Exploring the role of Integrin-linked kinase in placental development.
Introduction:
The success of pregnancy and the health of the newborn rely on the proper development of the placenta. One of the most critical phases of its formation is the differentiation of cytotrophoblasts into invasive extravillous trophoblasts (EVT). Trophoblasts are cells derived from the trophectoderm of the embryo that either fuse and form syncytiotrophoblast or proliferate and differentiate into invasive cells. The invasive cells that reach the decidua are known as interstitial trophoblasts and those that replace the endothelium of the maternal arteries are denominated endovascular trophoblasts. It has been well reported that if invasion is not adequate the patient can develop preeclampsia and the baby can suffer intrauterine growth restriction.
The ability of trophoblast cells to adhere to and sense the surrounding extracellular environment and to produce intracellular signals is crucial to their proper differentiation. Integrin-linked kinase (ILK) is a serine/threonine kinase that is able to regulate outside-inside signaling pathways by binding to integrins in the cellular membrane and bridging with adapter proteins that regulate cytoskeleton organization, as well as with growth factor receptors coordinating important activities such as motility and cell survival. Based on work in the literature demonstrating ILK over expression in invasive cancers, we hypothesize that ILK regulates that differentiation of cytotrophoblasts into invasive extravillous trophoblasts and that there is a downregulation of ILK expression or activity in preeclamptic patients.
Objectives:
-To define the role of ILK in EVT invasion
-To identify components of the ILK signaling pathway responsible for human trophoblast
differentiation
-To study the levels of ILK expression in preeclamptic patients and compare them with normal
patients
Materials and Methods:
Upon approval from Ethics Committee, human placentas from elective terminations and term deliveries were collected to study ILK expression by immunohistochemistry and immunoblot. Human placental explant-derived HTR8 SVneo cells were used for in vitro experiments. Several constructs such as pEGFP-E359K-ILK (dominant negative form), pEGFP-WT-ILK, pEGFP- S343D-ILK (constitutively active form), and pEGFP empty vector (as a control) were transiently transfected into a HTR8 to study the importance of ILK in the differentiation of trophoblasts. Immunoblots were performed on those cells to study ILK expression and other signaling partners such as pAKT, pGSK3β and pERK. Migration was assessed by wound assays and invasion was studied culturing HTR8 cells on matrigel-coated transwell inserts.
The effect of oxygen on ILK expression and activity was measured by immunoblot after culturing the cells with CoC12 or desferrioxamine as well as in a Proox culture chamber system. With this equipment we were able to culture the cells under a 3% 02 environment. We also had the opportunity to test the effect of oxygen on human placental explants cultured on matrigel.
ILK expression was also studied by immunohistochemistry in placentas from preeclamptic patient and compared to age-matched controls.
In order to study the expression of ILK in mouse placenta we collected samples from C57BL/6 mice at different time points of gestation from E7.5 to E17.5 and studied them by immunohistochemistry utilizing different markers such as cytokeratin, laminin, placental alkaline phosphatase, PAS staining, connexin 43 and connexin 31.
Results and Discussion:
ILK was expressed mainly in the villous and EVT of the human placenta as shown by the co-localization of ILK with cytokeratin 7 and α5-integrin in late first, early second trimester as well as in term placenta. The immunoblot analysis showed that the level of expression is maintained throughout gestation. Since the villous and EVT give origin to the cells that invade the maternal uterus we decided to explore if ILK had a role in migration and invasion. Transient transfection of the HTR8 SVneo with a dominant negative form of ILK impaired the motility of the cells as seen by a significant reduction of transfected cells able to migrate into a wound. In the same way when the cells were transfected with the dominant negative form of ILK, invasion through the matrigel-coated transwell inserts was significantly lower compared to cells transfected with the empty vector. A higher amount of cells invaded the matrigel when they were trasfected with the constitutively active form of ILK. These results strongly suggest that ILK might be playing an important role in the regulation of those events. So far there is no evidence of which signaling partners are regulated by ILK in migration and invasion as there is no change in the levels of pAKT, pGSK3β or pERK when studied by immunoblotting.
