bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1286: Neuronal Nitric Oxide Synthase Regulates Basal Vascular Tone in the Healthy Human Forearm
in vivo.
Authors: Seddon Mike , Chowienczyk Phil , Casadei Barbara , Shah Ajay
Published: July 2021

Nitric oxide (NO) has an established role in the maintenance of vascular tone, generally assumed to be mediated by endothelial NO synthase (eNOS). Previous studies using the non-selective NOS inhibitor N
G
monomethyl-L-arginine (L-NMMA) in humans confirmed the
in vivo
importance of NO but the contribution of neuronal NO synthase (nNOS) is unknown due to the lack of available selective NOS inhibitors for human use. In this study, we investigated for the first time in humans the effects of S-methyl-L-thiocitrulline (SMTC), a competitive nNOS-selective inhibitor with 17-fold selectivity over eNOS. SMTC or L-NMMA were infused into the brachial artery of healthy male volunteers and forearm blood flow was measured by venous occlusion plethysmography. SMTC 0.025, 0.05, 0.1 and 0.2 μmol/min caused a dose-dependent reduction in basal blood flow in the infused arm of 9.2±1.9, 16.2±2.9, 22.9±3.9 and 30.1±3.8% respectively (n=10; mean±SE; all P<0.01). Substantially higher doses of L-NMMA of 0.5, 1, 2 and 4 μmol/min were required to reduce basal flow by 11.5±3.0, 25.1±3.0, 33.7±3.0 and 37.4±3.1% respectively (n=10). The highest dose of SMTC (ie, 0.2 μmol/min) tested had no significant effect on the vasodilator response to acetylcholine (ACh): Ach 40 and 80nmol/min increased blood flow by 3.93±0.64 and 5.54±0.69 ml/min/100mls tissue above baseline during saline co-infusion versus 3.95±0.69 and 4.90±0.71 ml/min/100mls tissue during SMTC co-infusion (n=10; P=NS). In contrast, L-NMMA significantly reduced the response to these doses of ACh by 64±9.9 and 60±10% (n=10; both P<0.01). The effect of SMTC on basal blood flow was completely abolished in the presence of the NOS substrate L-arginine (n=6; P<0.001) but was unaffected by the stereoisomer D-arginine (n=6). SMTC had no effect on the vasodilator response to sodium nitroprusside (n=5).

In conclusion
, SMTC reduced basal blood flow by stereospecific inhibition of the L-arginine:NO pathway but did not affect the eNOS-mediated vasodilator response to ACh. These results indicate that nNOS has a crucial role in the regulation of basal vascular tone in the human forearm
in vivo
.
Abstract 1311: Bone Marrow Angiotensin II Type 1 Receptor Augments Atherosclerosis by Promoting Monocyte/Macrophage Lineage Differentiation from Hematopoietic Stem Cells
Authors: Tsubakimoto Yoshinori , Yamada Hiroyuki , Yokoi Hirokazu , Takata Hiroki , Kawahito Hiroyuki , Hirai Hideyo , Imanishi Jiro , Ashihara Eishi , Maekawa Taira , Takahashi Tomosaburo , Okigaki Mitsuhiko , Matsubara Hiroaki
Published: July 2021

[BACKGROUND]
The angiotensin II (Ang II) type 1 (AT
1
) receptor is crucially involved in atherogenesis, but bone marrow (BM) AT
1
-mediated proatherogenic action remains undefined-

.
[METHOD AND RESULT]
BM-derived mononuclear cells (BM-MNCs) more abundantly express AT
1
than VSMCs (2.2 fold) with similar affinities to Ang II. BM cells in 8-week-old apoE-deficient
−/−
) or wild-type (Agtr1
+
/
+
) cells. Four (apoE-KO) mice were replaced with AT
1
-deficient (Agtr1 weeks after the initiation of western diet and Ang II infusion (500ng/kg/min), atherosclerotic lesion area in aortic root was examined. ApoE-KO mice reconstituted with Agtr1
−/−
marrow (apoE-KO/BM-Agtr1
−/−
) showed a significant reduction in atherosclerotic lesions compared with apoE-KO/BM-Agtr1
+
/
+
mice (55%,
P
<0.05). The accumulation of macrophages was attenuated in apoE-KO/BM-Agtr1
−/−
mice (55%,
P
<0.05), concomitant with a decrease in the number of circulating Ly-6C
hi
monocytes (76%,
P
<0.01). The numbers of circulating CCR2
+
and CX3CR1
+
monocytes were also reduced in apoE-KO/BM-Agtr1
−/−
mice (87±16 vs 298±66; CCR2
+
monocyte, 72±12 vs 550±119 cells/μl; CX3CR1
+
monocyte, respectively,
P
<0.01). Furthermore, the number of macrophage progenitor cells defined by M-CSF stimulated macrophage colony-forming unit was markedly reduced by 82 % (p<0.01) in Agtr1
−/−
compared with Agtr1
+
/
+
mice. We next examined the effect of BM-AT
1
on the number of hematopoietic stem cells (HSCs), common myeloid progenitors (CMP), and granulocyte/macrophage progenitors (GMP) to determine at which point of the lineage pathway, BM-AT
1
is involved. The number of HSCs did not differ between the two groups (3.6±0.8 vs 2.9±0.6 × 10
3
cells/tibia,
P
=n.s.), whereas the numbers of CMP and GMP were much lower in apoE-KO/BM-Agtr1
−/−
mice (4.7±0.7 vs 10.2±1.4 ×10
3
cells/tibia, 9.9±2.7 vs 20.2±2.8 × 10
3
cells/tibia, respectively,
P
<0.05).

