bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Transfer of Dominant-Negative Mutants of Extracellular Signal–Regulated Kinase and c-Jun NH2-Terminal Kinase Prevents Neointimal Formation in Balloon-Injured Rat Artery
Authors: Izumi Yasukatsu , Kim Shokei , Namba Masashi , Yasumoto Hideo , Miyazaki Hitoshi , Hoshiga Masaaki , Kaneda Yasufumi , Morishita Ryuichi , Zhan Yumei , Iwao Hiroshi
Published: September 2007

Abstract
—We previously reported that extracellular signal–regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), belonging to mitogen-activated protein kinases, are rapidly activated in balloon-injured artery. Therefore, we examined the role of these kinase activations in neointimal formation by using an in vivo gene transfer technique. We made the dominant-negative mutants of ERK (DN-ERK) and JNK (DN-JNK) to specifically inhibit endogenous ERK and JNK activation, respectively. Before balloon injury, these mutants were transfected into rat carotid artery using the hemagglutinating virus of Japan liposome method. In vivo transfection of DN-ERK and DN-JNK significantly suppressed the activation of ERK and JNK, respectively, after balloon injury, confirming successful expression of the transfected genes. Neointimal formation at 14 and 28 days after injury was prevented by gene transfer of DN-ERK or DN-JNK. Furthermore, bromodeoxyuridine labeling index and total cell–counting analysis at 7 days showed that either DN-ERK or DN-JNK remarkably suppressed smooth muscle cell (SMC) proliferation in both the intima and the media after injury. Gene transfer of wild-type ERK (W-ERK) or JNK (W-JNK) significantly enhanced neointimal hyperplasia at 14 days after injury. Furthermore, DN-ERK and DN-JNK significantly suppressed serum-induced SMC proliferation in vitro. We obtained the first evidence that in vivo gene transfer of DN-ERK or DN-JNK prevented neointimal formation in balloon-injured artery by inhibiting SMC proliferation. Thus, ERK and JNK activation triggers SMC proliferation, leading to neointimal formation. These kinases may be the new therapeutic targets for prevention of vascular diseases.
Distinct Pathways of Ca2+Sensitization in Porcine Coronary Artery
Authors: Nobe Koji , Paul Richard J.
Published: September 2007

Abstract—Alterations of the Ca2+sensitivity of contraction have been reported for porcine coronary artery, but the mechanisms are not clearly understood. We investigated the mechanism(s) of Ca2+sensitization in response to the thromboxane A2analogue (U46619). Our hypothesis is that different mechanisms of Ca2+sensitization could be distinguished by their distinct time courses. Therefore, we measured the time course of [Ca2+]iand isometric force simultaneously in an intact artery after a single addition of U46619. The initial transient phase was associated with Ca2+release from the sarcoplasmic reticulum, whereas the maintained phase was associated with Ca2+influx. Two distinct types of Ca2+sensitization characterized these phases with either protein kinase C (PKC)-mediated or Rho-kinase–mediated mechanisms. Their effects were quite distinct on the basis of the time courses over which the sensitization was effective. PKC inhibition (1 μmol/L calphostin C) had a much greater effect in the initial phase, diminishing the size of the transient and prolonging the rise in force and the decline in [Ca2+]i. There were limited effects on the sustained force. Rho-kinase inhibition (10 μmol/L Y27632), in contrast, nearly abolished the sustained force but had a lesser effect on the transient phase. Neither inhibitor had any effect on the force versus [Ca2+]irelations for KCl contractures. Our evidence suggests that both PKC-mediated and Rho-kinase–mediated Ca2+sensitizations are present in coronary arteries, but the latter is dominant in thromboxane A2receptor–mediated contraction.
Sinoatrial Nodal Cell Ryanodine Receptor and Na
+
-Ca
2+
Exchanger
Authors: Bogdanov Konstantin Y. , Vinogradova Tatiana M. , Lakatta Edward G.
Published: September 2007

