bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3565: Regulation of Myocardial Creatine Uptake by Intra- and Extracellular Creatine Levels
Authors: Makinen Kimmo , Hove Michiel ten , Sebag-Montefiore Liam , Hunyor Imre , Wallis Julie , Lygate Craig , Neubauer Stefan
Published: July 2021

Creatine plays an important role in energy metabolism in the heart. Cardiomyocytes accumulate creatine via a specific creatine transporter (CrT), the capacity of which is reduced in the failing heart, resulting in lower myocardial creatine concentration. To gain insight into how the CrT is regulated by intra- and extracellular creatine concentrations, we studied two mouse models of severely altered myocardial creatine levels, guanidinoacetate-N-methyl transferase knock out (GAMT−/−) mice and mice overexpressing the myocardial CrT (CrT-OE). We measured cardiac creatine uptake level in isolated creatine free GAMT−/− mouse hearts from using 14C-radiolabeled creatine, and found that creatine uptake capacity was massively elevated, with a 7-fold increase in Vmax. Cardiac creatine uptake levels in CrT-OE mice were increased on average 2.7-fold, showing a considerable variation, in line with a similar variation in creatine content as measured with HPLC. To understand whether the elevated CrT uptake was due to transcriptional regulation, we measured CrT mRNA levels in the GAMT−/− mouse hearts, but found only a 1.4-fold increase relative to WT values, and no significant difference relative to CrT mRNA levels in the CrT-OE mice. However, the increase in Cr uptake and in CrT mRNA levels in the GAMT−/− mice was prevented when mice were fed a creatine-supplemented diet, indicating that creatine uptake is subject to negative feedback regulation. To determine whether high intracellular creatine concentrations could downregulate CrT transcription, we correlated the expression of total and endogenous CrT mRNA with myocardial creatine levels in the CrT-OE mouse hearts. Total CrT mRNA (transgenic CrT + endogenous CrT) levels correlated well with myocardial creatine content (r=0.67; p<0.0001), but endogenous CrT mRNA levels did not correlate at all with myocardial creatine content (r=0.01;p=0.96), suggesting that intracellular creatine levels did not influence CrT transcription. In summary, creatine uptake can be massively upregulated in the heart and this upregulation is subject to feedback inhibition. In addition, our results strongly suggest that CrT activity is predominantly regulated by mechanisms other than alterations in gene expression.
Abstract 3571: Evidence for Rho-Kinase Activation in Patients with Pulmonary Hypertension
Authors: Doe Zhulanqiqige , Fukumoto Yoshihiro , Takaki Aya , Tawara Shunsuke , Ohashi Junko , Nakano Makoto , Tada Tomohiro , Saji Kenya , Sugimura Kohichiro , Fujita Hiroshi , Nawata Jun , Hoshikawa Yasushi , Kondo Takashi , Shimokawa Hiroaki
Published: July 2021

Pulmonary hypertension (PH) still remains a fatal disease characterized by hyperconstriction and remodeling of pulmonary arteries (PA). We and others have previously demonstrated that long-term inhibition of Rho-kinase is useful for treatment of PH in animal models, however, it remains to be examined whether Rho-kinase is actually activated in patients with PH. First, we examined whether Rho-kinase activity is enhanced in circulating neutrophils from 40 healthy age- and gender-matched controls and 40 PH patients with various etiologies, including idiopathic PAH (n=18), and PH associated with connective tissue diseases (n=8), congenital heart diseases (n=7), or chronic thromboembolism (n=7). We measured total and phosphorylated forms of myosin binding subunit (MBS), a substrate of Rho-kinase, by Western blotting, and defined the p-MBS/t-MBS ratio as an index of systemic Rho-kinaes activity. Next, we examined Rho-kinase activity by immunostaining in lung tissues from 5 controls and 5 IPAH obtained during lung surgery and transplantation, respectively. Finally, we examined vascular responses of isolated small PA from those subjects in vitro. Systemic Rho-kinase activity was significantly increased in the PAH patients compared with the controls (P<0.0001). Among the 4 subgroups of PH, Rho-kinase activity was significantly increased in all except for PH with thromboembolism (P<0.05). Significant correlations were noted between Rho-kinase activity and mean PA pressure, pulmonary vascular resistance, and duration of the disorder in the PH patients (all P<0.05). Rho-kinase expression in small PA and Rho-kinase activity in the lung tissue also were significantly increased in the PAH patients compared the controls (both P<0.0001). Endothelium-dependent relaxations were markedly impaired and serotonin-induced contractions were markedly enhanced in the PAH patients compared with the controls, and the hypercontractions were abolished in the presence of hydroxyfasudil, a specific Rho-kinase inhibitor (all P<0.01). These results provide the first direct evidence for Rho-kinase activation in patients with PH, confirming the therapeutic importance of Rho-kinase in the treatment of PH in humans.
Abstract 3574: A Unique Model and Mechanisms of Hemolysis-Associated Pulmonary Hypertension
Authors: Hu Weiguo , Jin Richard , Halperin Jose A , Loscalzo Joseph , Qin Xuebin
Published: July 2021

