bluuebunny/crossref_metadata_2025 huggingface dataset:
Tie2 Receptor Ligands, Angiopoietin-1 and Angiopoietin-2, Modulate VEGF-Induced Postnatal Neovascularization
Authors: Asahara Takayuki , Chen Donghui , Takahashi Tomono , Fujikawa Koshi , Kearney Marianne , Magner Meredith , Yancopoulos George D. , Isner Jeffrey M.
Published: June 2012

Abstract
—Angiopoietin-1 (Ang1) has been recently identified as the major physiological ligand for the tyrosine kinase receptor Tie2 and assigned responsibility for recruiting and sustaining periendothelial support cells. Angiopoietin-2 (Ang2) was found to disrupt blood vessel formation in the developing embryo by antagonizing the effects of Ang1 and Tie2 and was thus considered to represent a natural Ang1/Tie2 inhibitor. In vivo effects of either angiopoietin on postnatal neovascularization, however, have not been previously described. Accordingly, we used the cornea micropocket assay of neovascularization to investigate the impact of angiopoietins on neovascularization in vivo. Neither Ang1 nor Ang2 alone promoted neovascularization. Pellets containing vascular endothelial growth factor (VEGF) alone induced corneal neovascularity extending from the limbus across the cornea. Addition of Ang1 to VEGF (Ang1+VEGF) produced an increase in macroscopically evident perfusion of the corneal neovasculature without affecting macroscopic measurements of length (0.58±0.03 mm) or circumferential neovascularity (136±10°). In contrast, pellets containing Ang2+VEGF promoted significantly longer (0.67±0.05 mm) and more circumferential (160±15°) neovascularity than VEGF alone or Ang1+VEGF (
P
<0.05). Excess soluble Tie2 receptor (sTie2-Fc) precluded modulation of VEGF-induced neovascularization by both Ang2 and Ang1. Fluorescent microscopic findings demonstrated enhanced capillary density (fluorescence intensity, 2.55±0.23 e
+9
versus 1.23±0.17 e
+9
,
P
<0.01) and increased luminal diameter of the basal limbus artery (39.0±2.8 versus 27.9±1.3 μm,
P
<0.01) for Ang1+VEGF compared with VEGF alone. In contrast to Ang1+VEGF, Ang2+VEGF produced longer vessels and, at the tip of the developing capillaries, frequent isolated sprouting cells. In the case of Ang2+VEGF, however, luminal diameter of the basal limbus artery was not increased (26.7±1.9 versus 27.9±1.3,
P
=NS). These findings constitute what is to our knowledge the first direct demonstration of postnatal bioactivity associated with either angiopoietin. In particular, these results indicate that angiopoietins may potentiate the effects of other angiogenic cytokines. Moreover, these findings provide in vivo evidence that Ang1 promotes vascular network maturation, whereas Ang2 works to initiate neovascularization.
AML1-like Transcription Factor Induces Serine Elastase Activity in Ovine Pulmonary Artery Smooth Muscle Cells
Authors: Wigle Dennis A. , Thompson Karen E. , Yablonsky Shmuel , Zaidi Syed H. E. , Coulber Claire , Jones Peter L. , Rabinovitch Marlene
Published: June 2012

Abstract
—In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of ≈2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells
.
Inducible Expression of Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells
Authors: Kume Noriaki , Murase Takatoshi , Moriwaki Hideaki , Aoyama Takuma , Sawamura Tatsuya , Masaki Tomoh , Kita Toru
Published: June 2012

Abstract
—Endothelial dysfunction, or activation, elicited by oxidized LDL (Ox-LDL) or its lipid constituent, has been implicated in the initiation and progression of atherosclerosis. We have recently identified a C-type lectin-like molecule, designated lectin-like Ox-LDL receptor-1 (LOX-1), which acts as a cell-surface receptor for Ox-LDL in cultured vascular endothelial cells. In this study, we provide evidence that LOX-1 expression can be upregulated by tumor necrosis factor-α (TNF-α) and phorbol 12-myristate 13-acetate (PMA) in cultured bovine aortic endothelial cells. TNF-α and PMA upregulated LOX-1 protein and mRNA in a time- and dose-dependent manner. Nuclear runoff assay revealed that TNF-α stimulates transcription of the LOX-1 gene. Chinese hamster ovary K1 cells stably expressing LOX-1 internalized 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled Ox-LDL but did not significantly internalize acetylated LDL (Ac-LDL), which was effectively suppressed by excess amounts of unlabeled Ox-LDL but not by Ac-LDL. Upregulated expression of LOX-1 by TNF-α and PMA was associated with increased uptake of DiI-Ox-LDL that cannot be blocked by excess amounts of unlabeled Ac-LDL. Taken together, LOX-1 is a receptor specific for Ox-LDL, and enhanced uptake of Ox-LDL via this novel receptor on vascular endothelial cells may play an important role in endothelial activation in atherogenesis.
Fluid Shear Stress Stimulates Phosphorylation of Akt in Human Endothelial Cells
Authors: Dimmeler Stefanie , Assmus Birgit , Hermann Corinna , Haendeler Judith , Zeiher Andreas M.
Published: June 2012

