bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence That Phospholipid Oxidation Products and/or Platelet-Activating Factor Play an Important Role in Early Atherogenesis
Authors: Subbanagounder Ganesamoorthy , Leitinger Norbert , Shih Peggy T. , Faull Kym F. , Berliner Judith A.
Published: June 2012

Abstract
—The goal of the present studies was to determine whether phospholipid oxidation products and/or platelet-activating factor (PAF) are mediators of early atherogenesis in vivo. Monocyte–endothelial cell interactions have been shown to play an important role in early atherogenesis. We and others have demonstrated that PAF and phospholipid oxidation products, present in atherosclerotic lesions, including 1-palmitoyl-2-(5-oxovaleroyl)-
sn
-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-
sn
-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-epoxyisoprostane E
2
-
sn
-glycero-3-phosphocholine (PEIPC), mediate the activation of monocytes and/or endothelial cells in vitro. Previous studies have shown that the action of PAF and PAF-like ether-containing phospholipids was inhibited by WEB 2086. We now demonstrate that pretreatment of human aortic endothelial cells with WEB 2086 (10 μmol/L) and several other PAF antagonists before treatment with POVPC and PEIPC but not PGPC prevented the activation of the endothelial cells to bind monocytes. We present evidence to suggest that this inhibition is not mediated by the PAF receptor. The role of bioactive oxidized phospholipids in fatty streak formation was tested using C57BL/6J LDL R−/− mice fed a chow or Western diet for 5 weeks with or without WEB 2086 mixed with drinking water. Quantitative electrospray ionization mass spectrometry showed similar concentrations of WEB 2086 in the plasma of mice on both diets (≈4 to 5 μmol/L); this concentration was inhibitory in vitro. Administration of WEB 2086 did not affect the lipid composition of mouse plasma. However, fatty streak formation was reduced by 62% in animals fed a Western diet, whereas no change was observed in the small lesions of mice on a chow diet. These studies provide evidence that PAF and/or PAF-like phospholipid oxidation products are important mediators of atherosclerotic lesion development in vivo and that specific receptor antagonists for these molecules may represent a novel therapeutic modality.
Peroxisome Proliferator-Activated Receptor Activators Inhibit Thrombin-Induced Endothelin-1 Production in Human Vascular Endothelial Cells by Inhibiting the Activator Protein-1 Signaling Pathway
Authors: Delerive Philippe , Martin-Nizard Françoise , Chinetti Giulia , Trottein François , Fruchart Jean-Charles , Najib Jamila , Duriez Patrick , Staels Bart
Published: June 2012

Abstract
—Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription–polymerase chain reaction analyses demonstrated that both PPARα and PPARγ are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARα and PPARγ ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription–polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARα and PPARγ are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.
Subcellular [Ca
2+
]
i
Gradients During Excitation-Contraction Coupling in Newborn Rabbit Ventricular Myocytes
Authors: Haddock Peter S. , Coetzee William A. , Cho Emily , Porter Lisa , Katoh Hideki , Bers Donald M. , Jafri M. Saleet , Artman Michael
Published: June 2012

Abstract
—The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca
2+
]
i
transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca
2+
]
i
occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca
2+
]
i
rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 μmol/L thapsigargin) slowed the rate of rise and decline of Ca
2+
in AD myocytes but did not alter Ca
2+
transient kinetics in NB myocytes. In contrast to adults, localized SR Ca
2+
release events (“Ca
2+
sparks”) occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na
+
-Ca
2+
exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS–treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca
2+
dynamics predicts that Ca
2+
flux via the Na
+
-Ca
2+
exchanger during an action potential can account for the subsarcolemmal Ca
2+
gradients in NB myocytes. Spatial separation of sarcolemmal Ca
2+
entry from SR Ca
2+
release channels may minimize the role of SR Ca
2+
release during normal EC coupling in NB ventricular myocytes.
Chronic Exposure of Smooth Muscle Cells to Minimally Oxidized LDL Results in Depressed Inositol 1,4,5-Trisphosphate Receptor Density and Ca
2+
Transients
Authors: Massaeli Hamid , Austria J. Alejandro , Pierce Grant N.
Published: June 2012