Pulmonary hypertension is a common complication of intravascular hemolysis and is suspected to be a strong mortality determinant of hemolytic disorders. However, the pathophysiologic mechanisms leading to hemolytic anemia-associated pulmonary hypertension remains unclear. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by
Streptococcus intermedius
that lyses only human cells due to its unique receptor specificity for human CD59 (hCD59). We recently generated a novel intravascular hemolysis mouse model in which ILY exclusively lyses erythrocytes transgenically expressing hCD59 (
ThCD59RBC
mice) (
Nature Medicine, 2008, 14: 98 –103
). Using this model, we investigated the pathogenesis of hemolytic anemia-associated pulmonary hypertension. In
ThCD59RBC
mice, but not wild type mice, intravenous injection of ILY with a 1.5-fold lethal dose induced rapid intravascular hemolysis with acute death (less than 1 minute) that was preceded by a sharp increase in the pulmonary pressure (10.40 ± 0. 89 mmHg increase, n = 5) with 0.65 ± 0.36 mmHg/second rate of rise of systolic pulmonary artery pressure as measured by right-heart catheterization. We also observed decreased NO levels indirectly measured by plasma nitrate + nitrite concentration [10.01 ± 2.47μM in ThCD59RBC (n=3) vs 22.16 ± 2.20 μM in WT (n=3);
P
<0.001] and increased plasma P-selectin levels [445.65 ± 54.85 ng/ml in ThCD59RBC (n=4) vs 186.95 ± 18.64 ng/ml in WT (n=4);
P
<0.001]. These results suggest that the lethality of acute intravascular hemolysis may result from a sudden increase in pulmonary pressure likely a consequence of the reduced bioavailability of NO and resulting pulmonary vasoconstriction and platelet activation.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3594: Nogo-B is Essential for Macrophage Dependent Inflammatory Arteriogenesis and Angiogenesis
Authors: Yu Jun , Fernandez-Hernando Carlos , Suarez Yajaira , Suarez Yajaira , Schleicher Michael , Hao Zhengrong , Wright Paulette L , Kyriakides Themis R , Sessa William C
Published: July 2021

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial, vascular smooth muscle and inflammatory cells. We previously have shown Nogo-B plays a critical role in vascular remodeling after injury
in vivo
. In mice and rabbits, neointimal expansion of injured blood vessels is associated with a marked reduction in Nogo-B, and, in humans, the loss of Nogo-B correlates with stenotic lesions and plaque rupture. However, the role of Nogo-B in regulating arteriogenesis or angiogenesis is not known. In the present study, we investigated the role of Nogo-B in arteriogenesis/angiogenesis using a mouse hindlimb ischemia model in Nogo-A/B KO (NgKO) and wild-type (WT) littermate control mice. Nogo-B mRNA and protein levels increased in the ischemic leg relative to the contralateral leg in WT. Hindlimb ischemia reduces tissue perfusion by 80% followed by a complete recovery of flow to pre-surgical values in WT after 4 weeks. In NgKO, there was a marked decrease in blood flow recovery post-ischemia, which was accompanied by a reduction in capillary density (assessed by isolectin staining), a decrease in smooth muscle/pericyte recruitment and an impaired collateral artery remodeling (assessed by quantitative angiography) compared to WT. Blood vessel formation and remodeling during ischemia requires coordination of the inflammatory response with regulated gene expression that promotes blood vessel assembly. Thus, we assessed the presence of tissue macrophages (F4/80 immunochemistry) 3 days after ischemia and found a 76% reduction of F4/80 positive cells in ischemic tissue in NgKO compared to WT. Mechanistically, the impaired recruitment of tissue macrophages was associated with reduced spreading, impairment of chemotaxis in response to monocyte chemokines compared to WT cells (76±4 vs. 113±8 cells/field in NgKO vs. WT), but not adhesion, of isolated bone marrow derived monocytes from NgKO compared to controls. Moreover, NgKO macrophage demonstrated impaired Rac activation, translocation and cytoskeletal organization upon stimulation of with chemokines. In conclusion, our data suggests that endogenous Nogo coordinates macrophage mediated inflammation with angiogenesis, arteriogenesis and blood flow control.
Abstract 3595: Chloride Intracellular Channel-4 Determines Native Collateral Density
Authors: Chalothorn Dan , Zhang Hua , Smith Jennifer E , Gordon Nicarter , Edwards John C , Faber James E
Published: July 2021

The density of native (pre-existing) collaterals in healthy tissue and their capacity to enlarge in occlusive disease (arteriogenesis) are major determinants of tissue injury in ischemia. Mechanisms regulating arteriogenesis remain unclear. Moreover, nothing is known about what specifies formation of native collaterals, except our report that VEGF contributes to native collateral formation (Clayton et al FASEB J 21:LB352, 2007). Recently, chloride intracellular channel proteins (CLICs) have been implicated in cellular processes including cell hollowing. CLIC1 and CLIC4 are expressed by endothelial cells, and VEGF-induced cell hollowing is critical for vascular formation in the embryo and angiogenesis in ischemia. Therefore, we examined CLIC1−/− and CLIC4−/− (null) mice for effects on native collateral density in healthy tissues and on arteriogenesis and angiogenesis in a hindlimb model of ischemia. Responses of CLIC1−/−mice did not differ from wild-type in any experiment. In contrast, CLIC4−/− showed a greater reduction in perfusion immediately after femoral ligation (p<0.05 here and all statements below). Since baseline capillary density and collateral diameter were not different from wild-type, this suggested CLIC4−/− have reduced native collateral density. This was confirmed angiographically. In agreement, during 10 days after ligation CLIC4−/− mice had worse recovery of perfusion, greater ischemia and atrophy, and more impaired limb use, whereas collateral remodeling was unaffected. While ischemia increased expression of CLIC1 and CLIC4 similarly in wild-type mice, CLIC1 was augmented 3-fold in CLIC4−/−, suggesting CLIC1 may compensate for absence of CLIC4. Despite greater ischemia in CLIC4−/− mice, HIF1α and VEGF increased less in CLIC4−/−, suggesting CLIC4 may reside upstream of Hif1α-VEGF signaling. Native collateral density in the cerebral circulation of CLIC4−/− mice (but not CLIC1−/−) was also greatly reduced, resulting in 4-fold larger infarctions. This reduced collateral density in adults was associated with reduced formation and stabilization of nascent collaterals in the newborn. Hence, CLIC4 represents the second gene identified, besides VEGF, that specifies formation of the native collateral circulation.
Abstract 3625: Targeted Ultra-Small Iron Oxide Particles for In Vivo MR Detection of Atherosclerotic Lesions Using Antibodies against Oxidized Low Density Lipoprotein
Authors: Briley-Saebo Karen , Shaw Peter X , Calcagno Claudia , Choi Seung-Hyuk , Fuster Valentin , Tsimikas Sotirios , Fayad Zahi A
Published: July 2021

