Sphingolipids are major constituents of the cell membrane and are involved in cell

**Figure 9.** Interconversion of sphingolipids. (DAG, diacylglycerol; PKC, protein kinase C). **Figure 9.** Interconversion of sphingolipids. (DAG, diacylglycerol; PKC, protein kinase C).

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**Figure 10.** Increases in sphingolipids related to the development of preeclampsia, \* *p* < 0.05, \*\* *p* < 0.01.

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Abnormal placental sphingolipid production may contribute to several features of preeclampsia. For example, ceramide induces apoptosis, which may contribute to placental cell death in preeclampsia [69], and S1P inhibits extravillous trophoblast migration [70], and so may contribute to failure of extravillous trophoblasts to effectively remodel

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**Figure 9.** Interconversion of sphingolipids. (DAG, diacylglycerol; PKC, protein kinase C).

**Figure 10.** Increases in sphingolipids related to the development of preeclampsia, \* *p* < 0.05, \*\* *p* < 0.01. **Figure 10.** Increases in sphingolipids related to the development of preeclampsia, \* *p* < 0.05, \*\* *p* < 0.01.

Abnormal placental sphingolipid production may contribute to several features of preeclampsia. For example, ceramide induces apoptosis, which may contribute to placental cell death in preeclampsia [69], and S1P inhibits extravillous trophoblast migration [70], and so may contribute to failure of extravillous trophoblasts to effectively remodel Abnormal placental sphingolipid production may contribute to several features of preeclampsia. For example, ceramide induces apoptosis, which may contribute to placental cell death in preeclampsia [69], and S1P inhibits extravillous trophoblast migration [70], and so may contribute to failure of extravillous trophoblasts to effectively remodel the spiral arteries in preeclampsia. S1P is also involved in inflammation, vascular permeability, and the immune response. S1P is an intracellular second messenger, but it is also a bloodborne lipid mediator, and as such, has extracellular actions by binding to S1P receptors. Placental secretion of S1P could be responsible for abnormalities in the maternal circulation. Very little information is available about sphingolipids in pregnancy, but maternal levels of ceramide and S1P have been reported to be elevated in preeclampsia and linked to a placental source [71,72].

### **4. Other Considerations Involving Neutrophils and Pregnancy-Specific Expression of Protease-Activated Receptor 1**

Normal pregnancy is characterized by leukocytosis caused by proliferation of neutrophils in the 2nd and 3rd trimesters. The number of neutrophils increases 2.5-fold by 30 weeks of gestation in normal pregnancy [73], and the number increases further in preeclampsia [74]. Neutrophils are usually thought of as part of the innate immune system and the first line of defense against infection. A role for neutrophils in non-infectious disease has not been widely considered, but accumulating evidence indicates a role for neutrophils in "sterile" inflammatory diseases [75].

For neutrophils to manifest their inflammatory effects, they need to infiltrate tissue, and in women with preeclampsia there is extensive neutrophil infiltration into the maternal systemic blood vessels (Figure 11) [76–79]. In preeclamptic women, 80–90% of vessels in subcutaneous and omental fat are infiltrated and, although all classes of leukocytes are activated [80,81], vascular infiltration is restricted to neutrophils [77,78]. Neutrophil infiltration is associated with a significant increase in inflammatory markers, e.g., interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB), thromboxane synthase (TBXAS1), and myeloper-

oxidase (MPO) [76,79,82,83]. The finding of neutrophil infiltration provides a basis for a new way of thinking about vascular dysfunction in preeclampsia. It does not discount the potential role of plasma factors but adds a new dimension to the understanding of the underlying mechanisms of the vascular phenotype. [76,79,82,83]. The finding of neutrophil infiltration provides a basis for a new way of thinking about vascular dysfunction in preeclampsia. It does not discount the potential role of plasma factors but adds a new dimension to the understanding of the underlying mechanisms of the vascular phenotype.

the spiral arteries in preeclampsia. S1P is also involved in inflammation, vascular permeability, and the immune response. S1P is an intracellular second messenger, but it is also a blood-borne lipid mediator, and as such, has extracellular actions by binding to S1P receptors. Placental secretion of S1P could be responsible for abnormalities in the maternal circulation. Very little information is available about sphingolipids in pregnancy, but maternal levels of ceramide and S1P have been reported to be elevated in preeclampsia and

**4. Other Considerations Involving Neutrophils and Pregnancy-Specific Expression of** 

Normal pregnancy is characterized by leukocytosis caused by proliferation of neutrophils in the 2nd and 3rd trimesters. The number of neutrophils increases 2.5-fold by 30 weeks of gestation in normal pregnancy [73], and the number increases further in preeclampsia [74]. Neutrophils are usually thought of as part of the innate immune system and the first line of defense against infection. A role for neutrophils in non-infectious disease has not been widely considered, but accumulating evidence indicates a role for neu-

For neutrophils to manifest their inflammatory effects, they need to infiltrate tissue, and in women with preeclampsia there is extensive neutrophil infiltration into the maternal systemic blood vessels (Figure 11) [76–79]. In preeclamptic women, 80–90% of vessels in subcutaneous and omental fat are infiltrated and, although all classes of leukocytes are activated [80,81], vascular infiltration is restricted to neutrophils [77,78]. Neutrophil infiltration is associated with a significant increase in inflammatory markers, e.g., interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB), thromboxane synthase (TBXAS1), and myeloperoxidase (MPO)

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 10 of 23

linked to a placental source [71,72].

trophils in "sterile" inflammatory diseases [75].

**Protease-Activated Receptor 1** 

**Figure 11.** Neutrophils (brown) in omental fat arteries. **Figure 11.** Neutrophils (brown) in omental fat arteries.

### *4.1. Pregnancy-Specific Expression of PAR-1 4.1. Pregnancy-Specific Expression of PAR-1*

Protease-activated receptor 1 (PAR-1), originally known as thrombin receptor, is activated by serine proteases, such as thrombin, neutrophil elastase, and matrix metalloproteinase-1 (MMP-1) [84–86]. Activation leads to downstream signaling mechanisms that include the RhoA kinase (ROCK) phosphorylation pathway. ROCK is a recognized mediator of enhanced vascular reactivity, and also regulates the shape and movement of cells. Protease-activated receptor 1 (PAR-1), originally known as thrombin receptor, is activated by serine proteases, such as thrombin, neutrophil elastase, and matrix metalloproteinase-1 (MMP-1) [84–86]. Activation leads to downstream signaling mechanisms that include the RhoA kinase (ROCK) phosphorylation pathway. ROCK is a recognized mediator of enhanced vascular reactivity, and also regulates the shape and movement of cells. There is pregnancy-specific expression of PAR-1. Wang et al. showed that PAR-1 is expressed on neutrophils, but only during pregnancy [87,88]. This suggests that something associated with the placenta is causing the expression of PAR-1 on circulating neutrophils. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 11 of 23 There is pregnancy-specific expression of PAR-1. Wang et al. showed that PAR-1 is expressed on neutrophils, but only during pregnancy [87,88]. This suggests that something associated with the placenta is causing the expression of PAR-1 on circulating neutrophils.

Figure 12 shows omental fat vessels of preeclamptic and normal pregnant women immunostained for PAR-1. In preeclampsia, PAR-1 is expressed in endothelial cells (EC), vascular smooth muscle (VSM), and in neutrophils flattened and adherent to the endothelium, infiltrated into the vessel, and present in the lumen of the vessel. In normal pregnancy, weak staining is present in the endothelium and neutrophils in the vessel lumen. There is an 8-fold increase in gene and protein expression of PAR-1 in blood vessels of women with preeclampsia [89]. Figure 12 shows omental fat vessels of preeclamptic and normal pregnant women immunostained for PAR-1. In preeclampsia, PAR-1 is expressed in endothelial cells (EC), vascular smooth muscle (VSM), and in neutrophils flattened and adherent to the endothelium, infiltrated into the vessel, and present in the lumen of the vessel. In normal pregnancy, weak staining is present in the endothelium and neutrophils in the vessel lumen. There is an 8-fold increase in gene and protein expression of PAR-1 in blood vessels of women with preeclampsia [89].

