For years, researchers and clinicians thought that heart valve disease was simply more common in men than in women, but recent research has identified clear sex differences in the disease. Researchers are now probing the molecular mechanisms that drive these cellular-scale sex differences to identify the best drugs to give people based on their sex.
Host: Stephanie DeMarco, PhD, Associate Editor, Team Lead
Kristi Anseth, University of Colorado, Boulder
Kristyn Masters, University of Colorado, Denver
Brian Aguado, University of California, San Diego
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Transcript
Stephanie DeMarco: Hi everyone! Welcome back to a new episode of DDN Dialogues! I’m your host, Stephanie DeMarco.
In today’s story, we’re taking a trip inside the human heart. To get blood to the rest of the body, the heart pushes blood from the left ventricle into the aorta, the body’s main artery. But, to move out of the left ventricle, the blood has to pass through the aortic valve. Next to the pulsing cardiac muscle, the three flaps of the aortic valve look like tiny little leaves fluttering in the wind, but Kristi Anseth, a bioengineer at the University of Colorado Boulder, told me that there is much more to these tiny tissues than that.
Kristi Anseth: When you look at your heart valves, there's cells that reside throughout the whole leaflet of a valve, and they're embedded in a matrix. As our heart beats 60 to 80 times a minute, and there can be really large forces and pressure drops across that valve, so those cells sitting in that thin little tissue or leaflet are constantly having to repair and regrow that surrounding matrix.
DeMarco: To make general repairs to the aortic valve, the valve cells become activated and turn into myofibroblasts.
Anseth: When they're activated, that's their wound healing state. They're repairing the matrix and regrowing it.
DeMarco: But, the problem is, sometimes the valve cells get stuck in this wound healing state.
Anseth: So, they keep making more and more of this matrix, which makes the valve tissue stiffer and stiffer, and then that makes it harder for it to open and close, which causes more stress on your heart. Left unchecked, the disease progresses, and those cells can even begin to calcify and make a really hard tissue.
DeMarco: This hardening of the aortic valve is called aortic valve disease or aortic stenosis. While the condition is rare in people younger than 75, about 13 percent of people over age 75 have it. If doctors don’t catch it in time, the calcification of the valve can progress and be fatal within two to five years. While clinicians treat aortic valve disease with valve replacement surgery, there are no drugs that can slow or reverse the disease yet. Kristyn Masters, a bioengineer who studies aortic valve disease at the University of Colorado, Denver and Anschutz Medical Campus, told me more.
Kristyn Masters: Heart valve disease is so fascinating, in that it lags behind some of the other cardiovascular diseases in our knowledge because for a very long time, there were two important misconceptions about it. One, that it was just a consequence of aging, like, 'Hey, you get old, you know, like wrinkles, right? It's normal, right? So, there's this feeling that well, your heart valve's getting calcified because it happens to so many people. It was viewed as like, well, this is just aging. We now know that's not true. It's an active disease process.
Two, there was the thought that well, it's just like atherosclerosis. That was another misconception. It's not. Valvular cells are different from vascular cells, and so both of these misconceptions, though, really held back the field for quite a long time.
DeMarco: But for many years, there was another feature of heart valve disease that clinicians and researchers didn’t realize was incredibly important. Doctors noticed that while women got aortic valve disease, men seemed to get it more often. But when women did get diagnosed, often their disease severity was higher than their male counterparts. Something wasn’t adding up. It turned out that the path to new drugs to treat or prevent heart valve disease would start with many trips to the butcher shop and a single spark of inspiration.
Masters: So, I love telling this story because it involves two separate events of serendipity and following the science. When I was first a junior faculty, many moons ago, I wanted to make tissue engineered heart valves for the purpose of making healthy tissue replacements, as many tissue engineers want to do, and we were getting very frustrated because my students would come to me and say that all their cells are just forming calcific nodules. How are we ever going to make this healthy valve when we can't maintain healthy cells? And this was a significant problem. Students were in despair. Me trying to get tenure was in despair, and then we did have that “aha moment” one day, where it's like, you know what? We're making disease cells really well. Maybe that's what we should do on purpose? Maybe our goal here isn't to make a tissue engineered heart valve replacement, but rather a tissue engineered model of heart valve disease.
Our second serendipitous moment was after we decided to start studying disease, my students would come to me and say, “Okay, now none of the cells are becoming diseased.” And, you know, commence bang-head-against-the-wall moments. So then we really tried to tease apart all the variables. What's happening with these batches of cells? And so, almost on a whim, I said, “You know what, ask the butcher to separate the female hearts from the male hearts because these were from pigs, and so they did that. And then what we saw is that the batches from females did not calcify, and the batches from males did, and we went back, and we ran some PCR on our previous batches that we'd been using. It turns out all of those batches where my students had come to me and said, “They're not getting disease,” were all from female pigs.
