PRECIS: The levels of TGF-β, TGFBR1 and 2, SMAD2 were increased in the preeclampsia placenta, but conversely the thrombospondin1 mRNA relative expression, it considered that there was no correlation between TGF-β and its receptors.

Introduction

In Indonesia, maternal mortality rate (MMR) is one indicator of success to achieve Sustainable Development Goals in 2030⁽¹⁾. The MMR in Indonesia was still relatively high, equal to 133 per 100,000 live birth⁽²⁾. Among them, 27.1% with hypertension in pregnancy⁽¹⁾. Maternal hypertension in pregnancy can be caused by the presence of preeclampsia, and characterized by hypertension and proteinuria at over 20 weeks of gestation⁽³⁾. Every year there was approximately 10 million pregnant women in the world with preeclampsia and 76,000 cases were death⁽⁴⁾.

One theory developed about the cause of preeclampsia was the presence of an ischemic placenta, placental failured to supply oxygen and nutrients that result in a pathophysiological disorder. The failure of the placenta was due to the failure of the placental development process, both in the invasion of trophoblastic cells in the maternal decidua during implantation and spiral artery remodeling. Both of these processes involved many proteins that play a role in angiogenesis, proangiogenesis, and antiangiogenesis^(5,6).

One protein that plays a role in angiogenesis in the placenta is TGF-β, a multifunctional signal that activates the intracellular signal transduction pathway by binding to its serine/threonin transmembrane receptor, TGF-β receptor 1 (TGFBR1) and TGF-β receptor 2 (TGFBR2)^(7,8). Some previous studies, TGF-β has been shown to be antiangiogenic and its expression is increased in preeclampsia^(9,10,11). The association between TGF-β with both of TGFBR1 and TGFBR2 triggers the activation of the SMAD proteins, one of them is the SMAD2, and signaling continues into to the nucleus and plays a role on regulation of gene expression^(12,13). Various tissues demonstrated the role of SMAD2, as an antiantiogenic peptide, it regulates the expression of the thrombospondin-1 (TSp-1) and sFLT-1 genes, an antiangiogenesis factor⁽¹⁴⁾. Therefore, it is considered that an increase in SMAD2 mRNA expression because of TGF-β signaling via TGF-β receptor 2 failured the placental vascular development in preeclampsia. This study was conducted to observe wether any different expression and correlation of TGF-β, TGFBR1 and 2, SMAD2, and Tsp-1 expression between normal and preeclampsia placentas, also to examine the possibility of TGF-β signal transduction disturbed through SMAD2 relative expression.

Materials and Methods

This observational study was a cross-sectional design, conducted in Molecular Biology Laboratory for Oxidative Stress, Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia. The samples used were biological material stored as many as 33 normal cases and 33 cases of pre-eclampsia. This research has been approved by the Ethic Committe of the Faculty of Medicine, University of Indonesia, number 0878/UN2.F1/ETIK/2018.

The examination of protein concentration: The first placenta was made to be homogenate. A total of 100 mg of placental tissue was balanced (Sartorius) and placed into a 1.5 mL microtube and then added with 500 µL PBS to be homogenized. After the tissue was completely blended, 500 µL PBS was added again and the homogenization process was resumed. The homogenate was then centrifuged for 10 min at 5.000 g, the supernatant formed was taken for the total protein content measured by spectrofluorophotometer (Varioscan) and compared to the standard curve formula of bovine serum albumin.

Examination of TGF-β and TGF-β protein receptor 1 and 2 levels was performed using the ELISA method. A total of 100 µL of standard solutions of all dilutions and samples were put into the microplate wells, sealed and incubated for 90 min at 37 °C. After that, the sample and standard solutions were removed from the wells, and filled with 100 µL Biotinylated Detection Ab Working Solution that was already diluted 100 times, and then incubated for 60 min at 37 °C. After, the solution was discharged, the well was washed using a 350 mL Wash Buffer (diluted 25 times), and repeated 3 times. In each standard and samples, 100 mL HRP Conjutae Working Solution (diluted 100 times) were put, and incubated for 30 min at temperature 37 °C. Then, after the solution was removed, the wells washed again with 350 µL wash buffer and repeated 5 times. A total of 90 µL Substrate Reagent was added to each well and incubated for 15-30 min to develop color, and the last, reaction was discontinued by adding 50 µL Stop Solution. The target protein concentration was examined by measuring the absorbance at a 450 nm wavelength and the result is calculated using the equation formula of the standard curve to obtain the concentration.

Measurement of SMAD2 and TSp-1 mRNA relative expression by reverse transcription polymerase chain reaction method (RT-PCR) method was performed using Sensifast^TM SYBR No-ROX One Step Kit RT-PCR (Meridian Bioscience). Before performing the relative expression, total RNA isolation was measured using a total RNA minikit (Geneaid). The primers used are forward primer and reverse primer: SMAD2 Forward primer: 5’-ACC-GAA-ATG-CCA-CGG-TAG-AA-3’, Reverse primer: 5’-TGG-GGC-TCT-GCA-CAA-AGA-T-3’; TSp-1 Forward primer: 5’-AGC-ATG-GTC-CTG-GAA-CTC-AG-3’, Reverse primer: 5’-CAG-CTC-ATT-GGC-CAA-CTC-TT-3’; 18s rRNA Forward primer: 5’-AAA-CGG-CTA-CCA-CAT-CCA-AG-3’, Reverse primer: 5’-CCT-CCA-ATG-GAT-CCT-CGT-TA-3’.

To obtain the relative expressions of mRNAs, optimization of the primary attachment temperature was prepared. The protocol of mRNA relative expression was in the following steps: Into each well the mix solution was added, it consisted of 10 µL Sensifast^TM SYBR No-ROX One Step mix, 0.8 µL forward primer, 0.8 µL reverse primer, 0.4 µL Ribosafe RNase Inhibitor, 0.2 µL Reverse Transcriptase, 4 µL mRNA template, and DEPC-H₂O maximum 16 µL. Each sample was run in triplo and using NTC as a negative control. The samples were then incubated on a qRT-PCR machine (PCR max Ecosains) with the appropriate protocol kit temperature.

The result of one-step qRT-PCR is then analyzed further to determine the relative expression of the gene using the following formula:

Statistical Analysis

Statistical analysis was performed using the statistical package for social science (SPSS) software for window version 2.0. All data were tested for their normality using Kolmogorov-Smirnov test. Furthermore, homogeneity tes, the normal and homogeneous data will be presented in mean and standard deviation using the unpaired t-test. Besides, the data that were not normally distributed, the Mann-Whitney test was used. Assessment of the correlation between two variables, the Pearson correlation was used for normally distributed data and the Spearman test was used for the data thet were not normally distributed. The significant limit used was p<0.05.

Results

Protein Level of TGF-β

Before calculating the TGF-β concentration, the total protein of the placentas was measured. There was a significant different between normal and preeclampsia placenta protein contents (Figure 1). Measurement of TGF-β using the ELISA sandwich method showed that using the Mann-Whitney test it was proffed a significantly different between the relative expression of normal and preeclampsia markers. TGF-β levels in the preeclampsia placenta was 2.69 (0.906-10.252) ng/mg, it was increased significantly compared with normal placenta, 1.19 (0.713-2.051) ng/mg, p=0.0001, (Table 1, and Figure 2).

Protein Level of TGF-β Receptor 1

Measurement of TGF-BR1 showed that its concentration was increased significantly in preeclampsia 1.2239 (0.372-2.553 ng/mg) compared to normal placenta 1.025 (0.622-1.402 ng/mg), p=0.002, (Figure 3 and Table 1).

Protein Level of TGF-β Receptor 2

TGF-β receptors 2 (TGFBR2) protein level were examined using the sandwich ELISA method (Figure 4). It showed that levels of TGF-β receptor 2 in the preeclamptic placenta 1.490 (0.775-3.645) ng/mg, it was higher significantly than normal 0.959 (0.644-1.6340) ng/mg. The Mann-Whitney test results showed a significant difference between the TGFBR2 relative expression of normal and preeclampsia with p=0.0001 (p<0.05), (Table 1).

Relative Expression of SMAD2 mRNA

The SMAD2 mRNA relative expression of preeclampsia was not significantly different compared to normal placentas, (Mann-Whitney test, p=0.075), Figure 5.

Protein Level of SMAD2

SMAD 2 protein concentration showed that its concentration in the preeclampsia placenta 3.508 (1.842-22.489) ng/mg was significantly higher than normal 2.087 (1.279-4.300) ng/mg, (Mann-Whitney, p=0.0001), (Table 1, and Figure 6).

Thrombospondin mRNA Relative Expression

The relative expression of TSp-1 mRNA was decreased significantly in the preeclampsia placenta compared to normal, (Mann-Whitney, p=0.0001), (Table 1, and Figure 7).

Comparison of Protein Markers Correlation Between Preeclampsia and Normal Placentas

The correlation between TGF-β and TGF-BR1 in the normal placenta was strong, (Spearman correlation, R=0.789, p=0.0001), but in the preeclampsia placenta, there was no correlation between TGF-β and TGF-BR1 in the normal placenta, (Spearman correlation, R=0.053, p=0.76), (Table 2). A similar results shown in the correlation between TGF-β and TGF-BR2. In the preeclampsia placenta, there was no correlation between TGF-β and TGF-BR2 (Spearman correlation R=0.028, p=0.878), but in the normal placenta it showed a strong correlation (Spearman correlation R=0.623, p=0.0001), (Table 2). Correlation between TGF-BR1 and TGF-BR2 showed strong correlation both in normal (Spearman correlation, R=0.799) and preeclampsia placenta, (Table 2).

Discussion

Preeclampsia is a disorder in pregnancy characterized by the presence of hypertension (blood pressure ≥140/90 mmHg) and proteinuria (urinary protein level ≥300 mg/24 h) at gestational age above 20 weeks^(15,16). Preeclampsia has a negative impact on the mother may lead to eclampsia and lead to HELLP syndrome, a set of syndromes characterized by microangiopathy, elevated liver enzymes (liver dysfunction) and low platelets^(16,17). Fetus of preeclampsia, may suffered to retardation during fetal growth, premature birth, low birth weight, and neonatal death^(18,19).

There were several developing theories about the pathophysiology of preeclampsia, but in developmental research on pre-eclampsia was focused on the placenta. Placental ischemia in preeclampsia is placental failure in supplying oxygen and nutrients that results in pathophysiological disorders and affects fetal development and maternal health. The failure of the placenta is due to an interruption of the placental development process. In the process of placental development, during the invasion, trophoblasts induce spiral artery remodeling to form a low -resistance vascular system and it is essential for fetal development. This developmental stage is characterized by a physiological change in oxygen pressure at the base of the intervillous region⁽²⁰⁾.

During the first few weeks of pregnancy, the trophoblast is in a relatively low-oxygen environment. Maternal blood flow to the placenta tends to be low for trophoblast invasion, although this low oxygen pressure state is important for normal embryo and placental development. As soon as the spiral artery remodeling is completed, maternal blood flow to the fetus increases and hypoxia may be discontinued⁽²⁰⁾. However, this condition does not occur in the placenta with impaired in trophoblast invasion, such as preeclampsia. Lack of optimum trophoblast invasion, cause the spiral artery remodeling process does not occur properly, leading to a decrease in maternal blood flow to the fetus, and hypoxia in pregnancy continues, leading to preeclampsia⁽³⁾.

The trophoblastic invasion process involved tissues, multiple cytokines, growth factors, and angiogenesis factors that are mutually coordinated. These molecules interact with each other to provide biological effects, by triggering the transcription of certain proteins. Therefore, failure in coordination and interaction between cytokines and growth factors may impact the disruption of trophoblast invasion that triggers placental abruption. A cytokine that play a role in the development of the placenta is TGF-β. In this study, we found that there was an increase in TGF-β levels of placental pre-eclampsia. This agreed with previous studies suggesting that TGF-β protein levels are higher in placenta preeclampsia because TGF-β has an anti-angiogenesis effect through smad2 but proangiogenesis through smad3^(21,22). Additionally, Extra Villous Trophoblastic cells treated with TGF-β1, TGF-β2, and TGF-β3 showed decreased invasion by decreasing protease activity^(22,23).

