Yong-Won Park, Department of Obstetrics and Gynecology, Yonsei University College of Medicine, 134 Shinchondong Seodaemoon gu, 120-752, Seoul, Korea. E-mail: email@example.com
Objectives. To evaluate the effect of nicotine on the production of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) in human umbilical vein endothelial cells (HUVECs) and trophoblast cells, and to assess the involvement of alpha 7 nicotinic acetylcholine receptor (α7 nAChR) in this process. Methods. Commercially available full-term placental trophoblasts and HUVECs derived from the umbilical cord of a normal pregnancy were used. The expression of α7 nAChR was assessed by immunostaining, RT-PCR, and western blotting. The expression of sFlt-1 and sEng protein in the cell media after 6 and 24 hours of treatment with nicotine was evaluated using a commercially available ELISA. To determine the involvement of α7 nAChR in the nicotinic effect, cells were treated with the α7 nAChR antagonist α-bungarotoxin (α-BGT) prior to the nicotine exposure. Levels of significance were determined using the Student's t-test and one-way ANOVA, and a p-value < 0.05 was considered significant. Main outcome measures. The levels of sFlt-1 and sEng protein were evaluated before and after the nicotine treatment with or without α-BGT pre-treatment. Results. In trophoblast cells, a significant reduction of sFlt-1 and sEng protein was observed after 24 hours of nicotine treatment as compared to the untreated group (p = 0.002, 0.000). In HUVECs, nicotine only had a suppressive effect on the expression of sEng at 6 hours (p = 0.03); there was no effect on sFlt-1 expression. However, pre-treatment with α-BGT did not reverse the nicotine-induced suppressive effect on the expression of sFlt-1 and sEng in trophoblasts and HUVECs. Conclusions. Nicotine reduced the production of sFlt-1 and sEng in trophoblasts and sEng in HUVECs. This effect was not mediated by α7 nAChR.
Although smoking has detrimental effects on pregnancy including preterm delivery and fetal growth restriction, it is the only factor that is associated with a reduced risk of preeclampsia (1–3). In a large population study by Hammoud et al., the incidence of preeclampsia was significantly reduced in smoking pregnant women, as compared to non-smokers (1). Furthermore, the rate of preeclampsia inversely correlated with the number of cigarettes smoked per day. In relation to whether smoking before pregnancy has any protective effect against pregnancy-related hypertension, England et al. observed that women who smoked throughout pregnancy had the lowest incidence of preeclampsia followed by women who had quit smoking during their pregnancy. The incidence was highest in women who had never smoked, as well as in women who had quit smoking before pregnancy (2). However, the exact mechanism by which smoking reduces the risk of preeclampsia is still unknown.
Many studies have reported that excessive amounts of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), which are most likely released from placental tissue, are involved in mediating endothelial cell dysfunction. This process contributes to the pathogenesis of the maternal syndrome preeclampsia (4–6). This proposed role for sFlt-1 and sEng is plausible as sFlt-1 was demonstrated to inhibit endothelial cell tube formation in vitro, and sEng was shown to induce vascular damage. In addition, sFlt-1 expression was significantly increased in the preeclamptic placenta, as compared to normal placenta (5, 7), and sFlt-1 and sEng serum levels were determined to be significantly increased in preeclamptic patients (4, 8–10). The strongest evidence for this hypothesis is from the animal study conducted by Maynard et al., which demonstrated that adenoviral delivery of sFlt-1 to normal rats resulted in characteristic clinical symptoms of preeclampsia including hypertension, proteinuria, and glomerular endotheliosis (5). Concomitant adenoviral overexpression of sFlt-1 and sEng induced hypertension, proteinuria, HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and fetal growth restriction in rodents (4). The results from these studies strongly support that sFlt-1 and sEng work synergistically to induce severe preeclampsia.
Recent reports on the significant difference in the serum anti-angiogenic factors and smoking status are interesting. In relation to sFlt-1, Schmidt-Lucke et al. reported that the level of circulating sFlt-1 was decreased in healthy smokers, as compared to healthy non-smokers (11). Levine et al. recently reported a similar pattern of reduced sFlt-1 serum levels, in addition to decreased sEng in pregnant women who had smoked during their pregnancy, as compared to non-smokers (8). This observation suggested that nicotine or another compound in cigarettes suppressed the production of sFlt-1 and sEng. From this standpoint, smoking may confer a benefit in protecting pregnant women against developing preeclampsia by suppressing the placental production of sFlt-1 and sEng.