Background: Oxidized low-density lipoprotein (OxLDL) plays a key role in the progression and destabilization of atherosclerotic plaque. Preliminary studies indicate that gadolinium (Gd) micelles labeled with oxidation-specific antibodies allow for specific targeting of vulnerable plaque. Due to issues associated with Gd toxicity (caused by retention and bio-transformation of the micelles), clinical translation of this platform may be limited. Iron oxides are recognized as safe and effective contrast agents for MRI. As a result, it was hypothesized that lipid coated ultra-small iron oxide particles (LUSPIOs) containing murine antibodies that bind malondialde-hyde lysine (MDA2) or oxidized phospholipids (EO6) associated with oxLDL may allow for the development of a safe platform for in vivo detection of vulnerable plaque.

Materials and Results: Untargeted, MDA2, and EO6 labeled LUSPIOs were prepared and characterized with respect to efficacy, pharmacokinetics, and biodistribution in Apolipoprotein deficient mice (ApoE−/−). MRI was performed at 9.4T prior to and 24 hrs after LUSPIO administration (4 mg Fe/kg) using both gradient echo and positive contrast GRASP sequences. MDA2 and EO6 increased the particle size, blood half-life, and MR efficacy of the LUSPIOs, relative to untargeted particles. Only limited signal attenuation was observed following administration of untargeted LUSPIO. MRI and microscopy revealed that MDA2 and EO6 LUSPIOs accumulate within oxLDL rich foam cells (Fig.1

).

Conclusions: This study suggests that biocompatible LUSPIOs may provide a clinically translatable platform for the MR detection of oxLDL rich foam cells in humans.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3628: Neuronal Nitric Oxide Synthase (nNOS) Regulates Basal Flow in the Human Coronary Circulation I
n Vivo
Authors: Seddon Mike , Chowienczyk Phil , Melikian Narbeh , Dworakowski Rafal , Casadei Barbara , Shah Ajay
Published: July 2021

Endothelial NO synthase (eNOS) is thought to be the major source of nitric oxide (NO) involved in the local regulation of human vascular tone. However, in studies using a selective neuronal NOS (nNOS) inhibitor S-methyl-L-thiocitrulline (SMTC), we recently reported that basal human forearm blood flow is regulated by nNOS. SMTC had no effect on acetylcholine-induced vasodilatation which however was inhibited by the non-selective NOS inhibitor N
G
monomethyl-L-arginine (L-NMMA). This study investigated the effects of nNOS in the human coronary circulation
in vivo
. We studied patients undergoing diagnostic cardiac catheterisation who had angiographically normal coronary arteries. Coronary flow velocity was measured by an intracoronary Doppler wire and epicardial artery diameter by QCA. We compared the effects of intracoronary SMTC or L-NMMA infusion on basal flow and the responses to substance P and isosorbide dinitrate (endothelium-dependent and -independent dilators, respectively). L-NMMA (25 μmol/min) reduced basal coronary flow by 22.3±5.3% and inhibited dilation to substance P (20 pmol/min) by 57±5.7% (n=8; both P<0.01). SMTC (0.625 μmol/min) also reduced basal flow (−34.8±6.3%; n=8; P<0.01), but had no effect on the response to substance P (inhibited by −2±14%; P=NS). The effects of SMTC were abolished by L-arginine (240μmol/ min; n=3). Both L-NMMA and SMTC reduced epicardial artery diameter (−2.5±0.6% and −2.8±0.9% respectively; P<0.05) but only L-NMMA reduced dilatation to substance P (5.6±1.3% before versus 3.0±0.8% after L-NMMA; P<0.05). These data indicate that local nNOS-derived NO regulates basal coronary blood flow in humans
in vivo
, whereas substance P-stimulated vasodilatation is eNOS-mediated. Our results indicate that nNOS and eNOS have distinct local roles in the physiological regulation of human coronary vascular tone
in vivo
.
Abstract 3629: Crucial Role of Nitric Oxide Synthases System in Endothelium-Dependent Hyperpolarization in Mice
Authors: Takaki Aya , Morikawa Keiko , Murayama Yoshinori , Tekes Ender , Yamagishi Hiroto , Ohashi Junko , Tsutsui Masato , Yada Toyotaka , Yanagihara Nobuyuki , Shimokawa Hiroaki
Published: July 2021

We have previously demonstrated that endothelium-derived hydrogen peroxide (H
2
O
2
) is an endothelium-derived hyperpolarizing factor (EDHF) in mouse and human mesenteric arteries and porcine coronary microvessels. We also have demonstrated that endothelial NO synthase (eNOS) is a major source of EDHF/H
2
O
2
, where Cu,Zn-superoxide dismutase (SOD) plays an important role to dismutate eNOS-derived superoxide anions to EDHF/H
2
O
2
in animals and humans. However, the mechanism for the endothelial production of H
2
O
2
as an endogenous EDHF remains to be elucidated. Indeed, some EDHF-mediated responses still remain in singly eNOS
−/−
mice and the remaining responses are also sensitive to catalase that dismutates H
2
O
2
to form water and oxygen. It is widely known that 3 NOS isoforms (neuronal, inducible, and endothelial) compensate each other. In this study, we examined the effects of genetic disruption of all NOS isoforms (n/i/eNOS
−/−
) on EDHF responses in mice. We examined the contribution of the whole NOS system to EDHF-mediated responses in eNOS
−/−
, n/eNOS
−/−
and n/i/eNOS
−/−
mice that we have recently generated. Isometric tensions and membrane potentials were recorded by organ chamber experiments and microelectrode technique, respectively. EDHF-mediated relaxations and hyperporalizations in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased (n=6 each), whereas vascular smooth muscle functions were preserved (n=6 each). Expressions of endothelial NOS isoforms in the NOSs
−/−
mice were compensated by NOS gene that had not been disrupted (n=5 each). Laser confocal microscopic examination demonstrated that endothelial H
2
O
2
and superoxide production was absent in n/i/eNOS
−/−
mice (n=3–5), whereas antihypertensive treatment with hydralazine failed to improve the EDHF-mediated responses (n= 4). Involvement of NOS uncoupling was ruled out as modulation of BH
4
synthesis had no effects (n=6–7) and BH
4
/BH
2
ratio (an index of BH
4
bioavailability) was preserved (n=4). These results provide the first direct evidence that EDHF-mediated responses are totally dependent on endothelial NOSs system in mouse mesenteric arteries.
Abstract 3645: RAGE Impairs Angiogenesis Consequent to Femoral Artery Ligation in Diabetic Mice
Authors: Shen Xiaoping , Belov Dmitri , Rosario Rosa , Naka Yoshifumi , Schmidt Ann Marie , Yan Shi Fang
Published: July 2021

The Receptor for Advanced Glycation Endproducts (RAGE) is implicated in the pathogenesis of diabetic complications. Previous studies illustrated that blockade/genetic deletion of RAGE prevented aberrant neointimal expansion in atherosclerosis and carotid artery balloon injury. Here, we tested the hypothesis that RAGE would contribute to impaired revascularization in the setting of acute femoral artery (FA) ligation, especially in diabetes. Wild type (WT) and RAGE
−/−
mice were rendered diabetic (D) with streptozotocin (stz); non-diabetic (ND) cohorts were treated with buffer alone. RAGE antigen (Western blotting) was upregulated ≈2.1 fold in the ipsilateral muscle tissue of animals subjected to arterial ligation beginning on day 3 and persisting through 28 days post-operatively; confocal microscopy localized RAGE expression to cells expressing CD31 (EC) and SM actin (SMC). A key proinflammatory RAGE ligand, S100B, was highly upregulated (Western blot) in the ipsilateral muscle tissue from day 1 through 14 after ligation and localized to cells expressing CD45 (inflammatory) and vascular cells (expressing CD31 and SMA). To test the role of RAGE in FA ligation, ND and D mice were subjected to surgery; immediately after surgery, animals received either RAGE ligand-binding decoy soluble RAGE (sRAGE), 200 μg, i/p per day for 7 days) or vehicle, PBS. In D mice, administration of sRAGE resulted in improved blood flow ratios on days 3 and 7 after FA ligation vs. vehicle (
p
<0.01). To dissect the particular role of RAGE, we tested homozygous RAGE null mice. Both ND and D RAGE
−/−
mice displayed improved blood flow ratios (laser Doppler) on days 28 after FA ligation compared to ND and D WT mice (
p
<0.05 &
p
<0.01). Immunohistochemistry staining with CD31 on ipsilateral muscle tissue demonstrated significantly increased microvessel density in D RAGE
−/−
mice on days 28 after FA ligation compared to D WT mice (
p
<0.001). Together, these data link the ligand/RAGE axis to impaired vascular responses consequent to severe tissue ischemia in the peripheral vasculature, particularly in diabetes.
Abstract 3662: Involvement Of The Small Gtpase Rap1 In Integrin Signaling In Endothelial Cells And Postnatal Neovascularization
Authors: Carmona Guillaume , Orlandi Alessia , Reinhard Henschler , Zeiher Andreas. M , Dimmeler Stefanie , Chavakis Emmanouil
Published: July 2021

Ras associated protein 1 (Rap1), a small GTPase of the Ras family, has attracted much attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes for integrin signaling in endothelial cells (EC) and angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVEC) express Rap1a and Rap1b mRNA as assessed by RT-PCR. In order to determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase activating protein, which specifically inhibits the activity of both Rap1a and Rap1b. Overexpression of Rap1GAP1 led to a significant inhibition of angiogenic sprouting of HUVEC under basal conditions and bFGF stimulation by 44 ± 5 % in a 3-dimensional spheroidal system and blocked tube formation in a matrigel assay, migration and adhesion. In order to separately investigate the role of Rap1a and Rap1b genes in angiogenesis, we performed gene silencing with siRNA. Silencing of either Rap1a or Rap1b significantly and additively blocked the sprouting of HUVEC under basal and bFGF-stimulated conditions (Rap1a-siRNA: 55 ± 5 %, Rap1b-siRNA: 61 ± 9 % and Rap1a+Rap1b siRNA: 73 ± 5% inhibition) and significantly reduced HUVEC migration and adhesion on fibronectin and collagen. Moreover, silencing of Rap1a and Rap1b reduced beta1-integrin affinity in HUVEC, suggesting the importance of Rap1a and Rap1b for inside-out integrin activation in EC. In addition, silencing of Rap1a and Rap1b prevented VEGF-induced PKB/Akt1 activation. These data prompted us to investigate the
in vivo
role of Rap1a using Rap1a-deficient mice. Interestingly, Rap1a
−/−
mice are born with a substantially reduced mendelian ratio. Rap1a
+/−
heterozygote mice displayed decreased microvessel density in comparison to wild-type mice (Rap1a
+/+
) in a matrigel plug assay. Moreover Rap1a
+/−
and Rap1a
−/−
displayed significantly reduced microvessel density in ischemic muscles in the model of hind limb ischemia in comparison to wild-type mice (Rap1a
+/−
: 32 ± 3 % ; Rap1a
−/−
: 43 ± 3 % inhibition). Thus, our data demonstrated a critical role of Rap1 in the regulation of β1-integrin signaling in endothelial cells and for postnatal neovascularization.
Abstract 3682: Heterozygous Knockout of the Insulin-like Growth Factor 1 Receptor in Mice Leads to Enhanced Insulin Sensitivity and Increased Endothelial Nitric Oxide Production
Authors: Abbas Afroze , Imrie Helen , Rajwani Adil , Viswambharan Hema , Cubbon Richard M , Kahn Matthew B , Wheatcroft Stephen B , Grant Peter J , Kearney Mark T
Published: July 2021

