bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 601: Mechanistic Dichotomy Of Resveratrol-mediated Inhibition Of Vascular Smooth Muscle Cell Proliferation And Restenosis
Authors: Khandelwal Alok R , Ekshyyan Viktoriya , Hebert Valeria , Dugas Tammy
Published: July 2021

Angioplasty is a widely used therapy for coronary artery stenosis, but its application is limited by restenosis occuring in 30 – 40% of patients within 3– 6 months of surgery. Restenosis is due to intimal hyperplasia resulting from migration to and proliferation of vascular smooth muscle cells (VSMC) within the intima. Recent epidemiological evidence suggests red wine exerts greater cardioprotection in women than men. Our hypothesis was that the anti-proliferative effects of resveratrol are mediated by estrogen receptor stimulation. We thus determined effects of resveratrol in VSMC isolated from female rats, compared to female VSMC treated with siRNA for estrogen receptor- (ER-)α. At low doses (0.5–25 μM) resveratrol inhibited VSMC proliferation through an ER-α dependent increase in inducible nitric oxide synthase (iNOS) activity and inhibited the cells in S phase, but at high doses (50–100 μM) the mechanism was NF-κB dependent, with inhibition of cells in G
0
/G
1
phase. At all doses, resveratrol inhibited IKK activity, an upstream kinase for NF-κB activation, but its effect at low doses was partially reversed by ER-α blockade. We also found a mechanistic link between ER-α receptors, cyclooxygenase-2 (COX-2), a known modulator of VSMC proliferation, and resveratrol. At low doses, resveratrol induced COX-2 expression, consistent with ER-α stimulation, while at high doses it inhibited COX-2 protein levels, likely due to an inhibition of NF-kB. To confirm these findings in an
in vivo
model for restenosis, B6.129 (n=8 per group) mice were fed with a high fat diet +/− 50 mg/kg resveratrol for 2 weeks. A carotid artery endothelial denudation procedure was conducted and after recovery, the mice were again fed a high fat diet +/−resveratrol for 2 weeks. Resveratrol increased arterial iNOS activity (p<0.05) and total NO production (p<0.05) but inhibited NF-κB activation (p<0.01). Studies in progress utilizing ER-α (−/−) mice are determining the role of ER-α in resveratrol- mediated effects and whether resveratrol prevents neointimal hyperplasia in this model. In conclusion, though resveratrol clearly inhibited NF-κB activation, it appears that it’s predominate therapeutic effect at low pharmacologically relevant concentrations is via ERα modulation.
Abstract 605: High Molecular Weight Kininogen Prevents Vascular Smooth Muscle Progenitor Cell Accumulation to Sites of Vascular Injury and Prevents Neointima Formation
Authors: Sedding Daniel , Reich Fabian , Preissner Klaus , Tillmanns Harald
Published: July 2021

Background:

We previously described the anti-adhesive properties of endogenous high molecular weight kininogen (HKa) and its peptide domain 5 (D5) during inflammatory cell recruitment. Since bone marrow-derived vascular progenitor cells substantially contribute to the pathogenesis of vasculoproliferative diseases, this study was aimed to evaluate the effect of HKa and D5 on the accumulation of circulating cells, the function of resident cells, and the resulting vascular remodeling in a mouse model of femoral artery injury.

Methods and Results:

6 weeks after lethal irradiation and bone marrow transplantation from GFP-transgenic into C57/BL6 background mice, the left femoral artery was dilated. Local peri-arterial application of HKa or D5 in a thermoresponsive polymer-gel to the injured artery reduced the accumulation of monocytes /macrophages (HKa: 9.8% ± 1.6%; D5: 4.7% ± 1.4% vs. control: 14.4% ± 1.6%) and prevented the accumulation of bone marrow-derived VSMC (GFP+/SMA+) in the neointima (HK: 16.1% ± 4.7%; D5: 6.6% ± 8.8% vs. control: 25.4% ± 3.4%). HKa and D5 also significantly reduced the number of proliferating (PCNA+) VSMC (HKa: 7.5% ± 1.9%; D5: 4.6% ± 1.9% vs. 11.9% ± 1.9%) and significantly increased the number of apoptotic VSMC in the neointima (HKa: 2.5% ± 2%; D5: 4.2% ± 2.2% vs. 1.6% ± 1.3%), resulting in a significant reduction of neointimal thickening 21 days after vascular injury, (neointima/media ratio HKa: 0.981 ± 0.174; D5: 0.549 ± 0.076 vs. 1.54 ± 0.150; n=6;
P
<0.05).

Conclusion:

Our data indicate that HKa and D5 due to their antiadhesive properties decrease the inflammatory response, the accumulation of bone marrow-derived cells and the proliferation of local neointimal VSMC, resulting in reduced neointima formation. Thus, these data add substantially to the understanding of the role of endogenous HKa, and therapeutic application of HKa may represent a novel approach to prevent vascular proliferative disease.
Abstract 635: Inhibiting Reactive Apoptosis by Cardiac-specific Nix Ablation Prevents Decompensation of Pathological Hypertrophy
Authors: Diwan Abhinav , Wansapura Janaka , Matkovich Scot , Syed Faisal , Li Hairong , Marreez Yehia , Dorn Gerald W
Published: July 2021

Background
: Compensated pathological hypertrophy will inevitably remodel, leading to functional decompensation. Postulated mechanisms include cardiomyocyte dropout, changes in myocardial matrix, and altered cardiac metabolism. Transcriptional modulation of apoptosis-regulating Bcl2 family members is associated with myocardial hypertrophy, but a role for programmed cardiomyocyte death in left ventricular (LV) remodeling has not been established.

