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Preeclampsia: A Plethora of Biomarkers with an Incomplete Mechanism Eshani Nandita Copyright May, 2013

Preeclampsia was discovered and defined 2000 years ago. It is also referred to as toxemia or pregnancy-induced hypertension (PIH). This medical condition develops in women who have high blood pressure and elevated levels of protein in urine during their second or third trimester of pregnancy. It is known to affect 5-10% of pregnancies, causing substantial complications (Haig, Karumanchi, & Yuan 2005). According to the Preeclampsia Foundation, this disease affects both the mother and the baby and can lead to eclampsia if left undetected. Most patients discover symptoms such as blurred vision, upper abdominal pain, headache, or unexplained anxiety (Preeclampsia Foundation). Due to lack of evidence regarding the development of preeclampsia, there is no known way to prevent it from causing pregnancy issues. Studies indicate that microRNAs (miRNAs) are involved in the pathogenesis of preeclampsia. MicroRNAs are small non-coding RNA molecules about ~22 nucleotide RNA sequences in length, involved in gene expression. These particular types of RNAs are used as nucleic acid markers in the maternal plasma. Placental tissues were collected after delivery from several patients and RNA was extracted from the tissues (Enquobahrie, 2011). Microarray experiments were used to conduct mRNA profiling and to measure the expression levels of different miRNAs. In the RNA microarray, cDNA copies were made from the isolated RNAs and tagged with fluorescent probes. These were spotted on a glass slide and the microarray was scanned under laser light. To further validate the data obtained from the microarray experiments, scientists used Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). This procedure requires an oligonucleotide probe to hybridize with the target sequence so that the


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cleavage of the probe during the process of PCR can enable the detection of the amplified target product. The probe is used for the specific detection of the sequences that were isolated from the preeclamptic patients. When the DNA polymerase comes across the probe, its exonuclease activity breaks down the probe causing an increase in fluorescence. The detection of fluorescence is what determines if the target sequence was amplified or not (Enquobahrie, 2011). To see if the two different types of measurements, RNA microarray and qRT-PCR, were consistent with each other, the correlation coefficient (R2) of fold changes was calculated. Fold change is used in microarray experiments to measure the change in the expression of a gene. When compared, the expression measurements gave an R2 value of 0.9396 (Enquobahrie, 2011). This indicates that there were similar fold change differences in both experiments, and both showed that miRNAs were differentially expressed in preeclamptic patients when compared with the controls (Enquobahrie, 2011). Pathways that might be affected due to the differentially expressed miRNAs include: cell adhesion, immune system, organ development, signaling pathway, and cell cycle (Enquobahrie, 2011). Proteins that are involved in the pathogenesis of preeclampsia are encoded by differentially expressed miRNAs. However, researchers are now finding that there is more to the story than just the differentiation in gene expression of miRNAs (Enquobahrie, 2011). Scientists are trying to link oxidative stress to preeclampsia whereby they are testing for biomarkers for oxidative damage in maternal and fetal proteins in the amniotic fluid. Five patients with preeclampsia and five controls underwent amniocentesis where their amniotic fluid was tested for any chromosomal abnormalities or fetal infections (Vascotto, 2007). Proteomic maps were generated from the collected amniotic fluids. Matrix-assisted laser ionization with time-of-flight mass spectrometry (MALDI-TOF-MS) was used to analyze the peptides. It was


