POPULARITY
3 episodes with ccr2
2 episodes with ccr2
2 episodes with ccr2
2 episodes with ccr2
2 episodes with ccr2
2 episodes with ccr2
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550077v1?rss=1 Authors: Santos, F. M., Melo, V. C., Araujo, S., Sousa, C. D. F., Moreira, T. P., Goncalves, M. R., Santos, A. C., Seabra, H. A., Costa, P. A. C., Barrioni, B. R., Souza, P. B., Pereira, M. d. M., Nogueira, M. L., Souza, D. G., Guimaraes, P. P., Texeira, M. M., Queiroz-Junior, C. M., Costa, V. V. Abstract: Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, WT C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF and CCL2. Higher levels of CCL2 at the local and systemic levels, was followed by significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying MAYV's bone pathology in which: i) MAYV infection promoted a pro-osteoclastogenic microenvironment mediated by IL-6, TNF and CCL2 and ii) migration of osteoclast precursors was dependent on the CCR2/CCL2 axis. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and to the identification future of specific therapeutic targets in MAYV-induced disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535167v1?rss=1 Authors: Ye, Q., Jo, J., Wang, C.-Y., Oh, H., Choy, T., Kim, K., D'Alessandro, A., Reshetnyak, Y. K., Jung, S. Y., Chen, Z., Marrelli, S. P., Lee, H. K. Abstract: Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests pH homeostasis is a new cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption and reactive gliosis, which were both rescued by pharmacological or genetic inhibition of the NO-CCL2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-NO-CCL2 axis as a pivotal mechanism controlling BBB integrity and repair, while providing insights for a novel therapeutic approach against BBB-related CNS disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
“Novel role of chemokines in the ovulatory cascade and their potential application for nonhormonal contraceptive development” The Anne Klibanski Visiting Lecture Series was created to support and advance the careers of women. These lectures offer the opportunity for women faculty from outside institutions that have hosted Anne Klibanski Scholars to present on their expertise, either alone or in tandem with an Anne Klibanski Scholar. Presenter: Marina Cinthia Peluffo, PhD, Independent Scientist, CONICET, División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina Learning Objectives: Upon completion of this activity, participants were able to: Describe the periovulatory events and the increasing evidence for the role of chemokines in regulating these crucial events in different species Assess our laboratory evidence regarding the chemokine receptor CCR2 and its chemokine ligands in the ovarian follicles and cumulus-oocyte complex (COC) in the rhesus monkeys and domestic cats Analyze the direct effect of triggering or inhibiting the CCR2 receptor within the COC using a feline model in vitro Evaluate the interaction between the CCR2 and epidermal growth factor receptor (EGFR, a crucial intermediate in the ovulatory cascade) systems within the feline COC Review and discuss the overall course of this research Click here to watch webinar.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526724v1?rss=1 Authors: HE, Y., Heng, Y., Qin, Z., Wei, X., Wu, Z., Qu, J. Abstract: Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. However, due to technical limitations, it remains unclear how MuSCs dynamically interact with non-myogenic cells, especially myeloid cells, in live animals. In this work, we developed a dual-laser multimodal nonlinear optical microscope platform to serve as an effective tool for studying the real-time interaction between MuSCs and non-myogenic cells during the early phase of muscle regeneration. Increased cell volume and mitochondrial mass, cell density, and myotube formation are indicative of MuSCs activation/growth, proliferation, and differentiation, respectively. Using 3D time-lapse imaging on live reporter mice containing both red fluorescence protein (RFP)-labeled macrophages and yellow fluorescence protein (YFP)-labeled MuSCs, and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we monitored the real-time spatiotemporal interaction between RFP+ macrophages/RFP- non-myogenic cells and YFP+ muscle stem/progenitor cells during the activation and the proliferation stages of regeneration. Our results indicated that their cell-cell contact was transient in nature. By inhibiting macrophage infiltration, we further showed that direct cell-cell contact between macrophages and MuSCs was not required for early activation of MuSCs before the proliferation stage. However, decreased macrophage infiltration impeded the proliferation and differentiation of MuSCs and also led to intramuscular fibrosis. Besides, neutrophil depletion in the CCR2 deficient mice did not delay the initial growth of MuSCs. These findings provide a new perspective on the role of myeloid cells during muscle regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
