Objective
To investigate placental protein 13 (PP13) localization in relation to cytoskeleton and lipid rafts in preeclampsia and HELLP syndrome.
Study Design
Placental cryosections from patients with preeclampsia and HELLP, and controls were stained for PP13, actin, PLAP (lipid raft marker), and CD71 (nonraft marker). BeWo cells exposed to stress conditions were stained for PP13 and actin. Protein localizations were investigated by confocal microscopy, PP13 concentrations by ELISA.
Results
PP13-actin colocalization was increased in syncytiotrophoblast juxtamembrane regions in term/preterm preeclampsia and HELLP. PP13-CD71 colocalization was decreased and PP13-PLAP proximity was increased in preterm but not term preeclampsia and HELLP. PP13-release from BeWo cells was inhibited by cytoskeleton disruption, and augmented by Ca 2+ -influx and ischemic stress.
Conclusion
The actin cytoskeleton, probably in connection with lipid rafts, controls trophoblastic “nonclassical” PP13 export. PP13 is released from the syncytiotrophoblast in preterm preeclampsia and HELLP, mimicked in BeWo cells by ischemic stress, suggesting PP13 is a placental alarmin.
Preeclampsia, one of the “great obstetric syndromes,” affects approximately 3-5% of pregnant women and is a major cause of maternal and perinatal morbidity and mortality. It is characterized by the new onset of hypertension and proteinuria after 20 weeks of gestation. In the most severe cases, seizures (eclampsia), multiorgan damage including cerebrovascular, hepatic or renal failure, and placental abruption and HELLP (Hemolysis, Elevated Liver enzymes and Low Platelets) syndrome may develop. HELLP syndrome, which affects 0.5-0.9% of all pregnancies and 2-12% of pregnancies complicated by preeclampsia, is thought to be a more severe form of preeclampsia although approximately 15-20% of the patients do not develop hypertension and/or proteinuria.
Both preeclampsia and HELLP syndrome are heterogeneous. The involvement of the placenta in the different pathogenic pathways is well-established because the only definitive treatment of these syndromes is delivery of the fetus and placenta. Preterm preeclampsia and HELLP syndrome are associated with abnormal placentation, impaired trophoblast invasion, and remodeling of the uterine spiral arteries. Failure of physiologic transformation of the spiral arteries in these cases is thought to result in intermittent, turbulent flow, damage of the placental architecture, fluctuations in placental oxygenation, and placental endoplasmic reticulum-, oxidative-, and nitrative stress. Placental ischemia-reperfusion injury triggers an increased release of apoptotic-necrotic syncytiotrophoblast microparticles (MPs), proinflammatory cytokines, and antiangiogenic factors, which in turn, lead to generalized maternal endothelial cell dysfunction and an exaggerated maternal systemic inflammatory response.
In normal pregnancy, MPs continuously shed from the syncytiotrophoblast, the outermost fused cell layer of the human placenta, to the maternal circulation as part of the natural renewal process of placental microvilli. The amount of circulating MPs increases with advancing gestational age in normal pregnancies and is significantly elevated in the blood of women with preterm preeclampsia. Of note, we have previously shown that syncytiotrophoblast cytoplasm protrusions, membrane blebs, and MPs shed from the syncytiotrophoblast apical membrane have intense placental protein 13 (PP13) immunostaining in preterm preeclampsia and HELLP syndrome. This is consistent with the findings that maternal serum PP13 concentration increases during normal pregnancy with a peak in the third trimester, and it is higher in patients presenting with preterm preeclampsia and HELLP syndrome than in preterm controls. Surprisingly, we did not find such difference in placental PP13 staining and maternal serum PP13 concentrations in patients with term preeclampsia when compared with normal term control patients.
In the context of these observations, it is important to note that PP13 is specifically expressed by the placenta in anthropoid primates, primarily in the syncytiotrophoblast. PP13 is a member of the evolutionarily conserved family of galectins (galectin-13), which are key regulatory proteins of immune-homeostasis and inflammation. Galectins are synthesized in the cytosol and are alternatively transported to the plasma membrane, avoiding the endoplasmic reticulum (ER) and Golgi vesicles, through a so-called “nonclassical” secretory pathway. On the cell surface and in the extracellular matrix, galectins can initiate leukocyte signaling and modify cell fate on binding to their cell-surface ligands, leading to either apoptosis or cell survival. Galectins can also have various effects on cell growth, cytokine secretion, cell-cell and cell-extracellular matrix interactions.
