bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective Role of Pulmonary Nitric Oxide in the Acute Phase of Endotoxemia in Rats
Authors: Gryglewski Ryszard J. , Wolkow Pawel P. , Uracz Wojciech , Janowska Ewa , Bartus Joanna B. , Balbatun Oleg , Patton Stephen , Brovkovych Viktor , Malinski Tadeusz
Published: June 2012

Abstract
—We present for the first time direct continuous assay of NO concentration (porphyrinic sensor) in the lung parenchyma of Sprague-Dawley rats in vivo during endotoxemia. Intravenous infusion of lipopolysaccharide (LPS, 2 mg · kg
−1
· min
−1
for 10 minutes) stimulated an acute burst of NO from constitutive NO synthase (NOS) that peaked 10 to 15 minutes after the start of LPS infusion, mirroring a coincident peak drop in arterial pressure. NO concentration declined over the next hour to twice above pre-LPS infusion NO levels, where it remained until the rats died, 5 to 6 hours after LPS infusion. The chronic drop in arterial pressure observed from 70 minutes to 6 hours after the start of LPS infusion was not convincingly mirrored by a chronic increase in NO concentration, even though indirect NO assay (Griess method, assaying NO decay products NO
2
−
/NO
3
−
) showed that NO production was increasing as a result of continuous NO release by inducible NOS. A NOS inhibitor,
N
ω
-nitro-
l
-arginine (L-NNA, 10 mg/kg IV) injected 45 minutes before LPS infusion, resulted in sudden death accompanied by macroscopically/microscopically diagnosed symptoms similar to acute respiratory distress syndrome <25 minutes after the start of LPS infusion. Pharmacological analysis of this L-NNA+LPS model by replacing L-NNA with 1-amino-2-hydroxy-guanidine (selective inhibitor of inducible NOS) or by pretreatment with
S
-nitroso-
N
-acetyl-penicillamine (NO donor), camonagrel (thromboxane synthase inhibitor), or WEB2170 (platelet-activating factor receptor antagonist) indicated that in the early acute phase of endotoxemia, LPS stimulated the production of cytoprotective NO, cytotoxic thromboxane A
2
, and platelet-activating factor.
ATP-Dependent Activation of the Atrial Acetylcholine-Induced K
+
Channel Does Not Require Nucleoside Diphosphate Kinase Activity
Authors: Sorota Steve , Chlenov Michael , Du Xiao-Yi , Kagan Michael
Published: June 2012

Abstract
—Prior reports by others have shown that cytoplasmically applied ATP can activate the acetylcholine-induced K
+
channel in inside-out atrial membrane patches when no guanine nucleotides are present in the solution bathing the cytosolic face of the membrane. A nucleoside diphosphate kinase mechanism was proposed to explain the activation by ATP. We show in the present study that cytoplasmic adenylylimidodiphosphate mimics the activation by ATP. Unlike ATP, the activation by adenylylimidodiphosphate does not subside on washout. Although commercially available adenylylimidodiphosphate is contaminated by guanylylimidodiphosphate, the activation by adenylylimidodiphosphate still occurs after HPLC purification to remove guanine nucleotide contamination. Adenylylimidodiphosphate does not support phosphotransferase activity by nucleoside diphosphate kinase. Therefore, nucleoside diphosphate kinase activity cannot explain the activation of atrial acetylcholine-induced K
+
current by ATP and adenylylimidodiphosphate. We hypothesize that the activation by millimolar concentrations of ATP is due to binding of adenine nucleotide to the guanine nucleotide binding site of the G protein(s) responsible for stimulating the acetylcholine-induced K
+
current.
Modulation of Mitochondrial ATP-Dependent K
+
Channels by Protein Kinase C
Authors: Sato Toshiaki , O’Rourke Brian , Marbán Eduardo
Published: June 2012

Abstract
—Pharmacological openers of mitochondrial ATP-dependent K
+
(mitoK
ATP
) channels mimic ischemic preconditioning, and such cardioprotection can be prevented by mitoK
ATP
channel blockers. It is also known that protein kinase C (PKC) plays a key role in the induction and maintenance of preconditioning. To look for possible mechanistic links between these 2 sets of observations, we measured mitochondrial matrix redox potential as an index of mitoK
ATP
channel activity in rabbit ventricular myocytes. The mitoK
ATP
channel opener diazoxide (100 μmol/L) partially oxidized the matrix redox potential. Exposure to phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) potentiated and accelerated the effect of diazoxide. These effects of PMA were blocked by the mitoK
ATP
channel blocker 5-hydroxydecanoate, which we verified to be a selective blocker of the mitoK
ATP
channel in simultaneous recordings of membrane current and flavoprotein fluorescence. The inactive control compound 4α-phorbol (100 nmol/L) did not alter the effects of diazoxide. We conclude that the activity of mitoK
ATP
channels can be regulated by PKC in intact heart cells. Potentiation of mitoK
ATP
channel opening by PKC provides a direct mechanistic link between the signal transduction of ischemic preconditioning and pharmacological cardioprotection targeted at ATP-dependent K
+
channels.
Independent and Exclusive Modulation of Cardiac Delayed Rectifying K
+
Current by Protein Kinase C and Protein Kinase A
Authors: Lo C. Frederick , Numann Randal
Published: June 2012