We previously demonstrated a difference in the placental expression of alpha 7 nicotinic acetylcholine receptor (α7 nAChR) between normal and preeclamptic women at term, which suggested that this receptor may play a role in the pathophysiology of the disease (12). In vitro studies demonstrated that α7 nAChR exerts proangiogenic, anti-inflammatory, and antiapoptotic effects, which could mediate the protective effects against preeclampsia conferred by smoking (13–15). It has never been studied whether nAChR plays a role in suppressing the antiangiogenic factors produced by the placenta, which is another possible mechanism by which smoking could reduce preeclampsia. In this study, we evaluated the effect of nicotine on the expression of sFlt-1 and sEng in human umbilical vein endothelial cells (HUVECs) and trophoblasts, and we assessed the role of α7 nAChR in mediating the nicotinic effect.
Material and methods
Third trimester umbilical cords were obtained from elective cesarean sections of women with normal pregnancies at the Department of Obstetrics and Gynaecology, Yonsei University College of Medicine, Seoul, Korea. All patients consented to the use of the tissue, and all protocols were Institutional Review Board-approved (4-2007-0012).
Nicotine hydrogen tartrate and α-bungarotoxin (α-BGT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunofluorescence staining and western blotting for α7 nAChR were performed using the monoclonal anti-nicotinic acetylcholine receptor, subunit α7, clone mAb 306 (Sigma, St. Louis, MO, USA). Levels of sFlt-1 and sEng in the culture media were measured using a commercial human sFlt-1 ELISA assay kit (R&D systems, Minneapolis, MN, USA).
The human cytotrophoblast cell line 3A, which was transformed by SV40ts30, was purchased from American Type Culture Collection (Manassas, VA, USA). The cell line was cultured in minimum essential medium (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 33°C in a humidified 5% CO2/95% air incubator. HUVECs were isolated from human umbilical cord veins as previously described by Jaffe et al. (16). The cells used in all experiments were between passages 4 and 5. Cells were cultured in M199 medium (Invitrogen) supplemented with 20% FBS and penicillin/streptomycin at 37°C in a humidified 5% CO2/95% air incubator.
RNA isolation, reverse transcription-PCR, and real-time PCR
Total RNA was extracted using an RNA extraction kit (iNtRON Biotechnology, Gyeonggi, Korea), according to the manufacturer's instructions. RT-PCR was performed to evaluate the mRNA expression of α7 nAChR in HUVECs and 3A cells. The RT parameters for cDNA synthesis were as follows: SuperScript™ III CellsDirect cDNA Synthesis System (Invitrogen, http://www.invitrogen.com) was used for reverse transcriptase-polymerase chain reaction (RT-PCR). About 2 μg of total RNA and 1 μl of Oligo (dT) 20 (50 μM) and 1 μl of 10 mM dNTP were mixed and then incubated again at 65°C for 5 minutes. Tubes were placed on ice for 2 minutes 2 μl 10 × RT Buffer, 1 μl RNaseOUT™, 1 μl SuperScript™ III RT (200 U/μl) and 1 μl mM DTT were added to each tube before incubated for 50 minutes at 50°C. The reaction was inactivated at 85°C for 5 minutes 1 μl of RNase H (2 U/μl) was added to each tube which was then incubated at 37°C for 20 minutes. The reactions were chilled on ice and stored at −20°C until PCR amplification.
The generated cDNA was subjected to PCR amplification in a GeneAmp PCR System 9700 (PerkinElmer, Waltham, MA, USA) amplification cycler. PCR reactions were carried out in a total volume of 20 μl containing 2 μg of cDNA, 0.4 mM dNTPs, each primer set, and 0.2 units of GoTaq polymerase (Promega, Madison, WI, USA). The PCR conditions were: 5 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and 1 minute at 72°C, followed by 72°C for 7 minutes. The PCR products were subjected to electrophoresis in 2% agarose gels. The sequences of the PCR primers for α7 nAChR and β-actin, which was used as an internal control, were as follows: α7 nAChR 5′-CCT GGC CAG TGT GGA G-3′ (sense primer) and 5′-TAC GCA AAG TCT TTG GAC AC-3′ (antisense primer; 414 bp product) and β-actin: 5′-AGG CCA ACC GCG AGA AGA TGA CC-3′ (sense primer) and 5′-GAA GTC CAG GGC GAC GTA GCA C-3′ (antisense primer; 313-bp product).
