Thank you, [INAUDIBLE]. And thank you, everyone, for your time to listen to some of the updates that I am very honored to present to you about our work in COPD pathogenesis and especially potential future [INAUDIBLE] fixing this very common disease. I would really want to also acknowledge Dean Wilkes. 

David has been a mentor for me at Indiana University, where I spent nine years. He was instrumental in recruiting me there and showing me the ropes, both of how to become an independent physician scientist, as well as an effective, hopefully, administrator. So I've moved about three years ago to National Jewish Health, affiliated with University of Colorado, where I am serving as division chief for Pulmonary Critical Care and Sleep Medicine. And so I'm really thankful to all my mentors that were mentioned, as well as Dean Wilkes, for the hospitality. 

So the question is can the lung be repaired in emphysema? And this is something that we all want to address in the clinic. The task is pretty tall, because emphysema is a component of COPD, a spectrum of diseases which encompasses a wide variety of phenotypes. And I'm showing here the two classical phenotypes of COPD, chronic bronchitis, characterized by thickening and inflammation of the large airways and mucous hyper production and cough. And ranging to emphysema, which is characterized, in contrast, by destruction and net loss of alveolar tissue and loss of gas exchange areas and hypoxemia and shortness of breath, both initially with exercise and then at rest. 

And as you all know, asthma features in the gray overlap area. We have many overlap syndromes of asthma and COPD. And as we learn more and more about the pathogenesis of asthma, we may import some of that knowledge into our understanding of COPD and therefore, the treatment options for COPD. 

So for the patients in the room and to understand the modeling of the disease, one needs to know what are the primary reasons why folks develop COPD. And this is a condition that takes decades to develop. And it's a chronic disease, as its name says. But in addition to a type heterogeneity, it has a clear both predisposition in this [INAUDIBLE] environmentally sourced, but also predisposition in relation to genetic background. 

So while the most common etiology is related to exposure to cigarette smoking in the developed world, exposure to other biomass fuels and environmental pollutants is clearly playing a role. And the most common genetic predisposition factor is that of alpha-1 antitrypsin deficiency, where a single mutation in these alpha-1 antitrypsin [INAUDIBLE] gene causes a misfolded protein retained in the liver, and the lung becomes more predisposed, about ten-fold higher risk of developing COPD in response to smoking. Otherwise, only about 25% of folks who smoke develop emphysema and develop COPD. And aging and age is an independent risk factor for this condition, as well. 

I wanted to point attention of, especially residents, of the association of HIV with COPD. These may come up on the boards and it may come up in your clinical practice, as well, as individuals with HIV live longer, they're at risk for chronic degenerative conditions. Many have inflammatory phenotype or a vascular phenotype. In some cases, then we are recognizing that HIV-infected individuals are presenting with a low DLCO irrespective of their exposure history to smoking or environmental pollutants. 

So as we mentioned yesterday, I'm going to refer twice to what happened yesterday. Because many of you were not there, so it's unfair to refer to yesterday, but I don't want to repeat myself too much from those who were there at my talk. This is a very simplistic view of COPD. And it's even with the simplistic view, when we enroll patients in trials, we don't really differentiate between emphysema and chronic bronchitis, which I think stalls a little bit of progress of identifying effective therapy. 

And the reality is that the disease is even more complicated, in that we have multiple sub phenotypes with small airway involvement, those with or without the presence of collateral, which makes a difference. Many systemic manifestations of the disease that pose a huge burden on the morbidity and even mortality of our patients. So we clearly need to have a phenotype and even endotype specific approach to understanding its pathogenesis and designing therapy. 

So I'm going to show you, since today the focus is going to be primarily on therapy, on repair and regeneration, I have to mention what are the current well-established mechanisms by which the lungs gets injured in COPD, and especially in emphysema. 

So in emphysema, as I mentioned, we have a destruction of this area called the alveolar unit, which I'm showing here. This is the alveolar septum. We have the airway-- that doesn't project-- with an alveolar type 2 cell, shown there, facing the airway. The lining of the alveolus is formed of type 1 epithelial cells. And then the capillaries are lined with endothelial cells there in the middle. 

