bluuebunny/crossref_metadata_2025 huggingface dataset:
Cosegregation Analysis in Genetic Crosses Suggests a Protective Role for Atrial Natriuretic Factor Against Ventricular Hypertrophy
Authors: Masciotra S. , Picard S. , Deschepper C. F.
Published: June 2012

Abstract
—In most rat models studied to date, increased ventricular mass is associated with high ventricular expression of the atrial natriuretic factor (ANF) gene. However, it is unknown whether ANF plays a beneficial or detrimental role in the course of left ventricular hypertrophy or whether ANF gene expression could be genetically linked to cardiac mass. To address such questions, we performed a cosegregation analysis in genetic crosses of inbred strains of rats. To select strains with the appropriate phenotypic characteristics, we first compared the ventricular abundance of ANF mRNA to ventricular mass (corrected for body weight) in 2 recombinant inbred strains derived from Wistar-Kyoto (WKY)/spontaneously hypertensive rat (SHR) hybrid crosses, ie, WKY-derived hyperactive (WKHA) and WKY-derived hypertensive (WKHT) rats, as well as in their parental inbred strains. In the 2 such strains that were normotensive, we observed that ventricular mass was higher in WKHA than in WKY rats, yet ventricular ANF mRNA was less abundant in WKHA than in WKY rats. Within a segregating population of F2 animals generated from a cross between WKY and WKHA genitors, the abundance of ventricular ANF mRNA and peptide correlated inversely with left ventricular mass, in contrast to the positive correlation observed with β-myosin heavy chain mRNA. Finally, in the equally hypertensive SHR and WKHT strains, we found that ventricular mass was higher in SHR than in WKHT, yet ventricular ANF mRNA was less abundant in SHR than in WKHT. These results demonstrate for the first time that low ventricular ANF gene expression can be linked genetically to high cardiac mass independently of blood pressure and are consistent with a protective role for ANF against left ventricular hypertrophy.
Ca
2+
Waves During Triggered Propagated Contractions in Intact Trabeculae
Authors: Miura Masahito , Boyden Penelope A. , Keurs Henk E. D. J. ter
Published: June 2012

Abstract
—During triggered propagated contractions, Ca
2+
waves travel along cardiac trabeculae with a constant velocity (V
prop
) ranging from 0.34 to 5.47 mm/s. To explore the determinants of V
prop
, we studied (1) the relationship between [Ca
2+
]
i
and V
prop
and (2) the effect of low concentrations of caffeine on V
prop
. Trabeculae were dissected from the right ventricle of rat hearts. [Ca
2+
]
i
was measured using electrophoretically injected fura-2 and an image-intensified CCD camera. Force was measured using a silicon strain gauge, and sarcomere length was measured using laser diffraction techniques. After induction of reproducible Ca
2+
waves by trains of electrical stimuli (2.5 Hz) at 21.9±0.2°C, the number of stimuli or [Ca
2+
]
o
was varied in 9 trabeculae. In 5 trabeculae, the effects of caffeine (0.1 to 1.0 mmol/L) at [Ca
2+
]
o
of 2.2±0.3 mmol/L were determined. All images were recorded under stable conditions of wave propagation. The increment in [Ca
2+
]
i
during the last electrically stimulated transient (ΔCa
T
) and [Ca
2+
]
i
just before onset of the Ca
2+
waves (Ca
D
) were used to estimate the Ca
2+
loading of the sarcoplasmic reticulum (SR) and the myoplasm, respectively. The ratio (ΔCa
W
/ΔCa
T
) of the [Ca
2+
]
i
increment during the waves (ΔCa
W
) to ΔCa
T
was used to estimate the probability of opening of the SR-Ca
2+
release channel during wave propagation. As a result of an increase of the number of stimuli or [Ca
2+
]
o
, V
prop
increased in proportion to (1) ΔCa
T
(
r
=0.82); (2) Ca
D
(
r
=0.88); (3) ΔCa
W
(
r
=0.85); and (4) ΔCa
W
/ΔCa
T
(
r
=0.74). The addition of caffeine (≤0.3 mmol/L) increased V
prop
for any ΔCa
T
and any ΔCa
W
, revealing an increased sensitivity of V
prop
to ΔCa
T
and ΔCa
W
. In contrast, caffeine had little effect on the relationship between V
prop
and Ca
D
and no effect on that between V
prop
and ΔCa
W
/ΔCa
T
. These results suggest that both the cellular Ca
2+
loading and open probability of the SR-Ca
2+
release channels determine the velocity of propagation of Ca
2+
waves.
Estrogen and Tamoxifen Metabolites Protect Smooth Muscle Cell Membrane Phospholipids Against Peroxidation and Inhibit Cell Growth
Authors: Dubey Raghvendra K. , Tyurina Yulia Y. , Tyurin Vladimir A. , Gillespie Delbert G. , Branch Robert A. , Jackson Edwin K. , Kagan Valerian E.
Published: June 2012

