bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization
Authors: Su E. J. , Cioffi C. L. , Stefansson S. , Mittereder N. , Garay M. , Hreniuk D. , Liau G.
Published: March 2015

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of ∼80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.
Central interleukin-1β antibody increases renal and splenic sympathetic nerve discharge
Authors: Lu Ning , Wang Yan , Blecha Frank , Fels Richard J. , Hoch Heather P. , Kenney Michael J.
Published: March 2015

We tested the hypothesis that intracerebroventricular (lateral ventricle) administration of interleukin-1β (IL-1β) antibody increases the level of sympathetic nerve discharge (SND) in α-chloralose-anesthetized rats. Mean arterial pressure (MAP), heart rate (HR), and SND (splenic and renal) were recorded before (Preinfusion), during (25 min), and for 45 min after infusion of IL-1β antibody (15 μg, 50 μl icv) in baroreceptor-intact (intact) and sinoaortic-denervated (SAD) rats. The following observations were made. First, intracerebroventricular infusion of IL-1β antibody (but not saline and IgG) significantly increased MAP and the pressor response was higher in SAD compared with intact rats. Second, renal and splenic SND were significantly increased during and after intracerebroventricular IL-1β antibody infusion and sympathoexcitatory responses were higher in SAD compared with intact rats. Third, intracerebroventricular administration of a single dose of IL-1β antibody (15 μg, 5 μl for 2 min) significantly increased splenic and renal SND in intact rats. These results suggest that under the conditions of the present experiments central neural IL-1β plays a role in the tonic regulation of SND and arterial blood pressure.
Glutathione and K+ channel remodeling in postinfarction rat heart
Authors: Rozanski George J. , Xu Zhi
Published: March 2015

Electrical remodeling of the diseased ventricle is characterized by downregulation of K+ channels that control action potential repolarization. Recent studies suggest that this shift in electrophysiological phenotype involves oxidative stress and changes in intracellular glutathione (GSH), a key regulator of redox-sensitive cell functions. This study examined the role of GSH in regulating K+ currents in ventricular myocytes from rat hearts 8 wk after myocardial infarction (MI). Colorimetric analysis of tissue extracts showed that endogenous GSH levels were significantly less in post-MI hearts compared with controls, which is indicative of oxidative stress. This change in GSH status correlated with significant decreases in activities of glutathione reductase and γ-glutamylcysteine synthetase. Voltage-clamp studies of isolated myocytes from post-MI hearts demonstrated that downregulation of the transient outward K+ current ( I to) could be reversed by pretreatment with exogenous GSH or N-acetylcysteine, a precursor of GSH. Upregulation of I to was also elicited by dichloroacetate, which increases glycolytic flux through the GSH-related pentose pathway. This metabolic effect was blocked by inhibitors of glutathione reductase and the pentose pathway. These data indicate that oxidative stress-induced alteration in the GSH redox state plays an important role in I to channel remodeling and that GSH homeostasis is influenced by pathways of glucose metabolism.
Rapid modification of the glycocalyx caused by ischemia-reperfusion is inhibited by adenosine A2Areceptor activation
Authors: Platts Steven H. , Linden Joel , Duling Brian R.
Published: March 2015

Ischemia-reperfusion (I/R) has been shown to cause microvascular dysfunction and to alter the appearance of the glycocalyx in electron micrographs. We hypothesized that I/R injury might alter the structure and/or permeability of the glycocalyx. Prior work had shown a role for adenosine in protection from I/R injury, and, therefore, we also explored the idea that activation of the adenosine A2Areceptor would attenuate I/R glycocalyx injury. Here, we report that I/R causes a rapid and dramatic decrease in the ability of the glycocalyx to exclude FITC-Dextran 70 (Dex70). Over a reperfusion period of 45 min, the glycocalyx dye exclusion zone for Dex70 decreased by one-half in capillaries and postcapillary venules, whereas the red blood cell exclusion zone was very slightly reduced in capillaries only. Pretreatment with the A2Aagonist ATL-146e significantly inhibited the changes in both vessel types. The modifications of the glycocalyx appear to be an early step in the inflammatory cascade typically associated with reperfusion injury, and adenosine A2Areceptor activation may play a role in protection from this injury.
Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell
Authors: Kurata Yasutaka , Hisatome Ichiro , Imanishi Sunao , Shibamoto Toshishige
Published: March 2015