A previous study showed that, similar to other galectins, PP13 accumulates in the cytoplasmic side of the plasma membrane, leading us to hypothesize that it is secreted to the syncytiotrophoblast surface by the actin filamental network. It has also been demonstrated that PP13 specifically binds to and colocalizes with annexin-II at the apical membrane of the syncytiotrophoblast. Of note, similar to placental alkaline phosphatase (PLAP), a protein highly expressed in the syncytiotrophoblast apical membrane, annexin-II is also associated with lipid rafts, which are highly dynamic, cholesterol- and sphingolipid-rich 10-200 nm microdomains in the mammalian cell membrane. Lipid rafts can grow to 1 μm and provide a stable platform for lipid-lipid and protein-lipid interactions on various stimuli, such as receptor cross-linking during signaling or endocytosis.
Based on these findings, we hypothesized that: (1) the actin filamental network and lipid rafts in the syncytiotrophoblast apical membrane may play a role in the normal secretory and shedding mechanisms of PP13, and (2) the pathophysiologic processes affecting the syncytiotrophoblast in various phenotypes of preeclampsia and HELLP syndrome may lead to the increased secretion and/or shedding of PP13 from the villous tree. Therefore, this study was designed to investigate whether changes in the placental localization of PP13 and its spatial relationship to lipid rafts and the actin cytoskeleton could be detected in the placentas obtained from women with preeclampsia or HELLP syndrome and gestational age-matched controls using confocal imaging. To get insight into the possible cellular mechanisms of PP13-release from the trophoblast, the role of the actin cytoskeleton and different stress factors on PP13-release was studied in vitro in BeWo cells.
Materials and Methods
Sample collection and patient groups
The study was approved by the Health Science Board of Hungary (ad.22-164/2007-1018EKU) and the Human Investigation Committee of Wayne State University (036410M1X). Written informed consent was obtained from women before sample collection; specimens were coded and data were stored anonymously. Placental villous tissues were collected at the First Department of Obstetrics and Gynecology, Semmelweis University (Budapest, Hungary, Federalwide Assurance: FWA00002527) in the following gestational age-matched groups (n = 5 in each): (1) preterm preeclampsia (≤35 weeks), (2) preterm HELLP syndrome (≤35 weeks), (3) term preeclampsia (>37 weeks), (4) preterm (≤35 weeks), and (5) term (>37 weeks) controls. Patients with multiple pregnancies or with fetuses having congenital or chromosomal abnormalities were excluded. Demographic and clinical characteristics of the patient groups are shown in the Table .
| Groups | Term controls | Term preeclampsia | Preterm controls | Preterm preeclampsia | Preterm HELLP syndrome |
|---|---|---|---|---|---|
| Number of cases a | 5 | 5 | 5 | 5 | 5 |
| Maternal age, y b | 30.8 (30.6–31.5) | 32.2 (30.3–33.6) | 31.6 (31.5–34.3) | 34.0 (30.2–34.5) | 28.1 (24.1–29.2) |
| Gestational age at delivery, wk b | 38.9 (38.7–39.0) | 38.4 (37.7–39.9) | 31.0 (30.9–34.0) | 32.6 (30.3–34.9) | 32.0 (29.3–33.1) |
| Primiparity c | 40 | 60 | 40 | 80 | 60 |
| Smoking c | 0 | 0 | 20 | 0 | 0 |
| Systolic blood pressure, mm Hg b | 130 (120–135) | 160 (153–170) d | 120 (120–120) | 160 (155–163) e | 170 (170–170) f |
| Diastolic blood pressure, mm Hg b | 80 (78–89) | 90 (90–100) | 80 (70–80) | 100 (98–101) e | 100 (90–110) f |
| Proteinuria c | 0 | 100 | 0 | 100 | 100 |
| Maternal BMI, kg/m 2 b | 26.7 (25.2–28.0) | 22.0 (20.0–23.3) | 23.4 (21.6–24.6) | 24.2 (22.6–24.9) | 21.4 (21.2–26.0) |
| Neonatal birthweight, g b | 3440 (3400–4030) | 2810 (2620–3200) d | 1990 (1640–2210) | 1100 (990–1200) | 1480 (990–1610) |
| Cesarean section c | 100 | 100 | 100 | 100 | 100 |
| Placental weight, g b | 518 (458–665) | 458 (431–476) | 301 (294–322) | 217 (211–224) | 302 (255–305) |
| Thrombocytes, M/mm 3 b | 178 (173–238) | 206 (191–250) | 227 (170–298) | 233 (187–360) | 80 (73–93) e |
| Hgb, g/100 mL | 10 (10–11) | 12 (12–12) | 11 (11–11) | 13 (13–13) f | 13 (12–14) |
| SGOT, U/L b , g | 25 (22–32) | 31 (25–80) | 333 (318–439) | ||