Abstract
—Expression of minK in
Xenopus
oocytes results in a current similar to the cardiac slow delayed rectifying K
+
(
I
Ks
) current. Modulation of the
I
Ks
current in cardiac myocytes has been studied extensively because of its role in shaping the cardiac action potential. The human and cat minK cDNA have been cloned, but their regulation by protein kinases has not been characterized. We report here on the complex modulation of human and cat
I
Ks
currents by protein kinase C (PKC) and protein kinase A (PKA). Activation of PKC by phorbol ester (100 nmol/L phorbol 12,13-didecanoate [PDD]) produces an increase in
I
Ks
current that peaks after 20 minutes and then subsequently decreases to ≈50% of the control level after 1 hour. PKA activation only produces a sustained increase in
I
Ks
current. Interestingly, premodulation by PKC prevents
I
Ks
current modulation by PKA, and PKC has no effect on
I
Ks
current after potentiation by PKA. This shows that the
I
Ks
current is modulated by PKC and PKA in a mutually exclusive manner and suggests that multiple interacting phosphorylation sites are involved. Activation of PKC by diacylglycerol analogues only produces a slow decrease in
I
Ks
current. The biphasic effects of PKC on
I
Ks
current activated by PDD can also be separated by dose and duration. Low doses of PDD (5 nmol/L) or brief applications (5 minutes) of 100 nmol/L PDD only produces
I
Ks
current activation. These data suggest that there are at least 2 independent PKC phosphorylation sites in the minK-KvLQT1 channel. Additionally, long-term activation of PKC strongly attenuates the
I
Ks
current expression even when the corresponding changes in capacitance are taken into account.
Expression of Inducible Nitric Oxide Synthase After Endothelial Denudation of the Rat Carotid Artery
Authors: González-Fernández Fernando , López-Farré Antonio , Rodríguez-Feo Juan A. , Farré Jerónimo , Guerra José , Fortes Jose , Millás Inmaculada , García-Durán Margarita , Rico Luis , Mata Pedro , de Miguel Lourdes Sánchez , Casado Santos
Published: June 2012

Abstract
—There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.
Kidney Is the Only Source of Human Plasma Renin in 45-kb Human Renin Transgenic Mice
Authors: Yan Yan , Chen Rong , Pitarresi Tina , Sigmund Curt D. , Gross Kenneth W. , Sealey Jean E. , Laragh John H. , Catanzaro Daniel F.
Published: June 2012

Abstract
—Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5′-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5′-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5′- and 8-kb 3′-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.
Heme Oxygenase-1–Derived Carbon Monoxide Contributes to the Suppression of Acute Hypertensive Responses In Vivo
Authors: Motterlini Roberto , Gonzales Armando , Foresti Roberta , Clark James E. , Green Colin J. , Winslow Robert M.
Published: June 2012

Abstract
—The enzyme heme oxygenase, which exists in inducible (HO-1) and constitutive (HO-2) isoforms, catalyzes the degradation of heme to biliverdin and CO in mammalian tissues. CO has been implicated in the control of vascular tone in a manner similar to that for NO. In the present study, we investigated the contribution of the heme oxygenase/CO pathway to the modulation of acute hypertensive responses in vivo induced by (1) ααHb, a chemically modified hemoglobin known to scavenge NO, and (2)
N
G
-nitro-
l
-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Experiments were carried out in conscious rats in which femoral arteries and veins were surgically catheterized 1 or 5 days before treatment with the vasoconstrictor agents. Intravenous infusion of ααHb (8% solution) or L-NAME (30 mmol/kg) produced an acute and significant increase in mean arterial pressure (
P
<0.05) in rats at 5 days after catheter implantation. In contrast, no change in blood pressure was observed when ααHb or L-NAME was infused 1 day after the surgical intervention. The suppression of the hypertensive response observed at 1 day after surgery correlated with a significant (
P
<0.05) HO-1 expression in aorta, heart, and liver as well as increased aortic CO production and cGMP levels. At 1 day after surgery, pretreatment of animals with the heme oxygenase inhibitor zinc protoporphyrin IX (50 μmol/kg IP) markedly decreased aortic CO and cGMP levels and completely restored the vasoconstrictor effects of both ααHb and L-NAME. These results provide evidence for a crucial role of the heme oxygenase/CO pathway in the regulation of blood pressure under stress conditions in vivo.
Caldesmon Inhibits Active Crossbridges in Unstimulated Vascular Smooth Muscle
Authors: Earley James J. , Su Xiaoling , Moreland Robert S.
Published: June 2012