[CONCLUSION]
BM-MNCs expressed the abundant densities of AT1, and AT1-mediated signals on BM-MNCs exaggerated atherosclerotic lesion development. BM-AT
1
is closely implicated in the differentiation of HSCs into macrophage progenitors and the behavior of monocytes/macrophages, indicating that BM-AT
1
could be a promising therapeutic target for the prevention of cardiovascular events.
Abstract 1312: Dietary Supplementation With Sodium Valproate Attenuates Lesion Development In Hyperglycemic Apolipoprotein E-deficient Mice
Authors: Bowes Anna J , Khan Mohammad I , Shi Yuanyuan , Robertson Lindsie A , Werstuck Geoff H
Published: July 2021

Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke however the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. We have shown that conditions of hyperglycemia are associated with accumulation of glucosamine, a down stream metabolite of glucose, in specific cell types. Our findings indicate that elevated levels of intracellular glucosamine can promote endoplasmic reticulum (ER) stress in vascular cells leading to inflammation and lipid accumulation - the hallmark features of atherosclerosis. Treatment with valproic acid, a small branch chain fatty acid, can protect cultured cells from glucosamine-induced lipid accumulation by a mechanism that appears to involve inhibition of the signaling factor glycogen synthase kinase (GSK)-3. Here we evaluate the hypothesis that valproic acid can reduce the development and progression of atherosclerotic lesions in a hyperglycemic mouse model of accelerated atherosclerosis. Female apoE
−/−
mice were made hyperglycemic with multiple low dose injections of streptozotocin. At 15 weeks of age, hyperglycemic mice had accelerated the development of atherosclerotic lesions at the aortic root compared to normoglycemic mice. Mice fed a diet supplemented with 625 mg/kg sodium valproate had significantly reduced lesion volume relative to unsupplemented controls (4.98±0.76 versus 8.62±1.23 μm
3
,
P
<0.05,
n
=9). Valproate supplementation had no apparent effect on plasma glucose or plasma lipid levels or on the expression of diagnostic markers of ER stress in the atherosclerotic plaque. Significant reductions were observed in total hepatic lipids (>50.4%,
P
<0.05,
n
=3) and hepatic GSK-3β activity (>55.8%,
P
<0.05,
n
=3) in mice fed the valproate diet. In conclusion, dietary supplementation with low levels of sodium valproate significantly attenuates atherogenesis in hyperglycemic apoE
−/−
mice. This effect may involve the previously observed ability of valproate to attenuate the downstream effects of ER stress, such as GSK-3β activity.
Abstract 1316: Preclinical Atherosclerosis in Primary Antiphospholipid Syndrome: The Impact of Oxidative Properties of High Density Lipopoprotein
Authors: Charakida Marietta , Halcox Julian , Batuca Joanna , Donald Ann , Sangle Shirish , Alves Jose Delgado , Cruz David , Mackworth-Young Charles , Hughes Graham , Deanfield John
Published: July 2021

Background:
Antiphospholipid syndrome (APS) is characterised by increased thrombogenicity and/or pregnancy morbidity in the presence of raised levels of antiphospholipid antibodies (aPL). Increased oxidative properties of high density lipoprotein (HDL)(decreased activity of paraoxonase (PON)) is associated with increased risk for atherosclerosis and has been described in APS. The impact of PON on atherosclerotic disease progression in APS is unclear. We therefore examined the effect of PON on intima media thickness (IMT), and pulse wave velocity (PWV) in patients with positive aPL.

Methods:
We studied 77 women with positive aPL (aPL) aged 46.6±1.2 yrs (mean±SE) and a control group of 77 women aged 47.5±1.2 yrs matched for traditional cardiovascular risk factors. High resolution ultrasound was used to determine carotid IMT. Arterial stiffness was assessed non-invasively by carotid-radial PWV. PON activity was assessed by measuring p-nitrophenol formation and activity expressed as nmoles p-nitrophenol/ml serum/minute.

Results:
APL patients had significantly increased IMT and PWV compared to controls (0.75±0.02mm vs 0.65±0.01mm, p<0.001 and 9.14±0.18 m/s vs 8.56±0.21m/s, p<0.05 respectively). PON activity was significantly reduced in aPL compared to controls (91.5[64.3, 05.1]mmol/ml/min, median[IQR] vs 103.1[80.4, 111.5] mmol/ml/min, p<0.006). Although PON activity was not associated with vascular measures in controls, an inverse association was noted in aPL patients (r=−0.26 [cIMT] and r=−0.23 [PWV], both p<0.05). In multivariate analysis, accounting for cardiovascular risk factors, PON activity (β=−0.42, p<0.001), age (β=0.33, p<0.001) and systolic blood pressure (β=0.24, p<0.05) were independent determinants of cIMT while PON activity (β=−0.32, p<0.01) and systolic blood pressure (β=0.28, p<0.05) remained the only independent predictors of PWV in aPL positive patients.

Conclusions:
APS is associated with increased arterial stiffness and carotid intimal thickening. Paraoxonase activity is inversely associated with IMT and PWV in ApL positve patients. These findings indicate that oxidative stress may play an important role in the development of atherosclerosis in patients with primary antiphospholipid syndrome.
Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex
Authors: Mohamed Tamer M , Oceandy Delvac , Alatwi Nasser , Baudoin Florence , Cartwright Elizabeth J , Neyses Ludwig
Published: July 2021

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca
2+
/calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca
2+
/Calmodulin-dependent Ca
2+
-pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness
in vivo
via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca
2+
-pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).
Abstract 1344: Biphasic Effect of Electrotonic Fibroblast-Myocyte Coupling on 2D Wave Conduction Velocity and Reentry
Authors: Zlochiver Sharon , Munoz Viviana , Oxford Eva M , Vikstrom Karen L , Berenfeld Omer , Jalife Jose
Published: July 2021

Fibroblast proliferation is common to various cardiac diseases. Differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that fibroblast proliferation and varying heterocellular coupling significantly alter 2-dimensional cardiac wave propagation and reentry dynamics.