The effect of low oxygen tension in ILK expression was studied in HTR8SVneo cultured at 20% with compounds such as CoCl2 and desferrioxamine that mimic hypoxia conditions. We also cultured the HTR8 under low oxygen tension conditions (3%02/92 %N2/5% C02) and compared them with cells cultured at 20% 02. No detectable changes in ILK protein expression were observed under these conditions. Even though ILK levels of expression did not change under low oxygen conditions, ILK activity might still be altered so we will evaluate the effect of such hypoxic environment on ILK activity with an in vitro kinase assay using GSK3β fusion protein as a substrate. In addition, although ILK expression in HTR8-SVneo seemed unaffected by hypoxia conditions in vitro when compared with a model more similar to in vivo conditions like hypoxic placental explants, ILK expression seemed to be reduced. In support of these findings, in preeclamptic tissues, where there is a poor perfusion of the placental tissue, ILK expression is reduced. This might be the reflection of regulatory factors that exist in the body that are not present in the cell culture system or the diverse cell types present in placental tissues compared to cell monolayers.
Although the data highly suggests that ILK might be involved in the regulation of invasion, further experiments are needed to study the role of ILK in the differentiation of trophoblast cells into an invasive phenotype. For that purpose we initiated studies on mouse placenta. We found by immunohistochemistry and the use of different markers such as PAS, laminin, placental alkaline phosphatase, cytokeratin and connexin 43 and 31, that ILK is expressed in those cells that invade the maternal decidua and arteries such as glycogen cells and trophoblasts giant cells. We are planning to conditionally knock down ILK expression in the spongiotrophoblasts (cells that give origin to glycogen cells) and evaluate if those cells are still able to differentiate and invade. We will continue exploring the signaling partners of ILK in order to try to understand the mechanisms that might be contributing to the development of preeclampsia with the hope that we could find a good early predictive marker of this condition.
Transdiciplinary aspect of the project:
Although I am involved in a basic science project to understand human placental development and thus potential molecular mechanisms that might trigger preeclampsia, my goal is to try to find a marker for the early diagnosis of preeclampsia. Preeclampsia is one of the major reasons for premature delivery and recently it has been reported that it can also cause long term effects on the mother's cardiovascular and renal health. If we were able to diagnose preeclampsia early, it would enable the development of a support program for the expectant mothers that would help them prepare to face a preterm labour and all the complications that accompany having a premature baby. Ideally if we could find a treatment for preeclampsia we might be able to lower the number of preterm births.
Working in a health science center in continuous interaction with Obstetricians and other disciplines such as community health, genetics, and immunology gives me the opportunity of amplifying the scope of my research to direct my future research towards solving concrete problems of the local community and also participate in the developing of training programs for this community. For example because I am working on human placental development, a psychologist approached me with the idea of organizing a small informative session with the collaboration of nurses, psychologists and nutritionists for new moms explaining the importance of breastfeeding. The mother needs to understand how the fetus was protected during pregnancy, how the placenta helps in this process and how important it is for the baby to breastfeed after birth.
Questions:
Since the availability of human samples is restricted, how valid do you think is the mouse model to study placental formation? Compare to the sheep or the rat?
Considering that during placental formation the first 10 weeks of gestation occur under low oxygen environment (around 3% O2) how long would you expose your cells and/or explants to recapitulate the in vivo situation?
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Thank you Pia,very
Thank you Pia,very interesting
Preeclampsia is the leading cause of the maternal morbidity and mortality,it is well known that placental insufficiency plays a very important role for the occurrence and development of preeclmpsia.While the differentiation of cytotrophoblasts into invasive extravillous trophoblasts is the critical stage for the placental development.Integrin-linked kinase has been associated with multiple cellular functions including cell migration, cell proliferation, and signal transduction.Pia examined that ILK disregulates invasive extravillous trophoblasts and there is a less expression in PE using cell line,mouse placenta and human placental tissue,this is very novel and interesting.
As this study was to explore the effect of ILK on placental development,if we study the placental tissue from 8 gestational weeks will be more interesting,also I wonder if there is any difference for the level of ILK expression in different trimesters?
Good job,Pia,congratulations!
