bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II Type 2 Receptor Is Upregulated in Human Heart With Interstitial Fibrosis, and Cardiac Fibroblasts Are the Major Cell Type for Its Expression
Authors: Tsutsumi Yoshiaki , Matsubara Hiroaki , Ohkubo Naohiko , Mori Yasukiyo , Nozawa Yoshihisa , Murasawa Satoshi , Kijima Kazuhisa , Maruyama Katsuya , Masaki Hiroya , Moriguchi Yasutaka , Shibasaki Yasunobu , Kamihata Hiroshi , Inada Mitsuo , Iwasaka Toshiji
Published: June 2012

Abstract
—The expression pattern of angiotensin (Ang) II type 2 receptor (AT
2
-R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT
1
-R and AT
2
-R was 59% and 41%, respectively. The expression of AT
2
-R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT
1
-R protein and mRNA levels in AMI hearts showed 1.5- and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT
1
-R expression was significantly downregulated. AT
1
-R–mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT
2
-R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT
1
-R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT
2
-R antagonist caused an additional significant increase in Ang II–induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT
2
-R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT
2
-R present in the fibroblasts exerts an inhibitory effect on Ang II–induced mitogen signals, and (3) AT
1
-R in atrial and LV tissues was downregulated during chronic heart failure, and AT
1
-R–mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT
2
-R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT
1
-R and AT
2
-R are differentially regulated in failing human hearts.
The Brain Renin-Angiotensin System Contributes to the Hypertension in Mice Containing Both the Human Renin and Human Angiotensinogen Transgenes
Authors: Davisson Robin L. , Yang Gongyu , Beltz Terry G. , Cassell Martin D. , Johnson Alan Kim , Sigmund Curt D.
Published: June 2012

Abstract
—We have previously shown that mice transgenic for both the human renin and human angiotensinogen genes (RA+) exhibit appropriate tissue- and cell-specific expression of both transgenes, have 4-fold higher plasma angiotensin II (AII) levels, and are chronically hypertensive. However, the relative contribution of circulating and tissue-derived AII in causing hypertension in these animals is not known. We hypothesized that the brain renin-angiotensin system contributes to the elevated blood pressure in this model. To address this hypothesis, mean arterial pressure (MAP) and heart rate were measured in conscious, unrestrained mice after they were instrumented with intracerebroventricular cannulae and carotid arterial and jugular vein catheters. Intracerebroventricular administration of the selective AII type 1 (AT-1) receptor antagonist losartan (10 μg, 1 μL) caused a significantly greater peak fall in MAP in RA+ mice than in nontransgenic RA− controls (−29±4 versus −4±2 mm Hg,
P
<0.01). To explore the mechanism of a central renin-angiotensin system–dependent hypertension in RA+ mice, we determined the relative depressor responses to intravenous administration of the ganglionic blocking agent hexamethonium (5 mg/kg) or an arginine vasopressin (AVP) V
1
receptor antagonist (AVPX, 10 μg/kg). Hexamethonium caused equal lowering of MAP in RA+ mice and controls (−46±3 versus −52±3,
P
>0.05), whereas AVPX caused a significantly greater fall in MAP in RA+ compared with RA− mice (−24±2 versus −6±1,
P
<0.01). Consistent with this was the observation that circulating AVP was 3-fold higher in RA+ mice than in control mice. These results suggest that increased activation of central AT-1 receptors, perhaps those located at sites involved in AVP release from the posterior pituitary gland, plays a role in the hypertension in RA+ mice. Furthermore, our finding that both human transgenes are expressed in brain regions of RA+ mice known to be involved in cardiovascular regulation raises the possibility that augmented local production of AII and increased activation of AT-1 receptors at these sites is involved.
Nitric Oxide-20–Hydroxyeicosatetraenoic Acid Interaction in the Regulation of K
+
Channel Activity and Vascular Tone in Renal Arterioles
Authors: Sun Cheng-Wen , Alonso-Galicia Magdalena , Taheri M. Reza , Falck John R. , Harder David R. , Roman Richard J.
Published: June 2012

