Describe the mechanisms involved in spiral artery remodeling and discuss how failure in the process may result in common pregnancy complications
Background
Spiral arteries mature in the second half of the menstrual cycle with the aid of progesterone(Ferenczy et al. 1979) and develop from radial arteries. In the absence of blastocyst implantation, the spiral arteries regress and are lost during menstruation. If implantation does occur spiral arteries remodeling occurs in the first 22 weeks of gestation and the main mechanisms involved are changes in cellular adhesion and sensitivity to death inducing stimuli, migration, vascular de-differentiation, migration, and extracellular matrix restructuring. Firstly, the trophoblast independent changes are thought to mediate priming of vessels that occur before complete invasion and transformation of the spiral arteries.
Following priming of the vessel, the process is trophoblast dependent. Extravillious trophoblasts made from cytotrophoblast stem cells can form two subpopulations where the interstitial extravillious trophoblasts invade the uterine wall and the endovascular extravilllous trophoblast migrate into the lumen of the spiral artery to progress to the myometrium and the endothelium is also replaced by cytotrophoblast(Pijnenborg et al. 1980). The remodeling leads to an increased vessel diameter and the total blood delivered to the intervillous space rises by 3-4 fold but at a lower pressure(Thaler et al. 1990; Kliman et al. 2000). These changes meet the demands of the growing fetus for nutrients, respiratory gases and removal of metabolic waste. However, failure of normal spiral artery remodeling results in common pregnancy complications.
Main mechanisms of spiral artery remodeling
Changes in cellular adhesion and sensitivity to death inducing stimuli
The maintenance of endothelial cell(EC) and vascular smooth muscle(VSMC) in the lumen of spiral arteries could be dependent on the proportion of pro and anti-apoptotic stimuli. Vascular cell loss in the spiral artery is not achieved solely by modulation of the vessel but also other numerous factors, implying the process is tightly regulated. Moreover, it has been proposed that invading trophoblasts can interfere with the maintenance of vascular cell death by a direct pathway, through the release of apoptotic factors or by an indirect route, by promoting loss of cellular adhesion where the endothelial cells are taken off the basement membrane and is lost in the circulation, the process by which the EC die is known as anoikis.
The apoptotic factors that promote vascular cell apoptosis are tumour necrosis factor-α (TNF-α), TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand(FasL) are synthesized and expressed by trophoblasts(King et al. 1995), therefore TNF-α is highly likely to affect trophoblast derived death and differentiation via their receptors found on trophoblasts.
A recent study conveyed fetal trophoblast invading from the placenta instigates remodeling by triggering cell death in VSMC(Koegh et al. 2007). TRAIL was detected by immunohistochemistry in the spiral artery VSMC. Trophoblasts that had been isolated from the first trimester placenta expressed TRAIL and was capable of inducing apoptosis of VSMC(Keogh et al. 2007). The original source of TRAIL is not clear as TRAIL could be produced by trophoblasts or from VSMC in an autocrine manner. Apoptosis of the VSMC was achieved in the presence of cytotrophoblasts; still pictures indicated a co-culture of trophoblasts and VSMC where the trophoblasts interacts with the VSMC and after 9 hours the trophoblast moves away and after 12 hours the VSMC undergoes apoptosis, suggesting the trophoblasts produces the apoptotic factor TRAIL and instigates apoptosis of VSMC via a TRAIL dependent mechanisms, contributing to spiral artery remodeling in pregnancy.
There are several TRAIL receptors that regulate apoptosis via the extrinsic pathway. Preliminary data shows human spiral artery endothelial cells specifically express TRAIL-R2 at term and TRAIL-R1 and TRAIL-R2 are expressed in some populations of spiral artery smooth muscle cells(Koegh et al. 2007). TRAIL-R1 and TRAIL-R2 transduce apoptosis upon binding with TRAIL but receptors such as TRAIL-R3 and TRAIL-R4 lack intracellular death domain and cannot initiate apoptosis therefore are classed as decoy receptors protecting EC from apoptotic signals(Secchiero 2003). The decoy receptors are expressed on endothelial cells(Zhang et al. 2000) and another TRAIL-R osteoprotegrin inhibits apoptosis by competing with TRAIL for binding with death receptors and blocks binding of TRAIL with TRAIL-R(Emery et al. 1998).
When the TRAIL/TRAIL receptor complex is activated, an adaptor molecule named Fas-associated death domain (FADD) binds to the intracellular death domain of the receptors. FADD contains a death domain and can bind to DDs of the Fas receptor complex(Chinnaiyan 1995), this activates caspase-8 which activates downstream caspases such as casapase-3 leading to apoptosis(Kimberley & Screaton 2004). Additionally, caspase inhibitor VI (zVAD) and anti-Fas blocking antibody also used in the culture confirmed apoptosis was occurring in VSMCs(Ashton 2004). The blockage of apoptosis shows the process is regulated by caspases and the reduction in the apoptotic potential by anti-Fas blocking antibody implies trophoblasts can use Fas/FasL to induce apoptosis.
Uterine spiral artery remodeling also involves apoptosis of endothelial cells induced by extravillious trophoblasts through Fas/FasL interactions. The spiral arteries used were dissected from non-placental bed biopsies obtained at Caesarean section and were cultured with trophoblasts in the lumen. Apoptotic changes in the endothelial layer were detected after 20 hours as arteries with trophoblasts in the lumen. There is Fas expression on spiral artery endothelial and smooth muscle cells, the presence of trophoblasts led to a 1.91 fold increase in SGHEC-7 apoptosis, human umbilical vein EC derived cell line, and a 2.04 fold increase in decidual EC apoptosis(Ashton 2004). In vitro experiments showed apoptotic events of endothelial cells, which was blocked by FasL blocking antibody (Fig. 1), suggesting apoptosis is mediated via the FasL pathway in EC. Correspondingly, when EC and TC are co-cultured there is an apoptotic end marker which is cleaved PARP shown in figure 2 ref ashton. These results illustrate that multiple factors are produced by trophoblasts that interact with EC and VSMCs to induce cell death and may be one way in which they remodel spiral arteries.
Figure 1 Figure 2
In contrasts, some studies have illustrated Fas/FasL interactions is not vital in inducing apoptosis in mice pregnancy(Chaouat & Clark 2001), spiral artery remodeling may occur both in mammals and mice but some studies emphasize uterine natural killers cells play a greater role than other factors (Croy et al. 2003). Natural killer cells are present as clusters in spiral arteries undergoing spiral artery remodeling. Decidual natural killer cells(dNK) have shown to interact with VSMCs to induce apoptosis and incubation of dNK cells with EC lead to 23.5% increase in apoptosis after 50hours. The caspase inhibitor zVAD-fmk inhibited the dNK-induced VSMC apoptosis to basal levels(Fraser et al. 2012). The dNK cells may modulate VSMC phenotype since in a normal spiral artery the VSMC is contractile, differentiated and non-migratory whereas when remodeling occurs the VSMC is dedifferentiated, produce ECM components and have migratory behaviours(Bulmer et al. 2012).
Migratory behaviours
During early pregnancy trophoblasts migrate to remodel the maternal spiral arteries. Numerous methods have been utilized to study the migratory behaviours of trophoblasts, the effective method is microscopy with an attached image acquisition system that has overcome the usage of in vitro assays as it can capture behaviours of individual cells. In the study by Hamszic et al. 2008 time-lapse-phase-contrast microscopy was utilized to illustrate the trophoblasts are highly motile and change appearance and brightness when the TC induces apoptosis of VSMCs. The results suggest that when VSMCs were co-cultured, the TC has more motile behavior, travelled longer distances and were more directional. The TC specific movement towards VSMC to induce apoptosis was evident(Hamzic et al. 2008), as the percentage of apoptotic cells at 60h with VSMC +TC was significantly higher than VSMC alone. Therefore this could show that the spiral artery remodeling is based upon the chemotactic sensing between TC and VSMC where the VSMC are susceptible to pro-apoptotic stimuli. Besides the VSMC undergoing apoptosis following contact with TC, observations by Bulmer et al. 2012 suggests that more VSMCs in the spiral arteries had migrated away from the vessels; these were associated with endovascular EVT cells. As a result EVTS are required for the final stages of spiral artery remodeling including the loss of VSMCs however it is not clear whether the separated VSMCs undergo apoptosis or de-differentiation.
Another contributor to migration are elastin derived peptides(VGVAPG) produced when walls of the spiral arteries breakdown during early pregnancy. VGVAPG is made via MMP-9 and MMP-12, expressed by EVT, mediated cleavage of elastin(Taddese et al. 2009; Heinz et al. 2010). FasL can be cleaved by MMPs in order to regulate apoptosis of endothelial cells within the spiral artery(Ashton 2004). In this study invasion assays across Matrigel-coated Transwell inserts were carried out using first trimester CTB exposed to VGVAPG for 48 hrs. EVT migration from the trophoblast cell columns of placental villous explants was increased after incubation with VGVAPG. The collected data demonstrates that migratory behavior induced by VGVAPG is mediated via phosphorylation of eNOS and activation of the MAPK pathway. It has been proposed that the ECM breakdown amid spiral artery remodeling can have a positive feedback loop through generation of EDP to promote further migration of EVT elastase which influences EVT invasion(Desforges et al. 2015).
Vascular cell de-differentiation
The changes in ECM composition could influence the dedifferentiation of VSMC. Amid pregnancy trophoblasts can interfere with the EC and VSMC interactions in spiral arteries by de-differentiating the vascular cells in the process of remodeling(Whitley & Cartwright 2009).
Recently a major factor (cysteine cathepsin 8), belonging to the proteolytic breakdown machinery of lysozymes, was implicated in vascular de-differentiation using mice studies. Cts8 causes smooth muscle reduction at a protein level by proteolytic degradation leading to de-differentiation of VSM(Screen et al. 2008). This finding shows spiral arteries are lacking smooth muscle when in contact with Cts8 expressing trophoblast giant cells. This illustrates that perivascular smooth muscles are undergoing de-differentiation under the influence of Cts8 is essential in the mechanism of spiral artery remodeling ability of invasive trophoblast giant cells.
Figure 3
The differentiation status of VSMC was investigated by co-culturing EC and VSMCs in hanging droplets to form vascular spheroids. When the spheroid RNA was analyzed, there was a statistically significant up regulation of C-X-C motif chemokine 10 at 7.9 fold, P<0.05 (Wallace et al. 2013). Previous studies had also shown EVT secrete IFN-γ ref 29 in article, the study had proven that IFN- γ is involved in the induction of CXCL10 in spiral arteries and that EVT conditioned medium and CXCL contribute toward VSMC dedifferentiation and motility. Both the EVT conditioned medium and C-X-C motif chemokine 10increased motility of VSMC conveying dedifferentiation of VSMC could have occurred.
Another study provided evidence that most media of smooth muscle cells of uteroplacental arteries of the guinea pig is not destroyed during trophoblast invasion but will dedifferentiate during arterial dilatation induced by pregnancy hence giving rise to mesenchyme like myoblasts(Nanaev et al. 2000). The purpose of the de-differentiation of VSMCs is to initiate mural disruption before permitting the colonization by EVTs for further remodeling. This is illustrated by the emergence of a population of roundish to polygonal dedifferentiated smooth muscle cells emerges which are immune negative for cytokeratin, desmin and smooth muscle myosin(Nanaev et al. 1995). There is also histochemical and ultrastructural data that suggests the increasing quantity of differentiated smooth muscle cells post partum is due to re-differentiation of previously de-differentiated myoblasts during pregnancy induced arterial dilatation, since there is no evidence for the proliferation of myoblastic stem cells giving rise to the differentiating smooth muscle means the de-differentiated cells could be the potential source. Despite the data being collected from pregnant guinea pigs, studies have demonstrated alpha-smooth muscle actin immunoreactivitities is in walls of degenerated uteroplacental arteries suggesting de-differentiation could also occur in humans (Craven and Ward 1996, Craven and Ward 1998). Further research is needed due to differences among the species because the guinea pig immobilization of uteroplacental arteries is not confined to the uterine wall as in the human (Verkeste et al. 1998).
Extracellular matrix restructuring
The extracellular matrix (ECM) provides structural support for the spiral arterial wall. The some ECM components found in the spiral artery walls are collagen and elastic fibers synthesized by endothelial and smooth muscle cells. A non-remodeled spiral artery is made up of three layers with the cells embedded in the matrix. Firstly, the intima is predominantly a single layer of endothelial cells present on top of the basement membrane made of collagen type-IV and laminins(Whitley & Cartwright 2010). Secondly, the elastic lamina forms a tight woven layer constituting of elastin and collagen type-IV fibres with fibronectin that separates the endothelial cells and the vascular smooth muscle cell to permit support and recoil of the vessel wall. There is found to be a second prominent layer of elastic fires surrounding the medial vascular smooth muscle cells (VSMC) forming the external elastic lamina. Thirdly, the adventitial layer will surround the second prominent layer with surrounding VSMCs(McGrath et al. 2005; Arribas et al. 2006 ).
Modifications in the spiral artery can be regulated before extravillious trophoblasts invade. These changes are followed by interstitial trophoblast invasion, which is thought to prepare the cells of the vessel wall for subsequent endovascular invasion(Pijnenborg et al. 1983, cited in Whitley et al. 2010). Upon trophoblast arrival there is transformation of myometrial segment of the spiral artery, which includes dilation of the lumen and alterations to the elastic lamina(De Wolf et al. 1980, cited in Whitley et al. 2010). Continued ECM restructuring is achieved by further invasion of endovascular trophoblasts. The attachment of EVTs to the internal elastic lamina of vessels was present at sites of disrupted endothelial connections illustrated using semi-thin sections of late first trimester placental bed specimens, which were viewed using electron microscopy(Crocker et al. 2005). At this stage the EVTs may endorse degradation of elastin.
Elastin an important constituent of arterial extracellular matrix and the mechanism of elastin catabolism is vital in remodeling of the spiral artery. Researchers have proposed that degradation of elastin fibres in the arterial wall is the rate determining step in the remodeling pathway(Harris 2011). The reason being is that permanent vasodilation cannot be present unless elastin of a vessel has been removed.
The evidence suggests EVTs migrate through the breaks. When the EVTs are cultured with Congo red-labeled elastin, the trophoblasts can be observed to engulf and degrade elastin fibres, as the EVTs engulf the elastin at the beginning and in the final stages the elastin becomes completely degraded(Harris et al. 2010). Also the distal regions of the spiral artery are abundant in elastin and are the location where impaired remodeling was observed(Pijnenborg et al. 1999).
EVTs and VSMCs express elastolytic proteases: MMP-2, MMP-7, MMP-9 and MMP-12(Laszio et al. 1990; Staun-Ram et al. 2004; Smith et al. 2009). MMP2 stimulates cleavage of collagen which releases endostatin to stimulate apoptosis of endostatin(Dhanabal et al. 1999).The protealytic enzymes are released to remodel the matrix and degrade all components of the ECM. The activity of MMP-2 can stimulate release of transforming growth factor-beta bound to the ECM(Mott & Werb 2004) and regulates trophoblast invasion(Tse et al. 2002) however VEGF released after actions of MMP-9 could have apoptotic influences on trophoblasts, so these two factors could be essential in modulating the process of remodeling. The interaction of EC and VSMC with the ECM and the production of essential intracellular modulators determines the existence of the vascular cells. As a consequence of the ECM breakdown there is loss of matrix derived factors which may result in apoptosis or VSMC migration.
The stability in the quantity of MMPs and TIMPs, TIMPs inhibit the action of MMPs, is highly likely to play a significant role in remodeling of spiral arteries as there were elevated expression of MMP-2, MT1MMP, MMP-3 and TIMP1 (P=0.01) in spiral arteries of early pregnant rats(Kelly et al. 2003). Recent evidence has verified that significantly greater quantities of MMP-2 and TIMP-1 in preeclampsia(Montagnana et al. 2009). The imbalance between the MMPs and TIMPs contributes greatly to the abnormal vascular changes of women with complicated pregnancies. It has been previously emphasized that the high MMP activity can induce endothelial dysfunction which is the key to the pathophysiology of preeclampsia(Karthikeyan et al. 2012).
Pre-eclampsia- defined by hypertension and proteinuria which affects 3-7% of pregnancies and is a major contributor to maternal fetal mortality.
To produce a blood supply that increases till term, the invading trophoblasts will cause modifications to the maternal arteries, in normal pregnancy these progresses from the ends of spiral arteries to the myometrium. However, in pre-eclampsia the modification is confined to the decidual terminations of the spiral artery and does not reach the myometrium. The reduced invasion of the fetal EVT cells and restricted remodelling of the spiral arteries is due to failure of maternal immune tolerance to antigens expressed by paternally derived EVT(Warning et al. 2011). This eventually results in dysfunctional uteroplacental circulation. Furthermore, decidua-associated changes to the inner myometrium could be deficient in those who develop pre-eclampsia amid pregnancy, these changes and trophoblast associated vascular remodelling mentioned afore are vital for subsequent trophoblast invasion and further remodelling of the spiral arteries(Pijnenborg et al. 2006). The initiation of pre-eclampsia can lead to placental ischaemia hence leading to dysfunctional maternal vascular endothelium causing hypertension by impairing the renal pressure and increasing total peripheral resistance.
Kadyrov et al. 2003(Kadyrov et al. 2003) found that the reduced interstitial trophoblasts in pre-eclampsia was not associated with high rate of apoptosis but there was a lower rate of apoptosis. Since uterine NK cells have been shown to directly influence vascular remodelling and the association between vascular smooth muscle destabilization, a uterine NK cell deficiency could also contribute to impaired remodelling of spiral arteries leading to poor fetal growth while in the mother’s womb at pregnancy, which is referred to as intrauterine growth restriction.
In conclusion, there are numerous mechanisms involved in spiral artery remodeling. Some mechanisms have been described independently of each other and some have greater contributions to the process. Several mechanisms are interlinked; apoptosis of VSMCs is affected by factors such as CXCL-10, which affects dedifferentiation and affects the apoptotic potential. Apoptosis of VSMCs can also be initiated upon changes to or breakdown of ECM due to the absence of matrix-derived factors. Ultimately, invading trophoblasts in the initial stages of spiral artery remodeling influences most of the events in spiral remodeling and any abnormalities are associated with common pregnancy complications such as pre-eclampsia and intra-uterine growth restriction.