Abstract
The phospholipase A2 (PLA2) family is a very diverse group of enzymes, all serving in the cleavage of phospholipids, thereby releasing high amounts of arachidonic acid (AA) and lysophospholipids. AA serves as a substrate for prostaglandin production, which is of special importance in pregnancy for the onset of parturition. Novel research demonstrates that PLA2 action affects the immune response of the mother toward the child and is therefore probably implied in the tolerance of the fetus and prevention of miscarriage. This review presents data on the biochemical and enzymatic properties of PLA2 during gestation with a special emphasis on its role for the placental function and development of the fetus. We also critically discuss the possible pathophysiological significance of PLA2 alterations and its possible functional consequences. These alterations are often associated with pregnancy pathologies such as preeclampsia and villitis or pregnancy complications such as obesity and diabetes in the mother as well as preterm onset of labor.
Introduction to the PLA2 superfamily
The phospholipase A2 (PLA2) superfamily consists of enzymes of very diverse nature with respect to their cellular localization, dependency on cofactors, and substrate preference. As a consequence of this diversity, PLA2 enzymes have been implicated in various biochemical processes (Kudo & Murakami 2002, Quach et al. 2014). Common to all these enzymes, however, is that all of them catalyze the hydrolysis of an ester bond at the second carbon group (sn-2 position) of a glycerophospholipid releasing a free fatty acid (FFA) and a lysophospholipid. The fatty acid freed from the lipid backbone is in many cases arachidonic acid (AA; C20:4), which is used within the cyclooxygenase (COX) or the lipoxygenase (LOX) pathway to form prostaglandin (PG) and thromboxane or leukotriene respectively. These metabolites are important mediators of inflammation. However, more importantly, eicosanoic lipid mediators generated within the COX pathway are crucial for the coordinated and timely onset of labor. Moreover, PGs are essential to ovulation, fertilization, and implantation, and endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. Additionally, phospholipases also play a role in the remodeling and the stability of lipid membranes (Robichaud et al. 2015), therefore affecting placental and fetal development during pregnancy. In the following, an overview of PLA2 family members will be provided, with special emphasis on those family members that are important in pregnancy, pregnancy pathologies, and (preterm) parturition.
Classification of human PLA2 family members
Many different aspects are used to subdivide PLA2 family members into four groups, with the most commonly found classification being that according to Dennis (Schaloske & Dennis 2006). This system uses cellular localization as well as calcium dependency to distinguish PLA2 groups. An overview of PLA2 family members is provided in Table 1. Important structural features of each PLA2 group are depicted in Fig. 1, whereas Fig. 2 summarizes the action of PLA2 isozymes in maternal and fetal circulation as well as reproductive tissues.
Schematic structure of PLA2 enzymes. (A) Representative secretory PLA2 isozyme, group IIA. Green box indicates the calcium binding domain and turquoise, the catalytic domain. The red box shows the typical short C-terminal extension. Two structurally and functionally important cysteine residues are indicated as well. (B) Cytosolic PLA2 group IVA. As before, green and blue boxes indicate calcium binding and catalytic domains respectively. Asterisks indicate amino acid residues important to active site formation. (C) Calcium-independent PLA2-VIA (PNPLA9). Orange boxes represent the seven characteristic ankyrin repeats, the red box indicates the patatin domain with its active site serine residue being highlighted. (D) Plasma PAF–AH. The blue box indicates the catalytic site with the serine residue of the G-X-S-X-G lipase motif being highlighted. Modified scheme from Murakami et al. (2002).
Citation: Reproduction 151, 6; 10.1530/REP-15-0519
Classification of PLA2 isozymes according to Schaloske and Dennis (2006).
| PLA2 subfamily | Enzymes | Other names | Protein size (kDa) | Ca2+ dependency (molar range) | Substrate preference | FFA released |
|---|---|---|---|---|---|---|
| sPLA2 | IB | Pancreatic lipase | 14 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA |
| IIA | Synovial lipase | 14 | mM | PS+PE>PC | AA, EPA, DHA, OA, LA | |
| IIC | 15 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA | ||
| IID | 14 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA | ||
| IIE | 14 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA | ||
| IIF | 16 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA | ||
| V | 14 | mM | PS+PE+PC | AA, EPA, DHA, OA, LA | ||
| X | 14 | mM | PS+PE+PC | AA, EPA, DHA, OA, LA | ||
| III | 55 | mM | PS, PE, PC | AA, EPA, DHA, OA, LA | ||
| XII | 19 | mM | PC>PS+PE | AA, EPA, DHA, OA, LA | ||
| cPLA2 | IV-A | cPLA2α | 85 | <µM | PC>PS | AA, EPA |
| IV-B | cPLA2β | 110 | <µM | Sn-1 position cleavage, PC | Little AA | |
| IV-C | cPLAγ | 60 | None | Sn-1 and sn-2 position cleavage, PC | Little AA | |
| iPLA2 | VI-A | iPLA2β, PNPLA9 | 85–88 | None | Sn-1 and sn-2 position cleavage, PC | No strict preference |
| VI-B | iPLA2γ, PNPLA8 | 90 | None | Sn-1 and sn-2 position cleavage, PC | No strict preference | |
| NA | iPLAζ, ATGL, PNPLA2 | 55 | None | Dietary TG | No strict preference | |
| NA | iPLA2δ, NTE, PNPLA6 | 155 | None | Lysophospholipids, LPC, LPA | No strict preference | |
| NA | iPLA2ε, PNPLA3 | 58 | None | Unclear | Unclear | |
| NA | iPLA2η, PNPLA4 | 27 | None | Retinoic acid | NA | |
| NA | NRE, PNPLA7 | 146 | None | Lysophospholipids, LPC, LPA | No strict preference | |
| PAF–AH | VII-A | Plasma PAF–AH, LpPLA2 | 45 | None | PC-TG, PAF, oxidized side chain | AA |
| VII-B | PAF–AH-II | 40 | None | PC-TG, PAF, oxidized side chain | AA | |
| VIII-A | PAF–AH 1 a1 subunit | 29 | None | PAF | Acetate | |
| VII-B | PAF–AH 1 a2 subunit | 30 | None | PAF | Acetate |
The official numbering system using Latin numbers as well as commonly used trivial names are given. Protein size (kDa), Ca2 + dependency, and major substrates are indicated. In addition to the mentioned lipases, recently two lysosomal PLA2s (aiPLA2 and LPLA2) as well as an adipose tissue-specific PLA2 (AdPLA2) have been discovered. PLA2 isozymes highlighted in bold are those playing a major part in pregnancy and therefore discussed in this review. sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; iPLA2, intracellular, calcium-independent PLA2; PNPLA, patatin-like phospholipase; ATGL, adipocyte triglyceride lipase; NTE, neuropathy target esterase; NRE, neuropathy target-related esterase; PAF–AH, platelet-activating factor–acetyl hydrolase.
Secretory PLA2
Secretory PLA2s (sPLA2s) form the largest group within the calcium-dependent PLA2 family (Murakami et al. 2011 a,b. They are typically very small enzymes ranging between 14 and 19 kDa, with the exception of PLA2-III (55 kDa). Apart from PLA2-XII, all group members are closely related and share significant sequence homology. All family members share six absolutely conserved disulfide bonds as well as some additional ones (Kudo & Murakami 2002), which are specific to enzyme. In close proximity to one another, a histidine–aspartic acid catalytic dyad and a calcium-binding loop are found in all the sPLA2s. It is also noteworthy that many sPLA2 family members tightly bind to heparan sulfate proteoglycans as the cationic cargo of the enzyme. Therefore, rather than being secreted, they are often located in cell membranes within the extracellular matrix (Murakami et al. 1996).
Overview of PLA2 family members in maternal circulation, uterine layers, placenta, placental membranes, and fetal circulation. Only the most important PLA2 isozymes and pregnancy pathologies are highlighted in this schematic overview. Factors regulating PLA2 isozymes, e.g. cytokines, chemokines, and phospholipids (not indicated in the scheme), are present in both maternal and fetal circulation.
Citation: Reproduction 151, 6; 10.1530/REP-15-0519
sPLA2-IIA (synovial lipase)
sPLA2-IIA acts as a pro-inflammatory enzyme, which is constitutively expressed within various tissues and cells. Both neutrophils and macrophages are able to store the enzyme in granules and secrete it upon activation (Balsinde et al. 1996). sPLA2-IIA is up-regulated by pro-inflammatory cytokines; down-regulated by glucocorticoids, transforming growth factor-b, and interleukin 10 (IL10; Kuwata et al. 1998); and additionally holds antimicrobial and anticoagulant properties (Mounier et al. 1996, Qu & Lehrer 1998).
High levels of plasma sPLA2-IIA are associated with pathologies characterized by a high degree of inflammation, such as septic shock (Pruzanski & Vadas 1991), rheumatoid arthritis (Seilhamer et al. 1998), atherosclerosis (Webb 2005), and respiratory distress syndrome (Masuda et al. 2005), and more importantly during labor (Lappas et al. 2004).
sPLA2-V
sPLA2-V is another important member of the secretory phospholipase group, likely owed to the fact that it can substitute for sPLA2-IIA activity. It is highly expressed in myocytes, but also in immune cells such as mast cells, macrophages, and Th2 cells (Balboa et al. 1996). Similar to sPLA2-IIA, it is up-regulated by pro-inflammatory stimuli such as IL1; however, it cannot be down-regulated by glucocorticoids (van der Helm et al. 1996, Thomas et al. 2000).
In addition to cytokines and hormones, receptors for sPLA2s (PLA2R) have been found to provide a regulatory mechanism for sPLA2 enzymes. PLA2R forms may be produced by the action of metalloproteinases and are highly expressed in the kidney, but also expressed in pancreas, amnion, chorion–decidua, and placenta (Moses et al. 1998). PLA2R1 binds sPLA2 groups I, II, V, and X but not III and XII. It is an 180 kDa integral membrane receptor with a very large extracellular and a very small intracellular domain. Although its precise function remains unclear, binding of sPLA2 to its receptor participates in both positive and negative regulation of sPLA2 functions as well as clearance of sPLA2. PLA2R1 seems to serve pleiotropic effects apart from fatty acid metabolism, regulate cell senescence, aging, and cancer (Lambeau 1999, Augert et al. 2013), and to be the major auto-antigen in nephropathy (Stanescu et al. 2011).
Cytosolic PLA2s
The cytosolic PLA2 (cPLA2) subfamily consists of three isozymes that are expressed in the cytosol of most cell types ubiquitously and constitutively (Leslie 1997, Hirabayashi & Shimizu 2000). This includes cell types of the uterus and trophoblasts of the placenta, which makes cPLA2 enzymes important in fertility, ovulation, implantation, and placentation (Uozomi et al. 1997, Song et al. 2002). The activity of cPLA2 and sPLA2 enzymes seems to be interconnected (Murakami et al. 1998).
Intracellular, calcium-independent PLA2s
Intracellular, calcium-independent PLA2 (iPLA2) isozymes are often also referred to as patatin-like phospholipases (PNPLAs). Although there are nine members of human PNPLAs, not all these enzymes have been assigned an abbreviation within the PLA2 classification system because most PNPLA/iPLA2 family members do not elicit classical PLA2 activity. Some PNPLAs/iPLA2s also have triglyceride (TG) lipase, lysophospholipase, esterase, or trans-acetylase activity, but no phospholipase activity at all (Murakami et al. 2011a,b). The most important member of this group is adipose TG lipase (ATGL). ATGL mainly hydrolyzes dietary TG and activates TG stored in lipid droplets and white adipose tissue (Zechner et al. 2009). ATGL has also been shown to be expressed in human and rodent placental tissue (Barrett et al. 2014).
