Placentation and the role of the trophoblast

Question 1

What is the placenta?

Answer 1

• An organ unique to pregnancy. Only present during pregnancy
• Forms the interface between the mother and the fetus. The placenta is foetal tissue that interacts with the wall of the uterus (maternal/foetal interface).
• The placenta is fetal in origin. At term, it weighs 500-1000g
• Acts as the lungs, gut and kidneys of the fetus
• Acts as an endocrine organ releasing hormones into the maternal circulation, such as hCG and progesterone. It acts as an important endocrine organ, releasing lots of growth factors and cytokines.
• The placenta is a semi-allograft, the cells have genetic material from both the mother and the father. Semi-allograft = the cells have genetic material from both the mother and the father.
• In a human pregnancy, fetal cells are in direct contact with maternal blood
• This requires mechanisms to evade the maternal immune system
• However the fetal and maternal circulations do not mix. Although foetal cells and maternal cells are in direct contact, maternal and foetal circulations themselves are separate.

Question 2

How does the placenta develop (implantation)?

Answer 2

• Implantation is the multistep process by which the free-floating blastocyst attaches to the endometrium, invades through the epithelium and into the stroma beneath and begins to establish the placenta.
• The blastocyst begins by attaching and adhering to the epithelial layer of the uterine wall.
• The trophoectoderm cells divide and migrate between the epithelial cells, through the basement membrane and into the underlying uterine wall.
• Trophoectoderm proliferates and these cells fuse to form a primitive syncytium (PS) beneath the implanted embryo =
  syncytiotrophoblast.
• These will continue to migrate or invade into the decidium. They will form an increasing primitive syncytium. Within that syncytium, they will form holes (known as lacunae).
• Lacunae (L) form by the action of proteases which later develop into the intervillous space. These lacunae will ultimately develop into the intervillous space. Behind the primitive syncytium, cells will divide. These are cytotrophoblasts; they start to divide and migrate through the primitive syncytium and into the uterine wall. These form the anchoring villi.

Question 3

Describe the structure of the placenta.

Answer 3

• Highly branched with a large surface area for exchange
• Outer layer of fused cells- the syncytium
• Underlying cytotrophoblast stem cells
• Diffusion distance to vessels small
• Growth is regulated by a number of factors including IGF I and II
• Can see the decidua basalis right underneath the placenta.
• The anchoring villi can be seen starting to branch as the placenta develops. The branching allows them to form secondary and tertiary villi. Within these villi are the blood vessels that supply the placenta from the foetus and they are responsible for exchanging nutrients and respiratory gases that come from the mother and are delivered back from the foetus.
• Also important to note the presence of the spiral arteries (vessels) that deliver maternal blood to the intervillous space. There are also veins that help to drain blood from the intervillous space back into the maternal circulation.
• Highly branched; increases the surface area available for exchange of nutrients and respiratory gases.
• The outer surface of the villi is formed from a fused syncytium. Underlying these are the cytotrophoblast stem cells.

Question 4

Describe the villous structure.

Answer 4

This is a floating villi; floats freely in the intervillous space.

• Within each of these villi, there is a highly branched vascular network of arteries and veins that take blood to and from the developing foetus.
• A cut section of the villi shows a fused layer of cells on the outside forming the syncytium (the cytotrophoblast).
• Directly underneath are the cytotrophoblast stem cells.
• Important for the delivery and exchange of nutrients is the fact that the vessels are very close to the surface of the line. This enables effective and rapid exchange of material.
• Also within the villi are macrophages, known as Hofbauer cells. The function of these cells is not entirely known, but it has been suggested that they may be involved in immune protection of the placenta with other potential roles, such as regulating formation and branching of these particular vessels.

Question 5

What are the two subtypes derived from cytotrophoblast cell columns and shell in the extravillous pathway?

Answer 5

• As well as the villous pathway (villous cytotrophoblast, syncytiotrophoblast, transport, protective and endocrine functions), there is the extravillous pathway.
• These cells are derived from the cytotrophoblast cell columns and cytotrophoblastic shell.
• They form two different subtypes = the endovascular extravillous trophoblasts and the interstitial extravillous trophoblasts. These two cell types work together to ultimately remodel the maternal spiral arteries.
• In the initial stages of pregnancy, the endovascular extravillous trophoblasts form what is called this trophoblast plug. This prevents maternal blood from entering into the intervillous space. The interstitial extravillous trophoblasts invade into the decidua and migrate towards maternal spiral arteries.
  1) Endovascular trophoblast = spiral artery plugs and remodelling
  2) Interstitial trophoblast = spiral artery remodelling

Question 6

How do the extravillous cytotrophoblasts invade?