Introduction Insulin like growth factor-1 (IGF-1) enhances glucose uptake and nitric oxide (NO) production, acting via similar signalling pathways to insulin. Several cross sectional and longitudinal studies have demonstrated an inverse relationship between IGF-1 bioactivity and cardiovascular disease. We assessed the hypothesis that a putative mechanism for these findings may be the modulation of endothelial NO bioavailability by IGF-1.

Methods and Results We investigated the effect of reduced IGF-1 bioactivity on metabolic and endothelial function in male mice, heterozygous for knockout of the IGF-1 receptor (IGF1RKO) and their wildtype littermates (WT). Glucose tolerance tests revealed impaired glucose handling in IGF1RKO mice compared to WT (mean AUC for IGF1RKO mice=1086 ± 26.82(mmol/L)minutes, mean AUC for WT mice=950.5 ± 22.5(mmol/L)minutes, n=5 in each group, p=0.005). However, using an insulin tolerance test, IGF1RKO mice were more insulin sensitive than controls (mean AUC for IGF1RKO=552.2 ± 25.18(mmol/L)minutes, n=10; mean AUC for WT=651.4 ± 22.26(mmol/ L)minutes, n=11, p=0.008). Ex-vivo assessment of vascular function was performed using thoracic aortic rings in an organ bath. Aortic rings from IGF1RKO mice were hypocontractile to phenylepherine compared to those from WT (mean Emax for IGF1RKO mice=0.60 ± 0.05g n=11 versus mean Emax for WT mice=0.79 ± 0.06g, n=10, p=0.03). Addition of the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), led to a 65.40 ± 13.40% (n=9) increase in mean Emax in IGF1RKO mice compared to a 15.78 ± 12.00% (n=9) increase in WT mice (p=0.01). In addition IGF1RKO aortic rings were resistant to IGF-1 exposure compared to WT (percentage change in mean Emax for IGF1RKO mice=32.80 ± 11.01%, n=9 versus 66.28 ± 10.60% in WT mice, n=8, p=0.05), but maintained sensitivity to insulin.

Conclusion IGF1RKO mice demonstrate impaired glucose handling but enhanced sensitivity to insulin compared to WT. Aortic rings from IGF1RKO mice are hypocontractile to phenylepherine and have increased basal NO production. These data raise the possibility that reduced IGF-1 action in the endothelium may have a favourable effect on NO bioavailability, possibly by a novel interaction with insulin signalling.
Abstract 3710: Estrogen Does not Account for Accelerated Atherosclerosis in LDLr−/− Mice
Authors: King Victoria L , Hatch Nicholas , Zhang Xuan , Tannock Lisa R
Published: July 2021

Clinical studies demonstrate less atherosclerosis in pre-menopausal women compared to age-matched men, but an equalization in atherosclerosis burden and cardiovascular event rates between genders following menopause. Conversely, using the LDLR−/− mouse model we and others have previously demonstrated that young female mice develop accelerated atherosclerosis compared to age matched males. Whether this difference is due to sex hormones or differences in metabolic factors is not clear. To determine if estrogen mediated the alterations in atherosclerosis in the female mice, female mice were ovariectomized (Ovx). Ovx mice had a marked reduction in uterus weight (Sham: 86 ± 1 vs Ovx: 26 ± 1mg, P < 0.001) and both Ovx females and males had greater body weight gain when fed a lard-enriched diet (10% kcal from fat,
D12451
, Research Diets) for 17 weeks compared to sham females. Ovariectomy resulted in an increase in fasting glucose concentrations, which was comparable to males. Cholesterol and triglyceride concentrations were higher in both sham and Ovx females compared to males; primarily distributed in increased VLDL and LDL particles. Interestingly, ovariectomy had no significant effect on extent of atherosclerotic lesion formation in female mice and the extent of atherosclerotic lesion area in both female groups was significantly increased compared to male mice. These data suggest that gender specific differences in lipids and atherosclerotic lesion formation in female LDL-R deficient mice fed a diet enriched in lard are not mediated by estrogen.
Abstract 3711: Increased Expression of Plasma Granzyme B in the Patients with Coronary Artery Disease Complicated with Chronic Kidney Disease
Authors: Ikemoto Tomokazu , Hojo Yukihiro , Kondo Hideyuki , Takahashi Nozomu , Hirose Masahiro , Ohya Ken-ichi , Katsuki Takaaki , Shimada Kazuyuki
Published: July 2021

Background:
Recent clinical studies have revealed that chronic kidney disease (CKD) is a significant independent risk factor for major cardiovascular events. However, the mechanisms in which CKD increases the incidence of cardiovascular diseases are still not fully understood.

Hypothesis:
Granzyme B is a member of serine protease family released from cytotoxic T lymphocytes and plays an important role in cellular apoptosis via an activation of caspases. We hypothesized that granzyme B is involved in formation of coronary artery lesions in patients with CKD.

