Podcasts about integrins

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.26.525682v1?rss=1 Authors: Bachmann, M., Kessler, J., Burri, E., Wehrle-Haller, B. Abstract: Transforming growth factor beta (TGFbeta) 1 regulates cell differentiation and proliferation in different physiological settings, but is also involved in fibrotic progression and protects tumors from the immune system. Integrin alphaVbeta6 has been shown to activate latent TGFbeta1 by applying mechanical forces onto the latency-associated peptide (LAP). While the extracellular binding between alpha;beta6 and LAP1 is well characterized, less is known about the cytoplasmic adaptations that enable alphaVbeta6 to apply such forces. Here, we generated new tools to facilitate the analysis of this interaction. We combined the integrin-binding part of LAP1 with a GFP and the Fc chain of human IgG. This chimeric protein, sLAP1, revealed a mechanical rearrangement of immobilized sLAP1 by alphaVbeta6 integrin. This unique interaction was not observed between sLAP1 and other integrins. We also analyzed alphaVbeta6 integrin binding to LAP2 and LAP3 by creating respective sLAPs. Compared to sLAP1, integrin alphaVbeta6 showed less binding to sLAP3 and no rearrangement. These observations indicate differences in the binding of alphaVbeta6 to LAP1 and LAP3 that have not been appreciated so far. Finally, alphaVbeta-sLAP1 interaction was maintained even at strongly reduced cellular contractility, highlighting the special mechanical connection between alphaVbeta6 integrin and latent TGFbeta1. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Praveen Tipirneni, CEO of Morphic Therapeutic, on targeting integrins with small molecules.

Integrins are a diverse family of proteins that play an essential role in many cellular biological processes. They also have been implicated a number of autoimmune, cardiovascular, and metabolic diseases, as well as fibrosis and cancer. While a number of biologics have come to market that target integrins, drug developers have been stymied in efforts to develop oral therapies that can target these proteins. Morphic Therapeutic is developing a new generation of oral integrin drugs it believes can transform the treatment model for a range of serious medical conditions. We spoke to Praveen Tipirneni, president and CEO of Morphic Therapeutic, about intgerins, the challenges of using small molecule therapies to target them, and why this can have a dramatic effect on how patients with a range of conditions get treated.

Watch the new season of MinuteBody - and get access to both CuriosityStream and Nebula - at https://curiositystream.com/minuteearth. Fevers are one of our best weapons against infections, but they don't work like you might think. We'd like to give a big thank you to Sharon Evans from the Roswell Park Comprehensive Cancer Center for sharing her expertise for this video! LEARN MORE ************** To learn more about this topic, start your googling with these keywords: Fever: a rise in body temperature, usually caused by infection Heat shock proteins (HSP): a family of proteins that are produced by cells in response to exposure to stressful conditions Neutrophils: white blood cells that play an important role in inflammation and are early responders to pathogens. T cells: major components of the adaptive immune system whose roles include regulating the immune response to pathogens. SUPPORT MINUTEEARTH ************************** If you like what we do, you can help us!: - Become our patron: https://patreon.com/MinuteEarth - Share this video with your friends and family - Leave us a comment (we read them!) CREDITS ********* Kate Yoshida | Script Writer, Narrator and Director Arcadi Garcia i Rius (@garirius) | Illustration, Video Editing and Animation Nathaniel Schroeder | Music MinuteEarth is produced by Neptune Studios LLC https://neptunestudios.info OUR STAFF ************ Sarah Berman • Arcadi Garcia i Rius David Goldenberg • Julián Gustavo Gómez Melissa Hayes • Alex Reich • Henry Reich • Peter Reich Ever Salazar • Leonardo Souza • Kate Yoshida OUR LINKS ************ Youtube | https://youtube.com/MinuteEarth TikTok | https://tiktok.com/@minuteearth Twitter | https://twitter.com/MinuteEarth Instagram | https://instagram.com/minute_earth Facebook | https://facebook.com/Minuteearth Website | https://minuteearth.com Apple Podcasts| https://podcasts.apple.com/us/podcast/minuteearth/id649211176 REFERENCES ************** Appenheimer, M.M. & Evans, S.S. (2018) Temperature and adaptive immunity. Handbook of Clinical Neurology 156: 397–415. https://linkinghub.elsevier.com/retrieve/pii/B978-0-444-63912-7.00024-2 Casadevall, A. (2016) Thermal Restriction as an Antimicrobial Function of Fever. PLoS Pathog 12(5): e1005577. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005577 ChangDong L. et al. (2019). Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins. Immunity 50(1):137-151. https://www.cell.com/immunity/fulltext/S1074-7613(18)30495-3 Evans, S.S. et al. (2015) Fever and the thermal regulation of immunity: the immune system feels the heat. Nature Reviews Immunology 15(6): 335+. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786079/ Hasday, J.D., Thompson, C., Singh, I.S. (2014) Fever, immunity, and molecular adaptations. Comprehensive Physiology 4:109–48. https://onlinelibrary.wiley.com/doi/10.1002/cphy.c130019 Hasday, J.D. & Singh, I.S. (2000) Fever and the heat shock response: distinct, partially overlapping processes. Cell Stress Chaperones 5:471–480. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC312879/ Javid, B., MacAry, P.A. & Lehner, P.J. (2007) Structure and function: heat shock proteins and adaptive immunity. Journal of Immunology 179:2035–2040. https://www.jimmunol.org/content/179/4/2035 Mackowiak, P. A.: Direct effects of hyperthermia on pathogenic microorganisms: teleological implications with regard to fever. Reviews of Infectious Diseases 1981, 3: 508–518. https://academic.oup.com/cid/article-abstract/3/3/508/307776

Naim Alkhouri and Jörn Schattenberg join Roger and Donna to review the closing session of NASH-TAG 2021, a potpourri of basic science and review of promising drugs earlier in development.The Saturday afternoon closing session of NASH-TAG 2021 spends half its time discussing basic science issues and the other half with BioPharma company drug developers discussing their promising drugs in development. Last year, this session yielded some real fireworks! On Sunday, join SurfingNASH to sort out how what the data means, how the pieces fit together and what questions will require more exploration. If you send us any questions emerging from Session #4 or the podcast, we will review them in our NASH-TAG wrap-up episode next week.

This week, join author authors John J.V. McMurrary and Milton Packer, and Associate Editor as they discuss their Perspective article "How Should We Sequence the Treatments for Heart Failure and a Reduced Ejection Fraction? A Redefinition of Evidence-Based Medicine." TRANSCRIPT BELOW Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, this feature discussion is going to knock you off your seat, because it did me. It's about treatment sequencing in HFrEF and discussing it with some luminaries on the field, Dr. John McMurray and Dr. Milton Packer. You are going to love it. I loved it. But I'm going to make you wait. How about you grab some coffee and let's start with some of the other papers in today's issue first. Dr. Greg Hundley: All right, Carolyn. How about if I go first? I'm going to start with a paper from Dr. Liam Brunham from the University of British Columbia. Well, Carolyn, the high density lipoprotein or HDL hypothesis of atherosclerosis has been challenged by clinical trials of cholesterol ester transfer protein, or CETP inhibitors, which failed to show significant reductions in cardiovascular events. Plasma levels of HDL cholesterol, or HDL-C, declined drastically during sepsis. And this phenomenon is explained in part by the activity of CETP, a major determinant of plasma HDL-C levels. So Carolyn, these authors tested the hypothesis that genetic or pharmacologic inhibition of CETP would preserve HDL levels and decrease mortality in clinical cohorts in animal models of sepsis. Dr. Carolyn Lam: Huh. Interesting. And what did they find? Dr. Greg Hundley: Well, Carolyn, results from both the human UK Biobank and the mouse model experiments suggested that inhibiting CETP may preserve HDL levels and improve outcomes for individuals with sepsis. Dr. Carolyn Lam: Wow. So is this ready for clinical applications somehow? Dr. Greg Hundley: Well, Carolyn, two conclusions from this work. First, high density, lipoprotein cholesterol, a commonly used biomarker for cardiovascular risk assessment, may also predict risk of death from sepsis. And then second, cholesterol ester transfer protein inhibitors that have been tested in clinical trials of cardiovascular disease could be repurposed and studied in clinical trials of sepsis. Dr. Carolyn Lam: Ooh, exciting. Well, Greg, for my paper, I'm going to ask you a question. Have you ever thought about what the temporal changes in medical prevention and adverse outcomes are in patients with symptomatic peripheral artery disease after revascularization? Well, wait no longer. Our next paper addresses that. It's from Dr. Sogaard from Aalborg University Hospital in Denmark and colleagues who identified all patients with a first open surgical or endovascular revascularization procedure in the lower extremities or abdomen in Denmark from 2000 to 2016. And this is what they found. Dr. Carolyn Lam: First, the profile of patients with PAD who underwent lower extremity revascularization changed towards older age and a higher prevalence of comorbidity. Despite increases in age and co-morbidity, medical prevention of adverse events improved substantially over time, particularly in the first part of the study period and among patients who used medications chronically. Dr. Carolyn Lam: Now in contrast, initiating treatment after revascularization increased modestly among treatment-naive patients. Now concurrently, prognosis improved for almost all adverse outcomes in patients of both sexes, all age groups, and in all high-risk co-morbidities. In particular, the risks of myocardial infarction and cardiovascular death declined by more than 40%. Dr. Greg Hundley: Well, Carolyn, are there any other findings with clinical implications here? Dr. Carolyn Lam: Yes. So that was the good news earlier. But despite overall improvements, significant disparities remain. Less than 40% of treatment-naive patients initiated cardioprotective therapy after revascularization, underscoring the need for raising levels of awareness and education in the vascular community, general practitioners and patients of this. Major amputations also remained unchanged and thus more work is needed to understand relationships between the preventive measures, revascularization and amputation. Dr. Greg Hundley: Great summary, Carolyn. My next paper comes from Dr. Rachael Cordina from the Royal Prince Alfred Hospital, University of Sydney. Neurocognitive outcomes beyond childhood in people with a Fontan circulation are not well-defined. So the investigators here aim to study neurocognitive functioning in adolescents and adults with a Fontan circulation and associations with structural brain injury, brain volumetry and postnatal clinical factors. Dr. Carolyn Lam: Okay, you got our attention. What did they find, Greg? Dr. Greg Hundley: Thanks, Carolyn. So participants with a Fontan circulation, without a pre-existing major neurological disability, were prospectively recruited from the Australia and New Zealand Fontan registry. And the investigators found that neurocognitive impairment is common in adolescents and adults with a Fontan circulation and is associated with smaller grey and white matter brain volume. Understanding, therefore Carolyn, modifiable factors that contribute to brain injury to optimize neurocognitive function is paramount. Dr. Carolyn Lam: Indeed. Well, this next paper I want to talk about is the first detailed endothelial cell cysteine-S self-hydrome. Dr. Greg Hundley: Self? S self-hydrome? What is that, Carolyn? Dr. Carolyn Lam: Good. I needed to catch your attention. Let me tell you about it. So in vascular endothelial cells, cysteine metabolism by cystathionine gamma-lyase, or CSE, generates hydrogen sulfide- related sulfane sulfur compounds. And these exert their biological actions via cysteine-S self-hydration of target proteins. So the paper we're talking about today by Dr. Fleming from Goethe University in Germany and colleagues, they aimed to map the S self-hydrome, which is the spectrum of proteins targeted by this hydrogen sulfide-related sulfane sulfur compounds, or H2Sn, in human endothelial cells. And they did this using liquid chromatography and tandem mass spectrometry. Dr. Carolyn Lam: So here's what they found: vascular disease was associated with mark changes in the S self-hydration of endothelial cell proteins involved in mediating responses to flow. Integrins were most effected by S self-hydration and the modification of beta-3 integrin resulted in reshuffling of the intramolecular disulfite bonds to preserve its extended and open confirmation. Loss of beta-3 integrin self-hydration, on the other hand, inhibited endothelial cell adhesion, impaired mechanosensing and attenuated flow induced phase with dilation. Thus, short term H2Sn supplementation could improve vascular reactivity in humans, highlighting the potential of interfering with this possibly to treat vascular disease. Dr. Greg Hundley: Very nice, Carolyn. You know, just more from the world of hydrogen sulfide and endothelial function. Thanks so much. Well, the next paper I have comes to us from Dr. John McEvoy from Johns Hopkins University School of Medicine. So Carolyn, recent clinical guidelines support intensive blood pressure treatment targets. However, observational data suggests that excessive diastolic blood pressure lowering might increase the risk of myocardial infarction. Therefore reflecting, does a J or U-shaped relationship exist when we're following the treatment of diastolic blood pressure? So Carolyn, these authors analyzed 47,407 participants from five cohorts with a median age of 60 years. First to corroborate prior observational analysis, the authors used traditional statistical methods to test the shape of association between diastolic blood pressure and cardiovascular disease. Dr. Carolyn Lam: Okay. So was it J or U? Dr. Greg Hundley: Okay, Carolyn. So interesting, traditional observational analysis of the cohorts suggested a J-shaped association between diastolic blood pressure and myocardial infarction. However by contrast, linear MRI analyses demonstrated an adverse effect of increasing diastolic blood pressure increments on cardiovascular disease outcomes, including myocardial infarction. Furthermore non-linear MRI analyses found no evidence for a J-shaped relationship. Instead confirming that myocardial infarction risk decreases consistently per unit decrease in diastolic blood pressure, even among individuals with low values of baseline diastolic blood pressure. So Carolyn, in answer to you, no, the J or U-shaped curve does not exist. Dr. Carolyn Lam: I suppose depending which way you look at it. Very interesting. Well, let's finish up with what else is in today's issue. There's an AJ update by Dr. Elkin on COVID-19 at one year, the American Heart Association president reflect on the pandemic. A white paper by Dr. Zannad on challenges of cardio kidney composite outcomes in large scale clinical trials. A research letter by Dr. Kass on  the reduced right ventricular sarcomere contractility in HFpEF with severe obesity. Another research letter by Dr. Messas on the feasibility and performance of non-invasive ultrasound therapy in patients with severe symptomatic aortic valve stenosis. A first in human study. Dr. Greg Hundley: Right, Carolyn. So I've got an exchange of letters from Dr. Vazgiourakis  addressing a prior publication entitled Right Heart Dysfunction in COVID-19 Patients: Does Mechanical Ventilation Play an Additional Role? And then finally, an exchange of letters from Drs. Carrizales-Sepúlveda and Topalisky regarding the prior paper, The Spectrum of Cardiac Manifestations in COVID-19. Well, Carolyn, I'm really excited to get to that feature that you explained to us right at the beginning. Very exciting. Dr. Carolyn Lam: So am I. So am I. Thanks, Greg. Dr. Carolyn Lam: Wow. Today's feature discussion could not be more star-studded in my point of view. We are talking about the very, very hot topic of how do we sequence treatments in heart failure with reduced ejection fraction now? A really hot topic because just last year in 2020, we suddenly got a bonanza of positive trials and everybody's grappling with how to put it all together. Dr. Carolyn Lam: Now who better than the two authors I'm going to talk to today, Professor John McMurray from University of Glasgow and Professor Milton Packer from Baylor University Medical Center in Texas. So welcome both. John, Milton, I'm almost tripping over myself to talk about this because this is an amazing perspective piece. Everybody must get your hands on it and even look at the figure while you're listening to this. We're going to divide today's discussion into just three simple questions. Why do we need a new sequencing approach? How in the world do you come up with a new sequencing approach? Based on what? And finally, what is that new approach that you're both proposing? So maybe I'll start off with you, John. What's wrong with what we've been doing? Dr. John McMurray: So Carolyn, I think we've maybe neglected the fact that while we think of, for example, cancer as something that's incredibly urgent to diagnose and to treat as fast as possible, to give the patient all those life-saving therapies, we haven't had the same urgency in our treatment with heart failure. And our existing approaches, as you know, being largely one of start with the first treatment that was ever tested in the trial, up titrate to the pill dose, take your time, then on the second, third and so on. And of course, what that means is that it takes months for patients to be treated with all of the fantastic life-saving options that we have available for them. And we know that that's failing. Dr. John McMurray: We've seen from numerous registries, CHAMP registry in particular springs to mind, where that's simply not happening. It's probably taking too long. It's too complicated for both the doctor and the patient, and we need to change it. And I suppose Milton will tell you his view, but I think my view and I think his as well, was that the SGLT2 inhibitor story really brought this question, I think, to the fore because here is our fourth life-saving drug that if we do things the same way might not get started for six months. And we really felt that we need to rethink what we're doing. Milton, I'm sure, will say whether he agrees with that. But I think that was sort of where the starting point was. Dr. Carolyn Lam: Great. But if I could interject a bit, so now we're talking about that left side of the panel, where in your beautiful article where you're showing, we start with ACE inhibitors and ARBs, and then go on to beta blockers and mineralocorticoid receptor antagonists, and so on. I would love to know, and Milton I'm sure you'll add, is it the sequence that's wrong? Or is it really just the timing? Or the fact that we're just all too lazy? What do you say to people who go, "But that's how the trials were done." Especially because you guys both led those amazing trials of ARNIs and SGLT2 inhibitors. It's just awesome. Dr. Milton Packer: So Carolyn, what's really amazing is that everyone assumes that that's how the trials were done. But two things, one, just because we did things in a certain way, developed things in a certain way, doesn't mean we have to prescribe them in a certain way. I mean, we developed digitalis before all of them and so does that mean we need to use digitalis in everyone? But a lot of the early trials, all the patients were, or most of the patients were, on cardiac glycosides. Dr. Milton Packer: There are four things that we've learned from the large-scale clinical trials. One is the order of drugs does not matter with respect to efficacy. The beta blockers work the same whether people are getting ACE inhibitors or not, MRAs are not effected by background therapy. Neither is neprilysin inhibitors. They work pretty much the same regardless of background therapy, so you don't have to sequence them in the order in which they were developed. Dr. Milton Packer: Two is low doses, low starting doses of these drugs seem to work amazingly well, perhaps surprisingly well. And the third thing is that they work very early. So in a lot of the clinical trials, nearly all the trials that were carried out, there was a meaningful separation of the curves and in effect size in the first 30 days of all of these trials. And in many of the trials, in the first 30 days, patients were still on the starting dose. Hadn't been uptitrated. Dr. Milton Packer: The last point is that these drugs can influence each other's safety profiles. So the result of all of this was for us to rethink what the sequence should be based not on how the drugs were developed, but how they might be most logically used with respect to relative efficacy, safety and ease of use. Dr. John McMurray: So, Carolyn, to go back to your question then is sort of what Milton is saying is that it's a bit of both of the things you asked about. It is about timing, but it's also about the order of the drug. And that last point Milton made is very important about the potential synergies in terms of making it easier to use treatments, but timing is critically important as well. I mean, we do tend in the conventional approach to therapy recommended in the guidelines to perhaps spend too much time trying to reach that target dose, and then doing that before moving on to the second drug. So again as Milton pointed out, if you're getting early benefit from all of these treatments, fundamentally what you want is as many of these treatments started as quickly as possible as you can do safely. And that may be facilitated by some of the synergies between treatments as we, I think, rather provocatively suggested in the new algorithm, might even be possible to start two treatments at once. Dr. Carolyn Lam: Okay. Now I know everybody's really, really wondering what that new algorithm is, but I'm going to lengthen the pain a little bit more because this is critically important. You've already started discussing the how did you come up with an algorithm. It seems a lot of, yeah, very reasonable approaches, but could you give us specific examples of actual scientific interrogation of the data from the trials that you've led, frankly, to show us these points, that maybe support that we can come up with a reasonable new approach? Those points that Milton very rightly put, the treatment benefit of each class is independent. Give us some examples of that. The dose issue, the side effects, how one could help in that too. Could you give us some examples? Dr. Milton Packer: Oh, my God. So let me say that there's so many pieces of evidence and please read the article. We try to summarize as much of them as possible. But in all of the major clinical trials, there was a separation that occurs within 30 days. That's true across every single major trial. Anyone who thinks that the treatment effects of these drugs are delayed, that it takes months to evolve, we're getting statistical significance within two to four weeks across all of the drugs. Dr. Milton Packer: Second is, in many of the trials, for example, COPERNICUS trial with carvedilol, the trials with MRAs, even the trials with ACE inhibitors, during that first 30 days when the curves were separating, patients hadn't been uptitrated. They started on low doses and remained on relatively low doses and the curves were separating. So we knew that the drugs had early effects at low doses, low starting doses. And we also have randomized trials that really tell us that if you go to high doses for some of these drugs, you get a little bit more benefit, but you don't get as much benefit as starting another drug with a different mechanism at a low dose. Dr. Milton Packer: And lastly, we know that some of these drugs actually prevent the side effects of others. There's evidence that neprilysin inhibitors and SGLT2 inhibitors mitigate the hyperkalemia produced by spironolactone and aplerno. So these are just a few examples. Dr. John McMurray: Sorry, Carolyn. To add a couple more, we obviously know that the treatments work independently. We primarily knew that from subgroup analyses, but also from trials like RALES for example, where spironolactone was tested in addition to an ACE inhibitor, but very, very few patients were on a beta blocker. Subsequently we tested different a MRA in patients who were taking both an ACE inhibitor and a beta blocker, and the benefit was essentially the same. And of course, our very first trial with an ACE inhibitor, the CONSENSUS trial, was actually done in a population where more than half of the patients were on a very large dose of an MRA. So you can sort of put all the trials together in a type of jigsaw and figure out that these drugs all clearly work independently. Dr. John McMurray: And then maybe the only other thing I would mention, because it's perhaps relevant to the new algorithm, is that we do have another key trial, which is, a trial I think often forgotten about, the CIBIS III, which was a study that tested whether or not you could start treatment with either a beta blocker or with and ACE inhibitor in patients with HFrEF, showing that you could start with a beta blocker in patients who had not yet received an ACE inhibitor and do that safely and efficaciously. So there's a lot of material out there that you can sort of put together to answer all of these questions. Dr. Carolyn Lam: Great. And now drum roll. Okay. What is the new algorithm? John, you want to introduce it? Or Milton? Up to you. Dr. Milton Packer: John can start. That's fine. Dr. Carolyn Lam: Well, which one, Carolyn? I suppose the one in the Circulation article is a three-step algorithm. It starts with the combination of a beta blocker, based as I mentioned, so there's three plus an SGLT2 inhibitor. So again, thinking about synergies, thinking about tolerability, thinking about size of effect and thinking about repetity of onset of benefit. So I think most of us would agree, beta blockers are incredibly effective treatments, life-saving treatments, reduce the risk of sudden death. We know that you can start a beta blocker safely as first-line therapy. We do know that there may be more intolerance in patients who are volume overloaded. So why not give a treatment that has a modest, initial diarrhetic effect when you're starting the beta blocker? In other words, the SGLT2 inhibitor. SGLT2 inhibitors work extremely quickly. There's no dose up titration needed. So they seem like the perfect combination to start with. Dr. Carolyn Lam: In step two, we suggested moving then to sacubitril valsartan, which in itself is two more drugs combination of an angiotensin receptor blocker and their prolines inhibitor. And then there's the third and final step. We suggested using a mineralocorticoid receptor antagonist. But Milton and I have had a lot of discussion about this. I think we're not saying that all those are necessarily the three steps for all patients. There may be different approaches in different people depending on patient's characteristics. But really the point here was, the provocative statement was we should be able to do this quickly in all patients. And this in fact was an approach to get all four of those drugs started potentially within four weeks. Dr. Milton Packer: So Carolyn, the mantra here, our motto going forwards, is four drugs in four weeks. Dr. Carolyn Lam: Okay. Dr. Milton Packer: An angiotensin receptor blocker, a beta blocker, an MRA, an SGLT2 inhibitor. Four drugs in four weeks. And if you're going to start all four drugs in four weeks, in all honesty, the order probably doesn't matter that much. John and I happen to think that if you have to define a first step, a combination of a beta blocker and an SGLT2 inhibitor simultaneously as step one makes a lot of sense. And then you can follow up with sacubitril valsartan and an MRA. Dr. Milton Packer: But here's the thing that's really important: do not take months to follow up. What we're proposing in this algorithm is you start a beta blocker and an SGLT2 inhibitor on day one, and you then follow through with sacubitril valsartan and an MRA within the next couple of weeks. But here's what's really important and we really need to emphasize this: this is a algorithm that assumes that someone's not on any of these drugs already. And of course, most of these patients are taking some of these drugs already. But the other thing that's really important is that we're also assuming that physicians are being very meticulous about background use of diuretics, so that patients really have to be maintained in a clinically euvolemic state in order to make this algorithm work. Dr. Carolyn Lam: Okay, well, I'm picking myself off the floor because it certainly was provocative. I love it. I love it for that. It's the first time I've ever seen any algorithm start with a beta blocker and SGLT2 inhibitor. You first go, "Where's the ACE and how come the new kid on the block is right on top?" So I really like that because it must challenge our current thinking. In other words, if we just look at the data for what it is, let's see how we could think it over. So salute you for that. But let me just press on a little bit. So four drugs in four weeks. That's really great. Are there any particular patients you may say the ARNIs come on top or the MRAs? Specific situations or...? Dr. Milton Packer: Well, Carolyn, as John has already said, the physicians need to understand the principles, but the application of those principles have to be individualized. So if a patient has a borderline blood pressure, you would probably be well advised to put the MRA before sacubitril valsartan. Depending on renal function, you may decide to advocate one drug a little bit earlier or preferentially compared to another. There are hundreds of individualized nuances, but to get tied up in these is to miss the point of our paper. The point of our paper is that we need to do things quickly... Four drugs, four weeks... And we need to not rely on our historical testing in order to determine the optimal sequence. If you embrace those conclusions, then patients and physicians can individualize their care to the greatest optimal degree. But our current approach, which is a historically-driven algorithm that takes six months to execute, it doesn't work. Dr. John McMurray: Carolyn, we obviously did give a lot of thought to the initial treatments, and we did realize that it would potentially be a surprise to people. But just to reiterate, I don't think there's much debate about the incredible benefits of beta blockers, the size of that benefit. We know that the benefit is apparent within 30 days. So Milton and we had Henry Krum's very nice paper about that in JAMA from the COPERNICUS trial, but we're seen it in the other trials. You know that SGLT2 inhibitors have had early benefit. You think about these two drugs being used in a newly presenting patient with HFrEF, probably don't even need to do any electrolyte monitoring, provided your patients not volume overloaded or recently decompensated. That patient's very unlikely to have any significant intolerance to these two treatments. Dr. John McMurray: They don't, in those sorts of patients, substantially reduced blood pressure in either drug, beta blockers certainly don't affect kidney function. SGLT2 inhibitors have minimal effect on kidney function. If your GFR is relatively normal, you probably don't even need to check it. And of course, there's no effect on potassium. So in terms of getting two treatments onboard quickly that will have a rapid benefit, are likely to be well-tolerated in the type of patient that we just described, and where they might have monitoring necessary is minimal, then this seemed to be the best option. And as Milton said thereafter, it's maybe less important what order you do it in as opposed to the speed with which you do it. And you're right, you would definitely probably tailor this approach according to the patient characteristics, but this was a general starting point to stimulate debate, which it seems to have done. Dr. Milton Packer: So Carolyn, there's something important. If you believe in what we've proposed, then at the end of four weeks, every patient with heart failure and reduced ejection fraction would be on low starting doses or four foundational drugs. Our estimate is if doing that would provide them with a substantial benefit, maybe 70, 75% of the benefit of bringing all of those doses to target doses. And if you can do that, you can do all four drugs at starting doses at four weeks and provide that magnitude of benefit really quickly? That has a big impact on patients. And that has a big impact on public health. Dr. Carolyn Lam: Wow. Just thank you so much for igniting this debate. I wish we could go on forever. I just had to share that when we editors looked at this paper, it did spur a very robust debate. But as you can see, we're publishing it as you've proposed it because we do see where you're coming from. It is very interesting. And I just want to reiterate what you both just said, to listeners out there, remember this is referring to a patient who is compensated. Diuretics are still part of it. Remember that the key message is to get everyone on the four foundational therapies within four weeks. And I just love the way you pushed the boundaries with this. Really, really appreciate it. Milton, you look like you want to say something else. If you'd like closing words, I'd love to... Dr. Milton Packer: We really thank Circulation for having the courage to do this. And please understand, John and I strongly feel that this is the start of a debate. This is the start of a discussion. This algorithm is a proposal to get people to start thinking differently. This is not the final word on the subject by far. We think this is the beginning of a very important discourse that will evolve over the next year or more. And we just wanted to remind people what the clinical trial evidence actually shows about these drugs, because we think it has been frequently misunderstood much to the detriment of patients with heart failure. Dr. Carolyn Lam: Yes. And John, any last words? Dr. John McMurray: I would go back to where I started, Carolyn. In a way, what's important here is to inject a sense of urgency back into the way in which we treat patients with heart failure and reduced ejection fraction. It deserves that sense of urgency, as I mentioned that, for example, cancer does. And also thank you for summarizing, I think, what we tried to get across absolutely accurately. Dr. Carolyn Lam: Okay. So John, Milton, so far I take it. I take your points well, but as a practitioner, what I would do, frankly, is if I have a patient that I'm starting a beta blocker and an SGLT2 inhibitor, I would surely just start an ACE inhibitor perhaps, or ARNI, at the same time. I don't see why I need to delay it. How about that? Even faster? Dr. John McMurray: Okay, well, I'll let Milton answer the faster, but I would say one thing, Carolyn, the new algorithm doesn't mention ACE inhibitors or angiotensin receptor blockers as a monotherapy. Because I think those days are gone. I really do think that we shouldn't go through that cycle of starting a RAS blocker, uptitrating it, then switching to an ARNI because that's a waste of time. You're delaying the introduction of life-saving therapy. And this is the whole point to, again, get that sense of urgency across in implementing all of these treatments as quickly as possible. Dr. Milton Packer: And Carolyn, if you want to go faster, that would be fine. Maybe we shouldn't have proposed four drugs in four weeks. We should have proposed four drugs in four days. But Carolyn, I think that changing the way people think is a gradual process. Perhaps four drugs in four weeks is a good starting point. If everyone feels comfortable with that and understands why we are proposing that, then in another six months or so, Circulation can invite John and I to come back and propose four drugs in four days. But let's see what happens. Dr. Carolyn Lam: Kudos. Thank you so much. Well, thank you once again, John and Milton. That was an incredible discussion. A beautiful paper. Dr. Carolyn Lam: Thank you so much, listeners. I'm sure you enjoyed that as much as I have or probably more. But thank you and please don't forget to tune in again next week. From Greg and I, here's Circulation on the Run. Dr. Greg Hundley This program is copyright of the American Heart Association, 2021.  