We developed an improved mathematical model for a single primary pacemaker cell of the rabbit sinoatrial node. Original features of our model include 1) incorporation of the sustained inward current ( I st) recently identified in primary pacemaker cells, 2) reformulation of voltage- and Ca2+-dependent inactivation of the L-type Ca2+channel current ( I Ca,L), 3) new expressions for activation kinetics of the rapidly activating delayed rectifier K+ channel current ( I Kr), and 4) incorporation of the subsarcolemmal space as a diffusion barrier for Ca2+. We compared the simulated dynamics of our model with those of previous models, as well as with experimental data, and examined whether the models could accurately simulate the effects of modulating sarcolemmal ionic currents or intracellular Ca2+ dynamics on pacemaker activity. Our model represents significant improvements over the previous models, because it can 1) simulate whole cell voltage-clamp data for I Ca,L, I Kr, and I st; 2) reproduce the waveshapes of spontaneous action potentials and ionic currents during action potential clamp recordings; and 3) mimic the effects of channel blockers or Ca2+ buffers on pacemaker activity more accurately than the previous models.
Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection
Authors: Pieper Galen M. , Nilakantan Vani , Hilton Gail , Halligan Nadine L. N. , Felix Christopher C. , Kampalath Bal , Khanna Ashwani K. , Roza Allan M. , Johnson Christopher P. , Adams Mark B.
Published: March 2015

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions ( g tensor factors for perpendicular and parallel components, respectively g ⊥ = 2.038 and g ∥ = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-κB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-κB by DETC-Fe was associated with increased IκBα inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-γ was also decreased by DETC-Fe. Thus DETC-Fe limits NF-κB-dependent gene expression and possesses significant immunosuppressive properties.
Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II
Authors: Wang Hui Di , Johns Douglas G. , Xu Shanqin , Cohen Richard A.
Published: March 2015

Our purpose was to address the role of NAPDH oxidase-derived superoxide anion in the vascular response to ANG II. Blood pressure, aortic superoxide anion, 3-nitrotyrosine, and medial cross-sectional area were compared in wild-type mice and in mice that overexpress human superoxide dismutase (hSOD). The pressor response to ANG II was significantly less in hSOD mice. Superoxide anion levels were increased twofold in ANG II-treated wild-type mice but not in hSOD mice. 3-Nitrotyrosine increased in aortic endothelium and adventitia in wild-type but not hSOD mice. In contrast, aortic medial cross-sectional area increased 50% with ANG II in hSOD mice, comparable to wild-type mice. The lower pressor response to ANG II in the mice expressing hSOD is consistent with a pressor role of superoxide anion in wild-type mice, most likely because it reacts with nitric oxide. Despite preventing the increase in superoxide anion and 3-nitrotyrosine, the aortic hypertrophic response to ANG II in vivo was unaffected by hSOD.
Coronary blood flow regulation in exercising swine involves parallel rather than redundant vasodilator pathways
Authors: Merkus Daphne , Haitsma David B. , Fung Tse-Yeung , Assen Yvette J. , Verdouw Pieter D. , Duncker Dirk J.
Published: March 2015

In dogs, only combined blockade of vasodilator pathways [via adenosine receptors, nitric oxide synthase (NOS) and ATP-sensitive K+ (KATP) channels] results in impairment of metabolic vasodilation, which suggests a redundancy design of coronary flow regulation. Conversely, in swine and humans, blocking KATP channels, adenosine receptors, or NOS each impairs coronary blood flow (CBF) at rest and during exercise. Consequently, we hypothesized that these vasodilators act in parallel rather than in redundancy to regulate CBF in swine. Swine exercised on a treadmill (0–5 km/h), during control and after blockade of KATP channels (with glibenclamide), adenosine receptors [with 8-phenyltheophylline (8-PT)], and/or NOS [with Nω-nitro-l-arginine (l-NNA)]. l-NNA, 8-PT, and glibenclamide each reduced myocardial O2 delivery and coronary venous O2 tension. These effects of l-NNA, 8-PT, and glibenclamide were not modified by simultaneous blockade of the other vasodilators. Combined blockade of KATP channels and adenosine receptors with or without NOS inhibition was associated with increased H+ production and impaired myocardial function. However, despite an increase in O2 extraction to >90% during administration of l-NNA + 8-PT + glibenclamide, vasodilator reserve could still be recruited during exercise. Thus in awake swine, loss of KATP channels, adenosine, or NO is not compensated for by increased participation of the other two vasodilator mechanisms. These findings suggest a parallel rather than a redundancy design of CBF regulation in the porcine circulation.
Integrin shedding as a mechanism of cellular adaptation during cardiac growth
Authors: Goldsmith Edie C. , Carver Wayne , McFadden Alex , Goldsmith Jack G. , Price Robert L. , Sussman Mark , Lorell Beverly H. , Cooper Garth , Borg Thomas K.
Published: March 2015

Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of β1-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of β1-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The β1-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a β1-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.
20-HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat
Authors: Kehl Franz , Cambj-Sapunar Liana , Maier Kristopher G. , Miyata Noriyuki , Kametani Shunishi , Okamoto Hirotsugu , Hudetz Anthony G. , Schulte Marie L. , Zagorac Drazen , Harder David R. , Roman Richard J.
Published: March 2015

This study examined the effects of blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) on the acute fall in cerebral blood flow after subarachnoid hemorrhage (SAH) in the rat. In vehicle-treated rats, regional cerebral blood flow (rCBF) measured with laser-Doppler flowmetry fell by 30% 10 min after the injection of 0.3 ml of arterial blood into the cisterna magna, and it remained at this level for 2 h. Pretreatment with inhibitors of the formation of 20-HETE, 17-octadecynoic acid (17-ODYA; 1.5 nmol intrathecally) and N-hydroxy- N′-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 mg/kg iv), reduced the initial fall in rCBF by 40%, and rCBF fully recovered 1 h after induction of SAH. The concentration of 20-HETE in the cerebrospinal fluid rose from 12 ± 2 to 199 ± 17 ng/ml after SAH in vehicle-treated rats. 20-HETE levels averaged only 15 ± 11 and 39 ± 13 ng/ml in rats pretreated with 17-ODYA or HET0016, respectively. HET0016 selectively inhibited the formation of 20-HETE in rat renal microsomes with an IC50of <15 nM and human recombinant CYP4A11, CYP4F2, and CYP4F3 enzymes with an IC50 of 42, 125, and 100 nM, respectively. These results indicate that 20-HETE contributes to the acute fall in rCBF after SAH in rats.
Regulation of cardiac β1-adrenergic receptor transcription during the developmental transition
Authors: Wadhawan Rajan , Tseng Yi-Tang , Stabila Joan , McGonnigal Bethany , Sarkar Sumita , Padbury James
Published: March 2015

The β1-adrenergic receptor (β1AR) gene contains binding sites for myc/max proteins within a glucocorticoid response element. Transcriptional activation of the β1AR is the result of cooperative binding between c- myc and the glucocorticoid receptor on the β1AR promoter. The transcriptional regulation of both β1AR and c- myc are developmentally regulated. We used transcription rate assays of nuclei isolated from fetal hearts to demonstrate a fivefold increase in the transcription rate of β1AR vs. postnatal hearts ( P < 0.01). This was associated with a fourfold increase in c- myctranscription. Transcription rate assays performed in a rat fibroblast cell line that overexpresses c- myc (myc+/+) showed similarly increased β1AR expression compared with the wild-type cell line. Transient transfection experiments in the myc+/+cells demonstrated robust expression of β1AR promoter constructs, which was abrogated by mutation of the myc/max binding site or by cotransfection with a c- myc antisense expression vector. These results suggest that the regulation of cardiac β1AR transcription and the expression of c- myc are tightly integrated.
Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function
Authors: Hyndman Mathew Eric , Verma Subodh , Rosenfeld Robin J. , Anderson Todd J. , Parsons Howard G.
Published: March 2015