Methods
: Monolayers of co-cultured fibroblasts and myocytes from neonatal rat ventricles were optically mapped. The junctional coupling was reduced using Cx43 RNA silencer in the fibroblasts. Simulations were used to quantify the effects of heterocellular coupling variation. Conduction velocity (CV) was measured when pacing at 2 Hz.

Results
: Experiments and simulations show linear CV and reentry frequency reduction with increased number of wavebreaks as fibroblast/myocyte mixed area ratio increases. However, simulations show that the effect of fibroblast-myocyte coupling on CV and reentry frequency is biphasic: as coupling increases reentry frequency and CV initially decrease and then increase. Panel A shows that for a 1/4 fibroblasts/myocytes mixture the minimum CV is at ~10% of the myocytes coupling. In both experiments and simulations, low heterocellular coupling can result in similar CV and frequency of rotation (p<0.05), but more fragmented wavefronts as compared with high coupling (panel B).

Conclusion:
Fibroblasts may have a dual role as current “sinks” and “transmitters”, depending on the level of heterocellular coupling. This study provides novel insight into the mechanisms whereby electrical interactions between myocytes and fibroblasts modify wave propagation and the propensity to arrhythmias.
Abstract 1346: Rac1 GTPase Activtiy is Associated with Atrial Fibrosis and Fibrillation
Authors: Adam Oliver , Lavall Daniel , Schäfers Hans-Joachim , Böhm Michael , Laufs Ulrich
Published: July 2021

Patients with atrial fibrillation (AF) are characterized by increased atrial fibrosis. The underlying signal transduction is incompletely understood. We hypothesized that activation of Rac1 GTPase contributes to increased fibrosis and atrial fibrillation via activation of NADPH oxidase and production of reactive oxygen species.

Methods and Results:
Samples of the left atrial appendage were analyzed in 8 patients with AF undergoing mitral valve surgery and 7 patients with sinus rhythm (SR) matched for atrial diameter. Despite same size of the atria, collagen content was significantly higher in AF compared to SR (14.9±2.1% vs. 8.5±1.3%). Expression analysis showed that AF was associated with upregulation of connective tissue growth factor (CTGF). Atria of patients with AF were characterized by significant upregulation of Rac1 total protein expression (Western blot), Rac1 activity (PAK pull-down assays) and a 20-fold upregulation of NADPH oxidase activity compared to SR (2225±500%). In order to test whether Rac1 plays a causal role in the pathogenesis in AF, transgenic mice with cardiac overexpres-sion (αMHC promoter) of Rac1 (RacET) were compared to wildtype (WT) and WT undergoing transaortic constriction (TAC, 360 μm). After 16 months, echocardiography showed similar left ventricular hypertrophy in RacET and TAC. RacET but not TAC exhibited atrial enlargement; 75% of RacET but no WT or TAC showed AF. Interstitial collagen content was significantly increased in the atria of RacET (44±1% of area vs 19±5% in WT). In contrast, interstitial fibrosis in TAC atria did not significantly differ from WT (31±6%). In the left ventricle, both RacET and TAC mice showed an elevated collagen content compared to the control group (WT 9±2%, RacET 29±3%; TAC 24±4%). In all mice, atrial collagen content exceeded ventricular collagen. All effects are significant with p<0.05.

Conclusion:
Left atria of patients with AF exhibit upregulation of Rac1, increased NADPH oxidase activity and interstitial fibrosis. Cardiac-specific overexpression of Rac1 in mice results atrial fibrosis and fibrillation independent of left ventricular hypertrophy. Rac1-GTPase mediated activation of left atrial NADPH-oxidase may represent a novel target for the prevention of atrial fibrosis.
Abstract 1356: Is Human Ventricular Fibrillation Different From Experimental Models?
Authors: Nash Martyn P , Mourad Ayman , Bradley Chris P , Paterson David J , Clayton Richard H , Sutton Peter M , Hayward Martin , Taggart Peter
Published: July 2021

Introduction:
There is a paucity of data on wavefront dynamics during human VF. Some experimental models show multiple small wavelets, whilst others implicate a single stationary rotor. We have studied human VF using phase and wavefront analysis based on global epicardial mapping.

Methods:
VF was induced by burst pacing in 10 patients undergoing routine cardiac surgery. For each subject, a 20 – 40 s episode of VF activity was sampled at 1 kHz using an epicardial sock containing 256 unipolar contact electrodes connected to a UnEmap system. Wavefronts were determined from the phase plane (based on de-trended voltage versus its Hilbert transform) using an active-edge algorithm to track the isolines of zero phase. Wavefront sizes and rotor statistics were tallied across all patients.

Results:
As illustrated, one or more wavefronts of at least 10 cm in size were present for 90% of the VF duration. Insert shows a polar projection of wavefronts between pairs of phase singularities. Large convoluted wavefronts were predominant with at least one wavefront of size > 20 cm present for over half of the VF duration. Persistent epicardial rotors (defined as those with lifetimes greater than 1000 ms, or approximately 5 rotations) were present for more than 78% of the total VF duration, whilst two or more persistent rotors were present more than half of the time.

