Daisy Shu, PhD
Postdoctoral Fellow at Schepens Eye Research Institute at Harvard Medical School, Boston, MA, USA
Daisy Shu received her BOptom and BSc degrees from the University of New South Wales (UNSW) Sydney, Australia in 2012. She then worked in clinical practice for two years before starting her PhD in Ophthalmology at the University of Sydney, Australia under the supervision of Professors Frank Lovicu and John McAvoy. Her research explored growth factor signalling pathways activated during the formation of fibrotic forms of cataract with a focus on transforming growth factor-beta (TGFβ)-driven epithelial-mesenchymal transition. She is currently a postdoctoral research fellow at Schepens Eye Research Institute, Harvard Medical School, Boston, MA in the Magali Saint-Geniez Laboratory studying the role of TGFβ in retinal diseases. Daisy currently serves on the Association for Research in Vision and Ophthalmology (ARVO) Advocacy and Outreach Committee (since 2018). Daisy has presented at numerous local, national and international conferences including ARVO, American Academy of Optometry Annual Meeting and the International Society for Eye Research (ISER). She is a recipient of American Academy of Optometry 2018 Irvin M. Borish Ezell Fellow, The University of Sydney John Irvine Hunter Prize for best publication in 2018, Australian Society for Medical Research (ASMR) 2017 Best Student Oral Presentation Prize and the ARVO 2016 Members-in-training Poster Prize in Lens. Daisy is passionate about science communication and outreach. She was part of the first cohort to complete the ARVO Science Communication Training Fellowship in 2017. She is a co-host of “The Peer Review”, a podcast about science, research and academia. She successfully crowdfunded her PhD research project on cataract formation and prevention on Experiment.com in 2017. She enjoys updating her followers on her science adventures on social media via the handle @EyeDaisyShu.
Learn about Daisy’s research
Title: Utilizing the lens epithelial explant culture system to investigate cataract formation
- Understand basic ocular lens anatomy and how disruption of this anatomy can result in lens pathology known as cataract
- Understand how lens epithelial explants are generated and how they can be applied to explore lens epithelial cell behavior and how cataract forms
- Understand the role of transforming growth factor-beta (TGFβ) and its downstream signaling pathways in cataract formation
Cataract, a clouding of the ocular lens, is the leading cause of blindness worldwide. Currently the only means of treatment is through surgical intervention. Given the sheer prevalence of cataract worldwide, surgical intervention places a significant financial burden on the health-care system. Hence, there is a need to develop pharmacological treatments to maintain the transparency of the lens. This webinar will explore the molecular and cellular basis of how cataract forms with a particular focus on the role of transforming growth factor-beta (TGFβ) and its downstream signaling pathways. The Lens Research Laboratory at the University of Sydney led by Professor Frank Lovicu seeks to unravel the complex interplay of growth factor signaling pathways involved in the formation of cataract and in doing so, find novel drug targets to combat cataract. The lens epithelial explant culture system was developed in the Lens Research Laboratory in the 1980s and has enabled the discovery of many now well-accepted phenomena about lens epithelial cell behavior. Using this model, observations can be made while primary lens epithelial cells are adherent to their native basement membrane, known as the lens capsule, thus enabling a closer representation of the in vivo situation compared to. This webinar will explain how lens epithelial explants are generated and utilized for experiments.
Watch the webinar
Moderator: Hello and welcome everyone. Thank you for joining us for today's webinar, "Utilizing the lens epithelial explant culture system to investigate cataract formation," presented by Daisy Shu, Ph.D. student at the University of Sydney, Australia. I'm Christy Jewel of LabRoots and I'll be your moderator for today's event. (00:25) We're delighted to bring you this educational web seminar presented by LabRoots and sponsored by Thermo Fisher Scientific. To learn more about our sponsor, please visit ThermoFisher.com/CellCultureHeroes.
Before we begin, I would like to remind everyone that this event is interactive. We encourage you to participate by submitting as many questions as you want at any time you want during the presentation. Simply click on the Ask a Question box, type in your question and click Send. We'll answer as many questions as we have time for at the end of the presentation.(01:00) Additionally, if you are viewing this webinar on-demand, you may continue to submit your questions and they will be answered via email through the contact information you provided at the time of registration.
If you have any trouble seeing or hearing the presentation, please use the Ask a Question box to let us know you're experiencing a problem. This presentation is educational and thus offers continuing education credits. Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits.(01:31) Our speaker today is Daisy Shu. For a complete biography on your speaker, please visit the Biography tab at the top of your screen.
Now without further ado, please join me in welcoming Miss Daisy Shu. Daisy, you may now begin your presentation.
Daisy Shu: Okay, hi everyone. Thank you so much and to Christy and Chelsea and Gibco Cell Culture Heroes for giving me this opportunity to share my research with you all and in particular, share a really novel explant culture system that we use in our lab. So, that's the purpose of my talk today, is really to highlight how we create these lens epithelial explant cultures to study how cataract forms.
So, a bit of background about me—and you can read more about it in the link in the bio—but I am actually an optometrist by training and I worked as a clinical optometrist for two years before I started my Ph.D. And I am currently working on my Ph.D. revisions, so soon enough, I will have that Ph.D. which is really exciting. But I have already started work as a post-doc in Boston at Schepens Eye Research Institute, and it's quite a different sort of project in itself. (02:59) Like I'm looking at the retina now. But I know a lot about the lens, having dedicated four years to it. So, that's what I'll be talking about today.
