Biomedical subjects

J L Zweier

Publications and source records attributed to J L Zweier.

At least 109 records · Page 6Linked to original sources

PubMed1995

Enzyme-independent formation of nitric oxide in biological tissues.

The gaseous free radical nitric oxide (NO.) is an important regulator of a variety of biological functions and also has a role in the pathogenesis of cellular injury. It has been generally accepted that NO. is solely generated in biological tissues by specific nitric oxide synthases, NOSs, which metabolize arginine to citrulline with the formation of NO.. We report that NO. can also be generated in the ischaemic heart by direct reduction of nitrite to NO. under the acidotic and highly reduced conditions that occur. This NO. formation is not blocked by NOS inhibitors, and with long periods of ischaemia progressing to necrosis, this mechanism of NO. formation predominates. We observe that enzyme-independent NO. generation results in myocardial injury with a loss of contractile function. The existence of this enzyme-independent mechanism of NO. formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Animals↗

PubMed1995

Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport.

To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca(2+)-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.

Adenosine Triphosphate↗

PubMed1995

Spontaneous oxygen radical production at sites of antigen challenge in allergic subjects.

Allergic reactions in the lung are characterized by the production of mediators, an influx of inflammatory cells, increased vascular permeability, and changes in airway mechanics. The mechanisms responsible for these airway changes have not been fully defined but may involve the production of reactive oxygen species (ROS) produced by the inflammatory cells. To examine whether ROS are produced by inflammatory cells at sites of antigen exposure, bronchoalveolar lavage (BAL) was performed in airway segments 19 h after challenge with saline or antigen in 14 allergic subjects. Antigen challenge increased cell recovery, predominantly as a result of an influx of eosinophils. Using electron paramagnetic resonance (EPR) spectroscopy with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), BAL cells from saline-challenged sites produced minimal ROS. Cells from antigen-challenged sites spontaneously produced a prominent DMPO-OH signal that was inhibited by superoxide dismutase (SOD), indicating the production of superoxide anions (O2-.). Reduction of ferricytochrome c and production of luminol-dependent chemiluminescence via SOD-inhibitable reactions confirmed the spontaneous production of O2-. Following density gradient separation of the antigen-challenged BAL cells, the granulocytic cells, which were predominantly eosinophils, not the mononuclear cells, were the major source of the ROS. At the sites of antigen challenge, the degree of airway permeability as assessed by albumin concentration in BAL fluid was correlated with O2- production by BAL cells measured by EPR spectroscopy. These results demonstrate that cells at sites of antigen challenge generate ROS that may contribute to the airway injury associated with allergic inflammation.

Adult↗

PubMed1994

Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation.

Endothelial cell-derived oxygen free radicals are important mediators of postischemic injury; however, the mechanisms that trigger this radical generation are not known, and it is not known if this process can occur in human cells and tissues. The enzyme xanthine oxidase can be an important source of radical generation; however, it has been reported that this enzyme may not be present in human endothelium. To determine the presence and mechanisms of radical generation in human vascular endothelial cells subjected to anoxia and reoxygenation, electron paramagnetic resonance measurements were performed on cultured human aortic endothelial cells using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). These measurements were correlated with cellular injury, xanthine oxidase activity, and alterations in cellular nucleotides. Upon reoxygenation after 60 min of anoxia, large DMPO-OH (aN = aH = 14.9 G) and smaller DMPO-R (aN = 15.8 G, aH = 22.8 G) signals were seen. Superoxide dismutase totally quenched this radical generation. The ferric iron chelator deferoxamine prevented cell death and totally quenched the DMPO-R signal with a 40% decrease in the DMPO-OH signal. Xanthine oxidase was shown to be present in these cells and to be the primary source of free radicals. While the concentration of this enzyme did not change after anoxia, the concentration of its substrate, hypoxanthine, markedly increased, resulting in increased free radical generation upon reoxygenation. Thus, reoxygenated human vascular endothelial cells generate superoxide free radicals, which further react with iron to form the reactive hydroxyl radical, which in turn causes cell death. Xanthine oxidase was the primary source of radical generation with this process triggered by the breakdown of ATP to the substrate hypoxanthine during anoxia.

Aorta↗

PubMed1994

Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation.

It has been hypothesized that free radical metabolism and oxygenation in living organs and tissues such as the heart may vary over the spatially defined tissue structure. In an effort to study these spatially defined differences, we have developed electron paramagnetic resonance imaging instrumentation enabling the performance of three-dimensional spectral-spatial images of free radicals infused into the heart and large vessels. Using this instrumentation, high-quality three-dimensional spectral-spatial images of isolated perfused rat hearts and rabbit aortas are obtained. In the isolated aorta, it is shown that spatially and spectrally accurate images of the vessel lumen and wall could be obtained in this living vascular tissue. In the isolated rat heart, imaging experiments were performed to determine the kinetics of radical clearance at different spatial locations within the heart during myocardial ischemia. The kinetic data show the existence of regional and transmural differences in myocardial free radical clearance. It is further demonstrated that EPR imaging can be used to noninvasively measure spatially localized oxygen concentrations in the heart. Thus, the technique of spectral-spatial EPR imaging is shown to be a powerful tool in providing spatial information regarding the free radical distribution, metabolism, and tissue oxygenation in living biological organs and tissues.

Animals↗

PubMed1994

Measurement of the intracellular concentration of oxygen in a cell perfusion system.

[O2] was measured in the embedding material (alginate) in a typical apparatus for conducting studies of viable cells with NMR, using low frequency EPR. In suspension cultures respiration was independent of [O2] in the perfusing media down to about 1 microM while in alginate beads, the comparable value was 70 microM, indicating that the alginate was a very substantial barrier to the free diffusion of oxygen. With knowledge of [O2] in the various compartments, [O2] in the perfusing medium can be increased and the full power of NMR can be used to provide information on metabolism under various conditions. These results also provide evidence supporting the feasibility and usefulness of EPR techniques using nitroxides to measure [O2] in macroscopic samples such as NMR perfusion tubes. This technique is rapid, apparently nonperturbing, and enables one to differentiate between the concentrations of oxygen in different compartments.

Alginates↗

PubMed1994

Development of a highly sensitive probe for measuring oxygen in biological tissues.

The development of a new highly sensitive electron paramagnetic resonance probe suitable for measuring low oxygen concentrations in biological cells and tissues is reported. This probe consists of a char prepared from glucose. This char exhibits an EPR spectrum with marked oxygen-dependent linewidth broadening with a linewidth of 0.145 G under anaerobic conditions and a marked oxygen-dependent linewidth broadening of 14.5 G per percent oxygen. This marked oxygen-dependent broadening allows oxygen tensions to be measured down to mTorr values which was not previously possible with any other technique. This oximetry probe was applied to perform measurements to determine the severity of hypoxia in the ischemic rat heart. It was observed that after 10 minutes of ischemia, the oxygen concentration was 440 mTorr and it continued to fall over a 70 minute period to a value of 120 mTorr. Thus, absolute anoxia did not occur in the ischemic heart. The glucose char oximetry probe enables the measurement of oxygen in hypoxic cells and tissues with higher sensitivity than previously possible and therefore it can serve as a new important tool in characterizing the effects of low oxygen tensions in metabolic control and cellular injury.

Animals↗

PubMed1994

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions.

Superoxide anions (O2.-) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O2.-. Mixtures of xanthine, xanthine oxidase, and DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O2.-, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O2.-, generated by concentrations of xanthine as low as 0.05 microM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 microM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.

Cyclic N-Oxides↗

PubMed1994

Measurement and characterization of free radical generation in reoxygenated human endothelial cells.