So I'm going to go back, and I'm going to try this again. There we go. All right. So it's a big, big area of highlight. So this is a capillary lined with the endothelial cells. And this was the [INAUDIBLE] cell. 

So how does this unit get destroyed? Can we regenerate it? Can we repair it? So the mechanisms of destruction, the earliest understood were related to oxidative stress and protease-antiprotease imbalance. Barrier dysfunction of both the epithelium and the endothelium are now clearly on the map, put there by others in our laboratory. Cell damage, cell injury, cell stresses are clearly recognized in response to cigarette smoking. And they include cell autophagy, including mitophagy, cell senescence, and death by various mechanisms. 

However, the airways, the larger airways, may have different mechanism of damage and inflammation. There is an early loss of ciliary [INAUDIBLE], of ciliary faction. And interestingly, that interfaces with autophagy as Choi's lab has recently shown. There's abnormal mucus production and obvious inflammation and autoimmune responses that are playing a huge role in this disease. 

But both the distal alveolar area, the distal parenchyma as well as the proximal airways, they both seem to be associated with an ineffective repair and the failure of stem cell or reparative cells to do their job. And the responses of the body to continuous injury may be maladaptive to eventually result in this disease. 

So how can we repair this chronic condition? So the logical laundry list of reparative approaches would include trying to interrupt the destructive processes, to remove the damaged cells that are lingering around, spilling their pro inflammatory and injurious content, to engage molecular and cellular repair through autophagy that recycles damaged proteins or damaged organelles, to DNA repair of mutated DNA. And as we recently learned, we also have a very robust mechanism of repairing the plasma membrane. Finally, engaging cellular repair and through maintenance programs such as cell regeneration, engaging local cell survival, proliferation, and matrix rejuvenation. 

So can we do this with a single drug? It's very unlikely that we can find a single targeted therapy and cure COPD. So therefore, we reasoned that a more general approach may be needed, and kind of throwing the kitchen sink at it. And what I'm going to talk to you about would be about using stem progenitor cells, either by exogenously administering them or improve the vigor of the endogenous lung or bone marrow pool of regenerative cells or progenitor cells. 

So the approaches in general to respiratory disease using regenerative approaches can also be categorized, as I said, by endogenous repair mechanisms. And in the exogenous repair, people typically include gene therapy and cell engraftment. Functional repair is often cited through engaging of autophagy, immunomodulation, erythrocytosis. 

And an interesting area that I'm not going to talk to you about, but I want to point this to you so you remember to follow the progress, is using organ bioengineering ex vivo, where one takes decellularized lungs and populate these decellularized lungs ex vivo with engineered cells or progenitor cells. And there's an interest in using these IPF cells to really repopulate the lung and perhaps use it for transplantation, since we have such a shortage of organ. 

There is less of a talk in the field about using these mini lung structures to reimplant them somehow endoscopically and see if they take and they participate in gas exchange. That would be more of a science fiction realm at this point. So most of this work is meant not only to understand pathogenesis, but to maybe create in the lab organs for future transplantation. 

So our interest in this field stemmed in Indiana when we came, our lab came with a vascular background. We thought that improving capillary health in the distal lung will prevent or repair emphysema. So there we talked with another lab in cardiology, led by Keith March, who was using adipose progenitor cells to help the heart or the peripheral circulation. And they were very frustrated that these cells would end up in the lung following systemic injection, intravascular injection, especially intravenous injection. 

So one thing led to another. And we decided to study this concept of administering exogenously these adult adipose trauma cells, which is not a stem cell, it's a stromal progenitor cell. I'm going to call it ASC from now on. But it's coming from this name, rather than stem. 

So it's been shown by their group and others to have angiogenic anti-apoptotic potential and stabilize blood vessels, like parasites. So we reasoned that if they end up in the lung, and if we want to improve blood vessels in the periphery of the lung, these should work. 

I want to preface that this is not how they work. We got some very favorable results, which I'm going to show you. But actually their lung homing was very brief, was maximal at one day following the intravenous injection. And then it just disappeared. And we tried to track it throughout the body and couldn't find them homed anywhere. By this time, there was more discussion about the paracrine effect that we followed up in the future experiments that I'm going to show you. But they did do their job that we were asking them to do. So despite their very transient entrapment, they did exert an anti-apoptotic effect even months following cigarette smoke exposure. 

So the experimental design here was to take mice and expose them to cigarette smoke in the black bar, and to look at the apoptosis after one week, several months of smoking, in their lung parenchyma. And the cigarette smoking model in mice involves exposing a mouse cage's whole body to research grade cigarettes from the University of Kentucky in a machine that's well ventilated for the workers. 

But the mice are having a blast. They get addicted after only two days of exposure, and they anticipate when they're coming to that smoking room, which smells like an ashtray, by the way, and they get really angry when you take them out. So we've learned not to do any procedures following smoke exposure, but rather to do anything prior to smoke exposure. 

So we do that. There are various protocols so that you avoid acute impact patients and you actually model a chronic disease. So we do five days a week, about five hours a day, about two cigarettes are burned into this chamber. And we can extract both sidestream and mainstream cigarette smoke to the vacuum tubing. And we give them off, which probably drives them crazy. 

But we have technicians that we have to keep. And about four to six months, depending on the mouse strain susceptibility, we obtain a modest emphysema phenotype. So it's a pretty gruesome model for trainees. Because they need to graduate. And these take four to six months, and a negative experiment can be really tough to tolerate. 

So anyways, despite that, this is a growing field and you should all do research in COPD, because it's an unfunded area, despite it being the third to fourth leading cause of mortality, both in the US and worldwide. So that's my counter pitch. 

So what we noticed is that when we give these cells during the last two months of smoking exposure, we obtain this anti-apoptotic effect. But then we asked what happens with airspace enlargement. So when we give these cells, as I mentioned, the design is schematized here, so smoking continued, we figured that people, if we mimic human disease, folks may be noticing lung damage here, but they would refuse to stop smoking, like most of our patients. And then we could start therapy and mitigate some of the damage or repair some of the damage with this design. 

So we noticed that smoking, as you would expect, in black here, causes a decrease in alveolar surface area. This is the surface available for gas exchange. So it's decreased because you have airspace destruction. So when you start destroying your alveolar septa, then the surface area available for gas exchange goes down. And when we injected these ASCs, we actually were able to preserve quite nicely the lung architecture. 

So then we asked, is this through direct, this one-day day homing, or is it a true paracrine effect? So to look into the paracrine action, we changed the mode of administration and compared IV with intraperitoneal, that would not be homing into the lung, they would stay in the peritoneum. 

So this is a busy slide, so bear with me. So what I am showing on the left side, this is the experimental design first. So we smoked the mice for six months, in this case, and then we treated them for two months with these intraperitoneal adipose ASCs. And so on the left in green are our controls. Six months of expose just to ambient air control, or eight months, and we're measuring the lung compliance, which is a functional measure of emphysema. 

So with smoking in a blue circled bars, at six months of smoking, so when animals were harvested right here after smoking, or when animals were harvested after smoking cessation for two months, we noticed increased lung compliance that persisted. And this is what we're expecting to see, that the lung compliance is the inverse of lung elastins. So the lungs of emphysematous animals are [INAUDIBLE]. So they're more compliant. They have less elastin. So the model worked. 

And then these animals were given, either intraperitoneally or intravenously, ASCs. And the administration led to a decrease in the lung compliance when they were given IP, just as much, if not even better, than when given IV. So that gave us a clue that these cells have a protective effect. The protective effect is probably reparative, not only preventative, because it works even after smoking cessation is initiated and it's associated with a paracrine mechanism. 

What our astute technicians-- and it's great to have inquisitive technicians in the lab-- what they've noted was that these animals looked better, too. So when we tried to figure out how they looked better, they actually preserved their weight and healthy fat, or the mouse cage sedentary fat. We didn't exercise these mice, unfortunately. So cigarette smoke decreased weight, as expected. They have access to food, but they still lose weight. And ASC receiving mice did not. And then we measure the fat content in the visceral fat area, and we saw preservation of that, as well. 

