bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction between adenosine and flow-induced dilation in coronary microvascular network
Authors: Liao J. C. , Kuo L.
Published: December 2017

Previous studies have demonstrated that coronary microvessels are regulated by at least three possible means: metabolite-induced, shear-induced, and pressure-induced (myogenic) mechanisms. Adenosine, a putative metabolic vasodilator, preferentially dilates downstream coronary microvessels, whereas the shear-sensitive mechanism is detected predominantly in upstream larger microvessels. However, the interaction of these mechanisms and the significance of the heterogeneous vascular responsiveness in flow regulation have not been evaluated. These tasks cannot be performed experimentally because of several confounding factors that cannot be separated. Therefore, the present study employed a data-based modeling approach to investigate the role of response heterogeneity in a coronary vascular network and to test the hypothesis that shear-sensitive mechanism or the myogenic mechanisms will enhance the vascular sensitivity to adenosine due to the heterogeneity of the vascular responsiveness. We obtained necessary data and developed empirical models for the responsiveness of single vessels to pressure, shear stress, and adenosine. With the single-vessel models, a network model was established based on the branching pattern of coronary microvessels, mass balance, and fluid mechanics laws. Model simulation predicted an enhanced vascular response to adenosine in the network. Such an enhancement is caused by the heterogeneous vascular response to adenosine and the predominant flow-induced dilation in the large arterioles. Preferential dilation of the downstream small arterioles to adenosine initiates an increase in flow and a decrease in pressure at upstream vessels. The increased flow activates the shear-sensitive mechanism of the upstream large arterioles and further enhances the flow. This hemodynamic interaction contributes up to approximately 20% of the adenosine-induced flow increase and also reduces the adenosine-induced pressure drop. In contrast to the shear-sensitive mechanism, myogenic response contributes relatively little to the vascular response to adenosine. These results suggest that various vascular regulation mechanisms and the response heterogeneity of vessels of different sizes may act in an integrative fashion for the optimal control of microvascular perfusion.
Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide
Authors: D'Angelo G. , Mogford J. E. , Davis G. E. , Davis M. J. , Meininger G. A.
Published: December 2017

It has previously been shown that synthetic peptides containing the sequence arginine-glycine-aspartic acid (RGD) cause vasodilation by activation of alpha(v)beta3-integrin present on vascular smooth muscle (VSM) cells. The purpose of this study was to determine whether this dilatory effect is mediated by a reduction in VSM cytosolic Ca2+ concentration ([Ca2+]i). First-order arterioles from the rat cremaster were isolated, cannulated, and pressurized. [Ca2+]i was quantitated from the ratio of emitted fluorescence intensity during alternate excitation of fura 2-loaded vessels at 340 and 380 nm. Cyclo(-Arg-Gly-Asp-D-Phe-Val) (cycloRGD; 0.21-210 microM) produced a concentration-dependent dilation of arterioles that had developed basal myogenic tone. Over the entire concentration range tested, [Ca2+]i decreased from 91 +/- 6 to 27 +/- 4 nM (69.7 +/- 5.0% reduction). In association with the decrease in [Ca2+]i, arteriolar lumen diameter increased from 89 +/- 8 to 184 +/- 8 pm (89.8 +/- 1.8% dilation). At intermediate concentrations, cycloRGD induced rhythmic spiking of Ca2+ superimposed on the concentration-dependent lowering of basal [Ca2+]i. These data directly link integrin activation with alterations in Ca2+ regulation, the net effect of which is a reduction in [Ca2+]i. These data further suggest that integrins, through their role in mediating cellular attachment to the extracellular matrix and in cellular signaling involving Ca2+, could provide a logical link to mechanotransduction and myogenic phenomena.
Inhibition by external Cd2+ of Na/Ca exchange and L-type Ca channel in rabbit ventricular myocytes
Authors: Hobai I. A. , Bates J. A. , Howarth F. C. , Levi A. J.
Published: December 2017

We investigated the effect of external Cd2+ on the Na/Ca exchange and the L-type Ca channel current (ICa,L) in whole cell patch-clamped rabbit ventricular myocytes at 36 degrees C. After the interfering ion channels and the Na/K pump were blocked, the exchange current was measured as the membrane current that was inhibited by 5 mM nickel. External Cd2+ inhibited Na/Ca exchange with a dissociation constant (KD) of 320.6 +/- 12.4 microM and a Hill coefficient of 1.5 +/- 0.09 (n = 13 cells) and ICa,L with a KD of 2.14 +/- 0.15 microM and a Hill coefficient of 0.74 +/- 0.03 (n = 11 cells). We observed some overlap in the Cd2+ concentration that blocked each mechanism. Cd2+ (100-500 microM) is used commonly to block ICa,L completely. However, 100 microM Cd2+ also inhibits 20% of the Na/Ca exchange activity, whereas 500 microM Cd2+ inhibits 60%.
Cardiac contractile function during coronary stenosis in dogs: association of adenosine in glycolytic dependence
Authors: Fang H. K. , Sturgeon C. , Segil L. J. , Ripper R. L. , Law W. R.
Published: December 2017