Proteases, such as MMP-1, neutrophil elastase, and thrombin, are elevated in women

so its activation by elevated proteases in preeclampsia activates an inflammatory mechanism unique to pregnancy. In normal pregnancy, it makes sense that the expression of inflammatory genes would be silenced. A mechanism for this could be DNA methylation to mask binding sites for inflammatory transcription factors, such a NF-κB. However, if the methylation marks were erased, it would open these sites, possibly leading to increased gene expression. One mechanism for erasing methylation marks involves the recently discovered TET proteins (ten-eleven translocation proteins, aka tet methylcytosine dioxygenases). TET proteins regulate gene expression by enzymatic de-methylation of DNA. They catalyze the conversion of 5-methycytosine (5-mC) to 5-hydroxy-methylcytosine (5-hmC) [91–94], which is further oxidized and then removed by the DNA base excision repair enzyme, thymine-DNA glycosylase, and replaced with unmodified cytosine [95]. TET enzymes were first discovered in 2009 [93], and little is known about their regulation or role in disease. TET2 is the main TET protein expressed in leukocytes, and its activation has been shown to play an essential role in regulating hematopoietic differentiation, which proceeds in mature cells without cell division normally during emigration

**Figure 12.** Expression of PAR-1 in omental vessels. **Figure 12.** Expression of PAR-1 in omental vessels.

from the circulation into tissue [96–98].

*4.2. PAR-1 Mediates Neutrophil Inflammatory Response in Pregnancy* 

### *4.2. PAR-1 Mediates Neutrophil Inflammatory Response in Pregnancy*

Proteases, such as MMP-1, neutrophil elastase, and thrombin, are elevated in women with preeclampsia [90]. The expression of PAR-1 on neutrophils is specific to pregnancy, so its activation by elevated proteases in preeclampsia activates an inflammatory mechanism unique to pregnancy. In normal pregnancy, it makes sense that the expression of inflammatory genes would be silenced. A mechanism for this could be DNA methylation to mask binding sites for inflammatory transcription factors, such a NF-κB. However, if the methylation marks were erased, it would open these sites, possibly leading to increased gene expression. One mechanism for erasing methylation marks involves the recently discovered TET proteins (ten-eleven translocation proteins, aka tet methylcytosine dioxygenases). TET proteins regulate gene expression by enzymatic de-methylation of DNA. They catalyze the conversion of 5-methycytosine (5-mC) to 5-hydroxy-methylcytosine (5-hmC) [91–94], which is further oxidized and then removed by the DNA base excision repair enzyme, thymine-DNA glycosylase, and replaced with unmodified cytosine [95]. TET enzymes were first discovered in 2009 [93], and little is known about their regulation or role in disease. TET2 is the main TET protein expressed in leukocytes, and its activation has been shown to play an essential role in regulating hematopoietic differentiation, which proceeds in mature cells without cell division normally during emigration from the circulation into tissue [96–98].