Abstract
—The goal of this study was to test the hypothesis that antioxidant estrogens, by a mechanism independent of the estrogen receptor, protect phospholipids residing in the plasma membrane of vascular smooth muscle cells from peroxidation and peroxidation-induced cell growth and migration. Peroxidation of membrane phospholipids was assessed by HPLC analysis of phospholipids extracted from rat aortic vascular smooth muscle cells prelabeled with
cis
-parinaric acid (a fatty acid that is susceptible to peroxidation, which quenches its fluorescent properties). Incubation of cells for 2 hours with the peroxyl radical donor 2,2′-azobis-2,4-dimethylvaleronitrile (AMVN) caused peroxidation of all measured membrane phospholipids. This effect was attenuated by pretreating cells for 15 minutes with 50 to 5000 ng/mL of 2-hydroxyestradiol (strong antioxidant but weak estrogen-receptor ligand) or 4-hydroxytamoxifen (strong antioxidant and potent estrogen-receptor ligand), but not by estrone or droloxifene (both weak antioxidants but potent estrogen-receptor ligands). Moreover, pretreatment of cells for 20 hours with physiological concentrations (0.3 ng/mL) of 2-hydroxyestradiol or pharmacologically relevant concentrations of 4-hydroxytamoxifen (40 ng/mL) also decreased AMVN-induced phospholipid peroxidation. Both 2-hydroxyestradiol and 4-hydroxytamoxifen were as effective as 2,2,5,7,8-pentamethyl-6-hydrochromane (an antioxidant homolog of vitamin E) in attenuating AMVN-induced peroxidation of membrane phospholipids. Also, physiological concentrations of 2-hydroxyestradiol, but not estrone, and pharmacologically relevant concentrations of 4-hydroxytamoxifen attenuated AMVM-induced DNA synthesis, cell proliferation, and cell migration. These studies demonstrate in vascular smooth muscle cells that antioxidant estrogens via a non-estrogen receptor–dependent mechanism attenuate peroxidation of membrane phospholipids and peroxidation-induced cell growth and migration.
Angiotensin II Type 1 Receptor–Mediated Inhibition of K
+
Channel Subunit Kv2.2 in Brain Stem and Hypothalamic Neurons
Authors: Gelband Craig H. , Warth John D. , Mason Helen S. , Zhu Mingyan , Moore Jennifer M. , Kenyon James L. , Horowitz Burton , Sumners Colin
Published: June 2012

Abstract
—Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT
1
) receptor–mediated inhibition of total outward K
+
current that contributes to an increase in neuronal firing rate. However, the exact K
+
conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K
+
current revealed a component of K
+
current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC
50
values of 21.7 μmol/L, 1.49 mmol/L, and 890 μmol/L, respectively, and insensitive to α-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 μmol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K
+
current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT
2
receptor blocker PD123,319, elicited an inhibition of neuronal K
+
current that was abolished by quinine (50 μmol/L). Reverse transcriptase–polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT
1
receptor in
Xenopus
oocytes demonstrated an Ang II–induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT
1
receptor–mediated reduction of neuronal K
+
current and subsequently to the modulation of cardiovascular function.
Regulation of Myocardial Blood Flow by Oxygen Consumption Is Maintained in the Failing Heart During Exercise
Authors: Traverse Jay H. , Melchert Peter , Pierpont Gordon L. , Jones Bryan , Crampton Melanie , Bache Robert J.
Published: June 2012

Abstract
—The hemodynamic abnormalities and neurohumoral activation that accompany congestive heart failure (CHF) might be expected to impair the increase in coronary blood flow that occurs during exercise. This study was performed to determine the effects of CHF on myocardial oxygen consumption and coronary blood flow during exercise. Coronary blood flow was measured in chronically instrumented dogs at rest, during 2 stages of graded treadmill exercise under control conditions (n=10), and after the development of CHF produced by 3 weeks of rapid ventricular pacing (n=9). In the normal dogs, coronary blood flow increased during exercise in proportion to the increase in the heart rate×the left ventricular systolic blood pressure product (RPP). After the development of CHF, resting myocardial blood flow was 25% lower than normal (
P
<0.05). Myocardial blood flow increased during the first stage of exercise, but then failed to increase further during the second stage of exercise despite an additional increase in the RPP. Myocardial oxygen consumption during exercise was significantly lower in animals with CHF and paralleled coronary flow. Despite the lower values for coronary blood flow in animals with CHF, there was no evidence for myocardial ischemia. Thus, even during the second level of exercise when coronary flow failed to increase, myocardial lactate consumption continued and coronary venous pH did not fall. In addition, the failure of coronary flow to increase as the exercise level was increased from stage 1 to stage 2 was not associated with a further increase in myocardial oxygen extraction. Thus, cardiac failure was associated with decreased myocardial oxygen consumption and failure of oxygen consumption to increase with an increase in the level of exercise. This abnormality did not appear to result from inadequate oxygen availability, but more likely represented a reduction of myocardial oxygen usage with a secondary decrease in metabolic coronary vasodilation.
Regulation of Sympathetic Nerve Activity in Heart Failure
Authors: Liu Jun-Li , Zucker Irving H.
Published: June 2012

