statin Treatment Ameliorates Metabolic Syndrome in Mice Lacking All Nitric Oxide Synthase Isoforms
Authors: Nakata Sei , Tsutsui Masato , Shimokawa Hiroaki , Suda Osamu , Sabanai Ken , Yatera Yasuko , Shibata Kiyoko , Morishita Tsuyoshi , Tasaki Hiromi , Nakashima Yasuhide , Yanagihara Nobuyuki , Otsuji Yutaka
Published: July 2021

Background

: We have recently succeeded in developing mice in which all three nitric oxide synthase (NOS) genes are completely disrupted (triply n/i/eNOS
−/−
[KO] mice) (
PNAS
2005). Our preliminary study has revealed that those mice manifest metabolic syndrome. In this study, we thus examined whether or not statin treatment ameliorates those metabolic phenotypes.

Methods

: Experiments regarding metabolic phenotypes were performed in 3-month-old male wild-type (WT) and triply-KO mice treated with or without atorvastatin (30 mg/kg/day, SC) for 1.5 months. Experiments regarding coronary vascular lesion formation were carried out in 5-month-old mice treated with or without atorvastatin for 2 months.

Results

: Hypertension (HT) (140 ± 3 vs. 110 ± 3 mmHg), hypertriglyceridemia (TG) (267 ± 15 vs. 179 ± 12 mg/dl), impaired glucose tolerance (IGT) (plasma glucose after intravenous glucose, 493 ± 38 vs. 238 ± 16 mg/dl), insulin resistance (IR) (insulin-induced [
3
H]-glucose uptake in isolated soleus muscle, 106 ± 8 vs. 204 ± 30%), and visceral obesity (VO) (epididymal white adipose tissue weight, 0.25 ± 0.01 vs. 0.18 ± 0.01 g), were significantly noted in untreated triply-KO mice as compared with untreated WT mice (all
P <
0.05, n = 6 –12). Long-term treatment with atorvastatin significantly alleviated all the metabolic phenotypes in the triply-KO mice; HT (129 ± 1), TG (176 ± 11), IGT (342 ± 47), IR (120 ± 6) and VO (0.17 ± 0.01) (all
P <
0.05, n = 6 –12). Importantly, significant coronary vascular lesions, as characterized by neointimal formation (NF) (intima/media ratio, 0.15 ± 0.01 vs. 0.01 ± 0.01), medial thickening (MT) (media/vascular ratio, 0.43 ± 0.07 vs. 0.13 ± 0.01), and perivascular fibrosis (PF) (fibrosis/vascular ratio, 0.38 ± 0.05 vs. 0.18 ± 0.03) (all
P <
0.05), were spontaneously developed in the untreated triply-KO mice as compared with the untreated WT mice, all of which changes were again significantly suppressed by the atorvastatin treatment; NF (0.06 ± 0.02), MT (0.32 ± 0.02), and PF (0.17 ± 0.02) (all
P <
0.05, n = 6 each).

Conclusions

: These results provide the first evidence that statin treatment ameliorates metabolic syndrome as well as coronary arteriosclerosis in the presence of defective NOSs system, suggesting a novel therapeutic strategy for the disorder.
Abstract 910: Catecholaminergic Polymorphic Ventricular Tachycardia: Genetics, Natural History and Response to Therapy
Authors: Rossenbacker Tom , Bloise Raffaela , De Giuli Luciana , Raytcheva-Buono Emilia V , Theilade Juliane , Keegan Roberto , Drago Fabrizio , Gasparini Maurizio , Napolitano Carlo , Priori Silvia G
Published: July 2021

Background:
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmo-genic cardiac disease predisposing affected individuals to sudden cardiac death (SCD).

Methods and Results:
Here we provide data on the largest CPVT population thus far described. The study population consisted of 177 patients (64% female) of 93 families. All 93 probands (pbs) were screened for RYR2 mutations: 54 (58%) pbs carried a heterozygous RYR2 mutation, 1 pb a compound heterozygous RYR2 mutation and 3 pbs a homozygous (1), compound heterozygous (1) or heterozygous (1) CASQ2 mutation. One KCNJ2 mutation (T305I) was detected in a total of 15 pbs. In family members, another 40 RYR2 mutation carriers were identified. Only 29 of 95 gene carriers were phenotypically silent, therefore the penetrance of a RYR2 mutation was 69%. Of note, female sex was predominant among CPVT probands without a RYR2 mutation. Before the initiation of any treatment related to CPVT, 77 of 93 (83%) pbs experienced a cardiac e vent. Mean age of first event was 12±9 years. Neither sex nor the presence/absence of a RYR2 mutation was associated with a higher risk of developing events or with a younger age at onset of symptoms. Before therapy, 70% and 40% of 177 patients remained free of cardiac arrest and SCD before therapy by age 20 and 40 years, respectively. A history of juvenile SCD (<40 yrs) was present in 36% of families: the mean age of the lethal event was 18±9 yrs. One-hundred CPVT patients were initiated on β-blockers. On top, 41 patients were implanted with an ICD. During a mean follow-up time of 4.5±3 years, 28% of patients experienced a syncope (n=11), cardiac arrest (n=5) or appropriate ICD shock (n=12). Neither sex nor genotype was associated with a worse response to therapy.

Conclusions:
In this largest CPVT population reported so far, the presence/absence of a RYR2 mutation wasn’t associated with different clinical characteristics or response to therapy as compared to non-mutation carriers. CPVT remains a highly malignant disease with only a moderate response to β-blockers.