DeMarco to Masters: Did you have any sort of inkling that there might be differences between male and females?
Masters: No, not at all. Actually, you know, the only sex differences that had really been noted at that point were that males got the disease more frequently than females. But lab research, sometimes you are trying to find anything, right? You're looking at the lot number of your FBS, you're looking at everything!
And you know, at the time, everyone doing valve research, 100 percent, we were all just getting mixed-sex bags of hearts, basically from the butcher. Or, you know, if you were getting human ones, you weren't really reporting it by sex because cellular-scale sex wasn't really a thing most people were thinking about back then.
DeMarco: Masters and her team published their observations of the differences between male and female heart valve cells back in 2012, and at first, they got a fair amount of pushback.
Masters: I then went out to conferences, and I'd be saying, “Everyone please sex separate your cells so we can actually compare results across papers! And you know another thing that came up was, is this just an in vitro culture phenomenon? So, was this only arising because we've pulled the cells out of your native environment and put them on plastic, and cells do crazy things on plastic. My response to that was A) well, we're all putting these cells on plastic, so even if it's only in vitro we still need to know, because it affects how we interpret our results. B) We did end up doing experiments to show that it wasn't just an in vitro phenomenon as well. But, even if it had been, still super important to account for.
DeMarco: Soon enough, more researchers began to spot differences between male and female cells in heart valve disease.
Masters: A couple years after our paper, one of my favorite papers in the field was a clinical paper showing that women essentially get a different type of heart valve disease than men. Women tend to have more fibrosis, and men tend to have more calcification. And that was really validating on multiple levels. The cellular level things that we're seeing connect to actual clinical observations. I feel like that paper was a real game changer for the field because it opens up so many new questions about, are we talking about a different process that varies by sex? Do men and women start at the same point and then somewhere along the disease process, they diverge? Where are they diverging? Or because this does tend to happen in mostly older individuals, do we actually start at a different starting point? Is the aging process different. There's so many cool questions that opens up!
DeMarco: Masters and her team engineer hydrogels to model the heart valve tissue as a way to better understand this disease. As she and her colleagues have continued to study sex differences at the cellular level of aortic valve disease, they keep finding more examples of differences between male and female cells.
Masters: Pretty much every cellular behavior we look at, we find cellular-scale sex differences. A year or so ago, we published a paper on cellular-scale sex differences in the angiogenic behaviors of these valve cells, so how much they are secreting factors that will affect the formation of new blood vessels, which is pathological in valves. It turns out that extracellular matrix secretion by sex differs. A lot of what we're still putting out is somewhat phenomenological, so I can't tell you why they're different, but we're starting to dig into that a little bit more now.
I do frequently get the question of, is it sex hormones, and those do play a role, but they're not necessary for the sex differences. You know, I've talked to various people, they ask, is it an epigenetic thing? I said, probably! They say, is it what's called chromosome compliment? I say probably! So…
DeMarco: There are lots of potential explanations for the sex differences in heart valve disease, and it’s very likely that there are many contributing factors. To dive deeper into some of the molecular mechanisms behind these sex differences I spoke with University of California, San Diego, bioengineer Brian Aguado.
Brian Aguado: One of the central research questions of my research group is trying to understand how biological sex can contribute to the sex dimorphisms observed in valve disease. It's a personal motivator for me. My grandma, right around the time when I started my postdoc research, she passed away from aortic valve stenosis, and she was one of these patients that was under-recommended for valve replacements simply because of male bias cut offs for deciding whether or not a patient receives a valve replacement. So, the issue of gender inequities in medicine hits close to home to me, and this is why I've set the mission of my laboratory to really start to address gender inequities in medicine through our research.
DeMarco: Like Masters and Anseth, Aguado uses hydrogels to model the physical environment of the aortic valve to study the disease. He and his colleagues were studying male and female valve cells using this system when they made an exciting new discovery in the mechanisms involved in these sex differences.
Aguado: When we were sex separating our cells and given the same exact microenvironmental input, we were observing sex differences in cellular behavior, which suggested an intracellular mechanism. So that intracellular mechanism could have been two things: so, since we use human samples, chronic exposure of differing hormone levels on the body could lead to different epigenetic changes within the cells. The other explanation could be X chromosome and Y chromosome dosage. So, from an experimental perspective, I decided to start to investigate how sex chromosome biology might be contributing to these sex dependent effects.
DeMarco: Aguado and his team performed transcriptomics on the activated male and female valve cells, and to their surprise, in the female cell sample, they saw increased expression of a subset of genes that are known to escape X chromosome inactivation.
Because female mammals have two copies of the X chromosome while males only have one, each cell in a female body inactivates one of its X chromosomes. This ensures that females have a similar gene expression dosage to males. But, there are about 15 percent of genes encoded on the X chromosome that escape inactivation, meaning that female cells express these genes at higher levels than males.
Aguado: With that discovery showcasing that there are specific subsets of genes coded on the X chromosome that are also upregulated in female myofibroblasts, that gave us a catalogue of targets that we could then intervene with traditional silencing techniques to assess whether or not these genes had an impact on myofibroblast signaling, which they did.
We chose inhibitors that are currently being used in different types of cancer treatments, so I know that these drugs have been used clinically in the past. The exciting thing about these drugs is that if these drugs can work in combination with other drugs known to target different myofibroblasts activation pathways, then combination therapies might potentially be a path forward for treating fibrosis versus calcification in the valve tissue. We're trying to target earlier stage valve disease where there could still be potential for inhibiting myofibroblasts activation to prevent substantial fibrosis and calcification.
DeMarco: Now, Aguado is diving deeper into chromosome biology to understand these sex differences further.
Aguado: By and large, I was surprised at how robust the sex-specific phenotypes were. If you knock down a X linked gene that escapes X chromosome inactivation, the effect on the cells was unique to the females. Some early work that hasn't been published yet is that when we knock down genes on the Y chromosome, the effects are male specific, so we're starting to tease out how sex chromosome genes impact overall cell signaling of a variety of different canonical signaling pathways. We've honestly just opened up Pandora's box of all sorts of different interactions between these sex chromosome linked genes and overall signaling networks.
DeMarco: So many more questions remain about how sex influences heart valve disease, but Anseth, Aguado, and Masters as well as their colleagues can’t wait to find answers.
Anseth: We're looking at drugs that could target why these cells get stuck in their wound healing activated state. Once it gets stiff, and it senses this environment, it causes it to activate more. So, we're learning which drugs could be used to get those cells to think they're in a soft, healthy environment and to stop making more matrix and making it stiffer.
Masters: The current grant that I have is looking at the intersection of sex, sex hormones, and age, in terms of what are the influential factors in aortic valve disease onset? It would honestly make sense if men and women start at, at least slightly different starting points because sex hormones, like estrogen, are really influential in our cellular processes and the way our tissues look.
I think this is where the power of in vitro models can really come in because in any in vivo model, it's really hard to separate these factors. Like, I can't have an animal that has young cells and old extracellular matrix. You can do any wacky combination you want in an in vitro model, so if I want something that has old tissue stiffness and cells, but young hormones, yeah, I can do that. It's not just combining things for the sake of combining things. It's that you can start to isolate these variables and their independent effects. In particular for women, with aging when we are simultaneously aging and having this really dramatic change in our biochemistry, essentially, what is driving disease development more?
Aguado: Inflammation is associated with the progression of a number of different diseases, including aortic valve stenosis, and we've collaborated with the CU Anschutz Medical School in addition to the UC San Diego Cardiovascular Center to collect blood from aortic valve disease patients, and use their serum as a tool to culture our aortic valve cells. We have found that the myofibroblasts activation levels can be quite different from patient to patient and correlate to patient measures of disease severity. So, what's really interesting is that we found that patients with more severe stenosis might have a different inflammatory signature relative to patients with less severe stenosis. And when you collect bloods from these patients and use the serum to culture the cells, the cells respond based on disease severity, so we think that using serum as a tool can start to bridge in vitro disease modeling with in vivo patient outcomes. We're doing a large cohort study right now where we're trying to collect serum samples for male and female patients to identify sex specific inflammatory signatures that might lead to sex-specific valve disease.
I'm still just completely fascinated by the idea that these sex chromosome-linked genes can have such potent impacts on how cells do anything in culture, from how they respond to a hydrogel environment, how they respond to drugs, to how they interpret inflammatory factors. There's just a whole host of things that I can't wait to see where our results take us.
DeMarco: That’s it for this episode of DDN Dialogues. Thank you so much to Kristyn Masters, Brian Aguado, and Kristi Anseth for talking with me. And thanks to all of you for listening! Until next time, I’m your host Stephanie DeMarco.
This episode of DDN Dialogues was reported, written, and produced by me with additional audio editing by Jessica Smart. To never miss an episode, subscribe to DDN Dialogues wherever you get your podcasts. And if you like the show, please rate us five stars and leave a review on your favorite podcasting platform. If you’d like to get in touch, you can send me an email at sdemarco@drugdiscoverynews.com.
And if your experiments are giving you trouble, consider the sex of your cells — it may just make all the difference.
