bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduction in Atherosclerotic Lesion Size in Pigs by αVβ3 Inhibitors Is Associated With Inhibition of Insulin-Like Growth Factor-I–Mediated Signaling
Authors: Nichols Timothy C. , Laney Tracey du , Zheng Bo , Bellinger Dwight A. , Nickols G. Allen , Engleman Wayne , Clemmons David R.
Published: June 2012

Abstract
—Insulin-like growth factor-I (IGF-I) is a potent stimulant of smooth muscle cell (SMC) migration and proliferation and has been implicated in the development of experimental atherosclerotic lesions. Because optimal stimulation of SMC in vitro by IGF-I requires ligand occupancy of αVβ3, these studies were conducted to determine whether αVβ3 antagonists would result in a change in lesion size and whether they could alter IGF-I-mediated actions. Clamps were placed on the carotid and femoral arteries of normal pigs that had been fed a high-cholesterol diet for 4 weeks. αVβ3 inhibitors (SC-69000, SC-65811) (10
−6
mol/L) or saline were infused for 2 weeks into the peristenotic area. Lesion area, the number of SMC layers, and proliferating cell nuclear antigen positive cells were determined in a 1.2-mm segment of each artery. Lesion areas were 304 788±113 453 μ
2
(saline), compared with 149 799±35 456 μ
2
(SC-69000) (
P
<0.01). Lesion areas in arteries treated with SC-64258, a compound that does not bind to αVβ3, were 310 284±160 467 μ
2
,
P
=not significant. In a second experiment, lesion areas were 110 391±17 347 μ
2
(saline) and 59 533±17 568 μ
2
(SC-65811,
P
<0.001). Neointimal SMC layers were reduced by SC-65811 from 7.4±4.5 to 3.0±0.4 (
P
<0.001). To determine whether IGF-I action was altered, IGF binding protein-5, which is synthesized in response to IGF-I, was analyzed. IGF-I binding protein-5 mRNA abundance was reduced by 67±8% in the 6 lesions treated with SRL-69000 compared with saline controls (
P
<0.001). We conclude that αVβ3 antagonists block the development of lesions in pigs that have been induced by a high-cholesterol diet and stenosis, and the effect of these compounds is associated with their ability to inhibit IGF-I–mediated signaling.
Adrenomedullin Receptor Antagonism by Calcitonin Gene-Related Peptide(8-37) Inhibits Carotid Artery Neointimal Hyperplasia After Balloon Injury
Authors: Shimizu Koichi , Tanaka Hiroyuki , Sunamori Makoto , Marumo Fumiaki , Shichiri Masayoshi
Published: June 2012

Abstract
—Intimal injury by angioplasty results in a series of changes, including smooth muscle cell hyperplasia, that lead to vascular restenosis. Adrenomedullin, a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. Adrenomedullin is secreted by endothelial and vascular smooth muscle cells, but its role in neointimal hyperplasia after balloon injury has not been previously described. We investigated the role of endogenous adrenomedullin in neointimal hyperplasia using an in vivo rat model of postinjury vascular restenosis. In the injured rats, bromodeoxyuridine-labeled nuclei in the media of untreated common carotid arteries were increased 2 days after injury, which were suppressed by in vivo treatment with the adrenomedullin receptor antagonist calcitonin gene-related peptide (CGRP)(8-37). Inhibition of neointimal hyperplasia by CGRP(8-37) was distinct at 7 and 14 days, whereas CGRP(1-37) had no effect. The expression of adrenomedullin in the media of both untreated and treated common carotid arteries was elevated at 2 days and further enhanced in hyperplastic intima of untreated common carotid arteries at 7 days. Our findings suggest a novel role for endogenous adrenomedullin in balloon injury–induced restenosis and indicate that CGRP(8-37) may be useful for the prevention of vascular restenosis.
Bone Marrow Origin of Endothelial Progenitor Cells Responsible for Postnatal Vasculogenesis in Physiological and Pathological Neovascularization
Authors: Asahara Takayuki , Masuda Haruchika , Takahashi Tomono , Kalka Christoph , Pastore Christopher , Silver Marcy , Kearne Marianne , Magner Meredith , Isner Jeffrey M.
Published: June 2012