Abstract
—Fluid shear stress alters the morphology and function of the endothelium by activating several kinases. Furthermore, shear stress potently inhibits apoptosis of endothelial cells. Since activation of Akt kinase has been shown to prevent cell death, we investigated the effects of shear stress on Akt phosphorylation. To test the hypothesis that shear stress interacts with the Akt kinase pathway, human umbilical venous endothelial cells were exposed to laminar shear stress (15 dyne/cm
2
). Western blotting with specific antibodies against the phosphorylated Akt demonstrated a time-dependent stimulation of Akt phosphorylation by shear stress with a maximal increase up to 6-fold after 1 hour of shear stress exposure. The stimulation of Akt phosphorylation by shear stress thereby seemed to be mediated by the phosphoinositide 3-OH kinase (PI3K), as evidenced by the significant inhibition of shear stress–induced Akt phosphorylation by the PI3K inhibitors wortmannin (20 nmol/L) and Ly294002 (10 μmol/L). In addition, pharmacological inhibition of PI3K reduced the antiapoptotic effect of shear stress against growth factor depletion–induced apoptosis. Most important, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of shear stress against serum depletion–induced apoptosis, thus indicating the direct involvement of shear stress–induced Akt phosphorylation for inhibition of endothelial cell apoptosis. These results define a novel shear stress–stimulated signal transduction pathway, namely, activation of the serine/threonine kinase Akt, which may contribute to the profound changes in endothelial morphology and function by shear stress.
Vitamin E Combined With Selenium Inhibits Atherosclerosis in Hypercholesterolemic Rabbits Independently of Effects on Plasma Cholesterol Concentrations
Authors: Schwenke Dawn C. , Behr Stephen R.
Published: June 2012

Abstract
—Several antioxidants inhibit atherosclerosis. This study investigated the hypothesis that combining vitamin E, a lipophilic antioxidant, with vitamin C, a hydrophilic antioxidant, and/or selenium, a cofactor of peroxidases that detoxify lipid peroxides, would inhibit atherosclerosis more effectively than vitamin E alone. We also considered whether regional variation in inhibition of atherosclerosis by antioxidants would be associated with regional variation in aortic lipophilic antioxidants. Rabbits were fed an atherogenic diet (control) or an atherogenic diet supplemented with vitamin E, vitamins E and C, vitamin E+selenium, vitamins E and C+selenium, or probucol (positive control). Supplements were as follows: vitamin E, 146 IU/d; vitamin C, 791 mg/d; selenium, 22 μg/d; or probucol, 406 mg/d. Vitamin C did not influence atherosclerosis. After 22 weeks of treatment, rank order of aortic atherosclerosis was control>vitamin E (with or without vitamin C)>vitamin E+selenium (with or without vitamin C)>probucol. Antioxidant treatment reduced aortic cholesterol concentrations 21% to 56%, 29% to 86%, and 19% to 75% for the aortic arch, descending thoracic aorta, and abdominal aorta, respectively (
P
<0.025 to
P
<0.0003 by ANOVA), with slightly greatly reductions for areas of atherosclerotic lesions. Some treatments reduced plasma cholesterol concentrations, but none altered the distribution of cholesterol among lipoproteins. Corrected for differences in plasma cholesterol concentrations, aortic cholesterol concentrations were reduced up to 72% (
P
<0.02) by the antioxidant treatments, with equal reductions by vitamin E+selenium and by probucol. Aortic α-tocopherol standardized by aortic cholesterol as a measure of aortic lipids was lower in the abdominal aorta than in the aortic arch of rabbits not given α-tocopherol and increased relatively more in the abdominal aorta than in the aortic arch with α-tocopherol supplementation. The results of this study suggest that vitamin E+selenium inhibited atherosclerosis as effectively as an equally hypocholesterolemic dose of probucol by a mechanism(s) that is in part independent of effects on plasma and lipoprotein cholesterol concentrations. The tendency for greater efficacy of antioxidant treatments in the abdominal aorta than aortic arch may relate to the lower concentrations of α-tocopherol in the abdominal aorta of unsupplemented rabbits.
Induction of Heme Oxygenase-1 During Endotoxemia Is Downregulated by Transforming Growth Factor-β1
Authors: Pellacani Andrea , Wiesel Philippe , Sharma Arunabh , Foster Lauren C. , Huggins Gordon S. , Yet Shaw-Fang , Perrella Mark A.
Published: June 2012