### *4.3. Proteases Activate Neutrophil TET2 and NF-κB to Mediate Inflammatory Response*

Protease activation of PAR-1 causes translocation of TET2 from the cytosol into the nucleus in neutrophils obtained from pregnant women as evidenced by immunofluorescence and confocal microscopy (Figure 13) [90]. TET2 (green) is localized to the cytosol in control cells of normal pregnant women. Protease treatment with MMP-1 or elastase results in translocation of TET2 into the nucleus (location identified by DAPI blue) in as early as 15 min, which is consistent for proteins containing a nuclear localization signal (NLS)**.** Nuclear translocation of TET2 coincides with activation of NF-κB. Similar to TET2, protease stimulation of pregnancy neutrophils causes translocation of the p65 subunit of NF-kB (red) from the cytosol to the nucleus. Inhibition of PAR-1, as well as inhibition of ROCK, prevents protease-induced translocation of TET2 and p65 into the nucleus (Figure 13). Inhibition of PAR-1 or ROCK also inhibits inflammatory response as measured by the production of IL-8 and TXB2, which are regulated by NF-κB. Protease treatment of neutrophils from normal pregnant women significantly increases IL-8 and TXB2, demonstrating that proteases stimulate inflammatory response, but when cells are pretreated with PAR-1 or ROCK inhibitors, protease-induced increases in IL-8 and TXB2 are prevented.

Expression and activation of neutrophil TET2 are increased in preeclampsia. Immunohistochemical staining reveals significantly more staining in omental vessels of preeclamptic women than in omental vessels of normal pregnant women (Figure 14) [90]. In preeclampsia, almost 90% of vessels stain for TET2 with neutrophils infiltrated into the vessel wall, as compared to only 16% of vessels in normal pregnancy with staining. When neutrophils are present in normal vessels, they are usually in the lumen of the vessel. High magnification images reveal dark staining of the polymorphonuclear nuclei of neutrophils in preeclampsia (Panel D), as opposed to diffuse staining in normal pregnancy (Panel C). Nuclear staining suggests TET2 is active in preeclampsia, and activation involves translocation from the cytosol to the nucleus just as observed for TET2 translocation induced by protease activation of PAR-1. Staining for TET2 in preeclamptic vessels mirrors the staining for PAR-1 with staining present in endothelium and vascular smooth muscle (VSM), as well as in neutrophils [90]. This close relationship between PAR-1 and TET2 likely has important implications for vascular inflammation in preeclampsia.


**Figure 13.** Confocal images of TET2 and p65 immuno-fluorescence staining in neutrophils of normal pregnant women. Nuclear localization induced by protease treatment was prevented by inhibition of PAR-1. Images were taken with a Zeiss LSM 700 using x63 lens and then cropped. **Figure 13.** Confocal images of TET2 and p65 immuno-fluorescence staining in neutrophils of normal pregnant women. Nuclear localization induced by protease treatment was prevented by inhibition of PAR-1. Images were taken with a Zeiss LSM 700 using x63 lens and then cropped.

**Figure 14.** TET2 in omental vessels. **Figure 14.** TET2 in omental vessels (**A**–**D**).

**Figure 15.** Molecular mechanism for protease activation of pregnancy neutrophils. Figure 15 summarizes the molecular mechanisms for protease activation of pregnancy neutrophils. In normal pregnancy, circulating proteases are not elevated, TET2 and NF-κB are localized to the cytosol, and inflammatory genes are not expressed. In preeclampsia, circulating proteases are elevated and activate neutrophils due to their pregnancy- specific expression of PAR-1. Activation of PAR-1 results in the movement of TET2 and NF-κB from the cytosol to the nucleus and the expression of inflammatory genes. The PAR-1 pathway involves ROCK phosphorylation because inhibition of either PAR-1 or ROCK blocks the movement of TET2 and NF-κB from the cytosol to the nucleus and the inflammatory response. To summarize, elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1. PAR-1 activates ROCK, which phosphorylates TET2 and NF-κB, causing their translocation from the cytosol to the nucleus. The fact that TET2 translocation into the nucleus coincides with movement of NF-κB implicates epigenetic mechanisms and suggests that TET2 may enzymatically de-methylate DNA, opening up transcription factor binding sites for NF-κB, resulting in the expression of inflammatory genes. **Figure 14.** TET2 in omental vessels.

oxidative stress, which drives the imbalance of increased thromboxane and decreased **Figure 15.** Molecular mechanism for protease activation of pregnancy neutrophils.

### *4.4. Expression of PAR-1 in the Placenta*

prostacyclin production [25].

core.

