Altered decorin leads to disrupted endothelial cell function: A possible mechanism in the pathogenesis of fetal growth restriction?
Introduction
Fetal growth restriction (FGR) is defined as a neonatal birth-weight less than 10th percentile for gestation together with evidence of fetal welfare compromise such as reduced amniotic fluid volume, increased head to abdomen circumference ratio and abnormal umbilical artery blood flow patterns [1]. FGR greatly increases the risk of perinatal complications including: fetal compromise in labour, fetal death in utero, neonatal morbidity and neonatal death [2], [3], [4]. Live born infants from pregnancies complicated by FGR have an increased risk of paediatric disorders such as cerebral dysfunction and learning difficulties, and of developing chronic adult onset diseases including: cardiovascular complications, type II diabetes, hypertension and ischaemic heart disease [5], [6], [7]. Idiopathic FGR accounts for 70% of all cases of FGR and is believed to be associated with uteroplacental insufficiency [8], abnormal umbilical artery Doppler velocimetry [9], oligohydramnios [10] and fetal growth asymmetry [11]. Placental insufficiency may result from various factors including: constriction of the placental blood vessels due to reduction in vasodilator activity [12], incomplete cytotrophoblastic invasion of the maternal spiral arteries [13] or maldevelopment of the placental villous structures [14]. These factors result in increased resistance to blood flow within the placenta in both the maternal and fetal circulations, ultimately resulting in fetal hypoxia and acidosis.
Normal pregnancy represents a hypercoagulable state characterised by profound changes in haemostasis, such as, increased concentration of pro-coagulant factors, decreased anticoagulant activity and diminished fibrinolytic activity [15]. These changes result in increased thrombin generation in maternal plasma and ultimately, increased fibrin formation. These changes in haemostasis ensure the rapid and effective control of bleeding at the time of placental separation during delivery [15]. On the other hand, these changes may also predispose pregnant women to thrombosis and placental vascular complications. Despite this, thrombotic events are rare in uncomplicated pregnancies [16], indicating that thrombin generation must be tightly regulated in this scenario. In contrast, histological examinations of placentae from FGR pregnancies demonstrate increased fibrin deposition and thrombi in the vasculature, including uteroplacental and intervillous thrombosis, perivillous fibrin deposits and villous stem artery thrombosis [16], indicating an increase in overall thrombin activation [17]. The cause of the coagulation disturbance and increased placental thrombosis observed in idiopathic FGR pregnancies is unknown. However, since thrombin generation is significantly increased during normal pregnancy compared to the non-pregnant state [18], the excess thrombin is likely to be generated predominantly by the placenta, as demonstrated by decreased thrombin generation following placental separation during delivery [19].
Proteoglycans (PGs) are macromolecules comprising a core protein and at least one negatively charged polysaccharide glycosaminoglycan (GAG) side chain. The small leucine-rich proteoglycan (SLRP) family constitutes a network of signal regulation: being mostly extracellular, they are upstream of multiple signalling cascades, a major conduit of information for cellular responses and modulators of distinct pathways [20]. Decorin (DCN) belongs to the Class I SLRPs and can be substituted with one of either chondroitin or dermatan sulphate glycosaminoglycan (GAG) side chains. Previously, we have demonstrated an association between reduced placental DCN expression and FGR [21], and propose that due to the many actions of DCN in vivo, this reduction contributes to the pathogenesis of FGR [22]. DCN and its side chain are involved in multiple biological functions such as, anticoagulation by binding to heparan cofactor II through a highly charged sequence [23], regulation of angiogenic growth factors such as, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) [24] as well as regulation of basic cellular functions such as proliferation, migration and invasion [25], [26]. Mouse knockout models of DCN demonstrate a range of pregnancy disorders including: pre-term birth [27], dystocia and delayed labour onset [28], as well as developmental anomalies in the offspring including: osteoporosis, osteoarthritis and corneal disease [29].
Since disturbances in many of these biological functions have been demonstrated in the pathogenesis of FGR, DCN may in fact play a major role in the pathogenesis of FGR. Therefore, in the present study, we investigated the effect of reduced DCN gene expression on the function of microvascular endothelial cells. We also determined the down-stream target genes of DCN, in a placental microvascular endothelial cell environment and further validated the expression of these targets in control and FGR-affected placentae.
Section snippets
Cell lines
The human microvascular endothelial primary cells from neonatal foreskin (HMVEC) were a kind gift from A/Prof. Grant Drummond (Department of Pharmacology, Monash University).
Reduction of DCN expression by siRNA
Four independent DCN siRNA oligonucleotides were obtained as “4-For-Silencing siRNA Duplexes”™ (Qiagen, Victoria, Australia). The DCN siRNAs showed no significant DNA sequence similarity to other genes in GenBank cDNA databases (data not shown).
RNA extraction and cDNA preparation
Total RNA was extracted from cultured HMVECs using PureLink RNA Mini-kits
Reduced DCN mRNA and protein expression following siRNA transfection in HMVECs
Four independent siRNAs (designated as siRNA1-4), were designed to reduce DCN expression in HMVECs. A non-siRNA transfected control (Mock) and a non-specific siRNA transfected control were used as negative controls. Fig 1A revealed that treatment with siRNA2 and siRNA3 significantly reduced DCN mRNA expression compared to both the Mock and NC controls (Mock: 0.90 ± 0.14 and NC: 0.59 ± 0.02 vs. s2: 0.01 ± 0.001 and s3: 0.05 ± 0.01, p < 0.005, n = 18, One-Way ANOVA) at 48 h after transfection. A
Discussion
In this current study we focused on DCN, a small leucine-rich proteoglycan, and demonstrate for the first time that reduction of DCN gene expression in a primary human microvascular endothelial cell type (HMVEC) results in a significant decrease in HMVEC proliferation, network formation and thrombin generation. We also revealed differential expression of DCN target genes in FGR-affected primary placental microvascular endothelial cells (PLECs). The results reveal a consistency in the expression
Acknowledgements
a) The authors would like to thank Diagnostica Stago (Australia) for the loan of the Calibrated Automated Thrombogram.
b) Sources of funding: This work was supported by National Health and Medical Research Council (NH&MRC) Project Grants (1004952 and 1042239), Australia.
References (47)
- et al.
Fetal nutrition and adult disease
Am J Clin Nutr
(2000) - et al.
Placental pathology of absent and reversed end-diastolic flow in growth-restricted fetuses
Obstet Gynecol
(1997) Placental insufficiency and its consequences
Eur J Obstet Gynecol Reprod Biol
(2003)- et al.
Development of the placental villous tree and its consequences for fetal growth
Eur J Obstet Gynecol Reprod Biol
(2000) Haemostatic changes in pregnancy
Thromb Res
(2004)- et al.
Haemostatic mechanisms in human placenta
Best Pract Res Clin Haematol
(2003) - et al.
Oxygen and placental villous development: origins of fetal hypoxia
Placenta
(1997) Generation of thrombin in blood plasma of non-pregnant and pregnant women studied through concentration of thrombin-antithrombin III complexes
Eur J Obstet Gynecol Reprod Biol
(1997)- et al.
Anticoagulant dermatan sulfate proteoglycan (decorin) in the term human placenta
Thromb Res
(1998) - et al.
Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction
J Biol Chem
(2008)
Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5
Biochim Biophys Acta
Beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules
J Biol Chem
hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization
Cell
Transformation of cultured human vascular endothelium by SV40 DNA
Cell
Placental syndecan expression is altered in human idiopathic fetal growth restriction
Am J Pathol
A distinct microvascular endothelial gene expression profile in severe IUGR placentas
Placenta
Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor 1alpha, vascular endothelial growth factor A, and induction of thrombospondin-1 and TIMP3
J Biol Chem
Decorin: a guardian from the matrix
Am J Pathol
Decorin is produced by capillary endothelial cells in inflammation-associated angiogenesis
Am J Pathol
Sustained down-regulation of the epidermal growth factor receptor by decorin. A mechanism for controlling tumor growth in vivo
J Biol Chem
Decorin is a biological ligand for the epidermal growth factor receptor
J Biol Chem
Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells
J Biol Chem
Decorin binds to a narrow region of the epidermal growth factor (EGF) receptor, partially overlapping but distinct from the EGF-binding epitope
J Biol Chem
Cited by (21)
Antiangiogenic effects of decorin restored by unfractionated, low molecular weight, and nonanticoagulant heparins
2017, Blood AdvancesCitation Excerpt :In addition, the risk of adverse heparin-induced thrombocytopenia and osteoporosis after LMWH treatment is greatly reduced compared with UFH.20-25 In our previous work, we showed that the messenger RNA and protein expression of the proteoglycan decorin (DCN) was reduced in PE- and FGR-affected placentae compared with controls.26-28 Furthermore, we also showed that downregulation of DCN in human microvascular endothelial cells resulted in decreased cell growth and proliferation, decreased network formation (angiogenesis), and a modest increase in thrombin generation.26
Pivotal role for decorin in angiogenesis
2015, Matrix BiologyThe Contribution of Vascular Proteoglycans to Atherothrombosis: Clinical Implications
2023, International Journal of Molecular SciencesThe potential role of the ESRRG pathway in placental dysfunction
2021, Reproduction