bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Cytokine-Induced Cardiac Myocyte Apoptosis by Nitric Oxide, Bak, and Bcl-x
Authors: Ing Douglas J. , Zang Jie , Dzau Victor J. , Webster Keith A. , Bishopric Nanette H.
Published: June 2012

Abstract
—Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ. These cytokines caused a time-dependent induction of cardiac myocyte apoptosis, but not necrosis, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase, reflecting caspase activation. Apoptosis was preceded by a >50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/μg protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (
N
-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor
S
-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1β; interferon-γ and tumor necrosis factor-α had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species.
Apoptosis of Cardiac Myocytes in Gsα Transgenic Mice
Authors: Geng Yong-Jian , Ishikawa Yoshihiro , Vatner Dorothy E. , Wagner Thomas E. , Bishop Sanford P. , Vatner Stephen F. , Homcy Charles J.
Published: June 2012

Abstract
—The stimulatory GTP-binding protein Gsα transmits signals from catecholamine receptors to activate adenylyl cyclase and thereby initiate a cascade leading to cardiac chronotropy and inotropy. Transgenic mice overexpressing the Gs α subunit (Gsα) selectively in their hearts exhibit increased cardiac contractility in response to β-adrenergic receptor stimulation. However, with aging, these mice develop a cardiomyopathy. This study sought morphological and biochemical evidence that overexpression of Gsα is associated with increased myocyte apoptosis in the older animals and to determine whether such overexpression can promote apoptosis of isolated neonatal cardiac myocytes exposed to β-adrenergic receptor agonists. In the hearts of 15- to 18-month-old Gsα transgenic mice, histochemistry and electron microscopy illustrated the existence of numerous myocytes with abnormal nuclei embedded in collagen-rich connective tissue. Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL, for in situ labeling of DNA breaks) demonstrated that ≈0.6% of myocyte nuclei contained fragmented DNA. Agarose gel electrophoresis provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation. Cultured cardiac myocytes from newborn Gsα transgenic mice showed increased TUNEL staining and internucleosomal DNA fragmentation compared with wild-type controls when treated with the β-agonist isoproterenol. Thus, enhanced activation of β-adrenergic signaling by overexpression of Gsα in the hearts of transgenic mice induces apoptosis of cardiac myocytes. This represents a potential mechanism that may contribute to the development of cardiomyopathy in this model.
Localized Expression of Aromatase in Human Vascular Tissues
Authors: Harada Nobuhiro , Sasano Hironobu , Murakami Hiroshi , Ohkuma Tsuneo , Nagura Hiroshi , Takagi Yasuyuki
Published: June 2012

Abstract
—The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/μg RNA of aromatase mRNA and 12.9 to 122.3 fmol · h
−1
· mg
−1
protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.
Mechanically Driven Contour-Length Adjustment in Rat Cardiac Titin’s Unique N2B Sequence
Authors: Helmes M. , Trombitás K. , Centner T. , Kellermayer M. , Labeit S. , Linke W. A. , Granzier H.
Published: June 2012

Abstract
—The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force–sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above ≈2.2 μm SL titin’s elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin’s extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable.
Neuregulin Signaling in the Heart
Authors: Zhao You-Yang , Feron Olivier , Dessy Chantal , Han Xinqiang , Marchionni Mark A. , Kelly Ralph A.
Published: June 2012

Abstract
—Two of the neuregulins (NRG1 and NRG2) and their receptors (erbB2 and erbB4) are essential for normal cardiac development and can mediate hypertrophic growth and enhance survival of embryonic, postnatal, and adult rat ventricular myocytes. The expression of erbB4, the predominant NRG receptor in postnatal rat ventricular muscle, declines after midembryogenesis, and its expression is limited to cardiac myocytes. A full-length erbB4 rat cDNA isolated from neonatal ventricular muscle was found to be highly homologous to human erbB4 and contained a caveolin binding motif within the cytoplasmic kinase domain. Using the complementary techniques of detergent-free density-gradient ultracentrifugation of myocyte lysates and coimmunoprecipitation of erbB4 and caveolin-3, the caveolin isoform expressed in cardiac myocytes, erbB4 could be localized (using both approaches) to caveolar microdomains. Moreover, addition of a soluble NRG1, recombinant human glial growth factor 2, resulted in rapid (2-minute) translocation of erbB4 out of caveolar microdomain in cardiac myocytes. Thus, erbB4 is dynamically targeted to caveolar microdomains within cardiac myocytes. Its rapid translocation after NRG1 binding may contribute to receptor desensitization in the continuous presence of ligand.
Intracellular Sodium Accumulation During Ischemia as the Substrate for Reperfusion Injury
Authors: Imahashi Kenichi , Kusuoka Hideo , Hashimoto Katsuji , Yoshioka Jun , Yamaguchi Hitoshi , Nishimura Tsunehiko
Published: June 2012