Abstract
—The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart’s pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca
2+
release during the DD produces a localized subsarcolemmal Ca
2+
increase that spreads in a wavelike manner by Ca
2+
-induced Ca
2+
release and produces an inward current via the Na
+
-Ca
2+
exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulum Ca
2+
release channel, in a dose-dependent manner reduces the SANC beating rate with an IC
50
of 2.6 μmol/L and abolishes the local Ca
2+
transients that precede the AP upstroke. In voltage-clamped cells in which the DD was simulated by voltage ramp, 3 μmol/L ryanodine decreased an inward current during the voltage ramp by 1.6±0.3 pA/pF (SEM, n=4) leaving the peak of L-type Ca
2+
current unchanged. Likewise, acute blockade of the NCX (via rapid substitution of bath Na
+
by Li
+
) abolished SANC beating and reduced the inward current to a similar extent (1.7±0.4 pA/pF, n=4), as did ryanodine. Thus, in addition to activation/inactivation of multiple ion channels, Ca
2+
activation of the NCX, because of localized sarcoplasmic reticulum Ca
2+
release, is a critical element in a chain of molecular interactions that permits the heartbeat to occur and determines its beating rate.
Acute Regulation of Fatty Acid Oxidation and AMP-Activated Protein Kinase in Human Umbilical Vein Endothelial Cells
Authors: Dagher Zeina , Ruderman Neil , Tornheim Keith , Ido Yasuo
Published: September 2007

Abstract
—It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of
3
H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with
3
H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK–ACC–malonyl CoA–carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.
Phosphatidylinositol 3-Kinase/Akt Signaling Controls Endothelial Cell Sensitivity to Fas-Mediated Apoptosis via Regulation of FLICE-Inhibitory Protein (FLIP)
Authors: Suhara Toshimitsu , Mano Toshiaki , Oliveira Beatriz Enes , Walsh Kenneth
Published: September 2007

Abstract
—Fas is constitutively expressed on endothelial cells, but in contrast to smooth muscle and other cell types, endothelial cells are highly resistant to Fas-mediated apoptosis. In this study, we examined the role of the serine/threonine kinase Akt/PKB in controlling the sensitivity of endothelial cells to Fas-mediated apoptosis. Serum deprivation inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP), which functions downstream from Fas. FLIP expression levels were restored when serum-depleted cells were treated with vascular endothelial growth factor. Treatment with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 or infection of the adenoviral construct expressing dominant-negative Akt (Adeno-dnAkt) also inhibited the expression of FLIP in endothelial cells, whereas the MEK inhibitor PD98059 had no effect. Conversely, adenovirus-mediated transfection of a constitutively-active
Akt
gene abolished the wortmannin- and LY294002-mediated downregulation of FLIP. Suppression of PI 3-kinase signaling sensitized endothelial cells to Fas-mediated apoptosis. Under conditions of suppressed PI 3-kinase signaling, restoration of FLIP expression reversed the induced sensitivity of endothelial cells to Fas-mediated apoptosis. These data suggest that inhibition of Fas-mediated apoptosis, via promotion of FLIP expression, is a mechanism through which Akt signaling can promote endothelial cell survival.
Energetic Crosstalk Between Organelles
Authors: Kaasik Allen , Veksler Vladimir , Boehm Ernest , Novotova Marta , Minajeva Ave , Ventura-Clapier Renée
Published: September 2007

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.
Smooth Muscle Cell Changes During Flow-Related Remodeling of Rat Mesenteric Resistance Arteries
Authors: Buus C. L. , Pourageaud F. , Fazzi G. E. , Janssen G. , Mulvany M. J. , De Mey J. G.R.
Published: September 2007

To obtain information on the molecular and cellular mechanisms of flow-induced arterial remodeling, we analyzed the morphology and smooth muscle cell (SMC) characteristics in rat mesenteric resistance arteries after interventions that decreased and increased flow. Juvenile male Wistar Kyoto rats were subjected to surgery that, compared with control arteries, provided arteries with chronic low flow and chronic high flow. Low flow resulted in a decreased passive lumen diameter, hypotrophy of the artery wall, and both loss and decreased size of SMCs. Time course studies, with intervention length ranging from 2 to 32 days of altered blood flow, showed that the narrowing of the lumen diameter in low-flow arteries appeared within 2 days and that an early dedifferentiation of SMC phenotype was indicated by markedly reduced levels of desmin mRNA. High flow resulted in an increased passive lumen diameter and in hypertrophy of the artery wall. The hypertrophy resulted from SMC proliferation because SMC number, measured by the 3D-dissector technique, was increased and immunohistochemical assessment of proliferating cell nuclear antigen also showed an increase. The widening of high-flow arteries required 16 days to become established, at which time desmin mRNA was reduced. This time was also required to establish changed wall mass in both low-flow and high-flow arteries. Apoptotic cells detected by TdT-mediated dUTP-biotin nick end labeling staining were mainly located in the medial layer, and evaluation of DNA fragmentation indicated that increased apoptosis occurred in both low flow and high flow. This study shows for the first time direct evidence that reduced and elevated blood flow in resistance arteries produce, respectively, decrease and increase in SMC number, with dedifferentiation of the SMCs in both cases.
Regulation of Vascular Bed–Specific Prothrombotic Potential
Authors: Edelberg Jay M. , Christie Patricia D. , Rosenberg Robert D.
Published: September 2007

Hemostasis is the result of interdependent and complex systemic and local endothelial pathways that govern vascular integrity and rheology. A striking feature of hypercoagulable conditions is the focal nature of the resultant thrombotic pathology. Such disorders in hemostasis may be associated with distinct vascular beds, thus implying that the relative combined contribution of individual regulatory pathways may be specific and/or unique to a particular locale in the vasculature. Systemic factors and platelets mediate the formation of fibrin deposition; however, it is the diverse interrelationships in the interaction of these systemic elements with the local endothelial components that dictate vascular bed–specific hemostatic regulation. Indeed, the local activation of coagulation cascades, rather than increases in systemic thrombotic potential, is what leads to fibrin formation in different vascular beds. Hence, the propensity for congenital or acquired disorders to result in local thrombotic pathology is based on the relative contribution of the various hemostatic regulatory pathways in individual vascular beds. The present review highlights the role of local endothelial regulation in the interaction between local and systemic elements that contribute to vascular bed–specific prothrombotic potential.
Abnormal Balance in the Angiopoietin-Tie2 System in Human Brain Arteriovenous Malformations
Authors: Hashimoto Tomoki , Lam Tiffany , Boudreau Nancy J. , Bollen Andrew W. , Lawton Michael T. , Young William L.
Published: September 2007

Brain arteriovenous malformations (BAVMs) are congenital vascular lesions that often present as cerebral hemorrhage in young adults. The variable nature of the clinical course, especially with respect to spontaneous hemorrhage, recurrence, growth, and regression, suggests that BAVMs are lesions with active angiogenesis and vascular remodeling. We examined mRNA and protein expression of angiopoietin 1 (Ang1) and Ang2 by semiquantitative reverse transcriptase–polymerase chain reaction, in situ hybridization, and Western blot in BAVMs and control brains obtained from temporal lobectomy for medically intractable seizures. Although Ang1 mRNA levels were similar in BAVMs and controls, Ang1 protein levels were 30% lower in BAVMs than in controls. Ang2 mRNA levels were 40% higher and Ang2 protein levels were 8-fold higher in BAVMs than in controls. In situ hybridization showed that the Ang2 mRNA was localized to the perivascular area in BAVMs. This abnormal balance in the Ang-Tie2 system may, in part, explain the aberrant vascular phenotype in BAVMs.
Oxidative Stress–Mediated Cardiac Cell Death Is a Major Determinant of Ventricular Dysfunction and Failure in Dog Dilated Cardiomyopathy
Authors: Cesselli Daniela , Jakoniuk Igor , Barlucchi Laura , Beltrami Antonio P. , Hintze Thomas H. , Nadal-Ginard Bernardo , Kajstura Jan , Leri Annarosa , Anversa Piero
Published: September 2007

Cell death has been questioned as a mechanism of ventricular failure. In this report, we tested the hypothesis that apoptotic death of myocytes, endothelial cells, and fibroblasts is implicated in the development of the dilated myopathy induced by ventricular pacing. Accumulation of reactive oxygen products such as nitrotyrosine, potentiation of the oxidative stress response by p66
shc
expression, formation of p53 fragments, release of cytochrome
c
, and caspase activation were examined to establish whether these events were coupled with apoptotic cell death in the paced dog heart. Myocyte, endothelial cell, and fibroblast apoptosis was detected before indices of severe impairment of cardiac function became apparent. Cell death increased with the duration of pacing, and myocyte death exceeded endothelial cell and fibroblast death throughout. Nitrotyrosine formation and p66
shc
levels progressively increased with pacing and were associated with cell apoptosis. Similarly, p50 (ΔN) fragments augmented paralleling the degree of cell death in the failing heart. Moreover, cytochrome
c
release and activation of caspase-9 and -3 increased from 1 to 4 weeks of pacing. In conclusion, cardiac cell death precedes ventricular decompensation and correlates with the time-dependent deterioration of function in this model. Oxidative stress may be critical for activation of apoptosis in the overloaded heart.
Regulation of Matrix Metalloproteinase Activity in Ischemic Tissue by Interleukin-10
Authors: Silvestre Jean-Sébastien , Mallat Ziad , Tamarat Radia , Duriez Micheline , Tedgui Alain , Levy Bernard I.
Published: September 2007

