Robert Taylor - In the beginning: identification and treatment of nascent endometriosis

Robert Taylor - In the beginning: identification and treatment of nascent endometriosis

Endofound’s Sixth Annual Medical Conference: Ending Endometriosis Starts at the Beginning

In the beginning: identification and treatment of nascent endometriosis

Robert Taylor, MD

 

What a wonderful lecture we just heard about pain and endometriosis. I am so thankful Tamer for your invitation to come here and proposing this very, sort of, cosmological view of endometriosis. Ending endometriosis starts at the beginning. As soon as Tamer suggested that title for the conference I was – in the beginning idea got into my head. I guess the thought that the big bang might really explain all of endometriosis I really like it. Plus, I thought it worked pretty well for Stephen Hawking, maybe our own little textbook we might be able to turn it into a sort of phenomenal, international bestseller as well. But I am not necessarily looking forward to that.

 

There have been some issues that we have discussed over the last several days regarding this kind of early versus later stages of endometriosis. Obviously there is a stage basis for this as shown to your right where you have got large kissing ovaries and a lot of pelvic inflammation and then stage wise the earlier lesions. There is also, and we will talk more about these kind of subtle lesions as we go along, but there is also a life course temporal early versus late and I think that we have all been in the last couple of days talking a lot about endometriosis and the adolescent population and how important that is. These are some pictures that I took from Deborah’s Endometriosis New Zealand project. Again, it is actually as Dr. As-Sanie mentioned we do sadly see these kind of repetitive surgical cases and we really I think do sometimes have to reconsider what is really the best way to understand how to treat, eliminate, mollify this disease processes early in the life course as we possibly can.

 

One of the questions that we have been asking ourselves in the laboratory a little better to try to understand how to model some of this in the laboratory is what really are the visual phenotypes of the nascent lesion, the new, early onset lesion? If we are going to nip this in the bud, what is the bud and how can we identify it? These are some reports over the years. One, these red, inflammatory looking lesions really predominate in early life stage endometriosis in the adolescent. Nisolle and Donnez several years ago really talked about these red, flame-like lesions representing the early stages of endometriosis after implantation. I will come back and talk a little bit too about Ron Batt’s very nice presentation yesterday. I am kind of losing track of the days here – yesterday. A lot of what I am going to be discussing here a little bit is more of acquired endometriosis as opposed to congenital but I thought that that was a really important distinction to make.

 

For a lot of years we were early into thinking about these, these hyperemic endometriosis lesions with this very neo-angiogenic response around them. A vasculature that you have all been looking at through the laparoscope for all these years really reflects these endothelial cell responses, this angiogenesis new blood vessel formation that we feel really is critical to the establishment of the lesion. Those might be important targets for understanding how to interrupt the disease process very early on.

 

We are going to hear a little bit later on from Asgi Fazleabas. He has done some really beautiful work in the baboon model where he actually takes healthy female baboons and introduces menstrual endometrium into their peritoneal cavity to establish endometriosis by inoculating endometrium there in a couple of cycles, menstrual cycles. Then he can follow in the baboon model with repetitive laparoscopies every three months. There are a lot of different types of lesions that develop over the course of the disease process but what I think should be most interesting to us is that there is a trend for early in the disease process to have a lot of these red flame like lesions that we see clinically. Over a period of time the number of those lesions seems to either dissipate or evolve into more lesions with more complex appearances. This also I think supports the idea that these early lesions are inflammatory, vascular and might give us some ideas about how we could potentially intervene in those lesions.

 

We have talked a lot about some of the great history about endometriosis. I bring John Sampson’s picture back here again. This is a diagram from his original, early 1920s work on the theories of endometriosis. Here is a more recent cartoon kind of showing the bleeding through the fallopian tube, fragments of endometrium falling through the peritoneal cavity on these gravitationally dependent surfaces giving rise to the kinds of lesions we think about. It was quite interesting to me because we have now heard three talks that actually describe Ivo Brosens’ new paper – it has only been out for a couple of weeks. Some of us have heard these at meetings so had a chance to think about it a little bit but this idea that the first menstrual period might in fact occur in the neo-natal period rather than at puberty is really quite an interesting concept. Ron talked about this yesterday and here are some of the data dating back to 1955 where pathology, typically autopsies of new born neonatal deaths have given rise to this idea that there really is menstrual like endometrium due to the stimulation with placental hormones. Uterine bleeding does occur in some newborns, more likely in term babies than in pre-term babies. This might explain some of the early causes of what Ron has referred to as acquired endometriosis. I think that that is actually a really interesting concept.

