bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 699: Intracoronary Infusion Of Bone Marrow-derived Progenitor Cells In Patients With Non-ischemic Dilated Cardiomyopathy Is Associated With An Increase Of Coronary Blood Flow In The Target Vessel
Authors: Fischer-Rasokat Ulrich , Assmus Birgit , Seeger Florian , Honold Joerg , Schaechinger Volker , Fichtlscherer Stephan , Martin Hans , Dimmeler Stefanie , Zeiher Andreas M.
Published: July 2021

We recently could demonstrate that intracoronary administration of bonemarrow-derived progenitor cells (BMC) is associated with improved target area contractility and enhanced cardiac function in patients (pts) with nonischemic dilated cardiomyopathy (DCM). The influence of coronary microvascular dysfunction on development and progression of DCM in the absence of epicardial artery atherosclerosis has been demonstrated in experimental studies. Therefore, to get insights into potential mechanisms of BMC therapy in pts with DCM, we performed a substudy of the TOPCARE-DCM trial, assessing coronary blood flow (CBF) by intracoronary Doppler measurements.

Methods
13 pts (age 31 to 64 yrs; ejection fraction 28 ± 14 %) were included. A mean number of 353 ± 119 * 10
6
BMC were infused into the left anterior descending (LAD) artery using the stop-flow technique. Doppler-derived CBF velocities in the target (LAD) as well as in a reference vessel (left circumflex artery, ref) were obtained at baseline prior to BMC administration and at 3 months’ angiographic follow up (FU).

Results
Heart rate, blood pressure and coronary artery lumen diameter did not differ between initial and FU measurements. Basal coronary vascular resistance (VR) decreased after 3 months in the target and in the ref vessel (target 6.32 ± 2.43 vs 5.43 ± 2.28, p < 0.01; ref 6.72 ± 2.15 vs 5.41 ± 1.94 mmHg*s/cm, p = 0.03). In contrast, adenosine-induced minimal VR decreased only in the target vessel but not in the ref vessel (target 1.53 ± 0.60 vs 1.31 ± 0.65, p < 0.01; ref 1.60 ± 0.45 vs 1.49 ± 0.45 mmHg*s/cm, p = ns). Due to the selective decrease in adenosine-induced minimal VR of the target vessel, the coronary flow reserve normalized for the ref vessel increased from 1.0 ± 0.2 to 1.2 ± 0.3 (p < 0.05). Likewise, the CBF increased in the target vessel (from 48 ± 18 to 62 ± 25 ml/min, p = 0.02), but remained unchanged in the ref vessel (from 40 ± 21 to 48 ± 18 ml/min, p = ns).

Conclusions
In pts with nonischemic DCM, intracoronary BMC infusion is associated with a significant improvement in microvascular function of the target area. Whether improved microvascular function as a potential mechanism underlying the beneficial effects of BMC is causally linked to an improved cardiac contractile function has to be analysed in larger studies.
Abstract 703: HIF-1α Dependent Expression of iNOS in Bone Marrow Stem Cells Mediates Protection of Cardiomyocytes against Ischemia
Authors: Dai Ying , Ashraf Muhammad , Wang Yigang , Xu Meifeng
Published: July 2021

We hypothesized that inducible nitric oxide synthase (iNOS) upregulated in bone marrow stem cells (BMSCs) protect ischemic cardiomyocytes via hypoxia inducible factor-1α (HIF-1α) pathway.

Methods:
Isolated BMSCs were exposed to hypoxia for 24 hours. The level of HIF-1α protein and its activated form were measured by ELISA. The expression of iNOS and HIF-1α were analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry. Cardiomyocytes in co-culture with BMSCs were subjected to hypoxia and H
2
O
2
(200 μmol). LDH release, DNA fragmentation and annexin-V positive cells were used as injury markers.

Results
: HIF-1α protein and its activated form were markedly increased (Fig. A, B

) and translocated to the nucleus or peri-nuclear area of BMSCs subjected to hypoxia, in parallel with increased expression of HIF-1α target gene iNOS, which was blocked by pretreating cells with neutralizing HIF-1α antibody (Fig. C

). Co-culture of cardiomyocytes with BMSCs not only prevented and reduced cardiomyocyte apoptosis induced by hypoxia and H
2
O
2
but also significantly reduced LDH release from cardiomyocytes (Fig. D–G

). The cardiac protection by BMSC was abolished by neutralizing HIF-1α antibodies, and by the selective iNOS inhibitor, 1400W (10 mg/L) as well as a potent competitive inhibitor of NOS, L-NAME (200 μmol/L). However, no effects of neutralizing HIF-1α antibody, L-NAME or 1400W on cardiomyocytes alone were seen. SNAP (300 μmol/L), a NO donor had no synergistic effect on the cardioprotective effect of BMSC.

Conclusion:
iNOS upregulated in bone marrow stem cells by hypoxia protects cardiomyocytes against ischemic injury via activating HIF-1α.
Abstract 714: Targeting Coagulation Factor XII to Protect from Cardiac Ischemia Reperfusion Injury
Authors: Frantz Stefan , Bauersachs Johann , Adamek Anna , Blomer Nadja , Ertl Georg , Renne Thomas
Published: July 2021