Abstract
—To elucidate the role of intracellular Na
+
kinetics during ischemia and reperfusion in postischemic contractile dysfunction, intracellular Na
+
concentration ([Na
+
]
i
) was measured in isolated perfused rat hearts using
23
Na nuclear magnetic resonance spectroscopy. The extension of the ischemic period from 9 minutes to 15, 21, and 27 minutes (at 37°C) increased [Na
+
]
i
at the end of ischemia from 270.0±10.4% of preischemic level (mean±SE, n=5) to 348.4±12.0% (n=5), 491.0±34.0% (n=7), and 505.3±12.1% (n=5), respectively, whereas the recovery of developed pressure worsened with the prolongation of the ischemic period (95.1±4.2%, 84.3±1.2%, 52.8±13.7%, and 16.9±6.4% of preischemic level). The kinetics of [Na
+
]
i
recovery during reperfusion was analyzed by the fitting of a monoexponential function. When the hearts were reperfused with low–[Ca]
o
(0.15 mmol/L) solution, the time constants of the recovery (τ) after 15-minute (8.07±0.85 minutes, n=5) and 21-minute ischemia (6.44±0.90, n=5) were significantly extended, with better functional recovery (98.5±1.4% for 15-minute [
P
<0.05]; 98.0±1.0% for 21-minute [
P
<0.05]) compared with standard reperfusion ([Ca]
o
=2.0 mmol/L, τ=3.58±0.28 minutes for 15-minute [
P
<0.0001]; τ=3.02±0.20 for 21-minute [
P
<0.0001]). A selective inhibitor of Na
+
/Ca
2+
exchanger also decelerated the [Na
+
]
i
recovery, which suggests that the recovery reflects the Na
+
/Ca
2+
exchange activity. In contrast, high-[Ca]
o
reperfusion (5 mmol/L) accelerated the [Na
+
]
i
recovery after 9-minute ischemia (τ=2.48±0.11 minute, n=5 [
P
<0.0001]) and 15-minute ischemia (τ=2.10±0.07, n=6 [
P
<0.05]), but functional recovery deteriorated only in the hearts with 15-minute ischemia (29.8±9.4% [
P
<0.05]). [Na
+
]
i
recovery after 27-minute ischemia was incomplete and decelerated by low-[Ca]
o
reperfusion, with limited improvement of functional recovery (42.5±7.9%, n=5 [
P
<0.05]). These results indicate that intracellular Na
+
accumulation during ischemia is the substrate for reperfusion injury and that the [Na
+
]
i
kinetics during reperfusion, which is coupled with Ca
2+
influx, also determines the degree of injury.
Gene Transfer of Endothelial Nitric Oxide Synthase to the Lung of the Mouse In Vivo
Authors: Champion Hunter C. , Bivalacqua Trinity J. , D’Souza Fiona M. , Ortiz Luis A. , Jeter James R. , Toyoda Kazunori , Heistad Donald D. , Hyman Albert L. , Kadowitz Philip J.
Published: June 2012

Abstract
—The effects of transfer of the endothelial nitric oxide synthase (eNOS) gene to the lung were studied in mice. After intratracheal administration of AdCMVβgal, expression of the β-galactosidase reporter gene was detected in pulmonary airway cells, in alveolar cells, and in small pulmonary arteries. Gene expression with AdCMVβgal peaked 1 day after administration and decayed over a 7- to 14-day period, whereas gene expression after AdRSVβgal transfection peaked on day 5 and was sustained over a 21- to 28-day period. One day after administration of AdCMVeNOS, eNOS protein levels were increased, and there was a small reduction in mean pulmonary arterial pressure and pulmonary vascular resistance. The pressure-flow relationship in the pulmonary vascular bed was shifted to the right in animals transfected with eNOS, and pulmonary vasodepressor responses to bradykinin and the type V cGMP-selective phosphodiesterase inhibitor zaprinast were enhanced, whereas systemic responses were not altered. Pulmonary vasopressor responses to endothelin-1 (ET-1), angiotensin II, and ventilatory hypoxia were reduced significantly in animals transfected with the eNOS gene, whereas pressor responses to norepinephrine and U46619 were not changed. Systemic pressor responses to ET-1 and angiotensin II were similar in eNOS-transfected mice and in control mice. Intratracheal administration of AdRSVeNOS attenuated the increase in pulmonary arterial pressure in mice exposed to the fibrogenic anticancer agent bleomycin. These data suggest that transfer of the eNOS gene in vivo can selectively reduce pulmonary vascular resistance and pulmonary pressor responses to ET-1, angiotensin II, and hypoxia; enhance pulmonary depressor responses; and attenuate pulmonary hypertension induced by bleomycin. Moreover, these data suggest that in vivo gene transfer may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders.
HIRA, a DiGeorge Syndrome Candidate Gene, Is Required for Cardiac Outflow Tract Septation
Authors: Farrell Michael J. , Stadt Harriett , Wallis Kathleen T. , Scambler Peter , Hixon R. Lester , Wolfe Raymond , Leatherbury Linda , Kirby Margaret L.
Published: June 2012

Abstract
—DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene
HIRA
are expressed in the neural crest and in neural crest–derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end–protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of
cHIRA
in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract.
Calcification of Vascular Smooth Muscle Cell Cultures
Authors: Wada Takeo , McKee Marc D. , Steitz Susie , Giachelli Cecilia M.
Published: June 2012

Abstract
—Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase–dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 μg/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.
Mechanism of Nitric Oxide–Induced Vasodilatation
Authors: Cohen Richard A. , Weisbrod Robert M. , Gericke Marion , Yaghoubi Mohammad , Bierl Charlene , Bolotina Victoria M.
Published: June 2012

Abstract
—The precise mechanisms by which nitric oxide (NO) decreases free [Ca
2+
]
i
, inhibits Ca
2+
influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca
2+
]
i
were resistant to nifedipine, suggesting Ca
2+
entry through non–L-type Ca
2+
channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca
2+
]
i
and the rate of Mn
2+
influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca
2+
from the cytoplasm, decreased [Ca
2+
]
i
, and inhibited Ca
2+
and Mn
2+
influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na
+
/Ca
2+
exchanger or the plasma membrane Ca
2+
ATPase had no influence on the ability of NO to decrease [Ca
2+
]
i
. NO maximally decreased [Ca
2+
]
i
within 5 s, whereas significant inhibition of AII-induced Ca
2+
and Mn
2+
influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca
2+
]
o
and functional SERCA, suggesting that during the delay before NO inhibits Ca
2+
influx, the influx of Ca
2+
and the uptake into intracellular stores are required. In the absence of [Ca
2+
]
o
, NO diminished the AII-induced [Ca
2+
]
i
transient by a SERCA-dependent mechanism and increased the amount of Ca
2+
in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca
2+
]
i
caused by NO in vascular smooth muscle is accounted for by the uptake of Ca
2+
by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca
2+
influx through non–L-type Ca
2+
conducting ion channels and that this maintains the decrease in [Ca
2+
]
i
and NO-induced relaxation.
Distinct Renin Isoforms Generated by Tissue-Specific Transcription Initiation and Alternative Splicing
Authors: Lee-Kirsch Min Ae , Gaudet Francois , Cardoso M. Cristina , Lindpaintner Klaus
Published: June 2012

