W. Kossenjans

Oxidative Stress Causes Vascular Dysfunction in the Placenta

Increased production of superoxide and nitric oxide may produce oxidative stress in the placenta by formation of the prooxidant peroxynitrite, which itself causes vascular dysfunction. Nitrotyrosine residues, which are a marker of peroxynitrite formation and action, are found in placental vessels of preeclamptic and diabetic pregnancies, indicating oxidative stress. Treatment of the placental vasculature with authentic peroxynitrite in vitro attenuates responses both to vasoconstrictors such as the thromboxane mimetic U46619 and to vasodilators, including glyceryl trinitrate and prostacyclin, indicating it has caused vascular dysfunction. Further, the responses of the fetal-placental vasculature of diabetic and preeclamptic placentae to these same vasoconstrictor and vasodilator agents are significantly attenuated when compared to responses in normal control placentae. Together these data suggest there may be a cause and effect relationship between formation and action of peroxynitrite and vascular dysfunction in the placenta of both preeclamptic and diabetic pregnancies. The presence of such attenuated vascular responses indicates that perhaps the placenta may not be able to adequately respond to demands for altered blood flow in situations where this is necessary in preeclamptic or diabetic pregnancies, thus leading to further fetal compromise.

Comparative localization of endothelial and inducible nitric oxide synthase isoforms in haemochorial and epitheliochorial placentae.

The presence and immunolocalization of type II (inducible or macrophage) and type III (endothelial) nitric oxide synthase (NOS) isoforms were compared in the term placentae of humans, rhesus monkeys, baboons, guinea-pigs, rats and sheep using isoform specific antibodies. In the human placenta, intense immunohistochemical staining for type III NOS was seen in syncytiotrophoblast with weaker staining in vascular endothelial cells. Only vascular endothelial cells showed positive type III NOS staining in rhesus monkey, baboon, guinea-pig, rat and sheep placentae. No positive type III NOS immunostaining was seen in trophoblast from any non-human placentae. Western blotting revealed a 135-kDa type III NOS species in placental homogenates, semi-purified by ADP-sepharose affinity chromatography, from all the species tested confirming antibody specificity. Type II NOS immunostaining was localized to certain villous stromal cells which also stained for CD14 (a monocyte/macrophage marker) in the placenta of humans, rhesus monkeys, baboons and sheep. No specific immunohistochemical staining for type II NOS or CD14 was noted in the two rodent species, guinea-pig and rat. On Western blots, a 130-kDa type II NOS species was identified in semi-purified placental homogenates of every species except guinea-pig, although weak bands were seen for rhesus monkey and baboon. The failure of the antibodies to show type II NOS in the rat placenta by immunohistochemistry may be due to a difference in antigen conformation from Western blots. As only human placental syncytiotrophoblast expresses type III NOS, the putative functions ascribed to this isoform in syncytiotrophoblast, i.e. to prevent platelet and leucocyte aggregation in the intervillous space and adhesion to the trophoblast surface or to mediate peptide hormone release from trophoblast, may be unique to humans. Alternatively, syncytiotrophoblast-derived NO may fulfil some other unknown function. The similar pattern of expression of type II NOS in those species with villous fetomaternal interdigitation and multivillous fetomaternal blood flow interrelations may represent a more universal role in surveillance and/or protection against maternal insults or pathogens by immunologic activation and subsequent synthesis of nitric oxide which exerts a cytostatic/cytotoxic response.

Differential localization of superoxide dismutase isoforms in placental villous tissue of normotensive, pre-eclamptic, and intrauterine growth- restricted pregnancies

Several isoforms of superoxide dismutase (SOD), including copper/zinc (cytosolic) and manganese (mitochondrial), exist. In the human placenta, SOD may prevent excessive superoxide accumulation and any potential deleterious oxidative effects. In pre-eclampsia, increased levels of lipid peroxide and decreased SOD activity have been described in the placenta. Oxidative stress such as occurs in pre-eclampsia can alter expression of SOD isoforms. The objective of this study was to localize the copper/zinc and manganese SOD isoforms in the placenta using immunohistochemistry and to compare localization and intensity of immunostaining in tissues from normotensive pregnancies with those from pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR). Western blotting with specific antibodies recognized a 17-kD copper/zinc and a 23-kD manganese SOD subunit in placental homogenates. Intense immunostaining for the manganese SOD isoform was seen in villous vascular endothelium, but only faint staining was found in the syncytiotrophoblast or villous stroma. In serial sections, intense immunostaining for copper/zinc SOD was seen in certain cells of the villous stroma but only faint immunostaining in syncytiotrophoblast and vascular endothelium. No apparent differences in localization or intensity of immunostaining for either isoform were seen between tissues of normotensive or pre-eclamptic pregnancies, with or without IUGR. The different cellular localizations of the SOD isoforms suggest that they fulfill different functional roles within the placenta.

