bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined Inhibition of Endothelin and Angiotensin II Receptors Blocks Volume Load–Induced Cardiac Hormone Release
Authors: Leskinen Hanna , Vuolteenaho Olli , Ruskoaho Heikki
Published: June 2012

Volume expansion has been shown to increase plasma atrial natriuretic peptide (ANP) levels, but the precise role of paracrine and autocrine factors in stretch-induced cardiac hormone release is not clear. In the present study, we report the effects of endothelin (ET) and angiotensin receptor (AT receptor) antagonists on baseline and atrial stretch–induced immunoreactive ANP (IR-ANP) and immunoreactive N-terminal ANP (IR-NT-ANP) release in vivo by using BQ-123 (ET
A
receptor antagonist), bosentan (ET
A
and ET
B
receptor antagonist), and losartan (AT
1
receptor antagonist). Intravenous administration of BQ-123 had no significant effect on baseline hemodynamics in conscious rats, whereas bosentan (10 mg/kg) and losartan (10 mg/kg) decreased slightly (4 to 7 mm Hg,
P
<.05 to .001) the mean arterial pressure. Both the ET
A
receptor antagonist BQ-123 and ET
A
/ET
B
receptor antagonist bosentan decreased plasma ANP and NT-ANP responses to volume load (
P
<.05 to .001), whereas the AT
1
receptor antagonist losartan had no significant effect on this response. The relative increase in plasma IR-ANP corresponding to a 3 mm Hg increase in right atrial pressure was 2.7-fold in the vehicle-treated group. BQ-123 (0.3 and 1.0 mg/kg) decreased this response 2.5- and 2.1-fold (
P
<.05); bosentan (3 and 10 mg/kg), 1.7-fold (
P
<.001) and 1.9-fold (
P
<.05); and bosentan (10 mg/kg)+losartan (10 mg/kg), 1.6-fold (
P
<.001). The responses in plasma IR-NT-ANP decreased simultaneously. These results indicate that combined inhibition of ET
A/B
and AT
1
receptors almost completely blocks ANP response to acute volume load. Therefore, our study shows that endogenous paracrine and/or autocrine factors liberated in response to atrial wall stretch rather than myocyte stretch itself are responsible for the activation of ANP peptide secretion in response to acute volume load. Our results also show that ET
A
receptors are more important in the regulation of mechanical stretch–induced changes in cardiac hormone secretion than AT
1
receptors.
The Effects of Hypoxia on pH
i
in Porcine Coronary Artery Endothelium and Smooth Muscle
Authors: Foy Robyn A. , Shimizu Shunichi , Paul Richard J.
Published: June 2012

Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pH
i
may modulate the release of the endothelium-derived relaxing factor. We tested the hypothesis that hypoxia-induced attenuation of endothelium-dependent relaxation is mediated by alterations in pH
i
. We developed a novel method for loading surface cells, whereby endothelial cell pH
i
could be measured in situ on the intact porcine coronary artery. Endothelial cells of arterial ring segments were selectively loaded with the fluorescent indicator BCECF-AM. Differential loading of the endothelial cell layer was verified by confocal microscopy. pH
i
of the endothelial cells in situ and of endothelium-denuded arteries was measured with a Photon Technology International spectrofluorimeter. The functional integrity of the endothelium was assessed by the endothelium-dependent relaxation to substance P in a paired adjacent ring. In the experimental protocol for pH
i
measurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCl (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pH
i
in endothelial cells on the intact six arteries was 6.92±0.07. Addition of KCl to the perfusion medium decreased (
P
=.025) pH
i
to 6.79±0.07. Subsequent bubbling with N
2
increased (
P
=.009) pH
i
to 7.00±0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pH
i
of the smooth muscle was not significantly altered from its basal value of 7.24±0.06 (n=5) by either KCl or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pH
i
may be an important mechanism of signal transduction in endothelial cells.
Dissociated Spatial Patterning of Gap Junctions and Cell Adhesion Junctions During Postnatal Differentiation of Ventricular Myocardium
Authors: Angst Brigitt D. , Khan Laeeq U.R. , Severs Nicholas J. , Whitely Kate , Rothery Stephen , Thompson Robert P. , Magee Anthony I. , Gourdie Robert G.
Published: June 2012

Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction–rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (
P
<.01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (
P
<.05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (
P
>.05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.
Na
+
-H
+
Exchange Inhibition Protects Against Mechanical, Ultrastructural, and Biochemical Impairment Induced by Low Concentrations of Lysophosphatidylcholine in Isolated Rat Hearts
Authors: Hoque A.N. Ehsanul , Haist James V. , Karmazyn Morris
Published: June 2012