Abstract
—The aspartyl protease renin catalyzes the initial and rate-limiting step in the formation of the biologically active peptide angiotensin II. It is mainly synthesized in the kidney as a preprohormone and secreted via constitutive and regulated pathways. We identified a novel transcript of the rat renin gene, renin b, characterized by the presence of an alternative first exon (exon 1b) that is spliced to exon 2 of the known transcript, termed renin a. We demonstrated that renin b is exclusively expressed in the brain. In contrast, renin a was not expressed in the brain. Using primer extension assays, we mapped the transcriptional start site of this novel mRNA within intron 1 of the rat genomic sequence, suggesting the presence of a brain-specific promoter within intron 1. The presence of a brain-specific renin isoform is evolutionally conserved, as demonstrated by the finding of renin b isoforms in mice and humans. The predicted protein renin b lacks the prefragment as well as a significant portion of the profragment and is therefore predicted not to be a secreted protein, unlike the classically described isoform renin a. As shown by in vitro translation of full-length renin b mRNA in the presence of microsomal membranes, renin b was not targeted into the endoplasmatic reticulum and remained intracellularly in transiently transfected AtT-20 cells. These findings provide evidence for a novel pathway of intracellular angiotensin generation that occurs exclusively in the brain.
A Concise Promoter Region of the Heart Fatty Acid–Binding Protein Gene Dictates Tissue-Appropriate Expression
Authors: Qian Qinghua , Kuo Lyn , Yu Yie-Teh , Rottman Jeffrey N.
Published: June 2012

Abstract
—The heart fatty acid–binding protein (HFABP) is a member of a family of binding proteins with distinct tissue distributions and diverse roles in fatty acid metabolism, trafficking, and signaling. Other members of this family have been shown to possess concise promoter regions that direct appropriate tissue-specific expression. The basis for the specific expression of the HFABP has not been previously evaluated, and the mechanisms governing expression of metabolic genes in the heart are not completely understood. We used transient and permanent transfections in ventricular myocytes, skeletal myocytes, and nonmyocytic cells to map regulatory elements in the HFABP promoter, and audited results in transgenic mice. Appropriate tissue-specific expression in cell culture and in transgenic mice was dictated by 1.2 kb of the 5′-flanking sequence of
FABP3
, the HFABP gene. Comparison of orthologous murine and human genomic sequences demonstrated multiple regions of near-identity within this promoter region, including a CArG-like element close to the TATA box. Binding and transactivation studies demonstrated that this element can function as an atypical myocyte enhancer–binding factor 2 site. Interactions with adjacent sites are likely to be necessary for fully appropriate, tissue-specific, developmental and metabolic regulation.
Anti–Monocyte Chemoattractant Protein-1/Monocyte Chemotactic and Activating Factor Antibody Inhibits Neointimal Hyperplasia in Injured Rat Carotid Arteries
Authors: Furukawa Yutaka , Matsumori Akira , Ohashi Naohiro , Shioi Tetsuo , Ono Koh , Harada Akihisa , Matsushima Kouji , Sasayama Shigetake
Published: June 2012

Abstract
—Monocyte chemoattractant protein-1 (MCP-1)/monocyte chemotactic and activating factor (MCAF) has been suggested to promote atherogenesis. The effects of in vivo neutralization of MCP-1 in a rat model were examined in an effort to clarify the role of MCP-1 in the development of neointimal hyperplasia. Competitive polymerase chain reaction analysis revealed maximum MCP-1 mRNA expression at 4 hours after carotid arterial injury. Increased immunoreactivities of MCP-1 were also detected at 2 and 8 hours after injury. Either anti–MCP-1 antibody or nonimmunized goat IgG (10 mg/kg) was then administered every 12 hours to rats that had undergone carotid arterial injury. Treatment with 3 consecutive doses of anti–MCP-1 antibody within 24 hours (experiment 1) and every 12 hours for 5 days (experiment 2) significantly inhibited neointimal hyperplasia at day 14, resulting in a 27.8% reduction of the mean intima/media ratio (
P
<0.05) in experiment 1 and a 43.6% reduction (
P
<0.01) in experiment 2. This effect was still apparent at day 56 (55.6% inhibition;
P
<0.05). The number of vascular smooth muscle cells in the neointima at day 4 was significantly reduced by anti–MCP-1 treatment, demonstrating the important role of MCP-1 in early neointimal lesion formation. However, recombinant MCP-1 did not stimulate chemotaxis of vascular smooth muscle cells in an in vitro migration assay. These results suggest that MCP-1 promotes neointimal hyperplasia in early neointimal lesion formation and that neutralization of MCP-1 before, and immediately after, arterial injury may be effective in preventing restenosis after angioplasty. Further studies are needed to clarify the mechanism underlying the promotion of neointimal hyperplasia by MCP-1.
Exposure of Human Vascular Endothelial Cells to Sustained Hydrostatic Pressure Stimulates Proliferation
Authors: Schwartz Eric A. , Bizios Rena , Medow Marvin S. , Gerritsen Mary E.
Published: June 2012