The action of two natriuretic peptides (atrial natriuretic peptide and brain natriuretic peptide) in the human placental vasculature

OBJECTIVEnOur purpose was to compare the actions of atrial natriuretic peptide and brain natriuretic peptide in the human placental vasculature.nnnSTUDY DESIGNnIsolated placental cotyledons were dually perfused with fetal perfusion pressure used as an index of vascular response. The effect of angiotensin II (10(-10) to 10(-6) mol/L bolus injection) was established in the absence or presence of atrial natriuretic peptide (10(-8) mol/L) or brain natriuretic peptide (10(-8) mol/L final concentration). The role of nitric oxide as a mediator of natriuretic peptide action was investigated by perfusion of n-nitro-L-arginine (10(-3) mol/L), an inhibitor of nitric oxide synthase. Attenuation of the action of atrial natriuretic peptide by placental peptidases was studied by perfusion with the peptidase inhibitor benzamidine (2 x 10(-2) mol/L). Statistical significance was determined by analysis of variance and paired t test.nnnRESULTSnSignificant attenuation of vasoconstrictor responses to angiotensin II occurred within both atrial natriuretic peptide and brain natriuretic peptide; however, brain natriuretic peptide was more effective. n-Nitro-L-arginine did not affect the attenuation of angiotensin II-induced vasoconstriction by atrial or brain natriuretic peptides. In the presence of benzamidine atrial natriuretic peptide exerted a significantly greater vasodilator effect.nnnCONCLUSIONnBrain natriuretic peptide is a more potent vasodilator of the placental vasculature than is atrial natriuretic peptide. The low efficacy of atrial natriuretic peptide may be related to placental peptidases. Nitric oxide does not mediate the action of atrial natriuretic peptide or brain natriuretic peptide.

Menadione-Induced Oxidative Stress in Bovine Heart Microvascular Endothelial Cellsa

Objective: Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione‐induced oxidative stress in bovine heart microvascular endothelial cells.

Selective Vasodilator Effects of Atrial Natriuretic Peptide in the Human Placental Vasculature

Objective: To determine whether atrial natriuretic peptide (ANP) attenuates the vasocon-strictor effects of angiotensin II (AII), a thromboxane mimetic (U46619), and endothelin-1 in the human fetal-placental vasculature and to determine whether nitric oxide (NO) has a role in the vasodilator activity of ANP. Methods: Isolated placental cotyledons were dually perfused, with fetal perfusion pressure used as an index of vascular response. The effects of AII (10-10-10-6 mol/L bolus injection), endothelin-1 (10-7 mol/L bolus), and U46619 (10-9-10-6 mol/L bolus or 10-8 mol/L infusion) were established in the absence or presence of ANP (10-8 mol/L). The role of NO as a mediator of ANP action was investigated by perfusion with n-nitro-L-arginine (NNLA, 10-3 mol/L), an inhibitor of NO synthase. Statistical significance was determined by analysis of variance. Results: Atrial nutriuretic peptide caused significant attenuation of vasoconstrictor responses to AII, but weak attenuation of endothelin-1 and no attenuation of U46619. Use of NNLA did not affect the attenuation of AII-induced vasoconstriction by ANP. Conclusion: Atrial natriuretic peptide is a vasodilator of the fetal-placental vasculature constricted with AII and endothelin-1, but not with U46619. Nitric oxide does not mediate the action of ANP.

Inhibition of nucleoside transport by reactive oxygen species in bovine heart microvascular endothelial cells.