Methods and Results
: Proapoptotic Nix, which is transcriptionally upregulated in pressure overload and Gαq-dependent hypertrophies, was subjected to germ-line or cardiac-specific gene targeting (KO) using Cre-lox technology and conditional cardiac-specific overexpression (TG) using tetracycline suppressible bitransgenic system in mice. Conditional forced Nix expression in neonatal and adult hearts was well tolerated, but acted synergistically with the pro-hypertrophic Gαq transgene to increase cardiomyocyte apoptosis (TUNEL %: 0.8±0.1 in Gαq TG vs 7.8±0.6 in Gαq+Nix TG) and caused lethal cardiomyopathy with LV dilation (LVEDD mm: 3.7±0.2 vs 4.6±0.2) and depressed systolic function (% fractional shortening (%FS): 39±4 vs 23±4).
Nix
KO combined with Gαq TG significantly reduced cardiomyocyte apoptosis (TUNEL %: 4.8±0.2 in GαqTG+
Nix
KO vs 8.4±0.5 in GαqTG), improved systolic function (LV%FS: 43±3 vs 27±3), attenuated LV remodeling (r/h:2.4±0.1 vs 4.0±0.6), and largely prevented lethality of the Gαq peripartum model of apoptotic cardiomyopathy. Cardiac-specific (Nkx2.5-Cre)
Nix
KO mice were subjected to transverse aortic constriction (TAC) (gradients: 89±4 vs 83±5 mmHg in controls, n=12 each,
P
=NS). In cardiac
Nix
KO, serial echocardiograms and terminal invasive hemodynamic studies at 9 weeks after TAC revealed significantly less LV dilation (LVEDD mm: 3.3±0.1 vs 3.8±0.2), maintained concentric remodeling (r/h:1.8±0.1 vs 2.3±0.3), and preserved function (%FS: 63±3 vs 51±4; +dP/dt40 mmHg/s: 11519±1211 vs 8499±399) as compared with controls.

Conclusions
: Nix-induced cardiomyocyte apoptosis is a major determinant of adverse remodeling in pathological hypertrophies, and suggests therapeutic value for apoptosis inhibition to prevent cardiomyopathic decompensation.
Abstract 640: Common Genetic Variation in the Promotor of the Ankyrin-2 Gene Modifies QT Interval
Authors: Sedlacek Kamil , Stark Klaus , Weber Stefan , Berger Iris , Pfeufer Arne , Wichmann H.-Erich , Hengstenberg Christian , Jeron Andreas
Published: July 2021

Cardiac QT interval represents both ventricular depolarization and repolarization events. Previous studies suggested significant genetic contribution to the normal QT interval length variation. In a candidate gene approach, we sought to identify novel genetic determinants of the normal QT interval length within the Ankyrin-2 (Ank-2) gene.

Population and Methods: The study population consisted of 1492 participants of the WHO MONICA population survey. A standard measurement of the QT interval from the 12-lead ECG was available in 1212 individuals. The QT interval was corrected for covariates using different general and population specific formulas (QTc). Altogether, 22 SNPs within the Ank-2 gene were genotyped using the TaqMan technology. Statistical analyses included ANOVA and linear regression based trend test for comparisons of the QTc means.

Results: In the promotor region of the alternative splicing Ank-2 variant, several SNPs and haplotypes were highly significantly associated with the QTc interval length. Specifically, a SNP rs1979086 (minor allele frequency f=0.44) was associated with the QTc length in the whole population (P=0.004 for ANOVA and P<0.001 for the trend test). The QTc difference between the genotype groups was 2.87 ms (95%CI 1.18 – 4.56) and more pronounced in men where the QTc difference between the genotype groups was 4.65 ms (95% CI 2.18 – 7.12; P=0.0006 for ANOVA and P=0.0004 for the trend test). The results remained robust when QTc extreme quartiles and quintiles, respectively, were tested in a case-control pattern. Highly significant association results were obtained for all genetic models used (OR 1.58; P=0.00009; trend test for extreme QTc quartiles). A frequent haplotype (f=0.28) spanning approximately 20 kb in this region was highly significantly associated with the QTc length in the whole study population (QTc difference vs. most prevalent haplotype 3.66 ms, 95%CI 3.14 – 4.17, P<0.0001).

Conclusion: A small genomic region in the promotor of the Ank-2 gene contains SNPs and haplotypes that significantly influence the QTc interval in the normal population of German descent. A gender-specific effect with greater influence in male participants requires further evaluation.
Abstract 173: Protein Kinase Cϵ Inhibits Vascular K
Atp
Channels By Caveolae-dependent Mechanism
Authors: Jiao Jundong , Garg Vivek , Yang Baofeng , Hu Keli
Published: July 2021

Vascular ATP-sensitive K
+
(K
ATP
) channels are critical regulators of arterial tone, thus blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is well known that protein kinase C (PKC) activation inhibits K
ATP
channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Since the K
ATP
channel pore-forming subunit Kir6.1 has been localized in caveolar microdomains of arterial smooth muscles, we tested the hypothesis that PKC-induced inhibition of vascular K
ATP
channels is through caveolae-dependent mechanism. The effect of caveolae/caveolin on PKC-mediated inhibition of recombinant K
ATP
channels was investigated in HEK293 cells transfected with Kir6.1 and SUR2B. As expected, whole-cell K
ATP
current density was significantly reduced by activation of PKC with phorbol-12-myristate-13-acetate (PMA, 1μM), but not its inactive congener 4α-PMA (1 μM). The PMA effect was prevented by application of PKCϵ inhibitor peptide Myristoylated PKCϵ V1–2 (Myr PKCϵ V1–2, 200 μM). Interestingly, the inhibitory effect of Myr PKCϵ V1–2 on K
ATP
channels was significantly reduced by disrupting caveolae with cholesterol-depleting agent methyl-β-cyclodextrin (10 mM, 51.9 ± 10.2% vs. 17.2 ± 5.6%, p<0.01, n=5), and further enhanced by overexpression of caveolin-1 (36.8 ± 4.3% vs. 68.3 ± 8.5%, p<0.05, n=5), indicating that integrity of caveolae is essential for PKC-dependent inhibition of K
ATP
channels. Further, Kir6.1 was localized in caveolar fraction by detergent-free sucrose gradient centrifugation. Immunofluorescence images showed that there was substantial colocalization between Kir6.1 and caveolin-1 on the sarcolemmal membrane as punctuate staining pattern. We also show that an antibody against caveolin-1 co-immunoprecipited Kir6.1, suggesting that Kir6.1 and caveolin-1 exist together in a complex. These results indicate that PKC-induced K
ATP
channel inhibition by PMA occurs within caveoar lipid raft microdomains. This study provides first evidence of caveolae-dependent mechanism involved in PKC-mediated K
ATP
channel modulation. Further studies in arterial smooth muscles are under investigation.
Abstract 174: Biophysical Basis of Protein Kinase C Inhibition of the Novel alpha1D Calcium Channel
Authors: Chahine Mohamed , Qu Yongxia , Boutjdir Mohamed
Published: July 2021