Platelet-activating factor–acetyl hydrolases
The family of platelet-activating factor–acetyl hydrolases (PAF–AH), which degrades pro-inflammatory phospholipids (PAF), consists of two intracellular isoforms (Ib and II) and one secreted isoform (plasma). The plasma and tissue concentration of PAF is determined by a balance between its biosynthesis and degradation (Arai et al. 2002).
Plasma PAF–AH
The more commonly termed lipoprotein-associated PLA2 (LpPLA2) is an extracellular enzyme produced and secreted mainly from differentiated macrophages (Tjoelker et al. 1995). In systemic circulation, this enzyme attaches to LDL (80%) and HDL (20%) particles. LpPLA2 possesses a unique substrate preference for oxidized phospholipids, chemically resembling PAF, which is the principal substrate of LpPLA2. The exact role of the enzyme, pro- or anti-inflammatory, remains enigmatic (Marathe et al. 2014) and might depend on association with either LDL or HDL (Lagos et al. 2009, Tellis & Tselepis 2009). Serum LpPLA2 levels are elevated in a number of inflammatory conditions (Vickers et al. 2009), but also familiar to hypercholesterolemia and diabetes (Serban et al. 2002, Tsimihodimos et al. 2002, Silva et al. 2011). In pregnancy, elevated serum LpPLA2 levels have been measured in mothers suffering from pregnancy hypertension, preeclampsia (PE), and gestational diabetes mellitus (GDM) – all are pregnancy-associated diseases.
PAF–AH-II
PAF–AH-II is an intracellular enzyme that shares about 41% sequence homology with the plasma form, LpPLA2 (Matsuzawa et al. 1997). It exerts a substrate preference similar to LpPLA2 but acts intracellularly with not only hydrolase but also trans-acetylase activity. High levels of intracellular PAF–AH-II activity have been detected in placental trophoblast cells, suggesting its possible relationship with labor induction (Gu et al. 2006).
PAF–AH-Ib
PAF–AH-Ib is an intracellular, hetero-trimeric enzyme with substrate preference for PAF, but not oxidized phospholipids. The tissue-specific enzyme distinctly reveals the importance in the development of fetal brain and spinal cord; however, apart from that, no further known association with pregnancy has been described so far (Hattori et al. 1994).
In addition, recently two lysosomal PLA2s (aiPLA2 and LPLA2) as well as an adipose tissue-specific PLA2 (AdPLA2) have been discovered but are not yet extensively studied (Murakami et al. 2011a,b).
Serum PLA2 levels in pregnancy
The best investigated PLA2 family member in general, but also in pregnancy, is sPLA2-IIA (synovial phospholipase). Although the cellular origin of sPLA2-IIA seems to be still unknown, in pregnant women, several uterine tissues, the placenta, and fetal membranes express high amounts of sPLA2-IIA. This is corroborated by increased activity of PLA2 during late gestation, which could be likely due to the impact of hormones such as estrogen, which is elevated during pregnancy. Oxytocin is another potential factor that could be contributing to modulating PLA2 levels. It is the most potent uterotonic agent known so far, and Lefebvre et al. (1992) showed that during gestation, oxytocin mRNA increases at term, supporting the idea that it may act as a local mediator rather than a circulating hormone.
PLA2 levels in the first trimester
Hayashi et al. (2000) investigated serum sPLA2-IIA levels in non-pregnant women during their menstrual cycle, as well as in pregnant women throughout the pregnancy. In non-pregnant women, serum sPLA2-IIA levels were lowest in the luteal phase and comparably high in the menstrual and follicular phases. In pregnant women, serum sPLA2-IIA levels were about twice as high as in the menstrual and follicular phases; however, this did not reach statistical significance. Also, among the three trimesters, no further changes in serum sPLA2-IIA levels were observed. There was also no significant difference for circulating PLA2-IIA levels between maternal and fetal serum; however, interestingly, serum PLA2-IIA concentrations were significantly higher in postpartum women than in normal pregnant women. These results suggest that a regulatory mechanism of PLA2-IIA may exist during the normal menstrual cycle and at puerperium. However, this study contradicted the work of Pulkinnen et al. (1993) that clearly showed low levels of sPLA2-IIA during the first trimester, likely to allow for proper implantation, and that sPLA2 is in turn induced significantly during labor, to allow parturition. In this study, not only sPLA2-IIA concentration but also total PLA2 activity was assessed. Serum concentration and activity correlated significantly, which can be explained by sPLA2-IIA, making up for about 80% of total sPLA2 activity.
PLA2 levels at term and in preterm labor
The induction of parturition is dependent on three rate-limiting steps: first, the release of AA, which is catalyzed by the action of both secretory and cPLA2 family members. Secondly, the released AA converts to lipid mediators by COXs (COX1 and COX2), and in the last step, specific PG synthases produce PGs, which is needed for the onset of labor from these precursors. Despite the progress in this field, the lack of identification of the mechanisms of human parturition has limited the specific and effective diagnosis and treatment of preterm labor and subsequent birth. Therefore, sPLA2-IIA was also extensively investigated in the serum of preterm labored women. A study on 38 women delivering preterm showed that their serum sPLA2-IIA concentration and activity were significantly higher than those in a control group delivering at term. Within the preterm group, 19 women were suffering from chorioamnionitis, an infection associated with severe inflammation. Within this subgroup, sPLA2-IIA concentration and activity were even higher compared with the general preterm group. Also inflammatory markers such as CRP, IL8, and IL6 levels were significantly elevated in the sera of women with amnionitis compared with those delivering preterm without infection or the control group. In total, these observations suggest that sPLA2-IIA concentration may be a useful indicator for preterm labor, and phospholipid metabolism is certainly activated both in preterm labor and in apparent inflammatory diseases (Koyama et al. 2000).