 

If we think about endometrial reflux and Sampson’s hypothesis – this is the cartoon that CY showed yesterday as well that Nisolle and Donnez actually demonstrated. You had this idea that there are viable fragments of endometrial tissue that are retrogradely shed and those can attach to the surface of the peritoneum. You start to get these early lesions and we believe that this is probably going to be a temporal process. It is very difficult to demonstrate that in vivo obviously unless you have a model like Asgi has. We will hear a little bit more about that.

 

At the University of Texas in San Antonio, Craig Witz and some of his colleagues did some very nice experiments where they actually – if you were here on Saturday Dr. Seckin showed us how he pins out these little fragments of peritoneum that he excises when he does his surgery for pathological sectioning. Actually this is the same kind of approach that Craig used in the laboratory to actually put these fragments, basically sheets of peritoneal tissue basically in a little flask and then add endometrial fragments to that. Those fragments will actually attach to the surface, to the mesothelial surface of the peritoneum and over a period of time he was actually able to observe the invasion of endometrial cells actually through the surface epithelium. Now this is very difficult obviously to demonstrate in vivo and there has been a lot of controversy at this meeting I am sure and almost every other endometriosis as to does this in fact happen in vivo.

 

Through these models we can see – and these are some really clever experiments that Craig did with confocal video microscopy over a period of time. You can label the endometrial cells with a red dye then watch them over time. Over a period of about 24 hours those cells actually invade through the mesothelium and find their way into the sub-peritoneal space, which is where the endometriotic lesions actually, typically grow. Again this is not an in vivo demonstration of this process that supports Sampson’s hypothesis for acquired endometriosis but it certainly is very compelling laboratory based data.

 

In San Francisco, and actually you saw one of Dan’s slides in Dr. As-Sanie’s lovely presentation too and we will talk a little bit more about that, but we had the idea that while attachment and implantation are critical steps in the establishment of an endometriosis lesion that they really are not sufficient for that lesion to grow and to develop and actually become potentially clinically problematic. We really invoked this angiogenic hypothesis, which was consistent with findings that Judah Folkman had made several years before in terms of cancer metastasis. He is really the father of angiogenesis in this regard. That this angiogenic response not only was consistent with what we see through the laparoscope but also with the biology of the early lesion. Here is a nice example of this. Which peritoneum would you prefer to have? I guess even with a little bit of sub-peritoneal fat I would probably rather have this one over here. You can see the dramatic, spider-like vascular responses that I think we all recognize now. At the time it a little bit of a new concept.

 

I have to digress for a second and give credit to this particular individual, Napo Ferrara was a post-doctoral fellow with me at UC San Francisco. He is from Catania in Sicily and was interested in doing an OBGYN residency and came to work in our laboratory and managed to get a little bit sidetracked. He actually discovered the family of proteins vascular endothelial growth factor. He won the 2010 Lasker Award, which is the United States’ version of the Nobel prize. A couple of years ago he won this Life Sciences Breakthrough.

 

The nature of science is such that you guys will recognize this person much more than you do Napo but we can live that. This is Mark Zuckerberg who, I think, put up a lot of the funding actually for this Life Sciences Breakthrough.

 

Napo made this brilliant observation of a new family of proteins that he thought was going to be very important in terms of angiogenesis. These are members of the vascular endothelial growth factor family – VEGF as we refer to it. These are a series of proteins that tend to dimerize. They bind receptors on a variety of different tissues. There are a lot of different isoforms and you can see there are a variety of different VEGF and placental growth factors, actually a variant or a member of this family. But these have the ability through different splice variants and isoforms that are produced to either stimulate blood vessel growth kind of classical angiogenesis, nerve growth and you have seen from Dr. As-Sanie’s discussion how important that is in the original probably innervation of the lesion before it starts doing all the complex sort of central signaling. And also lymph vessel growth and we are beginning to focus more and more on that.

 

The model that we have tended to use in my own laboratory is one in which we take endometriosis and normal endometrial samples from surgery. Here, endometriotic lesions will actually take this as an example of an ovarian lesion where we can excise part of that cyst wall that we saw very nicely removed in several of the lectures yesterday. Those cells can actually be isolated, put into culture and they have some advantages for drug discovery and a variety of other things. They are sort of normal, chromosomally normal. They come from women who have a normal karyotype. They are replication competent so we can actually expand the cells and get enough to experiment with. They retain their hormone responsivity because they are freshly out of these women who actually have hormone responsive cells so they respond estrogens, progestins, androgens and glucocorticoids. They have all the normal receptors. They are actually responsive to things that are in the environment that are not really natural hormones but do have receptors that allow them to respond to things like dioxin bisphenol A. They make inflammatory cytokines and we saw, again, in the cartoon that Dr. As-Sanie showed you how important those are in this process. They secrete factors probably relevant to her talk that stimulate nerves, blood vessels and lymphatics as I have mentioned. And in vitro we can actually test drugs and vitamins and even cigarette smoke extract if you are interested in the influence of cigarettes. It has actually been quite a convenient model for us.

 

This is just to show that using cells like this, cells from normal endometrium versus cells from endometriosis we can see quite a difference. In 24 hours the endometriotic cells make a lot more VEGF than do normal cells and at 48 hours you see the same sort of thing. These cell lines, cell culture models can actually be really quite helpful for trying to better understand the cell biology of endometriosis.

 

As we have all discussed over the last several days these lesions, they look very different, they are really quite highly complex in terms of the number of different cell types that are there. We are all aware that our histopathologists would like to see epithelium and stroma to actually validate and verify that these are endometriosis implants. We have a lot of leukocytes, immune cells but there are a lot of immune cells and we will talk a little bit more about the importance of immune cell infiltration. There are blood vessels and lymphatic vessels that actually are brought in to these lesions, and very importantly with respect to the last presentation is the innervation here. In this particular case we have stained these nerves with a chemical stain for PGP 9.5 which shows these little brown nerve fibers. These are actually growing into the endometriotic lesion itself.

 

This gives us the opportunity to ask how do these things get here and why are they different in women with endometriosis? What are some of the mediators of this? Here is an example of work that we did a couple of years ago. Amy Brown, when she was in the laboratory, looking with a hypothesis that maybe there are more nerve growth factors that are present in endometriosis samples than in normal. And that those nerve growth factors are what are attracting these nerves and if we knew what those nerve growth factors were we could possibly interfere with those. The classic, everybody’s most popular nerve growth factor, we actually did not find any differences between normal and endometriosis. But, a couple of other examples, neurotrophin 4 which is a member of that family, and brain derived neurotrophin factor, which we focused a number of our studies on because its level of secretion is quite a bit higher. These are nanogram amounts versus piktogram amounts. We tried to make our lives a little bit easier by looking at this. But there are actually dramatic differences in terms of this particular nerve growth factor expression in endometriosis samples.

 

Here are some examples of that actually in an endometriotic lesion. Here you can see the endometriosis glands and stroma with some muscle areas that are being invaded by this lesion. This is looking for the immunolocalization of brain derived neurotrophic factor, originally named because it was found in the brain but obviously it is found in a lot of other tissues as well now. You can see that both the epithelium and the stromal cells here make this pink protein in these slides, whereas the muscle tissue that is being invaded really does not have very much BDNF expressed. These are nerve fibers actually, again, stained with this PGP 9.5 immunostain. These are nerves fibers coming out toward you. We do not have any, sort of have to reorient the tissue to get nice linear pictures. But you can see a number of these nerve fibers, again, seem to be very heavily localized within the lesion with some in the region of the muscle but not nearly as dense in those areas. So those are the nerves.

 

Here are some data from Sylvia Mechsner’s laboratory in the Charité Hospital in Berlin. She has done some really, really wonderful work on lymphatics in endometriosis. I am calling this reciprocal lymphatic involvement because what she has demonstrated here are these are sentinel lymph nodes, actually pelvic lymph nodes in patients that have endometriosis resected. You can see that endometriotic lesions have migrated, essentially metastasized along lymphatic channels to find their way into these lymph nodes. Here they are stained with cytokeratin and epithelial stain which would not normally be present in a lymph node and with progesterone receptor. So these are endometriosis cells, stromal and epithelial cells. These are the epithelial cells at the endometriosis lesion. This kind of found its way into a lymph node.