Abstract
—The present study investigated the effects of sustained hydrostatic pressure (SHP; up to 4 cm H
2
O) on human umbilical vein endothelial cell (HUVEC) proliferation, focal adhesion plaque (FAP) organization, and integrin expression. Exposure of HUVECs to SHP stimulated cell proliferation and a selective increase in the expression of integrin subunit α
V
. The increase in α
V
was observed as early as 4 hours after exposure to pressure and preceded detectable increases in the bromodeoxyuridine labeling index. Laser confocal microscopy studies demonstrated colocalization of the α
V
integrin to FAPs. The individual FAPs in pressure-treated cells demonstrated a reduced area and increased aspect ratio and were localized to both peripheral and more central regions of the cells, in contrast to the predilection for the cell periphery in cells maintained under control pressure conditions. The pressure-induced changes in α
V
distribution had functional consequences on the cells: adhesivity of the cells to vitronectin was increased, and α
V
antagonists blocked the pressure-induced proliferative response. Thus, the present study suggests a role for α
V
integrins in the mechanotransduction of pressure by endothelial cells.
Vascular Remodeling in Response to Altered Blood Flow Is Mediated by Fibroblast Growth Factor-2
Authors: Bryant Shane R. , Bjercke Robert J. , Erichsen David A. , Rege Ajay , Lindner Volkhard
Published: June 2012

Abstract
—Vascular structures adapt to changes in blood flow by adjusting their diameter accordingly. The factors mediating this process are only beginning to be identified. We have recently established a mouse model of arterial remodeling in which flow in the common carotid artery is interrupted by ligation of the vessel near the carotid bifurcation, resulting in a dramatic reduction in vessel diameter as a consequence of inward remodeling and intimal lesion formation. In the present study, we used this model to determine the role of fibroblast growth factor-2 (FGF-2) in the remodeling response by maintaining neutralizing serum levels of a mouse monoclonal antibody against FGF-2 for 4 weeks. Morphometric analysis revealed that intimal lesion formation was not affected by the antibody. However, lumen narrowing was significantly inhibited, resulting in a greater than 3-fold increase in lumen area in anti–FGF-2–treated animals compared with controls. Treatment with anti–FGF-2 antibody significantly inhibited the reduction in vessel diameter (inward remodeling) and shortening of the internal elastic lamina in the ligated vessel. In addition, anti–FGF-2 treatment also caused outward remodeling of the contralateral carotid artery. These findings identify FGF-2 as an important factor in vascular remodeling, and its effects are likely to be mediated by increasing vascular tone. The results are consistent with the recent observation of reduced vascular tone in the FGF-2–deficient mouse.
Blocking Very Late Antigen-4 Integrin Decreases Leukocyte Entry and Fatty Streak Formation in Mice Fed an Atherogenic Diet
Authors: Shih Peggy T. , Brennan Marie-Luise , Vora Devendra K. , Territo Mary C. , Strahl Dana , Elices Mariano J. , Lusis Aldons J. , Berliner Judith A.
Published: June 2012