This week, please join author Kory Lavine and Associate Editor Thomas Eschenhagen as they discuss the article "Donor Macrophages Modulate Rejection After Heart Transplantation." 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: I'm Dr. Greg Hundley, associate editor and director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn, this week's feature, we are going to the world of preclinical science and we are going to learn about a very important new finding pertaining to heart transplant rejection, and macrophages may modulate this, but before we get to that feature, how about we grab a cup of coffee and go through some of the other articles in the issue? Dr. Carolyn Lam: I got mine. Would you like to go first, Greg? Dr. Greg Hundley: You bet, Carolyn. Well, my first study comes to us from Dr. Michael Pencino from Duke University. Carolyn, this study was performed to understand the predictive utility of a previously derived polygenic risk score for long-term risk of coronary heart disease and its additive value beyond traditional risk factors and how that might be able to inform prevention strategies. To accomplish this, data from adults aged 20 to 59 free of cardiovascular health disease from the Framingham Offspring Study and the Atherosclerosis Risk in Communities, or ARIC Study, were analyzed. Now, since the polygenic risk score was derived from people of predominantly European ancestry, individuals who self-reported white race were those that were included. Dr. Carolyn Lam: Oh, interesting, so what did they find, Greg? Dr. Greg Hundley: Right, Carolyn. Somewhat surprisingly, they found that, among 9,757 participants, both the traditional risk factor score and the polygenic risk score where significantly associated with incident cardiovascular heart disease in young, early midlife, and late midlife. Now, the delta C index, when the polygenic risk score was added to the traditional risk factor, score was 0.03, 0.02, and 0.002 in the young, the early midlife, and the late-midlife participants, respectively. Carolyn, despite a statistically significant association between the polygenic risk score and the 30-year risk of cardiovascular heart disease, the C index improved only marginally with the addition of the polygenic risk score to the traditional risk factor model among young adults and did not improve among midlife adults and, thus, Carolyn, the polygenic risk score, an immutable factor, has limited clinical utility for long-term cardiovascular heart disease prediction when added to a traditional risk factor model. Dr. Carolyn Lam: I really like that, Greg, because I think it also tells us that the traditional risk factors, which we can do something about, are still very important. Isn't that great? Well, the next paper is about POTS. Remember what that is? Should I give you a quiz? All right. It's okay. POTS, or Postural Orthostatic Tachycardia Syndrome, is a disorder of orthostatic intolerance that primarily affects females of childbearing age. While the underlying pathophysiology of POTS is not fully understood, it has been suggested that autoimmunity may play a role. Now, the aim of this study was to compare concentrations of autoantibodies to cardiovascular G protein-coupled receptors between 116 POTS patients and 81 healthy controls, and they were from Calgary, Canada, and Malmo, Sweden. Dr. Greg Hundley: Carolyn, really interesting, so what did they find here? Dr. Carolyn Lam: The investigators, led by Dr. Raj from University of Calgary in Canada, found that commercially available autoantibody concentrations to G protein-coupled receptors were not increased or altered in POTS patients relative to healthy controls as assessed using ELISA. Now, while this study suggests that these G protein-coupled receptor autoantibody concentrations alone cannot explain the pathophysiology of POTS, autoantibody activity and signals not picked up by ELISA should still be explored as these results may provide more insights into the pathophysiology of POTS. Dr. Greg Hundley: Very nice, Carolyn. Well, my next study comes to us from the world of pulmonary arterial hypertension. Carolyn, clinical worsening is commonly used as an endpoint in pulmonary arterial hypertension trials. These authors, led by Dr. Steeve Provencher from the Institut Universitaire de Cardiologie Pneumologie de Quebec, aimed to assess the trial-level surrogacy of clinical worsening for mortality in pulmonary artery hypertension trials and whether the various clinical worsening components were similar in terms of frequency of occurrence, treatment-related relative risk reduction and importance to patients. Dr. Carolyn Lam: Okay, so what did they find? Dr. Greg Hundley: Right, Carolyn, so they searched MEDLINE, Embase and the Cochrane Library for trials evaluating the effects of pulmonary arterial hypertension on clinical worsening and, among 35 independent cohorts, so 9,450 patients, the effects of pulmonary arterial hypertension-specific therapies on clinical worsening modestly correlated with mortality. Additionally, study-level clinical worsening was not found to be a surrogate for mortality in pulmonary arterial-hypertension trials. Moreover, components of clinical worsening largely vary in frequency, response to therapy and importance to patients and, thus, are not necessarily interchangeable. Dr. Carolyn Lam: Thank you, Greg. Can I tell you about some other papers in today's issue? There's a Research Letter from Dr. Cosentino on cardiorenal outcomes with ertugliflozin by baseline metformin use, and this is a post hoc analysis of the VERTIS CV trial. Dr. Greg Hundley: Oh, very good, Carolyn. Well, I've got an exchange of letters from Professors Boriani and Steinberg regarding the article “Driving Restrictions and Early Arrhythmias in Patients Receiving a Secondary Prevention Implantable Cardioverter-Defibrillator, the DREAM-ICD-II Study.” There's also an ECG Challenge from Professor Gao entitled “Syncope in a 3-Year-Old Child During the Perioperative Period. What is the diagnosis? What Signs Point Toward Impending Life-threatening Event?” Then, finally, there's a nice, On My Mind piece from Professor Greenland entitled “Insurance Payers Should Cover Selective Coronary Artery Calcium Testing in Intermediate Risk Primary Prevention Patients.” Well, Carolyn, how about we get on to that feature discussion and dive into the world of rejection after heart transplantation? Dr. Carolyn Lam: Yay. Here we go. Dr. Greg Hundley: Welcome, listeners, to this feature discussion on August 23rd. We have a very interesting article today to discuss with our author and associate editor pertaining to preclinical science and cardiac transplant rejection. Our author today is Dr. Kory Lavine from Washington University in St. Louis and our associate editor today is Dr. Thomas Eschenhagen from Hamburg, Germany. Welcome gentlemen. Kory, we'll start with you. Can you describe for us some of the background information pertaining to the construct of your study and what was the hypothesis that you wanted to address? Dr. Kory Lavine: Well, thank you for having me. Our study focused on heart transplant rejection, which remains a major clinical challenge that limits both the survival of heart transplant recipients as well as availability of donor hearts. Current clinical practice really focuses on suppressing the immune system in a global way, and that is somewhat effective, but carries important risks that include infection and life-threatening malignancies. Many studies have appropriately focused on immune cells that infiltrate the transplanted heart that come from the recipient to search for new ways to suppress the immune system safely. What we've understood and learned over the past several years is that the donor heart has its own immune system and its own immune cells, and the majority of those immune cells that come with the donor heart are macrophages that can be broadly divided into two distinct lineages with different functions, tissue-resident macrophages, which lack the cell surface receptors CCR2, and monocyte-derived macrophages with expressed cell surface receptors CCR2. We tested the hypothesis in this study that these macrophages that come with the donor heart remain active for a period of time after transplantation and play important roles in either suppressing or accelerating heart transplant rejection. Dr. Greg Hundley: What was the hypothesis that you wanted to address with your study? Dr. Kory Lavine: Yeah, so our prior work and others' work within this field had suggested that tissue-resident macrophages, CCR2-negative macrophages, are inflammatory, and CCR2-positive macrophages have the opposite functions being inflammatory and play roles in potentiating and initiating inflammation in the heart. In this study, we hypothesized that CCR2-negative macrophages would protect from rejection, while CCR2-positive macrophages may promote heart transplant rejection and could serve as a new therapeutic target to prevent rejection in transplant recipients. Dr. Greg Hundley: Excellent. Kory, can you describe for us the study design that you used to test your hypothesis? Dr. Kory Lavine: Yeah. The study design and approach we used involved a mouse model of heart transplantation where we transplant a donor heart into a recipient mouse that's fully mismatched at all the MHC loci, and this serves as a nice model for both cellular and antibody-mediated rejection. To facilitate tracking these donor macrophages, we used various genetic lineage tracing systems and, to study their phenotypes, we used single-cell RNA sequencing and, to understand their function, we used mouse models that allow us to specifically deplete each of the donor macrophage populations as well as genetic models to manipulate their activation and signaling. Dr. Greg Hundley: The outcome measures were going to be what? Dr. Kory Lavine: Yeah. The outcome measures for transplant rejection in this mouse model are allograph survival, so the survival of the transplanted heart. We're able to directly look at how much rejection is present by histopathology, and then we're able to observe various mechanistic features using detailed phenotyping such as single-cell RNA sequencing and T-cell activation assays. Dr. Greg Hundley: Very nice, Kory. Well, all, our listeners, we're very excited to hear what were your study results? Dr. Kory Lavine: We learned that donor macrophages are dynamic and they survive for a period of time after transplantation or eventually lost due to transplant rejection. When we phenotyped the macrophages that came from the donor heart, we learned that they remained transcriptionally distinct from immune cells that enter the heart that were derived from the recipients, and they had important and distinct functions. If we depleted the tissue-resident macrophages that were CCR2-negative, we observed reduced allograph survival and increased rejection. If we depleted CCR2-positive macrophages that came from the donor heart, we observed improved allograph survival and reduced rejection. Mechanistically, we learned that CCR2-positive macrophages are activated through a MyD88-dependent pathway and, if we inhibited MyD88 cytokines which controls the expression of pro-inflammatory cytokines and chemokines, we could prolong the survival of the donor heart for a very significant period of time, reduce rejection and prevent the development of T-cells that would attack the donor heart. From a mechanistic aspect, what we uncovered is that this signaling pathway in CCR2-positive macrophages regulated the recruitment of an activation of antigen-presenting cells which played important roles in generating T-cells that would target the transplanted heart. Dr. Greg Hundley: It sounds like a really informative and leap forward in the whole sphere of transplant rejection. Well, listeners, now we're going to turn to our associate editor, Dr. Thomas Eschenhagen. Thomas, you have many papers come across your desk. What attracted you to this particular paper and then, secondly, how do you put the results of this study really in the context of other research examining heart transplant rejection? Dr. Thomas Eschenhagen: Yeah, thanks for having me. I mean, first, we got attracted by this paper because it's somewhat an out-of-the-box approach. It's not the standard approach to improve the systemic immunosuppression as many studies did and with actually a lot of success over the last 30 years, survivor got much better. There had been a lot of progress in the field of transplantation medicine as we all know, but as Kory said already, we still have 30% rejection, and these immunosuppressions come at a price. Having this study which turns around somehow the argumentation and looks at the donor organ was something which really attracted us. It uses advanced methods and it applies somewhat in a practical way a concept which emerged over the last, I don't know, maybe decade this concept that macrophages are really very different kind of cells. They're all called macrophages, but they're quite different and even maybe in certain