Annie Eis

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.

Role of autophagy in angiogenesis in aortic endothelial cells.

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

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.

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.

Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with impaired pulmonary vasodilation at birth. Previous studies demonstrated that a decrease in angiogenesis contributes to this failure of postnatal adaptation. We investigated the hypothesis that oxidative stress from NADPH oxidase (Nox) contributes to impaired angiogenesis in PPHN. PPHN was induced in fetal lambs by ductus arteriosus ligation at 85% of term gestation. Pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were compared for their angiogenic activities and superoxide production. HTFL-PAEC had decreased tube formation, cell proliferation, scratch recovery, and cell invasion and increased cell apoptosis. Superoxide (O(2)(-)) production, measured by dihydroethidium epifluorescence and HPLC, were increased in HTFL-PAEC compared with NFL-PAEC. The mRNA levels for Nox2, Rac1, p47(phox), and Nox4, protein levels of p67(phox) and Rac1, and NADPH oxidase activity were increased in HTFL-PAEC. NADPH oxidase inhibitor, apocynin (Apo), and antioxidant, N-acetyl-cysteine (NAC), improved angiogenic measures in HTFL-PAEC. Apo and NAC also reduced apoptosis in HTFL-PAEC. Our data suggest that PPHN is associated with increased O(2)(-) production from NADPH oxidase in PAEC. Increased oxidative stress from NADPH oxidase contributes to the impaired angiogenesis of PAEC in PPHN.

Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension.

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91(phox) and p47(phox), NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis.

Changes in activity of cytosolic phospholipase A2 in human amnion at parturition

OBJECTIVE The purpose of this study was to determine whether increased cytosolic phospholipase A2 activity mediated arachidonic acid mobilization for prostaglandin synthesis in amnion at parturition. STUDY DESIGN Amnion was collected immediately after delivery from four groups of patients: preterm (<37 weeks) with no labor or labor and term (>37 weeks) with no labor or labor and stored at -70 degrees C. Tissues were homogenized and centrifuged for 1 hour at 100,000 g, and cytosol was assayed for cytosolic phospholipase A2 activity with use of carbon 14-labeled 1-stearoyl-2 arachidonyl phosphatidylcholine plus 10 micromol/L unlabeled substrate and 5 mmol/L calcium in 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4. Incubations were performed in duplicate +/- 10 micromol/L arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic phospholipase A2 activity, at 30 degrees C for 45 minutes. RESULTS Total cytosolic phospholipase A2 activity (in picomoles of arachidonic acid per minute per milligram of protein) calculated as the difference between the activity in the presence and absence of arachidonyl trifluoromethyl ketone was (mean +/- SE) as follows: preterm no labor (n = 7) 8.94 +/- 3.08, preterm with labor (n = 6) 6.79 +/- 2.31, term no labor (n = 7) 14.85 +/- 1.66, and term with labor (n = 5) 5.51 +/- 1.52. Enzyme activity increased with gestational age and was highest in the term no labor group. A significant decrease in cytosolic phospholipase A2 activity occurred with labor (p < 0.05). The greatest decrease in activity was in the term group (p < 0.05). CONCLUSION Total cellular cytosolic phospholipase A2 activity in amnion is highest in anticipation of labor but during labor total activity is depleted, resulting in the low activity measured after delivery of the placenta. The substrate specificity and changes in amnion total cytosolic phospholipase A2 activity with labor strongly suggests a role in mediation of arachidonic acid mobilization and prostaglandin synthesis at labor.

Cellular Redistribution of Inducible Hsp70 Protein in the Human and Rabbit Heart in Response to the Stress of Chronic Hypoxia: Role of protein kinases

Many infants who undergo cardiac surgery have a congenital cyanotic defect where the heart is chronically perfused with hypoxemic blood. Infant hearts adapt to chronic hypoxemia by activation of intracellular protein kinase signal transduction pathways. However, the involvement of heat shock protein 70 in adaptation to chronic hypoxemia and its role in protein kinase signaling pathways is unknown. We determined expression of message and subcellular protein distribution for inducible (Hsp70i) and constitutive heat shock protein 70 (Hsc70) in chronically hypoxic and normoxic infant human and rabbit hearts and their relationship to protein kinases. In chronically hypoxic human and rabbit hearts message levels for Hsp70i were elevated 4- to 5-fold compared with normoxic hearts, Hsp70i protein was redistributed from the particulate to the cytosolic fraction. In normoxic infants Hsp70i protein was distributed almost equally between the cytosolic and particulate fractions. Hsc70 message and subcellular distribution of Hsc70 protein were unaffected by chronic hypoxia. We then determined if protein kinases influence Hsp70i protein subcellular distribution. In rabbit hearts SB203580 and chelerythrine reduced Hsp70i message levels, whereas SB203580, chelerythrine, and curcumin reversed the subcellular redistribution of Hsp70i protein caused by chronic hypoxia, with no effect in normoxic hearts, indicating regulation of Hsp70i message and subcellular distribution of Hsp70i protein in chronically hypoxic rabbit hearts is influenced by protein kinase C and mitogen-activated protein kinases, specifically p38 MAPK and JNK. We conclude the Hsp70 signal transduction pathway plays an important role in adaptation of infant human and rabbit hearts to chronic hypoxemia.