stirrhs (en) - Exploring the role of Integrin-linked kinase in placental development. - Comments

Thanks to everybody that

elustondop — Fri, 17 Oct 2008 22:44:38 -0400

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

Hi Eric, I expect a big

elustondop — Fri, 17 Oct 2008 22:33:09 -0400

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

Hi Heather, This is an

elustondop — Fri, 17 Oct 2008 22:15:03 -0400

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 Martin, I agree with you

elustondop — Fri, 17 Oct 2008 22:01:17 -0400

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

Hi Alex, I hope some day we

elustondop — Fri, 17 Oct 2008 21:46:51 -0400

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

Thank you Eva, your comments

elustondop — Fri, 17 Oct 2008 14:48:35 -0400

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 would first like

boucher — Fri, 17 Oct 2008 12:41:49 -0400

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 Pia, I was contemplating

Allaway — Fri, 17 Oct 2008 11:58:39 -0400

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

Honestly it is one of better

bresson — Fri, 17 Oct 2008 09:39:13 -0400

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

Hi Pia, Great response -

mcintyre — Fri, 17 Oct 2008 08:26:11 -0400

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.

Thank you very much for the

frasch — Fri, 17 Oct 2008 01:50:11 -0400

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 Sandy, Thank you very

elustondop — Thu, 16 Oct 2008 21:55:15 -0400

Hi Sandy,
Thank you very much for the nice comments and for facilitating this session
Kind regards
Pia

Me again!! I just realize

elustondop — Thu, 16 Oct 2008 21:08:06 -0400

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 Martin, thank you for the

elustondop — Thu, 16 Oct 2008 21:05:56 -0400

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

Dear Pia, Excellent work!!

davidge — Thu, 16 Oct 2008 19:43:24 -0400

Dear Pia,

Excellent work!! This was an exciting forum!!I wish you the best in your future endeavors!

Sandy

Exploring the role of Integrin-linked kinase in placental development.

elustondop — Mon, 13 Oct 2008 00:06:48 -0400

Introduction: