The Association between Constitutional Knee Alignment and the Horizontal Orientation of the Ankle Joint Line.

PURPOSE OF THE STUDY To characterize constitutional frontal alignment of the ankle in genua vara, valga, and norma. MATERIAL AND METHODS Long-leg standing radiographs of 589 patients presenting between 2011 and 2020 for knee-complaints because of any reason were chosen from our database. Cases with fractures or history of bony-realignment-surgeries were excluded. The Hip-Knee-Ankle angle (HKA), the mechanical Lateral Distal Tibia Angle (mLDTA), and the Tibia-Plafond-Horizontal-Orientation angle (TPHA) were measured in 354 patients. For this study, neutral frontal alignment of the leg was defined as HKA between -3.0° and +4.0°. HKA-values <-3.0° were defined as genua valga and values >4.0° were defined as genua vara. According to these cutoffs, data was categorized into the following three patient groups: genua vara (n=157), genua norma (n=106), genua valga (n=91). For each group, the ankle alignment in the frontal plane was compared to the HKA. Finally, the three groups were compared to each other. RESULTS In the varus-group, the HKA-value was 6.9°±2.4°, the TPHA-value was 4.7°±3.5°, and the mLDTA-value was 87.4°±4.8°. In the neutral-group, the HKA-value was 1.8°±2.0°, the TPHA-value was 2.5°±2.9°, and the mLDTA-value was 87.2°±4.6°. In the valgus-group, the HKA-value was -6.0°±2.7°, the TPHA-value was -0.2°±4.7°, and the mLDTA-value was 85.0°±4.7°. DISCUSSION The frontal alignment of the ankle joint line depends on the overall frontal alignment of the leg. The TPHA correlates with varus or valgus alignment of the knee, but the mLDTA does not. In patients with valgus-aligned long-leg axis, the TPHA demonstrated less valgus alignment than in patients with varus-aligned long-leg axis. This knowledge is especially useful when planning osteotomies for correction of lower extremity malalignment. CONCLUSIONS During the planning process of osteotomies around the knee, the TPHA should be appreciated because it correlates with the constitutional knee alignment. Key words: valgus, varus, frontal alignment, coronal alignment, osteotomy.

Determination of non-bilayer phospholipid arrangements and their antibodies in placentae and sera of patients with hypertensive disorders of pregnancy.

Studies suggest that preeclampsia (PE) originates in the placenta and is associated with deficient trophoblast invasion of spiral arteries. The direct cause remains unknown, but preeclampsia is often associated with circulating factors that can induce generalized endothelial dysfunction. Antiphospholipid antibodies (APA) in circulation are also associated with vascular diseases. Although the quantification of APA is not currently used as a prognostic of the risk of PE, studies suggest that thrombophilias play a role in PE pathogenesis. In fact, the pathology of placentae from PE and Antiphospholipid syndrome patients is similar; atherosis, thrombosis and infarction, and endothelium activation represent the pathological mechanisms. We identified a new antibody which recognizes non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, and which triggered an autoimmune-like disease in mice. We evaluated the presence of NPA in the placentae and in sera, and whether NPA induced NPA antibodies in patients with hypertensive disorders of pregnancy (HDP). Results showed increased levels of NPA in the syncytiotrophoblast, extravillous cytotrophoblast, syncytial knots and the amnion epithelial cell membranes of the placenta, as well as increases in NPA and NPA antibodies in sera from HDP patients, when compared with controls. This suggests that NPA derived from placenta could be one of multiple factors associated with pregnancy pathologies.

Increased nitrotyrosine in the diabetic placenta: evidence for oxidative stress.

OBJECTIVE: To evaluate the presence of nitrotyrosine (NT) residues in placental villous tissue of diabetic pregnancies as an index of vascular damage linked to oxidative stress. RESEARCH DESIGN AND METHODS: Villous tissue was collected and flash frozen after delivery from 10 class C and D IDDM patients (37.9+/-3.2 weeks) and 10 normotensive pregnant individuals (37.5+/-3.8 weeks). Serial sections of tissue were immunostained with specific antibodies to NT, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and manganese superoxide dismutase (MnSOD). Sections were scored for intensity of immunostaining (0-3) by three observers blinded to the identity of tissue. RESULTS: All tissues demonstrated immunostaining for eNOS in both syncytiotrophoblast and stem villous vascular endothelium with no apparent differences between groups. Immunostaining for iNOS was seen in the villous stroma, but again was not different between the two groups. Significantly more intense NT staining was apparent in vascular endothelium and villous stroma (both P < 0.02) of diabetic placentas. The endothelium of large villous vessels of diabetic tissues also showed more intense immunostaining for MnSOD (P < 0.01). CONCLUSIONS: In these diabetic pregnancies, we were unable to show increased eNOS, unlike previous findings in preeclamptic pregnancies. The presence of NT may indicate vascular damage in the diabetic placenta due to peroxynitrite action formed from increased synthesis/interaction of nitric oxide and superoxide. The apparently paradoxical increase in MnSOD expression may be an adaptive response to increased superoxide generation.

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.

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.

Ontogeny of the expression and regulation of interleukin-6 (IL-6) and IL-1 mRNAs by human trophoblast cells during differentiation in vitro.

During human placental differentiation, mononuclear cytotrophoblast cells fuse and differentiate into syncytiotrophoblast cells. Although syncytiotrophoblast cells have been shown to express interleukin-1 alpha (IL-1 alpha), IL-1 beta and IL-6, the pattern of expression of these cytokines during placental differentiation is unknown. We have examined the expression of IL-1 alpha, IL-1 beta and IL-6 mRNA during differentiation of cytotrophoblast cells in culture. IL-1 alpha, IL-1 beta and IL-6 mRNA levels were determined by semiquantitative reverse transcription-PCR analysis using glyceraldehyde phosphate dehydrogenase as an internal control. All three cytokine mRNA levels decreased markedly during trophoblast differentiation. After 6 days in culture, when almost all the cytotrophoblast cells had fused and differentiated into syncytiotrophoblast cells, the amounts of IL-1 alpha, IL-1 beta and IL-6 mRNA were decreased by 87.1, 72.1 and 60.9% respectively. Exogenous IL-6 had differential effects on cytokine mRNA expression. When added to placental cultures during the first 6 days of culture, IL-6 markedly inhibited IL-6, IL-1 alpha and IL-1 beta mRNA expression. However, when added to the cells during days 6-9 of culture, when most of the cells were syncytiotrophoblast cells, IL-6 stimulated IL-1 alpha and IL-1 beta mRNA expression. The results of these studies indicate that IL-1 alpha, IL-1 beta and IL-6 mRNA expression decreases markedly during cytotrophoblast differentiation in vitro and that the regulation of trophoblast cytokine mRNA levels changes during differentiation.

Nitrotyrosine immunostaining correlates with increased extracellular matrix: evidence of postplacental hypoxia.

Nitrotyrosine residues (NT), an index of oxidative stress arising from peroxynitrite formation and action, are found in placental vasculature of pregnancies complicated by pre-eclampsia (PE) or pregestational insulin-dependent diabetes mellitus (IDDM). This study correlates conventional placental pathology with NT immunostaining in 20 cases of perinatal mortality (13 stillbirths and seven cases of neonatal mortality) associated with PE, IDDM, amniotic fluid infection syndrome (AFIS), or from fetal/neonatal demise not related to these conditions (congenital anomalies) (n = five/group). Patients with PE have more decidual arteriolopathy and Tenney-Parker change, while patients with IDDM and ascending infection have more villous cytotrophoblastic hyperplasia. Archival paraffin-embedded placental sections were immunostained for NT for correlation with clinical features and H&E histological findings. The intensity of immunostaining for NT varied from absent (n = 7) to 1+ (n = 5) or 2+ (n = 8). All eight placentae with 2+ staining showed increased villous extracellular matrix (ECM), compared to none of five with 1+ staining and two of seven with no staining (chi2 = 14.3, P = 0.001). There was no statistically significant difference in the percentage of stem villi with luminal vascular abnormalities (5.7 vs 10 vs 35.7 per cent, F = 2.3, P = 0.1). Our data show that increased production of reactive oxygen species by placental tissue may be associated with increased extracellular matrix, itself produced by fibroblasts under the influence of oxygen. NT immunostaining may therefore help differentiate those cases of perinatal morbidity/mortality associated with post-placental hypoxia provided that the secondary impact of intrauterine fetal death can be excluded by future studies.

Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro.

We have examined the distribution of the endothelial isoform of nitric oxide synthase (eNOS) in villous and extravillous trophoblast populations by immunohistochemistry and have further studied expression of eNOS during differentiation of cytotrophoblast into syncytiotrophoblast in culture. In first trimester villous tissue, NADPH diaphorase activity and eNOS immunostaining were present in syncytiotrophoblast but not the progenitor cytotrophoblast layer. Extravillous trophoblast in the basal plate of the placenta was identified by anticytokeratin immunostaining and displayed NADPH diaphorase activity, but not eNOS immunostaining. Both amnion epithelial cells and chorion cytotrophoblast had NADPH diaphorase activity but no eNOS immunostaining, whereas eNOS immunostaining was seen in the fibroblast layer of amnion. Purified villous cytotrophoblast cells from term placentae aggregated and fused to form a syncytium with increasing time in culture as assessed by antidesmosomal protein and antinuclear antibody immunostaining. Following 24 h in culture, the majority of cells were still mononucleate cytotrophoblast which did not display eNOS immunostaining, whereas a few syncytial aggregates had formed which were both eNOS positive and hPL positive. By 3 to 5 days in culture, the majority of cells were present as syncytiotrophoblast. However, eNOS and hPL immunostaining was more diffuse and not all syncytial aggregates were positive. Of the trophoblast populations, only syncytiotrophoblast appears to express eNOS. Differentiation of cytotrophoblast into syncytiotrophoblast is associated with eNOS expression.

Immunohistochemical localization of nitric oxide synthase in the human placenta.

We have studied the distribution of the endothelial isoform of nitric oxide synthase (NOS) through the term human umbilical cord and placenta by immunohistochemistry. Histochemistry with the NADPH diaphorase substrate nitroblue tetrazolium (NBT) has also been used to establish if other isoforms of NOS may be present in these tissues. Positive immunofluorescence for endothelial NOS was found in umbilical cord artery and vein endothelium, although positive staining was only found in approximately 50% of veins. The endothelium of stem villous vessels dissected from beneath the chorionic plate was also intensely immunostained. In the terminal villi punctate immunostaining was found at the basal aspect and around nuclei of syncytiotrophoblast, but was absent from stroma and endothelium of terminal villous vessels. A positive histochemical stain for NBT was found in cord artery and vein endothelium and stem villous vessel endothelium. Intense diffuse staining with NBT was found in syncytiotrophoblast, but no other cell types in the terminal villi stained with NBT. The endothelial NOS isoform appears to be localized in the resistance vasculature of the placenta, but not in the capillary endothelium of terminal villi where there is no underlying smooth muscle. It may contribute to the 'endothelial' function of syncytiotrophoblast if secreted towards the intervillous space or alternatively fulfil other signal transduction roles. The pattern of staining with NBT was similar to that with endothelial NOS and suggests that other isoforms of NOS are not present in the placental unit.

Inducible (type II) nitric oxide synthase in human placental villous tissue of normotensive, pre-eclamptic and intrauterine growth-restricted pregnancies.