Western blotting analysis
Proteins were extracted from the cells using lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The extracted proteins were loaded onto a 10% mini-SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane after electrophoresis. Blocking was carried out with 5% non-fat dry milk/PBS and 0.2% Tween-20 for 1.5 hours at room temperature (RT). The membrane was incubated with primary antibody overnight at 4°C with gentle shaking. After washing with PBS and 0.1% Tween-20, the membrane was exposed to anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated antibody (Amersham Life Science, Buckinghamshire, UK) for 1 hour at RT. The membrane was incubated with enhanced chemiluminescence (ECL) reagents (Amersham Life Science) and exposed to Hyperfilm ECL (Amersham Life Science). β-actin was used as an internal control for protein loading, so data could be compared across different films and exposures.
Cells grown on a glass slides were fixed in 2% paraformaldehyde for 30 minutes and then washed with cold PBS. Cells were incubated for 1 hour at RT in blocking solution containing 0.9 g of bovine serum albumin in 30 ml of PBS. They were then incubated with the primary antibody α7 nAChR (1:100 in PBS) for 2 hours at RT. After rinsing, the cells were incubated with FITC-conjugated anti-mouse IgG (1:100 in PBS, Allexis Biochemicals, San Diego, CA, USA) for 1 hour at RT. The cells were incubated with DAPI for 20 minutes at RT, and the coverslips were mounted onto glass slides. The staining was examined using an epifluorescence microscope.
The cell viability was evaluated using a commercially available MTT cell growth assay kit (CT02, Millipore Corp., Bedford, MA, USA), according to the manufacturer's instructions. Briefly, cells were seeded into a 96-well plate at a concentration of 1 × 104 cells/well, and cells were incubated overnight. After 6 and 24 hours, MTT solution was added to control and nicotine-treated, α-BGT-treated wells and incubated for 4 hours to allow the MTT to be metabolized. Medium and MTT were removed. The formazan product was then solubilized in 100 μl HCl in isopropanol. The absorbance was measured at a wavelength of 570 nm and a reference wavelength of 630 nm.
Analysis of level of sFlt-1 and sEng protein in culture media
Briefly, 2 × 104 cells were seeded onto a 24-well plate and cultured overnight until they were 80–90% confluent. Cells were treated with nicotine with or without pre-treatment with α-BGT (1 nM). The culture media was obtained at specific time points and assayed for sFlt-1 and sEng expression using commercial ELISA kits. Cells were harvested, and the total protein was extracted using lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The protein concentration was quantified using the Bradford assay. The concentration of sFlt-1 and sEng in the media was expressed in relation to the total amount of protein per well.
The MTT assay and ELISA results were expressed as means ± standard error of the mean. All experiments were performed in triplicate. Statistical significance was determined by performing a one-way ANOVA on the MTT assay results and a Student's t-test on the ELISA results. A confidence level of greater than 95% was used to establish significance.
The 3A cells, human placenta-derived cytotrophoblasts, and HUVECs were evaluated for the expression of α7 nAChR by immunofluorescence staining. The staining was positive in 3A cells and HUVECs. The specificity of the α7 nAChR staining was confirmed by RT-PCR and western blotting analysis (Figure 1).
The cell viability of cultured HUVECs and 3A cells was assessed using a MTT assay. Treatment of HUVECs and 3A cells with 10 μM of nicotine or 1 nM of α-BGT did not significantly reduce the cell viability at 6 and 24 hours when compared to the control cells (Figure 2).
The levels of sFlt-1 and sEng secreted into the culture media following treatment with nicotine were measured and expressed in relation to the total level of protein per well (Figure 3). There was no significant effect of nicotine on the sFlt-1 protein-to-total protein ratio at 6 and 24 hours in HUVECs (p = 0.04 and 0.28, respectively). However, the level of sEng was significantly reduced after 6 hours of nicotine treatment when compared to the control cells (0.9 ± 0.1 and 1.3 ± 0.1, respectively, p = 0.03). This reduction was not reversed by pre-treatment with the α7 nAChR inhibitor α-BGT.
In 3A cells, the protein-to-total protein ratio for sFlt-1 and sEng was significantly reduced at 24 hours, as compared to the control cells (38.5 ± 0.4 and 47.8 ± 1.1, p = 0.002; 40.5 ± 1.1 and 52.4 ± 1.3, p = 0.000). Similarly to HUVECs, the suppressive effect of nicotine on the production of sFlt-1 and sEng in 3A cells was not inhibited by pre-treatment with α-BGT.
