Podcasts about xbp1

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.18.516150v1?rss=1 Authors: Ursem, S. R., Diepenbroek, C., Kool, T., Eggels, L., Heijboer, A. C., la Fleur, S. E. Abstract: Fibroblast growth factor 23 (FGF23) is a key regulator of systemic phosphate homeostasis, but also an interplay with glucose metabolism has been suggested. Several studies implicate a function of FGF23 in the brain, and indeed we have recently identified FGF23 protein in several brain areas in rats, such as the hypothalamus, third ventricle and choroid plexus. In the current study, we aimed to determine the effect of an intracerebroventricular (icv) injection of FGF23 in the third ventricle of rats on hypothalamic genes involved in glucose regulation. In addition, we assessed whether glycerol can be used safely for icv injections as glycerol is used as a stabilizing compound for FGF23 protein. Adult Wistar rats received an icv injection of recombinant rat FGF23 or vehicle. Dose dependent behavioral changes, suggestive of stress, were observed directly after infusion of FGF23. After 60 min animals were sacrificed and the arcuate nucleus, lateral hypothalamus and choroid plexus were isolated. In these brain regions gene expression was determined of the FGF23 receptor complex (FGFR1, Klotho), NPY, POMC, phosphate transporters (SLC20 and SLC34 families) and markers of cellular ER stress (ATF4 and the ratio of spliced/unspliced XBP1). We showed that glycerol is well tolerated as stabilizer for icv injections. In FGF23-treated animals, cellular ER stress markers were increased in the arcuate nucleus. FGF23 injection did not affect expression of its receptor complex, NPY, POMC, or phosphate transporters. Future studies are warranted to investigate the effect of FGF23 in the brain on the protein level and on neuronal activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This month on Episode 39 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 5th and 19th issues of the journal. This episode also features an interview with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University on their article, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   Article highlights:   Jain, et al. Role of UPR in Platelets   Orlich et al: SRF Function in Mural Cells of the CNS   Xue et al: Gut Microbial IPA Inhibits Atherosclerosis   Wang et al: Endothelial ETS1 on Heart Development   Cindy St. Hilaire:        Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our August 5th and August 19th issues of Circulation Research. I'm also going to have a chat with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University about their study, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   But before I get to the interview, I first want to share an article from our August 5th issue, and that article is titled, Unfolded Protein Response Differentially Modulates the Platelet Phenotype. The first author of this study is Kanika Jain and the corresponding author is John Hwa from Yale University. Self-stress can lead to protein misfolding, and the accumulation of misfolded proteins can lead to a reduction in protein translation and may alter gene transcription, a process collectively known as the unfolded protein response, or UPR. UPR is well documented in nucleated cells; however, it has not been studied in platelets, which are anuclear, but do have a rapid response to cellular stress. In this study, they investigated the UPR in anucleate platelets and explore its role, if any, in platelet physiology and function.   They found that treating human and mouse platelets with various stressors caused aggregations of misfolded proteins and induction of UPR-specific factors. Oxidative stress, for example, induced the UPR kinase PERK, while an endoplasmic reticulum stressor induced the transcription of the UPR factor XBP1. The team went on to study the UPR in platelets from people with type II diabetes, which is a population in which platelet mediated thrombosis is a major complication. They showed that protein aggregation and upregulation of the XBP1 pathway in diabetic patient platelets correlated with disease severity. Furthermore, treating the diabetic patient platelets with a chemical chaperone that helps to correct protein misfolding reduced protein aggregations and prevented the cells prothrombotic activation. This work confirms that even without transcription, platelets display stress-induced UPR, and that targeting this response may be a way to reduce thrombotic risk in diabetic patients.   Cindy St. Hilaire:        The second article I want to share with you is from our August 5th issue and is titled, Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow, and it was led by Michael Orlich from Uppsala University in Sweden. Blood vessels are lined with endothelial cells and surrounded by mural cells. Vascular smooth muscle cells are the mural cells in the case of veins and arteries, and pericytes are the mural cells in the case of capillaries. In the capillaries, pericytes maintain blood-brain and blood-retina barrier function and can mediate vascular tone, similar to smooth muscle cells. While these pericytes and smooth muscle cells are related, they have distinct roles and characteristics.   To learn more about the similarities and the differences between pericytes and smooth muscle cells, this group examined how each would be affected by the absence of SRF in the other. SRF is a transcription factor, essential for nonvascular or visceral smooth muscle cell function. In visceral smooth muscle cells, SRF drives expression of smooth muscle actin and other smooth muscle genes. Using mice engineered to lack SRF in mural cells, they show that SRF drives smooth muscle gene expression in these pericytes and smooth muscle cells, and its loss from smooth muscle cells causes atrial venous malformations and diminishes vascular tone. In pericytes, loss of SRF impaired cell migration in angiogenic sprouting. In a mouse model of retinopathy, activation of SRF drove pathological growth of pericytes. This work not only highlights the various functions of SRF in mural cell biology, but it also suggests that it has a role in pathological capillary patterning.   Cindy St. Hilaire:        The third article I want to share is from our August 19th issue of Circulation Research and is titled, Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease. The first authors are Hongliang Xue and Xu Chen, and the corresponding author is Wenhua Ling from Sun Yat-Sen University in Guangzhou, China. Recent studies provide evidence that disorders in the gut microbiota and gut microbiome derived metabolites affect the development of atherosclerosis. However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of these alterations remain unclear. Gut microbiome derived metabolites, such as short-chain fatty acids and trimethylamine N-oxide, or TMAO, have been found to correlate with atherosclerotic disease severity.   This study has now found that serum levels of indole-3-propionic acid, or IPA, are lower in atherosclerosis patients than controls. The team performed unbiased metagenomic and metabolomic analyses on fecal and serum samples from 30 coronary artery disease patients and found that, compared with controls, patients with atherosclerosis had lower gut bacterial diversity, depletion of species that commonly produce IPA and lower levels of IPA in their blood. Examination of a second larger cohort of atherosclerosis patients confirmed this IPA disease correlation. The team also showed serum IPA was reduced in a mouse model of atherosclerosis, and that supplementing such mice with dietary IPA could slow disease progression. Analysis of the macrophages from these mice showed that IPA increased cholesterol efflux, and the team went on to elucidate the molecular steps involved. The results of this study not only unraveled the details of IPA's influence on atherosclerosis, but suggest boosting levels of this metabolite could slow atherosclerotic disease progression.   