bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Observations In Vivo on the Role of Endothelial Selectins and α
4
Integrin in Cytokine-Induced Leukocyte-Endothelium Interactions in the Mouse Aorta
Authors: Eriksson Einar E. , Werr Joachim , Guo Yancai , Thoren Peter , Lindbom Lennart
Published: June 2012

Abstract
—The molecular mechanisms underlying leukocyte recruitment in large arteries have been extensively studied using histological techniques on fixed tissues. However, there are no reports that address the dynamics of leukocyte recruitment in large arteries in vivo. We developed an intravital microscopy technique for direct observation of leukocyte-endothelium interactions in the mouse aorta. Circulating leukocytes were labeled intravasally with rhodamine 6G and microscopically visualized within the aorta, allowing direct analysis of leukocyte rolling and adhesion. In untreated vessels, leukocyte-endothelium interactions were virtually absent. However, local pretreatment with cytokines interleukin-1β and tumor necrosis factor-α induced clear-cut leukocyte rolling and adhesion, compatible with normal blood flow and wall shear rate. High shear decreased rolling leukocyte flux and increased leukocyte rolling velocity, thus decreasing the tendency for firm adhesion. Leukocyte rolling was almost abolished by an antibody blocking the function of P-selectin, whereas function-blocking antibodies against E-selectin and the α
4
-integrin subunit decreased rolling leukocyte flux to 51±34% (mean±SD) and 59±11% of the value before antibody treatment, respectively. In addition, inhibition of E-selectin function, but not of α
4
integrin, resulted in increased leukocyte rolling velocity (from 48±32 to 71±32 μm per second). Taken together, we introduce the first model for direct studies of leukocyte-endothelium interactions in a large artery in vivo and demonstrate cytokine-induced shear-sensitive leukocyte rolling that is critically dependent on P-selectin and modulated by E-selectin and α
4
integrin.
Relation Between Renal Interstitial ATP Concentrations and Autoregulation-Mediated Changes in Renal Vascular Resistance
Authors: Nishiyama Akira , Majid Dewan S. A. , Taher Khandaker A. , Miyatake Akira , Navar L. Gabriel
Published: June 2012

Abstract
—The present study was performed to examine the hypothesis that autoregulation-related changes in renal vascular resistance (RVR) are mediated by extracellular ATP. By use of a microdialysis method, renal interstitial concentrations of ATP and adenosine were measured at different renal arterial pressures (RAPs) within the autoregulatory range in anesthetized dogs (n=12). RAP was reduced in steps from the ambient pressure (131±4 mm Hg) to 105±3 mm Hg (step 1) and 80±2 mm Hg (step 2). Renal blood flow and glomerular filtration rate exhibited efficient autoregulation in response to these changes in RAP. RVR decreased by 22±2% in step 1 (
P
<0.01) and 38±3% in step 2 (
P
<0.01). The control renal interstitial concentration of ATP was 6.51±0.71 nmol/L and decreased to 4.51±0.55 nmol/L in step 1 (
P
<0.01) and 2.77±0.47 nmol/L in step 2 (
P
<0.01). In contrast, the adenosine concentrations (117±6 nmol/L) were not altered significantly. Changes in ATP levels were highly correlated with changes in RVR (
r
=0.88,
P
<0.0001). Further studies demonstrated that stimulation of the tubuloglomerular feedback (TGF) mechanism by increasing distal volume delivery elicited with acetazolamide also led to increases in renal interstitial ATP concentrations, whereas furosemide, which is known to block TGF responses, reduced renal interstitial fluid ATP concentrations. The data demonstrate a positive relation between renal interstitial fluid ATP concentrations and both autoregulation- and TGF-dependent changes in RVR and thus support the hypothesis that changes in extracellular ATP contribute to the RVR adjustments responsible for the mechanism of renal autoregulation.
Molecular Basis for the Association of Group IIA Phospholipase A
2
and Decorin in Human Atherosclerotic Lesions
Authors: Sartipy Peter , Johansen Berit , Gåsvik Kathrine , Hurt-Camejo Eva
Published: June 2012

Abstract
—Group IIA secretory nonpancreatic phospholipase A
2
(snpPLA
2
) is associated with collagen fibers in the extracellular matrix of human atherosclerotic plaques. Decorin, a small proteoglycan (PG) carrying chondroitin/dermatan sulfate glycosaminoglycans (GAGs), forms part of the collagen network in human arteries. To explore whether snpPLA
2
may be associated with collagen fibers via interaction with decorin, we performed (1) immunohistochemistry to compare the relative in vivo localization of snpPLA
2
and decorin in human atherosclerotic tissue and (2) in vitro experiments to study the interaction between snpPLA
2
and decorin. In atherosclerotic lesions, decorin was detected within the snpPLA
2
-positive part of the intima close to the media. Electrophoretic mobility shift assay showed that snpPLA
2
binds to decorin synthesized by human fibroblasts. Native and GAG-depleted decorin enhanced the association of snpPLA
2
to collagen types I and VI in a solid-phase binding assay. Furthermore, snpPLA
2
bound efficiently to a recombinant decorin core protein fragment B/E (Asp45-Lys359). This binding was competed with soluble decorin and inhibited at NaCl concentrations >150 mmol/L. The decorin core protein fragment B/E competed better than dermatan sulfate for binding of snpPLA
2
to decorin-coated microtiter wells. The enzymatic activity of snpPLA
2
increased 2- to 3-fold in the presence of decorin or GAG-depleted decorin. The results show that snpPLA
2
binds preferentially to the decorin protein core rather than to the GAG chain and that this interaction enhances snpPLA
2
activity. As a consequence, this active extracellular enzyme may contribute to the pathogenesis of atherosclerosis by modifying lipoproteins and releasing inflammatory lipid mediators at places of lipoprotein retention in the arterial wall.
Effect of Platelet-Derived Growth Factor Receptor-α and -β Blockade on Flow-Induced Neointimal Formation in Endothelialized Baboon Vascular Grafts
Authors: Davies Mark G. , Owens Erik L. , Mason David P. , Lea Holly , Tran P. Kiet , Vergel Selina , Hawkins Suzanne A. , Hart Charles E. , Clowes Alexander W.
Published: June 2012

Abstract
—The growth of neointima and neointimal smooth muscle cells in baboon polytetrafluoroethylene grafts is regulated by blood flow. Because neointimal smooth muscle cells express both platelet-derived growth factor receptor-α and -β (PDGFR-α and -β), we designed this study to test the hypothesis that inhibiting either PDGFR-α or PDGFR-β with a specific mouse/human chimeric antibody will modulate flow-induced neointimal formation. Bilateral aortoiliac grafts and distal femoral arteriovenous fistulae were placed in 17 baboons. After 8 weeks, 1 arteriovenous fistulae was ligated, normalizing flow through the ipsilateral graft while maintaining high flow in the contralateral graft. The experimental groups received a blocking antibody to PDGFR-α (Ab-PDGFR-α; 10 mg/kg; n=5) or PDGFR-β (Ab-PDGFR-β; 10 mg/kg; n=6) by pulsed intravenous administration 30 minutes before ligation and at 4, 8, 15, and 22 days after ligation. Controls received carrier medium alone (n=8). Serum antibody concentrations were followed. Grafts were harvested after 28 days and analyzed by videomorphometry. Serum Ab-PDGFR-α concentrations fell rapidly after day 7 to 0, whereas serum Ab-PDGFR-β concentrations were maintained at the target levels (>50 μg/mL). Compared with controls (3.7±0.3), the ratio of the intimal areas (normalized flow/high flow) was significantly reduced in Ab-PDGFR-β (1.2±0.2,
P
<0.01) but not in Ab-PDGFR-α (2.2±0.4). Ab-PDGFR-α decreased significantly the overall smooth muscle cell nuclear density of the neointima (
P
<0.01) compared with either the control or Ab-PDGFR-β treated groups. PDGFR-β is necessary for flow-induced neointimal formation in prosthetic grafts. Targeting PDGFR-β may be an effective pharmacological strategy for suppressing graft neointimal development.
Determinants of the Cardiomyopathic Phenotype in Chimeric Mice Overexpressing Cardiac Gsα
Authors: Vatner Dorothy E. , Yang Gui-Ping , Geng Yong-Jian , Asai Kuniya , Yun Jeung S. , Wagner Thomas E. , Ishikawa Yoshihiro , Bishop Sanford P. , Homcy Charles J. , Vatner Stephen F.
Published: June 2012