Abstract
—The mechanisms by which sympathetic function is augmented in chronic heart failure (CHF) are not well understood. A previous study from this laboratory (
Circ Res
. 1998;82:496–502) indicated that blockade of nitric oxide (NO) synthesis resulted in only an increase in renal sympathetic nerve activity (RSNA) when plasma angiotensin II (Ang II) levels were elevated. The present study was undertaken to determine if NO reduces RSNA in rabbits with CHF when Ang II receptors are blocked. Twenty-four New Zealand White rabbits were instrumented with cardiac dimension crystals, a left ventricular pacing lead, and a pacemaker. After pacing at 360 to 380 bpm for approximately 3 weeks, a renal sympathetic nerve electrode and arterial and venous catheters were implanted. Studies were carried out in the conscious state 3 to 7 days after electrode implantation. The effects of a 1-hour infusion of sodium nitroprusside (SNP; 3 μg · kg
−1
· min
−1
) on RSNA and mean arterial pressure (MAP) were determined before and after Ang II blockade with losartan (5 mg/kg) in normal and CHF rabbits. Changes in MAP were readjusted to normal with phenylephrine. Before losartan, SNP evoked a decrease in MAP and an increase in RSNA in both groups that was baroreflex-mediated, because both MAP and RSNA returned to control when phenylephrine was administered. In the normal group, losartan plus SNP caused a reduction in MAP and an increase in RSNA that was 152.6±9.8% of control. Phenylephrine returned both MAP and RSNA back to the control levels. However, in the CHF group, losartan plus SNP evoked a smaller change in RSNA for equivalent changes in MAP (117.1±4.1% of control). On returning MAP to the control level with phenylephrine, RSNA was reduced to 65.2±2.9% of control (
P
<0.0001). These data suggest that endogenous Ang II contributes to the sympathoexcitation in the CHF state and that blockade of Ang II receptors plus providing an exogenous source of NO reduces RSNA below the elevated baseline levels. We conclude that both a loss of NO and an increase in Ang II are necessary for sustained increases in sympathetic nerve activity in the CHF state.
Activation of Receptor for Advanced Glycation End Products
Authors: Schmidt Ann Marie , Yan Shi Du , Wautier Jean-Luc , Stern David
Published: June 2012

Abstract
—Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules and engages diverse ligands relevant to distinct pathological processes. One class of RAGE ligands includes glycoxidation products, termed advanced glycation end products, which occur in diabetes, at sites of oxidant stress in tissues, and in renal failure and amyloidoses. RAGE also functions as a signal transduction receptor for amyloid β peptide, known to accumulate in Alzheimer disease in both affected brain parenchyma and cerebral vasculature. Interaction of RAGE with these ligands enhances receptor expression and initiates a positive feedback loop whereby receptor occupancy triggers increased RAGE expression, thereby perpetuating another wave of cellular activation. Sustained expression of RAGE by critical target cells, including endothelium, smooth muscle cells, mononuclear phagocytes, and neurons, in proximity to these ligands, sets the stage for chronic cellular activation and tissue damage. In a model of accelerated atherosclerosis associated with diabetes in genetically manipulated mice, blockade of cell surface RAGE by infusion of a soluble, truncated form of the receptor completely suppressed enhanced formation of vascular lesions. Amelioration of atherosclerosis in these diabetic/atherosclerotic animals by soluble RAGE occurred in the absence of changes in plasma lipids or glycemia, emphasizing the contribution of a lipid- and glycemia-independent mechanism(s) to atherogenesis, which we postulate to be interaction of RAGE with its ligands. Future studies using mice in which RAGE expression has been genetically manipulated and with selective low molecular weight RAGE inhibitors will be required to definitively assign a critical role for RAGE activation in diabetic vasculopathy. However, sustained receptor expression in a microenvironment with a plethora of ligand makes possible prolonged receptor stimulation, suggesting that interaction of cellular RAGE with its ligands could be a factor contributing to a range of important chronic disorders.
Molecular Mechanisms of Neutrophil-Endothelial Cell Adhesion Induced by Redox Imbalance
Authors: Kokura Satoshi , Wolf Robert E. , Yoshikawa Toshikazu , Granger D. Neil , Aw Tak Yee
Published: June 2012

Abstract
—Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-κB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO–induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2).
N
-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin–specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti–E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing
ds
-oligonucleotides that contained nuclear factor-κB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil–endothelial adhesion response.
Structure of the Type B Human Natriuretic Peptide Receptor Gene and Association of a Novel Microsatellite Polymorphism With Essential Hypertension
Authors: Rehemudula Duolikun , Nakayama Tomohiro , Soma Masayoshi , Takahashi Yukie , Uwabo Jiro , Sato Mikano , Izumi Yoichi , Kanmatsuse Katsuo , Ozawa Yukio
Published: June 2012