*4.4. Expression of PAR-1 in the Placenta*  Several studies show PAR-1 is expressed in the placenta [99–102], which is a tissue prostacyclin production [25]. **Figure 15.** Molecular mechanism for protease activation of pregnancy neutrophils. Several studies show PAR-1 is expressed in the placenta [99–102], which is a tissue specific to pregnancy and dysfunctional in preeclampsia. Figure 16 shows staining for PAR-1

specific to pregnancy and dysfunctional in preeclampsia. Figure 16 shows staining for PAR-1 in a placental villus. PAR-1 is expressed in the syncytiotrophoblast cells, which are directly bathed by maternal blood. PAR-1 is also present in macrophages of the villous

cause PAR-1 is expressed in the syncytiotrophoblast, elevated levels of proteases in the intervillous space could activate PAR-1, leading to placental dysfunction. For example, protease stimulation of trophoblast PAR-1 causes increased release of the angiogenic factor, sFlt [100,103], by activation of placental NADPH oxidase to generate reactive oxygen species [99]. Activation of NADPH oxidase via PAR-1 could be responsible for placental oxidative stress, which drives the imbalance of increased thromboxane and decreased

in a placental villus. PAR-1 is expressed in the syncytiotrophoblast cells, which are directly bathed by maternal blood. PAR-1 is also present in macrophages of the villous core. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 15 of 23

**Figure 16.** Expression of PAR-1 in syncytiotrophoblasts and macrophages in the placenta (dark brown staining). **Figure 16.** Expression of PAR-1 in syncytiotrophoblasts and macrophages in the placenta (dark brown staining).

A protease activating mechanism of neutrophil and placental PAR-1 could explain why preeclampsia only occurs in pregnant women, and a protease feed-forward scenario could explain why clinical symptoms progressively worsen. Protease activation of PAR-1 could explain other features of preeclampsia. For example, because neutrophils have a limited life span of about 8 days, their rapid turnover would explain why maternal symptoms clear shortly after delivery because new neutrophils not expressing PAR-1 enter the circulation. Some women develop preeclampsia in the immediate post-partum period. Labor is recognized to be an inflammatory process, and even in normal term labor, there There is evidence that PAR-1 mediates placental dysfunction in preeclampsia. Because PAR-1 is expressed in the syncytiotrophoblast, elevated levels of proteases in the intervillous space could activate PAR-1, leading to placental dysfunction. For example, protease stimulation of trophoblast PAR-1 causes increased release of the angiogenic factor, sFlt [100,103], by activation of placental NADPH oxidase to generate reactive oxygen species [99]. Activation of NADPH oxidase via PAR-1 could be responsible for placental oxidative stress, which drives the imbalance of increased thromboxane and decreased prostacyclin production [25].

is extensive infiltration of neutrophils into maternal systemic vasculature [104]. Women who develop post-partum preeclampsia might have been on the verge of developing preeclampsia, and then neutrophil infiltration with labor pushed them over the edge. *4.5. Central Role for PAR-1 in the Clinical Manifestations of Preeclampsia*  Protease activation of PAR-1 may play a central role in the pathology of preeclampsia (Figure 17). Protease activation is involved in the neutrophil TET2 inflammatory response, neutrophil activation, and enhanced vascular reactivity [90,105]. Activation of PAR-1 may explain other pathologic features as well because PAR-1 mediates coagulation abnormalities, platelet aggregation, and thromboxane generation. Protease activation of endothelial PAR-1 activates NF-κB, upregulates cell adhesion molecules (ICAM-1), triggers produc-A protease activating mechanism of neutrophil and placental PAR-1 could explain why preeclampsia only occurs in pregnant women, and a protease feed-forward scenario could explain why clinical symptoms progressively worsen. Protease activation of PAR-1 could explain other features of preeclampsia. For example, because neutrophils have a limited life span of about 8 days, their rapid turnover would explain why maternal symptoms clear shortly after delivery because new neutrophils not expressing PAR-1 enter the circulation. Some women develop preeclampsia in the immediate post-partum period. Labor is recognized to be an inflammatory process, and even in normal term labor, there is extensive infiltration of neutrophils into maternal systemic vasculature [104]. Women who develop post-partum preeclampsia might have been on the verge of developing preeclampsia, and then neutrophil infiltration with labor pushed them over the edge.

