Leslie Myatt

Inflammation and Pregnancy

Inflammation is a process by which tissues respond to various insults. It is characterized by upregulation of chemokines, cytokines, and pattern recognition receptors that sense microbes and tissue breakdown products. During pregnancy, the balance of Th1 (cell-mediated immunity) and Th2 (humoral immunity) cytokines is characterized by an initial prevalence of Th2 cytokines, followed by a progressive shift toward Th1 predominance late in gestation, that when is abnormal, may initiate and intensify the cascade of inflammatory cytokine production involved in adverse pregnancy outcomes. Maternal and placental hormones may affect the inflammatory pathway. Hypoxia and the innate immune response are 2 adaptive mechanisms by which organisms respond to perturbation in organ function, playing a major role in spontaneous abortion, intrauterine growth restriction, preeclampsia, and preterm delivery. The interaction between tissue remodeling factors, like matrix metalloproteinases, and vasoactive/hemostatic factors, like prostaglandin and coagulation factors, mediates this adaptive response.

Oxidative stress in the placenta

Pregnancy is a state of oxidative stress arising from increased placental mitochondrial activity and production of reactive oxygen species (ROS), mainly superoxide anion. The placenta also produces other ROS including nitric oxide, carbon monoxide, and peroxynitrite which have pronounced effects on placental function including trophoblast proliferation and differentiation and vascular reactivity. Excessive production of ROS may occur at certain windows in placental development and in pathologic pregnancies, such as those complicated by preeclampsia and/or IUGR, overpowering antioxidant defenses with deleterious outcome. In the first trimester, establishment of blood flow into the intervillous space is associated with a burst of oxidative stress. The inability to mount an effective antioxidant defense against this results in early pregnancy loss. In late gestation increased oxidative stress is seen in pregnancies complicated by diabetes, IUGR, and preeclampsia in association with increased trophoblast apoptosis and deportation and altered placental vascular reactivity. Evidence for this oxidative stress includes increased lipid peroxides and isoprostanes and decreased expression and activity of antioxidants. The interaction of nitric oxide and superoxide produces peroxynitrite, a powerful prooxidant with diverse deleterious effects including nitration of tyrosine residues on proteins thus altering function. Nitrative stress, subsequent to oxidative stress is seen in the placenta in preeclampsia and diabetes in association with altered placental function.

Placental adaptive responses and fetal programming

Fetal programming occurs when the normal pattern of fetal development is disrupted by an abnormal stimulus or ‘insult’ applied at a critical point in in utero development. This then leads to an effect, for example diabetes or hypertension, which manifests itself in adult life. As the placenta is the regulator of nutrient composition and supply from mother to fetus and the source of hormonal signals that affect maternal and fetal metabolism, appropriate development of the placenta is crucial to normal fetal development. Placental function evolves in a carefully orchestrated developmental cascade throughout gestation. Disruption of this cascade can lead to abnormal development of the placental vasculature or of the trophoblast. Timing of a developmental ‘insult’ will be critical in consequent placental function and hence programming of the fetus. The ‘insults’ that alter placental development include hypoxia and abnormal maternal nutrient status, to which the placenta may adapt by alterations in transporter expression and activity to maintain fetal growth or by epigenetic regulation of placental gene expression. Hypoxia is physiological for organogenesis and placental tissue normally exists in a relatively hypoxic environment, but intrauterine growth restriction (IUGR) and pre‐eclampsia are associated with a greater degree of trophoblast hypoxia. The metabolic activity of placental mitochondria leads to oxidative stress even in normal pregnancy which is exacerbated further in IUGR, diabetic and pre‐eclamptic pregnancies and may also give nitrative stress known to lead to covalent modification and hence altered activity of proteins. Hypoxia, oxidative and nitrative stress all alter placenta development and may be a general underlying mechanism that links altered placental function to fetal programming.

Vitamins C and E to prevent complications of pregnancy-associated hypertension

BACKGROUND Oxidative stress has been proposed as a mechanism linking the poor placental perfusion characteristic of preeclampsia with the clinical manifestations of the disorder. We assessed the effects of antioxidant supplementation with vitamins C and E, initiated early in pregnancy, on the risk of serious adverse maternal, fetal, and neonatal outcomes related to pregnancy-associated hypertension. METHODS We conducted a multicenter, randomized, double-blind trial involving nulliparous women who were at low risk for preeclampsia. Women were randomly assigned to begin daily supplementation with 1000 mg of vitamin C and 400 IU of vitamin E or matching placebo between the 9th and 16th weeks of pregnancy. The primary outcome was severe pregnancy-associated hypertension alone or severe or mild hypertension with elevated liver-enzyme levels, thrombocytopenia, elevated serum creatinine levels, eclamptic seizure, medically indicated preterm birth, fetal-growth restriction, or perinatal death. RESULTS A total of 10,154 women underwent randomization. The two groups were similar with respect to baseline characteristics and adherence to the study drug. Outcome data were available for 9969 women. There was no significant difference between the vitamin and placebo groups in the rates of the primary outcome (6.1% and 5.7%, respectively; relative risk in the vitamin group, 1.07; 95% confidence interval [CI], 0.91 to 1.25) or in the rates of preeclampsia (7.2% and 6.7%, respectively; relative risk, 1.07; 95% CI, 0.93 to 1.24). Rates of adverse perinatal outcomes did not differ significantly between the groups. CONCLUSIONS Vitamin C and E supplementation initiated in the 9th to 16th week of pregnancy in an unselected cohort of low-risk, nulliparous women did not reduce the rate of adverse maternal or perinatal outcomes related to pregnancy-associated hypertension (ClinicalTrials.gov number, NCT00135707).