We tested the hypothesis that during critical coronary stenosis, endogenous adenosine alters myocardial glucose utilization to support myocardial contractile function (MCF). Anesthetized mongrel dogs were instrumented to measure hemodynamic variables, regional MCF (sonomicrometry), and substrate uptakes. Critical coronary artery stenosis was established with a screw clamp on the left circumflex coronary artery (LCX). Either 8-phenyltheophylline (3 x 10(-7) mol/min; adenosine-receptor blockade), iodoacetate (1 x 10(-5) mol/min; glycolysis blockade), or vehicle was infused into the LCX and the left anterior descending coronary artery (LAD). Critical coronary stenosis caused small decreases in arterial blood pressure and LCX blood flow, but no significant changes in MCF or other hemodynamics. There was a significant decrease in the O2 supply-to-consumption ratio in the stenotic region and an increased glucose uptake. Infusion of either 8-phenyltheophylline or iodoacetate caused a decrease in MCF in the stenotic LCX region concomitant with a decreased glucose uptake and without further changes in blood flow. This was not seen in the nonstenotic (LAD) region. These data support the hypothesis, indicating that glycolysis is vital for maintaining regional MCF during a decrease in the myocardial O2 supply-to-consumption ratio and that adenosine is important in this regard, independent of its vasoactive properties.
Significance of TNF in hemorrhage-related hemodynamic alterations, organ injury, and mortality in rats
Authors: Bahrami S. , Yao Y. M. , Leichtfried G. , Redl H. , Marzi I. , Schlag G.
Published: December 2017

To evaluate the role of tumor necrosis factor-alpha (TNF-alpha) in hemodynamic alterations, multiple organ damage, and mortality caused by hemorrhagic shock, we employed a monoclonal antibody to TNF-alpha (TNF-alpha MAb) in anesthetized rats subjected to prolonged hemorrhagic shock (mean arterial pressure of 30–x35 mmHg for 180 min) followed by resuscitation over 50 min. Treatment of rats with 20.0 mg/kg TNF-alpha MAb 15 min after the end of resuscitation significantly decreased the total peripheral resistance index (P = 0.031) and provided remarkable protection from multiple organ damage compared with controls. The 48-h survival rate was significantly higher in the treatment group (73.3%) than in the control group (26.7%; P = 0.029). The results suggest that TNF-alpha induced by hemorrhagic shock in rats is an important mediator of pathophysiological alterations associated with cardiovascular abnormalities, multiple organ injury, and even lethality. Postresuscitation treatment with TNF-alpha MAb, even after an initial TNF-alpha formation had occurred, significantly attenuated the cardiovascular consequences and improved the survival rate. Thus monoclonal antibodies to TNF-alpha might provide new prospects in the treatment of hemorrhage-related disorders.
Endothelin-1 in rat endotoxemia: mRNA expression and vasoreactivity in pulmonary and systemic circulations
Authors: Curzen N. P. , Kaddoura S. , Griffiths M. J. , Evans T. W.
Published: December 2017

Endothelin-1 (ET-1) is a vasoconstrictor and proinflammatory peptide, but its role in the vascular response to sepsis is unknown. After intraperitoneal injection of male Wistar rats (300 g) with 20 mg/kg of Salmonella enteritidis lipopolysaccharide (LPS), the expression of ET-1 mRNA was significantly increased in pulmonary artery and aorta within 1 h and arterial ET-1 concentration was elevated. Despite this increase in ET-1 production, there was no difference in baseline systemic or pulmonary arterial pressures between control and endotoxin-treated rats, and, furthermore, combined ETA/ETB receptor antagonism using bosentan produced reductions in systemic and pulmonary arterial pressures that were not greater than the modest fall seen in controls. However, bosentan completely antagonized the hemodynamic effects of exogenous ET-1 in controls but only weakly antagonized its effects in LPS animals. After LPS the initial (ETB-mediated) systemic hypotensive responses to ET-1 were attenuated, but the subsequent systemic pressor responses were not. By contrast, the increases in pulmonary arterial pressure in response to ET-1 and the ETB receptor agonist sarafotoxin S6c were significantly reduced in LPS animals. Vascular ET-1 mRNA expression and arterial ET-1 concentration are elevated after LPS treatment in rats, but the functional activity of ET-1 cannot be exposed by combined ETA/ETB receptor antagonism, possibly because of an alteration in the functional status of ET receptors.
A chloride current component induced by hypertrophy in rat ventricular myocytes
Authors: Benitah J. P. , Gomez A. M. , Delgado C. , Lorente P. , Lederer W. J.
Published: December 2017

The effect of hypertrophy on membrane currents of rat left ventricular myocytes was studied with the whole cell voltage-clamp method. We found that the slope of the total time-independent current density-voltage relationship was increased in hypertrophied cells. No change in the zero-current potential was observed. Surprisingly, the dominant time-independent current, the inward rectifier K+ current (measured as the Ba(2+)-sensitive current density) was unchanged. We therefore investigated the identity of the outwardly rectifying Ba(2+)-resistant current seen in the hypertrophied rat ventricular myocytes but not present in control cells. We found that this current 1) was not carried by monovalent cations, 2) was partially blocked by anthracene-9-carboxylic acid (9-AC), and 3) was sensitive to variations in extracellular Cl concentration. These findings are consistent with the current being carried at least partially by Cl-. The presence of an additional Cl(-)-dependent component in hypertrophied cells is supported by the actions of 9-AC on the measured action potentials (APs). 9-AC had no effect on control cells APs but prolonged hypertrophied cell APs. We conclude that a Cl- current component develops in hypertrophied rat heart cells. This component appears to shorten the AP duration and might thus provide protection from cardiac arrhythmias.
Release of nitric oxide and prostaglandin H2 to acetylcholine in skeletal muscle venules
Authors: Dornyei G. , Kaley G. , Koller A.
Published: December 2017