The present study was designed to investigate the interaction between 5-methyltetrahydrofolate and tetrahydrobiopterin in modulating endothelial function. Tetrahydrobiopterin is a critical cofactor for nitric oxide synthase and maintains this enzyme as a nitric oxide- versus superoxide-producing enzyme. The structure of 5-methyltetrahydrofolate is similar to tetrahydrobiopterin and both agents have been shown to improve endothelium-dependent vasodilatation. We hypothesized that 5-methyltetrahydrofolate interacts with nitric oxide synthase in a fashion analogous, yet independent, of tetrahydrobiopterin to improve endothelial function. We demonstrate that 5-methyltetrahydrofolate binds the active site of nitric oxide synthase and mimics the orientation of tetrahydrobiopterin. Furthermore, 5-methyltetrahydrofolate attenuates superoxide production (induced by inhibition of tetrahydrobiopterin synthesis) and improves endothelial function in aortae isolated from tetrahydrobiopterin-deficient rats. We suggest that 5-methyltetrahydrofolate directly interacts with nitric oxide synthase to promote nitric oxide (vs. superoxide) production and improve endothelial function. 5-Methyltetrahydrofolate may represent an important strategy for intervention aimed at improving tetrahydrobiopterin bioavailability.
Nitric oxide and endothelin in oxygen-dependent regulation of vascular tone of human umbilical vein
Authors: Mildenberger Eva , Biesel Beatrix , Siegel Günter , Versmold Hans T.
Published: March 2015

We investigated the possible contribution of nitric oxide (NO) and endothelin (ET) to oxygen-dependent regulation of human umbilical vein vascular tone by simultaneous registration of intracellular membrane potential and isometric tension of vessel strips with and without NO synthase inhibition [10–4 M Nω-nitro-l-arginine methyl ester (l-NAME)], ETA receptor blockade (10–5 M BQ-123), or ETB receptor blockade (10–7 M BQ-788) at Po2 values in the bath solution between 5 and 104 mmHg. Increasing Po2 above the physiological intrauterine range resulted in depolarization and an increase of isometric tension, whereas lowering Po2 resulted in hyperpolarization and a decrease in isometric tension. Removal of the endothelium reversed these effects. At Po2 values below 39 mmHg, intact preparations treated with either l-NAME, BQ-788, or BQ-123 were more depolarized than controls. In the case of treatment with l-NAME or BQ-123, this was accompanied by an increase in isometric tension. We conclude that it is NO that mediates the hypoxic hyperpolarization and vasodilatation of the human umbilical vein and that ET, via activation of ETB1 receptors on endothelial cells, contributes to this effect.
E-Tmod capping of actin filaments at the slow-growing end is required to establish mouse embryonic circulation
Authors: Chu Xin , Chen Ju , Reedy Mary C. , Vera Carlos , Sung K.-L. Paul , Sung Lanping Amy
Published: March 2015

Tropomodulins are a family of proteins that cap the slow-growing end of actin filaments. Erythrocyte tropomodulin (E-Tmod) stabilizes short actin protofilaments in erythrocytes and caps longer sarcomeric actin filaments in striated muscles. We report the knockin of the β-galactosidase gene ( LacZ) under the control of the endogenous E-Tmodpromoter and the knockout of E-Tmod in mouse embryonic stem cells. E-Tmod−/−embryos die around embryonic day 10 and exhibit a noncontractile heart tube with disorganized myofibrils and underdevelopment of the right ventricle, accumulation of mechanically weakened primitive erythroid cells in the yolk sac, and failure of primary capillary plexuses to remodel into vitelline vessels, all required to establish blood circulation between the yolk sac and the embryo proper. We propose a hemodynamic “plexus channel selection” mechanism as the basis for vitelline vascular remodeling. The defects in cardiac contractility, vitelline circulation, and hematopoiesis reflect an essential role for E-Tmod capping of the actin filaments in both assembly of cardiac sarcomeres and of the membrane skeleton in erythroid cells that is not compensated for by other proteins.
Cardiac contractility modulation by electric currents applied during the refractory period
Authors: Mohri Satoshi , He Kun-Lun , Dickstein Marc , Mika Yuval , Shimizu Juichiro , Shemer Itzhak , Yi Geng-Hua , Wang Jie , Ben-Haim Shlomo , Burkhoff Daniel
Published: March 2015