Objectives:
To elucidate the role of granzyme B in coronary artery disease (CAD) in patients complicated with CKD.

Subjects and methods:
We employed 141 patients (116 men and 25 women, mean age: 64.2±9.6 years old ). Diagnosis of CAD was confirmed by selective coronary angiography. CKD was defined as sustained decrease in estimated glomerular filtration rate less than 60 mL/min/1.73m
2
over 3 months. We assigned patients into three groups; CAD without CKD (CAD group, n=46), CKD without CAD (CKD group, n=17) and CAD with CKD (CAD/CKD group, n=78). Patients with acute coronary syndrome, immunological disorder, malignancy and acute inflammatory diseases were excluded from the study.

Results:
Analysis of variance showed that plasma levels of high-sensitive CRP (hsCRP) and granzyme B in CAD/CKD group were significantly higher than those in the other groups (hsCRP: CAD; 2420±763, CKD; 1802±584, CAD/CKD: 7419±2171 ng/mL, p<0.05, granzyme B: CAD: 9.1±2.3, CKD: 42.2±2.4, CAD/CKD: 159±51.8 pg/mL, p<0.01). A significant positive correlation was observed between plasma hsCRP and granzyme B levels (r=+0.30, p<0.05). A significant negative correlation was observed between eGFR and granzyme B levels (r=−0.32, p<0.01). Multiple regression analysis revealed that granzyme B and hs CRP levels were independent predicting variables for the number of stenosis in major coronary arteries (F=5.35, p=0.002, beta coefficients: granzyme B=+0.24, p=0.006, hsCRP=+0.18, p=0.03).

Conclusions:
These results indicate that granzyme B might be a critical enzyme for formation of coronary atherosclerosis by inducing apoptosis of vascular tissues in patients with CKD.
Abstract 3752: Mineralocorticoid Receptor Antagonism Prevents Aldosterone Mediated Dysfunction of Human Endothelial Progenitor Cells in Vitro, in Vivo and in Patients
Authors: Thum Thomas , Schmitter Kerstin , Fraccarollo Daniela , Widder Julian , Georg Ertl , Bauersachs Johann
Published: July 2021

Hyperaldosteronism has been associated with vascular injury. Bone marrow derived endothelial progenitor cells (EPC) play an important role in endothelial repair and vascular homeostasis. We therefore examined the effects of aldosterone and its blockade by the mineralocorticoid receptor (MR) antagonist eplerenone on EPC number and function
in vitro
,
in vivo
and a
clinical trial
. EPCs expressed the MR at the gene and protein level. Aldosterone treatment of human EPC led to a translocation of the MR into the nucleus and impaired multiple cellular functions, such as differentiation, migration, proliferation and incorporation in vascular networks. Impaired EPC function was rescued by pharmacological antagonism or genetic ablation of the MR. Aldosterone increased the intracellular production of reactive oxygen species (ROS), which was attenuated by MR blockade. Aldosterone-mediated increase of ROS was mainly protein kinase A-dependent and resulted in cAMP activation with subsequent impairment of EPC migration. Chronic aldosterone infusion in mice (implanted minipumps, 50mg/kg/d for 14d) impaired EPC functionality and resulted in endothelial dysfunction. MR antagonism prevented those effects. In patients with hyperaldosteronism, EPC displayed reduced migratory potential compared with age-matched healthy controls. Impaired function of EPC from patients with hyperaldosteronism significantly correlated with endothelial dysfunction assessed by peripheral arterial tonometry. Importantly, MR antagonism in patients with hyperaldosteronism improved EPC as well as endothelial function. EPC function is impaired in hyperaldosteronism by increased intracellular ROS production and cAMP activation. Beneficial effects of MR antagonists in cardiovascular disease prevention and therapy especially during hyperaldosteronism may be mediated in part by improved EPC biology.
Abstract 3758: Preventive Effects of Heregulin-Beta1 on Macrophage Foam Cell Formation and Atherosclerosis
Authors: Watanabe Takuya , Iso Yoshitaka , Koba Shinji , Sakai Tetsuo , Xu Gang , Arita Shigeko , Hongo Shigeki , Hirano Tsutomu , Kobayashi Youichi , Miyazaki Akira
Published: July 2021

Human heregulins, neuregulin-1 type I polypeptides known to activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, were recently found to be expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester (CE) accumulation, is modulated by scavenger receptor class A (SR-A), acyl-CoA:cholesterol acyltransferase-1 (ACAT1), and ATP-binding cassette transporter A1 (ABCA1). The present study clarified the functional roles of heregulins in macrophage foam cell formation and atherosclerosis. Plasma heregulin-beta1 levels were significantly decreased in 31 patients with acute coronary syndrome (ACS) and 33 patients with stable angina pectoris as compared with 34 mild hypertensive patients and 40 healthy volunteers (1.3+/−0.3, 2.0+/−0.4 versus 7.6+/−1.4, 8.2+/−1.2 ng/mL; at least
P
< 0.01). Immunoreactive heregulins and these receptor c-erbB3 were detectable within human coronary atherothrombosis obtained from ACS patients. In primary cultured human monocyte-macrophages, the expression of endogenous heregulins, heregulin-beta1, and c-erbB3 increased during monocytic differentiation into macrophages. In human macrophages differentiated by 7-day culture, exogenous heregulin-beta1, but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein (acLDL)-induced CE accumulation by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta1 significantly decreased endocytic uptake of [
125
I]acLDL and increased cholesterol efflux by apolipoprotein A1 from human macrophages. Chronic infusion of heregulin-beta1 by osmotic mini-pumps into apolipoprotein E-deficient mice significantly suppressed the progression of macrophage-driven atherosclerotic lesions by 64%. Our study provides the first evidence that heregulin-beta1 may participate in anti-atherogenesis by suppressing macrophage foam cell formation
via
SR-A and ACAT1 down-regulation and ABCA1 up-regulation.
Abstract 3781: Identification Of AS160 As An Insulin, Ischemia, And Exercise Regulated Signaling Protein In The Heart
Authors: An Ding , Toyoda Taro , Taylor Eric B , Hirshman Michael F , Goodyear Laurie J
Published: July 2021