Conclusion:
Epicardial mapping in humans suggests that neither multiple wavelets nor a single persistent stationary rotor, as seen in experimental models, drive VF. Instead, in all subjects the predominant characteristic was multiple mobile rotors of moderate duration (1000 –7000 ms), which generated large make and break wavefronts.
Abstract 1371: NCS-1 is a Potential Therapeutic Target for Avoiding Bradycardia and Optimizing Cancer Chemotherapy with Paclitaxel
Authors: Zhang Kun , Heidrich Felix M , Ehrlich Barbara
Published: July 2021

We hypothesize that the inositol 1,4,5-trisphosphate receptor (InsP3R) signaling pathway is crucial for the development of cardiac arrhythmia caused by paclitaxel treatment. Paclitaxel, a well established drug used for cancer chemotherapy, constrains tumor growth through a microtubule stabilizing mechanism. However, use of paclitaxel is limited by serious side effects including bradycardia, which cannot be explained by its effect on microtubules. Recently, a new binding partner of paclitaxel was described - neuronal calcium sensor-1 (NCS-1), a protein that modulates the function of the InsP3R and that we found to be expressed in cardiomoycytes. After treating neonatal ventricular cardiomyocytes from rats with a therapeutic dose of paclitaxel for several hours, NCS-1 levels were markedly elevated. The same effect was also detectable in vivo, in hearts extracted from adult mice chronically injected with paclitaxel. Using live cell imaging, we observed an increase in the peak of the intracellular calcium signal and a significant decrease in the spontaneous calcium oscillation frequency in paclitaxel treated cardiomyocytes compared to control. The changes could not be explained by the function of ryanodine receptors (RyR) because there is no functional interaction between NCS-1 and RyR, as previously shown in bilayer experiments, and no paclitaxel-dependent change in RyR expression. However, co-immunoprecipitation confirmed a direct interaction between NCS-1 and InsP3R type 2, the predominant isoform in cardiomyocytes. To test whether this interaction is functionally relevant, we stimulated cardiomyocytes with endothelin, an activator of the InsP3 signaling cascade and found a difference in calcium transients between paclitaxel-treated and untreated cardiomyocytes. NCS-1 knockdown in cardiomyocytes by shRNA validated our findings. Taken together, our results suggest that paclitaxel-induced cardiac arrhythmia in patients is mediated by the interaction of NCS-1 and the InsP3R. This gives us a new understanding of the pathomechanism and provides a new approach for reducing the side effects of paclitaxel treatment and optimizing cancer chemotherapy.
Abstract 1380: Antioxidants Decrease Neuronal Angiotensin II Type 1 Receptor in Heart Failure: Inhibition of Activator Protein 1 and Jun N-Terminal Kinase
Authors: Liu Dongmei , Gao Lie , Cornish Kurtis G , Zucker Irving H
Published: July 2021

In a previous study, we showed that Ang II type I receptor (AT1R) expression increased in the rostral ventrolateral medulla (RVLM) of chronic heart failure (CHF) rabbits and in normal rabbits infused with intracerebroventricular (ICV) Angiotensin II (AngII). The present study investigated if oxidative stress plays a role in Ang II induced AT1R upregulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In neuronal cell cultures, Ang II significantly increased AT1R mRNA by 153 ± 22%,
P
<0.01; c-Jun mRNA by 90 ± 10%,
P
< 0.01; NADPH oxidase activity by 126 ± 43%,
P
< 0.01 versus untreated cells; Tempol, Apocynin and the AP 1 inhibitor Tanshinone II reversed the increased AT1R, c-Jun expression and NADPH oxidase activity induced by AngII. We examined the effect of ICV Tempol on expression of these proteins in the RVLM of CHF rabbits. Compared to untreated CHF rabbits Tempol significantly decreased AT1R protein expression (0.88±0.16 vs. 1.6±0.29,
P
<0.05), phosphorylated Jnk protein (0.10±0.02 vs. 0.31±0.10,
P
<0.05), and phosphorylated c-Jun (0.02±0.001 vs. 0.14±0.05,
P
<0.05). These data suggest that Ang II induces AT1R upregulation at the transcriptional level by activation of oxidative stress and AP1 in both cultured cells and in intact brain. Antioxidant agents may be beneficial in CHF by decreasing AT1R expression through the Jnk and AP1 pathway.
Abstract 1391: Atherogenic Dyslipidemia Predicts Accelerated Progression of Atherosclerosis in Patients with Coronary Artery Disease
Authors: Galla John M , Tuzcu E. M , Wolski Kathy , Kalidindi Srinivasa , Nissen Steven E , Nicholls Stephen J
Published: July 2021

Background: Atherogenic dyslipidemia (AD) is characterized by the combination of elevated serum triglycerides, low levels of high-density lipoprotein cholesterol (HDL-C) and small-dense low-density lipoprotein cholesterol (LDL-C) and has emerged as a powerful risk factor of coronary disease. The effect of AD on the progression of plaque burden remains incompletely defined. This analysis aimed to determine the impact of AD on measures of atherosclerotic progression in patients receiving intensive lipid lowering therapy.

Methods: 910 patients with angiographic coronary artery disease treated with atorvastatin to a goal LDL-C of 100 ± 15mg/dl received torcetrapib or placebo. Atheroma burden was determined by intravascular ultrasound performed at baseline and following 24 months of treatment. Baseline and changes in atheroma burden were investigated in patients stratified according to the presence or absence of an AD phenotype (HDL-C < 45 mg/dL for men and < 50mg/dL for women, triglycerides 150 – 600mg/dL and LDL-C < 160mg/dL)

Results: 20.4% of patients met the criteria for AD. Patients with AD were younger (55.5 v 58 years, p=0.003), had higher body mass index (30.9 v 29.3 kg/m2, p<0.001) and were more likely to fit the definition of the metabolic syndrome (93 v 30.3%, p<0.001). Accelerated progression of percent atheroma volume (PAV, +0.76 v +0.01%, p=0.001) and total atheroma volume (TAV, −4.75 v −7.18 mm3, p=0.022) was observed in patients with AD. Patients with AD were less likely to undergo substantial regression (reduction in PAV >5%, 12.9% v 24.2%, p=0.001) and more likely to undergo substantial progression (increase in PAV >5%, 34.9% v 23.2%, p=0.001) No differences were observed between treatment groups with regard to changes in PAV (0.59 v 0.99%, p=0.727) or TAV (−5.7 v −3.8 mm3, p=0.461) in patients with AD. On multivariate analysis, the presence of atherogenic dyslipidemia was an independent predictor of increase in PAV (p=0.004) and TAV (p=0.045).