 

The reciprocal side of this is if we actually look at an endometriotic lesion, and here she has got some examples up here, and stain that lesion for markers that detect lymph channels, so not blood vessels but lymph vessels, you can see that there is actually quite a rich, plexus rich, supply of these lymphatic vessels that infiltrate into the lesion itself. We have got lymph vessels growing into the lesion and cells, essentially metastatic endometriosis cells, finding their way into the lymph nodes. The reason that we think that these lymph channels are being attracted to that is that the endometriosis lesions are expressing the VEGF family’s proteins C and D, which are the ones that are really quite specific for lymphatic vessel growth. Some of the other VEGFs are better for nerve growth, some are better for blood vessel growth. These are the ones that are most prominent in lymph growth and you can see them in the epithelium for sure, stroma around that and also in macrophages near these lesions, which are quite rich sources. This suggests that if you have an inflammatory response with macrophages they can be making factors that attract these vascular channels.

 

What about the role of the immune cell response? This has obviously been a really, really important development over the last 20 years in the endometriosis field. We know that the innate and adaptive immune response play very, very critical roles in this and they are highly complicated. I am just going to, in the interests of time, focus on macrophages, which seems to be one of the predominant responses and one that we have probably studied most. It should not eliminate our focus on some of these other things in the future.

 

This is one of our original versions of the same, the more complex cartoon that Dr. As-Sanie showed you from Dan Lebovic. This is a little bit of a complex and circular rationale but I would like – in these case of acquired endometriosis we still, like the Sampson concept that retrograde menses will give rise to endometriotic implants. Those implants actually we think this development of nerves, lymphatics and blood vessels – this is now getting to be complicated. I cannot give that word too much longer than this and be ever able to say it but we do see these things as being linked. That really comes from a process of recruitment of macrophages and probably other immune cells as well. There are a whole variety of biochemical factors that allow both lesions to talk to the immune cells, recruit the immune cells and blood vessels and nerves and lymphatics to the lesion and they get these complex lesions that give rise to the pathogenesis. Pain and infertility is how we have viewed this.

 

The macrophages we believe get there through the secretion of these chemotactic factors and one of the ones we have studied quite a bit is called RANTES. You can see that in more advanced stage of endometriosis, and this is quite old work some of the earlier stuff that we have done in the endometriosis field, that high levels of this protein are actually secreted into the peritoneal fluid and attract these inflammatory cells, macrophages. The more endometriosis you have the more that factor seems to be present.

 

Rather conversely, the macrophages themselves that we can get either from the peritoneal fluid around these lesions or the lesions themselves have an up-regulated receptor for this chemokine. So there is a chemoattractant substance that is being secreted. The cells that are actually in the endometriosis and for reasons that we really do not understand actually seem to be hypersensitized to respond to that. So you really create this vicious cycle in which you are attracting immune cells into an area that is inflamed causing more of an inflammatory response and this essentially positive feedback that gives rise to the symptoms. When we look at peritoneal macrophages, in this case from women undergoing laparoscopy for tubal ligation they have quite a bit less of this receptor protein expressed on their surface. These cells do not seem to really be as responsive. To be honest we do not really understand that part of the biology as yet.  

 

Now, this neurolymphangiogenesis as we are now starting to call it and which were viewed as one of the strategies that the nascent lesion might co-opt to establish this growing endometrial implant caused me to have some flashbacks. I will apologize to those of you who did not have the pleasure of spending a year in the gross anatomy lab as a first year medical student. I remember being quite puzzled and struggling in the brachial plexus, which is where the nerves, lymphatics and blood vessels actually come out of the lower part of neck and go into the axilla, into the armpit where they give rise to all the vessels and nerves and lymphatics in the arm. I thought who would have ever designed something that was so complex as this and why does it have all these Latin names that I have to remember? I still wake up with cold sweats and nightmares about the examination on that particular topic. That one has not really left me.

 

It was not really a particularly new idea in the 16th century Vesalius did these beautiful drawings of what the blood vessels do and what nerves do. A few years ago Carmeliet and Tessier-Lavigne from Belgium did these very nice co-localization studies and you really get the sense that nerves, lymphatics and blood vessels, and all of those of you who are surgeons have known this forever, are really coordinately attracted and grow. So this is an important embryologic phenomenon that recapitulated essentially by these little lesions that are growing on the peritoneum. It seems that if we could understand how all those things are happening maybe we could do something about it.

