the Liver Cell Biology in Hepatic Disease SIG Symposium entitled Cellular Senescence in Liver Disease and Aging. I'm Yasuko Iwakiri from Yale University, the current chair of the SIG and the co-chair of this session with Dr. Jordi Gracia-Sancho. Cellular senescence is a cell cycle arrest program often accompanied by secretion of pro-inflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degradation and may be involved in the pathogenesis of HCC fibrosis and cholangiopathies. This symposium will explore how experts are working to translate basic science discoveries into emerging therapeutic strategies for several liver diseases. Session format is pre-recorded presentation. We have absolutely fabulous speaker lineups. Dr. Diana Dirk will talk about basic concepts of cellular senescence. Dr. Jordi Gracia-Sancho will talk about impact of aging on liver cells and liver disease. Dr. Gianfranco Alpini will discuss cellular senescence in biliary disease. Dr. Jessica Zucmarosi will talk about telomere length, aging and development of liver tumors. Finally, Dr. Tatyana Keseleva will talk about the role of senescence in liver cells in the pathogenesis of liver fibrosis. Welcome and enjoy our symposium. Thank you for inviting me to this excellent meeting. I'm delighted to be here today. Today, I'm going to talk about the basic concepts of cellular senescence, its role in aging and liver disease. I have nothing to disclose. What is cellular senescence? Cellular senescence is a cell fate that involves an irreversible cell cycle arrest and is characterized by the activation of tumor suppressor pathways, profound chromatin changes, mitochondrial dysfunction and apoptosis resistance. It was first described by Leonard Haeflig in 1961. When he observed that human fibroblasts in culture could only divide for fixed number of divisions before they reach a plateau and become irreversible arrested. This has been termed the Haeflig limit. What are the mechanisms of cellular senescence? Since Haeflig's discovery, we have greatly advanced our understanding of the mechanisms underlying senescence. We know now that senescence can be induced by different type of stresses, including DNA damage, telomere damage, oxidative stress, genotoxic stress, amongst others. It engages the P16 and P21 pathway, which result in the inhibition of the cell cycle. It also characterized by the activation of senescence cell anti-apoptotic pathways, short SCAPs, which render the cells resistant to apoptosis. Finally, senescence is characterized by the secretion of pro-inflammatory cytokines and chemokines, which is commonly known as the senescence associated secretory phenotype or short SASP. The SASP can have paracrine effects impacting on neighboring cells, but also autocrine effects which help reinforce the senescent phenotype. Physiologically, senescent cells can have beneficial or detrimental effects depending on the context. Senescence can be beneficial in the short term as tumor suppressor. It can limit tissue damage. It contributes to wound healing, but also impacts in embryonic development. However, the accumulation of senescent cells during aging can contribute to tissue damage, promote tumor genesis and impact on age-related diseases. So how does senescence lead to tissue dysfunction? Senescent cells can spread senescence to otherwise healthy cells via the SASP, so paracrine senescence. Senescent cells can also, via the SASP, disrupt the stem cell niche and thereby impairing tissue homostasis and regeneration. Thirdly, senescent cells can also contribute to the degeneration of extracellular matrix and contribute to tissue fibrosis. And finally, the release of cytokines and chemokines of senescent cells can promote demigration of immune cells and as a result exacerbate tissue inflammation. Senescent cells are widespread. They accumulate during aging in all organs and tissues and they have been associated with many age-related diseases like Alzheimer's disease, Parkinson's, COPD, liver fibrosis or type 2 diabetes. In liver, senescent cells have been found in different cell types such as hepatocytes, cholangiocytes and hepatic-related cells. Furthermore, senescence has been associated with several liver diseases such as non-alcoholic and alcoholic fatty liver disease, chronic hepatitis and liver cancer amongst others. Currently, the field has adopted two different therapeutic strategies to counteract the negative effects of senescent cells. One involves finding drugs which target the senescent cell anti-apoptotic pathways and thereby inducing apoptosis specifically in senescent cells. This strategy is commonly known as senolytic. The second way of interacting with senescent cells is to inhibit pathways responsible for the SASP, the senescence-associated secretory phenotype, a strategy commonly known as xenomorphic or xenostatic. Consistent with the hypothesis that senescent cells are major players in aging and age-related diseases, current studies, mostly conducted in mice, have shown that the clearance of senescent cells both genetically or pharmacologically is able to improve a number of age-related conditions which you can see here in this