The maintenance of glucose utilization is necessary for normal cardiac function, yet the distal signaling mechanisms mediating myocardial glucose uptake are not fully understood. Studies using adipocytes and skeletal muscle demonstrate that the Akt substrate of 160 kDa (AS160) is essential for GLUT4 translocation and glucose uptake. AS160 is a Rab GTPase-activating protein (GAP) containing 6 consensus Akt phosphorylation sites collectively referred to as “PAS” sites. Phosphorylation of PAS sites inactivates the GAP domain, resulting in GLUT4 translocation. Despite the importance of AS160 in glucose uptake, there have been no studies of AS160 in the heart. Using phosphoproteomics we identified a novel phosphorylation site, Ser
711
, which is an AMPK consensus sequence. We next determined if AS160 is phosphorylated on PAS and Ser
711
sites in response to 3 stimulators of glucose uptake in the heart, insulin (max dose, ip, 10 min), ischemia (global, 3 min), and exercise (treadmill running, 30 min). In addition, we used Akt2 knockout (KO) and AMPKα2 inactive transgenic mice (AMPKα2i TG) to assess upstream signaling to AS160. Insulin robustly increased AS160 PAS phosphorylation (450% above basal; p<0.0001), an effect partially inhibited in Akt2KO mice (P<0.05). Surprisingly, despite being an AMPK consensus sequence, insulin increased phosphorylation of Ser
711
(30%; P<0.01), an increase abolished in Akt2KO mice. Neither ischemia nor exercise increased AS160 PAS phosphorylation. However, ischemia increased Ser
711
phosphorylation (70%; P<0.01), an effect partially inhibited in AMPKα2i TG mice. Exercise slightly increased Ser
711
phosphorylation (20%; P<0.02), which was abolished in AMPKα2i TG mice. To determine if a model of decreased cardiac glucose uptake has altered AS160 signaling, we used the db/db mouse and found a significant impairment in insulin-stimulated PAS and Ser
711
phosphorylation. In conclusion, this is the first report showing that insulin, ischemia, and exercise increase AS160 phosphorylation in the heart. These stimuli elicit distinct patterns of phosphorylation on PAS and the novel Ser
711
site. Akt2 and AMPKα2 are upstream signals mediating AS160 phosphorylation. AS160 is likely to be a critical signaling mechanism in the heart.
Abstract 3829: Endothelial Cell Response to Hypoxia is Modulated by Microrna-210 Direct Targeting of Ephrin-A3
Authors: Fasanaro Pasquale , D’Alessandra Yuri , Di Stefano Valeria , Melchionna Roberta , Romani Sveva , Capogrossi Maurizio C , Martelli Fabio
Published: July 2021

MicroRNAs (miRNAs) are small non-protein-coding RNAs that negatively modulate gene expression. In this study, miRNAs role in endothelial cell response to hypoxia was investigated. It was found that the expression of miR-210 progressively increased upon exposure to hypoxia. Interestingly, HIF1-alpha was necessary and sufficient for miR-210 induction, while HIF2-alpha was not necessary. miR-210 over-expression in normoxic endothelial cells increased the formation of capillary-like structures on Matrigel and VEGF-induced cell migration. Conversely, miR-210 blockade via LNA-anti-miRNA transfection decreased the formation of capillary-like structures stimulated by hypoxia and inhibited cell migration in response to VEGF. miR-210 over-expression did not affect endothelial cell growth in both hypoxia and normoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, both in normoxia and in hypoxia. We found that one relevant target of miR-210 in hypoxia was Ephrin-A3, since miR-210 was necessary and sufficient to down-regulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences: indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210, prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. A combination of proteomic and transcriptomic approaches has been undertaken, identifying potential direct and indirect targets of miR-210 action. We conclude that miR-210 induction is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration and differentiation.
Abstract 3832: Mir429 Regulates NO-dependent Differentiation Of Mouse Embryonic Stem Cells
Authors: Rosati Jessica , Spallotta Francesco , Vincenti Sara , Arcelli Diego , Presutti Carlo , Capogrossi Maurizio C , Gaetano Carlo , Illi Barbara
Published: July 2021

BACKGROUND: microRNAs (miRs) are emerging as important regulators of many cell functions and, possibly, also embryonic stem cells (ES) differentiation. Recently, it has been shown that specific microRNAs may allow ES to discriminate among different developmental lineages. Nitric oxide is a key regulator of vascular function and induces cardiogenesis in embryonic stem cells; however, no data are available about the effect of NO on microRNAs expression patterns, neither in adult nor in stem cells.

