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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY
Departments of Anatomy and Cell Biology and Obstetrics and Gynecology, Queen's University, Kingston, Ontario, Canada
Submitted 17 July 2006 ; accepted in final form 17 May 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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heme oxygenase; antioxidants; cigarette smoking; placenta; preeclampsia; cell culture
Placental oxidative stress appears to be critical for the development of PE (16, 20, 21, 22, 27). Therefore, any increase in the levels of endogenous antioxidants may prevent the development of PE. Several antioxidant systems have been identified within the placenta, including copper/zinc superoxide dismutase [Cu/Zn superoxide dismutase (SOD); inactivates O2
–] (29), catalase (CAT; inactivates H2O2) (29), glutathione peroxidase (GPx; inactivates H2O2 and lipid peroxides) (29) and the heme oxygenase enzymes heme oxygenase (HO)-1 and HO-2 (18). HO enzymes act as antioxidants by degrading heme (a prooxidant) into equimolar quantities of CO, biliverdin (bilirubin), and Fe2+ (3, 4); biliverdin and bilirubin are potent physiological antioxidants. HO-1 (32 kDa) is the inducible form of the enzyme, while HO-2 (36 kDa) is constitutively active (17).
A better understanding of the mechanism(s) by which cigarette smoking decreases the risk of developing PE will increase our understanding of the PE disease process and may lead to therapeutic options; delivery is currently the only known cure for PE. The objective of this study was to determine in vivo the effect of smoking on placental antioxidant enzyme expression, as well as the in vitro effect of cigarette smoke extract (CSE) treatment on trophoblast antioxidant systems. We hypothesized that exposure to cigarette smoke increases the expression of antioxidants within placental tissue and that this increase in placental antioxidant enzymes is linked to the decreased incidence of PE in smokers.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Tissue and cell preparation. Placental tissues were blunt dissected and random samples from the basal plate, anchoring villi (AV) and chorionic villi (CV) were obtained. The tissue was flash frozen in liquid nitrogen and stored at –80°C for future analysis.
Cells of the immortalized human trophoblast choriocarcinoma cell line HTR-8SVneo were a gift from Dr. C. H. Graham (Queen's University). The cells were maintained at 37°C in 75 cm2 flasks containing RPMI (Sigma-Aldrich, Oakville, Ontario, Canada) + 5% FBS (Sigma-Aldrich) (10). Confluent monolayers were trypsinized (Sigma-Aldrich), and then 5 x 105 cells were plated in 60-mm dishes for experimental procedures.
Cigarette smoke extract preparation and cell reatment. A vacuum-driven smoking device was adapted from the protocol used by Lee et al. (15). Briefly, the smoke from three cigarettes was drawn through 15 ml of RPMI media. This was established as 100% CSE. To ensure reproducibility from one batch of CSE to the next, dissolved CO2 levels were measured using the ABL5 gas monitor (Radiometer, London, Ontario, Canada) The 100% CSE was then diluted using RPMI containing 5% FBS.
Previous work with pulmonary artery endothelial cells demonstrated that treatment with CSE concentrations of greater than 10% resulted in significant cell death (15). The HTR-8SVneo cells were incubated for 48 h with 0.1%, 0.5%, 1%, 2%, 5%, 10%, and 20% dilutions of CSE or control RPMI.
Protein extraction. Placental samples were thawed and washed in PBS to remove excess blood. Four milliliters of protein extraction buffer (2% SDS, 10 mM Tris, 0.15 M NaCl, pH 7.5) was added for every 1 g of tissue; the tissue was homogenized, and DNA was sheared by passing the homogenate 12 times through an 18-gauge needle. Protein was extracted from the HTR-8SVneo cells using 70 µl of a protein extraction buffer (2% SDS, 10 mM Tris, 0.15 M NaCl, pH 7.5). The samples were then sonicated for 15 s to denature nucleic acids. Tissue and cell culture homogenates were boiled for 5 min and then centrifuged at 14,000 g at 22°C for 15 min to separate out cellular fragments. The supernatant was collected and stored at –80°C. The protein concentration of each sample was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA).