Learn about how a fever helps you get better; why the first reliable and widely available pregnancy test was the African clawed frog; and a potential breakthrough in the shockingly complicated traveling salesperson problem.  How Exactly Does Fever Help You Get Better? by Ashley Hamer  TED-Ed. (2016). The surprising reason you feel awful when you’re sick - Marco A. Sotomayor [YouTube Video]. In YouTube. https://www.youtube.com/watch?v=gVdY9KXF_Sg  ‌Mace, T. A., Zhong, L., Kilpatrick, C., Zynda, E., Lee, C.-T., Capitano, M., Minderman, H., & Repasky, E. A. (2011). Differentiation of CD8+ T cells into effector cells is enhanced by physiological range hyperthermia. Journal of Leukocyte Biology, 90(5), 951–962. https://doi.org/10.1189/jlb.0511229  The first reliable and widely available pregnancy test was... a frog? by Cameron Duke Tyssowski, K. (2018, August 31). Pee is for Pregnant: The history and science of urine-based pregnancy tests. Science in the News. http://sitn.hms.harvard.edu/flash/2018/pee-pregnant-history-science-urine-based-pregnancy-tests/#:~:text=The%20late%201920s%20marked%20the VAITUKAITIS, J. L. (2004). Development of the Home Pregnancy Test. Annals of the New York Academy of Sciences, 1038(1), 220–222. https://doi.org/10.1196/annals.1315.030 Wells, G. P. (1978). Lancelot Thomas Hogben, 9 December 1895 - 22 August 1975. Biographical Memoirs of Fellows of the Royal Society, 24, 183–221. https://doi.org/10.1098/rsbm.1978.0007 Yong, E. (2017, May 4). How a Frog Became the First Mainstream Pregnancy Test. The Atlantic; The Atlantic. https://www.theatlantic.com/science/archive/2017/05/how-a-frog-became-the-first-mainstream-pregnancy-test/525285/ Traveling salesperson update by Cody Gough Computer Scientists Break Traveling Salesperson Record. (2020). Quanta Magazine. https://www.quantamagazine.org/computer-scientists-break-traveling-salesperson-record-20201008/  Karlin, A. R., Klein, N., & Gharan, Shayan Oveis. (2020). A (Slightly) Improved Approximation Algorithm for Metric TSP. ArXiv.org. https://arxiv.org/abs/2007.01409  Curiosity Daily: Weirdest Types of Lightning, Spotting Audio Lies Easily, and the Traveling Salesman Problem https://omny.fm/shows/curiosity-daily/weirdest-types-of-lightning-spotting-audio-lies-ea  Subscribe to Curiosity Daily to learn something new every day with Cody Gough and Ashley Hamer. You can also listen to our podcast as part of your Alexa Flash Briefing; Amazon smart speakers users, click/tap “enable” here: https://www.amazon.com/Curiosity-com-Curiosity-Daily-from/dp/B07CP17DJY See omnystudio.com/listener for privacy information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.23.350959v1?rss=1 Authors: Jaddivada, S., Gundiah, N. Abstract: Mechanosensitive focal adhesion complexes mediate the dynamic interactions between cells and substrates, and regulate cellular function. Integrins in adhesion complexes link substrate ligands to stress fibers in the cytoskeleton, and aid in load transfer and traction generation during cell adhesion and migration. A repertoire of signaling molecules, including calcium, facilitate this process. We develop a novel one-dimensional, multi-scale, stochastic finite element model of a fibroblast on a substrate which includes calcium signaling, stress fiber remodeling, and focal adhesion dynamics that describes the formation and clustering of integrins to substrate ligands. We link the stochastic dynamics involving motor-clutches at focal adhesions to continuum level stress fiber contractility at various locations along the cell length. The stochastic module links to a calcium signaling module, via IP3 generation, and adaptor protein dyanamics through feedback. We use the model to quantify changes in cellular responses with substrate stiffness, ligand density, and cyclic stretch. Results show that tractions and integrin recruitments vary along the cell length and depend critically on interactions between the stress fiber and reversibly engaging adaptor proteins. Maximum tractions and integrin recruitments were present at the lamellar regions. Cytosolic calcium increased with substrate stiffness and ligand density. The optimal substrate stiffness, based on maximum tractions exerted by the cell, shifted towards stiffer substrates at high ligand densities. Cyclic stretch increased the cytosolic calcium and tractions at lamellipodial and intermediate cell regions. Tractions and integrin recruitments showed biphasic responses with substrate stiffness that increased with ligand density under stretch. The optimal substrate stiffness under stretch shifted towards compliant substrates at a given ligand density. Cells deadhere under stretch, characterized by near-zero recruitments and tractions, beyond a critical substrate stiffness. The coupling of stress fiber contractility to adhesion dynamics is essential in determining cellular responses under external mechanical perturbations. Copy rights belong to original authors. Visit the link for more info

