Do Serum Angiopoietin-1,Angiopoietin-2,and Their Receptor Tie-2 and 4G/5G Variant of PAI-1 Gene Have a Role in the Pathogenesis of Preeclampsia?

Preeclampsia is characterized by the development of proteinuria and hypertension ¹ and is associated with altered angiogenic factors. ² Abnormal regulation in the fibrinolytic system may play an important role in mediating preeclampsia. Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of fibrinolysis. When PAI-1 is high, fibrinolytic activity is depressed, and there is an increased risk for arterial and venous thrombosis.^3–5 It is known that the PAI-1 protein levels are increased in placentas of preeclamptic subjects. ⁶

It was previously demonstrated that the 4G allele of common 4/5 Guanine tract (−675 4G/5G) polymorphism in the PAI-1 gene promotor region is associated with higher plasma PAI-1 activity.^7,8 The purpose of this prospective case-control study was to determine if there is any abnormality in plasma indices of angiogenesis, such as angiopoietins (Ang-1 and Ang-2) and their soluble receptor Tie-2 present in women with preeclampsia. In addition, we tried to find out if there is any association between PAI-1 −675 4G/5G polymorphism and the development of preeclampsia.

Materials and Methods

The present study was conducted at Kasr El Aini hospital, Cairo University, between January and May 2010. Participants were recruited from the outpatient antenatal care clinic of the Obstetrics and Gynecology Hospital.

The study was approved by the institutional review board, and informed consent was taken from all participants in the study.

Participants were divided into 3 groups: group 1, pregnant women with preeclampsia (n = 68); group 2, 35 normotensive pregnant women with matched parity and duration of pregnancy; and group 3, healthy nonpregnant females (n = 24). Both groups 2 and 3 served as control group.

Exclusion Criteria

Women were excluded from entry into the study if they had a history of hypertension, renal disease, diabetes, or collagen vascular disease.

Assay

Blood on plain tubes were obtained at the time of delivery from pregnant women and randomly from the nonpregnant women. Sera were separated and stored at −80°C until analysis. Stored serum was thawed, and commercial enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) kits were used to assay of Ang-1, Ang-2, and Tie-2. The interassay coefficient of variation (CV [%]) of ELISA Ang-1 was 5.6%, and the intra-assay CV was 3.3%; whereas for Ang-2, the interassay CV was 7.4%, and the intra-assay CV was 4.2%. The interassay CV for ELISA Tie-2 was 5.2%, and the intra-assay CV was 5.0%.

Blood was collected on sterile EDTA tubes and then was subjected to extraction of DNA by salting out technique. The PAI-1 −6754G/5G genotype polymorphism was determined by polymerase chain reaction (PCR) amplification of genomic DNA using allele-specific primers using the protocol mentioned by Falk et al. ¹⁰ : (1) insertion 5G allele 5′-GTC TGG ACA CGT GGG GG-3′ and (2) deletion 4G allele 5′-GTC TGG ACA CGT GGG GA-3′. Each in combination with a common downstream primer, 5′-TGC AGC CAG CCA CGT GAT TGT CTA G-3′, gave rise to a 139-base pair (bp) DNA fragment. All reagents required for PCR were purchased from Fermentas (Burlington, Ontario, Canada). The thermal cycling conditions were 94°C for 45 seconds, 67°C for 45 seconds, and 72°C for 75 seconds for 30 cycles. The PCR products were separated by gel electrophoresis in 2% agarose gel, which had been stained with ethidium bromide and viewed under ultraviolet irradiation. Each subject was classified into 4G4G, 4G5G, or 5G5G according to the presence of the 139-bp PCR product (Fig. 1).

OPEN IN VIEWER

Figure 1

Determining PAI-1 gene polymorphism using insertion 5G (bottom panel) and deletion 4G (top panel) allele-specific primers. Results of seven patients are shown as examples (lane 2-8). Lane 2,6,8: 4G4G; Lane 4 and 7: 4G4G; Lane 3 and 5: 4G5G. Lane 1 shows 50bp ladder.

Results

Table 1 shows the demographic data of the patients and controls including gestational age, Apgar score, and blood pressure. Medical and obstetrical history and clinical data from prenatal visits and delivery were collected. There were no differences between the 3 groups regarding the age and parity, and the preeclamptic and normotensive pregnant groups had the same duration of pregnancy.

OPEN IN VIEWER

Table 1

Demographic Data, Clinical Characteristics, and Concentration of Angiogenic Markers

Serum levels of Ang-1 and Tie-2 were significantly lower in the preeclamptic group than in the other groups (P = 0.001 and P = 0.025, respectively), whereas the 2 normotensive groups did not differ from each other. In addition, the ratio of Ang-1 with each of Ang-2 and Tie-2 was lower in the same group (P = 0.013 and P = 0.039, respectively; Table 1).

