So, I will try to give some of the basic aspects of regeneration, but also to make some connections between regeneration and liver disease, because this is something which is really often not covered very well, and also point on the things that we believe we reasonably will understand, but also other things that we really do not understand very well. I don't know if you can see my cursor over here. Probably not. Oops. I have a laser pointer, but I have to go back and get it. Excuse me. Smoke if you got them. We're going to give you, we'll give you a pass on that one, George. Oh, by the way, if anybody's got a cell phone, make sure they got it turned off, or silenced, because mine will surely go off. So, in my first slide, kind of a funny angle over here, but the point I'm trying to make over here is this, that in the mythology of regeneration, where Prometheus' liver that was in the chest cavity in the ancient times, it moved down later, was eaten by the bird, it says, in essence, that the next day, the liver was coming to the same size as the day before. And the point I'm trying to make over here, and I'll try to emphasize it in the second half of my talk, is that liver is a very obsessive-compulsive organ. It always strives to be 100% of the liver-to-body weight ratio under optimal conditions. So other organs, if they lose part of their mass, like you lose half your pancreas, you can lose half your pancreas, and if you lose one lung, the other gets a little bigger, but doesn't make 200%, and if you lose one kidney, the other kidney gets a little bigger. But liver has to go to exactly where it needs to be to provide the optimal functions. So that forces a continual regeneration if there's a continual damage. So how does regeneration start? In the model, I will try to spend more time today, because we don't have too much time to discuss things, but it is liver regeneration after partial hepatectomy. As you know, in the rodents, liver has multiple lobes. Think of it as a stem with five grapes. You can take three grapes out, and the other two grapes grow to size to become the size of the original five. The advantage of this model is that it does not have any necrosis and any inflammatory components. So you can precisely determine when does regeneration process start, and there's no inflammatory components to really get mingled with the signaling pathways that control the process. So the most first and important thing which actually happens is that the entire portal vein flow now goes through only one-third of the capillary volume and surface, because you've done a two-thirds hepatectomy, and you have one-third left. That increases the pressure of the portal vein and has impact on the cells from which the flow goes through. And you can see over here, the pressure in this particular experiment, these investigators diverted part of the portal vein flow, not entirely, and they noticed that in the diversion, the pressure was kept constant, whereas in the absence, the pressure went up. And the cell volume was normally going up, and it stayed the same, and the activation of hepatocyte growth factor also stayed at a lower level. So there's some things that are associated with the flow, and we do not really fully understand that. And I'll try to give back some speculation. But within a minute after this happens, urokinase activity comes up, and urokinase starts the breaking of the extracellular matrix, activates HGF, activates plasminosine-2-plasmin, metalloproteinases, and breaking down the extracellular matrix, releasing more HGF. This is a very rapid process in one minute. Other things happen very quickly. For example, beta-catenin, which is shown right here under the plasma membrane, it migrates into the nucleus in about five minutes. The red and the green of the nuclei becomes yellow, and it stays on for several hours until it finally goes down very, very quickly. If you're looking to notch the intracellular domain, that also, after hypotectomy, migrates into the nuclei, whereas in sham operations, it stays in the plasma. And the dependent genes, HES-1 and HES-2, if I have it over there, they are also going up at the same time. So how can things happen so quickly? It's impossible to explain that on releasing of a humoral factor of some sort. So what I have proposed now for the last couple of years is this particular situation, this green cell with all these projections, is the most understudied cell of the liver, the normal stellate cell of the normal liver. We think of the activated stellate cell, which is small, makes connective tissue, contributes to cirrhosis, and all of that. But in the normal liver, you have all these processes coming in, connecting endothelial cells on one side and hepatocytes on the other. And Kendra Wake from Japan gave a very good description, published about three years ago, of these stellate cells. They make special contacts with