Abstract
—Oxidized LDL (oxLDL) (0.1 mg/mL) increased [Ca
2+
]
i
in vascular smooth muscle cells (VSMCs) within 5 to 10 seconds of incubation. This increase was mediated via an inositol 1,4,5-trisphosphate (IP
3
)-dependent release of Ca
2+
from the sarcoplasmic reticulum. However, atherosclerosis is a gradual process in which VSMCs are more likely exposed to low concentrations of oxLDL over extended periods rather than acute exposures. It is very possible, therefore, that lower [oxLDL] and longer exposure times may induce a very different response with regard to regulation of [Ca
2+
]
i
. VSMCs were incubated with 4- to 100-fold lower [oxLDL] for up to 6 days. The conditions were not cytotoxic. Basal [Ca
2+
]
i
was not altered. Surprisingly, however, after chronic exposure to oxLDL, a brief addition of oxLDL (0.1 mg/mL) or norepinephrine failed to elicit the expected rise in Ca
2+
i
. Because the acute effects of oxLDL on control cells were mediated through an IP
3
-dependent pathway, we investigated the integrity of the VSMC IP
3
receptors. Immunocytochemical analysis and Western blots revealed a depression in the density of IP
3
receptors after chronic exposure of VSMCs to oxLDL. These changes in IP
3
receptors have significance under atherosclerotic conditions as well. Immunocytochemical analysis revealed a decrease in IP
3
receptor density in the medial layer under atherosclerotic plaques in situ. Our data, therefore, demonstrate a striking difference between the acute and chronic effects of oxLDL on VSMC calcium. Whereas acute exposure to oxLDL stimulates [Ca
2+
]
i
, chronic exposure results in depressed Ca
2+
transients, apparently through a decrease in IP
3
receptor density. These changes have functional implications for the atherosclerotic vessel in vivo, and our data implicates oxLDL in this process.
Overexpression of Human Catalase Inhibits Proliferation and Promotes Apoptosis in Vascular Smooth Muscle Cells
Authors: Brown Michael R. , Miller Francis J. , Li Wei-Gen , Ellingson Andy N. , Mozena Jonathan D. , Chatterjee Papri , Engelhardt John F. , Zwacka Ralf M. , Oberley Larry W. , Fang Xiang , Spector Arthur A. , Weintraub Neal L.
Published: June 2012

Abstract
—The role of reactive oxygen species, such as superoxide anions (O
2
·
−
) and hydrogen peroxide (H
2
O
2
), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H
2
O
2
, rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (Ad
Cat
) or a control gene, β-galactosidase (Ad
LacZ
). Infection with Ad
Cat
resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of Ad
Cat
, cellular catalase activity was increased by 50- to 100-fold, and intracellular H
2
O
2
concentration was reduced, as compared with control. Infection with Ad
Cat
reduced [
3
H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.37±0.09 disintegrations per minute per cell [Ad
LacZ
] versus 0.22±0.08 disintegrations per minute per cell [Ad
Cat
],
P
<0.05). Five days after infection with 100 MOI of Ad
Cat
, cell numbers were reduced as compared with noninfected or Ad
LacZ
-infected cells (157 780±8413 [Ad
Cat
],
P
<0.05 versus 233 700±3032 [noninfected] or 222 410±5332 [Ad
LacZ
]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of Ad
Cat
as compared with control. Infection with Ad
Cat
resulted in induction of cyclooxygenase (COX)–2, and treatment with a COX-2 inhibitor overcame the Ad
Cat
-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2–dependent mechanism. Our results suggest that endogenously produced H
2
O
2
importantly modulates survival and proliferation of vascular smooth muscle cells.
Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth
Authors: Searles Charles D. , Miwa Yoichi , Harrison David G. , Ramasamy Santhini
Published: June 2012