Increased levels of TGF-β in the placenta pre-eclampsia are also triggered by the presence of placental hypoxia. In a hypoxic state, the cells produce HIF-1 proteins that regulate the transcription of certain genes in response to their environment. This increase in HIF-1 also triggers an increased expression of TGF-β mRNA in trophoblasts⁽²⁴⁾. Increased levels of TGF-β by HIF-1 may be intended to trigger angiogenesis to reduce hypoxi; it is considered that TGF-β via another receptor signaling molecule may also be proangiogenic. However, when the state of hypoxia did not goes down, the TGF-β levels increased excessively and eliminated its proangiogenic effec; also, the increased in SMAD2 led to suppress the trophoblast ability to make network⁽²⁵⁾.

Similar results were found in the examination of levels of TGF-β receptor 2 protein, which were higher in the preeclampsia than in the normal placenta. The interaction of the TGF-β ligand with its receptor triggers both of these receptor types for association, which are then followed by the phosphorylation of type 1 receptors by the type 2 receptors in the domain of the serine-threonin kinase. This activated type 1 receptor followed by passing the signal into an intracellular mediator called SMADs⁽⁶⁾. In line with elevated levels of TGF-β and TGF-β receptor 2 proteins, the relative expression of SMAD2 mRNA in preeclamptic placentas also significantly increased compared to normal placentas. This increase in SMAD2 mRNA expression was not very high compared with elevated levels of TGF-β and TGF-β receptor 2 proteins, it was considered that may be other factors affecting the regulation of SMAD2 mRNA expression.

In this study, the correlation analysis showed a weak positive correlation relationship between TGF-β protein and the relative expression of SMAD2 mRNA in the normal placenta. Conversely, a correlation analysis of both proteins in the preeclamptic placenta groups did not show any correlation. In the other side, a correlation test also performed on TGF-β receptor 2 protein levels with SMAD2 mRNA expression, it also showed no correlation between both parameters, either on normal or pre-eclampsia placentas.

In various studies on the TGF-β signaling pathway in preeclampsia, it mentioned that this protein will bind to both receptors, which can then give a pleiotropic effect: Proangiogenesis or antiangiogenesis⁽²⁶⁾. It was also mentioned that TGF-β signaling via TGF-β receptor 1 (ALK5) and TGF-β Receptor 2 phosphorylates the antiangiogenesis SMAD2 protein^(27,28). This suggests that the TGF-β signaling pathway was more influential on the activation of latent SMAD2 protein, compared with its mRNA expression. We consider that any other molecule affects SMAD2 mRNA expression, based on the result that there was no correlation between TGF-β protein levels and their receptor with SMAD2 mRNA expression, presented in this study. Although, both groups of these parameters were increased in the preeclampsia placenta compared with normal.

Study Limitation

There was a limitation in this study due to using storaged placentas that not yet be parafined therefore the immunohystochemistry preparation couldnot be performed to observe TSp-1, and fibrotic area due to the high level of the TGF-β.

Conclusion

TGF-β protein level, TGF-β receptors and SMAD2 were increased in the preeclampsia placenta. Additionally, there was a mild positive correlation between TGF-β protein and relative expression of SMAD2 mRNA in the normal placenta, but was not found in the preeclamptic placenta. There may be other factors contributing to the regulation of SMAD2 mRNA expression.

Ethics

Ethics Committee Approval: This research has been approved by the Ethic Committe of the Faculty of Medicine, University of Indonesia, number 0878/UN2.F1/ETIK/2018.

Informed Consent: Not necessary.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: A.R.P., N.T.O., F.C.I., N.M., Y.P., Concept: A.R.P., N.T.O., F.C.I., N.M., Y.P., Design: A.R.P., N.T.O., F.C.I., N.M., Y.P., Data Collection or Processing: A.R.P., N.T.O., F.C.I., N.M., Y.P., Analysis or Interpretation: A.R.P., N.T.O., F.C.I., N.M., Y.P., Literature Search: A.R.P., N.T.O., F.C.I., N.M., Y.P., Writing: A.R.P., N.T.O., F.C.I., N.M., Y.P.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.