Abstract
—The natriuretic peptide (NP) system may play a crucial role in development of essential hypertension (EH). C-type NP dilates arteries and lowers blood pressure and inhibits proliferation of vascular smooth muscle cells via the type B NP receptor (NPR-B). However, the association of the human NPR-B gene with EH has not been studied, because little is known about the genomic organization of this gene. We designed oligonucleotide primers based on the cDNA sequence of the human NPR-B gene, and long-range polymerase chain reaction (PCR) was performed. The amplified fragments were sequenced directly, and the exon/intron organization of the human NPR-B gene was determined. The gene, which spans ≈16.5 kbp, is composed of 22 exons, and the intron-exon junctions follow the GT-AG rule. Seven hundred fifty base pairs of the 5′-flanking region were sequenced using a thermal asymmetric interlaced–PCR (TAIL-PCR) method. This region contains 10 potential Sp1 binding sites and lacks a TATA box. Rapid amplification of cDNA ends (RACE) revealed the transcriptional start site at −14 bp. A CA/GT microsatellite repeat was identified with a hybridization-based method and was converted to a sequence-tagged site (STS). The GT microsatellite repeat was localized to intron 2 ≈150 bp downstream of the exon-intron junction. Two alleles, (GT)10 and (GT)11, were detected in both EH patients and age-matched normotensive (NT) controls. Multiple logistic linear regression analysis indicated that the NPR-B genotype is associated significantly with EH (odds ratio 1.55; 95% confidence interval, 1.02 to 2.35). The (GT)11 frequency was 0.316 (65/206) for the EH group and 0.218 (44/202) for the NT group and differed significantly between the EH and NT groups (χ
2
=4.97,
P
=0.026). The structural organization of the human NPR-B gene was determined, and a novel GT repeat polymorphism, which associated with EH, was identified. These results suggest that one cause of EH is a mutation in this gene or a closely related gene or region.
Nitric Oxide and C-Type Atrial Natriuretic Peptide Stimulate Primary Aortic Smooth Muscle Cell Migration via a cGMP-Dependent Mechanism
Authors: Brown Claire , Pan Xiaolei , Hassid Aviv
Published: June 2012

Abstract
—Migration of aortic smooth muscle cells is thought to be of essential importance in vascular restenosis, remodeling, and angiogenesis. Recent studies have shown that NO donors inhibit the migration of subcultured aortic smooth muscle cells. However, there is evidence that NO elicits opposite effects on cell proliferation in primary versus subcultured cells, indicating fundamental differences among different models of aortic smooth muscle cell cultures. The purpose of the current study was to investigate the effect of NO donors on migration of primary cultures of rat aortic smooth muscle cells and to compare and contrast their response with those in subcultured cells. A second purpose was to investigate some of the underlying mechanisms associated with NO-induced effects on cell migration. We report that 2 NO donors,
S
-nitroso-
N
-acetylpenicillamine (SNAP) and 2,2-(hydroxynitrosohydrazino)bis-ethanamine, stimulated the migration of primary cells in a wounded-culture model as well as in a transwell migration model. The effect of NO donors was mimicked by 2 cGMP analogues and C-type natriuretic peptide and blocked by a specific inhibitor of guanyl cyclase, 1H-(1,2,4)oxadiazolo[4,3,-a]quinoxalin-1-one, indicating the involvement of cGMP as second messenger. Moreover, neither NO donors nor cGMP analogues altered migration of primary cultures stimulated by either FBS or angiotensin II. In contrast to its effect in primary cultures, SNAP did not alter basal or stimulated migration of subcultured cells, except at a relatively high concentration of 1 mmol/L, at which migration was inhibited. The migration-stimulatory effect of NO donors and cGMP was associated with altered cell morphology and dissociation of actin filaments, consistent with recent studies indicating that cell morphology and cytoskeletal organization influence cell migration. The results suggest the possible involvement of NO-induced cell migration in vascular injury or remodeling, representing conditions in which vascular NO levels would be expected to be elevated.
Pressure Overload Induces Severe Hypertrophy in Mice Treated With Cyclosporine, an Inhibitor of Calcineurin
Authors: Ding Bo , Price Robert L. , Borg Thomas K. , Weinberg Ellen O. , Halloran Philip F. , Lorell Beverly H.
Published: June 2012

