bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of MAP Kinase In Vivo Follows Balloon Overstretch Injury of Porcine Coronary and Carotid Arteries
Authors: Pyles J. M. , March K. L. , Franklin M. , Mehdi K. , Wilensky R. L. , Adam L. P.
Published: June 2012

Abstract
Vascular restenosis involves contraction, proliferation, and remodeling of the arterial wall in response to overstretch injury. Mitogen-activated protein kinases (MAPKs) are implicated in both contraction and proliferation of vascular smooth muscle (VSM), and studies of porcine carotid arterial muscle strips have shown that mechanical stretch leads to the activation of the extracellular signal–regulated kinase (ERK) family of MAPKs in vivo. We, therefore, analyzed the acute effect of mechanical overstretch injury on ERK-MAPK (herein referred to simply as MAPK) activity in porcine coronary and carotid arteries in vivo. Balloon angioplasty catheters were inflated to 6 atm three times over 5 minutes at a balloon-artery ratio of 1.2:1 in either porcine coronary or carotid arteries. The arteries were snap-frozen after angioplasty, and MAPK activity was measured. Angioplasty of the left anterior descending (LAD, n=5), left circumflex (LCx, n=5), and carotid (n=5) arteries effected an increase in MAPK activity compared with the activity in uninstrumented right coronary arteries (RCAs) or carotid arteries from the same animals used for controls. Balloon angioplasty of carotid arteries led to an increase in MAPK activity that was 7.7-fold over the activity in control arteries and comparable to the activity in stretched carotid arterial muscle strips in vivo. The increase in coronary artery kinase activity on angioplasty was variable from animal to animal. The increase in MAPK activity over that in control arteries ranged from 4.5- to 31.7-fold (mean±SEM, 10.7±5.3) in the LAD and 1.8- to 31.3-fold (mean±SEM, 9.7±5.7) in the LCx. There were no apparent inherent differences in the levels of MAPK activity in the three different types of coronary arteries (RCA, LAD, and LCx) without instrumentation. MAPK activation occurs rapidly during angioplasty, suggesting that this kinase may play an early role in initiating the injury response in both porcine coronary and carotid arteries. MAPKs may be key enzymes targeted to treat or prevent restenosis.
Angiotensin II Induces Apoptosis of Human Endothelial Cells
Authors: Dimmeler Stefanie , Rippmann Volker , Weiland Ulrike , Haendeler Judith , Zeiher Andreas M.
Published: June 2012

Abstract
Angiotensin II (Ang II) importantly contributes to the pathobiology of atherosclerosis. Since endothelial injury is a key event early in the pathogenesis of atherosclerosis, we tested the hypothesis that Ang II may injure endothelial cells by activation of cellular suicide pathways leading to apoptosis. Human umbilical venous endothelial cells (HUVECs) were incubated with increasing doses of Ang II for 18 hours. Apoptosis of HUVECs was measured by ELISA specific for histone-associated DNA fragments and confirmed by DNA laddering and nuclear staining. Ang II dose-dependently induced apoptosis of HUVECs. Simultaneous blockade of both the AT
1
and AT
2
receptor prevented Ang II–induced apoptosis, whereas each individual receptor blocker alone was not effective. Selective agonistic stimulation of the AT
2
receptor also dose-dependently induced apoptosis. Ang II–mediated as well as selective AT
2
receptor stimulation–mediated apoptosis was associated with the activation of caspase-3, a central downstream effector of the caspase cascade executing the cell death program. Specific inhibition of caspase-3 activity abrogated Ang II–induced apoptosis. In addition, the NO donors sodium nitroprusside and
S
-nitrosopenicillamine completely inhibited Ang II–induced apoptosis and eliminated caspase-3 activity. Thus, Ang II induces apoptosis of HUVECs via activation of the caspase cascade, the central downstream effector arm executing the cell death program. NO completely abrogated Ang II–induced apoptosis by interfering with the activation of the caspase cascade.
Local Angiotensin II Generation in the Rat Heart
Authors: Müller Dominik N. , Fischli Walter , Clozel Jean-Paul , Hilgers Karl F. , Bohlender Jürgen , Ménard Joël , Busjahn Andreas , Ganten Detlev , Luft Friedrich C.
Published: June 2012