We have previously shown that deficiency in the anti-inflammatory cytokine interleukin-10 (IL-10) is responsible for enhanced angiogenesis after hindlimb ischemia. This study examined the putative involvement of matrix metalloproteinase (MMP) activation in this process. Ischemia was produced by artery femoral occlusion in both C57BL6 IL-10
+/+
and IL-10
−/−
mice. Angiographic vessel density and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/nonischemic leg ratio by, respectively, 1.8-fold and 1.4-fold in IL-10
−/−
mice compared with IL-10
+/+
mice. This was associated with an increase in vascular endothelial growth factor (VEGF) protein content in the ischemic hindlimb. Three days after ischemia, gelatin zymography showed a significant increase in both pro- and active forms of MMP-2 and MMP-9 in ischemic hindlimbs of IL-10
−/−
mice compared with IL-10
+/+
mice (
P
<0.01). This increase in MMP activity in IL-10
−/−
mice was completely inhibited by treatment with BB-94 (5 mg/kg IP), a specific MMP inhibitor. Furthermore, increases in both vessel density and blood perfusion indexes at day 28 in IL-10
−/−
mice were abolished after treatment with BB-94 (0.78±0.06 versus 1.17±0.09 and 0.62±0.02 versus 0.88±0.04, for vessel density and blood perfusion ratio, respectively, in IL-10
−/−
mice treated with BB-94 versus untreated IL-10
−/−
mice,
P
<0.05). In contrast, BB-94 treatment did not affect the rise in VEGF protein content. These findings in IL-10
−/−
mice underscore the critical role of MMP activation, in a context of increased VEGF expression, in promoting ischemia-induced angiogenesis.
Methods of Detection of Vascular Reactive Species
Authors: Tarpey Margaret M. , Fridovich Irwin
Published: September 2007

The evanescent nature of reactive oxygen and nitrogen species, the multiple cellular mechanisms evolved to maintain these substances at low (submicromolar) concentrations within the vascular system, and the often multifaceted nature of their reactivities have made measurement of these compounds within the vasculature problematic. This review attempts to provide a critical description of some of the most common approaches to quantification of nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite, with attention to key issues that may influence the utility of a particular assay when adapted for use in vascular cells and tissues.
p27-p16 Fusion Gene Inhibits Angioplasty-Induced Neointimal Hyperplasia and Coronary Artery Occlusion
Authors: Tsui Lisa V. , Camrud Allan , Mondesire Jean , Carlson Paula , Zayek Nathalie , Camrud LaDonna , Donahue Brian , Bauer Scott , Lin Andy , Frey David , Rivkin Marianne , Subramanian Ajit , Falotico Robert , Gyuris Jeno , Schwartz Robert , McArthur James G.
Published: September 2007

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury–induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10
12
viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus–treated vessels. The average intimal area of the AV-W9–treated group 10 days after balloon injury and treatment was 0.42±0.36 mm
2
, whereas the AV-null group demonstrated an intimal area of 0.70±0.52 mm
2
. At day 10 the average intimal thickness of the AV-W9–treated vessels was 9.1 μm (n=5, ×20 magnification) compared with 21.2 μm (n=5, ×20 magnification) in control virus–treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9–treated vessels demonstrated an average intimal thickness of 4.7 μm (n=8, ×20 magnification) compared with 13.3 μm (n=3, ×20 magnification) in control virus–treated vessels and 7.3 μm (n=5, ×20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty–induced intimal hyperplasia in the coronary artery.
Clinical Imaging of the High-Risk or Vulnerable Atherosclerotic Plaque
Authors: Fayad Z. A. , Fuster V.
Published: September 2007

The study of atherosclerotic disease during its natural history and after therapeutic intervention will enhance our understanding of disease progression and regression and aid in selecting appropriate treatments. Several invasive and noninvasive imaging techniques are available to assess atherosclerotic vessels. Most of the standard techniques identify luminal diameter, stenosis, wall thickness, and plaque volume; however, none can characterize plaque composition and therefore identify the high-risk plaques. We will present the different imaging modalities that have been used for the direct assessment of the carotid, aortic, and coronary atherosclerotic plaques. We will review in detail the use of high-resolution, multicontrast magnetic resonance for the noninvasive imaging of vulnerable plaques and the characterization of plaques in terms of their various components (ie, lipid, fibrous, calcium, or thrombus).
Phospholipase A2in Vascular Disease
Authors: Hurt-Camejo Eva , Camejo Germán , Peilot Helena , Öörni Katarina , Kovanen Petri
Published: September 2007

Secretory phospholipase A2(PLA2) can be proatherogenic both in the circulation and in the arterial wall. In blood plasma, PLA2can modify the circulating lipoproteins and so induce formation of small dense LDL particles, which are associated with increased risk for cardiovascular disease. In the arterial wall, PLA2can hydrolyze lipoproteins. The PLA2-modified lipoproteins bind tightly to extracellular proteoglycans, which may lead to their enhanced retention in the arterial wall. The modified lipoproteins may also aggregate and fuse, which can lead to accumulation of their lipids within the extracellular matrix. The PLA2-modified particles are more susceptible to further modifications by other enzymes and agents and can be taken up by macrophages, leading to accumulation of intracellular lipids. In addition, lysophospholipids and free fatty acids, the hydrolysis products of PLA2, promote atherogenesis. Thus, these lipid mediators can be carried, either by the PLA2-modified lipoproteins themselves or by albumin, into the arterial cells, which then undergo functional alterations. This may, in turn, lead to specific changes in the extracellular matrix, which increase the retention and accumulation of lipoproteins within the matrix. In the present article, we discuss the possible actions of PLA2enzymes, especially PLA2-IIA, in the arterial wall during atherogenesis.
O
2
Modulates Large-Conductance Ca
2+
-Dependent K
+
Channels of Rat Chemoreceptor Cells by a Membrane-Restricted and CO-Sensitive Mechanism
Authors: Riesco-Fagundo A. M. , Pérez-García M. T. , González C. , López-López J. R.
Published: September 2007

Hypoxic inhibition of large-conductance Ca
2+
-dependent K
+
channels (maxiK) of rat carotid body type I cells is a well-established fact. However, the molecular mechanisms of such inhibition and the role of these channels in the process of hypoxic transduction remain unclear. We have examined the mechanisms of interaction of O
2
with maxiK channels exploring the effect of hypoxia on maxiK currents recorded with the whole-cell and the inside-out configuration of the patch-clamp technique. Hypoxia inhibits channel activity both in whole-cell and in excised membrane patches. This effect is strongly voltage- and Ca
2+
-dependent, being maximal at low [Ca
2+
] and low membrane potential. The analysis of single-channel kinetics reveals a gating scheme comprising three open and five closed states. Hypoxia inhibits channel activity increasing the time the channel spends in the longest closed states, an effect that could be explained by a decrease in the Ca
2+
sensitivity of those closed states. Reducing maxiK channels with dithiothreitol (DTT) increases channel open probability, whereas oxidizing the channels with 2,2′-dithiopyridine (DTDP) has the opposite effect. These results suggest that hypoxic inhibition is not related with a reduction of channel thiol groups. However, CO, a competitive inhibitor of O
2
binding to hemoproteins, fully reverts hypoxic inhibition, both at the whole-cell and the single-channel level. We conclude that O
2
interaction with maxiK channels does not require cytoplasmic mediators. Such interaction could be mediated by a membrane hemoprotein that, as an O
2
sensor, would modulate channel activity.
Gender-Based Differences in Cardiac Repolarization in Mouse Ventricle
Authors: Trépanier-Boulay Véronique , St-Michel Chantale , Tremblay Annie , Fiset Céline
Published: September 2007