| SGPT, U/L b , g | 17 (13–19) | 33 (23–80) | 337 (315–448) | ||
| LDH, U/L b , g | 246 (196–332) | 293 (217–350) | 572 (510–646) | ||
| Bilirubin, μmol/L b , g | 15 (12–15) | 5 (4–12) | 19 (17–21) |
a Values are presented as number;
b Values are presented as median (interquartile [IQR] range);
c Values are presented as percentage;
d P < .05 compared with gestational age matched, term controls;
e P < .05 compared with gestational age matched, preterm controls;
Term and preterm controls had no medical complications or clinical or histologic signs of chorioamnionitis, and delivered neonates of appropriate weight for gestational age. Preeclampsia was defined as hypertension that developed after 20 weeks of gestation (systolic or diastolic blood pressure ≥140 or ≥90 mm Hg, respectively, measured at 2 different time points, 4 hours to 1 week apart) coupled with proteinuria (≥300 mg in a 24-hour urine collection, or 2 random urine specimens obtained 4 hours to 1 week apart containing ≥1+ by dipstick or 1 dipstick of ≥2+ protein). HELLP syndrome was defined as hemolysis (serum LDH >600 IU/L; bilirubin >1.2 mg/dL; presence of schistocytes in peripheral blood), elevated liver enzymes (serum ALT and/or AST >70 IU/L), and thrombocytopenia (platelet count <100,000/mm 3 ).
None of the patients had regular uterine contractions; cesarean delivery was performed in all cases because of previous cesarean section (term controls), or preterm premature rupture of the membranes and breech presentation (preterm controls), or severe clinical symptoms (patients with preeclampsia or HELLP syndrome).
Fluorescence immunohistochemistry
Fresh placental specimens were frozen in isopentane (Sigma-Aldrich, St. Louis, MO); subsequently, 10 μm thick cryosections (2-4 cryosections/sample) were cut and placed on Superfrost Plus slides (Thermo Scientific, Walthem, MA), fixed with 4% paraformaldehyde and then kept at 4°C in phosphate-buffered saline (PBS) until staining. Nonspecific antibody binding was blocked with 5% bovine serum albumin (BSA) in PBS (30 minutes at room temperature). Samples were incubated with monoclonal anti-PP13 antibody (clone: 27-2-3; 5 μg/mL; Diagnostic Technologies Ltd, Yokneam, Israel) alone or in combination with anti-PLAP (1 μg/mL; Sigma-Aldrich), anti-CD71 (5 μg/mL; Biolegend, San Diego, CA), or antiactin (8 μg/mL; Sigma-Aldrich) antibodies in PBS containing 1% BSA. Samples were stained with anti-PP13 overnight, whereas in the case of other primary antibodies, the incubation time was 45 minutes. Washing was followed by incubation (45 minutes at room temperature) with Alexa Fluor488-conjugated antimouse IgG1 (2 μg/mL; Invitrogen-Molecular Probes, Eugene, OR) for anti-PP13, Alexa Fluor633-conjugated antimouse IgG2a (2 μg/mL; Invitrogen-Molecular Probes) for anti-PLAP and anti-CD71, or Alexa Fluor647-conjugated antirabbit IgG (2 μg/mL; Invitrogen-Molecular Probes) for antiactin. Isotype control antibodies were used as negative controls: mouse IgG1 (clone MOPC-21; BD Pharmingen, San Jose, CA) and mouse IgG2a (clone HOPC-1; Southern Biotech, Birmingham, AL). Nuclei were counterstained with propidium iodide (0.2 μg/mL; Sigma-Aldrich). Washed samples were assayed with an Olympus Fluoview 500 confocal microscope (Hamburg, Germany) equipped with 3 lasers and 4 optical channels, using a 60× (N.A.: 1.1) oil-immersion objective.
BeWo cell transfection
BeWo cells (American Type Culture Collection, Manassas, VA) were incubated in 2 T-75 flasks with F12 medium (Invitrogen-Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) until reaching 80% confluency. PP13 vector DNA (9 μg; OriGene, Rockville, MD) was diluted in 450μL of serum-free Opti-MEM I medium (Invitrogen-Gibco). Then, 36 μL FuGENE HD transfection reagent (Promega, Madison, WI) was added to the mix, which was incubated for 15 minutes at room temperature. Ten milliliters of fresh Opti-MEM I medium were added to 1 of the T-75 flasks after the cells were washed once with PBS. The transfection complex was then added to this flask and incubated for 6 hours before replacing the medium to F12 (supplemented with 10% FBS and 1% P/S). BeWo cells without PP13 vector transfection were used as control.
