bluuebunny/crossref_metadata_2025 huggingface dataset:
NADH Oxidase Activation Is Involved in Arsenite-Induced Oxidative DNA Damage in Human Vascular Smooth Muscle Cells
Authors: Lynn Shugene , Gurr Jia-Ran , Lai Hsien-Tsung , Jan Kun-Yan
Published: June 2012

Abstract
—Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 μmol/L. DNA strand breaks of arsenite-treated cells were increased by
Escherichia coli
formamidopyrimidine–DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or
d
-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1,2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis.
Atrial but Not Ventricular Fibrosis in Mice Expressing a Mutant Transforming Growth Factor-β
1
Transgene in the Heart
Authors: Nakajima Hidehiro , Nakajima Hisako O. , Salcher Olga , Dittiè Andrea S. , Dembowsky Klaus , Jing Shaoliang , Field Loren J.
Published: June 2012

Abstract
—Increased transforming growth factor (TGF)–β
1
activity has been observed during pathologic cardiac remodeling in a variety of animal models. In an effort to establish a causal role of TGF-β
1
in this process, transgenic mice with elevated levels of active myocardial TGF-β
1
were generated. The cardiac-restricted α–myosin heavy chain promoter was used to target expression of a mutant TGF-β
1
cDNA harboring a cysteine-to-serine substitution at amino acid residue 33. This alteration blocks covalent tethering of the TGF-β
1
latent complex to the extracellular matrix, thereby rendering a large proportion (>60%) of the transgene-encoded TGF-β
1
constitutively active. Although similar levels of active TGF-β
1
were present in the transgenic atria and ventricles, overt fibrosis was observed only in the atria. Surprisingly, increased active TGF-β
1
levels inhibited ventricular fibroblast DNA synthesis in uninjured hearts and delayed wound healing after myocardial injury. These data suggest that increased TGF-β
1
activity by itself is insufficient to promote ventricular fibrosis in the adult mouse ventricle.
The Antianginal Drug Trimetazidine Shifts Cardiac Energy Metabolism From Fatty Acid Oxidation to Glucose Oxidation by Inhibiting Mitochondrial Long-Chain 3-Ketoacyl Coenzyme A Thiolase
Authors: Kantor Paul F. , Lucien Arnaud , Kozak Raymond , Lopaschuk Gary D.
Published: June 2012

Abstract
—Trimetazidine is a clinically effective antianginal agent that has no negative inotropic or vasodilator properties. Although it is thought to have direct cytoprotective actions on the myocardium, the mechanism(s) by which this occurs is as yet undefined. In this study, we determined what effects trimetazidine has on both fatty acid and glucose metabolism in isolated working rat hearts and on the activities of various enzymes involved in fatty acid oxidation. Hearts were perfused with Krebs-Henseleit solution containing 100 μU/mL insulin, 3% albumin, 5 mmol/L glucose, and fatty acids of different chain lengths. Both glucose and fatty acids were appropriately radiolabeled with either
3
H or
14
C for measurement of glycolysis, glucose oxidation, and fatty acid oxidation. Trimetazidine had no effect on myocardial oxygen consumption or cardiac work under any aerobic perfusion condition used. In hearts perfused with 5 mmol/L glucose and 0.4 mmol/L palmitate, trimetazidine decreased the rate of palmitate oxidation from 488±24 to 408±15 nmol · g dry weight
−1
· minute
−1
(
P
<0.05), whereas it increased rates of glucose oxidation from 1889±119 to 2378±166 nmol · g dry weight
−1
· minute
−1
(
P
<0.05). In hearts subjected to low-flow ischemia, trimetazidine resulted in a 210% increase in glucose oxidation rates. In both aerobic and ischemic hearts, glycolytic rates were unaltered by trimetazidine. The effects of trimetazidine on glucose oxidation were accompanied by a 37% increase in the active form of pyruvate dehydrogenase, the rate-limiting enzyme for glucose oxidation. No effect of trimetazidine was observed on glycolysis, glucose oxidation, fatty acid oxidation, or active pyruvate dehydrogenase when palmitate was substituted with 0.8 mmol/L octanoate or 1.6 mmol/L butyrate, suggesting that trimetazidine directly inhibits long-chain fatty acid oxidation. This reduction in fatty acid oxidation was accompanied by a significant decrease in the activity of the long-chain isoform of the last enzyme involved in fatty acid β-oxidation, 3-ketoacyl coenzyme A (CoA) thiolase activity (IC
50
of 75 nmol/L). In contrast, concentrations of trimetazidine in excess of 10 and 100 μmol/L were needed to inhibit the medium- and short-chain forms of 3-ketoacyl CoA thiolase, respectively. Previous studies have shown that inhibition of fatty acid oxidation and stimulation of glucose oxidation can protect the ischemic heart. Therefore, our data suggest that the antianginal effects of trimetazidine may occur because of an inhibition of long-chain 3-ketoacyl CoA thiolase activity, which results in a reduction in fatty acid oxidation and a stimulation of glucose oxidation.
Biphasic Actions of Prostaglandin E
2
on the Renal Afferent Arteriole
Authors: Tang Lilong , Loutzenhiser Kathy , Loutzenhiser Rodger
Published: June 2012