Abstract
—The expression of the endothelial NO synthase (eNOS) is dramatically influenced by the state of cell growth. In proliferating cells, mRNA levels are increased 4-fold compared with postconfluent, nonproliferating cells. Nuclear run-on analysis indicated that there is no difference in the transcriptional rate of eNOS in proliferating versus postconfluent cells. The half-life of eNOS mRNA, measured after actinomycin D transcriptional arrest, was 3-fold greater in preconfluent compared with confluent endothelial cells. Using UV–cross-linking analysis, a cytoplasmic protein with an apparent molecular mass of 51 kDa was found to bind to terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in proliferating cells. Further characterization of the eNOS mRNA indicated that a 43-nt sequence at the origin of the 3′-untranslated region (UTR) is critical in binding of this protein. Endothelial cells were stably transfected with a chimeric cDNA plasmid containing chloramphenicol acetyl transferase (CAT) ligated to the eNOS coding region and either the wild-type 3′-UTR (pcDNACAT/eNOS
(wtUTR)
) or a mutant 3′-UTR lacking the 43 nt found to bind the 51-kDa protein (pcDNACAT/eNOS
(ΔUTR)
). The CAT/eNOS mRNA half-life was dramatically stabilized in these latter cells as compared with cells transfected with pcDNACAT/eNOS
(wtUTR)
). Thus, this 43-nt region plays a critical role in destabilizing eNOS mRNA. These studies demonstrate a mechanism for modulation of eNOS expression during cell growth via interactions between the proximal 3′-UTR and a novel ≈51-kDa cytosolic protein.
Self-Protection by Cardiac Myocytes Against Hypoxia and Hyperoxia
Authors: Winegrad Saul , Henrion Daniel , Rappaport Lydie , Samuel Jane Lise
Published: June 2012

Abstract
—Cardiac muscle must maintain a continuous balance between its energy supply and work performed. An important mechanism involved in achievement of this balance is cross talk via chemical signals between cardiac myocytes and the cardiac muscle vascular system . This has been demonstrated by incubating isolated cardiac myocytes in different concentrations of oxygen and then assaying the conditioned media for vasoactive substances on isolated aortic rings and small-resistance arteries. With increasing oxygen concentrations above 6%, cardiac myocytes produce increasing amounts of angiotensin I, which is converted to angiotensin II by the blood vessel. The angiotensin II stimulates vascular endothelial cells to secrete endothelin and increase vascular tone. Below 6% oxygen, cardiac myocytes secrete adenosine, which acts directly on vascular smooth muscle to block the effect of α-adrenergic agonists and reduce vascular tone. In an intact heart, the net effect of these 2 regulatory systems would be the maintenance of oxygen concentration within a narrow range at the cardiac myocytes. By acting as oxygen sensors, cardiac myocytes modulate vascular tone according to the needs of the myocytes and reduce potential problems of hypoxia and extensive formation of reactive oxygen species.
Activation of Mitochondrial ATP-Sensitive K
+
Channel for Cardiac Protection Against Ischemic Injury Is Dependent on Protein Kinase C Activity
Authors: Wang Yigang , Hirai Kyoji , Ashraf Muhammad
Published: June 2012

Abstract
—Protein kinase C (PKC) is involved in the second messenger signaling cascade during ischemic and Ca
2+
preconditioning. Given that the pharmacological activation of mitochondrial ATP-sensitive K
+
(mitoK
ATP
) channels also mimics preconditioning, the mechanisms linking PKC activation and mitoK
ATP
channels remain to be established. We hypothesize that PKC activity is important for the opening of the mitoK
ATP
channel. To examine this, a specific opener of the mitoK
ATP
channel, diazoxide, was used in conjunction with subcellular distribution of PKC in a model of ischemia/reperfusion (I/R). Langendorff-perfused rat hearts were subjected to 40-minute ischemia followed by 30-minute reperfusion. Effects of activation of the mitoK
ATP
channel and other interventions on functional, biochemical, and pathological changes in ischemic hearts were assessed. In hearts treated with diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly improved after I/R; lactate dehydrogenase release was also significantly decreased. The morphology was well preserved in diazoxide-treated hearts compared with nontreated ischemic control hearts. The salutary effects of diazoxide on the ischemic injury were similar to those of Ca
2+
preconditioning. Administration of sodium 5-hydroxydecanoate, an effective blocker of the mitoK
ATP
channel, or chelerythrine or calphostin C, an inhibitor of PKC, during diazoxide pretreatment or during continuous presence of diazoxide in the ischemic period, completely abolished the beneficial effects of the diazoxide on the I/R injury. Blockade of Ca
2+
entry during diazoxide treatment by inhibiting the L-type Ca
2+
channel with verapamil also completely reversed the beneficial effect of diazoxide during I/R. PKC-α was translocated to sarcolemma, whereas PKC-δ was translocated to the mitochondria and intercalated disc, and PKC-ε was translocated to the intercalated disc of the diazoxide-pretreated hearts. Colocalization studies for mitochondrial distribution with tetramethylrhodamine ethyl ester (TMRE) and PKC isoforms by immunoconfocal microscopy revealed that PKC-δ antibody specifically stained the mitochondria. ATP was significantly increased in the diazoxide-treated hearts. Moreover, the data suggest that activation and translocation of PKC to mitochondria appear to be important for the protection mediated by mitoK
ATP
channel.
Oxidative Stress as a Regulator of Gene Expression in the Vasculature
Authors: Kunsch Charles , Medford Russell M.
Published: June 2012

