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;
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