#### tion of neutrophil chemokines (IL-8), and increases permeability of the endothelium to *4.5. Central Role for PAR-1 in the Clinical Manifestations of Preeclampsia*

trigger edema formation [106–112]. PAR-1 may explain the elevation in angiogenic factors because trophoblast and decidual production of sFlt is stimulated by protease activation of PAR-1 [100,103]. Placental oxidative stress may be explained by protease stimulation of trophoblast PAR-1, which activates NADPH oxidase to generate reactive oxygen species, resulting in the release of sFlt [99]. Activation of NADPH oxidase could also explain the placental imbalance of increased thromboxane and decreased prostacyclin characteristic of preeclampsia because oxidative stress drives this imbalance [25]. The effect of aspirin on PAR-1 signaling should be evaluated. If aspirin interferes with downstream signaling of PAR-1, this would be another action to account for its beneficial effects. Protease activation of PAR-1 may play a central role in the pathology of preeclampsia (Figure 17). Protease activation is involved in the neutrophil TET2 inflammatory response, neutrophil activation, and enhanced vascular reactivity [90,105]. Activation of PAR-1 may explain other pathologic features as well because PAR-1 mediates coagulation abnormalities, platelet aggregation, and thromboxane generation. Protease activation of endothelial PAR-1 activates NF-κB, upregulates cell adhesion molecules (ICAM-1), triggers production of neutrophil chemokines (IL-8), and increases permeability of the endothelium to trigger edema formation [106–112]. PAR-1 may explain the elevation in angiogenic factors because trophoblast and decidual production of sFlt is stimulated by protease activation of

PAR-1 [100,103]. Placental oxidative stress may be explained by protease stimulation of trophoblast PAR-1, which activates NADPH oxidase to generate reactive oxygen species, resulting in the release of sFlt [99]. Activation of NADPH oxidase could also explain the placental imbalance of increased thromboxane and decreased prostacyclin characteristic of preeclampsia because oxidative stress drives this imbalance [25]. The effect of aspirin on PAR-1 signaling should be evaluated. If aspirin interferes with downstream signaling of PAR-1, this would be another action to account for its beneficial effects. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 16 of 23

**Figure 17.** Clinical manifestations of preeclampsia that can be explained by protease activation of PAR-1. **Figure 17.** Clinical manifestations of preeclampsia that can be explained by protease activation of PAR-1.

### *4.6. Placental Activation of Neutrophils*

*4.6. Placental Activation of Neutrophils*  Although all classes of leukocytes are activated in the circulation of women with preeclampsia [80,81,113–115], only neutrophils extensively infiltrate maternal blood vessels [76–78]. The extensive infiltration of activated neutrophils into blood vessels of women with preeclampsia [76,78,79] could explain systemic vascular inflammation and why multiple organs are affected. The question arises as to how neutrophils are activated. The placenta would seem to be a source for the activator because preeclampsia only occurs in the presence of placental tissue. Lipid peroxides are potent activators of leukocytes [116–118], and the human placenta produces lipid peroxides and secretes them into the maternal circulation [13,42,46,119]. In women with preeclampsia, placental production of lipid peroxides is significantly higher than in women with normal pregnancy [13,42,46]. Therefore, it is plausible that activation occurs as neutrophils circulate through the intervillous space and are exposed to lipid peroxides released by the placenta [51,120,121]. Although all classes of leukocytes are activated in the circulation of women with preeclampsia [80,81,113–115], only neutrophils extensively infiltrate maternal blood vessels [76–78]. The extensive infiltration of activated neutrophils into blood vessels of women with preeclampsia [76,78,79] could explain systemic vascular inflammation and why multiple organs are affected. The question arises as to how neutrophils are activated. The placenta would seem to be a source for the activator because preeclampsia only occurs in the presence of placental tissue. Lipid peroxides are potent activators of leukocytes [116–118], and the human placenta produces lipid peroxides and secretes them into the maternal circulation [13,42,46,119]. In women with preeclampsia, placental production of lipid peroxides is significantly higher than in women with normal pregnancy [13,42,46]. Therefore, it is plausible that activation occurs as neutrophils circulate through the intervillous space and are exposed to lipid peroxides released by the placenta [51,120,121].