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis and thus exaggerate myocardial ischemia/reperfusion injury. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, but can reduce thrombus formation. Therefore, we studied the contribution of FXII for myocardial ischemia reperfusion injury. Following 30 minutes of ischemia and 24 hours of reperfusion infarct size in FXII deficient mice was significantly reduced as compared to wild type controls (infarct/area at risk, WT vs. FXII−/−, 73.5%±4.4% vs. 42.3±1.6%, p<0.001) despite similar area at risk (WT vs. FXII−/−, 28.3%±1.7% vs. 29.9±1.0%, p=n.s.). There was no difference in bleeding complications and bleeding time. Mice deficient in the FXII substrate factor XI were similarly protected from ischemia reperfusion injury (infarct/area at risk, FXI−/−, 16.9%±4.1%, vs. WT p<0.001). The phenotype of FXII−/−mice could be rescued by intravenous application of human FXII (infarct/area at risk, FXII−/−+FXII, 77.3%±8.4%, p<0.001 vs. FXIIKO). WT mice treated with the FXII inhibitor PCK (Pro-Phe-Arg-chloromethylketone) were similarly protected from myocardial ischemia/reperfusion injury (infarct/area at risk, 46.5% ±3.7%, vs. WT p<0.001). The data suggest that the intrinsic pathway is crucial for pathological clotting in myocardial ischemia reperfusion injury. FXII inhibition may offer a selective and safe strategy to reduce ischemic injury.
Abstract 744: Arrhythmogenesis in Heart Failure: Role of Interstitial Fibrosis
Authors: Massare Jorge E , Naseem R. Haris , Berry Jeff M , Rob Farhana , Hill Joseph A
Published: July 2021

Background:
Sudden cardiac death due to ventricular tachyarrhythmia (VT) accounts for a large number of deaths in patients with heart failure. Several cellular events which occur during pathological remodeling of the failing ventricle are implicated in the genesis of VT, including action potential prolongation, dysregulation of intercellular coupling, and fibrosis. Interestingly, transgenic mice over-expressing constitutively active PKD (caPKD) develop severe heart failure without interstitial fibrosis, an otherwise prominent feature of the disease. The goal here was to define the role of interstitial fibrosis in the proarrhythmic phenotype of failing myocardium.

Methods and Results:
We performed echocardiographic, electrocardiographic, and in vivo electrophysiologic studies in 8 –10 week old caPKD mice (n=12). Similar studies were performed in mice with load-induced heart failure induced by surgical pressure overload (sTAB, n=10), a model of heart failure with prominent interstitial fibrosis. caPKD and sTAB mice showed similar degrees of ventricular dilation (LV systolic dimension caPKD 2.4±0.8 mm vs 3.0±0.9 sTAB, p=0.18) and severe systolic dysfunction (% fractional shortening caPKD 25±11 vs 28±11 sTAB, p=0.62). Yet, caPKD mice showed minimal interstitial fibrosis, comparable to unoperated controls. With the exception of ventricular refractory period, which was higher in caPKD (48±11 msec vs 36±7 TAB and 40±8 WT, p<0.05), other electrocardiographic and electrophysiologic variables were similar among the 3 groups (p=NS), including heart rate, QT duration, and mean VT threshold. As expected, VT (≥3beats) was readily inducible by programmed stimulation in sTAB mice (7/10). By contrast, VT was less inducible in caPKD mice (4/12; p=0.1 vs TAB and <0.05 vs WT), and uninducible in unoperated controls (0/12). VT was polymorphic in both models, but episodes of VT were both slower (VT cycle length caPKD 58±4.0 msec vs 48±1 sTAB, p=0.016) and longer in caPKD mice (caPKD 1.8±0.7 sec vs 0.47±0.3 sTAB, p=0.038).

Conclusion:
Interstitial fibrosis contributes to the inducibility, maintenance, and rate of VT in heart failure. These findings highlight the importance of anti-remodeling therapies known to target fibrosis in heart disease.
Abstract 753: Effects of Na,K-ATPase Beta-Subunit Knockout on Heart Function
Authors: Barwe Sonali P , Jordan Maria C , Skay Anna , Inge Landon , Rajasekaran Sigrid A , Wolle Daniel , Johnson Christina L , Neco Patricia , Fang Kun , Rozengurt Nora , Goldhaber Joshua , Roos Kenneth P , Rajasekaran Ayyappan K
Published: July 2021

Na,K-ATPase also known as the sodium pump, is an oligomeric membrane bound enzyme that catalyzes ATP-dependent transport of three Na
+
ions out and two K
+
ions into the cell. It plays an important role in cardiac function by influencing the intracellular Ca
2+
levels via the Na
+
/Ca
2+
exchanger and thereby cardiac contractility. Na,K-ATPase is composed of two essential subunits, alpha-subunit and beta-subunit, both of which have multiple isoforms. The alpha subunit, which is also the catalytic subunit, is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. Evidence indicates that Na,K-ATPase alpha1, alpha2 and beta1 isoforms as well as the Na,K-ATPase enzymatic activity is reduced in the failing human heart. Heterozygous knockout mice of the alpha-subunit isoforms had altered cardiac contractility. However, nothing much is known about the role of the beta-subunit in cardiac function. To investigate the role of beta-subunit in the myocardium, we used Cre/loxP technology to inactivate the beta-subunit gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte beta-subunit gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 9 months of age. These animals had ~20% and ~50% of control levels of beta-subunit and alpha-subunit protein in the heart respectively. At 13–14 months of age, these mice had 24% higher heart/body weight ratios, elevated levels of markers of cardiac hypertrophy such as ANP, BNP, alpha MHC, alpha skeletal actin, and reduced contractility by echocardiography (56.7% versus 66.4% EF in the WT, n = 7 each) as compared to their WT littermates. Pressure overload by transverse aortic constriction in younger mice, resulted in compensated hypertrophy in WT mice, but 50% of Na,K-beta knockout mice died soon after TAC. The few survivors exhibited decreased contractile function (50% as compared to 71% EF in WT, n = 4 each) and mimicked the effects of Na,K-beta knockout in old mice. In conclusion, our results suggest that loss of beta-subunit leads to significant pathophysiology from altered ionic homeostasis in the heart.
Abstract 760: Heme Oxygenase-1 expression in Dendritic Cells determines the Outcome of Transplantation Arteriosclerosis
Authors: Noordeloos Annemarie , van Deel Elza , Hermes Denise , Simoons Maarten L , Duncker Dirk J , Cheng Caroline , Duckers Henricus J
Published: July 2021

Introduction:
Although expression of heme oxygenase-1 (HO1) attenuates transplantation arteriosclerosis, the mechanism by which HO1 exerts its protective effect remains unclear. We studied the effect of HO1-deficient vs. wildtype (WT) dendritic cells (DCs) on the T-cell priming response and outcome in a murine transplant arteriosclerosis model.

