bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Endothelial Cell Binding Sites on the Laminin γ1 Chain
Authors: Ponce M. Lourdes , Nomizu Motoyoshi , Delgado Mucio C. , Kuratomi Yuichiro , Hoffman Matthew P. , Powell Sharon , Yamada Yoshihiko , Kleinman Hynda K. , Malinda Katherine M.
Published: June 2012

Abstract
—The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like structures when plated on a laminin-1–rich basement membrane matrix, Matrigel. Laminin-1 is composed of 3 chains, α1, β1, and γ1. Because laminin-1 is known to contain multiple biologically active sites, we have screened 156 synthetic overlapping peptides spanning the entire laminin γ1 chain for potential angiogenic sequences. Only 7 of these peptides, designated as C16, C25, C30, C38, C64, C75, and C102, disrupted the formation of capillary-like structures by human umbilical vein endothelial cells on Matrigel. Dose-response experiments in the presence of 50 to 200 μg/mL showed that tube formation was prevented by most peptides at 150 and 200 μg/mL, except for C16, which showed strong activity at all concentrations. Active peptides promoted vessel sprouting from aorta rings and angiogenesis in the chick chorioallantoic membrane assay. In addition, the active peptides also promoted endothelial cell adhesion to dishes coated with 0.1 μg of peptide and inhibited attachment to laminin-1 but not to plastic or fibronectin. Four of the active peptides, C25, C38, C75, and C102, may have cell-type specificity with endothelial cells, since they did not promote PC12 neurite outgrowth or adhesion of B16-F10 melanoma and human submandibular gland cells. These results suggest that specific laminin γ1–chain peptides have angiogenic activity with potential therapeutic applications.
Chloride Channel Inhibition Blocks the Protection of Ischemic Preconditioning and Hypo-Osmotic Stress in Rabbit Ventricular Myocardium
Authors: Diaz Roberto J. , Losito Vito A. , Mao Gou D. , Ford Meredith K. , Backx Peter H. , Wilson Gregory J.
Published: June 2012

Abstract
—The objective of this study was to examine the role of chloride (Cl
–
) channels in the myocardial protection of ischemic preconditioning (IP). Isolated rabbit ventricular myocytes were preconditioned with 10-minute simulated ischemia (SI) and 20-minute simulated reperfusion (SR) or not preconditioned (control). The myocytes then received 180-minute SI or 45-minute SI/120-minute SR. Indanyloxyacetic acid 94 (IAA-94, 10 μmol/L) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 1 μmol/L) was administered before IP or before SI or SI/SR to inhibit Cl
–
channels. Electrophysiological studies indicate that these drugs, at the concentrations used, selectively abolished Cl
–
currents activated under hypo-osmotic conditions (215 versus 290 mOsm). IP significantly (
P
<0.001) reduced the percentage of dead myocytes after 60-minute (30.8±1.3%, mean±SEM), 90-minute (35.3±1.3%), and 120-minute (39.2±1.7%) SI compared with controls (44.7±1.6%, 54.5±1.3%, and 58.9±1.8%, respectively) and after 45-minute SI/120-minute SR (36.3±0.6%) compared with control (56.6±2.2%). Hypo-osmotic stress also produced protection similar to IP. IAA-94 or NPPB abolished the protection of both IP and hypo-osmotic stress. In buffer-perfused rabbit hearts preconditioned with three 5-minute ischemia/10-minute reperfusion cycles given before the 40-minute long ischemia and 60-minute reperfusion, IP significantly
(P
<0.0001) reduced infarct size (IP+vehicle, 4.7±0.9%, versus control+vehicle, 26.6±3.3%; mean±SEM). Again, IAA-94 or NPPB abolished the protection of IP. Our results implicate Cl
–
channels in the IP protection of the myocardium against ischemic/reperfusion injury and demonstrate that hypo-osmotic stress is capable of preconditioning cardiomyocytes.
Molecular Mechanisms Underlying Ionic Remodeling in a Dog Model of Atrial Fibrillation
Authors: Yue Lixia , Melnyk Peter , Gaspo Rania , Wang Zhiguo , Nattel Stanley
Published: June 2012