table which includes cardiac aging, diabetes, fatty liver disease, neurodegeneration, and osteoarthritis. In my lab, we have been interested in the hypothesis that senescence is a driver of liver aging and may contribute to steatosis. This stems from an observation I made very early on in my career where I observed that senescent markers such as p16 and p21 increase with age in mouse livers. Additionally, by performing an immunofish which allows the detection of DNA damage foci, we use gamma H2X as marker. At telomere regions, which you can see here, these are telomeres here in red, we look for colloquialization between both of them and they are called telomere-associated foci. We actually found that these senescent hepatocytes increase during aging. And over the years, we found the telomere-associated DNA damage foci is a very robust and reliable marker for senescent cells. We have also found that factors known to accelerate aging increase hepatocyte senescence. For instance, by enhancing chronic inflammation, we used for this a mouse model with increased NF-kappa B activity, we saw increased frequency of senescent hepatocytes during aging, as well as an impaired regenerative capacity in the liver. We also have shown that obesity is associated with increased hepatocyte senescence. And finally, in a collaboration with the lab of Masashi Narita in Cambridge, we have shown that impaired autophagy also exacerbates hepatocyte senescence. Then we asked if cellular senescence was associated with hepatic steatosis. We did the experiment where we kept two cohorts of mice. One cohort was at lipidome and the second cohort was dietary restricted. So these mice were only allowed to eat 60% of the control group. It has been known so far that dietary restriction leads to an increase of a lifespan about 30 to 40%. It reduces cancer rates up to 55% and it improves glucose tolerance. What we found is, after these animals have been restricted for one year, that there is an increase of lipids accumulating in the at lipidome animals, here seen by staining for oil red oil, but we don't see this increase in animals under dietary restriction. We confirmed this also by staining with Nile red, but more interestingly, it was that at the same time, we see that there is this increase of numbers of senescence in the at lipidome group, but we don't see this increase in the mice under dietary restriction. This led us to ask if hepatocyte senescence could be directly contributing to steatosis or if this was a mere correlation. In order to answer this question, we used the InkaTag mouse model. This mouse can be treated with a drug which is called AP2187. And upon administration of this drug, we can eliminate highly expressing P16 positive senescent cells. So we use this drug to treat mice which have been under normal diet or under a high-fat diet from six months of age. At the age of 11 months, we started treatment until they were 15 months. As expected, we saw there was a significant increase of P16 positive senescent hepatocytes in mice under high-fat diet. And after treatment with AP2187, we saw a significant reduction. But more surprisingly, at the same time, we saw that this increase in lipid accumulation in hepatocytes could be reversed by clearing or eliminating senescent cells. We next used again the InkaTag mouse model and we wanted to investigate if clearance of senescent cells can also decrease liver steatosis in aged animals. Here we did not only use the drug to clear P16 positive cells, we also used a senolytic drug cocktail which is Dasatinib and Quercetin. And we found again that we were successfully able to decrease the amount of senescent cells with both treatment and at the same time, we reduced hepatic steatosis. Next, we asked the question if fat accumulation in hepatocytes was a direct consequence of the induction of the senescence program. In order to do so, we used the premature aging mouse model, the XPG knockout mouse, which is characterized by deficient nucleotide excision repair. This mouse model was developed by Jan Heumarkers and Rotterdam. These animals show an increased DNA damage respond, they have increased senescence as well as a premature aging and reduced lifespan. For this experiment, we generated mice which have a liver specific inactivation of the DNA repair gene XPG. We found that these mice showed an age dependent increase in expression of senescent markers in hepatocytes such as TAF, P21, but they also showed an increase in hepatic fat deposition. Further supporting a relationship between fat content and senescence, we found that markers of senescence such as telomere associated foci and P21 did correlate with fat content in NAFLD patients. Altogether, our work shows an important causal relationship between cellular senescence and lipid accumulation. However, an important question remains, why do senescent cells accumulate lipids? Firstly, our work shows that this phenotype is not exclusive to hepatocytes. We have also found it in other senescent cell types like fibroblasts or brain astrocytes. Given its universal nature, we named it ALICE, the accumulation of lipids in senescence. Secondly, we