Inotropic effects of electric currents applied during the refractory period have been reported in cardiac muscle in vitro using voltage-clamp techniques. We investigated how electric currents modulate cardiac contractility in normal canine hearts in vivo. Six dogs were instrumented to measure regional segment length, ventricular volume (sonomicrometry), and ventricular pressure. Cardiac contractility modulating (CCM) electric currents (biphasic square pulses, amplitude ±20 mA, total duration 30 ms) were delivered during the refractory period between pairs of electrodes placed on anterior and posterior walls. CCM significantly increased index of global contractility ( E es) from 5.9 ± 2.9 to 8.3 ± 4.6 mmHg/ml with anterior CCM, from 5.3 ± 1.8 to 8.9 ± 4.0 mmHg/ml with posterior CCM, and from 6.1 ± 2.6 to 11.0 ± 7.0 mmHg/ml with combined CCM ( P < 0.01, no significant change in volume axis intercept). End-systolic pressure-segment length relations showed contractility enhancement near CCM delivery sites, but not remotely. Relaxation was not influenced. CCM increased mean aortic pressure, but did not change peripheral resistance. Locally applied electrical currents enhanced global cardiac contractility via regional changes in myocardial contractility without impairing relaxation in situ.
Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction
Authors: Earley Scott , Walker Benjimen R.
Published: March 2015

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca2+]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100–200 μM ID) abluminally loaded with the Ca2+indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca2+] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca2+] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca2+] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca2+] contributes to attenuated reactivity following CH by decreasing VSM cell Ca2+sensitivity.
Effect of hyperglycemia and fatty acid oxidation inhibition during aerobic conditions and demand-induced ischemia
Authors: Chavez Pedro N. , Stanley William C. , McElfresh Tracy A. , Huang Hazel , Sterk Joseph P. , Chandler Margaret P.
Published: March 2015

Metabolic interventions improve performance during demand-induced ischemia by reducing myocardial lactate production and improving regional systolic function. We tested the hypotheses that 1) stimulation of glycolysis would increase lactate production and improve ventricular wall motion, and 2) the addition of fatty acid oxidation inhibition would reduce lactate production and further improve contractile function. Measurements were made in anesthetized open-chest swine hearts. Three groups, hyperglycemia (HG), HG + oxfenicine (HG + Oxf), and control (CTRL), were treated under aerobic conditions and during demand-induced ischemia. During demand-induced ischemia, HG resulted in greater lactate production and tissue lactate content but had no significant effect on glucose oxidation. HG + Oxf significantly lowered lactate production and increased glucose oxidation compared with both the CTRL and HG groups. Myocardial energy efficiency was greater in the HG and HG + Oxf groups under aerobic conditions but did not change during demand-induced ischemia. Thus enhanced glycolysis resulted in increased energy efficiency under aerobic conditions but significantly enhanced lactate production with no further improvement in function during demand-induced ischemia. Partial inhibition of free fatty acid oxidation in the presence of accelerated glycolysis increased energy efficiency under aerobic conditions and significantly reduced lactate production and enhanced glucose oxidation during demand-induced ischemia.
Dual upregulation of Fas and Bax promotes alloreactive T cell apoptosis in IL-10 gene targeting of cardiac allografts
Authors: Tung Thomas C. , Oshima Kiyohiro , Cui Guanggen , Laks Hillel , Sen Luyi
Published: March 2015

Activation-induced cell death and cytokine deprivation are demonstrated by peripheral T cell populations at the conclusion of natural immune responses, and each of these processes is modulated by the immunosuppressive cytokine interleukin (IL)-10 in vitro. This study employs a clinically relevant in vivo model of IL-10 gene transfer with heterotopically transplanted cardiac allografts to determine the mechanisms of the effects of IL-10 on T cell survival. IL-10 protein overexpression within allografts 4–5 days after gene transfer augments apoptosis of CD4+and CD8+graft-infiltrating lymphocytes by 7.1-fold ( P < 0.001) and 6.0-fold ( P < 0.001), respectively. Graft-infiltrating T cells express 10-fold more proapoptotic Fas ( P < 0.01) and 30-fold more Bax ( P < 0.01) than controls. The fractions of activated caspase-8 (FADD-like IL-1β-converting enzyme) and activated caspase-9 were increased 7- and 2.3-fold, respectively, in IL-10 gene-treated allografts at postoperative day 4–5. These changes in the Fas-Fas ligand pathway and Bcl-2 mitochondrial apoptosis regulation are enhanced by complete suppression of antiapoptotic FADD-like IL-1β-converting enzyme inhibitory protein (FLIP) (from 30.5 to 0.0%, P < 0.01) and Bcl-xL (from 22.5 to 0.1%, P = 0.03) expression among these cells from the earliest days after gene transfer. Although changes in proteins of Fas- and Bcl-2-mediated apoptosis signaling occur, only the levels of Fas and FLIP correlate to the rate of apoptosis of graft-infiltrating CD3 lymphocytes and histological rejection scores. These results indicate that dichotomous apoptosis-regulatory pathways are affected by IL-10 gene therapy, but Fas-mediated mechanisms of activation-induced cell death more substantially contribute to the greater cell death of graft-infiltrating T cells after ex vivo IL-10 gene transfer.
Hemodynamics of anesthetized ventilated mouse models: aspects of anesthetics, fluid support, and strain
Authors: Zuurbier Coert J. , Emons Vanessa M. , Ince Can
Published: March 2015