 

One of the things that we can do about it certainly is to surgically excise these lesions and so there is laparoscopy and the surgical approaches to endometriosis have really evolved dramatically. We get to see the peritoneal environment in its entirety essentially and we can excise these lesions. There are some issues that certainly require a lot of surgical skill. This was discussed in great detail yesterday. There are probably sub-clinical lesions that we do not get the opportunity to see. There are adhesion formations, high costs and post-operative recovery. Then, again, I am sure this is related to the adequacy of the initial surgery but there is certainly a risk of recurrence. What are the roles then for medical therapy either as an adjunct to surgery at the time or certainly in the post-operative period or as an alternative to surgery.

 

This is the way the reproductive endocrinologists view the universe. I apologize a little bit for that. This is normal endometrial response but we believe that endometriosis in many ways responds similarly. This is the axis and the medications that we currently have available would include oral contraceptives, pills, GnRH analogues and hopefully in the future maybe some selective progesterone receptor modulators, some of that data looks promising although there are some endometrial response effects. Levonorgestrel IUD has been quite a useful tool in this setting. Aromatase inhibitors also are being used more and more now, not FDA approved for endometriosis but eventually will be quite effective there. Obviously non-steroidal anti-inflammatory drugs probably have broad effects both centrally as well as locally. Opiates, and we have heard a little bit about those certainly have much more of a central effect. The central part is absolutely critical as Dr. As-Sanie has really convinced us all. And then we have a variety of other things, Danazol and medroxyprogesterone acetate. I am going to talk a little bit in a second about serums because I think we have some promising ideas about that.

 

A couple of days ago I talked a little bit about natural, botanical approaches potentially as anti-inflammatory drugs that might be developed for endometriosis. I will not go into a lot of detail with this but one of the compounds that we have been quite interested in is curcumin and I am going to show you a couple examples of some of the effects that curcumin can have on this in vitro endometriosis cell model that I described to you a little bit earlier. Here we are taking normal endometrial stromal cells and these endometriotic stromal cells are from lesions. You can see that in terms of their ability to secrete interleukin 1 beta there is more coming from the endometriotic stromal cells and with increasing doses of curcumin that we can put into solution in the cell culture medium we can actually inhibit some of those inflammatory proteins. Interleukin 1 beta here, tumor necrosis factor-alpha RANTES, the chemokine that we believe is important in retracting macrophages and monocytes to the lesion and here is the vascular endothelial growth factor. In each of these we see a nice inhibitory effect of curcumin bringing the levels of these proteins down into the more normal range that normal cells make. We are hopeful that this might be a source for relatively safe, well-tolerated medications that might be used chronically.

 

We also talked a little bit about using other herbal therapies. This is in a rat model of endometriosis where we can take rat endometrium, sew it in the peritoneal cavity and over a period of months those grow into lesions that do not look exactly like human endometriosis unless you want to call that a vesicular lesion. You can see here where this – you can actually see one of the little sutures where we have sewed this tissue. Herbal combinations that we can make as a tea that we feed these rats or a GnRH analogue in this case, which works also very well at reducing these lesions. Again, this suggests that this might be a promising approach. 

 

But what I would like to talk to you about just for the last couple of slides is some very exciting work that we have done in collaboration with the group at the University of Illinois, Drs. Benita and John Katzenellenbogen - a recently published paper using novel selective estrogen, two really novel drugs that they are developing, selective estrogen receptor modulators, or SERMs. This is in a mouse model. Here we can take syngeneic strains of mice, so they are all related and can transplant tissues from one mouse to another and it will not be rejected because these are genetically similar mice. We can take an intact donor mouse, take pieces of her uterus and sew them into the peritoneal cavity of a recipient mouse that has been ovariectomized so we can control the estrogen effects in that animal.  These lesions will establish over a two week period of time as lesions very much like in the rat that I showed you. Those animals can then be treated with hormones and various drugs over that 14 day period. Then we look to see what the effect of these endometriotic like drugs are.

 

Here is the chemical structure of estradiol and these were the two SERMs that John Katzenellenbogen has synthesized. I will call them OBHS and CLI just to make it easier. These are drugs that have some sort of selective properties for the estrogen receptor, alpha and beta receptors respectively. I will not go into a lot of detail about that but just so you can see what the biological effects of these compounds are.

 

This shows the lesion growth over a two week period of time and animals that are treated with vehicle, and remember these are ovariectomized oophorectomized mice so they do not make any estrogen, their lesions are not actually stimulated to grow so the lesions do not really grow that much in the absence of estrogen. The vehicle here is purple you cannot really see what is under there. If you treat the cells with estradiol these endometriotic lesions grow, as you would expect, minimally over that period of time but not in the presence of either of these SERMs. Both of the SERMs block that growth. VEGF messenger RNA is inhibited by those stimulated by estradiol and inhibited by those as you might anticipate because VEGF is an estrogen responsive gene. 