Lysophophatidylcholine (LysoPC) accumulates rapidly in the ischemic myocardium and is an important mediator of ischemia-induced cell injury. Na
+
-H
+
exchange (NHE) inhibition has been demonstrated to protect the ischemic and reperfused myocardium. We determined whether NHE inhibition can also modulate cardiotoxicity produced by LysoPC (3 and 5 μmol/L) in isolated rat hearts. At 3 μmol/L, LysoPC produced a depression in left ventricular developed pressure (LVDP) and elevation in left ventricular end-diastolic pressure (LVEDP), which were 19±7% and 1290±205% of pre-LysoPC values, respectively, after 30 minutes of treatment. In the presence of the NHE inhibitor 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642, 5 μmol/L), LVDP was reduced to only 80.8±8.6%, and LVEDP increased to 270±32% (
P
<.05 for both parameters). LysoPC significantly depressed tissue ATP, creatine phosphate, and glycogen contents and increased lactate levels, all of which were significantly attenuated by HOE 642. Moreover, marked LysoPC-induced ultrastructural abnormalities, including mitochondrial and myofibrillar disruption, were totally prevented by HOE 642. This protection was mimicked by another NHE inhibitor, methylisobutylamiloride (5 μmol/L). HOE 642 was also effective against injury produced by 5 μmol/L LysoPC although, generally, the protection was less marked than that observed against 3 μmol/L; LVDP depression after 30 minutes was 10.1±4.3% and 41.4±10.4% of pre-LysoPC values in control and HOE 642-treated hearts, respectively (
P
<.05), whereas corresponding LVEDP elevations were 1629±393% and 990±144% (
P
>.05). In myocytes superfused with bicarbonate-free buffer subjected to acid loading by NH
4
Cl pulsing, pH recovery (as measured by acid flux) was significantly stimulated by 3 μmol/L LysoPC, indicative of NHE activation. Our study shows that cardiac injury produced by low concentrations of LysoPC can be effectively attenuated by NHE inhibition. The results also suggest that the beneficial effects of NHE inhibitors on the ischemic myocardium may be, at least partially, mediated by inhibiting the deleterious effects of LysoPC.
Role of Ca
2+
in Metabolic Inhibition–Induced Norepinephrine Release in Rat Brain Synaptosomes
Authors: Du Xiao-Jun , Bobik Alex , Little Peter J. , Esler Murray D. , Dart Anthony M.
Published: June 2012

Ischemia and simulated ischemic conditions induce enhanced release of norepinephrine (NE) in the brain and the heart. Although studies with neuronal preparations demonstrated a rise in [Ca
2+
]
i
under energy-depleted conditions, such release of NE in the heart appears to be predominantly Ca
2+
independent. Since Ca
2+
overload occurs in ischemia or energy depletion and since a rise in [Ca
2+
]
i
triggers exocytosis without membrane depolarization, we tested the possibility, using brain synaptosomes, that increased NE release could be, at least in part, a consequence of raised [Ca
2+
]
i
. Brain synaptosomes were incubated with Krebs-Henseleit medium, and ischemia was mimicked by treatment with metabolic inhibitors. NE content in incubation medium (supernatant) and synaptosomes was analyzed chromatographically. Treatment with metabolic inhibitors reduced ATP content by 75% and increased [Ca
2+
]
i
by more than fourfold within minutes. Metabolic inhibition elicited NE release, which started within 10 minutes and reached a maximum after 30 minutes, with a corresponding 55% reduction in synaptosomal NE content after 40 minutes. NE release, together with a marked increase in [Ca
2+
]
i
, was also induced in energy-depleted synaptosomes by Ca
2+
repletion after incubation with the Ca
2+
-free medium. Effects on NE release of various interventions to prevent Ca
2+
overload were tested. Omission of Ca
2+
from the incubation medium or loading synaptosomes with the Ca
2+
chelator BAPTA-AM (20 and 100 μmol/L) prevented NE release, indicating a Ca
2+
-dependent mechanism. Inhibition of Ca
2+
channels with ω-conotoxin, cadmium, or nifedipine had no effect on NE release during energy depletion. In contrast, nickel and 3,4-dichlorobenzamil, Na
+
-Ca
2+
exchange inhibitors, dose-dependently inhibited NE release. In conclusion, this study provides evidence that under energy-depleted conditions, Ca
2+
overload in synaptosomes of noradrenergic neurons from the brain is an important mechanism for the enhanced release of NE and that a reversal of Na
+
-Ca
2+
exchange may be the key pathway leading to intraneuronal Ca
2+
overload.
Functional and Biochemical Analysis of Angiotensin II–Forming Pathways in the Human Heart
Authors: Wolny Arlene , Clozel Jean-Paul , Rein Josiane , Mory Paul , Vogt Paul , Turino Marko , Kiowski Wolfgang , Fischli Walter
Published: June 2012