The role of endothelium in regulating venular resistance is not well characterized. Thus we aimed to elucidate the endothelium-derived factors involved in the mediation of responses of rat gracilis muscle venules to acetylcholine (ACh) and other vasoactive agents. Changes in diameter of perfusion pressure (7.5 mmHg)- and norepinephrine (10(-6) M)-constricted venules (approximately 225 microns in diam) to cumulative doses of ACh (10(-9) to 10(-4) M) and sodium nitroprusside (SNP, 10(-9) to 10(-4) M), before and after endothelium removal or application of various inhibitors, were measured. Lower doses of ACh elicited dilations (up to 42.1 +/- 4.7%), whereas higher doses of ACh resulted in smaller dilations or even constrictions. Endothelium removal abolished both ACh-induced dilation and constriction. In the presence of indomethacin (2.8 x 10(-5) M), a cyclooxygenase blocker, or SQ-29548 (10(-6) M), a thromboxane A2-prostaglandin H2 (PGH2) receptor antagonist, higher doses of ACh caused further dilation (up to 72.7 +/- 7%) instead of constriction. Similarly, lower doses of arachidonic acid (10(-9) to 10(-6) M) elicited dilations that were diminished at higher doses. These reduced responses were, however, reversed to substantial dilation by SQ-29548. The nitric oxide (NO) synthase blocker, N omega-nitro-L-arginine (L-NNA, 10(-4) M), significantly reduced the dilation to ACh (from 30.6 +/- 5.5 to 5.4 +/- 1.4% at 10(-6) M ACh). In contrast, L-NNA did not affect dilation to SNP. Thus ACh elicits the release of both NO and PGH2 from the venular endothelium.
Open-system kinetics of myocardial phosphoenergetics during coronary underperfusion
Authors: Kroll K. , Kinzie D. J. , Gustafson L. A.
Published: December 2017

A novel hypothesis is proposed and tested describing open-system kinetics for myocardial phosphoenergetics. The hypothesis is that during severe coronary underperfusion there is precise matching of the rates of ATP synthesis and hydrolysis, but despite the precise balance of ATP rates, there is a decrease in the concentration of ATP and an increase in the concentration of phosphocreatine (PCr) caused by the hydrolysis of AMP to adenosine. Isolated rabbit hearts were perfused using a crystalloid medium, and coronary flow was reduced by 95% from baseline for 45 min followed by reperfusion. Phosphorus nuclear magnetic resonance spectroscopy showed a rapid decrease in PCr concentration to 25% of baseline at the onset of underperfusion followed by a gradual increase in PCr to 42% of baseline, while ATP decreased continuously to 65% of baseline. The kinetics of PCr and ATP could only be described by the precise matching of the rates of ATP synthesis and ATP hydrolysis and an open adenylate system that included the decrease in cytosolic AMP concentration via the production and efflux of adenosine. To confirm the hypothesis of open-system kinetics, two independent predictions were tested in separate experiments: 1) total coronary venous purine efflux (adenosine+inosine+hypoxanthine) during underperfusion was equal to the decrease in ATP concentration, and 2) there was no increase in PCr during moderate coronary underperfusion (80% flow reduction). In conclusion, the open nature of the myocardial adenylate system causes mass action effects that exert novel control over PCr and ATP concentrations during coronary underperfusion. The open-system kinetics cause ATP to decrease and PCr to increase, even though there is precise matching of the rates of ATP synthesis and hydrolysis. Finally, the hydrolysis of AMP to adenosine may benefit tissue survival during ischemia by improving the free energy of ATP hydrolysis, thereby delaying or preventing calcium overload.
Glibenclamide-induced blockade of ischemic preconditioning is time dependent in intact rat heart
Authors: Schultz J. E. , Yao Z. , Cavero I. , Gross G. J.
Published: December 2017

The ATP-sensitive potassium (KATP) channel has been demonstrated to be a potential mediator of ischemic preconditioning (PC) in most species, with the exception of the rat. This conclusion is based on the failure of the KATP channel antagonist glibenclamide (Glib) to block PC in this species. However, previous studies did not take into consideration the kinetic properties of Glib binding to its receptor in the rat myocardium. Therefore, the purpose of the present study was to test the hypothesis that ischemic PC is mediated by the myocardial KATP channel in the rat and that blockade of PC by Glib is a time-dependent phenomenon. Barbiturate-anesthetized, open-chest male Wistar rats were subjected to 30 min of occlusion and 2 h of reperfusion. Ischemic PC was elicited by three 5-min occlusion periods interspersed with 5 min of reperfusion. Infarct size (IS) as a percentage of the area at risk (AAR; IS/AAR) was determined with triphenyltetrazolium staining. PC and the KATP channel opener nicorandil (50 micrograms.kg-1.min-1 i.v.) resulted in marked reductions in IS/AAR from 53 +/- 3% to 8 +/- 1% and 17 +/- 5%, respectively (P < 0.05). Two doses of Glib (1 or 0.3 mg/kg i.v.) given 30 min before PC abolished the protective effect of PC; however, Glib (0.3 mg/kg i.v.) given 5 min before PC failed to block the effect. Glib administered 30 min before the 15-min nicorandil infusion blocked the cardioprotective effect of NC but did not block its transient hypotensive effect. These results demonstrate that the KATP channel is involved in ischemic PC in the intact rat heart and that the inhibitory effect of Glib to block PC is time dependent.
CD18-dependent leukocyte adherence and vascular injury in pig cerebral circulation after ischemia
Authors: Gidday J. M. , Park T. S. , Gonzales E. R. , Beetsch J. W.
Published: December 2017

