Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy.

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

Biological aspects of reactive nitrogen species.

Nitric oxide (NO) plays an important role as a cell-signalling molecule, anti-infective agent and, as most recently recognised, an antioxidant. The metabolic fate of NO gives rise to a further series of compounds, collectively known as the reactive nitrogen species (RNS), which possess their own unique characteristics. In this review we discuss this emerging aspect of the NO field in the context of the formation of the RNS and what is known about their effects on biological systems. While much of the insight into the RNS has been gained from the extensive chemical characterisation of these species, to reveal biological consequences this approach must be complemented by direct measures of physiological function. Although we do not know the consequences of many of the dominant chemical reactions of RNS an intriguing aspect is now emerging. This review will illustrate how, when specificity and amplification through cell signalling mechanisms are taken into account, the less significant reactions, in terms of yield or rates, can explain many of the biological responses of exposure of cells or physiological systems to RNS.

Vascular endothelium viability and function after total cardiopulmonary bypass in neonatal piglets.

Endothelium dysfunction with severe pulmonary hypertension may occur after total cardiopulmonary bypass (CPB) in infants as a result of a widespread inflammatory response. The aim of this study was to separate out the effects of lung ischemia-reperfusion from membrane oxygenator-induced activation of leukocytes on the function and viability of the pulmonary and systemic endothelia in neonatal piglets submitted to 90-min total CPB followed by 60-min reperfusion or in sham animals. Hemodynamics, gas exchange, endothelial-dependent relaxation in pulmonary and femoral arteries, and lung and skeletal muscle myeloperoxidase activity were assessed before, during, and after CPB, i.e., after reperfusion. Pulmonary and aortic endothelial cells and circulating leukocytes were harvested to assess reperfusion-induced changes in endothelial cells' viability and proliferation, and leukocyte-endothelial cell adhesion and cytotoxicity. Gas exchange worsened after reperfusion with pulmonary hypertension, increase in lung but not skeletal myeloperoxidase, and reduction of endothelial-dependent relaxation in pulmonary but not femoral arteries. After reperfusion, viabilities of pulmonary and aortic endothelial cells were reduced to 50%, endothelial cell growths were faster in pulmonary arteries than aorta, and leukocyte-pulmonary endothelial cell adhesion and cytotoxicity increased. These results suggest that in total CPB lung ischemia-reperfusion aggravates the inflammatory response and predisposes the lung endothelium to leukocyte-mediated injury.

Inhaled nitric oxide attenuates reperfusion injury in non-heartbeating-donor lung transplantation. Paris-Sud University Lung Transplantation Group.

BACKGROUND: Non-heartbeating-donor (NHBD) lung transplantation could help reduce the current organ shortage. Polymorphonuclear neutrophil (PMN) activation plays a pivotal role in ischemia-reperfusion injury (I-R), and can be inhibited by nitric oxide (NO). We hypothesized that inhaled NO might be beneficial in NHBD lung transplantation. METHODS: The effect of inhaled NO on PMNs was studied by measuring in vivo PMN lung sequestration (myeloperoxidase activity) and adhesion of recipient circulating PMNs to cultured pulmonary artery endothelial cells (PAECs) in vitro. Pigs were randomly assigned to an NO or a control group (n=9 each). In the NO group, cadavers and recipients were ventilated with oxygen and 30 parts per million of NO. After 3 hr of postmortem in situ warm ischemia and 2 hr of cold ischemia, left allotransplantation was performed. The right pulmonary artery was ligated, and hemodynamic and gas exchange data were recorded hourly for 9 hr. Recipient PMN adherence to tumor necrosis factor-alpha- and calcium ionophore-stimulated PAECs was measured before and after reperfusion, and lung PMN sequestration was determined after death. RESULTS: NO-treated animals exhibited lowered pulmonary vascular resistance (P<0.01), as well as improved oxygenation (P<0.01) and survival (P<0.05). Adhesion of PMNs to PAECs was inhibited in the NO group before (P<0.001) and after reperfusion (P<0.0001). Lung PMN sequestration was reduced by NO (P<0.05). CONCLUSIONS: Inhaled NO attenuates I-R injury after NHBD lung transplantation. This is likely due to the prevention of I-R-induced pulmonary vasoconstriction and to the direct effect on peripheral blood PMN adhesion to endothelium, which results in reduced sequestration and tissue injury.