The mouse heart has become a widely used model for genetic studies of heart diseases. Thus, understanding gender differences in mouse cardiac repolarization is crucial to the interpretation of such studies. The objective of this study was to evaluate whether there are gender differences in cardiac repolarization in mouse ventricle and to gain insights into the ionic and molecular mechanisms underlying these differences. Action potential durations (APDs) and K+currents in male and female ventricular myocytes were compared using a patch-clamp technique. APD20, APD50, and APD90were found to be significantly longer in females than males. Examination of the different K+currents revealed that a significantly lower current density exists in female ventricular myocytes compared with male myocytes for the ultrarapid delayed rectifier K+current,IKur(at +30 mV, male, 33.2±2.9 pA/pF [n= 22]; female, 20.9±1.73 pA/pF [n= 19],P<0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding forIKur) in female compared with male ventricle. The additional K+currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression levels for the K+channels underlying these currents were detected between both sexes. This study demonstrates that adult mice exhibit gender differences in cardiac repolarization. The expression of Kv1.5 and of its corresponding K+current,IKur, is significantly lower in female mouse ventricle, and as a result, the APD is lengthened.
Glucagon-Like Peptide-1 Increases cAMP but Fails to Augment Contraction in Adult Rat Cardiac Myocytes
Authors: Vila Petroff Martín G. , Egan Josephine M. , Wang Xiaolin , Sollott Steven J.
Published: September 2007

The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A–mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7±0.5 to 13.1±0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca
2+
transient (CaT), and pH
i
in indo-1 and seminaphthorhodafluor (SNARF)–1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160±34%) and CaT (70±5%), GLP-1 induced a significant decrease in contraction amplitude (−27±5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1–loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (ΔpH
i
−0.09±0.02 and −0.08±0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1–induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to β-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin–sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca
2+
responsiveness probably resulting from intracellular acidification.
Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subjects
Authors: Isner Jeffrey M. , Vale Peter R. , Symes James F. , Losordo Douglas W.
Published: September 2007

Clinical trials of cardiovascular gene therapy, whether using viral (53%) or nonviral (47%) vectors, have thus far disclosed no evidence indicative of inflammatory or other complications, including death, directly attributable to the vector used. Indeed, despite the fact that initial trials of cardiovascular gene therapy targeted patients with end-stage vascular disease, including critical limb ischemia and refractory myocardial ischemia, the mortality for patients enrolled in clinical trials of cardiovascular gene therapy reported to date compares favorably with mortality for similar groups of patients in contemporary controlled studies of medical or interventional therapies. The most common morbidity reported after cardiovascular gene transfer is lower extremity edema; in contrast to data involving genetically engineered mice, however, evidence of life- or limb-threatening edema has not been described in any patients, including patients after gene transfer for myocardial ischemia. Concerns regarding the potential for angiogenic cytokines to promote the progression of atherosclerosis are not supported by angiographic follow-up of patients with coronary or peripheral vascular disease. The levels and duration of gene expression investigated for therapeutic angiogenesis transfer have been unassociated with hemangioma formation. Likewise, there is little evidence from either preclinical or clinical studies to support the notion that the administration of angiogenic growth factors, per se, is sufficient to stimulate the growth of neoplasms. Patients enrolled in clinical studies of angiogenic cytokines, including patients with diabetes and a previous history of retinopathy, have disclosed no evidence to suggest that ocular pathology is a risk of angiogenic growth factor gene transfer.
Differential Regulation of Matrix Metalloproteinase-9 by Monocytes Adherent to Collagen and Platelets
Authors: Galt Spencer W. , Lindemann Stephan , Medd Don , Allen Loren L. , Kraiss Larry W. , Harris Estelle S. , Prescott Stephen M. , McIntyre Thomas M. , Weyrich Andrew S. , Zimmerman Guy A.
Published: September 2007

Abstract—

Circulating monocytes adhere to platelets and matrix proteins at sites of vascular injury, where engagement of specific surface tethering molecules mediates outside-in signaling and synthesis of gene products by the leukocytes. Here we demonstrate that interaction of isolated human monocytes with collagen induces matrix metalloproteinase-9 (MMP-9; gelatinase B) synthesis by monocytes, a process that is greatly enhanced in the presence of platelets. MMP-9 is a potent matrix degrading enzyme implicated in atherosclerotic plaque rupture, aneurysm formation, and other vascular syndromes. Synthesis of MMP-9 by monocytes is tightly regulated and synergistically increased following adhesion to collagen and platelets. Adhesion to control matrix proteins alone did not result in MMP-9 protein production and, similarly, adhesion of monocytes to platelets activated with thrombin in suspension was not sufficient to induce MMP-9 synthesis in the absence of monocyte adhesion to collagen. Interruption of intercellular contact between platelets and monocytes dramatically inhibited MMP-9 synthesis. These observations demonstrate that discrete adhesion-dependent signaling pathways govern MMP-9 synthesis by monocytes. The synthesis of MMP-9 by monocytes may be critical in vascular syndromes and other pathological processes that are dependent on dysregulated cell-cell and cell-matrix interactions.
Identification of Genes Potentially Involved in Rupture of Human Atherosclerotic Plaques
Authors: Faber Birgit C.G. , Cleutjens Kitty B.J.M. , Niessen Ron L.J. , Aarts Petra L.J.W. , Boon Wendy , Greenberg Andrew S. , Kitslaar Peter J.E.H.M. , Tordoir Jan H.M. , Daemen Mat J.A.P.
Published: September 2007