This study evaluates the effects of anesthesia and fluid support on hemodynamic parameters of the mechanically ventilated mouse of four different strains. All experiments were performed at a similar surgical level of anesthesia, as indicated by the probing of the pedal withdrawal reflex. Three anesthetic regimens [fentanyl-fluanisone-midazolam (FFM), ketamine-medetomidine-atropine (KMA), and isoflurane (ISO)], four commonly used mouse strains (Swiss, CD-1, BalbC, and C57Bl6), and three different fluid support strategies (no fluid, 0.2 ml · h−1· 10 g−1of 6% polystarch solution, and 0.5 ml · h−1· 10 g−1saline) were studied. Mean arterial pressure (MAP) or heart rate (HR) was similar among the four strains of mice except a trend toward lower HR for the BalbC mice. In terms of MAP, KMA is the preferred anesthetic for the Swiss and CD-1 mice, whereas KMA or ISO are recommended for BalbC or C57Bl6 mice. In terms of HR, ISO is the preferred anesthetic for the Swiss, CD-1, and C57Bl6 strains. No differences in HR for the three anesthetics were observed for the BalbC strain. Compared with administration of no fluid, both saline and polystarch administration similarly increased MAP by 7 ± 2, 10 ± 2, and 11 ± 2 mmHg at t = 1, 2, and 3 h, respectively, whereas fluid administration was without effect on HR. Saline supplementation resulted in an increased dry-to-wet ratio of the heart and both fluid regimens decreased total hemoglobin in the blood from 12.6 ± 0.5 to 10.4 ± 0.5 g/100 ml. Saline administration was associated with blood acidosis (pH 7.20 ± 0.03) compared with the Haes (pH 7.29 ± 0.02) or no-fluid group (pH 7.34 ± 0.03), whereas Pco2was ∼30 mmHg for all groups. We conclude that at similar surgical levels of anesthesia, the preferable type of anesthesia (ISO or KMA, but never FFM) depends on the strain used and whether MAP or HR is the focus of study. Additional fluid support is beneficial in terms of raising arterial blood pressure, although this is at the cost of changes in organ water content and increased anemia.
Enhancement of reperfusion injury by elevation of microvascular pressures
Authors: Takase Shinya , Lerond Laurence , Bergan John J. , Schmid-Schönbein Geert W.
Published: March 2015

Elevated venous pressure can be associated with severe tissue injury. Few links, however, between venous hypertension and tissue damage have been established. We examined here the effects of micropressure elevation on the outcome of venular occlusion/reperfusion in the mesenteric microvasculature of male Wistar rats. One hour of venular occlusion (diameter ∼50 μm) by micropipette occlusion followed by reperfusion were carried out with sham surgery without occlusion as control. Leukocyte rolling, adhesion, and migration, oxygen radicals detected by dichlorofluorescein (DCF), and parenchymal cell death detected by propidium iodide (PI) were recorded simultaneously in the same vessel at a location upstream of the occlusion site with elevated micropressure and at a downstream location with low micropressure. The number of rolling, adhering, and migrating leukocytes increased on the upstream side of the occlusion to a higher level than downstream of the occlusion site. During occlusion, DCF intensity on the venular endothelium was greater on the upstream side than in the downstream side, but there were no differences during reperfusion. The number of PI-positive cells adjacent to the venules increased significantly compared with controls, and it remained greater on the upstream higher-pressure side than the downstream side. Leukocyte adhesion and transvascular migration in postcapillary venules as well as parenchymal cell death could be significantly reduced by the hydroxyl radical scavenger dimethylthiourea. Microhemorrhages of blood cells into the mesentery interstitium were observed only on the upstream side of the occlusion. These results indicate that an elevation of the venular blood pressure during occlusion/reperfusion exacerbates the inflammatory cascade and tissue injury. Venous occlusion may constitute an important mechanism for tissue injury.
Cholinergic regulation of pericyte-containing retinal microvessels
Authors: Wu David M. , Kawamura Hajime , Sakagami Kenji , Kobayashi Masato , Puro Donald G.
Published: March 2015