Abstract
—To elucidate the local effects of renin in the coronary circulation, we examined local angiotensin (Ang) I and II formation, as well as coronary vasoconstriction in response to renin administration, and compared the effects with exogenous infused Ang I. We perfused isolated hearts from rats overexpressing the human angiotensinogen gene in a Langendorff preparation and measured the hemodynamic effects and the released products. We also investigated cardiac Ang I conversion, including the contribution of non-angiotensin-converting enzyme–dependent Ang II–generating pathways. Finally, we studied Ang I conversion in vitro in heart homogenates. Renin and Ang I infusion both generated Ang II. Ang II release and vasoconstriction continued after renin infusion was stopped, even though renin disappeared immediately from the perfusate. In contrast, after Ang I infusion, Ang II release and coronary flow returned to basal levels. Ang I conversion (Ang II/Ang I ratio) was higher after renin infusion (0.109±0.027 versus 0.026±0.003, 15 minutes,
P
<.02) compared with infused Ang I. Remikiren added to the renin infusion abolished Ang I and II; captopril suppressed only Ang II, whereas an AT
1
receptor blocker did not affect Ang I and II formation. All the drugs prevented renin-induced coronary flow changes. Total cardiac Ang II–forming activity was only partially inhibited by cilazaprilat (4.1±0.1 fmol · min
−1
· mg
−1
) and on a larger extent by chymostatin (2.6±0.3 fmol · min
−1
· mg
−1
) compared with control values (5.6±0.4 fmol · min
−1
· mg
−1
). We conclude that renin can be taken up by cardiac or coronary vascular tissue and induces long-lasting local Ang II generation and vasoconstriction. Locally formed Ang I was converted more effectively than infused Ang I. Furthermore, the comparison of in vivo and in vitro Ang I conversion suggests that in vitro assays may underestimate the functional contribution of angiotensin-converting enzyme to intracardiac Ang II formation.
Role of Primary and Secondary Capture for Leukocyte Accumulation In Vivo
Authors: Kunkel Eric J. , Chomas James E. , Ley Klaus
Published: June 2012

Abstract
—Leukocyte accumulation during inflammation depends on the concerted action of selectin and integrin adhesion molecules, which promote capture, rolling, and arrest of these cells on activated endothelium. In addition to interacting with endothelial cells, leukocytes can also adhere to already adherent leukocytes through an L-selectin–dependent mechanism. Initiation of adhesion through this mechanism has been called nucleation and leads to characteristic geometric patterns (ie, clusters and strings) of adherent leukocytes in flow chambers. We have used intravital microscopy of tumor necrosis factor-α (TNF-α)–treated mouse cremaster muscles to quantitatively investigate the potential role of leukocyte-leukocyte adhesion in initiating and maintaining the leukocyte clusters that are commonly observed in inflamed venules. Our data show that in TNF-α–treated venules with diameters between 23 and 108 μm, leukocyte adhesion occurs in clusters that are 19 to 50 μm long and 8 to 44 μm wide. They are almost entirely made up of slow-rolling leukocytes. Of all leukocytes recruited into a cluster (100%), the majority enter the cluster rolling along the endothelium and sharply reduce their velocity in the absence (59%) or presence (15%) of other leukocytes in proximity (one cell diameter). Some of the rolling leukocytes (17%) pass through the cluster without reducing their velocity. Recruitment of leukocytes from the free flow regime into a cluster is a rare event and accounts for only 7 (1.2%) of 476 leukocytes arriving in the cluster. However, of the leukocytes captured from the free flow, 6 initiated contact with a slow-rolling leukocyte rather than making direct contact with the endothelium. Our data show that leukocyte-leukocyte interactions can occur in vivo but are not important for cluster formation. This is confirmed by the observation of normal cluster formation in L-selectin–deficient mice, in which leukocyte-leukocyte interactions under flow are abolished. We conclude that leukocyte-mediated nucleation contributes little to leukocyte recruitment during inflammation in vivo. Cluster formation appears to be dominated by areas of endothelium with a higher expression of E-selectin, because cluster formation is greatly reduced in E-selectin–deficient mice.
Hydrogen Peroxide Activates Mitogen-Activated Protein Kinases and Na
+
-H
+
Exchange in Neonatal Rat Cardiac Myocytes
Authors: Sabri Abdelkarim , Byron Kenneth L. , Samarel Allen M. , Bell Jeremy , Lucchesi Pamela A.
Published: June 2012

Abstract
—Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na
+
-H
+
exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 μmol/L hydrogen peroxide (H
2
O
2
) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H
2
O
2
(100 μmol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H
2
O
2
. Pretreatment with chelerythrine (10 μmol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 μmol/L, 45 minutes) or herbimycin A (3 μmol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H
2
O
2
-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H
2
O
2
also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 μmol/L, 60 minutes). These results suggest that low doses of H
2
O
2
induce MAP kinase–dependent pathways that regulate NHE activity during reperfusion injury.
Angiotensin II Stimulation In Vitro Induces Hypertrophy of Normal and Postinfarcted Ventricular Myocytes
Authors: Liu Yu , Leri Annarosa , Li Baosheng , Wang Xiaowei , Cheng Wei , Kajstura Jan , Anversa Piero
Published: June 2012

Abstract
—To determine whether angiotensin II (Ang II) stimulation of adult ventricular myocytes in vitro results in cellular hypertrophy, the changes in myocyte volume and protein content per cell were examined by confocal microscopy. Moreover, the possibility was considered that the upregulation of Ang II receptors on myocytes after infarction may potentiate and/or accelerate Ang II–mediated myocyte growth. Left ventricular myocytes isolated from control and failing hearts 3 days after infarction were cultured for 3 and 7 days in the presence of Ang II. Normal myocytes did not show an increase in volume and protein content at 3 days, but a 16% and 20% increase in these respective parameters was found at 7 days. Cell growth was faster and greater in myocytes from postinfarcted hearts. In these cells, myocyte volume increased 23% and protein content increased 28% at 3 days after Ang II administration. The higher hypertrophic reaction of myocytes from infarcted hearts occurred in spite of a 19% larger volume at isolation. In both groups of myocytes, the AT
1
receptor blocker losartan completely inhibited the consequences of Ang II. Conversely, the AT
2
receptor antagonist PD123319 had no effect on Ang II–induced hypertrophy. In conclusion, Ang II promotes myocyte growth through the activation of AT
1
receptors, which modulate the time and magnitude of this cellular response.
Phosphorylation of p130
Cas
by Angiotensin II Is Dependent on c-Src, Intracellular Ca
2+
, and Protein Kinase C
Authors: Sayeski Peter P. , Ali M. Showkat , Harp Joyce B. , Marrero Mario B. , Bernstein Kenneth E.
Published: June 2012

Abstract
—p130
Cas
is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130
Cas
tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130
Cas
tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca
2+
chelator BAPTA-AM but not the Ca
2+
channel blocker verapamil. The Ang II–induced tyrosine phosphorylation of p130
Cas
was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130
Cas
to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Cα and the cell adhesion signaling molecule pp120 formed temporal associations with p130
Cas
in response to Ang II. c-Src was found to associate with p130
Cas
in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II–induced tyrosine phosphorylation of p130
Cas
. These results are the first to demonstrate that the tyrosine phosphorylation of p130
Cas
by Ang II is transduced by the Src, intracellular Ca
2+
, protein kinase C signaling pathway.
Overexpression of Human Superoxide Dismutase Inhibits Oxidation of Low-Density Lipoprotein by Endothelial Cells
Authors: Fang Xiang , Weintraub Neal L. , Rios C. David , Chappell David A. , Zwacka Ralf M. , Engelhardt John F. , Oberley Larry W. , Yan Tao , Heistad Donald D. , Spector Arthur A.
Published: June 2012

Abstract
—Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O
2
.−
) and oxidize LDL in vitro; however, the role of O
2
.−
in endothelial cell–induced LDL oxidation is unclear. Incubation of human LDL (200 μg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5±1.1 versus 6.3±0.2 [mean±SEM] nmol malondialdehyde/mg LDL protein, respectively;
P
<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 μg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O
2
.−
release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O
2
.−
contributes to endothelial cell–induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.
Connexin45 Expression Is Preferentially Associated With the Ventricular Conduction System in Mouse and Rat Heart
Authors: Coppen Steven R. , Dupont Emmanuel , Rothery Stephen , Severs Nicholas J.
Published: June 2012

Abstract
—Cardiac myocytes are electrically coupled by gap junctions, clusters of low-resistance intercellular channels composed of connexins. Variations in the quantities and spatial distribution of different connexin types have been implicated in regional differentiation of electrophysiological properties in the heart. Although independent studies have demonstrated that connexin43 is abundant in working ventricular myocardium and that connexin40 is preferentially expressed in the atrioventricular conduction system of a number of species, information on the spatial distribution of connexin45 in the heart is limited to data obtained using an antibody raised to a single peptide sequence. In the present study, we report on the production and characterization of a new anti-connexin45 antibody and its application to the investigation of connexin45 expression in mouse and rat myocardium. The affinity-purified antiserum, raised in guinea pig to residues 354 to 367 of human connexin45, recognized a single 45-kD band on Western blots of HeLa cells transfected to express connexin45 and gave punctate immunolabeling at the cell borders, demonstrated by freeze-fracture cytochemistry to represent gap junctions. Only low levels of connexin45 mRNA were detected on Northern blots of mouse and rat cardiac tissues, and connexin45 protein levels were below the limit of detection on Western blots. Confocal microscopy of immunolabeled ventricular tissue revealed that the major part of the working myocardium was immunonegative for connexin45. A clearly defined zone containing connexin45-expressing cells was, however, localized to the endocardial surface, overlapping with connexin40-expressing myocytes of the conduction system. As these results contrast with the prevailing view that connexin45 is widely distributed in working ventricular myocytes, we compared the immunolabeling pattern obtained with a commercially supplied anti-connexin45 antiserum raised against the same peptide that was used in previous studies. The commercial connexin45 antiserum gave widespread labeling throughout the ventricular myocardium, but this labeling was inhibited by a six–amino acid peptide matching part of the connexin43 sequence, indicating cross-reaction of the commercial connexin45 antiserum with connexin43 in the tissue. Further evidence for such cross-reactivity came from observations on connexin43-transfected cells, which gave positive immunolabeling with the commercial anti-connexin45 antiserum. Our demonstration of a specific association of connexin45 with connexin40-expressing myocytes in rat and mouse ventricle raises the possibility that connexin45 contributes to the modulation of electrophysiological properties in the ventricular conduction system and highlights the need for reappraisal of the distribution and role of connexin45 in other species.
Biphasic Activation of the JAK/STAT Pathway by Angiotensin II in Rat Cardiomyocytes
Authors: Kodama Hiroaki , Fukuda Keiichi , Pan Jing , Makino Shinji , Sano Motoaki , Takahashi Toshiyuki , Hori Shingo , Ogawa Satoshi
Published: June 2012

Abstract
—This study was designed to demonstrate the characteristic pattern of angiotensin II–induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. Angiotensin II (10
−7
mol/
l
) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the
sis
-inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type-1 (AT
1
) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II–induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation.
Transcriptional Activation of the p34
cdc2
Gene by cdc2 Promoter Binding Factor/Nuclear Factor-Y in Fetal Rat Ventricular Myocytes
Authors: Liu Qingquan , Yan Huajun , Dawes Nicola J. , Lu Yujuan , Zhu Hong
Published: June 2012

Abstract
—To determine how myocardial terminal differentiation is regulated by cell cycle control genes, we studied cdc2 expression in rat cardiac muscle and found that cdc2 mRNA and protein levels were reduced in neonatal compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression assay was performed using the progressively truncated 6.2-, 1.8-, 1.1-, 0.7-, and 0.1-kb human cdc2 5′ flanking regions. All five fragments activated reporter expression in fetal myocytes and were significantly less active in neonatal myocytes. The 0.1-kb fragment showed 65% of the activity of the 6.2-kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1-kb fragment by DNase I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1- and 6.2-kb promoter activities. Competition and antibody supershift assays suggested that CPBF was identical or related to the transcription factor, nuclear factor Y (NF-Y). The 0.1-kb promoter activity was suppressed by a dominant-negative NF-Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off when the juvenile stage is attained. A 0.1-kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation and suppression in fetal and neonatal myocytes, respectively. NF-Y or its related factor plays a critical role in activating the 0.1-kb cdc2 promoter.
Characteristics of the Temporal and Spatial Excitable Gap in Anisotropic Reentrant Circuits Causing Sustained Ventricular Tachycardia
Authors: Peters Nicholas S. , Coromilas James , Hanna Michael S. , Josephson Mark E. , Costeas Constantinos , Wit Andrew L.
Published: June 2012