Abstract
—Cardiac hypertrophy is the fundamental adaptation of the adult heart to mechanical load. Recent work has shown that inhibition of calcineurin activity with cyclosporine suppresses the development of hypertrophy in calcineurin transgenic mice and in in vitro systems of neonatal rat cardiocytes stimulated with peptide growth factors. To test the hypothesis that the calcineurin signaling pathway is critical for load-induced hypertrophy in vivo, we examined the effects of cyclosporine treatment on left ventricular hypertrophy induced by experimental ascending aortic stenosis for 4 weeks in mice. Left ventricular systolic pressure was elevated to a similar level in aortic stenosis mice that were treated with cyclosporine versus no drug. Left ventricular mass and myocyte size were similar in treated and untreated aortic stenosis animals and significantly greater than control animals, showing that cyclosporine treatment does not suppress hypertrophic growth. Both treated and untreated animals showed increased left ventricular expression of the load-sensitive gene atrial natriuretic factor. Calcineurin activity was measured in the left ventricle and the spleen from control mice and aortic stenosis mice treated with cyclosporine versus no drug. Levels of calcineurin activity were similar in the spleens of control and untreated aortic stenosis mice. However, calcineurin activity was severely depressed in left ventricular tissue of untreated aortic stenosis mice compared with control mice and was further reduced by cyclosporine treatment. Thus, pathological hypertrophy and cardiac-restricted gene expression induced by pressure overload in vivo are not suppressed by treatment with cyclosporine and do not appear to depend on the elevation of left ventricular calcineurin activity.
Stimulation of Different Subtypes of Angiotensin II Receptors, AT
1
and AT
2
Receptors, Regulates STAT Activation by Negative Crosstalk
Authors: Horiuchi Masatsugu , Hayashida Wataru , Akishita Masahiro , Tamura Kouichi , Daviet Laurent , Lehtonen Jukka Y. A. , Dzau Victor J.
Published: June 2012

Abstract
—Angiotensin II type 2 (AT
2
) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT
2
receptor in AT
2
receptor cDNA–transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT
1
) receptor–mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1α/β, STAT2, and STAT3 without influence on Janus kinase. AT
2
receptor activation also inhibited the tyrosine phosphorylation of STAT1α/β induced by interferon-γ, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT
2
receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT
2
receptor. Moreover, AT
2
receptor inhibited serine phosphorylation of STAT1α and STAT3 via the inhibition of extracellular signal–regulated kinase (ERK) activation. Stimulation of AT
2
receptor inhibited the binding of STATs with
sis
-inducing element in c-
fos
promoter, resulting in decreased c-
fos
expression. Taken together, our results suggest that AT
2
receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation.
Evidence for Intracellular Endothelin-Converting Enzyme-2 Expression in Cultured Human Vascular Endothelial Cells
Authors: Russell Fraser D. , Davenport Anthony P.
Published: June 2012

Abstract
—We have previously reported the intracellular localization of the endothelin-converting enzyme-1 (ECE-1) in human umbilical vein endothelial cells. In the present study, we provide the first immunocytochemical and biochemical evidence for the presence of ECE-2 in human cells. ECE activity was determined by conversion of exogenously added big endothelin-1 (big ET-1) to ET-1 in subcellular fractions obtained by sucrose density gradient centrifugation of human umbilical vein endothelial cell homogenates. ECE-1 and ECE-2 can be differentiated by pH dependence for optimal activity and by sensitivity to phosphoramidon, which shows selectivity for ECE-2 over ECE-1 and PD159790, a novel ECE-1 selective inhibitor. Optimal ECE activity was measured at pH 6.0, a value intermediate between that reported for ECE-1 (pH 6.8) and ECE-2 (pH 5.5), indicating expression of both enzymes. At pH 6.9, conversion of big ET-1 was inhibited markedly by 30 μmol/L PD159790 and by 100 μmol/L phosphoramidon but not by 0.1 μmol/L phosphoramidon. In contrast, ECE activity was unaffected by 30 μmol/L PD159790 but was inhibited markedly by 0.1 and 100 μmol/L phosphoramidon at pH 5.4 (IC
50
1.5 nmol/L), consistent with ECE-2 activity. Confocal microscopy revealed a punctate pattern of ECE-2–like immunoreactive staining in the cell cytosol, suggesting localization to secretory vesicles with a possible role in processing big ET-1 while in transit to the cell surface via the constitutive secretory pathway.
Direct Demonstration of Exocytosis and Endocytosis in Single Mouse Juxtaglomerular Cells
Authors: Friis Ulla G. , Jensen Boye L. , Aas Jeanette K. , Skøtt Ole
Published: June 2012