This is part 1 of a 2-part masterclass on sleep. In this episode, I discuss why sleep is so crucial to enjoying vibrant health and vitality. We'll go over what happens to our bodies during sleep, how to reduce the risk for many common health challenges, and the consequences of a lack of adequate, quality rest. 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.26.133553v1?rss=1 Authors: Heller, J. P., Heintz, T. G., Kwok, J. C., Martin, K. R., Fawcett, J. W. Abstract: Retinal pigment epithelial (RPE) cells have been used in disease modelling and transplantation studies in retinal diseases. Several types of RPE cells have been trialed, ranging from primary cells and immortalized cell lines to stem cell-derived RPE cells. During aging and in disease, the extracellular environment of the RPE cells changes, interfering with RPE cell adhesion. We hypothesize that this could be a key problem in transplantation studies that have reported lack of adhesion and survival of the transplanted RPE cells. Integrins are essential for the proper function of the RPE, mediating adhesion to Bruch's membrane, and the binding and subsequent phagocytosis of photoreceptor outer segments. Variability that has been found in clinical trials might be due to the variability of cell types used and their expression profiles of surface molecules. Here, we set out to analyze integrin expression in primary rat RPE cells and in the human cell line ARPE-19 using immunochemistry. We found that both cell types express integrins to varying degrees. After long-term culturing, ARPE-19 cells resemble mature RPE cells, and increase integrin expression. We hence argue that it is important to test the properties of these cells prior to transplantation to avoid failure of adhesion and to facilitate correct function. Copy rights belong to original authors. Visit the link for more info