Cindy St. Hilaire:        The last article I want to share is also from our August 19th issue, and it's titled, Endothelial Loss of ETS1 Impairs Coronary Vascular Development and Leads to Ventricular Non-Compaction. The first author is Lu Wang and the corresponding author is Paul Grossfeld, and they are at UCSD. Congenital heart defects, or CHDs, are present in nearly 1% of the human population. In some cases, the heart defects result from a genetic error, which can give researchers clues to its etiology. Jacobson syndrome is a complex condition caused by deletions from one end of chromosome 11, and the occurrence of a congenital heart defect in this syndrome has been associated with the loss of the gene ETS1. ETS1 is an angiogenesis promoting transcription factor, but how ETS1 functions in heart development was not known.   Wang and colleagues now show that both global or endothelial-specific loss of ETS1 in mice caused differences in embryonic heart development that ultimately led to a muscular wall defect known as ventricular non-compaction. The mice also had defective coronary vasculogenesis associated with decreased abundance of endothelial cells in the ventricular myocardium. RNA sequencing of ventricular tissue revealed that, compared with controls, mice lacking ETS1 had reduced expression of several important angiogenesis genes and upregulation of extracellular matrix factors, which together contributed to the muscular and vascular defects.   Cindy St. Hilaire:        Today I have with me, Dr Annet Kirabo and Dr Ashley Pitzer, both from Vanderbilt University, and we're going to talk about their paper, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension. This article is in our August 5th issue of Circulation Research. Thank you both so much for joining me today.   Annet Kirabo:             Yeah, thank you so much for having us.   Ashley Pitzer:              Yeah, thank you for having us.   Cindy St. Hilaire:        Yeah, it's a great paper. I think we're all familiar with hypertension and this idea that too much salt is bad for our cardiovascular system. When I was a kid, my grandparents had those salt replacements on their kitchen table, Mrs. Dash and whatever. But, like you said in the start of your paper, the exact mechanism by which salt intake increases blood pressure and also increases cardiovascular risk, it's not really well understood, and you guys are focusing on the contribution of immune responses in this process or in this pathogenesis. Before we dig into the details of your paper, I was wondering if you could give us a little bit of background about what's known regarding the role of inflammation in this salt-sensitive hypertension pathogenesis.   Annet Kirabo:             Yeah. It's difficult to know where begin to from, but the role of inflammation in cardiovascular disease have been known for many, many decades. Right now, Dr David Harrison showed more than 10 years ago that T cells contribute to hypertension, but the mechanisms were not known. Back when I was a post doc in David Harrison's lab, we discovered a new mechanism, how immune cells are activated in inflammation and hypertension, whereby we found that there is increased oxidative stress in antigen-presenting cells. This leads to formation of oxidative products known as arachidonic acid or lipid products known as isolevuglandin, or IsoLGs. These IsoLGs are highly, highly reactive and they adapt to lysines on proteins. This is a covalent binding, which leads to permanent alteration of proteins, and so these proteins act as neoantigens that are presented as self-antigens to T cells, leading to an autoimmune-like state in hypertension.   Annet Kirabo:             We found that these antigen-presenting cells are activated and they start producing a lot of cytokines that paralyze T cells to IL-17 producing T cells that contribute to hypertension. And so, when I started my lab back in 2016, we discovered that excess dietary salt profoundly activates this pathway, and we found for the first time that these antigen-presenting cells, they express ENaC, the epithelial sodium channel, and sodium goes into these antigen-presenting cells and activates the NADPH oxidase, which is an enzyme which produces this reactive oxygen species, leading to this IsoLG formation, which I've talked about, and leading to inflammation.   So, three years ago when Ashley joined my lab, she had extensively studied the inflammasome in her PhD program, and she suggested why don't we look at the role of the inflammasome in this pathway and how IsoLG may contribute to this. In her paper that we are discussing right now, she found that in a dependent manner, sodium enters the cell and activates this pathway, and the NLRP3 inflammasome is involved in this process.   Cindy St. Hilaire:        That's such a wonderful story that fits together so many pieces. One of the things you talk about, which I guess I didn't even appreciate myself is, there are certain individuals out there who are more salt-sensitive than others.   Annet Kirabo:             Yeah.   Cindy St. Hilaire:        What is that difference? Do we know the root cause of that? And then also, how many individuals are we talking about are salt-sensitive?   Annet Kirabo:             Salt-sensitive blood pressure, it is a variable trait and it's normally distributed in the population, but it happens more in some individuals than others. It happens even in 25% of people without any hypertension. These people go to that doctor, that doctor thinks they're normal, they don't have any hypertension, but these people can be at a risk of sudden heart attack or cardiovascular risk or even a stroke, simply because when they eat a salty meal, their blood pressure will go up.   Cindy St. Hilaire:        Yeah, that's one of my questions. How much salt are we talking about here? And not only how much in a meal, but a sustained amount? How bad is a miso soup a day?   Annet Kirabo:             Yes. The American Heart Association and the World Health Organization have recommendations. American Heart Association recommends one spoon per day. We have refused to adapt to this recommendation, but that is the recommendation that they have recommended per day to eat. But this is difficult because most of the salt, as you know, is already in our food through processing in our processed foods and we don't have any control over how much salt we have, and there's also a lot of adding of salt at a table.   Cindy St. Hilaire:        Ashley, your background was more the inflammasome. What were your thoughts entering into this project? Did you have much of a hypertension background?   Ashley Pitzer:              No. My graduate thesis focused mainly on endothelial dysfunction and cardiovascular disease, and so it was a pretty easy segue. But it was just with Annet, so excited about the project and showing me all the data and this robust IL-1 beta production that she was seeing after these immune cells being exposed to high salt, I, with my inflammasome background, was immediately like, this could be playing a role. And so it was, like I said, a pretty easy transition and, as is in the paper, we're doing human studies. All of my research back in grad school was very basic research, so it was very exciting to see how our research was being translated with people having this condition and potentially finding mechanisms where we can target this to help actual people.   Cindy St. Hilaire:        I think a lot of us who are not in the hypertension field, and maybe this was you before you joined Annet's lab, we really only kind of think of the kidneys and the blood vessels when we think about hypertension, but studies like this are changing that. And I think a lot of Annet's earlier work, as well as the work of others, have shown a role for this epithelial sodium channel as an important player in this salt-induced hypertension. New to me, it's not just found in the kidney, which I totally did not appreciate that. And it's this channel sensing the salt that can trigger this IL-1 beta production that does a whole bunch of other things.   