Conclusions: Despite aggressive lipid lowering therapy, AD is an independent predictor of accelerated progression of atherosclerosis. With a rise in the prevalence of obesity and its metabolic complications there is an increasing need to intensively modify factors that place these patients at high risk.
Abstract 1396: Mitigation Of Cardiac Injury By Sulfaphenazole, A CYP2C9 Inhibitor Through Involvement Of Inducible Nitric Oxide Synthase (iNOS) in Post-ischemic Heart
Authors: Khan Mahmood , Mohan Iyyapu K , Kumbala Damodhar , Kutala Vijay K , Kuppusamy Periannan
Published: July 2021

Cytochrome P450 enzymes play a significant role in ischemia-reperfusion (I/R) injury. Sulfaphenazole (SFZ), a potent CYP2C9 inhibitor, is known to reduce I/R injury. However, the mechanism of its cardioprotective effects and the role of nitric oxide (NO) is not clear.

Objective
: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important mediators of I/R injury. The objective of this study was to determine how SFZ treatment modulates myocardial tissue oxygenation (pO
2
) and RNS generation
in vivo
.

Methods:
Myocardial infarction was induced in rats by ligating the left anterior descending coronary artery (LAD) for 30 min, followed by 24 h of reperfusion. The study was divided into 4 groups: Untreated I/R control, SFZ, L-NAME, and SFZ+L-NAME. L-NAME (100 mg/kg/day), a nitric oxide synthase inhibitor, was given orally for 3 days prior to LAD ligation. SFZ (1.5 mg/kg, ip) was injected 30 min prior to LAD ligation. Oxygen-sensing crystals were implanted into the LV wall and the rat was placed in an L-band (1.2 GHz) electron paramagnetic resonance (EPR) spectrometer for measurement of myocardial tissue pO
2
during I/R injury. Hemodynamic data was collected with a microtip catheter. Infarct size was measured after 24 h of reperfusion. Superoxide generation was determined by dihydroethidium fluorescence imaging. Immunohistological staining was performed for nitrotyrosine and iNOS.

Results:
After LAD ligation, pO
2
decreased from 18 mmHg baseline to <2 mmHg. At reperfusion, there was a significant myocardial hyperoxygenation in SFZ-treated rats compared to control group (45.0±1.3 vs. 34.0±2.0 mmHg, P<0.05). In L-NAME and L-NAME+SFZ-treated rats, there was a significant reduction in pO
2
(24.0±1.6 and 26.0±2.3 mmHg, respectively). Compared to control, SFZ treatment significantly improved the left ventricular developed pressure (94.0±4.7 vs. 69.0±6.5 mmHg, P<0.05), decreased infarct size % (35.0±4.2 vs. 16.0±2.5, P<0.05), decreased superoxide generation and nitrotyrosine production.

Conclusions:
These findings demonstrate that SFZ may provide potent cardioprotection by attenuating post-ischemic ROS and RNS generation, and could serve as an attractive adjuvant therapy in the clinical setting of myocardial I/R injury.
Abstract 1410: Clarithromycin Inhibited MMP-9 Activation and Attenuated Acute and Chronic Rejection in Murine Cardiac Allografts
Authors: Suzuki Jun-ichi , Ogawa Masahito , Takayama Kiyoshi , Isobe Mitsuaki
Published: July 2021

Background: Matrix metalloproteinases (MMPs) are critical in the development of inflammation and tissue remodeling. MMP-9 is a key member of MMPs and the level is associated with the rejection of heart transplantation. Clarithromysin (CAM), a major macrolide antibiotic, has been many biological functions including MMP regulation. However, little is known about the effect of CAM in heart transplantation via MMP-9.

Methods and Results: To clarify the role of MMPs regulated by CAM in the progression of rejection, we orally administered CAM (100mg/kg) into murine cardiac allograft recipients. CAM improved acute rejection judged by graft survival (control: 7.3+/−0.2 days n=8; CAM: 9.7+/−0.2 days n=8; P<0.05) and by myocardial cell infiltrating area (control: 35.7+/−1.9% n=9; CAM: 19.7+/−3.0% n=9, P<0.05) in a total allomismatch combination. 8 weeks after transplantation, CAM significantly suppressed the development of graft arterial disease (5.1+/−3.0%, n=6) compared to that of non-treatment (25.1+/−6.3%, n=6; P<0.05) in the class II mismatch combination. Immunohistochemically, non-treated allografts showed enhanced expression of T-cells, macrophages, MMP-9 and NF-kappaB while CAM decreased these positive cell numbers. RPA also revealed that CAM-treated allografts significantly attenuated the expression of proinflammatory cytokines such as IFN-gamma, IL-6, IL-10 and IL-15 compared with non-treated grafts. In situ zymography indicated that enhanced MMPs activities were observed in non-treated hearts, while CAM suppressed the levels. In vitro study showed that CAM inhibited the expression of MMP-9 using Western blot and RT-PCR in cytokine stimulated macrophages and SMCs. The effects of inhibited MMP-9 by CAM suppressed SMCs migration (native: 23.5+/−4.5 cells, control: 97.0+/− 5.0, CAM: 53.4+/−4.5, P<0.05) and proliferation (native: 0.44+/−0.01OD, control: 0.56+/−0.03, CAM: 0.44+/−0.004, P<0.05) compared with control.