Abstract
—Circulating endothelial progenitor cells (EPCs) have been isolated in peripheral blood of adult species. To determine the origin and role of EPCs contributing to postnatal vasculogenesis, transgenic mice constitutively expressing β-galactosidase under the transcriptional regulation of an endothelial cell–specific promoter (Flk-1/LZ or Tie-2/LZ) were used as transplant donors. Localization of EPCs, indicated by
flk-1
or
tie-2/lacZ
fusion transcripts, were identified in corpus luteal and endometrial neovasculature after inductive ovulation. Mouse syngeneic colon cancer cells (MCA38) were implanted subcutaneously into Flk-1/LZ/BMT (bone marrow transplantation) and Tie-2/LZ/BMT mice; tumor samples harvested at 1 week disclosed abundant
flk-1/lacZ
and
tie-2/lacZ
fusion transcripts, and sections stained with X-gal demonstrated that the neovasculature of the developing tumor frequently comprised Flk-1– or Tie-2–expressing EPCs. Cutaneous wounds examined at 4 days and 7 days after skin removal by punch biopsy disclosed EPCs incorporated into foci of neovascularization at high frequency. One week after the onset of hindlimb ischemia, lacZ-positive EPCs were identified incorporated into capillaries among skeletal myocytes. After permanent ligation of the left anterior descending coronary artery, histological samples from sites of myocardial infarction demonstrated incorporation of EPCs into foci of neovascularization at the border of the infarct. These findings indicate that postnatal neovascularization does not rely exclusively on sprouting from preexisting blood vessels (angiogenesis); instead, EPCs circulate from bone marrow to incorporate into and thus contribute to postnatal physiological and pathological neovascularization, which is consistent with postnatal vasculogenesis.
Angiotensinogen Gene–Activating Elements Regulate Blood Pressure in the Brain
Authors: Nishii Tadahiko , Moriguchi Atsushi , Morishita Ryuichi , Yamada Kazuo , Nakamura Shigefumi , Tomita Naruya , Kaneda Yasufumi , Fukamizu Akiyoshi , Mikami Hiroshi , Higaki Jitsuo , Ogihara Toshio
Published: June 2012

Abstract
—Although the angiotensinogen gene is a possible candidate as a determinant of hypertension, the molecular mechanisms of tissue angiotensinogen gene regulation have yet to be clarified. We identified essential transcription regulators of angiotensinogen production in the central nervous system using synthetic double-stranded oligodeoxynucleotides (ODNs) as “decoy”
cis
elements to block the binding of nuclear factors to promoter regions of the targeted gene. Using a gel mobility shift assay, angiotensinogen gene–activating element (AGE) 2 binding protein was detected in the brain nuclear extracts of both spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs). Importantly, the binding activity of AGE 2 and angiotensinogen mRNA level were significantly higher in the brain of SHRs than in that of WKYs. Using the decoy approach, we demonstrated a significant decrease in the blood pressure of SHRs by transfection of AGE 2 decoy, but not mismatched, ODNs into the lateral cerebroventricle, accompanied by a significant decrease in brain angiotensinogen concentration and mRNA, and angiotensin II level. That these effects, demonstrated herein, are due to central effects is confirmed by the fact that no changes in circulating levels of angiotensinogen or angiotensin II concentrations were observed. Notably, AGE 2 decoy ODNs did not decrease the blood pressure of WKYs. We conclude that the abnormal expression of AGE 2 binding protein in the central nervous system plays a crucial role in high blood pressure of a genetically hypertensive rat model.
Species-Specific Differences in Positive and Negative Regulatory Elements in the Renin Gene Enhancer
Authors: Shi Qi , Black Thomas A. , Gross Kenneth W. , Sigmund Curt D.
Published: June 2012

Abstract
—A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4.1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.
Sustained Inhibition of Angiotensin I–Converting Enzyme (ACE) Expression and Long-Term Antihypertensive Action by Virally Mediated Delivery of ACE Antisense cDNA
Authors: Wang Hongwei , Katovich Michael J. , Gelband Craig H. , Reaves Phyllis Y. , Phillips M. Ian , Raizada Mohan K.
Published: June 2012

Abstract
—Angiotensin I–converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of ≈70% to 75% in ACE expression and a 50% increase in the B
max
for the AT
1
s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca
2+
levels in both ACE-S– and ACE-AS–expressing cells, the stimulation was significantly higher in ACE-AS–expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by ≈15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.
Small Guanine Nucleotide-Binding Proteins and Myocardial Hypertrophy
Authors: Clerk Angela , Sugden Peter H.
Published: June 2012

Abstract
—The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.
Coupling Function of Endogenous α
1
- and β-Adrenergic Receptors in Mouse Cardiomyocytes
Authors: Sabri Abdelkarim , Pak Elena , Alcott Sasha A. , Wilson Brenda A. , Steinberg Susan F.
Published: June 2012

Abstract
—Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. β
1
-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective β
2
-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. β
2
-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. α
1
-ARs fail to activate phospholipase C, the extracellular signal–regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by
Pasteurella multocida
toxin (a G
q
α-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via β-ARs in mouse cardiomyocytes, but β-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in α
1
-AR signaling and a defect in β
2
-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.
Atrial Natriuretic Factor Binding to Its Receptor Is Dependent on Chloride Concentration
Authors: Misono Kunio S.
Published: June 2012

Abstract
—Although considerable evidence indicates a role for atrial natriuretic factor (ANF) in renal salt regulation, other studies have found a lack of natriuretic response to high-plasma ANF under certain physiological and pathophysiological conditions. The mechanism for this apparent insensitivity to ANF is unknown. In the present study, it was found that ANF binding to its receptor requires the presence of chloride and occurs in a chloride concentration–dependent manner. ANF binding was measured using the purified recombinant hormone-binding domain of the ANF receptor in the presence of 0.1 mol/L NaCl or other selected salt. High specific binding was detected in the presence of NaCl, KCl, or NH
4
Cl. However, binding was undetectable when the salt was replaced with NaHCO
3
, CH
3
COONa, or CH
3
COONH
4
, indicating that binding requires the presence of chloride. Chloride dependence was also found with the native receptor in bovine adrenocortical membrane preparations. ANF binding to the recombinant protein was chloride concentration–dependent over a range from 0.05 to 10 mmol/L, and a half-maximum binding was attained at ≈0.6 mmol/L equivalent chloride concentration. Competitive-binding assays at several fixed concentrations of NaCl showed that lowering chloride concentration caused a decrease in maximum binding but did not alter
K
d
values, suggesting that a loss of chloride turns off ANF binding rather than reducing affinity for ANF. Saturation-binding studies showed that excess ANF cannot overcome loss of binding caused by low chloride. Chloride-dependent ANF-receptor binding may function as a feedback-control mechanism regulating the ANF-receptor action and, hence, renal sodium excretion.
Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH
Authors: Wittstein Ilan S. , Qiu Weiping , Ziegelstein Roy C. , Hu Qinghua , Kass David A.
Published: June 2012

Abstract
—Endothelial cytosolic pH (pH
i
) modulates ion channel function, vascular tone, and cell proliferation. Steady shear induces rapid acidification in bicarbonate buffer. However, in vivo shear is typically pulsatile, potentially altering this response. We tested effects and mechanisms of pH
i
modulation by flow pulsatility, comparing pressurized steady versus pulse-flow responses in bovine aortic endothelial cells cultured within glass capillary tubes. Cells were loaded with the fluorescent pH
i
indicator carboxy seminaphthorhodafluor-1 and perfused with physiological pulsatile pressure and flow generated by a custom servo-control system. Raising mean pressure from 0 to 90 mm Hg at 0.5 mL/min steady flow in bicarbonate buffer induced sustained acidification (−0.33±0.09 pH units,
P
<0.01). A subsequent increase in steady flow resulted in further acidification. In contrast, if mean pressure and flow were unchanged but perfusion made pulsatile, pH
i
rose +0.3±0.03 (
P
<0.0001) over 30 to 60 minutes. HCO
3
−
removal and use of acid/base exchange inhibitors 5-(
N
-ethyl-
N
-isopropyl)amiloride or diisothiocyanato stilbene disulfonic acid identified both extracellular Na
+
–independent Cl
−
-HCO
3
−
and Na
+
-H
+
exchangers as activated by static pressure, whereas pulsatility activated extracellular Na
+
–dependent Cl
−
-HCO
3
−
and Na
+
-H
+
exchangers to raise pH
i
. Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca
2+
-free buffer. Inhibition of c-Src tyrosine kinase (4-amino-5-[4-chlorophenyl]-7-[
t
-butyl]pyrazolo [3,4-
d
]pyrimidine; PP2) or MEK-1 (mitogen-activated protein kinase [MAP]/extracellular signal–regulated kinase [ERK]–1) (PD98059, blocking ERK1/2) blocked or reversed the pulsatile-flow pH
i
change to acidification. In contrast, PP2 had no effect on steady flow acidification, whereas MEK-1 inhibition converted it to alkalinization. Thus, pulsatile and steady flow trigger opposite effects on endothelial pH
i
by differential activation of acid/base exchangers linked to c-Src and MAP kinase phosphorylation, but not to Ca
2+
. These data highlight specific signaling responses triggered by phasic shear profiles.
Changes in Ca
2+
Cycling Proteins Underlie Cardiac Action Potential Prolongation in a Pressure-Overloaded Guinea Pig Model With Cardiac Hypertrophy and Failure
Authors: Ahmmed Gias U. , Dong Pei Hong , Song Guoji , Ball Nancy A. , Xu Yanfang , Walsh Richard A. , Chiamvimonvat Nipavan
Published: June 2012