Abstract
—Oxidative stress and the production of intracellular reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases. In excess, ROS and their byproducts that are capable of causing oxidative damage may be cytotoxic to cells. However, it is now well established that moderate amounts of ROS play a role in signal transduction processes such as cell growth and posttranslational modification of proteins. Oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state play an important role in the regulation of gene expression. Recent insights into the etiology and pathogenesis of atherosclerosis suggest that this disease may be viewed as an inflammatory disease linked to an abnormality in oxidation-mediated signals in the vasculature. In this review, we summarize the evidence supporting the notion that oxidative stress and the production of ROS function as physiological regulators of vascular gene expression mediated via specific redox-sensitive signal transduction pathways and transcriptional regulatory networks. Elucidating, at the molecular level, the regulatory processes involved in redox-sensitive vascular gene expression represents a foundation not only for understanding the pathogenesis of atherosclerosis and other inflammatory diseases but also for the development of novel therapeutic treatment strategies.
Transcriptional Activation of the Zinc Finger Transcription Factor BTEB2 Gene by Egr-1 Through Mitogen-Activated Protein Kinase Pathways in Vascular Smooth Muscle Cells
Authors: Kawai-Kowase Keiko , Kurabayashi Masahiko , Hoshino Yoichi , Ohyama Yoshio , Nagai Ryozo
Published: June 2012

Abstract
—We have recently demonstrated that a developmentally regulated zinc finger protein, basic transcription regulatory element binding protein 2 (BTEB2), is induced in neointimal smooth muscle in response to vascular injury. In this study, we investigated the molecular mechanisms regulating BTEB2 expression in vascular smooth muscle cells (SMCs) in vitro. BTEB2 mRNA expression is rapidly and persistently induced in SMCs by phorbol 12-myristate 13-acetate (PMA) and basic fibroblast growth factor. We have isolated and characterized the promoter region of the human BTEB2 gene to determine the regulatory network controlling expression of this gene in vascular SMCs. Functional studies on the BTEB2 promoter coupled to a luciferase reporter gene demonstrated activation of the promoter by PMA and basic fibroblast growth factor. Both characterization of DNA-protein complexes in vitro and site-specific mutation analysis of the BTEB2 promoter have defined a 9-bp sequence, 5′-CGCCCGCGC-3′, located at −25, as the Egr-1 binding site mediating an induction of the BTEB2 promoter activity by PMA. In addition, we show that this site mediates inducible expression through the mitogen-activated protein kinase pathways. These results indicate that BTEB2 is a target of the early-response gene Egr-1, and mitogen-activated protein kinase pathways directly or indirectly activate BTEB2 expression. Given a rapid induction of Egr-1 on stimulation with growth factors or injury, these findings may represent at least one of the molecular mechanisms underlying phenotypic modulation of smooth muscles after vascular injury.
Hypercholesterolemia Causes Mechanical Weakening of Rabbit Atheroma
Authors: Rekhter Mark D. , Hicks Gary W. , Brammer David W. , Hallak Hussein , Kindt Erick , Chen Jing , Rosebury Wendy S. , Anderson Maureen K. , Kuipers Paul J. , Ryan Michael J.
Published: June 2012