Abstract
—The excitable gap of a reentrant circuit has both temporal (time during the cycle length that the circuit is excitable) and spatial (length of the circuit that is excitable at a given time) properties. We determined the temporal and spatial properties of the excitable gap in reentrant circuits caused by nonuniform anisotropy. Myocardial infarction was produced in canine hearts by ligation of the left anterior descending coronary artery. Four days later, reentrant circuits were mapped in the epicardial border zone of the infarcts with a multielectrode array during sustained ventricular tachycardia induced by programmed stimulation. During tachycardia, premature impulses were initiated by stimulation at sites around and in the reentrant circuits, and their conduction characteristics in the circuit were mapped. All circuits had a temporal excitable gap in at least part of the circuit, which allowed premature impulses to enter the circuit. Completely and partially excitable segments of the temporal gap were identified by measuring conduction velocity of the premature impulses; conduction was equal to the native reentrant wave front in completely excitable regions and slower than the reentrant wave front in partially excitable regions. In some circuits, a temporal gap existed throughout the circuit, permitting the entire circuit to be reset over a range of premature coupling intervals, although the size of the gap varied at different sites. In other circuits, the gap became so small at local sites that even though premature impulses could enter the circuit, the circuit could not be reset. Premature impulses could terminate reentry in circuits that could be reset or not. We also found a significant spatial gap, which was identified by determining the distance between the head of the circulating wave front, which could be located on the activation map, and its tail, which was the site most distal from the head as located by the site of entry of the premature wave front into the circuit. The spatial gap could also vary in different parts of the circuit. Therefore, nonuniform anisotropic reentrant circuits have both a temporal and spatial excitable gap with fully and partially excitable components that change in different parts of the circuit.
Normal Development of the Outflow Tract in the Rat
Authors: Ya Jing , van den Hoff Maurice J. B. , de Boer Piet A. J. , Tesink-Taekema Sabina , Franco Diego , Moorman Antoon F. M. , Lamers Wouter H.
Published: June 2012

Abstract
—The outflow tract (OFT) provides the structural components forming the ventriculoarterial connection. The prevailing concept that this junction “rotates” to acquire its definitive topography also requires a concept of “counterrotation” and is difficult to reconcile with cell-marking studies. Rats between 10 embryonic days (EDs) and 2 postnatal days were stained immunohistochemically and by in situ hybridization. DNA replication was determined by incorporation of bromodeoxyuridine and apoptosis by the annexin V binding and terminal deoxynucleotidyl transferase–mediated dUTP-X nick end labeling (TUNEL) assays. Starting at ED12, cardiomyocytes in the distal (truncal) part of the OFT begin to shed their myocardial phenotype without proceeding into apoptosis, suggesting transdifferentiation. Myocardial regression is most pronounced on the dextroposterior side and continues until after birth, as revealed by the disappearance of the myocardial cuff surrounding the coronary roots and semilunar sinuses and by the establishment of fibrous continuity between mitral and aortic semilunar valves. Fusion of the endocardial ridges of the truncus on late ED13 is accompanied by the organization of α-smooth muscle actin–and nonmuscle myosin heavy chain–positive myofibroblasts into a central whorl and the appearance of the semilunar valve anlagen at their definitive topographical position within the proximal portion of the truncus. After fusion of the proximal (conal) portion of the endocardial ridges, many of the resident myofibroblasts undergo apoptosis and are replaced by cardiomyocytes. The distal myocardial boundary of the OFT is not a stable landmark but moves proximally over the spiraling course of the aortic and pulmonary routes, so that the semilunar valves develop at their definitive topographic position. After septation, the distal boundary of the OFT continues to regress, particularly in its subaortic portion. The myocardializing conus septum, on the other hand, becomes largely incorporated into the right ventricle. These opposite developments account for the pronounced asymmetry of the subaortic and subpulmonary outlets in the formed heart.
Lactosylceramide Stimulates Human Neutrophils to Upregulate Mac-1, Adhere to Endothelium, and Generate Reactive Oxygen Metabolites In Vitro
Authors: Arai Toshiyuki , Bhunia Anil Kumar , Chatterjee Subroto , Bulkley Gregory B.
Published: June 2012