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found that endoprotease AspN digestion, which cleaves the amino side of aspartate residues, delivered the most spectral differences when compared to trypsin digestion. This demonstrated that they will have a variety of proteins to analyze if they performed the AspN digestion. It was found that the protein, transthyretin (TTR) was different in patients who were suffering from preeclampsia. Patients from both categories were found to have TTR (Vascotto, 2007). However, in preeclamptic women, there was a greater number of these monomeric forms of proteins when compared to the control group. This means that preeclampsia affected the amount of the different TTR forms but did not play a role in structural modification of the TTR dimer. The modified monomeric forms of TTR are due to different oxidizing reactions. This indicates that a higher oxidative stress in preeclamptic patients has been a major factor in the destabilization of the TTR dimeric form (Vascotto, 2007). Immunohistochemical analysis was performed to determine the chemical variations found in preeclamptic TTR. Data from this study showed that infarction was more prevalent in preeclamptic placentas than normal placentas. Areas with infarction were stained and the antibodies were selective for TTR. Preeclamptic placentas did not show any signs of TTR amyloidosis when tested using the immunohistochemical analysis. TTR Amyloidosis is when TTR misfolds and aggregates, thus forming insoluble fibrous proteins. Therefore, the high accumulation of preeclamptic TTR does not lead to TTR amyloidosis, where insoluble deposits promote organ dysfunction. The entire process of the accumulation of preeclamptic TTR is largely dependent on the extent of oxidative stress. Therefore, scientists are now searching for a monoclonal antibody that can recognize oxidized forms of TTR via Western blotting or ELISA analysis (Vascotto, 2007). This can promote a better understanding of the chemical reactions


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associated with preeclamptic TTR and the role of oxidative stress on the pathogenesis of preeclampsia. TTR is not the only biomarker when it comes to the development of preeclampsia. Scientists have also found isoprostanes to be related to oxidative damage and vasoconstriction of the maternal vascular system. Isoprostanes are prostaglandin products which are generated in vivo by the free radical-catalyzed peroxidation of essential fatty acids, particularly arachidonic acid. Examples of isoprostanes include: F2-isoprostanes which are present in the human plasma and F-ring isoprostanes which are associated with myelin. The formation of isoprostanes takes place without the cyclooxygenase enzyme (COX). Isoprostanes are markers of oxidative damage and stress, which are highly prominent in preeclamptic patients. Placenta samples were collected from normal and preeclamptic patients immediately after delivery. These samples were tested using the enzyme immunoassay (EIA) and gas chromatography combined with mass spectrometry (GC-MS) (Walsh, Vaughan, & Roberts II, 2000). Isoprostane concentration was measured by an enzyme immunoassay (EIA). In this procedure, the wells are coated with antibodies and filled with the sample. Only the antigen of interest from the sample binds to the antibody while the rest of the molecules are washed out of the well. Another antibody is introduced, which is covalently attached to an enzyme that binds to the antigen of interest. After a second wash, the well is filled with a colorgenic enzyme-substrate solution, generating a visible color in the solution. This indicates that the second antibody is bound to the antigen of interest. The data from the EIA experiment revealed that the concentration of malondialdehyde (MDA) was substantially higher for the preeclamptic patients than for the normal patients. MDA is the product that is formed after lipid peroxidation. Results from the EIA were further confirmed by GC-MS. The experiment indicated that isoprostane


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concentration was higher in preeclamptic women than in normal women. Scientists also found out that isoprostanes were more pronounced in the maternal side of the placental cotyledon; the secretion rate was discovered to be eightfold greater in the maternal side than in the fetal side (Walsh, Vaughan, & Roberts II, 2000). Isoprostanes, in general, could be the cause of placental vasoconstriction in preeclamptic patients as a result of oxidative stress. The study of oxidative stress generated the idea to test markers such as hydrogen peroxide, superoxide dismutase, and superoxide anion to determine the pathophysiological alterations they induce in preeclamptic patients. Scientists wanted to determine whether the accumulation of isoprostanes was tied to the introduction of oxidative stress. Tissue samples from normal patients were incubated with xanthine (X) and xanthine oxidase (XO) while normal tissue samples were incubated without X and XO. Xanthine oxidase produces superoxide anions when xanthine is converted to uric acid. After 24 hours, it was noted that the control samples (without X and XO) had accumulated 6.3 ng/mg of isoprostane, while the X+XO sample had about 14.1 ng/mg of isoprostane. However, a period of 48 hours produced 10.6 ng/mg for the control sample and 19.7 ng/mg for the X+XO sample (Walsh, Vaughan, & Roberts II, 2000). Both the control and the experimental group showed an increase in isoprostane production with time. The enzyme (xanthine oxidase) and substrate (xanthine) combination promoted the generation of oxidative stress in the tissue by releasing superoxide anions. When researchers compared this data to the isoprostane concentrations they had obtained from the preeclamptic tissues, they saw that the data was similar in the concentration level of isoprostane (Walsh, Vaughan, & Roberts II, 2000). Therefore, oxidative stress is responsible for the accumulation of superoxide anions in preeclamptic patients, which leads to placental vasoconstriction. Isoprostanes produced in the placenta could combine with the maternal