Abstract
—The rapid atrial rate during atrial fibrillation (AF) decreases the ionic current density of transient outward K
+
current, L-type Ca
2+
current, and Na
+
current, thereby altering cardiac electrophysiology and promoting arrhythmia maintenance. To assess possible underlying changes in cardiac gene expression, we applied competitive reverse transcriptase–polymerase chain reaction to quantify mRNA concentrations in dogs subjected to 7 (group P7 dogs) or 42 (group P42 dogs) days of atrial pacing at 400 bpm and in sham controls. Rapid pacing reduced mRNA concentrations of Kv4.3 (putative gene encoding transient outward K
+
current; by 60% in P7 and 74% in P42 dogs;
P
<0.01 and
P
<0.001, respectively, versus shams), the α
1c
subunit of L-type Ca
2+
channels (by 57% in P7 and 72% in P42 dogs;
P
<0.01 versus shams for each) and the α subunit of cardiac Na
+
channels (by 18% in P7 and 42% in P42;
P
=NS and
P
<0.01, respectively, versus shams) genes. The observed changes in ion channel mRNA concentrations paralleled previously measured changes in corresponding atrial ionic current densities. Atrial tachycardia did not affect mRNA concentrations of genes encoding delayed or Kir2.1 inward rectifier K
+
currents (of which the densities are unchanged by atrial tachycardia) or of the Na
+
,Ca
2+
exchanger. Western blot techniques were used to quantify protein expression for Kv4.3 and Na
+
channel α subunits, which were decreased by 72% and 47%, respectively, in P42 dogs (
P
<0.001 versus control for each), in a manner quantitatively similar to measured changes in mRNA and currents, whereas Na
+
,Ca
2+
exchanger protein concentration was unchanged. We conclude that chronic atrial tachycardia alters atrial ion channel gene expression, thereby altering ionic currents in a fashion that promotes the occurrence of AF. These observations provide a potential molecular basis for the self-perpetuating nature of AF.
Modulation of Ras/Raf/Extracellular Signal–Regulated Kinase Pathway by Reactive Oxygen Species Is Involved in Cyclic Strain–Induced Early Growth Response-1 Gene Expression in Endothelial Cells
Authors: Wung B. S. , Cheng J. J. , Chao Y. J. , Hsieh H. J. , Wang D. L.
Published: June 2012

Abstract
—Endothelial cells (ECs) exposed to cyclic strain induce gene expression. To elucidate the signaling mechanisms involved, we studied the effects of cyclic strain on ECs by using early growth response-1 (Egr-1) as a target gene. Cyclic strain induced a transient increase of Egr-1 mRNA levels that resulted in an increase of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. ECs subjected to strain enhanced Egr-1 transcription as revealed by promoter activities. Catalase pretreatment inhibited this induction. ECs, transfected with a dominant positive mutant of Ras (RasL61), increased Egr-1 promoter activities. In contrast, transfection with a dominant negative mutant of Ras (RasN17) attenuated this strain inducibility. ECs transfected with a dominant negative mutant of Raf-1 (Raf301) or the catalytically inactive mutant of extracellular signal–regulated kinase (ERK)-2 (mERK2) diminished strain-induced promoter activities. However, little effect on strain inducibility was observed in ECs transfected with a dominant negative mutant of Rac (RacN17) or a catalytically inactive mutant of JNK (JNK[K-R]). Consistently, strain-induced Egr-1 expression was inhibited after ECs were treated with a specific inhibitor (PD98059) to mitogen-activated protein kinase kinase. Moreover, strain to ECs induced mitogen-activated protein kinase/ERK activity. The activation of the ERK pathway was further substantiated by an increase of strain-induced transcriptional activity of Elk1, an ERK substrate. This strain-induced ERK activity was attenuated after ECs were treated with
N
-acetylcysteine or catalase. Consequently, this Egr-1 gene induction was abolished after ECs were treated with
N
-acetylcysteine or catalase. Deletion analyses of the promoter region (–698 bp) indicated that cyclic strain and H
2
O
2
shared a common serum response element. Our data clearly indicate that cyclic strain–induced Egr-1 expression is mediated mainly via the Ras/Raf-1/ERK pathway and that strain-induced reactive oxygen species can modulate Egr-1 expression at least partially via this signaling pathway.
Natural Androgens Inhibit Male Atherosclerosis
Authors: Alexandersen Peter , Haarbo Jens , Byrjalsen Inger , Lawaetz Henrik , Christiansen Claus
Published: June 2012