Abstract
—Caldesmon is a thin-filament–associated protein believed to be important in the regulation of smooth muscle contraction, although the precise mechanism is unknown. We used antisense oligodeoxynucleotides to produce intact swine carotid smooth muscle tissue deficient in
h
-caldesmon. Caldesmon content was decreased by 78% after 7 days in culture with antisense oligodeoxynucleotides but was unchanged in tissues in the presence of sense oligodeoxynucleotides or vehicle. Antisense oligodeoxynucleotides produced a significant decrease in the caldesmon/actin ratio, but no change was measured in the calponin/actin ratio, suggesting that the effect was specific to caldesmon and not other thin-filament–associated proteins. Basal and KCl-stimulated levels of myosin light chain phosphorylation were not different among tissues from all 3 groups. In contrast,
h
-caldesmon–deficient tissues produced 62% less KCl-induced force than controls. Unstimulated
h
-caldesmon–deficient smooth muscle tissues stretched and then released, redeveloped force, demonstrating active crossbridge cycling; strips containing normal
h
-caldesmon content did not redevelop force on release. We suggest that in resting vascular smooth muscle, active crossbridges are inhibited by caldesmon. Therefore, regulation of smooth muscle includes a thin-filament–based disinhibition component.
Interferon Induction of TAP1
Authors: Min Wang , Pober Jordan S. , Johnson David R.
Published: June 2012

Abstract
—Interferon (IFN)-γ and IFN-α/β induction of the transporter associated with antigen processing-1 (TAP1) promoter was compared in HeLa cells and endothelial cells (ECs). In HeLa cells, IFN-γ acts through Stat1α/Stat1α homodimers binding to the gamma activating sequence (GAS) and IFN-α/β acts through Stat1/Stat2/p48 binding to the IFN-stimulated response element (ISRE). In ECs, however, IFN-γ and IFN-α/β act through both the GAS and ISRE. The basis of the IFN signaling crossover in ECs was investigated. HeLa and ECs contain similar ratios of Stat1α to Stat2 proteins, and IFN-α/β also activates the same Janus kinases (JAKs) (Jak1 and tyrosine kinase (Tyk) 2 but not Jak2). However, IFN-α/β activates more Stat1α than does IFN-γ in ECs, whereas the reverse occurs in HeLa, and expression of the IFN-α/β receptor-associated phosphatase SHP-1 is much lower in ECs than HeLa cells. Overexpression of SHP-1 in ECs blocks IFN-α/β signaling through GAS, and expression of a dominant negative SHP-1 in HeLa cells permits IFN-α/β signaling through GAS, demonstrating a role for SHP-1 in regulating crossovers between the IFN-α/β and IFN-γ signaling pathways.
Emigrated Neutrophils Regulate Ventricular Contractility via α
4
Integrin
Authors: Poon B. Y. , Ward C. A. , Giles W. R. , Kubes P.
Published: June 2012

Abstract
—We have previously shown that CD18 and α
4
integrin were important in the adherence of emigrated neutrophils to cardiac myocytes. Whether either of these molecules is important in myocyte dysfunction is unclear. In this study, we measured contractility as an index of myocyte function. Control contractility was compared with shortening response in myocytes exposed to neutrophils in the presence and absence of anti-CD18 or anti-α
4
antibodies. Control unloaded cell shortening, expressed as a percentage of resting cell length, measured 10.06±1.16% (n=10) at 5 minutes. Circulating neutrophils caused a 35% reduction in cell shortening, an event prevented by anti-CD18, but not by anti-α
4
antibody. When emigrated neutrophils were added to the myocytes, a profound reduction (50%) in unloaded cell shortening was noted. A significant increase in CD18 and α
4
integrin was found on emigrated neutrophils. Addition of anti-CD18 antibody did not protect the myocyte from the emigrated neutrophils, whereas the addition of an anti-α
4
antibody significantly reduced neutrophil-induced cell shortening, despite some neutrophils still adhering to the myocytes. Furthermore, emigrated neutrophils were able to cause myocytes to go into contracture within 5 minutes in the presence of neutrophils with or without anti-CD18 antibody. In addition to the impairment in unloaded cell shortening, at later times (10 minutes), neutrophils also caused a 40% reduction in the rate of contraction and relaxation. The addition of either anti-CD18 or anti-α
4
antibody protected the myocytes from these changes. The data suggest that immunosuppression of CD18 on emigrated neutrophils was only partially effective in reducing myocyte dysfunction. In contrast, immunosuppression of the α
4
integrin alone was sufficient to dramatically reduce all parameters of cell dysfunction measured in this study.
Vitaxin, a Humanized Monoclonal Antibody to the Vitronectin Receptor (α
v
β
3
), Reduces Neointimal Hyperplasia and Total Vessel Area After Balloon Injury in Hypercholesterolemic Rabbits
Authors: Coleman Kimberly R. , Braden Gregory A. , Willingham Mark C. , Sane David C.
Published: June 2012