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circulation and cause the narrowing of blood vessels in the maternal vascular system. This mechanism could trigger the early symptoms of preeclamptic pregnancy and further elicit the development of eclampsia. Researchers also looked into superoxide anions as they are a substantial part of oxidative stress and damage in humans. When the activities of superoxide dismutase and catalase were measured, they found that the superoxide dismutase and catalase concentration was higher in normal women than in preeclamptic women (Ðorđevića, et al., 2008). However, the concentration of hydrogen peroxide was higher in women suffering from preeclampsia than in the control group of subjects (Ðorđevića, et al., 2008). Elevated amounts of hydrogen peroxide in preeclamptic women were due to the conversion of the increased levels of superoxide anion via superoxide dismutase. Therefore, vasoconstriction can be further enhanced by the excretion of such compounds as a result of oxidative stress. Scientists have found that it is likely that high levels of Na/K-ATPase (NKA) inhibitors and cardiotonic steroids (CTSs) like Marinobufagenin (MBG) promote the pathogenesis of preeclampsia. MBGs are chemicals present in the blood that constricts blood vessels and has been found in high levels in preeclamptic patients. Evidence shows that NKAs and CTSs trigger cell signaling cascades that cause oxidative stress to occur (Ishkaraeva-Yakovleva, 2012). These factors were used to serve as targets for immunoneutralization by DigiFab, which is a drug substitute for Digibind (no longer commercially available). Patients who were suffering from preeclampsia had an increase in MBG and an increase in the level of inhibition of NKA activity. Several ex vivo experiments indicated that DigiFab can substantially restore the NKA activity that was previously suppressed by elevated blood pressure in preeclamptic patients. To test if


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DigiFab could be a good substitute for Digibind, researchers tested placental samples from normal and preeclamptic patients (Ishkaraeva-Yakovleva, 2012). High Performance Liquid Chromatography (HPLC) was used to determine the amount of MBG in preeclamptic and control samples. It indicated that the preeclamptic plasma had a 2.5fold greater MBG concentration than in the control samples. Monoclonal antibodies were used to detect the elevated levels of MBG in the preeclamptic plasma (Ishkaraeva-Yakovleva, 2012). In normal patients, CTSs are used to regulate the excretion of Na+ by inhibiting NKA in renal tubules. However, in a preeclamptic patient, CTSs inhibit the Na+ pump that induces vasoconstriction. Thus, CTS has a significant role in the pathogenesis of preeclampsia and is responsible for the Na+/K+-ATPase inhibition. There is evidence to suggest that preeclampsia is tied predominantly to oxidative stress. Many biomarkers are available to further learn about the different mechanisms that are initiated in preeclamptic patients, such as miRNAs, TTRs, CTSs, and isoprostanes. The qRT-PCR and the microarray experiment determined that there was a similar fold change seen in both. The two experiments clarified that miRNAs were differentially expressed in preeclamptic patients than in normal patients. Once these miRNAs are expressed, they create impaired proteins which cause the first few signs of preeclampsia in pregnant women (Enquobahrie, 2011). Using MALDITOF-MS, TTR was found to be structurally different in preeclamptic patients. Oxidizing reactions modified the TTR protein into monomers rather than dimers, causing it to become destabilized. Immunohistochemical analysis did not show any chemical variations in preeclamptic patients, which indicates that TTR amyloidosis will not arise in those patients (Vascotto, 2007).