So, firstly, I'd like to really highlight the anatomy of the eye, and that's something that we kind of—we probably don’t know much about, and I honestly had to learn all these different structures. And it's a very complex organ. At the front of the eye, you've got the cornea, which is a transparent tissue at the front. (03:34) At the back of the eye, you've got the retina, and that's really important for getting all the signals in our world to be converted to electrical signals for the brain to interpret. And inside the eye—towards the front part of the eye as you can see there—is a really beautiful structure called the lens. And that is actually really important for focusing images from our world onto the retina. (04:04)
So, if we didn’t have a lens and the retina still worked, we still wouldn't be able to see, because we really need the light rays to be focused onto the retina. And so, you can see here, in the zoomed up version of the lens, that the lens actually is quite a complicated structure. There are different cells and they're arranged in a really, really organized way.
There's the lens epithelial cells, that's at the front of the lens, and you can see that they have this cuboidal structure. They're like little bricks, if you will, that they form a monolayer of cells at the front of the lens.
Then we have lens fiber cells and they are actually differentiated forms of lens epithelial cells. And these lens fiber cells are really elongated and they form this curved shape, as you an see. And actually, the highly ordered architecture of these lens fiber cells, that's what allows the lens to be able to focus light onto the retina and also maintain the transparency of the lens, which is super important. As you can imagine, if you don’t have the transparent lens then you wouldn't be able to focus light onto the retina.
And now that's all encased within the basement membrane which is known as the lens capsule.And that goes all around the whole lens, at the front and the back, all around, and that encapsulates the lens. And that's really interesting because the lens is actually—all the cells within the lens never gets exposed to any other cell in the rest of the body. So, it's like it's its own thing inside the eye.
And in fact, if you were to put a hole in the lens capsule and all of a sudden, the other ocular tissues got exposed to lens epithelial cells or lens fiber cells or those proteins inside the lens, they would probably not react very well to that. (06:07) Because that environment is very contained within the lens capsule, and that's actually really important to know about the lens, because it keeps growing and growing within that lens capsule and it never (sees) anything outside of the lens.
Okay, so now my research is all about epithelial cells. As you noticed, there were epithelial cells at the front surface of the lens. Now what can happen to these cells, as I described, they were kind of like bricks or cuboidal-shaped cells. What can happen is that they can actually undergo transformations into the cells called mesenchymal cells. Now that's a completely different cell types. (06:51) You're going from cuboidal square-shaped, brick-like cells into these spindle-shaped cells that are mesenchymal.
And what happens during that process is that the cells—as I described as bricks, they're like the epithelial cells—they have these really tight adhesions to each other. And they're also tightly adhered to the basement membrane which I've described as the lens capsule. That's known as the lens capsule.(07:17) So, they are really solid in where they are. But when they differentiate into mesenchymal cells, they transform into these really irregular strange looking cells that are spindle-shaped. They're elongated. They've lost their cell-to-cell adhesion, so they're no longer stuck to each other. And they've lost their cell-to-basement membrane adhesions, as well, so they're no longer stuck to the lens capsule. And they also start to secrete what is called extracellular matrix, which is...it's what the basement membrane is made of, as well, but it's basically like a scar tissue, to be honest. (07:53) It's spindle-shaped cells mixed with this extracellular matrix.
This process is known as EMT, epithelial to mesenchymal transition. And a really important growth factor that is known to induce this EMT process is called transforming growth factor beta. And you may have heard about this in a lot of cancer research, because it is a really hot topic right now. TGFβ-induced EMT is actually a really prominent cause of why cancer spreads to different parts of the body.
So, there's three types of EMT. There's Type 1, which is embryogenesis. So, that's when we're kind of in the womb and we're developing into the complex human beings that we become eventually. But basically, if you can imagine, we start out as a single cell. So that needs to actually become all the different types of cells that we are today. (08:57) So, in order to do that, it needs to actually undergo this EMT process. So, that's actually a good type of EMT, where we're becoming more complex organisms by having an epithelial cell differentiate into mesenchymal cells. So, that's good.
But there's also Type 3 EMT, which I described as metastasis, which means the spreading of cancer. And what happens there is that—so, say you have an epithelial type of tumor. (09:29) So, for example, like an epithelial type of breast cancer—and that cancer wants to spread to a different part of the body. Well, in order for it to move to a different part of the body, it has to undergo EMT. It has to break away from the basement membrane, break away from all the cells that they're attached to and actually migrate to a different part of the body. And mesenchymal cells are cells that can migrate, whereas epithelial cells just stay put. So, that's why EMT is so important when cancer wants to spread to a different part of the body. (10:02) So, that breast cancer could potentially be a brain tumor, for example, and it needs to undergo that EMT process.
Now my research is looking at fibrosis, which is the fancy way of saying scarring. So, when you think about it, like if you cut your hand and you get a really deep wound, then your skin can scar and that can be like a permanent thing. And the scar tissue, as you see, it's often quite shiny and that's that extracellular matrix I was talking about. (10:33) That's actually really important for closing up the wound. So, obviously, you made a big cut and so the cells will start to transform into these mesenchymal cells, because they want to close up the wound. So, they need to migrate to the wound site and kind of contract, and that contraction process allows the wound to seal up. (10:56)
But then in addition to that, the cells will secrete that extracellular matrix and that’s kind of like padding, if you will. It's basically kind of like a little bit of a band aid situation,and that shiny stuff is there to really heal up that spot that's open and basically close it up so the skin is protected. So, that’s in the context of the skin. Now I'm going to talk about that now in the context of the lens.
And this is a paper that I published. So if you're interested in learning more about TGFβ-induced EMT in the eye, this is a really great paper to look at. Because I pretty much talk about how TGFβ-induced EMT isn't actually just relevant to the lens—which is the topic of this talk today—but it's actually important for all different types of ocular tissues. As I described, it's important in the cornea. (11:55) It's also important in glaucoma, which is another eye disease. It's important for the retina, and that's actually currently kind of what I'm researching now in my post-doc. So, if you're interested in that topic, do check that paper out.
So, back to the eye and the lens. So, I pretty much described the lens epithelial cells. Now what happens is they can transform into the spindle-shaped mesenchymal cells. And as I described, instead of having these really perfectly cuboidal cells, we have this mass of mesenchymal cells.
And I've depicted that as a red circle, but that's basically a cataract. So, if you think about it, if we have a scab, like a scar on our skin, that is not too bad. It's maybe cosmetically not that attractive, but it's not too bad for the function of the skin. If it's on your hand, it's still going to pretty much work. But if you have a scab in your eye and particularly in your lens, that's not a good thing. (13:06) Because you won't be able to see anymore, because the purpose of a lens is to maintain that transparency, so that it can bend light, to be focused onto the retina. And if light can't even get through the lens, then you won't be able to see.
And so that's why when it comes to scarring in the lens, research into that is really important, because we really want to figure out how we can maintain that transparency. And one such cataract that is caused by EMT is called an anterior subcapsular cataract. And that's the basis for a lot of the work that I do.
This is a picture of a patient with an anterior subcapsular cataract. You can see that it's pretty much localized in the A panel, you can see that it's dead in the center of the eye. And that (plaque) so when light hits, obviously it's going to hit in the middle of the eye and the cataract is right there, so that's not a good thing. And the picture in Panel B is basically showing how light is hitting it and it's getting all scattered. (14:14) And that's what happens when people have these types of cataracts is that light just hits the eye, but it doesn't translate into any sort of useful visual image.
So, another type of cataract is an interesting one. It also is involved in the EMT process. It's something that happens after cataract surgery. So, the good thing about cataracts is that there is a treatment for it. You can remove the cataract. But the problem with this surgery is that there's complications. As with all surgeries, something can go wrong later down the track. So, the surgeon will basically remove the massive lens fiber cells and they'll only remove a circular portion of the front part of the lens.
So, you can see there are some cells left behind. Then we'll implant an intraocular lens into that and that provides the focusing ability. So, it replaces the function of the lens, so that light can still be focused onto the retina.
But what happens is the residual lens epithelial cells that are left behind at the (equator)—and as hard as this surgeon wants to try to remove all the lens epithelial cells, it's impossible. There's always some cells left behind. And what happens to these cells is they undergo the EMT process.
And they start to migrate and form these cataracts onto the back of the lens. And you can see an example of that, that's basically all these little cells that have formed a secondary cataract on the back of the lens and sometimes also on the implant. And that requires a secondary laser procedure to remove. So, research into EMT and the lens is really trying to tackle these two types of cataracts, posterior capsular opacification and interior subcapsular cataracts. So, we're trying to figure out how these form and by doing that, we can pretty much find a novel drug to block these processes.
So, the way in which our lab looks at these processes is using a culture system that is a primary culture system and it's called the rat lens epithelial explant system. And what we do is we take 21-day-old Wistar rats and we isolate their eyes. We take their lenses. (16:49) We tear open the posterior capsule, so the back of the lens.--so we don't tear at the front, because that's where all the precious lens epithelial cells are. So we tear that open and what we do from there is we pin down this monolayer of cells onto a 35-mm culture dish. And you can see basically an example of that, we've got two explants pinned down on a 35-mm culture dish. (17:24)
And I've now got some videos that I'd like to show you, and they basically depict what it's like when I'm at the dissecting microscope. It is a bit of a challenge to really kind of figure out how to move your hands while you're looking through a really magnified view through the microscope. So, this technique actually took me, I think, probably like six months to really get a handle of. So, it does take a bit of time to really perfect the formation of these explants, but once you get it done, it's really exciting. (18:00) And the lenses are—I've got a video in there, as well, of the lenses and they're really, really pretty. They're transparent little gems, I would—like they're really pretty. So, we'll play the video now, so you can see what it's like.
[VIDEO PLAYS FROM 18:21 TO 18:52]
Daisy Shu: [on video] And this is the microscope that I use, and you can see that I've already got some lenses that I've prepared and they look really cool. They're really cute lenses. [INTERFERENCE]
Daisy Shu: Okay, great. I hope you enjoyed that video.
So I'm going to move onto just showing you a paper that if you want to learn more about creating lens epithelial explant systems, this is a really great paper. And my supervisor, Frank Lovicu, contributed to this. And it's just a really great way to summarize exactly how these explants are created and how they can be useful.
Alright, so back to this diagram. Now what we can do with these explants is we can add different growth factors into the pink media. As you can see, the cells are pinned down and we put this pink culture media into it. And from there, we can basically add different growth factors or inhibitors of these growth factors and observe how these lens epithelial cells change over time. (19:58) So, we've got TFG-β now, and as I've described TFG-β is a really important and very potent inducer of EMT.
And these pictures will demonstrate that. So, in the control state—so if we zoom in on these little, tiny circle explants here, if we zoom in on that with the phase-contrast microscope, you can see that the cells form a really beautiful monolayer of cells. They're like really organized. They are what we describe as a cobblestone-like arrangement. So, it's a very nice sheet of cells. (20:33)
And just below that image is a confocal microscopy image labeling for alpha smooth muscle actin. And alpha smooth muscle actin is actually a protein that is upregulated when you have EMT. So, if the cells become mesenchymal, they will start to express alpha smooth muscle actin. And that's really important for the contractile ability of the cells. (20:59)
So, alpha smooth muscle actin is actually also found in the heart. So, you can see some similarities there. There's a lot of contraction going on when the heart is pumping blood. And surprisingly, these contractile cells get upregulated in the eye, in the lens in fact. So, that's what I'm going t show you now.
So, we've got a TGF-β added to these explants, and you can see that they're no longer cuboidal and organized. They're starting to elongate into these spindle-shaped cells and α-SMA, that gets really significantly upregulated. And the really interesting thing is that α-SMA gets incorporated into the stress fibers. So you can see that there's lots of lines of α-SMA and that's really important for its contractile properties.
So, the thing is with my research, it's all about how TGF-β does this EMT process. And what's really interesting is that TGF-β isn't the only growth factor that is in the eye. There's actually lots of different growth factors that float around in the eye.