Western Blot analysis. The protein extraction samples (30–60 µg) were separated by electrophoresis on 12% SDS-PAGE gels at 165V for 2.5 h. The separated proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA) at 110 V for 1 h. The membranes were blocked overnight in PBS-Tween (PBST) with 5% skim milk and 1% bovine serum albumin to prevent nonspecific binding. Membranes were then incubated for 1 h with primary antibody (HO-1, RCH Antibodies, Sydenham, Ontario, Canada; HO-2, and GPx, Stressgen, Victoria, British Columbia, Canada; SOD, Santa Cruz Biotechnology, Santa Cruz, CA; and CAT, Sigma-Aldrich). Following incubation with the primary antibody, the membranes were washed five times for 5 min in PBST and then incubated in the appropriate secondary antibody [1:15,000 goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for HO-1, HO-2, and SOD; and 1:25,000 goat anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA) for CAT and GPx] for 1 h, and washed as after the primary antibody. Following the second set of washes, secondary antibodies were detected by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA) and exposed onto X-OMAT Blue XB-1 film (Kodak, Rochester, NY).
As an internal control, all membranes were stripped of bound antibody (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7) at 50°C for 30 min and reprobed for the housekeeping protein 
-actin (1:8,000, Sigma-Aldrich). The membranes were processed as above.
Immunofluorescence staining of CSE-treated HTR8 SVneo cells. HTR8 SVneo cells were seeded on glass coverslips in 35-mm cell culture dishes and allowed to adhere overnight in 37°C incubator at 5% CO2 in complete RPMI (RPMI +10% FBS) (Sigma-Aldrich). Media were replaced with complete RPMI, RPMI + 0.1%, or RPMI + 0.5% CSE, and cells were incubated for a further 24 h. Cells were fixed in 4% PFA on ice for 15 min, washed twice in cold PBS, and permeabilized in 0.5% Triton x 100 PBS for 15 min on ice. Cells were washed twice in PBS 01% Triton x 100 and incubated in 200 µg/ml RNAse A (Sigma-Aldrich) at 37°C for 1 h. Cells were then blocked in 5% normal goat serun PBST for 1 h, incubated with anti HO-1 chicken antibody(RCH Antibodies) for 1 h followed by incubation for 1 h in anti-chicken Alexa 488 secondary antibody (Molecular Probes Eugene OR), and washed and incubated in propidium iodide 2 µg/ml. (Sigma-Aldrich). The cells were mounted on slides and photographed on a Leica TCS SP2 Confocal Microscope.
Data analysis.
The band densities were determined using the AlphaEase software (Alpha Innotech, San Leandro, CA). The data are expressed as the ratio of the protein density of each lane to the -actin density value of the same lane.
Statistical analysis. Results were expressed as the means ± SE. The human placenta data were analyzed using a two-tailed paired Students t-test, and results were considered statistically significant when P < 0.05. The HTR-8SVneo data were analyzed using a one-way ANOVA, and the data were subjected to a Tukey post hoc test.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The HO-1 (Fig. 1, A–C) and HO-2 (Fig. 2, A and C) expression was increased (P < 0.05) in the smoking group compared with the nonsmoking group in the basal plate but not (P > 0.05) in either the AV or CV. Placental HO-1 protein expression was lower than that of HO-2. 50 µg/ul of protein was required to visualize the 32 kDa band corresponding to HO-1, while 30 µg/ul of protein was sufficient for the visualization of HO-2, CAT, SOD, and GPx.
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Cell culture data. A reproducible CO2 concentration of 80–100 ppm was used to demonstrate consistency and accepted for use in CSE preparations. A "fresh" pack of cigarettes was used for each experiment as using "stale" cigarettes resulted in elevated CO2 concentrations that were not within this 80–100 ppm working range. Although it is not known which component(s) of cigarette smoke are captured in this CSE, CO levels are undetectable as measured by gas chromatography (data not shown).
All concentrations of CSE above 2% were associated with 100% HTR-8SVneo human cytotrophoblast cell death. The 2% dilution was also associated with cell death, although sufficient protein could still be collected to allow for Western blot analysis. Concentrations less than 2% did not alter the rate of cell growth and division nor were they associated with visible morphological changes in the HTR-8SVneo cell line.