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Through EIA and GC-MS it was determined that isoprostane concentration was higher in preeclamptic patients as a result of oxidative stress. The increase in isoprostanes could cause the narrowing of blood vessels in the patient which ultimately leads to the development of preeclampsia (Walsh, Vaughan, & Roberts II, 2000). Superoxide dismutase and catalase were shown to appear more in normal women. Conversely, hydrogen peroxide showed an increase in preeclamptic women. Since the level of superoxide anions are high in preeclamptic women, superoxide dismutase ends up converting it to hydrogen peroxide. Thus, an increase in hydrogen peroxide is noted in patients suffering from preeclampsia (Ðorđevića, et al., 2008). MBGs are involved in the constriction of blood vessels and are present in high levels in preeclamptic patients. HPLC confirmed that there was a higher amount of MBGs in preeclamptic patients and as a result, the NKA is inhibited. The repression of NKA causes vasoconstriction which triggers the development of preeclampsia (Ishkaraeva-Yakovleva, 2012). Researchers have discovered many biomarkers; each revolving around the idea of oxidative damage. However, these biomarkers have not fully explained the mechanisms of the pathogenesis of preeclampsia. Similar experiments are being carried out all over the world to figure out the true cause of this disease. Mechanisms related to preeclampsia are still under constant research; ongoing experiments are searching for new discoveries in order to lower the mortality rate of preeclamptic patients. Although oxidative damage seems like a promising cause, there is much more to this disease than meets the eye.


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Works Cited Ðorđevića, N., Babićb, G., Markovića, S., Ognjanovića, B., Štajna, A., Žikića, R., et al. (2008). Oxidative stress and changes in antioxidative defense system in erythrocytes of preeclampsia in women. Reproductive Toxicology, 213-218. Enquobahrie, D., Abetew, D., Sorensen, T., Willoughby, D., Chidambaram, K., & Williams, M. (2011). Placental microRNA expression in pregnancies complicated by preeclampsia. American Journal of Obstetrics & Gynecology, 178.e12-178.e21. Haig, D., Karumanchi, S., & Yuan, H. (2005). Angiogenic Factors in the Pathogenesis of Preeclampsia. Current Topics in Development Biology, 297-312. Ishkaraeva-Yakovleva, V., Fedorova, O., Solodovnikova, N., Frolova, E., Bzhelyansky, A., Emelyanov, I., et al. (2012). DigiFab Interacts With Endogenous Cardiotonic Steroids and Reverses Preeclampsia-Induced Na/K-ATPase Inhibition. Reproductive Sciences, 1260-1267. Preeclampsia Fact Sheet. (n.d.). Retrieved January 21, 2013, from Preeclampsia Foundation: https://docs.google.com/viewer?a=v&q=cache:96N5UFVESh8J:www.preeclampsia.org/ pdf/Preeclampsia%2520Fact%2520sheet%2520v2.pdf+&hl=en&gl=us&pid=bl&srcid=A DGEESgkSpUobw_sGkTQyRqBOVLdh6b3ka0rzGVBne0ef_k0822WS59kACyT9Tghq X1FshO86g1vCYB6N_EGck6uR_1EwYCfpRy5GM879 Vascotto, C., Salzano, A., Ambrosio, C., Fruscalzo, A., Marchesoni, D., Loreto, C., et al. (2007). Oxidized Transthyretin in Amniotic Fluid as an Early Marker of Preeclampsia. Journal of Proteome Research, 160-170.


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Walsh, S., Vaughan, J., & Roberts II, L. (2000). Placental Isoprostane Is Significantly Increased In Preeclampsia. The FASEB Journal, 1289-1296.


Preeclampsia: A Plethora of Biomarkers with an Incomplete Mechanism