And these are just a few of them that I found in the eye. And so, my Ph.D. project is really trying to figure out how these different growth factors interact with TGF-β. Because in the in vivo situation—so in our actual eyes—we're not just really studying TGF-β in isolation. (22:33) There's actually lots of interactions there. So, that was my question. How do different growth factors impact on TGF-β? So, today I'm going to start with EGF and then I'll move onto BMP later.
So, the interesting thing is that what I've found is that the growth factors that float around in the aqueous humour—which is basically the liquid inside our eye—that they actually do different things to TGF-β. So, firstly, EGF which is epidermal growth factor, it appears to augment the effects of TGF-β. So, these are the images I showed previously.
And so, you can see now that if we add EGF to the mix, you can see that α-SMA is getting upregulated, but you can see in the phase contrast that more of the cells are looking elongated. So, we've got, instead of just a few cells elongating to these mesenchymal cells, you've got lots more. And that was really intriguing. So, the question really was how is it doing that?
So then I looked at—so this is just to confirm—we looked at alpha smooth muscle actin, and you can see that it goes with TGF-β. This is a western blot and a western blot just looks at total protein and looks at whether or not it's there or not there. Whereas the immunofluorescence is really useful because if it's there, we can see what kind of pattern it takes on. And as I described, α-SMA does take on that stress fiber like pattern. (24:14)
But with the western blot, we can really quantify things. So, that's why it's so useful to do a western blot, as well, because then we can get some statistics on whether or not this is a significant difference or not. So, you can see if we don't have any growth factors—no EGF, no TGF-β—then there's pretty much no α-SMA. So, that's the control state.
Now if we add TGF-β, you can see α-SMA goes up and that's what we saw in the immunofluorescence. Or if we add EGF and TGF-β, then we could see even more alpha smooth muscle actin. (24:44) And tropomyosin just below that is another kind of contractile protein, and that also showed a similar trend. So, really this is what this is telling us is that EGF—epidermal growth factor—is somehow exacerbating the effects of TGF-β. So, we're essentially seeing a more exaggerated cataract form.
So, the question really was how is it doing this? And so, that brings me to signaling pathways, and that's pretty much the gist of what I do. I look at signaling pathways inside the cells. So, as you can imagine, the cells are kind of contained within this membrane. So, if something is outside of the membrane, how is it going to communicate a signal to the inside of the cell, so the cell can do something about that signal? (25:35) And so, obviously those growth factors like TGF-β outside of the cell, how does it actually impact on the cell in the end?
Well, the really cool thing is these cells have these things called receptors that are stuck onto the membrane. And TGF-β can bind to (inaudible) receptor, which is the TGF-β receptor. There's Types 1 and 2 and from there, it can activate that receptor. (26:01) And then from that activation of the receptor, it can then activate different signaling pathways inside the cell. So, TGF-β doesn't ever go inside the cell, it just activates a receptor at the membrane of the cell. And then you get all these crazy things happen, so eventually ending up in the cataract.
So, TGF-β activates these SMAD signaling pathways. And that's a really funny name, I know, when I first learned about this, I definitely thought SMADs sounded really weird. (26:34) And in fact, what it stands for is Small Mothers Against Decapentaplegic, I think. So, that's also even stranger. But the SMAD pathway is actually the canonical pathway for TGF-β. So, it gets activated and in particular, it activates SMAD2 and SMAD3. And when I talk about activation, I'm really talking about phosphorylation, so that's why there's a little P group that's stuck there. And so then that SMAD2 and 3 then forms a complex with SMAD4. That gets activated, too, and the whole complex goes into the nucleus, and that can activate different genes to be switched on or off. (27:12) In this case, it activates the EMT genes.
Now work in our laboratory also looks at different signaling pathways that aren't SMAD. So, if it's not SMAD, then it's called non-canonical. So, we've got ERK, which is the Extracellular Regulated Kinase, that basically that's a really important MAP kinase, which is activated by TGF-β. Work in our lab has shown that TGF-β can indeed activate that, and if you block ERK signaling, you can block the EMT process. (27:45)So, it's very important for TGF-β to activate ERK.
Now the question was what about epidermal growth factor receptor? Because we found that EGF—which is epidermal growth factor—can augment the effects of TGF-β, maybe epidermal growth factor receptor signaling is somehow involved. So, that was sort of our take on this. And nobody had looked at this before, so we were like, "Okay, we're not sure." But there was some studies in cancer research that showed that TGF-β could activate EGFR, so that kind of inspired it.
Alright, so what we found was really exciting. We found that TGF-β actually did activate the phosphorylation of EGFR. So, here we can see another western blot, labeling for phosphorylated form of EGFR and the GAPDH there. That's always there because that's a housekeeping protein, and so that enables us to know whether or not we had equal loading of our proteins. So, we always compare all the groups to GAPDH.(28:52)
So, with just TGF-β at one minute we don't see any phospho-EGFR, but at 18 hours we see an upregulation of EGFR signaling. And that was really interesting. So, TGF-β did indeed activate EGFR, but it was a delayed sort of response. Whereas EGF on the other hand, activated its own receptor signaling very rapidly at one minute and also at 18 hours. So, we can see here that something is going on, definitely that TGF-β does activate EGFR, but it wasn't immediate.
And then we introduced an inhibitor of EGFR signaling, so this will block EGFR signaling. And you can see here that if you add this into the mix, then TGF-β can't activate EGFR at 18 hours anymore.
And the other interesting thing we did was we looked at the gene expression of EGFR using a real-time PCR. So, you see in the control state, there's just a basal level of EGFR. But if you add TGF-β to the mix, then there's a significant upregulation of EGFR gene expression. So, this is all really interesting, because as I was saying, TGF-β is something that activates its own receptor and that then translates into all these things that change in the cell. (30:16) But why is it hijacking EGFR signaling? Because that belongs to EGF, if you think about it. It's something that EGF uses to activate certain receptors. Somehow TGF-β was activating EGF receptor signaling. So, that was very strange, but really interesting. So, the next question was okay, we know that TGF-β activates EGFR, but is it even important in the EMT process?
So, these are the images I showed earlier with the control. And then we add TGF-β, we get lots of α-SMA, which is that mesenchymal marker. Now what happens if we add the inhibitor of EGFR signaling with TGF-β?
You can see that we block α-SMA. So, we no longer see that EMT marker come up. And the inhibitor by itself just as another control; it doesn't induce EMT. So you don't see much α-SMA getting into those stress fibers there. So, we can see here that TGF-β requires EGFR signaling in order to get the EMT. So, it's actually really important to have EGFR signaling, so it's playing a big role.
So, now we're back to this diagram again, where I show that TGF-β activates SMADs, ERK, and now I'm showing it does activate EGFR.
And we did a whole series of western blots to look at whether or not there was some sort of crosstalk between SMADs, ERK and EGFR. Because we thought that there probably was some sort of network that formed between all these signaling pathways. They probably talk to each other.
And just to summarize all of that, I did indeed find that and I found that there was some sort of crosstalk and feedback loop between EGFR and ERK signaling. So, if you block ERK with OU126 (32:04) you reduce EGFR signaling. And similarly, if you block EGFR signaling, you reduce ERK. So, they were pretty much talking to each other,so that was really interesting.
And you can read more about it in this paper here about ERK-mediated EGFR signaling in TGF-β-induced EMT. That will take you through all the data which was quite complex.
Okay, so the next question was—okay, so I've shown that there's some sort of crosstalk between the non-canonical pathways, EGFR, and ERK. What about does EGFR talk to SMAD signaling, for example, which is the canonical signaling pathway?
So, what we decided to do was look at SMAD nuclear translocation. So, as I told you, the SMAD2/3 proteins actually have to—that forms a complex that translocates into the nucleus of the cell, in order to activate any sort of genes. So, at two hours—these are all images taken at two hours and these are immunofluorescence images that label for SMAD 2/3.(33:12) And you can see here that in green the SMAD 2/3 in the control state is not in the nucleus. And the nucleus is these black circles here. So, they're not in the nucleus, but if you add TGF-β at two hours, the SMAD goes into the nucleus. And that just shows that TGF-β is activating SMADs and that's going to change the genes and lead to EMT.
But what happens if you have TGF-β with the inhibitor of EGFR signaling? Well, nothing much happens. It's pretty much the same. (33:44 )It looks the same as TGF-β. The cells still have SMAD 2/3 in the nucleus. So, that was interesting.
So, we decided, okay, what about later, at 18 hours? Well, we actually found that at 18 hours, while the control and TGF-β look the same, if you have the inhibitor with TGF-β, then you don't see that translocation occurring. So, the cytoplasm, which is outside the nucleus, that now has the SMAD 2/3. So, that was really interesting. So, in order for TGF-β to activate SMADs and get that into the nucleus, you do need EGFR signaling. Because if you block that, then it's not going to happen. So, definitely some crosstalk between the canonical and non-canonical signaling pathways.
So, the next thing we decided to do was look at whether or not TFG-β could upregulate other EGF ligands. So, I showed this earlier, where EGFR gets upregulated by TGF-β. But you can see here with TGF-β plus the inhibitor—which is PD153035—if we introduce that inhibitor, we reduce TGF-β's ability to upregulate EGFR and may inhibit itself as a control. It doesn’t do anything.(35:04) It looks like the control.
And now what was really interesting is that heparin-binding EGF-like growth factor—which is part of the EGF family of growth factors and can activate EGF-receptor signaling—it was really highly upregulated by TGF-β. So, that was really interesting that TGF-β was somehow increasing another growth factor, in this case HB-EGF. And that could be blocked if you had the inhibitor, EGFR signaling and EGFR signaling, the inhibitor itself didn't do anything. (35:37) So, this really is showing off that there's something going on where TGF-β is upregulating EGF ligands, so that was interesting.
So, that brings me onto the next slide, which is looking at the family of genes, which is known as the Adams family. Not the kooky kind, but a different kind. They are a disintegrin metalloproteinase family.So, what they do is they cut things. They do what is called ectodomain shedding, and they basically cleave EGF ligands, so they can become the active form. (36:18)
So, sometimes when growth factors are floating around—because they can act so potently on cells and actually cause a lot of damage or good for the cell—sometimes they're not in their active form. So, that's called the pro form and they'll just float around and not be able to do anything, until they're cleaved. So they're cut in a way that will activate them into the functionally active form. (36:46)
And so, that's what happens to the EGF ligands. Like HB-EGF, they actually need to be cleaved in order to have the functional version floating around, to then activate the EGF receptor. So Adams do that, and I thought this would be a really great mechanism to tie together the TGF-β and EGF story.
So, I decided to look at all these different Adams. (37:12) There's actually a lot of them and you can see there's Adam 9, 10, 17, 19 that I looked at. But the most prominent result that I found was Adam19. You can see in the control state, there's not much there. And then that's really highly upregulated with the addition of TGF-β. And that gets suppressed all the way down to basal levels, when you have the TGF-β plus the inhibitor of EGFR. And EGFR inhibitor by itself doesn't do anything. So, we can see here that something's going on with Adam19. TGF-β is upregulating it.
So, that brings me to the next slide which is really kind of putting it all together. So, this is what I'm proposing that is happening inside the cell. So, as we showed, TGF-β activates its own receptor that activates the canonical SMAD signaling pathway, the ERK signaling pathway and now my research was like, "Okay, how is EGFR involved?" Well, the really telling thing was that TGF-β didn't activate EGFR in one minute, but it was a delayed response. (38:16) So, that really suggests that maybe it wasn't directly activating EGFR receptors. So the TGF-β ligands weren't actually going onto the EGFR receptor and activating it, but maybe it was some sort of indirect response.
So, what I'm proposing is that possibly TGF-β is activating the SMADs and ERK signaling pathways, and that then goes on to activate different genes, one of which is Adam19. (38:46) And Adam19 then gets made into a protein that can then cleave the HB-EGF and release that soluble functionally active form of HB-EGF that then binds onto EGFR. And then from there, then there's that crosstalk that happens between ERK and EGFR. So, possibly that is what's happening, and we really need to delve more into that relationship to really figure it out. But that's kind of an exciting finding from my research.
And you can read more about this in a paper that I published this year. As I mentioned, it will detail all these—especially all the western blots. There were many, many western blots.
Okay, so back to these growth factors. Now what I mentioned earlier is that there's lots of them and another one is the BMP.
And the BMP, as I described different growth factors can do different things to TGF-β, and what I found was that BMP actually played a different role.
So, BMP actually blocked TGF-β-induced EMT.So, here's the control state. With the phase-contrast image you can see the cells are beautiful, monolayer, very nice cobblestone-like arrangement, minimal α-SMA expressions, which is that mesenchymal marker. We add the TGF-β, the cells get elongated and then α-SMA gets expressed very strongly and also incorporated into these spindle-shaped cells in the form of stress fibers.
Now you add the BMP to the mix and you block it. So, no longer are the cells expressing α-SMA and they're also not getting incorporated into the stress factors. And you can see in the phase contrast that the cells are maintained in this nice sheet of epithelial cells that aren't elongated.
So, the next question, of course, was what sort of dosage? Is it a dose-dependent response? And indeed, we found that with the control state, α-SMA is minimally expressed in this western blot. When you add the TGF-β, α-SMA goes up. You add 1 ng of BMP and you suppress it a little bit, but not to the point of the control state, and you add higher doses, you can see that we suppressed α-SMA pretty much down to basal levels. (41:14) So, definitely there was some sort of relationship between the dose of BMP-7 and how effective it is at blocking TFGβ-induced EMT.
So, now back to this signaling diagram. So, as I described, we've got these TGF-β molecules that will bind onto its TGF-β receptor. But you've also got these BMP and it binds onto the BMP receptor, and that will activate the receptor or phosphorylate it. Now the interesting thing about BMP and TGF-β is they actually belong to the same family. So, I was very surprised to find that they were having very opposing effects, because they're all part of the TGF-β super family of growth factors. (42:04)And as such, they actually share some commonalities. So, they both use SMAD signaling. TGF-β though uses SMAD2 and 3 and BMP uses SMAD1, 5 and 8. So, a little bit different in that sense.
So, what I found was that there was some sort of differential activation of these SMAD proteins. So, we can see here, this is a western blot and you can see that TGF-β—we don’t have any TGF-β just BMP-7 in the first panel. So, I looked at the time points of 20 minutes, two hours, four hours and six hours. You can see that if you just have BMP-7 alone, it will activate SMAD1 and 5, and that's pretty strong at all the time points. (42:45)
Now if we just have TGF-β alone in the second panel and no BMP-7, we can see that TGF-β activates SMAD2 and 3 very strongly at 20 minutes and two hours. But what happens if we combine the two? Well, if we combine the two, you can see that now TGF-β induced SMAD2/3, so the SMAD2/3 that is the TGF-β form of SMAD2/3 isn't upregulated. And the BMP version of the SMAD1 and 5 is strongly upregulated. (43:19)
So, that was really interesting. So, if we have the two growth factors at the same time, then BMP is going to activate its own signaling, SMAD1 and 5, really strongly and that actually suppresses the TGF-β SMADs, 2 and 3. So, there was some sort of rapid thing happening at 20 minutes that was causing this blocking effect of BMP.
And we went to look at this further on the gene level. This is a real-time PCR looking at the ID 2 and 3 genes which are the inhibitors of differentiation. So, those are the target genes of BMP-7 and you can see that in the first panel, in the white box, here we can see that in the control state, there's pretty high levels of ID-2 and ID-3, as well. If you add TGF-β, that suppresses ID-2 and 3 levels. (44:17) So, it looks like when TGF-β is downregulating these genes, but if you have a TGF-β plus BMP then you can restore ID-2 and 3 levels back to the basal level. So, if anything, it seems like BMP-7 is sort of protecting the cells from TGF-β's suppression of ID-2 and 3, which appear to be important in making sure that EMT is not occurring, perhaps, on a basal level.
So, that's a mechanism that we're proposing. So, the interesting thing here is that TGF-β activates SMAD2 and 3. BMP activates SMADs 1, 5 and 8. But they all share this common SMAD4. So, they both need SMAD4 to form that complex, that then translocates into the nucleus. So, we believe that possibly what's happening in that BMP can block TGF-β because it is competing for SMAD4. And so, it takes more of the SMAD4 and therefore it can then go on to activate the genes in the nucleus and upregulate ID-2 and 3 or promote the upregulation of these genes. (45:35) So, potentially, that could be what's happening in the blocking effect of BMP-7 could be competition for SMAD4 and in addition to maintaining those levels of ID-2 and 3 to protect the cells from EMT.
Okay, so to summarize everything—which was quite a lot on all the growth factor signaling pathways—firstly, the important thing here is that we're really looking at anterior subcapsular cataracts and posterior capsular opacification following cataract surgery, and they're both really important forms of fibrotic cataract. (46:14) And that's that scarring response I was talking about, where it just clouds the lens and we get that loss of transparency.