found that it was associated with decreased mitochondrial function and importantly, decreased fatty acid oxidation. We have also confirmed our findings by doing lipidomics analysis of senescent cells. We found that specific lipid species such as triglycerides, ceramides, and sphingomyelins were more abundant in senescent cells than in proliferating cells. We have also used holotomographic microscopy. This type of microscopy allows long-term imaging of organelles in cells without any labels. This means it is less toxic to the cells. Our goal was to measure the dynamics of lipid droplets and its interactions with mitochondria. So what can you see here? We did manually color these organelles. You can see blue is the mitochondria and purple or dark pink, we have lipid droplets. And what we have found is that lipid droplets in senescent cells show decreased motility and decreased interaction with mitochondria. However, we are still not sure what is the physiological relevance of this finding. Finally, we tested the impact of the allies phenotype in the development of senescence. We are doing this by using x-ray radiation. So we irradiate proliferating young cells and then we are checking the establishment of the senescent phenotype seven days, 14 days, and three weeks after irradiation. And what we normally see, we see an increase in DNA damage response, we get a cell cycle arrest, but we also see an increase of lipids accumulation, which you can see here in green. And we see the establishment of this senescence-associated secretory phenotype. Here represented by the increase of IL-6, IP10, and KC. We then wanted to test what is the impact of lipids. So we used lipid-deprived media and we are doing this by using a lipid-deprived FBS. And what we can see is the cells do start to develop a senescent phenotype, they start proliferating, they have increased DNA damage, but we see a decrease in the accumulation of lipid droplets. And interestingly, at the same time, we can see that the SASP is not going to be established. We have a decrease in IL-6, we have a decreased levels of IP10, and we also don't see an increase in KC. So we have a suppression of the senescence-associated secretory phenotype. This observation has led my lab to ask the following questions, which we are now investigating. Are specific lipids regulating the senescence-associated secretory phenotype? And if so, what are the mechanisms involved? And secondly, can we interfere with lipid metabolism to counteract the SASP? And now I would like to conclude my talk and I hope I was able to convince you today that senescence is important for liver aging and during liver disease. I showed you that obesity, aging, and DNA damage can contribute to an increase of senescent SARS, especially hepatocytes. this leads to mitochondrial dysfunction, which then has an impact on fat accumulation and steatosis. I also have shown you that clearance of senescent cells could be a therapeutic strategy to reduce steatosis, reduce senescent cell burden, and restore liver function. And, of course, I would like to thank all the people which have contributed over the years to our stories. First of all, Mikovai Okvortnik, who just started his own lab in Vienna this month. I also would like to thank Edward, Thomas and João, and our collaborators at Newcastle University in Rotterdam, Glasgow, San Diego, and, of course, here at Mayo Clinic. And, of course, not to forget, I would like also to thank all the funding bodies which have supported. Dear colleagues and friends, it is for me a pleasure to talk in this special interest group program about our recent works on aging and liver disease. These are my disclosures. Today's agenda is going to be divided in three main topics. First, I will talk about the liver sinusoid and its relevance in liver diseases, following by the role of sinusoidal cells and the impact of aging, and, finally, how aging impacts on sinusoidal cells in the context of liver disease. As you very well know, the hepatic microcirculation is quite unique and it has a dual blood inflow coming from the portal vein and the hepatic artery, where blood mixes and diffuses through the hepatic microcirculation in a vascular bed called the liver sinusoid. Three main cell types are recognized in the liver sinusoid. Liver sinusoidal endothelial cells, which are discontinuous, they present fenestra and they lack basal membrane. They play key roles in homeostasis, inflammation, toxicants clearance, and the regulation of hepatic vascular tone. Hepatic stellate cells, which are the contractile cells within the sinusoid and they are the esterage of vitamin A. And, finally, Kapher cells, which are the resident macrophage in the liver, and they play key roles in defense, inflammation, and tissue remodeling. It's well known that during the progression of chronic liver disease and from a scenario where all liver cells are in a healthy situation, they react to liver injury and they change their phenotype, going through a normal phenotype to a disease phenotype, where in advanced stages of the disease we observe dysfunctional or capillarized L-sec, activated Kapher cells, and