 

Consistent with that the vascularization of these lesions, I do not know how well this shows, but this pink staining is blood vessels in the microscopic lesions. This is in vehicle, so there is no estrogen there, lesions do not grow very much. You see the occasional endothelial cell but they are not really very well organized. Here you can see nice blood vessels in the estrogen treated animals. If they are treated with estrogen in the presence of either of these two SERMs there is an inhibition of that micro vessel. Here we have counted the micro vessels per field and you get a lot of them with estradiol - this sort of organization. But in the presence of these SERMs that process is blocked.

 

There is also an anti-inflammatory effect of these drugs. Here are the control animals making RANTES over a six week period of time. This is a more chronic response so we have looked at this over a longer period of time. Tumor necrosis factor-alpha is also increased early, or two weeks at least, and then falls back to baseline a little bit later but in the presence of these drugs there is quite an inhibition of this inflammatory response. We not see very many macrophages infiltrating into the lesion like we do in the vehicle treated animals. Here we have counted macrophages and you can see that both of those compounds actually inhibit that process.

 

So to summarize, we believe that these nascent endometriosis implants are more likely to be red, flame-like lesions and we think with this prominent neurolymphangiogenesis, this sort of vascular inflammatory response, it really is required to get those lesions to be viable, grow and be able to expand and invade. They appear to be really likely, very likely, dependent on a different VEGF isoforms of these proteins, as an example, probably lots of other growth factors as well but for growth. This gives us some ideas about targets. Superficial lesions are very effectively treated by laparoscopic surgery, but also may be amendable to medical therapies, possibly in combination with surgery. Ideally, in the long term, what we would like to have are preventative therapies. Again, if we are able to intervene early enough we might be able to prevent acquired endometriosis. We probably will not be able to do very much about congenital versions.

 

Current FDA approved medications for the treatment of endometriosis and other off-label approaches really have focused almost completely on the hypothalamic-pituitary-ovarian axis. These all have very predictable side effects that we are familiar with. The idea of maybe focusing more on the inflammatory aspects of endometriosis might give us some new insights as to how to develop new treatments. Some of these might be natural botanical preparations, which we assume or hope would be quite safe. But also the idea that these novel estrogen receptor modulating drugs such as the ones that John and Benita have helped us study in this model might really be promising in applications in the future.

 

I want to thank a variety of peopled, particularly Benita and John Katzenellenbogen, and Yuechao Zhao who has actually done all the experiments that you saw in those really nice papers, and a whole variety of collaborators over the years that have really contributed to these studies.

 

I will stop here but I am going to leave you with one of my favorite poems from T.S. Eliot that I thought once I got over the brachial plexus nightmare and started to relax a little bit I thought about this poem. I will leave it up here. It reminds me of “In my beginning is my end, in my end is my beginning” and the cyclical, monthly if I could, aspects of regeneration that is so important to not only the endometrium but the way we think about this disease process.

 

Thanks a lot for your attention and thank you very much for inviting me here.

 

 

Tamer Seckin, MD:  You first showed a slide of the big bang and I just want your opinion in public with respect to where was the big bang in neurovascular lymphangiogenesis? I just want you to make some comments on stem cells – your thought process.

 

Rob Taylor, MD:  The stem cell hypothesis, there is no questions that, I mean that one of the great things about, I think one of the lucky things really for our field is this. This is actually one of the reasons that I had the opportunity to get involved in the California Institute of Regenerative Medicine in the early stages, Proposition 71 I think it was, was passed in California under the Bush administration when there was a moratorium on stem cell work nationally. The State of California pushed through this proposition to study stem cells there. What was nice was that they came to us in reproductive medicine because at that time we were the ones that knew the embryo biology. We knew the reproductive biology that led to the early studies. I would say now that we can make induced pluripotent stem cells out of tissues from everywhere our ability as reproductive medicine folks to really be as influential might be a little bit reduced. But it was great to get in on the front end of that.

 

We have to appreciate that what we know about embryology and endometrial biology with its regenerative capacity, which exceeds any other healthy organ, there are tissues that grow rapidly but we do not want them to mostly, gives us that advantage. I would say for us the big bang came from working on these regenerative medicine issues, embryo growth development and endometrial growth development gave us some early insights into this.

 

I do not know if that was precisely the question. But there is no question that endometrium contains within it and I think Caroline Gargett’s work is beautiful in this area and Hugh Taylor has done some very, very nice work as well. He has invoked a bit more of a bone marrow derived cell, but there is clearly a stem cell population within the endometrium itself, perivascular probably according to Caroline’s work, that have cells that really have this multi potent ability to generate a new endometrium and for sure these endometriotic lesions as well. I think if we knew exactly what the markers were on that, and she is working quite hard on identifying those, we might be able to develop some very focused, specific therapies toward that stem cell population. Again, it is going to be hard to target that just to the endometriosis lesion and not whack the endometrial stem cell population. But that would be the holy grail.

 

Linda Griffith, PhD:  Rob, beautiful presentation as always. I am curious what you think about possible heterogeneity in mechanisms among different patients. When you look at your individual cytokine data like RANTES some patients, on average a group with disease will have the higher level of meme, but there will be a distribution around that. Some of them that may be of really operative mechanism are involved and others not and you think about related diseases like rheumatory arthritis where TNF-alpha inhibitors really help some patients and others are refractory. Could you give us some perspective because you have such a long and broad perspective on this, on possible heterogeneities that some of these molecular mechanisms of – certainly lymphangiogenesis, neuroangiogenesis do occur by these processes but whether they are dominant or not as dominant in particular patients versus others would be interesting to know from you.

 

Rob Taylor, MD:  Those are great questions. Heterogeneity and the presentations, symptoms, visual appearance of the lesions themselves that is really part and parcel of our work. I guess in keeping with the theme of this “in the beginning” I would say that I think there is also a really important temporal pattern. We have seen, at least in our rodent models, that there is a progressive regulation of these chemokines. For example, MCP-1, monocyte chemo-attractant protein-1, is a much more acute response when we actually induce the lesions than RANTES, which actually comes up at a later period of time. I think that part of the heterogeneity is where we catch the disease. I think that asking everybody, and this is a way over-simplification, but I think that one of the reasons we are seeing heterogeneity is actually where those women are in their life course what the inflammatory responses might be. There is a pretty well-orchestrated, progressive I think, in a very controlled laboratory setting, I think we can see that some of these things really are quite progressive. Part of that heterogeneity is catching people at different points in time. I am sure that there are more explanations than that but I think this temporal idea – beginning and is actually a pretty powerful way of thinking about this.

 

Audience Member:  I know you are excited about the SERMs and really focusing on the inflammatory aspect of endo and I completely appreciate that. But I am curious about your thoughts on GnRH antagonists in development, where you think they might play eventually in this, your thoughts.

 

Rob Taylor, MD:  There are now, actually the first one that I became familiar with was synthesized and really developed by Y. Lee Viejo who died a few years ago, a wonderful endocrinologist that some of us knew and really loved and miss quite a bit. The idea was once – so GnRH, the structure of GnRH was a Nobel-agenic discovery, this ten amino acid peptide that actually regulates the whole reproductive axis was pretty exciting. When people figured out the three dimensional structure of that peptide then they started to be able to screen for small molecules because peptides are not so easy to administer clinically. That is why these things are sub-q and long term injections or nasal sprays, things like that. The idea was to develop chemical compounds, small chemical compounds that could be orally active and there are a couple of those that are available now under study. Elagolix being, I believe, the first one to be analyzed. These are very good drugs, very potent, actually I think they are going to be pretty simple to administer. I think there is going to be a lot of interest in people taking an oral medication rather than getting a monthly injection or three-monthly injection. But the mechanism of action I do not think is going to be a whole lot different and some of the side effects I think are not going to be dramatically different. What is exciting to me about the oral GnRH antagonists is that they might be titratable so women can maybe take enough to get symptom relief but have some markers so they can dose adjust. I know when I have talked to the pharmaceutical companies about that they get a little bit nervous because they would rather have something that is simple for people to take and they can follow the directions. Having people do a lot of really creative things with their medications I think makes them, probably appropriately, a little bit nervous. But for me that is one of the things that seems like it would be really quite an advantage to that that you might over the currently available things. Again, I think that the side effects of hypoestrogenism, bone loss, those kinds of issues are going to be fairly similar, even though it is oral rather than a subcutaneous route or whatever.

 

Thank you so much!