Abstract
—Ventricular arrhythmias are common in both cardiac hypertrophy and failure; cardiac failure in particular is associated with a significant increase in the risk of sudden cardiac death. We studied the electrophysiologic changes in a guinea pig model with aortic banding resulting in cardiac hypertrophy at 4 weeks and progressing to cardiac failure at 8 weeks using whole-cell patch-clamp and biochemical techniques. Action potential durations (APDs) were significantly prolonged in banded animals at 4 and 8 weeks compared with age-matched sham-operated animals. APDs at 50% and 90% repolarization (APD
50
and APD
90
in ms) were the following: 4 week, banded, 208±51 and 248±49 (n=15); 4 week, sham, 189±68 and 213±69 (n=16); 8 week, banded, 197±40 and 226±40 (n=21); and 8 week, sham, 156±42 and 189±45 (n=22), respectively;
P
<0.05 comparing banded versus sham-operated animals. We observed no significant differences in the K
+
currents between the 2 groups of animals at 4 and 8 weeks. However, banded animals exhibited a significant increase in Na
+
and Na
+
-Ca
2+
exchange current densities compared with controls. Furthermore, we have found a significant attenuation in the Ca
2+
-dependent inactivation of the L-type Ca
2+
current in the banded compared with sham-operated animals, likely as a result of the significant downregulation of the sarcoplasmic reticulum Ca
2+
ATPase, which has been documented previously in the heart failure animals. Our data provide an alternate mechanism for APD prolongation in cardiac hypertrophy and failure and support the notion that there is close interaction between Ca
2+
handling and action potential profile.
Interdependence of Modulated Dispersion and Tissue Structure in the Mechanism of Unidirectional Block
Authors: Laurita Kenneth R. , Rosenbaum David S.
Published: June 2012

Abstract
—We previously showed that a premature stimulus can significantly alter vulnerability to arrhythmias by modulating spatial gradients of ventricular repolarization (ie, modulated dispersion). However, it is not clear if such changes in arrhythmia vulnerability can be attributed to the formation of an electrophysiological substrate for unidirectional block and what the potential role is of tissue structure in this process. Therefore, the main objective of the present study was to examine the concomitant effect repolarization gradients and tissue structure have on unidirectional block. Optical action potentials were recorded from 128 ventricular sites (1 cm
2
) in 8 Langendorff-perfused guinea pig hearts. Propagation was confined to the epicardial surface using an endocardial cryoablation procedure, and a 12-mm barrier with a 1.5-mm isthmus was etched with a laser onto the epicardium. A premature stimulus (S2) was delivered over a range of S1S2 coupling intervals to modulate repolarization gradients in a predictable fashion. When a second premature stimulus (S3) was delivered from the center of the isthmus, the occurrence and orientation of unidirectional block were highly dependent on repolarization gradients created by the S2 beat. In this model, a local repolarization gradient of 3.2 ms/mm was required for unidirectional block at this isthmus. In addition, the formation of unidirectional block was critically dependent on the presence of the source-sink mismatch imposed by the isthmus. These results may explain how the interplay between spatial heterogeneities of repolarization and tissue structure form a substrate for unidirectional block and reentry.
Maternal Hypercholesterolemia Enhances Atherogenesis in Normocholesterolemic Rabbits, Which Is Inhibited by Antioxidant or Lipid-Lowering Intervention During Pregnancy
Authors: Napoli Claudio , Witztum Joseph L. , Calara Federico , de Nigris Filomena , Palinski Wulf
Published: June 2012