The endothelial cell is thought to be an important site of free radical generation in ischemic tissues. It has been demonstrated that endothelial cells from several species generate a burst of free radical generation upon reoxygenation; however, it has been suggested that human endothelial cells are not similarly capable of generating free radicals on reoxygenation. In view of the central importance of revascularization with accompanying reoxygenation in the clinical treatment of tissue ischemia/infarction, we have performed studies to determine the presence, mechanism, and kinetics of free radical generation in human endothelial cells. Therefore, we subjected cultured human umbilical vein endothelial cells to anoxia followed by reoxygenation. Cell suspensions of 10(7) cells/ml were subjected to varying periods of anoxia and reoxygenation. On reoxygenation with addition of a 50 mM concentration of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), after 90 min of anoxia an electron paramagnetic resonance (EPR) signal was observed consisting of 2 components: a quartet 1:2:2:1 DMPO-OH signal, aN = aH = 14.9 G, and a six-peaked DMPO-R signal, aN = 15.6 G aH = 22.9 G, whereas cells in air gave no signal. The observed signal was quenched by superoxide dismutase (SOD) or catalase. Deferoxamine decreased the measured radical signals by 40%. Cyclooxygenase blockers did not decrease radical generation, but the xanthine oxidase blocker oxypurinol did decrease radical generation by 60%.

Animals↗

PubMed1994

Coenzyme Q10 enhances cardiac functional and metabolic recovery and reduces Ca2+ overload during postischemic reperfusion.

The effects of coenzyme Q10 (CoQ) were studied in isolated, isovolumic rat hearts during a 30-min period of global ischemia followed by 40 min of reperfusion. After reperfusion 1) the relative recovery of developed pressure (DP) was increased by CoQ (75 vs. 40% of the preischemic value for 20 microM CoQ and control hearts, respectively, P < 0.001); 2) diastolic pressure elevation was decreased by CoQ (20 vs. 50 mmHg in CoQ vs. control hearts, respectively, P < 0.001); and 3) recovery of high-energy phosphates and reduction of inorganic phosphate were approximately twofold greater in CoQ vs. control hearts (P < 0.001 for each parameter). The beneficial effects of CoQ were not observed when CoQ was added at the onset of reperfusion. The total free generation during reperfusion was not affected by CoQ. In unpaced hearts, in the presence of verapamil to prevent spontaneous beating, spontaneous Ca2+ oscillations were measured as scattered laser light intensity fluctuations (SLIF). The transient rise in SLIF in the postischemic reperfused myocardium, which previously has been shown to predict the extent of Ca2+ overload, was suppressed by CoQ (P < 0.001). These results suggest that while early CoQ treatment does not scavenge the primary burst of superoxide or hydroxy radical generation, which occurs on reperfusion, it markedly improves the functional recovery during reperfusion by enhancing the recovery of high-energy phosphates and preventing Ca2+ overload.

Animals↗

PubMed1994

Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store.

BACKGROUND: Increases in both leukocyte and endothelial cytosolic free [Ca2+] may be involved in intercellular adhesion by regulating the affinity of surface adhesion molecules or by facilitating transendothelial leukocyte migration. The purpose of this study was to examine the effect of initial contact and subsequent adhesion of human neutrophils or monocytes on human aortic endothelial [Ca2+]. METHODS AND RESULTS: Endothelial monolayers were loaded with the fluorescent Ca2+ indicator indo 1 and exposed to isolated human peripheral blood neutrophils or to a cultured human monocyte cell line. A rapid, fourfold to fivefold increase in endothelial cytosolic [Ca2+] occurred within seconds of leukocyte contact. No increase in endothelial [Ca2+] occurred on contact of 18.25-microns inert microspheres, isolated red blood cells, or suspensions of cultured human aortic endothelial cells. In experiments performed on monolayers grown in 1-mm2 capillary flow tubes, the increase in endothelial cytosolic [Ca2+] on initial leukocyte contact was found to be related to the subsequent resistance to leukocyte detachment during exposure to arterial levels of shear stress (13.4 dyne.cm-2). The increase in endothelial cytosolic [Ca2+] during leukocyte contact was not inhibited in Ca(2+)-free buffer but was abolished by prior depletion of an endoplasmic reticulum Ca2+ store by thapsigargin. Pretreatment of neutrophils with R15.7, a specific monoclonal antibody to the adhesion protein CD-18, inhibited the increase in endothelial cytosolic [Ca2+] on neutrophil contact. CONCLUSIONS: Initial contact leading to subsequent adhesion of human leukocytes to human aortic endothelial cells releases an endothelial intracellular Ca2+ store. This may, in part, be mediated by specific adhesion proteins and may in turn regulate the affinity of surface adhesion molecules or facilitate transendothelial migration of leukocytes.

Animals↗

PubMed1994

In vivo EPR spectroscopy of free radicals in the heart.