Cindy St. Hilaire:        What are those other things? What are those cells that are affected and where is this happening? Obviously it's not just kidney cells, but is it only in the kidney or are these systemic cells? What do we think is happening?   Ashley Pitzer:              That's the question, is, where is this happening? There's been studies at Vanderbilt by Jens Titze and his lab showing, where are these immune cells sensing the salt? And so they've shown that sodium accumulates in the skin, a huge argument is for they're sensing the sodium in the kidney because that's where a lot of it is being processed. But these immune cells travel through the whole body, so they're seeing it where there are the highest amounts of sodium concentration, and so I would argue it's in the kidney.   Annet Kirabo:             Indeed, because we're now collaborating with Tina Kon, and we have recently published with her a paper in the International Journal of Science, where we have done sodium MRI and we find this accumulation of sodium in the kidney even much more than in the skin. And we know that the kidney is where sodium is highly concentrated. So the working hypothesis in the lab is that these immune cells can be activated wherever they are, in the lymph nodes or not, in other tissues, but they can travel to the kidney.   We find that in high salt, if you feed high salt to the mouse, the endothelium in the kidney becomes dysfunctional and it expresses molecules, chemoattractants, that attract these immune cells in the kidney. We think that the high salt accumulation in the kidney can activate these, and then these immune cells are activated and they produce cytokines. Dr Steve Crowley showed that they can produce IL-1 beta, which induces activation of sodium channels that can be induced. We have also actually found that even IL-17 can be produced by these immune cells in the kidney and they can activate sodium channels in the kidney, leading retention of sodium and water and hypertension.   Cindy St. Hilaire:        Very cool. You used a lot of mice in this paper. Can you tell us, I just want to know a little bit about the models you chose to use, but also how similar is hypertension in mouse and humans? Obviously for atherosclerosis, we have to do lots of things to get them to form a plaque. Is hypertension similar in a mouse and do mice also show this salt-sensitive phenotype?   Annet Kirabo:             That is an extremely important point. If you read our paper, we use a slightly different approach. Most people do benchside to bed approach. We did the opposite. We did a bed to benchside approach.   Cindy St. Hilaire:        Always smart.   Annet Kirabo:             Yeah. We first started humans, and then with some references, we went to the mice, because I think when it comes to salt-sensitive blood pressure, mice are different from humans. In fact, if we look in the lab, we find that female mice are protected from salt-sensitive blood pressure, but we find that in the humans, it's the opposite. Females are more prone to salt-sensitive hypertension. Those are studies that we are doing right now. We haven't published. But we know that it can be different.   The model we use most of the time in the lab, the C57 mice, are resistant to salt-sensitive hypertension. These C57 mice would rather die before they raise their blood pressure in response to salt. We can induce salt-sensitivity in these mice like in the paper that we are discussing. When we induce the endothelial dysfunction using L-NAME and we wash it out, then these mice, when you give them, subsequently, salt, suggests that they become salt-sensitive. But we also have a salt-sensitive mouse model that we use, the 129/SV mouse. So we use several models to kind of prove the same thing over and over again with the findings that we found in humans.   Cindy St. Hilaire:        And you used a technique, which I'm a little bit familiar with, but I'd like to hear, A, about it from you, but also your experience in using it, and that is CITE-seq. So, how does that work?   Ashley Pitzer:              That was with our human study where we actually had patients come in, who were hypertensive, took them off medication for 2 weeks. They come in, we get baseline samples, we give them a salt load on one day, and then the next day we completely salt deplete them.   Cindy St. Hilaire:        How much is a salt load? Like a Big Mac? What's a salt load?   Ashley Pitzer:              Yeah, it's pretty much just like eating Lays chips all day. It's a lot of salt. It's a very salty meal.   Annet Kirabo:             And then in addition, we also infuse saline too.   Cindy St. Hilaire:        Oh, wow.   Annet Kirabo:             Because these people, when they come into the hospital, some them have already eating high salt. This approach is to just maximize the whole system so that then when we sort deplete everybody, it's at the same level and it's just to unify the whole process. But sorry, Ashley, you go ahead.   Ashley Pitzer:              With the CITE-seq, we're able to take different patients on different days. So we take samples each day, and we can give each sample a barcode, basically. Give them a barcode, we can pool them all together, process them, and we can sequence their RNA, we can probe for a certain amount of protein expression as well. So then when we analyze, we can look at protein expression, so you get the translation and the transcription for each person on each day, and then you're able to compare. And so you get this huge picture and it's a lot of data.   Cindy St. Hilaire:        How long did it take you to sort through?   Ashley Pitzer:              Well, we have a statistician who does all of that, because my wheelhouse is here and it is on a different planet. So we have somebody who helps us with that who does an unbiased approach. And then once he does an analysis, gives us back what are the things that are changing the most, and one of those was IL-1 beta.   Annet Kirabo:             As you can see, our list is huge, this is a massive input of so many collaborators. We have computational people on there that help us with this. I can't even begin to learn these techniques, but with all this collaboration and the resources at Vanderbilt, these things are possible. And so, this is a really powerful approach where you can combine protein expression and you get the specific cells that express the genes and you couple the channel type to the gene expression.   Annet Kirabo:             We actually found that not all monocytes are the same. There's a specific class that of monocytes, A small class of monocytes that is so angry, and the inflammasome is activated and producing this IL-1 beta, and that is enough to contribute to this phenotype of salt-sensitive hypertension, which dynamically changed according to blood pressure, suggesting that this is a targetable salt-sensitive blood pressure, even in normotensive people, is a targetable trait. And because these monocytes are in blood, can we get a blood sample and routinely diagnose salt-sensitive blood pressure so that doctors are aware and they can appropriately advise patients.   Cindy St. Hilaire:        This was samples obviously taken from a blood draw, right? So they're circulating.   Annet Kirabo:             It was a blood draw, yes.   Cindy St. Hilaire:        What do you think about these immune cells, perhaps, native in the kidney? Do you think the small population of angry cells, like you said, is escaping from the kidney environment? What do you think?   