Abstract—

Although rupture of an atherosclerotic plaque is the major cause of acute vascular occlusion, the exact molecular mechanisms underlying this process are still poorly understood. In this study, we used suppression subtractive hybridization to make an inventory of genes that are differentially expressed in whole-mount human stable and ruptured plaques. Two libraries were generated, one containing 3000 clones upregulated and one containing 2000 clones downregulated in ruptured plaques. Macroarray analysis of 500 randomly chosen clones showed differential expression of 45 clones. Among the 25 clones that showed at least a 2-fold difference in expression was the gene of perilipin, upregulated in ruptured plaques, and the genes coding for fibronectin and immunoglobulin λ chain, which were downregulated in ruptured plaques. Reverse transcriptase–polymerase chain reaction analysis on 10 individual ruptured and 10 individual stable plaques showed a striking consistency of expression for the clones SSH6, present in 8 ruptured and 2 stable plaques, and perilipin, expressed in 8 ruptured plaques and completely absent in stable plaques. Localization studies of both perilipin mRNA and protein revealed expression in cells surrounding the cholesterol clefts and in foam cells of ruptured atherosclerotic plaques. No expression was observed in nondiseased artery, and only a few cells in the shoulder region of stable plaques tested positive for perilipin. In conclusion, this study shows that it is possible to identify genes that are differentially expressed in whole-mount stable or ruptured atherosclerotic plaques. This approach may yield several potential regulators of plaque destabilization.
Ascorbate Attenuates Atrial Pacing-Induced Peroxynitrite Formation and Electrical Remodeling and Decreases the Incidence of Postoperative Atrial Fibrillation
Authors: Carnes Cynthia A. , Chung Mina K. , Nakayama Tomohiro , Nakayama Hitomi , Baliga Reshma S. , Piao Shengfu , Kanderian Anne , Pavia Steven , Hamlin Robert L. , McCarthy Patrick M. , Bauer John A. , Van Wagoner David R.
Published: September 2007

Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min−1) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org.
Thrombin Suppresses Endothelial Nitric Oxide Synthase and Upregulates Endothelin-Converting Enzyme-1 Expression by Distinct Pathways
Authors: Eto Masato , Barandiér Christine , Rathgeb Lisa , Kozai Toshiyuki , Joch Hana , Yang Zhihong , Lüscher Thomas F.
Published: September 2007

Abstract—

An imbalance of nitric oxide and endothelin plays an important role in cardiovascular disease. Thrombin exerts profound effects on endothelial function. The present study investigated the molecular mechanisms by which thrombin regulates endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE)-1 expression in human endothelial cells. Incubation of human umbilical vein endothelial cells with thrombin (0.01 to 4 U/mL) for 15 to 24 hours markedly downregulated eNOS and increased ECE-1 protein level in a dose-dependent manner. Thrombin also decreased eNOS mRNA and increased ECE-1 mRNA level. In mRNA stability assay, thrombin shortened the half-life of eNOS mRNA but not that of ECE-1 mRNA. Activation of protease-activated receptor 1 by the agonist (SFLLRN, 10 to 100 μmol/L) had no effect on eNOS expression but increased ECE-1 level as thrombin. Thrombin activated Rho A and extracellular signal–regulated kinase (ERK)1 and ERK2. Inhibition of Rho A by C3 exoenzyme (20 μg/mL) and ROCK by Y-27632 (10 μmol/L) prevented the downregulation of eNOS expression by thrombin. Y-27632 also prevented the reduction in NOS activity induced by prolonged incubation with thrombin. On the other hand, inhibition of ERK1 and ERK2 activation by PD98059 (50 μmol/L) prevented the upregulation of ECE-1 expression by thrombin as well as the increase in ECE activity and ET-1 accumulation in the medium. Treatment of rat aorta with thrombin overnight impaired endothelium-dependent relaxations but not endothelium-independent relaxations. Thus, thrombin suppresses eNOS and upregulates ECE-1 expression via Rho/ROCK and ERK pathway, respectively. These effects of thrombin may be important for endothelial dysfunction in cardiovascular disease, particularly during acute coronary episodes.
Differential Expression of T- and L-Type Voltage-Dependent Calcium Channels in Renal Resistance Vessels
Authors: Hansen Pernille B. , Jensen Boye L. , Andreasen Ditte , Skøtt Ole
Published: September 2007