Abstract
—The present study examined whether inhibition of P4504A enzyme activity and the formation of 20-HETE contributes to the activation of K
+
channels and vasodilator effects of nitric oxide (NO) in renal arterioles. Addition of an NO donor to the P4504A2 enzyme that produces 20-HETE increased visible light absorbance at 440 nm indicating that NO binds to heme in this enzyme. NO donors also dose-dependently inhibited the formation of 20-HETE in microsomes prepared from renal arterioles. In patch-clamp experiments, NO donors increased the open-state probability of a voltage-sensitive, large-conductance (195±9 pS) K
+
channel recorded with cell-attached patches on renal arteriolar smooth muscle cells. Blockade of guanylyl cyclase with [1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one] (ODQ, 10 μmol/L), or cGMP-dependent kinase with 8
R
,9
S
,11
S
-(−)-9-methoxycarbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1
H
,8
H
,11
H
-2,7
b
,11
a
-trizadibenzo-(
a
,
g
)-cy-cloocta-(
c
,
d
,
e
)-trinden-1-one (KT-5823) (1 μmol/L) did not alter the effects of NO on this channel. In contrast, inhibition of the formation of 20-HETE with 17-octadecynoic acid (1 μmol/L) activated this channel and masked the response to NO. Preventing the NO-induced reduction in intracellular 20-HETE levels also blocked the effects of NO on this channel. Sodium nitroprusside (SNP) increased the diameter of renal interlobular arteries preconstricted with phenylephrine to 80±4% of control. Blockade of guanylyl cyclase with ODQ (10 μmol/L) attenuated the response to SNP by 26±2%; however, fixing 20-HETE levels at 100 nmol/L reduced the response by 67±8%. Blockade of both pathways eliminated the response to SNP. These results indicate that inhibition of the formation of 20-HETE contributes to the activation of K
+
channels and the vasodilator effects of NO in the renal microcirculation.
Roles of Ca
2+
and Crossbridge Kinetics in Determining the Maximum Rates of Ca
2+
Activation and Relaxation in Rat and Guinea Pig Skinned Trabeculae
Authors: Palmer Sue , Kentish Jonathan C.
Published: June 2012

Abstract
—We examined the influences of Ca
2+
and crossbridge kinetics on the maximum rate of force development during Ca
2+
activation of cardiac myofibrils and on the maximum rate of relaxation. Flash photolysis of diazo-2 or nitrophenyl-EGTA was used to produce a sudden decrease or increase, respectively, in [Ca
2+
] within Triton-skinned trabeculae from rat and guinea pig hearts (22°C). Trabeculae from both species had similar Ca
2+
sensitivities, suggesting that the rate of dissociation of Ca
2+
from troponin C (
k
off
) is similar in the 2 species. However, the rate of relaxation after diazo-2 photolysis was 5 times faster in the rat (16.1±0.9 s
−1
, mean±SEM, n=11) than in the guinea pig (2.99±0.26 s
−1
, n=7). This indicates that the maximum relaxation rate is limited by crossbridge kinetics rather than by
k
off
. The maximum rates of rapid activation by Ca
2+
after nitrophenyl-EGTA photolysis (
k
act
) and of force redevelopment after forcible crossbridge dissociation (
k
tr
) were similar and were ≈5-fold faster in rat (
k
act
=14.4±0.9 s
−1
,
k
tr
=13.0±0.6 s
−1
) than in guinea pig (
k
act
=2.57±0.14 s
−1
,
k
tr
=2.69±0.30 s
−1
) trabeculae. This too may be mainly due to species differences in crossbridge kinetics. Both
k
act
and
k
tr
increased as [Ca
2+
] increased. This Ca
2+
dependence of the rates of force development is consistent with current models for the Ca
2+
activation of the crossbridge cycle, but these models do not explain the similarity in the maximal rates of activation and relaxation within a given species.
Expression of Tissue Inhibitor of Metalloproteinases-3 in Human Atheroma and Regulation in Lesion-Associated Cells
Authors: Fabunmi Rosalind P. , Sukhova Galina K. , Sugiyama Seigo , Libby Peter
Published: June 2012

Abstract
—Atherosclerotic plaque stability depends on the structural integrity of its extracellular matrix skeleton. The balance between degradation by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may regulate plaque stability. Although MMP expression in atheroma is well documented, localization and control of expression of TIMPs in these lesions is incomplete. Extracts of atheroma (n=14) had 5-fold higher levels of TIMP-3 than nonatherosclerotic tissue (n=10). Plaques (n=24) contained abundant TIMP-1, -2, and -3 in macrophages in plaque shoulders, intimal-medial borders, and areas overlying the lipid core, as well as in medial smooth muscle cells, albeit in lesser amounts. These observations suggested that macrophages, a cell type not heretofore known to express TIMP-3, did so in atheroma in vivo. Further studies in vitro established the human macrophage as a novel source of TIMP-3 mRNA and protein. Human smooth muscle cells constitutively expressed TIMP-1, -2 and -3 proteins; platelet-derived growth factor and transforming growth factor-β augmented levels of TIMP-1 and TIMP-3 but not TIMP-2. These findings suggest that regulated expression of TIMP-3, in addition to the presence of TIMP-1 and TIMP-2, counteracts MMP activity in atheroma and hence influences plaque stability.
Importance of E-Selectin for Firm Leukocyte Adhesion In Vivo
Authors: Ley Klaus , Allietta Margaretta , Bullard Daniel C. , Morgan Sharon
Published: June 2012

Abstract
—Leukocyte adhesion under flow is preferentially mediated by the selectins. In this study we used intravital microscopy to investigate whether E-selectin may promote firm leukocyte adhesion in vivo. E-Selectin is expressed by endothelial cells activated with tumor necrosis factor-α (TNF-α) and causes slow leukocyte rolling. Microinjection of formyl-methionyl-leucyl-phenylalanine (fMLP) or macrophage inflammatory protein-2 (MIP-2) next to a venule of the TNF-α–treated mouse cremaster muscle significantly increased the number of adherent leukocytes. In gene-targeted mice homozygous for a null mutation in the E-selectin gene or in wild-type mice treated with an E-selectin monoclonal antibody (mAb), this response was significantly attenuated (by >80%). No such defect was seen in intercellular adhesion molecule-1 (ICAM-1)–deficient mice. E-Selectin–null mice showed more rapid leukocyte rolling than wild-type or ICAM-1–deficient mice, resulting in significantly shortened leukocyte transit times through venules. Topical application of fMLP onto the whole cremaster muscle generated the same number of adherent leukocytes in wild-type and E-selectin–deficient mice. We conclude that slow leukocyte rolling through E-selectin results in long transit times, which are essential for efficient leukocyte adhesion in response to a local chemotactic stimulus.
Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal
Authors: Kockx Mark M. , De Meyer Guido R. Y. , Buyssens Norbert , Knaapen Michiel W. M. , Bult Hidde , Herman Arnold G.
Published: June 2012

Abstract
—Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8±0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.
Microtubule Involvement in Translational Regulation of Fibronectin Expression by Light Chain 3 of Microtubule-Associated Protein 1 in Vascular Smooth Muscle Cells
Authors: Zhou Bin , Rabinovitch Marlene
Published: June 2012

Abstract
—Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3′ untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.
Lysophosphatidylcholine Enhances Cytokine-Induced Interferon Gamma Expression in Human T Lymphocytes
Authors: Nishi Eiichiro , Kume Noriaki , Ueno Yasushi , Ochi Hiroshi , Moriwaki Hideaki , Kita Toru
Published: June 2012

Abstract
—Accumulation of substantial numbers of activated T lymphocytes, as well as monocyte/macrophages, in focal areas of arterial intima appears to be a hallmark of atherogenesis. Our previous report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate the expression of heparin-binding epidermal growth factor–like growth factor and the interleukin (IL)-2 receptor in cultured human peripheral T lymphocytes. In this study, we show that lyso-PC can also enhance interferon gamma (IFN-γ) secretion and gene expression in human T lymphocytes. Lyso-PC–induced upregulation of IFN-γ depended on the presence of IL-2, IL-12, or phytohemagglutinin in culture media and was similarly observed in both CD4
+
and CD8
+
subsets. Actinomycin D chase by Northern blotting showed that lyso-PC significantly prolonged IFN-γ mRNA half-lives in human T cells. Transient transfection of IFN-γ promoter-reporter gene construct in the human T-cell line Jurkat cells demonstrated that lyso-PC stimulated the transcription of IFN-γ promoter-driven luciferase gene. Analyses of serial deletion mutations of IFN-γ promoter revealed that the lyso-PC–responsive element is located between base pairs −102 and −78 of the transcription initiation site of the IFN-γ gene. Enhanced expression of IFN-γ in T lymphocytes by lyso-PC may play a crucial role in atherogenesis.
Connexin43 Is Highly Localized to Sites of Disturbed Flow in Rat Aortic Endothelium but Connexin37 and Connexin40 Are More Uniformly Distributed
Authors: Gabriels Joseph E. , Paul David L.
Published: June 2012

Abstract
—Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.
Inhibition of Cardiac Delayed Rectifier K
+
Current by Overexpression of the Long-QT Syndrome HERG G628S Mutation in Transgenic Mice
Authors: Babij Philip , Askew G. Roger , Nieuwenhuijsen Bart , Su Chien-Min , Bridal Terry R. , Jow Brian , Argentieri Thomas M. , Kulik John , DeGennaro Louis J. , Spinelli Walter , Colatsky Thomas J.
Published: June 2012