Abstract
—Heme oxygenase (HO)-1 generates CO, a gas with vasodilatory properties, during heme metabolism. HO-1 is expressed highly in vascular tissue after endotoxin stimulation, and generation of CO through the HO-1 pathway contributes to the hemodynamic compromise of endotoxic shock. Shock related to endotoxemia is an immune-mediated process that involves the generation of proinflammatory cytokines such as interleukin (IL)-1β. Because transforming growth factor (TGF)-β1 is a modulator of immune-mediated inflammatory responses and it blocks the hypotension of endotoxic shock, we determined whether TGF-β1 could be used to reduce expression of HO-1 in vascular tissue and smooth muscle cells. In a rat model of endotoxic shock, lipopolysaccharide-induced HO-1 mRNA and protein expression was reduced by TGF-β1 in highly vascularized tissue, such as heart and lung, by Northern and Western analysis. Furthermore, TGF-β1 downregulated HO-1 mRNA after its induction by IL-1β in vascular smooth muscle cells in culture. TGF-β1 also decreased HO-1 but not HO-2 protein expression in these cells. TGF-β1 decreased HO enzyme activity induced in IL-1β–treated vascular smooth muscle cells to a level not different from that in vehicle-treated cells. These studies suggest that this downregulation of HO-1 mRNA and protein expression and decrease in IL-1β–induced HO enzyme activity may contribute to the beneficial effect of TGF-β1 on endotoxic shock.
Novel Mechanism of HERG Current Suppression in LQT2
Authors: Nakajima Tadashi , Furukawa Tetsushi , Tanaka Toshihiro , Katayama Yoshifumi , Nagai Ryozo , Nakamura Yusuke , Hiraoka Masayasu
Published: June 2012

Abstract
—In a
Xenopus
oocyte heterologous expression system, we characterized the electrophysiology of 3 novel missense mutations of
HERG
identified in Japanese LQT2 families: T474I (within the S2-S3 linker), A614V, and V630L (in the outer mouth of pore-forming region). For each of the 3 mutations, injection of mutant cRNA alone did not express detectable currents. Coinjection of wild-type (WT) along with each mutant cRNA (T474I/WT, A614V/WT, and V630L/WT) suppressed HERG current in a dominant-negative manner, and the order of magnitude of current suppression was V630L/WT>A614V/WT>T474I/WT. In addition to decreases in slope conductance for all 3 mutants, the voltage dependence of steady-state inactivation was shifted to negative potentials for V630L/WT and A614V/WT. Consequently, channel availability at positive potentials was diminished, and inward rectification was enhanced for these 2 mutants. Thus, missense mutations of
HERG
caused dominant-negative suppression through multiple mechanisms. The shift in voltage dependence of HERG inactivation and the resulting enhanced inward rectification in A614V/WT and V630L/WT provide a novel mechanism for suppression of the HERG current carrying outward current during the repolarization phase of the action potential.
Lidocaine Action on Na
+
Currents in Ventricular Myocytes From the Epicardial Border Zone of the Infarcted Heart
Authors: Pu Jielin , Balser Jeffrey R. , Boyden Penelope A.
Published: June 2012

Abstract
—Myocytes overlying a zone of infarction form the primary substrate for serious reentrant ventricular arrhythmias. In vitro and in vivo studies suggest that antiarrhythmic agents affect Na
+
channels of cells from the epicardial border zone (EBZ) of the 5-day infarcted heart differently than they affect those of normal muscle. However, the mechanisms responsible for this difference remain unclear. Previous studies have revealed differences in Na
+
current (
I
Na
) density and inactivation gating kinetics in myocytes dispersed from the EBZ (IZs). Since changes in inactivation gating could influence lidocaine action, we examined the effects of lidocaine on
I
Na
of IZs (n=38) and epicardial myocytes from the noninfarcted heart (NZs) (n=50) using the whole-cell variation of the patch-clamp technique. In drug-free conditions, the voltage dependence of steady-state inactivation of IZs was shifted negative to that of NZs, causing greater inactivation of IZ channels at depolarized (≥−100-mV) holding potentials. Consistent with a high affinity for the inactivated channel conformation, lidocaine produced more tonic block in IZs than NZs at depolarized holding potentials. Additionally, in drug-free conditions, IZ
I
Na
exhibited an enhanced rate of inactivation from closed states, a delay in recovery from inactivation, and increased use-dependent reduction in amplitude during rapid (1- to 3-Hz) pulse trains. In both IZs and NZs, lidocaine (20 to 120 μmol/L) accelerated the rate of time-dependent loss of availability and markedly delayed recovery from availability, inducing significant use-dependent reduction of
I
Na
. However, at drug concentrations ≥60 μmol/L, the difference in use-dependent current reduction between IZs and NZs was minimized. The action of lidocaine to render Na
+
channel inactivation in NZs more similar to that of IZs may be central to its (pro)antiarrhythmic effects.
Versican Expression Is Associated With Chamber Specification, Septation, and Valvulogenesis in the Developing Mouse Heart
Authors: Henderson Deborah J. , Copp Andrew J.
Published: June 2012