Recent accumulating evidence indicates that leukocytes contribute importantly to ischemic brain injury. Although large numbers of leukocytes are present in the ischemic territory of reperfused brain 24-48 h after the ischemic insult, little is known of the acute inflammatory response to cerebral ischemia, particularly regarding the time course and magnitude of leukocyte adherence to cerebrovascular endothelium and the functional consequences of such adherence. To study these issues, we developed an epifluorescence videomicroscopy system for observing and quantifying the dynamic behavior of rhodamine-labeled leukocytes in the cerebrovascular microcirculation. Anesthetized piglets equipped with closed cranial windows were used in these investigations. During the initial 2 h of reperfusion after 9 min of asphyxia (n = 6), a marked, progressive increase in adherent leukocytes was noted in cerebral postcapillary venules that was significantly greater in magnitude than that seen in nonasphyxic, time-matched controls (n = 8). A similar response was observed after complete global ischemia of 10 min duration. A significant increase in sodium fluorescein permeability was also measured at 2 h of reperfusion in asphyxic animals. Pretreating a separate asphyxic animal group (n = 7) with a monoclonal antibody to the leukocyte adhesion glycoprotein complex CD11/CD18 severely attenuated both leukocyte adherence and the increase in vascular permeability. These results provide evidence that adherent leukocytes contribute to disruption of endothelial integrity during early reperfusion after global ischemic insults, the inhibition of which may reduce the vasogenic edema that occurs early during reperfusion after birth asphyxia, stroke, and cardiac arrest.
LPS-induced delayed myocardial adaptation enhances acute preconditioning to optimize postischemic cardiac function
Authors: Rowland R. T. , Meng X. , Cleveland J. C. , Meldrum D. R. , Harken A. H. , Brown J. M.
Published: December 2017

Myocardial tolerance to ischemia and reperfusion (I/R) injury can be achieved by either acute or delayed cardioprotective mechanisms. Ischemic preconditioning has been demonstrated to be a powerful acute cardioprotective stimulus. We have reported that lipopolysaccharide (LPS) pretreatment induces delayed myocardial adaptation to I/R injury. To optimize myocardial protection, we examined the ability of delayed myocardial adaptation to enhance acute ischemic preconditioning in the isolated working rat heart. Male Sprague-Dawley rats were divided into control, acute [transient ischemia (TI); 5-min global ischemia, 37 degrees C], delayed (LPS; 500 micrograms/kg i.p.), or combined (LPS + TI) cardioprotective groups. Delayed cardioprotection involved LPS injection 72 h before heart isolation. All hearts were subjected to 20-min global ischemia (37 degrees C) and 30-min reperfusion. Coronary effluent collected during reperfusion was assayed for creatine kinase (CK) activity. Both TI and LPS treatment improved postischemic aortic flow recovery (29 +/- 4.5 and 44 +/- 4.0%, respectively; P < 0.05, LPS vs. TI) compared with control hearts (11 +/- 2.2%; P < 0.05, TI or LPS vs. control). When TI was applied to LPS-treated hearts (LPS + TI), aortic flow recovery was further enhanced (57 +/- 3.8%; P < 0.05 vs. TI or LPS alone). CK release during 20 and 30 min of reperfusion was decreased in all treated hearts compared with control hearts (P < 0.05). These results indicate that delayed myocardial adaptation and acute ischemic preconditioning independently activate protective mechanisms against ischemia. Enhanced protection occurs when induced delayed mechanisms are combined with acute cardioprotective stimuli, which optimize postischemic myocardial function and reduce myocellular necrosis.
VEGF effect on vascular permeability is mediated by synthesis of platelet-activating factor
Authors: Sirois M. G. , Edelman E. R.
Published: December 2017

Vascular endothelial growth factor (VEGF), a protein synthesized and secreted by a variety of normal and neoplastic cells, serves as an endothelial cell-specific mitogen and a potent angiogenic factor. Intradermal injection of VEGF increases vascular permeability, a critical event in inflammation and angiogenesis. We sought to identify which tissues are responsive to the inflammatory effect of VEGF, to investigate the mechanisms by which VEGF increases vascular permeability, and to establish whether mediators of inflammation such as platelet-activating factor (PAF) play a role in this regard. Intravenous injection of VEGF (0.001-0.1 nmol/kg) into rats induced dose-dependent protein extravasation in vascular beds of certain tissues up to 177% over control levels. In some tissues, the elevation in vascular permeability in vivo was abolished completely by selective PAF-receptor antagonists. Moreover, VEGF (10(-11) to 10(-9) M) was found to increase PAF synthesis in cultured bovine aortic endothelial cells up to 20-fold. Our results suggest that VEGF increases vascular permeability by inducing PAF synthesis.
Apoptosis in circulating PMN: increased susceptibility in L-selectin-deficient PMN
Authors: Matsuba K. T. , Van Eeden S. F. , Bicknell S. G. , Walker B. A. , Hayashi S. , Hogg J. C.
Published: December 2017