We have utilized two distinct monospecific antibodies against the type II (macrophage or inducible) nitric oxide synthase (NOS) isoform to localize the distribution of the enzyme within the human placenta in tissues from normotensive pregnancies and those complicated by pre-eclampsia and/or intrauterine growth restriction. Both antibodies immunolocalize to cells in the villous stroma on frozen sections of villous tissue. Colocalization studies with anti-CD14 or anti-CD45 antibodies that recognize cells of leucocyte or monocyte/macrophage lineage indicate that Hofbauer cells are expressing type II NOS. This is in contrast to expression of type III (endothelial) NOS which is seen in syncytiotrophoblast and in villous vascular endothelium. In some, but not all, normotensive and pathologic placental tissue, some type II NOS immunostaining could be seen in syncytiotrophoblast and vascular endothelium; however, no differences could be discerned between groups of tissues. Expression of type II NOS by Hofbauer cells may indicate they are involved in surveillance against maternal immune insult or maternal pathogens whereby they secrete nitric oxide to exert a cytostatic/cytotoxic effect.

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 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.

Endothelin transfer and endothelin effects on water transfer in human fetal membranes.

Although endothelin-1 is synthesized by human amnion, its physiologic role and its ability to be transferred to myometrium for oxytocic action remain unclear. We investigated the transfer of endothelin-1 itself and the effects of endothelin-1 on the transfer of tritiated water across human amnion or amnion/chorion/decidua in vitro with an Ussing chamber technique. Permeability coefficients (mean +/- standard error) for 3H2O across amnion/chorion/decidua in the fetal to maternal direction were 1.26 +/- 0.21 and 1.25 +/- 0.10 x 10(-4) cm/second (N = 6) and in the maternal to fetal direction 0.90 +/- 0.07 and 0.98 +/- 0.15 x 10(-4) cm/second (N = 5) in the absence or presence, respectively, of 10(-9) mol/L endothelin-1 in the fetal reservoir. Comparable values were found in either direction with 10(-9) mol/L endothelin-1 in the maternal reservoir. Apparent permeability coefficients for 125I endothelin-1 in the fetal to maternal direction were 3.34 +/- 0.79 and 2.43 +/- 0.68 x 10(-5) cm/second (N = 5) for amnion or amnion/chorion/decidua. However, appreciable trapping of 125I endothelin-1 by the fetal membranes was apparent. Endothelin-1 does not appear to affect water movement across the human fetal membranes, and only a small proportion of endothelin-1 itself is transferred across the membranes.

Pathogenesis of genital herpes simplex virus infection in mice. IV. Quantitative aspects of viral latency.

Experiments in the mouse model of herpes simplex virus (HSV) infection involving the intact genital mucous membranes as inoculation site yielded the following results. In untreated mice the extent of latency was correlated with the degree of peripheral virus replication. This correlation could not be observed when the course of infection was interrupted by chemotherapy, interferon, or passive immunization. Acyclovir had little effect on peripheral virus multiplication, but markedly reduced latent ganglionic infection. As acute ganglionic infection and virus concentration in the spinal nerves were already reduced, acyclovir is assumed to inhibit either virus penetration into the nerve endings or virus replication in the ganglia. Interferon apparently has an active role in the elimination of virus infected cells from the ganglia, as its effect was restricted to a reduced rate of latency and of lethality. Passive immunization with antiserum led to similar results as ACV-treatment. While lacking a pronounced effect on virus replication in the mucous membranes, specific antibody was found to influence both virus elimination from the ganglia, and conversion from productive to latent ganglionic infection. Immune lymphocytes proved to be the only agent capable of suppressing peripheral infection, thereby inhibiting the neural spread of the virus. These results suggest that the decrease in latency may result from modulations occurring at different stages in the course of infection.

Immunolocalization of the inducible nitric oxide synthase isoform in human fetal membranes.