The recently reported α
1D
calcium channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single channel level is not known. Therefore, the effect of PKC activation was studied on α
1D
calcium channel expressed in tsA201 cells using cell-attached method. Unitary currents were recorded in the presence of 70 mM Ba
2+
as the charge carrier. Unitary currents were evoked by 500 ms depolarizing pulses from a holding potential of −80 mV every 0.5 Hz. Under basal condition, channel activity was rare and infrequent, however Bay K 8644 (1 μM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by PMA (10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the α
1D
calcium channel. This is consistent with a decreased entry of α
1D
Ca channel into open states in the presence of PMA. These data show, for the fist time, 1) the α
1D
calcium channel activity at the single channel level and 2) the biophysical basis of by which PKC activation inhibits the α
1D
calcium channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the α
1D
Ca-channel activity and the reduction in whole-cell α
1D
Ca current previously reported. Altogether, these data are relevant to the understanding of the patho-physiology of α
1D
calcium channel and its regulation by the autonomics.
Abstract 655: Paradoxical Reduction in Glycosylation Sensitizes the Diabetic Heart to Mitochondrial Permeability Transition
Authors: Watson Lewis J , Ngoh Gladys A , Zhu Yanqing , Campbell Ashley L , Xuan Yu-Ting , Jones Steven P
Published: July 2021

Hexosamine biosynthesis, an accessory pathway for glucose metabolism, culminates in the formation of UDP-GlcNAc, which is the monosaccharide donor for the post-translational modification, O-linked beta-N-acetylglucosamine (O-GlcNAc). Augmented hexosamine biosynthesis has been implicated in the pathogenesis of diabetes in many peripheral tissues. Yet, glucose transporters in cardiac myocytes are predominantly insulin dependent, thereby limiting intracellular glucose availability in diabetic cardiac myocytes. Driven by such paradoxes, we hypothesized that unlike other tissues, intracellular O-GlcNAc signaling would actually be reduced in diabetic hearts. HPLC revealed a 24% reduction in UDP-GlcNAc levels in db/db diabetic (2.2 +/−0.5 nmol) compared to nondiabetic (2.9 +/−0.5 nmol) mouse hearts. Immunoblots showed a reduced expression of O-GlcNAc transferase (61 +/−14% of nondiabetic), which is the enzyme that adds the O-GlcNAc modification to proteins, and reduced O-GlcNAc levels (86 +/−8% of nondiabetic) in diabetic hearts (n = 8/group). We recently reported an interaction between O-GlcNAc signaling and mitochondrial-controlled cell death and, accordingly, evaluated the sensitivity to mitochondrial permeability transition pore (mPTP) formation here. Cardiac mitochondria from diabetic hearts were more sensitive to calcium-induced mitochondrial swelling (138 +/−15% of nondiabetic) compared with mitochondria from nondiabetics (n = 3/group). Because diabetes is multi-faceted, we distilled the system to one variable, reduced O-GlcNAc levels, by injecting additional groups of nondiabetic mice with a drug (TT04) to reduce O-GlcNAc levels, or Vehicle (n =3/group). Cardiac mitochondria from nondiabetic mice with reduced O-GlcNAc levels (TT04) were also more sensitive to calcium-induced swelling (178 +/−12% of Vehicle) than cardiac mitochondria from Vehicle treated mice, thus recapitulating one element of the diabetic phenotype and supporting the idea that reduction of O-GlcNAc levels is sufficient to sensitize cardiac mPTP formation. We conclude that the paradoxical reduction in O-GlcNAc signaling in diabetic hearts may be responsible for heightened sensitivity to cardiac injury, particularly in terms of mPTP formation.
Abstract 658: Suppressed Hypoxic Pulmonary Vasoconstriction in Malonyl-CoA-Decarboxylase Knock-Out Mice
Authors: Sutendra Gopinath , Bonnet Sebastien , Haromy Alois , Lopaschuk Gary , Dyck Jason , Michelakis Evangelos D
Published: July 2021

Background
: Hypoxic pulmonary vasoconstriction (HPV) is intrinsic to the pulmonary artery smooth muscle cells (PASMC). A recently proposed mechanism for HPV suggests that hypoxia is sensed within the mitochondria, altering the production of activated oxygen species (AOS) in response to changes in PO
2
. Decreased AOS (like H
2
O
2
) inhibit Kv channels, depolarize PASMC, increase influx of Ca
++
, causing PASMC contraction. Malonyl-CoA-Decarboxylase (MCD) is a metabolic enzyme, that when inhibited suppresses the mitochondrial-based fatty acid oxidation, promoting glucose oxidation. We have shown that knockout mice lacking MCD (KO-MCD) are resistant to chronic-hypoxia-induced pulmonary hypertension (CH-PHT) but they have a normal phenotype in normoxia. We hypothesized that KO-MCD mice have suppressed HPV.