the hepatocytes and the endothelial cells, almost like synaptic contacts. This is a stellate cell after birth. In three months, it looks like this, and it becomes very, very big. This is a stereotactic reconstruction. And what he proposed is that, actually, in the liver, there are these units called stellons, where you have a stellate cell connecting two hepatocytes and five endothelial cells. Now, if this is the case, and you increase the strain and the stress of the endothelial cells, conceivably, a signal can be coming from the stellate cells that can be not a humoral one, but an electrochemical one, because stellate cells make catecholamines. They have all the catecholamines synthesizing enzymes, a lot of neurotrophic receptors, and they're very peculiar cells. They come from a cardiac mesenchyme, but they look more like the astrocytes of the brain. That is just a speculative situation, but beyond that, things we've been able to study, because I started, let's say, 10 minutes later on, is the story with AGF, hepatic extracellular matrix, urokinase, matrix metalloproteinases. And the protein itself is heavily localized in the extracellular matrix. This activates a single chain AGF to the active heterodimer, and then the urokinase activity rises very quickly. And I should also point out, in this particular slide over here, this is the AGF coming up in one hour in the plasma, and this is the hepatocyte DNA synthesis, and this is the heavy chain of the heterodimer, so this AGF is activated when it goes to the blood. The mRNA for AGF, however, doesn't come up until about three hours later. So this is from pre-existing stores, from the degradation of the extracellular matrix. The receptors for AGF, the MET receptor in EGFR, all become activated within about 30 minutes after a hepatectomy, and this is sort of associated with the beginning of the whole process. Now, there's a lot of things that happen in the first hour that not all of them are explained by AGF and EGF, but there's a decrease, there's a translocation of beta-catenin, we talked about that, there's the receptors being activated, but there's also increased not just AGF, but also norepinephrine, interleukin-6, TNF-alpha, and TGF-beta-1, and hyaluronic acid, which is the fact that the extracellular matrix breaks down, and the glue that holds it together, the hyaluronic acid, is just pumped up into the blood. I forgot to mention that when we say that the mRNA goes up in the liver for AGF, it goes also up in the lungs, and it goes up in the kidney, and it goes up in the spleen. What transmits the signal to those other organs to make more AGF when the liver is regenerating? We really don't have a clue. Norepinephrine can stimulate production of AGF or fibroblasts, in fact, Dr. Peterson was associated with a particular paper, and it can seemingly be the factor, since it rises in the blood, that turns it on, but nobody has actually proven the point. Now, through the years, over the last two and a half, maybe three decades, a lot of signals have come up with regeneration. There was a time in the 90s and the early 2000s where somebody would take out one signal, see the regeneration delay, and say, aha, this controls liver regeneration. There's about 20 papers of that type. But if you look at them all, there's quite a few of these signals, very valid, and I've been separating them into auxiliary mitogens, because these do not turn on hepatocyte DNA synthesis in cells in culture, and if you inject them to the whole animal, you do not get DNA synthesis in a normal mouse, contrasting those to the complete mitogens, which they can stimulate hepatocyte proliferation in culture, and if you inject them to a normal mouse, you can get liver enlargement and proliferation, and those are AGF in this receptor and EGFR, and these ligands are the ones that have been actually investigated in that regard. But since that time, other pathways, complete, complex pathways have come up, and it's difficult to assess those in culture, when, for example, all the ligands are very hydrophobic and they really don't do very well in cell culture, and these are complex pathways that should be looked at from kind of an intermediate perspective. The fact that they're auxiliary mitogens does not diminish their importance. If you have lack of regeneration, and you have a massive liver failure, missing any of these signals and delay of regeneration can be lethal to the patient that suffers from it. Now, once the process starts, hepatocytes in the brown line over here are the first ones to go into DNA synthesis, this is the rat chronology at 24 hours, the mouse is about 36 to 40 hours, cholangiocytes go at about the same time, and macrophages, copper cells, not too far from there. The endothelial cells, however, have a very, very, very prolonged regeneration, and there's now evidence that we cannot cover from several investigators that for