Abstract
—Prostaglandin (PG) E
2
is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE
2
and the EP receptor subtypes involved. Ibuprofen potentiated Ang II–induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE
2
, which reversed Ang II–induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE
2
-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE
1
(0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP
2
receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE
2
elicited vasodilation, higher concentrations (1 to 10 μmol/L) restored Ang II–induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 μg/mL) and was mimicked by the EP
1
/EP
3
agonist sulprostone (1 to 300 nmol/L). Reverse transcription–polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP
4
and all 3 EP
3
splice variants (α, β, and γ) but not EP
1
or EP
2
. Our findings thus indicate that PGE
2
elicits both vasodilatory and vasoconstrictor actions on the afferent arteriole. The vasodilation is mediated by EP
4
receptors coupled to cAMP, presumably via G
αs
. The vasoconstriction is mediated by an EP
3
receptor coupled to G
αi
and appears to reflect a functional antagonism of the EP
4
-induced vasodilation.
Inhibition of Extracellular Signal–Regulated Kinase Enhances Ischemia/Reoxygenation–Induced Apoptosis in Cultured Cardiac Myocytes and Exaggerates Reperfusion Injury in Isolated Perfused Heart
Authors: Yue Tian-Li , Wang Chuanlin , Gu Juan-Li , Ma Xin-Ling , Kumar Sanjay , Lee John C. , Feuerstein Giora Z. , Thomas Heath , Maleeff Beverly , Ohlstein Eliot H.
Published: June 2012

Abstract
—Three major mammalian mitogen-activated protein kinases, extracellular signal–regulated kinase (ERK), p38, and c-Jun NH
2
-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)–induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase–mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.
Connexin Expression and Turnover
Authors: Saffitz Jeffrey E. , Laing James G. , Yamada Kathryn A.
Published: June 2012

Abstract
—Electrical activation of the heart requires current transfer from one cell to another via gap junctions, arrays of densely packed intercellular channels. The extent to which cardiac myocytes are coupled is determined by multiple mechanisms, including tissue-specific patterns of expression of diverse gap junction channel proteins (connexins), and regulatory pathways that control connexin synthesis, intracellular trafficking, assembly into channels, and degradation. Many connexins, including those expressed in the heart, have been found to turn over rapidly. Recent studies in the intact adult heart suggest that connexin43, the principal cardiac connexin, is surprisingly short-lived (half-life ≈1.3 hours). Both the proteasome and the lysosome participate in connexin43 degradation. Other ion channel proteins, such as those forming selected voltage-gated K
+
channels, may also exhibit rapid turnover kinetics. Regulation of connexin degradation may be an important mechanism for adjusting intercellular coupling in the heart under normal and pathophysiological conditions.
Single-Molecule Mechanics of R403Q Cardiac Myosin Isolated From the Mouse Model of Familial Hypertrophic Cardiomyopathy
Authors: Tyska M. J. , Hayes E. , Giewat M. , Seidman C. E. , Seidman J. G. , Warshaw D. M.
Published: June 2012

Abstract
—Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that can result in sudden death in the absence of any overt symptoms. Many of the cases documented to date have been linked with missense mutations in the β-myosin heavy chain gene. Here we present data detailing the functional impact of one of the most deadly mutations, R403Q, on myosin motor function. Experiments were performed on whole cardiac myosin purified from a mouse model of FHC to eliminate potential uncertainties associated with protein expression systems. The R403Q mutant myosin demonstrated 2.3-fold higher actin-activated ATPase activity, 2.2-fold greater average force generation, and 1.6-fold faster actin filament sliding in the motility assay. The force- and displacement-generating capacities of both the normal and mutant myosin were also characterized at the single molecule level in the laser trap assay. Both control and mutant generated similar unitary forces (≈1 pN) and displacements (≈7 nm) without any differences in event durations. On the basis of the distribution of mean unitary displacements, this mutation may possibly perturb the mechanical coordination between the 2 heads of cardiac myosin. Any of these observations could, alone or possibly in combination, result in abnormal power output and potentially a stimulus for the hypertrophic response.
Rapid Displacement of Vimentin Intermediate Filaments in Living Endothelial Cells Exposed to Flow
Authors: Helmke Brian P. , Goldman Robert D. , Davies Peter F.
Published: June 2012