Abstract
—Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031–sensitive K
+
current resembling the rapidly activating cardiac delayed rectifier K
+
current (
I
kr
) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26°C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37°C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of
I
Kr
in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous
I
Kr
in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.
3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Increase Fibrinolytic Activity in Rat Aortic Endothelial Cells
Authors: Essig Marie , Nguyen Geneviève , Prié Dominique , Escoubet Brigitte , Sraer Jean-Daniel , Friedlander Gérard
Published: June 2012

Abstract
—3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HRIs) have been recently shown to prevent atherosclerosis progression. Clinical benefit results from combined actions on various components of the atherosclerotic lesion. This study was designed to identify the effects of HRI on one of these components, the endothelial fibrinolytic system. Aortas isolated from rats treated for 2 days with lovastatin (4 mg/kg body wt per day) showed a 3-fold increase in tissue plasminogen activator (tPA) activity. In a rat aortic endothelial cell line (SVARECs) and in human nontransformed endothelial cells (HUVECs), HRI induced an increase in tPA activity and antigen in a time- and concentration-dependent manner. In SVARECs, the maximal response was observed when cells were incubated for 48 hours with 50 μmol/L HRI. An increase of tPA mRNA was also in evidence. In contrast, HRI inhibited plasminogen activator inhibitor-1 activity and mRNA. The effects of HRI were reversed by mevalonate and geranylgeranyl pyrophosphate, but not by LDL cholesterol and farnesyl pyrophosphate, and were not induced by α-hydroxyfarnesyl phosphonic acid, an inhibitor of protein farnesyl transferase. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and plasminogen activator inhibitor-1 activity and blocked its reversal by geranylgeranyl pyrophosphate. The effect of HRI was associated with a disruption of cellular actin filaments without modification of microtubules. A disrupter of actin filaments, cytochalasin D, induced the same effect as lovastatin on tPA, whereas a disrupter of microtubules, nocodazole, did not. In conclusion, HRI can modify the fibrinolytic potential of endothelial cells, likely via inhibition of geranylgeranylated Rho protein and disruption of the actin filaments. The resulting increase of fibrinolytic activity of endothelial cells may contribute to the beneficial effects of HRI in the progression of atherosclerosis.
Retinal Arterial Tone Is Controlled by a Retinal-Derived Relaxing Factor
Authors: Delaey Christophe , Van de Voorde Johan
Published: June 2012

Abstract
—The present study provides evidence that retinal tissue may profoundly influence the retinal arterial smooth muscle cell tone by releasing an unknown retinal relaxing factor. Isolated bovine retinal arteries with and without adhering retinal tissue were mounted in a wire myograph for isometric tension recordings. The maximal contraction induced by prostaglandin F
2α
was 0.95±0.7 mN (n=6) in the presence and 5.15±0.76 mN (n=6) in the absence of adhering retinal tissue. The contractions induced by U-46619, serotonin, and endothelin-1 were similarly blocked in the presence of retinal tissue. The K
+
120 mmol/L-induced contraction was not significantly affected (2.8±0.7 mN, n=6, in the presence and 3.6±0.7 mN, n=6, in the absence of retinal tissue). Placing a piece of bovine retinal tissue in the proximity of a contracted (ie, with prostaglandin F
2α
) retinal artery induced a complete relaxation of the retinal vessel, suggesting the involvement of a diffusible chemical vasorelaxant. Also porcine, canine, and ovine retinal tissue completely relaxed the contracted (with prostaglandin F
2α
) bovine retinal artery. Other smooth muscle preparations, including rat mesenteric and renal arteries and rat main bronchi, also relaxed with the application of a piece of bovine retinal tissue. Incubation of bovine retinas in a Krebs-Ringer bicarbonate solution yielded a solution that relaxed isolated precontracted bovine retinal arteries, confirming the involvement of a diffusible chemical messenger. Hexane extraction, heating the solution to 70°C, or treatment with trypsin did not alter the relaxing properties of the incubation solution. The characteristics of the retinal relaxing factor do not correspond with those of nitric oxide, prostanoids, adenosine, acetylcholine, or any other of the known vasoactive neurotransmitters released from the retina. Our results suggest that retinal arterial tone is controlled by a diffusible, hydrophilic, and heat-stable relaxing factor that does not correspond with a known vasoactive molecule formed within the retina.
A Major Role for Prostacyclin in Nitric Oxide–Induced Ocular Vasorelaxation in the Piglet
Authors: Hardy Pierre , Abran Daniel , Hou Xin , Lahaie Isabelle , Peri Krishna G. , Asselin Pierre , Varma Daya R. , Chemtob Sylvain
Published: June 2012

Abstract
—We studied the mechanisms of retinal and choroidal vasorelaxation elicited by nitric oxide (NO) using piglet eyes. The NO donors sodium nitroprusside (SNP) and diethylamine-NONOate caused comparable concentration–dependent relaxation that was partially (≈40%) attenuated by the guanylate cyclase inhibitors methylene blue and LY83583 and reduced to a lesser extent (≈25%) by the inhibitor of cGMP–dependent kinase, KT 5823. In contrast, NO-induced dilatation (by NO donors and endogenous NO after stimulation with bradykinin) was substantially (≈70%) diminished by the K
Ca
channel blockers tetraethylammonium (TEA), charybdotoxin, and iberiotoxin; by the cyclooxygenase inhibitors indomethacin and ibuprofen; by the prostaglandin I (PGI
2
) synthase inhibitor
trans
-2-phenyl cyclopropylamine (TPC); and by the removal of endothelium; whereas relaxation of endothelium-denuded vasculature to SNP was unaltered by indomethacin, TPC, and charybdotoxin but was nearly nullified by methylene blue and the K
v
channel blocker 4-aminopyridine. NO donors significantly increased PGI
2
synthesis and the putative PGI
2
receptor–coupled second messenger cAMP, from ocular vasculature (retinal microvessels and choroidal perfusate), and this increase in PGI
2
formation was markedly reduced by TPC, tetraethylammonium, charybdotoxin, and/or the removal of endothelium, but it was only slightly reduced by methylene blue and LY83583. Also, SNP and K
Ca
channel openers NS1619 and NS004 caused an increase in PGI
2
synthesis in cultured endothelial cells, which was virtually abolished by K
Ca
blockers. Finally, vasorelaxation to a cGMP analogue, 8-bromo cGMP, and protein kinase G stimulant β-phenyl-1,
N
2
-etheno-8-bromoguanosine 3′:5′-cyclic monophosphate was mostly K
v
dependent and, in contrast to NO, largely unrelated to PGI
2
formation. In conclusion, data indicate that NO-induced ocular vasorelaxation is partly mediated by cGMP through its action on smooth muscle, and more importantly, by stimulating PGI
2
formation of endothelial origin via a mechanism mostly independent of guanylate cyclase, which involves the opening of a K
Ca
channel.
Infection of Human Fetal Cardiac Myocytes by a Human Immunodeficiency Virus-1–Derived Vector
Authors: Rebolledo Michael A. , Krogstad Paul , Chen Fuhua , Shannon Kevin M. , Klitzner Thomas S.
Published: June 2012

Abstract
—Cardiomyopathy associated with HIV-1 infection is a well-recognized complication. However, it is unknown whether direct cardiomyocyte infection is involved in the pathogenesis of the cardiomyopathy. An HIV-1–based lentiviral vector and wild-type HIV-1 were used to infect human fetal cardiac myocytes in a primary culture. Quantitative polymerase chain reaction, viral p24 antigen determination, and immunofluorescence were used to detect the synthesis of HIV-1 DNA and proteins after the infection. High-efficiency infection occurred using the HIV-1–based lentiviral vector, although no infection occurred with the wild-type HIV-1 strain. Dual-labeling immunofluorescence for HIV-1 proteins and myosin confirmed that cardiomyocytes were infected. This in vitro analysis suggests that direct myocyte infection with wild-type HIV-1 may not be involved in the pathogenesis of HIV-1 cardiomyopathy. However, HIV-1–based vectors may prove useful for ex vivo cardiovascular gene therapy.
Characterization of a Slowly Inactivating Outward Current in Adult Mouse Ventricular Myocytes
Authors: Zhou Jun , Jeron Andreas , London Barry , Han Xingqiang , Koren Gideon
Published: June 2012

Abstract
—We recently have reported that suppression of the slowly inactivating component of the outward current, I
slow
, in ventricular myocytes of transgenic mice (long QT mice) overexpressing the N-terminal fragment and S1 segment of Kv1.1 resulted in a significant prolongation of action potential duration and the QT interval. Here we describe the detailed biophysical properties and physiological role of I
slow
by applying the whole-cell patch-clamp technique at both room temperature and 37°C. This current activates rapidly with time constants ranging from 3.8±0.8 ms at –20 mV to 2.1±0.5 ms at 50 mV at room temperature. The half-activation voltage and slope factor are –12.5±2.6 mV and 7.7±1.0 mV, respectively. The inactivation of this current is slow compared with the fast inactivating component I
to
, with time constants of ≈100 ms at 37°C. The steady-state inactivation of I
slow
is not temperature-dependent, with half-inactivation voltages and slope factors of –35.1±1.3 and –5.4±0.4 mV at 37°C, and –37.6±1.8 and –5.8±0.6 mV at room temperature. Double exponentials were required to describe the time-dependent recovery of I
slow
from steady-state inactivation, with time constants of 233±34 and 3730±702 ms at 37°C, and 830±240 and 8680±2410 ms at room temperature. I
slow
is highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, α-dendrotoxin, and E-4031. Stimulation with action-potential waveforms under voltage-clamp mode revealed that this current plays an important role in the early and middle phases of repolarization of the cardiac action potential. We conclude that the biophysical properties and pharmacological profiles of I
slow
are similar to those of Kv1.5-encoded currents.
Human Heart Generates Complement Proteins That Are Upregulated and Activated After Myocardial Infarction
Authors: Yasojima Koji , Schwab Claudia , McGeer Edith G. , McGeer Patrick L.
Published: June 2012