Annet Kirabo:             When I was a post-doc in David Harrison's lab, we found that the most angry dendritic cells that contribute to this inflammation and hypertension are monocyte-derived. So that's why in the human study we focused on monocytes, because there are so many subtypes of dendritic cells, plasmacytoid dendritic, classical dendritic cells. We have studied all of these subtypes, and we have focused on monocyte-derived dendritic cells because they're the ones that seem to be contributing to this phenotype the most.                                     Cindy St. Hilaire:        You guys focused in on the NLRP3 inflammasome, which, obviously it's a really critical component broadly for the innate immune system. Do you think that this is going to be a targetable approach that can be leveraged for hypertension? Or do you think it's too broad? What do you think about that as a therapeutic potential?   Ashley Pitzer:              Even when you look in our paper, and we use a knockout model, where we use a completely global knockout model, put them on high salt, and we give them back only dendritic cells that are from wild-type mice, so they have that NLRP3, that have been exposed to high salt. We were able to increase blood pressure, but I also did, in mice, where I gave them an IL-1 beta neutralizing antibody, similar to canakinumab, which is the CANTOS trial, and there's not much of a difference. There is, but it's minor. It's very minor.   Ashley Pitzer:              So, to be able to target in specific cell types in humans one thing, it's very difficult, and maybe one day we can get there. But I think it at least gives us a better idea of what is the full picture, what's the big mechanism going on with immune cells? In part of our human study, we are looking at something to try and be able to identify who is salt sensitive. So if anything, we're able to sit here and potentially have a way of identifying salt-sensitive patients, where, right now, all we can do is have them come in like we do and do a 3-day study, and not everybody can do that.   Annet Kirabo:             To add onto that, perhaps you know, we are talking about precision medicine. This is an era of precision medicine where you need to really tailor treatments if we can get there, and I think this is one way. CANTOS trial. They had no way of knowing who is salt-sensitive and who is not, it was a global approach, and the lack of differences in blood pressure might be explained that this IL-1 beta pathway is targetable in a specific population whose blood pressure is probably driven by inflammation. There are so many, many mechanisms that drive hypertension, and so perhaps we need to focus this on salt-sensitive people, and maybe we can really use this approach to target. Plus, this is ENaC-dependent.   As you know, amiloride has lost favor in the clinic as a treatment of hypertension, because in the majority, it's not effective. But studies have shown that in Black men, for example, who had been categorized salt-resistant, when they give them amiloride, their blood pressure went down, and yet it's not effective in the majority of the people.   So, can we bring back, can we take another look at amiloride. As our studies indicate that blockade of ENaC is anti-inflammatory and it's also antioxidant agent, can we at least bring back amiloride and look at it again and we focus it for specific populations of people that may be more prone to salt-sensitive hypertension?   Here we have so many targets for potential precision treatment of salt-sensitive potential in this paper. You can target SGK1, which we know is possible, we listed a number of clinical trials that they have used NLRP3 inflammasome inhibitors, you can use amiloride for these people, and you can also potentially scavenge IsoLGs.     Cindy St. Hilaire:        What was the most challenging aspect of this study? There's a lot of moving parts, so what was the biggest challenge? And then, also, what was the most surprising part or the most pleasantly surprising part?   Ashley Pitzer:              You have to think, most of this was going on right when the pandemic hit. And right before that, we had started our human recruitment for the human study. And so that put a little bit of a time damper on it.   Ashley Pitzer:              Other than that, it was just, we were finding one thing, developing a new experiment, doing it again, doing it again. And honestly, what was the most surprising and rewarding was just seeing the same thing in, because we took just PBMCs from normotensive patients, treated them with high salt, and saw the changes that we did with the inflammasome. And to see that exactly again in an in vivo model of giving patients high salt and seeing the same thing, it was very rewarding and confirmed that, okay, we're on the right path. Seeing the same thing over and over and over again, it kind of reaffirms that you had a good idea.   Annet Kirabo:             I might add, one of the most challenging was, initially, the computational. Oh, part of the pandemic I was, the pandemic hit, I had a baby during the pandemic, and it was my time to leave my home, and then all these things were going on. We had a clinical trial where patients had to come in. Vanderbilt was so super supportive ,even checking for COVID-19. Our patients could not have COVID-19. We needed to check them.   Cindy St. Hilaire:        Yeah.   Annet Kirabo:             They also had to check for COVID-19. And so during that time, I realized, wait, I need learn computation analysis. I realized I cannot learn, and then reached out to collaborators that helped. That was extremely challenging. And then the other challenging thing that we faced later during the pandemic is vaccinations. In our criteria, these people cannot be vaccinated for reasons. We've studied inflammation, hypertension, and so vaccination was confounding. And even COVID-19 is even more for confounding. So we had this exclusion criteria where we could not recruit anyone.   Annet Kirabo:             Everybody was having COVID, everybody was being vaccinated, and everybody was in that exclusion criteria, so it was difficult to get people. We have had some slow down, but right now it's beginning to build up.   Cindy St. Hilaire:        So, what's next? What's the next question?   Annet Kirabo:             We have so many.   Cindy St. Hilaire:        That means it was a great study. If you have more, that means it was a great study.   Annet Kirabo:             Yeah. This study and us, it kind of warms. The inside seat just opened up, we have primary data in the genetic regulation of ENaC, we have primary data where we found. We are trying to figure out the specific ENaC channel in these antigen-presenting cells. We don't know. We found that ENaC delta, for example, it's not found in a kidney or you talked about a kidney contribution versus immune cells. ENaC delta is not found in the kidney, but we have primary data that show that ENaC delta is the most correlated with cardiovascular risk, is the most correlated with kidney disease and all forms of hypertension. So now we're like, ENaC delta expressed in the immune cells, not in the kidney, it is the one that is most involved in cardiovascular disease, so how are we going to tell the world that.   Cindy St. Hilaire:        Yeah, very cool.   Annet Kirabo:             Those cells, not necessarily the kidney. The kidney plays a part because the cells are going there, but it's very, very exciting. Plus a number of other lines that we are investigating.   Cindy St. Hilaire:        It's great. Well, congratulations, again, on this publication, on just getting all this done with what sounds like extremely difficult patient recruitment. So, Dr Kirabo and Dr Pitzer, thank you so much for joining me today and I'm looking forward to these next studies on maybe ENaC delta.   Annet Kirabo:             Thank you. Thank you so much.   Ashley Pitzer:              Thank you for having us.   Cindy St. Hilaire:        That's it for the highlights from the August 5th and August 19th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and hashtag Discover CircRes. Thank you to our guests, Dr Annet Kirabo and Dr Ashley Pitzer.   This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. Opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