Abstract
—The rate of renin secretion from renal juxtaglomerular (JG) cells is the major determinant of the activity of the renin-angiotensin system. However, the mechanisms involved in the excretion and turnover of secretory granules in the JG cells remain obscure. Therefore, in the present study, the whole-cell patch-clamp technique was applied to single JG cells from the mouse kidney to measure changes in cell membrane capacitance (
C
m
) as an index of secretory activity. Resting JG cell
C
m
was stable, on average 3.13±0.13 pF (SEM, n=106). In isotonic solutions,
C
m
was unaffected by [Cl
−
]
i
.
C
m
was consistently increased (7.0±1.3% and 7.2±3.1%) by intracellular cAMP (1 to 10 μmol/L). This effect was mimicked by extracellular application of the β-agonist isoproterenol to the JG cells (9.4±3.1%). At 100 μmol/L, cAMP induced a paradoxical decrease in
C
m
of ≤20%, which was mimicked by forskolin. Cell swelling induced by a 7% reduction in osmolality increased
C
m
with no significant additional effects to [Cl
−
]
i
and cAMP. cAMP increased whole-cell outward current 2- to 4-fold in all groups, but no correlation between changes in whole-cell currents and
C
m
existed. We conclude that the whole-cell patch-clamp method allows the study of exocytosis and endocytosis in JG cells. Renin release induced by the cAMP pathway and by cell swelling is exocytotic, and high-intracellular cAMP levels activate membrane retrieval mechanisms.
Expression of Human Scavenger Receptor Class B Type I in Cultured Human Monocyte-Derived Macrophages and Atherosclerotic Lesions
Authors: Hirano Ken-ichi , Yamashita Shizuya , Nakagawa Yumiko , Ohya Takeshi , Matsuura Fumihiko , Tsukamoto Kosuke , Okamoto Yoshihisa , Matsuyama Akifumi , Matsumoto Kengo , Miyagawa Jun-ichiro , Matsuzawa Yuji
Published: June 2012

Abstract
—The scavenger receptor class B type I (SR-BI) and its human homologue CLA-1 (
C
D36 and
L
IMPII
A
nalogous-
1
) have recently been identified to bind HDL and mediate the selective uptake of HDL lipids. Tissue distribution of both murine and human receptors is quite similar, in that they are expressed abundantly in liver and steroidogenic tissues. However, expression and function of the human SR-BI (hSR-BI), in the periphery of reverse cholesterol transport such as macrophages, are still unclear. In the present study, we have raised two different kinds of anti–hSR-BI polypeptide antibodies (Abs): one against the extracellular domain and the other against the intracellular domain. We have investigated the expression of hSR-BI mRNA and immunoreactive mass in freshly isolated cultured human monocyte-derived macrophages (hMφ) and in atherosclerotic lesions. Contrary to the earlier report, hSR-BI mRNA was expressed in cultured hMφ and markedly upregulated with differentiation, determined by Northern blot and reverse transcriptase–based polymerase chain reaction analyses. The mRNA expression pattern during differentiation of hMφ was very similar to those of SR class A and another member of SR class B, CD36. Protein expression was confirmed by Western blot analyses with the above Abs to show a major 83-kDa band. Modified lipoproteins such as oxidized LDL and acetylated LDL induced a 5-fold increase in mRNA and protein expression of hSR-BI. Confocal immunofluorescence microscopy demonstrated that hSR-BI immunoreactive mass was detectable as a heterogeneous, punctate staining pattern. Furthermore, immunohistochemical analysis showed that immunoreactive mass of hSR-BI was detected in foam cells in human aortic atherosclerotic lesions and that there was no significant difference of staining patterns between the two Abs. This study clearly demonstrates that hSR-BI is expressed in the lipid-laden macrophages in human atherosclerotic lesions, suggesting that it is very important to know its function and regulation in hMφ to understand the biological utility of this molecule.
A Dual Inhibitor of Platelet-Derived Growth Factor β-Receptor and Src Kinase Activity Potently Interferes With Motogenic and Mitogenic Responses to PDGF in Vascular Smooth Muscle Cells
Authors: Waltenberger Johannes , Uecker Andrea , Kroll Jens , Frank Hedwig , Mayr Ulrike , Bjorge Jeffrey D. , Fujita Donald , Gazit Aviv , Hombach Vinzenz , Levitzki Alexander , Böhmer Frank-D.
Published: June 2012

Abstract
—PP1 has previously been described as an inhibitor of the
Src
-family kinases p56
Lck
and FynT. We have therefore decided to use PP1 to determine the functional role of
Src
in platelet-derived growth factor (PDGF)–induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against
Src
was examined. PP1/AGL1872 potently inhibited recombinant p60
c-src
in vitro and
Src
-dependent tyrosine phosphorylation in p60
c-srcF572
-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of
Src
kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor–stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF α-receptor was susceptible, albeit to a lesser extent than PDGF β-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the
Src
family of tyrosine kinases. It is also a potent inhibitor of the PDGF β-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.
Sphingosylphosphorylcholine Induces a Hypertrophic Growth Response Through the Mitogen-Activated Protein Kinase Signaling Cascade in Rat Neonatal Cardiac Myocytes
Authors: Sekiguchi Kenichi , Yokoyama Tomoyuki , Kurabayashi Masahiko , Okajima Fumikazu , Nagai Ryozo
Published: June 2012

Abstract
—The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 μmol/L), S1P (1 to 10 μmol/L), or SPC (0.1 to 10 μmol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein–coupled receptor and a MAPK signaling cascade.
Upregulation of Na
+
/Ca
2+
Exchanger Expression and Function in an Arrhythmogenic Rabbit Model of Heart Failure
Authors: Pogwizd Steven M. , Qi Ming , Yuan Weilong , Samarel Allen M. , Bers Donald M.
Published: June 2012

Abstract
—Three-dimensional cardiac mapping in rabbits with nonischemic cardiomyopathy has shown that ventricular arrhythmias initiate by a nonreentrant mechanism that may be due to triggered activity from delayed afterdepolarizations. Delayed afterdepolarizations are thought to be due to spontaneous release of Ca
2+
from the sarcoplasmic reticulum (SR) and consequent activation of an inward Na
+
/Ca
2+
exchange (NaCaX) current. The goal of this study was to determine whether there is enhanced NaCaX gene expression and functional activity that may contribute to nonreentrant activation. Heart failure (HF) was induced in rabbits by combined aortic insufficiency and aortic constriction. HF rabbits had left ventricular enlargement (left ventricular end-diastolic dimension increased from 1.43±0.03 to 1.97±0.05 cm) and severely depressed function (fractional shortening reduced from 37% to 26%,
P
<0.02). Heart-to-body weight was increased by 79% in HF. Western blots showed a 93% increase in NaCaX protein in HF (
P
<0.04). NaCaX mRNA (7-kb transcript) was increased by 104% relative to the 18S rRNA in HF. A 14-kb NaCaX transcript was also seen in the HF rabbits, raising total NaCaX mRNA to 2.7-fold compared with controls. The amplitude of caffeine-induced contractures, used to assess SR Ca
2+
load, was not significantly different in HF. Relaxation and [Ca
2+
]
i
decline during caffeine-induced contractures is attributable to Ca
2+
transport by NaCaX and was 61% and 45% faster in HF (
P
<0.05), respectively. NaCaX current measured under controlled voltage clamp conditions was also 2-fold higher in HF cells. SR Ca
2+
-ATPase mRNA and protein levels and Ca
2+
current density were not significantly altered in HF. Twitch amplitudes from HF myocytes were 26% smaller compared with control (
P
<0.02), but twitch relaxation and [Ca
2+
]
i
decline (due largely to SR Ca
2+
-ATPase) were not altered. Thus myocytes and myocardium from HF rabbits exhibit enhanced NaCaX expression and function. The enhanced NaCaX activity may contribute to depressed contractions, increased transient inward current (for a given SR Ca
2+
release), delayed afterdepolarizations, and nonreentrant initiation of ventricular tachycardia in this arrhythmogenic model of HF.
A Flavoprotein Mechanism Appears to Prevent an Oxygen-Dependent Inhibition of cGMP-Associated Nitric Oxide–Elicited Relaxation of Bovine Coronary Arteries
Authors: Iesaki Takafumi , Gupte Sachin A. , Wolin Michael S.
Published: June 2012

Abstract
—The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donor
S
-nitroso-
N
-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 μmol/L DPI under an atmosphere of 21% O
2
(5% CO
2
). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N
2
(5% CO
2
). If BCAs are treated with DPI under 21% O
2
and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N
2
, there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O
2
but not under N
2
. Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 μmol/L DPI under 21% O
2
, but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O
2
-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.
Recent Advances in Cardiac β
2
-Adrenergic Signal Transduction
Authors: Xiao Rui-Ping , Cheng Heping , Zhou Ying-Ying , Kuschel Meike , Lakatta Edward G.
Published: June 2012

Abstract
—Recent studies have added complexities to the conceptual framework of cardiac β-adrenergic receptor (β-AR) signal transduction. Whereas the classical linear G
s
–adenylyl cyclase–cAMP–protein kinase A (PKA) signaling cascade has been corroborated for β
1
-AR stimulation, the β
2
-AR signaling pathway bifurcates at the very first postreceptor step, the G protein level. In addition to G
s
, β
2
-AR couples to pertussis toxin–sensitive G
i
proteins, G
i2
and G
i3
. The coupling of β
2
-AR to G
i
proteins mediates, to a large extent, the differential actions of the β-AR subtypes on cardiac Ca
2+
handling, contractility, cAMP accumulation, and PKA-mediated protein phosphorylation. The extent of G
i
coupling in ventricular myocytes appears to be the basis of the substantial species-to-species diversity in β
2
-AR–mediated cardiac responses. There is an apparent dissociation of β
2
-AR–induced augmentations of the intracellular Ca
2+
(Ca
i
) transient and contractility from cAMP production and PKA-dependent cytoplasmic protein phosphorylation. This can be largely explained by G
i
-dependent functional compartmentalization of the β
2
-AR–directed cAMP/PKA signaling to the sarcolemmal microdomain. This compartmentalization allows the common second messenger, cAMP, to perform selective functions during β-AR subtype stimulation. Emerging evidence also points to distinctly different roles of these β-AR subtypes in modulating noncontractile cellular processes. These recent findings not only reveal the diversity and specificity of β-AR and G protein interactions but also provide new insights for understanding the differential regulation and functionality of β-AR subtypes in healthy and diseased hearts.
Disruption of Cadherin-Related Junctions Triggers Autocrine Expression of Vascular Endothelial Growth Factor in Bovine Aortic Endothelial Cells
Authors: Castilla M. Angeles , Arroyo Maria Victoria Alvarez , Aceituno Esther , Aragoncillo Paloma , González-Pacheco Francisco R. , Texeiro Emma , Bragado Rafael , Caramelo Carlos
Published: June 2012

Abstract
—The mechanisms involved in the blockade of proliferation in confluent endothelial cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of endothelial monolayer cell growth. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both endothelial cell proliferation and autocrine production of growth factors. With this purpose, we assessed the changes in growth, death resistance, and expression of vascular endothelial growth factor (VEGF) under conditions of disruption of the intercellular junctions between endothelial cells. Disruption of cell junctions was produced by means of a specific anti–vascular endothelial cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the 121–, 165–, and 189–amino acid isoforms of VEGF. Further evidence of the relationship between endothelial cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical endothelial injury and in endothelial cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with growth-promoting and cytoprotective effects, as assessed by [
3
H]thymidine uptake,
51
Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic interendothelial junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of endothelial cells.
E2F-1 Overexpression in Cardiomyocytes Induces Downregulation of p21
CIP1
and p27
KIP1
and Release of Active Cyclin-Dependent Kinases in the Presence of Insulin-Like Growth Factor I
Authors: von Harsdorf Rüdiger , Hauck Ludger , Mehrhof Felix , Wegenka Ursula , Cardoso M. Cristina , Dietz Rainer
Published: June 2012

Abstract
—The heart is a postmitotic organ unable to regenerate after injury. The mechanisms controlling cell cycle arrest in cardiomyocytes are still unknown. Adenoviral delivery of E2F-1 to primary rat cardiomyocytes resulted in an increase in the expression of key cell cycle activators and apoptosis in >90% of the cells. However, insulin-like growth factor I (IGF-I) rescued cardiomyocytes from E2F-1–induced apoptosis. Furthermore, overexpression of E2F-1 in the presence of IGF-I induced the specific downregulation of total p21
CIP1
and p27
KIP1
protein levels and their dissociation from cyclin-dependent kinases (cdks). In contrast, p16
INK4
and p57
KIP2
protein levels and their association with cdks remained unaltered. The dissociation of p21
CIP1
and p27
KIP1
from their cdk complexes correlated well with the activation of cdk2, cdk4, and cdk6 and the release from cell cycle arrest. Under these circumstances, the number of cardiomyocytes in S phase rose from 1.2% to 23%. These results indicate that IGF-I renders cardiomyocytes permissive for cell cycle reentry. Finally, the specific downregulation of p21
CIP1
and p27
KIP1
further suggests their key role in the maintenance of cell cycle arrest in cardiomyocytes.
Patterns of Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 Expression in Rabbit and Mouse Atherosclerotic Lesions and at Sites Predisposed to Lesion Formation
Authors: Iiyama Kaeko , Hajra Leena , Iiyama Motoi , Li Hongmei , DiChiara Maria , Medoff Benjamin D. , Cybulsky Myron I.
Published: June 2012

Abstract
—The recruitment of mononuclear leukocytes and formation of intimal macrophage-rich lesions at specific sites of the arterial tree are key events in atherogenesis. Inducible endothelial cell adhesion molecules may participate in this process. In aortas of normal chow-fed wild-type mice and rabbits, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), but not E-selectin, were expressed by endothelial cells in regions predisposed to atherosclerotic lesion formation. En face confocal microscopy of the mouse ascending aorta and proximal arch demonstrated that VCAM-1 expression was increased on the endothelial cell surface in lesion-prone areas. ICAM-1 expression extended into areas protected from lesion formation. Hypercholesterolemia induced atherosclerotic lesion formation in rabbits, LDL receptor and apolipoprotein E knockout mice, and Northern blot analysis demonstrated increased steady-state mRNA levels of VCAM-1 and ICAM-1, but not of E-selectin. Immunohistochemical staining revealed that VCAM-1 and ICAM-1 were expressed predominantly by endothelium in early lesions and by intimal cells in more advanced lesions. In early and advanced lesions, staining was most intense in endothelial cells at and adjacent to lesion borders. ICAM-1 staining extended into the uninvolved aorta. These expression patterns were highly reproducible in both species. The only difference was that VCAM-1 expression in endothelium over the central portions of lesions was found frequently in rabbits and rarely in mice. The expression of VCAM-1 by arterial endothelium in normal animals may represent a pathogenic mechanism or a phenotypic marker of predisposition to atherogenesis.