BeWo cell treatments
After 24 hours in culture, transfected and nontransfected BeWo cells were trypsinized and plated onto 6-well plates (2 × 10 5 cells/well) and 4-well Lab-Tek chamber slides (5 × 10 4 cells/well; Thermo Scientific), and were cultured for 24 hours in F12 medium supplemented with 10% FBS and 1% P/S. After medium change, 60-hour treatments started in the following regimes: (1) to inhibit actin polymerization, cells were treated with Latrunculin B (0.125 and 0.25 μM; EMD Chemicals, Gibbstown, NJ) between hours 48-60; (2) to stimulate nonclassical protein secretion by elevated cytoplasmic Ca 2+ -level, cells were treated with ionophore A23187 (1 μM; Sigma-Aldrich) between hours 48-60; and (3) to induce cellular stress, cells were treated with either human recombinant TNFα (4 ng/mL; R&D Systems, Minneapolis, MN) between hours 0-60, or kept under hypoxic (1%O 2 ) or ischemic conditions (1% and 20% O 2 in alternating, 12 hour cycles) between hours 0-60. Control cells were treated with vehicle (DMSO; Sigma-Aldrich).
RNA isolation and quantitative real-time reverse transcribed-polymerase chain reaction
After collecting supernatants, BeWo cells were harvested from 6-well plates and total RNA was isolated using QuickGene-Mini80 (Fujifilm, Allendale, NJ) according to the manufacturer’s protocol. Five hundred nanograms of total RNA was reverse transcribed with TaqMan Reverse Transcription Reagent kit using random hexamers (Applied Biosystems, Foster City, CA). Quantitative reverse transcribed-polymerase chain reaction (qRT-PCR) was run in triplicates with LGALS13 TaqMan assay (Hs00747811_m1) and RPLPO TaqMan Endogenous Control (4326314E) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Ct values for LGALS13 and RPLPO were averaged over 3 technical replicates. -DCt value, a surrogate of log 2 mRNA concentration, was obtained for each sample as: -DCt ( LGALS13 ) = Ct (RPLPO) − Ct (LGALS13) .
Protein isolation
After collecting supernatants, BeWo cells were harvested from 6-well plates, homogenized with 120 μL lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 5 mM EDTA) and incubated for 1 hour on ice. Homogenates were centrifuged (15 minutes at 10,000 rpm) to pellet nonsoluble debris; supernatants containing soluble or solubilized proteins were collected, and their total protein contents were measured by BCA assay (Thermo Scientific).
PP13 immunoassay
PP13 content of BeWo cell lysates and supernatants was measured using a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (Diagnostic Technologies Ltd) with a pair of PP13-specific monoclonal antibodies, marked with amplified biotin-extravidin-horseradish-peroxidase complex, and developed with tetramethylbenzidine substrate, as described previously. The optical density was measured at 450 nm against a 650 nm background. PP13 concentrations were determined by extrapolation from a standard curve constructed using recombinant PP13 standards (0-500 pg/mL), and were normalized to total protein in cell lysates. The sensitivity of the assay was 9.6 pg/mL. The laboratory staff performing the assays was blinded to sample information.
Fluorescence immunocytochemistry
PP13-transfected, treated or untreated BeWo cells on chamber slides were simultaneously fixed and permeabilized in PBS supplemented with 4% paraformaldehyde and 0.1% Triton X-100 (Sigma-Aldrich), followed by washing with PBS supplemented with 1% FBS and 0.1% sodium-azide. Nonspecific antibody binding was blocked with 5% BSA in PBS (30 minutes at room temperature). Samples were incubated overnight with monoclonal anti-PP13 antibody (clone: 27-2-3; 20 μg/mL) alone or in combination with antiactin (7 μg/mL) antibody in PBS containing 5% BSA. Washing was followed by incubation (2 hours at room temperature) with Alexa Fluor488-conjugated antimouse IgG1 (2 μg/mL) for anti-PP13 and Alexa Fluor555-conjugated antirabbit IgG (2 μg/mL) for antiactin. Isotype control antibodies were used as negative controls. Alternatively, cells were stained with Alexa Fluor555-conjugated phalloidin (2 U/mL; Invitrogen-Molecular probes) to visualize filamentous actin. Nuclei were counterstained with DRAQ5 (1 μM; Biostatus, Leicestershire, UK). Washed samples were assayed with an Olympus Fluoview 500 confocal microscope equipped with 4 lasers and 4 optical channels, using 20× (N.A.: 0.7) or 40× (N.A.: 0.85) objectives.