Methods:
At day 0 C57bl6 mice received either WT (n=6) or HO1-knockout DCs (n=6) pre-sensitized with Balb/c splenocytes lysate to accelerate the development of arteriosclerosis. At day 10 an aorta segment from Balb/c mice was transplanted into the carotid artery position of C57Bl6 mice.14 days after transplantation allografts were excised and processed for immunohistochemical analysis.

Results:
HO1-deficient DCs significantly increased neointimal hyperplasia as compared to WT DCs (116995 vs. 46114μm
2
P<0.05) and incidence of intima formation (83 vs. 50% in WT DC). HO1 deficient DCs also increased medial thickeness (15936 vs.12034 μm
2
P<0.05) and intimal VSMCs content (76 vs. 46% P<0.05) and resulted in more prominent medial cell infiltration (461μm
2
vs. 232μm
2
P<0.05). Although HO1 deficient and WT DCs could be detected in allografts, HO1-nullizygous DCs induced an increase in CD4+ T-cell infiltration (9.5 vs. 0.1% in WT P<0.05) concomitant to a decrease of CD8+ T cell infiltration (8 vs.14%, P<0.05). In line with these observations Affymetrix microarray analysis confirmed that HO1 deletion in DCs was associated with a significant downregulation of MHCII-H2A expression (associated with CD4+T-cell activation) and induction of inhibitors of MHCII expression (including IK protein) whereas MHC I expression remained unchanged.

Conclusions:
HO1 expression in dendritic cells increases vascular cell infiltration with a higher CD8+/CD4+ T-cell ratio by stabilizing MHCII expression in vascular allografts resulting in inhibition of neointima formation and hence improved allograft survival.
Abstract 777: Caveolin-1 Regulates NADPH Oxidase Subcellular Location And Activity In Endothelial Cells.
Authors: Yang Baohua , Rizzo Victor J
Published: July 2021

Background:
The biological setting or location where reactive oxygen species (ROS) are produced is a new concept which may provide a mechanistic explanation for redox signaling specificity. In this regard, we recently reported that NADPH oxidase, a major superoxide producing enzyme system, is localized and functional in endothelial cell caveolae. Here, we investigate the role of caveolin-1, the main structural protein of caveolae, in the organization and regulation of NADPH oxidase activity and ROS mediated responses to cytokine challenge.

Methods and Results:
Superoxide production was measured by DHE assay in endothelial cells derived from wild type (wt) and caveolin-1 knockout (Cav−/ −) mouse lungs (MLEC). TNFα failed to induced superoxide production in Cav−/ − endothelial cell cultures (MOI = 0). However, infection of knockout cells with full-length caveolin-1 containing Adenovirus at MOI of 10 and 50 progressively restored TNFα effect on superoxide production with MOI 50 nearly reaching those observed in wt cells. The distribution of NADPH oxidase within the plasma membrane showed localization to membrane rafts fractions in wt cells which shifted to the non-raft membrane fractions in Cav-1−/ − MLEC. Re-expression of caveolin-1 (MOI 50) restored raft targeting of NADPH oxidase and both gp91 and p22phox co-precipitated with newly expressed caveolin-1 immunoprecipitated from raft fractions. To evaluate the cellular consequences of a potential redox signaling relay involving caveolin-1, we examined an NADPH oxidase dependent pathway involving activation of JNK1/2 MAP kinases and downstream upregulation of ICAM. We found that phosphorylation of JNK1/2 observed in wt MLEC in response to TNFα was significantly attenuated in Cav−/ − MLEC. In addition, TNFα did not induce upregulation of ICAM expression in caveolin-1 knockout MLEC or wt cells pretreated with DPI.

Conclusions:
Collectively, these results indicate that caveolin-1 is a necessary component of the molecular machinery for proper subcellular positioning required for cytokine activation of NADPH oxidase and superoxide production in endothelial cells. Thus, caveolin-1 may constitute a potential therapeutic target for altering ROS associated with cardiovascular pathology.
Abstract 780: Atorvastatin Activates A Novel Nbs1-dependent Mechanism Of Accelerating Dna Repair In Atherosclerosis
Authors: Mahmoudi Melli M , Gorenne Isabelle C , Mercer John R , Figg Nichola L , Bennett Martin R
Published: July 2021

There is increasing evidence that reactive oxidant species (ROS) and DNA damage promote the development and complications of atherosclerosis. Although statin therapy reduces both ROS and DNA damage in atherosclerosis, the mechanism of this effect is unknown. We first examined expression of DNA damage and repair markers in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaques. With increasing disease severity, there was increased VSMC expression of the DNA repair markers P-ATM/ATR substrate and P-H2AX from 2.7%Â±2.2 and 0.5Â±0.71 [meanÂ±SEM] (AHA Grade I/II), to 21%Â±3.5 and 36.5Â±2.1 (Grade III) lesions, and 86.5%Â±0.7 and 69.3Â±7.6 (Grade IV/V). Cultured plaque VSMCs also showed a 1.5 fold increased oxidant stress; a 4.4 fold increased double-stranded DNA breaks, and expression of P-H2AX by Western blots. ROS analogues induced a robust DNA damage response in VSMCs, characterised by lengthening of tails on COMET assay, and activation of ATM and P-H2AX, with completion of repair by 6 hours. Atorvastatin pre-treatment accelerated DNA repair by approximately 2 hours without inhibiting ROS induction or DNA damage, and markedly accelerated the kinetics of nibrin (NBS-1) and P-H2AX activation, both proteins recruited to sites of DNA damage, by preventing degradation of NBS-1. Atorvastatin induced phosphorylation of HDM2, an E3 ligase and putative regulator of NBS-1 stability, and siRNA knockdown of HDM2 replicated the effect of atorvastatin on NBS-1. The ability of atorvastatin to accelerate repair was completely dependent upon NBS-1, as atorvastatin was ineffective in cells either null or expressing constitutively active NBS-I. In summary, we have demonstrated a novel NBS-1-dependent mechanism by which statins accelerate DNA repair in atherosclerosis, through HDM2 phosphorylation and stabilisation of NBS-1. We believe that both NBS-1 and HDM2 are critical to DNA repair in atherosclerosis.
Abstract 795: Enhanced Sensitivity Of The Cardiac Ryanodine Receptor To Activation By Luminal Ca
2+
As A Primary Cause Of Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Uchinoumi Hitoshi , Yano Masafumi , Ohno Makoto , Xu Xiaojuan , Tateishi Hiroki , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Matsuzaki Masunori
Published: July 2021

