High serum angiopoietin-like protein-4 levels are associated with gestational hypertension and preeclampsia: a case-control study

Introduction

Preeclampsia (PE), a serious complication of pregnancy, affects 8.5 million women every year in the world, with a prevalence ranging from 3 to 8 %. PE, which is a multisystemic disease, is responsible for 18 % of pregnancy-related maternal deaths and 40 % of fetal deaths. Currently, there is no definitive diagnostic tool and effective treatment method that can be used in the early diagnosis of PE [1]. Hypertension diagnosis criteria were used if a pregnant woman had a systolic blood pressure exceeding 140 mmHg or a diastolic blood pressure exceeding 90 mmHg, which was measured at least twice with a 4 h interval while at rest. On the other hand, if the amount of protein detected in the 24 h total urine is over 300 mg/dL, it was accepted as the diagnostic criterion for preeclampsia [2]. Detection of hypertension after 20 weeks of gestation in a previously normotensive pregnant is defined as gestational hypertension (GH). In these cases, hypertension resolves after delivery [3]. According to the American College of Obstetricians and Gynecologist (ACOG) in 2013, proteinuria is not mandatory for the diagnosis of preeclampsia [4].

Conditions such as inflammation, oxidative stress, some angiogenic and/or antiangiogenic and endothelial damaging agents, enzymes associated with hormone and lipid metabolism, and fetal distress are among the currently used and investigated markers for the early diagnosis of PE [5]. The effectiveness of biomarkers is determined by the ability to diagnose the disease long before the appearance of clinical symptoms, and it would be an advantage if possible changes could be detected through minimally invasive blood tests performed as part of routine controls [6]. However, when they are used together rather than alone, these markers increase their effectiveness in the early diagnosis of PE [7]. Placental growth factor (PIGF) is angiogenic, fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are antiangiogenic proteins. Antiangiogenic factors are increased in PE [8]. Before the clinical feature of preeclampsia appears, maternal PIGF levels decrease, whereas maternal sFlt-1 and sEng levels increase [9]. Serum neutrophil gelatinase-related lipocalin levels were found to be higher in patients with both early- and late-onset preeclampsia than in patients without preeclampsia [10]. It has been reported that fractalkine, a chemokine involved in the inflammatory process, is increased in the serum of patients with premature rupture of membranes and preeclampsia [11, 12]. Angiopoietin-like protein 4 (ANGPTL-4) is involved in the regulation of angiogenesis as well as in energy, lipid, and glucose metabolism [13]. It has been shown that increased ANGPTL-4 levels are associated with hypertension [14], atherosclerosis [15], and coronary artery disease [16]. ANGPTL-4 can act as an antiangiogenic protein as well as showing a proangiogenic effect. Although ANGPTL-4 has been shown to be effective in protecting endothelial cell integrity, it has also been shown to play a role in the development of atherosclerosis by causing endothelial damage [17]. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and lymphangiogenesis [18]. VEGF-A induces endothelial cell proliferation and increased vascular permeability [19]. VEGF-A, which is expressed in the placenta during pregnancy, plays an important role in the regulation of placental angiogenesis and maternal spiral artery remodeling [20].

Although many markers have been examined for the possible diagnosis and follow-up of PE, we investigated the maternal serum values of ANGPTL-4 and VEGF-A, which are known to have important roles in the maintenance of angiogenesis and endothelial functions, with the thought that they may have a role in the pathophysiology of PE.

Materials and methods

This case-control study was carried out in the Obstetrics Clinic of Fırat University, Faculty of Medicine, after obtaining the approval of the Ethics Committee (Ethics Committee decision no: 2021/02-10). The study was conducted in accordance with the Helsinki principles. Three groups (G) were formed with a total of 90 patients included in the study.

• G1 (n=30)=control group consisting of healthy pregnant women between 37 and 41 weeks.

• G2 (n=30)=patients diagnosed with GH between 20 and 37 weeks.

• G3 (n=30)=patients diagnosed with PE between 20 and 37 weeks.

Age, number of pregnancies, gestational week, systolic, diastolic and mean arterial blood pressure were recorded.

Results

Mean age of both groups was similar (p>0.03). The number of nulliparous pregnant was higher in G2 and G3 compared to G1 (p<0.001). Gestational week was lower in G2 and G3 than G1 (p<0.001). Systolic, diastolic and mean arterial pressure (MAP) values were higher in G2 and G3 than in G1 (p<0.001) (Table 1).

While ANGPTL-4 and VEGF-A levels were similar in G2 and G3 (p>0.03), they were significantly higher than G1 in both groups (p<0.001) (Table 1).

Discussion

In this study, we evaluated serum concentrations of ANGPL-4 and VEGF-A in maternal serum. As a result of our study, the mean values of ANGPL-4 and VEGF-A serum levels were found to be significantly higher in the PE and GH group than in normal pregnant women. We think that it can be used as an early marker in the development of PE by establishing the cut-off value for ANGPI-4 and VEGF-A serum levels by studying in larger patient groups.

PE and GH are more common in primigravids [21]. In our study, we found the number of primigravid pregnant women to be high in the GH and PE groups.