Electron paramagnetic resonance (EPR) spectroscopy can be applied to directly measure free radicals; however, it has not been possible to measure important biologic radicals in situ because conventional spectrometer designs are not suitable for the performance of measurements on large aqueous structures such as whole organs or tissues. We describe the design, construction, and application of instrumentation developed in an effort to obtain optimum performance in measuring free radicals in intact biologic organs or tissues. This spectrometer consists of a 1- to 2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed recessed gap, loop-gap resonator. The principles of resonator design and construction are analyzed and described. Using this spectrometer radical concentrations as low as 0.4 microM in aqueous solutions could be measured. Studies of isolated beating hearts involving simultaneous real time measurements of free radicals and cardiac contractile function are performed. This in vivo EPR technique is applied to study the kinetics of free radical uptake and metabolism in normally perfused and globally ischemic hearts. In addition, it is demonstrated that this technique can be used to noninvasively measure tissue oxygen consumption. Thus, low frequency EPR spectroscopy offers great promise in the study of in vivo free radical generation and the effects of this radical generation on whole biologic tissues.

Animals↗

PubMed1993

Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow.

Previous in vitro studies have shown that isolated mitochondria can generate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present study, we tested the hypothesis that resumption of mitochondrial respiration upon reperfusion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitors of mitochondrial respiration might prevent this phenomenon. Three groups of Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia at 37 degrees C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mM sodium amobarbital (Amytal, which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 5 mM potassium cyanide (to block mitochondrial respiration distally, at the level of cytochrome c oxidase). The hearts were then processed to directly evaluate oxygen radical generation by electron paramagnetic resonance spectroscopy, or to measure oxygen radical-induced membrane lipid peroxidation by malonyl dialdehyde (MDA) content of subcellular fractions. Severity of ischemia, as assessed by 31P-nuclear magnetic resonance measurements of cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered free radical concentration averaged 3.84 +/- 0.54 microM in reperfused control hearts, and it was significantly reduced by Amytal treatment (1.98 +/- 0.26; p < 0.05), but not by KCN (2.58 +/- 0.96 microM; p = not significant (NS)), consistent with oxygen radicals being formed in the mitochondrial respiratory chain at Site I. Membrane lipid peroxidation of reperfused hearts was also reduced by treatment with Amytal, but not with KCN. MDA content of the mitochondrial fraction averaged 0.75 +/- 0.06 nM/mg protein in controls, 0.72 +/- 0.06 in KCN-treated hearts, and 0.54 +/- 0.05 in Amytal-treated hearts (p < 0.05 versus both groups). Similarly, MDA content of lysosomal membrane fraction was 0.64 +/- 0.09 nM/mg protein in controls, 0.79 +/- 0.15 in KCN-treated hearts, and 0.43 +/- 0.06 in Amytal-treated hearts (p < 0.05 versus both groups). Since the effects of Amytal are known to be reversible, in a second series of experiments we investigated whether transient mitochondrial inhibition during the initial 10 min of reperfusion was also associated with beneficial effects on subsequent recovery of cardiac function after wash-out of the drug. At the end of the experiment, recovery of left ventricular end-diastolic and of developed pressure was significantly greater in those hearts that had been treated with Amytal during ischemia and early reflow, as compared to untreated hearts.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1993

Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Evidence for plasma-mediated leukocyte activation.

BACKGROUND: It has been hypothesized that chemotaxis and activation of polymorphonuclear leukocytes (PMNs) occur upon reperfusion of ischemic myocardium. Questions remain, however, regarding the mechanisms by which PMNs are chemotaxed and activated and how this process causes contractile failure. METHODS AND RESULTS: Studies were performed in an isolated rat heart model in which the effects of isolated cellular or humoral factors could be studied. Isolated rat hearts were perfused by the method of Langendorff, subjected to 20 minutes of global ischemia, and reperfused with perfusate alone or with perfusate containing PMNs, plasma, PMNs plus plasma, or PMNs plus inactivated plasma (preheated to 56 degrees C for 30 minutes to denature complement) (n = 10 in each group). Left ventricular developed pressure (LVDP) was measured during 1 minute of preischemic control infusion and on reflow after a 20-minute period of global ischemia. Additional measurements of free-radical generation were also performed on the coronary effluent by electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). During control infusion, no significant alterations in LVDP were observed, and there was no measurable free-radical generation. Reperfusion with plasma or PMNs alone did not alter postischemic LVDP, whereas plasma and PMNs together caused marked injury. LVDP after 45 minutes of reflow with PMNs plus plasma was 31.9 +/- 6.1% of control compared with 60.6 +/- 9.9% with plasma, 64.5 +/- 8.8% with PMNs, and 63.6 +/- 7.2% with perfusate alone (p < 0.01). With plasma, which was preheated to deplete complement, this injury was not seen; LVDP was 70.8 +/- 10.9%. EPR measurements with the spin trap DMPO in the absence of PMNs demonstrated that oxygen free-radical generation is observed only during the first 1-2 minutes of reflow. Upon reperfusion with PMNs and plasma, however, radical generation persisted for more than 10 minutes. Increased neutrophil accumulation was observed in the postischemic heart in the absence of plasma; however, plasma factors were required for neutrophil-mediated contractile failure. C5a alone did not cause significant injury, but in the presence of PMNs it effectively substituted for plasma, causing marked injury. CONCLUSIONS: Thus, plasma factors, most likely complement, are required for neutrophil activation with oxygen free-radical generation and secondary contractile dysfunction.

Animals↗

PubMed1993

Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia-reperfusion injury.

BACKGROUND: Previous studies have demonstrated that polymorphonuclear leukocytes (PMNs) are locally activated in reperfused myocardium and contribute to the myocardial cell injury associated with reperfusion. It has been suggested that the adhesion of activated PMNs in reperfused myocardium is mediated by the PMN adhesion molecule CD-18. In the present study, we performed experiments to determine if the specific anti-CD-18 monoclonal antibody (MAb) R15.7 can prevent PMN adhesion and PMN-mediated reperfusion injury in the heart. METHODS AND RESULTS: Studies were performed with isolated, Langendorff-perfused rat hearts (nine per group) in which the hearts were subjected to 20 minutes of global ischemia followed by 45 minutes of reperfusion. Human PMNs (50 million) and rat plasma (HNRP) were infused directly into the coronary circulation of nonischemic and postischemic hearts. When HNRP was administered to nonischemic hearts, no significant alterations in coronary flow, left ventricular developed pressure, or left ventricular end-diastolic pressure were observed. When hearts were reperfused in the presence of HNRP, however, marked impairment of contractile function was observed with more than 90% reduction in coronary flow throughout the reperfusion period (P < .001 versus baseline). In addition, left ventricular developed pressure was significantly depressed (P < .001 versus baseline) throughout the reperfusion period in the HNRP group and recovered to only 13.0 +/- 3.0% at 45 minutes of reperfusion. Moreover, left ventricular end-diastolic pressure was significantly elevated (P < .001) in the HNRP group throughout the reperfusion period. Treatment with the anti-CD-18 monoclonal antibody MAb R15.7 (20 micrograms/mL) at the time of reperfusion resulted in a 92.9 +/- 4.9% recovery of coronary flow (P < .001 versus HNRP) as well as a 71.0 +/- 10.1% recovery of left ventricular developed pressure (P < .001 versus HNRP). Administration of MAb R15.7 also very significantly attenuated the elevation in left ventricular end-diastolic pressure that was observed in the untreated HNRP (30.2 +/- 7.8 versus 110.3 +/- 10.3 mm Hg, P < .001) at 45 minutes of reperfusion. Cardiac myeloperoxidase activity, an index of PMN accumulation, was markedly reduced in the MAb R15.7 group at 45 minutes of reperfusion compared with the HNRP group (0.03 +/- 0.01 versus 0.3 +/- 0.05, P < .001). To determine that the protective effect of MAb R15.7 was based on functional blocking of CD-18, additional experiments were performed with identical concentrations of MAb 3.1, which binds to the alpha-subunit of LFA-1. This PMN-binding but non-CD-18-blocking antibody had little effect on the recovery of postischemic function or coronary flow and did not reduce tissue myeloperoxidase activity. CONCLUSIONS: The administration of a specific anti-CD-18 monoclonal antibody, MAb R15.7, attenuates much of the PMN-mediated contractile dysfunction associated with this in vitro model of myocardial ischemia-reperfusion injury by limiting PMN accumulation. We conclude that CD-18-mediated adhesion may play a critical role in the pathogenesis of PMN-induced myocardial injury.