Abstract
—Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0.5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (
P
<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.
Cardiovascular Overexpression of Transforming Growth Factor-β
1
Causes Abnormal Yolk Sac Vasculogenesis and Early Embryonic Death
Authors: Agah Ramtin , Prasad K. S. Srinivasa , Linnemann Ruth , Firpo Meri T. , Quertermous Thomas , Dichek David A.
Published: June 2012

Abstract
—Transforming growth factor-β
1
(TGF-β
1
) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-β
1
expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-β
1
in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-β
1
is expressed from the SM22α promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22α-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22α–β-galactosidase transgene. SM22α– β-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22α–TGF-β
1
transgenic mice died before E11.5. SM22α–TGF-β
1
transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-β
1
from the SM22α promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-β
1
expression in adults may require a conditional transgenic approach. Moreover, because the SM22α promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22α promoter to drive expression of “vasculoactive” transgenes may be particularly likely to cause embryonic death.
Three Distinct Types of Ca
2+
Waves in Langendorff-Perfused Rat Heart Revealed by Real-Time Confocal Microscopy
Authors: Kaneko Tomoyuki , Tanaka Hideo , Oyamada Masahito , Kawata Satoshi , Takamatsu Tetsuro
Published: June 2012

Abstract
—Although Ca
2+
waves in cardiac myocytes are regarded as arrhythmogenic substrates, their properties in the heart in situ are poorly understood. On the hypothesis that Ca
2+
waves in the heart behave diversely and some of them influence the cardiac function, we analyzed their incidence, propagation velocity, and intercellular propagation at the subepicardial myocardium of fluo 3–loaded rat whole hearts using real-time laser scanning confocal microscopy. We classified Ca
2+
waves into 3 types. In intact regions showing homogeneous Ca
2+
transients under sinus rhythm (2 mmol/L [Ca
2+
]
o
), Ca
2+
waves did not occur. Under quiescence, the waves occurred sporadically (3.8 waves · min
−1
· cell
−1
), with a velocity of 84 μm/s, a decline half-time (t
1/2
) of 0.16 seconds, and rare intercellular propagation (propagation ratio <0.06) (sporadic wave). In contrast, in presumably Ca
2+
-overloaded regions showing higher fluorescent intensity (113% versus the intact regions), Ca
2+
waves occurred at 28 waves · min
−1
· cell
−1
under quiescence with a higher velocity (116 μm/s), longer decline time (t
1/2
=0.41 second), and occasional intercellular propagation (propagation ratio=0.23) (Ca
2+
-overloaded wave). In regions with much higher fluorescent intensity (124% versus the intact region), Ca
2+
waves occurred with a high incidence (133 waves · min
−1
· cell
−1
) and little intercellular propagation (agonal wave). We conclude that the spatiotemporal properties of Ca
2+
waves in the heart are diverse and modulated by the Ca
2+
-loading state. The sporadic waves would not affect cardiac function, but prevalent Ca
2+
-overloaded and agonal waves may induce contractile failure and arrhythmias.
Coexpression of Connexins 40 and 43 Enhances the pH Sensitivity of Gap Junctions
Authors: Gu Hong , Ek-Vitorin Jose F. , Taffet Steven M. , Delmar Mario
Published: June 2012

Abstract
—Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype.
Xenopus
oocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from ≈6.7 when cells expressed only Cx40 or only Cx43 to ≈7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined. The full text of this article is available at http://www.circresaha.org.
Mechanical Stress–Induced Heat Shock Protein 70 Expression in Vascular Smooth Muscle Cells Is Regulated by Rac and Ras Small G Proteins but Not Mitogen-Activated Protein Kinases
Authors: Xu Qingbo , Schett Georg , Li Chaohong , Hu Yanhua , Wick Georg
Published: June 2012