PLA2 levels in PE and GDM
Increased serum levels of sPLA2-IIA have been reported in pregnant women suffering from both mild and severe PE (Pulkkinen et al. 1993, Lim et al. 1995); however, other studies contradicted these findings (Tempfer et al. 2001). Conclusions drawn from studies on PE have to be interpreted carefully as children from PE pregnancies are often delivered pre-maturely and control groups would have to be matched for gestational age. Appropriate control groups were not indicated in all the mentioned studies. However, as PE is characterized as a highly inflammatory condition, increases in sPLA2-IIA appear probable, knowing the relationship between sPLA2-IIA and inflammation.
In the last few years, LpPLA2 (plasma PAF–AH) has emerged as a biomarker for cardiovascular risk (Castro FariaNeto et al. 2004) and has extensively been studied in atherosclerosis, hyperlipidemia, diabetes, and metabolic syndrome (MetS). Throughout the course of pregnancy, mothers adapt to a lot of metabolic challenges such as insulin resistance, hyperlipidemia, and possibly hypertension. Some pregnancies are therefore affected by conditions such as GDM, cholestasis, or hypertension, which are considered transient and disappear after delivery. Nevertheless, there is an increased risk for cardiovascular disease in these mothers later in their lives. Three recent studies investigated LpPLA2 levels in postpartum women. Derbent et al. investigated LpPLA2 levels in women with a history of GDM with respect to their future risk of developing MetS. This is the first study which found that women – who had delivered up to 5 years before the study was conducted – still presented with significantly lower HDL levels but higher insulin resistance index, BMI, and most importantly higher serum LpPLA2 levels compared with controls (Derbent et al. 2011). Within the case group, 50% of women showed signs of MetS and 25% were diagnosed with type 2 diabetes mellitus (T2DM). Another study investigating LpPLA2 levels in women with a history of GDM showed that LpPLA2 levels in maternal serum remained significantly higher compared with control women for up to 2 years after delivery. In this study, 10% of women who had GDM later developed T2DM, 20% had signs of MetS, and 5% became hypertensive (Mai et al. 2014). Furthermore, Zhou et al. found that LpPLA2 levels as well as LDL-C, TG, and blood pressure were higher in women with a history of PE than in controls. LpPLA2 mass was correlated with the development of postpartum hypertension (systolic blood pressure) as well as LDL-C levels (Zhou et al. 2014).
Another study that investigated LpPLA2 in pregnancy hypertension was conducted prospectively in 51 pregnant women, whose LpPLA2 levels were measured in the first, second, and third trimester of pregnancy (Okumura et al. 1999). Of these, ten women developed pregnancy-induced hypertension (PIH) later in the third trimester. The general observation among the study population was that LpPLA2 levels were higher in the first than in the second and third trimesters. However, this was not true for all women and some still had increasing LpPLA2 levels over the second and third trimesters. Of the total women, 43% who had high levels of LpPLA2 in the second trimester also developed PIH, whereas only 8% of women who had low levels of LpPLA2 in the second trimester developed PIH. The authors therefore suggested LpPLA2 levels along the course of pregnancy as one potential risk marker for PIH.
PLA2 in the human placenta
AA mobilization by PLA2 and subsequent PG synthesis is thought to be a pivotal event in the onset and/or maintenance of human labor. Although PLA2 isozymes have been extensively studied within uterine tissues, different placental cell types, and fetal membranes, their role in homeostasis and pathophysiology during pregnancy has not been clearly established. The placenta as the connective organ between mother and fetus acts as the separating barrier between the maternal and fetal circulations and ensures nutrient and oxygen supply from the mother to the growing infant. In this context, PLA2 action within the placenta is not only important in parturition (Lappas et al. 2004) but also for catabolism and transport of TG from the mother to the fetus across the placenta (Freed et al. 1997).
So far, three PLA2 isozymes have been shown to be especially important for placental AA release and onset of parturition: sPLA2-IIA, sPLA2-V, and cPLA2-IV, although the mRNA expression levels of these enzymes within the tissue appears to be diverse. sPLA2-IIA expression in placental tissue and chorion–decidua was the highest, but undetectable in amnion. sPLA2-V mRNA was highly expressed in the placenta and amnion, but undetectable in decidual tissue. cPLA2-IV mRNA was expressed in all the three tissue types at similar levels (Johansen et al. 2000). A study focusing on sPLA2-IIA and cPLA2-IV in the human myometrium (Slater et al. 2014), which is the actual contractile layer within the uterine wall, investigated PLA2 function and its association with labor and onset at term and preterm deliveries. In this study, myometrial samples were collected from four different groups: a term/no labor, term/in labor, preterm/no labor, and a preterm/in labor group. Both in the preterm and in the term groups, spontaneous onset of labor significantly induced sPLA2-IIA expression in the myometrium, whereas cPLA2-IV levels remained constant. Therefore, one can speculate that sPLA2-IIA plays a more important role in inducing myometrial contractions and labor onset than cPLA2-IV.
Placental PLA2 in term and preterm labor
sPLA2-IIA and its role in preterm labor was also investigated by Lappas et al. (2001) using preterm and term groups with subgroups being in labor or without previous labor. Decidua, amnion, and placenta samples were assayed for both sPLA2-IIA concentration and total PLA2 activity. The main outcome of this study was a significant increase in sPLA2-IIA in amnion and decidua in the preterm compared with the term group. Furthermore, increased levels of sPLA2-IIA in amnion in the preterm group were associated with premature rupture of membranes. Interestingly, although infection is often associated with preterm labor, in a subset of women in this study, amnionitis was not characterized by increased levels of sPLA2-IIA in decidua, amnion, or placenta compared with controls. A similarly designed study, however, did not find any significant changes in sPLA2-IIA neither between preterm and term groups nor between labor and non-labor groups (Munns et al. 1999).