Abstract
—The vitronectin receptor (α
v
β
3
) mediates several biological processes that are critical to the formation of a neointima after coronary interventions. Blockade of α
v
β
3
could reduce neointima formation by inhibiting smooth muscle cell migration, decreasing transforming growth factor-β
1
expression, enhancing apoptosis, or reducing neovasculature. The effects of short-term administration of Vitaxin, a humanized monoclonal antibody to α
v
β
3
, on the responses to balloon injury were tested in hyperlipidemic rabbits. Balloon angioplasty was performed on the iliac arteries of male New Zealand White rabbits that were fed an atherogenic diet for 1 week before injury and until euthanization at 4 weeks. Rabbits were given either saline (control) or 1 of 2 dosing regimens of Vitaxin (high dose, 5.0 mg/kg, and low dose, 0.5 mg/kg), which were administered intra-arterially before injury and intramuscularly on days 2 and 3. High-dose and low-dose Vitaxin were equally effective in decreasing neointima formation even in the presence of hypercholesterolemia, a stimulus to α
v
β
3
expression. Vitaxin reduced transforming growth factor-β
1
and enhanced apoptosis in injured arteries. Despite these positive effects, Vitaxin administration was associated with a reduction in artery size, indicating a negative effect on remodeling. Vitaxin has a potential role in preventing intimal hyperplasia, especially if the negative effects on remodeling can be overcome, by dose adjustment or other strategies.
Multiple Connexins Form Gap Junction Channels in Rat Basilar Artery Smooth Muscle Cells
Authors: Li Xing , Simard J. Marc
Published: June 2012

Abstract
—Three connexins, Cx43, Cx40, and Cx37, have been found by protein or mRNA analysis to be prominent in mammalian blood vessels, but electrophysiological characterization of gap junction channels in freshly isolated vascular smooth muscle cells (SMCs) has not previously been reported. We used a dual-perforated patch-clamp technique to study gap junction conductances in SMC pairs from rat basilar arteries. Macroscopic junctional conductance (
G
j
) measured in 98 cell pairs with either Cs
+
or K
+
ranged between 0.68 and 24.8 nS. In weakly coupled cells (
G
j
<5 nS), single-channel currents were readily resolved without pharmacological uncoupling agents, allowing identification of 4 major unitary conductances. Two of these conductances, 80 to 120 pS and 150 to 200 pS, corresponded to the major conductance states for homotypic channels formed from Cx43 or Cx40, which we confirmed were present in smooth muscle by immunofluorescence analysis. Two other conductances, 220 to 280 pS and >300 pS, were identified that have not been previously reported in vascular SMCs. Macroscopic recordings revealed currents that deactivated incompletely over a broad range of transjunctional potentials. In about half of the pairs, we identified macroscopic as well as single-channel currents that exhibited marked voltage asymmetry, consistent with nonhomotypic, ie, either heterotypic or heteromeric channels. Our data indicate that basilar artery SMCs are coupled in vivo in a richly complex manner, involving Cx43, Cx40, and other large-conductance channels, and that a significant number of couplings involve putative nonhomotypic channels.
Structural Plasticity of the Cardiac Nuclear Pore Complex in Response to Regulators of Nuclear Import
Authors: Perez-Terzic Carmen , Gacy A. Marquis , Bortolon Ryan , Dzeja Petras P. , Puceat Michel , Jaconi Marisa , Prendergast Franklyn G. , Terzic Andre
Published: June 2012

Abstract
—Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca
2+
stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca
2+
indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca
2+
homeostasis.
Spatiotemporal Heterogeneity in the Induction of Ventricular Fibrillation by Rapid Pacing
Authors: Cao Ji-Min , Qu Zhilin , Kim Young-Hoon , Wu Tsu-Juey , Garfinkel Alan , Weiss James N. , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: June 2012

Abstract
—The mechanism by which rapid pacing induces ventricular fibrillation (VF) is unclear. We performed computerized epicardial mapping studies in 10 dogs, using 19-beat pacing trains. The pacing interval (PI) of the first train was 300 ms and then was progressively shortened until VF was induced. For each PI, we constructed restitution curves for the effective refractory period (ERP). When the PI was long, the activation cycle length (CL) was constant throughout the mapped region. However, as the PI shortened, there was an increase in the spatiotemporal complexity of the CL variations and an increase in the slope of the ERP restitution curve. In 5 dogs, we documented the initiation of VF by wavebreak at the site of long-short CL variations. Computer simulation studies using the Luo-Rudy I ventricular action potential model in simulated 2-dimensional tissue reproduced the experimental results when normal ERP and conduction velocity (CV) restitution properties were intact. By altering CV and ERP restitutions in this model, we found that CV restitution creates spatial CL variations, whereas ERP restitution underlies temporal, beat-to-beat variations in refractoriness during rapid pacing. Together, the interaction of CV and ERP restitutions produces spatiotemporal oscillations in cardiac activation that increase in amplitude as the PI decreases, ultimately causing wavebreak at the site of intrinsic heterogeneity. This initial wavebreak then leads to the formation of spiral waves and VF. These findings support a key role for both CV and ERP restitutions in the initiation of VF by rapid pacing.
Identification and Cloning of a Secreted Protein Related to the Cysteine-Rich Domain of Frizzled
Authors: Duplàa Cécile , Jaspard Béatrice , Moreau Catherine , D’Amore Patricia A.
Published: June 2012