Inflammatory bowel disease, which includes ulcerative colitis (UC) and Crohn’s disease, affects an estimated 3 million adults in the United States. Although numerous therapies are available for the treatment of moderate to severe UC, many patients still suffer poor quality of life and suboptimal outcomes. Therefore, safer and more effective UC therapies are needed. In this CME podcast activity, Dr. David Rubin and Dr. Peter Higgins will discuss investigational anti-integrin therapies for UC. For the transcript of this podcast, click here To obtain CME credit, click here

Cancer-associated fibroblasts lay the tracks for directional migration Cancer-associated fibroblasts, or CAFs, regulate tumor progression by secreting chemokines and remodeling the extracellular matrix. Erdogan et al. reveal that the CAF-dependent alignment of fibronectin promotes directional cancer cell migration. This biosights episode presents the paper by Erdogan et al. from the November 6th, 2017, issue of The Journal of Cell Biology and includes an interview with two of the paper's authors, Begum Erdogan and Mingfang Ao (Vanderbilt University, Nashville, TN). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Dr Cordes speaks with ecancertv at EACR 2016 about the link between integrins and DNA repair. Integrins represent a family of cell adhesion molecules which, when inhibited, can aid in radiotherapeutic targeting of cancer cells. Dr Cordes reports on the overlap between this radiosensitizing capability and therapies which directly prevent the repair of damaged DNA in cancer cells. He describes how models and results from other tumours have shaped his research in cell adhesion, including the emergence of radio-resistance and combination therapies.

Background Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. Results Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. Conclusions Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Integrins are ubiquitously expressed adhesion receptors with important functions in cellular adhesion, proliferation, migration and signaling. These functions are determined by integrin trafficking through endosomal compartments and receptor affinity regulation. In this thesis, we identified the distal NxxY motif of the β1 integrin cytoplasmic tail as a molecular switch modulating a spatiotemporally controlled binding of two FERM-domain proteins in different cellular compartments. Kindlins mediate integrin activation at the plasma membrane and they dislodge upon internalization. In the endosomal compartment, the free cytoplasmic domain is subsequently bound by sorting nexin 17 (SNX17) to inhibit integrin degradation. We identified SNX17 as a new β1 integrin adaptor protein, which uses the kindlin-binding site in endosomal compartments to stabilize integrins and to promote their recycling back to the plasma membrane.

Ventral lamellipodia plug the gaps When leukocytes enter or exit the blood stream, they leave small wounds both in and between the vascular endothelial cells, which must be quickly repaired so that the endothelium can maintain its barrier function. Martinelli et al. reveal that wounding causes a loss of tension in endothelial cells, inducing the formation of ventral lamellipodia that close the holes left by transmigrating leukocytes. This biosights episode presents the paper by Martinelli et al. from the April 29, 2013, issue of The Journal of Cell Biology and includes an interview with authors Roberta Martinelli and Christopher Carman (Harvard Medical School, Boston, MA). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Integrins constitute an important class of cell adhesion receptors, as they bidirectionally transduce information between the cytoplasm of biological cells and the surrounding extracellular matrix. By means of atomic force microscopy, spectroscopic measurements of the specific interactions of integrins with their corresponding ligands were performed. Basically, these experiments allow deep insights into cellular signal transduction, but despite sophisticated vibration isolation systems the acquired data exhibit very low signal-to-noise ratios that impair an accurate analysis. This drawback was overcome by a novel post-processing algorithm, which significantly reduces the noise and thus improves the signal-to-noise ratio. Thereby, previously invisible signal features can be revealed. Another important task when evaluating this kind of experiments is the identification of steplike transitions corresponding to unbinding events between the receptor-ligand bonds. To this end, a technique has been developed that can be adjusted to detect very low or narrow steps even if they are smooth and hidden by noise. By applying the noise reduction algorithm to force spectroscopy data obtained with living T lymphocytes, the onset force required for the extraction of a membrane tether could be observed for the first time. Using the step detection method, strong evidence of sub-10-pN steps was found. Moreover, it was shown that the chemokine SDF-1α leads to a strengthening of individual bonds between VLA-4, one type of integrins primarily involved in the early stages of chemokine-induced lymphocyte adhesion, and its ligand VCAM-1. The adhesion strengthening is accompanied by a stiffening of the integrins’ environment. It is independent of an intracellular binding site of VLA-4 to talin, the major intracellular factor involved in integrin affinity modulation. Further, the functional role of the integrin trans-membrane domains in receptor-ligand interactions was explored by analyzing the effects of two mutations of the integrin αvβ3 on cellular adhesion: a chimera encompassing the strongly dimerizing trans-membrane domain of glycophorin A and a point mutation known to induce trans-membrane domain dissociation. The results show that both constructs provoke strong cell adhesion. They correspond well to a three-state model of integrin activation. A resting state is activated by intracellular ligands to an intermediate state without trans-membrane domain separation. The dimerizing chimera mimics the intermediate state, which strengthens cellular adhesion.

The scope of this work is to gain insight and a deeper understanding of exploring and controlling molecular devices like proteins and rotors by fine tuned manipulation via mechanical or electrical energies. I focus on three main topics. First, I investigate vectorial forces as a tool to explore the energy landscape of protein complexes. Second, I apply this method to a biologically important force transduction complex, the integrin-talin complex. Third, I use Terahertz electric fields to manipulate the energy landscape of a molecular rotor on a gold surface and drive their effective rotation bidirectionally. Force is by nature a vector and depends on its three parameters: magnitude, direction and attachment point. Here, the impact of different force protocols varying these parameters is shown for an antibody-antigen complex and the ribonuclease-inhibitor complex barnase-barstar. Antibodies are essential for our adaptive immune system in their function to bind specific antigens. Here, the binding of an antibody to a peptide is probed with varying attachment points. Different attachment points clearly change the dissociation pathways. The barriers identified using experimental atomic force microscopy (AFM) and molecular dynamics (MD) simulations are in excellent agreement. I determine the molecular interactions of two main barriers for each setup. This results in a common outer barrier of the complex and different inner barriers probed by AFM. The ribonuclease barnase and its inhibitor barstar form an evolutionary optimized complex. Different force protocols are shown to determine the hierarchy of relative stability within a protein complex. For the barnase-barstar complex, the internal fold of the barstar is identified to be less stable than the barnase-barstar binding interaction. High velocities probe the lability or barriers of the system while low velocities probe the stability or energy wells of this system. Forces impact biological life on totally different length scales which range from whole organisms to individual proteins. Integrins are the major cell adhesion receptors binding to the extracellular matrix and talin. Talin activates the integrins and creates the initial connection to the actin cytoskeleton of the cell. Here, I have chosen to investigate the integrin-talin complex as a biologically important force transduction complex. The force dependence of the system is probed by constant force MD simulations. The two main results include the activation of the complex and its force response. I demonstrate, that the binding of talin to integrin does not disrupt the integrin's transmembrane helix interactions sterically. Since, this disruption is necessary for integrin activation, a modified activation mechanism requiring a small force application is proposed. The response of the integrin-talin complex normal and parallel to the cell membrane is analyzed. The complete dissociation pathways generated for both directions identify a force-induced formation of a stabilizing beta strand between integrin and talin only for normal forces. Furthermore, the complex tries to rotate such that the external force aligns with the more force resistant axis of the complex. In nature, molecular rotors are essential building blocks of many molecular machines and brownian motors like the F1-ATPase or the flagellum of a bacterium. The direction of rotation often steers different processes in clockwise and counterclockwise directions. Rotation on the nanoscopic level in artificial devices is still very limited and requires a deeper understanding. In my last project, I study the switching and driving of a molecular diethylsulfid rotor on a gold (111) surface by Terahertz electric fields. The response of the rotational energy landscape to static and oscillation electric fields is analyzed. Varying the Terahertz driving frequency, the rotation direction and frequency are controlled. A theoretical framework is presented to describe the behavior of the molecular rotor. This can be seen as the first step into the direction of man-made controllable nano-devices driven and controlled by energy from the electric wall-socket.

Introduction: Human mesenchymal stem cells (hMSC) are easily obtainable from bone mar-row and possess the ability to differentiate into osteoblasts. Therefore, they have been sug-gested as a suitable source for bone regeneration. HMSC are equipped with a variety of in-tegrins that mediate essential cell-matrix interactions. Collagen I represent approximately 90% of the bone protein content. Cell attachment to collagen I is mediated by three members of the integrin receptor family named a1b1, a2b1 and a11b1 integrins. The main aim of this doctoral thesis was to investigate the basal expression of those integrins in hMSC and to func-tionally analyze the knockdown effect of a single collagen I-binding integrin on hMSC behav-ior in vitro. Materials and methods: HMSC were cultured on collagen I-coated surface. A lentiviral trans-fer of a1-, a2- and a11-specific shRNA was applied for downregulation of the corresponding integrin mRNA. Quantitative PCR and western blot analysis were used to assess the basal ex-pression, knockdown efficiency and integrin compensation. Colorimetric adhesion assay was used for estimation of the extent of cells attachment. HMSC spreading and migration was ob-served by time lapse experiments. JC-1 staining was used for investigation of the initiation of apoptosis. Results: Quantitative PCR were used to assess the basal expression of collagen I-binding integrins in three hMSC donors. We found that these integrins are differently expressed as integrin a11 had the highest and integrin a2 the lowest expression. Next, we applied lentiviral delivery of target-specific short hairpin RNA (shRNA) in order to knockdown each of the collagen I-binding integrins and compared them to the hMSC transduced with a sequence against a non-human gene abbreviated as shRNA control. We achieved significant downregulation (> 80%) of the collagen I-binding integrin mRNA and protein. Subsequently to the transduction, we did not noticed pronounce morphological cell changes, however, a clear decrease of a2- and a11-knockdown hMSC numbers was observed during cultivation. Using a quantitative adhesion assay, we estimated that 120 min after plating only 30% of integrin a11-deficent cells were able to attach to collagen I. In contrast, at the same time point, 70% of integrin a2-knockdown hMSC were attached while integrin a1- and shRNA control hMSC have already reached 100% cell adhesion. Furthermore, a time lapse-based investigation showed that integrin a1- and shRNA control hMSC need approximately 35 min to fully spread on collagen I. In contrast, integrin a2- and a11-knockdown hMSC took approximately double more time for spreading in comparison to shRNA control hMSC. Additionally, we analyzed the migration capability of the four different hMSC lines. The average path which integrin a1- and shRNA control hMSC passed was approximately 170 µm with mean speed of 11.5 µm/h. In parallel integrin a2 and a11-deficient hMSC migrated to a distance of approximately 70 µm with a velocity of 5 µm/h. Since it was observed a lost of a2- and a11-deficient hMSC, next we performed JC-1 staining that visualizes mitochondrial leakage, a hallmark of apoptosis. The majority of integrin a2- and a11-knockdown hMSC exhibited mitochondrial leakage whereas integrin a1- and shRNA control hMSC showed intact mitochondria. Finally, we used quantitative PCR to investigate whether there were compensatory effects between the three integrin receptors. We detect that knockdown of integrin a1 led to upregulation of a2 and a11. Similarly, when integrin a2 was downregulated, integrin a1 and a11 expression increased. Interestingly, knockdown of integrin a11 caused only a slight increase in integrin a1 but not in a2 expression. We also observed that upon osteogenic stimulation, integrin a2 and a11-deficient hMSC further reduced in number and did not mineralize the matrix even on a single cell level. Moreover, our preliminary investigation in hMSC-derived from osteoporosis suffering patients showed a tremendous downregulation of integrin a2. Conclusions: Our results strongly suggested that integrins a2b1 and a11b1 mediate an indis-pensible signaling for hMSC. Once these receptors were ablated from cell surface, hMSC re-duced their spreading, adhesion, migration and survival rates. Our integrin knockdown mod-els can be used for further investigations and understanding of the integrins a2b1 and a11b1 importance and signaling in hMSC and hOB since we observed a strong downregulation of integrin a2 expression in osteoporosis.

Wed, 2 Feb 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16065/ https://edoc.ub.uni-muenchen.de/16065/1/Azimifar_Seyed_Babak.pdf Azimifar, Seyed Babak ddc:540, ddc:500, Fakultät für Ch

Microtubules align along the apical-basal axis of epithelial cells with their plus ends pointing to the basal side. Hotta et al. reveal how signals from the extracellular matrix establish this arrangement by recruiting the microtubule-anchoring factors LL5s to the basal cell cortex. This biosights episode presents the paper by Hotta et al. from the May 31, 2010 issue of The Journal of Cell Biology, and includes an interview with senior author Yuko Mimori-Kiyosue. Produced by Caitlin Sedwick and Ben Short.   Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Thu, 28 May 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10202/ https://edoc.ub.uni-muenchen.de/10202/1/Laemmermann_Tim.pdf Lämmermann, Tim ddc:540, ddc:500, Fakultät für Chemie und

The extracellular matrix (ECM) provides the structural frame for the development of tissues and organs. The ECM is bound by numerous membranous matrix-adhesion molecules and thereby triggers intracellular signals that control various cellular functions such as survival, polarity, proliferation and differentiation. Integrins represent an important family of ECM adhesion molecules which link the ECM with the intracellular actin-cytoskeleton. Integrin mediated adhesion structures also serve as important signaling platforms, although the integrin itself does not harbors any catalytic domains. Therefore integrin signaling depends on the recruitment of a number of cytoplasmic proteins that directly or indirectly bind to the short cytoplasmic integrin tails. During my PhD thesis I worked on three of these molecules, ILK, Kindlins and Palladin, and used the mouse as a model system to address their in vivo function. First, I investigated the role of integrin-linked kinase (ILK) in skeletal muscle. Loss of ILK expression in mice leads to peri-implantation lethality due to a cell polarization defect of the early embryo and abnormal actin accumulations. Studies in Caenorhabditis elegans and Drosophila melanogaster revealed an essential function for ILK in the attachment of actin filaments to the membrane of muscle cells and lack of ILK expression results in early lethality during embryogenesis. We generated mice with a skeletal muscle-restricted deletion of ILK that developed a mild, but progressive muscular dystrophy. This phenotype is predominantly restricted to myotendinous junctions (MTJs). Ultrastructural analyses showed muscle cell detachment from the basement membranes, and an accumulation of extracellular matrix. Endurance exercise training enhances the defect leading to disturbed subsarcolemmal myofiber architecture and an abrogation of the phosphorylation of Ser473 as well as Thr308 of protein kinase B (PKB)/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Second, I studied the expression and in vivo function of a further integrin- and actin- associated protein, palladin. Palladin belongs to the palladin/myotilin/myopalladin protein family. Palladin represents a phosphoprotein which plays an important role in cell adhesion and motility. Initially, I characterized the gene structure and the expression pattern of palladin. The palladin gene spans about 400 kb, with 25 exons and 3 alternative promoters resulting in at least three different isoforms (200 kDa, 140 kDa and 90-92 kDa) in mice. Using RT-PCR and in situ hybridizations of embryonic and adult tissues, I could show that the 200kDa isoform is predominantly expressed in heart and skeletal muscle. In contrast, the 140kDa isoform is expressed in various tissues and represents the major palladin isoform of the brain. The 90-92 kDa isoform is almost ubiquitously expressed with highest levels in tissues rich in smooth muscle, like bladder, uterus, small intestine and colon. The expression of the 200kDa isoform was characterized in more detail with a polyclonal antibody showing that this isoform localizes to the Z-discs of heart and skeletal muscle cells. In vitro differentiation experiments with a mouse myoblast cell line revealed an induction of the 200kDa isoform during myoblast fusion and differentiation suggesting that the biggest palladin isoform may serve as a molecular scaffold during myogenesis. Third, I specifically inactivated the largest palladin isoform in mice. Lack of the 200 kDa palladin isoform has no impact on the development, viability and fertility of mice. However ultrastructural analyses by transmission electron microscopy (TEM) showed a mild cardiac myopathy due to disintegration of myofibrils. In collaboration with the group of Olli Carpén, we generated palladin 200 kDa isoform/ myotilin double knockout mice. Myotilin is also expressed in heart and skeletal muscle. Ablation of both myotilin and palladin 200 kDa isoform in mouse revealed in addition to the mild cardiac myopathy a structural and functional impairment of skeletal muscle. Finally, I was also involved in the characterization of the expression and subcellular localization of a novel family of integrin associated proteins: the Kindlins. The Kindlin family consists of three members, Kindlin-1, -2 and -3. Mutations in Kindlin-1 cause a human disease, called Kindler Syndrome, which represents a skin blistering disease affecting the actin cytoskeleton of basal keratinocytes. Kindlin gene expression was first analyzed at the mRNA level by RT-PCR and Northern Blot studies. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia. Kindlin-2 is expressed in all tissues with highest levels in striated and smooth muscle cells. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. In contrast, Kindlin-3 expression is restricted to hematopoietic cells. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in podosomes, which are specialized adhesion structures of hematopoietic cells.