Abstract
—The versican (PG-M) gene encodes a chondroitin sulfate proteoglycan that is nonpermissive for cell migration and appears in association with slow cell proliferation and cytodifferentiation. Using the techniques of in situ hybridization and immunocytochemistry on sectioned mouse embryos, we found that the mRNA and protein for versican show similar distributions and are expressed in a dynamic pattern during development of the heart. Versican exhibits generalized expression in the tubular heart but becomes rapidly downregulated in the atrium and exhibits higher transcript levels on the right side of the ventricular chamber than the left, before the onset of ventricular septation. Versican is expressed strongly in the trabeculated ventricular myocardium, whereas the compact proliferative zone has lower transcript abundance. It is expressed in the outer layers and on the crest of the ventricular septum and is prominent on the mesenchymal cap of the primary atrial septum. Versican is particularly strongly expressed in the endocardial cushions of the atrioventricular and outflow tract regions and in the atrioventricular, semilunar, and venous valves. This study raises the possibility that versican may be involved in specification of the ventricular chambers, in growth and fusion of the atrial and ventricular septa, and in the transformation from epithelium to mesenchyme that characterizes development of the endocardial cushions. Versican may be a key participant in cardiogenesis, responding to the many diffusible signals that mediate interactions between the developing endocardium and myocardium.
Plasmalogen-Derived Lysolipid Induces a Depolarizing Cation Current in Rabbit Ventricular Myocytes
Authors: Caldwell Ray A. , Baumgarten Clive M.
Published: June 2012

Abstract
—Plasmalogen rather than diacyl phospholipids are the preferred substrate for the cardiac phospholipase A
2
(PLA
2
) isoform activated during ischemia. The diacyl metabolite, lysophosphatidylcholine, is arrhythmogenic, but the effects of the plasmalogen metabolite, lysoplasmenylcholine (LPLC), are essentially unknown. We found that 2.5 and 5 μmol/L LPLC induced spontaneous contractions of intact isolated rabbit ventricular myocytes (median times, 27.4 and 16.4 minutes, respectively) significantly faster than lysophosphatidylcholine (>60 and 37.8 minutes, respectively). Whole-cell recordings revealed that LPLC depolarized the resting membrane potential from –83.5±0.2 to –21.5±1.0 mV. Depolarization was due to a guanidinium toxin–insensitive Na
+
influx. The LPLC-induced current reversed at –18.5±0.9 mV and was shifted 26.7±4.2 mV negative by a 10-fold reduction of bath Na
+
(Na
+
/K
+
permeability ratio, ≈ 0.12±0.06). In contrast, block of Ca
2+
channels with Cd
2+
and reducing bath Cl
–
failed to affect the current. The actions of LPLC were opposed by lanthanides. Gd
3+
and La
3+
were equally effective inhibitors of the LPLC-induced current and equally delayed the onset of spontaneous contractions. However, the characteristics of lanthanide block imply that Gd
3+
-sensitive, poorly selective, stretch-activated channels were not involved. Instead, the data are consistent with the view that lanthanides increase phospholipid ordering and may thereby oppose membrane perturbations caused by LPLC. Plasmalogens constitute a significant fraction of cardiac sarcolemmal choline phospholipids. In light of their subclass-specific catabolism by phospholipase A
2
and the present results, it is suggested that LPLC accumulation may contribute to ventricular dysrhythmias during ischemia.
Functional Knockout of the Transient Outward Current, Long-QT Syndrome, and Cardiac Remodeling in Mice Expressing a Dominant-Negative Kv4 α Subunit
Authors: Barry Dianne M. , Xu Haodong , Schuessler Richard B. , Nerbonne Jeanne M.
Published: June 2012

Abstract
—A novel in vivo experimental strategy, involving cell type–specific expression of a dominant-negative K
+
channel pore-forming α subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K
+
current (
I
to
). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the α-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that
I
to
is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies
I
to
in the mammalian heart. Functional knockout of
I
to
leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K
+
current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K
+
channels is altered.
Electrophysiological Basis of Arrhythmogenicity of QT/T Alternans in the Long-QT Syndrome
Authors: Chinushi Masaomi , Restivo Mark , Caref Edward B. , El-Sherif Nabil
Published: June 2012