the macrophages, some of them come from the bone marrow, and they mix with the copper cells that are there. Same thing for the endothelial cells, VEGF comes up in the blood, and progenitors of endothelial cells come into the liver and mingle with the sinusoidal endothelial cells to establish the new pathways. Now, during regeneration, a proliferating hepatocyte loses a lot of its differentiation. So if all hepatocytes are going to regenerate at the same time, you may have a liver failure. So regeneration actually proceeds as a wave from the periportal down to the central lobular regions, and you look here, one of these metalloproteinases, it was metalloproteinase 9, did immunohistochemical stain, just one periportal cell at about two to four hours, but you go to 24 hours, and about two-thirds of the lobules are now positive, and at 48 hours in the rat, it's only pericentral. And this wave allows the organ to deliver the essential functions without having liver failure coming in, with every cell losing its differentiation. As things proceed, in about a day or two, hepatocytes really make a lot of other growth-related signals to the adjacent cells. They make GMC-SF for the copper cells. They get back AGF and TNF-alpha-angioleukin-6. The stellar cells send back inactive AGF, and they get EGF-alpha from the hepatocytes. And for the blood, there comes EGF, and we'll talk about that a little bit more. So currently, the liver regeneration is the ultimate successful and very willful and very wily process, because you can never stop it by stopping one single signaling pathway. And anything that has been tried so far, it just delays the process. In fact, when we took out hepatocyte, the AGF gene, we actually waited for about a month, and we did a hepatectomy, and we found out, actually, that the proliferation of hepatocytes in the mice that had the gene of AGF inactivated was perfectly normal, and the protein levels were also normal. It hadn't gone down, because there's so much of it in the hepatic extracellular matrix. So we had to do two chemical hypotectomies with carbon tetrachloride to be able to diminish and eliminate the existing AGF in the exocellular matrix over here, and then we noticed that there was actually an effect on regeneration. So think of AGF in the exocellular matrix as a charged battery reserved for the liver to be able to always have enough to use it for whatever process it may need. If you inject AGF in the tail vein, not in the portal vein, most of the AGF goes to the liver. It goes to the periportal areas. It is normally made by the stellate cells, not by the hepatocytes. But when we took both the signals for the AGF receptor and the EGF receptor, we stopped liver regeneration. And I was so happy because I think I've been studying all my life, I was finally able to block it. And that may sound paradoxical, but nonetheless, what we found was that the peak of the control in terms of labeling index, you can see it over here. You take out the EGF receptor, it goes down. You take out MET, it goes down quite a bit. But MET, you have more coming up at day four. And if you take out both of them, then it goes down even further, and down there is zero actually at day 14. But if you look into the liver-to-body weight ratio, which is I think more instructive, this is the, in our minds, the normal liver-to-body weight ratio. And with the hepatectomy, drops down to here, and if you stretch the line to the double inhibited at day 14, there's no increase. So we wanted to know what is the reason for that, and what happens in these very suppressed livers. And we found some very interesting things. This is a glutamine synthetase stain, which is positive for one cell layer around the central veins. So you can see the central veins, the distance, kind of the size of the lobule by looking at them. The same, this is day 14 control mites. This is day 14 double inhibited. The lobules shrink. It's the same magnification. And the hepatocytes over here in terms of size, they come down to about I think 35% of a normal hepatocyte. They're much smaller here compared to the normal at day 14 hepatocytes over there. So a lot of things collapse, and I'm not going to have a chance in a time to go through all of those things. But as an example, the lipid synthesizing enzymes, you can see in the different categories, the black is the double inhibited. They're very, very much down, all of these lipid synthesizing enzymes. And at day two after hepatectomy, there's lipid accumulation normally in the normal hepatocytes. They last for about a day or two, and then it goes away. And that happens in the control. But in the EGFR inhibited, there's no lipid accumulation. With met, there's some. And a double inhibited looks like the EGFR receptor. We pursued that, and I'm not going to talk about it today, but we just published a paper in hepatology. It finally came out in print after