Previous work from our laboratory has shown that polymorphonuclear leukocytes (PMN) lose L-selectin as they age in the circulation. The present study was designed to examine the relationship between PMN age and susceptibility to apoptosis in the circulation using L-selectin as a marker of PMN age in rabbits. L-selectin-deficient leukocytes were separated from a mixed population of PMN in leukocyte-rich plasma using magnetic beads. Apoptosis was measured both with both morphological criteria and by determining the level of DNA fragmentation. The L-selectin-deficient cells separated in vitro showed morphological features of apoptosis (P < 0.01) and had higher levels of DNA fragmentation (P < 0.01) than the mixed population of PMN from which they were obtained. To determine if aging had a similar effect in vivo, PMN were labeled in the bone marrow with 5'-bromo-2'-deoxyuridine (BrdU) and L-selectin levels (immunocytochemistry) and DNA fragmentation (sandwich enzyme-linked immunosorbent assay) were measured in BrdU-labeled PMN in peripheral blood. The results showed that the peak release of BrdU-labeled PMN from the bone marrow into peripheral blood was associated with high levels of L-selectin expression, and these PMN had the lowest levels of DNA fragmentation. These results confirm that the level of L-selectin expression can be used as a marker of cell age and extend this observation by showing that aging in the circulation is associated with an increased susceptibility to apoptosis.
Prolongation of bleeding time by acute hemolysis in rats: a role for nitric oxide
Authors: Wollny T. , Iacoviello L. , Buczko W. , de Gaetano G. , Donati M. B.
Published: December 2017

The present study was aimed at clarifying the interaction between red blood cell trauma and bleeding observed in some clinical conditions. Acute hemolysis provoked by distilled water injection was followed by a significant prolongation of the "template" bleeding time in rats. Comparable effects were observed after injection of an isotonic lysate of washed red blood cells. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) formation from L-arginine, normalized bleeding time when given to rats before hemolysis induction. The occurrence of hemolysis decreased ex vivo platelet adhesion to collagen without affecting platelet aggregation and induced a transient drop in blood pressure, the latter occurring during the first minute after injection. L-NAME pretreatment increased ex vivo platelet adhesion but did not affect either platelet aggregation or fall in blood pressure. All the effects of L-NAME were blunted by treating the animals with the NO precursor L-arginine but not D-arginine. Incubation of the erythrocyte lysate with apyrase prevented the prolongation of bleeding time induced by the hemolysate. Moreover, ADP administration, at doses that did not increase hemoglobin levels, induced effects similar to those observed after hemolysis (on template bleeding time and ex vivo platelet adhesion), which were also reversed by L-NAME and restored by L-arginine. ADP is abundantly released from (hemo)lysed red blood cells and is known to stimulate release of NO, a potent vasodilator and inhibitor of platelet adhesion. ADP-dependent NO release could be responsible for bleeding time prolongation, due to abnormalities in platelet-vessel wall interaction, during acute hemolysis. Lysis of white blood cells may also contribute to prolongation of bleeding time. Because ADP could not be detected in these cells, we postulate that other mechanisms also can be involved in bleeding time prolongation after blood cell activation in vivo.
Lipopolysaccharide induces cell shrinkage in rabbit ventricular cardiac myocytes
Authors: Lew W. Y. , Ryan J. , Yasuda S.
Published: December 2017

The effects of 10 ng/ml of lipopolysaccharide (LPS) on cell volume were examined in rabbit left ventricular myocytes. The myocytes were isolated with depyrogenated digestive enzymes (< 0.7 ng/ml of LPS) to minimize tolerance. Myocyte cross-sectional area (CSA) did not change after 1 h of LPS. However after 8 h, the CSA decreased to 0.93 +/- 0.01 (SE) of the baseline CSA (time = 0) in 19 LPS-exposed myocytes compared with 1.00 +/- 0.01 in 13 control myocytes (P = 0.0015). LPS-induced cell shrinkage was completely blocked by coincubation with 1 mM N-monomethyl-L-arginine, indicating a nitric oxide-mediated mechanism. Cardiac guanosine 3',5'-cyclic monophosphate (cGMP) did not change after 1 h but increased 6 h after LPS (548 +/- 31 vs. 312 +/- 20 fmol/mg protein in control cells; P < 0.05). After 8 h, bumetanide (10 microM for 30 min), a Na+/K+/2Cl- cotransport inhibitor, decreased the CSA in 15 control myocytes to 0.92 +/- 0.02 of the baseline CSA. However, in 19 myocytes with a CSA of 0.93 +/- 0.01 of baseline after 8 h of LPS, the addition of bumetanide caused no additional cell shrinkage. We conclude that low levels of LPS increase cardiac cGMP to inhibit Na+/K+/2Cl- cotransport, causing significant cell shrinkage in cardiac myocytes.
Response of resistance arteries to reduced Po2 and vasodilators during hypertension and elevated salt intake
Authors: Liu Yanping , Fredricks Kim T. , Roman Richard J. , Lombard Julian H.
Published: December 2017

Pages H869–H877: Yanping Liu, Kim T. Fredricks, Richard J. Roman, and Julian H. Lombard. “Response of resistance arteries to reduced Po2 and vasodilators during hypertension and elevated salt intake.” On page H873, Fig. 7 should appear as below (units for iloprost concentration corrected on x-axis label).
Mechanism of enhancement of slow delayed rectifier current by extracellular sulfhydryl modification
Authors: Yao J. A. , Jiang M. , Tseng G. N.
Published: December 2017