Abstract
—The effect of natural androgens on serum lipids and atherosclerosis is controversial. We therefore studied this important issue prospectively in an animal model of atherosclerosis. Eighty male rabbits were randomized to bilateral castration, and 20 animals were sham operated. The castrated rabbits were randomized to 500 mg oral dehydroepiandrosterone (DHEA) daily, 80 mg oral testosterone undecanoate (TU) daily, or 25-mg intramuscular injection of testosterone enanthate (TE) twice weekly, whereas the fourth castrated group (placebo) and the sham-operated rabbits did not receive any hormones. All animals were fed a cholesterol-rich diet during the 30-week treatment period. Average serum lipids and atherogenic lipoproteins were higher in the placebo group than in the other groups (ANOVA,
P
<0.0001). Aortic atherosclerosis, as evaluated by the cholesterol content (nmol/mg protein), was also highest in the placebo group (308±39) and lowest in the TE group (61±12), but was intermediate in the DHEA (155±30), TU (191±43), and sham operation (162±29) groups (ANOVA,
P
<0.0001). ANCOVA indicated that the androgen effect on aortic atherosclerosis was only in part explained by the changes in lipoproteins. Aortic estrogen receptor contents were significantly lower in the androgen-treated groups than in the control groups, whereas there was no difference in aortic androgen receptor contents between groups. Natural androgens inhibit aortic atherosclerosis in castrated male rabbits only partly through a lipid-mediated effect.
Activation of p38 Mitogen-Activated Protein Kinase by Oxidized LDL in Vascular Smooth Muscle Cells
Authors: Jing Qing , Xin Sun-Mei , Cheng Zhi-Jie , Zhang Wen-Bo , Zhang Ru , Qin Yong-Wen , Pei Gang
Published: June 2012

Abstract
—Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 μg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [
3
H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin–sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.
Endogenous Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Is a Physiological Regulator of Myocardial Oxygen Consumption
Authors: Loke Kit E. , McConnell Patrick I. , Tuzman Joshua M. , Shesely Edward G. , Smith Carolyn J. , Stackpole Christopher J. , Thompson Carl I. , Kaley Gabor , Wolin Michael S. , Hintze Thomas H.
Published: June 2012

Abstract
—Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O
2
consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O
2
consumption in mice with defects in the expression of eNOS [eNOS (−/−)] and inducible NOS [iNOS (−/−)] was measured with a Clark-type O
2
electrode. The rate of decreases in O
2
concentration was expressed as a percentage of the baseline. Baseline O
2
consumption was not significantly different between groups of mice. Bradykinin (10
−4
mol/L) induced significant decreases in O
2
consumption in tissues taken from iNOS (−/−) (−28±4%), wild-type eNOS (+/+) (−22±4%), and heterozygous eNOS(+/−) (−22±5%) but not homozygous eNOS (−/−) (−3±4%) mice. Responses to bradykinin in iNOS (−/−) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with
N
-nitro-
l
-arginine methyl ester (L-NAME) (−2±5%, −3±2%, and −6±5%, respectively,
P
<0.05). In contrast,
S
-nitroso-
N
-acetyl-penicillamine (SNAP, 10
−4
mol/L), which releases NO spontaneously, induced decreases in myocardial O
2
consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O
2
consumption in tissues taken from normal but not iNOS (−/−)–deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O
2
consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered.
Mechanisms of Deficient Cardiac Septation in the Mouse With Trisomy 16
Authors: Webb Sandra , Anderson Robert H. , Lamers Wouter H. , Brown Nigel A.
Published: June 2012

Abstract
—It used to be thought that the atrioventricular septum was predominantly the product of the atrioventricular endocardial cushions. In a previous study, we have shown that multiple developmental primordia are of importance in its formation. With this in mind, we have evaluated cardiac morphogenesis in the mouse with trisomy 16, an animal model with a high incidence of atrioventricular septal defects. Normal and trisomic fetuses from an Rb(11.16)2H/Rb(16.17)7Bnr×C57BL/6J cross were collected on days 10 to 15 of gestation and examined by scanning electron microscopy and histological serial sectioning. No evidence was found to suggest that atrioventricular septal defect could be explained simply on the basis of “failure of fusion” between the atrioventricular endocardial cushions. Rather, our findings supported two other developmental elements as being important in the genesis of atrioventricular septal defect. The first is an alteration in the configuration of the heart tube, with inadequate remodeling of the inner heart curvature. This resulted in the failure of the atrioventricular junction to expand to the right, with subsequent malalignment of the atrioventricular endocardial cushions with the proximal outflow cushions. The second is a variability in the connection of the primary atrial cardiac segment to the body of the embryo, the so-called dorsal mesocardium, which influences its relationship to the extracardiac mediastinal mesoderm. There appeared little difference in the connection between normal and trisomic embryos at the stage of 20 to 25 somites, but the area subsequently showed marked changes. In most trisomic embryos, the connection with the mediastinal mesoderm of the body was over a larger area than seen in normal embryos. As this area of attachment encloses the pulmonary pit, the entry point of the pulmonary vein, this gives potential for variation in the connection of the pulmonary vein. In addition, in the majority of trisomic embryos, the right pulmonary ridge (the spina vestibuli) did not accumulate extracardiac mesoderm, nor did it undergo the pronounced forward growth seen in normal embryos of equivalent stages. Consequently, the trisomic embryos show incomplete formation of both the atrial and the atrioventricular septal structures.
In Vivo Enzymatic Assay Reveals Catalytic Activity of the Human Renin Precursor in Tissues
Authors: Methot Danielle , Silversides David W. , Reudelhuber Timothy L.
Published: June 2012

Abstract
—The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.
Sarcolemmal Versus Mitochondrial ATP-Sensitive K
+
Channels and Myocardial Preconditioning
Authors: Gross Garrett J. , Fryer Ryan M.
Published: June 2012

Abstract
—Ischemic preconditioning (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. Although a number of substances and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, the overwhelming majority of evidence suggests that the ATP-sensitive potassium channel (K
ATP
channel) is an important component of this phenomenon and may serve as the end effector in this process. Initially, it was hypothesized that the surface or sarcolemmal K
ATP
(sarc K
ATP
) channel mediated protection observed after IPC; however, subsequent evidence suggested that the recently identified mitochondrial K
ATP
channel (mito K
ATP
) may be the potassium channel mediating IPC-induced cardioprotection. In this review, evidence will be presented supporting a role for either the sarc K
ATP
or the mito K
ATP
in IPC and potential mechanisms by which opening these channels may produce cardioprotection; additionally, we will address important questions that still need to be investigated to define the role of the sarc or mito K
ATP
channel, or both, in cardiac pathophysiology.
Distribution and Prevalence of Hyperpolarization-Activated Cation Channel (HCN) mRNA Expression in Cardiac Tissues
Authors: Shi Wenmei , Wymore Randy , Yu Hangang , Wu Jiying , Wymore Rigel T. , Pan Zongming , Robinson Richard B. , Dixon Jane E. , McKinnon David , Cohen Ira S.
Published: June 2012

Abstract
—HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (
I
f
) exist in rabbit Purkinje fibers, and we could not record
I
f
in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of
I
f
, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of
I
f
to be ≈−70 mV in neonatal rat ventricle and −113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is ≈5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for
I
f
activation, whereas the HCN2 isoform is associated with a more negative activation curve. The full text of this article is available at http://www.circresaha.org.
Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes
Authors: Cook Stuart A. , Sugden Peter H. , Clerk Angela
Published: June 2012

Abstract
—Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress–induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H
2
O
2
induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H
2
O
2
stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from ≈30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H
2
O
2
also induced translocation of cytochrome
c
from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H
2
O
2
showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by ≈1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress–induced cardiac myocyte apoptosis.
Formation of Nitric Oxide–Derived Oxidants by Myeloperoxidase in Monocytes
Authors: Hazen Stanley L. , Zhang Renliang , Shen Zhongzhou , Wu Weijia , Podrez Eugene A. , MacPherson Jennifer C. , Schmitt David , Mitra Shome N. , Mukhopadhyay Chaitali , Chen Yonghong , Cohen Peter A. , Hoff Henry F. , Abu-Soud Husam M.
Published: June 2012

Abstract
—Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO
2
−
), a major end product of nitric oxide (
•
NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography–mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO
2
−
; occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous
•
NO generator PAPA NONOate (
Z
-[
N
-{3-aminopropyl}-
N
-{
n
-propyl}amino]diazen-1-ium-1,2-diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of
•
NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of
•
NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadienoic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating
•
NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.
Permanent Cardiovascular Protection From Hypertension by the AT
1
Receptor Antisense Gene Therapy in Hypertensive Rat Offspring
Authors: Reaves Phyllis Y. , Gelband Craig H. , Wang Hongwei , Yang Hong , Lu Di , Berecek Kathleen H. , Katovich Michael J. , Raizada Mohan K.
Published: June 2012

Abstract
—Our previous studies have demonstrated that the introduction of angiotensin II type I receptor antisense (AT
1
R-AS) cDNA by a retrovirally mediated delivery system prevents the development of hypertension in the spontaneously hypertensive rat (SHR), an animal model for primary hypertension in humans. These results have led us to propose the hypothesis that an interruption of the renin-angiotensin system (RAS) activity at a genetic level would prevent hypertension on a permanent basis. F
1
and F
2
generations of offspring from a retroviral vector, LNSV- and LNSV-AT
1
R-AS–treated SHR, were generated, and various physiological parameters indicative of hypertension were studied and compared with those of their parents to investigate this hypothesis. Both F
1
and F
2
generations of LNSV-AT
1
R-AS–treated SHR expressed a persistently lower blood pressure, decreased cardiac hypertrophy and fibrosis, decreased medial thickness, and normalization of renal artery excitation-contraction coupling, Ca
2+
current, and [Ca
2+
]
i
when compared with offspring derived from the LNSV-treated SHR. In fact, the magnitude of the prevention of these pathophysiological alterations was similar to that observed in the LNSV-AT
1
R-AS–treated SHR parent. The prevention of cardiovascular pathophysiology and expression of normotensive phenotypes are, at least in part, a result of integration and subsequent transmission of AT
1
R-AS from the SHR parents to offspring. These data demonstrate that a single intracardiac injection of LNSV-AT
1
R-AS causes a permanent cardiovascular protection against hypertension as a result of a genomic integration and germ line transmission of the AT
1
R-AS in the SHR offspring. The full text of this article is available at http://www.circresaha.org.
A Subpopulation of Cardiomyocytes Expressing α-Skeletal Actin Is Identified by a Specific Polyclonal Antibody
Authors: Clément Sophie , Chaponnier Christine , Gabbiani Giulio
Published: June 2012

Abstract
—The NH
2
-terminal decapeptide of α-skeletal actin that contains a primary sequence specific for this isoform was used to raise a polyclonal antibody in rabbits. Using sequential affinity chromatography, we recovered from serum antibodies reacting exclusively with α-skeletal actin when tested by immunoblotting and immunofluorescence. Epitope mapping by means of competition assays with synthetic peptides indicated that the acetyl group and the first 9 amino acids are essential for specificity. The monospecific antibody was then used to investigate the distribution of α-skeletal actin in the myocardium of newborn and normal or hypertensive (with or without fibrotic areas) adult rats. Immunostaining of normal heart revealed that α-skeletal actin is diffusely distributed within practically all myocardial fibers of the newborn rat, whereas it is restricted to a small proportion of adult rat cardiomyocytes, which appear intensely stained. A correlation, albeit not complete, was found between the distribution of α-skeletal actin and β-myosin heavy chain. During cardiac hypertrophy induced by aortic ligature between the renal arteries, the expressions of α-skeletal actin mRNA and protein were increased. The distribution of immunostaining had a focal pattern similar to that of normal adult rats, reactive fibers being more numerous and more intensely stained compared with normal myocardium. Positive fibers were particularly abundant at the periphery of fibrotic areas. Using this antibody, we have demonstrated for the first time the differential distribution of α-skeletal actin in heart tissues. Changes in the distribution of this isoform in hypertrophic heart provide new insight into the mechanisms by which the heart adapts to work overload. This antibody will prove useful in exploring the mechanisms of expression of α-skeletal actin and in defining its role in physiological and pathological situations. The full text of this article is available at http://www.circresaha.org.
Remodeling of Cell-Cell and Cell–Extracellular Matrix Interactions at the Border Zone of Rat Myocardial Infarcts
Authors: Matsushita Tsutomu , Oyamada Masahito , Fujimoto Kazushi , Yasuda Yuko , Masuda Shinsuke , Wada Yukio , Oka Takahiro , Takamatsu Tetsuro
Published: June 2012

Abstract
—At the border zone of myocardial infarcts, surviving cardiomyocytes achieve drastic remodeling of cell-cell and cell–extracellular matrix interactions. Spatiotemporal changes in these interactions are likely related to each other and possibly have significant impact on cardiac function. To elucidate the changes, we conducted experimental infarction in rats and performed 3-dimensional analysis of the localization of gap junctions (connexin43), desmosomes (desmoplakin), adherens junctions (cadherin), and integrins (β
1
-integrin) by immunoconfocal microscopy. After myocardial infarction, changes in the distribution of gap junctions, desmosomes, and adherens junctions showed a similar but nonidentical tendency. In the early phase, gap junctions almost disappeared at stumps (longitudinal edges of cardiomyocytes facing the infarct), and, although desmosomes and adherens junctions decreased, they still remained. In the healing phase, at stumps, connexin43, desmoplakin, and cadherin were closely associated between multiple cell processes originating from a single cardiomyocyte. Electron microscopy confirmed the presence of junctional complexes between the cell processes. β
1
-Integrin at the cell process increased during the formation of papillary myotendinous junction–like structures. Abnormal localization of connexin43 was often accompanied by desmoplakin and cadherin on lateral surfaces of surviving cardiomyocytes. These findings suggested that remodeling of gap junction distribution was closely linked to changes in desmosomes and adherens junctions and that temporary formation of intracellular junctional complexes was an element of the remodeling of cell-cell and cell–extracellular matrix interactions after myocardial infarction. Moreover, the remodeling of the intercalated disk region at the myocardial interface with area of scar tissues was associated with the acquisition of extracellular matrix and β
1
-integrin.
Oncostatin M Induces Interleukin-6 and Cyclooxygenase-2 Expression in Human Vascular Smooth Muscle Cells
Authors: Bernard Catherine , Merval Régine , Lebret Marilyne , Delerive Philippe , Dusanter-Fourt Isabelle , Lehoux Stéphanie , Créminon Christophe , Staels Bart , Maclouf Jacques , Tedgui Alain
Published: June 2012

Abstract
—Oncostatin M (OSM), a cytokine first identified from activated monocytes and T lymphocytes, is one of the most potent autocrine growth factor for AIDS and Kaposi’s sarcoma. Little is known about the effects of OSM on normal vascular cells. We thus exposed human aortic smooth muscle cells (hASMCs) to OSM, examined cell proliferation and morphology, and determined interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) expression. OSM had a weak antiproliferative effect. After a 4-day incubation with 100 ng/mL OSM, cell count decreased to 69±3% of control. However, OSM induced striking changes in hASMC morphology, characterized by a polyclonal shape, in contrast to the spindle morphological feature of control hASMCs. OSM stimulated the release of IL-6 by hASMCs in a dose-dependent way; after a 48-hour exposure, values were 8.5±0.7, 29.7±3.5, 50.9±4.4, and 73.8±7.6×10
3
U/mL (n=6) at OSM concentrations of 0, 1, 10, and 100 ng/mL, respectively. OSM induced marked expression of COX-2 protein and mRNA. Leukemia inhibitory factor had no effect on hASMCs, indicating that OSM effects on hASMCs were mediated by the OSM type II receptor and not by the leukemia inhibitory factor receptor. OSM used the JAK/STAT signaling pathway, as demonstrated by rapid phosphorylation of JAK1 and specific activation of STAT1. Interestingly, OSM acted in synergy with IL-1β on IL-6 production and COX-2 expression. In conclusion, OSM is a novel regulator of human smooth muscle cell functions, acting in concert with IL-1β, and OSM may play a role in major vascular diseases such as atherosclerosis.
Activation of Mitochondrial K
ATP
Channel Elicits Late Preconditioning Against Myocardial Infarction via Protein Kinase C Signaling Pathway
Authors: Takashi En , Wang Yigang , Ashraf Muhammad
Published: June 2012

Abstract
—Activation of mitochondrial K
ATP
(mitoK
ATP
) channel induces acute ischemic preconditioning (PC) against ischemic injury. The ability of this channel to elicit late PC remains unknown. The present study tests the hypothesis that stimulation of mitoK
ATP
channel induces late PC via the protein kinase C (PKC) signaling pathway. Rats were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion (I/R). In other groups, rats were pretreated with diazoxide, a specific opener of the mitoK
ATP
channel (7 mg/kg, IV), 12, 24, 48, and 72 hours before they were subjected to I/R. A maximum reduction in infarct size was observed after 24 hours (33.3±2.2% versus I/R group, 62.1 ±2.4%). Pretreatment with diazoxide did not reduce the infarct size significantly after 12, 48, and 72 hours (50.2±4.3%, 50.5±4.6%, and 58.2±4.9%) compared with the I/R group. The protection was blocked with 5-hydroxydecanoic acid (5-HD, 5 mg/kg IV), a relatively selective mitoK
ATP
channel blocker (56.5±2.7%), and chelerythrine (5 mg/kg IV), an effective PKC inhibitor (57.1±3.4%) administered either on the first day before diazoxide pretreatment or 10 minutes before I/R on the second day. Cell necrosis was decreased by ≈50% in the diazoxide preconditioned hearts compared with control I/R hearts. Cell death by apoptosis was also significantly decreased in diazoxide pretreated hearts (3.2%) as compared with I/R (11.3%). In conclusion, activation of mitoK
ATP
channel with diazoxide produces late PC against reperfusion injury. The effect of mitoK
ATP
channel appears to be dependent on the PKC-mediated signal pathway.
Differential Regulation of p90 Ribosomal S6 Kinase and Big Mitogen–Activated Protein Kinase 1 by Ischemia/Reperfusion and Oxidative Stress in Perfused Guinea Pig Hearts
Authors: Takeishi Yasuchika , Abe Jun-ichi , Lee Jiing-Dwan , Kawakatsu Hisaaki , Walsh Richard A. , Berk Bradford C.
Published: June 2012

Abstract
—Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na
+
/H
+
exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H
2
O
2
, ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H
2
O
2
(100 μmol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(
t
-butyl)pyrazolo[3,4-
d
]pyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant
N
-2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.
Novel Vascular Graft Grown Within Recipient’s Own Peritoneal Cavity
Authors: Campbell Julie H. , Efendy Johnny L. , Campbell Gordon R.
Published: June 2012

Abstract
—A method by which to overcome the clinical symptoms of atherosclerosis is the insertion of a graft to bypass an artery blocked or impeded by plaque. However, there may be insufficient autologous mammary artery for multiple or repeat bypass, saphenous vein may have varicose degenerative alterations that can lead to aneurysm in high-pressure sites, and small-caliber synthetic grafts are prone to thrombus induction and occlusion. Therefore, the aim of the present study was to develop an artificial blood conduit of any required length and diameter from the cells of the host for autologous transplantation. Silastic tubing, of variable length and diameter, was inserted into the peritoneal cavity of rats or rabbits. By 2 weeks, it had become covered by several layers of myofibroblasts, collagen matrix, and a single layer of mesothelium. The Silastic tubing was removed from the harvested implants, and the tube of living tissue was everted such that it now resembled a blood vessel with an inner lining of nonthrombotic mesothelial cells (the “intima”), with a “media” of smooth muscle–like cells (myofibroblasts), collagen, and elastin, and with an outer collagenous “adventitia.” The tube of tissue (10 to 20 mm long) was successfully grafted by end-to-end anastomoses into the severed carotid artery or abdominal aorta of the same animal in which they were grown. The transplant remained patent for at least 4 months and developed structures resembling elastic lamellae. The myofibroblasts gained a higher volume fraction of myofilaments and became responsive to contractile agonists, similar to the vessel into which they had been grafted. It is suggested that these nonthrombogenic tubes of living tissue, grown in the peritoneal cavity of the host, may be developed as autologous coronary artery bypass grafts or as arteriovenous access fistulae for hemodialysis patients.
Decreased Neointimal Thickening After Arterial Wall Injury in Inducible Nitric Oxide Synthase Knockout Mice
Authors: Chyu Kuang-Yuh , Dimayuga Paul , Zhu Jenny , Nilsson Jan , Kaul Sanjay , Shah Prediman K. , Cercek Bojan
Published: June 2012