about a year of being accepted that shows that if you inhibit the EGFR receptor, you actually can stop NAFLD. And if you have NAFLD and you inhibit the EGFR receptor, you reverse it by about 90%. We're trying to understand the mechanisms, and the paper describes more of what happens when that receptor is inhibited, PPR, gamma is involved, and many other things. But we started all the work from this particular finding that there's something happening with EGFR that the met doesn't exactly do. But there were more things of interest. We talked about this. Urea synthesis enzymes, two of them are mitochondrial. And those are the purple lines over here. They're very, very suppressed. So the urea cycle does not work. And the mice at day 14 have a lot of ammonia and low glucose. But this is, to me, a paradoxical situation. These are all the proteins that the hepatocytes send out to the peripheral blood, the plasma proteins, all of them. And the pink bars are those small, shrunken hepatocytes that have lost all other functions, but they will not abandon that function, which says send proteins to the blood. And that's almost like a computer program. There's a bit of a cybernetic situation here that everything else can be shut down, mTOR was inhibited, AKT was inhibited. But that particular program, in fact, was intensified compared to any of the other normal categories for things which we truly, at this point, do not understand. But it's impressive how much the cybernetics of gene expression are really something which we need to understand a little bit more. The mice predictably died about day 14. An upset mouse over there, as you can see, with, again, low glucose and high ammonia. And it would do the same thing to normal mice, not hepatectomy. They also died day 14. So there's different plasma, different gene expression changes for those. Now, if you look into the liver, we gave EGFR inhibitor in the food so that the intestinal epithelium should also be inhibited, and the MET elimination was systemic. And yet, the KS67 in the crypts of the intestine, all this brown, is perfectly normal. Intestine was not affected. Lungs were not affected, or kidneys, for that matter, only liver. So why is liver so dependent on those two receptors? Well, from this cartoon, as I said before, AGF is in the extracellular matrix, heavily embedded, and actually, the AGF receptor and the EGF receptor are activated in the mouse all the time. But where is this EGF coming from? And this took a while to figure out because it came to a lot of neglected publications, if you want. That is the mucosa of the duodenum there, and underneath it, there are the brunner glands of the duodenum, which are like salivary glands. As you know, salivary glands make EGF. This is why animals leak their wounds, because there's EGF in the saliva. So this EGF, as the team from Olsen, et cetera, from Sweden showed, was injected intact into the lumen of the intestine. It was not broken down. It was absorbed intact from the duodenum and went back to the parotid vein into the liver. So there's a constant supply of EGF from the brunner glands of the duodenum going back to the liver, so the liver is constantly exposed to EGF and AGF. And I think that that's a supply form of signaling that, if collapsed, most of the other signaling pathways, as we understand them, are actually collapsing right along with it. Now, we talked about the fact the liver tends to regenerate to 100% of its original volume, and that creates a problem, because if you have chronic liver disease, there's continual hepatocyte proliferation that takes place. But there's more than that. Hepatocytes normally increase ploidy after birth, and you can get to be in the mouse octoploid, hexadecaploid, all that. If you have continual proliferation because of inflammation or whatever, then there's a reversal of the ploidy, and you end up with diploid cells, and this has been published by several groups. And that's not too bad. What's bad is that in the normal hepatocytes, there's polyploid, randomly missing chromosomes. So you have an octoploid cell that is missing, let's say, a chromosome 13, and it makes eventually four diploid cells, of which one of these diploid cells has only one chromosome 13. But this proliferation does not take place in the normal environment. It is an environment of a chronic inflammation with peroxidized lipids, with reactive oxygen radicals, a lot of genotoxic electrophiles. So there's, at the end of the day, loss of heterozygosity in some of these diploid cells, and have accumulated mutations which are not balanced by the genes of the normal chromosome. This is why every chronic liver disease, every one of them, increases the chance of getting liver cancer. There's no special mechanisms. This is fundamentally one of the causes for that. And this is a diagram describing the whole concept. It was published in Hepatology about