Positive significant correlation was found between Ang-2 and Tie-2, (r = 0.26, P = 0.024). The genotypic distribution of the PAI-1 polymorphism for both cases and controls is shown in Table 2. The frequency of the genotypes differed between the preeclamptic and the normotensive groups (P = 0.001). There was a tendency toward risk of development of preeclampsia in those bearing 4G/4G and 4G/5G genotypes than in carriers of 5G/5G genotype (OR, 2.9; 95% CI, 0.95-8.9; P = 0.056), Table 3.

OPEN IN VIEWER

Table 2

Genotype Frequency of the PAI-1 Polymorphism in Normotensive Pregnant and Nonpregnant Women Versus Preeclamptic Ones

OPEN IN VIEWER

Table 3

Risk Estimate of PAI-1 4G Genotype in the Development of Preeclampsia

Discussion

The present study showed that serum levels of Ang-1 and Tie-2 were significantly different among preeclamptic women and that the blood pressure correlates well with the PAI-1 genotype being higher in those bearing the 4G allele.

Although the cause of preeclampsia remains elusive, the origin of the condition is recognized as lying in the placenta. This is because preeclampsia occurs only in pregnancy; it resolves after delivery of the placenta. It can also occur in the absence of a viable fetus, for example, in molar pregnancies. Various agents have been evaluated to see whether they influence the development of preeclampsia. ¹¹

Angiopoietins are integral to vasculogenesis and angiogenesis. This process of neovascularization is initiated by angiogenic factors, which play a crucial role in the growth and development of the placenta. ¹² Disturbance in this process is associated with preeclampsia. ¹³

The study of Nadar et al. ¹⁴ revealed that plasma Ang-1 was highest in the preeclampsia group (P < 0.001), whereas Ang-2 was highest in the normotensive pregnant group (P = 0.018) and plasma Tie-2 was highest in the pregnancy-induced hypertension group. In concordance with this was the study done by Vuorela et al. ¹⁵ This was in contrast with our study, as we found that serum levels of Ang-1 and Tie-2 were significantly lower in the preeclamptic group than the other groups, and Ang-2 did not differ significantly between groups.

The discrepancy between our results and others’ may be attributed to the time of sampling, as these previous authors did not mention the date of sampling. In addition, our study differed from the study of Hirokoshi et al. ¹⁶ who reported a decrease in serum level of Ang-2 in preeclampsia. This may be attributed to their small number of cases with severe preeclampsia (5 of 26 cases), which differed from ours (38 of 68 preeclamptic cases had severe preeclampsia).

However, our findings were comparable to the findings of Bolin et al. ¹⁷ who reported that although the median of Ang-1/Ang-2 increased during pregnancy in all women, the ratios were significantly lower at gestational weeks 25 and 28 in women who later developed preeclampsia than in healthy pregnant women. In addition, Gotsch et al. ¹⁸ found that patients with preeclampsia and those who delivered small for gestational age neonates had a lower median maternal plasma concentration of Tie-2 than those with a normal pregnancy.

A number of gene polymorphisms have been found to be associated with the risk of developing preeclampsia. A deletion/insertion polymorphism (4G or 5G) in the promoter of the PAI-1 gene is suggested to be involved in regulating the synthesis of the inhibitor, 4G allele, being associated with the enhanced gene expression and plasma PAI-1 levels, which is an important inhibitor of the fibrinolytic system. Therefore, it is biologically plausible that elevated levels could suppress fibrinolysis and result in an increased risk of thrombosis. Preeclampsia is associated with thrombosis of the intervillous or spiral artery. ¹⁹ Accordingly, in our study, the frequency of 4G/4G and 4G/5G genotype was higher in the preeclamptic group than in the control group.

The distribution of PAI-1 polymorphisms in our cases was found to be concordant with those of cases in other studies.^19,20 In addition, we found that there was a tendency toward the risk of development of preeclampsia in those carrying the 4G allele.

This finding supports the genetic role of preeclampsia. However, other investigators found that similar allelic distribution of PAI-1 4G/5G polymorphism was observed in the preeclamptic and normotensive groups.^5,6

Because of a previous report by Festa et al. ²¹ that there is ethnic variation in PAI-1 4G/5G polymorphism on their studying the frequency in black, Hispanics, and non-Hispanic whites, this explains the discrepancy between our results (which was on Egyptian women) and others'.

In conclusion, the present study demonstrated that angiogenesis markers as Ang-1, Ang-2, and Tie-2 as well as variants of 4G/5G genotype of the PAI-1 gene have positive implications in the pathogenesis of preeclampsia. The availability of highly sensitive and specific physiologic and biochemical markers would allow early identification of patients at increased risk for development of preeclampsia.

Because preeclampsia is a multifaceted disorder, therefore, there is a need for high-quality, large-scale, multicenter trials that enroll patients with different risks for developing the syndrome and throughout multiethnic background to assess the predictive value of different markers and finally propose the best marker combination for a routine use in clinical settings.