Conclusion: CAM is useful to suppress allograft remodeling because it is critically involved in the prevention of cardiac rejection through the suppression of MMP-9.
Abstract 1412: Critical Role Of Extracellular Sod For Protecting The Heart Against Myocardial Hypertrophy And Dysfunction After Myocardial Infarction
Authors: Deel Elza D , LU Zhongbing , Xu Xin , Zhu Guangshuo , Hu Xinli , Fassett John , Zhang Ping , Oury Tim D , Bache Robert J , Duncker Dirk J , Chen Yingjie
Published: July 2021

Extracellular SOD (
SOD3
) contributes only a small fraction to total SOD activity in the normal heart, but is strategically located to scavenge free radicals in the extracellular compartment. SOD3 expression is decreased in the failing heart, but whether SOD3 can abrogate oxidative stress or modify left ventricular (LV) remodeling following myocardial infarction (MI) is unclear. To examine this question, we studied LV remodeling in SOD3 KO mice and wild type mice following MI. Under unstressed conditions, SOD3 KO had no effect on myocardial total SOD activity, SOD1 or SOD2 protein content, or myocardial nitrotyrosine or superoxide anion production, and caused no change in LV ejection fraction. However, 4 weeks or 8 weeks after MI, SOD3 KO mice developed more LV hypertrophy (8 weeks after MI, ventricular mass increased 1.64-fold in KO mice as compared to 1.35-fold in wild type mice, p<0.01) and had a greater reduction of LV ejection fraction (8 weeks after MI, LV ejection fraction was 35±2.4% in wild type mice as compared to 30±2.0% in KO mice, p<0.01). As compared with wild type mice, SOD3 KO mice had significantly greater increases of myocardial nitrotyrosine and superoxide anion production, a significantly greater decrease of ANP in the peri-infarct zone, and a significant more decrease of SERCA2a in both the peri-infarct and remote zones. In addition, MI caused greater activation of mitogen-activated protein kinase (MAPK) signaling pathways in SOD3 KO mice, as demonstrated by significantly greater increases of p-p38
Thr180/
Tyr182
, p-Erk
Thr202/Tyr204
and p-JNK
Thr183/Tyr185
in SOD3 KO mice 8 weeks after MI. The finding that SOD3 KO had no effect on myocardial total SOD activity, but significantly exacerbated MI induced LV remodeling implies that the specific extracellular location of SOD3 is more important than its contribution to overall SOD activity in protecting the heart against contractile dysfunction following myocardial infarct.
Abstract 1436: CYP2J2 Endothelial Overexpression Increases Nitric Oxide and Cyclooxygenase Independent Renal Dilation and Attenuates Endothelin-1 Vasoconstriction
Authors: Imig John D , Lee Craig R , Bradbury Alyce , Graves Joan P , DeGraff Laura M , Foley Julie , Zeldin Darryl C
Published: July 2021

Human CYP2J2 is expressed in endothelial cells and active in the biosynthesis of epoxyeico-satrienoic acids (EETs). However, the functional role of CYP2J2 and its products in the renal vasculature remain poorly characterized. To address this, we developed transgenic (Tr) mice with constitutive, endothelial cell-specific expression of human CYP2J2 (
Tie2
promoter and full enhancer) and enhanced EET biosynthesis. Experiments were conducted in the juxtamedullary nephron preparation to determine renal microvascular responses to acetylcholine (ACh) and endothelin-1 in
Tie2
-CYP2J2 Tr mice and wild type (Wt) littermate controls. Administration of phenylephrine to kidney perfusate decreased the diameter of afferent arterioles from 20.1±0.5 to 13.9±0.6 μm (n=21) in Wt mice and 19.4±0.6 to 13.5±0.6 μm (n=23) in
Tie2
-CYP2J2 Tr mice. Following phenylephrine, the afferent arteriole diameter response to ACh (0.01nM-10μM) was determined. There was a leftward shift in the logEC50 in
Tie2
-CYP2J2 Tr mice (−6.5±0.2, n=13) compared to Wt mice (−6.1±0.2, n=11). However, the maximal afferent arteriolar relaxation to ACh was decreased in
Tie2
-CYP2J2 Tr mice (59±6%) compared to Wt mice (70±7%, p=0.12). Endothelial expression of CYP2J2 increased the maximal renal vascular response to ACh in the presence of nitric oxide synthase (100μM L-NAME) and cyclooxygenase (10μM indomethacin) inhibition. Afferent arterioles relaxed by 27±4% (n=12) in Wt mice and 44±6% (n=10, p=0.018) in
Tie2
-CYP2J2 Tr mice (10μM ACh). The afferent arteriolar dose response curve to endothelin-1 (0.001–10nM) was significantly attenuated in
Tie2
-CYP2J2 Tr compared to Wt mice. Afferent arteriolar diameter decreased by 24±4% (n=6) in Wt mice and 13±2% (n=5, p=0.023) in
Tie2
-CYP2J2 Tr mice (3nM endothelin-1). These results demonstrate that the nitric oxide- and cyclooxygenase-independent afferent arteriolar dilation to ACh is enhanced by endothelial overexpression of CYP2J2, and endothelin-1 mediated constriction is attenuated. In conclusion, endothelial overexpression of CYP2J2 can oppose renal vascular constrictor responses and enhance dilator responses in mice, implicating the important role of CYP2J2-derived eicosanoids in the regulation of vascular tone.
Abstract 1437: Polymorphisms in the Estrogen Receptor Genes and Endothelium-Dependent Vasodilation in Elderly
Authors: Ingelsson Erik , Syvänen Ann-Christine , Lind Lars
Published: July 2021

Background:
Single nucleotide polymorphisms (SNPs) in the estrogen receptor 1 (
ESR1
) and 2 (
ESR2
) genes have been associated with blood pressure, subclinical and overt cardiovascular disease, but their relation to endothelium-dependent vasodilation is unknown.