Nitrotyrosine Residues in Placenta: Evidence of Peroxynitrite Formation and Action

The interaction of nitric oxide and superoxide produces peroxynitrite anion, a strong, long-lived oxidant with pronounced deleterious effects that may cause vascular damage. The formation and action of peroxynitrite can be detected by immunohistochemical localization of nitrotyrosine residues. We compared the presence and localization of nitrotyrosine and of the endothelial isoform of nitric oxide synthase in placental villous tissue from normotensive pregnancies (n = 5) with pregnancies complicated by preeclampsia (n = 5), intrauterine growth restriction (n = 5), and preeclampsia plus intrauterine growth restriction (n = 4), conditions characterized by increases in fetoplacental vascular resistance, fetal platelet consumption, and fetal morbidity and mortality. In all tissues, absent or faint nitrotyrosine immunostaining but prominent nitric oxide synthase immunostaining were found in syncytiotrophoblast. In tissues from normotensive pregnancies, faint nitrotyrosine immunostaining was found in vascular endothelium, and nitric oxide synthase was present in stem villous endothelium but not in the terminal villous capillary endothelium. In contrast, in preeclampsia and/or intrauterine growth restriction, moderate to intense nitrotyrosine immunostaining was seen in villous vascular endothelium, and immunostaining was also seen in surrounding vascular smooth muscle and villous stroma. The intensity of nitrotyrosine immunostaining in preeclampsia (with or without intrauterine growth restriction) was significantly greater than that of controls. Intense nitric oxide synthase staining was seen in endothelium of stem villous vessels and the small muscular arteries of the terminal villous region in these tissues and may be an adaptive response to the increased resistance. The presence of nitrotyrosine residues, particularly in the endothelium, may indicate the formation and action of peroxynitrite, resulting in vascular damage that contributes to the increased placental vascular resistance.

Review: Reactive oxygen and nitrogen species and functional adaptation of the placenta

The placenta regulates fetal growth and development via transport of nutrients and gases, and synthesis and secretion of steroid and peptide hormones. These functions are determined by vascular development and blood flow and by growth and differentiation of the trophoblast, which contains receptors, transporters and enzymes. The placenta generates reactive oxygen species which may contribute to the oxidative stress seen even in normal pregnancy but this is increased in pregnancies complicated by preeclampsia, IUGR and pregestational diabetes where oxidative and nitrative stress have been clearly documented. Nitrative stress is the covalent modification of proteins and DNA by peroxynitrite formed by the interaction of superoxide and nitric oxide. We have demonstrated nitrative stress by localizing nitrotyrosine residues in these placentas and found increased expression of NADPH oxidase (NOX) enzyme isoforms 1 and 5 as a potential source of superoxide generation. The presence of nitrative stress was associated with diminished vascular reactivity of the fetal placental circulation, a situation that could be reproduced by treatment with peroxynitrite in vitro. We find many nitrated proteins in the placenta, including p38 MAP kinase which has a role in development of the villous vasculature. Nitration of p38 MAPK was increased in the preeclamptic placenta and associated with loss of catalytic activity. We hypothesize that nitration of proteins in the placenta including receptors, transporters, enzymes and structural proteins can alter protein and placental function and this influences fetal growth and development. Increasing nitrative stress but a decrease in oxidative stress, measured as protein carbonylation, is found in the placenta with increasing BMI. Formation of peroxynitrite may then consume superoxide, decreasing nitrative stress. As protein carbonylation is a covalent modification at Lys, Arg, Pro and Thr residues the switch from carbonylation to nitration at tyrosine residues may alter protein function and hence placental function.

THE ACTION OF NITRIC OXIDE IN THE PERFUSED HUMAN FETAL-PLACENTAL CIRCULATION

Nitric oxide is thought to be the endogenous endothelium-derived relaxing factor. We investigated the effects of compounds that either generate nitric oxide intracellularly or inhibit its action on the vasculature of the human placental villus. Addition to perfusion medium of methylene blue (10(5) mol/L), which is an inhibitor of activation of guanylate cyclase by nitric oxide, significantly increased perfusion pressure of the fetal-placental circulation over a range of flow rates (1 to 10 ml/min) compared with the perfusion pressures seen in the absence of methylene blue. This suggests basal release of nitric oxide may contribute to maintenance of resting vascular tone. Both glyceryl trinitrate (10(-9) to 5 x 10(6) mol/L) and S-nitroso-N-acetylpenicillamine (10(-8) to 10(-4) mol/L), which generate nitric oxide intracellularly, were able to significantly vasodilate the fetal-placental circulation preconstricted with the thromboxane mimetic U46619 (1 to 5 x 10(-8) mol/L) in a concentration-dependent manner. These compounds had no effect in the absence of the vasoconstrictor. Thus it appears that the placental villus tree has the ability to both generate and respond to nitric oxide.

Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation

OBJECTIVE We hypothesized that the endothelial-derived relaxing factor nitric oxide may contribute to low resting vascular tone and may attenuate vasoconstrictor action in the human fetal-placental circulation. STUDY DESIGN Isolated human placental cotyledons were dually perfused in vitro, and the effects of N-monomethyl-L-arginine and N-nitro-L-arginine (3 x 10(-4) mol/L), which are nonmetabolizable analogs of L-arginine, the substrate for nitric oxide synthase, on resting perfusion pressure and on the fetal-placental circulation preconstricted with U46619 (10(-8) mol/L) or endothelin-1 (10(-8) mol/L) were established. Responses before and after inhibition were compared by paired t test. The effects of glyceryl trinitrate (10(-6) mol/L), acetylcholine (10(-4) mol/L), the calcium ionophore A23187 (10(-6) mol/L), and histamine (10(-8) to 10(-4) mol/L) were also determined in the preconstricted fetal-placental circulation. RESULTS Both N-monomethyl-L-arginine and N-nitro-L-arginine (3 x 10(-4) mol/L) increased resting perfusion pressure (p less than 0.06), and N-nitro-L-arginine promptly and significantly increased perfusion pressure in the fetal-placental circulation preconstricted with U46619 (p less than 0.0004) or endothelin-1 (p less than 0.06). Nitric oxide generated by addition of glyceryl trinitrate (10(-6) mol/L) attenuated the vasoconstrictor effects of U46619 (p less than 0.026) or endothelin-1 (p less than 0.01). Neither acetylcholine nor the calcium ionophore A23187 had an effect on the fetal-placental circulation, whereas bradykinin further increased perfusion pressure. Histamine only relaxed the preconstricted preparations at concentrations (10(-6) to 10(-4) mol/L) above those shown to release nitric oxide in other systems. CONCLUSION The stimulus to nitric oxide generation in the fetal-placental circulation may be hydrodynamic. Nitric oxide appears to contribute to maintenance of basal vascular tone and to attenuate the actions of vasoconstrictors in this circulation.

Role of placenta in preeclampsia

Preeclampsia, which manifests itself as hypertension, proteinuria, and edema in pregnancy, requires the presence of trophoblast tissue but not a fetus. It is characterized by abnormal trophoblast invasion of the spiral arteries of the decidua and myometrium leading to a failure to establish an adequate uteroplacental blood flow and, therefore, is thought to give rise to relatively hypoxic trophoblast tissue. This, in turn, may promote an exaggerated state of oxidative stress in the placenta. This hypoxia/oxidative stress may then further attenuate trophoblast invasion but also alters placental villous angiogenesis leading to a poorly developed fetoplacental vasculature with abnormal reactivity. Oxidative stress per se may also affect vascular reactivity, blood flow, and oxygen and nutrient delivery to the fetus, which ultimately may be compromised. The synthetic and transport functions of the syncytiotrophoblast may also be altered, and there is an increased rate of trophoblast apoptosis. The linkage among abnormal trophoblast invasion, trophoblast dysfunction, and the maternal disease remains unidentified. The presumptive humoral factor that is released by the preeclamptic placenta to cause maternal disease remains elusive. Current therapies to prevent preeclampsia aim toward preventing the maternal syndrome, not preventing the primary pathophysiology.

Vascular biology of preeclampsia

Summary.  Preeclampsia, a pregnancy‐specific syndrome characterized by hypertension, proteinuria and edema, resolves on delivery of the placenta. Normal pregnancy is itself characterized by systemic inflammation, oxidative stress and alterations in levels of angiogenic factors and vascular reactivity. This is exacerbated in preeclampsia with an associated breakdown of compensatory mechanisms, eventually leading to placental and vascular dysfunction. The underlying pathology of preeclampsia is thought to be a relatively hypoxic or ischemic placenta. Both the placenta and maternal vasculatures are major sources of reactive oxygen and nitrogen species which can interact to produce peroxynitrite a powerful prooxidant that covalently modifies proteins by nitration of tyrosine residues, to possibly alter vascular function in preeclampsia. The linkage between placental hypoxia and maternal vascular dysfunction has been proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta that bind vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) in the maternal circulation. There is also abundant evidence of altered reactivity of the maternal and placental vasculature and of the altered production of autocoids in preeclampsia. The occurrence of preeclampsia is increased in women with preexisting vascular disease and confers a long‐term risk for development of cardiovascular disease. The vascular stress test of pregnancy thus identifies those women with a previously unrecognized at risk vascular system and promotes the development of preeclampsia. Preexisting maternal vascular dysfunction intensified by placental factors is possibly responsible for the individual pathologies of preeclampsia.