Abstract
—Mechanical injury in vivo results in the expression of the inducible form of nitric oxide synthase (iNOS) in vascular smooth muscle cells. However, the role of iNOS in modulating neointima formation after arterial wall injury is not clear. To determine whether the induction of iNOS gene expression promotes or attenuates the neointimal response to injury, we used a murine model of perivascular injury induced by placing a periadventitial collar around the carotid arteries in both wild-type and iNOS knockout mice (iNOS-KO mice). Periadventitial injury induced iNOS expression in the wild-type but not the iNOS-KO mice. Neointimal area and the intima/media ratio were significantly less in the iNOS-KO mice compared with the wild-type mice at 21 days. Injury-induced proliferation of medial cells and vascular cell adhesion molecule-1 expression were also attenuated in iNOS-KO mice compared with wild-type mice. The induction of iNOS and the activation of the nuclear factor-κB–mediated pathway were also demonstrated in an in vitro injury model. We conclude that mechanical injury in vivo and in vitro induces iNOS expression and that lack of iNOS expression attenuates neointima formation after perivascular arterial injury. Taken together, these findings suggest that iNOS expression after vascular injury may promote neointima formation.
Inhibition of Copper-Zinc Superoxide Dismutase Induces Cell Growth, Hypertrophic Phenotype, and Apoptosis in Neonatal Rat Cardiac Myocytes In Vitro
Authors: Siwik Deborah A. , Tzortzis John D. , Pimental David R. , Chang Donny L.-F. , Pagano Patrick J. , Singh Krishna , Sawyer Douglas B. , Colucci Wilson S.
Published: June 2012

Abstract
—Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 μmol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro–atrial natriuretic peptide, and c-
fos
mRNAs and decreased sarcoplasmic reticulum Ca
2+
ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 μmol/L) stimulated myocyte apoptosis, as evidenced by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase–mediated nick end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in the pathogenesis of myocardial remodeling and failure.
Very Low Density Lipoprotein–Mediated Signal Transduction and Plasminogen Activator Inhibitor Type 1 in Cultured HepG2 Cells
Authors: Banfi Cristina , Mussoni Luciana , Risé Patrizia , Cattaneo Maria Grazia , Vicentini Lucia , Battaini Fiorenzo , Galli Claudio , Tremoli Elena
Published: June 2012

Abstract
—In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[
3
H]inositol–labeled HepG2 cells, VLDL (100 μg/mL) caused a time-dependent increase in [
3
H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all
P
<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca
2+
release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (
P
<0.05), and the MAP kinase/extracellular signal–regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.
Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration and Proliferation Depend on a Nitric Oxide–Mediated Decrease in Protein Kinase Cδ Activity
Authors: Shizukuda Yukitaka , Tang Shaoqing , Yokota Ryoji , Ware J. Anthony
Published: June 2012

Abstract
—Vascular endothelial growth factor (VEGF) promotes angiogenesis and endothelial cell (EC) migration and proliferation by affecting intracellular mediators, only some of which are known, distal to its receptors. Protein kinase C (PKC) participates in the function of VEGF, but the role of individual PKC isoenzymes is unknown. In this study, we tested the importance of the activity of specific PKC isoenzymes in human EC migration and proliferation in response to VEGF. PKCδ specific activity was depressed by the addition of VEGF (by 41±8% [P<0.05] at 24 hours) in human umbilical vein ECs (HUVECs) and in a HUVEC-derived EC line, ECV, without changing the total amount of either protein or mRNA encoding PKCδ. Neither basic fibroblast growth factor (FGF-2) nor serum altered PKCδ specific activity. The VEGF-induced decrease of PKCδ activity, which began at 8 hours after stimulation, was strongly blocked by pretreatment with the nitric oxide (NO) synthase inhibitor
N
G
-monomethyl-
l
-arginine in HUVECs; NO release peaked within 2 hours after stimulation. An exogenous NO donor, sodium nitroprusside, also decreased PKCδ activity. The inhibition by
N
G
-monomethyl-
l
-arginine of VEGF-induced HUVEC migration and proliferation, but not that induced by FGF-2 or serum, suggested that the decrease in PKCδ via NO pathway is required for VEGF-induced EC migration and proliferation. Overexpression of PKCδ in ECV cells specifically prevented EC response to VEGF but not to FGF-2 or serum. Thus, we conclude that suppression of PKCδ activity via a NO synthase mechanism is required for VEGF-induced EC migration and proliferation, but not for that induced by FGF-2 or serum.