Methods:
We evaluated 959 70-year-old participants of the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study, using invasive forearm technique with intra-brachial infusion of acetylcholine (EDV; reflecting vasodilation in resistance arteries), and brachial artery ultrasound to assess flow-mediated vasodilation (FMD; reflecting vasodilation in conduit arteries). We analyzed 25 common SNPs in the
ESR1
gene and 5 in the
ESR2
gene by minisequencing, and related them to EDV and FMD using multivariable linear regression, adjusting for sex, systolic blood pressure, diastolic blood pressure, pulse rate, anti-hypertensive treatment, total cholesterol, high-density cholesterol, lipid-lowering medication, fasting glucose, anti-diabetic medication, body mass index, current smoking, and prior diagnosis of cardiovascular disease. Haplotypes were estimated using the PHASE software.

Results:
We observed an association between rs1709183 in the
ESR1
gene and EDV (nominal P=0.0012), with a lower EDV in carriers of the minor allele (C). This association remained significant after adjustment for multiple testing (empirical P=0.031, obtained using bootstrap re-sampling). Two
ESR1
haplotypes in the block containing rs1709183 were associated with EDV (individual haplotype P=0.0015 and P=0.025); the directions of effect were consistent with the individual SNP analyses. There were no associations between SNPs in the
ESR2
gene and EDV with a nominal P=0.01. FMD was not associated with either
ESR1
or
ESR2
.

Conclusions:
In our community-based study of elderly, a polymorphism in the
ESR1
gene was associated with endothelium-dependent vasodilation in resistance arteries, but not in conduit arteries. Our findings should stimulate to replication and further exploration in other settings.
Abstract 1443: Angiotensin Ii Type 2 Receptor-dependent Dilation Is Reduced In Type Ii Diabetes Due To Cox-2 Generated Thromboxane A2 And Reactive Oxygen Species.
Authors: Retaileau Kevin , de Chantemele Eric Belin , guihot annelaure , Vessieres Emilie , Chanoine Sebastien , Jardel Alain , henrion daniel , Loufrani Laurent
Published: July 2021

The role of angiotensin II type 2 receptors (AT2R) is not fully understood, especially in vascular diseases. Although its vasodilatory and antitrophic properties are well accepted, in hypertensive rats AT2R stimulation induces vasoconstriction. Abnormalities in the renin-angiotensin system have a key role in cardiovascular disorders associated with the evolution of type II diabetes. Thus, we hypothesized that AT2R function might be altered in diabetic rats resistance arteries. Mesenteric resistance arteries (250 μm diameter) were isolated from type II diabetic Zucker (ZDF) and control rats (LZ) rats receiving the antioxidant tempol (16 mg/kg/day) or water for 3 weeks. Dilation to angiotensin II (due AT2R stimulation) in the presence of candesartan (angiotensin II type 1 receptor blocker) was studied in ring segment of arteries using a wire-myograph mesenteric wall force. AT2R induced endothelium and NO-dependent dilation in LZ (inhibited by L-NAME). It was not affected by cyclooxygenase (indomethacin) or reactive oxygen species (ROS) inhibition (TEMPOL). In ZDF rats AT2R-induced dilation was lower than in LZ rats and independent of NO (not inhibited by L-NAME). It was restored to control level after ROS (tempol) and cycloxygenase inhibition (indomethacin). Cycloxygenase-2 (NS398) and Thromboxane A2 receptor (SQ29548) inhibition also restored AT2R-dilation in ZDF rats to control (LZ) level. Cycloxygenase-2 and ROS were overexpressed (immuno-histological detection using confocal microscopy) in ZDF rats arteries. In ZDF rats chronically treated with the antioxydant Tempol (SOD mimetic) AT2R-induced dilation was equivalent to that in LZ (control) rats and it involved NO and cycloxygenase-2-derivatives in equivalent proportion (dilation inhibited in part by L-NAME and by NS398). In tempol-treated rats SQ29548 had no effect. We conclude that in type II diabetic rats AT2R induced dilation is reduced due to ROS and thromboxane A2-dependent vasoconstriction due to COX-2 activation. Chronic treatment of diabetic rats with tempol restored AT2R-dependent dilation through the suppression of ROS and TxA2 production. These findings might be important to consider in the choice of vasoactive drugs in diabetic patients.
Abstract 251: Endogenous Endocrine Function of Cardiac-derived Nitric Oxide Yields Distant Organ Protection Against Ischemic Injury
Authors: Elrod John W , Bryan Nathan S , Calvert John W , Gundewar Susheel , Lefer David J
Published: July 2021

Background

: eNOS-derived nitric oxide (NO) has long been considered a paracrine signaling molecule only capable of affecting nearby cells due to its short half-life in blood (<2ms). We have recently shown that nitrite, previously thought to be an inert oxidation product of NO, is an important storage form converted to NO under hypoxic conditions via reductase activity. Currently, no studies have revealed endogenously generated NO to possess a clearly defined endocrine function, we therefore evaluated whether generation of NO in the heart could modulate remote physiological actions.

