Anti-angiogenic collagen fragment arresten is increased from 16 weeks' gestation in pre-eclamptic plasma

Highlights

• Anti-angiogenic type IV collagen fragments arresten is significantly increased in pre-eclamptic plasma.

• Increases in plasma seen before the onset of pre-eclampsia from 16 weeks' gestation.

• Increased arresten levels are associated with greater disease severity.

• Maternal decidua may be a source of these collagen fragments.

Abstract

Introduction

Arresten and canstatin are endogenous anti-angiogenic factors derived from type IV collagen α-chains COL4A1 and COL4A2 respectively. While their functions are explored in cancer studies, little is known about their role in pregnancy. Pre-eclampsia (PE) is a common, serious hypertensive disorder of pregnancy that is characterised by systemic endothelial dysfunction. COL4A1 and COL4A2 are maternal PE susceptibility genes that have increased mRNA expression in PE decidua. Our study aim was to determine the levels of arresten and canstatin in plasma and decidua from PE and gestational age matched normotensive patients.

Methods

Plasma was collected from normotensive (n = 44) and PE (n = 39) women during the second and third trimesters of pregnancy. Third trimester decidua was collected at delivery from normotensive and PE women (n = 4 each). Levels of arresten and canstatin were determined by Western immunoblotting.

Results

Arresten levels were significantly increased in second and third trimester PE plasma, and in third trimester PE decidua (p < 0.05). Third trimester PE plasma arresten levels also significantly correlated with the need for MgSO₄ treatment, where a 1.7 fold increase was observed in patients requiring MgSO₄ treatment (p < 0.05). No significant change in canstatin levels was observed between normotensive and PE patients.

Discussion

This is the first study to report significant increases in the levels of collagen fragment arresten in PE plasma and decidua. Given its significant increase before the onset of clinical disease and associations with clinical severity in the third trimester, arresten may be a useful biomarker for predicting PE and monitoring its severity.

Introduction

Pre-eclampsia (PE) is a serious pregnancy complication with no known cause. PE is characterised by new onset hypertension and proteinuria after 20 weeks' gestation in a previously normotensive woman [1], [2]. PE affects between 2 and 8% of all pregnancies and is a leading cause of maternal and fetal morbidity and mortality worldwide [3]. The only cure for PE is the removal of the placenta [4]. Approximately 5% of women with PE also develop the haemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, which can progress rapidly to become life-threatening [5], [6]. Another complication that may develop from PE is eclampsia, where the woman suffers from seizures that may lead to death [5]. Depending on the severity of the mother's condition, the fetus may need to be delivered prematurely. Over 40% of medically indicated preterm births are attributed to PE [7]. Preterm births are associated with adverse outcomes including increased risks of neonatal death, poor long term health and delayed mental development outcomes [8].

Our previous genetic linkage studies identified COL4A1 and COL4A2 as maternal PE susceptibility genes [9]. COL4A1 and COL4A2 code for type IV collagen α chain 1 and α chain 2 respectively. The mRNA expression levels of these genes are significantly increased in PE decidua and these increased levels correlate with an earlier onset of PE [10]. The α1 and α2 chains form a triple helix isoform [α1(IV)]2α2(IV), which is expressed in all basement membranes [11]. Type IV collagen forms the scaffolding of basement membranes in tissues, allowing other basement membrane proteins to be incorporated and for cell interaction to occur with the basement membrane [11]. Type IV collagen is detectable in maternal blood and is significantly increased in third trimester PE serum [12]. Serum collagen levels reflect the degradation of the basement membrane and damage to the endothelium [12].

Degradation of type IV collagen generates many protein fragments [13]. A study by Bieglmayer and Hofer [14] showed an increase of type IV collagen non-collagenous (NC1) domain fragments in third trimester PE serum. Circulating NC1 fragment levels are also increased in patients with autoimmune, metastatic tumour, liver and renal disease [15], [16]. These studies did not identify which of the six different α chains of type IV collagen was responsible for the observed increases. The six α chains of type IV collagen produce six distinct NC1 fragments, which are derived from the carboxyl terminus of the α chains [17]. NC1 fragments arresten (COL4A1), canstatin (COL4A2) and tumstatin (COL4A3) have anti-angiogenic effects that increase endothelial cell apoptosis and decrease endothelial cell proliferation, migration and tube formation [17], [18], [19]. Research on these fragments is currently focused on their potential as anti-cancer therapeutics by targeting tumour angiogenesis [20]. To date, the levels of each NC1 fragment of type IV collagen have not been investigated and their roles in pregnancy are unknown.

Our earlier findings showed increased decidual expression of COL4A1 and COL4A2 in PE, which correlated with an earlier onset of PE, a marker of clinical severity [10]. Having shown a link between PE and increased collagen expression, the study aims were to measure and compare the second and third trimesters levels of arresten and canstatin in the plasma of PE women compared with normotensive controls, to correlate these levels with clinical parameters relating to the severity of PE and to determine whether the maternal decidua was a source of these fragments.