Endometriosis Foundation of America
Medical Conference 2019
From Biomarkers to Precision Surgery
March 8-9, 2019 - Lenox Hill Hospital, NYC
So, I feel like I'm standing on the shoulder of giants, and I really want to say thank you for asking me to be a part of this and asking me to be here. And I also want to say that the article that the Endometriosis Foundation published about two weeks ago on my story of why I'm doing what I'm doing has made me famous. It's unbelievable. So thank you. The first thing I want to talk about is how we in my lab, really think about the initiation and development of endometriosis. And so we think about it as an initiation phase, which is immune predominant. So there are two different phases that we think about, the initiation phase, and then the progression phase. The initiation phase we think is really predominantly due to the neutrophils and the macrophages. And then the proliferation is due to hormone dependence.
But within all of this, this is all due to an interconnected series of stages through attachment, development of a blood supply. There are patterning and immune modulation. Because as we notice, we start out with tissue that doesn't really look very glandular when we first begin in developing these lesions. These reorganize and develop what is known as then the proliferative lesion, the established lesion, and then these respond to hormones. So probably not all of you think about environmental toxicants all day as I do. So I wanted to give you a little bit of a definition. And we think about endocrine disrupting chemicals and how they act on the endocrine system by mimicking, blocking and/or interfering with the natural hormone system.
And secondly ... oops ... secondly thinking about immunotoxicants. These are chemicals that act on the immune system by disrupting the normal processes and functioning of the normal immune regulation. So that leads us to our question. How do environmental toxicants that are in our environment ... and as you noticed, my water bottle that I bring with me has no plastics in it, and I'm going to show you that on purpose. So, the first chemicals that we decided to look at are the bisphenols. So, bisphenol A and bisphenol AF. So the structures are shown here but ... oops ... I'm not good at this. So typically, years ago, three and seven were the plastics that showed the high levels of BPA. And these were used to make the clear, hard plastics.
And years ago they found that BPA was more abundant in the serum with endometriosis. However, what is now being called regrettable substitutions, in your hard, clear plastics, we're finding that these things are not BPA free. They are BPA free but they're not bisphenol free. And they're replacing them with other compounds that may be more estrogenic than their parent compound. So, in the literature, they're called regrettable substitutions. And some of my work that I did as a postdoc, we looked at a whole series of environmental endocrine disrupters and found the BPAF was one that was much more estrogenic than it's parent compound.
So we used a mouse model in my lab, where we take a uterus from a mouse that is GFP positive. We remove that. We take the uterus out, carefully peel away the outer myometrium. We split the uterus longitudinally, and then mince it into tiny little pieces. From there then, in this experiment, we are using host mice that were overactive mice so they have no ovaries. So the only chemicals that we see then that are able to act estrogenically are what we give them. We also use a mouse model where they have the intact reproductive system and a complete HPG axis. We then treat with the environmental endocrine disrupters for six weeks. And then what I say is we do a scavenger hunt to try and find where those lesions are in the mouse.
So, we first hypothesized that BPA and BPAF are able to act as endocrine disrupting chemicals on the development of endometriosis or promotion of endometriosis. So to do this, we wanted to make sure that we were within limits that are presented by the EPA. And the EPA publishes the no-observed-adverse-effect level, which is the NOAEL. And that is at 5 mg/kg per day. And the LOAEL is published at 50 mg/kg per day. So our diets are here at 3 mg/kg per day, 50 mg/kg, and then 90 mg/kg. And then based on a series of publications that have been very well documented, so that human exposure levels are completely within this range. So we are trying to demonstrate that we're getting the right doses.