Adhäsionsvorgänge von Leukozyten spielen eine wichtige Rolle bei den verschiedensten biologischen Prozessen. Die Adhäsionsinteraktionen können Signalkaskaden aktivieren, die Funktionen wie die Zellmigration, Proliferation und Reifung von T-Lymphozyten steuern. Die Zellen des Immunsystems müssen schnell auf körperfremde Eindringlinge reagieren können und Adhäsionsvorgänge zwischen Zellen bzw. zwischen Zellen und der extrazellulären Matrix effektiv regulieren. Um jeden Infektionsherd im Körper zu erreichen, benutzen die Immunzellen die Lymph- und Blutbahnen, können diese Systeme aber verlassen (Diapedese) und durch Gewebe migrieren. Am Infektionsort interagieren die Immunzellen mit infizierten Zellen und starten Vernichtungsprogramme. Weiterhin präsentieren antigenpräsentierende Zellen im Lymphknoten ihre Antigene vorbeiziehenden T-Zellen, die bei korrekter Antigenerkennung zu T-Effektorzellen proliferieren. Bei all diesen regulierten Adhäsionsreaktionen spielen besonders die Integrine eine große Rolle. Von besonderem Interesse ist hierbei das Heterodimer LFA-1 (CD11a/CD18). LFA-1 wird nur auf Leukozyten exprimiert und bindet an die Liganden ICAM-1,-2,-3 der Immunglobulinsuperfamilie. Die kontrollierte Adhäsion bzw. Deadhäsion von Leukozyten bedarf einer spezifischen Regulation des LFA-1-Integrins und die Aufklärung der molekularen Grundlagen dieser Vorgänge ist von großem Interesse. Die LFA-1-vermittelte Zelladhäsion kann über den intrazellulären Guanin-Nukleotid-Austauschfaktor Cytohesin-1 aktiviert werden. Die Aktivierung wird dabei u.a. über Inositid-abhängige Membranrekrutierung von Cytohesin-1 kontrolliert. In dieser Arbeit wurde ein mit Cytohesin-1 interagierendes Protein, CYTIP, identifiziert, welches durch Cytokine in hämatopoetischen Zellen vermehrt exprimiert wird. CYTIP interagiert über seine „coiled-coil“-Proteininteraktionsdomäne direkt mit der N-terminalen „coiled-coil“-Domäne von Cytohesin-1 und inhibiert vollständig die Zelladhäsion auf Integrinliganden. Aufgrund der zwei Proteininteraktionselemente („coiled-coil“-Domäne, PDZ-Domäne) stellt CYTIP ein Adaptermolekül dar, um verschiedene Signalkomponenten in einem Multiproteinkomplex zu koppeln. CYTIP (Cytohesin-1 interacting protein) stellt eine neue Molekülklasse dar, die durch direkte Interaktion mit Cytohesin-1 die LFA-1 vermittelte Zelladhäsion negativ regulieren kann.

Die Aktivierung von T-Lymphozyten ist ein essentieller Prozeß für die Regulation der adaptiven Immunantwort. Hierbei werden in Folge einer Antigen-Exposition über verschiedene Oberflächenrezeptoren intrazelluläre Signaltransduktionsprozesse initiiert, welche in einer klonalen Expansion von T-Lymphozyten sowie deren Differenzierung zu Effektor- oder Gedächtniszellen resultieren. Die Spezifität dieser Prozesse wird durch die Wechselwirkung zwischen dem jeweiligen T-Zell-Antigenrezeptor (TCR) und einem Antigen-beladenen MHC-Molekül vermittelt, einer Interaktion, die allerdings nicht hinreichend für eine vollständige und dauerhafte Aktivierung der T-Zelle ist. Hierzu ist die Aktivierung sogenannter akzessorischer Oberflächenrezeptoren nötig, welche sowohl zur Verstärkung der Zell-Zell-Adhäsion als auch zur Induktion intrazellulärer Signalwege beitragen. Ein costimulatorisches Signal für naive T-Lymphozyten vermittelt zum Beispiel das CD28-Molekül, wodurch der Übergang dieser Zellen in einen anergischen Zustand verhindert und Zellproliferation ermöglicht wird. Zur Verstärkung der Zell-Zell-Interaktion tragen demgegenüber vor allem Adhäsionsmoleküle wie Selektine, Cadherine und Integrine bei. Der einzige Vertreter der β2-Integrinfamilie auf Lymphozyten, LFA-1, stand im Zentrum der im Folgenden beschriebenen Analysen. Es gab bereits Anhaltspunkte dafür, daß LFA-1 neben seiner Adhäsionsfunktion auch in Signaltransduktionsprozesse involviert ist. Allerdings sind die molekularen Mechanismen der LFA-1-vermittelten Signalwege bislang unvollständig charakterisiert. Dies liegt vor allem darin begründet, daß eine hochaffine Ligandenbindung erst nach einer Konformationsänderung des Integrins erfolgen kann. Eine solche Aktivierung des Integrins wird beispielsweise durch Stimulation der T-Lymphozyten über den T-Zell-Antigenrezeptor induziert, wodurch sich jedoch LFA-1-abhängige Signale mit denen des TCRs überlagern. Daher wurde im Verlauf der hier vorliegenden Arbeit ein auf Fusionsproteinen basierendes experimentelles System etabliert, welches eine aktivierungsunabhängige Untersuchung der LFA-1-vermittelten Signaltransduktion ermöglicht. Hierdurch wurde gezeigt, daß in humanen T-Lymphozyten die Aggregation der zytoplasmatischen Domäne der Integrin β2-Kette (CD18-Untereinheit) zur Erhöhung der intrazellulären Calciumkonzentration, zur IL-2-Promotoraktivierung sowie zum Anstiegs des Phosphotyrosingehalts zellulärer Proteine führte. Die Modulation dieser charakteristischen T-Zell- Aktivierungsparameter wird durch die zytoplasmatische Domäne der β2-Untereinheit von LFA-1 induziert, nicht aber durch die intrazellulären Anteile der Integrin αL- bzw. β1-Kette. Weiterhin konnte demonstriert werden, daß ein für die β2-Untereinheit spezifisches NPXFSequenzmotiv für deren Signalfunktion erforderlich ist. Dieselbe Region ist ebenso für die Signaltransduktion des nativen LFA-1-Gesamtmoleküls von elementarer Bedeutung. Überdies zeigten Untersuchungen in TCR-defizienten T-Zellen, daß die Oberflächenexpression des T-Zell-Rezeptors für den LFA-1-induzierten Signalweg essentiell ist. Demgegenüber erfolgte die intrazelluläre Calciummobilisierung durch das CD28-Molekül auch unabhängig von einer Expression des TCRs. Insgesamt konnte durch die vorliegenden Analysen die wesentliche Bedeutung des zytoplasmatischen Anteils der β2-Untereinheit des LFA-1-Integrins für den Prozeß der T-Zell-Aktivierung dokumentiert werden. Darüberhinaus wurde eine bisher unbekannte funktionelle Kooperativität zwischen LFA-1 und Komponenten des T-Zell-Antigenrezeptors aufgezeigt.

Das Leukocyten-spezifische Integrin LFA-1 spielt eine wichtige Rolle bei der Immunantwort, durch die Vermittlung dynamischer Zell-Zell- bzw. Zell-Matrix-Interaktionen. Die kontrollierte Adhäsion bzw. Deadhäsion von Leukocyten bedarf einer spezifischen Regulation des LFA-1-Integrins und die Aufklärung der molekularen Grundlagen dieser Vorgänge ist von großem Interesse. Cytohesin-1 war unmittelbar vor Beginn dieser Arbeit als cytoplasmatischer Regulationsfaktor der durch LFA-1 vermittelten Zelladhäsion identifiziert worden und seine spezifische Interaktion mit der cytoplasmatischen Domäne von CD18 konnte in vitro dokumentiert werden. Im Rahmen dieser Arbeit gelang es zunächst, die Assoziation von Cytohesin-1 und LFA-1 auch endogen, im intakten Zellverband, mittels Kolokalisationsstudien in der lymphoblastoiden B-Zellinie LCL-721, zu demonstrieren. Ferner konnte mit Hilfe von Mutationsanalysen die, für die Interaktion kritische Region in der cytoplasmatischen Domäne von CD18 lokalisiert werden. Sie befindet sich im aminoterminalen Bereich und umfaßt die Aminosäuren WKA(723 - 725). Die Mutation dieser Aminosäurereste nach TRG resultierte in einem vollständigen Interaktionsverlust mit Cytohesin-1. Die Inhibition der Cytohesin-1/CD18-Bindung konnte dabei sowohl durch Protein-Protein-Interaktionsanalysen in Hefe als auch durch biochemische Bindungsstudien in vitro dokumentiert werden, wobei jeweils Fusionsproteine der cytoplasmatischen Domäne von CD18 charakterisiert wurden. Funktionale Analysen der WKA(723-725)-Region von CD18 ergaben, daß die Mutation von WKA(723-725) nach TRG im intakten LFA-1-Molekül eine signifikante Reduktion der Integrin- Aktivität zur Folge hatte. Sowohl T-Zellklone als auch nicht hämatopoetische Zellen, wie HeLa, wiesen nach Expression von LFA-1(TRG), mit Hilfe rekombinanter Vaccinia- Viren, eine stark reduzierte Adhäsionsfähigkeit an immobilisiertes ICAM-1 auf. Ferner ergaben funktionale Studien mit HeLa-Zellen, die LFA-1 stabil exprimierten, daß Cytohesin-1 nur dann eine gesteigerte Adhäsion dieser Zellen an ICAM-1 induzierte, wenn sie Wildtyp-LFA-1 exprimierten. HeLa-Zellen, die LFA-1(TRG) exprimierten, ließen sich durch Cytohesin-1 zu keiner verstärkten Adhäsion aktivieren. Diese Ergebnisse demonstrierten die Bedeutsamkeit der Cytohesin-1/CD18-Interaktion für eine effiziente, durch LFA-1 vermittelte Zelladhäsion. Unklar war jedoch der Mechanismus, durch den Cytohesin-1 die Integrin/Liganden-Bindung regulierte. Studien mit dem Reporterantikörper 24 ließen darauf schließen, daß Cytohesin-1 durch die Bindung an CD18 eine Konformationsänderung in der extrazellulären Domäne des LFA-1-Integrins induzieren konnte, die möglicherweise die Affinität des Rezeptors modulierte. Diese Modulation der LFA-1-Konformation schien jedoch nicht hinreichend für eine stabile Bindung an ICAM-1 zu sein, wie eingehendere Analysen von Dr. W. Kolanus zeigten. Vielmehr erforderte eine effiziente Zelladhäsion zusätzlich die Guaninnukleotid-Austauschfunktion (GEF-Funktion) von Cytohesin-1, da die GEF-defekte Punktmutante, Cytohesin-1(E157K), nicht mehr in der Lage war, die Adhäsion von Jurkat E6-Zellen an ICAM-1 stabil zu induzieren. Biochemische Interaktionsstudien konnten dabei zeigen, daß die Mutante weiterhin fähig war, die cytoplasmatische Domäne von CD18 zu binden. Diese und weitere Ergebnisse von Dr. W. Nagel, die einen Zusammenhang zwischen der GEF-Funktion von Cytohesin-1 und dem „Spreading“ von adhärenten Jurkat E6-Zellen aufzeigten, legen die Vermutung nahe, daß Cytohesin-1 durch einen dualen Mechanismus in die LFA-1-Regulation involviert ist. Sowohl die direkte Interaktion von Cytohesin-1 und dem Integrin als auch seine GEF-Funktion stellen essentielle Faktoren für eine stabile Zelladhäsion, die durch LFA-1 vermittelt wird, dar. Welche funktionalen Mechanismen dabei durch den Guaninnukleotid-Austausch und der damit verbundenen Aktivierung einer GTPase induziert werden, ist noch unklar. Primär wäre eine Modulation des Aktin-Cytoskelettes und eine damit verbundene erhöhte laterale Mobilität der Integrine denkbar, die eine verstärkte Rezeptormultimerisierung und dadurch eine Aviditätsänderung des Integrins ermöglicht. Weitere Studien dieser Arbeit analysierten die Regulation von Cytohesin-1 selbst. Es konnte gezeigt werden, daß PI3-Kinase in die Kontrolle der Cytohesin-1-Funktion involviert war. Die Überexpression einer konstitutiv aktiven Form dieser Kinase (P110*) führte zu einer gesteigerten Adhäsion von Jurkat E6-Zellen an ICAM-1. Eine Inkubation dieser Zellen mit dem PI3-Kinase-spezifischen Inhibitor Wortmannin resultierte dagegen in einer signifikanten Reduktion der Zelladhäsion. Weitere funktionale Analysen, die die Zelladhäsion von Jurkat E6-Zellen nach Koexpression von P110* und der PH-Domäne von Cytohesin-1 untersuchten, sowie eingehendere Studien von Dr. W. Nagel, ermöglichten die Entwicklung eines Modells zur Regulation von Cytohesin- 1. Demzufolge führt die Aktivierung der PI3-Kinase zu einer verstärkten Rekrutierung von Cytohesin-1 an die Plasmamembran. Als Rekrutierungsmodul fungiert dabei die PHDomäne, die durch Bindung von PtdIns(3,4,5)P3, einem Produkt der PI3-Kinase, die Assoziation mit der Membran gewährleistet. Die Rekrutierung von Cytohesin-1 an die Plasmamembran führt zur Aktivierung von LFA-1 und der damit verbundenen stabilen Zelladhäsion an ICAM-1.