Abstract
—In human heart, we detected mRNAs and proteins for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 with the use of reverse transcriptase–polymerase chain reaction, Western blotting, and immunohistochemical techniques. We found an upregulation of both mRNAs and proteins in areas of recent and old myocardial infarctions. In both situations, the classical complement pathway was activated, with C4d, C3d, and the membrane attack complex (C5b-9) being deposited on damaged cardiac myocytes. These activated complement components were also identified on Western blots of infarcted tissue. Complement mRNAs in infarcted heart tissue were higher than those in liver, and liver complement mRNAs were not upregulated in cases with infarcted hearts. Our results establish that (1) complement proteins are endogenously produced by human heart; (2) the classical complement pathway is fully activated after myocardial infarction; (3) complement activation is directly involved in myocardial damage after ischemic insults; and (4) damage from complement activation may be chronically sustained. These data suggest that inhibition of the complement system should be effective in treating myocardial infarction.
Modulation of Arachidonic Acid Release and Membrane Fluidity by Albumin in Vascular Smooth Muscle and Endothelial Cells
Authors: Beck Richard , Bertolino Sophie , Abbot Stewart E. , Aaronson Philip I. , Smirnov Sergey V.
Published: June 2012

Abstract
—Albumin is the major plasma protein circulating in blood. Albumin potently decreases capillary permeability, although the mechanisms are not understood completely. Albumin also effectively binds arachidonic acid (AA), which increases capillary permeability. To investigate the interactions of BSA and AA with the cell membrane, the effect of these substances on [
3
H]AA release and membrane fluidity was studied in vascular myocytes and endothelial cells. BSA (0.2 and 1 mg · mL
−1
) stimulated a significant release of [
3
H]AA from both intact rat aorta and cultured smooth muscle cells. This effect was not mimicked by γ-globulin or myoglobin (both 1 mg · mL
−1
) in intact tissue. BSA, but not γ-globulin and myoglobin, decreased the membrane fluidity (assessed as changes in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene) in a concentration-dependent manner with a half-maximum concentration between 0.007 and 0.4 mg · mL
−1
in both freshly isolated and cultured rat aortic myocytes and human umbilical vein endothelial cells. AA (1 to 200 μmol/L) caused the opposite effect, increasing membrane fluidity and antagonizing the effect of BSA. BSA modified at its arginine residues, which are thought to be important in AA binding, did not stimulate [
3
H]AA release and was significantly less potent than native BSA in altering the membrane fluidity. The effect of BSA can be explained by a high-affinity binding of AA to the protein and extraction of AA from the cell membrane. The interaction between BSA and AA could play a role in the regulation of vascular permeability.
Angiotensin II Induces Monocyte Chemoattractant Protein-1 Gene Expression in Rat Vascular Smooth Muscle Cells
Authors: Chen Xi-Lin , Tummala Pradyumna E. , Olbrych Matthew T. , Alexander R. Wayne , Medford Russell M.
Published: June 2012

Abstract
—Monocyte infiltration into the vessel wall, a key initial step in the process of atherosclerosis, is mediated in part by monocyte chemoattractant protein-1 (MCP-1). Hypertension, particularly in the presence of an activated renin-angiotensin system, is a major risk factor for the development of atherosclerosis. To investigate a potential molecular basis for a link between hypertension and atherosclerosis, we studied the effects of angiotensin II (Ang II) on MCP-1 gene expression in rat aortic smooth muscle cells. Rat smooth muscle cells treated with Ang II exhibited a dose-dependent increase in MCP-1 mRNA accumulation that was prevented by the AT
1
receptor antagonist losartan. Ang II also activated MCP-1 gene transcription. Inhibition of NADH/NADPH oxidase, which generates superoxide and H
2
O
2
, with diphenylene iodonium or apocynin decreased Ang II–induced MCP-1 mRNA accumulation. Induction of MCP-1 gene expression by Ang II was inhibited by catalase, suggesting a second messenger role for H
2
O
2
. The tyrosine kinase inhibitor genistein and the mitogen-activated protein kinase kinase inhibitor PD098059 inhibited Ang II–induced MCP-1 gene expression, consistent with a mitogen-activated protein kinase-dependent signaling mechanism. Ang II may thus promote atherogenesis by direct activation of MCP-1 gene expression in vascular smooth muscle cells.
Superoxide Released to High Intra-arteriolar Pressure Reduces Nitric Oxide–Mediated Shear Stress– and Agonist-Induced Dilations
Authors: Huang An , Sun Dong , Kaley Gabor , Koller Akos
Published: June 2012