PROBLEM: Nitric oxide (NO) synthesized by fetal membranes may protect the fetus from maternal infection or immune challenge or have a tocolytic effect on myometrium. The sites of synthesis and enzymes responsible for NO production in human fetal membranes remain unidentified. METHOD OF STUDY: Fetal membranes were obtained from four groups of patients: term (> 37 weeks gestation) or preterm (< 37 weeks gestation), both either in labor or not in labor. Frozen sections of membrane rolls were immunostained for inducible (iNOS) and endothelial (eNOS) nitric oxide synthase isoforms and the monocyte/macrophage marker CD14. RESULTS: Positive iNOS immunostaining was found in fibroblasts of amnionic and chorionic mesenchyme and in decidual macrophages identified by CD14 from all four groups of tissues. No iNOS immunostaining was seen in amnion epithelium or chorion trophoblast. Very intense iNOS staining was seen with evidence of monocyte/macrophage invasion of membranes. eNOS immunostaining was only found in decidual vascular endothelium. CONCLUSIONS: Constitutive expression of iNOS in decidual macrophages and fetal membrane fibroblasts may form an immune barrier against maternal insult. In chorioamnionitis, macrophage recruitment and NO expression may be part of the maternal immune response.

Transfer of aspirin across the perfused human placental cotyledon.

Pregnancy-induced hypertension is associated with a reduction in prostacyclin synthesis that is relative to normotensive pregnancy, whereas thromboxane A2 synthesis is unchanged or increased. The net effect is a decreased prostacyclin/thromboxane ratio that may result in the reduced fetal-placental blood flow seen in pregnancy-induced hypertension because thromboxane is known to constrict this circulation. Low-dose aspirin (acetylsalicylic acid), which is used to treat pregnancy-induced hypertension, selectively inhibits thromboxane synthesis and therefore may alter fetal-placental blood flow. We have investigated the transfer of acetylsalicylic acid in the perfused human placental cotyledon and its effects on fetal-placental perfusion pressure. Human placental cotyledons were perfused with tissue culture medium 199 plus 5% polyvinylpyrrolidone that was gassed with 95% oxygen/5% carbon dioxide at flow rates of 10 ml/min (maternal) and 4 ml/min (fetal). Acetylsalicylic acid (10(-5) mol/L) was added to the maternal circuit, and cotyledons were perfused for 1 hour with aliquots taken from a closed fetal circuit every 5 minutes. Acetylsalicylic acid was assayed by spectrofluorometry at 306/412 nm. Our data indicate an initial rapid transfer of acetylsalicylic acid during the first 5 minutes into the fetal-placental circulation, the concentration then decreased to a steady state at 0.4 x 10(-5) mol/L. Resting perfusion pressure of both maternal and fetal circulation did not change after the addition of acetylsalicylic acid to maternal perfusate and transfer to the fetal circulation.

Immunohistochemical characterization of placental nitric oxide synthase expression in preeclampsia.

OBJECTIVE: Our purpose was to compare the expression of endothelial nitric oxide synthase in the placenta and umbilical cord of preeclamptic placenta with that of the normotensive placenta. STUDY DESIGN: We compared placental endothelial nitric oxide synthase expression in preeclamptic (n = 3) with that in normal (n = 3) pregnancies. Frozen sections of umbilical cords, chorionic plate vessels, and terminal villi were immunostained with a monoclonal endothelial nitric oxide synthase antibody (H32). RESULTS: No difference in endothelial nitric oxide synthase immunostaining in the endothelium of the umbilical cord artery and vein, chorionic plate vessels, and stem villous vessels was found between preeclamptic and normotensive pregnancies. In contrast, in the preeclamptic placentas endothelial nitric oxide synthase immunostaining was seen in the small terminal villous vessels with underlying smooth muscle layer. In the syncytiotrophoblast endothelial nitric oxide synthase immunostaining appeared primarily apical in location and diffuse in distribution in the preeclamptic placentas but primarily basal and punctate in the normotensive placentas. CONCLUSIONS: Differences in endothelial nitric oxide synthase expression in terminal villous vessels and in syncytiotrophoblast may be a result of vascular alterations or damage that take place in the placenta in preeclampsia. These differences may alter the regulation of blood flow in the fetal and maternal placental vasculatures in preeclampsia.

Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies.