So the first question we asked is, because if the BPA is acting as an environmental immunotoxicant, we would potentially see, or likely see a higher number of lesions that are formed. So one of the things that I want to point out, is looking at ovariectomized model versus the intact model, looking at the lesion numbers per mouse. This is our vehicle. Our BPA doses, our BPAF doses, and then our ethinyl estradiol dose. We actually find that there's no change in lesions. Then, we see a whole completely different story, when we ask if these are endocrine disrupting chemicals. So looking at the ovariectomized model, looking at lesion volume as a surrogate for the growth of these lesions, we find a classical non-monotonic dose response for BPA. And we see a significant increase with BPAF at 30 and 90 mg/kg, which is very similar to the ethinyl estradiol.
And surprisingly with our intact model, we see BPAF at lower levels than the lowest-observed-adverse-effect level. Show that we see a distinct progression of the disease. So from this, I want to just summarize and say that BPA and BPAF act differentially based on the hormonal status of the mouse. BPAF does seem to have stronger estrogenic effects. And the relevant human doses that we are probably exposed to have an effect on disease. So the next thing I wanted to point out is phthalates, and this is going to be a greater portion of the talk because this is what has been funded by the Endometriosis Foundation. So phthalates are also known as plasticizers, and they are found to make plastics more mobile and more movable. And they are found ... also a very nice study that was done by Pat Hunt's group and in 2016, shows that there are higher levels of DEHP in women with endometriosis.
They're found in a number of different places, and you can read them. But what is completely shocking to me, is that the FDA does not regulate anything that has to with cosmetics, lotions, shampoos, those types of things. And DEHP can be listed in ingredients as a fragrance. So what are these fragrances in? Products that are mostly used by women. So there's a very nice paper that shows that women after they've taken their shower, done their grooming processes, have a really high spike in the level of DEHP in their serum. So more importantly, DEHP has been known to be an endocrine disrupter, and also an immunotoxicant. So with this model, and based on some of the data that's already been shown today, I don't have to preface it as much as I thought I would, is that, we hypothesized that DEHP is acting as an immunotoxicant through IL6-mediated trans-signaling in the mouse model to look at the development of disease.
So the first question we wanted to ask with DEHP is, does DEHP have an effect on the uterine weight? Because uterine weight is also a surrogate to look at estrogenic properties. And we're finding that there is no significant difference in the uterine weight. We also looked at mouse weight, because we wanted to make sure that the dose we chose for DEHP was not toxic. And one thing I should point out is that the dose of DEHP that we use does seem high. However, mice metabolize DEHP much more quickly than humans. So we had to make sure that we had a five to eight-fold higher dose of DEHP in the mice than we would see in humans. So, looking at what happens when we treat mice with DEHP and endometriosis, we see that there's a significant increase, about a two-fold increase in lesion weight with DEHP.
But here's my favorite and most exciting piece of data for me, to be able to dig into this deeper is, that lesion number in this mouse model significantly increases with DEHP. So we want to ask, why does this happen, and what happens. So first, initially one of the things that we wanted to look at is to look at the uterus of these animals that had been on DEHP for six weeks. And looking at, this is the message level of interleukin 6, and these are genes that are all we've talked about already today that are involved in endometriosis. And then lactoferrin which is a classical estrogen receptor target gene, to show endocrine disrupting activity. So we're seeing a significant increase in the levels of IL6 in the uterus, about a 20-fold increase in COX-2 or prostaglandin synthase. No change in the MMP9, but about a 10-fold increase in MMP7, and no change in lactoferrin.
Now, if we look at the lesions that are the lesions that we removed from the same mouse, we see that there's differential regulation of IL6. We also see then an increase in the lesions themselves with COX-2, and that is augmented with the EHP. We see a very high level of MMP9 and the same with MMP7. And now what's interesting is, one of the things that we are also looking at is the lactoferrin. And lactoferrin is also involved in secretions, and we know that these lesions are definitely secreting things. So this is something we are definitely looking into. The next question we wanted to look at and to dig deeper was, how is DEHP affecting the IL6 signaling. So IL6 signaling is very complex. It's both pro and anti-inflammatory. And it signals through classical signaling which is in white blood cells and hepatocytes.