Abstract
—Maternal hypercholesterolemia during pregnancy is associated with a marked increase in aortic fatty streak formation in human fetuses and faster progression of atherosclerosis during normocholesterolemic childhood. However, the mechanisms responsible are unknown, and the contribution of genetic differences is difficult to assess in humans. The goal of this study was to determine whether maternal hypercholesterolemia per se may cause enhanced fatty streak formation in offspring and whether interventions during pregnancy can reduce it. During pregnancy, 1 group of New Zealand White rabbits was fed control chow and 8 groups were fed hypercholesterolemic diets Chol 1 (yielding plasma cholesterol of 153 mg/dL) or Chol 2 (yielding 359 mg/dL) without or with cholestyramine, vitamin E, or both. Offspring (n=15 to 25 per group) were killed at birth. Maternal hypercholesterolemia enhanced mean lesion size in the aorta of their offspring at birth from 44±18×10
3
μm
2
per section in controls to 85±26×10
3
in Chol 1 and 156±49×10
3
in Chol 2 groups (
P
<0.0001 for both). Cholestyramine or vitamin E treatment of mothers significantly reduced atherosclerosis at birth by up to 39% compared with controls on the same diet. Oxidized fatty acids and malondialdehyde in aortic atherosclerotic lesions and plasma were similarly affected by diets and treatment as atherosclerosis. Our results establish the causal role of hypercholesterolemia and peroxidation in fetal atherogenesis and demonstrate that both lipid-lowering and antioxidant interventions during pregnancy can reduce it. If it can be established that interventions in mothers also affect progression of lesions after birth, this may indicate a novel approach for the prevention of atherosclerosis.
Competition for p300 Regulates Transcription by Estrogen Receptors and Nuclear Factor-κB in Human Coronary Smooth Muscle Cells
Authors: Speir Edith , Yu Zu-Xi , Takeda Kazuyo , Ferrans Victor J. , Cannon Richard O.
Published: June 2012

Abstract
—Previous studies suggest that estrogen may prevent expression of cell adhesion molecules implicated in vascular inflammation associated with atherosclerosis. We demonstrate the interaction and reciprocal interference of estrogen receptors (ERs) with p65, the nuclear factor-κB component, in smooth muscle cells that express ERα and ERβ after exposure to 17β-estradiol for 48 to 72 hours. ER and p65 do not associate directly, as shown by lack of coprecipitation, but instead compete for limiting amounts of p300, a close relative of the CREB-binding protein. Overexpressed p300 significantly reduced the inhibitory effect of ER on p65-dependent transcription as well as the inhibitory effect of p65 on ER-dependent transcription. These actions were ligand-dependent. The expression of both ER and nuclear factor-κB–dependent reporter genes was partially rescued from ER/p65 mutual inhibition by transient transfection of smooth muscle cells with a p300 expression vector. These actions of 17β-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.
Gene Therapy for Attenuating Cardiac Allograft Arteriopathy Using Ex Vivo E2F Decoy Transfection by HVJ-AVE–Liposome Method in Mice and Nonhuman Primates
Authors: Kawauchi Motohiro , Suzuki Jun-ichi , Morishita Ryuichi , Wada Yuko , Izawa Atsushi , Tomita Naruya , Amano Jun , Kaneda Yasufumi , Ogihara Toshio , Takamoto Shinichi , Isobe Mitsuaki
Published: June 2012

Abstract
—Cardiac allograft arteriopathy, which limits the long-term survival of recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. The transcription factor E2F plays a pivotal role in the coordinated transcription of cell-cycle regulatory genes. To test the hypothesis that double-stranded DNA with specific affinity for E2F (E2F decoy) is effective in preventing intimal hyperplasia, we performed ex vivo single intraluminal delivery of E2F decoy into cardiac allografts of mice and Japanese monkeys using the hemagglutinating virus of Japan (HVJ) artificial viral envelope–liposome method. In murine models, antisense cyclin-dependent kinase 2 (cdk2) kinase oligodeoxynucleotide (ODN) and no transfers were performed to compare the effects. Severe intimal thickening was observed, and multiple cell-cycle regulatory genes were enhanced in untreated allografts. E2F decoy prevented neointimal formation and suppressed these genes for up to 8 weeks, whereas antisense cdk2 kinase ODN had limited effects. In primate models, E2F decoy dramatically prevented neointimal thickening and suppressed multiple cell-cycle regulatory genes, whereas intimal thickening developed in the nontransfected or mismatch decoy-transfected allografts. Gel mobility shift assay proved the specific effects of E2F decoy, and reverse transcriptase–polymerase chain reaction documented that neither complication nor dissemination of HVJ into other organs was observed. We demonstrate that ex vivo gene delivery to allografts is a potent strategy to modify allograft gene expression, resulting in prevention of graft arteriopathy without systemic adverse effects.
Cardiomyocyte Apoptosis Induced by Gαq Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway
Authors: Adams John W. , Pagel Amy L. , Means Christopher K. , Oksenberg Donna , Armstrong Robert C. , Brown Joan Heller
Published: June 2012