respects having opposing effect. I think many people know about this M1/M2 concept. It's CCR2 receptor positive and negative. It's criticized by some people, but here we see that it really seems to be really important and, of course, then the third argument why we really like the story is that it has a specific, clear translation impact. I mean, looking at the heart, the donor heart, and potentially even treating the donor heart before transplanting it is something which comes immediately out of the story, and that's something which we found super attractive. Dr. Greg Hundley: Really interesting, so really understanding the mechanism and focusing on donor hearts. Well, listeners, let's circle back with Kory. Kory, given that, what do you think is the next study that really needs to be performed in this sphere of research? Dr. Kory Lavine: I think Thomas said it exactly as we're thinking about it, so the next area that we're really excited to attack and we're hopeful that the field will focus on is ways to build methods and technologies to treat the donor heart between the time of procurement and the time of transplant, when it's being transported and potentially even being perfused for a period of time. We're really interested in finding approaches to identify small molecules and other potential biologic therapies that could be used to prevent the activation of donor CCR2-positive macrophages. It's a really attractive approach because treating the donor heart ex vivo decreases the risk of adversely affecting other organs that may be transplanted if you're treating the donor, for instance, and it may decrease the risk of immunosuppression and infection by not having to treat the recipient and we're catching the heart in this window where the risks are much lower. The other area that we're really excited to focus on is trying to identify the exact mediators that are generated from donor CCR2-positive macrophages that mediate the recruitment and activation of antigen-presenting cells because that would represent another potential therapeutic target. Dr. Greg Hundley: Very nice. Thomas, what are your thoughts about what might be the next study to be performed really in this sphere of research? Dr. Thomas Eschenhagen: It's obviously something rather a question to Kory than to me, but I agree to what he said. I think it is pretty obvious what are the next steps mechanistically on the one hand, but practically on the other hand. I mean, at this point, we are at the mouse level, so the question is to which extent can this concept be translated into larger animals and then finally in humans? I was wondering, given these newer methods to keep donor hearts alive for long, extended periods, I was wondering which extent you are already collaborating with the respective groups who develop this approach because that obviously would increase the window of opportunity here for drugs. I think it's really an exciting and pretty visible next steps which we see here, and I can just hope that you're going this path and that it will be successful. Dr. Greg Hundley: Kory, any thoughts on those collaborations that Thomas just spoke of? Dr. Kory Lavine: We're definitely establishing collaborations to focus on ex vivo profusion of donor hearts because that's, as Thomas mentioned, is a perfect window to manipulate the immune populations that are within the donor heart. Those studies have to be team science, they have to be collaborative and they have to have a focus on large animals and then moving into clinic. We're definitely forming those collaborations and excited to work as a group. Dr. Greg Hundley: Very nice. Well, listeners, what an exciting paper to discuss here as part of this feature discussion from the world of preclinical science. We want to thank Dr. Kory Lavine from Washington University in St. Louis, Missouri, and also our own associate editor, Dr. Thomas Eschenhagen from Hamburg Germany, for really bringing us this research study highlighting that distinct populations of donor and recipient macrophages coexist within the transplanted heart, and donor CCR2-positive macrophages are key mediators of allograph rejection and deletion of MyD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograph survival. Well, on behalf of Carolyn and myself, we want to wish you a great week, and we will catch you next week on the run. Dr. Greg Hundley: This program is copyright of the American Heart association, 2022. The opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, please visit ahajournals.org.