Abstract
—Hemodynamic shear stress at the endothelial cell surface induces acute and chronic intracellular responses that regulate vessel wall biology. The cytoskeleton is implicated by acting both as a direct connector to local surface deformation and as a distribution network for mechanical forces throughout the cell; however, direct observation and measurement of its position during flow have only recently become possible. In this study, we directly demonstrate rapid deformation of the intermediate filament (IF) network in living endothelial cells subjected to changes in hemodynamic shear stress. Time-lapse optical sectioning and deconvolution microscopy were performed within the first 3 minutes after the introduction of flow (shear stress, 12 dyn/cm
2
). Spatial and temporal dynamics of green fluorescent protein–vimentin IFs in confluent endothelial cells were analyzed. The imposition of shear stress significantly increased the variability of IF movement throughout the cell in the
x
-,
y
-, and
z
-directions compared with the constitutive dynamics noted in the absence of flow. Acute polymerization and depolymerization of the IF network were absent. The magnitude and direction of flow-induced IF displacement were heterogeneous at the subcellular level. These qualitative and quantitative data demonstrate that shear stress acting at the luminal surface of the endothelium results in rapid deformation of a stable IF network.
Coupling of Ca
2+
to CREB Activation and Gene Expression in Intact Cerebral Arteries From Mouse
Authors: Cartin L. , Lounsbury K. M. , Nelson M. T.
Published: June 2012

Abstract
—Pathological changes of the vasculature are characterized by changes in Ca
2+
handling and alterations in gene expression. In neurons and other cell types, [Ca
2+
]
i
often drives changes in gene expression. However, the relationship between Ca
2+
signaling and gene expression in vascular smooth muscle is not well understood. This study examines the ability of Ca
2+
influx through voltage-dependent, L-type Ca
2+
channels (VDCCs) and Ca
2+
release through ryanodine receptors (RyRs) to activate the transcription factor, cAMP-responsive element binding protein (CREB), and increase c-
fos
levels in intact cerebral arteries. Membrane depolarization increased the fraction of nuclei staining for phosphorylated CREB (P-CREB) and levels of c-
fos
mRNA in intact mouse cerebral arteries. Ryanodine, which inhibits RyRs, increased P-CREB staining and c-
fos
levels. Forskolin, an activator of adenylyl cyclase, and sodium nitroprusside, an NO donor, increased P-CREB and c-
fos
levels. Nisoldipine, an inhibitor of VDCCs, reversed the effects of depolarization and ryanodine on P-CREB and c-
fos
levels, but not the effects of forskolin or sodium nitroprusside. Inhibition of Ca
2+
/calmodulin-dependent protein kinase (CaM kinase) blocked increases in P-CREB and c-
fos
levels seen with membrane depolarization, suggesting that CaM kinase has an important role in the pathway leading from Ca
2+
influx to CREB-mediated changes in c-
fos
levels. Our data suggest that membrane depolarization increases [Ca
2+
]
i
through activation of VDCCs, leading to increased P-CREB and c-
fos
, and that RyRs have a profound effect on this pathway by indirectly regulating Ca
2+
entry through VDCCs. These results provide the first evidence of Ca
2+
regulation of CREB and c-
fos
in arterial smooth muscle.
Nerve Sprouting and Sudden Cardiac Death
Authors: Cao Ji-Min , Chen Lan S. , KenKnight Bruce H. , Ohara Toshihiko , Lee Moon-Hyoung , Tsai Jerome , Lai William W. , Karagueuzian Hrayr S. , Wolf Paul L. , Fishbein Michael C. , Chen Peng-Sheng
Published: June 2012

