 So good morning, everybody. And thank you very much for attending the grand rounds. I would like to introduce Dr. Shizuya Saika. He is the professor and chairman of the Department of Ophthalmology at the Wakayama Medical University School, and this is in Wakayama, Japan. Here, you have a summary of his other activities. He was an IZO-Oftalmic In Plans Committee member. He is a member of the editorial board of different research journals. He was invited, as a guest speaker, worldwide. And his major research interests involve capsule bag of pacification, intraocular lens biocompatibility, and coronary research. And he published more than 190 publications on this subject. So I had the pleasure to collaborate with him in a book on posterior capsule pacification that involves basic science and clinical applications. If you're interested, this book is in the library. So the book was published last year. And I had the pleasure to be invited by him to lecture in his department last year. Had a wonderful time in the department. Had a wonderful time visiting some sites around Japan with his himself associates. This was during fall, and it was absolutely gorgeous. Yesterday, he delivered a great lecture on the research seminar. And today, he's going to present on the epithelium mesenchymal transition, the key phenomenon in ocular fibrotic disease. So this is very important research with applications in different areas of ophthalmology. So thank you again for coming. Thank you very much for being here, and to me. Good morning, good morning, good morning. It's a big honor to be here at the front of you. I have a chance to have a talk about my research data in terms of the EMT-epithelium mesenchymal transition. Actually, let me introduce myself. I don't be interested in the wound healing response in the eye, especially in the corneal and the lens. Very fortunately, wound healing response in lens. But not in corneal, much related to the Greek phenomenon of the PCO development. So today, I'd like to talk about with this title, hopefully, much fit to the Lydianus interest. This is the Vakaya prefecture in the center of my city. Let me start. Before that, let me introduce my city, Vakayama. Here we are. Here is Japan. My city, my city, Japan, Vakayama is one hour south from Osaka by car. OK. Today, my talks are much related to the generation and the role of the myofibroblast. The key cell type involving the tissue fibrosis of the scarring of the tissue in the corneal and the skin and, of course, in the PCO. This myofibroblast expresses a lot of the mixed components. And also, they exert a contraction by expressing the alfalfa snusmus reacting. This contraction may contract the newly regenerated scarring tissue. And also, the cell expresses a lot of the chemo that are capable of recruiting the inflammatory cells in the injured tissue. In corneal, one of my major interests, the myofibroblast is, as you know, generated from the kerosites during the healing after injury. However, in length, we know there is no mesenchymal cell. Still, but still, the PCO contains the myofibroblast. So in this case, we can say the myofibroblast in PCO tissue is different to derived from the Psyria cells. That is, then the Psyria cells. We say this by the process from the Psyria cell to fibroblastic mesenchymal cells. We say EMT, Psyria mesenchymal transition. Here, you can see a typical cell culture phenomenon. Psyria cell types express the e-card healing between the among the cell border. However, after EMT process, such as under the condition with the TG beta, the cells lose the e-card healing expression and the up-regulate the alfalfa snusmus acting, the hallmark of the myofibroblast. OK, DI is susceptible to the fibrolytic scarring disease. Among them, today's topic includes the EMT-related tissue fibrosis in the first in the capsule of a situation. And in the later part of my talk, I will briefly touch about the PBR. And also, if we have time, I'd like to touch about the corneal and Psyria cell EMT and the Psyria tissue fibrosis. First, as we know, then the Psyria ectoderma origin. You can see clearly the first ectoderma in virginate to the eye cap. In mice, in mice, this picture, actually this H.E. staining specimen was cut by myself. They are 10.5 days after you can see here a Psyria alfalfa ectoderma in virginate to the eye cap. Then grow the mice during the growing the Psyria cell form, like this. Here is the corneal. Here is the eyelid. So this is also there. It's very familiar to you all. During the heating after eye oil surgery, then the Psyria cells migrate behind the intraocular lens. During this process, the Psyria cells undergo