In this MEGAphone episode we will be shouting out one of the most informative people to follow on social media that I know about, he specializes in power lifting and biomechanics and injury prevention and physical therapy. His Instagram handle is @SquatUniversity and his name is Dr. Aaron Horschig. As a physical therapist & strength coach, Aaron helps athletes move better, decrease their aches & pains associated with training, and find their true athletic potential.Dr. Horschig wrote an extremely informative and also controversial article that I love and want to share. This information addresses a well known injury protocol… A protocol that I used when I was an athlete in high school in college. A protocol that I used with clients and family members of mine when they got injured… A protocol that I believed in whole heartedly and taught every chance I could. A protocol my college courses taught me, straight out of the text books. It was simple… when you get hurt… the solution is RICE. Rest, Ice, Compression, Elevation. We all knew RICE. And it turns out… we were all wrong. References: Dr. Aaron Horschig IG: @SquatUniversitywww.squatuniversity.com Book: Rebuilding MiloArticle Citations:1. Mirkin G, Hoffman M. The Sports Medicine Book. 1978. Little Brown & Co2. Lu H, Huang D, Saederup N, et al. Macrophages recruited via CCR2 produce insulin-like growth factor-1 to repair acute skeletal muscle injury. FASEB J. 2011;25(1):358-69.3. Summan M, Warren GL, Mercer RR, Chapman R, et al. Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study. Am J Physiol Regul Integr Comp Physiol. 2006;290(6):R1488-954. Pelosi L, Giacinti C, Nardis C, Borsellino G, et al. (2007) Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J. 21, 1393–14025. Singh DP, Lonbani ZB, Woodruff MA, Parker JP, et al. Effects of topical icing on inflammation, angiogenesis, revascularization, and myofiber regeneration in skeletal muscle following contusion injury. Front Physiol. 2017;8:93.6. Takagi R., Fujita N., Arakawa T., Kawada S., Ishii N., Miki A. (2011). Influence of icing on muscle regeneration after crush injury to skeletal muscles in rats. J. Appl. Physiol. 110, 382–388.7. Tiidus PM. Alternative traetments for muscle injury: massage, cryotherapy, and hyperbaric oxygen. Current reviews in musculoskeletal medicine. 2015;8(2):162-78. Reinl G. Iced! The illusionary treatment option. 2nd Edition. Gary Reinl. 2014.9. Khoshnevis S, Kraik NK, Diller KR. Cold-induced vasoconstriction may persist long after cooling ends: an evaluation of multiple cryotherapy units. Knee Surg Sports Traumatol Arthrosc. 2015;23(9):2475-238310. Dirks ML, Wall BT, van Loon L CJ. Interventional strategies to combat muscle disuse atrophy in humans: focus on neuromuscular electoral stimulation and dietary protein. J Appl Physiol. 2018;125:850-86111. Raynor MC, Pietrobon R, Guller U, Higgins LD. Cryotherapy after ACL reconstruction: a meta-analysis. J Knee Surg. 2005;18(2):123-912. Spencer JD, Hayes KC, Alexander IJ. Knee joint effusion and quadriceps reflex inhibition in man. Arch Phys Rehabil. 1984;65:171-17713. Buckwalter JA, Grodzinsky AJ. Loading of healing bone, fibrous tissue, and muscle: implications for orthopaedic practice. J Am Acad Orthop Surg. 1999;7(5):291-9.14. Silveria EM, Rodrigues MF, Krause MS, et al. Acute exercise stimulates macrophage function: possible role of NF-kappaB pathways. Cell Biochem Funct. 2007;25(1):63-7315. Teixeira E, Duarte JA. Skeletal muscle loading changes its regenerative capacity. Sports Med.