recruited macrophages, activated hepatic stellate cells, and dysfunctional and necropoptotic hepatocytes. All this has a deep impact in the progression and the clinical complications of liver diseases. Just to give you two examples, in chronic liver disease and its main complication, portal hypertension, we know that different vasoactive molecules and underlying mechanisms occurring in liver sinusoidal endothelial cells, in stellate cells, and in Kapher cells, they contribute to the development of hepatic microcirculatory dysfunction, which at the end leads to an increase in the hepatic vascular resistance and an elevation in portal pressure, thus resulting in portal hypertension. And, similarly, also liver sinusoidal cells, L-sec, Kapher cells, and hepatic stellate cells, play also a key role in acute liver injury, and in this picture you see the main underlying mechanisms contributing to ischemia and reperfusion injury, therefore confirming the key role of sinusoidal cells in this acute liver injury, in addition also, as I explained, to chronic liver disease. On the other hand, we know that the population in our world is getting older due to the increased median age and life expectancy, and this has an impact and it has been described different structural and functional abnormalities in the liver due to aging. Indeed, it has been described a reduction in liver mass and hepatic blood inflow, a decreased liver detoxification capacity in aging, an increment in oxidative stress, reduction in proliferation, and also a partial loss of L-sec fenestra, which is a term called pseudocapillarization of L-sec. And this indeed may have also a role in the context of liver diseases, because it is well known from different epidemiological studies that the incidence of liver disease increases with age. So, with this data, a few years ago we started different research avenues in our lab to understand the impact of aging on sinusoidal cells, both in healthy situations but also in chronic liver disease and acute liver injury. And this was elegantly developed by two PhD students at that time, Raquel Maeso and Diana Haidt, which now are postdoctoral fellows in other laboratories, and also, obviously, in collaboration with different people from our team and also from the clinical unit. So, in the first study, we aimed to understand which was the impact of a healthy aging on the liver sinusoid, and for that we used a preclinical model of healthy aging, rats with 20 months old. We compared these rats with younger rats, and we analyzed the hemodynamic characteristics of these animals, the function of the liver microcirculation, and the phenotype of the different liver cells. And also, we wanted to move a little bit to the bedside, and in collaboration with the surgeons in our hospital, we could do some array analysis in liver tissue from young and old individuals going surgery. In this scenario, we are talking about non-cirrhotic healthy individuals. So, the results of this study show us that old animals exhibit a slight increase in mean arterial pressure, which was already described. But, interestingly, we also observed a trend to an increase in portal pressure. This was not pathological, this was not a clinical relevant increase in portal pressure, but there was a trend. And, interestingly, when we analyzed the hepatic vascular resistance and portal blood inflow, we confirmed the reduction in portal blood inflow, and, interestingly, we observed an increase in the hepatic vascular resistance of these old animals in comparison to young. So, to understand how this hepatic vascular resistance may be increased due to aging, we characterize the main cellular components of the liver cell site. Analyzing LSEC, we confirm in our preclinical model a reduction in the number of fenestra, the percentage of porosity in these animals, also a trend in a reduction in the diameter. These data were already demonstrated and described by different groups in previous years. Nevertheless, a deeper characterization of LSEC, we observe a reduction in different markers of healthy LSEC, like CD32B, a dramatic decrease in INOS, a dramatic increase not only in the INOS protein expression, but also in the nitric oxide levels, where we observe a reduction in cyclic GMP as a secondary messenger of nitric oxide in liver tissue, and also a statistically significant decrease in nitric oxide levels in LSEC primarily isolated from young versus old. You can see here the old animals, all LSEC from this preclinical model of aging. Analyzing hepatic stellate cells, we observe a trend, maybe to an increase in the number of hepatic stellate cells, but also a dramatic and significant increase in the number of lipid droplets contained in these hepatic stellate cells, and this was already also validated by other groups. And a deeper characterization confirms a slight activation of these cells, we observe an increase in the expression of alpha sma, an increase in the expression of desmin, also at mRNA level increments in alpha sma, collagens and PDG receptor beta in old in comparison to young hepatic stellate cells, and also oxidative stress seems to be deregulated in hepatic stellate cells from old individuals in comparison to young. When analyzing macrophages and myeloid cells, firstly we observe an increase in the number of recruited neutrophils in the old animals, and also an increment in the neutrophil activity, measured as net score, also was increased in old animals, and this was accompanied by an increment in the number of CD68 positive cells without changes in CD163 positive cells, and interestingly there were no major changes in the phenotype, maybe a slight increase in some pro-inflammatory phenotype, in some pro-inflammatory markers, but not very dramatic. Finally, in this study we wanted to analyze how aging may impact on these molecular mechanisms and molecular markers of liver sinusoid, and analyzing different genes in human livers from young versus old humans, we observed deregulation in endothelial markers, deregulation in hepatic stellate cell markers, in addition to some deregulation in lipid metabolism markers, so confirming that indeed there was a global deregulation in sinusoidal pathways in human livers, in old human livers, in comparison to young. In the second study we aim at analyzing the impact of aging in the context of chronic liver disease and portal hypertension, so in this study we developed a preclinical model of chronic liver disease due to chronic CCL4 inhalation in old animals, and we compared the same preclinical model in young animals with chronic liver disease, and we analyzed the hemodynamics, liver microvascular function, and liver cells phenotype, and similarly to the previous study we also took advantage of our collaborators in the clinical unit, and we could analyze during some RNA analysis, liver tissue from young individuals with chronic liver disease, here you have the age of these patients, and liver tissue from patients, older patients with chronic liver disease, and as I said we did some array analysis. So the results of this study show us that aged animals with chronic liver disease exhibit a more dramatic increase in portal pressure, so the disease seems to be more aggravated when animals are old in comparison to young, so you can see here the value of portal pressure which is higher than in young animals with cirrhosis, and this increase in the portal pressure would be due to an increment, a further increment, in the hepatic vascular resistance in age in comparison to young. Analyzing the different cellular compartments, we observe a further decrease in endothelial defenestration in old advanced chronic liver disease animals in compared to young cirrhotic animals, and this was accompanied also by a decrease in different angiocrine, typical angiocrine markers of L-sex phenotypes, so this L-sex were further capillarized in the context of age and chronic liver disease. Hepatic stellate cells also, when we analyze cirrus red as a marker of of collagen deposition, we observe an exacerbation of fibrosis deposition, so it seems that these animals exhibit a higher fibrotic content in comparison to young cirrhotic animals, and this was accompanied by an increment of different markers of hepatic stellate cells activation, collagen, alpha-sma, and phosphomoesin, therefore suggesting an over-activation of hepatic stellate cells in the context of age and chronic liver disease. The analysis of human data, here you can see a representative heat map where you can clearly define two different populations, this is coming from liver tissue, so you can see how the human tissue of young individuals with chronic liver disease is different from the human tissue coming from old individuals with chronic liver disease, and indeed, analyzing, doing some enrichment analysis and and and ingenuity pathway analysis, we observe that indeed there were a significant number of genes that were differently upregulated or downregulated in old individuals in comparison to young, therefore suggesting that this disease may have a different molecular or underlying molecular mechanism depending on the age of the patient. In this study, we also wanted to to analyze if a possible therapeutic could be applied in aging chronic liver disease and portal hypertension, and we focus on statins. Obviously, and we focus on that considering the preclinical data showing benefits of statins in different preclinical models, but also considering the data, the evidence that is available in different clinical trials and retrospective analysis, analyzing the benefits or showing the benefits of statins in chronic liver disease. So, we add new groups in this preclinical model of chronic liver disease in all the animals, and we treated these animals, we had two groups treated with synvastatin, low dose for 15 days, or treated with vehicle, and we analyzed the effects on on major descriptors of portal hypertension. So, interestingly, you can see how the old animals with cirrhosis treated with synvastatin exhibited a portal pressure that was significantly lower than those treated with vehicle, and this was accompanied by a dramatic restoration in L-sex phenotype, as you can see, in an increment in all the parameters analyzing fenestration in these livers. Also, an improvement