Abstract
—The factors that contribute to the occurrence of sudden cardiac death (SCD) in patients with chronic myocardial infarction (MI) are not entirely clear. The present study tests the hypothesis that augmented sympathetic nerve regeneration (nerve sprouting) increases the probability of ventricular tachycardia (VT), ventricular fibrillation (VF), and SCD in chronic MI. In dogs with MI and complete atrioventricular (AV) block, we induced cardiac sympathetic nerve sprouting by infusing nerve growth factor (NGF) to the left stellate ganglion (experimental group, n=9). Another 6 dogs with MI and complete AV block but without NGF infusion served as controls (n=6). Immunocytochemical staining revealed a greater magnitude of sympathetic nerve sprouting in the experimental group than in the control group. After MI, all dogs showed spontaneous VT that persisted for 5.8±2.0 days (phase 1 VT). Spontaneous VT reappeared 13.1±6.0 days after surgery (phase 2 VT). The frequency of phase 2 VT was 10-fold higher in the experimental group (2.0±2.0/d) than in the control group (0.2±0.2/d,
P
<0.05). Four dogs in the experimental group but none in the control group died suddenly of spontaneous VF. We conclude that MI results in sympathetic nerve sprouting. NGF infusion to the left stellate ganglion in dogs with chronic MI and AV block augments sympathetic nerve sprouting and creates a high-yield model of spontaneous VT, VF, and SCD. The magnitude of sympathetic nerve sprouting may be an important determinant of SCD in chronic MI.
Polymorphisms of the β
2
-Adrenergic Receptor Determine Exercise Capacity in Patients With Heart Failure
Authors: Wagoner Lynne E. , Craft Laura L. , Singh Balkrishna , Suresh Damodhar P. , Zengel Paul W. , McGuire Nancy , Abraham William T. , Chenier Thomas C. , Dorn Gerald W. , Liggett Stephen B.
Published: June 2012

Abstract
—The β
2
-adrenergic receptor (β
2
AR) exists in multiple polymorphic forms with different characteristics. Their relevance to heart failure (HF) physiology is unknown. Cardiopulmonary exercise testing was performed on 232 compensated HF patients with a defined β
2
AR genotype. Patients with the uncommon Ile164 polymorphism had a lower peak V̇
o
2
(15.0±0.9 mL · kg
−1
· min
−1
) than did patients with Thr164 (17.9±0.9 mL · kg
−1
· min
−1
,
P
<0.0001). The percentage achieved of predicted peak V̇
o
2
was also lower in patients with Ile164 (62.3±4.5% versus 71.5±5.1%,
P
=0.045). The relative risk of a patient having a V̇
o
2
≤14 mL · kg
−1
· min
−1
who had Ile164 was 8.0 (
P
=0.009). Catheterization-based invasive exercise testing revealed depressed changes in the exercise-induced cardiac index, systemic vascular resistance, stroke volume, and V̇
o
2
in patients with Ile164. The polymorphisms at position 16 also impacted exercise capacity: peak V̇
o
2
for Arg16 versus Gly16 was 17.0±0.8 versus 15.6±0.5 mL · kg
−1
· min
−1
, respectively (
P
=0.03). Because the polymorphisms at loci 16 and 27 can occur together, 4 homozygous combinations exist. Patients with Arg16/Glu27 had the highest percentage achieved of predicted peak V̇
o
2
(75.7±6.4%), whereas those with Gly16/Gln27 had the lowest (55.3±2.8%,
P
=0.0032). The above findings were not confounded by baseline clinical characteristics, including β-blocker usage. We conclude that the β
2
AR polymorphisms Ile164, Gly16, and the combination of Gly16 and Gln27 are associated with depressed exercise performance in HF and represent a genetically determined factor in the pathophysiology of HF.
Functional Deletion Mutation of the 5′-Flanking Region of Type A Human Natriuretic Peptide Receptor Gene and Its Association With Essential Hypertension and Left Ventricular Hypertrophy in the Japanese
Authors: Nakayama Tomohiro , Soma Masayoshi , Takahashi Yukie , Rehemudula Duolikun , Kanmatsuse Katsuo , Furuya Kiyohide
Published: June 2012

Abstract
—The natriuretic peptide (NP) family is involved in the regulation of blood pressure and fluid volume. We isolated the 5′-flanking region of the type A human NP receptor gene and identified an insertion/deletion mutation in this region. We then assessed whether there is a genetic association between this mutation and essential hypertension (EH). The deletion allele lacks 8 nucleotides and alters binding sites for the activator protein-2 (AP-2) and Zeste transcriptional factors. We genotyped 200 EH and 200 normotensive (NT) individuals and found 9 subjects with the deletion (8 in the EH group and 1 in the NT group). All 9 individuals were heterozygous. The NT subject with the mutation had left ventricular hypertrophy without hypertension. Transcriptional activity of the deletion allele was <30% that of the wild-type allele. The plasma levels of brain NP in EH patients with the deleted allele were significantly higher than the levels in the EH patients with the wild-type allele, and plasma brain NP levels were significantly higher in subjects with the deleted allele than in subjects with the wild-type allele, despite comparable blood pressures. These findings suggest that in Japanese individuals, this deletion in the human NP receptor gene reduces receptor activity and may confer increased susceptibility to developing EH or left ventricular hypertrophy.
Vascular Endothelial Growth Factor–Stimulated Actin Reorganization and Migration of Endothelial Cells Is Regulated via the Serine/Threonine Kinase Akt
Authors: Morales-Ruiz Manuel , Fulton David , Sowa Grzegorz , Languino Lucia R. , Fujio Yasushi , Walsh Kenneth , Sessa William C.
Published: June 2012