the EMT Psyria mesenchymal transition. And also, they express out the matrix components. They induce the fibro-type tissue fibrosis. During this process, the EMT-derived myofibrosis exerts the contractile action. The lens capsules shrink around the implanted lens. You see here, here's the anterior capsules of the application part. You see here, all the matrix components consists of the carcinoma and the other matrix is formed under the anterior capsules. Here is the CCCH. And the cells no longer have a Psyria-shaped back. They express the alfalfa mesenchymal acting. And we can say no longer they are Psyria cells, but they are myofibro-type. When we think about the behavior of the one cell, you are in the heating up to some external stimuli or injury. Mainly two similar streams modulate the cell behavior, such as proliferation, migration EMT, or ECM expression. These major two similar streams include the one is from the gross factor cytokine signal. The other one is the signaling stream derived from the ECM through the integrin binding. First, I'd like to talk a touch about the gross factor signal. As we know, many gross factors are involved in tissue wound, the tissue repair of the regeneration. Today I'd like to talk about mainly on TGF at all. The reason why is the TGF beta predominates the IACUS humor and also the major gross factor, as you know, involving the EMT process. OK. Let me quickly review the TGF signaling. TGF beta has also the MAP kinase signaling, like other gross factors. Besides the classical MAP kinase signaling, MAP kinase, P3A, JMK, the TGF beta 5B member also has the signaling cascades, consisting of the SMED molecule group. You can see here, after binding the TGF beta to the specific receptor, SMED 2 or SMED 3 activate and bind with the SMED 4 make a complex. This complex turned into the nuclei for gene expression regulation. Later, I'd like to talk about the sum law of the SMED 7. Just let me quickly tell you the SMED 7 is inhibitory SMED molecule that blocks the SMED signaling at this phosphorylation process. OK. First, a long time ago, I have checked the intra-cellular localization of the SMED molecule in the epithelial cells. This is a CCC specimen, which we believe during the carotid surgery. You can see here, SMED molecule, in this case, SMED 3 is located to the site plus, but not in the nuclei. But after 10 days, the patient was very unfortunately had to receive the extraction of the IOL with a capsule. With some presumably infection disease, you can see here, the SMED 3 has already finalized a nuclear translocation in the CCC cells in 10 days. Then I had a chance to check the PCO specimens in terms of the SMED localization. You can see here, in these specimens, SMED molecules are detected in the nuclei of the PCO tissue. That means that these cells are under the motivational regulation of the TG-hibita signaling, especially the SMED molecules are there in the nuclei of the lead epithelial cells surrounding the intraocular lens. OK. Yesterday, I had a chance to talk about the cell culture study to uncover the sub-mechanism of the EMT. But today, I'd like to skip them. And then, final conclusion of our previous study, the TG-hibita SMED signaling, up-regulated snail. This snail is one of the major gatekeeper of the EMT process. The snail transmission factor is followed by the PI3 kinase activation. And the alpha-synosomax acting is up-regulated to finalize the process of the EMT. And that's right. Just I have skipped some experiment data to figure out these cascades. OK. Then, at that time, I tried to reproduce this lens epithelial cell response in response to the injury, our carousel injury. I developed a very simple model of the lens puncture in mice. You can go. After lens puncture in mice, the fibrosis tissue due to the EMT fibrosis is formed inside the lens. This experimental model is very simple but very useful because once we see a fibrosis tissue in the lens, 100% there's EMT fibroplasts. Not without any contamination of the fibroplastic cells from outside the lens. That means that, still in vivo, but the system is pretty much like the in-vitro condition. In case of, in terms of the cell lineage purity. OK. First, it's OK. No problem. The first, in this red puncture model, I have checked the expression time schedule of the smear signaling. You see here, an injured healthy lens. In this case, I used a smart phone as a molecule of the indicator of the smear localization in the cell. You see here, smart phone locates such positive like human cases in healthy lens. However, just after, in after 12 hours, puncture, post puncture, already smear molecule finalize the translocation to the nuclei. This translocation nuclei between these 12 hours was completely averaged by the injection of Tg greater than the antibody into the eye at the time of the puncture. Then still 20 hours, smear molecule are there in the nuclei. But the cells are still of serial shape, but not the mesenchymal, without any expression of the smear signaling. Then, in day five after lens puncture in mice, the cells transform to the shape into the myobrastic with the expression, the alfalfa smear. So it's a very simple story. To check to this smear signaling involved in the EMT or not, just I had a chance to use the smear three-knocker mice. Actually, I got this from my mentor, the doctor, the late Dr. Antlovertz. She was the founder of the Tg-hubator a long time ago. It's a very simple experiment. Just puncture lens, puncture lens, then see a wounding process in smear three-knocker. In the absence of smear three, lens cells never perform the EMT. Still, they can migrate out through the puncture site, but no fibros tissue as compared to the wild-type tissue. Then these cells, in the absence of the smear three-signaling, they fail to express the snail, they fail to express the fibros matrix components. Here, you can see here, just look at these two pictures. In the absence of the smear three, the cells do not express up irregularity in the snail. They are gatekeeper of the EMT, but in the wild-type, you can see here the inside to hybridize as your signal is there in the EMT cells in your injured lens. This is also the case of the alpha-synosmastic expression. There is no expression of the alpha-synosmastic acting in smear three-knocker mice lens have to injure it. Then when we think about the future strategy to block the EMT processing in EMT fibros disease, PCO, other disease, later I will talk about. First, we can block the TG-hubaito at the ligand level, like a VGF story, or block the receptor, or block the signaling cascades. Actually, when I worked with Dr. Robert's about 10 years before at the National Counseling Institute, I had a chance to get a patent in here, in this country, to blocking this system may be beneficial to block the disease with her. Today, I'd like to show some piece of data blocking the smear signaling by adenoviral gene transfer to the lens. Before that, I'd like to summarize the blocking system, inhibitory system smear signaling by the other transmission factor. Already, I told you the SMAR-7 is a strong block molecule against the smear signaling. Another one is the BMP-derived transmission factor, ID-2, and ID-3 also, of capable of inhibition smear signaling. This is one piece of data. Gene transfer of the SMAR-7 to the eye at a time of the lens injury by carrying the adenoviral vector. You can see here, with the gene transfer, the cells maintained of epithelial cells, still the cells can proliferate, but they show the much more epithelial shape without the expression of the SNA and the alpha-synosctene. Just I'd like to skip the data, but the overexpression of the ID-2 or the 3 also showed a similar effect. They were published in American Journal of Physiology. I'd like to move on to the next signaling system that's involved in the cell behavior modulation. This is the, this another one is the, as already I told you, the ECM-inch system. From now, I'd like to show some piece of data. This inch-bring ECM-molecule signaling attenuate the SMAR-synosctene. Today, I'd like to talk about my two molecules to your pointy and lumina. Just, I'd like to skip the data of the tenacity, see a tenacity X to save my time for corneal talk. Before the, before I show you the, I will show you the data. The summary is like this, the already I told you the STIGI of the SMAR signaling is the major modulator of the EMT process in the PCR cells after injury of the carousel surgery. And the PCR cells transform into the myofibrous, inducing PCO. This system is much modulated, promoted by a TG with SMAR-synosctene. The father, up to regulate these metric cellular proteins, these proteins overall support the SMAR-synosctene. For example, osteoponding. Osteoponding is one of the metric cellular protein that is detected in the inflammatory side, such as the arthritis or some other inflammatory immune disease tissue. Here's the, you can see here's a thrombin-cleavage site. The, after the thrombin-cleavage site, after this site is cleaved by thrombin in the inflammatory site, newly exposed C-terminal, SVB-G-O-O, this terminal can exert a variety of the biological function modulating the SMAR-synosctene and other modulations of the ingramatory process. This osteoponding is also detected in the PCO tissue, like other myometrics molecules. Then the story is very easy. Just I checked here, how EMT is modulated in the absence of this osteoponding by using NACA mice. The story is very simple. In the absence of osteoponding, expression of the alpha-synosctene in the cluster of the LEMSA-PCR cell proliferation have been easily untold capsule. After puncture was much more attenuated on day five. It starts the highest tissue in mice. However, in a wild-type mice, here's the anterior capsule and per puncture, LEMSA-PCR cells cluster proliferation are to regulate the abundance of alpha-synosctene. But this effect was not seen at the end. That means the loss of osteoponding does not completely block the EMT or B-synosctene, but also the tertiary attenuation process. To uncover the mechanism, underwrite this enduro phenomenon, I conduct some self-cultured study. It's very interesting. You see here, I got the self-cultured from the wild-type of NACA cells mice. The adding tissue beta. You can see here, just blocking the osteoponding in NACA mice cells, the SMAS signaling for SMAS-3 oscillation is much more impaired, blocked. This is also cases of p-38 activation, also blocked in the absence of the, by blocking the osteoponding gene in cells. And finally, I go back to the in vivo study and immunostated and gained the tissue. They use it here. This is also case in vivo. You can see here, in wild-type mice, after day one, then puncture, you see here of the SMAS-2 oscillation in the lens. However, in osteoponding NACA mice, such SMAS-2 activation was observed firstly at day five. After that, please see, I published this in the 2007, but after that, in 2015, one Japanese group showed here one paper, published one paper, how osteoponding SVIVA gene fragment activated tissue beta in the paper. But very unfortunately, I am so sad, they do not cite my papers. Then I tried to go ahead on the same story in the aluminum. Before I talk about the lens, firstly, I had a chance to generate the aluminum NACA mice in seasonality at the Winsun Kowloff. Then I developed the NACA mice. The cornea is, the cornea become opaque in the absence of the aluminum with a disarrangement of carbon fibers. Time I got in 2000. After that, I had a chance to check the lens injury response in my aluminum NACA mice. You see here, same as osteoponding NACA mice, most of the aluminum can also disturb the EMT-alpha-smooth mass-acting expression in lens. I published this paper in 2003. Remember, my osteoponding paper came out in 2007. At that time, in 2003, I had no idea this impairment of EMT is involved in the blocking or impairment of TGC. Just I saw this, I saw this on the publish which I'll tell you in detail, in deep. However, after that, I proposed one idea to my mentor, Gao, in Cincinnati University. A loss of aluminum may have some function in moderating the TGC beta after seeing this. After, I actually, after seeing the osteoponding results, I proposed this to the Winston. Then, okay, Winston told me, okay, she's there. This is quite possible. So in this case, you can send me a fellow. I send a fellow to the Winston lab at Cincinnati, and this guy's now associate professor in my department. He spent four and a half years. Four and a half years, finally, he found a new mechanism of the lumiculomoderation, a TGF signaling. Details you can refer in this publication. But quick summarizer, the final results, the lumiculomoderectri bind to the TGF, the receptor molecule. The TGF receptor molecule bind with the lumiculomoderectri and the C-terminal peptide directly moving to the nuclear and regular gene transfer. This is a very, very new model of the matrix modulation of the TGF signaling. This is the in-silicone molecule complex, the lumiculomoderectri-terminal ILK5 TGF receptor peptide. So this is the same picture, same scheme. I'd like to emphasize the EMT process toward the PCO formation. It's mainly modulated by the TGF, the smart signaling. But this smart signaling is further affected by these matrix components signaling. The other story is, other EMT related by disease is PBO, as you know, briefly. This is a PBO tissue extracted by myself during build-up surgery. You see here, in H-standing, all of the cells contain the pigment. That means they are dispersed, they are pigmented, they are cells clustered. But some of them are expressed, some of them express the alphasmus acting that suggests some of the retinopigmented PSEA cells and the EMT, thank you very much. Thank you. The, these red colors are just auto fluorescence of the pigment in pigmented PSEA cells. Then when we go back to the cell culture study, I'd like to quickly show the activation system that TGF is mad in the pigmented PSEA cells. You see, these are just from the cell line of the pigmented PSEA cells. And they are porcine pigmented PSEA cells. You see here, after TGF beta addition, the smell is so quickly activated. Then the porcine pigmented PSEA cells, the cells continuously undergo EMT without exogenous TGF beta. But now TGF beta neutralizes antibody block the EMT. And you can see here still the cells remain pigment, more pigment in the cypressum. So again, story is very simple. I go, I go back to the SMAS3 story. I prepare, I develop the retinol detachment model in the SMAS in NACA, SMAS in NACA mice. But the difference for human cases after long-standing retinol detachment in mice, fibrous tissue is not formed on the retina, but on the original pigment PSEA cell layer. You can see here, this EMT fibrous tissue formed on the pigment PSEA after long-standing retinol detachment a week and a week eight. However, this formation of the fibrous tissue on the pigment PSEA is blocked. In the absence of the SMAS3. The story is very same as the PCEO. So in this case, gene transverse SMAS7 recaptuate the SMAS3 absence. They block the SMAS signaling. SMAS2, SMAS3 signaling are blocked. And the formation of the fibrous tissue is much attenuated with the SMAS7 gene transverse compared to the empty back to that menstruation with the suppression of the expression of the alpha SMAS3 acting and the collagen expression. Okay, the final part of my talk is composed of the cornea and the cerium. Cornea and the cerium is also susceptible for the EMT disease, labiravitae and cerium. The endocelular cell dysfunction led to the buous catabash, as we know, so well. This is a case of the human syphilis catatitis. Perform the pentatonic capacity. Then, in the tissue, you can see here, many strands of tissue beneath the endocelium in the anterior chamber. Then, when we immunostain for the collagen one in this tissue, you can see here, collagen one is located through the inside the fibrous tissue but also the surface and the cereal cells. However, when other piece of the tissue observed under the scanning microscopy, the endocelular cells no longer show the endocelular cell shape but they show the fibro-plastic shape. When I saw this picture, ah, it should be, it should be our national, maybe a process of the EMT. To reproduce this EMT-like phenomenon in animals, this is misspearing. I prepare, I make an alkali-brown model in that. Just I make an alkali-brown in latconia. The alkali penetrate to the endocelium also the induce the disarrangement of the endocelium. Like this. You'll totally, I strongly hypothesize it involved in the EMT. So first, I conduct some sort of organ culture study to uncover which signals involved in the endocelial wound healing. I gather the latconia, they are prepared of some pieces and the scraping the half endocelium gone. They put into cell culture, organ culture. There you can see here, endocelium start to migrate to recover the denuded disarrangement membrane. Here's the migration areas. First, I put the Tg-hubate antibody into the culture region. Broken Tg-hubate endocelial cell recovery in our own culture. But still, from these results, we cannot say which signals is much involved in. Then I, so in this case, when we think about the Tg-hubate signaling again, besides the canonical mass signaling, Tg-hubate also activate the classical map kinase on JNK and P38. So which signals is much involved in the cell migration process? This is my passion at this time. So and also, when we think about the blocking EMT, tissue fibrosis in corneal endocelium, still we would maintain this signaling, the beneficial to recovering the endocelial function and healing. So I conduct the organ culture experiment again with each signaling inhibitors. You can see here, P38 inhibitor block the migration, and map kinase inhibitor also block the migration. JNK inhibitor doesn't have the blocking effect on cell migration. Also the LK5 inhibitor, that block the canonical mass signaling. Canonical mass signaling inhibition also doesn't, doesn't have the inhibitory effect cell migration. So I think blocking LK5 signaling, that means SNAS signaling may or may not inhibitory effect of the cell endocelial recovering, but they might have the inhibition on EMT process in corneal endocelium. I summarize this organ culture experiment like this. Map kinase signaling are involved in the cell proliferation and P38 expression, is similarly involved in cell proliferation. These two phenomenon is favorable over the endocelial healing regeneration. So I'd like to keep this, keep this, keep these phenomena healthy. Still one block, one block the EMT fibrotic process in corneal endocelium. Just as simple as this shows, very same. Just I introduced the SMAS7 inhibitor SMAD gene to the corneal endocelium of alkali-burned left corneal. I first prepared the alkali-burned in the left corneal, then I might prepare the mixture of the hyaluronic and the adenoviral vector. Actually the adenoviral gene to us is worked well in the corneal endocelium. In the preliminary experiment, the SMAS7 gene transfer led to the over expression SMAS7 in the corneal endocelium, but none with the entity vector. This is out. After making alkali-burned, corneal endocelium become thick with the ECM accumulation. However, with the SMAS7 gene transfer, endocelium maintain their monoreal morphology without the accumulation of the abnormal metric components. Scanning microscopy also shows the EMT-like endocelial cells here. However, you can see here the original-like endocelial cells here. They are inflammatory cell adhesions. However, among inflammatory cells, you can see here very nicely organized endocelium with the SMAS7 gene transfer. I further analyze the components in these conditions. You can see here SMAS activation is blocked. Alpha-acid-morphoctin is much expressed in the control endocelial cells. However, none with the SMAS7, causing one accumulation and much more interesting here. With the SMAS7, you can see here that the cell proliferation is up-regulated as compared to the empty vector. That means the SMAS7 gene transfer blocked the EMT by blocking the SMAS signaling also activates the cell proliferation. It should be favorable for the regeneration of endocelium. Normal-like endocelium. Okay, we can summarize this. In corneal and cerium, the cell also undergo the EMT-like process, express the alpha-acid-morphoctin and the metric components, after some severe injury or inflammation in corneal tissue, corneal storm. However, SMAS7 gene transfer blocked these processes. The EMT and the matrix component accumulation and also in turn, up-regulate the cell proliferation to restoration of their normal, healthy-like endocelium. So this is kind of the final slide. EMT process is much modulated by TG-Veta signaling. Block this, my pulse gone, the ECM gone. It should be, we have the very favorable valve. Maybe a partner slide. Then my future, my dream is to block these diseases. Okay, thank you very much. Preferably in Japanese. Yeah, by manually? By mechanically, yes. By mechanically, mechanically. Yeah. Actually, but in turn, last year, I had expressed one patient with the unknown reason, in that patient after my surgery, the CAHPS neighbor cross. As we know, CAHPS closure is promoting factor of the PCO. In turn, I don't know exactly the reason, but however, remaining CAHPS open after surgery with the lens, finally, totally ends up going, in this case, the fixation of the interocular lens was not so good. The mild CAHPS shrinkage should be beneficial to fix up the IOL in eye. In eye? I have never, in PCO study? In PCO study, I always use the, the TGV-2, but my other major interest, corneal healing, I always use the TGV-1 in experiments. Yeah, very similar, very similar, very similar. But as you know, the effect of TGV-3 still under argument. So I don't want to touch about the TGV-3. Please speak more slowly, I am Japanese, not native, native speaker, English speaker. Oh, nice, Japanese. I'm not sure, I could follow well your English. I see, I see. Oh, in these experiments here, I inject the adenobiorgine transfer to the eye, but this is not my, this is not for the clinical application. This experiment just performed to clarify the mechanism. Once I got the mechanism, I think we will be able to think about the multiple administrative routes. For example, I'm working on the inhibitory effect also made with some, just chemical extract from the Chinese harvon mason. Some of the Chinese harvon mason component, just a real chemical, has an inhibitory effect on smear. So when we use this kind of harvon mason intake, it may be beneficial with less stomach side effect. This can be an answer to your question. I'm not sure, yeah, yeah, yeah. Until then maybe, maybe in one month, maybe in one month. Oh, one month. Badumba? Oh, yeah, yeah, yeah. Generation, very good. I see, I see. I think that I could follow your, what you wanna say is if not, very sorry. In animals, especially mice, restoration of the fibrosis tissue back to normal, this phenomenon is very common in animals, especially mice as compared to rats. But sometimes the fibrosis tissue stands longer, stands longer, but in mice, I think mice are very strong. They are very against to the fibrosis disease, whatever tissue.