Mobilisé dans le cadre de l'opération Résilience, l'A400M Atlas a également réalisé des missions de transfert de patients lourdement médicalisés d'Île-de-France vers des hôpitaux en région. Dès le jeudi 2 avril au soir, il est venu renforcer le plot avancé mis en place sur la base aérienne 107 de Villacoublay. Retour sur l'engagement de l'aéronef et de son équipage ainsi que sur les perspectives de cette capacité sanitaire inédite. Un renfort essentiel dans la lutte contre le Covid-19. Air actualités du mois de juin 2020 : https://fr.calameo.com/read/000014334eaf98000ca8f Vidéo : https://youtu.be/xBp1_ja_GkA

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.07.191262v1?rss=1 Authors: Weightman Potter, P. G., Washer, S., Jeffries, A. R., Holley, J. E., Gutowski, N. J., Dempster, E. L., Beall, C. Abstract: Aims/hypothesis: Recurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure. Methods: Human primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform. Results: 24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following LG exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures. Conclusions/interpretation: These data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be mediated by metabolic adaptations to better preserve intracellular and/or ER ATP levels, but this requires further investigation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.28.119164v1?rss=1 Authors: Garcia-Huerta, P., Troncoso-Escudero, P., Wu, D., Thiruvalluvan, A., Cisternas, M., Henriquez, D. R., Plate, L., Chana-Cuevas, P., Sequel, C., Thielen, P., Longo, K. A., Geddes, B. J., Lederkremer, G., Sharma, N., Shenkman, M., Naphade, S., Sardi, P., Spichiger, C., Richter, H. G., Court, F., Ellerby, L., Wiseman, L., Gonzalez-Billault, C., Bergink, S., Vidal, R. L., Hetz, C. Abstract: Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease-modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here we studied the impact of IGF2 signaling on protein aggregation in models of Huntington disease (HD) as proof-of-concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 were independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human post-mortem brain tissue, and blood samples from HD patients showed a reduction of IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein aggregates. Copy rights belong to original authors. Visit the link for more info

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 cohosts. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr Greg Hundley:             And I'm Greg Hundley, associate editor from the Poly Heart Center at VCU health in Richmond, Virginia. Carolyn, oh, this is going to be an exciting featured article today, and we're going to discuss the combination of agents or their administration et al that are best suited for managing both anticoagulation and antiplatelet therapy and those with coronary disease, peripheral arterial disease and heart failure. And, we'll speak with Dr Kelley Branch from the University of Washington. Dr Carolyn Lam:                And me! Dr Greg Hundley:             Yes. How am I going to interview you? And, we'll discuss the utility of Rivaroxaban with or without aspirin in patients with heart failure or peripheral arterial disease from the compass trial. Dr Carolyn Lam:                Well, I'm not going to let you get there until I tell you about this first basic paper I've chosen because it focuses on the unfolded protein response. Dr Greg Hundley:             What's that? Dr Carolyn Lam:                Well, Greg, I was really hoping you'd ask. The unfolded protein response is a cellular adaptive process to cope with protein folding stress. Now, approximately 40% of human proteins are predicted to be either transmembrane or secretory. The synthesis, the folding, the cellular transportation and location of these proteins rely on proper functioning of this secretory pathway. Numerous studies have established that the unfolded protein response plays versatile roles during development and under physiologic and pathophysiologic conditions. However, the role of this unfolded protein response in the regulation of cardiomyocyte growth is unclear. Dr Greg Hundley:             That's fantastic, Carolyn. I've already learned something here. So, what did this paper show? Dr Carolyn Lam:                This is from Dr Wang and colleagues from UT Southwestern, and basically, they use both gain and loss of function approaches to genetically manipulate spliced X-box binding protein one or XBP1, which is the most conserved signaling branch of the unfolded protein response in the heart. In addition, primary cardiomyocyte cultures were employed to address the role of XBP1S in cell growth in a cell autonomous manner. They found that XBP1S expression was reduced in both human and Rhode and cardiac tissues with heart failure deficiency of XBP1S lead to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac restricted over expression of XBP1S prevented the development of cardiac dysfunction. Mechanistically, they found that XBP1S stimulated adaptive cardiac growth, your activation of mechanistic target of rapamycin or MTOR signaling which is mediated via the FK-506 binding protein 11, which is a novel transcriptional target of XBP1S. So in conclusion, this study really showed a critical role of the XBP1S FKB or FK-506 binding protein 11 and MTOR axis in coupling the unfolded protein response and cardiac cell growth regulation. Dr Greg Hundley:             Boy Carolyn, you explained that so well, and I learned a lot from that. I hope I can do as well with this next article from Professor Johann Backs from the University of Heidelberg. Now paradoxically, some glucose lowering drugs have been shown to worsen heart failure, raising the question of how glucose mediates protective versus detrimental cardiac signaling, and this study from his group focused on one of the class two histone deacetylases or HDAC's namely HDAC-4, which functions as an important epigenetic regulator by responding to upstream stress signals, and linking them to downstream gene regulatory programs involved in among other things, metabolic regulation. Dr Carolyn Lam:                Very interesting. So what did they find? Dr Greg Hundley:             What they found is that HDAC4 acts as an important maintenance factor of cardiac function in diabetes and O-glycine-N0acetylglucosamine of HDAC4 at searing 642 induces the production of cardio-protective HDAC F-end terminal fragment and attenuates cardio detrimental Cam kinase two mediated phosphorylation of HDAC4 at searing 632. Vice versa, Cam kinase two mediated phosphorylation of HDAC4 at searing 632 attenuates HDAC-4 n terminal production. Thus, these findings lay the ground for the development of novel therapeutic strategies for diabetic patients with heart failure by inhibiting Cam kinase phosphorylation at CIHR 632 or enhancing o-glycine and escalation at searing 642. Dr Carolyn Lam:                Fascinating, Greg. Well, my next paper is a subgroup analysis of EUCLID and is the first to assess acute limb ischemia in the context of a large-scale clinical trial studying a primary peripheral artery disease population. Dr Greg Hundley:             So Carolyn, reminded us what was the EUCLID trial. Dr Carolyn Lam:                Okay, so EUCLID stands for Examining Use of Ticagrelor in Peripheral Artery Disease, and this was a randomized clinical trial that included acute limb ischemia as an adjudicated outcome in a primary peripheral artery disease population randomized to ticagrelor versus clopidogrel. Now in EUCLID ticagrelor was not superior to Clopidogrel for the prevention of cardiovascular events in patients with stable peripheral artery disease. However, a EUCLID subgroup analysis of patients with and without prior limb revascularization demonstrated significantly higher risk for acute limb ischemia hospitalization in patients with prior low extremity revascularization. Dr Greg Hundley:             So Carolyn, that's interesting. So, what did they find related in this study that focused on the acute limb ischemia? Dr Carolyn Lam:                Right. So, today's paper is from Dr Hess and colleagues at University of Colorado School of Medicine and CPC, clinical research in Aurora, Colorado. And, they found that acute limb ischemia occurred in 1.7% of almost 13,900 randomized patients with a median time to hospitalization for acute limb Ischemia of 320 days after randomization. In this population, prior lower extremity revascularization, atrial fibrillation and lower ankle brachial index identified patients at higher risk for acute limb ischemia. Hospitalization for acute limb ischemia was associated with subsequent cardiovascular and limb ischemic events. So, the take home message is providers should monitor for signs and symptoms of acute limb ischemia in patients with stable symptomatic peripheral artery disease, particularly those with prior lower extremity revascularization, atrial fibrillation, and lower ankle brachial index. Dr Greg Hundley:             That's very instructive, Carolyn. Fantastic message. So, I'm going to ask you if you could select one lipid biomarker to forecast future adverse cardiovascular events, which would you select? Total cholesterol, HTLC, non-HTLC, direct and calculated LDLC, APO-A1, or APO-B? Dr Carolyn Lam:                Well, I'm traditional. I would have chosen LDL. Dr Greg Hundley:             Okay. Well, the authors of this study led by Dr Paul Welsh at the University of Glasgow attempted to answer this question by studying participants from the UK Biobank without baseline cardiovascular disease and not taking statins with relevant lipid measurements. They had 346,686 participants. An incident fatal or nonfatal cardiovascular event occurred in 6,200 participants of which 1,656 were fatal, and they occurred over a median time of 8.9 years. So, the associations of non-fasting lipid measurements, total cholesterol, HDLC, non HDLC, direct and calculated LDLC, APO-a1, and APO-B with cardiovascular disease were compared using Cox models, adjusting for classical risk factors and predictive utility was determined by the C-index and net reclassification index. Also, prediction was tested in 68,649 participants taking a statin with or without baseline cardiovascular disease, and that group experienced 3,515 cardiovascular events. Dr Carolyn Lam:                Okay, so drum roll. What did they find? Dr Greg Hundley:             So, measurement of total cholesterol and HDLC in the non-fasted state is sufficient or was sufficient to capture the lipid associated risk in the cardiovascular disease prediction with no meaningful improvement from addition of APO lipoproteins, direct or calculated LDLC. And, similar findings were reproduced in those taking a statin at baseline.                                                 As such, the authors feel like calls for widespread use of APO lipoproteins are not warranted given the negligible difference in risk prediction beyond total cholesterol in HDLC. And, direct LDLC is also not required for risk prediction. Non HDLC is a cheaper or equivalent predictor of risk on and off statins without the requirement of one of us being fasting. This is an excellent article for our listeners to review or download. Dr Carolyn Lam:                Wow, that is so cool. So, from one excellent paper to another excellent paper in our feature discussion. Let's go, shall we? Dr Greg Hundley:             Welcome everyone to discussion of our featured article. We have Dr Kelley Branch from the University of Washington and our own Carolyn Lam, and they're going to be discussing the compass trial. So Kelley, could you tell us a little bit about the rationale for compass as opposed to the previously published commander study? Dr Kelley Branch:              So, in order to understand compass and compare it to commanders, we're going to have to go back a little bit in time here. And recall, you know well over 20 years ago that when we used anticoagulants in coronary artery disease, that was actually shown to be more beneficial than aspirin alone, but because of the excess bleeding risk, warfarin or vitamin K antagonists not used, and aspirin won. Fast forward a number of years, and now we have the non-vitamin K anticoagulants, and the was potentially that we could find the goldilocks, if you will, the good balance of benefit as well as less bleeding maybe used to these new agents. So, the compass trial was really born from an atlas ACS one and Atlas ACS two, which found that a low dose of, in this case, Rivaroxaban 2.5 milligrams VAB as well as five milligrams VAB were shown to be beneficial in patients after acute coronary syndrome.                                                 And then, it was thought what happens if we treat these patients with now chronic coronary disease as well as arterial disease? And from this 27,000 patients, 47,395 patients were tested, and our study very specifically looked at patients with a baseline or a history of heart failure when they answered compass. Compass were shown to be beneficial with specifically the use of aspirin plus Rivaroxaban, 2.5 milligrams BAD. And, our idea was to test this in patients with this baseline or history of heart failure. Now, this is in real contradistinction to what the commander tried to do. And the reason why encompass, we actually excluded patients with severe heart failure. This was defined as a New York Heart Association class three or four or an ejection fraction less than 30%. Now if you looked at patients with commander, these patients had ejection fraction less than 40%. That was a criteria to get in. And of course, these patients had to have a recent hospitalization for heart failure. So, these are very different patient populations. Well, both of them, yes, they did have coronary artery disease, but really very different patient populations. Dr Greg Hundley:             Very good. So Kelley, tell us specifically, what were your treatment group assignments and the doses and the outcomes that you were going to follow, and then lead us into what did you find? What were the outcomes of your study? Dr Kelley Branch:              Sure, so compass was actually developed as a partial three by two factorial. The arm that we're going to be talking about is the rivaroxaban arm. There was also another arm that tested the use of Proton pump inhibitors, and that actually was shown to not be as beneficial as we thought to decreased bleeding. But specifically for rivaroxaban, the baseline was aspirin, and this was on top of guideline based medical therapy. And then patients were randomized to either aspirin alone plus placebo or Rivaroxaban, five milligrams BAD, plus placebo. So, no aspirin at all or aspirin, a hundred milligrams daily, plus Rivaroxaban, 2.5 milligrams BAD. Those were really the three treatments. Patients were going to be followed for about three to four years. That's what we expected to get our 2200 events , an event-driven trial. But, because of the overwhelming benefits at 23 months median follow up, this trial was actually stopped early, so we only had a little over 1300 events at that time.                                                 And with that we saw substantial reduction in major adverse cardiovascular events, about 24% mortality was reduced 18%, and there was a bleeding risk along with this, major bleeding, little different way of actually measuring major bleeding, but that was increased by about 70%, and that was the overall trial results. So, looking at the patients with heart failure, though, there was actually a relatively large proportion of patients, so 5,902 patients, about 22% of patients, actually had either baseline heart failure or had a history of heart failure coming in. Now, this was defined specifically by the PI's. These were not rigorously defined as compared to say commander, but these were patients where the PI said this patient has history or has chronic heart failure. So, with these 5,902 patients, we looked specifically at the outcomes of major adverse cardiovascular events similar to what we saw with compass and that is cardiovascular death, myocardial infarction, or any stroke, that combination. And then, looked at some others exploratory analysis like mortality.                                                 And, what we found is that in patients with heart failure, the baseline rate was substantially higher for a mate's. Not too surprising because this tends to be a higher risk patient population. But, what we found is that the hazard ratio was about 0.68, so pretty similar to what we've seen the 24% relative risk. In this case, this was a 32% relative risk reduction in those patients with heart failure. Now, if we looked at a patients without heart failure, the hazard ratio is 0.79, so fairly similar and the [conference intervals 00:16:33] overlap. No statistical heterogeneity or no difference between those, but what we did see if we looked at the absolute risk reduction, was an absolute risk reduction in heart failure of 2.4% reduction. That means a number needed to treat of about 42. If you look at the absolute risk reduction for those patients without heart failure, that was 0.9 to 1.0 depending on what the rounding was. We took 1.0 so that means the number needed to treat of 103. So, these were slightly different relative risks, but overall, what we saw is that the hazard ratio is very consistent with the overall effect of compass in the same direction.                                                 Interestingly, and actually I think even for me it was surprisingly, we actually looked at the hazard ratios for bleeding, and when we looked at the hazard ratios for bleeding, we fully expected that because it's the higher risk patient population, we actually expected that to go up. What we saw is that the bleeding actually was no difference at all, and if anything in the heart failure population was slightly lower. And, this was fairly surprising to us because we thought that the patients with heart failure, the bleeding would actually trend up because this was a higher risk patient population. So it looks like it's something can be used and really no substantial increase in bleeding. Dr Greg Hundley:             Very good. Well Carolyn, as someone that's managing patients with heart failure, what do you see are the clinical implications of this study? Dr Carolyn Lam:                That is a beautifully simple, direct question but is not as easy to answer as I may have thought. And, that's because the commander trial that Kelley did describe a bit earlier was neutral on its primary outcome. And, the commander trial is what we would traditionally think of as a heart failure trial. And why? Because those were patients that we rigorously define heart failure, including a naturally acid peptide inclusion criteria. And, because we really wanted these to be severe heart failure patients, we recruited them very close to their hospitalization or decompensation event. So, I just want to reiterate what Kelley has already so beautifully described that commander was neutral, whereas this heart failure subset of compass showed very impressive results that were consistent with the very impressive positive results of the overall compass trial.                                                 So, how do we reconcile all of it? Well, first of all, I have to humbly remind myself that this heart failure subset of compass, the entire subset was actually bigger in numbers than the entire of the commander trials. So, this is not a small little subgroup analysis. This is a huge subgroup analysis. And that's why a paper like this, we're so proud to be publishing in circulation.                                                 So, how do I apply it? Well, when I have a compass like patient, which means it's a stable coronary artery disease or peripheral artery disease patient who happens to have some mild heart failure. I think of this patient as a compass patient and I think that the combination of aspirin and low dose Rivaroxaban has been shown to be effective in these patients. So, in such a patient, I continue the aspirin rivaroxaban combination. However, if I have a new patient coming in with decompensated heart failure, a very low ejection fraction and has some coronary artery disease, by the way, I see that as a commander patient, and I just want to make sure that in such a patient I'm not trying to reduce their overall mortality by treating them with a combination of aspirin Rivaroxaban because commander has shown that I don't impact their overall survival with this combination, even though we may still have beneficial effects on their thromboembolic thrombotic events.                                                 Kelley, would you agree? Dr Kelley Branch:              I would completely agree. That was actually born out very, very well by Barry Greenberg who had a really a wonderful sub analysis which he looked at the thrombotic events published in Jama cardiology and really showing that yes, you can affect the thrombotic events, but I mean really what it comes down to is we want to save lives. We want people to be better. There's just an overwhelming risk for these patients with heart failure that is really non thrombotic, primarily. And so, you're really not going to move the needle very much. You may prevent a stroke here, you may prevent some cardiovascular death from a thrombotic problem, but overwhelmingly pump failure, arrhythmia, et cetera. Those are really going to be the drivers for the commander like population. Dr Carolyn Lam:                But Kelley, this comes up a lot when we've chatted, but if you have a compass patient who has heart failure and then gets admitted with heart failure, what would you do then? Dr Kelley Branch:              That's a really interesting question, right? It depends on what the overall goal is. So, if the patient gets admitted for heart failure, now has it decreased ejection fraction sick. So has an MI, now decreased the ejection fraction. What's the end game? Right? Well you know, you may not be affecting mortality in this case because there's now competing events. However, if the goal was to decrease stroke, we've seen that. Still this goal is to decrease MI to some extent than we see that also. So, it would be reasonable to continue in order to prevent those events. But, just knowing full well that there's many other medications which actually do much better for the patients with decreased ejection fraction. And, those would probably be considered first line, but it's reasonable to continue. But, I would never start it. Dr Carolyn Lam:                Kelley, I couldn't agree more. And here I think the, your data showing that the bleeding risk is not significantly increased in this patient matters a lot. So, if I had a patient, a compass patient who was already on the combination and then gets admitted with heart failure, I too, if there's no additional bleeding risk, I would continue the combination as well. Dr Kelley Branch:              Couldn't agree more. Dr Greg Hundley:             Well listeners, this was a fantastic discussion, and we look forward to seeing you next week. Have a great week. Dr Carolyn Lam:                This program is copyright American Heart Association 2019.  