Die Identifizierung von Genen, deren Expression Einfluß auf die Metastasierung von Tumoren nimmt, stellt eine Möglichkeit zur Verbesserung sowohl diagnostischer als auch therapeutischer Ansätze in der Behandlung von Krebs dar. Jede achte Frau in westlichen Industrienationen erkrankt an Brustkrebs, wobei die Entwicklung neuer Methoden zur frühzeitigen Erkennung von Metastasen und deren zielgerichtete Behandlung entscheidend ist, um eine Therapie von Patientinnen mit progressivem Mammakarzinom zu ermöglichen. Entwickelt sich eine Zelle eines Primärtumors zu einer invasiven metastasierungsfähigen Zelle, so ist für diese Veränderung des Phänotyps eine grundlegende Modifizierung in der Expression zahlreicher Gene zu erwarten. In einem zellulären Modellsystem für die Progression des Mammakarzinoms wurde in der invasiven Zellinie MCF-7ADR das „Progressions-assoziierte Protein“ (PAP) identifiziert, in der nicht-invasiven Zellinie MCF-7 konnte dagegen keine Expression nachgewiesen werden. Die Aufgabenstellung dieser Arbeit ist die Klärung der Bedeutung der Expression dieses Gens für die Veränderung einer Zelle von einem nicht invasiven hin zu einem invasiven Phänotyp. PAP stellt ein Protein mit 157 Aminosäuren dar und gehört zur PMP22-Genfamilie, deren Mitglieder putative Viertransmembran-Rezeptoren sind. Neben der Hypothese der Einflußnahme von PAP auf die Metastasierungsfähigkeit einer Zelle werden für die Homologen eine Vielzahl zellulärer Funktionen postuliert, wie z.B. die Ausbildung von Zell-Zell-Kontakten, Adhäsionsvermittlung, Zellzyklusregulation, Tumorgenese und Apoptose. Die in dieser Arbeit durchgeführten Expressionsstudien zeigten, daß PAP in einer Vielzahl von Normalgeweben exprimiert wird, mit Ausnahme von Geweben des Zentralen Nervensystems (ZNS) und peripherer Blutlymphozyten. Erste vergleichende Prävalenzstudien mittels Northern-Blot- Analysen zwischen Tumor- und Normalgewebe einzelner Patienten wiesen im Fall von Gewebeproben aus Organen des Zentralnervensystems eine positive Korrelation der PAP-Expression mit den Tumorproben auf. Eine Untersuchung von Mammakarzinom-Zellinien mit unterschiedlichem Metastasierungsgrad in der Nacktmaus belegte, daß PAP lediglich in den als metastasierend eingestuften Zellen exprimiert wurde. Über gekoppelte in vitro Transkription/Translation konnte gezeigt werden, daß die in einen Expressionsvektor klonierte PAP-cDNS für ein Protein mit einer Größe von etwa 18 kDa kodierte. Auch mittels Immunfluoreszenzstudien transient transfizierter COS-7-Zellen konnte die Expression eines Epitop-markierten Proteins und die Lokalisierung an der Zellmembran nachgewiesen werden. PAP exprimierende Zellen waren nicht apoptotisch, jedoch oft auffallend abgerundet. Einzelne Klone stabil transfizierter MCF-7-Zellen, die PAP konstitutiv exprimierten, zeigten kein anderes Wachstumsverhalten in Proliferationstests gegenüber der untransfizierten oder den mocktransfizierten MCF-7-Zellen. Auch ihr Verhalten in in-vitro-Invasionstests unterschied sich nicht von dem der Ursprungszellen, während MCF-7ADR hier starke Invasivität aufwies. Eine endgültige Aussage über eine Funktion von PAP bei der Invasion von Tumoren kann jedoch erst nach der Auswertung von Experimenten in Nacktmäusen gemacht werden. Durch Serumentzug wachstumsarretierte humane Primärzellen zeigten für PAP eine inverse Regulation im Vergleich zu dem homologen Protein PMP22. PAP wurde in proliferierenden Zellen stärker exprimiert als in arretierten, während für PMP22 ein Anstieg der RNS in arretierten Zellen zu beobachteten war. Die Ergebnisse dieser Arbeit zeigen, daß alleine die Expression von PAP nicht ausreicht, um MCF-7- Zellen in vitro zur Invasion zu befähigen. Dafür könnten allerdings sowohl extrazelluläre Stimuli, als auch intrazelluläre Interaktionspartner fehlen, die zur Änderung des Phänotyps der Zellen und zur Invasion notwendig sein könnten. Da Rezeptoren jedoch in allen Schritten der Metastasierung von grundlegender Bedeutung sind, kann auch für PAP nicht ausgeschlossen werden, daß es in diesen komplexen zellulären Mechanismen eine Rolle spielt. Ein Einfluß auf die Proliferationsfähigkeit von Zellen konnte durch die konstitutive Expression von PAP nicht nachgewiesen werden. Eindeutig belegt werden konnte aber eine Korrelation mit dem Zellzyklus. Durch Serumentzug arretierte primäre Zellen zeigten eine verminderte PAP-Expression im Vergleich zu proliferierenden Zellen. Die Überexpression von PAP in COS-7-Zellen läßt allerdings die Vermutung zu, daß PAP, ebenso wie das homologe PMP22, einen Einfluß auf die Zellmorphologie und auf die Adhäsion von Zellen haben könnte. PAP könnte dabei in einen Adhäsion-regulierenden Mechanismus eingebunden sein, der bei einer Überexpression von PAP zu einem Abrunden der Zellen und einem Substratkontaktverlust führen könnte. Unter physiologischen Bedingungen könnte dies für das Loslösen der Zellen während der G2-Phase des Zellzyklus notwendig sein. Bei einer fehlerhaften Regulation (einer gesteigerten Expression von PAP) unter pathologischen Bedingungen könnte eine leichtere Loslösung von Tumorzellen die Metastasierung begünstigen. Denkbar wäre eine Interaktion von PAP mit Integrin- Rezeptoren, wodurch die Affinität des Integrins beeinflußt werden könnte. Diese Hypothese bietet einen Ansatzpunkt für weitere Studien bezüglich des Einflusses von PAP auf zelluläre Vorgänge, wie Zellzyklus-Regulation und Zellwanderung.