Abstract
—It is thought that elevated levels of reactive oxygen metabolites contribute to the dysfunction of vascular endothelium in hypertension. We hypothesized that high intravascular pressure itself elicits production of superoxide, which then interferes with nitric oxide (NO)–mediated responses of arterioles. Thus, isolated arterioles (≈80 μm in diameter) from gracilis muscle of normotensive Wistar rats were cannulated and exposed to 140 mm Hg perfusion pressure for 30 minutes (in the absence of perfusate flow). After high intravascular pressure treatment, dilations to increases in perfusate flow (0 to 30 μL/min) were significantly reduced (from 39±2.2 to 19±2.1 μm at 30 μL/min), eliciting an increase in wall shear stress from ≈20 to ≈60 dyne/cm
2
. N
ω
-nitro-
l
-arginine (10
−4
mol/L) did not affect, whereas indomethacin eliminated, flow-induced dilations after pressure treatment. In control, substance P (SP, 10
−9
to 5×10
−8
mol/L), sodium nitroprusside (SNP, 10
−8
to 10
−6
mol/L), and adenosine (ADO, 10
−6
to 5×10
−5
mol/L) elicited dilations (SP: 31.5±1.9 μm, SNP: 45.6±4 μm, and ADO: 37.2±4.1 μm, at maximum concentrations, respectively). After pressure treatment, maximum dilations to SP and SNP were significantly reduced (by 49% and 39%, respectively), whereas responses to ADO were not affected. Presence of superoxide dismutase (120 U/mL) and catalase (80 U/mL), but not catalase alone, in the perfusate solution prevented the reduction in dilation of arterioles to flow and agonists after pressure treatment by restoring NO mediation. We conclude that high intravascular pressure per se elicits the release of superoxide, which then interferes with NO, a mechanism that contributes to the elevation of wall shear stress and peripheral resistance in hypertension.
Local Overexpression of Thrombomodulin for In Vivo Prevention of Arterial Thrombosis in a Rabbit Model
Authors: Waugh J. M. , Yuksel E. , Li J. , Kuo M. D. , Kattash M. , Saxena R. , Geske R. , Thung S. N. , Shenaq S. M. , Woo S. L. C.
Published: June 2012

Abstract
—Endothelial thrombomodulin plays a critical role in hemostasis by binding thrombin and subsequently converting protein C to its active form, a powerful anticoagulant. Thrombomodulin thus represents a central mechanism by which patency is maintained in normal vessels. However, thrombomodulin expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion. An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on in vivo thrombus formation was subsequently examined in a stasis/injury model of arterial thrombosis. The construct prevented arterial thrombosis formation in all animals, while viral and nonviral controls typically developed occluding thrombi. By histological analysis, nonviral controls exhibited intravascular thrombus occluding a mean of 70.52±3.72% of available lumen, while viral controls reached 86.85±2.82% thrombotic occlusion; in contrast, Adv/RSV-THM reduced thrombosis to 28.61±3.31% of lumen in cross section. No significant intima-to-media ratio was observed in the thrombomodulin group relative to controls. Local infiltration of granulocytes and macrophages significantly decreased in the Adv/RSV-THM group relative to controls, while neutrophilic infiltration increased in viral controls relative to nonviral controls. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery, without the inflammatory damage that has limited many other adenoviral applications.
Cardiac Myosin Binding Protein C
Authors: Winegrad Saul
Published: June 2012