Methods and Results
: We compared KO-MCD to wild mice (W-MCD) using PASMC patch clamping, intracellular Ca
++
(fura-2), mitochondrial membrane potential (ΔΨm, using TMRM and confocal microscopy), mitochondrial AOS production (mitosox and confocal microscopy) and isolated intact resistance PA rings; we also studied exercise tolerance (distance covered in a treadmill) during normoxia and acute hypoxia (n =7–10 mice/group for all studies). As expected, in W-MCD mice acute hypoxia inhibited K
+
current (Ik), increased intracellular Ca
++
and mitochondrial ΔΨm, decreased mitochondrial AOS production, increased PA tone and decreased exercise tolerance (due to the rapid increase in PA pressure and decrease in cardiac output). In contrast, KO-MCD showed an impressive lack of response to acute hypoxia in all the parameters studied (table

).

Conclusions
: Our data show that acute hypoxia is not sensed in W-MCD mice and might offer at least a partial explanation for the resistance to CH-PHT in these mice. They also offer a novel insight into the metabolic basis of pulmonary vascular reactivity and HPV.
Abstract 669: ET
A
Receptor Activation and ET
B
Receptor Deficiency Reduce Circulating Endothelial Progenitor Cells in Salt-Sensitive Hypertension
Authors: Chen Dan-Dan , Dai Xiao-Ling , Li Melissa W , Galligan James J , Fink Gregory D , Chen Alex F
Published: July 2021

Background:
Circulating endothelial progenitor cells (EPCs) are reduced in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. DOCA-salt and ET
B
receptor deficiency rats (ET
B
−/ −) have salt-sensitive hypertension, characterized by increased ET-1 and oxidative stress levels. We hypothesized that ET
A
receptor activation and ET
B
receptor deficiency reduce circulating EPCs in adult male DOCA-salt rats.

Methods and Results:
Systolic blood pressure (SBP) was higher in DOCA-salt (4-wk regimen) vs. Sham rats (199.3 ± 4.8 vs. 133.4 ± 4.6 mmHg, n =8 –15, p<.05), which was significantly reduced by in vivo treatment with ET
A
antagonist ABT-627 (5 mg/kg/d) or NADPH oxidase inhibitor apocynin (1.5 mmol/L) (167.8 ± 6.8 and 156.9 ± 16.6 mmHg, respectively). Flow cytometry showed that circulating CD34- and Flk-1-positive EPCs were significantly lower in DOCA vs. Sham rats (31.5 ± 0.2 vs. 18.3 ± 0.8% and 21.1 ± 0.9 vs. 16.3 ± 0.7%, respectively, n=7– 8, p<.05). Dil-acLDL and isolectin double-stainings also showed reduced EPCs in DOCA vs. Sham rats (4.5 ± 0.4 vs. 10.2 ± 0.3 cells/hpf, n=5–15, p<.01), which was rescued by in vivo treatment with ABT-627 (10.1 ± 1.7) or apocynin (8.9 ± 0.8). Real-time RT-PCR and fluorescence confocal microscopy showed that ET
A
and ET
B
receptor mRNA and proteins were present in EPCs of SD rats. Intracellular ROS level was increased by >50% in EPCs of DOCA vs. Sham rats (DCF fluorescence, n = 9 –11, p<.05). Apoptosis was increased by >80% in EPCs of DOCA vs. Sham rats, which was reversed by ABT-627 or apocynin (TUNEL assay, n=5– 6, p<.05). EPCs were significantly lower in ET
B
−/ − vs. ET
B
+/+ rats (6.3 ± 0.6 cells/hpf, n =7– 8), paralleled with higher SBP (telemetry 151 ± 1.2 vs. 129 ± 0.9 mmHg, n = 6), plasma ET-1 level (5.2 ± 0.3 vs. 3.6 ± 0.1 pg/ml, n = 5–12), and suppressed telomerase activity (354.6 ± 105.5 vs. 83.3 ± 26.2%, n = 3) (all p<.05). ET-1 treatment (10 nM, 48 hrs) also reduced EPCs of normal rats (6.9 ± 0.1 vs. 10.3 ± 0.3 cells/hpf, n = 5– 6, p<.05), which was rescued by ABT-627 (9.9 ± 0.3), but not by ET
B
antagonist BQ788 (7.6 ± 0.4).

Conclusion:
ET
A
activation and ET
B
deficiency reduce circulating EPCs in DOCA-salt and ET
B
−/ − rats, in part, due to NADPH oxidase-induced oxidative stress, apoptosis, and telomerase inactivation.
Abstract 670: Immediate And Sustained Blood Pressure Lowering by CRF-receptor Stimulation: A Novel Approach To Antihypertensive Therapy?
Authors: Dieterle Thomas , Meili-Butz Silvia , Buehler Katrin , Morandi Christian , John Dietlinde , Pfisterer Matthias , Buser Peter T , Vale Wylie W , Peterson Kirk L , Brink Marijke
Published: July 2021

Background:
Recently, novel corticotropin-releasing factor (CRF)-related peptides, named urocortin I (UcnI), UcnII, and UcnIII were described. Available data suggest that the Ucns are part of a peripheral CRF system modulating cardiovascular function and mediating cardiovascular responses to stress. Blood pressure (BP) lowering effects have been described after administration of UcnI. However, no data are available on effects of UcnII on BP in an animal model of systemic arterial hypertension.