Shuqin Wei
University of Montreal
Hi Shuqin Wei: Thank you for
Hi Shuqin Wei: Thank you for your comments. I think I was not very clear when I stated my results, sorry!! I studied the levels of ILK expression in placentas as early as 7 weeks of gestation to week 12 (late first trimester), I also had samples from week 13, 14 and 15 on the second trimester, and I studied placentas at term (week 38-42): all from normal pregnancies. In those samples I found neither any difference in the levels of expression, as seen by immunoblot analysis, nor any difference in the pattern of expression in the immnuhistochemistry experiments. Both the villous and the extravillous cytotrophoblasts expressed ILK in all the samples. When I compared the expression of ILK in the placenta of preeclamptic patients with normal placentas it seemed to be a reduction in the level of ILK. I only had access to fixed tissues so I only studied the placentas by immunohistochemistry which is not a quantitative method. I am planning to evaluate the level of expression of ILK in preeclamptic placentas by immunoblot analysis. I think that it would be very interesting to compare the serum levels of ILK in normal pregnancies with preeclamptic patients. If the levels in preeclamptic patients are lower I would like to collect serum from early stages of pregnancies and evaluate the predictive value of ILK as a marker for preeclampsia.
Thanks a lot for the feedback,
Kind regards, Pia
Dear Pia, Thanks for your
Dear Pia,
Thanks for your very interesting presentation. There is no doubt that the basic sciences have made enormous contributions to understanding normal human physiological functions as well as disease processes. As a clinician with no formation in basic science, it is a challenge to understand all the complex techniques, methods of analysis, animal models, etc, used in basic science. For this reason, I find this forum a good opportunity to explore different domains and learn from sharing ideas with other disciplines.
Regarding your question about which animal model should be used to better approach human placentation, I did a quick search for literature reviews who have addressed this topic. I found one that seems interesting (1).
According to this review, the mouse model is less than optimal for studies of trophoblast invasion and vascular remodeling. The reason the author gives are that trophoblast invasion is very limited in the mouse and transformation of uterine arteries depends on maternal factors such as natural killer cells and not in the trophoblast itself like in humans. However the author says that analogies can be made between mouse and human in placental cell types and genes controlling placental development. Thus, in your case I think it is valid to study ILK expression in mouse cells, however I don’t know how should be addressed the ability to invade given the divergences between mouse and human placentation.
Regarding the sheep, this review says that this model is well established for fetal physiology but is of limited value for placental research given that here is no trophoblast invasion of uterine vessels. Moreover, it is very expensive to house.
The author presents the guinea pig as a good alternative rodent model. This is a well-established model for the study of placental transfer and fetal growth restriction. It seems that pregnancy toxemia (preeclampsia) occurs in these animals and can be experimentally induced. Further studies are needed to confirm this model as useful for preeclampsia.
Finally the author proposes non-human primates as important models to study preeclampsia and fetal growth.
As I said, this was a quick review, it may have some bias that I am not aware of. I would like to know your opinion about the difficulties on studying trophoblast invasion on mouse models.
Thank you again. Congratulations for your work and for your PhD.
Regards,
Maria del Pilar Vélez
1. Carter AM. Animal models of human placentation--a review. Placenta. 2007 Apr; 28 Suppl A:S41-7.
Hi Maria del Pilar, I agree
Hi Maria del Pilar, I agree with you that this is a great opportunity for all of us to read about new areas, and I see you took it very seriously, thanks it enriches the discussion and I also learn!!
Although the trophoblast invasion has been describe as shallow when compare with how deep the trophoblasts reach in the human system, that ivasion is enough in for proper nutrition, gas exchange and wate removal. My idea of utilizing the mouse model comes into play because we will conditionally knock down ILK in spongiotrophoblasts and we will study the impact of that mutation in the ability of those cells to invade compare to non mutated mice. I am not planning on using them as model for preeclmapsia. So far I had only done the characterization of the expression of ILK and what I found sometimes complicated is to find the adequate markers to identify the different cells in a specific way. I might have to switch to in situ hibrydization which is more complicated than immunohistochemistry
I hope this answers the question if not I would be glad to contiue the discussion, kin regards, thank you,
Pia
Hi Pia, Thank you for the
Hi Pia,
Thank you for the concise summary of your research. I am a psychology student and so I know little about the specific methodology you were using, so I have some questions I’d like to ask to get a better understanding of your methodological approach.
When you compare the PE tissues with normal control tissues, how do you determine that there are differences (i.e., in infection with a dominant negative form of ILK impaired the motility of the cells, invasion of the matrigel-coated transwell inserts was slower, etc.). Do you visually compare the differences? Do you perform statistical analyses? I’m curious about how that process works.