METHODS AND RESULTS: mouse ES were deprived of Leukemia Inhibitory Factor (LIF) and treated with the NO donor diethylenetriamine nitric oxide adduct (DETA/NO) for 90 minutes and microRNA microarray analysis was performed. Among the differentially expressed microRNAs, we focused on miR429, belonging to the miR200 mirRNA family, which regulates epithelial to mesenchymal transition, targeting ZEB transcription factors. ES cells (5x10
5
) were exposed from 90 minutes to 24 hours to DETA/NO in the absence of LIF and real time PCR experiments, were performed. We found that miR429 progressively increased along the whole time course, in the presence of DETA/NO, to be upregulated 8 fold with respect to control cells at the 24 hours time point. Moreover, we found that, consistently with miR429 upregulation, NO treatment down-modulated ZEB2/Sip1 transcription factor expression in ES cells. Both antagomir429 and ZEB2/Sip1 overexpression blocked NO-dependent expression of cardiovascular markers, such as Flk-1 and SM22α, in ES, while inducing ES proliferation, indicating the mir429/ZEB circuitry as an important regulator of ES cell fate. To our knowledge, these data identify the first, NO-dependent, microRNA-based mechanism regulating ES development and indicate ZEB2/Sip1 as a novel, potential “stem” gene.
Abstract 3857: Structural Heterogeneities are a Substrate for Re-excitation, Reflection, and Arrhythmogenesis in Cardiac Muscle
Authors: Auerbach David S , Mironov Sergey , Jalife Jose
Published: July 2021

Background:
Heart diseases predispose to arrhythmias and sudden cardiac death by mechanisms that are poorly understood. We tested the hypothesis that in fully excitable tissue abrupt geometrical expansions resulting from varying wall thickness, replacement fibrosis, ischemia, or accessory pathways, set the stage for to-and-fro propagation (reflection) of electrical impulses over the same pathway, leading to premature excitation and reentry initiation.

Methods:
We used patterned monolayers of cultured neonatal rat ventricular myocytes consisting of two wide regions connected by a fully excitable, thin isthmus (0.1, 0.5, 1, & 2 mm wide, 6 mm long). We compared control monolayers with those overexpressing Na channels (NaCh, Ad-hSCN5a). Impulse propagation was optically imaged (Di-8-ANEPPS) at high resolution.

Results:
Impulses initiated proximally in a wide region propagated 1:1 through relatively broad isthmuses (2 mm) and then into the distal expansion at higher frequencies than through narrower isthmuses (0.1 mm). In control monolayers, with relatively low excitability, the prevalence of reflection was small (15%, n=61). NaCh overexpression increased excitability. It also increased the incidence of reflection (38%, n=26). In homogeneous monolayers, NaCh overexpression increased the conduction velocity (15–17%) and prolonged the action potential duration (APD, 21–26%) at the frequencies (2– 4 Hz) at which reflection occurred. During reflection, the APD at the distal expansion was prolonged, compared to APDs within the isthmus or distally when there was no reflection. APD prolongation provided a substrate for local phase-3 re-excitation at the isthmus, and thus reflection. In some cases, reflection was sustained over several consecutive beats. Reflection also triggered the initiation of reentry; as the beat reflected back, a small distal region was re-excited, resulting in unidirectional propagation and the initiation of reentry. Reflection was never observed in structurally homogeneous monolayers, whether with or without the presence of NaCh overexpression.

Conclusion:
Source-to-sink mismatch in areas of cardiac muscle expansion creates APD heterogeneities, which may serve as a substrate for reflection and arrhythmogenesis.
Abstract 3880: GRK2 Inhibition Prevents Development Of Cardiac Insulin Resistance And Impaired Glucose Uptake In Post Ischemic Heart Failure
Authors: Ciccarelli Michele , Rengo Giuseppe , Chuprun Kurt , Santulli Gaetano , Trimarco Bruno , Iaccarino Guido , Koch Walter J
Published: July 2021

The beta adrenergic receptor (βAR) kinase, GRK2, is upregulated and participates to the evolution of heart failure (HF) through downregulation and desensitization of βARs. Recent studies showed that this molecule affects insulin signaling and reduce glucose uptake in hepatocytes and adipocytes. We hypothesized that in HF, GRK2 reduces cardiac performance also through inhibition of cardiac glucose metabolism. In 12 week old Sprague/Dawley rats, we measured cardiac glucose uptake by PET 3 days, 3 and 6 weeks after myocardial infarction (MI). Function and cardiac dimensions were measured by echocardiography. We observed that glucose uptake was reduced in animal post-MI at 3 and 6 weeks respect to healthy animals (3
rd
week: 1.3±0.22 vs 2.1±0.3; 6
th
week: 1±0.1 vs 2.4±0.2, ml/min/g, p<0.05). No difference was observed in glucose uptake acutely after surgery. Echo showed cardiac dilation and reduced function at 6 weeks (LVD: 9.2± 0.3 vs 7.2± 0.4 mm; EF: 42%±1.1 vs 66%±2.2, p<0.05, Sham vs MI). To inhibit GRK2 in the heart during post-ischemic HF, we delivered the GRK2 inhibitor βARKct by adeno-associated type 6 virus (AAV6) to the left ventricle before induction of the MI. As a control we treated rats with AAV6 encoding for the green fluorescent protein (GFP). Cardiac dilation and function were preserved after 6 weeks post MI in AAV6 βARKct respect to AAV6GFP rats (LVD: 7.73 ±0.25 vs 9.9 ±0.8 mm; EF: 55%±2.25 vs 44%±2, p<0.05). Glucose uptake was better preserved in AAV6βARKct rats after 3 and 6 weeks post MI respect to AAV6GFP group (3rd week: 2.3±0.3 vs 1.2±0.2; 6th week: 1.8±0.2 vs 1.1±0.05, ml/min/g, p<0.05). Since Akt mediates most of the anabolic effects of insulin in cells, we evaluated the effects of GRK2 overexpression by adenovirus (ADGRK2) in neonatal cardiomyocytes (NRVMs) on Akt phosphorylation later on insulin stimulation (ins, 10 – 6 M). As control we induced overexpression of GFP by adenovirus (ADGFP). We observed reduced activation of Akt in presence of GRK2 overexpression as compared to the ADGFP treated cells (1.2±0.2- vs. 3.5±0.4- fold activation over basal, p<0.05). Our data show that post MI, impaired glucose extraction precedes development of HF, and that early GRK2 inhibition prevents impaired myocardial glucose uptake and HF development.