Abstract
—Previous studies have documented that acute elevation in blood pressure results in heat shock protein (hsp) 70–mRNA expression followed by hsp70-protein production in rat aortas. In this article, we provide evidence that mechanical forces evoke rapid activation of heat shock transcription factor (HSF) and hsp70 accumulation. In our study, Western blot analysis demonstrated that hsp70-protein induction peaked between 6 and 12 hours after treatment with cyclic stain stress (60 cycles/minute, up to 30% elongation). Elevated protein levels were preceded by hsp70-mRNA transcription, which was associated with HSF1 phosphorylation and activation stimulated by mechanical forces, suggesting that the response was regulated at the transcriptional level. Conditioned medium from cyclic strain–stressed vascular smooth muscle cells (VSMCs) did not result in HSF-DNA–binding activation. Furthermore, mitogen-activated protein kinases (MAPKs), including extracellular signal–regulated kinases, c-Jun NH
2
-terminal protein kinases or stress-activated protein kinases, and p38 MAPKs, were also highly activated in response to cyclic strain stress. Inhibition of extracellular signal–regulated kinase and p38-MAPK activation by their specific inhibitors (PD 98059 and SB 202190) did not influence HSF1 activation. Interestingly, VSMC lines stably expressing dominant-negative rac (rac N17) abolished hsp-protein production and HSF1 activation induced by cyclic strain stress, whereas a significant reduction of hsp70 expression was seen in ras N17–transfected VSMC lines. Thus, our findings demonstrate that cyclic strain stress–induced hsp70 expression is mediated by HSF1 activation and regulated by rac and ras GTP–binding proteins. Induction of hsp70 could be important in maintaining VSMC homeostasis during vascular remodeling in response to hemodynamic stimulation.
Power-Law Behavior of Beat-Rate Variability in Monolayer Cultures of Neonatal Rat Ventricular Myocytes
Authors: Kucera Jan P. , Heuschkel Marc O. , Renaud Philippe , Rohr Stephan
Published: June 2012

Abstract
—It is known that extracardiac factors (nervous, humoral, and hemodynamic) participate in the power-law behavior of heart-rate variability. To assess whether intrinsic properties of cardiac tissue might also be involved, beat-rate variability was studied in spontaneously beating cell cultures devoid of extracardiac influences. Extracellular electrograms were recorded from monolayer cultures of neonatal rat ventricular myocytes under stable incubating conditions for up to 9 hours. The beat-rate time series of these recordings were examined in terms of their Fourier spectra and their Hurst scaling exponents. A non-0 Hurst exponent was found in 21 of 22 preparations (0.29±0.09; range, 0.11 to 0.45), indicating the presence of fractal self-similarity in the beat-rate time series. The same preparations exhibited power-law behavior of the power spectra with a power-law exponent of −1.36±0.24 (range, −1.04 to −1.96) in the frequency range of 0.001 to 1 Hz. Furthermore, it was found that the power-law exponent was nonstationary over time. These results indicate that the power-law behavior of heart-rate variability is determined not only by extracardiac influences but also by components intrinsic to cardiac tissue. Furthermore, the presence of power-law behavior in monolayer cultures of cardiomyocytes suggests that beat-rate variability might be determined by the complex nonlinear dynamics of processes occurring at the level of the cellular network, eg, interactions among a large number of cell oscillators or metabolic regulatory systems.
ADP-Ribosyl Cyclase in Rat Vascular Smooth Muscle Cells
Authors: de Toledo Frederico G. S. , Cheng Jingfei , Liang Mingyu , Chini Eduardo N. , Dousa Thomas P.
Published: June 2012

Abstract
—We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 μmol/L) GT
1B
, GD
1
, and GM
3
. Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by
N-
endoglycosidase F and to thermal inactivation at 45°C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn
2+
and Cu
2+
ions; the inhibition by Zn
2+
was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn
2+
stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-
trans
retinoic acid (atRA), and panagonist 9-
cis
-retinoic acid–upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)
2
–Vitamin D
3
(calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED
50
≅56 pmol/
l
). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta (≅+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3′-triiodothyronine (T
3
) to rats resulted in an increase of ADPR-cyclase activity in aorta (≅+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from “classic” CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn
2+
and Cu
2+
; (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T
3
in vitro; and (3) administration of atRA and T
3
increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.
Prevention of Hypoxia-Induced Pulmonary Hypertension by Enhancement of Endogenous Heme Oxygenase-1 in the Rat
Authors: Christou Helen , Morita Toshisuke , Hsieh Chung-Ming , Koike Hideo , Arkonac Burak , Perrella Mark A. , Kourembanas Stella
Published: June 2012

Abstract
—We investigated the role of heme oxygenase (HO)–1 in the development of hypoxia-induced pulmonary hypertension. HO catalyzes the breakdown of heme to the antioxidant bilirubin and the vasodilator carbon monoxide. Hypoxia is a potent but transient inducer of HO-1 in vascular smooth muscle cells in vitro and in the lung in vivo. By using agonists of HO-1, we sustained a high expression of HO-1 in the lungs of rats for 1 week. We report that this in vivo enhancement of HO-1 in the lung prevented the development of hypoxic pulmonary hypertension and inhibited the structural remodeling of the pulmonary vessels. The mechanism(s) underlying this effect may involve a direct vasodilating and antiproliferative action of endogenous carbon monoxide, as well as an indirect effect of carbon monoxide on the production of vasoconstrictors. These results provide evidence that enhancement of endogenous adaptive responses may be used to prevent hypoxia-induced pulmonary hypertension.
Human Vascular Adhesion Protein-1 (VAP-1) Plays a Critical Role in Lymphocyte–Endothelial Cell Adhesion Cascade Under Shear
Authors: Salmi Marko , Tohka Sami , Jalkanen Sirpa
Published: June 2012

Abstract
—Lymphocyte binding to vascular endothelium is a prerequisite for the movement of immune cells from the blood into lymphoid tissues and into sites of inflammation. Human vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein involved in this interaction. It also displays an enzymatic (monoamine oxidase) activity. Here we examined how recombinant human VAP-1 mediates lymphocyte binding using rotatory and flow chamber binding assays. VAP-1 cDNA transfected into an endothelial cell line, which does not bind lymphocytes, renders the cell line capable of binding lymphocytes in a shear-dependent manner. VAP-1 transfectants bound lymphocytes 5 times better than monocytes with a preference for T killer cells, and no specific granulocyte adherence was detectable. The binding is partially inhibited by anti–VAP-1 monoclonal antibodies or by blocking lymphocyte L-selectin and CD18 integrins, but not by inhibition of several other homing-associated molecules. In contrast, CD44 ligation on lymphocytes markedly upregulates their VAP-1–dependent adhesion, suggesting that the VAP-1 counterreceptor can be activated via CD44. The transfectant model also allowed us to perform detailed structure-function analyses of VAP-1. We show that the exposed integrin-binding motif RGD or the enzymatic activity is not indispensable for VAP-1–dependent adhesion. Together, these data show that VAP-1 can reconstitute the lymphocyte-endothelial adhesion cascade under shear and propose a critical role for VAP-1 in lymphocyte emigration from the blood.
Laminar Shear Stress Inhibits Vascular Endothelial Cell Proliferation by Inducing Cyclin-Dependent Kinase Inhibitor p21

Sdi1/Cip1/Waf1
Authors: Akimoto Shigeo , Mitsumata Masako , Sasaguri Toshiyuki , Yoshida Yoji
Published: June 2012

Abstract
—Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and human umbilical vein ECs, determined by [
3
H]thymidine incorporation, was inhibited by steady laminar shear stress (5 and 30 dyne/cm
2
). This growth inhibition due to shear stress was associated with suppression of cell transition from the G
1
to S phase of the cell cycle. Therefore, we studied G
1
-phase events to find the molecules responsible for this cell cycle arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21

Sdi1/Cip1/Waf1

protein, but not that of p27

Kip1

, increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis through the induction of EC stability disruption.
Roles of Mitogen-Activated Protein Kinases and Protein Kinase C in α
1A
-Adrenoceptor–Mediated Stimulation of the Sarcolemmal Na
+
-H
+
Exchanger
Authors: Snabaitis Andrew K. , Yokoyama Hiroyuki , Avkiran Metin
Published: June 2012

Abstract
—Activation of the sarcolemmal Na
+
-H
+
exchanger (NHE) has been implicated as a mechanism of inotropic, arrhythmogenic, antiacidotic, and hypertrophic effects of α
1
-adrenoceptor (AR) stimulation. Although such regulation of sarcolemmal NHE activity has been shown to be selectively mediated through the α
1A
-AR subtype, distal signaling mechanisms remain poorly defined. We investigated the roles of various kinase pathways in α
1A
-AR–mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. As an index of NHE activity,
trans
-sarcolemmal acid efflux rate (
J
H
) was determined through microepifluorescence in single cells, during recovery from intracellular acidosis in bicarbonate-free conditions. Extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (MAPK), and p90
rsk
activities were indexed on the basis of analysis of their phosphorylation status. In control cells, there was no change in
J
H
in response to vehicle. Phenylephrine and A61603, an α
1A
-AR subtype–selective agonist, increased
J
H
, as well as cellular ERK and p90
rsk
activities. Neither agonist affected p38 activity, which was increased with sorbitol. The MAPK kinase inhibitor PD98059 abolished phenylephrine- and A61603-induced increases in
J
H
and cellular ERK and p90
rsk
activities. In contrast, the PKC inhibitor GF109203X abolished phenylephrine- and A61603-induced increases in
J
H
but failed to prevent the increases in ERK and p90
rsk
activities. Our findings suggest that α
1A
-AR–mediated stimulation of sarcolemmal NHE activity in rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade and that the ERK-mediated effect may occur via p90
rsk
. Activation of PKC is also required for α
1A
-AR–mediated NHE stimulation, but such regulation occurs through an ERK-independent pathway.
Myocardial Glucose Uptake Is Regulated by Nitric Oxide via Endothelial Nitric Oxide Synthase in Langendorff Mouse Heart
Authors: Tada Hideo , Thompson Carl I. , Recchia Fabio A. , Loke Kit E. , Ochoa Manuel , Smith Carolyne J. , Shesely Edward G. , Kaley Gabor , Hintze Thomas H.
Published: June 2012

Abstract
—Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (−/−)], and its heterozygote [ecNOS (+/−)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of
N
ω
-nitro-
l
-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (−/−), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/−) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/−) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g · beats/min, L-NAME increased glucose uptake from 0.1±0.1 to 3±0.4 μg/min · mg in ecNOS (+/−) mice and from 0.2±0.1 to 2.7±0.7 μg/min · mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (−/−) mice, 8-bromoguanosine 3′:5′-cyclic monophosphate (8-Br-cGMP), a cGMP analog or
S
-nitroso-
N
-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/−) mice, 1
H
-[1,2,4]oxadiazolo[4,3,-
a
]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.
Immunoglobulin Isotype Determines Pathogenicity in Antibody-Mediated Myocarditis in Naïve Mice
Authors: Kuan Anita P. , Zuckier Lionel , Liao Li , Factor Stephen M. , Diamond Betty
Published: June 2012

Abstract
—Antimyosin reactivity is associated with cardiac damage in autoimmune myocarditis, an inflammatory heart disease characterized by a cellular infiltrate in the myocardium and myocyte necrosis. We are interested in the pathogenicity of antimyosin antibodies and their ability to cause autoimmune myocarditis. We have shown that antimyosin antibodies of the IgG isotype will induce disease in the DBA/2 mouse. In the present study, we show that IgM antimyosin antibodies do not induce myocarditis; however, these same antibodies become pathogenic when converted to the IgG isotype. Although IgM antibodies can penetrate the myocardium during cardiac inflammation, they are usually less able to leave the vascular compartment and penetrate cardiac tissue, thus accounting for their lack of pathogenicity. Thus, antimyosin B cells may be potentially pathogenic only after antigen activation and heavy chain class switching or under conditions that alter vascular permeability in the heart.
The ε Subtype of Protein Kinase C Is Required for Cardiomyocyte Connexin-43 Phosphorylation
Authors: Doble Bradley W. , Ping Peipei , Kardami Elissavet
Published: June 2012

Abstract
—Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the ε-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2–induced Cx43 phosphorylation. In nonstimulated myocytes, PKCε displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCε distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCε. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCε but not PKCα coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCε, suggesting increased association between PKCε and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCε decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2–induced effects on cardiac GJs and that PKCε likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.