Abstract
—Glycosphingolipids (GSLs) and their metabolites play important roles in a variety of biological processes. We have previously reported that lactosylceramide (LacCer), a ubiquitous GSL, stimulates NADPH oxidase–dependent superoxide generation by aortic smooth muscle cells and their consequent proliferation. We postulated that LacCer may upregulate adhesion molecules on human polymorphonuclear leukocytes (hPMNs), perhaps also via NADPH oxidase–dependent reactive oxygen metabolite (ROM) generation. Incubation of hPMNs with LacCer upregulated CD11b/CD18 (Mac-1) and CD11c/CD18, as determined by fluorescence-automated cell sorting. LacCer also stimulated these hPMNs to generate superoxide via NADPH oxidase, as determined by lucigenin-enhanced chemiluminescence. However, the upregulation of Mac-1 by LacCer did not itself appear to be mediated by ROMs, since neither an antioxidant nor an NADPH oxidase inhibitor substantially inhibited the Mac-1 upregulation. However, this Mac-1 upregulation was significantly inhibited by two disparate phospholipase A
2
(PLA
2
) inhibitors. Moreover, LacCer induced arachidonic acid metabolism, which was inhibited by the PLA
2
inhibitors, but not by an NADPH oxidase inhibitor. To evaluate the effect of LacCer on hPMN adhesion to endothelium, hPMNs stimulated with LacCer were allowed to adhere to unstimulated human endothelial cell monolayers. LacCer stimulated hPMN adhesion to endothelial cells, which was blocked by anti-CD18 and by the PLA
2
inhibitors. We conclude that LacCer stimulates both Mac-1 upregulation and superoxide generation in hPMNs but that ROMs are not the upstream signal for Mac-1 upregulation. This mechanism may well be relevant to acute endothelial injury in inflammation and other pathological conditions.
Evolutionarily Conserved Promoter Region Containing CArG*-Like Elements Is Crucial for Smooth Muscle Myosin Heavy Chain Gene Expression
Authors: Zilberman A. , Dave V. , Miano J. , Olson E. N. , Periasamy M.
Published: June 2012

Abstract
—In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle–specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5′ flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between −1208 and −1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between −967 and −1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1–like element, an Nkx2–5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac α-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.
Effects of Constitutive Overexpression of Insulin-Like Growth Factor-1 on the Mechanical Characteristics and Molecular Properties of Ventricular Myocytes
Authors: Redaelli Giorgio , Malhotra Ashwani , Li Baosheng , Li Peng , Sonnenblick Edmund H. , Hofmann Polly A. , Anversa Piero
Published: June 2012

Abstract
—Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat α-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca
2+
sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca
2+
levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca
2+
sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V
1
in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.
Effects of Overexpression of the Na
+
-Ca
2+
Exchanger on [Ca
2+
]
i
Transients in Murine Ventricular Myocytes
Authors: Yao Atsushi , Su Zhi , Nonaka Akihiko , Zubair Iram , Lu Liyan , Philipson Kenneth D. , Bridge John H. B. , Barry William H.
Published: June 2012

Abstract
—We measured [Ca
2+
]
i
and [Na
+
]
i
in isolated transgenic (TG) mouse myocytes overexpressing the Na
+
-Ca
2+
exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca
2+
]
i
transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca
2+
]
i
was significantly prolonged. The decline of ES [Ca
2+
]
i
transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca
2+
]
i
transient was markedly faster in TG myocytes, and [Na
+
]
i
was identical in TG and WT myocytes, indicating that the overexpressed Na
+
-Ca
2+
exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca
2+
]
i
transient in 0 Na
+
/0 Ca
2+
solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca
2+
-ATPase function is not altered by overexpression of the Na
+
-Ca
2+
exchanger. There was no difference in L-type Ca
2+
current density in WT and TG myocytes. However, the sensitivity of ES [Ca
2+
]
i
transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca
2+
]
i
transients in nifedipine was inhibited by Ni
2+
and required SR Ca
2+
content, consistent with enhanced Ca
2+
influx by reverse Na
+
-Ca
2+
exchange, and the resulting Ca
2+
-induced Ca
2+
release from SR. The rate of rise of [Ca
2+
]
i
transients in nifedipine in TG myocytes was much slower than when both the L-type Ca
2+
current and the Na
+
-Ca
2+
exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na
+
-Ca
2+
exchanger in TG myocytes accelerates the decline of [Ca
2+
]
i
during relaxation, indicating enhanced forward Na
+
-Ca
2+
exchanger function. Increased Ca
2+
influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na
+
-Ca
2+
exchange.
Cell Replication in the Arterial Wall
Authors: Koyama Hiroyuki , Olson N. Eric , Dastvan F. Frank , Reidy Michael A.
Published: June 2012