Mutations in cardiac ryanodine receptor (RyR2) was found to be linked with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of CPVT, we developed knock-in mice harboring the Arg-to-Ser (R2474S) mutation. The RyR2
R2474S/+
knock-in (KI) mice revealed no structural or histological abnormality in hearts. Echocardiography showed no contractile or relaxation dysfunction at rest. In all KI mice (n=6), bidirectional ventricular tachycardia (VT) was observed during or after exercise with treadmill, but never observed in wild-type (WT) mice (n=6). In intact cardiomyocytes, the frequency of Ca
2+
sparks (SpF; s
−1
·100μm
−1
) was significantly increased in KI mice, but not in WT mice (at 2 mM [Ca
2+
]; KI:6.4±0.7, WT:0.9±0.08, p<0.01). To investigate the sensitivity of the RyR2 channel to activation by luminal Ca
2+
{[Ca
2+
] in sarcoplasmic reticulum (SR)}, we measured cytoplasmic [Ca
2+
] ([Ca
2+
]
C
) and luminal [Ca
2+
] ([Ca
2+
]
L
) simultaneously in saponin-permeabilized cardiomyocytes, using Rhod-2 and Fluo-5N AM as Ca
2+
indicators, respectively. When [Ca
2+
]
C
was buffered at 100 nM (by 1 mM EGTA), the spontaneous Ca
2+
sparks were frequently observed both in KI and WT cardiomyocyts (SpF: KI:22.1±0.9, WT:22.0±0.8, p=ns). When we added thapsigargin (1 μM) to the cardiomyocytes under this condition ([Ca
2+
]
C
=100 nM), both SpF and [Ca
2+
]
L
gradually decreased due to a decrease in SR Ca
2+
content caused by an inhibition of SR Ca
2+
ATPase. The relationship curve between SpF and [Ca
2+
]
L
(SpF -[Ca
2+
]
L
) during the addition of thapsigargin was markedly shifted to the left in KI cardiomyocytes compared to WT cardiomyocytes, thereby lowering the threshold of [Ca
2+
]
L
to induce Ca
2+
sparks to approximately one-fifth in KI cardiomyocytes. In conclusion, the enhanced sensitivity of the RyR2 channel to activation by [Ca
2+
]
L
: i.e. decreased threshold [Ca
2+
]
L
to induce spontaneous Ca
2+
release, may be a primary cause of CPVT.
Abstract 828: Adiponectin Enhanced ApoA-1- and HDL- mediated Cellular Cholesterol Efflux from Human Monocyte-Derived Macrophages
Authors: Koseki Masahiro , Tsubakio-Yamamoto Kazumi , Matsuura Fumihiko , Oku Hiroyuki , Sandoval Jose C , Yuasa-Kawase Miyako , Masuda Daisaku , Nishida Makoto , Ishigami Masato , Hirano Ken-ichi , Kihara Shinji , Hori Masatsugu , Shimomura Iichiro , Yamashita Shizuya
Published: July 2021

(Background)
Plasma HDL-cholesterol levels are inversely correlated to the risk of CAD. HDL plays a crucial role as a shuttle carrying cholesterol derived from peripheral tissues and transporting it back to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) have been identified as important membrane receptors to generate HDL by removing cholesterol and phospholipids from macrophage foam cells in the arterial wall. On the other hand, adiponectin (APN) abundantly secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, and APN levels are decreased in patients with CAD. In the current study, we investigated the role of APN on cellular cholesterol efflux from human macrophages.

(Methods)
Recombinant human APN was prepared. Human monocyte-derived macrophages were isolated from whole blood of healthy volunteers and incubated with RPMI-1640 medium containing 10% type AB serum. After 7 days incubation, the indicated concentrations of recombinant APN were added to the medium containing 0.1% BSA and incubated for 24 hours. The expression of ABCA1 and ABCG1 was measured by real-time quantitative PCR and western blot analyses. Cholesterol efflux from macrophages, previously incubated with [
3
H]-labeled acetylated LDL, was measured with or without APN and expressed by a percentage of radioactivity of the medium to total radioactivity (cells plus medium). APN was added into the medium containing 0.1% BSA and apoA-1 (10 μg/ml) or HDL (50 μg/ml HDL protein) and incubated for 24 hours.

(Results)
APN significantly up-regulated the mRNA and protein levels of ABCA1 in human macrophages by 3.6- and 2-fold, respectively. ApoA-1-mediated cholesterol efflux from macrophages was 8.1-fold-increased by APN treatment. Furthermore, the expression of ABCG1 was also significantly increased by APN. HDL-mediated cholesterol efflux was slightly increasd by APN.

(Conclusions)
In conclusion, APN might act as a protective factor against atherosclerosis by increasing cholesterol efflux from human macrophages in both ABCA1- and ABCG1-dependent pathways.
Abstract 838: microRNA-210 Regulates Endothelial Cell Response to Hypoxia
Authors: Fasanaro Pasquale , D’Alessandra Yuri , Di Stefano Valeria , Melchionna Roberta , Romani Sveva , Pompilio Giulio , Capogrossi Maurizio C , Martelli Fabio
Published: July 2021

Background
. MicroRNAs (miRNAs) are small non-protein-coding RNAs that downregulate gene expression by inhibiting mRNA translation in mammalian cells. In this study, it was investigated miRNAs regulation and functional role in endothelial cell response to hypoxia.