Abstract
—Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because α4 null mice are not viable, a peptidomimetic was used to block VLA-4–mediated leukocyte binding. The ability of a synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to block the recruitment of leukocytes and the accumulation of lipid in the aortic sinus of either wild-type mice (strain C57BL/6J) or mice with a low-density lipoprotein null mutation (LDLR−/−) maintained on an atherogenic diet was assessed. The active (Ac) CS-1 peptide or scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice using a mini osmotic pump. Mice were exposed to the peptide for 24 to 36 hours before the onset of the atherogenic diet. In C57BL/6J mice, leukocyte entry into the aortic sinus, as assessed by en face preparations, was inhibited by the active peptide (Ac=28±4, Sc=54±6 monocytes/valve;
P
=0.004). Additionally, frozen sections stained with Oil Red O were analyzed to assess lipid accumulation in the aortic sinus. C57BL/6J mice that received the (Ac) compound demonstrated significantly reduced lesion areas as compared with mice that received the (Sc) peptide (Ac=4887±4438 μm
2
, Sc=15 009 ±5619 μm
2
;
P
<0.0001). In a separate study, LDLR−/− mice were implanted with pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR−/− mice fed a chow diet displayed small lesions at 14 weeks, the effects of the peptide seen in these animals represented a change in early lipid accumulation rather than initiation. By using whole-mount preparations, the (Ac) but not the (Sc) peptide significantly reduced the area of lipid accumulation in the aortic sinus, resulting in an approximate 66% decrease. Plasma analysis from all studies revealed concentrations of peptide to be present at levels previously determined by in vitro analysis to block adhesion. (Ac) CS-1 peptide, which blocks VLA-4 on the leukocyte surface, is effective in reducing leukocyte recruitment and lipid accumulation in the aortic sinus. The present study provides in vivo evidence that the VLA-4 integrin plays an important role in the initiation of the atherosclerotic lesion and lipid accumulation, and it suggests a potential therapeutic strategy for this disease.
Cellular Mechanisms of Altered Contractility in the Hypertrophied Heart
Authors: Shorofsky Stephen R. , Aggarwal Rajesh , Corretti Mary , Baffa Jeanne M. , Strum Judy M. , Al-Seikhan Badr A. , Kobayashi Yvonne M. , Jones Larry R. , Wier W. Gil , Balke C. William
Published: June 2012

Abstract
—To investigate the cellular mechanisms for altered Ca
2+
homeostasis and contractility in cardiac hypertrophy, we measured whole-cell L-type Ca
2+
currents (
I
Ca,L
), whole-cell Ca
2+
transients ([Ca
2+
]
i
), and Ca
2+
sparks in ventricular cells from 6-month-old spontaneously hypertensive rats (SHRs) and from age- and sex-matched Wistar-Kyoto and Sprague-Dawley control rats. By echocardiography, SHR hearts had cardiac hypertrophy and enhanced contractility (increased fractional shortening) and no signs of heart failure. SHR cells had a voltage-dependent increase in peak [Ca
2+
]
i
amplitude (at 0 mV, 1330±62 nmol/L [SHRs] versus 836±48 nmol/L [controls],
P
<0.05) that was not associated with changes in
I
Ca,L
density or kinetics, resting [Ca
2+
]
i
, or Ca
2+
content of the sarcoplasmic reticulum (SR). SHR cells had increased time of relaxation. Ca
2+
sparks from SHR cells had larger average amplitudes (173±192 nmol/L [SHRs] versus 109±64 nmol/L [control];
P
<0.05), which was due to redistribution of Ca
2+
sparks to a larger amplitude population. This change in Ca
2+
spark amplitude distribution was not associated with any change in the density of ryanodine receptors, calsequestrin, junctin, triadin 1, Ca
2+
-ATPase, or phospholamban. Therefore, SHRs with cardiac hypertrophy have increased contractility, [Ca
2+
]
i
amplitude, time to relaxation, and average Ca
2+
spark amplitude (“big sparks”). Importantly, big sparks occurred without alteration in the trigger for SR Ca
2+
release (
I
Ca,L
), SR Ca
2+
content, or the expression of several SR Ca
2+
-cycling proteins. Thus, cardiac hypertrophy in SHRs is linked with an alteration in the coupling of Ca
2+
entry through L-type Ca
2+
channels and the release of Ca
2+
from the SR, leading to big sparks and enhanced contractility. Alterations in the microdomain between L-type Ca
2+
channels and SR Ca
2+
release channels may underlie the changes in Ca
2+
homeostasis observed in cardiac hypertrophy. Modulation of SR Ca
2+
release may provide a new therapeutic strategy for cardiac hypertrophy and for its progression to heart failure and sudden death.
Altered Crossbridge Kinetics in the αMHC
403/+
Mouse Model of Familial Hypertrophic Cardiomyopathy
Authors: Blanchard Edward , Seidman Christine , Seidman J. G. , LeWinter Martin , Maughan David
Published: June 2012