Abstract
—We report the isolation of a cDNA,
FrzA
(
frizzled in aorta
; GenBank accession No.
U85945
), from bovine aortic endothelium. It is the bovine counterpart of the mouse
sFRP1
, which encodes for a secreted protein that is homologous to the cysteine-rich domain of frizzled. Members of the frizzled family of genes have been shown to be required for tissue polarity and to act as receptors for Wnt. The predicted protein product of this gene includes the cysteine-rich extracellular domain, but not the 7 putative transmembrane domains that are highly conserved among members of the frizzled family. Visualization of
FrzA
mRNA and protein revealed that it was widely distributed among adult tissues. FrzA is expressed by highly differentiated or polarized cells, eg, neurons, cardiocytes, or various epithelia. Analysis of its expression in endothelium revealed that
FrzA
mRNA levels were high in endothelial cells scraped from freshly obtained bovine aortas
,
decreased when cells were placed in culture and began to proliferate, but increased at confluence. Transient transfection assays and an assay using addition of purified protein indicate that FrzA reduces the proliferation of endothelial cells. These data demonstrate the existence of a secreted protein homologous to the extracellular domain of the fz receptor, which we speculate plays a role in controlling cell growth and differentiation, possibly by regulating accessibility to Wnt family members.
Vascular Injury Causes Neointimal Formation in Angiotensin II Type 1a Receptor Knockout Mice
Authors: Harada Koichiro , Komuro Issei , Sugaya Takeshi , Murakami Kazuo , Yazaki Yoshio
Published: June 2012

Abstract
—Many studies using small-animal models suggest that angiotensin II (Ang II) plays an important role in neointimal formation after vascular injury. In the present study, we examined whether Ang II type 1 receptor (AT
1
)-mediated Ang II signaling is indispensable for the development of injury-induced neointimal formation using AT
1a
knockout (KO) mice. Reverse transcriptase–polymerase chain reaction analysis revealed that AT
1
mRNA was not detectable in both uninjured and injured carotid arteries of KO mice, whereas the AT
1
gene was expressed in uninjured carotid arteries of wild-type (WT) mice. At 14 days after injury, AT
1
mRNA levels were increased by 1.5-fold in injured arteries of WT mice. Although AT
2
mRNA was not detectable in uninjured arteries, expression of AT
2
gene was induced in both animal groups at 2 weeks after injury. Vascular injury induced neointimal formation in KO mice as well as in WT mice. There were no significant differences between WT and KO mice in the extent of histological findings such as increased cross-sectional areas of the neointima and the media, the number of proliferating smooth muscle cells, and the amount of collagen and fibronectin. Treatment with subpressor doses of Ang II after injury enhanced the growth of neointima in WT mice but not in KO mice. Moreover, treatment with the selective AT
1
antagonist CV-11974 before injury significantly decreased the formation of neointima in only WT mice, whereas treatment with the selective AT
2
antagonist PD-123319 before injury had no effects in both animal groups. These results suggest that AT
1
-mediated Ang II signaling is not essential for the development of neointimal formation, although it may modify it.
Glucocorticoids Downregulate Cyclooxygenase-1 Gene Expression and Prostacyclin Synthesis in Fetal Pulmonary Artery Endothelium
Authors: Jun Sandy S. , Chen Zhong , Pace Margaret C. , Shaul Philip W.
Published: June 2012

Abstract
—Prostacyclin (prostaglandin I
2
[PGI
2
]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI
2
synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10
–10
to 10
–6
mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10
–6
mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17β-estradiol on COX-1 expression. DEX alone (10
–8
mol/L for 48 hours) caused a 93% fall in basal PGI
2
production, and bradykinin- and A23187-stimulated PGI
2
were diminished 96% and 94%, respectively. Similarly, PGI
2
synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10
–6
mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI
2
synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI
2
production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages.
Examination of the Transverse Tubular System in Living Cardiac Rat Myocytes by 2-Photon Microscopy and Digital Image–Processing Techniques
Authors: Soeller C. , Cannell M. B.
Published: June 2012