Abstract
—Mice with overexpressed cardiac Gsα develop cardiomyopathy, characterized by myocyte hypertrophy and extensive myocardial fibrosis. The cardiomyopathy likely involves chronically enhanced β-adrenergic signaling, because it can be blocked with long-term propranolol treatment. It remains unknown whether the genotype of the myocyte is solely responsible for the progressive pathological changes. A chimeric population in the heart should answer this question. Accordingly, we developed a chimeric animal, which combined cells from a transgenic overexpressed Gsα parent and a Rosa mouse containing the LacZ reporter gene, facilitating identification of the non–Gsα cells, which express a blue color with exposure to β-galactosidase. We studied these animals at 14 to 17 months of age (when cardiomyopathy should have been present), with the proportion of Gsα cells in the myocardium ranging from 5% to 88%. β-Galactosidase staining of the hearts demonstrated Gsα and Rosa cells, exhibiting a mosaic pattern. The fibrosis and hypertrophy, characteristic of the cardiomyopathy, were not distributed randomly. There was a direct correlation (
r
=0.85) between the extent of myocyte hypertrophy (determined by computer imaging) and the quantity of Gsα cells. The fibrosis, determined by picric acid Sirius red, was also more prominent in areas with the greatest Gsα cell density, with a correlation of
r
=0.88. Thus, the overexpressed Gsα can exert its action over the life of the animal, resulting in a local picture of cardiomyopathic damage in discrete regions of the heart, where clusters of the overexpressed Gsα cells reside, sparing the clusters of normal cells derived from the normal Rosa parent.
Reduced Myocardial Nerve Growth Factor Expression in Human and Experimental Heart Failure
Authors: Kaye David M. , Vaddadi Gautam , Gruskin Sara L. , Du Xiao-Jun , Esler Murray D.
Published: June 2012

Abstract
—Maintenance of cardiac performance is tightly controlled by the autonomic nervous system. In congestive heart failure (CHF), although the adverse pathophysiological effects of cardiac sympathetic overactivity are increasingly recognized, the paradoxical finding of reduced sympathetic innervation density in the failing heart remains unexplained. Given these observations, we tested the hypothesis that a reduction in the myocardial production of nerve growth factor (NGF), which is important for the maintenance of sympathetic neuronal survival, could explain the conflicting neurochemical and neuroanatomical profile of CHF. In healthy humans (n=11), there was a significantly greater transcardiac venoarterial plasma NGF gradient than in CHF patients (n=11,
P
<0.05). In a rat model of CHF, a 40% reduction (
P
<0.05) NGF mRNA expression was apparent in association with a 24% reduction in tissue NGF content (
P
<0.05). In conjunction, evidence of reduced sympathetic innervation in the failing heart was apparent, as measured histologically by catecholamine fluorescence and by expression of the neuronal NGF receptor trkA. Norepinephrine (10 μmol/L) exposure reduced both NGF mRNA and protein expression in isolated cardiomyocytes, suggesting that myocardial NGF downregulation may represent an adaptive response to sympathetic overactivity. These data indicate that NGF expression in the heart is dynamic and may be altered in cardiovascular disease states. In CHF, reduced NGF expression may account for alterations in sympathetic neuronal function and neuroanatomy. The full text of this article is available at http://www.circresaha.org.
Activation of Mitogen-Activated Protein Kinases in Human Heart During Cardiopulmonary Bypass
Authors: Talmor Daniel , Applebaum Azai , Rudich Assaf , Shapira Yoram , Tirosh Amir
Published: June 2012

Abstract
—Mitogen-activated protein kinases (MAPKs) have been shown to be activated in both in vitro and in vivo models of cardiac tissue in response to ischemia/reperfusion injury. We investigated whether MAPKs are activated in human heart during coronary artery bypass grafting (CABG) surgery. During elective CABG surgery of 8 patients, 3 right atrial appendage biopsies were obtained at baseline, at the end of cross-clamping, and after coronary reperfusion. The expression of the p38-MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal–regulated kinases (ERK1/2) MAPKs was not altered during CABG. The phosphorylation and activation of both ERK1/2 and p38-MAPK were increased ≈2-fold by ischemia and even more (8- and 4-fold, respectively) by reperfusion. Although the ischemic period did not result in a significant activation of JNK, an ≈6-fold increase in JNK activity could be observed after reperfusion. In conclusion, distinct activation patterns of ERK1/2, p38, and JNK MAPKs can be observed in human heart during CABG.
Hydrogen Peroxide– and Peroxynitrite-Induced Mitochondrial DNA Damage and Dysfunction in Vascular Endothelial and Smooth Muscle Cells
Authors: Ballinger Scott W. , Patterson Cam , Yan Chang-Ning , Doan Richard , Burow David L. , Young Christal G. , Yakes F. Michael , Van Houten Bennett , Ballinger Carol A. , Freeman Bruce A. , Runge Marschall S.
Published: June 2012

Abstract
—The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear β
-globin
gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO
−
, resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.
Estrogens and Glucocorticoids Inhibit Endothelial Vascular Cell Adhesion Molecule-1 Expression by Different Transcriptional Mechanisms
Authors: Simoncini Tommaso , Maffei Silvia , Basta Giuseppina , Barsacchi Giuseppina , Genazzani Andrea R. , Liao James K. , De Caterina Raffaele
Published: June 2012