Abstract
—Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding β-galactosidase (β-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with β-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of β-gal–infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.
Cyclooxygenase-2 Is Required for Tumor Necrosis Factor-α– and Angiotensin II–Mediated Proliferation of Vascular Smooth Muscle Cells
Authors: Young Wilson , Mahboubi Keyvan , Haider Asifa , Li Irene , Ferreri Nicholas R.
Published: June 2012

Abstract
—Tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) induced a transient increase in vascular smooth muscle cell (VSMC) cyclooxygenase-2 (COX-2) mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 mRNA accumulation were similar in VSMCs challenged with either TNF-α or Ang II; mRNA accumulation peaked at 2 hours and decreased to control levels by ≈6 hours. Accumulation of COX-2 mRNA was associated with a time-dependent increase of COX-2 protein expression that displayed similar kinetics in response to either TNF-α or Ang II. Both the increase in COX-2 mRNA accumulation and protein expression in response to either TNF-α or Ang II were inhibited by the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD098059. In addition, the AT
1
-selective receptor antagonist losartan attenuated the Ang II–mediated increase in COX-2 mRNA accumulation; the AT
2
-selective antagonist PD123319 had no effect. Prostacyclin I
2
synthesis was tightly coupled to expression of COX-2, whereas prostaglandin E
2
and thromboxane A
2
(TXA
2
) synthesis may be associated with differential usage of COX-1 and COX-2. The COX-2–selective inhibitors NS-398 and nimesulide and the TXA
2
receptor antagonist BMS 180,291 inhibited TNF-α– and Ang II–mediated increases in DNA content and cell number by ≈95%. These findings suggest that a prostanoid derived from COX-2, possibly TXA
2
, may contribute to VSMC hyperplasia in vessel injury or pathophysiological conditions associated with elevated levels of either TNF-α or Ang II.
Vascular Superoxide Production by NAD(P)H Oxidase
Authors: Guzik Tomasz J. , West Nick E. J. , Black Edward , McDonald Denise , Ratnatunga Chandi , Pillai Ravi , Channon Keith M.
Published: June 2012

Abstract
—Superoxide anion plays important roles in vascular disease states. Increased superoxide production contributes to reduced nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of vascular disease. We measured superoxide production by NAD(P)H oxidase in human blood vessels and examined the relationships between NAD(P)H oxidase activity, NO-mediated endothelial function, and clinical risk factors for atherosclerosis. Endothelium-dependent vasorelaxations and direct measurements of vascular superoxide production were determined in human saphenous veins obtained from 133 patients with coronary artery disease and identified risk factors. The predominant source of vascular superoxide production was an NAD(P)H-dependent oxidase. Increased vascular NAD(P)H oxidase activity was associated with reduced NO-mediated vasorelaxation. Furthermore, reduced endothelial vasorelaxations and increased vascular NAD(P)H oxidase activity were both associated with increased clinical risk factors for atherosclerosis. Diabetes and hypercholesterolemia were independently associated with increased NADH-dependent superoxide production. The association of increased vascular NAD(P)H oxidase activity with endothelial dysfunction and with clinical risk factors suggests an important role for NAD(P)H oxidase–mediated superoxide production in human atherosclerosis. The full text of this article is available at http://www.circresaha.org.
Nongenomic, ER-Mediated Activation of Endothelial Nitric Oxide Synthase:
Authors: Mendelsohn Michael E.
Published: June 2012

The administration of estrogens in animals tends to inhibit the development of atherosclerosis... [Therefore], attempts are being made in human males to prevent further progress of the disease in cases of angina or previous coronary thrombosis by long-range estrogen therapy. This work is in its infancy. It is hoped that, with further research, compounds might be found that eliminate the undesirable action of such hormones and yet retain its beneficial effects on arteries (Samuel A. Levine, 1958).

1
The Late Phase of Preconditioning
Authors: Bolli Roberto
Published: June 2012

Abstract
—Unlike the early phase of preconditioning (PC), which lasts 2 to 3 hours and protects against infarction but not against stunning, the late phase of PC lasts 3 to 4 days and protects against both infarction and stunning, suggesting that it may have greater clinical relevance. It is now clear that late PC is a polygenic phenomenon that requires the simultaneous activation of multiple stress-responsive genes. Chemical signals released by a sublethal ischemic stress (such as NO, reactive oxygen species, and adenosine) trigger a complex cascade of signaling events that includes the activation of protein kinase C, Src protein tyrosine kinases, and nuclear factor κB and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. An analogous sequence of events can be triggered by a variety of stimuli, such as heat stress, exercise, and cytokines. Thus, late PC appears to be a universal response of the heart to stress in general. Importantly, the cardioprotective effects of late PC can be reproduced pharmacologically with clinically relevant agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists), suggesting that this phenomenon might be exploited for therapeutic purposes. The purpose of this review is to summarize current information regarding the pathophysiology and mechanism of late PC.
Multiple Transcriptional Domains, With Distinct Left and Right Components, in the Atrial Chambers of the Developing Heart
Authors: Franco Diego , Campione Marina , Kelly Robert , Zammit Peter S. , Buckingham Margaret , Lamers Wouter H. , Moorman Antoon F. M.
Published: June 2012

Abstract
—During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and
Pitx-2
, and transgenic mouse lines expressing
nlacZ
under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of
Pitx-2
reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.
Ventricular Fibrillation
Authors: Weiss James N. , Chen Peng-Sheng , Qu Zhilin , Karagueuzian Hrayr S. , Garfinkel Alan
Published: June 2012

Abstract
—Combined experimental and theoretical developments have demonstrated that in addition to preexisting electrophysiological heterogeneities, cardiac electrical restitution properties contribute to breakup of reentrant wavefronts during cardiac fibrillation. Developing therapies that favorably alter electrical restitution properties have promise as a new paradigm for preventing fibrillation.
Current Perspectives on the Use of Gene Therapy for Hypertension
Authors: Gelband Craig H. , Katovich Michael J. , Raizada Mohan K.
Published: June 2012

Abstract
—Systemic hypertension is a pathophysiological state that is manifested as high blood pressure and is a major risk factor for stroke, ischemic heart disease, peripheral vascular disease, and progressive renal damage. Pulmonary hypertension occurs in 3 distinct forms: primary pulmonary hypertension, pulmonary hypertension of the newborn, or secondary pulmonary hypertension attributable to a variety of lung and cardiovascular diseases. This review discusses the use of gene therapy in the control of systemic and pulmonary hypertension. Overexpression of vasodilator genes as well as antisense knockdown of vasoconstrictor genes has been successfully used in animal models of both forms of hypertension. Furthermore, the use of viral vectors to deliver these constructs has achieved long-term control of hypertension. The successful establishment of gene therapy techniques in the animal models of hypertension coupled with the anticipated advances in the genetic aspects of this disease would make it highly feasible to attempt gene delivery in the control of human hypertension.
Myocardial Cell Death in Human Diabetes
Authors: Frustaci Andrea , Kajstura Jan , Chimenti Cristina , Jakoniuk Igor , Leri Annarosa , Maseri Attilio , Nadal-Ginard Bernardo , Anversa Piero
Published: June 2012

Abstract
—The renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of a dilated myopathy. Angiotensin II (Ang II) locally may lead to oxidative damage, activating cardiac cell death. Moreover, diabetes and hypertension could synergistically impair myocardial structure and function. Therefore, apoptosis and necrosis were measured in ventricular myocardial biopsies obtained from diabetic and diabetic-hypertensive patients. Accumulation of a marker of oxidative stress, nitrotyrosine, and Ang II labeling were evaluated quantitatively. The diabetic heart showed cardiac hypertrophy, cavitary dilation, and depressed ventricular performance. These alterations were more severe with diabetes and hypertension. Diabetes was characterized by an 85-fold, 61-fold, and 26-fold increase in apoptosis of myocytes, endothelial cells, and fibroblasts, respectively. Apoptosis in cardiac cells did not increase additionally with diabetes and hypertension. Diabetes increased necrosis by 4-fold in myocytes, 9-fold in endothelial cells, and 6-fold in fibroblasts. However, diabetes and hypertension increased necrosis by 7-fold in myocytes and 18-fold in endothelial cells. Similarly, Ang II labeling in myocytes and endothelial cells increased more with diabetes and hypertension than with diabetes alone. Nitrotyrosine localization in cardiac cells followed a comparable pattern. In spite of the difference in the number of nitrotyrosine-positive cells with diabetes and with diabetes and hypertension, apoptosis and necrosis of myocytes, endothelial cells, and fibroblasts were detected only in cells containing this modified amino acid. In conclusion, local increases in Ang II with diabetes and with diabetes and hypertension may enhance oxidative damage, activating cardiac cell apoptosis and necrosis.
The β
2
-Adrenergic Receptor Delivers an Antiapoptotic Signal to Cardiac Myocytes Through G
i
-Dependent Coupling to Phosphatidylinositol 3′-Kinase
Authors: Chesley Alan , Lundberg Martha S. , Asai Toshinobu , Xiao Rui-Ping , Ohtani Seiji , Lakatta Edward G. , Crow Michael T.
Published: June 2012

Abstract
—Recent studies have shown that chronic β-adrenergic receptor (β-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective β
1
- and β
2
-AR subtype stimulation on apoptosis induced by hypoxia or H
2
O
2
in rat neonatal cardiac myocytes. Although neither β
1
- nor β
2
-AR stimulation had any significant effect on the basal level of apoptosis, selective β
2
-AR stimulation protected myocytes from apoptosis. β
2
-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal–regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3′-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. β
1
-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked β
2
-AR–mediated protection from apoptosis as well as the β
2
-AR–stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked β
2
-AR–mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on β
2
-AR–mediated protection. These findings demonstrate that β
2
-ARs activate a PI-3K–dependent, pertussis toxin–sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.
Targeted Disruption of Kir2.1 and Kir2.2 Genes Reveals the Essential Role of the Inwardly Rectifying K
+
Current in K
+
-Mediated Vasodilation
Authors: Zaritsky Joshua J. , Eckman Delrae M. , Wellman George C. , Nelson Mark T. , Schwarz Thomas L.
Published: June 2012

Abstract
—The molecular bases of inwardly rectifying K
+
(Kir) currents and K
+
-induced dilations were examined in cerebral arteries of mice that lack the Kir2.1 and Kir2.2 genes. The complete absence of the open reading frame in animals homozygous for the targeted allele was confirmed. Kir2.1
−/−
animals die 8 to 12 hours after birth, apparently due to a complete cleft of the secondary palate. In contrast, Kir2.2
−/−
animals are viable and fertile. Kir currents were observed in cerebral artery myocytes isolated from control neonatal animals but were absent in myocytes from Kir2.1
−/−
animals. Voltage-dependent K
+
currents were similar in cells from neonatal control and Kir2.1
−/−
animals. An increase in the extracellular K
+
concentration from 6 to 15 mmol/L caused Ba
2+
-sensitive dilations in pressurized cerebral arteries from control and Kir2.2 mice. In contrast, arteries from Kir2.1
−/−
animals did not dilate when the extracellular K
+
concentration was increased to 15 mmol/L. In summary, Kir2.1 gene expression in arterial smooth muscle is required for Kir currents and K
+
-induced dilations in cerebral arteries. (Circ Res. 2000;87:160-166.)
LDL Cholesterol Upregulates Synthesis of Asymmetrical Dimethylarginine in Human Endothelial Cells
Authors: Böger Rainer H. , Sydow Karsten , Borlak Jürgen , Thum Thomas , Lenzen Henrike , Schubert Bibiana , Tsikas Dimitrios , Bode-Böger Stefanie M.
Published: June 2012

Abstract
—Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginine
N
-methyltransferases (PRMTs), which utilize
S
-adenosylmethionine as methyl group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitors
S
-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished by
S
-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-
14
C label is transferred from
S
-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated by
S
-adenosylmethionine–dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 μmol/L), ADMA significantly inhibited the formation of
15
N-nitrite from
l
-[
guanidino
-
15
N
2
]arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis. (Circ Res. 2000;87:99-105.)
Alterations in the Determinants of Diastolic Suction During Pacing Tachycardia
Authors: Bell Stephen P. , Nyland Lori , Tischler Marc D. , McNabb Mark , Granzier Henk , LeWinter Martin M.
Published: June 2012