Prevention of ischemia-reperfusion lung injury by sulfated Lewis(a) pentasaccharide. The Paris-Sud University Lung Transplantation Group.

Inhibition of polymorphonuclear neutrophil (PMN) adhesion to the pulmonary endothelium attenuates ischemia-reperfusion (I/R) lung injury. We hypothesized that 3'-sulfated Lewis(a) (SuLa), a potent ligand for the selectin adhesion molecules, may have a beneficial effect on I/R lung injury, as measured by the filtration coefficient (K(fc)), and reduce pulmonary sequestration of PMN as assessed by the lung myeloperoxidase (MPO) activity. Blood-perfused rat lungs were subjected to 30 min of perfusion, 60 min of warm ischemia, and 90 min of reperfusion after treatment with either SuLa (200 microg) or saline. Effects of SuLa on PMN adhesion to cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha and calcium ionophore were also investigated. Compared with preischemia conditions, I/R induced a significant increase in K(fc), which was attenuated with SuLa (80 +/- 8 vs. 30 +/- 5%; P < 0.001). SuLa reduced lung MPO and PMN adhesion to stimulated HUVEC. These results indicate that SuLa reduces I/R-induced lung injury and PMN accumulation in lung. This protective effect might be related to inhibition of PMN adhesion to endothelial cells.

Gene therapy in lung transplantation: feasibility of ex vivo adenovirus-mediated gene transfer to the graft.

Lung transplantation is associated with complications such as reperfusion injury and graft rejection. Gene therapy targeted to the graft offers a promising approach to the prevention of these complications. Because adenovirus vectors can transfer genes in vivo to the lung vasculature, we evaluated the feasibility of adenovirus-mediated gene transfer to the lung graft in a porcine model of left lung allotransplantation. Following removal of the donor lung, an adenovirus vector encoding the beta-galactosidase (beta-Gal) gene was injected ex vivo into the lumen of the upper lobe pulmonary artery of the graft. After 2 hr of incubation at 10 degrees C, the lung graft was implanted into the recipient animal. Three days later, the animals were sacrificed and the lung graft was evaluated for beta-Gal activity. No beta-Gal activity was detected in the left lower lobe used as a control. In contrast, beta-Gal activity was detected in endothelial cells of the left upper lobe pulmonary circulation, and was also observed in airway and alveoli epithelial cells. However, less than 1% of cells of the graft expressed beta-Gal. In vitro experiments showed that this may be explained in part by the low temperature and the short duration of adenovirus incubation within the graft, and by the low susceptibility of porcine cells to human adenovirus. Furthermore, expression of the exogenous gene occurred in several organs of recipient animals. Thus, adenovirus-mediated gene transfer to the lung graft is feasible ex vivo, but several parameters limit gene transfer efficiency and need to be improved before clinical application is attempted.

Inhibition of human neutrophil binding to hydrogen peroxide-treated endothelial cells by cAMP and hydroxyl radical scavengers.

Hydrogen peroxide (H2O2) increases adherence of human polymorphonuclear neutrophils (PMN) to cultured human umbilical vein endothelial cells (HUVEC). Catalase and HO. scavengers did not affect the increased PMN adherence to HUVEC stimulated by other compounds such as phorbol myristate acetate (PMA) and thrombin, showing that the observed effect was H2O2- and HO.-specific. This effect was inhibited by hydroxyl radicals (HO.) scavengers and not by iron-chelators that do not penetrate the cells, suggesting the involvement of intracellular HO. in the increased adherence mechanism. An increase in cAMP inhibited H2O2-induced adherence, as observed with isoproterenol, isobutylmethylxanthine, and dibutyryl-cAMP. Similarly, pentoxifylline (Ptx), an HO. scavenger that also increases cAMP, inhibited H2O2-mediated adherence but had no effect on that induced by PMA or thrombin. PKA inhibitors cancelled the Ptx-induced inhibition of H2O2-mediated adherence. However, PKA inhibitors or atrial natriuretic peptide that decreases cAMP did not increase adherence, showing that decrease in cAMP is not responsible for increased adherence. HO. scavengers did not alter the H2O2-induced reduction in cAMP levels, but did inhibit the effect of H2O2 on adherence. We conclude that HO. mediates the H2O2-induced increased in PMN adherence to HUVEC, and that the increase in cAMP that mediates PKA activation downregulates this effect.

Anti-vascular endothelial cell antibodies (AECA): comparison of two assay methods and clinical applications.