Cellular homeostasis depends on the structural and functional integrity of the membrane bilayer. Damage to the membrane can interfere with vital cellular processes, including signal transduction, molecular recognition, maintenance of the membrane potential, cellular metabolism and transport of molecules. Modification of the membrane bilayer by reactive oxidative species (ROS) is a major contributor to membrane damage and has been implicated in many pathological processes. In a number of diseases, injury to endothelial cells is mediated by oxidative species generated during ischemia/reperfusion or by activated neutrophils. During ischemia and oxidant injury, several metabolic events occur, including depletion of intracellular ATP and formation of a number of purine catabolites including inosine (Ino), hypoxanthine (Hyp) and adenosine (Ado) (Halliwell and Gutteridge, 1990). Accumulated Hyp can be oxidized by xanthine oxidase when the tissue is oxygenated, causing rapid generation of Superoxide and hydrogen peroxide. On the other hand, formation of Ado is especially important because it has vasodilatory and antiinflammatory activity. Alterations in nucleoside transport (NT) by ROS might have an effect on extracellular Ado concentration in vivo. Reduced Ado transport could elevate Ado concentrations, especially when Ado is formed extracellularly. Inhibition of NT could also diminish extracellular nucleoside uptake required for the reconstitution of intracellular nucleotide pools after cellular stress. Decreased nucleoside efflux might protect cells by allowing more efficient intracellular nucleoside salvage.

Interaction of angiotensin II and brain natriuretic peptide in the placentas of normal and diabetic women

Objective To evaluate the effects of angiotensin II and brain natriuretic peptide on the placental vasculature of diabetic women. Methods Term placentas from five diabetic women and five nondiabetic controls were collected. Isolated placental cotyledons were perfused dually with fetal perfusion pressure as an index of vascular response. The effect of angiotensin II (10−10–10−5 mol/L bolus injection) was established in the fetal-placental vasculature of all placentas in the absence or presence of brain natriuretic peptide (10−8 mol/L final concentration). Data were analyzed using repeated measures analysis of variance and paired t test where appropriate. Results A significant vasoconstrictor response to angiotensin II was achieved in placentas of both diabetic and nondiabetic women (P < .001); however, the angiotensin II-induced increase in perfusion pressure was significantly greater in the diabetic group (P < .01). Significant attenuation of vasoconstrictor response to angiotensin occurred in the presence of brain natriuretic peptide in placentas of both nondiabetic (P < .0025) and diabetic (P < .025) women, but the effect was more prominent in the diabetic group. Conclusion The in vitro placental vasculature of diabetic women is more sensitive to angiotensin II than is the in vitro placental vasculature of nondiabetic women. The attenuation exerted by brain natriuretic peptide on angiotensin II-induced vasoconstriction is more prominent in placentas from diabetic women compared to those from nondiabetic women.

Comparative localization of endothelial and inducible nitric oxide synthase isoforms in hemochorial and epitheliochorial placentas

The presence and immunolocalization of type II (inducible or macrophage) and type III (endothelial) nitric oxide synthase (NOS) isoforms were compared in the term placentae of humans, rhesus monkeys, baboons, guinea-pigs, rats and sheep using isoform specific antibodies. In the human placenta, intense immunohistochemical staining for type III NOS was seen in syncytiotrophoblast with weaker staining in vascular endothelial cells. Only vascular endothelial cells showed positive III NOS staining in rhesus monkey, baboon, guinea-pig, rat and sheep placentae. No positive type III NOS immunostaining was seen in trophoblast from any non-human placentae. Western blotting revealed a 135-kDa type III NOS species in placental homogenates, semi-purified by ADP-sepharose affinity chromatography, from all the species tested confirming antibody specificity. Type II NOS immunostaining was localized to certain villous stromal cells which also stained for CD14 (a monocyte/macrophage marker) in the placenta of humans, rhesus monkeys, baboons and sheep. No specific immunohistochemical staining for type II NOS or CD14 was noted in the two rodent species, guinea-pig and rat. On Western blots, a 130-kDa type II NOS species was identified in semi-purified placental homogenates of every species except guinea-pig, although weak bands were seen for rhesus monkey and baboon. The failure of the antibodies to show type II NOS in the rat placenta by immunohistochemistry may be due to a difference in antigen conformation from Western blots. As only human placental syncytiotrophoblast expresses type III NOS, the putative functions ascribed to this isoform in syncytiotrophoblast, i.e., to prevent platelet and leucocyte aggregation in the intervillous space and adhesion to the trophoblast surface or to mediate peptide hormone release from trophoblast, may be unique to humans. Alternatively, syncytiotrophoblast-derived NO may fulfill some other unknown function. The similar pattern of expression of type II NOS in those species with villous fetomaternal interdigitation and multivillous fetomaternal blood flow interrelations may represent a more universal role in surveillance and/or protection against maternal insults or pathogens by immunologic activation and subsequent synthesis of nitric oxide which exerts a cytostatic/cytotoxic response.