It has been established that cigarette smoking is associated with a reduced risk of preeclampsia. Women who smoked cigarettes throughout their pregnancy had a 33% reduced risk of developing preeclampsia (3, 17, 18). Therefore, it was useful to determine the mechanism by which smoking reduces this risk, as it could result in the identification of a novel therapeutic target to treat preeclampsia. Unfortunately, the exact mechanism for this nicotinic-associated risk reduction has not yet been elucidated.
North et al. hypothesized that smoking reduced the exaggerated immune response observed in preeclamptic patients, resulting in risk reduction (19). Other studies have suggested that the chronically-induced endothelial events caused by cigarette smoking may eventually down-regulate the sensitivity of endothelial cells to the signals that trigger the development of preeclampsia (20). Bainbridge et al. proposed that the carbon monoxide generated from cigarette smoking may directly act on the placenta to increase trophoblast invasion, placental flow, nitric oxide levels through hemoprotein activation, activation of the antioxidant system, and decrease the localized inflammatory response and syncytiotrophoblast apoptosis (21).
Levine et al. evaluated the association between smoking and circulating antiangiogenic factors in a nested case-control study of pregnant women, and they found that those who smoked throughout gestation had significantly lower levels of sFlt-1 and sEng, as compared to those who had never smoked or had quit before the last menstruation (8). This finding, along with other reports involving non-pregnant individuals, strongly suggests that smoking may modulate antiangiogenic factors to prevent pregnant women from developing preeclampsia (11, 22). Furthermore, Mehendale et al. observed that cigarette smoke extract significantly reduced the secretion of sFlt-1 from placental explants under hypoxia and normoxia in a dose-dependent manner, which recapitulated the nicotinic effect in vivo in smokers (23). However, their study could not identify which cell types were predominantly affected by the cigarette smoke or the specific mechanism(s) involved, since placental explants are comprised of various cell types including vessels, stromal cells, and trophoblasts.
In the present study, we demonstrated a nicotine-induced reduction in the secretion of sFlt-1 and sEng from a trophoblast cell line and a reduction in sEng secretion from HUVECs. Our results suggest that suppression of antiangiogenic factors by nicotine may be an important mechanism involved in mediating the reduction in the incidence of preeclampsia in pregnant smokers.
There have been various favorable effects reported for α7 nAChR in endothelial cells in response to nicotine. According to Saeed et al., stimulation of α7 nAChR by nicotine suppressed the activation of endothelial cells, inhibited TNF-induced adhesion molecule expression and chemokine production by endothelial cells, and blocked leukocyte migration during inflammation (24). In addition, stimulation of α7 nAChR protected cells from hypoxia-induced apoptosis (15, 25, 26). Since endothelial cell activation, inflammation, and apoptosis in placental tissue are suggested as part of the pathophysiological process involved in preeclampsia, the aforementioned effects of α7 nAChR may play a significant role in reducing the risk of preeclampsia in pregnant smokers. Thus, we hypothesized that this receptor may mediate the effect of nicotine on suppressing the secretion of antiangiogenic factors; however, pharmacological blockage of α7 nAChR did not inhibit the downregulatory effect of nicotine on sFlt-1 and sEng expressions.
There are potential limitations with the present study. First, the human trophoblast cell line used in this study was immortalized by tsA mutants of simian virus 40, which cannot fully recapitulate in vivo syncytiotrophoblasts. Thus, obtaining trophoblast cells that have not been manipulated and confirming our nicotinic effect in these unmanipulated cells is crucial to substantiate our findings. Second, HUVECs are endothelial cells from the umbilical vein; thus, they do not represent peripheral villous vessels. Therefore, the in vivo response of placental vessels to nicotine in relation to sFlt-1 production cannot be extrapolated because of the lack of response to nicotine by the HUVECs used in this study.
In summary, the present study introduced the potential proangiogenic role of nicotine as a suppressor of sFlt-1 and sEng expression in a trophoblast cell line and a suppressor of sEng expression in HUVECs. These results may explain the protective effect conferred by cigarette smoking in preventing preeclampsia. However, we failed to demonstrate a role for α7 nAChR in this phenomenon. Therefore, the mechanism by which nicotine decreases the secretion of antiangiogenic factors from trophoblasts and HUVECs requires further investigation.
The authors wish to thank Yong Son Maeng, Moung Hwa Kang, and Jeong Hye Hwang for technical assistance.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.