Vascular endothelial cells may be a target for autoantibodies (AECAs) against membrane antigens that are constitutively expressed, induced or bound to their surface. To test this hypothesis, we used an enzyme-linked immunosorbent assay (ELISA) with two types of human endothelial cells as the substrate, i.e., human umbilical cord vein endothelial cells (HUVECs) or the hybrid cell line EAhy-926 obtained by fusion of HUVECs with the bronchial carcinoma cell line A549. A comparative functional study of these two cell types demonstrated that EAhy-926 cells produced only small amounts of VIII von Willebrand factor and tissular factor, did not contain Weibel Palade bodies visible under the electron microscope, and expressed ICAM-1 and selectin E in levels of no more than 15% of those expressed by human umbilical cord vein endothelial cells both after stimulation by bacterial lipopolysaccharide and under basal conditions. However, the two assay methods yielded similar IgG AECA titers when used on sera from patients with rheumatoid vasculitis or antiphospholipid syndrome. These antibodies did not exhibit cytotoxicity for cord vein or EAhy-926 cells. They were not specific for endothelium, since their activity decreased by a mean of 40% after incubation of sera with the epithelial cell line A549. A cross-sectional study of 565 sera demonstrated that anti-vascular IgG and IgM AECAs reactive with EAhy-926 cells occurred mainly in patients with dermatomyositis (IgG, 58%; IgM, 22%), systemic scleroderma (IgG, 48%; IgM, 18%), primary Sjögren's syndrome (IgG, 44%; IgM, 12%) and secondary and primary systemic vasculitides (IgG, 38%; IgM, 18%) including Wegener's granulomatosis. A longitudinal study in patients with Wegener's granulomatosis showed that AECAS were predictive of disease activity.

Effects of pentoxifylline on the adherence of polymorphonuclear neutrophils to oxidant-stimulated human endothelial cells: involvement of cyclic AMP.

Cultured human umbilical vein endothelial cells (HUVECs) treated with reactive oxygen species (ROS) show increased adherence of polymorphonuclear leukocytes (PMNs). Because pentoxifylline (PTX) is known to inhibit cell interactions, we studied PMN adherence to ROS-stimulated HUVECs pretreated with PTX. ROS were generated by the oxidation of hypoxanthine by xanthine oxidase, giving rise to superoxide anion and hydrogen peroxide. Human PMNs were then added to HUVEC monolayers. After various times, the cultures were washed and the number of adherent PMNs was estimated by measuring myeloperoxidase in the total cell homogenate. PTX inhibited adherence in a concentration-dependent manner. Moreover, the increase in intracellular cAMP content varied with the PTX concentration. Isobutylmethylxanthine (IBMX) and isoproterenol (ISO) which increase intracellular cAMP content, also inhibited the adherence of PMNs to ROS-stimulated HUVECs. We conclude that cAMP is probably involved in the intracellular regulation of ROS-mediated PMN adherence to endothelial cells.

Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation.

We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N-acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM-1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.

Comparative sugar degradation by (OH). produced by the iron-driven Fenton reaction and gamma radiolysis.

We compared the attack of deoxyribose and mannitol by (OH). produced by gamma radiolysis or the iron-driven Fenton reaction. Deoxyribose (DR) oxidation by gamma radiolysis gave rise to thiobarbituric reactive substances (TBARS) with a yield of 17 mol (OH). per mole of TBARS. (OH). scavengers (benzoate, formate, and pentoxifylline) protected DR from oxidation. Mannitol was similarly oxidized by (OH). produced by gamma radiolysis, with a yield of 14 mol (OH). per mole of TBARS produced. A mixture containing both DR and mannitol gave rise to TBARS production with a yield of 9 mol (OH). per mole of TBARS superior to that of each product separately, suggesting the formation of secondary radicals responsible for the degradation of DR or mannitol. When (OH). was produced by the iron-driven Fenton reaction, DR gave rise to TBARS whereas mannitol did not. Furthermore, mannitol protected DR against degradation, apparently in the same way as desferrioxamine and diethylenetriaminepentaacetic acid, suggesting that it acts as an iron chelator. It can thus be assumed that, according to the site of (OH). production, sugar molecules are degraded as a function of their rate constant with (OH). or their iron chelating capacity.

Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers.

The hydroxyl radical (OH.) is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH. scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH.-induced alterations of a protein, lysozyme. Lysozyme was inactivated by OH. with a yield of 6.5 mol OH./mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH. scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH. with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.