Methods:

Mice with cardiac-specific (αMHC promoter) eNOS overexpression (CS-eNOS-Tg) and WT littermates were analyzed for NO metabolites in the heart, plasma, and liver (NO
2
−
/NO
3
−
- ion chromatography; nitroso/NO-heme - gas phase chemiluminescence). WT and CS-eNOS-Tg mice were subjected to 60 min hepatic ischemia and following 5 hr reperfusion (IR) serum ALT and AST were analyzed.

Results:

Overexpression of eNOS restricted to the heart resulted in a significant increase in nitrite in the heart (2.2 fold), plasma (3.2 fold) and liver (6.5 fold). CS-eNOS-Tg displayed a significant reduction in hepatic IR injury (4.3 fold reduction in ALT and a 3.5 fold reduction in AST).

Conclusions:

This represents the first report demonstrating that eNOS-derived NO is transported in the blood and stored in remote organs. Importantly, increases in nitrite levels resulted in protection against IR injury. These findings suggest NO possesses endocrine function via transport of nitrite in the blood to distant organs and that transported nitrite exhibits physiological activity.
Abstract 1460: Nox2 Signalling via p21
Cip1
and p53 in Endothelial Cell Cycle Regulation
Authors: Fan Lampson M , George Vinoj , Brooks Gavin , Li Jian-Mei
Published: July 2021

The abilities of endothelial cells to proliferate, to be quiescent in monolayer and to undergo apoptosis during remodeling are important determinants relating to angiogenesis, wound healing and many diseases including atherosclerosis. During ischemia, angioplasty or organ transplantation, endothelial cells experience sustained nutrient deprivation (starvation), which causes cell oxidative stress and apoptosis. Endothelial cells express constitutively an NADPH oxidase, which is a major source of superoxide production. The catalytic subunit of NADPH oxidase has several isoforms (Nox1–5). However, their individual roles in endothelial function remain unknown. In this study, we investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC (HMEC1), Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4~1:13), but was upregulated 24h after starvation and increased to 8±3.5-fold at 36h of starvation as detected by quantitative real-time PCR. Accompanying the upregulation of Nox2, there was a 2.28±0.18-fold increase in O
2
.−
production detected by tiron (O
2
−.
scavenger)-inhibitable lucigenin (5 μM)-chemiluminescence; a dramatic induction of p21
cip1
and p53 protein expression detected by immunoblotting; cell cycle arrest and the onset of cell apoptosis detected by propidium-iodide FACS analysis (all P<0.05). All these changes were inhibited significantly by adding apocynin (an NADPH oxidase inhibitor), or by
in vitro
deletion of Nox2 expression using full-length antisense Nox2 cDNA, or in coronary microvascular endothelial cells isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8±0.5-fold increase in Nox4 mRNA expression after 36h of starvation (P<0.01), neither O
2
.−
production nor the p21
cip1
and p53 expressions were increased significantly and only 0.46% of cells were apoptotic compared to ~12% apoptotic cells found in wild-type control cells cultured under the same condition. In conclusion, Nox2-derived O
2
.−
, through the modulation of p21
cip1
and p53 expression, participates in the regulation of endothelial cell cycle control and apoptosis.
Abstract 1471: Development and Characterization of Force Generating Human Engineered Heart Tissue from Embryonic Stem Cells
Authors: Rogge Christina , Didié Michael , Wettwer Erich , Ravens Ursula , Graichen Ralph , Xu Xiuqin Q , Colman Alan , Davidson Bruce , Zweigerdt Robert , Eschenhagen Thomas , Zimmermann Wolfram H
Published: July 2021

Engineered Heart Tissue (EHT) from neonatal rat cardiomyocytes has been used successfully as in vitro model and in cardiac repair. Here, we hypothesized that human embryonic stem cells (hESC) can be used to generate EHT with properties of native myocardium.

Methods:
hESC (hES3-ENVY) were differentiated in embryoid bodies, enzymatically dispersed, and subjected to EHT-generation in circular casting molds (1.5x10
6
cells, 0.4 mg collagen, 10% Matrigel/EHT; inner/outer diameter - 2/4 mm). Contractile function was assessed 10 days after casting under isometric conditions (37°C, 1.5 Hz, Tyrode’s solution). Action potentials (AP) were recorded in spontaneously contracting EHTs with intracellular electrodes (37°C, Tyrode’s solution). Calcium gradients were assessed by confocal laser scanning microscopy (CLSM) after rhod-2 loading. EHT-morphology was examined by CLSM and electron microscopy (EM).

Results:
hESC-EHTs contracted synchronously and spontaneously at 1.1±0.1 Hz (n=3). Increasing concentrations of extracellular calcium (0.2–2.4 mM) enhanced force of contraction from 53±8 to 199±22 μN (n=8, p<0.05; EC
50
: 0.8±0.04 mM). Isoprenaline (1 μM) at 0.4 mM calcium increased twitch tension from 61±7 to 108±15 μN (n=8, p<0.05) and shortened relaxation time from 111±6 to 87±4 ms (n=3, p<0.05). Cardiomyocytes within EHTs formed a functional syncytium composed of predominantly oriented muscle strands with a high degree of sarcomere differentiation (CLSM, EM). Cell-cell contacts through adherens junctions were identified by EM. Synchronous calcium gradient spread in spontaneously contracting EHTs indicated electrical coupling of individual cells within the multicellular constructs. AP recordings identified pacemaker cells (spontaneous diastolic depolarization) and cells with a flat phase 4 of the AP (working myocardium-like cells). Pharmacological studies demonstrated the presence and functional relevance of I
Na
(10–30 μM flecainide), I
Ca
(1 μM nisoldipine), and I
Kr
(1–5 μM E4031).

Conclusion:
Human force-generating EHT with functional and morphological properties of native myocardium can be generated. Ultimately, hESC-EHTs may constitute a model system for substance screening and could further be utilized in cardiac repair.