To explore the role of sulfhydryl (SH) groups in the function of cardiac slow delayed rectifier channels, we tested the effects of extracellular thimerosal (TMS, a hydrophilic SH modifier) on slow delayed rectifier current (IKs) induced by human IsK (hIsK) in oocytes and on the native IKs in canine ventricular myocytes. TMS (25 or 50 microM) had similar effects on the two currents: current amplitude increased, and there was an acceleration of activation and a slowing of deactivation. These effects showed little or no reversal after washout of TMS. The effects did not depend on intracellular Ca release or protein kinase activities but could be suppressed by dithiothreitol pretreatment. According to the current model of transmembrane topology, there is no cystein in the extracellular domain of hIsK. A likely candidate for TMS modification is the SH group on another subunit in oocyte cell membrane that interacts with IsK to form a functional channel. To explore the domain of hIsK involved in the interaction, extracellular serines of hIsK were mutated to cysteines at three locations: S37C (close to the transmembrane domain), S4C (close to the NH2-terminus), and S28C (in between). S37C and S28C mutations did not affect channel properties or hIsK response to TMS. On the other hand, S4C mutation reduced current expression even when S4C cRNA was injected at a quantity 50-fold higher than that of the other three proteins. Importantly, the response to TMS was markedly reduced in S4C compared with the other three proteins. Therefore, the NH2-terminus of hIsK may be involved in hIsK interaction with the SH-bearing subunit, and this interaction modulates slow delayed rectifier channel function.
Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase
Authors: Ooboshi H. , Chu Y. , Rios C. D. , Faraci F. M. , Davidson B. L. , Heistad D. D.
Published: December 2017

Gene transfer with replication-deficient adenovirus is a potentially useful tool to study vascular biology. We have constructed a replication-deficient adenovirus (AdRSVeNOS) that carries cDNA for endothelial nitric oxide synthase (eNOS). Transfection of COS-1 cells with AdRSVeNOS increased nitric oxide synthase activity (measured as production of L-citrulline from L-arginine) that was calcium dependent and inhibited by N omega-nitro-L-arginine methyl ester. To investigate effects of overexpression of eNOS on vascular function, we incubated common carotid arteries from rabbits in organ culture with AdRSVeNOS or AdRSV beta gal encoding beta-galactosidase. Transgene expression and responses to vasoactive agents were examined 1 day after transduction. Histochemical staining of beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in endothelium and adventitial cells. After precontraction with phenylephrine, vessels treated with AdRSVeNOS demonstrated greater relaxation to acetylcholine than vessels treated with vehicle or AdRSV beta gal. Relaxation to calcium ionophore A-23187 was much greater in vessels treated with AdRSVeNOS than in vessels treated with vehicle or AdRSV beta gal. Augmented relaxation in response to A-23187 was also observed after denudation of endothelium in vessels treated with AdRSVeNOS and was inhibited by N omega-nitro-L-arginine. Thus vasorelaxation in response to stimuli that release nitric oxide is augmented after adenovirus-mediated overexpression of eNOS. Transgene expression in adventitial cells appears to be sufficient to alter vasomotor function.
Desensitization of norepinephrine receptor function is associated with G protein uncoupling in the rat aorta
Authors: Seasholtz T. M. , Gurdal H. , Wang H. Y. , Johnson M. D. , Friedman E.
Published: December 2017

Infusion of norepinephrine (NE) in rats results in desensitization of NE-mediated aortic contraction and a reduction (55% at 1 and 10 microM) in NE-stimulated vascular inositol phosphate accumulations. The functional responses to angiotensin II (ANG II) were also reduced in the tissues of NE-infused animals. alpha 1-Adrenoceptor number determined by 2-[beta-(4-hydroxy-3-[125I]iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) binding and levels of G alpha or G beta proteins measured by immunoblot analyses were not changed in the aortic membranes of NE-infused animals. To determine whether desensitization is associated with receptor-G protein uncoupling, agonist-stimulated palmitoylation of G alpha proteins was measured. NE infusion decreased phenylephrine (1 microM)-stimulated [3H]palmitate incorporation into Gq alpha, Gs alpha, and Gi alpha proteins and ANG II (10 microM)-stimulated palmitoylation of Gq alpha and Gi alpha in aortic membranes. Phenylephrine- and ANG II-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) binding to Gq alpha was also decreased in the aortas of NE-infused animals. These results show that an infusion of NE causes heterologous desensitization of the contractile and inositol phosphate accumulation responses in the rat aorta and that these changes are mediated by an uncoupling of receptors from their G proteins.
Cumulative conducted vasodilation within a single arteriole and the maximum conducted response
Authors: Rivers R. J.
Published: December 2017

The vascular network functions to distribute blood flow to the tissues that require it, and conducted vasodilation may facilitate this function. Experiments on arterioles in anesthetized hamster cheek pouch modeled the conducted responses that may come from a series of neighboring capillary modules and determined whether cumulative conducted responses could thereby maximally dilate upstream arterioles. Methacholine (10(-5) M) was simultaneously microapplied on an arteriole (resting diameter, approximately 22 microns; maximum diameter, approximately 47 microns) from one to four micropipettes spaced 100 microns apart, and with each added pipette the conducted dilation increased (up to a maximum dilation of approximately 5 microns). Increasing the methacholine 10-fold (10(-4) M) did not further increase the conducted response. The conducted response could also not be increased by lengthening the duration of microapplication. Yet, dilations that were not cumulative along a single arteriole became cumulative when initiated instead on adjacent arterioles. Therefore, these data demonstrate that conducted dilation along a single arteriole is limited and, if this model is correct, suggest that neighboring capillary modules may communicate only a limited conducted response to the network.
Role of cytoskeleton in shear stress-induced endothelial nitric oxide production
Authors: Knudsen H. L. , Frangos J. A.
Published: December 2017