Methods:
Experiments were performed in Dahl salt-sensitive (DSS) and salt-resistant rats (DSR, control). Animals were fed a diet containing 4% NaCl (high salt) to induce arterial hypertension in DSS rats. At the end of week 2 of high salt diet, both DSS and DSR rats were randomly assigned to i.p. injections of either UcnII (2.5 μg/kg body weight) or vehicle b.i.d. for five weeks. Animals underwent repetitive tail cuff BP measurements at baseline (prior to first injection), at 5 and 15 minutes after the first injection and at week 1, 2, and 5 of b.i.d. treatment. At week 5 animals were sacrificed to determine heart weight /body weight ratio.

Results:
Systolic BP (SBP, mmHg) and heart rate (HR, min
−1
) are given in the following table

as mean ± SD (n=10 per group).

Conclusions:
In hypertensive DSS rats, acute CRF-receptor stimulation by UcnII immediately lowered BP to the range observed in DSR rats. Compared to vehicle-treated DSS rats, sustained BP reduction was observed with further chronic administration of UcnII. No severe reflex tachycardia was observed after administration of UcnII. Thus, CRF-receptor stimulation might represent a novel approach to the treatment of arterial hypertension.
Abstract 680: KC706, an Oral p38 MAP Kinase Inhibitor, Increases HDL-C
Authors: Miller Stephen G , Crowley Constance , Lundstrom Jan , Larson Christopher J , Prior Kent E , King Bernard D
Published: July 2021

Introduction: KC706 is a novel p38 MAP kinase inhibitor with anti-inflammatory properties. In rats and monkeys, KC706 administration was associated with a 50 – 60% ↑ in HDL-C. Because HDL-C is an important target for the treatment of CV disease a Phase 2a study was designed to determine whether KC706 administration would elevate HDL-C levels in patients with dyslipidemia.

Methods: A double-blind, placebo-controlled, 6-week study was conducted in patients with ↓ HDL-C and ↑ triglyceride (TG) levels. Patients (N = 115) were randomized to placebo or to KC706 (150 or 300 mg po qd). Standard chemistry and hematology parameters and lipid, metabolic, inflammatory, and thrombotic markers were monitored. The primary end point was an increase in HDL-C in treatment v. placebo groups.

Results: Patients were primarily male (59%) and white (97%). At baseline, 35% were receiving statins, 44% were hypertensive, 15% had CV disease, 70% fulfilled ATPIII criteria for metabolic syndrome, and 43% had CRP > 3 mg/L. The primary endpoint of the study was met at both dose levels. Compared to baseline, HDL-C ↓ 2.9% in the placebo group, ↑ 18.6% in the 150 mg group (p < 0.0001 v. placebo) and ↑ 11.6% in the 300 mg group (p = 0.0003 v. placebo) after 6 wks of treatment. Effects were noted in men and women, whether patients were receiving statins or not, and whether CRP was elevated or not at baseline. ApoAI ↑ significantly in the 150 mg group (p = 0.0056 v. placebo). TG, LDL-C, and VLDL-C were unchanged. CRP levels trended downward in patients with ↑ baseline CRP levels. HDL/Total cholesterol (+22%), ApoA1/ApoB100 (+14%), and HDL/LDL (+31%) increased in the KC706 150 mg group. Mean HDL particle size (measured in a subset of patients) increased from 8.4 to 8.7 nm in the KC706 150 mg group (p = 0.04), from 8.5 to 8.7 nm in the KC706 300 mg group (p = 0.01) but not in the placebo group (8.3 v. 8.4 nm, p = 0.66). No increases in either systolic or diastolic blood pressures were observed in any group. One patient in the 150 mg group and 4 in the 300 mg group had a reversible elevation in either AST and/or ALT of > 3X ULN. Adverse events were similar in the 3 treatment groups.

Conclusions: KC706, an oral p38 MAP kinase inhibitor with anti-inflammatory properties, increased HDL and ApoA1 in patients with dyslipidemia and was well tolerated.
Abstract 697: Mobilization of Oct-4
+
Bone Marrow-Derived Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction
Authors: Wojakowski Wojciech , Kucia Magda , Machalinski Boguslaw , Paczkowska Edyta , Ciosek Joanna , Kazmierski Maciej , Buszman Pawel , Wieczorek Piotr , Ratajczak Janina , Ratajczak Mariusz Z , Tendera Michal
Published: July 2021

Bone marrow-derived CD34
+
CXCR4
+
progenitor cells are mobilized into peripheral blood early in acute myocardial infarction (MI). Adult murine bone marrow contains population of small CD34
+
lin
−
CD45
−
CXCR4
+
cells expressing markers of pluripotent stem cells (PSC) SSEA, Oct-4 and Nanog. This population of very small embryonic-like cells (VSEL) has unique morphology (small size 2– 4 μm, large nucleus, euchromatin) and capability to form embrioid bodies (EB). Murine EB-derived cells can
in vitro
differentiate into cells from all three germ layers including cardiomyocytes. We hypothesized that in patients with acute MI small cells expressing the VSEL immunophenotype and PSC markers are present in bone marrow and mobilized into peripheral blood. Blood samples (20 mL) from 18 patients with acute MI were obtained after 12 hours, 2 and 5 days after symptoms onset. Bone marrow samples (20 mL) were obtained from 2 patients with acute MI and 3 healthy volunteers. Mononuclear cells were isolated using hypotonic lysis and samples were analyzed by FACS. Mobilization of following cell populations was confirmed: hematopoietic lin
−
CD45
+
CXCR4
+
, lin
−
CD45
+
CD133
+
, lin
−
CD45
+
CD34
+
and non-hematopoietic (VSEL) lin
−
CD45
−
CXCR4
+
, lin
−
CD45
−
CD133
+
, lin
−
CD45
−
CD34
+
. Analysis of the cell number using lymphocyte gate showed more significant increase of CD45
+
(hematopoietic) populations of lin
−
CD34
+
, lin
−
CD133
+
and lin
−
CXCR4
+
cells. After gating for small events (VSEL size range) we found more significant mobilization of small, non-hematopoietic populations of lin
−
CD34
+
, lin
−
CD133
+
and lin
−
CXCR4
+
cells (Table

). The expression of PSC markers (Oct-4, Nanog, SSEA-1) in VSEL was confirmed using real-time RT-PCR.