Abstract
—The transverse tubular system (t-system) of cardiac muscle is a structure that allows rapid propagation of excitation into the cell interior. Using 2-photon molecular excitation microscopy and digital image–processing methods, we have obtained a comprehensive overview of the t-system of rat ventricular myocytes in living cells. We show that it is possible to quantify the morphology of the t-system in terms of average local tubule diameter, branching pattern, and local abundance of the t-system by immersing living myocytes in a dextran-linked fluorescein solution. Our data suggest that previous electron microscopic examinations of t-system structure have underestimated both the geometric complexity of the t-system morphology and the fraction of cell volume occupied by the t-system (3.6% in this species). About 40% of tubules occur between Z-lines, and the t-tubule diameter is 255±0.85 nm (mean±SEM). The t-tubules leave the outer surface of the cell in an approximately rectangular array; however, at some points junctions between the t-tubules and the surface membrane are missing. In view of the complexity of the t-system apparent from our images, we propose that the t-system be renamed the “sarcolemmal Z rete.” The methods presented here are generally applicable to the quantification of the sarcolemmal Z rete and other structures within cells by fluorescence microscopy in a variety of cell types.
Reversal of GATA-6 Downregulation Promotes Smooth Muscle Differentiation and Inhibits Intimal Hyperplasia in Balloon-Injured Rat Carotid Artery
Authors: Mano Toshiaki , Luo Zhengyu , Malendowicz Slawomir L. , Evans Todd , Walsh Kenneth
Published: June 2012

Abstract
—The
GATA-6
transcription factor is expressed in quiescent vascular smooth muscle cells (VSMCs) in culture, and levels of its transcript are rapidly downregulated on mitogen stimulation. In this study, we demonstrate that the
GATA-6
transcript, protein, and DNA-binding activity are downregulated in rat carotid arteries on balloon injury. Downregulation was detected at 1 and 3 days after injury and recovered by 7 days. To assess the role of GATA-6 downregulation in injury-induced vascular lesion formation, adenoviral vectors were used to express wild-type human
GATA-6
cDNA (Ad-GATA6) or an inactive mutant cDNA that lacks a portion of the zinc-finger domain (Ad-GATA6ΔZF). Adenovirus-mediated
GATA-6
gene transfer to the vessel wall after balloon injury partially restored the levels of GATA-6 protein and DNA-binding activity to before injury levels. The local delivery of Ad-GATA6 but not Ad-GATA6ΔZF inhibited lesion formation by 46% relative to saline control and 50% relative to a control adenovirus that expressed
lacZ
. Local delivery of Ad-GATA6 also reversed changes in the expression patterns of smooth muscle myosin heavy chain, smooth muscle α-actin, calponin, vinculin, metavinculin, and proliferating cell nuclear antigen that are associated with injury-induced VSMC phenotypic modulation. These data indicate that the injury-induced downregulation of GATA-6 is an essential feature of VSMC phenotypic modulation that contributes to vessel lesion formation.
Rapid Induction and Translocation of Egr-1 in Response to Mechanical Strain in Vascular Smooth Muscle Cells
Authors: Morawietz Henning , Ma Yunn-Hwa , Vives Franklin , Wilson Emily , Sukhatme Vikas P. , Holtz Jürgen , Ives Harlan E.
Published: June 2012

Abstract
—The effect of mechanical strain on transcription and expression of the immediate-early genes, early growth response gene-1 (Egr-1), c-
jun
, and c-
fos
, was investigated in neonatal rat aortic vascular smooth muscle (VSM) cells. Cells grown on silicone elastomer plates were subjected to cyclic mechanical strain (1 Hz) at various durations and magnitudes. Egr-1 mRNA increased rapidly in response to cyclic strain, reached a maximum of 10-fold after 30 minutes, and returned to baseline after 4 hours. c-
jun
exhibited a similar pattern, whereas c-
fos
mRNA expression was unaffected by strain. Cycloheximide prolonged the increase in Egr-1 and c-
jun
mRNA and caused superinduction of both. The threshold level of continuous cyclic strain needed to induce expression was 5% for Egr-1 and c-
jun
. Even a single cycle of mechanical strain that lasted 1 second was sufficient to induce Egr-1 and c-
jun
mRNA. Strain also increased expression of a transiently transfected Egr-1 promoter-reporter construct. The effect of varying extracellular matrices on strain-induced Egr-1 and c-
jun
mRNA was examined. In contrast to collagen type 1- and pronectin-coated plates, strain did not significantly alter expression of Egr-1 and c-
jun
was less induced on laminin-coated plates. On collagen type 1, strain increased Egr-1 protein levels by 2.1-fold at 60 minutes. Immunofluorescence microscopy revealed translocation of Egr-1 to the nucleus in response to strain. These observations indicate that Egr-1 expression and translocation are sensitive to mechanical perturbation of the cell. c-
jun
is also induced by strain, but c-fos is not. The signal for this induction may involve specific cell-matrix interactions.
Regulation of Smooth Muscle α-Actin Expression In Vivo Is Dependent on CArG Elements Within the 5′ and First Intron Promoter Regions
Authors: Mack Christopher P. , Owens Gary K.
Published: June 2012

Abstract
—The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM) α-actin and to determine whether regulation of SM α-actin expression in vivo is dependent on CArG (CC(A/T)
6
GG)
cis
elements. Promoter deletions and site directed mutagenesis techniques were used to study gene regulation in transgenic mice as well as in smooth muscle cell (SMC) cultures. Results demonstrated that a Lac Z transgene that contained 547 bp of the 5′ rat SM α-actin promoter was sufficient to drive embryonic expression of SM α-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections into SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrated that the intronic CArG bound serum response factor. A transgene construct from −2600 through the first intron (p2600Int/Lac Z) was expressed in embryos and adults in a pattern that closely mimicked endogenous SM α-actin expression. Expression in adult mice was completely restricted to SMCs and was detected in esophagus, stomach, intestine, lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutation of the intronic CArG selectively abolished expression in SMCs, which suggests that it may act as an SMC-specific enhancer-like element. Taken together, these results provide the first in vivo evidence for the importance of multiple CArG
cis
elements in the regulation of SM α-actin expression.
Plasminogen Is Not Required for Neointima Formation in a Mouse Model of Vein Graft Stenosis
Authors: Shi Chengwei , Patel Anand , Zhang Dorothy , Wang Hong , Carmeliet Peter , Reed Guy L. , Lee Mu-En , Haber Edgar , Sibinga Nicholas E. S.
Published: June 2012

Abstract
—Recent studies of mice that lack plasminogen have identified a critical role for this zymogen in arterial remodeling. To permit the use of these (and other) genetically modified mice in the analysis of venous injury, we developed a model in which a patch cut from the external jugular vein of a mouse is grafted to repair a surgically created defect in its carotid artery. In wild-type mice, the venous graft showed initial endothelial denudation and formation of a neointima that progressively and reproducibly expanded in a manner analogous to human vein graft disease, albeit at an accelerated pace. This neointima occupied 37±4.6% of the vessel lumen at day 7 and 66±5.7% at day 20. The proliferative index of neointimal cells assessed by proliferating cell nuclear antigen staining was 50.6±3.6% at day 7 and 15.2±2.0% at day 20. CD45-positive leukocytes and α-actin–positive smooth muscle cells accounted for 9.5±1.0% and 9.9±1.1% of intimal area at day 7, respectively, with the latter increasing to 40.9±2.6% at day 20. Collagen accounted for 6.8±0.7% of intimal area at day 7 and 20.7±1.8% at day 20. Surprisingly, even though arterial neointima formation due to electrostatic and immune-mediated injury is impaired in plasminogen –/– mice, in our study vein graft neointima formation in these mice was not significantly different from that in controls (70.9±6.4 versus 65.6±4.4% luminal occlusion,
P
=NS). Thus, plasmin proteolysis, although critical in extracellular matrix degradation and cellular migration after arterial injury, does not appear to be so important in vein graft neointima formation, perhaps because of the relative lack of structural barriers to cellular migration in the normal vein wall. This novel model of vein graft injury should be useful for further studies of differences in the response to injury of arterial and venous tissues.
Upregulation of Endothelial Receptor for Oxidized Low-Density Lipoprotein (LOX-1) in Cultured Human Coronary Artery Endothelial Cells by Angiotensin II Type 1 Receptor Activation
Authors: Li D. Y. , Zhang Y. C. , Philips M. I. , Sawamura T. , Mehta J. L.
Published: June 2012

Abstract
—Cross talk between oxidized LDL (ox-LDL) and angiotensin II (Ang II) may be relevant in atherosclerosis. In this study, we examined the presence of a specific endothelial receptor for ox-LDL (LOX-1) and Ang II receptors in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of Ang II on LOX-1 gene and protein expression. LOX-1 was consistently identified in HCAECs by reverse transcriptase–polymerase chain reaction (RT-PCR), cDNA sequence, Western blot, and
125
I-labeled ox-LDL binding assay (B
max
, 29.7 ng/mg protein). The HCAECs also exhibited Ang II receptors (AT
1
>AT
2
), as determined by RT-PCR and
125
I-labeled Ang II binding assay (B
max
, 2.21 and 1.19 fmol/mg protein, respectively). Incubation of HCAECs with Ang II markedly increased LOX-1 mRNA (RT-PCR) and protein (Western blot) expression. The increase in LOX-1 expression was dependent on Ang II concentration (10
–12
to 10
–6
mol/L). Ang II caused a concentration-dependent increase in
125
I-labeled ox-LDL uptake by HCAECs and enhanced ox-LDL–mediated cell injury, as evident from an increase in LDH release and a decrease in cell viability. These effects of Ang II were completely blocked by pretreatment of HCAECs with losartan, a specific AT
1
blocker, but not by
PD123319
, a specific AT
2
blocker. These observations indicate the following: (1) HCAECs possess abundant LOX-1 as well as Ang II (AT
1
>AT
2
) receptors, (2) Ang II upregulates LOX-1 receptor and ox-LDL uptake, (3) the effects of Ang II are mediated by AT
1
activation, and (4) Ang II enhances ox-LDL–mediated injury to HCAECs.
Transcription Factor Decoy to Study the Molecular Mechanism of Negative Regulation of Renin Gene Expression in the Liver In Vivo
Authors: Tomita Sawako , Tomita Naruya , Yamada Takehiko , Zhang Lunan , Kaneda Yasufumi , Morishita Ryuichi , Ogihara Toshio , Dzau Victor J. , Horiuchi Masatsugu
Published: June 2012