Abstract
—In cardiomyocytes, generation of restoring forces (RFs) responsible for elastic recoil involves deformation of the sarcomeric protein titin in conjunction with shortening below slack length. At the left ventricular (LV) level, recoil and filling by suction require contraction to an end-systolic volume (ESV) below equilibrium volume (Veq) as well as large-scale deformations, for example, torsion or twist. Little is known about RFs and suction in the failing ventricle. We undertook a comparison of determinants of suction in open-chest dogs previously subjected to 2 weeks of pacing tachycardia (PT) and controls. To assess the ability of the LV to contract below Veq, we used a servomotor to clamp left atrial pressure and produce nonfilling diastoles, allowing measurement of fully relaxed pressure at varying volumes. We quantified twist with sonomicrometry. We also assessed transmural ratios of N2B to N2BA titin isoforms and total titin to myosin heavy chain (MHC) protein. In PT, the LV did not contract below Veq, even with marked reduction of volume (end-diastolic pressure [EDP], 1 to 2 mm Hg), whereas in controls ESV was less than Veq when EDP was less than ≈5 mm Hg. In PT, both systolic twist and diastolic untwisting rate were reduced, and there was exaggerated transmural variation in titin isoform and titin-to-MHC ratios, consistent with the more extensible N2BA being present in larger amounts in the subendocardium. Thus, in PT, determinants of suction at the level of the LV are markedly impaired. The altered transmural titin isoform gradient is consistent with a decrease in RFs and may contribute to these findings.
Pulsatile Stretch Remodels Cell-to-Cell Communication in Cultured Myocytes
Authors: Zhuang Jianping , Yamada Kathryn A. , Saffitz Jeffrey E. , Kléber André G.
Published: June 2012

Abstract
—Mechanical stretch is thought to play an important role in remodeling atrial and ventricular myocardium and may produce substrates that promote arrhythmogenesis. In the present work, neonatal rat ventricular myocytes were cultured for 4 days as confluent monolayers on thin silicone membranes and then subjected to linear pulsatile stretch for up to 6 hours. Action potential upstrokes and propagation velocity (Θ) were measured with multisite optical recording of transmembrane voltage of the cells stained with the voltage-sensitive dye RH237. Expression of the gap junction protein connexin43 (Cx43) and the fascia adherens junction protein N-cadherin was measured immunohistochemically in the same preparations. Pulsatile stretch caused dramatic upregulation of intercellular junction proteins after only 1 hour and a further increase after 6 hours (Cx43 signal increased from 0.73 to 1.86 and 2.02% cell area, and N-cadherin signal increased from 1.21 to 2.11 and 2.74% cell area after 1 and 6 hours, respectively). This was paralleled by an increase in Θ from 27 to 35 cm/s after 1 hour and 37 cm/s after 6 hours. No significant change in the upstroke velocity of the action potential or cell size was observed. Increased Θ and protein expression were not reversible after 24 hours of relaxation. Nonpulsatile (static) stretch produced qualitatively similar but significantly smaller changes than pulsatile stretch. Thus, pulsatile linear stretch in vitro causes marked upregulation of proteins that form electrical and mechanical junctions, as well as a concomitant increase in propagation velocity. These changes may contribute to arrhythmogenesis in myocardium exposed to acute stretch.
Tie2 Receptor Expression Is Stimulated by Hypoxia and Proinflammatory Cytokines in Human Endothelial Cells
Authors: Willam Carsten , Koehne Petra , Jürgensen Jan Steffen , Gräfe Michael , Wagner Kay Dietrich , Bachmann Sebastian , Frei Ulrich , Eckardt Kai-Uwe
Published: June 2012

Abstract
—The tyrosine kinase receptor Tie2 (also known as Tek) plays an important role in the development of the embryonic vasculature and persists in adult endothelial cells (ECs). Tie2 was shown to be upregulated in tumors and skin wounds, and its ligands angiopoietin-1 and -2, although they are not directly mitogenic, modulate neovascularization. To gain further insight into the regulation of Tie2, we have studied the effect of hypoxia and inflammatory cytokines, two conditions frequently associated with neoangiogenic processes, on Tie2 expression in human ECs. Exposure to 1% O
2
led to a time-dependent significant rise of Tie2 protein levels in human coronary microvascular endothelial cells (HCMECs) and dermal microvascular ECs (HMEC-1) (3.2- and 2.5-fold within 24 hours), which was reversible after reoxygenation, and induced a less marked increase in human umbilical vein ECs (HUVECs; 1.7-fold). Hypoxia-conditioned medium and
d
-deoxyglucose did not change Tie2 expression, but desferrioxamine and cobalt, which are known to mimic hypoxia-sensing mechanisms, induced Tie2 at ambient oxygen tensions. Tumor necrosis factor-α induced Tie2 in a time- and dose-dependent fashion in all 3 EC types (HUVEC, 2.3-fold; HMEC-1, 2.8-fold; and HCMEC, 3.0-fold; 10 ng/mL, 24 hours). Enhanced expression was also found after exposure to interleukin-1β (1 ng/mL). Changes in Tie2 protein levels were paralleled by changes in mRNA expression. In accordance with these in vitro findings, immunohistochemistry revealed focal upregulation of Tie2 in capillaries at the border of infarcted human and rat myocardium. In conclusion, the data show that hypoxia and inflammatory cytokines upregulate Tie2, which may contribute to the angiogenic response in ischemic tissues.