Methods and results.
Human Umbelical Vein Endothelial Cells (HUVEC) were cultured either in normoxic conditions or in the presence of 1% oxygen for 8 – 48 hours. Then, a selection of 157 miRNAs was measured using a real-time PCR assay designed to quantify only mature miRNAs. The expression of miR-210 and miR-150 progressively increased upon exposure to hypoxia. At 48 hours, miR-210 and miR-150 were 35±5 and 14±4 fold more expressed than control (p<0.001 and <0.007, respectively). Both responses were independent of cell confluency and were not limited to vascular cells. In additional experiments it was characterized miR-210 regulation and its the role on HUVEC function. Neither acidification nor oxidative stress, two components of the cell response to hypoxia, modulated miR-210. Further, miR-210 overexpression in normoxic HUVEC stimulated the formation of capillary-like structures on Matrigel and VEGF-driven cell migration. Conversely, miR-210 blockade via LNA-antimiR transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to VEGF. miR-210 overexpression did not affect HUVEC growth in both normoxia and hypoxia. Conversely, antimiR-210 transfection inhibited cell growth both in normoxia (66%±8 of control; p<0.003) and in hypoxia (50%±9 of control; p<0.001). While DNA synthesis was not affected, miR-210 blockade induced apoptosis, as assessed by measuring cytoplasmic nucleosomal DNA. One relevant target of miR-210 in hypoxia was Ephrin-A3, since miR-210 was necessary to downmodulate its expression upon hypoxia. Moreover, miR-210 overexpression in normoxic condition was sufficient to inhibit Ephrin-A3 protein level. Finally, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210.

Conclusions
. miR-210 upregulation is a crucial element of cell response to hypoxia, affecting cell survival, migration and differentiation. #
Abstract 843: Cardiomyocyte Overexpression of the Hydrogen Sulfide Producing Enzyme Cystathioine gamma-Lyase Attenuates Myocardial Ischemia-Reperfusion Injury
Authors: Elrod John W , Calvert John W , Chow Chi-Wing , Morrison Joanna , Doeller Jeannette E , Kraus David W , Kimura Hideo , Lefer David J
Published: July 2021

Background

: Hydrogen sulfide (H
2
S) was recently discovered to be an endogenously produced gaseous second messenger capable of modulating many physiological processes. We have previously demonstrated that administration of a H
2
S donor limits the extent of myocardial infarction. This prompted us to investigate the potential of endogenously generated H
2
S in acute cardioprotection utilizing mice with transgenic overexpression of an H
2
S producing enzyme.

Methods:

Mice with cardiac-specific overexpression of murine cystathionine γ-lyase (αMHC-CGL-Tg) were generated and analyzed for increased enzyme expression and H
2
S production utilizing a H
2
S specific polarographic electrode. αMHC-CGL-Tg and WT mice were then subjected to 45 min of
in vivo
LCA ischemia and 72 hr reperfusion and infarct size was evaluated using TTC staining.

Results:

αMHC-CGL-Tg mice displayed an increased level of myocardial CGL RNA, which translated into a (15 fold) increase in protein expression. This increase in CGL enzyme resulted in a significant (2 fold) increase in H
2
S production by myocardial homogenates of αMHC-CGL-Tg mice. αMHC-CGL-Tg mice were found to have a 47% reduction in infarct size per area-at-risk (INF/AAR) as compared to WT littermates. AAR was similar between both groups.

Conclusions:

This is the first evidence that overexpression of a H
2
S producing enzyme can decrease infarct size following MI-R injury. These findings demonstrate that modulation of endogenous H
2
S production may be of clinical benefit in ischemic disorders and that H
2
S generating enzymes may be viable therapeutic targets.
Abstract 862: Ser517:-phosphorylation Of Sirt1 Is Required For Its Pi3k/akt-mediated Nuclear Translocation And Cardiomyocyte Protection Against Oxidant Stress In Failing Hearts.
Authors: Tanno Masaya , Miura Tetsuji , Miki Takayuki , Yano Toshiyuki , Horio Yoshiyuki , Shimamoto Kazuaki
Published: July 2021

[Purpose] We recently found that SIRT1, a protein deacetylase, shuttles between the nucleus and cytoplasm. In this study, we examined the role of nuclear SIRT1 in cardiomyocyte protection against oxidant stress and involvement of PI3K/Akt in the nuclear translocation.

[Methods and Results] First, the critical intracellular location of SIRT1 for its anti-apoptotic function was examined. C2C12 cells were transfected with wild-type SIRT1 (WT) or SIRT1 with site-directed mutations in the nuclear localizing signal (mtNLS) and exposed to antimycin A (AA), an oxidative stressor. AA-induced apoptosis was suppressed in WT-transfected cells expressing SIRT1 in the nuclei compared with that in mtNLS-transfected cells expressing SIRT1 in the cytoplasm (TUNEL-positive cells = 4.4±0.7% vs. 34.6±8.0%). AA-induced apoptosis and also angiotensin II-(angII)-induced apoptosis in neonatal rat cardiomyocytes (NRCM) were suppressed by resveratrol, a SIRT1 activator. This protective effect of resveratrol was attenuated by transfection of SIRT1-siRNA but not by transfection of control siRNA. Next, we assessed the role of PI3K/Akt in nuclear translocation of SIRT1. SIRT1 in NRCM was localized in both the nucleus and cytoplasm under baseline conditions, and IGF-1 induced its nuclear translocation. This effect of IGF-1 was suppressed by LY294002 (LY), a PI3K inhibitor. Deletion mutagenesis study showed that LY-induced nuclear exclusion was observed for SIRT1[223–540] but not for SIRT1[223– 489]. Replacement of serine517 with alanine (S517A) increased cytoplasmic SIRT1, and S517A showed attenuated nuclear translocation in response to IGF-1, indicating that serine517 is the target site of PI3K/Akt. Finally, to confirm heart failure-associated SIRT1 translocation in vivo, myocardial infarction was induced in WKY rats. The number of ventricular cardiomyocytes with nuclear SIRT1 at 4 weeks after infarction was significantly larger than that in sham-operated hearts (10.2±2.9% vs. 0.7±0.2%).