Abstract
—This study examined intracellular signal events of arterial cells following balloon catheter injury to rat carotid artery. Within 30 minutes, a marked increase in extracellular signal–regulated kinase-1/2 (ERK1/2) activity was observed. This activity remained elevated for 12 hours but had decreased to control levels by day 1. No increase in ERK1/2 was detected at any later times. Injection of anti–fibroblast growth factor 2 antibody (60 mg IV) significantly inhibited the activation of ERK1/2 at 30 minutes after the injury. PD98059 (80 μmol/L), a selective inhibitor of mitogen-activated protein kinase/ERK kinase-1 (MEK1), decreased ERK1/2 activity in injured arteries and also reduced the medial cell replication. In contrast, PD98059 did not block the intimal cell replication at day 8. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was expressed within hours after injury but only weakly at later times; MKP-1 was again expressed after 7 and 14 days. The expression of MKP-1 was associated with an activation of c-Jun amino-terminal kinase. Injury to the arterial wall also stimulated the activity of p70 S6 kinase from 30 minutes to 12 hours, suggesting an alternative pathway in mitogenic signaling of early cell replication. These findings demonstrate that fibroblast growth factor 2–induced ERK1/2 activation promotes medial cell replication after balloon injury; however, signaling of intimal cell replication may not be linked to the MEK1-dependent ERK pathway.
Cytoskeletal Role in the Transition From Compensated to Decompensated Hypertrophy During Adult Canine Left Ventricular Pressure Overloading
Authors: Tagawa Hirofumi , Koide Masaaki , Sato Hiroshi , Zile Michael R. , Carabello Blase A. , Cooper George
Published: June 2012

Abstract
—Increased microtubule density causes cardiocyte contractile dysfunction in right ventricular (RV) pressure-overload hypertrophy, and these linked phenotypic and contractile abnormalities persist and progress during the transition to failure. Although more severe in cells from failing than hypertrophied RVs, the mechanical defects are normalized in each case by microtubule depolymerization. To define the role of increased microtubule density in left ventricular (LV) pressure-overload hypertrophy and failure, in a given LV we examined ventricular mechanics, sarcomere mechanics, and free tubulin and microtubule levels in control dogs and in dogs with aortic stenosis both with LV hypertrophy alone and with initially compensated hypertrophy that had progressed to LV muscle failure. In comparing initial values with those at study 8 weeks later, dogs with hypertrophy alone had a very substantial increase in LV mass but preservation of a normal ejection fraction and mean systolic wall stress. Dogs with hypertrophy and associated failure had a substantial but lesser increase in LV mass and a reduction in ejection fraction, as well as a marked increase in mean systolic wall stress. Cardiocyte contractile function was equivalent, and unaffected by microtubule depolymerization, in cells from control LVs and those with compensated hypertrophy. In contrast, cardiocyte contractile function in cells from failing LVs was quite depressed but was normalized by microtubule depolymerization. Microtubules were increased only in failing LVs. These contractile and cytoskeletal changes, when assayed longitudinally in a given dog by biopsy, appeared in failing ventricles only when wall stress began to increase and function began to decrease. Thus, the microtubule-based cardiocyte contractile dysfunction characteristic of pressure-hypertrophied myocardium, originally described in the RV, obtains equally in the LV but is shown here to have a specific association with increased wall stress.
Acute Pressure Overload Could Induce Hypertrophic Responses in the Heart of Angiotensin II Type 1a Knockout Mice
Authors: Harada Koichiro , Komuro Issei , Zou Yunzeng , Kudoh Sumiyo , Kijima Kazuhisa , Matsubara Hiroaki , Sugaya Takeshi , Murakami Kazuo , Yazaki Yoshio
Published: June 2012