in liver microvascular dysfunction, so these livers could dilate much better than those treated with vehicle, and this was accompanied by a significant decrease in fibrosis. You can see 30% reduction in serious red staining that was accompanied by a deactivation in hepatic stellar cells using markers of alpha-asthma, collagen-1, and phosphomoesin as a marker of contractivity. Also, and unfortunately due to the lack of time, I'm not able to explain all this study, but you can look for it that has been published during this year, there was another study developed by Diana Heidt in our lab, where we analyzed the impact of aging in the context of acute liver injury, and we compared ischemic reperfusion injury, partial warm ischemic reperfusion in young individuals and old individuals, and we observed that there was a dramatic change in hemodynamic data, in liver microvascular function, and also in the phenotype of cells, comparing these two animal groups in this context of acute liver injury, and indeed we had some groups treated with simvastatin, and we observed how simvastatin also is protective in this preclinical scenario of acute liver injury. Before wrapping up, I would like to acknowledge my team, both the people in Barcelona and the people in Bern, especially as I said the former members, now brilliant postdocs in other labs, Raquel Maeso and Diana Heidt, but also Anabel Fernández and Martí Ortega play key roles in this preclinical work. Also our collaborators, especially Juan Carlos García Pagán, Joan François Dufour and Elisabeth Gigotti, and Victoria Coger, our friends in Sydney, help a lot in this preclinical models. Obviously, the funding agencies that support our work. So, ladies and gentlemen, dear friends, just to finalize my talk, I hope I convince you that aging has a deep impact on sinusoidal cells in the context of liver disease. Aging is accompanied by insignificant liver sinusoidal deregulation, both in rodents and in humans, suggesting vulnerability of liver microcirculation in chronic or acute liver injury. In chronic liver disease, aging activates different or additional molecular mechanisms from those observed in young, so maybe the therapeutic window also should take in consideration the age of the patient. And also, it is important, I think, that considering the low translatability of therapies that we obtain in preclinical models to the clinical scenario, we propose using closer to real-life models to allow the development of more reliable therapeutics. Just to finalize, I would like to tell all of you that in our lab we have samples from different organs and blood from all these preclinical models, aging and aging plus liver disease, and these are available for collaboration, so just email me or send me a Twitter message and I will be more than happy to collaborate with you. With this I finalize and I will be happy to take any questions. Thank you and take care. So greetings from Dr. Alpini's virtual office. Thank you for the invitation to give this presentation at the SLD 2020, I have no financial disclosure. The objective of my lecture is to demonstrate the inhibition of biliary senescence in the model of PSC or other models of cholestasis, such as BDL, will decrease the biliary damage in liver fibrosis. Also the melatonin signaling, differential and modulated biliary senescence in the cholestatic model, and that melatonin inhibition of biliary senescence in liver fibrosis is mediated by the regulation of 200B angiogenic signaling. Also I will show data that prolonged treatment of MDMAs or melatonin ameliorated PSC phenotypes. Why am I going to have this link between P16 and melatonin? Because by PS software and other preliminary data, P16 modulates the melatonin signaling, but also melatonin regulates the senescence, and this link may happen through 200B. We don't have all the data yet, but I'm going to show you some of the data that we have. In addition to modulating the actual biosecretion, cholangiocytes are the target of a number of cholestatic liver diseases such as PSC, PBC, biliary atresia, etc. And the change in natural reaction contributed to change of senescence, or vice versa, and then to the release of biliary senescence factor called the SASPs, which may induce the activation of cell cells through paracrine pathway. There is a lot of studies showing there is an increase of biliary senescence in PBC, PSC, for Larusso group. We show in unpublished data an increase of biliary senescence in cholangiocytes from patients with alcoholic hepatitis. We also published data showing an increase of biliary senescence in PSC patients. Kennedy and Francis have shown in American Journal of Pathology an increase of biliary senescence in patients with NASH. So we postulated that when there is a liver injury, there is an increase of biliary senescence which induces a change of natural reaction, or vice versa, and an increase of senescence may induce activation of cell cells through the release of SASPs phenotypes such as cytokines interleukin-168-TGF-beta induce the activation of cell cells by paracrine loop. And the increase of senescence may induce the change of cholangiocytes senescence in other subset of cholangiocytes. In our model, we treat wild-type animals and MDR animals with P16 mismatch morpholino control, or saline, or a vivo-morpholino which reduces the immunoreactivity expression P16. Then we will measure liver section, isolated cholangiocytes, STLA cells, marco-senescence, fibrosis, and biliary proliferation. We show that there is an increase of P16 immunoreactivity in cholangiocytes from MDR mice and also an increase of P16 by PCR and a decrease of senescence in STLA cells from MDR animals. This is an established concept, increase of biary senescence induces activation of STLA cells, a decrease of STLA cells senescence. When we treat MDR animals with P16 vivo-morpholino, we found a decrease of P16 immunoreactivity. Compare the MDR controls. Same profile was observed when we treated the animal with P16 morpholino. We found a decrease of senescence gene expression in orange compared to the MDR animal in green. Similarly, when we treated MDR animals with P16 vivo-morpholino, we found a decrease of dachshund mass. Compare MDR animals. No change was seen in normal animals treated with vivo-morpholino. Similar profile was observed regarding fibrosis. We observed a decrease of fibrosis in MDR animals treated with morpholino for P16. Compare MDR animals. We found a decrease of collagen deposition. Compare MDR animals. And also, we found a decrease of fibrosis gene expressions in colangiosides from MDR animals treated with P16 vivo-morpholino in purple. Compare MDR in orange. Previously, we have identified an important factor, the TGF-beta, that is an important suspect which may mediate the interaction between colangiosides and their cells. As shown here, when you block senescence, we have a decrease in immunoreactivity of TGF-beta. In addition to that, we identified a number of other factors which are decreased by treatment of the MDR mice with a P16 vivo-morpholino. There is a decrease of number of suspects in green, such as interleukin, VGF, TNF-alpha resistant, compared to the colangiosides from MDR animals in red. Now, one of the potential links between senescence and melatonin signaling is maybe 200B. So, in addition to look how P16 may influence senescence, we will look how melatonin may influence senescence, maybe through 200B. We don't have all the data, but we have the data to show the melatonin effect is mediated by changing 200B and the angiogenic factors. Why 200B? Because it is an important haemoglobin, which is a particular and humble disease. In terms of background on melatonin, melatonin is not only synthesized by the pancreas and the brain, but a number of peripheral organs, including the liver. In the liver, the major producer of melatonin is the colangioside, which is especially ANAT, a synthesized melatonin. And we found a low expression of ANAT in stellar cells, very little, and no in hepatocytes. Regarding the expression of the receptor, we found expression in T1 and T2, mostly in colangiosides, very little in stellar cells, and no expression in hepatocytes. We are going to show you data on how the modulation of T1 and T2 may affect the proliferation of the actual reaction of colangiosides. We are not speaking about clocked genes, even though clocked genes are very important. Why? Because they are modulated by melatonin, and because the change in clocked genes may affect SNS and melatonin signaling. Now, we know there is an increased expression of MT1 in the PSC human sample, in bald-hatted animals, and also in MDR. But we don't know exactly about the distribution of MT2. We found an increased MT2 in bald-hatted animals, in PSC human sample, a decrease in MDR. So we are doing more experiments to pinpoint this issue. Why? Because potentially, if the MT1 is the better set, or if you block MT1 thoretically, we should have an immediate ratio of PSC phenotypes. And in fact, when we do BDL in MT1 knockout animals, total knockout, we found a decrease of senescence. Compare what type? We found an increase of senescence in MT2 BDL knockout animal. Compare the whole type. Similarly, in MT1 BDL knockout animal, we found a decrease of dactyl reaction. Compare to the whole type, an increase of dactyl reaction or proliferation in MT2 BDL knockout animal. Compare the BDL. Supporting the concept that MT1 is the better reset, MT2 maybe is the good reset. A similar profile was seen regarding fibrosis, because we found a decrease of collagen deposition in BDL MT1 knockout animals. Compare the BDL whole type, and a huge increase of collagen deposition in BDL MT2 knockout animal compared to the BDL whole type. Now, one of the potential mechanisms for this differential effect may be cyclic MPC granule. Why? Because CMP, PKA, HERC1 and HERC2 is a key mediator of biliary proliferation, and CMP may interact not only with cholangiocytes, but also with other cells, perhaps maybe through melatonin, MT1 and MT2. So, we are trying to put together a story how changes in the animal influence these downstream signaling. We don't have all the data. We have also some parts of the puzzle, but we know that when you block MT1 in baldactylic DNA animals, there is a decrease in