[Conclusion] The results suggest that phosphorylation of SIRT1 at Ser517 by PI3K/Akt is involved in nuclear translocation of SIRT1, which contributes to cardiomyocyte protection from oxidant stress-mediated injury in failing hearts.
Abstract 867: The Longevity Factor Sirt1 Exacerbates LV Dysfunction and Energy Depletion in Response to Pressure Overload
Authors: Alcendor Ralph , Hong Chull , Zhai Peiyong , Gao Shumin , Sadoshima Junichi
Published: July 2021

Sirt1, a class III histone deacetylase, extends the lifespan of many organisms. Longevity mechanisms usually confer stress resistance to organisms, and accumulation of stress resistance leads to lifespan extension. We have shown previously that Sirt1 is upregulated by stress up to 10 fold in the heart, and heart specific overexpression (up to 7.5 fold) of Sirt1 in mice not only suppresses histological/biochemical markers of aging, but also induces resistance to oxidative stress in the heart. We examined whether Sirt1 is protective against another pathologically relevant stimulus, namely pressure overload. Cardiac specific Sirt1 transgenic mice (Tg-Sirt1) from line #40, the line which has been shown to be protected against aging and oxidative stress, were subjected to transverse aortic constriction (TAC). Unexpectedly, at 10 days, the left ventricular (LV) ejection fraction (EF) in Tg-Sirt1 was significantly reduced (46 vs 71%, p<0.01), the LV end diastolic dimension was significantly increased (4.1 vs 3.4 mm, p<0.05), and the pressure gradient was reduced (92 vs 57 mmHg, p<0.05), possibly due to reduced LV contractility, in Tg-Sirt1 compared with non-transgenic (NTg) controls. At 4 weeks, LV weight/body weight (BW) (6.4 vs 4.7, p<0.05) and lung weight/BW (18.8 vs 7.0, p<0.05) were significantly increased in Tg-Sirt1, LV +dP/dt was significantly reduced (4617 vs 7513, p<0.05), and the LV end diastolic pressure was significantly elevated (13.6 vs 1.4 mmHg, p<0.05) in Tg-Sirt1 compared with NTg. These results suggest that Tg-Sirt1 mice develop more severe LV dysfunction than NTg in response to TAC. Tg-Sirt1 mice exhibited significantly less apoptosis (−50%, p<0.05) than NTg however, despite the development of LV dysfunction, suggesting that the LV dysfunction may be caused by apoptosis-independent mechanisms. The myocardial ATP content in Tg-Sirt1 was significantly less (−41%, p<0.05) than that in NTg after TAC. These results suggest that the cardioprotective effect of Sirt1 depends on the type of stress: although modest expression of Sirt1 confers resistance to aging and oxidative stress, it exacerbates heart failure in response to TAC through apoptosis-independent mechanisms possibly involving energy depletion.
Abstract 193: Lapaquistat Acetate, A Novel Squalene Synthase Inhibitor, Co-administered with Atorvastatin Reduces Plasma Lipids and C-Reactive Protein Levels in Subjects with Primary Hypercholesterolemia
Authors: Davidson Michael H , Maki Kevin C , Zavoral James H , Yu Shawn , Popovici Clement , Price Graham D
Published: July 2021

Background/Objective:
Lapaquistat acetate (LAPA), a novel lipid-lowering agent, inhibits squalene synthase, an enzyme distal to HMG-CoA reductase in the cholesterol biosynthetic pathway. We evaluated the effects of LAPA on LDL-C, other plasma lipid variables, and high-sensitivity C-reactive protein (hs-CRP) levels when co-administered with a stable dose of the HMG-CoA reductase inhibitor, atorvastatin (ATV), in subjects with primary hypercholesterolemia already on ATV therapy.

Methods:
This multicenter, randomized, double-blind, placebo-controlled, Phase III trial was conducted at 79 centers in the US. After a 4-wk dietary run-in and ATV (10, 20, or 40 mg/d) stabilization, 448 subjects (LDL-C ≥100 mg/dL, TG ≤400 mg/dL) were randomized to 1 of 3 treatments: LAPA 50 mg, 100 mg, or placebo co-administered with ongoing ATV dosed once daily for 24 wks. The primary endpoint was percent (%) change from baseline in direct LDL-C at 24 wks; secondary endpoints included % changes in calculated LDL-C, total cholesterol (TC), non-HDL-C, Apo B, TG, HDL-C, VLDL-C, Apo A1, and hs-CRP levels. Safety was monitored using clinical and laboratory examinations.

Results:
Baseline clinical and lipid characteristics were comparable among the 3 treatment groups. Compared with placebo, LAPA treatment resulted in significant reductions (see Table

) in LDL-C, other lipid variables, and hs-CRP. The enhanced LDL-C-lowering effect was independent of ATV dose. LAPA co-administration was well tolerated. Overall, adverse event (AE) incidences were similar in the 3 treatment groups. Serious AEs in 12 subjects (5 in placebo, 3 in 50 mg LAPA, and 4 in 100 mg LAPA) were reported.

Conclusion:
Lapaquistat acetate, a novel squalene synthase inhibitor, was well tolerated and effective in reducing LDL-C, hs-CRP, and in improving the atherogenic lipid profile when co-administered with atorvastatin. This combination may prove to be a useful therapeutic option for patients with hypercholesterolemia.