Abstract
—A mutation in the cardiac β-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the α-myosin heavy chain (αMHC
403/+
). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the αMHC
403/+
hearts. In resting strips (pCa 8), dynamic stiffness was ≈40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was ≈3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing (
b
) and work-absorbing (
c
) steps of the crossbridge were less in αMHC
403/+
strips than in wild-type strips (
b
=11±1 versus 15±1 Hz;
c
= 58±3 versus 66±3 Hz; 27°C). At maximal calcium activation, strip oscillatory power was reduced (0.53±0.25 versus 1.03±0.18 mW/mm
3
; 27°C), which is partly attributable to the reduced frequency
b
, at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.
Expression and Distribution of the Type 1 and Type 3 Inositol 1,4,5-Trisphosphate Receptor in Developing Vascular Smooth Muscle
Authors: Tasker Paul N. , Michelangeli Francesco , Nixon Graeme F.
Published: June 2012

Abstract
—The recent discoveries of inositol 1,4,5-trisphosphate (IP
3
) receptor subtypes with different affinities for IP
3
and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP
3
receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP
3
receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP
3
receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP
3
receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP
3
receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP
3
receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP
3
receptor during development from the type 3 with low affinity for IP
3
to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle.
Tranilast Inhibits Vascular Smooth Muscle Cell Growth and Intimal Hyperplasia by Induction of p21
waf1/cip1/sdi1
and p53
Authors: Takahashi Akihiro , Taniguchi Takahiro , Ishikawa Yuichi , Yokoyama Mitsuhiro
Published: June 2012

Abstract
—Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC
50
value, evaluated as cell number, was 100 μmol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21
waf1/cip1/sdi1
(p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G
1
/S transition. Tranilast suppressed their activities by >80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (
P
<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.
Mechanisms of Altered Excitation-Contraction Coupling in Canine Tachycardia-Induced Heart Failure, II
Authors: Winslow Raimond L. , Rice Jeremy , Jafri Saleet , Marbán Eduardo , O’Rourke Brian
Published: June 2012

Abstract
—Ca
2+
transients measured in failing human ventricular myocytes exhibit reduced amplitude, slowed relaxation, and blunted frequency dependence. In the companion article (O’Rourke B, Kass DA, Tomaselli GF, Kääb S, Tunin R, Marbán E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart, I: experimental studies.
Circ Res
. 1999;84:562–570), O’Rourke et al show that Ca
2+
transients recorded in myocytes isolated from canine hearts subjected to the tachycardia pacing protocol exhibit similar responses. Analyses of protein levels in these failing hearts reveal that both SR Ca
2+
ATPase and phospholamban are decreased on average by 28% and that Na
+
/Ca
2+
exchanger (NCX) protein is increased on average by 104%. In this article, we present a model of the canine midmyocardial ventricular action potential and Ca
2+
transient. The model is used to estimate the degree of functional upregulation and downregulation of NCX and SR Ca
2+
ATPase in heart failure using data obtained from 2 different experimental protocols. Model estimates of average SR Ca
2+
ATPase functional downregulation obtained using these experimental protocols are 49% and 62%. Model estimates of average NCX functional upregulation range are 38% and 75%. Simulation of voltage-clamp Ca
2+
transients indicates that such changes are sufficient to account for the reduced amplitude, altered shape, and slowed relaxation of Ca
2+
transients in the failing canine heart. Model analyses also suggest that altered expression of Ca
2+
handling proteins plays a significant role in prolongation of action potential duration in failing canine myocytes.
Nuclear Factor-κB Regulates Induction of Apoptosis and Inhibitor of Apoptosis Protein-1 Expression in Vascular Smooth Muscle Cells
Authors: Erl Wolfgang , Hansson Göran K. , de Martin Rainer , Draude Georg , Weber Kim S. C. , Weber Christian
Published: June 2012