three years ago. But what if you block liver proliferation, hepatocyte proliferation? This was done by Emanuel Farber, 1977, way back. I was a graduate student at the time. And if you block liver regeneration with AAF, as he tried, nothing happens. Liver cannot grow, because it makes adducts to the DNA, and all of that. If you use diethyl nitrosamine before that, and think of diethyl nitrosamine as a genomic bazooka, it will just really flood the whole thing with mutations. Then you get these nodules that come up, and they restore the weight of the normal liver by generating these nodules. And he called those resistant hepatocytes, that there's a random existence of hepatocytes with different properties that happen to be, some of them, resistant to something. And that's very important for human liver disease, because if you look at the hemochromatosis, accumulation of iron, these hepatocytes cannot proliferate very much. But all the hepatocellular carcinomas that come from there cannot store iron. They're the resistant hepatocytes that didn't have the defect, randomly. And glycosine storage disease, lipid storage disease, alpha-autotripsin deficiency, they're all missing the fundamental cause of the disease, and they have to proliferate because of the hepatostat to maintain the liver weight. And in doing so, they proliferate more intensely, because there's very, very few of them, and they tend to accumulate more mutations, compared to the situation of all hepatocytes proliferating. My last two slides over here, and this is something we published two years ago, and this is just one slide. And we showed, in essence, that the hippo pathway, which regulates YAP, is actually controlled by Glybicin-3, which binds to a protein called CD81, and when that binds there, then hippo pathway goes up, it's become more active, and YAP goes down. Now, CD81 is the portal of entry of the hepatitis C virus. And I didn't know that at the time. But the E2 protein of the hepatitis C virus, if you add it, in essence, YAP goes down, you have increased, in essence, decreased, rather, in essence, phosphorylation, and decrease in YAP, and activation of the kinases of the hippo pathway. The HCV would tend to suppress hepatocyte proliferation by allowing the hippo pathway to be active, and YAP to be going down. Think of the salt and fiber protocol, the AAF, you suppress all hepatocyte proliferation. Anything resistant will have to keep proliferating to maintain liver weight. Well, how does this apply to HCV? HCV is a major association with hepatocellular carcinomas in the U.S. today. So if you take the approach that HCV decreases hepatocyte proliferation, then you say, what are the resistant hepatocytes to HCV? And if you look at the hepatocellular carcinomas over here, 78% of them do not express CD81. And there's paper that came from Liang and NIDDK that if you don't have CD81, you're resistant to HCV infection. Now, I'm trying to understand how this HCV is associated with liver cancer, because it does not have an activating oncogene. It does not enter into the DNA. It causes chronic inflammation, and now we know that it suppresses YAP. It could be a salt and fiber protocol on slow motion, if you think about it that way. And I think that it may be more of a promoter for carcinogenesis than an initiator. And I think I have... You're out of time. Out of time? Okay. Very quickly, how does regeneration terminate? Well, you can say, how does it start? But if you look in the gene expression patterns over here, at day six, proliferation of hepatocytes stops, but the gene expression changes, go up to beyond day 14. Interglycline kinase is associated as a growth suppressor signal for hepatocytes. If you take it out, you do partial hepatectomy. Liver jumps above the beginning of its weight. Liver is bigger. You take interglycline kinase, and it's one of the things we think is associated with termination of regeneration, but there's obviously many others who don't fully understand it. What I'm trying to say over there is that regeneration overall is good for acute damage, and if it is a chronic proliferation, then we may have not so much a boon, but a bane, because you have hepatocytes in essence associated with chronic proliferation, fibrosis, and all that. And that is something which we need to put into perspective. And also the fact that there's some resistant hepatocytes that can proliferate more than needed to give rise to cancer. Thanking all my current collaborators and friends in the laboratory over here. Wendy Morris, who's here with us at the meetings, Bruce Forensic, who's here, Barad Bushan, Kelly Crowell could not make it, Yuhui Hu, but also my future collaborators, my grandchildren, which someday, if I'm still around, they may come and join me. Thank you very much. Thank you.

liver regeneration

liver disease

organ regeneration

partial hepatectomy

stellate cells

Hippo pathway