bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Smooth Muscle Myosin Heavy Chain B in Cardiac Vessels of Normotensive and Hypertensive Rats
Authors: Wetzel Ulrike , Lutsch Gudrun , Haase Hannelore , Ganten Ursula , Morano Ingo
Published: June 2012

Abstract
—We investigated expression of the 5′-spliced isoform of smooth muscle myosin heavy chain (SM-MHC-B) in smooth muscle cells of cardiac vessels of the left ventricle of normotensive (Wistar-Kyoto) and spontaneously hypertensive rats of the stroke-prone strain by immunofluorescence microscopy. In parallel, liver and bladder were studied for characterization of the nature of vessels expressing SM-MHC-B and for semiquantitative evaluation of its abundance. Smooth muscle cells were detected by staining with a monoclonal antibody specific for α-smooth muscle actin. Abundance of the SM-MHC-B isoform in these cells was evaluated by using an antibody raised against the seven–amino acid insert at the 25K/50K junction of the myosin head (a25K/50K) that specifically recognized SM-MHC-B. In the ventricle, a25K/50K immunoreactivity was observed in smooth muscle cells of precapillary arterioles but not in larger vessels or aorta. The a25K/50K immunoresponse of those vessels with the highest expression level of SM-MHC-B closely resembled the signal observed in the smooth muscle layer of urinary bladder known to preferentially express SM-MHC-B. Interestingly, in left ventricles of stroke-prone spontaneously hypertensive rats, there was a significantly reduced fraction of a25K/50K-positive precapillary arterioles compared with normotensive control rats.
Aspirin Attenuates Cytomegalovirus Infectivity and Gene Expression Mediated by Cyclooxygenase-2 in Coronary Artery Smooth Muscle Cells
Authors: Speir Edith , Yu Zu-Xi , Ferrans Victor J. , Huang Eng-Shang , Epstein Stephen E.
Published: June 2012

Abstract
—Human cytomegalovirus (CMV) infection of smooth muscle cells generates reactive oxygen species (ROS) and thereby activates nuclear factor κB (NFκB), which causes expression of viral and cellular genes involved in immune and inflammatory responses. These changes could account for the mounting evidence suggesting that CMV may contribute causally to restenosis and atherosclerosis. We found that CMV induces ROS, at least partly, through a cyclooxygenase-2 (COX-2)–dependent pathway. Moreover, the viral immediate-early (IE) gene products, IE72 and IE84, have the capacity to transactivate the COX-2 promoter. Aspirin and indomethacin, both cyclooxygenase inhibitors as well as direct ROS scavengers, reduce CMV-induced ROS, probably through both of these activities. Sodium salicylate also has antiviral effects as the result of its potent antioxidant properties. Furthermore, by reducing ROS, aspirin and sodium salicylate inhibit CMV-induced NFκB activation, the ability of IE72 to transactivate its promoter, CMV IE gene expression after infection of SMCs, and CMV replication in SMCs. This is the first time aspirin has been shown to have antiviral effects. Thus, it is possible that aspirin has previously unrecognized therapeutic effects in various clinical situations, such as in viral infections (when used as an antipyretic agent) and in atherosclerosis (when used as an antiplatelet agent).
Factor XIIIa Cross-links Lipoprotein(a) With Fibrinogen and Is Present in Human Atherosclerotic Lesions
Authors: Romanic Anne M. , Arleth Anthony J. , Willette Robert N. , Ohlstein Eliot H.
Published: June 2012

Abstract
—During the development of atherosclerotic lesions, lipoprotein(a) [Lp(a)], a highly atherogenic lipoprotein, accumulates within fibrin clots attached to blood vessel walls. As Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. It is not understood, however, which mechanisms are involved in the binding of Lp(a) to fibrin and, hence, the stable incorporation of Lp(a) into the fibrin clot. The results of the present study demonstrate that factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between endo-γ-glutaminyl and endo-ε-lysyl residues of proteins, is capable of cross-linking Lp(a) to fibrinogen, the soluble precursor of fibrin. Biochemical assays were conducted to demonstrate that factor XIIIa cross-links Lp(a) with fibrinogen in a time- and concentration-dependent manner. Additionally, immunohistochemical studies revealed that factor XIII protein expression colocalizes with Lp(a) expression in human atherosclerotic plaques. It is proposed that factor XIIIa–mediated cross-linking of Lp(a) to fibrin effectively increases the local concentration of Lp(a) within a fibrin clot. The accumulation of Lp(a) within the blood vessel promotes an antifibrinolytic environment, foam cell formation, the generation of a fatty streak, and an increase in smooth muscle cell content, all of which may contribute to the pathogenesis of atherosclerosis.
Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats
Authors: Cernadas María R. , de Miguel Lourdes Sánchez , García-Durán Margarita , González-Fernández Fernando , Millás Inmaculada , Montón Mercedes , Rodrigo José , Rico Luis , Fernández Patricia , de Frutos Trinidad , Rodríguez-Feo Juan A. , Guerra José , Caramelo Carlos , Casado Santos , López-Farré Antonio
Published: June 2012

Abstract
—Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca
2+
-dependent and Ca
2+
-independent conversion of [
14
C]
l
-arginine into [
14
C]
l
-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the
l
-arginine antagonist
l
-
N
ω
-nitro-
l
-arginine and a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging.
Human Endothelial Cell Storage Granules
Authors: Russell Fraser D. , Skepper Jeremy N. , Davenport Anthony P.
Published: June 2012

Abstract
—We have previously shown endothelin (ET)-like immunoreactive staining in Weibel-Palade bodies, storage granules that are an integral component of the regulated secretory pathway in endothelial cells. These structures degranulate after chemical or mechanical stimuli that result in cytosolic calcium influx. We therefore investigated whether the regulated pathway might be an intracellular site involved in the cleavage of big ET-1 to the biologically active peptide ET-1 by determining the ultrastructural localization of endothelin-converting enzyme (ECE)-1. A low level of ECE-like immunoreactivity was detected on the cell surface of human umbilical vein and coronary artery endothelial cells by scanning electron microscopy. Exogenous big ET-1 was added to permeabilized and nonpermeabilized cultured human umbilical vein endothelial cells, and ECE activity was measured by the detection of ET-like immunoreactivity in the culture supernatant. A marked increase in ECE activity was observed in permeabilized cells, indicating that ECE may also be expressed in intracellular compartments. Confocal microscopy revealed intense immunofluorescence staining for big ET-1 and the 2 isoforms of ECE-1 (ECE-1α and ECE-1β) in the perinuclear region and in Weibel-Palade bodies of the human umbilical vein endothelial cells. Stimulated degranulation of storage granules by the calcium ionophore A23187 caused release of ET into the culture supernatants. The findings of this study indicate that big ET-1 is processed to the mature vasoactive peptide by ECEs located within endothelial storage granules. We hypothesize that this activity may be important in the regulated mobilization of ET in human endothelial cells.
Estrogen Replacement Suppresses a Prostaglandin H Synthase–Dependent Vasoconstrictor in Rat Mesenteric Arteries
Authors: Davidge Sandra T. , Zhang Yunlong
Published: June 2012

Abstract
—There is evidence that estrogen can upregulate nitric oxide (NO) synthase expression. There is also evidence that NO increases the activity of prostaglandin H synthase (PGHS). Our initial hypothesis was that removal of ovarian steroids would decrease endothelium/NO-dependent relaxation responses but that estrogen replacement would increase NO and PGHS activity, leading to increased vasodilation. Resistance-sized (<250 μm) mesenteric arteries from ovariectomized Sprague-Dawley rats without and with 17β-estradiol replacement (0.15 or 0.5 mg/pellet, 60-day release) for 4 weeks were studied in a myograph system. The vasodilator response to methacholine, an endothelium-dependent muscarinic agonist, was reduced in the arteries of the ovariectomized rats compared with estradiol-replaced rats. In the presence of PGHS inhibitors (meclofenamate, valeryl salicylate, and NS-398) or a thromboxane A
2
(TxA
2
)/prostaglandin H
2
(PGH
2
) receptor blocker (SQ-29548), there was no longer a significant difference among the groups. Contrary to our initial hypothesis, inhibition of the PGHS pathway significantly enhanced the relaxation response in the arteries from the ovariectomized rats, which was similar to the response in the arteries from estradiol-replaced rats, indicating that a PGHS-dependent vasoconstrictor had modified the response to methacholine. Confirming these data, in response to exogenous arachidonic acid, arteries from ovariectomized rats exhibited constriction, whereas the arteries from the estradiol-replaced rats exhibited vasodilation. In the ovariectomized rats, pretreatment with inhibitors of the PGHS pathway reversed the vasoconstriction to a vasodilation. In addition, the vasoconstrictor response to the thromboxane mimetic U-46619 as well as PGH
2
was enhanced in endothelium-denuded arteries from the ovariectomized rats compared with the estradiol-replaced rats. These data demonstrate that removal of ovarian steroids increased endothelium-mediated PGHS-dependent vasoconstriction that was associated with augmented sensitivity of the TxA
2
/PGH
2
receptor. Chronic estrogen replacement in the ovariectomized rat suppressed this PGHS-dependent vasoconstrictor response.
Human Coronary Arteriolar Dilation to Arachidonic Acid Depends on Cytochrome P-450 Monooxygenase and Ca
2+
-Activated K
+
Channels
Authors: Miura Hiroto , Gutterman David D.
Published: June 2012

Abstract
—Endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMCs) plays a crucial role in regulating vascular tone, especially in resistance vessels. It has been proposed that metabolites of arachidonic acid (AA), formed by cytochrome P-450 monooxygenase (P450), are endothelium-derived hyperpolarizing factors (EDHFs). These metabolites have been reported to mediate dilation to endogenous vasoactive compounds, such as bradykinin and acetylcholine. However, it is not known whether these metabolites of AA contribute to dilation of human resistance vessels. This is important since it has been proposed that EDHF serves as a compensatory mechanism to maintain dilation in disease states. Therefore, we studied the effect of AA on vessel diameter and VSMC membrane potential in isolated human coronary microvessels. Arterioles (81±5 μm, n=70) were dissected from right atrial appendages at the time of cardiac surgery and cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in internal diameter were recorded with videomicroscopy. Some vessels were impaled with glass microelectrodes to measure membrane potential of VSMCs while internal diameters were simultaneously recorded. After constriction (47±2%) with endothelin-1, AA (10
−10
to 10
−5
mol/L) induced substantial dilation of human coronary microvessels, which was abolished by removal of the endothelium. Treatment with 17-octadecynoic acid (17-ODYA, 10
−5
mol/L; a P450 inhibitor) attenuated maximal dilation to AA (49±9% versus 91±4% [control];
P
<0.05 versus control), whereas indomethacin (INDO, 10
−5
mol/L; a cyclooxygenase inhibitor) and
N
ω
-nitro-
l
-arginine methyl ester (L-NAME, 10
−4
mol/
l
; a NO synthase inhibitor) were without effect. Both 17-ODYA and miconazole (10
−5
mol/L, a chemically distinct P450 inhibitor) further reduced the dilation to AA in the presence of INDO. The presence of 40 mmol/L KCl or charybdotoxin (10
−8
mol/L, a blocker of large-conductance Ca
2+
-activated K
+
channels) impaired dilation to AA (19±9% [KCl] versus 76±5% [control] and 47±6% [charybdotoxin] versus 91±3% [control];
P
<0.05 for both). After depolarization with endothelin-1 (−26±1 mV from −48±3 mV [before endothelin]), AA (10
−5
mol/L) in the presence of INDO and L-NAME induced hyperpolarization of VSMCs (−57±5 mV). In the presence of 17-ODYA together with INDO and L-NAME, endothelin produced similar depolarization (−26±2 mV from – 48±3 mV), but hyperpolarization to AA was reduced (−33±2 mV;
P
<0.05 versus absence of 17-ODYA). AA metabolites formed primarily by P450 produce potent endothelium-dependent dilation of human coronary arterioles via opening of Ca
2+
-activated K
+
channels and hyperpolarization of VSMCs. These findings support an important role for P450 metabolites in the regulation of human coronary arteriolar tone.
Expression of Proto-oncogenes and Gene Mutation of Sarcomeric Proteins in Patients With Hypertrophic Cardiomyopathy
Authors: Kai Hisashi , Muraishi Akihiko , Sugiu Yuji , Nishi Hirohumi , Seki Yukihiko , Kuwahara Fumitaka , Kimura Akinori , Kato Hirohisa , Imaizumi Tsutomu
Published: June 2012

Abstract
—Several mutations of cardiac β-myosin heavy chain (β-MHC) gene were reported in patients with hypertrophic cardiomyopathy (HCM). Involvement of proto-oncogenes has been shown in the mechanism of experimental cardiac hypertrophy. This study sought to examine the effects of c-H-
ras
and c-
myc
expression in the steady-state myocardium on hypertrophic changes and to evaluate the possible interaction between β-MHC mutation and proto-oncogene expression in HCM. Endomyocardial biopsy was performed in 17 HCM patients (5 β-MHC mutations and 1 troponin T mutation) and 7 control subjects (no mutation). Reverse transcription–polymerase chain reaction analysis revealed c-H-
ras
expression in all members of both groups. Cardiomyocyte size was correlated with the expression level of c-H-
ras
(
P
<0.001), and c-H-
ras
expression was upregulated in HCM patients (
P
<0.01). HCM patients with a β-MHC mutation had the higher c-H-
ras
expression than did control subjects or patients without a mutation (
P
<0.01). c-
myc
mRNA was expressed in 7 of 17 HCM patients but not in control subjects. Myocyte size was greater in c-
myc
–positive HCM patients than in control subjects and c-
myc
–negative HCM patients (
P
<0.001 and
P
<0.05, respectively). The proto-oncogene expression did not affect clinical findings, myocardial fibrosis, or disarray. In conclusion, c-H-
ras
and c-
myc
expression in the steady-state myocardium may play a role in the hypertrophic mechanism in HCM. It is possible that β-MHC gene mutation has some effect on the regulation of proto-oncogene expression in HCM.
Strong Binding of Myosin Modulates Length-Dependent Ca
2+
Activation of Rat Ventricular Myocytes
Authors: Fitzsimons Daniel P. , Moss Richard L.
Published: June 2012

Abstract
—Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca
2+
sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca
2+
sensitivity of tension (ie, pCa
50
) were measured in the absence and presence of
N
-ethylmaleimide–modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non–tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of ≈1.90 μm, application of NEM-S1 markedly increased submaximal Ca
2+
-activated tensions and thereby increased Ca
2+
sensitivity; ie, pCa
50
increased from 5.40±0.02 to 5.52±0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca
2+
sensitivity of tension, in that the ΔpCa
50
between short and long lengths was 0.02±0.01 pCa units in the presence of NEM-S1 compared with a ΔpCa
50
of 0.10±0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca
2+
sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.
Hydrogen Peroxide Decreases pH
i
in Human Aortic Endothelial Cells by Inhibiting Na
+
/H
+
Exchange
Authors: Hu Qinghua , Xia Yong , Corda Stefano , Zweier Jay L. , Ziegelstein Roy C.
Published: June 2012

Abstract
—Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pH
i
, the effect of hydrogen peroxide on endothelial pH
i
was examined. Hydrogen peroxide (100 μmol/L for 10 minutes) decreased pH
i
from 7.24±0.01 to 7.02±0.02 and inhibited recovery from an ammonium chloride–induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)–loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na
+
/H
+
exchange with 5-(
N
-ethyl-
N
-isopropyl)amiloride (10 μmol/L), by removal of extracellular Na
+
, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pH
i
. A 2-minute exposure to 100 μmol/L H
2
O
2
decreased intracellular ATP levels by ≈40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide–induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pH
i
in human endothelial cells by inhibiting Na
+
/H
+
exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pH
i
in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na
+
/H
+
exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.
Animal Model That Mimics Atherosclerotic Plaque Rupture
Authors: Rekhter Mark D. , Hicks Gary W. , Brammer David W. , Work Carolyn W. , Kim Jong-Seung , Gordon David , Keiser Joan A. , Ryan Michael J.
Published: June 2012

Abstract
—Atherosclerotic plaque rupture is the main cause of coronary thrombosis and myocardial infarcts. Currently, there is no animal model of plaque disruption. We have developed a rabbit model in which an atherosclerotic plaque can be ruptured at will after an inflatable balloon becomes embedded into the plaque. Furthermore, the pressure needed to inflate the plaque-covered balloon may be an index of overall plaque mechanical strength. The thoracic aorta of hypercholesterolemic rabbits underwent mechanical removal of endothelial cells, and then a specially designed balloon catheter was introduced into the lumen of the thoracic aorta. As early as 1 month after catheter placement, atherosclerotic plaque formed around the indwelling balloon. The plaques were reminiscent of human atherosclerotic lesions, in terms of cellular composition, patterns of lipid accumulation, and growth characteristics. Intraplaque balloons were inflated both ex vivo and in vivo, leading to plaque fissuring. The ex vivo strategy is designed to measure the mechanical strength of the surrounding plaque, while the in vivo scenario permits an analysis of the plaque rupture consequences, eg, thrombosis. In addition, our model allows local delivery of various substances into the plaque. The model can be used to study the pathogenesis of plaque instability and to design plaque stabilization therapy.
Differential Activation of Cardiac c-Jun Amino-Terminal Kinase and Extracellular Signal-Regulated Kinase in Angiotensin II–Mediated Hypertension
Authors: Yano Masahiko , Kim Shokei , Izumi Yasukatsu , Yamanaka Shinya , Iwao Hiroshi
Published: June 2012

Abstract
—Two subgroups of mitogen-activated protein kinases, c-jun NH
2
-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), are thought to be involved in cultured cardiac myocyte hypertrophy and gene expression. To examine the in vivo activation of these kinases, we measured cardiac JNK and ERK activities in conscious rats subjected to acute or chronic angiotensin II (Ang II) infusion, by using in-gel kinase methods. About 50 mm Hg rise in blood pressure by Ang II (1000 ng · kg
−1
· min
−1
) infusion caused larger activation of left ventricular JNK than ERK, via the AT
1
receptor. In spite of short duration (about 30 minutes) of maximal blood pressure elevation by Ang II, JNK sustained the peak value (more than 5-fold increase) from 15 minutes up to at least 3 hours. Similar activation of JNK was seen in the right ventricle. Thus, cardiac JNK activation by Ang II seems to be in part mediated by its direct action via the AT
1
receptor. The dose-response relationships for Ang II–induced rises in blood pressure and cardiac JNK and ERK activation indicated that cardiac JNK or ERK was not activated by a mild increase in blood pressure and that cardiac JNK was activated by Ang II–mediated hypertension in a more sensitive manner than ERK. Cardiac hypertrophy, induced by chronic Ang II infusion, was preceded by JNK activation without ERK activation. Furthermore, gel mobility shift analysis showed that cardiac JNK activation was followed by increased activator protein-1 DNA binding activity due to c-Fos and c-Jun. These results provided the first evidence for the preferential activation of cardiac JNK in Ang II–induced hypertension and suggested that JNK might play some role in Ang II–induced cardiac hypertrophic response in vivo. However, further study is needed to elucidate the role of JNK in cardiac hypertrophy in vivo.
Adenosine Mediates Sustained Adrenergic Desensitization in the Rat Heart via Activation of Protein Kinase C
Authors: Perlini Stefano , Khoury Edmon P. , Norton Gavin R. , Chung Eugene S. , Fenton Richard A. , Dobson James G. , Meyer Theo E.
Published: June 2012

Abstract
—Adenosine attenuates the myocardial metabolic and contractile responses induced by β-adrenergic stimulation. Our study was conducted to investigate the longevity of this antiadrenergic action after adenosine exposure. Adenosine (33 μmol/L) was infused into isolated perfused rat hearts for 1, 5, 30, or 60 minutes, and the adrenergic responsiveness (AR) to isoproterenol (10
−8
mol/L) was determined at the end of each infusion period and during a 45-minute adenosine washout period. Interstitial levels of adenosine, as determined from epicardial surface transudates, returned to preinfusion levels within 10 minutes of washout. The duration of adenosine infusion had no effect on the extent of attenuation of AR at the end of the infusion. Whereas AR returned to preadenosine levels with washout of shorter adenosine infusions (1 and 5 minutes), there was a slow and incomplete recovery of AR after the longer exposures (30 and 60 minutes) to adenosine. The magnitude of this persistent antiadrenergic effect (PAE) of adenosine at 15 minutes of washout was proportional to the epicardial concentration of adenosine during infusion of the nucleoside. Infusion of adenosine either with the nonselective adenosine receptor antagonist 8-
p
-sulfophenyl theophylline or with the selective A
1
-receptor antagonist 1,3-dipropyl, 8-cyclopentylxanthine, abolished the PAE during the washout period. In addition, the PAE could be demonstrated only with the selective A
1
-receptor agonist 2-chloro-N
6
-cyclopentyladenosine and not with the selective A
3
-receptor agonist 4-aminobenzyl-5′-N methylcarboxamido-adenosine. When the protein kinase C (PKC) inhibitor chelerythrine was coadministered with adenosine, the PAE of adenosine was not apparent during adenosine washout. A 30-minute infusion of phenylephrine, an α-adrenergic agonist that enhances PKC activity, produced a PAE that lasted for up to 30 minutes of washout. This effect was prevented by the coinfusion of chelerythrine. Thus, it is concluded that the PAE of adenosine is determined by the myocardial concentration of this nucleoside and is manifested when myocardial concentrations of adenosine returned to baseline levels. Moreover, a 5-minute duration of adenosine exposure is required for the expression of the PAE. This latter effect seems to be dependent on adenosine-induced PKC activation via A
1
-receptors.
Stretch-Induced Alkalinization of Feline Papillary Muscle
Authors: Cingolani H. E. , Alvarez B. V. , Ennis I. L. , Camilión de Hurtado M. C.
Published: June 2012

Abstract
—Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na
+
/H
+
exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pH
i
). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pH
i
was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (L
max
) and maintained in a length that was ≈92% L
max
(L
i
). During the “stretch protocol,” muscles were quickly stretched to L
max
for 10 minutes and then released to L
i
; pH
i
significantly increased during stretch and came back to the previous value when the muscle was released to L
i
. The increase in pH
i
was eliminated by (1) specific inhibition of the NHE (EIPA, 5 μmol/L), (2) AT
1
-receptor blockade (losartan, 10 μmol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 μmol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ET
A
antagonist (BQ-123, 300 nmol/L). The increase in pH
i
by exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pH
i
increases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles.
Vascular Endothelial Growth Factor Upregulates the Expression of Matrix Metalloproteinases in Vascular Smooth Muscle Cells
Authors: Wang He , Keiser Joan A.
Published: June 2012

Abstract
—Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain–containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase–polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.
Calcium- and Protein Kinase C–Dependent Activation of the Tyrosine Kinase PYK2 by Angiotensin II in Vascular Smooth Muscle
Authors: Sabri Abdelkarim , Govindarajan Geetha , Griffin Tina M. , Byron Kenneth L. , Samarel Allen M. , Lucchesi Pamela A.
Published: June 2012

Abstract
—Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating G
q
-protein–coupled AT
1
receptors, which leads to elevation of cytosolic Ca
2+
([Ca
2+
]
i
) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II–induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca
2+
]
i
or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle α-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT
1
receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca
2+
]
i
. Treatment with either phorbol ester or Ca
2+
ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca
2+
]
i
are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-
src
complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca
2+
]
i
and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.
Cardiac Troponin I Gene Knockout
Authors: Huang XuPei , Pi YeQing , Lee Kevin J. , Henkel Anne S. , Gregg Ronald G. , Powers Patricia A. , Walker Jeffery W.
Published: June 2012

Abstract
—Troponin I is a subunit of the thin filament–associated troponin-tropomyosin complex involved in calcium regulation of skeletal and cardiac muscle contraction. We deleted the cardiac isoform of troponin I by using gene targeting in murine embryonic stem cells to determine the developmental and physiological effects of the absence of this regulatory protein. Mice lacking cardiac troponin I were born healthy, with normal heart and body weight, because a fetal troponin I isoform (identical to slow skeletal troponin I) compensated for the absence of cardiac troponin I. Compensation was only temporary, however, as 15 days after birth slow skeletal troponin I expression began a steady decline, giving rise to a troponin I deficiency. Mice died of acute heart failure on day 18, demonstrating that some form of troponin I is required for normal cardiac function and survival. Ventricular myocytes isolated from these troponin I–depleted hearts displayed shortened sarcomeres and elevated resting tension measured under relaxing conditions and had a reduced myofilament Ca sensitivity under activating conditions. The results show that (1) developmental downregulation of slow skeletal troponin I occurs even in the absence of cardiac troponin I and (2) the resultant troponin I depletion alters specific mechanical properties of myocardium and can lead to a lethal form of acute heart failure.
Anoxia/Reoxygenation-Induced Tolerance With Respect to Polymorphonuclear Leukocyte Adhesion to Cultured Endothelial Cells
Authors: Cepinskas Gediminas , Lush Cameron W. , Kvietys Peter R.
Published: June 2012

Abstract
—Exposing human umbilical vein endothelial cells (HUVECs) to anoxia/reoxygenation (A/R) results in an increase in polymorphonuclear leukocyte (PMN) adhesion to HUVECs. This A/R-induced hyperadhesion is completely prevented by a previous (24 hours earlier) exposure of HUVECs to A/R. This phenomenon has been termed “A/R tolerance.” Exposing HUVECs to A/R induces an increase in nuclear factor κB (NF-κB) in HUVEC nuclei within 4 hours. Interfering with either NF-κB activation (proteasome inhibitor) or translocation (double-stranded oligonucleotides containing NF-κB binding sequence) prevents the development of A/R tolerance (ie, the increase in A/R-induced PMN adhesion to HUVECs is the same after the first and second A/R challenges). NO production by HUVECs is increased after the second A/R challenge, but not after the first A/R challenge. Inhibition of NO synthase (NOS) during the second A/R challenge prevents the development of A/R tolerance with respect to PMN adhesion. However, while HUVECs contained endothelial NOS protein, no inducible NOS was detected in either tolerant or nontolerant cells. Further studies indicated that inhibition of GTP-cyclohydrolase I (an enzyme involved in de novo synthesis of an important cofactor for NOS activity, tetrahydrobiopterin) prevented the generation of NO in A/R-tolerant cells. Extracellular generation of NO (NO donor) did not effect the hyperadhesion response induced by the initial A/R challenge. A/R also induced an oxidant stress in naive HUVECs, but not in A/R-tolerant HUVECs. Inhibition of NOS during the second A/R insult results in the generation of an oxidant stress similar to that observed after the first A/R challenge. Taken together, the findings of the present study are consistent with a role for NF-κB in the development of A/R tolerance (with respect to PMN adhesion), perhaps by transcriptional regulation of GTP-cyclohydrolase. The increased NO production during the second A/R insult reduces PMN adhesion most likely by reducing the intracellular oxidant stress induced by A/R.
Determinants of Vascular Smooth Muscle Cell Apoptosis After Balloon Angioplasty Injury
Authors: Pollman Matthew J. , Hall Jennifer L. , Gibbons Gary H.
Published: June 2012

Abstract
—We have observed that acute medial cell loss is an initial event in the response to vascular injury induced by balloon-catheter distention of the rabbit carotid artery. Numerous apoptotic medial cells were observed as early as 30 minutes after balloon inflation, and a 70% loss of cellularity was evident by 90 minutes. Balloon injury was associated with oxidative stress as reflected by a fall in glutathione levels by 63% within 30 minutes after injury. We hypothesized that balloon injury activated a redox-sensitive signaling pathway coupled to the regulation of apoptosis. Indeed, the activity of the proapoptotic signal mediator, stress-activated protein kinase, was increased severalfold within 10 minutes after injury. Moreover, modifying the vascular redox state by the administration of 1 of 2 structurally dissimilar antioxidants,
N
-acetyl cysteine or pyrrolidine dithiocarbamate, markedly attenuated both stress-activated protein kinase activation and the induction of apoptosis at 30 minutes. We hypothesized further that the induction of vascular smooth muscle cell apoptosis is modulated by phenotype. In contrast to medial cells, we observed that neointimal cells were relatively resistant to apoptotic death induced by angioplasty injury. This resistance to balloon injury–induced death was associated with an upregulation of the antiapoptotic mediator bcl-x
L
. This study suggests that acute apoptotic cell death after vascular injury is a highly regulated process governed by cellular redox state and the relative expression of antiapoptotic genes. Angioplasty-induced vascular cell apoptosis may be an important determinant of vascular remodeling and restenosis.
Coupling of β
2
-Adrenoceptor to G
i
Proteins and Its Physiological Relevance in Murine Cardiac Myocytes
Authors: Xiao Rui-Ping , Avdonin Pavel , Zhou Ying-Ying , Cheng Heping , Akhter Shahab A. , Eschenhagen Thomas , Lefkowitz Robert J. , Koch Walter J. , Lakatta Edward G.
Published: June 2012

Abstract
—Transgenic mouse models have been developed to manipulate β-adrenergic receptor (βAR) signal transduction. Although several of these models have altered βAR subtypes, the specific functional sequelae of βAR stimulation in murine heart, particularly those of β
2
-adrenergic receptor (β
2
AR) stimulation, have not been characterized. In the present study, we investigated effects of β
2
AR stimulation on contraction, [Ca
2+
]
i
transient, and L-type Ca
2+
currents (
I
Ca
) in single ventricular myocytes isolated from transgenic mice overexpressing human β
2
AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous β
2
AR activation. In contrast, β
2
AR stimulation by zinterol or isoproterenol plus a selective β
1
-adrenergic receptor (β
1
AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, β
2
AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the
I
Ca
, [Ca
2+
]
i
, and contractile responses to β
2
AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac β
2
AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced β
2
AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [γ-
32
P]GTP azidoanilide into α subunits of G
i2
and G
i3
after β
2
AR stimulation by zinterol or isoproterenol plus the β
1
AR blocker CGP 20712A. This effect to activate G
i
proteins was abolished by a selective β
2
AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) β
2
ARs in murine cardiac myocytes couple to concurrent G
s
and G
i
signaling, resulting in null inotropic response, unless the G
i
signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to β
2
AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of β
2
AR-coupled G
i
proteins; and (3) spontaneous β
2
AR activation may differ from agonist-stimulated β
2
AR signaling.
Role of Protein Kinase C in Mitochondrial K
ATP
Channel–Mediated Protection Against Ca
2+
Overload Injury in Rat Myocardium
Authors: Wang Yigang , Ashraf Muhammad
Published: June 2012

Abstract
—Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoK
ATP
channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoK
ATP
channel diazoxide attenuates the lethal injury associated with Ca
2+
overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoK
ATP
channels; whether diazoxide mimics the effects of Ca
2+
preconditioning; and whether diazoxide reduces Ca
2+
paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca
2+
PD (10 minutes of Ca
2+
depletion followed by 10 minutes of Ca
2+
repletion). The effects of the MitoK
ATP
channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca
2+
PD were assessed. In hearts treated with 80 μmol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca
2+
PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca
2+
PD hearts. The salutary effects of diazoxide on the Ca
2+
PD injury were similar to those in hearts that underwent Ca
2+
preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca
2+
PD. The addition of sodium 5-hydroxydecanoate, a specific MitoK
ATP
channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca
2+
PD. Blockade of Ca
2+
entry during diazoxide treatment by inhibiting L-type Ca
2+
channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca
2+
PD. PKC-δ was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-α and PKC-ε were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoK
ATP
channel is mediated by PKC-mediated signaling pathway.
EVEC, a Novel Epidermal Growth Factor–Like Repeat-Containing Protein Upregulated in Embryonic and Diseased Adult Vasculature
Authors: Kowal Robert C. , Richardson James A. , Miano Joseph M. , Olson Eric N.
Published: June 2012

Abstract
—A hallmark of vascular lesions is the phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a more primitive, proliferative phenotype with a more fetal pattern of gene expression. Using subtraction hybridization to identify genes that may regulate this transition, we cloned a novel gene named EVEC, an acronym for its expression in the
e
mbryonic
v
asculature and the presence of Ca
2+
binding
e
pidermal growth factor–like repeats
c
ontained in the predicted protein structure. Although these repeats are characteristic of the extracellular matrix proteins, fibrillin, fibulin, and the latent transforming growth factor-β binding proteins, EVEC most closely resembles the H411 and T16/S1-5 gene products, the latter of which are believed to regulate DNA synthesis in quiescent fibroblasts. Using in situ hybridization, we demonstrated that EVEC is expressed predominantly in the VSMCs of developing arteries in E11.5 through E16.5 mouse embryos. Lower levels of expression are also observed in endothelial cells, perichondrium, intestine, and mesenchyme of the face and kidney. EVEC mRNA expression is dramatically downregulated in adult arteries, except in the uterus, where cyclic angiogenesis continues; however, EVEC expression is reactivated in 2 independent rodent models of vascular injury. EVEC mRNA is observed in cellular elements of atherosclerotic plaques of LDL receptor–deficient, human apolipoprotein B transgenic mice and in VSMCs of the media and neointima of balloon-injured rat carotid arteries. These data suggest that EVEC may play an important role in the regulation of vascular growth and maturation during development and in lesions of injured vessels.
Rho and Rho Kinase Mediate Thrombin-Stimulated Vascular Smooth Muscle Cell DNA Synthesis and Migration
Authors: Seasholtz Tammy M. , Majumdar Mousumi , Kaplan Daniel D. , Brown Joan Heller
Published: June 2012

Abstract
—Aberrant regulation of smooth muscle cell proliferation and migration is associated with the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth muscle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thrombin peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [
3
H]thymidine incorporation. Both ligands also increased cell migration as measured by the Boyden chamber method.
l
-Phenylephrine failed to induce either of these responses but increased inositol phosphate accumulation and mitogen-activated protein kinase activation in these cells, which indicated that the cells were responsive to α
1
-adrenergic stimulation. The C3 exoenzyme, which ADP-ribosylates and inactivates Rho, fully inhibited both thrombin-stimulated proliferation and migration but had no effect on inositol phosphate accumulation. In addition, Y-27632, an inhibitor of the Rho effector p160ROCK/Rho kinase, decreased thrombin-stimulated DNA synthesis and migration. To directly examine Rho activation, Rho-[
35
S]GTPγS binding was measured. The addition of the thrombin peptide SFLLRNP, but not phenylephrine, to RASMC lysates resulted in a significant increase in Rho-[
35
S]GTPγS binding. Thrombin and SFLLRNP, but not phenylephrine, also increased membrane-associated Rho in intact RASMCs, consistent with selective activation of Rho by thrombin. These results indicate that thrombin activates Rho in RASMCs and establish Rho as a critical mediator of thrombin receptor effects on DNA synthesis and cell migration in these cells.