Least Squares Mean % Change from Baseline (±SE)
Abstract 194: Rosuvastatin Reduces Progression of Carotid Atherosclerosis within 12 Months of Treatment: The METEOR Trial
Authors: Bots Michiel L , Raichlen Joel S , Evans Gregory W , Palmer Mike K , O’Leary Daniel H , Grobbee Diederick E , Crouse John R
Published: July 2021

Background: In several statin trials, vascular event rates for treatment groups begin to separate 1 year after commencement of treatment. For atherosclerosis progression, the temporal sequence of the effect has not been defined. We sought to determine the earliest time point at which significant differences in atherosclerosis progression rates were detectable after initiation of statin therapy using data from the METEOR trial (Measuring Effects on intima media Thickness: an Evaluation Of Rosuvastatin).

Methods: METEOR was a double-blind, randomized, placebo-controlled trial among 984 low risk subjects, which studied the effect of LDL lowering with 40 mg rosuvastatin on the rate of change in carotid intima media thickness (CIMT) over time. Ultrasound assessments were made at 12 carotid artery sites at baseline and every 6 months up to two years. In these analyses, the data were cut at 6 months, 1 year, and 18 months, and compared with analysis of all data at 2 years, using the same statistical method.

Results: The difference in rate of maximum CIMT progression for all carotid artery sites (primary endpoint - near and far walls of the left and right common carotid artery [CCA], carotid bulb and internal carotid artery) between the rosuvastatin and placebo groups was apparent 6 months after baseline (0.0023 mm/yr and 0.0106 mm/yr, respectively p =0.36). After 12 months CIMT progression rates were significantly different between groups: 0.0032 mm/yr and 0.0133 mm/yr (p=0.049). This divergence grew with further follow-up: − 0.0009 mm/yr and 0.0131 mm/yr after 18 months (p<0.0001), and − 0.0014 mm/yr and 0.0131 mm/yr after 24 months of treatment (p<<178>0.0001). For the individual carotid artery segments, significant differences were seen at 12 months for the mean CIMT of the CCA, and at 18 months for the maximum CIMT of the bulb and the CCA.

Conclusion: Aggressive LDL lowering with rosuvastatin exerts its beneficial effect on atherosclerosis during the first year of treatment, which parallels the timing of event rate reduction seen in clinical trials. These findings suggest that, in trials examining the effects of treatment on CIMT progression, a duration of 12 months may be adequate, given sufficient sample size, high precision of measurements, and treatment effect.
Abstract 874: MLN1202, a Novel CCR2 Antagonist, Decreases C-reactive protein in Patients at Risk for Atherosclerotic Cardiovascular Disease in a Double Blind Placebo Controlled study
Authors: Davidson M. , Lekstrom-Himes J. , Gilbert J. , Donaldson D. , Lee Y. , Hu M. , Xu J.
Published: July 2021

Background:
Macrophages play a central role in atherosclerotic plaque formation. The CC chemokine receptor 2 (CCR2), expressed on the surface of circulating monocytes, and its ligand MCP-1 (CCL2), are present in atherosclerotic plaques and may play a critical part in endothelial monocyte recruitment and activation. MLN1202 is a humanized monoclonal antibody with high specificity to CCR2, which interrupts MCP-1 binding to CCR2. MLN1202 is being developed for the treatment of immune mediated diseases.

Hypothesis:
We tested the hypothesis that MLN1202 significantly influences disease activity in patients at risk for ASCVD as measured by a reduction in circulating levels of high sensitivity C-reactive protein (hsCRP), an established biomarker of inflammation.

Trial Design:
In this double-blind placebo controlled study patients with at least 2 or more risk factors for ASCVD, no history or symptoms of ASCVD disease, and circulating levels of hsCRP > 3mg/L, were randomized 1:1 to receive a single infusion of 10 mg/kg MLN1202 (n 56) or placebo (n = 56). Subjects with hypercholesterolemia on stable doses of lipid-lowering agents were included. Circulating levels of hsCRP were determined every 2 weeks, and clinical examination performed every 4 weeks for 16 weeks following treatment.

Results and Conclusion:
Patients were recruited from nine centers in the US. The study population had a mean age of 60.9 years and included subjects with hypertension (59%), hypercholesterolemia (70%), significant smoking history (28%), and type 2 diabetes (16%). At screening the median value CRP was 6.8 mg/L with interquartile range from 4.7–9.3 mg/L. PK/PD results showed that the plasma level of MLN1202 required for > 90% receptor saturation was maintained for 6 to 8 weeks. A between-group difference in reduction of hsCRP was statistically significant from week 4 through week 8 following dosing. The maximum difference in absolute median reduction was observed at week 8 and it was 1.6 mg/L (p = 0.0275; Wilcoxon); the observed median percent reduction of hsCRP was 24.2% for MLN1202 group versus 2.5% increase for placebo group at 8 weeks (p = 0.0089; Wilcoxon). These data indicate that blockade of CCR2 reduces a biomarker related to inflammation in patients at risk for ASCVD.
Abstract 883: Elevated Epidermal Growth factor Receptor Phosphorylation Induces Resistance Artery Dysfunction in Type 2 Diabetes
Authors: Matrougui Khalid , Palen Desiree I
Published: July 2021

Objective: We previously showed that epidermal growth factor receptor (EGFR) transactivation is a key mechanism in the regulation of resistance artery myogenic tone. Type 2 diabetes is associated with microvascular complications. We hypothesized that elevated EGFR phosphorylation contributes to resistance artery dysfunction in type 2 diabetes.

Methods and results: Type 2 diabetic db-/db- (diabetic) and non-diabetic db-/db+ (control) mice were treated with EGFR inhibitor (AG1478, 1 microM) for 2 weeks. Isolated coronary artery (CA) and mesenteric resistance artery (MRA) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was increased in MRA and CA from diabetic mice and normalized by AG1478 treatment. Phenylephrine-induced contraction and nitric oxide donor-induced relaxation were similar in all groups. Relaxation endothelium-dependent in response to shear-stress and acetylcholine of MRA and CA from diabetic mice were altered and associated with reduced eNOS expression and phosphorylation. The treatment of diabetic mice with AG1478 improved CA and MRA endothelial function and restored eNOS expression. Immunostaining and western blot analysis showed increased endothelial and SMC EGFR phosphorylation of MRA and coronary from diabetic mouse, which was reduced by AG1478 treatment. Primary cultured endothelial cells (EC), from resistance arteries, treated with high glucose for 48hrs showed an increase of EGFR phosphorylation associated with a decrease of eNOS expression and phosphorylation in response to calcium ionophore. Pretreatment of endothelial cells with AG1478 prevented the effect of high glucose.

Conclusion: This study provides evidence of the role of elevated EGFR phosphorylation in CA and MRA dysfunction in type 2 diabetes. Therefore, EGFR should be a potential target for overcoming diabetic small arteries complications.
Abstract 890: Feed-forward Signaling of TNF alpha and NFkappa B Produces Endothelial Dysfunction in Coronary Arterioles In Type Ii Diabetic Mice.
Authors: Yang Jiyeon , Xu Xiangbin , Laine Glen A , Zhang Cuihua
Published: July 2021

Nuclear factor-κB (NFκB) signaling reportedly increases tumor necrosis factor-alpha (TNF expression), and the oxidative stress induced by TNF may then lead to further increase NFκB expression, i.e., a feed-forward interaction. Accordingly, we hypothesized that this feed-forward interaction between TNF and NFκB may amplify one another toward the evolution of vascular disease in diabetes. To test this hypothesis, we assessed the role of NFκB in endothelial dysfunction in Lepr
db
mice by evaluation of endothelial function of isolated coronary resistance vessels of m Lepr
db
(heterozygote, normal) and Lepr
db
(homozygote, diabetic) mice. Coronary arterioles (40 –100 μm) were isolated and pressurized (60 cmH2O) without flow. Although dilation of vessels to the endothelium-independent vasodilator, sodium nitroprusside (SNP) was not different between Lepr
db
and m Lepr
db
mice (n = 6), dilation to the endothelium-dependent agonist, acetycholine (ACh) was reduced (n = 5, P < 0.05). Electron Paramagnetic Resonance (EPR) results show that superoxide production was reduced by NFκB antagonist (MG-132), or anti-TNF in Lepr
db
mice suggesting that NFκB and TNF were contributing to elevated oxidative stress. MG-132 (1 μM, n = 4) antagonized the inhibitory effect of Lepr
db
mice on ACh-induced dilation vs. Lepr
db
without treatment, but did not affect dilation in m Lepr
db
mice. To better understand the basis for enhanced contributions of TNF and NFκB in diabetes, we used Western analysis to assess expression of major proteins involved in TNF-mediated signaling. Previous studies have provided compelling evidence that IKK beta plays an essential role in NFκB activation in response to TNF, whereas IKK alpha appears to play a lesser role; therefore, we examined the expression levels of IKK alpha and IKK beta mRNA and protein in Lepr
db
null for TNF. The protein concentration and mRNA expression level of IKK alpha were increased in Lepr
db
mice null for TNF (db
TNF-
/db
TNF-
) mice. One intriguing finding of this study is that the roles of IKK alpha and IKK beta appear reversed in the inflammatory response in diabetic Lepr
db
mice. Furthermore, our results indicate that NFκB and TNF signaling interact to amplify the oxidative stress and induce endothelial dysfunction in type II diabetes.
Abstract 897: Alliance of Blood Derived Endothelial Cells and Adipose Stromal Cells in Human Vasculogenesis
Authors: Traktuev Dmitry O , Prater Daniel N , Sanjeevaiah Aravind R , Merfeld-Clauss Stephanie , Johnstone Brian H , Ingram David A , March Keith L
Published: July 2021

Introduction
Both Endothelial progenitor cells (EPC) and adipose stromal cells (ASC) are under investigation as therapies for cardiovascular diseases. Both cell types are capable of modulating vascular assembly and are, thereby, capable of directly promoting revascularization of ischemic tissues. We have shown that EPC differentiate into endothelial cells to form small vessels, whereas ASC have pericytic properties and naturally stabilize vessels. In this study we tested the possibility that ASC would interact with EPC to assemble de novo vessels in collagen in an
in vivo
chimeric implant.

Methods and Results
Collagen implants embedded with either umbilical cord blood EPC or adult ASC or a 4:1 mixture of both (2x10
6
cells/ml) were implanted subcutaneously into NOD/SCID mice. After 14 d implants were harvested and evaluated by immunohistochemistry. There was a pronounced difference among the groups in vascular network assembly. The majority of vessels formed in the EPC and ASC monocultures were small capillaries bounded by a single endothelial layer. Conversely, 100% of the plugs embedded with both cell types were highly invaded with multilayered arteriolar vessels. The density of the CD31
+
vessels in the EPC and co-culture plugs was 26.6 ± 5.8 and 122.4 ± 9.8 per mm
2
, respectively. No CD31
+
cells of human origin were detected in the ASC monocultures, indicating that ASC, which do not express this EC-specific marker, engage murine EC or form pseudovessels in this system. The density of α-SMA
+
vessels with lumens per mm
2
was 13.1 ± 3.6 (EPC), 10.2 ± 3.5 (ASC) and 124.7 ± 19.7 (co-culture). The total overlap of CD31
+
and SMA
+
vessels demonstrates that mature, multilayered conduits were formed with the co-culture. Moreover, the majority of these vessels were filled with erythrocytes (92.5 ± 16.2 per mm
2
), indicating inosculation with the native vasculature, which was confirmed by ultrasound with echogenic microbubbles and persisted to at least 4 months.

Conclusion
This study is the first to demonstrate that non-transformed human EPC and ASC cooperatively form mature and stable vasculature with subsequent functional integration into a host vasculature system.