Abstract
—Renin is synthesized in high quantities in the juxtaglomerular cells of the kidney, but little or none is synthesized in the liver. Our previous in vitro and biochemical studies have demonstrated that tissue-specific expression of the mouse renin gene is regulated by the specific interaction between negative regulatory element (NRE) in the 5′-flanking region of the renin gene and NRE binding protein (NREB). In this study, we examined the hypothesis that this interaction between the NRE in the promoter region of the rat renin gene and the NREB in the liver contributes to the suppressed renin gene expression in this tissue in vivo. We used in vivo transfection of NRE transcription factor decoy (TFD) double-stranded oligonucleotide into the rat liver via portal vein infusion. A gel mobility shift assay showed that transfected NRE TFD blocked endogenous NREB binding with the rat renin gene. This resulted in enhanced hepatic renin mRNA expression, immunohistochemical detection of renin in the liver, and consequently, increased plasma renin concentration. Taken together, these results document the importance of NREB in the inhibition of renin gene expression in rat liver in vivo and suggest the possibility of in vivo renin gene modulation by the TFD approach.
Very Low-Density Lipoprotein Activates Nuclear Factor-κB in Endothelial Cells
Authors: Dichtl Wolfgang , Nilsson Lennart , Goncalves Isabel , Ares Mikko P. S. , Banfi Cristina , Calara Federico , Hamsten Anders , Eriksson Per , Nilsson Jan
Published: June 2012

Abstract
—High plasma levels of VLDL are associated with increased risk for atherosclerosis. Here we show that VLDL (75 to 150 μg/mL) activates nuclear factor-κB (NF-κB), a transcription factor known to play a key role in regulation of inflammation. Oxidation of VLDL reduced its capacity to activate NF-κB in vitro, whereas free fatty acids such as linoleic and oleic acid activated NF-κB to the same extent as did VLDL. Intravenous injection of human VLDL (6 mg protein per kg) into rats resulted in arterial activation of NF-κB as assessed by electrophoretic mobility shift assay. Aortic endothelial cells showed positive nuclear staining for the activated RelA (p65) subunit of NF-κB at 6 to 24 hours after injection. There was also a parallel expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, as well as the cytokine tumor necrosis factor-α. Pretreatment of the rats with diet containing 1% of the antioxidant probucol for 8 weeks did not inhibit arterial activation of NF-κB in response to injection of VLDL. Moreover, injection of triglycerides (10% Intralipid, 5 mL/kg) activated arterial expression of NF-κB to the same extent as VLDL. Our results suggest that VLDL may promote the development of atherosclerotic lesions by activation of the proinflammatory transcription factor NF-κB. The effect appears to be mediated by a release of VLDL fatty acids but not to involve VLDL oxidation.
Intimal Hyperplasia of the Infant Parasellar Carotid Artery
Authors: Weninger Wolfgang J. , Müller Gerd B. , Reiter Christian , Meng Stefan , Rabl Silvi U.
Published: June 2012

Abstract
—Intimal cushions that project into the lumen of arteries are precursors of atherosclerotic plaque formation. The “carotid siphon,” although frequently affected by atherosclerosis, was never analyzed for the occurrence of neonatal intimal hyperplasia. This study provides a topographic and morphometric analysis of intimal cushions in the parasellar internal carotid artery (pICA) of the human infant. A total of 35 specimens were studied in detail, using both standard histological techniques and a new method of computer-aided 3D reconstruction. Intimal hyperplasia occurred at 3 characteristic locations of the pICA: (1) the convex side of the posterior knee (C5 cushion), (2) the bottom of the horizontal segment (C4 cushion), and (3) the concave side of the anterior knee (C3 cushion). The extension of the cushions and the degrees to which they occluded the vessel lumens were measured. The complex shape of the pICA required 3D computer models for exact topographical descriptions and precise measurements. Our results suggest that the occurrence and degree of intimal hyperplasia are related to shape changes of the pICA during postnatal development. We predict that individuals who retain the relatively straight course of the fetal pICA throughout their lives are less prone to develop atherosclerotic lesions at this portion of the carotid artery. A possible contribution of neonatal intimal cushions to the origin of sudden infant death syndrome is discussed.