Abstract
—Tachycardia-dependent QT/T alternans occurs in patients with the congenital or idiopathic form of long-QT syndrome (LQTS) and may presage the onset of polymorphic ventricular tachyarrhythmias. To examine the electrophysiological basis of arrhythmogenicity of QT/T alternans in LQTS, the tridimensional repolarization pattern of QT/T alternans was studied in the anthopleurin-A model of LQTS, a surrogate for LQT3. In 11 anesthetized mongrel puppies, tridimensional repolarization and activation patterns were analyzed from 256 to 384 unipolar electrograms. Cardiac repolarization was evaluated as the activation-recovery interval (ARI) of local electrograms. To induce QT/T alternans, the pacing cycle length (CL) was abruptly shortened in steps of 50 ms from a basic drive of 1000 ms. ARIs were calculated at epicardial (Epi), midmyocardial (Mid), and endocardial (End) sites. ARI restitution at each site was assessed by using a single premature stimulation delivered after the basic drive. ARI alternans occurred at longer CLs at Mid sites compared with End and Epi sites, and the magnitude of alternans at Mid sites was greater. Two factors contributed to the modulation of ARI during QT/T alternans: (1) differences in restitution kinetics at Mid sites, characterized by larger ΔARI and a slower time constant (τ), and (2) differences in diastolic intervals resulting in different input to restitution at the same constant CL. These 2 factors could explain not only the onset of alternans at Mid sites at longer CLs but also the critical observation that ARI dispersion between Epi and Mid sites during alternans was greater than during the slower basic CL. Marked ARI alternans could be present in local electrograms without manifest alternation of the QT/T segment in the surface ECG. The latter was seen at critically short CLs associated with reversal of the gradient of ARI between Epi and Mid sites, with a consequent reversal of polarity of the intramyocardial QT wave in alternate cycles. The arrhythmogenicity of QT/T alternans was primarily due to the greater degree of spatial dispersion of repolarization during alternans than during slower rates not associated with alternans. This could result in functional conduction block and reentrant ventricular tachyarrhythmias during the fixed drive associated with alternans.
Endothelial NADPH Oxidase as the Source of Oxidants in Lungs Exposed to Ischemia or High K
+
Authors: Al-Mehdi Abu B. , Zhao Guochang , Dodia Chandra , Tozawa Kasumi , Costa Karen , Muzykantov Vladimir , Ross Chris , Blecha Frank , Dinauer Mary , Fisher Aron B.
Published: June 2012

Abstract
—We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K
+
and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K
+
. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 μmol/L PR-39. ROS production in lungs exposed to high K
+
or ischemia was essentially abolished in mice with a “knockout” of gp91
phox
, a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control
.
PR-39 also inhibited ischemia and the high K
+
–mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47
phox
, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K
+
results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.
Slow Conduction in Cardiac Tissue, II
Authors: Kucera Jan P. , Kléber André G. , Rohr Stephan
Published: June 2012

Abstract
—In cardiac tissue, functional or structural current-to-load mismatches can induce local slow conduction or conduction block, which are important determinants of reentrant arrhythmias. This study tested whether spatially repetitive mismatches result in a steady-state slowing of conduction. Patterned growth of neonatal rat heart cells in culture was used to design unbranched cell strands or strands releasing branches from either a single point or multiple points at periodic intervals. Electrical activation was followed optically using voltage-sensitive dyes under control conditions and in elevated [K
+
]
o
(5.8 and 14.8 mmol/L, respectively; in the latter case, propagation was carried by the L-type Ca
2+
current). Preparations with multiple branch points exhibited discontinuous and slow conduction that became slower with increasing branch length and/or decreasing inter-branch distance. Compared with unbranched strands, conduction was maximally slowed by 63% under control conditions (from 44.9±3.4 to 16.7±1.0 cm/s) and by 93% in elevated [K
+
]
o
(from 15.7±2.3 to 1.1±0.2 cm/s). Local activation delays induced at a single branch point were significantly larger than the delays per branch point in multiple branching structures. Also, selective inactivation of inward currents in the branches induced conduction blocks. These 2 observations pointed to a dual role of the branches in propagation: whereas they acted as current sinks for the approaching activation thus slowing conduction (“pull” effect), they supplied, once excited, depolarizing current supporting downstream activation (“push” effect). This “pull and push” action resulted in a slowing of conduction in which the safety was largely preserved by the “push” effect. Thus, branching microarchitectures might contribute to slow conduction in tissue with discontinuous geometry, such as infarct scars and the atrioventricular node.
Overexpression of the Sarcolemmal Calcium Pump in the Myocardium of Transgenic Rats
Authors: Hammes Annette , Oberdorf-Maass Silke , Rother Tobias , Nething Katja , Gollnick Frank , Linz Klaus W. , Meyer Rainer , Hu Kai , Han Hong , Gaudron Peter , Ertl Georg , Hoffmann Sigrid , Ganten Ursula , Vetter Roland , Schuh Kai , Benkwitz Claudia , Zimmer Hans G. , Neyses Ludwig
Published: June 2012

Abstract
—The plasma membrane calmodulin–dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca
2+
homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na
+
/Ca
2+
exchanger. We approached the question of the physiological function of the calcium pump using a transgenic “gain of function” model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the
L
-type Ca
2+
current, or fast [Ca
2+
]
i
transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 μmol/L phenylephrine. A 1.4-fold– and 2.0-fold–higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the α- and β-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of α- and β-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca
2+
-pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.
Mechanical Stretch Activates the JAK/STAT Pathway in Rat Cardiomyocytes
Authors: Pan Jing , Fukuda Keiichi , Saito Mikiyoshi , Matsuzaki Junichi , Kodama Hiroaki , Sano Motoaki , Takahashi Toshiyuki , Kato Takahiro , Ogawa Satoshi
Published: June 2012