Answer 6

• Growth of the trophoblast column
• Regulated by factors such as IGF1 produced by the underlying mesenchymal cells
• As the cells from the column move into the decidua, they undergo epithelial-mesenchymal cell transition. During this process, cells lose whatever polarity they may have, they lose adherence and they become more motile, more invasive in nature. As this progresses, they also start to express different cell surface markers. Initially, they will start expressing α6β4, αVβ6 and E cadherin. As the cells migrate away from the cell column, they start to express different molecules in our cells surface, including αVβ3, α1β1 and VE-cadherin.

Question 7

What is the purpose of this trophoblast plug?

Answer 7

• Up until 12th week the uterine spiral arteries are plugged with trophoblasts
• Placental development therefore occurs under relative hypoxia 2-3% O2
• 8-10 weeks gestation = 2-3%, 12-13 weeks gestation = 7-8%.
• While the spiral arteries are plugged nutrition is histiotrophic nutrients being secreted by the glandular cells
• Following dissolution of the trophoblast plug, the placenta switches to haemotrophophic nutrition
• The main purpose is that it reduces the amount of oxygen that the villous tissue is exposed to in early gestation. Up until about the 12th week of gestation, the concentration of oxygen within the intervillous space is relatively hypoxic (2 to 3% oxygen). While the spiral arteries are plugged, the nutrition that is provided to the growing foetus is through a process of histiotrophic nutrition. This is provided by the secretions of glandular cells. However, following the dissolution of the plug, the placenta switches to a normal haemotrophic nutrition and this occurs around the 12th week. During this period, the oxygen concentration varies significantly. It is believed to be because during early gestation, the developing foetus is very sensitive to the oxygen concentration and this has to therefore be reduced.
• Low oxygen early in pregnancy is important normal pregnancy progression. Prolonged low oxygen leads to placental pathologies!

Question 8

What are the in vivo methods used to study human placental development, as there are clearly ethical issues as to why we can’t experiment on human pregnancies?

Answer 8

• Human studies are limited for ethical reasons
• To overcome this, experimenters have looked at animal models. However, there are significant differences in the way the placenta develops between mammals. Even the most commonly used laboratory animal, the mouse, is not always a suitable model for the events that take place at the foetal-maternal interface of a human pregnancy. There are, however, some similarities as highlighted. Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood. However, the extent of interstitial invasion of the decidua is significantly less in mice than in a human pregnancy. A particular drawback when looking at obstetric complications is that the mouse does not seem to exhibit the same ones as humans.
• The best model for human pregnancy is that of the great apes. They also express trophoblast invasion of the decidua and maternal arterial wall (interaction with the maternal spiral arteries). As in the human, the trophoblasts come into direct contact with maternal blood. Deep interstitial invasion of the decidua does occur. Some evidence that they do exhibit the same obstetric complications as humans. However, there are extreme ethical issues relating to the use of these animals (ethically unacceptable to experiment on these animals).

Question 9

If animal models are not appropriate, what are the in vitro methods used to study human placental development?

Answer 9

1) The major alternative is to use human tissue. Human tissue can only be obtained either in the first trimester from TOPs or at term. This can be obtained at two points in pregnancy = either after termination of pregnancy in the first trimester or at term upon delivery. Tissue can either be used whole or cells can be isolated from them. There are also a number of cell lines available which have proved useful, but there are some drawbacks to these which will be discussed.

2) Trophoblast cell lines derived from choriocarcinomas JEG3, Jar and BeWo
- grow well
- have lost some characteristics
- The first trophoblast cell lines to be used are those derived from choriocarcinomas, such as JEG3 cells, Jar and BeWo. These cells grow well in culture and have some of the characteristics of their original cell type. However, they have lost and do lose some of their characteristics. Some of this is due to the fact that these cells proliferate readily in culture.

3) Developed following transfection with oncogenes such as t- and T-antigen of SV40 or more recently hTERT
- grow well
- have lost some characteristics but this can depend on how they are cultured
- Alternatives have been used. Cell lines can be developed using transfection of viral oncogenes (carried out at St. George’s), such as the little and large T-antigen of SV40 or, more recently, the hTERT (the telomerase activity). These cells grow well but, like most cells in culture, they have lost some of their characteristics. However. this can be manipulated by growing them in the correct environment.

4) Human embryonic stem cell-derived trophoblast cells (hESCs)
- characterisation has proved problematic
- An approach that showed initial promise is the use of human embryonic stem cell-derived trophoblast cells. However, this early promise has proved problematic, and it is an approach that can’t be used in a number of regions due to legal considerations.

5) Human trophoblast stem cells (hTSCs) derived from the trophectoderm and first trimester placentae
- express characteristics of first trimester trophoblasts
- can be induced to differentiate along either syncytial or extravillous lineages
- difficult to prepare and grow
- The most recent development and one that shows initial promise is the use of trophoblast stem cells. These are derived from trophectoderm and first trimester placentae. Again, in some regions, this is not a viable alternative. They can be difficult to both prepare and grow but said to grow well once established.

• In all of these models, the phenotype of these cells is very much dependent on the culture conditions used. In the images shown, a trophoblast cell line ahs been developed that was first grown as a monolayer and then in a 3D culture system. The middle image shows a Western blot for MMP-2 and 9. A significant difference between the expression can be seen between the monolayer culture on the left and the 3D culture on the right. This illustrates a common problem in trophoblast research; studies performed on cells cultured in different ways.

Question 10

What are the different culture formats?

Answer 10

1) Simple mono layer cultures
2) Simple co-cultures = Co-cultural models contain various forms; either just mixing two cell types together and growing them in a monolayer or separating them using multi-cell inserts (the cells in the insert are grown on a mesh essentially, so factors produced by each cell type can freely diffuse between the two and responses of these cells in isolation can be monitored)
3) Addition of extracellular matrix = A more complex process can be used where cells are grown in the presence and absence of different extracellular matrices. The multi-cell insert approach can again be used, so communication can take place between cells (in a more direct fashion than in the previous model). It depends on what matrix is used, e.g. collagen, fibronectin or Matrigel (a mixture of many different extracellular matrices, including molecules like delaminate).
4) Effect of flow = This is something that has been done in relation to some of the studies looking at trophoblast remodelling.
5) 3D environment = There are many different sorts of 3D models, including a spheroid model or a perfusion model of dissected arteries (as shown in diagram).
6) Organoid cultures = A more recent and very promising approach is to use an organoid cultured approach. Cells are isolated but then the tissue is reconstructed.

Question 11

What are the different culture formats?

Answer 11

1) Simple mono layer cultures
2) Simple co-cultures = Co-cultural models contain various forms; either just mixing two cell types together and growing them in a monolayer or separating them using multi-cell inserts (the cells in the insert are grown on a mesh essentially, so factors produced by each cell type can freely diffuse between the two and responses of these cells in isolation can be monitored)
3) Addition of extracellular matrix = A more complex process can be used where cells are grown in the presence and absence of different extracellular matrices. The multi-cell insert approach can again be used, so communication can take place between cells (in a more direct fashion than in the previous model). It depends on what matrix is used, e.g. collagen, fibronectin or Matrigel (a mixture of many different extracellular matrices, including molecules like delaminate).
4) Effect of flow = This is something that has been done in relation to some of the studies looking at trophoblast remodelling.
5) 3D environment = There are many different sorts of 3D models, including a spheroid model or a perfusion model of dissected arteries (as shown in diagram).
6) Organoid cultures = A more recent and very promising approach is to use an organoid cultured approach. Cells are isolated but then the tissue is reconstructed.

Question 12

What 2 models have been used to look at factors that regulate trophoblast invasion?

Answer 12

• These are two very different but complementary models.

1) The first is an ex vivo model using human placental tissue. This is a piece of first trimester placental tissue and an anchoring villus. These can be identified and dissected, then place don an extracellular matrix. The migration of cells from this villus tissue can be monitored by time-lapse microscopy or just normal photography. These processes can be manipulated using both pharmacological and molecular techniques.
2) An alternative approach that can be used is an in vitro model using these inserts previously seen. On top of the extracellular matrix, there are seeded cells (extravillous trophoblast in this case). A stimulus can then be put into the lower chamber. If this stimulates the trophoblast, it may cause them to migrate through the matrix.
Using a combination of these approaches and many others, a number of factors that regulate trophoblast invasion have been identified. Some are listed on the slide. There are many factors that regulate trophoblast invasion, including hCG, EGF, HGF, IGF-1 and 2. This diagram also identifies some of the pathways that can be activated. There are a number of pathways that can be activated in a trophoblast that stimulates them to either migrate or invade. However, not all factors will.

Question 13

What factors influence trophoblast invasion?

Answer 13

• Many of the mechanisms that regulate invasion by trophoblast and cancer cells are common. An important difference between the invasion seen in a normal pregnancy and that seen in metastasis is that it is tightly regulated in pregnancy (there are both positive and negative regulators of trophoblast invasion.
• The positive regulators of trophoblast invasion include growth factors, cytokines and matric metalloproteases that digest the extracellular matrix. These are held in common with invasive cancer cells. However, the environment that trophoblasts find themselves in the decidua is also one where factors that may inhibit/regulate this process are found, e.g. tissue inhibitors of matrix metalloproteases, TIMPs, and inhibitory cytokines and growth factors.
• In order for a successful pregnancy to be established and maintained, there has to be a balance between the important stimulatory and inhibitory factors. If this balance is shifted, it can give rise to significant pregnancy complications.
  1) Growth factors and cytokine
• HGF
• IGF-1
• Prolactin
  2) Matrix proteases
• MMP-2, 9, 10, 12
  3) Tissue inhibitors matrix
  metalloproteinases
  4) Inhibitory factors
• TNF
• TGFβ
• IGFBP-1

Question 14

What role do maternal immune cells have in regulating trophoblast invasion?

Answer 14

• A major source of factors that regulate trophoblast invasion are the maternal immune cells
• The immune cells secrete a number of factors which have two roles; first, to prepare the spiral artery for the invading trophoblast and second, to release chemokines which will attract the invading trophoblast. The effects of their secretions on trophoblast migration and motility can be demonstrated using the methods that have been discussed. The image demonstrates the effects of these secretions on the migration of extravillous trophoblasts from the anchoring villous (from a piece of first trimester chorionic villi)
• The presence of immune cells can be demonstrated using immunohistochemistry. The image shows a spiral artery with surrounding uNK cells (little, brown dots).
• Immune cells in decidua basalis =
  1) 70% uterine natural killer cells (uNK cells)
  2) 20% are macrophages
• They are recruited following implantation
• They localise to maternal spiral arteries
• They precede the invasion trophoblasts
• They secrete factors that regulate trophoblast invasion?

Question 15

Why is development of the placenta important?

Answer 15

• Failure of the placental to develop normally can lead to common pregnancy disorders such as early pregnancy loss, pre-eclampsia and fetal growth restriction.
• Failure to thrive in utero has lifelong consequences including increased risk of developing hypotension and diabetes
• The extravillous trophoblasts have a role in the remodelling of the maternal spiral arteries. This is important because failure of the placenta to develop normally can lead to common pregnancy disorders. Long-term, a baby that is small for gestational age tends to be more prone to cardiovascular disease in later life and more, e.g. diabetes. Can be an important issue for long-term adult health.

Question 16

Summarise the function of extravillous trophoblasts.

Answer 16

• early in pregnancy cells of the placenta (trophoblasts) invade the decidua. In early pregnancy, extravillous trophoblasts will migrate from the anchoring villi into the decidua.
• they form plugs that block the maternal spiral arteries
• These two functions lead to two different cellular phenotypes, the interstitial extravillous trophoblasts and the endovascular extravillous trophoblasts. The invading interstitial trophoblasts migrate towards the spiral arteries and interact primarily with the vascular smooth muscle cells that line these vessels. They ultimately start to replace them. The endovascular trophoblasts, which will plug initially the spiral artery, will then migrate down the lumen of these vessels. They interact primarily with the endothelium cells and ultimately lead to their loss. The physiological reason for this interaction is that the spiral arteries under normal circumstances will respond to vasoconstrictors and dilators. These arteries will, under those circumstances, be under the control of maternal needs. By removing the ability of these vessels to contract, the foetus removes the ability of the mother to regulate the flow of blood into the intervillous space.
• they interact with and replace the cells of the vessel wall
• ultimately this will result in increased blood flow to the developing baby.

Question 17

What is the purpose of spiral artery remodelling?

Answer 17

• In a non-pregnant state, spiral arteries have a diameter of about 200 microns. Like most arterioles, they consist of an endothelial cell lumen surrounded by both connective tissue, whether that be elastin and/or collagen, and smooth muscle cells. Cross-section of a human maternal spiral artery.
• Following the remodelling process (interaction with trophoblast and stimulation by pregnancy-related hormones, the diameter of this vessel increases significantly. This results in a change of system from a low flow, high resistance vessel to a high flow, low resistance vessel in the remodelled state. A remodelled spiral artery can be seen (stained for cytokeratin-7, a marker of extravillous trophoblasts). This particular spiral artery has a large number of trophoblasts within the lumen and some trophoblasts can be seen surrounding the vessel.

Question 18

What are the two phases of spiral artery remodelling?

Answer 18

1) Trophoblast independent
- Immune cell
- Pregnancy hormones
- The trophoblast independent phase involves the maternal immune cells, specifically the uNK cells. But it is also influenced by pregnancy-related hormones, e.g. progesterone. Studies looking at ectopic pregnancies where the blastocyst implants in a place other than the uterine wall have shown a certain amount of spiral artery remodelling, showing there is a trophoblast independent phase.

2) Trophoblast dependent
- We know this by studying ectopic pregnancies

Question 19

How are vascular cells lost from spiral arteries in spiral artery remodelling?

Answer 19

• Mechanisms turn the contractile phenotype of smooth muscle cells to a highly modified vessel. There is a loss of smooth muscle cells and endothelial cells, which are replaced by foetal cells invading extravillous trophoblasts.
• Some influences include dNK cells, EVT, decidual macrophages may also play a role and also the stromal cells within the decidua. Now focusing on the extravillous trophoblasts.
• The extravillous trophoblasts will ultimately replace both the endothelial and the smooth muscle cells within the wall of the spiral artery.
• There are a number of mechanisms that have been implicated. One is that the cells could just induce vascular cells to migrate away from the vessel. The trophoblasts can try to induce de-differentiation of both the smooth muscle and the endothelial cells. There could be loss of adhesion and this might lead to a process within endothelial cells, called anoikis. This is a form of programmed cell death. There could also be vascular cell apoptosis, i.e.
  1) Migration
  2) De-differentiation
  3) Loss of adhesion (Anoikis)
  4) Vascular cell apoptosis

Question 20

The hypothesis is that extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling. What are four potential mechanisms that may be involved?

Answer 20

1) Vascular cell apoptosis
2) Migration
3) Dedifferentiation
4) Loss of adhesion

Question 21

What is apoptosis?

Answer 21

• Programmed cell death
• Cell death without the inflammatory response
• Characterised by distinct morphological changes
  1) Cell shrinkage
  2) Chromatin condensation
  3) DNA fragmentation
  4) Membrane blebs and blisters
• Characterised by distinct biochemical changes
  1) Cleavage of lamins and actin filaments in the cytoskeleton
  2) The breakdown of chromatin in the nucleus leading to nuclear condensation
  3) Translocation of phosphatidylserine to outer membrane
  4) Cleavage of key enzymes such as poly ADP ribose phosphate (PARP) involved in DNA repair
• Induced by cellular stress such as nutrient deprivation, hypoxia and viral infection
• Mediated by a family of enzymes called caspases. Although caspase independent apoptosis does occur
• Apoptosis is a phenomena which occurs between the digits of the finger during development. It is also the process by which tadpoles lose their tail during their transition into a frog/toad.
• This is a complex process that requires energy. It is also a process that has kinetics to it. The graph represents cardiomyocytes undergoing apoptosis. Can see that some of the markers of apoptosis occur at different times. One of the key markers, DNA fragmentation, is a relatively late phenomenon, whereas something like caspase activation can be seen within the first few hours. It is an important process that has quite well-defined kinetics.
• One of the other things that happens is that the cells redistribute the phospholipids within their plasma membrane. In particular, phosphatidyl serine (predominantly an intracellular phospholipid that is present on the inner leaflet of the plasma membrane, is moved to the outside of the cell. This is a characteristic marker of apoptosis in a number of cells.
• Can follow apoptosis in vitro using time-lapse microscopy. Image shows a smooth muscle cell undergoing apoptosis. Relatively prolonged process. The normal smooth muscle cell in culture undergoes characteristic blebbing and blistering. Experimentally, apoptosis is a complex process and usually requires examining a number of different biochemical markers in order to absolutely confirm that the processes that can be seen down the microscope are supported by the biological/biochemical reactions that are important.

Question 22

What is the nature of the factors produced by trophoblasts for apoptosis?

What is a specific example?

Answer 22

• Trophoblast use Fas/FasL to induce apoptosis in Endothelial Cells and Vascular smooth muscle cells
• There are a family of cytokines that induce apoptosis. This consists of members of the TNF-alpha family. This experiment aims to identify whether a particular member of that family is involved in trophoblast-induced vascular cell apoptosis. Looks at a molecules Fas ligand, which binds to Fas on either the endothelial or vascular smooth muscle cells. This experiment is very similar to the one showed previously, but it is carried out in the presence of a neutralizing antibody, called NOK2. NOK2 binds to FasL and prevents it from activating Fas. In the presence of this inhibitory antibody, there is a significant reduction in the ability of trophoblasts to induce both endothelial cell and, to a lesser extent, vascular smooth muscle cell apoptosis.

Question 23

What is an example of an in vivo model of human placentation?

Answer 23

• Red-Horse et al. 2006. Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation.
• The experiment shown is an in vivo model of human placentation. They took placental villi and transplanted them into the mammary fat pads of Scid mice (strain of mouse) for three weeks. These mice have a defective immune system, so they did not reject the human placental tissue (as a normal mouse would). It allows the placental villi to interact with the vessels of the mouse fat pad.
• CK+ve TC invaded and interacted with vessels.
• Induction of vascular cell apoptosis in vivo and in vitro co-cultures
• The series of panels illustrate the initial localization of both trophoblasts (CK7 in green) and endothelial cells (marker of endothelial cells = CD31). The vessels can be seen in red with the invading extravillous trophoblasts and some interaction can be seen. A tunnel assay can be used to detect apoptosis; one of the features of apoptosis is the cleavage of DNA and the ends of these fragments can be detected using this particular reagent. This is also shown in green in these experiments. F shows a stain for smooth muscle actin, a marker of smooth muscle cells. G shows CD31 again. Can see some co-incidence staining of both vascular cells and tunnel staining, suggesting that the data found in vitro can be replicated in an in vitro model of vascular remodelling.

Question 24

The hypothesis is that extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling. What mechanisms do data suggest are involved?

Answer 24

• The data presented has suggested that vascular cell apoptosis induced by invading extravillous trophoblasts may play a role in the remodelling process.
• There is also data that suggests vascular cell de-differentiation may also be involved in this process.

Question 25

What are the characteristics of a dedifferentiated vascular smooth muscle cell?

Answer 25

• Unlike many cells, vascular smooth muscle cells have a plastic phenotype and they can move from a contractile phenotype (seen in a normal functioning vessel) to a de-differentiated, non-contractile phenotype (primarily seen in pathological conditions). This switch from a differentiated to a de-differentiated phenotype and vice versa is stimulated by a number of factors. The two predominant factors involved in this process are TGF-beta and platelet derived growth factor. They can push the cells in the directions illustrated.
  TGF-beta will push a cell towards the differentiated phenotype, which are largely non-migratory, have a low proliferative rate and are contractile in nature (express a lot of contractile proteins). The platelet-derived growth factor has the opposite effect; results in de-differentiation. A dedifferentiated vascular smooth muscle cell is more migratory, has increased proliferated potential and it secretes extracellular matrix components. These changes in phenotype are characterised by loss of contractile proteins, such as actin, calponin and caldesmon. Is there any evidence for such a dedifferentiation in vascular smooth muscle cells in a remodelling spiral artery?
  The non-remodelled spiral artery on the left shows a tight ring of vascular smooth muscle cells in these vessels. In a spiral artery that is undergoing remodelling (right), although the smooth muscle cells are still present, they are much more loosely packed with gaps in the circumference of these vessels. There is even some presence of less well-stained cells that appear to have moved away from the vessel wall. This would suggest that in vivo there is some evidence for vascular smooth muscle cell dedifferentiation. Is there any evidence to support this from in vitro studies? Using trophoblast condition media (media that has been incubated with trophoblasts and contains all the factors that are secreted by the trophoblasts) to stimulate smooth muscle cells, a dose-dependent drop in the expression of smooth muscle actin can be seen, but also calponin, suggesting that there is dedifferentiation taking place. One of the markers of dedifferentiation is the fact that they are more motile. Looking at the motility of vascular smooth muscle cells in response to trophoblast condition media, a dose-dependent rise in cell motility can be seen. Again, this suggests that such a hypothesis is in fact a valid one.

Question 26

What is one approach that has been used to investigate trophoblast-dependent remodelling?

Answer 26

• To examine the effects of endovascular trophoblast on these vascular spheroids, we first simply conducted a gene expression study. Conditioned media from an extravillous trophoblast cell line, SGHPL-4 (also grown in 3-D) was added to our vascular spheroids. Changes in gene expression were examined by Illumina bead-chip microarray, and genes which were significantly altered by the trophoblast conditioned media were examined.
• Trophoblast conditioned media has been generated using a 3D culture of trophoblasts. Growing cells in 3D stimulates the expression of a much more physiological phenotype. This trophoblast condition media was then used to stimulate a 3D culture model where both endothelial cells, seen in green, and smooth muscle cells, seen in orange, are co-cultured together in what is called a hanging drop system. This allows the cells to form without adhering to a plate. When they are grown like this, the endothelial cells migrate to the outside of the small ball of cells that form. This forms what would appear to be an inside out lumen. If these spheroids are then stimulated with trophoblast conditioned media and a gene array is performed, the genes that are up or down regulated in response to stimulation by trophoblast can be identified. This was done and a number of genes were identified.
  1) Trophoblast conditioned media generated
  2) Control or conditioned media added to vascular spheroids for 24 hours
  3) RNA extracted from vascular spheroids
  4) Gene expression altered by trophoblast examined by Illumina Bead-chip array
• There are a group of molecules that are increased in this spheroid model in response to trophoblasts conditioned media, which are involved in vascular smooth muscle cell dedifferentiation. These include PDGF, KLF4 (a transcription factor which is expressed only in VSMC that are undergoing dedifferentiation) and there are also other cytokines., e.g. CXCL10. The other cytokines have been implicated in other studies in VSMC dedifferentiation. Other genes that are overexpressed or increased in expression in this model include MMP10 (a matrix metalloprotease that digests elastin, significantly affecting the structure of the vessel). One of its substrates is elastin, which is known to undergo degradation to form elastin-derived peptides. These have been shown to stimulate trophoblast invasion. It is possible that trophoblasts will stimulate the vascular smooth muscle and endothelial cells to produce MMP10. Helps to break down some of the matrix that forms the vessel wall and these, in addition to reducing the structure of the vessel, may also have a significant effect on recruiting more trophoblasts to the remodelling vessel (Matrix degradation and possibly elastin derived peptides which stimulate trophoblast invasion). Other factors that are synthesised in response to trophoblast conditioned media is the cytokine IL-8. It is again known to act as a chemokine to trophoblasts. It stimulates trophoblast invasion. Two more important chemokine molecules are IL-11 (Regulates VSMC phenotype) and CCL20 (chemokine).
• Looking at all of the genes that are altered in response to trophoblasts conditioned media, a number of them fall into categories which may be involved in vascular remodelling (certainly have been involved in vascular remodelling in other systems). These broad categories are likely to be important in the remodelling process seen in early pregnancy.
• Gene ontologies = Blood vessel morphogenesis, Inflammatory response, Angiogenesis, Blood vessel development, Vasculature development, Response to stress.

Question 27

Summary of a working model of how spiral arteries are remodelled by trophoblasts.

Answer 27

• In conclusion, a working model can be summarised of how it is thought spiral arteries are remodelled by trophoblasts.
• Initially, there will be communication between the vascular wall and the trophoblasts. It is likely that the trophoblasts will stimulate vascular smooth muscle cells to release factors which will begin to recruit more trophoblasts.
• Trophoblasts can induce vascular cell apoptosis, both smooth muscle cell and endothelial cells.
• At least Fas/FasL interactions are important for this.
• Endovascular trophoblasts will interact with the vascular endothelial cells primarily. The interstitial trophoblasts will produce factors that interact with the vascular smooth muscle cells.
• Trophoblast conditioned media stimulates the release of a number of factors, including PDGF which is important for regulating VSMC differentiated state (pushing it into a more de-differentiated, motile state).
• Trophoblast conditioned media also stimulates the release of matrix metalloproteases. These possibly have two functions; one is to break down the external matrix in the vessel wall and the second is to synthesise/generate these elastin-derived peptides which have been shown to be chemoattractant for trophoblasts.
• The second part of the lecture will introduce possible clinical implications of trophoblast invasion.