Conclusion: We report for the first time that acute MI is associated with mobilization of non-hematopoietic VSELs expressing pluripotent stem cells markers.
Abstract 704: Cardiac Progenitor Cell Aging Is Responsible for Organ Aging
Authors: Gonzalez Arantxa , Rota Marcello , Nurzynska Daria , Misao Yu , Ojaimi Caroline , Tillmanns Jochen , Padin-Iruegas Elena , Mueller Patrick , Esposito Grazia , Bearzi Claudia , Vitale Serena , Hintze Thomas H , Baker Mathue , Dawn Buddhadeb , Bolli Roberto , Urbanek Konrad , Hosoda Toru , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

To establish whether the aging cardiomyopathy is dictated by depletion of the cardiac progenitor cell (CPC) pool and accumulation of old myocytes, we measured first the number of CPCs in rats at 4, 12, 20 and 28 months of age. CPCs are lineage-negative cells that express the stem cell antigens c-kit, MDR-1 and/or Sca-1. Unexpectedly, from 4 to 28 months, the number of CPCs in the heart increased ~3-fold. The increase in mitotic index of CPCs in the old heart indicated enhanced activation of CPCs which resulted in an increased number of myocyte progenitors and precursors and ultimately differentiated cardiomyocytes. These observations were at variance with the concomitant accumulation of p16
INK4a
-positive-myocytes and the 6-fold increased rate of myocyte apoptosis in the senescent heart pointing to defects in the CPC compartment with aging. Activation and differentiation of old CPCs may generate a myocyte progeny that rapidly reaches senescence. To evaluate the biological age of CPCs, we measured two parameters of cellular aging: the senescence-associated protein p16
INK4a
and telomere length. From 4 to 28 month, p16
INK4a
-positive-CPCs increased from ~6% to ~81% resulting in a sharp decrease in the number of functionally-competent CPCs. Telomere length was measured by Q-FISH in cytospin preparations of c-kit-positive-cells collected from hearts at 3 and 27 months. In old hearts, the distribution of telomere length in CPCs, myocyte progenitors-precursors and developing myocytes was shifted to the left towards shorter telomeres. Average telomere length in old CPCs, myocyte progenitors-precursors and developing myocytes was 30%, 38% and 52% shorter than in the corresponding young cell populations, respectively. Importantly, 55% of old CPCs and 15% of young CPCs had telomeres less than 8 kbp and were p16
INK4a
-positive. Thus, telomere attrition in CPCs with age leads to the generation of a myocyte progeny that rapidly acquires the senescent phenotype. However, nearly 16% of the old CPC pool had telomeres greater than 14 kbp pointing to a relevant growth reserve of the senescent myocardium. In conclusion, CPC aging with reduction in telomeric length dictates the formation of a senescent progeny which conditions organ aging and failure.
Abstract 726: Nox4 Expression in Human Monocyte-Derived Macrophages: Upregulation by Oxidized LDL via a MEK-ERK1/2-dependent Pathway
Authors: Qiao Mu , Asmis Reto
Published: July 2021

Nox4 is member of the family of NADPH oxidase expressed in endothelium and smooth muscle cells, but has not previously been reported in human monocytes or monocyte-derived macrophages. We now identified Nox4 as a new Nox member in human monocytes and monocyte-derived macrophages. Nox4 and the phagocytic NADPH oxidase gp91/Nox2 were differentially regulated during monocyte differentiation. In contrast to Nox2, Nox4 protein expression was upregulated in mature human macrophages, and confocal microscopy revealed that Nox4 colocalized primarily with mitochondria. Exposure of macrophages to OxLDL for 24 h further increased Nox4 expression in a concentration-dependent manner. OxLDL-induced Nox4 expression was blocked by inhibitors of MEK1/2, UO126 and PD98059, but not by SB203580 and SP600125, inhibitors of p38MAPK or JNK, respectively. Furthermore, MEK inhibitors, but not inhibitors of p38MAPK or JNK, protected macrophages from OxLDL-induced cell injury, identifying a MEK/ERK1/2-dependent signaling pathway upstream of OxLDL-induced Nox4 expression that is involved in macrophage death. Previously, we showed that OxLDL stimulates the intracellular formation of reactive oxygen species (ROS) in human macrophages, but the source of these ROS remained elusive. We also demonstrated that the peroxyl radical scavenger Trolox protects macrophages from OxLDL-induced cell injury. Interestingly, Trolox did not prevent Nox4 expression induced by OxLDL, suggesting that Trolox acts downstream of OxLDL-induced Nox4 expression. These results not only identify Nox4 as a new intracellular source of ROS in macrophages, but also implicate Nox4 in OxLDL-induced ROS formation and macrophage death, which may have important implications for the pathogenesis of atherosclerosis.
Abstract 732: The P2Y
2
Receptor Selectively Binds to Filamin A to Regulate Spreading and Migration of Vascular Smooth Muscle Cells
Authors: Yu Ningpu , Fernandez Gerardo E , Weisman Gary A , Seye Cheikh I
Published: July 2021

The functional expression of the G protein-coupled P2Y
2
nucleotide receptor has been associated with the development of intimal lesions. Activation of this receptor also stimulates actin cytoskeleton reorganization and migration of vascular smooth muscle cells (SMCs). Since cell migration has been linked to the dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, the aim of the present study was to identify cytoskeletal proteins that bind to the P2Y
2
receptor and potentially regulate SMC migration. Using the yeast two-hybrid system, we isolated filamin A, a filamentous actin-cross linking protein that interacts with the C-terminal domain of the P2Y
2
receptor and we used deletion mapping to identify amino acid deletion in the P2Y
2
receptor that led to selective loss of filamin A binding.
Ex-vivo
treatment of aortic explants with the P2Y
2
receptor agonist UTP (10 μmol/L) significantly promoted migration of SMCs in wild type but not in P2Y
2
receptor
−/−
mice. Likewise, using a Transwell migration assay we showed that UTP increased migration of SMCs in wild type (3.5 folds, P<0.01 vs. untreated cells) but not P2Y
2
receptor
−/−
mice (P<0.4 vs. untreated cells). Adenoviral infection of the full length P2Y
2
receptor restored UTP-mediated cell migration in P2Y
2
receptor
−/−
mice whereas infection of a mutant P2Y
2
receptor that does not bind filamin A did not. UTP-induced migration was preceded by a rapid phosphorylation of filamin A that was not observed either in P2Y
2
receptor
−/−
SMCs or in P2Y
2
receptor
−/−
SMCs infected with the mutant P2Y
2
receptor that does not bind filamin A. Treatment of SMCs from wild type mice with UTP (10 μmol/L) caused a 4-fold increase in spreading to collagen I as compared to unstimulated cells. The UTP-mediated increase of SMC spreading was not found in P2Y
2
receptor
−/−
SMCs but was restored by adenoviral infection of the full length P2Y
2
receptor cDNA into these cells. However, adenoviral delivery of a mutant receptor
−/−
which does not bind to filamin A did not restore UTP-mediated spreading of P2Y
2
receptor SMCs to collagen. This study demonstrates that P2Y
2
-dependent modulation of the actin cytoskeleton selectively regulates spreading and migration of SMC.
Abstract 181: Overexpression of Arachidonic Acid Cytochrome P450 Expoxygenases Prevents the Development of High Blood Pressure via Enhancing Atrial Natriuretic Peptide in Spontaneously Hypertensive Rats
Authors: Wang Dao Wen , Xiao Bin , Wang Yong , Xiong Xiaojun , Zeldin Darryl C
Published: July 2021

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have potent vasodilatory and diuretic feature, and therefore potentially hypotensive effect. No
in vivo
studies, however, were performed to support it. This study investigated the hypothesis via overexpressing CYP epoxygense genes in spontaneously hypertensive rats (SHR). Recombinant adeno-associated virus vector (rAAV) was utilized to mediate long-term transfection of CYP2J2 and CYP2C11 genes, respectively, in adult SHR, and animal systolic blood pressure (SBP) was monitored using arterial caudilis indirect manometric method. Results showed that at 2 months the urinary excretion of stable hydrolysis metabolic product of 14, 15-EE, 14–15-DHET increased by 11 and 8.7 folds in rAAV-2J2 and rAAV-2C11 groups, respectively, compared with AAV-GFP-treated rats. (2) SBP in 2J2- and 2C11-treated rats decreased from 175.0 ± 2.8mHg to 163.5 ± 5.8mmHg and 161.2 ± 6.1 mmHg, respectively, (
p
<0.01) at month 2, and it is 165.0 ± 4.7 mmHg and 173.0 ± 12.8 mmHg at month 6 after gene injection (~30mmHg and ~23mmHg lowerer than that in control animals, respectively,
p
<0.001). (3) Before the rats were sacrificed, cardiac function tests with Pressure-Volume System showed that maximum intracardiac pressure was 202.1 ± 30.0 & 209.1 ± 17.1mmHg in two gene-treated rats, respectively, significantly lower than control (241.2 ± 18.2mmHg,
p
<0.01) and cardiac output in treatment rats were significantly higher than control (p<0.05). (4) Interestingly, atrial natriuretic peptide (ANP) mRNA were up-regulated 6–14 folds respectively in myocardium of 2J2 and 2C11 groups; furthermore, C-type receptor mRNA of ANP was increased in heart, lung, kidney and aorta. (5) in cultured atrial cells (HLB2G5), exogenous EETs stimulated ANP production. In conclusions, for first time our data indicates overexpression of CYP2J2 or CYP2C11 could prevent development of hypertension in SHR, improve cardiac functions, which may involve up-regulating ANP expression and its receptors in target tissues, which suppresses collagen deposition and cardiovascular remodeling.
Abstract 747: A Novel Cardiac Myosin Light Chain Kinase Regulates Sarcomere Assembly in the Vertebrate Heart
Authors: Seguchi Osamu , Takashima Seiji , Yamazaki Satoru , Asakura Masanori , Asano Yoshihiro , Shintani Yasunori , Wakeno Masakatsu , Minamino Tetsuo , Kitamura Soichiro , Tomoike Hitonobu , Mochizuki Naoki , Kitakaze Masafumi
Published: July 2021

Background
Cardiomyocytes mainly consist of sarcomere structure and marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing myocardium. Among several constituents of sarcomeres, myosin regulatory light chain 2 (MLC2v) affects physiological cardiac sarcomere formation and heart development through its phosphorylation. Exact upstream kinase of MLC2v in cardiomyocytes however, have not been elucidated so far.

Mathods and Results
We identified a novel cardiac-specific gene whose expression was upregulated in failing myocardium and was also correlated with the values of pulmonary arterial pressure of each patient. This gene has kinase domain and MLC2v was identified as a putative substrate by in vitro kinase reaction using mouse heart extracts and recombinant protein of this gene. Therefore, we termed this novel gene as a cardiac-specific myosin light chain kinase (cardiac-MLCK, gene symbol:
MYLK3
). To further investigate the function of cardiac-MLCK, we targeted cardiac-MLCK using specific siRNAs in cultured neonatal rat cardiomyocytes. Reduced expression of cardiac-MLCK caused the impairment of sarcomere reassembly on epinephrine treatment. Also knockdown of cardiac-MLCK expression in zebrafish embryos using specific morpholino antisense oligonucleotides led to dilation of the cardiac ventricle with incomplete sarcomere formation. We also analyzed the nature of cardiac-MLCK and MLC2v in mouse models of heart failure. Expression levels of cardiac-MLCK proteins and phosphorylation levels of MLC2v were severely reduced in the failing heart of trans-aortic constriction mice.