Abstract—

The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells showed coexpression of mRNAs for T-type subunits (Ca
V
3.1, Ca
V
3.2) and for an L-type subunit (Ca
V
1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca
V
1.2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed a significant increase of calcium in response to K
+
(100 mmol/L) in isolated afferent arterioles (140±25%) and in juxtamedullary efferent arterioles (118±21%). These calcium responses were attenuated by the L-type antagonist calciseptine and by the T-type antagonist mibefradil. Intracellular calcium increased in response to K
+
in cortical efferent arterioles (21±9%). Mibefradil and nickel concentration dependently blocked K
+
-induced contraction of perfused rabbit afferent arterioles. Calciseptine blocked the contraction mediated by K
+
(EC
50
8×10
−14
). S-(-)-Bay K 8644 had no effect on vascular diameter in the afferent arteriole. We conclude that voltage-dependent L- and T-type calcium channels are expressed and of functional significance in renal cortical preglomerular vessels, in juxtamedullary efferent arterioles, and in outer medullary vasa recta, but not in cortical efferent arterioles.
Upregulation of Functional β3-Adrenergic Receptor in the Failing Canine Myocardium
Authors: Cheng Heng-Jie , Zhang Zhu-Shan , Onishi Katsuya , Ukai Tomohiko , Sane David C. , Cheng Che-Ping
Published: September 2007

Abstract—Altered expression and functional responses to cardiac β3-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte β3-AR mRNA and protein levels and myocyte contractile, [Ca2+]itransient, and Ca2+current (ICa,L) responses to BRL-37344 (BRL, 10−8mol/L), a selective β3-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription–polymerase chain reaction, we detected β3-AR mRNA from myocyte total RNA in each animal. Using a cloned canine β3-AR cDNA probe and myocyte poly A+RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. β3-AR protein was detected by Western blot. β3-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced β3-AR–mediated negative modulation on myocyte contractile response and [Ca2+]iregulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca2+]itransient andICa,L. These responses were not modified by pretreating myocytes with metoprolol (a β1-AR antagonist) or nadolol (a β1- and β2-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (β3-AR antagonists). We conclude that in dogs with pacing-induced CHF, β3-AR gene expression and protein levels are upregulated, and the functional response to β3-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.
Vascular Morphogenesis and Remodeling in a Model of Tissue Repair
Authors: Patan Sybill , Munn Lance L. , Tanda Shigeru , Roberge Sylvie , Jain Rakesh K. , Jones Rosemary C.
Published: September 2007

To investigate mechanisms of vascular morphogenesis in tissue repair, we performed ovariectomy with resection of the corresponding branches of the ovarian vessels in nude mice. This induces a vascular network remodeling response in the healing ovarian pedicle. Reconstruction of 2000 histological serial sections demonstrated that a new vascular network composed of venous-venous loops forms in the wall of the dilated ovarian vein. Preexisting veins of all sizes, including a branch of the main artery, are subjected to segmentation. Loop formation and segmentation are based on intussusceptive microvascular growth. Loop formation is followed by elongation. Loop remodeling occurs also by intussusception and results in the formation of compound loop systems. All loop systems observed were completely patent. Blind-ending sprouts were extremely rare. Anastomoses between the preexisting vessels subjected to segmentation and the loop systems were established to include the newly formed vessels into the preexisting vascular network. The formation of an increasing number of patent loop systems likely decreases hypoxia and subsequently arrests angiogenesis with transformation of the granulation tissue into a scar. Loop formation also occurred inside a large thrombus that occluded a part of the lumen of the main vein.