Abstract
—Myosin binding protein C (MyBP-C) is one of a group of myosin binding proteins that are present in the myofibrils of all striated muscle. The protein is found at 43-nm repeats along 7 to 9 transverse lines in a portion of the A band where crossbridges are found (C zone). MyBP-C contains myosin and titin binding sites at the C terminus of the molecule in all 3 of the isoforms (slow skeletal, fast skeletal, and cardiac). The cardiac isoform also includes a series of residues that contain 3 phosphorylatable sites and an additional immunoglobulin module at the N terminus that are not present in skeletal isoforms. The following 2 major functions of MyBP-C have been suggested: (1) a role in the formation of the sarcomeric myofibril as a result of binding to myosin and titin and (2) in the case of the cardiac isoform, regulation of contraction through phosphorylation. The first is supported by the demonstrated effect of MyBP-C on the packing of myosin in the thick filament, the coincidence of appearance of sarcomeres and MyBP-C during myofibrillogenesis, and the defective formation of sarcomeres when the titin and/or myosin binding sites of MyBP-C are missing. The second is supported by the specific phosphorylation sites in cardiac MyBP-C, the presence in the thick filament of an enzyme specific for MyBP-C phosphorylation, the alteration of thick filament structure by MyBP-C phosphorylation, and the accompaniment of MyBP-C phosphorylation with all major physiological mechanisms of modulation of inotropy in the heart.
Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125
FAK
) in Cultured Rat Cardiac Myocytes
Authors: Takahashi Naoyuki , Seko Yoshinori , Noiri Eisei , Tobe Kazuyuki , Kadowaki Takashi , Sabe Hisataka , Yazaki Yoshio
Published: June 2012

Abstract
—Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125
FAK
), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125
FAK
as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125
FAK
from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125
FAK
was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125
FAK
was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60
c-src
. Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125
FAK
is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.
Effects of Myosin Heavy Chain Isoform Switching on Ca
2+
-Activated Tension Development in Single Adult Cardiac Myocytes
Authors: Metzger Joseph M. , Wahr Philip A. , Michele Daniel E. , Albayya Faris , Westfall Margaret V.
Published: June 2012

Abstract
—Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca
2+
sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the α-MHC isoform to exclusively the β-MHC isoform. We found a significant desensitization in the Ca
2+
sensitivity of tension development in β-MHC–expressing ventricular myocytes (pCa
50
=5.51±0.03, where pCa is –log[Ca
2+
], and pCa
50
is pCa at which tension is one-half maximal) compared with that in predominantly α-MHC–expressing myocytes (pCa
50
=5.68±0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca
2+
activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in β-MHC–expressing myocytes compared with the α-MHC–expressing cardiac myocytes (pCa
50
=5.47±0.05 versus 5.76±0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca
2+
sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.
Contribution of α-Adrenergic and β-Adrenergic Stimulation to Ischemia-Induced Glucose Transporter (GLUT) 4 and GLUT1 Translocation in the Isolated Perfused Rat Heart
Authors: Egert Silvia , Nguyen Ngoc , Schwaiger Markus
Published: June 2012

Abstract
—The intracellular signaling mechanism of the ischemia-stimulated glucose transporter (GLUT) translocation in the heart is not yet characterized. It has been suggested that catecholamines released during ischemia may be involved in this pathway. The purpose of this study was to evaluate the contribution of α-adrenoceptors and β-adrenoceptors to ischemia-mediated GLUT4 and GLUT1 translocation in the isolated, Langendorff-perfused rat heart. Additionally, GLUT translocation was studied in response to catecholamine stimulation with phenylephrine (Phy) and isoproterenol (Iso). The results were compared with myocardial uptake of glucose analogue [
18
F]fluorodeoxyglucose (FDG). Subcellular analysis of GLUT4 and GLUT1 protein on plasma membrane vesicles (PM) and intracellular membrane vesicles (IM) using membrane preparation and immunoblotting revealed that α- and β-receptor agonists stimulated GLUT4 translocation from IM to PM (2.5-fold for Phy and 2.1-fold for Iso,
P
<0.05 versus control), which was completely inhibited by phentolamine (Phe) and propranolol (Pro), respectively. Plasmalemmal GLUT1 moderately rose after Iso exposure, and this was prevented by Pro. In contrast, ischemia-stimulated GLUT4 translocation (2.2-fold,
P
<0.05 versus control) was only inhibited by α-adrenergic antagonist Phe but not by β-adrenergic antagonist Pro. Similarly, Phe but not Pro inhibited ischemia-stimulated GLUT1 translocation. GLUT data were confirmed by FDG uptake monitored using bismuth germanate detectors. The catecholamine-stimulated FDG uptake (6.9-fold for Phy and 8.9-fold for Iso) was significantly inhibited by Phe and Pro; however, only Phe but not Pro significantly reduced the ischemia-induced 2.5-fold increase in FDG uptake (
P
<0.05 versus ischemia). This study suggests that α-adrenoceptor stimulation may play a role in the ischemia-mediated increase in glucose transporter trafficking leading to the stimulation of FDG uptake in the isolated, perfused rat heart, whereas β-adrenergic activation does not participate in this signaling pathway.