Mosher et al. (2014) investigated another PLA2 family member, sPLA2-IID, in uterine tissue. Also this study compared groups at term with and without labor. sPLA2-IID mRNA was shown to be expressed in a very low amount in human myometrium, placenta, and amnion, but by far highest in decidua. mRNA levels are related to protein levels, and interestingly, sPLA2-IID mRNA and protein was lower in the labor group. The authors suggested that sPLA2-IID may not be important in labor onset but serves an important role in placental immune function and tolerance of the fetus as sPLA2-IID has been shown to be an effector for regulatory T-cells (Tregs). Within the decidual layer, Tregs have been shown to be important for proper implantation without rejecting the fetus (Jin et al. 2009).
M-type receptor for sPLA2s (PLA2R1) has been detected in human placenta (Moses et al. 1998) and could represent an additional mechanism regulating sPLA2 activity within this tissue. PLA2R1 was found to be expressed in chorion–decidua and placental tissue, but not in the amnion. Levels were generally higher in chorion–decidua than in placenta; however, placental expression increased with labor onset.
Placental PLA2 in pregnancy-related pathologies
The only iPLA2 member shown to play a role in placental tissue so far is iPLA2ζ, more commonly known as ATGL (PNPLA2). Barrett et al. (2014) showed the presence of ATGL in placental tissue for the first time. Immune histochemistry showed the presence of ATGL in placental stromal cells (e.g., Hofbauer cells (HBCs)), syncytiotrophoblast (SCT), and endothelial layer of placental vessels. ATGL (PNPLA2) mRNA, as well as mRNA of its co-activator CGI-58, were up-regulated in GDM placentae. However, this did not translate to enhanced ATGL protein abundancy in GDM placentae. Nevertheless, increased ATGL and CGI-58 could point toward increased lipolysis in GDM placentae. If so, fetuses would receive more TG, which explains the higher fetal fat mass accretion in late pregnancy. This may lead to macrosomia, which is frequently observed in neonates born from diabetic pregnancies (Herrera & Ortega-Senovilla 2010, Lawlor et al. 2010).
Also the cytosolic PAF–AH isoforms I and II have been shown to be expressed in pregnant and non-pregnant myometrium (Yasuda & Okumura 2001). PAF–AH-II is additionally expressed in placental trophoblast cells. A study on PE placentae showed that both the levels of PAF, a potent mediator of inflammation, and trophoblast PAF–AH-II, which is able to degrade PAF, were higher compared with controls (Gu et al. 2006). Induction of PAF–AH-II was suggested to be a compensatory mechanism to clear PAF and therefore to decrease the inflammatory component of PE.
Glucose intolerance and alterations in lipid metabolism are common features of pregnancy. Newborns of diabetic or obese mothers are often considerably bigger and have higher amounts of body fat than children born to normoglycemic or lean mothers (Durnwald et al. 2004). This reflects that the metabolic status of the mother affects the offspring’s outcome already in utero. As the action of PLA2s also gives rise to pro-inflammatory lipid mediators, they likely represent a link between inflammation and lipid metabolism. To the best of our knowledge, the only study investigating PLA2 family members in the placenta focusing on lipid catabolism between the mother and the fetus was conducted by Varastehpour et al. (2006). They investigated PLA2 mRNA levels in the placenta of normal and obese newborns and found that sPLA2-IIA, sPLA2-V, sPLA2-VI, and LpPLA2 mRNA was increased up to three-fold. Also, tumor necrosis factor alpha (TNFα) and leptin mRNA were increased in obese placentae. In vitro, they investigated the effect of TNFα and leptin on sPLA2-IIA and sPLA2-V and found that the expression of these PLA2 isozymes was induced. They concluded that increased sPLA2 action in the placenta of obese neonates may result in even more FFAs available for fetal adipogenesis but also for generation of pro-inflammatory mediators. For a better overview, all studies on PLA2 in the placenta and their principal results are summarized in Table 2.
Overview of important PLA2 family members in human placental tissue and fetal membranes.
| PLA2 isozyme | Tissues/fetal membranes | Results | Sample type | Detection technique | References | ||
|---|---|---|---|---|---|---|---|
| Placenta | Chorion–decidua | Amnion | |||||
| sPLA2-IIA | X | X | X | sPLA2 activity was highest in amnion during labor | Tissue homogenate | Activity assay, ELISA | Munns et al. (1999) |
| PLA2 unspecified | X | NI | NI | Correlation of secreted and intracellular PLA2 protein | Explant culture | Activity assay, ELISA | Farrugia et al. (1997) |
| sPLA2-IIA | X Preterm/term – labor/no labor groups | NI | NI | More sPLA2-IIA was present in preterm tissues than term | Tissue homogenate | ELISA | Lappas et al. (2001) |
| sPLA2-IIA and sPLA2-V; cPLA2-IV | X | X | X | All three expressed in placenta; amnion cPLA2-IV and sPLA2-V; chorion sPLA2-IIA and cPLA2-IV | RNA | RT-qPCR | Johansen et al. (2000) |
| sPLA2-IIA and cPLA2-IV | X | X | X | High amounts of sPLA2-IIA in placenta, high levels of cPLA2 in amnion | RNA | NB | Freed et al. (1997) |
| sPLA2-IIA and cPLA2-IV | X | X | X | cPLA2-IV was highest in amnion, sPLA2-IIA was highest in placenta | RNA, protein and tissue sections | RT-qPCR, WB, and IHC | Slater et al. (2004) |
| PLA2-VI, PLA2-IIA, PLA2-V, PLA2-IID, PLA2-IIE, PLA2-IIF, PLA2-XII, PLA2-VII, PLA2-IVa, and PLA2-IVb. PLA2-IVc, PLA2-X, and PLA2-IB | X Control and obese groups* | NI | NI | sPLA2-IIA and sPLA2-V were higher in obese placenta; leptin was shown to regulate these two PLA2s | Isolated cells and RNA | RT-qPCR | Varastehpour et al. (2006) |
| sPLA2-IID | X | X Non-labor vs labor* | X | sPLA2-IID mRNA was highest in decidua in the non-labor group and amnion of the labor group; IL1b-induced sPLA2-IID dose dependently | Decidual cells, tissue RNA and protein | RT-qPCR and WB | Mosher et al. (2014) |
The expression of PLA2 isozymes in the placenta and fetal membranes is marked with an X. For better understanding of different results from different studies, sample types and detection techniques used are indicated. RT-qPCR, RT-quantitative real-time PCR; WB, western blot; NB, northern blot; IHC, immunohistochemistry; NI, not investigated.
PLA2 expression in placental cell types
Using gene expression analysis, our research group compared the mRNA levels of various PLA2 family members in four distinct primary cell types of the human placenta, arterial endothelial cells (AEC), venous endothelial cells (VEC), cytotrophoblasts (CT), and SCT (data analyzed from Cvitic et al. (2013); Table 3). Comparing the mean expression intensity, PLA2G15 (encoding a lysosomal PLA2) was mostly expressed in AEC and VEC, PLA2G16 (encoding an AdPLA2) in CT, and PLA2G7 (encoding LpPLA2) in SCT. M-type PLA2 receptor, PLA2R1, was uniformly expressed in all placental cell types. Considering fold-change values, only PLA2G4F (encoding PLA2-IV-F, a cPLA2) showed differential expression between AEC and VEC with a 1.82-fold, whereas expression of PLA2 members in CT and SCT was similar (Table 3). On contrary, when comparing the expression of PLA2 members between placental endothelium (combined AEC and VEC expression intensities) and trophoblast (combined CT and SCT expression intensities), most members showed differential expression with an average of 1.71-fold. As the expression of PLA2R1 did not differ between endothelium and trophoblast (Table 4), it is likely that PLA2s are not regulated by the respective receptor in these two placental compartments.
Expression of PLA2 family members in various cell types of human term placenta.
| Gene symbol | AEC | VEC | FC | P value (AEC vs VEC) | CT | SCT | FC | P value (CT vs SCT) |
|---|---|---|---|---|---|---|---|---|
| PLA2G7 | 5.3±0.6 | 4.9±0.3 | 1.06 | NS | 10.0 ±1.2 | 10.7±1.4 | 0.94 | <0.05 |
| PLA2G15 | 9.8±0.3 | 10.0±0.2 | 0.98 | NS | 9.0±0.1 | 8.6±0.4 | 1.05 | NS |
| PLA2G6 | 6.7±0.1 | 6.8±0.2 | 0.99 | <0.01 | 7.9±0.2 | 7.3±0.3 | 1.07 | <0.01 |
| PLA2G16 | 8.2±0.2 | 7.7±0.3 | 1.05 | <0.05 | 10.1±0.4 | 9.1±0.5 | 1.11 | <0.01 |
| PAFAH2 | 8.2±0.1 | 8.3±0.1 | 0.98 | NS | 8.6±0.2 | 8.9±0.2 | 0.96 | NS |
| PLA2G12A | 8.9±0.1 | 8.9±0.1 | 1.00 | NS | 8.5±0.2 | 8.3±0.4 | 1.02 | NS |
| PLA2G2D | 8.0±0.1 | 8.1±0.8 | 0.99 | NS | 8.3±0.1 | 8.7±0.3 | 0.95 | <0.01 |
| PLA2G10 | 4.4±0.4 | 4.3±0.2 | 1.02 | NS | 4.8±0.4 | 5.2±0.5 | 0.91 | NS |
| PLA2G4D | 6.5±0.1 | 6.6±0.2 | 0.98 | NS | 6.4±0.1 | 6.3±0.2 | 1.00 | NS |
| PLA2G3 | 6.3±0.1 | 6.4±0.1 | 0.99 | NS | 6.4±0.1 | 6.6±0.2 | 0.97 | NS |
| PLA2G2A | 6.5±0.1 | 6.6±0.1 | 0.98 | NS | 6.6±0.1 | 6.8±0.2 | 0.98 | NS |
| PLA2G4F | 6.3±0.1 | 6.4±0.2 | 0.98 | <0.001 | 6.5±0.1 | 6.5±0.2 | 1.00 | NS |
| PLA2G4A | 7.5±0.6 | 4.1±0.2 | 1.82 | <0.05 | 4.5±0.3 | 5.2±0.3 | 0.86 | <0.01 |
| PLA2G5 | 7.2±0.7 | 6.5±0.1 | 1.10 | NS | 6.5±0.1 | 6.7±0.3 | 0.97 | NS |
| PLA2G1B | 6.6±0.1 | 6.3±0.1 | 0.98 | NS | 6.2±0.1 | 6.5±0.2 | 0.95 | <0.05 |
| PLA2G4E | 5.7±0.2 | 5.8±0.1 | 0.98 | NS | 5.8±0.1 | 5.9±0.1 | 0.97 | NS |
| PLA2G2E | 6.9±0.1 | 7.0±0.1 | 0.99 | <0.01 | 6.9±0.1 | 7.0±0.4 | 0.99 | NS |
| PLA2R1 | 8.4±0.7 | 7.7±0.8 | 1.10 | NS | 8.3±0.4 | 7.7±0.5 | 1.08 | <0.01 |
Values represent mean mRNA expression intensities from AEC (n = 9), VEC (n = 9), CT (n = 10), and SCT (n = 10) measured by Affymetrix GeneChip Human 1.0 ST arrays. Expression intensities range from 1 to 13. Data were used from Cvitic et al. (2013). FC, fold-change was calculated as the ratio of mean expression for AEC vs VEC and CT vs SCT respectively. AEC, arterial endothelial cells; VEC, venous endothelial cells; CT, cytotrophoblast; SCT, syncytiotrophoblast.