### *4.7. Inhibition of Neutrophils and Treatment of Preeclampsia with Aspirin*

*4.7. Inhibition of Neutrophils and Treatment of Preeclampsia with Aspirin*  Low-dose aspirin is currently standard of care for the prevention of preeclampsia in high-risk populations. Low-dose aspirin selectively inhibits maternal platelet thromboxane production without affecting prostacyclin production and, as shown above, it appears to also selectively inhibit placental thromboxane production, as well as placental oxidative stress. However, maternal platelets and placental trophoblasts may not be the only aspirin targets. Neutrophils may also be a target. The expression of cyclooxygenase-2 is increased in neutrophils of preeclamptic women [79,122], and aspirin inhibits neutrophil production of thromboxane, as well as the generation of reactive oxygen species [117,118]. Neutrophils could be a major source of thromboxane and oxidative stress due to the marked increase in their numbers during pregnancy. Aspirin treatment might also reduce the infiltration of neutrophils into the maternal blood vessels. Future studies are necessary to address the various mechanisms by which low-dose aspirin is able to reduce the incidence of preeclampsia. Low-dose aspirin is currently standard of care for the prevention of preeclampsia in high-risk populations. Low-dose aspirin selectively inhibits maternal platelet thromboxane production without affecting prostacyclin production and, as shown above, it appears to also selectively inhibit placental thromboxane production, as well as placental oxidative stress. However, maternal platelets and placental trophoblasts may not be the only aspirin targets. Neutrophils may also be a target. The expression of cyclooxygenase-2 is increased in neutrophils of preeclamptic women [79,122], and aspirin inhibits neutrophil production of thromboxane, as well as the generation of reactive oxygen species [117,118]. Neutrophils could be a major source of thromboxane and oxidative stress due to the marked increase in their numbers during pregnancy. Aspirin treatment might also reduce the infiltration of neutrophils into the maternal blood vessels. Future studies are necessary to address the various mechanisms by which low-dose aspirin is able to reduce the incidence of preeclampsia.

Low-dose aspirin is currently being used to prevent preeclampsia in women at risk, but given its effectiveness, consideration should be given to the use of aspirin in treating

cern that it might reduce amniotic fluid volume or cause closure of the ductus arteriosus. However, this concern may be unwarranted because only 30% of an aspirin dose crosses

Low-dose aspirin is currently being used to prevent preeclampsia in women at risk, but given its effectiveness, consideration should be given to the use of aspirin in treating women with preeclampsia. Aspirin was contraindicated for use in pregnancy due to concern that it might reduce amniotic fluid volume or cause closure of the ductus arteriosus. However, this concern may be unwarranted because only 30% of an aspirin dose crosses from the maternal to the fetal side of the placenta [43], and more importantly, the Collaborative Perinatal Project in the 1970s involving over 40,000 pregnant women and their offspring, over 24,000 of whom took aspirin during their pregnancy, 1500 of whom were heavily exposed, found no harmful effects of aspirin use on the neonates [123].

### **5. Mysterious Beneficial Effects of Low-Dose Aspirin—Is Cyclooxygenase Involved?**

The known mechanism of aspirin is to inhibit cyclooxygenase enzymes, the constitutive COX-1 and the inducible COX-2. However, reports are appearing that aspirin also affects non-cyclooxygenase products. For example, placental soluble fms-like tyrosine kinase 1 (sFlt-1) is elevated in the circulation of women with preeclampsia and implicated in preeclampsia pathology [124,125]. sFlit-1 is not a cyclooxygenase product, but low-dose aspirin reduces hypoxia-induced sFlt-1 release by cytotrophoblast cells in vitro [126,127]. Hypoxia causes oxidative stress and the induction of COX-2, so inhibition of sFlt-1 may be related to aspirin's ability to decrease ROS generated by COX-2 (Figure 7).

Aspirin has favorable effects through alterations in phosphoproteins, transcription factors, and microRNAs implicated in placental apoptosis and trophoblast migration [128–131]. Aspirin facilitates trophoblast invasion by regulating a family of microRNAs that inhibit trophoblast invasion [131]. Thus, aspirin may augment extracellular trophoblast remodeling of the spiral arteries, which is deficient in preeclampsia. COX-2 is elevated in the process of apoptosis [129], so aspirin may decrease placental apoptosis by inhibiting COX-2.

Another puzzling effect of low-dose aspirin is on the regulation of placenta-derived exosomes. Exosomes are lipid bilayer nano-vesicles released by many cells and are present in blood [132]. Their lipid makeup reflects their tissue of origin. Placental exosomes can be specifically identified because they contain microRNAs of the chromosome 19 miRNA cluster that are highly and exclusively expressed by the placenta throughout pregnancy [133–136]. The placenta releases exosomes throughout pregnancy into maternal blood and placental exosomes are higher in women with preeclampsia and may contribute to endothelial dysfunction [137]. Aspirin has been shown to inhibit exosome formation and shedding by platelets, erythrocytes, monocytes, and vascular smooth muscle cells, and it has been suggested that low-dose aspirin may have a similar beneficial effect on placental exosome shedding and content during pregnancy [137].

Most of the studies demonstrating beneficial effects of low-dose aspirin on noncyclooxygenase products were conducted in vitro or with animal models of preeclampsia. It remains to be shown that these effects occur in pregnant women in vivo. However, these studies expose how much there is to learn about how low-dose aspirin achieves its protective effect. Does aspirin affect non-cyclooxygenase pathways, or are cyclooxygenase metabolites involved in regulating these other pathways? Future studies are needed to address these issues.

### **6. Conclusions**

In summary, low-dose aspirin therapy for the prevention of preeclampsia began with the discovery of an imbalance of thromboxane and prostacyclin production by placentas of women with preeclampsia. Although the benefits of low-dose aspirin are generally attributed to inhibition of maternal platelet thromboxane, they must extend to the placenta. Maternal low-dose aspirin appears to attenuate placental oxidative stress and correct the thromboxane/prostacyclin imbalance. Abnormalities in eicosanoids and sphingolipids not affected by low-dose aspirin may explain why some aspirin-treated women develop preeclampsia.

Meta-analysis studies provide new considerations for low-dose aspirin therapy beyond those currently recommended by the American College of Obstetrics and Gynecology. These include the following: (1) a higher dose of aspirin of 150 mg/day (or 2 baby aspirin/day) is more effective, (2) aspirin should be started before 16 weeks of gestation, (3) obese women might need a higher dose, (4) low-dose aspirin is most effective in preventing preterm preeclampsia, and (5) compliance is very important and should be emphasized to the patient.

Neutrophils and the pregnancy-specific expression of PAR-1 also play significant roles in preeclampsia. Proteases are elevated in women with preeclampsia and protease activation of PAR-1 on neutrophils and placental trophoblasts can explain major clinical manifestations of preeclampsia. Additional mechanisms of action of aspirin to prevent preeclampsia should be explored, and consideration should be given to using a standard dose of aspirin and possible supplementation with L-arginine for treatment of women with preeclampsia.

**Author Contributions:** S.W.W.—conceptualization, writing—original draft preparation. J.F.S.III writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) grants 5R01 HD088386 (SWW), 1U01 HD087198 (SWW).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Office of Research Subjects Protection of Virginia Commonwealth University (HM20009145, approved 6/10/2021; HM20005160, 10/27/2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

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