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca2+levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca2+-activated chloride currents and caused pericytes to contract in a Ca2+-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.
Changes of RBC aggregation in oxygenation-deoxygenation: pH dependency and cell morphology
Authors: Cicha Iwona , Suzuki Yoji , Tateishi Norihiko , Maeda Nobuji
Published: March 2015

The effects of the oxygenation-deoxygenation process on red blood cell (RBC) aggregation were examined in relation to morphological changes in RBCs and the contribution of CO2. A low-shear rheoscope was used to measure the rate of rouleaux (one-dimensional aggregate) formation in diluted autologous plasma exposed to gas mixtures with different Po2and Pco2. RBC indexes and RBC suspension pH were measured for the oxygenated or the deoxygenated condition, and the cell shape was observed with a scanning electron microscope. In the oxygenation-deoxygenation process, the rate of rouleaux formation increased with rising pH of the RBC suspension, which was lowered in the presence of CO2. The rate increased with increasing mean corpuscular hemoglobin concentration (thus the cells shrank), which increased with rising pH and decreased in the presence of CO2. With rising pH, cell diameter increased and cell thickness decreased (thus the cell flattened). In addition, slight echinocytosis was induced in the presence of CO2, and the aggregation was reduced by the morphological change. In conclusion, RBC aggregation in the oxygenation-deoxygenation process is mainly influenced by the pH-dependent change in the surface area-to-volume ratio of the cells, and the aggregation is modified by CO2-induced acidification and the accompanying changes in mean corpuscular hemoglobin concentration and cell shape.
Cardiac preconditioning with 4-h, 17°C ischemia reduces [Ca2+]i load and damage in part via KATP channel opening
Authors: Chen Qun , Camara Amadou K. S. , An Jianzhong , Riess Matthias L. , Novalija Enis , Stowe David F.
Published: March 2015

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17°C hypothermic ischemia on cardiac cytosolic [Ca2+], mechanical and metabolic function, and infarct size, and 2) the potential role of KATP channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca2+] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 ± 2 (SE)% to 15 ± 1% with IPC. Bracketing ischemic pulses with 200 μM 5-hydroxydecanoic acid or 10 μM glibenclamide increased infarct size to 28 ± 3% and 26 ± 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca2+] loading and enhanced ATP-sensitive K channel opening.
Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition
Authors: Hampl Václav , Bı́bová Jana , Straňák Zbynek , Wu Xichen , Michelakis Evangelos D. , Hashimoto Kyoko , Archer Stephen L.
Published: March 2015

Fetal to maternal blood flow matching in the placenta, necessary for optimal fetal blood oxygenation, may occur via hypoxic fetoplacental vasoconstriction (HFPV). We hypothesized that HFPV is mediated by K+channel inhibition in fetoplacental vascular smooth muscle, as occurs in several other O2-sensitive tissues. With the use of an isolated human placental cotyledon perfused at a constant flow rate, we found that hypoxia reversibly increased perfusion pressure by >20%. HFPV was unaffected by cyclooxygenase or nitric oxide synthase inhibition. HFPV and 4-aminopyridine, an inhibitor of voltage-dependent K+(Kv) channels, increased pressure in a nonadditive manner, suggesting they act via a common mechanism. Iberiotoxin, a large conductance Ca2+-sensitive K+(BKCa) channel inhibitor, had little effect on normoxic pressure. Immunoblotting and RT-PCR showed expression of several putative O2-sensitive K+channels in peripheral fetoplacental vessels. In patch-clamp experiments with smooth muscle cells isolated from peripheral fetoplacental arteries, hypoxia reversibly inhibited Kvbut not BKCaor ATP-dependent currents. We conclude that human fetoplacental vessels constrict in response to hypoxia. This response is largely mediated by hypoxic inhibition of Kvchannels in the smooth muscle of small fetoplacental arteries.
Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor
Authors: Kirchhof Paulus , Fabritz Larissa , Fortmüller Lisa , Matherne G. Paul , Lankford Amy , Baba Hideo A. , Schmitz Wilhelm , Breithardt Günter , Neumann Joachim , Boknik Peter
Published: March 2015