Abstract
—Increasing evidence has suggested that locally produced angiotensin II (Ang II) plays an important role in the development of cardiac hypertrophy through the Ang II type 1 receptor (AT1). We and others have recently reported that Ang II is critical for mechanical stress–induced hypertrophic responses in vitro. Using AT1a knockout (KO) mice, we examined whether Ang II is indispensable for pressure overload–induced cardiac hypertrophy in the present study. Reverse-transcriptase polymerase chain reaction analysis revealed that AT1 mRNA levels were <10% in the heart of KO mice compared with wild-type (WT) mice, but the Ang II type 2 receptor gene was expressed at almost the same levels in the hearts of both mice. Intravenous infusion of subpressor dose of Ang II induced c-
fos
gene expression in the hearts of WT mice but not KO mice. Acute pressure overload, however, induced expressions of immediate-early response genes and activations of mitogen-activated protein kinases in the hearts of KO mice as well as WT mice. Both basal and activated levels of all these responses were significantly higher in KO mice than in WT mice. Pressure overload markedly increased the heart weight–to–body weight ratio in both mice strains at 14 days after aortic banding. These results suggest that acute hypertrophic responses could be induced by pressure overload in the in vivo heart without AT1 signaling.
Overexpression of Human Endothelial Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells and in Balloon-Injured Carotid Artery
Authors: Chen Lihua , Daum Günter , Forough Reza , Clowes Monika , Walter Ulrich , Clowes Alexander W.
Published: June 2012

Abstract
—Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium-specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus-mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS-expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs.
N
ω
-Nitro-
l
-arginine (L-NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon-injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3-fold increase in vessel diameter) at 2 weeks compared with LXSN SMC–seeded arteries. Orally administered L-NA blocked these changes. Phosphorylation of vasodilator-stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC–seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo.
Smooth Muscle–Specific Expression of the Smooth Muscle Myosin Heavy Chain Gene in Transgenic Mice Requires 5′-Flanking and First Intronic DNA Sequence
Authors: Madsen Cort S. , Regan Christopher P. , Hungerford Jill E. , White Sheryl L. , Manabe Ichiro , Owens Gary K.
Published: June 2012

Abstract
—The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle–specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-
lac
Z reporter construct containing ≈16 kb of the SM-MHC genomic region from −4.2 to +11.6 kb (within the first intron) expressed the
lac
Z transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5′-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.
A Particle-Receptor Model for the Insulin-Induced Closure of Connexin43 Channels
Authors: Homma Nobuo , Alvarado José Luis , Coombs Wanda , Stergiopoulos Kathleen , Taffet Steven M. , Lau Alan F. , Delmar Mario
Published: June 2012

Abstract
—Connexin43(Cx43) channels can be regulated by a variety of factors, including low pH
i
. Structure/function studies from this laboratory have demonstrated that pH gating follows a particle-receptor mechanism, similar to the “ball-and-chain” model of voltage-dependent inactivation of ion channels. The question whether the particle-receptor model is applicable only to pH gating or to other forms of Cx43 regulation as well remains. To address this question, we looked at the uncoupling effects of insulin and of insulin-like growth factor-1 (IGF) on Cx43 channels expressed in
Xenopus
oocytes. These agonists do not induce changes in pH
i
. Junctional conductance (Gj) was measured by the dual 2-electrode voltage-clamp technique. Control studies showed that relative Gj did not change spontaneously as a function of time. Continuous exposure of Cx43-expressing oocytes to insulin (10 μ/L) led to a decrease in Gj. After 80 minutes, Gj was 54±5% from control (n=12). Exposure of oocytes to IGF (10 nmol/L) caused an even more pronounced change in Gj (37±4% of control, n=6). The time course of the IGF-induced uncoupling was similar to that observed after insulin exposure. The effect of insulin was abolished by truncation of the carboxyl-terminal domain of Cx43 at amino acid 257 (M257). Interestingly, as in the case of pH gating, coexpression of the carboxyl-terminal domain (amino acids 258 to 282) together with M257 rescued the ability of insulin to reduce coupling (Gj, 39±12% from control; n=6). Structure/function experiments using various deletion mutants of the carboxyl-terminal domain showed that insulin treatment does not modify Gj if amino acids 261 to 280 are missing from the Cx43 sequence. Our results suggest that a particle-receptor (or ball-and-chain) mechanism, similar to that described for pH gating, also applies to chemical regulation of Cx43 by other factors.