PE has been shown to be associated with an imbalance between angiogenic and anti-angiogenic factors, also defined as “angiogenic imbalance”. It includes proangiogenic factors VEGF and PlGF, sEng [22], soluble VEGF receptor 1 (sVEGF-R1), and a soluble form of fms-like tyrosine kinase 1 (sFlt1) known as its generic splice variant [23]. Blocking VEGF and PLGF by sFlt1 inhibits angiogenic effects in the maternal circulation [24]. It has been reported that sFlt-1 levels increase in maternal serum five weeks before the onset of PE [25].

In a preeclamptic placenta, the remodeling of the spiral arteries is impaired, which reduces the oxygenation of the placenta, resulting in ischemia. Placental ischemia contributes to maternal endothelial damage by increasing the production of antiangiogenic factors [26]. Placental hypoxia causes endothelial damage in preeclampsia by activating hypoxia Inducible Factor-1α (HIF-1α). sFlt1 causes endothelial damage in preeclampsia due to its antiangiogenic property. However, ANGPTL-4 regulates inflammation, lipid and glucose metabolism, and increased vascular permeability [27, 28].

Hypoxia-induced expression of ANGPTL-4 in human endothelial cells elicits a potent VEGF-independent pro-angiogenic response [29]. HIF-1α causes placental disorder by inducing ANGPTL-4 in placental hypoxia and it also blocks trophoblast invasion because it increases TGF-β3, which is another target [30], and thus it is associated with PE [31]. It has been reported that hypoxia causes maternal glomeruloendotheliosis, proteinuria, maternal hypertension and fetal growth retardation in the pathophysiology of preeclampsia [32], decreased choroidal blood flow causes low oxygenation, causing the release of hyphae [33], causing the release of angiogenic factors such as VEGF [26] and ANGPTL-4 [34], thus leading to pathological macular neovascularization [35]. They reported that the hypoxic environment increased the production of ANGPTL-4 levels in human periodontal ligament fibroblasts via HIF-1 [36]. In contrast, Kubo H et al. showed that ANGPTL-4 expression in gastric cancer cells under hypoxia is regulated through a pathway independent of HIF-1α [37].

As a result of our study, we showed that ANGPL-4 serum levels in PE and GH cases were significantly higher than in healthy pregnant women. We think that this is the result of placental hypoxia in PE as stated in the literature above. It has been reported that serum ANGPTL-4 levels are greatly increased in patients with end-stage renal disease and nephritic syndrome. It has been shown that ANGPTL-4 can also be used as a kidney function marker due to its association with glomerular capillary wall damage and proteinuria [38]. We also think that high ANGPTL-4 serum levels in the PE group may be related to the proteinuria seen in patients with PE. We also showed that ANGPTL-4 level increased in our GH group, similar to the PE group. This supports the ACOG’s view that the presence of proteinuria is not essential for the diagnosis of PE in 2013. This is because we showed that ANGPTL-4 levels were increased in maternal serum before proteinuria in GH cases before the development of PE. This may indicate that maternal serum ANGPTL-4 measurement may be a candidate for a marker that can be used in the diagnosis of PE.

VEGF-A is a mitogen that induces proliferation of endothelial cells and vascular permeability [39]. In our study, we showed that serum VEGF-A values in our GH and PE group were higher than the control group. This may be the reason for the increase in VEGF level which increases angiogenesis in the placenta with the compensatory mechanisms that can be induced by hypoxia as a result of abnormal angiogenesis. It can be thought that VEGF-A may increase in GH cases even before PE develops, contributing to the development of edema and proteinuria in PE.

Some limitations of our study; the limited number of cases, the inability to study more advanced hypoxic biochemical markers, and the fact that ANGPTL-4 in the placenta has not been evaluated immunohistochemically. Another limitation of ours is that it could not be determined from which gestational week the ANGPTL-4 levels in maternal serum started to increase in cases with PE. Our other limitations are that our cases do not have body mass indexes, that the gestational weeks, pregnancy and birth numbers of our control group and case groups do not match, and that the cases receiving multivitamin, antihypertensive and aspirin treatment are not differentiated in our GH and PE groups.

In addition, the fact that ANGPTL-4 and VEGF-A levels were increased in both our PE and GH cases reduces the level of discrimination. We could not follow up the babies after birth. In this context, different studies can be designed by adding some other data such as IUGR development, preterm labor, birth weeks, normal spontaneous vaginal birth, and cesarean section rates.

One of the strengths of our study is that ANGPTL4 levels were evaluated for the first time in GH and PE cases. Another strength of ours is that it has been shown that maternal serum ANGPTL-4 and VEGF-A levels increase in pregnant women with GH even before the development of PE. Therefore, ANGPTL-4 may be a candidate for an easily available, effective biochemical marker that can be used in the early diagnosis of PE.

In conclusion, we showed that ANGPTL-4 and VEGF-A levels were increased in maternal serum in GH and PE cases. Increased maternal serum ANGPTL-4 levels may be a biomarker that can be used in the early diagnosis of PE.