To study the role of the cytoskeleton in mechanochemical transduction, human umbilical vein endothelial cells were exposed to cytoskeleton-disrupting or -stabilizing agents, and the flow-induced production of nitric oxide (NO) as monitored by intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) was examined. A shear stress of 20 dyn/cm2 elevated cGMP levels approximately twofold relative to basal (stationary) levels (1.9 +/- 0.1 pmol cGMP in stationary controls; P < 0.01). Treatment with 1 microM phalloidin and 0.5 microM cytochalasin D did not significantly affect the flow-induced response (1.77 +/- 0.23 and 2.89 +/- 0.18 pmol cGMP in stationary controls, respectively), whereas disruption of microtubules with 0.5 microM colchicine significantly elevated the response (3.64 +/- 0.18 pmol cGMP in stationary controls; P < 0.01). The NO synthase inhibitor NG-amino-L-arginine abrogated all flow-induced elevations of cGMP, indicating that increased cGMP levels were mediated by NO. Cytoskeletal disruption with 0.2 microM cytochalasin D or 0.5 microM colchicine did not alter cGMP levels in response to 10 nM bradykinin. The role of the plasma membrane in mechanochemical transduction was examined by treatment with cholesteryl hemisuccinate, which attenuated the flow-induced response in a dose-dependent manner. In conclusion, the pathways of flow- and bradykinin-mediated NO production in endothelial cells did not require actin filament turnover or intact actin or microtubule cytoskeletons, and cholesterol, possibly by stiffening the plasma membrane, attenuated the flow response.
17 beta-Estradiol attenuates endotoxin-induced excessive nitric oxide production in ovariectomized rats in vivo
Authors: Kauser K. , Sonnenberg D. , Tse J. , Rubanyi G. M.
Published: December 2017

Experiments were designed to examine the effect of 17 beta-estradiol on lipopolysaccharide (LPS)-induced excessive nitric oxide production in vivo. Ovariectomized and sham-operated female rats were injected with LPS (5 mg/kg ip), and different groups of ovariectomized, LPS-injected animals were treated with either 17 beta-estradiol, dexamethasone, or aminoguanidine. Nitric oxide generation was estimated by measuring plasma nitrite levels 12 h after LPS injection. LPS treatment of the rats resulted in a four- to fivefold increase in circulating plasma nitrite level, significantly higher in the ovariectomized animals compared with the sham-operated animals. The LPS-induced plasma nitrite increase could be prevented by dexamethasone (3 mg/kg ip) injected 1 h before LPS treatment. 17 beta-Estradiol (3 micrograms/rat sc), administered 48 h before LPS treatment, significantly attenuated the LPS-induced elevation in plasma nitrite levels. This effect was comparable to that achieved by aminoguanidine (200 microM/kg), an inhibitor of inducible nitric oxide synthase, injected 1 h after LPS treatment. The present findings suggest that estrogens may be beneficial in pathological conditions associated with excessive nitric oxide generation.
Contribution of asynchrony and nonuniformity to mechanical interaction in normal and stunned myocardium
Authors: Fan Dongsheng , Soei Loe Kie , Stubenitsky Rene , Boersma Eric , Duncker Dirk J. , Verdouw Pieter D. , Krams Rob
Published: December 2017

In anesthetized pigs, we investigated whether asynchrony (Δ T) and nonuniformity (regional differences) in contractility (Δ E) could describe the interaction between normal and stunned myocardium. Mechanical interaction was evaluated by regional postsystolic work (PSW) before and after production of stunning by a 5-min occlusion of the left circumflex coronary artery [LCX (LCX stunning)] and a subsequent 10-min occlusion of the left anterior descending coronary artery [LAD (LAD stunning)]. Δ T and Δ E were intensified by intracoronary (LAD) infusions of dobutamine. From regional end-systolic pressure-segment length relationships, systolic segment shortening (SS), end-systolic elastance ( E), external work (EW), and PSW were determined. LCX stunning decreased SSLCX from 14 ± 2 (mean ± SE, n = 9) to 10 ± 2% and E LCX from 103 ± 25 to 52 ± 7 mmHg/mm, whereas the LAD region was unaffected. EWLCX decreased from 165 ± 16 to 138 ± 20 mmHg ⋅ mm, whereas PSWLCX increased from −4 ± 6 to 8 ± 3 mmHg ⋅ mm. Additional LAD stunning reduced SSLAD from 16 ± 2 to 9 ± 3% and E LAD from 79 ± 10 to 31 ± 6 mmHg/mm, without affecting SSLCX and E LCX. In the normal myocardium, PSWLADincreased and PSWLCX decreased, but, during local LAD dobutamine infusions after stunning, both PSWLCX and PSWLAD increased. In normal myocardium, the changes in PSWLCXcould be described by Δ T (65 ± 11%) and Δ E(37 ± 15%). After stunning of the LAD area, the contribution of Δ E increased to 55 ± 14% at the expense of Δ T (37 ± 15%). Similar contributions of Δ E (54 ± 13%) and Δ T (57 ± 13%) were found when both the LCX and LAD distribution areas were stunned. In normal myocardium, both Δ T and Δ E modulate mechanical interaction, with the contribution of Δ Texceeding that of Δ E. In stunned myocardium, both factors contribute, but the contribution shifts in favor of Δ E.
Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells
Authors: Natarajan Rama , Bai Wei , Lanting Linda , Gonzales Noe , Nadler Jerry
Published: December 2017

Vascular endothelial growth factor (VEGF), in addition to its growth-promoting effects on endothelial cells, can also increase vascular permeability and monocyte migration. It has therefore been implicated in the pathogenic neovascularization associated with diabetic retinopathy and atherosclerosis. However, the factors regulating VEGF expression in the vascular wall are not fully understood. In this study, we examined the regulation of VEGF expression in vascular smooth muscle cells (VSMC) by hyperglycemia as well as by angiotensin II (ANG II). We also examined whether the 12-lipoxygenase (12-LO) product 12-hydroxyeicosatetraenoic acid (12-HETE) can alter VEGF expression, since 12-LO products of arachidonic acid have angiogenic properties, and ANG II as well as high glucose (HG, 25 mM) can increase 12-LO activity and expression in VSMC. Studies were carried out in human (HSMC) or porcine VSMC (PSMC), which were cultured for at least two passages under normal glucose (NG, 5.5 mM) or HG conditions. HG culture alone increased the expression of VEGF mRNA and protein in both HSMC and PSMC. Furthermore, ANG II treatment significantly induced VEGF mRNA and protein expression only in VSMC cultured in HG and not NG. In addition, 12-HETE significantly increased VEGF mRNA and protein expression in HSMC cultured in NG as well as in HG. Cells cultured in HG also secreted significantly greater amounts of VEGF into the culture medium. These results suggest that elevated VEGF production under HG conditions may play a role in the accelerated vascular disease observed in diabetes.
Protein, not adenosine or adenine nucleotides, mediates platelet decrease in endothelial permeability
Authors: Patil Sandeep , Kaplan John E. , Minnear Fred L.
Published: December 2017

Platelets and platelet-conditioned medium (PCM) decrease endothelial protein permeability in vitro. Adenosine and a >100-kDa protein have previously been implicated as the soluble factors released from platelets that decrease endothelial permeability. The objective of this study was to further investigate the role of adenosine in this platelet response. Measurements of adenosine and its precursor adenine nucleotides by high-performance liquid chromatography were correlated with the assessment of permeability by125I-labeled albumin clearance and electrical resistance across endothelial cell monolayers derived from the bovine pulmonary artery. PCM contained micromolar concentrations of AMP, ADP, and ATP, but adenosine was below detectable levels (≤0.1 μM). Adenosine deaminase, an enzyme that converts adenosine to inactive inosine, or an adenosine-receptor antagonist did not block the platelet- or PCM-mediated decrease in endothelial permeability. A <3-kDa fraction of PCM that contained micromolar concentrations of AMP and ADP did not affect endothelial permeability, whereas a >3-kDa fraction that contained much reduced levels of AMP and ADP significantly decreased permeability. This activity of PCM was sensitive to insoluble trypsin. This study rules out adenosine and adenine nucleotides as primary factors in the platelet-induced decrease in endothelial permeability and suggests that the active factor is a protein.
Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress
Authors: Schnittler Hans-Joachim , Püschel Bernd , Drenckhahn Detlev
Published: December 2017

The role of cadherins and the cadherin-binding cytosolic protein plakoglobin in intercellular adhesion was studied in cultured human umbilical venous endothelial cells exposed to fluid shear stress. Extracellular Ca2+depletion (<10−7 M) caused the disappearance of both cadherins and plakoglobin from junctions, whereas the distribution of platelet endothelial cell adhesion molecule 1 (PECAM-1) remained unchanged. Cells stayed fully attached to each other for several hours in low Ca2+ but began to dissociate under flow conditions. At the time of recalcification, vascular endothelial (VE) cadherin and β-catenin became first visible at junctions, followed by plakoglobin with a delay of ∼20 min. Full fluid shear stress stability of the junctions correlated with the time course of the reappearance of plakoglobin. Inhibition of plakoglobin expression by microinjection of antisense oligonucleotides did not interfere with the junctional association of VE-cadherin, PECAM-1, and β-catenin. The plakoglobin-deficient cells remained fully attached to each other under resting conditions but began to dissociate in response to flow. Shear stress-induced junctional dissociation was also observed in cultures of plakoglobin-depleted arterial endothelial cells of the porcine pulmonary trunk. These observations show that interendothelial adhesion under hydrodynamic but not resting conditions requires the junctional location of cadherins associated with plakoglobin. β-Catenin cannot functionally compensate for the junctional loss of plakoglobin, and PECAM-1-mediated adhesion is not sufficient for monolayer integrity under flow.
Heparin/heparan sulfate chelation inhibits control of vascular repair by tissue-engineered endothelial cells
Authors: Han Richard O. , Ettenson David S. , Koo Edward W. Y. , Edelman Elazer R.
Published: December 2017

The relative importance of heparin-like compounds in mediating vascular repair is unclear. We investigated how protamine, a chelator of heparin, affected endothelial cell inhibition of vascular smooth muscle cell growth and intimal hyperplasia. The 52% ( P < 0.001) reduction in smooth muscle cell proliferation produced by postconfluent endothelial cell-conditioned medium was entirely reversed by pretreatment of medium with heparinase and heparitinase and was inhibited in a dose-dependent fashion by the coadministration of protamine. Pretreatment of conditioned medium with heparinase and heparitinase largely prevented protamine’s mitogenic activity, suggesting that protamine affects growth by interacting with heparin-like compounds. Perivascular implantation of polymer-engrafted endothelial cells reduced neointima formation in denuded rat carotid arteries by 92% ( P < 0.001) and cell proliferation by 81% ( P < 0.001). Coadministration of protamine abolished the inhibitory potential of the cell implants, resulting in a nearly twofold exacerbation of intimal hyperplasia compared with controls ( P < 0.001). Thus heparin-like molecules are essential to the biochemical regulation of vascular repair provided by endothelial cells, and the continued routine clinical use of heparin chelators, like protamine, may be questionable.