Blockade of the renin-angiotensin system by inhibition of angiotensin-converting enzyme (ACE) is beneficial for the treatment of hypertension and congestive heart failure. However, it is unclear how complete the blockade by ACE inhibitors is and if there is continuing angiotensin II (Ang II) formation during chronic treatment with ACE inhibitors. Indeed chymase, a serine protease, which is able to form angiotensin II from angiotensin I (Ang I) and cannot be blocked by ACE inhibitors, has been shown to be present in human heart. The goal of the present study was to evaluate the extent of renin-angiotensin system blockade and the Ang II–forming pathways in cardiac tissue of patients chronically treated with ACE inhibitors or in patients without ACE inhibition therapy. Our studies indicate an incomplete ACE inhibition in human heart tissue after chronic ACE inhibitor therapy. Moreover, ACE contributes only a small portion to the total Ang I conversion, as shown in biochemical studies in ventricular and coronary homogenates or functionally as Ang I contractions in isolated rings of coronary arteries. A serine protease was responsible for the majority of Ang II production in both the membrane preparation and Ang I–induced contractions of isolated coronary arteries. In humans, the serine protease pathway is likely to play an important role in cardiac Ang II formation. Thus, drugs such as renin inhibitors and Ang II receptor blockers might be able to induce a more complete blockade of the renin-angiotensin system, providing a more efficacious therapy.
Functional Effects of Protein Kinase C Activation on the Human Cardiac Na sup + Channel
Authors: Murray Katherine T. , Hu NingNing , Daw J. Richard , Shin Hyeon-Gyu , Watson Marshall T. , Mashburn Amy B. , George Alfred L.
Published: June 2012

The cardiac Na sup + current plays an important role in determining normal and abnormal impulse propagation in the heart. We have investigated the effects of protein kinase C (PKC) activation on the recombinant human cardiac Na sup + channel (hH1) following heterologous expression in Xenopus laevis oocytes. Phorbol 12-myristate 13-acetate (PMA), which directly activates PKC, reduced current amplitude at all test potentials (43 +/- 12% at -10 mV). In contrast to the rat brain IIA (rBIIA) channel, there was no apparent change in either macroscopic Na sup + current decay or the voltage dependence of channel gating. Further experiments indicate that the effects of PMA were mediated by PKC activation: (1) an inactive stereoisomer, 4 alpha-PMA, had no effect; (2) preincubation with the protein kinase inhibitor chelerythrine prevented the PMA effects; and (3) a hydrolyzable diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol, also reduced current (22 +/- 5%). In addition, when the alpha
1B
-adrenergic receptor was coexpressed with hH1, the alpha-receptor agonist methoxamine reduced hH1 current (45 +/- 10%), an effect that could be eliminated by chelerythrine preincubation. When a conserved consensus PKC site (serine 1503) in the III-IV interdomain linker thought to be responsible for the PKC effects on rBIIA was mutated, PMA still reduced Na sup + current, but the magnitude of the effect was smaller compared with that for the wild-type channel. Similar findings were obtained with alpha
1
-receptor stimulation following receptor coexpression with the mutant channel. We conclude that activation of PKC modulates the human cardiac Na sup + channel by at least two mechanisms, one similar to that seen with rat brain channels, involving a conserved putative PKC site, and a second more specific to the cardiac isoform.
Embryonic Expression of the Gax Homeodomain Protein in Cardiac, Smooth, and Skeletal Muscle
Authors: Skopicki Hal A. , Lyons Gary E. , Schatteman Gina , Smith Roy C. , Andrés Vicente , Schirm Sabine , Isner Jeffrey , Walsh Kenneth
Published: June 2012

Abstract Gax
is a homeobox-containing gene that has been detected in adult cardiovascular tissues and exhibits a growth arrest–specific pattern of expression in cultured vascular myocytes. To study the regulation of
gax
during development, we performed immunohistochemistry and in situ hybridization on mouse embryos. Gax was present in mesodermally and, as with other homeobox genes, neuroectodermally derived tissues. Early mesodermal protein expression was limited to the lateral plate and somitic mesoderm. Gax in the cardiac muscle lineage exhibited a biphasic pattern of expression. Expression was prominent in the heart tube of the earliest cardiomyocytes and remained prominent through the looping stage (day 12.5 post coitum [pc]) but fell below the threshold of detection in atria and ventricles by day 13.5 pc. At day 15.5 pc, Gax protein was again detectable but restricted to cells within the compact layer of the ventricular myocardium. Gax expression was also noted in smooth muscle cells as early as day 9.5 pc. In the skeletal muscle lineage, Gax protein was expressed at the onset of somitogenesis before the expression of the myogenic basic helix-loop-helix and MEF2/RSRF family proteins. Subsequently, it was noted at day 9.5 pc in premyogenic cells migrating into head, trunk, and limb buds. Gax was detected in myotomes, premuscle masses, and mature muscle groups. These data suggest an important developmental role for Gax in all muscle lineages.
Plasminogen Activator Inhibitor Type 1 and Tissue Inhibitor of Metalloproteinases-2 Increase After Arterial Injury in Rats
Authors: Hasenstab David , Forough Reza , Clowes Alexander W.
Published: June 2012

Abstract
Vascular injury induced by angioplasty causes smooth muscle cells to migrate, proliferate, and form a neointima. The neointima is further enlarged by the accumulation of matrix molecules synthesized by smooth muscle cells. Smooth muscle cell migration and matrix accumulation are associated with an increase in the expression of matrix-degrading enzymes and might be regulated by the balance of protease and anti-protease activity. We have studied the inhibitors of two major classes of matrix-degrading enzymes, the plasminogen activators and the matrix metalloproteinases (MMPs) to understand better the regulation of proteolytic activity following balloon catheter injury in the rat carotid artery. At various times after injury, protease inhibitor expression was analyzed by Northern blotting, reverse zymography, immunohistochemistry, and Western blotting. During the first month after injury, we found that the expression of two proteinase inhibitors (plasminogen activator inhibitor type 1 [PAI-1] and tissue inhibitor of metalloproteinases-2 [TIMP-2]) was modulated. PAI-1 mRNA expression reached a maximum 6 hours after injury before tapering off to baseline levels by 3 days. PAI-1 activity, as measured by reverse zymography, followed the same temporal profile. PAI-1, localized by immunohistochemistry, was expressed at low levels in the media of control arteries and was increased after injury primarily in the medial smooth muscle cells. TIMP-2 mRNA levels began to increase 24 hours after injury and reached a maximum at day 7. TIMP-2 activity, measured by reverse zymography, peaked at day 3 after injury. TIMP-2 protein was increased in the intima compared with the media and adventitia at day 7 after injury. The increase of PAI-1 and TIMP-2 after injury supports the hypothesis that changes in the proteolytic balance play an important role in smooth muscle cell migration after arterial injury.
Vascular Smooth Muscle Cell Cytotoxicity and Sustained Inhibition of Neointimal Formation by Fibroblast Growth Factor 2–Saporin Fusion Protein
Authors: Farb Andrew , Lee Sue J. , Min Duk H. , Parandoosh Zahra , Cook Jennifer , McDonald John , Pierce Glenn F. , Virmani Renu
Published: June 2012

Abstract
Basic fibroblast growth factor (FGF2) is an important mediator of smooth muscle cell (SMC) proliferation following arterial injury that results in neointimal growth. The present study was designed to explore the effects of recombinant FGF2 linked to the ribosome-inactivating protein saporin-6 (rFGF2-SAP) on vascular SMC cytotoxicity and neointimal formation following arterial injury. Cultured rat aortic SMCs were exposed to various concentrations of rFGF2-SAP, FGF2, and saporin-6 (SAP). Incubation with rFGF2-SAP resulted in a decreased number of SMCs beginning at a concentration of 10
−9
mol/L. Significant cytotoxicity was observed with as little as a 30-minute exposure of SMCs to rFGF2-SAP. To evaluate the ability of rFGF2-SAP in an in vivo model to reduce neointimal formation, Sprague-Dawley rats underwent carotid artery balloon denudation and received an intravenous bolus of vehicle or 5, 10, 15, or 20 μg/kg rFGF2-SAP on 0, 3, 6, and 9 days after injury. Rats were euthanized at 14 days, and carotid arteries were analyzed by computerized morphometry. The threshold dose for a significant reduction in neointimal area by rFGF2-SAP was 15 μg/kg (47% reduction in neointima). When dosing was extended to include days 16, 19, and 22, the neointima was reduced 33% at 28 days (
P
=.048). rFGF2-SAP reduced neointima without associated medial thinning or arterial wall dilatation. To determine if rFGF2-SAP directly targets SMCs in vivo, rats underwent carotid injury and received either 15 μg/kg rFGF2-SAP or vehicle on day 0 and at 72 hours, with euthanasia at 78 hours after balloon denudation. Medial SMC number was reduced 46% in the rFGF2-SAP group. Tissue sections from arteries 3 days after balloon injury demonstrated rFGF2-SAP binding to medial SMCs and adventitial cells. Staining for fibroblast growth factor receptor 1 revealed a high level of expression in ballooned arteries 3 and 14 days after injury. Taken together, these results provide a molecular and cellular basis for the observed specificity. Prolonged delivery of rFGF2-SAP can affect the natural history of arterial repair after injury.
Nitric Oxide Induces Heme Oxygenase-1 Gene Expression and Carbon Monoxide Production in Vascular Smooth Muscle Cells
Authors: Durante William , Kroll Michael H. , Christodoulides Nick , Peyton Kelly J. , Schafer Andrew I.
Published: June 2012

Abstract
Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase–stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside,
S
-nitroso-
N
-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-1 (HO-1) mRNA and protein levels in a concentration- and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1β and tumor necrosis factor-α induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-
l
-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.
Oxygen Radicals Can Induce Preconditioning in Rabbit Hearts
Authors: Tritto Isabella , D’Andrea Davide , Eramo Nicola , Scognamiglio Annalisa , De Simone Ciro , Violante Anna , Esposito Annamaria , Chiariello Massimo , Ambrosio Giuseppe
Published: June 2012

Abstract
Indirect evidence suggests that oxygen radicals may contribute to ischemic preconditioning. We directly investigated whether exposure to oxygen radicals per se, in the absence of ischemia, could reproduce the beneficial effects of ischemic preconditioning on infarct size and on postischemic contractile dysfunction. In one branch of the study, isolated rabbit hearts underwent 30 minutes of total global ischemia and 45 minutes of reperfusion (n=6, control group). A second group, before ischemia/reperfusion, was exposed for 5 minutes to a low flux of oxygen radicals generated by purine/xanthine oxidase (P/XO), followed by a 15-minute washout (n=6). Oxygen radical pretreatment significantly improved postischemic recovery of contractile function. We then investigated in another branch of the study whether this preconditioning effect would also reduce infarct size and whether it was mediated by protein kinase C activation. Control hearts were subjected to coronary artery occlusion for 30 minutes, followed by 2.5 hours of reperfusion (n=6). A second group, before coronary occlusion, was exposed to oxygen radicals and washout as described (n=8). A third group was subjected to oxygen radical infusion, but an inhibitor of protein kinase C (polymyxin B, 50 μmol/L) was administered throughout subsequent ischemia (n=7). A fourth group was exposed to oxygen radicals in the presence of scavengers (superoxide dismutase, 250 U/mL; catalase 500, U/mL; n=8). Pretreatment with oxygen radicals markedly reduced infarct size, from 65±19% of risk region in controls to 12±4% (
P
<.05). Protein kinase C inhibition significantly attenuated this effect (infarct size, 37±9% of risk region;
P
<.05 versus P/XO;
P
=NS versus controls). Oxygen radical–induced preconditioning was prevented by scavengers (infarct size, 55±14% of risk region;
P
<.05 versus P/XO;
P
=NS versus P/XO+polymyxin B). Our data show that in the absence of ischemia, exposure to low concentrations of oxygen radicals can reproduce the beneficial effects of ischemic preconditioning on infarct size and postischemic recovery of left ventricular function. Thus, oxygen radicals might be potential contributors to ischemic preconditioning.
Vascular Thrombin Receptor Regulation in Hypertensive Rats
Authors: Capers Quinn , Laursen Jørn Bech , Fukui Toshiki , Rajagopalan Sanjay , Mori Ichiro , Lou Pingping , Freeman Bruce A. , Berrington William R. , Griendling Kathy K. , Harrison David G. , Runge Marschall S. , Alexander R. Wayne , Taylor W. Robert
Published: June 2012

Abstract
Thrombin has been implicated as an important mediator of vascular lesion formation in atherosclerosis and restenosis. To investigate a potential role for thrombin signaling in the vascular response to hypertension, we have studied thrombin receptor (TR) expression and regulation in hypertensive rats. Aortic TR mRNA was upregulated by angiotensin II (Ang II)–induced hypertension (10.7±2.5 times control,
P
<.02), which correlated with a 4-fold increase in thrombin-induced constriction in isolated endothelium-denuded aortic rings. The AT
1
receptor antagonist losartan normalized blood pressure and TR mRNA. Conversely, lowering blood pressure to the same degree with hydralazine did not abolish the upregulation of TR mRNA expression. When low-renin low–Ang II hypertension was induced in Dahl salt-sensitive rats, there was no detectable increase in the expression of aortic thrombin receptor mRNA. Finally, treatment with a chimeric heparin-binding form of the recombinant human Cu/Zn superoxide dismutase caused complete inhibition of TR mRNA upregulation, suggesting that an increased rate of superoxide anion production is an important signaling mechanism. Thus, increased TR expression via a redox-sensitive mechanism in the aortic smooth muscle of rats treated with Ang II represents a novel in vivo mechanism through which the hypertensive effects of Ang II are mediated.
Activity and Expression of Na
+
-H
+
Exchanger Isoforms 1 and 3 in Kidney Proximal Tubules of Hypertensive Rats
Authors: Kelly M. P. , Quinn P. A. , Davies J. E. , Ng L. L.
Published: June 2012

Abstract
Increased activity of the cellular Na
+
-H
+
exchangers (NHEs), especially isoform 1 (NHE-1), is a recognized intermediate phenotype of hypertension. NHE activity has been demonstrated to be increased in proximal tubules of the spontaneously hypertensive rat (SHR). However, with the recent cloning of other members of this family of transporters, it is unclear which isoforms may contribute to this increased activity. We have used specific antibodies raised against glutathione-S-transferase fusion proteins of rat NHE-1 and NHE-3 to determine the relative contributions of these isoforms to the NHE activity in freshly isolated and cultured proximal tubule cells from SHR and Wistar-Kyoto (WKY) normotensive control rats. In freshly isolated proximal tubule cells, NHE activity was elevated almost 3-fold in SHR cells (
P
<.001), and in both rat strains, the contribution from NHE-1 and NHE-3 was approximately equal. Western blots of membranes from these cells showed equal amounts of NHE-1 protein in SHR and WKY cells. However, NHE-3 protein expression was increased 50% in SHR cells (
P
<.001), and this may account for the elevated activity of this isoform in SHR. The effect of culturing these cells in vitro was then examined. Although total NHE activity in both cell types was decreased during culture, this was mainly due to loss of expression of NHE-3 protein. NHE-1 activity was persistently elevated in the SHR cells in culture. These findings suggest that elevated NHE activity in SHR proximal tubules could be mediated by two mechanisms: (1) increased NHE-1 activity without any increased NHE-1 protein content that persists despite culture and may resemble those changes described for extrarenal tissues and (2) increased NHE-3 activity due to increased expression of NHE-3 protein. Disappearance of NHE-3 during culture implies that our culture conditions did not replicate the in vivo environment and may have removed the factors contributing to the increased NHE-3 expression in SHR cells.
Expression of a Mutant (Arg
92
Gln) Human Cardiac Troponin T, Known to Cause Hypertrophic Cardiomyopathy, Impairs Adult Cardiac Myocyte Contractility
Authors: Marian Ali J. , Zhao Guiling , Seta Yukihiro , Roberts Robert , Yu Qun-tao
Published: June 2012

Abstract
The mechanism(s) by which mutations in sarcomeric proteins cause hypertrophic cardiomyopathy (HCM) remains unknown. A leading hypothesis proposes that mutant sarcomeric proteins impair cardiac myocyte contractility, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg
92
Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, on adult cardiac myocyte contractility. A full-length human cTnT cDNA was cloned, and the Arg
92
Gln mutation was induced. Recombinant adenoviruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg
92
Gln were generated through homologous recombination. Adult feline cardiac myocytes were infected with recombinant adenoviruses or a control viral vector (Ad5ΔE1) at a multiplicity of infection of 100. Expression levels of the full-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was also shown by immunocytochemistry and immunofluorescence, and their incorporation into myofibrils was confirmed by Western blotting on myofibrillar extracts. Electron microscopy showed intact sarcomere structure in rod-shaped cardiac myocytes in all groups. Cell fractional shortening and the peak velocity of shortening were not significantly different among the groups 24 hours after transduction. However, 48 hours after transduction, both fractional shortening and the peak velocity of shortening were significantly reduced (24% [
P
<.001] and 26% [
P
<.001], respectively) in cardiac myocytes in the Ad5/CMV/cTnT-Arg
92
Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was greater at 72 hours after transduction (45% and 39%, respectively;
P
<.001). Our results indicated that expression of the mutant (Arg
92
Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myocyte contractility. Our data also show that both normal and mutant cTnT were incorporated into myofibrils. These results provide a potential mechanism by which mutations in sarcomeric proteins cause HCM.
Growth Factor and Cytokine-Regulated Hyaluronan-Binding Protein TSG-6 Is Localized to the Injury-Induced Rat Neointima and Confers Enhanced Growth in Vascular Smooth Muscle Cells
Authors: Ye Li , Mora Rosalia , Akhayani Nahid , Haudenschild Christian C. , Liau Gene
Published: June 2012

Abstract
Hyaluronan (HA) and HA-binding proteins have been implicated in a diverse array of biological processes, including development, tissue repair, and tumor invasion. However, the role of HA and HA-binding proteins in atherosclerosis and restenosis is poorly understood. PS4 (TSG-6) is a HA-binding protein expressed by cultured vascular smooth muscle cells (SMCs) in response to serum and growth factor stimulation. To delineate a possible role for TSG-6 in vascular disease progression, we have characterized its expression in cultured SMCs and in a rat vascular injury model, and we have studied the effect of constitutive overexpression of TSG-6 on SMC behavior. We found that interleukin-1 (IL-1) but not tumor necrosis factor or interleukin-6 was able to stimulate TSG-6 expression in SMCs. The IL-1 pathway could be distinguished from the growth factor pathway by its insensitivity to protein synthesis inhibitors. Furthermore, epidermal growth factor, fibroblast growth factor-1, and transforming growth factor-β1 were all capable of augmenting maximum IL-1–induced expression of TSG-6. To gain further insight into the function of TSG-6 in SMCs, we examined the effect of constitutive overexpression of TSG-6 on these cells. We found that TSG-6–overexpressing cells grew >50% faster than control cells. Furthermore, this growth advantage became more evident in the absence of serum growth factors, with an average increase in cell number of 118% over control cells after 6 days. Consistent with these in vitro data, we observed intense immunostaining for TSG-6 in proliferating SMCs in the rat neointima after injury, whereas only an occasional cell was positive for TSG-6 in the medial layer and in nonballooned arteries. We conclude that the expression of TSG-6 is tightly controlled by growth factors and cytokines via two distinct pathways in SMCs and that overexpression of TSG-6 confers a growth advantage to these cells.
Estrogen Upregulates Endothelial Nitric Oxide Synthase Gene Expression in Fetal Pulmonary Artery Endothelium
Authors: MacRitchie Amy N. , Jun Sandy S. , Chen Zhong , German Zohre , Yuhanna Ivan S. , Sherman Todd S. , Shaul Philip W.
Published: June 2012

Abstract
NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17β caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10
−10
to 10
−6
mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.
Monomeric Phospholamban Overexpression in Transgenic Mouse Hearts
Authors: Chu Guoxiang , Dorn Gerald W. , Luo Wusheng , Harrer Judy M. , Kadambi Vivek J. , Walsh Richard A. , Kranias Evangelia G.
Published: June 2012

Abstract
Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca
2+
-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys
41
to Phe
41
, was shown to be as effective as pentameric phospholamban in inhibiting Ca
2+
transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys
41
→Phe
41
) phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with ≈50% of phospholamban migrating as monomers and ≈50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca
2+
-uptake assays revealed that the EC
50
values for Ca
2+
were as follows: 0.32±0.01 μmol/L in hearts overexpressing monomeric phospholamban, 0.49±0.05 μmol/L in hearts overexpressing wild-type phospholamban, and 0.26±0.01 μmol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca
2+
kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov), (4) amplitude of the Ca
2+
signal (21% mt versus 40% ov), and (5) time for decay of the Ca
2+
signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac myocyte mechanics and Ca
2+
transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.
Urokinase but Not Tissue Plasminogen Activator Mediates Arterial Neointima Formation in Mice
Authors: Carmeliet Peter , Moons Lieve , Herbert Jean-Marc , Crawley James , Lupu Florea , Lijnen Roger , Collen Désiré
Published: June 2012

Abstract
To define the role of the plasminogen activators (PAs) tissue PA (t-PA) and urokinase PA (u-PA) in vascular wound healing, neointima formation and reendothelialization were evaluated after electric or mechanical arterial injury in mice with a single or combined deficiency of t-PA (t-PA
−/−
) and/or u-PA (u-PA
−/−
). In both models, neointima formation and neointimal cell accumulation were reduced in u-PA
−/−
and in t-PA
−/−
/u-PA
−/−
arteries but not in t-PA
−/−
arteries. The electric injury model was used to characterize the underlying cellular mechanisms. Topographic analysis of vascular wound healing in electrically injured wild-type and t-PA
−/−
arteries revealed a similar degree of migration of smooth muscle cells from the noninjured borders into the necrotic center. In contrast, in u-PA
−/−
and t-PA
−/−
/u-PA
−/−
arteries, smooth muscle cells accumulated at the uninjured borders but failed to migrate into the necrotic center. Cultured u-PA
−/−
but not t-PA
−/−
smooth muscle cells also failed to migrate in vitro after scrape wounding. Proliferation of smooth muscle cells was not affected by PA deficiency. Reendothelialization after electric injury was similar in all genotypes. In situ analysis revealed markedly elevated u-PA zymographic activity, mRNA, and immunoreactivity in smooth muscle cells, endothelial cells, and leukocytes within 1 week after injury, eg, when cells migrated into the wound. Thus, u-PA plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.
Two Isoforms of the Mouse
Ether-a-go-go
–Related Gene Coassemble to Form Channels With Properties Similar to the Rapidly Activating Component of the Cardiac Delayed Rectifier K
+
Current
Authors: London Barry , Trudeau Matthew C. , Newton Kimberly P. , Beyer Anita K. , Copeland Neal G. , Gilbert Debra J. , enkins Nancy A. J , Satler Carol A. , Robertson Gail A.
Published: June 2012