Data analysis
Colocalization indices were determined from approximately 100 regions of interest (ROIs)/each group by the ImageJ software ( http://rsbweb.nih.gov.easyaccess1.lib.cuhk.edu.hk/ij . Accessed May 2, 2011.) using the Red Green Correlator plug-in ( http://www.uhnresearch.ca/facilities/wcif/imagej/colour_analysis.htm . Accessed May 2, 2011.). The Pearson’s colocalization index (CI) provides a reliable estimate on the extent of protein colocalization. A CI value of −1 means the exclusive appearance of the 2 labeled specimens, its value close to zero indicates a random appearance, whereas CI values between 0 and +1 reflect a continuously increasing degree of colocalization, while CI = 1 corresponds to a full overlap between the 2 colors in each pixels of the image.
Line scan analysis was carried out using the Fluoview software. Lines (approximately 200/group) were laid across placental microvilli stained for PP13 and actin or PLAP. Intensity values were sorted into membrane/juxtamembrane (3 μm at opposite sides of the villi) and cytoplasmic (internal) regions. Based on the staining pattern, 3 groups were formed for quantifying these intensity distributions: (1) PP13 and actin (or PLAP) were localized in the same region; (2) only PP13 was localized in the selected region; and (3) only actin (or PLAP) was localized in the selected region (based on the actual intensity exceeding the threshold level). The relative frequency of each staining pattern was then calculated from approximately 300 ROIs. Intensity distributions were also demonstrated on line scan histograms. The interactive 3-dimensional (3D) surface plot plug-in of ImageJ was also used to create another representation platform of the data on the different samples.
Statistical analysis
Demographic and clinical data were analyzed by Statistica8 (StatSoft Inc, Tulsa, OK). Comparisons among the groups were performed by χ 2 test and Fisher’s exact test for proportions and Kruskal-Wallis test, followed by Mann-Whitney test for continuous variables. GraphPad Prism 4 (GraphPad Software, La Jolla, CA) was used to analyze the differences in the colocalization, gene expression, and protein concentration values between the groups using Mann-Whitney test or t test. A P value of < .05 was considered statistically significant.
Results
Localization of PP13 in the syncytiotrophoblast in preeclampsia and HELLP syndrome
The localization of PP13 in placental villous tissues from patients with preeclampsia or HELLP syndrome and gestational age-matched controls was determined with confocal microscopy using immunohistochemical staining. Similar to earlier data, PP13 was present in the endothelium of fetal vessels, and it was found in the membrane and the cytoplasm of the syncytiotrophoblast ( Figure 1 ). There was a shift in the localization of PP13; stronger staining was observed in the plasma membrane rather than the cytoplasm in patients with term and preterm preeclampsia and HELLP syndrome in comparison to gestational age-matched controls ( Figure 1 ). This finding is consistent with our previous report.
Visualization of the interaction patterns of PP13 with cortical actin cytoskeleton and the raft marker PLAP or the nonraft protein CD71 in placental cryosections
Next, we analyzed the interaction patterns of PP13 with the cortical actin cytoskeleton and with 2 membrane proteins, PLAP and CD71. A relatively high colocalization was found between PP13 and actin in term and preterm controls (CIs; mean ± standard error of the mean [SEM]: 0.357 ± 0.009 and 0.357 ± 0.008, respectively). The colocalization of these 2 proteins (PP13 and actin) was significantly increased in all patient groups (CIs, 0.459 ± 0.008, P < .05 for term preeclampsia; 0.509 ± 0.009, P < .05; and 0.520 ± 0.010, P < .05 for preterm preeclampsia and HELLP syndrome, respectively) when compared with gestational age-matched controls ( Figures 2 , A and 3 , A). The membrane localization of PP13 was also altered in preeclampsia and HELLP syndrome. Although PP13 highly colocalized with the lipid raft-associated membrane protein PLAP in both control groups (CIs, 0.468 ± 0.007, 0.481 ± 0.012), no significant changes in the colocalization of PP13 and PLAP could be seen in term and preterm preeclampsia or HELLP syndrome (CIs, 0.468 ± 0.009, 0.440 ± 0.009, and 0.485 ± 0.010, respectively) ( Figures 2 , B and 3 , B). In contrast, PP13 highly colocalized with CD71 in term and preterm controls (CIs, 0.505 ± 0.009 and 0.466 ± 0.009, respectively) as well as in term preeclampsia (CI, 0.490 ± 0.010), but this colocalization was significantly reduced in preterm preeclampsia (CI, 0.327 ± 0.009, P < .05) and HELLP syndrome (CI, 0.350 ± 0.013, P < .05) ( Figures 2 , C and 3 , C).
