Chronic hypoxia in vivo reduces placental oxidative stress

Abstract

Decreased placental oxygenation and increased oxidative stress are implicated in the development of preeclampsia. Oxidative stress arises from imbalance between pro-versus anti-oxidants and can lead to biological oxidation and apoptosis. Because pregnant women living at high altitude (3100 m, HA) have lowered arterial PO2 and an increased incidence of preeclampsia, we hypothesized that HA placentas would have decreased anti-oxidant enzyme activity, increased oxidative stress (lipid peroxidation, protein oxidation and nitration) and greater trophoblast apoptosis than low-altitude (LA) placentas. We measured enzymatic activities, lipid and protein oxidation and co-factor concentrations by spectrophotometric techniques and ELISA in 12 LA and 18 HA placentas. Immunohistochemistry (IHC) was used to evaluate nitrated proteins and specific markers of apoptosis (activated caspase 3 and M30). Superoxide dismutase activity was marginally lower (p=0.05), while glutathione peroxidase activity (p<0.05), thioredoxin concentrations (p<0.005) and thioredoxin reductase activity p<0.01 were all reduced in HA placentas. Decreased anti-oxidant activity was not associated with increased oxidative stress: lipid peroxide content and protein carbonyl formation were lower at HA (p<0.01). We found greater nitrotyrosine residues in the syncytiotrophoblast at 3100 m (p<0.05), but apoptosis did not differ between altitudes. Our data suggest that hypoxia does not increase placental oxidative stress in vivo. Nitrative stress may be a consequence of hypoxia but does not appear to contribute to increased apoptosis. Lowered placental concentrations of anti-oxidants may contribute to the susceptibility of women living at HA to the development of preeclampsia, but are unlikely to be etiological.

Figure 1

A: Superoxide dismutase activity was marginally (p=0.05) greater in the low altitude (blue bar, range 1.19-2.99) than high altitude placentas (red bar, range 0.56 – 0.91). B: Glutathione peroxidase activity was greater (p = 0.01) in the low altitude (blue bar, range 11.63-21.89) than high altitude placentas (red bar, range 5.32 – 18.79). C. Thioredoxin content was greater (p = 0.002) in low altitude (blue bar, range 75.58 – 176.8) than high altitude placentas (red bar, range = 41.20-122.0). D. Thioredoxin reductase activity was also greater (p = 0.009) in low altitude (blue bar, range 6.22 – 27.01) than high altitude placentas (red bar, range 1.69 – 19.04).

Figure 2

A: Oxidative stress, as indicated by lipid peroxidation, was greater (p = 0.007) in low altitude placentas (blue bar, range 4.89 – 29.81) when compared with high altitude placentas (red bar, range 3.42 – 21.22). B: Oxidative stress, as reflected in protein carbonylation, was greater (p=0.002) in low altitude (blue bar, range 75.6 – 176.8) than high altitude placentas (red bar, range 41.2-122.0).

Figure 3

A: Kidney was used as positive control for nitrotyrosine staining (positive staining is a dark reddish brown). Note the intense staining in the renal tubules. B: Ischemic brain was used as a second positive control. C. Placenta with the primary antibody for nitrotyrosine omitted. D. A low altitude placenta showing staining of serum within blood vessels and the intervillous space, some stromal staining, but a relative absence of syncytiotrophoblast staining. E. A high altitude placenta showing intense syncytiotrophoblast staining. F. A high altitude placenta showing strong staining in the intermediate (extravillous) trophoblast.

Figure 4

A: Tonsil was used as a positive control tissue for immunohistochemical detection of Activtaed Caspase-3. Positive staining is indicated by the reddish brown color in the cytoplasm of positively stained cells. B: Placenta with primary antibody for caspase 3 omitted. Placenta with primary antibody omitted for M30 had the same appearance (data not shown). C: A low altitude placenta showing a rare positively stained cell in the syncytiotrophoblast. D: A high altitude placenta showing an equally rare positively stained cell in the syncytiotrophoblast. E: Adenocarcinoma of the colon, used as a positive control for M30, showed rare epithelial cells with strong staining (brownish red) with faint staining in the apical cyctoplasm of other epithelial cells. F. BeWo cells treated with camptothecin were used as an additional positive controls for M30 (note the much greater frequency of positive staining than in the normal placentas). G. A low altitude placenta showing rare positive syncytiotrophoblast staining for M30. H. A high altitude placenta showing rare positive staining for M30 in the syncytiotrophoblast.

Figure 5

The components of oxidative stress measured in the present study are shown in red. The hypothesis predicted that high altitude placentas would show decreased endogenous anti-oxidants such as total superoxide dismutase (SOD), a reduction in glutathione peroxidase and in thioredoxin reductase, and in thioredoxin, the substrate for thioredoxin reductase, predictions confirmed by our measurements. Due to reduced anti-oxidant protection we expected increased, but instead observed decreased lipid-peroxidation and protein carbonylation and no difference in apoptosis. Despite reduced lipid peroxidation and protein carbonylation, there was increased protein nitrosylation, specifically in the syncytiotrophoblast.