Mon, 1 Feb 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19111/ https://edoc.ub.uni-muenchen.de/19111/1/Hiekel_Anian.pdf Hiekel, Anian

Laurie H. Glimcher, M.D., Weill Cornell Medical College, New York, New York

Laurie H. Glimcher, M.D., Weill Cornell Medical College, New York, New Yor

Genome-wide association studies identified a PTGER4 expression-modulating region on chromosome 5p13.1 as Crohn's disease (CD) susceptibility region. The study aim was to test this association in a large cohort of patients with inflammatory bowel disease (IBD) and to elucidate genotypic and phenotypic interactions with other IBD genes. A total of 7073 patients and controls were genotyped: 844 CD and 471 patients with ulcerative colitis and 1488 controls were analyzed for the single nucleotide polymorphisms (SNPs) rs4495224 and rs7720838 on chromosome 5p13.1. The study included two replication cohorts of North American (CD: n = 684; controls: n = 1440) and of German origin (CD: n = 1098; controls: n = 1048). Genotype-phenotype, epistasis and transcription factor binding analyses were performed. In the discovery cohort, an association of rs4495224 (p = 4.10×10⁻⁵; 0.76 [0.67-0.87]) and of rs7720838 (p = 6.91×10⁻⁴; 0.81 [0.71-0.91]) with susceptibility to CD was demonstrated. These associations were confirmed in both replication cohorts. In silico analysis predicted rs4495224 and rs7720838 as essential parts of binding sites for the transcription factors NF-κB and XBP1 with higher binding scores for carriers of the CD risk alleles, providing an explanation of how these SNPs might contribute to increased PTGER4 expression. There was no association of the PTGER4 SNPs with IBD phenotypes. Epistasis detected between 5p13.1 and ATG16L1 for CD susceptibility in the discovery cohort (p = 5.99×10⁻⁷ for rs7720838 and rs2241880) could not be replicated in both replication cohorts arguing against a major role of this gene-gene interaction in the susceptibility to CD. We confirmed 5p13.1 as a major CD susceptibility locus and demonstrate by in silico analysis rs4495224 and rs7720838 as part of binding sites for NF-κB and XBP1. Further functional studies are necessary to confirm the results of our in silico analysis and to analyze if changes in PTGER4 expression modulate CD susceptibility.

Mutations in the gene coding for the ATP binding cassette protein A3 (ABCA3) are known as the most frequent genetic cause of fatal neonatal respiratory distress syndrome and chronic interstitial lung disease (ILD) of children. ABCA3 transporter is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells. It transports surfactant phospholipids into lamellar bodies and is essential for their biogenesis. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). This study showed previously not examined cellular dysfunction in cultured lung epithelial A549 cells overexpressing the three ABCA3 mutations R43L, R280C and L101P. All three mutations were found in children with ABCA3-associated lung disease either with fatal neonatal respiratory distress syndrome (L101P and R43L) or chronic pediatric ILD (R280C). Cell biology of R43L and R280C mutations was studied here for the first time. L101P mutation was used as a known example of the trafficking/folding defect leading to the ER retention of ABCA3 protein. Human lung epithelial A549 cells were transfected with vectors containing wild type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress- and apoptosis-proteins, Annexin V surface staining and determination of the intracellular glutathion level. Induction of epithelial-mesenchymal transition (EMT) was assessed by immunoblotting. It was demonstrated that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptosis in A549 cells. A549 cells expressing L101P additionally gain a mesenchymal phenotype. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. These data suggest that expression of partially or completely ER localized ABCA3 mutant proteins induce raised intracellular stress and apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD via a fatal ER-stress/apoptosis/fibrogenesis-axis.

Background: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. Results: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e. g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1. The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. Conclusions: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Background: ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). Methods: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. Results: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. Conclusion: Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.

Increased levels of reactive oxygen species (ROS) contribute to vascular diseases like pulmonary hypertension and atherosclerosis. Although a NOX2-containing NADPH oxidase similar to the neutrophil one has been described to be active in endothelial cells, the contribution of newly discovered NOX homologues (NOX1-NOX5) was still unclear. Therefore, the overall aim of this study was to better characterize the expression, regulation and function of NOX homologues in different endothelial cell models. First, we could demonstrate the presence of NOX1, NOX2, NOX4, NOX5 including NOX5S as well as p22phox mRNA and protein levels in Ea.Hy926 or HMEC-1 cells. Furthermore, NOX5 protein was also present in endothelial and smooth muscle cells in the vascular wall of spleen and lung tissue. We found that NOX2, NOX4 and NOX5 were present in an intracellular perinuclear compartment, whereby NOX2 and NOX4 could be localized simultaneously in one cell. NOX2, NOX4, NOX5 were able to interact with p22phox and overexpression of NOX2, NOX4 and NOX5 increased ROS generation, although NOX5-dependent ROS generation did not require the presence of p22phox. NOX2, NOX4 and NOX5 also increased endothelial proliferation while depletion of NOX2, NOX4 and NOX5 decreased ROS generation, proliferation and tube forming ability indicating angiogenic activity under basal conditions. NOX2- and NOX4-induced proliferation was mediated by p38 MAP kinase. Although NOX1 expression as well as the expression of its regulatory subunits NOXO1 and NOXA1 was detectable in endothelial cells, depletion of NOX1 did not significantly affect basal ROS generation or proliferation of endothelial cells. Second, we could demonstrate the upregulation of NOX2, NOX5 and NOX5S after thrombin stimulation in endothelial cells and the modulation of p22phox expression in an ATF4- and XBP1-dependent manner under ER-stress conditions. Cellular stress either by thrombin or UPR also induced ROS generation of endothelial cells. In addition, thrombin induced proliferation and enhanced the tube forming ability of endothelial cells. Thrombin-induced ROS generation, proliferation and tube forming ability were diminished by silencing NOX2 or NOX5, whereas UPR induced ROS generation was inhibited by silencing p22phox as well as by silencing ATF4 or XBP1. In summary, this work provides evidence that in endothelial cells, NOX2, NOX4 and NOX5, but not NOX1, contribute to basal ROS generation, proliferation and angiogenesis and that the NOX proteins NOX2 and NOX5 as well as p22phox play an important role in the response to thrombin and ER-stress providing new insights in endothelial function and redox signaling.