Abstract
—This study was designed to determine whether mechanical stretch activates the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and, if so, by what mechanism. Neonatal rat/murine cardiomyocytes were cultured on malleable silicone dishes and were stretched by 20%. Mechanical stretch induced rapid phosphorylation of JAK1, JAK2, Tyk2, STAT1, STAT3, and glycoprotein 130 as early as 2 minutes and peaked at 5 to 15 minutes. It also caused gel mobility shift of sis-inducing element, which was supershifted by preincubation with anti-STAT3 antibody. Preincubation with CV11974 (AT
1
blocker) partially inhibited the phosphorylation of STAT1, but not that of STAT3. Preincubation with TAK044 (endothelin-1-type A/B-receptor blocker) did not attenuate this pathway. RX435 (anti–glycoprotein 130 blocking antibody) inhibited the phosphorylation of STAT3 and partially inhibited that of STAT1. Phosphorylation of STAT1 and STAT3 was strongly inhibited by HOE642 (Na
+
/H
+
exchanger inhibitor) and BAPTA-AM (intracellular calcium chelator), but not by gadolinium (stretch-activated ion channel inhibitor), EGTA (extracellular Ca
2+
chelator), or KN62 (Ca
2+
/calmodulin kinase II inhibitor). Chelerythrine (protein kinase C inhibitor) partially inhibited the phosphorylation of STAT1 and STAT3. Mechanical stretch also augmented the mRNA expression of cardiotrophin-1, interleukin-6, and leukemia inhibitory factor at 60 to 120 minutes. These results indicated that the JAK/STAT pathway was activated by mechanical stretch, and that this activation was partially dependent on autocrine/paracrine–secreted angiotensin II and was mainly dependent on the interleukin-6 family of cytokines but was independent of endothelin-1. Moreover, certain levels of intracellular Ca
2+
were necessary for stretch-induced activation of this pathway, and protein kinase C was also partially involved in this activation.
Chemiluminescent Detection of Oxidants in Vascular Tissue
Authors: Tarpey Margaret M. , White C. Roger , Suarez Edward , Richardson Gloria , Radi Rafael , Freeman Bruce A.
Published: June 2012

Abstract
—Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to reaction mixtures of xanthine oxidase plus NADH resulted in increased oxygen consumption, as well as superoxide dismutase–inhibitable reduction of cytochrome
c
, indicative of enhanced rates of superoxide formation. Additionally, it was revealed that lucigenin stimulated oxidant formation by both cultured bovine aortic endothelial cells and isolated rings from rat aorta. Lucigenin treatment resulted in enhanced hydrogen peroxide release from endothelial cells, whereas exposure to lucigenin resulted in inhibition of endothelium-dependent relaxation in isolated aortic rings that was superoxide dismutase inhibitable. In contrast, the chemiluminescent probe coelenterazine had no significant effect on xanthine oxidase–dependent oxygen consumption, endothelial cell hydrogen peroxide release, or endothelium-dependent relaxation. Study of enzyme and vascular systems indicated that coelenterazine chemiluminescence is a sensitive marker for detecting both superoxide and peroxynitrite.
Direct Preconditioning of Cardiac Myocytes via Opioid Receptors and K
ATP
Channels
Authors: Liang Bruce T. , Gross Garrett J.
Published: June 2012

Abstract
—Previous studies demonstrated that opioid receptor activation mimics the cardioprotective effect of ischemic preconditioning via K
ATP
channels in the intact heart. However, it is unknown whether this beneficial effect is exerted at the level of the cardiac myocyte or coronary vasculature or is mediated via the sarcolemmal or the mitochondrial K
ATP
channel. Thus, the purpose of the present study was to investigate whether opioid receptor stimulation could mimic the cardioprotective effect of preconditioning in a cardiac myocyte model of simulated ischemia. Cardiac ventricular myocytes cultured from chick embryos 14 days in ovo were used as an in vitro model for ischemic preconditioning. A 5-minute exposure of the myocytes to the opioid receptor agonist morphine protected the myocytes during a subsequent 90-minute period of simulated ischemia, which was manifested as a pronounced reduction in the percentage of cardiac cells killed and the amount of creatine kinase released during ischemia. The preconditioning-like effect of morphine was concentration-dependent, reached a maximal effect at 1 μmol/L, and was reversed by naloxone (0.1 to 10 μmol/L). When K
ATP
channel antagonists, such as glibenclamide, or the mitochondrial selective inhibitor 5-hydroxydecanoic acid were present during preexposure to morphine, they abolished the protective effect of morphine. Thus, cardiac myocytes express functional opioid receptors, and their activation mimics the cardioprotective effect of ischemic preconditioning. These results provide direct evidence that the preconditioning-like effect of morphine in the intact heart can be exerted at the level of cardiac myocytes and is most likely the result of mitochondrial K
ATP
channel activation.
Deficient Platelet-Derived Nitric Oxide and Enhanced Hemostasis in Mice Lacking the NOSIII Gene
Authors: Freedman Jane E. , Sauter Ryan , Battinelli Elisabeth M. , Ault Kenneth , Knowles Cathy , Huang Paul L. , Loscalzo Joseph
Published: June 2012