phosphorylation of PKA that was increased in MT2 compared to BDL. Also, you can see an increase of PKA phosphorylation in MDR animal model PSC and human PSC samples. So, this is an important mechanism. We are looking at how this may be the downstream signaling which may go into interactivity maybe with melatonin signaling. Now, if you treat MDR2 animals with MT1 via morpholino, which is not a specific knockout model, but is a model which allows two degrees of specific on the leader MT1, you reduce not only suspected MT1 immunoreactivity expression compared to the mismatched MDR2 animals, more severe degrees of liver injury, dactyl mass, liver fibrosis, and inflammation compared to the mismatched MDR2 animals, a very drastic decrease of these phenotypes. Now, what we are doing now, we are treating MDR2 animals with melatonin for one week or three months, and we also treat the animal in the dark, which increases the melatonin release, and we are going to show some phenotypes. When you treat the MDR2 animal with melatonin for 12 weeks, you have a huge decrease of senescence. We found the same profile, of course, with the dark. This is shown by beta-gallons liver section and by PCR decrease or senescence gene expression compared to MDR2 animals. We found an increase of 200B in MDR2 animals, which was decreased by dark therapy or melatonin treatment. Similar profile was observed in vitro when we treated cholangiocytes with melatonin. And the validation of the model, we found an increase in melatonin when we treated MDR animals with melatonin in the dark. We don't understand why there is an increase of melatonin in MDR2 animals, maybe a compensatory mechanism. Again, if you treat MDR animals with dark therapy or melatonin, you have a huge decrease of dactyl mass. Compare the MDR animals. We treated the animal with melatonin for three months, and we found a huge decrease of liver inflammation and infiltration macrophages. Compare the MDR animals. When we treated MDR animals for one week with melatonin or dark therapy, we found a decrease of collagen deposition compared to MDR2 animals, suggesting that this is an important factor. Now, again, I show you data in p16, senescence and melatonin. We don't have the link completely, but we are working together to see how these feedback mechanisms may influence biliary damage, senescence, and liver fibrosis. We also show that when we treat the MDR2 animals with dark or melatonin therapy for one week or three months, there is a decrease of angiogenesis. Why I show you angiogenesis is because angiogenesis is an important component of PSC, and we have shown previously how angiogenesis increases in PSC in MDR animals. I'm going to show you some data on TGF-beta because it's an important trigger for angiogenesis. Presumably, this axis, melatonin 200B, angiogenesis is important, which may be added by TGF-beta 1. We don't have all the pieces because we are trying to put together the link between p16 and 200B. In our model, we propose that when there is a damage of cholangiocytes which undergoes senescence, they may affect the senescence of other neighboring cholangiocytes. Thus, it changes the biliary senescence of other subset of cholangiocytes and the senescence and their potential natural reaction. This may bring to a change to release a number of SASPs, such as interleukin, TGF-beta, TNF-alpha, NGF, BGF, etc., which may influence the activation of senescence by paracrine pathway, by the release of these SASPs. Of course, melatonin is an important component because it may block this p16. So, while p16 influences melatonin, melatonin signaling feedback will speak to the p16. We don't have all the pieces. In my opinion, this is a very interesting hypothesis, which may give a lot of information. Also, the downstream signaling mechanism, signaling MP mechanism, mechanism of repair. Now, I wouldn't be here without the support of the NIH, the VA, the PSC Partnership in Cure, Indiana University, especially from Texas A&M, but also from IU, Dr. Francis' group, which is invaluable asset to our program. Also, Boots in XR from IU, who is a transplant surgeon who helps us a lot tremendously on the translational study. I want to thank you more for my presentation, for my virtual lab, and I hope in STL we can see each other in person because this is very challenging, and also very difficult to try to give a lecture looking at yourself. And with this, I want to thank you again, and again, I hope I can see you in STL in person. This is a closing remark for Liver Cell Biology in Hepatic Disease-Sick Symposium, entitled Cellular Senescence in Liver Disease and Aging. I'm Yasuko Iwakiri, a current chair of Liver Cell Biology-Sick, and co-chair of this symposium with Dr. Gracia Sancho. I would like to thank all the speakers to make this symposium possible, and also thank all the attendees for joining this session. If you have any questions, please write in the chat area. Thank you again. Stay safe and healthy.

Liver Cell Biology

Hepatic Disease

SIG Symposium

Cellular Senescence

Pro-inflammatory Cytokine Secretion

Mitochondrial Dysfunction

Liver Diseases

HCC Fibrosis

Cholangiopathies