I was also thinking about your idea regarding markers for PE and support groups to prepare women for preterm labour and its associated complications. On the one hand, I think it could be good to inform women ahead of time in that it would help reduce anxiety and distress in the moment when the preterm labour happens, and on the other hand I wonder if it would increase anxiety during pregnancy, as it sounds like there is little they could do to prevent it. It’s kind of like being told that you will have a car accident later that day but there is nothing you can do to prevent it (an extreme example, I know). I’m playing devil’s advocate here and do not have my own mind made up, but I’m wondering how strong / consistent a marker it would have to be to be used in this way. Do you have any thoughts on this?
Thanks!
Alex.
P.S. I also liked the idea of an education group for staff and nurses about the role of the placenta during pregnancy and how breastfeeding can take on that protective role after birth…
Hi Alex: I added the
Hi Alex: I added the response in the "post a comment" section instead of the "reply" one ,so you'll find the response bellow Martin Frasch's comment. I hope this does not bring big inconveniences.
Sincerely,
Pia
Me again!! I just realize
Me again!! I just realize that the software automatically pushes my answer to the last comment, I hope you find it,
have a good day, Pia
Hi Pia, I am the facilitator
Hi Pia,
I am the facilitator for your discussion group. Your work has created a diverse discussion. I am very impressed with both you and your work but also with the comments you have received. The comments by Alex are interesting and I would just like to follow-up in regard to the use of ILK as a serum marker. Is serum ILK a good marker for the placental levels?
Keep up the great work and I look forward to your discussions with Alex.
Sincerely,
Sandy
Hi Sandy: Yes it’s getting
Hi Sandy:
Yes it’s getting interesting and transdisciplinar!!!
I still don’t know if ILK levels of placenta correlate with serum, it’s one of the things I would like to do as part of my project. There is a research group in Australia that studied serum levels of ILK. They showed that ILK was detectable in normal patients as well as in patients with benign tumors and that ILK levels were 6-9 fold higher in patients with grade 1, 2 or 3 ovarian cancers (Ahmed N et al. Clin Cancer Res. 2004 Apr 1;10(7):2415-20.). So I am especulating that if there is a serious problem in the invasion of the trophoblasts in preeclamptic placentas, as has been reported in the literature, it might be refelcted in a drop in the serum levels of ILK, but as I said this is a hypothesis and it needs to be tested. My plan is to compare the serum level of ILK in normal pregnant patients with those with preeclampsia.
Thank you very much,
Regards,
Pia
Dear Pia, thank you for
Dear Pia,
thank you for presenting this illuminating, exciting piece of research.
It is not directly my field, so what I would like to dare are some speculations, hopefully within what we can agree to be common sense ;-)
Many great comments were made in this forum. I hope what I contribute will be somewhat useful.
You state: 'ILK shows overexpression in invasive cancers, we hypothesize that ILK regulates that differentiation of cytotrophoblasts into invasive extravillous trophoblasts and that there is a downregulation of ILK expression or activity in preeclamptic patients.'
Why is ILK overexpressed in invasive cancers?
Is it ILK gene mutation, or external, e.g., niche factors promoting overexpression, perhaps by uncoupling some sort of negative feedback?
Extending this line of thought, probably similar questions are being asked in pre-eclampsia research. What is known there?
Consequently, it might be that ILK overexpression shows temporal profile and the question arises when is the time window when you would expect to be able to measure such overexpression and when, possibly, overexpression turns into an attempt of down-regulation when it is 'too late'?
As bottom line, it all boils down to the famous chicken/egg series of questions.
Other questions/thoughts:
1. Would you please highlight the reasons for the choices and roles of ILK's signaling partners pAKT, pGSK3β and pERK and whatever else you have been looking at?
2. Please explain the use of the three different experimental approaches you presented (human/cell culture/mouse). I think it would made the reader (me ;-) better appreciate each method used and what can be expected from it. You do say it to some extent in Results, but positioning it all jointly in the Methods would make reading / understanding easier I believe.
3. Do you have access to cord blood values of fetal oxygenation at birth from the fetuses whose placentas you study? If yes, you might correlate these values to the expression levels of ILK and its co-players. Then, from a methodical standpoint it would be interesting to compare what you find with your cell culture oxygen deprivation studies. Would they be consistent/comparable?
4. One more thought re: oxygen deprivation studies on ILK expression: Is there a reason to assume that such hypothesized expression decrease would show a certain temporal profile suggesting it matters when you measure it following the exposure to low pO2? - I just saw, that is your second question ;-) Again, maybe following up on Point 3 above might help to elucidate this question.
5. Are you accounting for fetal birth weight / maternal weight ratio in your studies to better delineate the fetal / placental growth pattern?
6. Maybe you find this URL interesting: http://sabiosciences.com/pathway.php?sn=Integrin_Pathway
A product search on their website for 'ILK' renders a series of assays that might be of interest to functionally knock-out ILK using shRNA (then you could use guinea pig!) and do pathway studies. Cf. e.g. http://sabiosciences.com/rt_pcr_product/HTML/PAHS-058A.html and http://sabiosciences.com/gene_array_product/HTML/OHS-058.html
Kind regards,
Martin
Hi Martin, thank you for the
Hi Martin, thank you for the interest you showed in the project and the good comments, I can see that you are a curious person, I don’t think I can answer some of the questions in big details because it might take me a week to answer all of them. I think my comprehensive exam had less questions, just joking, but I am open to continue the discussion.
The report that ILK was overexpressed in invasive cancers was the basis for our first working hypothesis. ILK has been reported in many invasive cancers as a good monitor of malignization, there are melanomas studies with patient where there is high correlation between the expression of ILK and the survival of the patients. In prostate cancers and in ovarian cancers the level of expression in serum and in the tumour, respectively, has been correlated with the invasiveness of the tumour. In melanomas the studies suggest that ILKAP a phosphatase that dephosphorylates ILK is deregulated. Studies in prostate cancer cell lines, a mutation in PTEN an inhibitor of the phosphorylation of PIP2 to PIP3 (ILK binds to PIP3 and is activated) apparently is the cause of ILK deregulation. PTEN is also frequently mutated in glyoblastomas. ILK expression and activity has also been shown to be elevated in adenomatous polyposis and colon carcinomas where the common mutation is a loss of function in adenomatous polyposis coli (APC) protein. I think the common denominator is an increase in function because of a loss of regulation. There is an ILK inhibitor in clinical trials as a anticancer drug.
Preeclampsia is a a broad topic but I can write a short, quick overview but for details I highly recommend to read a review, there are many good ones such as Redman CW, 2005, Science 308, 1592 or Huppertz, 2008, Hypertension;51:970-975
The exact mechanisms that lead to preeclampsia are still unknown. I think it’s like the literature reports: a multifactorial disease very difficult to study . It’s is believe that an insufficient invasion leads to a failure of the extravillous trophoblasts to adequately transform the uterine spiral arteries, that reduces the flow of maternal blood into the intervillous space with a consequence of periods of hypoxia followed by reoxygenation of the placenta . That hypoxia damages the villous trophoblasts by oxdative stress, there is release of syncytiotrophoblast to the maternal circulation and that triggers maternal inflammatory response resulting in the development of preeclampsia. Every time a group reports a marker they don’t know if it is the cause or a consequence of the local low oxygen. There is a group lead by Karumanchi who develop a rat model of preeclampsia by injecting soluble flt-1, a soluble form of the VEGF receptor, into the rats so this suggest that the increase secretion of sflt-1 might be one of the factors that starts the disease but the etiology still has not been discovered.
Regarding the overexpression of ILK and the time frame, so far I was only able to measure ILK expression from weeks 7 to 15 and at term and I did not observe a significant difference between the samples so, when the time comes I will have to test levels over gestation to see if the secretion is maintained or if there are fluctuations.
1. When I started the project there were a few proteins described as substrates of ILK , mainly AKT and GSK3b, now a days the list is long and I am planning to test most of them in the trophoblast. Akt is mainly involve in inhibition of apoptosis in normal cells, it is especially important in invasive proteins because it inhibits anoikis which is the apoptosis triggered by detachment. GSK3b is involved in glycogen methabolism and in most of the cells is important for cell proliferation. ERK is involved in cellular growth and proliferation. I used these ones because I wanted to study the signaling pathways controlled by ILK in the trophoblasts.
2.The human placentas were used to study where ILK was expressed. Because immunohistochemistry is not a quantitative method, I decided to use immunoblots throughout gestation to see if there was a difference in any stage of pregnancy. We did not find a difference in amounts but we could see that the villous and the extravillous expressed ILK and very little detection was found in the syncitiotrophoblasts.
The cell culture approach is very useful because it provides the opportunity to manipulate the conditions so I can use non- transfected cells and compare their behavior with cells transfected with vectors that expressed the mutated form of ILK, or the constitutively active form of ILK and see if it changes the opposite way the response. Cells that are invasive are able to migrate and invade in vitro.
We found that the human placentas were limited in numbers and in weeks of gestation. We wanted to confirm if ILK is involved in invasion and even though the invasion in mouse is not as extended as in humans there is invasion so if we can delete ILK gene in those cells that invade the maternal uterus by creating a conditional knockout mouse and study if they are still able to invade I think we can gain a lot of valuable information.
3. I don’t have access to cord blood, and I am not familiar with that topic. I only remember from my undergrad that the fetal hemoglobin has a much more higher affinity for oxygen than the maternal so this might bias the results. It would be interesting to review the literature to see what benefits could add to compare the levels of a maternal marker of a disease with the ones on the fetus, I cannot see any correlation , because or either is from the mother that passes through placenta or is fetal ILK which obviously we won’t know. I might investigate this aspect.
4. We are trying to confirm if ILK expression or activity is affected by oxygen, usually what I do is I fix the cells immediately after taking them out of the chamber. I would eventually compare the reaction regarding ILK activity or expression to low oxygen of explants of normal pregnant women compare to preeclamptic ones. My question was more methodological I only culture them for two days but strictly in the body they are expose to low oxygen for two weeks so I think maybe I can increase the days I culture them.
5. I am not accounting for fetal birth at the moment. I will when I start working with the patients
Thank you so much for the web pages
have a good day!!
Pia
Thank you very much for the
Thank you very much for the detailed reply!
I appreciate the quick synopsis.
Just briefly: if you look at the umbilical vein blood gases etc., they reflect maternal placental side. Umbilical arteries carry deoxygenated blood from the fetus to the placenta.
All the best,
Martin
Hi Martin, I agree with you
Hi Martin, I agree with you about the valuable information that the blood cord blood taken from the umbilical vein can provide, and for what I 've been reading (today! so it was quick look) the partial pressure of oxygen does not diverse significantly with the O2 tension in the maternal placenta, so it would not be a factor affecting the results, like I thought when I answered the previous questions. I think that although the maternal blood is reflected in the fetal blood vein not all the compounds cross placenta, so when evaluating the results we have to be careful. Anyways, it would be interesting to test blood cord but, considering my objectives I don't think is justifiable for this project.
Thanks again for the discussion I really appreciate your interest and the time you took to think about it .
Have a good day,
regards, Pia
Hallo Pia I find your
Hallo Pia
I find your project very interesting, (maybe because I’m interested myself in cell signalling ;)) and quite complete with your different approaches. I must say that I was impressed by the open minded people you are talking about. Our lab is working on premature children’s respiratory problems and we are confronted to the hostility of hospital workers who find our work no worthy and in general are not interested in clinician-fundamentalist dialogue. It is reassuring to know that there are some places were this kind of collaboration is possible.
Eva
Hi Eva , thank you for the
Hi Eva , thank you for the nice comments, I must say that I cannot complain about the people, here they are fantastic. Maybe because I just collect a sample that tey won't use (the placenta) makes thinkg easier here but I think that you don't have to loose your hope, keep trying you'll find open mind people. Sometimes we have to justify a lot more or show them facts, numbers, concrete cases in which the cooperation lead to a good result. Keep going!!!
Kind regards, Pia
Hallo Pia You say that your
Hallo Pia
You say that your goal is to try to find a marker for the early diagnosis of preeclampsia. I’m interested in that because I’m trying to do the same thing concerning the premature child lung, to assess his developmental state. The question I want to ask : it is possible to detect those markers in placenta ? Is there already this kind of procedure in use?
Eva
Hi Eva I already found ILK
Hi Eva
I already found ILK in the placenta utilizing immunohistochemistry and immunoblot analysis, I don't know if ILK will be a good marker because I still have to compare the preeclamptic patients with the normal ones. If your question is if there are other markers that are used to date several biochemical agents have been assessed as markers for predicting pre-eclampsia, but there is still a need for more studies to justify their use. The most promising biochemical markers, to date, are soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). These markers allow screening at a early stage show relatively high predictive values and improved diagnostic performance if combined with first trimester Doppler sonography.
I hope this answers your question,
have a good day,
Pia
Hi Alex, Your comments are
Hi Alex, Your comments are welcome!! I will first explain you the methodology aspect of your question. When I tested the preeclamptic patients I only had access to fixed tissues. Those tissues are cut in thin sections and stained with specific antibodies. What I evaluate is the presence of colour and the pattern of that colouring. As you can imagine that is a not a good technique for quantfying yet because I always add the proper controls and I run the normal samples and the preeclamptic samples at the same time and also because I repeated the experiment with several samples I can report what seems to be a decrease in ILK preeclamptic placentas, but is only preliminary data that needs to be coplemented with other methods and set the basis for the future work.
Regarding the markers for preecplamsia your questions is the perfect example that I should approach the project in a transdisciplinary way: talking to a psychology expert like you could help to make an action plan in the case that the test of a marker for preeclampsia is implemented. I am glad you aware me about this fact, sometimes we get too concentrated in the little molecules and forget about the individuals involve in the scientific problem (by focusing on the tree we might not see the forest..). My point of finding a marker even though we still don't have a successful treatment to cure preeclampsia comes from the idea that, maybe, we can take actions like suggesting a change in diet or exercise or habits that may improve the condition or at least allow the pregnancy to reach later weeks of gestation. The problem with preeclampsia is that the symptoms, hypertension and proteinuria, appear after week 20 but it is thought that the problems start earlier in gestation, so maybe by detecting those patients earlier we can at least try to help them deliver later with the benefit of lowering prematurity in the babies. Regarding the psychological impact that the news of a possible preterm labour can have on a patient I will/can only give you my personal opinion: I think that too much information with no responses is an unnecessary way of increasing the anxiety in a patient that has a lot on the plate. I totally agree with you that it cannot be good to tell them if there in nothing they can do, but the truth is that there is still a lot to discover in this field and maybe some day, by the time we discover a good marker, there will be a solution. To implement a marker it will have to show definitively that is robust and even if it is robust it might only become a marker of risk of developing preeclamsia. For example, there are certain lab tests combine with the age of the patients and the familiar history that can predict the risk of Down syndrome. Usually the doctor asks the patient if she would like to test herself, and at that point is up to the entire decision of the patient if she wants to deal with the news at that moment or if she prefers to wait until the delivery. The case is a little bit different in that once you gone through the lab test you can even have the option of undergoing an amniocentesis, but my point is that they ask the patient if they want to know. In the case of preeclamsia, I think that could be a possible way of approaching the patient with proper information and offering her support from the system.
Thank you so much, we should keep in touch to design the action plan!!!
Kind regards, Pia
Hi Pia, Great response -
Hi Pia,
Great response - thank you! I think the analogy to Down's syndrome makes a lot of sense and offering the patients choice about whether they want to be tested and counseling about outcome or positive things they could do to improve the health and the health of the baby makes sense. I also see the importance of identifying PE symptoms earlier than 20 weeks.
Thank you also for your explanation of your methodology. I guess the quantitative person in me always wonders how you know that you are actually finding a true difference between groups and it is not due to chance or seeing what you want to see (can still happen with quantitative methods :), but from your explanation of the methodology I can see how use of several different tissues helps to make your findings stronger.
You've answered a lot of interesting questions this week from multiple perspectives - like comps again, hey? :)
Hopefully I will see you at the conference in Montreal,
Alex.
Hi Alex, I hope some day we
Hi Alex, I hope some day we can really make plans to improve things!! Thanks again for including diversity in the discussion and stressing the importance of the people that one day might get involved in the studies.
Hopefully we can continue the discussion in Montreal,
kind regards, Pia
Dear Pia, Excellent work!!
Dear Pia,
Excellent work!! This was an exciting forum!!I wish you the best in your future endeavors!
Sandy
Hi Sandy, Thank you very
Hi Sandy,
Thank you very much for the nice comments and for facilitating this session
Kind regards
Pia
Honestly it is one of better
Honestly it is one of better presentations I’ve seen and one can feel you got a good control and an excellent knowledge of your research subject. I appreciate greatly this discussion and I learned much. Congratulations.
eva
Thank you Eva, your comments
Thank you Eva, your comments brought up an important point in basic research which is that we need the collaboration of different disciplines in order to achieve our goals. It's a big challenge for basic researchers to start the dialogue with the other professionals and convince them that working together there is a lot more that can be understand and maybe improve.
TThnaks a lot for the compliments, I hope you the best,
Kind regards, Pia
Hi Pia, I was contemplating
Hi Pia,
I was contemplating over the question you posed about the validity of models. Rodents do have the same discoid placenta as humans where as sheep have a cotyledonary placenta.
Though the sheep is a good model for fetal development however when discussing the placenta it is like discussing apples and oranges. The cotyledonary placenta is many small disc shaped placentas. These type is non invasive with no trophoblast invasion and are attached in the same place during every pregnancy. This is not like human placentation which to my limited knowledge is very invasive and does not occur at the same place during every pregnancy.
Rodents have a problem unto themselves where they are polyovulatory mammals who deliver immature young. The quick gestation period of rodents may be useful during these early stage since you would be able to perform multiple replications somewhat easily, quickly and cost effectively.
Once past these initial stages however the most appropriate model is the non-human primate. The most appropriate of the non human primates are the macaque and the baboon. The other non-human primates do not appear to have interstitial trophoblasts in the placentas therefore limiting their effectiveness as an appropriate model for placentation.
Thank you for sharing your findings with the group
Heather
Hi Heather, This is an
Hi Heather, This is an important piece of information, I think that we try to use the best model for what we are going to study but we also have to accommodate to the facilities, to the time frames, to the accessibility to engineer animals and to the expertise of the people with whom we are training. What is very important is to be aware of the advantages and limitations of each model at the time of concluding from the results and most important we have to be very careful if we decide to extrapolate to other species (particularly in placenta where there is so much diversity). Even if you are not working with animal models, knowing those limitations help us to interpret the results from the different publications.
I appreciate your feedback, I realized that I have got so used to the mouse and the human that sometimes I even skip the non-human primates publications and I shoud pay more attention to them.
Thank you very much,
Have a good day,
Kind regards,
Pia
Hi Pia, I would first like
Hi Pia,
I would first like to say that your presentation was very interesting and a joy to read.
Pre-eclampsia and placentation is a bit far from my field, so I will limit my comment on methological aspect of your presentation.
Conserning the use of immunoblots to detect variation during pregnacy or between PE and normal placentas. While I agree that immunoblots can be semi-quantitative, it is generaly agreed that they do not always detect low to moderate variations between samples and normalization of the results are often difficult. How big a difference do you expect? Maybe your experiment would benifit of the use of a more sensitive technique for quantification? If you have acces to the equipment, quantitative real-time PCR would be tailor-made for your experimental setup. It can detect subtle variation between many samples and results can be robustly normalized (utilisation of more than one control gene)for all the samples studied.
Best regards,
Eric
Hi Eric, I expect a big
Hi Eric, I expect a big difference (only pure speculation) but I am planning not only to study those placentas by immunohistochemistry but also by an ELISA based test in the blood, which is a more sensitive method. The immunoblot is only for the placenta samples (pieces of the tissue). We do have access to qRT-PCR and I am at the moment testing the probes for ILK (which are working well so soon I should be able to start studying samples) Like you said I found it's a very sensitive method so it might result very useful to detect small differences. My only concern is that even if I find a difference in mRNA levels this change might not be reflected by a change in protein levels, probably having no biological effect on the cell function.
I will still try the qRT-PCR because if there is change in ILK protein levels , mRNA detection might prove to be a more sensitive method of detecting the differences than the ELISA.
Thank you very much for the good and useful feedback,
Kind regards, Pia
Thanks to everybody that
Thanks to everybody that participated in the discussion, I received a lot of positive feedback full of information that I can use in the future in my research. I hope I was able to answer your questions appropriately, if you think you still would like to know more feel free to continue asking. Kind regards, Pia