Abstract
—Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2±3 versus 133.4±3 seconds,
P
<0.00005). To determine the contribution of endothelium- versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (Δbleeding time, −24.6±9.1 and −3.4±5.3 seconds, respectively;
P
<0.04). Platelet recruitment was studied by measuring serotonin release from a second recruitable population of platelets that were added to stimulated platelets at the peak of NO production. There was 40.3±3.7% and 52.0±2.1% serotonin release for platelets added to wild-type or eNOS-deficient platelets, respectively (
P
<0.05). In summary, mice that lacked eNOS had markedly decreased bleeding times even after endothelial NO production was controlled. These data suggest that the lack of platelet-derived NO alters in vivo hemostatic response by increasing platelet recruitment. Thus, these data support a role for platelet-derived NO production in the regulation of hemostasis.
Potential Role of a Membrane-Bound NADH Oxidoreductase in Nitric Oxide Release and Arterial Relaxation to Nitroprusside
Authors: Mohazzab-H. Kamal M. , Kaminski Pawel M. , Agarwal Ritu , Wolin Michael S.
Published: June 2012

Abstract
—The site of metabolism in vascular smooth muscle responsible for the release of nitric oxide (NO) from nitroprusside is not well established. In this study we observed that a membrane-bound NADH oxidoreductase in the pulmonary artery activates nitroprusside to release NO, and we examined whether this process could potentially participate in relaxation to nitroprusside. Relaxation to nitroprusside in bovine calf pulmonary artery is inhibited by a scavenger of NO and by an antagonist of NO stimulation of guanylate cyclase. A flavoprotein probe that inhibits pulmonary artery NADH oxidoreductase (1 μmol/L diphenyliodonium) and electron acceptors for NADH oxidoreductase (0.3 mmol/L nitroblue tetrazolium and 0.1 mmol/L ferricyanide) inhibited pulmonary artery relaxation to nitroprusside, but not to nitroglycerin. Pulmonary arteries were observed to promote the release of NO from nitroprusside in vitro, and NO release was inhibited by the presence of nitroblue tetrazolium, ferricyanide, and diphenyliodonium. In homogenates of pulmonary arteries, NADH (0.1 mmol/L) increased the release of NO from nitroprusside by ≈6-fold, whereas NADPH, mitochondrial substrates, and other redox cofactors had minimal effects on NO release, and the action of NADH on nitroprusside was inhibited by nitroblue tetrazolium, ferricyanide, and diphenyliodonium. A membrane fraction enriched in NADH oxidoreductase activity showed a NADH-dependent release of NO from nitroprusside; nitroprusside caused NADH consumption, and it also inhibited the NADH-dependent reduction of nitroblue tetrazolium. Thus, a membrane-bound NADH oxidoreductase appears to contribute to the release of NO from nitroprusside, but not nitroglycerin, in calf pulmonary artery.
Alteration of Endothelium-Dependent Hyperpolarizations in Porcine Coronary Arteries With Regenerated Endothelium
Authors: Thollon Catherine , Bidouard Jean P. , Cambarrat Christine , Delescluse Isabelle , Villeneuve Nicole , Vanhoutte Paul M. , Vilaine Jean P.
Published: June 2012

Abstract
—The present study was designed to test the ability of regenerated endothelium to evoke endothelium-dependent hyperpolarizations. Hyperpolarizations induced by serotonin and bradykinin were compared in isolated porcine coronary arteries with native or regenerated endothelium, 4 weeks after balloon endothelial denudation. The experiments were performed in the presence of inhibitors of nitric oxide synthase (
N
ω
-nitro-
l
-arginine) and cyclooxygenase (indomethacin). The transmembrane potential was measured using conventional glass microelectrodes. Smooth muscle cells from coronary arteries with regenerated endothelium were depolarized in comparison with control coronary arteries from the same hearts. Spontaneous membrane potential oscillations of small amplitude or spikes were observed in some of these arteries but never in arteries with native endothelium. In coronary arteries from control pigs, both serotonin and bradykinin induced concentration-dependent hyperpolarizations. In the presence of ketanserin, 10 μmol/L serotonin induced a transient hyperpolarization in control coronary arteries. Four weeks after balloon denudation, the response to serotonin was normal in arteries with native endothelium, but the hyperpolarization was significantly lower in coronary arteries with regenerated endothelium. In control arteries, the endothelium-dependent hyperpolarization obtained with bradykinin (30 nmol/L) was reproducible. Four weeks after balloon denudation, comparable hyperpolarizations were obtained in coronary arteries with native endothelium. By contrast, in arteries with regenerated endothelium, the hyperpolarization to bradykinin became voltage-dependent. In the most depolarized cells, the hyperpolarization to bradykinin was augmented. The changes in resting membrane potential and the alteration in endothelium-dependent hyperpolarizations observed in the coronary arteries with regenerated endothelium may contribute to the reduced response to serotonin and the unchanged relaxation to bradykinin described previously.
Balloon-Artery Interactions During Stent Placement
Authors: Rogers Campbell , Tseng David Y. , Squire James C. , Edelman Elazer R.
Published: June 2012