Abstract
—Expression of the wild-type α subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates phospholipase C, as does expression of a constitutively active mutant form of Gαq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity (≈4-fold), cytochrome
c
redistribution, and nuclear chromatin condensation in ≈12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome
c
release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome
c
redistribution to only ≈2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.
Sustained Inward Current During Pacemaker Depolarization in Mammalian Sinoatrial Node Cells
Authors: Mitsuiye Tamotsu , Shinagawa Yasuko , Noma Akinori
Published: June 2012

Abstract
—Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na
+
current, L- and T-type Ca
2+
currents, hyperpolarization-activated cation current, and various types of delayed rectifier K
+
currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (
I
st
). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na
+
current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na
+
or L-type Ca
2+
channels both in unitary conductance and gating kinetics. Because
I
st
was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of
I
st
for generation of intrinsic cardiac automaticity was suggested. (Circ Res. 2000;87:88-91.)
Heparin Blockade of Thrombin-Induced Smooth Muscle Cell Migration Involves Inhibition of Epidermal Growth Factor (EGF) Receptor Transactivation by Heparin-Binding EGF-Like Growth Factor
Authors: Kalmes Andreas , Vesti Beatrice R. , Daum Günter , Abraham Judith A. , Clowes Alexander W.
Published: June 2012

Abstract
—Agonists of G protein–coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein–coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF–dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF–mediated EGFR transactivation. (Circ Res. 2000;87:92-98.)
Stretch-Dependent Modulation of Contractility and Growth in Smooth Muscle of Rat Portal Vein
Authors: Zeidan As’ad , Nordström Ina , Dreja Karl , Malmqvist Ulf , Hellstrand Per
Published: June 2012

Abstract
—Increased intraluminal pressure of the rat portal vein in vivo causes hypertrophy and altered contractility in 1 to 7 days. We have used organ cultures to investigate mechanisms involved in this adaptation to mechanical load. Strips of rat portal vein were cultured for 3 days, either undistended or loaded by a weight. Length-force relations were shifted toward longer length in stretched cultured veins compared with freshly dissected veins, whereas the length-force relations of unstretched cultured veins were shifted in the opposite direction. This occurred after culture either with or without 10% FCS to promote growth. The wet weight of loaded veins increased by 56% in the presence of FCS, whereas that of undistended control veins increased by 24%. No weight increase was seen in serum-free culture. The dry/wet weight ratio decreased during culture with FCS but was not affected by stretch. Electron microscopy revealed increased cell cross-sectional area in stretched relative to unstretched veins, and protein contents were greater, as were [
3
H]thymidine and [
3
H]leucine incorporation rates. Growth responses were associated with the activation of stretch-sensitive extracellular signal–regulated kinases 1 and 2 and were inhibited by herbimycin A and PD 98059, inhibitors of extracellular signal–regulated kinases 1 and 2. The results demonstrate that by culture of whole vascular tissue, smooth muscle cells are maintained in the contractile phenotype and respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system, similar to that in vivo. Mechanical stimulation and growth factors are both required for functionally significant growth.
Protein Tyrosine Kinase Is Not Involved in the Infarct Size–Limiting Effect of Ischemic Preconditioning in Canine Hearts
Authors: Kitakaze Masafumi , Node Koichi , Asanuma Hiroshi , Takashima Seiji , Sakata Yasuhiko , Asakura Masanori , Sanada Shoji , Shinozaki Yoshiro , Mori Hidezo , Kuzuya Tsunehiko , Hori Masatsugu
Published: June 2012

Abstract
—Protein kinase C (PKC) plays an important role in ischemic preconditioning (IP). Because (1) tyrosine kinase is located at the downstream of PKC for IP in the rabbit hearts and (2) we have reported that ecto–5′-nucleotidase is the substrate for PKC and plays a crucial role for the infarct size–limiting effect, we tested whether tyrosine kinase activation contributes to either activation of ecto–5′-nucleotidase or the infarct size–limiting effect of the early phase of IP in the canine heart. In dogs, the IP procedure (4 cycles of 5-minute occlusion of coronary artery) and exposure to 12,13-phorbol myristate acetate (PMA) each activated myocardial ecto–5′-nucleotidase and Lck tyrosine kinase. Genistein (10, 30, and 100 μg · kg
−
1
· min
−
1
IC), an inhibitor of tyrosine kinase, attenuated the activation of Lck tyrosine kinase but did not attenuate the activation of ecto–5′-nucleotidase due to either IP or PMA. In the other canine hearts, IP attenuated infarct size (49±5 versus 11±3 or 16±3%,
P
<0.01) due to 90 minutes of coronary occlusion followed by 6 hours of reperfusion, which was not blunted by 3 or 2 (30 and 100 μg · kg
−
1
· min
−
1
) doses of genistein (infarct sizes, 15±4, 13±4, and 13±3%, respectively, and 17±3 and 15±4%, respectively) or lavendustin A. Tyrosine kinase does not activate ecto–5′-nucleotidase or trigger the infarct size–limiting effect of the early phase of IP in canine hearts.
Human Vascular Smooth Muscle Cells but Not Endothelial Cells Express Prostaglandin E Synthase
Authors: Soler Marta , Camacho Mercedes , Escudero José-Román , Iñiguez Miguel A. , Vila Luís
Published: June 2012

Abstract
—In a previous work, we postulated that endothelial cells possess only the following 2 enzymes involved in prostanoid synthesis: cyclooxygenase and prostacyclin synthase. The present work focused on investigating the expression of prostaglandin (PG) E synthase (PGES) in vascular cells. After incubation of vascular smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) with [
14
C]arachidonic acid, the profile of prostanoid synthesis was assessed by HPLC. Untransformed PGH
2
released by the cells was evaluated as the difference in the formation of PGF
2α
in the incubations performed in the presence and in the absence of SnCl
2
. Resting SMCs and SMCs stimulated with phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α formed PGE
2
and PGI
2
(evaluated as 6-oxo-PGF
1α
), and in the presence of SnCl
2
only a small amount of PGE
2
was deviated toward PGF
2α
. In contrast, resting and stimulated HUVECs produced PGI
2
, PGE
2
, PGF
2α
, and PGD
2
, and SnCl
2
completely diverted PGE
2
and PGD
2
toward PGF
2α
. Reverse transcriptase–polymerase chain reaction analysis shows that mRNA encoding for PGES was not present in HUVECs and in endothelial cells from saphenous vein. Nevertheless, PGES was expressed in SMCs and induced by IL-1β and TNF-α, and by PMA and LPS, although to a lesser extent. Whereas SMC stimulation led to an increase in the synthesis of PGE
2
and PGI
2
but not of untransformed PGH
2
, stimulation of endothelial cells resulted in an enhanced release of the vasoconstricting prostanoid PGH
2
.
Reversal of Angiogenesis In Vitro, Induction of Apoptosis, and Inhibition of Akt Phosphorylation in Endothelial Cells by Thromboxane A
2
Authors: Gao Yunling , Yokota Ryoji , Tang Shaoqing , Ashton Anthony W. , Ware J. Anthony
Published: June 2012

Abstract
—Thromboxane A
2
(TxA
2
) causes platelet aggregation, vasoconstriction, and inhibition of endothelial cell (EC) migration and prevents vascular tube formation via its specific receptors (TP), of which there are two isoforms (TPα and TPβ), both expressed in human ECs. In this study, we demonstrate that the TxA
2
mimetic IBOP increases apoptosis of human ECs and inhibits the phosphorylation of Akt kinase, an intracellular mediator required for cell survival. Treatment with IBOP destroyed EC networks formed on a basement membrane matrix in vitro. To distinguish the role of the TP isoforms, each isoform was expressed in TP-null ECs to create TPα and TPβ ECs. IBOP induced apoptosis and inhibited phosphorylation of Akt kinase in both TPα and TPβ. IBOP increased cAMP levels in TPα but not in TPβ. Apoptosis induced by IBOP in TPα was not affected by either the adenylyl cyclase activator forskolin or the protein kinase A inhibitor 14-22 amide or H-89, whereas that in TPβ was suppressed by forskolin and enhanced by the protein kinase A inhibitor 14-22 amide or H-89, suggesting that the TP isoforms differ in their signal pathways in mediating apoptosis. In conclusion, apoptosis may be the mechanism by which TxA
2
-mediated destruction of vascular structures in ECs occurs; although both TP isoforms induce apoptosis, possibly via inhibiting Akt phosphorylation, the signaling differs in each isoform, in that activation of the adenylyl cyclase pathway prevents apoptosis caused by TPβ, but not by TPα, stimulation.
Hyperinsulinemia Enhances Transcriptional Activity of Nuclear Factor-κB Induced by Angiotensin II, Hyperglycemia, and Advanced Glycosylation End Products in Vascular Smooth Muscle Cells
Authors: Golovchenko Inga , Goalstone Marc L. , Watson Peter , Brownlee Michael , Draznin Boris
Published: June 2012

Abstract—Pathogenesis of macrovascular complications of diabetes may involve an activation of the transcription factor nuclear factor-κB (NF-κB) by hyperglycemia and advanced glycosylation end products (AGEs). Activation of NF-κB is believed to be dependent on activation of the Rho family of GTPases. Although the precise mechanism of the Rho-mediated action is not completely understood, posttranslational modification of the Rho proteins by geranylgeranylation is required for their subsequent activation. We observed that in cultured vascular smooth muscle cells (VSMCs), insulin stimulated the activity of geranylgeranyltransferase (GGTase) I and increased the amounts of geranylgeranylated Rho-A from 47% to 60% (
P
<0.05). GGTI-286, an inhibitor of GGTase I, blocked both effects of insulin. Increased availability of prenylated Rho-A significantly augmented the abilities of angiotensin II (Ang II), hyperglycemia, and AGEs to activate NF-κB, as measured by NF-κB response-element luciferase reporter activity. Preincubations of VSMCs with insulin for 24 hours doubled NF-κB transactivation by Ang II, hyperglycemia, and AGEs. This priming effect of insulin was completely inhibited by GGTI-286. We demonstrate for the first time, to our knowledge, that insulin potentiates NF-κB–dependent transcriptional activity induced by hyperglycemia, AGEs, and Ang II in VSMCs by increasing the activity of GGTase I and the availability of geranylgeranylated Rho-A.
Autonomous and Growth Factor–Induced Hypertrophy in Cultured Neonatal Mouse Cardiac Myocytes
Authors: Deng Xing-Fei , Rokosh D. Gregg , Simpson Paul C.
Published: June 2012

Abstract
—Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a β-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [
3
H]phenylalanine-labeled protein content. T3 and NE also increased α-myosin heavy chain (MyHC) mRNA and reduced β-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including α
1
-adrenergic agonists, endothelin-1, prostaglandin F
2α
, interleukin 1β, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or β-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal–regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and β-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C–coupled agonists do not.
Overexpression of 12-Lipoxygenase Causes Cardiac Fibroblast Cell Growth
Authors: Wen Yeshao , Gu Jiali , Liu Yaxia , Wang Ping H. , Sun Yanyu , Nadler Jerry L.
Published: June 2012

Abstract
—Evidence suggests that leukocyte type 12-lipoxygenase (12-LO) plays an important role in cell growth. However, the role of 12-LO in cardiac cell growth has not been tested. We have now stably overexpressed 12-LO cDNA in rat fetal cardiac fibroblasts to evaluate the role of the 12-LO pathway in cardiac cell growth. Overexpression of 12-LO increased cell [
3
H]leucine incorporation by 2.1±0.1-fold (
P
<0.01) and cell protein content by 2.2±0.3-fold (
P
<0.01) over mock-transfected cells. These findings were confirmed in additional clones. Baicalein, a 12-LO enzyme inhibitor, dose-dependently inhibited serum-induced leucine incorporation in cardiac fibroblast cells as well as partially inhibited leucine incorporation in cells overexpressing 12-LO. 12-LO overexpression also caused cell [
3
H]thymidine incorporation to increase by 3.4±0.3-fold (
P
<0.01). Cell flow cytometry analysis showed that the size of 12-LO–overexpressing cells was markedly enlarged compared with that of mock-transfected cells. The fibronectin content of the 12-LO–overexpressing cardiac fibroblasts was also significantly increased. We next evaluated the effects of 12-LO RNA overexpression on kinase pathways linked to cellular growth. The overexpression of 12-LO enhanced extracellular signal-regulated kinase activity (4.1±0.5-fold), c-Jun NH
2
-terminal kinase activity (2.9±0.5-fold), and p38 mitogen-activated protein kinase activity (2.2±0.3-fold). Pretreatment with SB202190 (100 nmol/L), a specific inhibitor of p38, prevented the increases in protein content of 12-LO–overexpressing cardiac fibroblast cells. These data clearly demonstrate that the overexpression of 12-LO causes cell growth of cardiac fibroblasts, thus supporting the role of 12-LO as a novel growth-promoting pathway in the heart.