Conclusions
We identified a novel cardiac-MLCK which contributed mainly to cardiac MLC2v phosphorylation. Attenuation of cardiac-MLCK expression caused severe sarcomere disorganization both in vitro and in vivo implies its important roles in cardiac functions. Our results suggest that cardiac-MLCK mRNA expression may be upregulated to compensate for the lower expression and reduced phosphorylation of MLC2v in failing myocardium. As a possible therapeutic modality in patients with CHF, upregulation of cardiac-MLCK may promote sarcomere assembly and enhanced contractility of the failing heart
Abstract 776: Protein Disulfide Isomerase Is Involved In Redox Regulation Of Nitric Oxide Output During Laminar Shear Stress In Endothelial Cells
Authors: Fernandes Denise C , Santos Celio X , Jo Hanjoong , Laurindo Francisco R
Published: July 2021

While anti-atherogenic effects of sustained laminar shear (LS) involve NO release from eNOS, increases in LS trigger transient superoxide production via NADPH oxidase. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm
2
) in a cone-plate system for 18h exhibited (vs. static controls):

Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation);

Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay);

Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR);

Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5[SD] μM, NO Analyzer and Griess reaction);

Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with
c-myc
-tagged plasmid coding for wild-type (WT) PDI or PDI mutated in 4 thioredoxin-motif cysteine residues.

Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (18h) (Δmutated = 17.2±3.3 μM vs. ΔWT = 7.0±1.9 μM, n=3, p<0.02). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static conditions, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity.
Abstract 785: MesP1 Induces Vertebrate Cardiovascular Differentiationby Dkk-1 Mediated Blockage Of Wnt-signalling
Authors: David Robert , Brenner Christoph , Stieber Juliane , Brunner Stefan , Schwarz Florian , Rupp Ralph , Mueller-Hoecker Josef , Franz Wolfgang-Michael
Published: July 2021

Cardiovascular diseases are the most frequent cause of death in the western world. Stem cell based cardiovascular repair will require in-depth understanding of molecular mechanisms underlying cardiovascular ES cell differentiation
in vitro
. A candidate cardiovascular fate inducer is the bHLH transcription factor Mesp1. As one of the earliest cardiovascular markers it is specifically expressed in almost all precursors of the cardiovascular system and required for cardiac morphogenesis. However, the mode of action of MesP1 in the underlying processes is unknown. Here we show in overexpression experiments using
Xenopus
embryos that MesP1 is the first known single transcription factor sufficient to induce ectopic beating heart tissue in vertebrates. Furthermore, MesP1 overexpression in ES cells results in significantly increased cardiovasculogenesis. Electrophysiological analysis of isolated beating cardiomyocytes showed all subtypes described for cardiac ES cell differentiation. Our results from RT-PCRs reveal a prominent function of MesP1 within a gene regulatory cascade causing Dkk-1 mediated blockage of canonical wnt signaling non cell autonomously leading to upregulation of the endodermal transcription factor Hex. In ChiP and bandshift assays we confirmed the Dkk-1 promoter as a direct target for MesP1. We consider our studies as a starting point to further decipher the detailed molecular position of MesP1 within the complex networks involved in cardiovasculogenesis. This knowledge will be a prerequisite to program ES cells towards a cardiovascular fate for cell therapy and cardiovascular tissue engineering. Likewise, it will be of great interest to transfer this approach to various subpopulations of human adult stem cells which still have not definitively been shown to bear cardiac transdifferentiation potential in their native state.
Abstract 814: Atorvastatin Attenuates Oxidative Stress And Improves Neuronal Nitric Oxide Synthase In The Brain Stem Of Heart Failure Mice
Authors: Francis Joseph , Yu Li , Guggilam Anuradha , Sriramula Srinivas , Zucker Irving H
Published: July 2021

3-Hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to reduce the incidence of myocardial infarction independent of their lipid-lowering effects. Nitric oxide (NO) in the central nervous system contributes to cardiovascular regulatory mechanisms. Imbalance between nitric oxide (NO) and superoxide anion (O
2
. −
) in the brain may contribute to enhanced sympathetic drive in heart failure (HF). This study was done to determine whether treatment with atorvastatin (ATS) ameliorates the imbalance between NO and O
2
. −
production in the brain stem and contributes to improvement of left ventricular (LV) function.

Methods and Results:
Myocardial infarction (MI) was induced by ligation of the left coronary artery or sham surgery. Subsequently, mice were treated with ATS (10 μg/kg) (MI + ATS), or vehicle (MI + V). After 5 weeks, echocardiography revealed left ventricular dilatation in MI mice. Realtime RT-PCR indicated an increase in the mRNA expression of the LV hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Neuronal NOS (nNOS) and endothelial NOS (eNOS) mRNA expression were significantly reduced, while that of NAD(P)H oxidase subunit (gp91phox) expression was elevated in the brain stem of MI mice. Compared with sham-operated mice, ATS-treated mice showed reduced cardiac dilatation, decreased ANP and BNP in the LV. ATS also reduced gp91phox expression and increased nNOS mRNA expression in the brain stem, while no changes in eNOS and iNOS were observed.

Conclusion:
These findings suggest that ATS reduces oxidative stress and increases neuronal NOS in the brain stem, and improves left ventricular function in heart failure.