Expression of PLA2 family members in endothelium and trophoblast of human term placenta.
| Gene symbol | Endothelium | Trophoblast | FC (endothelium vs trophoblast) | P value (endothelium vs trophoblast) |
|---|---|---|---|---|
| PLA2G7 | 5.1±0.2 | 3.1±0.5 | 1.70 | <0.001 |
| PLA2G15 | 9.9±0.2 | 5.5±0.3 | 1.80 | <0.001 |
| PLA2G6 | 6.7±0.1 | 3.9±0.4 | 1.74 | <0.001 |
| PLA2G16 | 7.9±0.3 | 4.4±0.7 | 1.81 | <0.001 |
| PAFAH2 | 8.6±0.1 | 4.7±0.2 | 1.77 | <0.001 |
| PLA2G12A | 8.9±0.0 | 4.9±0.1 | 1.80 | <0.001 |
| PLA2G2D | 8.1±0.1 | 4.6±0.3 | 1.77 | <0.001 |
| PLA2G10 | 4.4±0.1 | 5.0±0.3 | 0.88 | <0.01 |
| PLA2G4D | 6.6±0.1 | 3.8±0.0 | 1.73 | <0.01 |
| PLA2G3 | 6.3±0.1 | 3.7±0.2 | 1.72 | <0.01 |
| PLA2G2A | 6.5±0.1 | 3.8±0.1 | 1.72 | <0.01 |
| PLA2G4F | 6.4±0.1 | 3.7±0.0 | 1.72 | <0.05 |
| PLA2G4A | 5.8±2.4 | 3.0±0.5 | 1.95 | <0.05 |
| PLA2G5 | 6.9±0.5 | 3.8±0.2 | 1.80 | NS |
| PLA2G1B | 6.2±0.1 | 3.6±0.2 | 1.71 | NS |
| PLA2G4E | 5.8±0.1 | 3.4±0.1 | 1.69 | NS |
| PLA2G2E | 6.9±0.1 | 4.0±0.1 | 1.74 | NS |
| PLA2R1 | 8.1±0.5 | 8.00±0.5 | 1.01 | NS |
Values represent mean mRNA expression intensities from endothelium (n = 18) and trophoblast (n = 20) measured by Affymetrix GeneChip Human 1.0 ST arrays. Expression intensities range from 1 to 13. Data were used from Cvitic et al. (2013). FC, fold-change was calculated as the ratio of mean expression for endothelium vs trophoblast.
PLA2 in neonates
Although PLA2 family members are well investigated in maternal circulation and even more comprehensively in placentae of different hosts, uterine tissue, and fetal membranes, very little is known about PLA2 levels, activity, and function in the neonatal circulation. Pulkinnen et al. investigated sPLA2-IB and -II (pancreatic and synovial phospholipase) levels in umbilical cord blood and found that high levels of sPLA2-IB in cord blood were reflected by poor Apgar scores in these neonates. Moreover, pregnancy-induced hypertensive diseases increased by four- to ten-fold the concentration of synovial-type PLA2; in eight out of 14 cases, the enzyme was increased if the fetus was to be delivered prematurely (Pulkkinen et al. 1990). However, apart from Apgar score and premature delivery, no other parameters of neonatal distress or inflammatory markers were investigated.
Neonates suffering from meconium aspiration syndrome (MAS) develop respiratory distress. On the one hand, this is caused by sPLA2-IB present in meconium itself, which is responsible for the degradation of surfactant and therefore harmful to the neonate’s lung development. On the other hand, investigation of pulmonary lavage fluids showed that local production of sPLA2-IIA is also contributing to inflammation in MAS (De Luca et al. 2011).
Additionally, sPLA2-IIA levels are well established in the serum of newborns suffering from sepsis, which is a common phenomenon in preterm infants. In a study on newborns admitted to ICU, 24 children with proven sepsis, 77 children with suspected infection, and 55 proven healthy children were investigated for their sPLA2-IIA activity. Enzyme activity was highest in the septic children and was also significantly elevated in the ‘suspected infection’ group compared with controls (Schrama et al. 2008). sPLA2 activity correlated positively with other markers of inflammation, such as CRP and IL6 levels as well as leukocyte count. Moreover, sPLA2-IIA levels were especially high in children experiencing respiratory distress syndrome in addition to sepsis. The authors suggested that this is not only due to high sPLA2 in serum but also locally high levels of sPLA2 in neonatal lungs, causing hydrolysis of lung surfactant phospholipids. In adults, sPLA2-IIA is already a valid marker of sepsis.
A study investigating LpPLA2 (plasma PAF–AH) activity and distribution among LDL and HDL particles within both maternal serum and neonatal cord blood in PE pregnancies showed that there was no significant alteration in LpPLA2 activity and distribution in maternal blood caused by PE (Fan et al. 2012). However, in offsprings born to PE pregnancies, LDL levels were significantly higher than in control neonates. Also LpPLA2 activity was higher in cord blood in the PE group and activity was elevated in LDL particles compared with controls. In summary, this study showed that the neonates from women with severe PE have higher plasma PAF–AH activities, higher ratio of LDL–PAF–AH to HDL–PAF–AH activities, and higher TG:HDL-C ratio than the neonates from women with normal pregnancies. Results suggest that the neonates of the patients might present a chronic inflammation status, and/or increased oxidative stress, in addition to an unfavorable lipid profile.
Conclusion
In general, most pregnancy pathologies are characterized by similar metabolic changes such as elevation of certain cytokines, derailed lipid metabolism, insulin resistance, or infiltration of immune cells into the placenta.
Insulin resistance is a necessary metabolic alteration in pregnancy to ensure energy supply of the fetus and to maintain a glucose gradient across the placenta. In GDM, insulin resistance is augmented, and it is also often observed in obese pregnant women as well as women suffering from PE (Rademacher et al. 2007). These three conditions have also been characterized by poor maternal lipid profiles, with high TG, high LDL-C, smaller proatherogenic LDL particles, and low HDL-C levels in many studies (Derbent et al. 2011, Fan et al. 2012, Mai et al. 2014). Also, in GDM, obesity, and PE, elevated levels of certain pro-inflammatory cytokines have been reported, e.g. TNFα, IL1b, IL2, and IL6. However, anti-inflammatory IL10 was shown to be down-regulated in these conditions (Gomes et al. 2013, Raghupathy 2013, Katzman 2015). TNFα, IL1, IL6, and IL10 are able to regulate sPLA2-IIA and sPLA2-V (van der Helm et al. 1996, Kuwata et al. 1998). Enhanced activity of these secretory enzymes could contribute to some of the observed symptoms of these conditions: e.g. preterm labor in PE or macrosomia of the fetus by enhanced lipid catabolism in GDM and obesity.
Inflammation and presence of pro-inflammatory cytokines also play an important role in other pregnancy conditions, e.g., chorioamnionitis (an infectious disease of the placenta) and villitis of unknown etiology (VUE, inflammation of the placenta without a detectable infectious agent). Both conditions have been characterized by massive infiltration of placental tissue with T-lymphocytes, macrophages, and also Tregs; Tamblyn et al. 2013, Faas et al. 2014). Moreover, an accumulation of placental macrophages (so-called HBCs) has also been reported in GDM and PE pregnancies (Evsen et al. 2013, Yu et al. 2013). These macrophages, especially if exposed to pro-inflammatory mediators, enhance the secretion of sPLA2s and may therefore cause alterations in PLA2 activity and changes in lipid metabolism within placental tissue.
PLA2s seem to be of tremendous importance throughout various stages of pregnancy as well as in a number of common pregnancy pathologies (as summarized in Table 5). Modulation of PLA2 activity could therefore be a strategy to treat preterm labor onset, GDM (and/or obesity), PE, or placental inflammation. However, treatment opportunities so far are limited: fine-tuned modulation of only certain PLA2s would be necessary; however, most inhibitors of PLA2 activity act rather unspecific on various PLA2 isozymes (Yedgar et al. 2006, Garcia-Garcia et al. 2009). For instance, inhibitors of sPLA2-IIA often also inhibit the closely related enzymes sPLA2-V and sPLA2-X. Therefore, so far it is more common to reduce PG and leukotriene production in certain inflammatory conditions, e.g. sepsis, by blocking COX and LOX pathways than by blocking sPLA2. However, liver toxicity is observed when blocking COX and LOX pathways, blocking sPLA2 would probably have fewer side effects (Martel-Pelletier et al. 2003). In addition, translating results from mouse models into humans is problematical: Varespladib and Darapladib compounds inhibiting sPLA2-IIA and LpPLA2, respectively, showed promising effects in the treatment of atherosclerosis and vascular disease in mice, but not in man, and phase II trials on both compounds were stopped (Rosenson et al. 2011, O’Donoghue 2014). Furthermore, passing the placental barrier and treating placental inflammation without affecting the fetus is challenging. Currently, methods to regulate sPLA2 action via site-directed delivery of small peptide molecules on nanoparticles and liposomes are being developed and show promising results (van den Hoven 2011, Zhu et al. 2011). These techniques could also be used in order to overcome the placental barrier, which can be passed by nanoparticles (Grafmüller et al. 2013, Lopalco et al. 2015). Nevertheless, in order to develop better treatment strategies, deeper understanding of the relationships between inflammation and lipases and lipid metabolism in the placenta, but also maternal and fetal circulation, is still needed. Future studies will hopefully help in gaining this knowledge and improve health care for mother and child.
PLA2s in pregnancy pathologies and maternal and fetal outcome.
| Pathology | PLA2 isozyme | Maternal outcome | Neonatal outcome | References |
|---|---|---|---|---|
| Preterm delivery | sPLA2-IIA | ↑ Serum levels | NA | Pulkinnen et al. (1993) |
| Severe preeclampsia | sPLA2-IIA | ↑ Serum levels | NA | Pulkinnen et al. (1993) |
| sPLA2-IIA | ↑ Serum levels | NA | Lim et al. (1995) | |
| sPLA2-IIA | Serum levels unchanged | NA | Tempfer et al. (2001) | |
| LpPLA2 | Serum levels unchanged, adverse lipid profile | ↑ Serum levels adverse lipid profile | Fang et al. (2012) | |
| Gestational diabetes | LpPLA2 | ↑ Serum levelsWomen developed T2DM, MetS after pregnancy | NA | Derbent et al. (2011) |
| LpPLA2 | ↑ Serum levelsWomen developed T2DM, MetS after pregnancy | NA | Mai et al. (2014) | |
| LpPLA2 | NA | ↑ Plasma levels | C Besenboeck, J Loegl, S Kopp, M Peinhaupt, U Lang, G Desoye and C Wadsack (unpublished observation) | |
| Cholestasis | sPLA2-IIA | Serum levels unchanged, no correlation with cholic acid levels | NA | Pulkinnen et al. (1993) |
| Pregnancy-induced hypertension (PIH) | LpPLA2 | Elevated serum LpPLA2 levels in second trimester appeared to serve as risk marker for PIH later in third trimester | NA | Okamura et al. (1999) |
| Sepsis | sPLA2-IIA | NA | ↑ Serum levels Corr. to CRP, IL6, and leukocyte count | Schrama et al. (2008) |
Results on maternal and neonatal outcome with respect to serum PLA2 isozyme levels are summarized and compared. Upward facing arrows indicate elevations of the respective isozyme within serum. Abbreviations: ↑, increased; NA, not applicable, is given if only maternal or neonatal outcome, respectively, was investigated; T2DM, type 2 diabetes mellitus; MetS, metabolic syndrome.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
C Besenboeck is funded by a training grant of the Austrian Science Fund, FWF, within the DK-MOLIN program (W1241). S Cvitic has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013), project Early Nutrition under grant agreement number 289346.
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