So what's different about these cell types, are they actually have the IL6 receptor that is membrane bound. Whereas trans-signaling happens in all cells. Because all cells have gp130. And then what's really interesting is in, when white blood cells actually die, there's cleavage that occurs, and you have soluble IL6 receptor that is secreted. So IL6 itself binds to the soluble IL6, and that mediates through signaling cascades inflammation. And we potentially want to ask about lesion formation. Whereas with classical signaling, this leads to an acute response and tissue clearance. As you see here, I have this balance. The balance is very important for tissue homeostasis and disease recovery. And so what we think, is that with DEHP we're pushing more towards this end of the spectrum.
So as [inaudible 00:12:55] mentioned, and some of the work that Bruce mentioned, they look at protein STAT3, and also then the IRK 1-2 pathway and the MAP kinase pathway. So looking at STAT3 phosphorylation in these uteri, and if we think about it, and we follow the retrograde menstruation hypothesis, this would be the tissue that would be flowing back into the peritoneal cavity. We see that now STAT3 is phosphorylated. And we see a huge increase in the phosphorylation of p44/42. Okay, so you may ask, "Okay fine, they both signal through different kinase signalings, who cares?" But we wanted to then look at specifically the IL6 receptor, and then the soluble IL6 receptor. And this a relatively messy blot. But, what happened is, we see similar levels of IL6 receptor, but we see a large increase in the soluble IL6 receptor alpha.
And more importantly, then, gp130 acts as a complex. That complex is gained in the ... and most treated with DEHP. And we actually lose then the soluble gp130 complex. And the soluble gp130 complex is thought to be inhibitory in this whole process to be a protective mechanism. So moving back and going to look at 48 hours after the initiation of disease, because if we think that this is acting as an immunotoxicant, we want to look early in the disease and what's happening in these lesions very early. So these experiments were done at 48 hours. We're looking again at the uterus, this is IL6. But now look at this response for IL6 in the early lesion development. It's now about a 300-fold increase. And this is further augmented with DEHP.
So we wanted to ask what cell types are being recruited. And that's one of the things that we looked at with the neutrophils and the macrophages. So overall, looking at the cells that are recruited into the peritoneal cavity, we find that with sham animals, and these are animals that had operations but were just injected with PBS, we see, just with the EHP alone, that there's is a different milieu of immune cells that are in the peritoneal cavity. And as we saw before and published before, there is a significant increase with endometriosis. But this actually did not increase with the DEHP. Looking specifically then at neutrophils which are Ly6G positive cells, we again find that there's a significant increase with DEHP. And this is where I'm really excited.
Because we see a large increase in endometriosis as we've seen before. But this is further augmented with DEHP. Now, data that we have that's too preliminary to put on a slide, but we're looking at specific types of neutrophils which are proangiogenic or proinflammatory. And we're actually starting to see that there are differences in those as well. Also data that is somewhat too preliminary to go into detail with are macrophages, but I'm just going to show you the monocytes. Because the monocytes are typically the monocyte-derived macrophages that lead to these cell types. So we're looking just at CD115, and we're again seeing very similar trends to what we saw with the neutrophils. Suggesting that there's a high potential that both of these are being misregulated and important in these lesion developments.
So hopefully I've been able to show that DEHP increases the lesion weight and the lesion number. DEHP increases the uterine and lesion inflammatory secretory and extracellular matrix factors that are important in endometriosis. And we've demonstrated a potential link between DEHP exposure, altered inflammatory signaling, and this increased lesion development. And with that, I want to make sure I thank the people that have been involved with this work. The people that are in my laboratory, my technician is Stephanie Lang. And I've had a number of ... a postdoc and also an REI fellow who have come through the lab, and some masters degree students. I started this work all in my postdoctoral laboratory with Dr. Ken Korach, and I'm collaborating with Romana Nowak's laboratory who's also looking at pain sensations with DEHP. and the funding sources, and again the DEHP work has really been supported by the Endometriosis Foundation. So thank you.