Animals↗

PubMed1993

Soluble complement receptor type 1 inhibits the complement pathway and prevents contractile failure in the postischemic heart. Evidence that complement activation is required for neutrophil-mediated reperfusion injury.

BACKGROUND: Complement-mediated neutrophil activation has been hypothesized to be an important mechanism of reperfusion injury. It has been proposed that soluble complement receptor 1 (sCR1), a potent inhibitor of both classical and alternative complement pathways, may prevent the complement-dependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium and thereby inhibit PMN-derived free radical generation and prevent postischemic contractile failure. Therefore, we performed studies to determine the effects of sCR1 on contractile function, PMN adhesion, complement deposition, and PMN-derived free radical generation in the postischemic heart. METHODS AND RESULTS: Studies were performed in an isolated rat heart model in which the isolated effects of given cellular or humoral factors could be determined. Plasma and PMNs were present to study the effects of sCR1 on contractile function, coronary flow, leukocyte adhesion, complement deposition, and PMN-derived free radical generation. Isolated rat hearts were perfused by the method of Langendorff (n = 10 in each group) and subjected to 20 minutes of global ischemia and reperfusion with PMNs and plasma in the presence or absence of sCR1. Left ventricular developed pressure (LVDP), coronary flow (CF), left ventricular end-diastolic pressure (LVEDP), and rate-pressure product (RPP) were measured during the preischemic period, during 1-minute control infusion of PMNs and plasma, and on reflow following 20 minutes of global ischemia. During the preischemic control infusion, no significant alterations in the physiologic parameters were observed, and there was no measurable free radical generation. Reperfusion with sCR1 markedly improved the recovery of postischemic contractile function. LVDP after 45 minutes of reperfusion was 76 +/- 9.8% compared with 32 +/- 6.2% (P < .001). In addition, significant improvements in LVEDP, RPP, and CF were observed in hearts treated with sCR1. Additional experiments were also performed to determine the effect of sCR1 on complement-mediated PMN activation. Measurements of PMN-derived free radical generation were performed in both isolated PMNs and the coronary effluent of hearts using electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR measurements in both isolated PMNs and coronary effluent demonstrated that sCR1 blocked complement-mediated free radical generation from the PMNs. Increased accumulation of PMNs was observed both in hearts treated with sCR1 and in those not treated with sCR1. Immunohistochemical staining of the postischemic myocardial tissue demonstrated marked complement deposition on the endothelial surface of small arterioles and capillaries, which was prevented by sCR1 treatment. Thus, sCR1 did not prevent PMN adhesion but did prevent complement deposition with activation of the PMN oxidative burst. CONCLUSIONS: The potent complement inhibitor sCR1 was found to be effective at preventing postischemic myocardial contractile dysfunction and enhancing the recovery of coronary flow. This study demonstrated that complement activation occurs in postischemic myocardium and is necessary for activation of the neutrophil oxidative burst with the generation of reactive oxygen free radicals. The process of neutrophil adhesion, however, was not affected by sCR1 and was independent of complement factors. These findings demonstrate the sCR1 is a highly potent agent at preventing complement-mediated PMN activation and secondary free radical generation in the postischemic heart. This genetically engineered protein appears to be a promising therapeutic agent in the prevention of myocardial reperfusion injury.

Animals↗

PubMed1993

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Animals↗

PubMed1992

Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase.

OBJECTIVES: The purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow. BACKGROUND: This phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts. METHODS: Thirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 degrees K. RESULTS: Three radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with g parallel = 2.033 and g perpendicular = 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and aN = 24 G suggestive of a nitrogen-centered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 +/- 0.8 mumol compared with 4.4 +/- 0.9 mumol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 +/- 2.5 mumol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 +/- 1.2 mumol (p < 0.05 vs. Group III). CONCLUSIONS: This study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.

Animals↗