Abstract
—Apoptosis is important in normal development as well as in diseases such as atherosclerosis. However, the regulation of apoptosis is still not completely understood. We now show that the transcription factor nuclear factor-κB (NF-κB) controls the induction of apoptosis in human and rat vascular smooth muscle cells (SMCs). SMCs in high-density culture exhibited a high NF-κB activity and were insensitive to induction of apoptosis. Inhibition of NF-κB by adenovirus-mediated overexpression of its inhibitor IκBα caused a marked increase in cell death at low but not high cell density. Elevating endogenous IκBα levels by inhibiting its degradation with proteasomal inhibitors resulted in induction of apoptosis in low-density SMCs, as detected by increased binding of annexin V, reduced mitochondrial membrane potential, and increased hypodiploid DNA. In high-density cultures, protection against apoptosis was associated with the expression of inhibitor of apoptosis protein-1 (IAP-1). Transfer of IκBα reduced human IAP-1 mRNA levels, which suggested that IAP-1 is transcriptionally regulated by NF-κB. This was confirmed through identification of a motif with NF-κB–like binding activity in the human IAP-1 promoter region. Moreover, antisense inhibition of IAP-1 sensitized high-density SMCs to the induction of cell death. Together, our data imply that SMCs at high density are protected by an antiapoptotic mechanism that involves increased expression of NF-κB and IAP-1. Interference with pathways that control the susceptibility to programmed cell death may be helpful in the treatment of diseases where dysregulation of apoptosis is involved, eg, atherosclerosis and restenosis.
Angiotensin II Induces Interleukin-6 Transcription in Vascular Smooth Muscle Cells Through Pleiotropic Activation of Nuclear Factor-κB Transcription Factors
Authors: Han Youqi , Runge Marschall S. , Brasier Allan R.
Published: June 2012

Abstract
—Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar
1
Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar
1
Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-κB (NF-κB) motif. Sar
1
Ang II induced cytoplasmic-to-nuclear translocation of the NF-κB subunits Rel A and NF-κB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-κB translocation was dependent on weak simultaneous proteolysis of the IκBα and β inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-κB1 form. Pretreatment with a potent inhibitor of IκBα proteolysis, TPCK, completely blocked Sar
1
Ang IIAng II-induced NF-κB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-κB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-κB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs.
Calmodulin Kinase Inhibition Prevents Development of the Arrhythmogenic Transient Inward Current
Authors: Wu Yuejin , Roden Dan M. , Anderson Mark E.
Published: June 2012

Abstract
—Although it is widely accepted that afterdepolarizations initiate arrhythmias when action potentials are prolonged, the underlying mechanisms are unclear. In this study, we tested the hypothesis that action potential prolongation would raise intracellular calcium and thereby activate the arrhythmogenic transient inward current (
I
ti
). Furthermore, given that
I
ti
can be activated by sarcoplasmic reticulum Ca
2+
release, we tested the hypothesis that inhibition of calmodulin (CaM) kinase would prevent
I
ti
. Isolated rabbit ventricular myocytes were studied with whole-cell–mode voltage clamp. Stimulation with a prolonged action potential clamp, under near-physiological conditions, increased [Ca
2+
]
i
.
I
ti
was reproducibly induced in 60 of 60 cells, but
I
ti
was not seen with the use of a shorter action potential waveform (n=12).
I
ti
was associated with a secondary elevation in [Ca
2+
]
i
. When [Ca
2+
]
i
buffering was enhanced by dialysis with BAPTA (20 mmol/L, n=9), no
I
ti
was present. The Na
+
/Ca
2+
exchanger was likely responsible for
I
ti
, because
I
ti
was inhibited by the Na
+
/Ca
2+
exchanger inhibitory peptide XIP (10 μmol/L, n=6), but not by an inactive scrambled peptide (10 μmol/L, n=5) or by the Cl
–
current antagonist niflumic acid (10 to 40 μmol/L, n=9). Activator Ca
2+
from the sarcoplasmic reticulum was essential for development of
I
ti
, because it was prevented by pretreatment with ryanodine (10 μmol/L, n=6) or thapsigargin (1 μmol/L, n=6). Two different CaM kinase inhibitory peptides (n=16) and a CaM inhibitory peptide (n=4) completely suppressed
I
ti
. These results are consistent with the hypothesis that CaM kinase plays a role in arrhythmias related to increased [Ca
2+
]
i
.