And lo and behold, we had a fabulous hematopoietic stem cell expert, Hal Broxmeyer at IU, and Hal was looking, do these cells home in the bone marrow? And the answer is no. But with that investigation, we found a totally unexpected result of this ASC administration. And that was on the bone marrow of these mice. So when mice were exposed to smoking, the number of progenitor cells, but not the total cellularity, just the progenitor cells, was decreased by smoking quite dramatically and quite dramatically improved by ASC administration. 

And this affected all lineages. I'm just showing here the CFU forming GM [INAUDIBLE] monocyte progenitors. They were totally down after smoking and improved by ASCs. And this is not only a mouse thing. We could go to the American Society of Hematologists-- and I know this is [INAUDIBLE], and I'm hoping we have hematologists here who can confirm-- one of the first things you see is that one of the risk factors for bone marrow dysfunction is smoking. So we think that this is real and significant. 

So we pursued that further. We thought, could there be a connection between bone marrow stem cells and progenitor cells and how the lung is faring? So when we pursued that, we noticed that the paracrine effect-- I need to go back one slide, I am sorry-- yeah. I wanted to mention one thing before I go there. So when we look further in detail into this, we noticed that this effect on the bone marrow that not only occurred after months of smoking, but occurs even quickly after days of smoking. So that gave us a very nice tool to investigate mechanisms. 

At the same time this paracrine theory was really generating a lot of papers in the field. And the [INAUDIBLE] lab, who's very interested in MSC and ASC function, has described this protein called TNF secreted gene 6, TSG-6. It's a protein that's secreted by cells when stimulated by TNF. So normally we have very low levels of this TSG-6, but we increase it when we get the inflammation. So he has shown that ASC secrete large amounts of this protein. And this protein is protective and it carries the past the job of ASCs in its anti-inflammatory effect. 

So we pursued that as a mechanism. We always want to go back to what are the mechanism of the effects that we're seeing. And we looked at TSG-6 as a mechanism. The way we did that, we looked at if ASC is exposed to smoke produced TSG-6. And they indeed expressed much more when exposed to cigarette smoke than to air control. So not only TNF can stimulate this production, but cigarette smoke can do it, too, in ASCs. 

And then we looked at this effects of smoke on the progenitor cells in the bone marrow. And as shown before, smoke decreases that number. When we injected ASCs that had non-targeted silencing RNA in their-- they were transected with non-targeted [INAUDIBLE] RNA, they did improve the cellularity of the progenitor cell number. 

When we injected ASCs in which this TSG-6 was silent, so they were incapable of producing this protein, shown here, they failed to improve the progenitor cell number here. And this is a scramble control, again. So these graphs suggest that ASCs do exert a protective effect on the bone marrow through a paracrine mechanism that is mediated in large part by this TSG-6. 

So what does TSG-6 and ASC do on the bone marrow? We did an array at that time. And just for the sake of time, just focus on this side. These are genes that were inhibited by smoke, compared to air control, but then brought back half by the ASC therapy. So these were genes that were involved in the following pathways that may modulate the survival or thickness of these progenitor cells. And then we went on and showed that it is a particularly lineage I'm going to show in a minute. 

So again, why are we so interested in these bone marrow stem cells is because they are the factory for a variety of progenitor cells in the body that may participate in the lung maintenance, lung regenerative effect. So I am not going to go into these, just to remind you how rich the bone marrow is in these. 

So then when I wanted to see which cells are affected by smoking in the bone marrow, we've looked at this acute effect that I mentioned before, that even after four days of smoking, we saw a decrease in bone marrow progenitor cellularity. And it persisted for a week after we removed them from cigarette smoking. And then using flow cytometry, we've shown that it is this LSK positive, [INAUDIBLE] positive, [INAUDIBLE] positive cells and reach for hematopoietic stem and progenitor cell population that was affected by smoke and improved by ASCs. 

So with this in mind, we thought, well, can we use, if we have a depletion from smoking of these cells that are supposed to help our lungs, can we use a methodology to mobilize them, to get them out? Because we're not able to increase them in the bone marrow. We're not going to do bone marrow transplant right now. Let's try to get what's there, get them out, whip them out of the bone marrow. 

And Hal Broxmeyer, that I mentioned to you, was one of the first in the field to investigate this drug called AMD3100, which is a molecule that interferes with this interaction between SDF-1s, stem cell-- I'm blanking the SDF abbreviation, I apologize for that, senior moment-- this factor that interacts with the CXCR4 receptor in the bone marrow. [INAUDIBLE] keeps the stem cells into the bone marrow. So the interaction between these SDF molecule on these cells and CXCR4 keeps bone marrow cells lodged in that niche. 

So when you interfere and block this interaction, ligand-receptor receptor interaction, you allow the cells to exit from the bone marrow. So AMD3100 is used clinically in patients in which you desire early mobilization of your cells from the bone marrow niche. So obviously, there are non-specific SDF1 ligand-receptor interactions in other parts of the body that we could interfere with. But we thought this would be a nice first step to see if our paradigm is in any way valid. 

So what we did here, this was a fellow, a junior fellow, that came to the lab and he was very excited to test this hypothesis. So what he did in cigarette smoke animals, he designed a methodology using the clinical strategy of AMD31 administration-- 3100 administration-- with five repetitive doses several weeks apart. And we saw a dramatic effect on both lung morphology, which I'm showing here on this side, when smoke increased airspace enlargement, and the AMD3100 helped that. And this is lung compliance increasing with smoke and being ameliorated by this drug. 

But what was shocking to us is what happens with the bone marrow. So this is hematopoetic progenitor cells in the bone marrow. They went down with smoke at 2 weeks. They went down with smoke at 24 weeks. And the drug ameliorated them. I was surprised to see this result, because I thought that we were going to take out more bone marrow cells from the bone marrow. So we, at best, will keep them low, if not maybe we might decrease them. 

So to our surprise, we actually improved even the [INAUDIBLE] of the bone marrow with this drug. And now the task is to follow this result with mechanistic studies, because it is one of the most effective results that we've seen. And it would be a pity not to follow it. Unfortunately, that fellow graduated, went to Indiana in practice, and we need new blood to study the blood progenitor cells. 

So what I've shown you so far is the effect that we started in our lab on these exogenous administration of progenitor. I would be remiss not to tell you that there was a clinical trial many years ago, maybe by now, seven years ago, done by Osiris in Maryland. Dan Weiss was the PI-- he's at University of Vermont-- administering these MSCs, so adipose progenitor cells are a subtype of [INAUDIBLE] stem progenitor cells. And this company used, based on favorable preclinical data on MSCs from bone marrow at that time-- and I'm not claiming that ASCs are better than MSCs from the bone marrow, I think they're the same, it's just easier to get them, win-win situation, liposuction and so on. 

But this MSC trial, they took MSCs from the bone marrow of a healthy young male and injected them intravenously into a population of mixed COPD'ers with mild to moderate disease. Nobody succumbed, which was great. This is really a progress. Because there was a worry about embolism. You put a bunch of cells that some of them may be large, and they may die of PE. They have pulmonary disease already. Nobody died. There were no major side effects. 

But the trial was negative from an [INAUDIBLE] perspective. And nobody looked at emphysema. Nobody's been [INAUDIBLE]. That means it was not long enough. So the only positive thing you can look at, the half full, is that the markers of inflammation, which was the surrogate with CRP, C-reactive protein, they did improve. But the function did not get better. 

So the field right now, I think, requires a large multi-center brainstorming to design, well design a trial for this type of regenerative or stem cell or progenitor cell therapy, in the right population of patients. Right now, mom and pop shops are sprouting, even in Florida, in Tijuana and Poland. No offense to any of those citizens of those places, but we don't know what they're doing. And recently, in lay press, there are side effects with blindness, et cetera, reported. So that's not the way to advance care. The way to advance care is to do a well-designed trial. 

But patients are desperate and they need therapy now. They cannot wait for us to brainstorm with a multi-center trial. So they do desperate things in desperate times. And you will be asked in clinic, what should I do, Doc? Should I go? I have the money. Should I go and pay for my stem cell infusion in Tijuana? And it's a very tough question to answer when you have the individual patient looking at you in the eye. 