Abstract
—The antiatherogenic effect of estrogen is mediated, in part, by inhibitory effects on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. To determine the mechanism by which estrogen regulates VCAM-1 expression, we compared the effect of 17β-estradiol (E
2
) and of the glucocorticoid dexamethasone (Dex) on lipopolysaccharide (LPS)–induced VCAM-1 expression in human endothelial cells. E
2
decreased LPS-induced VCAM-1 mRNA and protein expression to a greater extent than Dex. Dex, but not E
2
, stabilized VCAM-1 mRNA. This correlated with inhibition of monocytoid U937 cell adhesion to endothelial cells. Transfection of endothelial cells with a functional VCAM-1 promoter construct showed that E
2
inhibited LPS-induced VCAM-1 gene transcription more potently than did Dex. However, using a truncated construct containing only the nuclear factor-κB (NF-κB)–responsive elements but lacking the consensus sequences for activator protein-1 (AP-1) and GATA, E
2
and Dex had similar inhibitory effects. Consistently, gel-shift assays showed that E
2
and Dex comparably inhibit LPS-induced activation of NF-κB, whereas E
2
inhibited LPS-induced activation of AP-1 and GATA to a greater extent than Dex. E
2
inhibition of NF-κB after LPS treatment was associated with decreased inhibitor κB (IκB) kinase activity and with a stabilization of the NF-κB inhibitor IκBα. These results indicate that E
2
decreases VCAM-1 gene expression through the inhibition of NF-κB, AP-1, and GATA and suggest novel mechanisms for the antiatherogenic effect of estrogen on the vascular wall. (

Circ Res. 2000;87:19-25.)
β
2
-Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets
Authors: Queen Lindsay R. , Xu Biao , Horinouchi Kazumi , Fisher Ian , Ferro Albert
Published: June 2012

Abstract
—Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet β-adrenoceptors (βARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of
l
-[
3
H]-arginine to
l
-[
3
H]-citrulline, after βAR stimulation or cAMP elevation. Basal NOS activity was 0.11±0.03 pmol
l
-citrulline/10
8
platelets. The βAR agonist isoproterenol 1 μmol/L and the adenylyl cyclase activator forskolin 1 μmol/L each increased NOS activity, to 0.26±0.04 and 0.23±0.03 pmol
l
-citrulline/10
8
platelets, respectively (
P
<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 μmol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca
2+
. In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3±0.2% versus 2.6±0.2% respectively;
P
<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through β
2
ARs, as determined by coincubation with βAR subtype-selective antagonists. We conclude that β
2
ARs activate platelet NOS by increasing cAMP, and that this activation is Ca
2+
-independent. β
2
ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the
l
-arginine/NO system mediates the effects of β
2
ARs on adhesion but not aggregation. (

Circ Res. 2000;87:39-44.)
Cardiac Septal and Valvular Dysmorphogenesis in Mice Heterozygous for Mutations in the Homeobox Gene
Nkx2-5
Authors: Biben Christine , Weber Roberta , Kesteven Scott , Stanley Edouard , McDonald Lachlan , Elliott David A. , Barnett Louise , Köentgen Frank , Robb Lorraine , Feneley Michael , Harvey Richard P.
Published: June 2012

Abstract
—Heterozygous mutations in the cardiac homeobox gene,
NKX2-5
, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for
Nkx2-5
–null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of
Nkx2-5
heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the
Nkx2-5
heterozygous phenotype. Our data demonstrate that the complex effects of
Nkx2-5
haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human
NKX2-5
mutations may be modulated by interacting alleles.
Role of the Calcium-Independent Transient Outward Current
I
to1
in Shaping Action Potential Morphology and Duration
Authors: Greenstein Joseph L. , Wu Richard , Po Sunny , Tomaselli Gordon F. , Winslow Raimond L.
Published: June 2012

Abstract
—The Kv4.3-encoded current (
I
Kv4.3
) has been identified as the major component of the voltage-dependent Ca
2+
-independent transient outward current (
I
to1
) in human and canine ventricular cells. Experimental evidence supports a correlation between
I
to1
density and prominence of the phase 1 notch; however, the role of
I
to1
in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35°C are developed on the basis of experimental measurements. A model of canine
I
to1
is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca
2+
current and
I
Kv4.3
and predict a bimodal relationship between
I
Kv4.3
density and APD whereby perturbations in
I
Kv4.3
density may produce either prolongation or shortening of APD, depending on baseline
I
to1
current level.
Ignition of Calcium Sparks in Arterial and Cardiac Muscle Through Caveolae
Authors: Löhn Matthias , Fürstenau Michael , Sagach Victoriya , Elger Marlies , Schulze Wolfgang , Luft Friedrich C. , Haller Hermann , Gollasch Maik
Published: June 2012

Abstract
—Ca
2+
sparks are localized intracellular Ca
2+
events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca
2+
spark triggering depends on precise delivery of Ca
2+
ions through dihydropyridine (DHP)-sensitive Ca
2+
channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca
2+
influx channels and RyRs. Because caveolae contain DHP-sensitive Ca
2+
channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca
2+
sparks. Using methyl-β-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca
2+
sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca
2+
sparks were not significantly affected. We ruled out the possibility that the decreases in Ca
2+
spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca
2+
load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca
2+
sparks in a caveolae microdomain. The transient elevation in [Ca
2+
]
i
at the inner mouth of a single caveolemmal Ca
2+
channel induces simultaneous activation and thus opens several RyRs to generate a local Ca
2+
release event, a Ca
2+
spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca
2+
signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.
Dyssynchronous Ca
2+
Sparks in Myocytes From Infarcted Hearts
Authors: Litwin Sheldon E. , Zhang Dongfang , Bridge John H. B.
Published: June 2012

Abstract
—The kinetics of contractions and Ca
2+
transients are slowed in myocytes from failing hearts. The mechanisms accounting for these abnormalities remain unclear. Myocardial infarction (MI) was produced by ligation of the circumflex artery in rabbits. We used confocal microscopy to record spatially resolved Ca
2+
transients during field stimulation in left ventricular (LV) myocytes from control and infarcted hearts (3 weeks). Compared with controls, Ca
2+
transients in myocytes adjacent to the infarct had lower peak amplitudes and prolonged time courses. Control myocytes showed relatively uniform changes in [Ca
2+
] throughout the cell after electrical stimulation. In contrast, in MI myocytes [Ca
2+
] increased inhomogeneously and localized increases in [Ca
2+
] occurred throughout the rising and falling phases of the Ca
2+
transient. Ca
2+
content of the sarcoplasmic reticulum did not differ between MI and control myocytes. Peak L-type Ca
2+
current density was reduced in MI myocytes. The macroscopic gain function was not different in control and MI myocytes when calculated as the amplitude of the Ca
2+
transient/peak
I
Ca
. However, when calculated as the peak rate of rise of the Ca
2+
transient/peak
I
Ca
, the gain function was modestly decreased in the MI myocytes. Application of isoproterenol (100 nmol/L) improved the synchronization of Ca
2+
release in MI myocytes at both 0.5 and 1 Hz. The poorly coordinated production of Ca
2+
sparks in myocytes from infarcted rabbit hearts likely contributes to the diminished and slowed macroscopic Ca
2+
transient. These abnormalities can be largely overcome when phosphorylation of Ca
2+
cycling proteins is enhanced by β-adrenergic stimulation.
Soluble Epoxide Hydrolase Regulates Hydrolysis of Vasoactive Epoxyeicosatrienoic Acids
Authors: Yu Zhigang , Xu Fengyun , Huse Linn M. , Morisseau Christophe , Draper Alison J. , Newman John W. , Parker Carol , Graham LeRae , Engler Marguerite M. , Hammock Bruce D. , Zeldin Darryl C. , Kroetz Deanna L.
Published: June 2012

Abstract
—The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14,15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22±4 mm Hg (
P
<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor
N
,
N
′-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.
Regulation of Cardiac L-Type Calcium Channels by Protein Kinase A and Protein Kinase C
Authors: Kamp Timothy J. , Hell Johannes W.
Published: June 2012

Abstract
—Voltage-dependent L-type Ca
2+
channels are multisubunit transmembrane proteins, which allow the influx of Ca
2+
(
I
Ca
) essential for normal excitability and excitation-contraction coupling in cardiac myocytes. A variety of different receptors and signaling pathways provide dynamic regulation of
I
Ca
in the intact heart. The present review focuses on recent evidence describing the molecular details of regulation of L-type Ca
2+
channels by protein kinase A (PKA) and protein kinase C (PKC) pathways. Multiple G protein–coupled receptors act through cAMP/PKA pathways to regulate L-type channels. β-Adrenergic receptor stimulation results in a marked increase in
I
Ca
, which is mediated by a cAMP/PKA pathway. Growing evidence points to an important role of localized signaling complexes involved in the PKA-mediated regulation of
I
Ca
, including A-kinase anchor proteins and binding of phosphatase PP2a to the carboxyl terminus of the α
1C
(Ca
v
1.2) subunit. Both α
1C
and β
2a
subunits of the channel are substrates for PKA in vivo. The regulation of L-type Ca
2+
channels by Gq-linked receptors and associated PKC activation is complex, with both stimulation and inhibition of
I
Ca
being observed. The amino terminus of the α
1C
subunit is critically involved in PKC regulation. Crosstalk between PKA and PKC pathways occurs in the modulation of
I
Ca
. Ultimately, precise regulation of
I
Ca
is needed for normal cardiac function, and alterations in these regulatory pathways may prove important in heart disease.
Diabetes-Induced Vascular Hypertrophy Is Accompanied by Activation of Na
+
-H
+
Exchange and Prevented by Na
+
-H
+
Exchange Inhibition
Authors: Jandeleit-Dahm Karin , Hannan Katherine M. , Farrelly Caroline A. , Allen Terri J. , Rumble Jon R. , Gilbert Richard E. , Cooper Mark E. , Little Peter J.
Published: June 2012

Abstract
—Vascular disease often involves vessel hypertrophy with underlying cellular hypertrophy or hyperplasia. Experimental diabetes stimulates hypertrophy of the rat mesenteric vasculature, and we investigated the hypothesis that this hypertrophy is associated with activation of Na
+
-H
+
exchange (NHE) activity. We measured the NHE activity in isolated, intact blood vessels from control and streptozotocin-induced diabetic adult rats using concurrent myography and fluorescence spectroscopy. The role of inhibiting NHE activity in preventing the development of the mesenteric hypertrophy in streptozotocin-diabetic rats was investigated by administration of cariporide (100 mg/kg body weight per day in 3 doses by gavage) after induction of diabetes and subsequently determining vessel weight and structure. The weight of the mesenteric vasculature was not increased 1 week after streptozotocin treatment but was significantly increased by an average of 56% at 3 weeks. NHE activity in mesenteric arteries showed an enhanced maximal velocity (
V
max
) in diabetic vessels at 1 and 3 weeks (0.246±0.006 and 0.238±0.007 versus 0.198±0.007 pH U/min) with no change in the apparent
K
m
. Moreover, NHE-1 mRNA in mesenteric arterioles at 3 weeks after streptozotocin treatment was increased by >60% (55.8±6.4 versus 91.3±12.3 fg). Administration of cariporide significantly reduced mesenteric vascular weight, the wall/lumen ratio, and mesenteric extracellular matrix accumulation in the diabetic animals. Our study shows that diabetes in vivo correlates with elevated NHE activity and mRNA in the mesenteric vasculature and furthermore that inhibition of this system prevents the hypertrophic response. These data suggest that NHE may be a target for therapeutic modulation of vascular changes in diabetes.
De Novo Expression of Macrophage Migration Inhibitory Factor in Atherogenesis in Rabbits
Authors: Lin Shu-Guang , Yu Xi-Yong , Chen Yong-Xiong , Huang Xiao R. , Metz Christine , Bucala Richard , Lau Chu-Pak , Lan Hui Y.
Published: June 2012

Abstract
—Macrophage migration inhibitory factor (MIF) has been shown to play an important role in macrophage-mediated diseases. We investigate the potential role of MIF in atherogenesis using a hypercholesterolemic rabbit model. New Zealand White rabbits fed with a 2% cholesterol diet developed hypercholesterolemia and early fatty streaks at 1 month. The lesions became advanced at 3 months and were associated with de novo MIF expression by vascular endothelial cells (VECs) and smooth muscle cells (SMCs), as demonstrated by immunohistochemistry, reverse transcriptase–polymerase chain reaction, and in situ hybridization. By contrast, there was no increase in MIF levels in rabbits fed a normal diet. In early atherogenesis, marked upregulation of MIF mRNA and protein by VECs and some intimal cells were closely associated with CD68
+
monocyte adhesion onto and subsequent migration into subendothelial space. Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, which may be associated with the macrophage-rich fatty streak lesion formation. Upregulation of MIF by SMCs is transient during atherogenesis. Importantly, strong MIF expression by activated macrophages may be responsible for the development of foam cell-rich lesions. Finally, the ability of MIF to induce intercellular adhesion molecule-1 expression by VECs implicates its pathogenic role in atherogenesis. In conclusion, the present study provides the first demonstration that MIF is markedly upregulated during atherogenesis. Upregulation of MIF by VECs and SMCs may play a role in macrophage adhesion, transendothelial migration, accumulation, and, importantly, transformation into foam cells. Furthermore, strong MIF expression by macrophages may both initiate and amplify the atherogenesis process.
Endotoxemia in Transgenic Mice Overexpressing Human Glutathione Peroxidases
Authors: Mirochnitchenko Oleg , Prokopenko Olga , Palnitkar Urmila , Kister Ilya , Powell William S. , Inouye Masayori
Published: June 2012

Abstract
—In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra- and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1β and tumor necrosis factor-α) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury.
In Vivo Analysis of an Essential Myosin Light Chain Mutation Linked to Familial Hypertrophic Cardiomyopathy
Authors: Sanbe Atsushi , Nelson David , Gulick James , Setser Elizabeth , Osinska Hanna , Wang Xuejun , Hewett Timothy E. , Klevitsky Raisa , Hayes Eric , Warshaw David M. , Robbins Jeffrey
Published: June 2012

Abstract
—Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutations in both the regulatory (Glu22Lys) and essential light chains (Met149Val) result in an unusual pattern of hypertrophy, leading to obstruction of the midventricular cavity. When a human genomic fragment containing the Met149Val essential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguously establish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, we generated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the coding regions of the gene loci. Expression of the proteins did not lead to a hypertrophic response, even in senescent animals. Changes did occur at the myofilament and cellular levels, with the myofibrils showing increased Ca
2+
sensitivity and significant deficits in relaxation in a transgene dose–dependent manner. Clearly, mice do not always recapitulate important aspects of human hypertrophy. However, because of the discordance of these data with data obtained in transgenic mice containing the human genomic fragment, we believe that the concept that these point mutations by themselves can cause hypertrophic cardiomyopathy should be revisited.
Impaired Contractile Performance of Cultured Rabbit Ventricular Myocytes After Adenoviral Gene Transfer of Na
+
-Ca
2+
Exchanger
Authors: Schillinger Wolfgang , Janssen Paul M. L. , Emami Shahriyar , Henderson Scott A. , Ross Robert S. , Teucher Nils , Zeitz Oliver , Philipson Kenneth D. , Prestle Jürgen , Hasenfuss Gerd
Published: June 2012

Abstract
—Na
+
-Ca
2+
exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral β-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min
−1
, FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163,
P
<0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027±0.002 at 30 min
−1
to 0.037±0.002 at 120 min
−1
(
P
<0.05 versus 30 min
−1
) and to 0.040±0.002 at 180 min
−1
(
P
<0.05 versus 30 min
−1
). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024±0.002 at 30 min
−1
, 0.029±0.002 at 120 min
−1
(
P
<0.05 versus 30 min
−1
,
P
<0.05 versus control), and 0.026±0.002 at 180 min
−1
(NS versus 30 min
−1
,
P
<0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca
2+
load, were significantly reduced at 120 min
−1
in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78±4% in control and 65±3% in the Ad-NCX group (
P
<0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.
Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Synthase via the PI3-Kinase–Akt Pathway in Human Endothelial Cells
Authors: Haynes M. Page , Sinha Diviya , Russell Kerry Strong , Collinge Mark , Fulton David , Morales-Ruiz Manuel , Sessa William C. , Bender Jeffrey R.
Published: June 2012

Abstract
—17β-Estradiol (E
2
) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E
2
-stimulated endothelial NO release can occur without an increase in cytosolic Ca
2+
. Here we demonstrate for the first time, to our knowledge, that E
2
rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase–Akt pathway. E
2
treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E
2
stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E
2
-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca
2+
levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E
2
-stimulated NO release. The membrane-impermeant E
2
BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase–Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca
2+
fluxes and describes an important pathway relevant to cardiovascular pathophysiology.
Myocardial Ischemia/Reperfusion Injury in NADPH Oxidase–Deficient Mice
Authors: Hoffmeyer Michaela R. , Jones Steven P. , Ross Christopher R. , Sharp Brent , Grisham Matthew B. , Laroux F. Stephen , Stalker Timothy J. , Scalia Rosario , Lefer David J.
Published: June 2012

Abstract
—Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (−/−) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase
–/–
mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55±3%) and NADPH oxidase
–/–
hearts (61±4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42±4%) and NADPH oxidase
–/–
mice (34±5%) (
P
=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase
–/–
mice (n=8; 27±2.5%) and heterozygous control mice (n=10; 23.3±3.3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (
P
<0.01) in NADPH oxidase
–/–
mice (n=6) compared with heterozygous mice (n=7) (0.04±0.03 versus 2.2±0.08 nmol O
2
·min
–1
·10
6
cells
–1
). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase
–/–
mice (
P
=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.
Transforming Growth Factor-β/Smads Signaling Induces Transcription of the Cell Type–Restricted Ankyrin Repeat Protein CARP Gene Through CAGA Motif in Vascular Smooth Muscle Cells
Authors: Kanai Hiroyoshi , Tanaka Toru , Aihara Yasushi , Takeda Sin-ichi , Kawabata Masahiro , Miyazono Kohei , Nagai Ryozo , Kurabayashi Masahiko
Published: June 2012

Abstract
—Transforming growth factor (TGF)-β plays a major role in the development of vascular diseases. Despite the pleiotropic effects of TGF-β on vascular smooth muscle cells (VSMCs), only a few genes have been characterized as direct targets of TGF-β in VSMCs. Cardiac ankyrin repeat protein (CARP) has been thought to be expressed exclusively in the heart. In the present study, we showed that CARP is expressed in the vasculature after balloon injury and in cultured VSMCs in response to TGF-β. Analysis of a half-life of the cytoplasmic CARP mRNA levels and the transient transfection of the CARP promoter/luciferase gene indicates that the regulation of CARP expression is increased by TGF-β at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Deletion analysis and site-specific mutagenesis of the CARP promoter indicate that TGF-β response element is localized to CAGA motif at −108 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that the binding activity to the CAGA motif was increased in nuclear extracts of cultured VSMCs by TGF-β. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-β–mediated inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-β/Smad signaling in VSMCs and suggest a role of CARP in mediation of the inhibitory effects of TGF-β on the proliferation of VSMCs.
Ras Reduces L-Type Calcium Channel Current in Cardiac Myocytes
Authors: Ho Peter D. , Fan Jing-Song , Hayes Nicole L. , Saada Nehad , Palade Philip T. , Glembotski Christopher C. , McDonough Patrick M.
Published: June 2012

Abstract
—Heart failure is associated with dysregulation of intracellular calcium ([Ca
2+
]
i
), reduction in myofibrils, and increased activation of Ras, a regulator of signal-transduction pathways. To evaluate the potential effects of Ras on [Ca
2+
]
i
, we expressed constitutively active Ras (Ha-Ras
V12
) in cardiac myocytes and monitored [Ca
2+
]
i
via fluorescence and electrophysiological techniques. Ha-Ras
V12
reduced the magnitude of the contractile calcium transients. Unexpectedly, however, calcium loading of the sarcoplasmic reticulum was increased, suggesting that Ha-Ras
V12
introduces a defect in excitation-calcium release coupling. Consistent with this idea, L-channel calcium currents were reduced by Ha-Ras
V12
, which also downregulated the activity of the L-channel gene promoter. Coexpression of L-channels and SERCA2 largely corrected Ha-Ras
V12
–induced dysregulation of [Ca
2+
]
i
. Furthermore, whereas Ha-Ras
V12
downregulated myofibrils, this effect was blocked by coexpression of L-channels. These results suggest that Ras downregulates L-channel expression, which may play a pathophysiological role in cardiac disease.
Coordinated Control of Cell Ca
2+
Loading and Triggered Release From the Sarcoplasmic Reticulum Underlies the Rapid Inotropic Response to Increased L-Type Ca
2+
Current
Authors: Trafford A. W. , Díaz M. E. , Eisner D. A.
Published: June 2012

Abstract
—The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca
2+
content and systolic Ca
2+
are controlled when Ca
2+
entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca
2+
concentration ([Ca
2+
]
i
). Increasing external Ca
2+
concentration ([Ca
2+
]
o
) from 1 to 2 mmol/L increased the amplitude of the systolic Ca
2+
transient with no effect on SR Ca
2+
content. This constancy of SR content is shown to result because the larger Ca
2+
transient activates a larger Ca
2+
efflux from the cell that balances the increased influx. Decreasing [Ca
2+
]
o
to 0.2 mmol/L decreased systolic Ca
2+
but produced a small increase of SR Ca
2+
content. This increase of SR Ca
2+
content is due to a decreased release of Ca
2+
from the SR resulting in decreased loss of Ca
2+
from the cell. An increase of [Ca
2+
]
o
has two effects: (1) increasing the fraction of SR Ca
2+
content, which is released on depolarization and (2) increasing Ca
2+
entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca
2+
transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca
2+
]
o
than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca
2+
entry and greater fractional release of Ca
2+
is an important factor in regulating excitation-contraction coupling.