Abstract
HERG
, the human
ether-a-go-go
–related gene, encodes a K
+
-selective channel with properties similar to the rapidly activating component of the delayed rectifier K
+
current (
I
Kr
). Mutations of
HERG
cause the autosomal-dominant long-QT syndrome (LQTS), presumably by disrupting the normal function of
I
Kr
. The current produced by HERG is not identical to
I
Kr
, however, and the mechanism by which
HERG
mutations cause LQTS remains uncertain. To better define the role of
Erg
in the heart, we cloned
Merg1
from mouse genomic and cardiac cDNA libraries.
Merg1
has 16 exons and maps to mouse chromosome 5 in an area syntenic to human chromosome 7q, the map locus of
HERG
. We isolated three cardiac isoforms of
Merg1
: Merg1a is homologous to HERG and is expressed in heart, brain, and testes, Merg1a′ lacks the first 59 amino acids of Merg1a and is not expressed abundantly, and Merg1b has a markedly shorter divergent N-terminal cytoplasmic domain and is expressed specifically in the heart. The Merg1 isoforms, like HERG, produce inwardly rectifying E-4031–sensitive currents when heterologously expressed in
Xenopus
oocytes. Merg1a and HERG produce currents with slow deactivation kinetics, whereas Merg1a′ and Merg1b currents deactivate more rapidly. Merg1b coassembles with Merg1a to form channels with deactivation kinetics that are more rapid than those of Merg1a or HERG and nearly identical to
I
Kr
. In addition, a homologue of Merg1b is present in human cardiac and smooth muscle. Thus, we have identified a novel N-terminal Erg isoform that is expressed specifically in the heart, has rapid deactivation kinetics, and coassembles with the longer isoform in
Xenopus
oocytes. This N-terminal Erg isoform may determine the properties of
I
Kr
and contribute to the pathogenesis of LQTS.
17β-Estradiol Regulation of Human Endothelial Cell Basal Nitric Oxide Release, Independent of Cytosolic Ca
2+
Mobilization
Authors: Caulin-Glaser Teresa , García-Cardeña G. , Sarrel Phillip , Sessa William C. , Bender Jeffrey R.
Published: June 2012

Abstract
Estradiol retards the development of atherosclerosis. Animal models have suggested that NO may be a critical effector molecule in this cardiovascular protection. In this study, female human umbilical vein endothelial cells (HUVECs) were propagated in phenol red–free gonadal hormone–free medium and pretreated with 17β-estradiol (E
2
). Reduced NO
2
−
and NO
3
−
(NO
X
) concentration, determined by chemiluminescence, demonstrated a rapid increase in basal HUVEC NO release in response to physiological concentrations of E
2
. The estrogen receptor (ER) antagonist ICI 164,384 inhibited the augmented NO release, demonstrating an ER-mediated component of this response. Because endothelial NO synthase (eNOS) activity is largely regulated by cytosolic Ca
2+
, relative [Ca
2+
]
i
in response to E
2
was determined in a fluorometric assay. E
2
did not promote HUVEC Ca
2+
fluxes. Furthermore, eNOS activity in E
2
-pretreated endothelial whole-cell lysates was not dependent on additional Ca
2+
. Despite involving the ER, this is a nongenomic effect of E
2
, as demonstrated by maintained responses in transcriptionally inhibited cells and by the rapidity (10 minutes) of cGMP formation in an NO bioassay. We demonstrate, for the first time, that independent of cytosolic Ca
2+
mobilization, there is augmentation of eNOS activity with a resultant increase in HUVEC basal NO release in response to short-term estradiol exposure. Implications for the cardiovascular protective role of estrogen are discussed.
Application of Transcription Factor “Decoy” Strategy as Means of Gene Therapy and Study of Gene Expression in Cardiovascular Disease
Authors: Morishita Ryuichi , Higaki Jitsuo , Tomita Naruya , Ogihara Toshio
Published: June 2012

Abstract
—Recent progress in molecular biology has provided new techniques for inhibiting target gene expression. In particular, the application of DNA technology, such as antisense strategy to regulate the transcription of disease-related genes in vivo, has important therapeutic potential. Recently, transfection of
cis
-element double-stranded oligodeoxynucleotides (ODNs), referred to as “decoy” ODNs, has been reported to be a powerful tool in a new class of anti-gene strategies for gene therapy and in the study of transcriptional regulation. Transfection of double-stranded ODNs corresponding to the
cis
sequence will result in the attenuation of authentic
cis
-
trans
interaction, leading to the removal of
trans
factors from the endogenous
cis
elements with subsequent modulation of gene expression. This “decoy” strategy is not only a novel strategy for gene therapy as an anti-gene strategy but also a powerful tool for the study of endogenous gene regulation in vivo as well as in vitro. In this article, we reviewed (1) the mechanisms and (2) the potential applications of decoy strategy.