Abstract
—Endovascular stents expand the arterial lumen more than balloon angioplasty and reduce rates of restenosis after coronary angioplasty in selected patients. Understanding the factors involved in vascular injury imposed during stent deployment may allow optimization of stent design and stent-placement protocols so as to limit vascular injury and perhaps reduce restenosis. Addressing the hypothesis that a previously undescribed mechanism of vascular injury during stent deployment is balloon-artery interaction, we have used finite element analysis to model how balloon-artery contact stress and area depend on stent-strut geometry, balloon compliance, and inflation pressure. We also examined superficial injury during deployment of stents of varied design in vivo and in a phantom model ex vivo to show that balloon-induced damage can be modulated by altering stent design. Our results show that higher inflation pressures, wider stent-strut openings, and more compliant balloon materials cause markedly larger surface-contact areas and contact stresses between stent struts. Appreciating that the contact stress and contact area are functions of placement pressure, stent geometry, and balloon compliance may help direct development of novel stent designs and stent-deployment protocols so as to minimize vascular injury during stenting and perhaps to optimize long-term outcomes.
Homocysteine Enhances Neutrophil-Endothelial Interactions in Both Cultured Human Cells and Rats In Vivo
Authors: Dudman Nicholas P. B. , Temple Suzanna E. , Guo Xue Wei , Fu Weiyu , Perry Michael A.
Published: June 2012

Abstract
—Despite intense investigation, mechanisms linking the development of occlusive vascular disease with elevated levels of homocysteine (HCY) are still unclear. The vascular endothelium plays a key role in regulating thrombogenesis and thrombolysis. We hypothesized that vascular lesions in individuals with elevated plasma HCY may be related to a dysfunction of the endothelium triggered by HCY. We investigated the effect of HCY on human neutrophil adhesion to and migration through endothelial monolayers. We also examined the effect of HCY on leukocyte adhesion and migration in mesenteric venules of anesthetized rats. We found that pathophysiological concentrations of HCY in vitro induce increased adhesion between neutrophils and endothelial cells. This contact results in neutrophil migration across the endothelial layer, with concurrent damage and detachment of endothelial cells. In vivo, HCY infused in anesthetized rats caused parallel effects, increasing leukocyte adhesion to and extravasation from mesenteric venules. Our results suggest that extracellular H
2
O
2
, generated by adherent neutrophils and/or endothelial cells, is involved in the in vitro endothelial cell damage. The possibility exists that leukocyte-mediated changes in endothelial integrity and function may lead to the vascular disease seen in individuals with elevated plasma HCY.
Mechanical Stimulation Regulates Voltage-Gated Potassium Currents in Cardiac Microvascular Endothelial Cells
Authors: Fan Jinping , Walsh Kenneth B.
Published: June 2012

Abstract
—Vascular endothelial cells are constantly exposed to mechanical forces resulting from blood flow and transmural pressure. The goal of this study was to determine whether mechanical stimulation alters the properties of endothelial voltage-gated K
+
channels. Cardiac microvascular endothelial cells (CMECs) were isolated from rat ventricular muscle and cultured on thin sheets of silastic membranes. Membrane currents were measured with the use of the whole-cell arrangement of the patch-clamp technique in endothelial cells subjected to static stretch for 24 hours and compared with measurements from control, nonstretched cells. Voltage steps positive to −30 mV resulted in the activation of a time-dependent, delayed rectifier K
+
current (
I

K

) in the endothelial cells. Mechanically induced increases of 97%, 355%, and 106% at +30 mV were measured in the peak amplitude of
I

K

in cells stretched for 24 hours by 5%, 10%, and 15%, respectively. In addition, the half-maximal voltage required for
I

K

activation was shifted from +34 mV in the nonstretched cells to −5 mV in the stretched cells. Although
I

K

in both groups of CMECs was blocked to a similar extent by tetraethylammonium, currents in the stretched endothelial cells displayed an enhanced sensitivity to inhibition by charybdotoxin. Preincubation of the CMECs with either pertussis toxin or phorbol 12-myristate 13-acetate during the 24 hours of cell stretch did not prevent the increase in
I

K

. The application of phorbol 12-myristate 13-acetate and static stretch stimulated the proliferation of CMECs. Stretch-induced regulation of K
+
channels may be important to control the resting potential of the endothelium and may contribute to capillary growth during periods of mechanical perturbation.