Nitric oxide (NO) regulates blood flow in the human placenta. As increased resistance to blood flow is seen in the fetal-placental vasculature in pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR), we examined expression of endothelial nitric oxide synthase (eNOS) in these placentas. Placental villous tissue sections were obtained from normotensive control (n = 5), IUGR alone (n = 5) or pre-eclamptic (with or without IUGR (n = 9) patients, immunostained for eNOS and scored for localization, type (punctate or diffuse) and intensity of eNOS staining in syncytiotrophoblast and placental vessels. The significance of differences was calculated using the Mann-Whitney U-test. No differences in intensity or type of immunostaining in syncytiotrophoblast were seen. Placentas from patients with pre-eclampsia with or without IUGR had a significantly more basal distribution of eNOS in syncytiotrophoblast. eNOS immunostaining was absent in terminal villous capillary and faint in stem villous vessel endothelium of normal placentas, but was intense in the endothelium of both of these types of vessels in the IUGR and pre-eclampsia groups, with significantly greater staining seen in stem vessels of patients with IUGR alone. This increased eNOS expression and hence increased NO production in the fetal-placental vasculature may be an adaptive response to the increased resistance and poor perfusion in these pathological pregnancies.

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.

Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia.

Oxidative stress may increase production of superoxide and nitric oxide, leading to formation of prooxidant peroxynitrite to cause vascular dysfunction. Having found nitrotyrosine residues, a marker of peroxynitrite action, in placental vessels of preeclamptic and diabetic pregnancies, we determined whether vasoreactivity is altered in these placentas and treatment with peroxynitrite produces vascular dysfunction. The responses of diabetic, preeclamptic, and normal placentas to increasing concentrations of the vasoconstrictors U-46619 (10(-9)-10(-7) M) and ANG II (10(-9)-10(-7) M) and the vasodilators glyceryl trinitrate (10(-9)-10(-7) M) and prostacyclin (PGI(2); 10(-8)-10(-6) M) were compared as were responses to these agents in normal placentas before and after treatment with 3.16 x 10(-4) M peroxynitrite for 30 min. Responses to both vasoconstrictors and vasodilators were significantly attenuated in diabetic and preeclamptic placentas compared with controls. Similarly, responses to U-46619, nitroglycerin, and PGI(2), but not ANG II, were significantly attenuated following peroxynitrite treatment. The presence of nitrotyrosine residues confirmed peroxynitrite interaction with placental vessels. Overall, our data suggest that peroxynitrite formation is capable of attenuating vascular responses in the human placenta.

Immunohistochemical localization of phospholipase A2 isoforms in human myometrium during pregnancy and parturition.

OBJECTIVE: The purpose of this study was to localize secretory phospholipase A2 and cytosolic phospholipase A2 isoforms in pregnant human myometrium and to determine changes in expression with gestational age or parturition. STUDY DESIGN: Myometrium was collected at cesarean section at term (>37 weeks) or preterm (<37 weeks) from patients who were or were not in labor (n = 5 each group). Frozen sections were incubated with specific monoclonal antibodies against secretory phospholipase A2 or cytosolic phospholipase A2 and immunostaining visualized with the Vectastain ABC method. The intensity of immunostaining in different cellular localizations was scored by an investigator blinded to tissue identity and compared among tissues with use of the Mantel-Haenszel chi2 test. RESULTS: Secretory phospholipase A2 immunostaining was dispersed in the perinuclear region throughout the myometrial smooth muscle fibers and in vascular smooth muscle. Cytosolic phospholipase A2 immunostaining was predominantly localized to endothelial cells of myometrial blood vessels and weakly throughout myometrial fibers. There was no apparent change in intensity of immunostaining for either isoform with gestational age or with the absence or presence of labor. CONCLUSION: The differential localization of the two phospholipase A2 isoforms suggests different functions. The apparent lack of change in expression during late gestation or with labor possibly suggests changes in myometrial phospholipase A2 activity and hence local myometrial arachidonic acid mobilization and presumably prostaglandin synthesis may not be associated with the onset of or maintenance of parturition.