So, the aim of our research really is trying to prevent these diseases from happening, so we can promote lens transparency and protect the lens, so that it can do its job in focusing light onto the retina. And TFG-β is a really potent inducer of these fibrotic forms of cataracts and that activates a complex network of signaling pathways.(46:46) As you saw today, it activates the canonical SMADs signaling pathway and it also activates the non-canonical EGFR signaling pathway and ERK, as well. And they all seem to form this crosstalk, which is quite complex and it's still something we need to tease out. And in addition to all these pathways, in fact, there's actually many other pathways that are also activated by TGF-β. And so we're looking to all these other pathways to see if there's any connections there.(47:17)
What I'm showing also today is that there's different growth factors that float around in the aqueous humour of the eye, and they can impact on TGF-β differently. So, in one instance, we've got a growth factor like EGF that can augment TGF-β, and then we've got BMP that can block TGF-β. So, really as much as it's really interesting to study the effects of TGF-β alone, we have to be mindful that in the actual real life situation, there's lots of different growth factors. (47:53) And as I mentioned, there's actually even more than those three. So, if you put all of those in the mix, what actually happens—and that's something that our lab is working to understand further—is that what actually happens inside the eye, to impact on the activity and the fibrotic activity of TGF-β. But what I really want to highlight is that if we block EGFR signaling or if we promote BMP signaling, then we can really target cataracts.(48:24) We can potentially block the form of fibrotic cataracts, which is really exciting. And they can be quite novel strategies to cure cataracts.
And finally, I would like to acknowledge the members of my lab, in particular my supervisor, Professor Frank Lovicu and also, Donna and Sheng from the Bosch Institute at the University of Sydney for helping me, especially with the PCR.
And lastly, if you'd like to reach out to me, I'm more than happy to talk more about my research. I'm on Twitter, so you can follow me @EyeDaisyShu and also on Instagram. You can visit my website or you can email me. So, I'm more than happy to hear from you or any of your thoughts on my research or just anything, in general, about social media, I'm always happy to hear that. And finally, I'd be more than happy to hear any questions, so please feel free to ask any questions now.
Moderator: Thank you, Daisy, for that informative presentation. Now just a quick reminder to our audience on how to submit questions. Simply type them into the Ask a Question box and click on the Send button. We will answer as many questions as we have time for. Okay, Daisy, your first question. Could the expression of ERK-1, ERK-2 in the eye be a signal for MCI, mild cognitive impairment, as these markers are well-documented in MCI, or vice versa?
Daisy Shu: Right. Thanks very much for that question. I have actually never really thought about mild cognitive impairment, but that's a really interesting link, because mild cognitive impairment is actually more prevalent in the older population, as is cataracts. And so, there could definitely be a link there. In addition to that, I know that oxidative stress plays a big role in the type of cataracts that we're looking at. (50:32) In fact, work in our laboratory has shown that reactive oxygen species does get increased in our type of cataract. So, there could definitely be a link looking at ERK-1 and 2. And work in our lab has shown that if you block ERK, then you can block the cataract.
We haven't really tied the link between whether or not there are any brain abnormalities. (51:00) But that could be a really novel way of looking at sort of brain function, by using the eye as a sort of more easy to...like readily accessible means of looking at brain function. So, I'd be really curious to see if there's any sort of research out there. And if someone could really do that experiment, I'd be very curious to see that. So, thanks for that question.
Moderator: Thank you, Daisy. How do you avoid introducing infections into the petri dish?
Daisy Shu: That's a super important question and what we do is in our cell culture media, we've got antibiotics. So, we've got penicillin and streptomycin, and we've also got amphotericin-B. So, these things really do help to reduce any sort of contamination. In addition to that, we just try our best to be as sterile as possible. We do our experiments in the hood and also make sure that we UV the hood and UV the room, as well, before and after our experiments. (52:19) And we 70% ethanol everything, as well. So, those are some of the things we do.
Moderator: Now Daisy, have you used human LECs in your work?
Daisy Shu: Yes, I have actually. So, some of the newer work in my research is using—so as I showed in the presentation, the surgeon when they perform the cataract surgery, they actually have to remove all that material that has undergone that cataract (inaudible) change. So, when they remove it, they actually discard that material. (52:57) Now those cells that are part of that cataract can actually be used for experiments. So, having collaborated with a cataract surgeon, I was able to collect some of these cells and actually culture them, and they responded in a very similar way to the rat lenses. So, that was really cool and it's a really great way to use material that would typically have gone in the bin, to actually see if we can translate some of that animal work to the human situation.
Moderator: Daisy, is this system applicable to lenses of all species?
Daisy Shu: That's a great question. I've actually done lens epithelial explants on mice lenses. And that's actually a really great way to study transgenic mouse models and see if these lenses perform in the same way. Because it's really—yeah, often we don't really do sort of gene manipulation on rats, and it's usually mice. So, that's definitely a really great way of testing if we overexpress certain genes or knockout certain genes in mice, will these lens epithelial cells behave the same way when we add different growth factors? (54:21)
So, I have done this successfully in mice, although they are super, super tiny compared to rat eyeballs.So, it just requires a little bit more practice before you do it, but it is possible. And I've read in the literature people have used pig lenses, as well. And pig lenses are actually a lot larger, so they're probably a lot easier to use. And so, that's another very great model to use. (54:50) And other people have used embryonic chick lenses, and of course, the human lens epithelial cells you can get from donated lenses and also the discarded material after cataract surgery. So, those are the ones I've read in the literature, but it's definitely possible.
Moderator: Thank you, Daisy. Now given the link between oxidative stress and cataract formation, have you looked at the regulatory effect of oxidative stress-induced PTMs on TGF-β and EGFR-regulated pathways?
Daisy Shu: Okay, PTMs, could the person who asked that question just clarify what they mean by PTMs? I don't think I got that.
Moderator: Right, Brian, thank you for your question. If you could elaborate on what PTMs are and submit your question again, we'll be able to answer that after the next few questions. Thanks. Okay, we'll go to your next question. Daisy, what are some ways that the drug can be delivered to the eye, to stop cataract formation?
Daisy Shu: That's a good question. So, the way I envision this treatment to be administered to humans is probably during the time of cataract surgery. So, this is when we're talking about the secondary type of cataract. So, I believe at the time of surgery, it would be really ideal for the intraocular lens—so that's that implant that's put in place of the removed cataract—that lens can be loaded up with a drug that slowly releases. (56:29) And there's actually some papers in the literature on people who are trying to develop such an intraocular lens implant that slowly releases drugs. And I think that would be a really novel way to incorporate these inhibitors to block that secondary cataract from forming.(56:46)
The other really interesting ways could be looking at an eye drop formulation. And the only issue with that is how it can cross the layers of the eye, to actually reach the lens. Another way is to have an injection into the aqueous humour and that's another way—and because the surgeon during the time of cataract surgery is already going to be injecting antibiotics and anti-inflammatory agents, it makes sense to also inject something that will stop that fibrotic effect from forming. (57:23) So, those are the ways I see that these inhibitors could be incorporated in the real world.
Moderator: Thank you, Daisy. Now let's go back to that previous question. Given the link between oxidative stress and cataract formation, have you looked at the regulatory effect of oxidative stress-induced post-translational modifications—PTMs—on TGF-β and EFGR-regulated pathways?
Daisy Shu: Oh, okay, great. Thanks for clarifying that, Brian. I haven't actually looked at that, but work in our laboratory by another Ph.D. student has looked at oxidative stress—not post-translational modifications per se and not on EGFR—so, purely on the reactive oxygen species in TGF-β. (58:16) And specifically, he looked at the role of NOXs, and they're really interesting sort of players in reactive oxygen species, where they can actually produce reactive oxygen species. And he found that certain NOXs are upregulated during the addition of TGF-β. And if you block NOX4 then you can block TGF-β-induced EMT, which was really interesting. (58:46) And he's looking into that further, looking at mitochondrial stress and things like that. But that's definitely an area that is developing and it's a really interesting area, and I'm so curious to see how EGFR would fit into that. And it's not an area we've looked at, but I would be really curious about that.
Moderator: The LACs are present in a low-oxygen microenvironment. How do you factor that into your findings?
Daisy Shu: Wow, that's a really interesting question. The lens epithelial—I hadn't really considered oxygen availability or content in my study. We pretty much just used standard cell culture settings in the incubator, I guess. So I haven't quite thought about that, but I know that hypoxia plays a big role in lens epithelial cells, oxidative stress. (59:47) So, I'll be really curious to see whether or not any hypoxia upregulated genes would be involved in TGF-β-induced EMT. We haven't actually looked at that, but that's a really interesting next step to look into. Thanks.
Moderator: Thank you, Daisy. And we are almost out of time, so we're going to wrap with this final question. And a reminder to our audience that any questions we do not have time for today and those submitted during our on-demand period will be addressed via the contact information that you provided at the time of registration. Okay, Daisy, with mouse lens epithelial explants, how much protein RNA and DNA do you typically recover from one pair of mouse lens epithelial explants?
Daisy Shu: That's a very good question. It's like a tiny, tiny amount sadly, because the mouse eyeballs are tiny, and obviously, we're taking the lens epithelial cells, just a monolayer of cells from these mice. I haven't really worked that much with mouse lens epithelial explants. I've only kind of done a (flat mount) with them and stained them for immunofluorescence, which is a little bit easier to do. (61:05) But I have some lab (mates) who have looked at collecting them, and I would say that they typically need like five pairs of lenses in order to get a substantial amount of RNA and protein, so in order to run the PCRs and western blots.
I couldn't tell you exactly the concentration that they get, but they're getting proteins being expressed. (61:38) Like their western blots are actually showing (inaudible) with about five explants per treatment group. So yeah, it's definitely harder with these primary explants, but it's feasible. So yeah, I definitely—yeah, it's a great system to use if you're wanting to have the cells on their native basement membrane. So, that's probably the biggest advantage with these explants. (62:10)
But as I said, yeah, the protein and RNA that you can actually extract from each one is quite limited. For the rats, we can get away with about three. But typically I like to have five rat eyes per treatment group for one experiment, just to get as much of that protein RNA per group. Thanks.
Moderator: Thank you, Daisy, and thank you to our audience for joining us and for their interesting questions. Daisy, do you have any final comments for our audience?
Daisy Shu: I just want to say a huge thank you to LabRoots and Gibco for giving me this amazing opportunity to share my research with you all. I am so thankful to all the attendees that are actually tuning in and listening. And you all asked so many really insightful and amazing questions and have given me a lot of food for thought in terms of where I can take my research next. So, thank you so much for those.
Moderator: Thank you again, Daisy. I would also like to thank LabRoots and our sponsor Thermo Fisher Scientific for underwriting today's educational webcast. This webcast can be viewed on-demand. LabRoots will alert you via email when it's available for replay. We encourage you to share that email with your colleagues who may have missed today's live event. You will now be directed to our next upcoming webinar. We encourage you to register, and we hope to see you there. Goodbye.
Get to know Daisy
Why did you choose eye research?
The eye is such a beautiful organ and I’ve always been fascinated by how our eyes enable us to see.
What motivates you to succeed in your field?
Humans are very visual beings and not surprisingly, sight is one of our most precious senses. To lose sight is such a devastating experience. My motivation to succeed in eye research is driven by my mission to help restore vision and cure blindness.
“Where you stumble, there lies your treasure” – Joseph Campbell
If you didn’t have to sleep, what would you do with the extra time?
I would be reading more books, writing about science and travelling the world.
What are some small things that make your day better?
Fun chats with my friends/lab mates between experiments, ticking things off my to-do-list (however small the task may be) and seeing my plants alive and healthy.
I want to be the next Gibco Cell Culture Hero
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