So in the next few minutes-- I think I have till 1:30, is that true? So in the next few minutes-- what's that? 

[INAUDIBLE] 

1:15. Oh, perfect. So in the next one minute, I'm going to show you what I'm not going to show you. No, no, it's OK. I want to respect everyone's time. So we are also actively looking at improving fitness of the lung from the other side, which is to look at endogenous cells that are supposed to repair the lung and seeing if they are in any way impaired, how they're impaired, and making them better. 

So I'm going to go over all my data slides and just show you two things. OK, so bear with me. Just, no sphingolipids, no nothing. This is what I want to show you. 

So I have to precede it with this. So when we investigated lung endothelial cells isolated from humans with a history of smoking, shown here at the bottom, versus a history of non-smoking, we've shown that these micro vascular endothelial cells, which are thought to be an endogenous repair cell for the microvasculature, by Choi, Stevens et al. as opposed to macro vascular cells, which may not be able to do that, so these cells can be clonal for just a few generations. 

So cells from nonsmokers, if you hit them with something that's supposed to kill them, they die. And if you take the cells from smokers and you hit them with something that they're supposed to kill them, they don't die. They undergo this ER stress and autophagy. So we thought, wow, what an interesting finding. Let's look, is this resilience? Were we getting the cells from donors who had motor vehicle accidents, were smokers, and would never develop emphysema? Maybe this is a sign of the great cell. Or are these cells actually on their way, they are maladaptive, they're on their way of becoming disease causing cells? 

So one way that we started investigating them is that was to look at their function. And if you put the endothelial cells in [INAUDIBLE], they know how to form these tubes. They're not vessels, they're just tubes. So when we measure the tubes from cells isolated from nonsmokers, they form tubes with this number. But cells from smokers in the same condition, they did not. So they're not these super man or super woman cells. They're actually more feeble. They don't really do what they're supposed to do. 

But we could whip them into shape if we expose them to-- if we cold culture them with these circulating progenitor cells that Merv Yoder, who recently retired, unfortunately for the field, at Indiana University. He's a neonatologist who truly put order in the field of endothelial progenitor cells. It was a mess, and he made it not a mess. So he has identified and characterized both molecularly and functionally different progenitor cells that are circulating. So with his collaboration, we isolated from donor individuals these circulating hematopoietic progenitor cells that have the good guys that are pro androgen. Good guys when you want to repair vessels. 

So when we co-incubated cells from nonsmokers with these circulating cells, we didn't see much of a change. But when we incubated cells from smokers, compared to how they did at baseline, which was not so great, they did improve their function. So this is pilot data. We haven't published it yet. Because I moved, and I need to find the right collaborators to pursue this. 

But this suggests to us that endogenous cells from chronic smokers may not do their job to repair the lungs. But we can improve them. And this was a methodology of throwing the kitchen sink at them. But then we also have signaling mechanisms in the [INAUDIBLE] lipid arena that we're pursuing to improve their function through those means by improving the [INAUDIBLE] in one phosphate, responsiveness and signaling. 

So with that, let's go for a lot of data. So what was I thinking, right? Typical. Never learn. OK. So this is the mechanism that we're pursuing to invigorate their prosurvival and proproliferation by engaging S1P signaling. 

And to conclude, I think we have to repair the COPD lung in a phenotype specific way. Emphysema will require different measures than chronic bronchitis. Pulmonary hypertension will require different measures, et cetera. The cachectic person may need something else then the obese person, and the osteoporotic or the person with no bone marrow LSK cells will need something else. 

And we have to interrupt this self-perpetuating injury that I talked about yesterday and to reengage this maintenance repair program that our lungs have. And we know resilient lungs of the 100-year-old, who always smoked a pack a day and look, they're fine, and learn from that and engage you to help the young people who try to stop smoking, they can't, or the person who starts smoking and despite that, they continue to [INAUDIBLE] to get injured. 

So with that, I thank you for the attention. I want to thank my collaborators. Some of them I mentioned. Some of them are here. And I will take questions in the 30 seconds. 

[APPLAUSE]