HO-1 levels were increased in a dose-dependent manner following treatment with increasing concentrations of CSE (0.1%, 0.5%, 1.0% and 2.0%) (Fig. 3). HO-2, CAT, and SOD levels were unaffected by the CSE treatment (data not shown). Immunostaining for HO-1 confirmed the dose-dependent effect of increasing CSE treatment (representative data Fig. 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Smoking constitutes a systemic oxidative stress, as each inhalation of cigarette smoke contains 1014 oxidants in the tar phase and 1015 oxidants in the gas phase (11). Although acute oxidative stress (i.e., ischemia/reperfusion injury) is associated with tissue damage, chronic oxidative stresses such as that experienced by smokers may lead to an upregulation of the antioxidant enzymes. HO-1 is the inducible isoform of HO, and its expression is known to be increased in response to oxidative stress in the heart, kidneys, and liver (12).
While HO-1 is known to be induced by cigarette smoke in other tissues, the increased expression of HO-2 in the basal plate of smokers was unexpected as this isoform is constitutively active. However, HO-2 has recently been shown to be upregulated in nervous tissue following hyperthermic injury (9, 24, 25), which can be attenuated by antioxidant pretreatment (23), suggesting that this upregulation is at least in part due to oxidative stress.
The observed dose-dependent increase in HO-1 expression and immunoflourescence following CSE treatment in the HTR-8SVneo cells mimics the increased HO-1 expression seen in the placental basal plate of smoking women. However, the in vivo experiments did not examine a possible dose-dependent effect but compared HO expression in the placentas from nonsmokers with that from moderate to heavy smokers.
The in vivo effect of cigarette smoke on HO-1 and HO-2 expression was limited to the basal plate. The cells of the basal plate are mainly extravillous cytotrophoblast, while the AV and CV are composed of syncytiotrophoblast with scattered cytotrophoblast cells (Langerhans' cells) found only in 20% of the villous surface at term. The different structural and functional properties of the subdivisions of trophoblast could result in different responses to the stress of maternal cigarette smoking. The HTR-8SVneo cell line employed in this study is believed to be extravillous cytotrophoblast-like in that it possesses numerous characteristics that are limited to this trophoblast cell population, including a highly invasive phenotype (10) and an inability to fuse to form a syncytium.
Decreased HO expression has been observed in several pathological conditions in pregnancy, including spontaneous abortion, hydatiform mole, choriocarcinoma, and PE (30, 32). The decreased expression of HO in PE placentas may be the result of tissue damage, as HO-2 levels are decreased in infarcted and periinfarcted regions of the placenta (14), and the placentas from women with PE exhibit more areas of infarction. This decreased HO activity could contribute to the widespread oxidative stress that is present in PE.
Increased HO expression would result in increased biliverdin and bilirubin production locally. At physiological oxygen tensions, bilirubin has been found to be significantly more potent than vitamin E at protecting against lipid peroxidation (26); as little as 10 nM bilirubin is capable of protecting against a 10,000 fold higher concentration of H2O2 (5). Farrera et al. (8) compared antioxidants and found that bilirubin was the most potent superoxide and peroxyl radical scavenger. Bilirubin is also capable of preventing peroxynitrite-induced protein oxidation (19). Therefore, any upregulation in HO-1 expression in the basal plate of smokers could potentially lead to the generation of more bilirubin, which could then function at the maternal-fetal interface to decrease oxidative stress.
In summary, smoking throughout pregnancy is associated with an increase in placental basal plate HO activity, which is supported by the in vitro CSE-mediated dose-dependent increase in HO-1 in the placental cell line HTR-8SVneo. It is speculated that this upregulation of HO may explain, in part, the decreased incidence of PE in smokers by facilitating trophoblast invasion of the spiral arteries and decreasing placental oxidative damage, especially as the relationship between smoking and the development of PE is also dose dependent. The "protective" effect of smoking is likely multifactorial, representing a combination of the antiapoptotic and vasodilatory effects of CO, as well as the antioxidant actions of HO-1. Investigations into how cigarette smoking alters the HO enzyme system and the effects this has on endogenous oxidant/antioxidant balance and the development of PE could lead to the therapeutic use of inducers of this system in the prevention and/or treatment of PE.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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