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 ± 18 vs. 522 ± 24 beats/min, P < 0.05; exercise: 650 ± 13 vs. 765 ± 28 beats/min, P < 0.05; 1 h after exercise: 588 ± 18 vs. 720 ± 12 beats/min, P < 0.05; all values are means ± SE). Mean HR was lower during exercise (589 ± 16 vs. 698 ± 34 beats/min, P < 0.05) and after exercise (495 ± 16 vs. 592 ± 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% ( P < 0.05) in TG compared with WT mice. Pertussis toxin ( n = 4 pairs, 150 μg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 ± 2 vs. 37 ± 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.
Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits
Authors: Schulz Rainer , Aker Stephanie , Belosjorow Sergej , Konietzka Ina , Rauen Ursula , Heusch Gerd
Published: March 2015

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 ± 0.7 (means ± SE) to 18.8 ± 0.7 mm and a reduced shortening fraction from 31 ± 1to10 ± 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts ( n = 8). However, p38 MAPK phosphorylation [164,488 ± 29,323 vs. 43,565 ± 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-α and -β were increased in failing compared with normal hearts (149,441 ± 38,381 and 170,430 ± 32,952 vs. 68,815 ± 28,984 and 81,788 ± 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.
Delayed cardioprotection by isoflurane: role of KATP channels
Authors: Tonkovic-Capin Marija , Gross Garrett J. , Bosnjak Zeljko J. , Tweddell James S. , Fitzpatrick Colleen M. , Baker John E.
Published: March 2015

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (KATP) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7–10 days of age ( n = 5–16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means ± SD) by 50% (10 ± 5%) versus untreated controls (20 ± 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 ± 4%) versus untreated controls (54 ± 6%). The mitochondrial KATP channel blocker 5-hydroxydecanoate (5-HD) completely (55 ± 3%) and the sarcolemmal KATPchannel blocker HMR 1098 partially (62 ± 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 ± 5%). We conclude that both mitochondrial and sarcolemmal KATP channels contribute to isoflurane-induced delayed cardioprotection.
Electrophysiological response of rat ventricular myocytes to acidosis
Authors: Komukai Kimiaki , Brette Fabien , Pascarel Caroline , Orchard Clive H.
Published: March 2015

The effects of acidosis on the action potential, resting potential, L-type Ca2+( I Ca), inward rectifier potassium ( I K1), delayed rectifier potassium ( I K), steady-state ( I SS), and inwardly rectifying chloride ( I Cl,ir) currents of rat subepicardial (Epi) and subendocardial (Endo) ventricular myocytes were investigated using the patch-clamp technique. Action potential duration was shorter in Epi than in Endo cells. Acidosis (extracellular pH decreased from 7.4 to 6.5) depolarized the resting membrane potential and prolonged the time for 50% repolarization of the action potential in Epi and Endo cells, although the prolongation was larger in Endo cells. At control pH, I Ca, I K1, and I SS were not significantly different in Epi and Endo cells, but I K was larger in Epi cells. Acidosis did not alter I Ca, I K1, or I K but decreased I SS; this decrease was larger in Endo cells. It is suggested that the acidosis-induced decrease in I SS underlies the prolongation of the action potential. I Cl,ir at control pH was Cd2+ sensitive but 4,4′-disothiocyanato-stilbene-2,2′-disulfonic acid resistant. Acidosis increased I Cl,ir; it is suggested that the acidosis-induced increase in I Cl,ir underlies the depolarization of the resting membrane potential.
Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation
Authors: Bobe Régis , Yin Xing , Roussanne Marie-Cécile , Stepien Olivier , Polidano Evelyne , Faverdin Claude , Marche Pierre
Published: March 2015

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]ielevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.
Stimulation of perivascular nitric oxide synthesis by oxygen
Authors: Thom Stephen R. , Fisher Donald , Zhang Jie , Bhopale Veena M. , Ohnishi S. Tsuyoshi , Kotake Yashige , Ohnishi Tomoko , Buerk Donald G.
Published: March 2015

We hypothesized that elevated partial pressures of O2would increase perivascular nitric oxide (·NO) synthesis. Rodents with O2- and ·NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O2at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O2stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state ·NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in ·NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O2appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O2, but not 2.0 ATA O2, increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited ·NO elevation at 2.8 but not 2.0 ATA O2. Hyperoxia increased the concentration of ·NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen.