bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Apoptosis in the Disappearance of Infiltrated and Proliferated Interstitial Cells After Myocardial Infarction
Authors: Takemura Genzou , Ohno Michiya , Hayakawa Yukihiro , Misao Jun , Kanoh Motoo , Ohno Atsuko , Uno Yoshihiro , Minatoguchi Shinya , Fujiwara Takako , Fujiwara Hisayoshi
Published: June 2012

Abstract
—Myocardial infarction (MI) progresses from the acute death of myocytes and the infiltration of inflammatory cells into granulation, followed by scars. During the healing process, the myocardial interstitial cell population in the infarcted tissues increases markedly and then decreases. We postulated that apoptosis is responsible for this process. Twenty-four male Japanese white rabbits underwent a 30-minute occlusion of the left coronary artery followed by reperfusion for 2 days, 2 weeks, or 4 weeks (n=8 each). The histological features consisted of dead cardiomyocytes and marked leukocyte infiltration at 2 days after MI and granulation consisting of numerous α-smooth muscle actin–positive myofibroblasts, macrophage antigen–positive macrophages, and neovascularization at 2 weeks. At 4 weeks, the cellularity decreased markedly, and scars were evident. Interstitial cells with positive nick end labeling were significantly more frequent at the light microscopic level in the 2-day MI samples (5.3±3.6% in the center and 6.9±3.3% in the periphery of the infarct region) than in the 2-week (2.5±1.0%) and 4-week (0.5±0.5%) samples. DNA electrophoresis showed a clear ladder in tissues from the ischemic areas at 2 days after MI but not at 2 and 4 weeks after MI. Ultrastructurally, typical apoptotic figures, including apoptotic bodies and condensed nuclei without ruptured plasma membranes, were detected in leukocytes from all hearts with 2-day MI and in myofibroblasts, endothelial cells, and macrophages from all hearts with 2-week MI. In the electron microscopic in situ nick end labeling, immunogold particles intensely labeled the condensed chromatin of the typical apoptotic nuclei. These particles were also accumulated on nuclei of the interstitial cells showing homogeneous density but not definite condensation as typical apoptotic nuclei, suggesting an early stage of apoptosis. Thus, apoptosis plays an important role in the disappearance of both the infiltrated leukocytes and the proliferated interstitial cells after MI. This finding may have therapeutic implications for postinfarct ventricular remodeling through apoptosis handling during the healing stage of MI.
Acute Host-Mediated Endothelial Injury After Adenoviral Gene Transfer in Normal Rabbit Arteries
Authors: Channon Keith M. , Qian HuSheng , Youngblood Scot A. , Olmez Ercument , Shetty Geetha A. , Neplioueva Valentina , Blazing Michael A. , George Samuel E.
Published: June 2012

Abstract
—Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (≤days) in 52 New Zealand White rabbits undergoing gene transfer (β-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of ≥4×10

9

plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in β-galactosidase expression through 2×10

10

pfu/mL, whereas in in vivo arteries, titers above 4×10

9

pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time- and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1×10

11

pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury.
Possible Involvement of Stress-Activated Protein Kinase Signaling Pathway and Fas Receptor Expression in Prevention of Ischemia/Reperfusion-Induced Cardiomyocyte Apoptosis by Carvedilol
Authors: Yue Tian-Li , Ma Xin-Liang , Wang Xinkang , Romanic Anne M. , Liu Gao-lin , Louden Calvert , Gu Juan-Li , Kumar Sanjay , Poste George , Ruffolo Robert R. , Feuerstein Giora Z.
Published: June 2012

Abstract
—Carvedilol, a new vasodilating β-adrenoceptor antagonist and a potent antioxidant, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Recent clinical studies in patients with heart failure have demonstrated that carvedilol reduces morbidity and mortality and inhibits cardiac remodeling. The present study was designed to explore whether the protective effects of carvedilol on the ischemic myocardium include inhibition of apoptosis of cardiomyocytes and, if so, to determine its mechanism of action. Anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 4 hours of reperfusion. Detection of apoptosis of cardiomyocytes was based on the presence of nucleosomal DNA fragments on agarose gels (DNA ladder) and in situ nick end labeling. Carvedilol (1 mg/kg IV), administered 5 minutes before reperfusion, reduced the number of apoptotic myocytes in the ischemic area from 14.7±0.4% to 3.4±1.8% (77% reduction,
P
<.001). Propranolol, administered at equipotent β-blocking dosage, reduced the number of apoptotic myocytes to 8.9±2.1% (39% reduction,
P
<.05). DNA ladders were observed in the hearts of all six vehicle-treated rabbits but only one of six carvedilol-treated rabbits (
P
<.01). Immunocytochemical analysis of rabbit hearts demonstrated an upregulation of Fas protein in ischemic cardiomyocytes, and treatment with carvedilol reduced both the intensity of staining as well as the area stained. Myocardial ischemia/reperfusion led to a rapid activation of stress-activated protein kinase (SAPK) in the ischemic area but not in nonischemic regions. SAPK activity was increased from 2.1±0.3 mU/mg (basal) to 8.9±0.8 mU/mg after 30 minutes of ischemia followed by 20 minutes of reperfusion. Carvedilol inhibited the activation of SAPK by 53.4±6.5% (
P
<.05). Under the same conditions, propranolol (1 mg/kg) had no effect on SAPK activation. Taken together, these results suggest that carvedilol prevents myocardial ischemia/reperfusion–induced apoptosis in cardiomyocytes possibly by downregulation of the SAPK signaling pathway, by inhibition of Fas receptor expression, and by β-adrenergic blockade. The former two actions represent novel and important mechanisms that may contribute to the cardioprotective effects of carvedilol.
Prostaglandin D
2
Inhibits Inducible Nitric Oxide Synthase Expression in Rat Vascular Smooth Muscle Cells
Authors: Nagoshi Hiroshi , Uehara Yoshio , Kanai Fumihiko , Maeda Shin , Ogura Tsutomu , Goto Atsuo , Toyo-oka Teruhiko , Esumi Hiroyasu , Shimizu Takao , Omata Masao
Published: June 2012

Abstract
—Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D
2
(PGD
2
) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD
2
modulates NO metabolism in rat VSMCs. PGD
2
at a concentration of 10
–7
mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1β (IL-1β). In a dose-response analysis of IL-1β and nitrite accumulation, PGD
2
was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD
2
at cytokine receptors. Northern analysis showed that PGD
2
suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1β–stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A
2
(TXA
2
) analogue, U46619 (10
–5
mol/L), produced less inhibition of NO generation than did PGD
2
. Neither the PGI
2
analog carbaprostacyclin nor PGE
1
showed any inhibition. PGD
2
dose-dependently inhibited NO generation despite the addition of the TXA
2
antagonist SQ29548. PGJ
2
, Δ
12
-PGJ
2
, and 15-deoxy-Δ
12,14
-PGJ
2
, all metabolites of PGD
2
, were as potent as or slightly stronger than PGD
2
in the inhibition of NO generation. These data suggest that PGD
2
suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ
2
series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD
2
regulates NO metabolism in vascular lesions.
Mitogenic Effect of Angiotensin II on Rat Carotid Arteries and Type II or III Mesenteric Microvessels but Not Type I Mesenteric Microvessels Is Mediated by Endogenous Basic Fibroblast Growth Factor
Authors: Su E. J. , Lombardi D. M. , Wiener J. , Daemen M. J. A. P. , Reidy M. A. , Schwartz S. M.
Published: June 2012

Abstract
—In this study, anti–basic fibroblast growth factor (anti-bFGF) antibody was used to determine whether the mitogenic effect of angiotensin II in vivo could be blocked by neutralizing bFGF in the vessel wall. Animals, divided into six experimental groups, were given (1) angiotensin II, (2) angiotensin II+anti-bFGF antibody, (3) angiotensin II+normal goat IgG (ngIgG), (4) anti-bFGF antibody, (5) ngIgG, and (6) Ringer’s solution. Angiotensin II at 435 ng · kg
−1
· min
−1
was infused into rats continuously for 1 week to induce smooth muscle cell replication, and anti-bFGF antibody or ngIgG was injected intravenously 4 times over the 1-week period at a dose of 60 mg/injection. Bromodeoxyuridine (30 mg/mL) was also continuously infused during the 1-week period. The left carotid artery of all animals was balloon-injured on day 4 of the treatment, and all groups were killed for study on day 7. The results showed that angiotensin II significantly stimulated smooth muscle replication in the balloon-injured carotid artery, intact carotid artery, and three branch levels of the mesenteric vascular tree. Anti-bFGF was able to block the mitogenic effect of angiotensin II in larger vessels but not the smallest (type I) microvessels of the mesenteric arterial tree. This differential response may be attributable to the nature of the lesions in type I vessels versus larger vessels: the type I vascular lesion has a large component of proliferating macrophages, whereas the larger vessels show less injury, few macrophages, and varying levels of smooth muscle replication. Our data suggest that the vessel wall remodeling in the angiotensin II–treated larger vessels involves DNA replication that is dependent on the presence of bFGF.
ATP-Sensitive K
+
Channels, Adenosine, and Nitric Oxide–Mediated Mechanisms Account for Coronary Vasodilation During Exercise
Authors: Ishibashi Yutaka , Duncker Dirk J. , Zhang Jianyi , Bache Robert J.
Published: June 2012

Abstract
—We previously reported that combined blockade of adenosine receptors and ATP-sensitive K+ channels (K
+
ATP
channels) blunted but did not abolish the response of coronary blood flow to exercise. This study tested the hypothesis that the residual increase in coronary flow in response to exercise after adenosine receptor and K
+
ATP
channel blockade is dependent on endogenous NO. Dogs were studied at rest and during a four-stage treadmill exercise protocol under control conditions, during K
+
ATP
channel blockade with glibenclamide (50 μg · kg
−1
· min
−1
IC) in the presence of adenosine receptor blockade with 8-phenyltheophylline (8-PT, 5 mg/kg IV), and after the addition of the NO synthase inhibitor
N
G
-nitro-
l
-arginine (LNNA, 1.5 mg/kg IC). During control conditions, coronary blood flow was 49±3 mL/min at rest and increased to 92±8 mL/min at peak exercise. LNNA alone or in combination with 8-PT did not alter resting coronary flow and did not impair the normal increase in flow during exercise, indicating that when K
+
ATP
channels are intact, neither NO nor adenosine-dependent mechanisms are obligatory for maintaining coronary blood flow. Combined K
+
ATP
channel and adenosine blockade decreased resting coronary flow to 27±3 mL/min (
P
<.05), but exercise still increased flow to 45±5 mL/min (
P
<.05). The subsequent addition of LNNA further decreased resting coronary flow to 20±2 mL/min and markedly blunted exercise-induced coronary vasodilation (coronary vascular conductance, 0.20±0.03 mL · min
−1
· mm Hg
−1
at rest versus 0.24±0.04 mL · min
−1
· mm Hg
−1
during the heaviest level of exercise;
P
=.22), so that coronary flow both at rest and during exercise was below the control resting level. The findings suggest that K
+
ATP
channels are critical for maintaining coronary vasodilation at rest and during exercise but that when K
+
ATP
channels are blocked, both adenosine and NO act to increase coronary blood flow during exercise. In the presence of combined K
+
ATP
channel blockade and adenosine receptor blockade, NO was able to produce approximately one quarter of the coronary vasodilation that occurred in response to exercise when all vasodilator systems were intact.
Structural Domains in Phospholemman
Authors: Chen Zhenhui , Jones Larry R. , O’Brian Jeffrey J. , Moorman J. Randall , Cala Steven E.
Published: June 2012

Abstract
—Phospholemman (PLM) is a small (72–amino acid) transmembrane protein found in cardiac sarcolemma that is a major substrate for several protein kinases in vivo. Detailed structural data for PLM is lacking, but several studies have described an ion conductance that results from PLM expression in oocytes. Moreover, addition of purified PLM to lipid bilayers generates similar ion currents, suggesting that the PLM molecule itself might be sufficient for channel formation. To provide a framework for understanding the function of PLM, we investigated PLM topology and structure in sarcolemmal membrane vesicles and analyzed purified recombinant PLM. Immunoblot analyses with site-specific antibodies revealed that the extracellular segment (residues 1 to 17) exists in a protected configuration highly resistant to proteases, even in detergent solutions. The intracellular portion of the molecule (residues 38 to 72), in contrast, was highly susceptible to proteases. Trypsin treatment produced a limit peptide (residues 1 to 43), which showed little change in electrophoretic mobility in SDS gels and retained the ion-channel activity in lipid bilayers that is characteristic of the full-length protein. In addition, we found that conductance through PLM channels exhibited rapid inactivation during depolarizing ramps at voltages greater than ±50 mV, Channels formed by trypsinized PLM or recombinant PLM 1–43 exhibited dramatic reductions in voltage-dependent inactivations. Our data point to distinct domains within the PLM molecule that may correlate with functional properties of channel activity observed in oocytes and lipid bilayers.
Ca
2+
Sparks Triggered by Patch Depolarization in Rat Heart Cells
Authors: Shorofsky Stephen R. , Izu Leighton , Wier Withrow Gil , Balke C. William
Published: June 2012

Abstract
—The goal of this study was to examine the relationship between Ca
2+
entry through L-type Ca
2+
channels and local [Ca
2+
]
i
transients (Ca
2+
sparks) in single rat cardiac ventricular cells. L-type Ca
2+
channels were activated by depolarization of cell-attached membrane patches, and [Ca
2+
]
i
was measured simultaneously as fluo 3 fluorescence using laser scanning confocal microscopy. Patch depolarization with Ca
2+
as the charge carrier (10 or 110 mmol · L
–1
) significantly increased the probability of the occurrence of Ca
2+
sparks (Ca
2+
spark rate) only in the volume of cytoplasm located immediately beneath the membrane patch (basal Ca
2+
spark rate, 119 Ca
2+
sparks · cell
−1
· s
−1
; patch depolarization Ca
2+
spark rate, 610 Ca
2+
sparks · cell
−1
· s
−1
;
P
<.005). With Ba
2+
in the pipette solution (10 mmol · L
−1
), patch depolarization was not associated with an increased Ca
2+
spark rate at the position of the pipette or at any other sites distant from the pipette. Therefore, Ca
2+
entry and not voltage per se was a necessary event for the occurrence of Ca
2+
sparks. Under identical experimental conditions, patch depolarization experiments opened single L-type Ca
2+
channels with a single-channel conductance of 19 pS with Ba
2+
as the charge carrier. Although single-channel openings could not be resolved when Ca
2+
was the charge carrier, ensemble averages yielded an inward current of up to 0.75 pA. The results suggest that voltage-activated Ca
2+
entry through one or a small number of L type Ca
2+
channels triggers the release of Ca
2+
only from the sarcoplasmic reticulum in direct proximity to those L-type Ca
2+
channels. The relatively low probability of triggering Ca
2+
sparks may have resulted from some alteration of excitation-contraction coupling associated with the technique of the cell-attached patch clamp.
Angiotensin II Activates Na
+
-Independent Cl
−
-HCO
3
−
Exchange in Ventricular Myocardium
Authors: de Hurtado María C. Camilión , Alvarez Bernardo V. , Pérez Néstor G. , Ennis Irene L. , Cingolani Horacio E.
Published: June 2012

Abstract
—The effect of angiotensin II (Ang II) on the activity of the cardiac Na
+
-independent Cl
−
-HCO
3
−
exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pH
i
was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-(
N
-ethyl-
N
-isopropyl)amiloride–sensitive increase in pH
i
in the absence of external HCO
3
−
(HEPES buffer), consistent with its stimulatory action on Na
+
-H
+
exchange (NHE). This alkalinizing effect was not detected in the presence of a CO
2
-HCO
3
−
buffer (pH
i
7.07±0.02 and 7.08±0.02 before and after Ang II, respectively; n=17). Moreover, in Na
+
-free HCO
3
−
–buffered medium, in which neither NHE nor Na
+
-HCO
3
−
cotransport are acting, Ang II decreased pH
i
, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II–induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO
3
−
-dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO
3
−
buffer that were first exposed to 1 μmol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pH
i
from 7.05±0.05 to 7.22±0.05 (
P
<.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pH
i
recovery after the imposition of an intracellular alkali load in a HCO
3
−
-containing solution either with or without Ang II. The rate of myocardial pH
i
recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl
−
in a Na
+
-free solution. Under this condition, the rate of intracellular alkalinization increased from 0.053±0.016 to 0.108±0.026 pH unit/min (n=6,
P
<.05) in the presence of Ang II. This effect was canceled either by the presence of the AT
1
receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C–dependent regulatory pathway linked to the AT
1
receptors.
Synergistic Exacerbation of Diastolic Stiffness From Short-term Tachycardia–Induced Cardiodepression and Angiotensin II
Authors: Senzaki Hideaki , Gluzband Yehezkiel A. , Pak Peter H. , Crow Michael T. , Janicki Joseph S. , Kass David A.
Published: June 2012

Abstract
—Synergistic interaction between angiotensin II (Ang II) and evolving cardiodepression may play an important role in worsening chamber function, particularly in diastole. To test this hypothesis, Ang II was infused at 10 or 17 ng · kg
−1
· min
−1
in 18 conscious dogs 4 days before and during induction of subacute cardiodepression by 48-hour tachypacing. The lower dose yielded negligible systemic pressure changes. Twelve additional animals served as paced-only controls. Pressure-dimension relations were recorded, and serial endocardial biopsies were obtained to assess histological and metalloproteinase (MMP) changes. Forty-eight–hour pacing alone depressed systolic function but had little effect on diastolic stiffness. Ang II alone only modestly raised diastolic stiffness at both doses and enhanced contractility at the higher dose. These changes recovered toward baseline after a 7-day infusion. However, Ang II (at either dose) combined with 48-hour pacing markedly increased ventricular stiffness (110±26% over baseline) and end-diastolic pressure (22±1.7 mm Hg). In contrast, pacing-induced inotropic and relaxation abnormalities were not exacerbated by Ang II. Zymography revealed MMP activation (72- and 92-kD gelatinases and 52-kDa caseinase) after a 4-day Ang II infusion (at both doses), which persisted during pacing. Tachypacing initiated 24 hours after cessation of a 7-day Ang II infusion also resulted in diastolic stiffening and corresponded with MMP reactivation. Ang II also induced myocyte necrosis, inflammation, and subsequent interstitial fibrosis, but these changes correlated less with chamber mechanics. Thus, Ang II amplifies and accelerates diastolic dysfunction when combined with evolving cardiodepression. This phenomenon may also underlie Ang II influences in late-stage cardiomyopathy, when chamber distensibility declines.
Cloning and Functional Expression of a Novel Cardiac Two-Pore Background K
+
Channel (cTBAK-1)
Authors: Kim Donghee , Fujita Akikazu , Horio Yoshiyuki , Kurachi Yoshihisa
Published: June 2012

Abstract
—We have cloned from a mouse heart cDNA library a novel K
+
channel subunit that has two pore-forming domains and four transmembrane regions. Its amino acid sequence shares 25% identity with mouse TWIK-1, 22% with mouse TREK-1, and 33% with a putative K
+
channel of
Caenorhabditis elegans
(C40C9). Strikingly abundant mRNA for this clone was expressed in the heart. The mRNA was also detected in kidney, brain, skin, testis, lung, skeletal muscle, small intestine, and stomach but not in liver, thymus, or spleen. Reverse transcription–polymerase chain reaction analyses of single cells showed that the mRNA of the clone was expressed in both atrial and ventricular myocytes per se.
Xenopus
oocytes injected with the cRNA of the clone expressed a Ba
2+
-sensitive K
+
-selective current with an almost linear steady-state current-voltage relationship. In cell-attached patches, the expressed channel exhibited short-lasting openings with a mean open time of ≈2 milliseconds and a unitary conductance of ≈16 pS (150 mmol/L [K
+
]
o
). The K
+
current was insensitive to intracellular Na
+
(50 mmol/L), Ca
2+
(0.1 mmol/L), H
+
(pH 6.4), and arachidonic acid (10 μmol/L) in inside-out patches. Thus, the current flowing through the channel may contribute to the cardiac cellular electrical activity as a linear background K
+
conductance. Therefore, we designated the clone cTBAK (cardiac two-pore background K
+
channel).
Early Coronary Angiogenesis in Response to Thyroxine
Authors: Tomanek Robert J. , Doty Matthew K. , Sandra Alexander
Published: June 2012

Abstract
—Although a substantial coronary angiogenesis occurs after thyroid hormone treatment, its regulation and relationship to cardiac hypertrophy are not understood. This study was designed to determine (1) the onset of capillary proliferation, (2) the sites of capillary proliferation, and (3) whether basic fibroblast growth factor (bFGF) upregulation occurs in response to thyroxine administration. Male Sprague-Dawley rats were injected daily with
l
-thyroxine (T
4
, 0.2 mg/kg SC). Bromodeoxyuridine labeling of capillary endothelial cells increased during the first 24 hours of treatment and peaked after 2 days of treatment. Northern blot analysis revealed a slight increase in bFGF mRNA during this period, followed by a doubling of expression by 48 hours, at which time bFGF protein was also increased. In situ hybridization, used to localize bFGF mRNA, showed an increase in transcripts within 24 hours after T
4
. This enhancement was uniform in the epimyocardium and endomyocardium. Histochemical analysis (double staining for alkaline phosphatase and dipeptidyl peptidase) of frozen sections, used to discriminate capillary profiles as arteriolar and venular, respectively, showed that growth occurred in the latter, since the percentage of capillary profiles positive for dipeptidyl peptidase was higher than the control value after 4 days of T
4
administration. These data indicate that in the thyroxine model of cardiac hypertrophy (1) capillary DNA synthesis occurs after a single injection of thyroxine, (2) capillary growth coincides with an upregulation in bFGF mRNA and increase in bFGF protein, and (3) proliferation occurs in the venular capillaries.
Differential Expression of Gap Junction Proteins in the Canine Sinus Node
Authors: Kwong King F. , Schuessler Richard B. , Green Karen G. , Laing James G. , Beyer Eric C. , Boineau John P. , Saffitz Jeffrey E.
Published: June 2012

Abstract
—Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: ≈55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.
Effects of In Vivo Administration of Anti–B7-1/B7-2 Monoclonal Antibodies on Murine Acute Myocarditis Caused by Coxsackievirus B3
Authors: Seko Yoshinori , Takahashi Naoyuki , Azuma Miyuki , Yagita Hideo , Okumura Ko , Yazaki Yoshio
Published: June 2012

Abstract
—In viral myocarditis, we previously reported that antigen-specific T cells infiltrate the heart and play an important role in the pathogenesis of myocardial damage. For antigen-specific T-cell activation to occur, it is necessary for T cells to receive costimulatory signals provided by costimulatory molecules expressed on antigen-presenting cells as well as main signals provided by binding of T-cell receptors to antigens. To investigate the roles of costimulatory molecules B7-1 and B7-2 in the development of acute viral myocarditis, we first analyzed the expression of B7-1/B7-2 in the hearts of mice with acute viral myocarditis induced by coxsackievirus B3 (CVB3). Second, we evaluated the induction of B7-1/B7-2 in cultured cardiac myocytes treated with interferon gamma (IFN-γ). Third, we examined the effects of in vivo administration of anti–B7-1/B7-2 monoclonal antibodies (mAbs) on the development of acute viral myocarditis. We found that CVB3-induced murine acute myocarditis resulted in enhanced expression of B7-1/B7-2 in cardiac myocytes. The expression of B7-1/B7-2 in cardiac myocytes could be induced in vitro by IFN-γ. We found that in vivo anti–B7-1 mAb treatment markedly decreased myocardial inflammation, whereas anti–B7-2 mAb treatment abrogated the protective effect of anti–B7-1. Our findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of acute viral myocarditis and raise the possibility of immunotherapy with anti–B7-1 mAb to prevent T-cell–mediated myocardial damage in viral myocarditis.
Cardiac Graft Intercellular Adhesion Molecule-1 (ICAM-1) and Interleukin-1 Expression Mediate Primary Isograft Failure and Induction of ICAM-1 in Organs Remote From the Site of Transplantation
Authors: Wang Catherine Y. , Naka Yoshifumi , Liao Hui , Oz Mehmet C. , Springer Timothy A. , Gutierrez-Ramos Jose-Carlos , Pinsky David J.
Published: June 2012

Abstract
—During the first few hours after heart transplantation, the occurrence of graft failure is unpredictable and devastating. An explosive cascade of inflammatory events within the reperfused graft vasculature is likely to be mediated, at least in part, by the local expression of the leukocyte adhesion receptor intercellular adhesion molecule-1 (ICAM-1, CD54). Furthermore, although proinflammatory cytokines such as interleukin-1 (IL-1) are known to autoinduce their own (and ICAM-1) expression in vitro, there are no data to identify their functional in vivo cross talk in the setting of isograft transplantation. To determine the role of ICAM-1 in primary graft failure, we used an isogeneic vascularized model of heterotopic cardiac transplantation. ICAM-1 mRNA and protein increased in grafts during the early posttransplant period and were predominantly localized in the endothelium. The functional significance of this was established using donor hearts obtained from either ICAM-1–deficient (ICAM-1 −/−) or control (ICAM-1 +/+) mice. ICAM-1 +/+ grafts exhibited increased neutrophil infiltration, reduced left ventricular compliance, and poorer survival than did ICAM-1 −/− grafts. Increased ICAM-1 expression was not limited to ICAM-1 +/+ grafts but also occurred in unmanipulated recipient organs located remote from the site of surgery (but only after transplantation of ICAM-1 +/+, not ICAM-1 −/−, cardiac grafts). This expression of ICAM-1 in remote organs appeared to be triggered by IL-1α released from the graft, because (1) in situ hybridization revealed increased IL-1 mRNA within cells of the reperfused graft, including myocytes and endothelial cells; (2) ICAM-1 expression in remote organs coincided with a significant increase in serum levels of IL-1α after transplantation of ICAM-1 +/+ grafts; both remote organ ICAM-1 expression and IL-1α levels were blunted by implantation of ICAM-1 −/− grafts; and (3) remote organ ICAM-1 expression and neutrophil infiltration and IL-1 levels could be blocked by the administration of an IL-1 receptor antagonist. These data demonstrate an apparent positive-feedback loop in which local ICAM-1 and IL-1 expression leads to a mutual amplification of each other’s expression within the reperfused graft, promulgating inflammatory events that are likely to be an important cause of primary cardiac graft failure. Because IL-1 receptor blockade reduces the IL-1–mediated autoinduction of IL-1, reduces the expression of ICAM-1 in both the graft and remote organs, and improves graft survival, it may provide a new and effective strategy to prevent the occurrence of primary cardiac graft failure.
Cytosolic Alkalinization of Vascular Endothelial Cells Produced by an Abrupt Reduction in Fluid Shear Stress
Authors: Ziegelstein Roy C. , Blank Paul S. , Cheng Linda , Capogrossi Maurizio C.
Published: June 2012

Abstract
—Reductions in fluid shear stress produce endothelium-dependent vasoconstriction and promote neointimal hyperplasia, but the intracellular signaling mechanisms involved in these processes are poorly understood. To examine whether decreases in fluid shear stress affect endothelial cytosolic pH, carboxy-seminaphthorhodafluor-1–loaded rat aortic endothelial cells were cultured in glass microcapillary tubes and examined during abrupt reductions in laminar flow. After a 30-minute exposure to a shear stress of 2.7 dyne/cm
2
in bicarbonate buffer, the acute reduction of fluid shear stress from 2.7 to 0.3 dyne/cm
2
transiently increased cytosolic pH from 7.20±0.02 to 7.47±0.07 (mean±SEM,
P
<.05 versus control). This was not affected by prior inhibition of the Na
+
-H
+
exchanger with 10 μmol/L ethylisopropylamiloride but was abolished in bicarbonate-free buffer. Recovery from an ammonium chloride prepulse–induced acid load occurred more rapidly when fluid shear stress was abruptly reduced from 2.7 to 0.3 dyne/cm
2
after maximal acidification (+0.04±0.02 pH unit at 2 minutes) than when shear stress was maintained at 2.7 dyne/cm
2
continuously (0.00±0.00 pH unit at 2 minutes,
P
<.05). This accelerated cytosolic pH recovery was dependent on the presence of bicarbonate ion and was blocked by the addition of the exchange inhibitors DIDS (100 μmol/L) and ethylisopropylamiloride or by removal of buffer Na
+
, indicating that the acute reduction in fluid shear stress activates the extracellular Na
+
–dependent Cl
−
-HCO
3
−
exchanger and the Na
+
-H
+
exchanger and increases cytosolic pH in vascular endothelial cells.
Timing Is Everything in Life
Authors: Fishman Glenn I.
Published: June 2012

Abstract
—Manipulation of the mouse genome by traditional transgenic approaches has facilitated studies of gene function within the context of the intact organism and allowed for the creation of useful animal models of human disease. However, the timing of gene activation or repression is a critical determinant of phenotype, and the ability to regulate the temporal profile of transgene expression remains an important experimental goal. In this Mini Review, we describe the current status of systems to tightly regulate target gene expression in vivo, focusing on binary systems using chimeric transcription factors. Although experimental difficulties persist, regulated expression systems are beginning to produce conditional phenotypes with exciting experimental implications. We review the experience to date and examine the potential utility of these approaches within the context of cardiovascular medicine.
Expression of Extracellular Matrix Proteins Accompanies Lesion Growth in a Model of Intimal Reinjury
Authors: Koyama Hiroyuki , Reidy Michael A.
Published: June 2012

Abstract
—Reinjury of rat arterial lesions induces an increase in lesion size that is not associated with an increase in cell number. In this study, matrix volume was examined after reinjury to preexisting lesions, and the kinetics of matrix gene expression and activity of proteolytic enzymes in the lesion were evaluated. Volume densitometry in intima showed a significant increase in matrix volume 28 days after the reinjury, although no change was observed at 14 days. Three common vascular matrix molecules, α
1
(I)procollagen, tropoelastin, and fibronectin, were expressed highly at 7 days after the reinjury. Expression of tropoelastin remained upregulated for the entire 28 days after the reinjury, whereas α
1
(I)procollagen and fibronectin returned to the control level by 28 days. Protease activity was also increased after reinjury. Within days, a marked increase in urokinase plasminogen activator activity was observed in intima, and this activity decreased to control level by 14 days. The activity of tissue plasminogen activator did not change. The 95-kDa gelatinolytic activity was increased 1 to 2 days after the reinjury, but no change in other gelatinolytic activities was observed. These findings demonstrate that the accumulation of extracellular matrix is important in the increase in lesion size after reinjury and that a balance of matrix synthesis and degradation may explain why no change in matrix volume was detected until 28 days after the reinjury.
Genetic Disruption of Poly (ADP-Ribose) Synthetase Inhibits the Expression of P-Selectin and Intercellular Adhesion Molecule-1 in Myocardial Ischemia/Reperfusion Injury
Authors: Zingarelli Basilia , Salzman Andrew L. , Szabó Csaba
Published: June 2012

Abstract
—The nuclear enzyme poly (ADP-ribose) synthetase (PARS) has been shown to play an important role in the pathogenesis of ischemia/reperfusion injury and circulatory shock. The aim of this study was to investigate whether PARS activity may modulate endothelial-neutrophil interaction. We present evidence that genetic disruption of PARS provides protection against myocardial ischemia and reperfusion injury by inhibiting the expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) and, consequently, by inhibiting the recruitment of neutrophils into the jeopardized tissue. Furthermore, using in vitro studies, we demonstrate that in fibroblasts lacking a functional gene for PARS, cytokine-stimulated expression of ICAM-1 is significantly reduced compared with fibroblasts from animals with a normal genotype. Similarly, in cultured human endothelial cells, oxidative- or cytokine-dependent expression of P-selectin and ICAM-1 is reduced by pharmacological inhibition of PARS by 3-aminobenzamide. These findings provide the first direct evidence that PARS activation participates in neutrophil-mediated myocardial damage by regulating the expression of P-selectin and ICAM-1 in ischemic and reperfused myocardium, and they also provide the basis for a novel therapeutic approach for the treatment of reperfusion injury.
Leptin, the Product of Ob Gene, Promotes Angiogenesis
Authors: Bouloumié Anne , Drexler Hannes C. A. , Lafontan Max , Busse Rudi
Published: June 2012

Abstract
—The adipocyte-derived cytokine leptin is thought to play a key role in the control of satiety and energy expenditure. Because adipogenesis and angiogenesis are tightly correlated during the fat mass development, we tested the hypothesis that leptin is able to modulate the growth of the vasculature. Experiments were performed using cultured human umbilical venous endothelial cells (HUVECs) and porcine aortic endothelial cells. The presence of 170-kDa endothelial leptin receptor (Ob-R) was assessed in HUVECs by Western blot analysis. Reverse transcriptase–polymerase chain reaction analysis using specific oligonucleotides for the short and long Ob-R forms further revealed the expression of both Ob-R transcripts in endothelial cells. Moreover, leptin evoked a time-dependent tyrosine phosphorylation of a number of endothelial proteins, the most prominent of which were the mitogen-activated protein kinases Erk1/2. Treatment of HUVECs with leptin led to a concentration-dependent increase in cell number that was maximal at 10 ng/mL leptin and equivalent to that elicited by vascular endothelial growth factor. This effect was associated with an enhanced formation of capillary-like tubes in an in vitro angiogenesis assay and neovascularization in an in vivo model of angiogenesis. These results indicate that leptin, via activation of the endothelial Ob-R, generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We speculate that this leptin-mediated stimulation of angiogenesis might represent not only a key event in the settlement of obesity but also may contribute to the modulation of growth under physiological and pathophysiological conditions in other tissues.
Endogenous Interleukin-10 Regulates Hemodynamic Parameters, Leukocyte-Endothelial Cell Interactions, and Microvascular Permeability During Endotoxemia
Authors: Hickey Michael J. , Issekutz Andrew C. , Reinhardt Paul H. , Fedorak Richard N. , Kubes Paul
Published: June 2012

Abstract
—The objective of this study was to determine whether endogenous IL-10 is capable of regulating hemodynamic parameters, leukocyte recruitment, and microvascular permeability in response to endotoxin. Intravital microscopy was used to examine hemodynamic parameters, leukocyte rolling and adhesion, and microvascular permeability in cremasteric postcapillary venules in wild-type mice and in IL-10–deficient (IL-10
−/−
) mice exposed to lipopolysaccharide (LPS). Doses of LPS (3 or 30 μg/kg, IV), which did not reduce blood pressure and minimally altered microvascular hemodynamic factors in wild-type mice, caused significant reductions in these parameters in IL-10
−/−
mice, demonstrating at least a 10-fold increased sensitivity in IL-10
−/−
mice to LPS-induced hemodynamic alterations. Furthermore, in response to LPS (30 μg/kg, IV), leukocyte rolling, adhesion, and fluorescein isothiocyanate–albumin extravasation were increased in the IL-10
−/−
mice. Antibody blockade experiments showed that in both types of mice, leukocyte rolling was mediated by E-selectin and P-selectin. Leukocyte accumulation into other tissues, such as lung, also was enhanced greatly in IL-10
−/−
mice. This was specific to endotoxin, because acute chemotactic stimuli including N-formyl-methionyl-leucyl-phenylalanine elicited similar responses in IL-10
−/−
and wild-type mice. These results suggest that endogenous IL-10 may be a homeostatic regulator of hemodynamic parameters, leukocyte-endothelial cell interactions, and microvascular dysfunction in response to endotoxin and provide potential mechanisms to explain the protective effect of IL-10 against LPS-induced mortality.
T Cells With Similar T-Cell Receptor β-Chain Complementarity-Determining Region 3 Motifs Infiltrate Inflammatory Lesions of Synthetic Peptides Inducing Rat Autoimmune Myocarditis
Authors: Hanawa Haruo , Inomata Takayuki , Okura Yuji , Hirono Satoru , Ogawa Yusuke , Izumi Tohru , Kodama Makoto , Aizawa Yoshifusa
Published: June 2012

Abstract
—Experimental autoimmune myocarditis (EAM) resembles the giant cell myocarditis seen in humans, and recurrent forms lead to dilated cardiomyopathy. EAM has been shown to be a T cell–mediated autoimmune myocarditis. We have previously shown that cDNA encoding Vβ complementarity-determining region (CDR) 3 from heart– and pericardial space–infiltrating T cells in EAM induced by rod cardiac myosin contains more restricted sequences than that from normal spleen T cells. Recently, it has become apparent that several epitopes of EAM exist in rod cardiac myosin; therefore, T cells infiltrating into lesions may recognize certain epitopes in EAM induced by rod cardiac myosin. In this study, we examined heart– and pericardial space–infiltrating T-cell clonotypes in EAM induced by synthetic peptides of cardiac myosin. EAM was produced by immunization with synthetic peptides corresponding to N-terminally acetylated amino acids 1539 to 1555 of rat cardiac myosin α heavy chain. Five of 12 rats receiving synthetic peptides developed macroscopic signs of myocarditis. To examine T-cell receptor (TCR) Vβ expression and CDR3 of the TCR β chain of lesion-infiltrating T cells in EAM, total RNA was isolated from heart, pericardial effusion, spleen, lymph node, and peripheral blood. TCR Vβ expression of the T cells infiltrating the lesions revealed a predominance of Vβ4. On the basis of single-strand conformation polymorphism analysis for CDR3 of the TCR Vβ4 chain, heart– and pericardial space–infiltrating T cells were considered to be oligoclonal, whereas spleen, lymph node, and peripheral blood in a rat with EAM and spleen in a native rat were considered to be polyclonal. In the same rat, clonotypes of heart-infiltrating T cells were almost the same as those of pericardial space–infiltrating T cells. Furthermore, on sequence analysis for CDR3 of the TCR Vβ4 chain, the amino acid motifs were similar among T cells infiltrating into lesions of different EAM rats. In the present study, TCR β chains of heart– and pericardial space–infiltrating T cells in EAM induced by synthesized peptide consisting of 17 amino acids were examined. Vβ4+ T cells with similar Vβ CDR3 motifs that infiltrate the heart and pericardial space may recognize the same epitope.
Tyrosine Kinase and c-Jun NH
2
-Terminal Kinase Mediate Hypertrophic Responses to Prostaglandin F
2α
in Cultured Neonatal Rat Ventricular Myocytes
Authors: Adams John W. , Sah Valerie P. , Henderson Scott A. , Brown Joan Heller
Published: June 2012

Abstract
—Myocardial infarction results in focal areas of ischemia, hypoxia, necrosis, and decreased contractile function. To compensate for loss of contractile function, remaining viable myocytes undergo hypertrophic growth. Prostaglandin F
2α
(PGF
2α
), which is released from cells of the myocardium during periods of stress such as hypoxia or ischemia/reperfusion, has recently been shown to stimulate hypertrophic growth in neonatal rat ventricular myocytes. In the present study, we determine which growth-related intracellular pathways are required for PGF
2α
to induce morphological and genetic features characteristic of the hypertrophic phenotype. In cardiomyocytes, PGF
2α
increases the hydrolysis of inositol phosphates and induces the translocation of protein kinase Cε to the myocyte membrane, consistent with PGF
2α
receptor coupling to G
q
. PGF
2α
also activates the extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase pathways. Surprisingly, studies using pharmacological inhibitors and transfection of dominant-interfering proteins demonstrate that PGF
2α
-induced myocyte hypertrophy occurs independent of either PKC, p38, or ERK pathways. Additional studies demonstrate that PGF
2α
stimulates protein tyrosine phosphorylation and activates c-Jun NH
2
-terminal kinase and suggest that these pathways mediate hypertrophic growth in response to PGF
2α
.