This week, please join author Andrew Stokes as he and Greg Hundley discuss the Research Letter "E-Cigarette Use and Risk of Cardiovascular Disease: A Longitudinal Analysis of the PATH Study (2013–2019)." Dr. Greg Hundley: Well, listeners, welcome to this May 17th issue of Circulation on the Run. And I am Dr. Greg Hundley, associate editor, director of the poly heart center at VCU Health in Richmond, Virginia. And this week, Carolyn is away out on vacation and we are going to go through the summaries together. We have a great feature today on e-cigarette use and the risk of cardiovascular disease. But before we get to that, how about we grab a cup of coffee and jump into some of the other articles in the issue? And the first one comes to us from the world of clinical science and Dr. Jiaqi Huang from the National Cancer Institute. Listeners, the objective of this study was to examine overall and cause-specific mortality in relation to dietary and serum cholesterol, as well as egg consumption through the prospective analysis of 27,000 men in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention or ATBC Study, and also a systematic review and meta-analysis of several other cohort studies. Dr. Greg Hundley: So, what did the investigators find? Well, first, based on 482,000 person-years of follow-up, the authors identified 22,000 deaths, including 9,110 deaths from cardiovascular disease. Now, greater dietary cholesterol and egg consumption were associated with increased risk of overall and cardiovascular disease mortality. Now, second, from the meta-analysis component of the study, overall consumption of one additional 50-gram egg per day was associated with an increased cardiovascular disease risk with a pooled relative risk of 1.04 with a higher risk of cardiovascular disease among those from us cohorts where their pooled relative risk ratio was 1.88, a borderline higher cardiovascular disease risk in European cohorts with a pooled relative risk of 1.05, but not an increased cardiovascular disease risk in the Asian cohorts. So, the results from this study, which includes an updated meta-analysis, suggest that there is support for restricted consumption of dietary cholesterol as really a means to improve long-term health and longevity. Dr. Greg Hundley: Well, let's go to our next article. So, in this next study, we are going to move from cholesterol risk now to salt substitution. And this article comes to us from Professor Maoyi Tian from the Harbin Medical University. Listeners, the Salt Substitute and Stroke Study, or SSaSS is a five-year cluster randomized controlled trial and demonstrated that replacing regular salt with a reduced sodium added or potassium salt substitute reduced the risk of stroke, major cardiovascular events, and premature death among individuals with prior stroke or uncontrolled high blood pressure that lived in rural China. So, this particular study, a substudy, assessed the cost-effectiveness profile of this particular intervention. Dr. Greg Hundley: So, listeners, what did the study find? Well, there was a mean follow-up of 20,995 participants that was conducted a little over four years, and over the period, replacing regular salt with salt substitute reduced the risk of stroke by 14% and the salt substitute group had on average 0.054 more quality-adjusted life years per person. The average costs were lower in the salt substitute group, and this intervention was dominant. That is better outcomes at a lower cost for prevention of stroke as well as for quality-adjusted life-years gained. Now, interestingly sensitivity analyses showed that these conclusions were robust except when the price of the salt substitute was increased to the median and highest market prices identified in China. The salt substitute intervention had a 95% probability of being cost-saving and a greater than 99.9% probability of being cost-effective. A really interesting article. Dr. Greg Hundley: Well, now, let's turn our attention to the world of population science. And in this study, these authors led by Dr. Steven Lubitz from Massachusetts General Hospital performed a Genome-Wide Association Study or GWAS of the QT corrected interval among 84,630 United Kingdom Biobank participants. And they created a polygenic risk score. Now, among 26,976 participants with whole-genome sequencing and electrocardiogram data in the Trans-Omics for Precision Medicine or TOPMed program, they identified 160 carriers of punitive pathogenic, rare variants in 10 genes known to be associated with the QT interval. Dr. Greg Hundley: So, the authors here examined the QTC corrected associations with the polygenic risk score and with rare variants from the TOPMed cohort. So, what did they find? They found 54 independent loci by GWAS in the UK Biobank. 21 loci were novel of which 12 were replicated in TOPMed. The polygenic risk score comprising over a million common variants was significantly associated with the QTC in TOPMed, and carriers of punitive pathogenic rare variants had longer QTC intervals than non-carriers. Now, 23.7% of individuals with a QT corrected of greater than 480 milliseconds carried either a monogenic rare variant or had a polygenic risk score in the top decile. 3.4% for monogenic and 21% for the top decile of the polygenic risk score. Dr. Greg Hundley: So, listeners, the findings of this study indicate that the QTC duration in the population is influenced by both rare variants in genes, underlying cardiac repolarization and polygenic risk, with a sizeable additional contribution from polygenic risk. And therefore, comprehensive assessment of the genetic determinants of QTC prolongation should include incorporation of both polygenic and monogenic risk. Dr. Greg Hundley: Well, listeners, let's turn our attention to the world of preclinical science. And this next article comes to us from Professor Junbo Ge from the Department of Cardiology in Zhongshan Hospital in Fudan University. Well, listeners, after myocardial infarction, cardiac resident macrophages, which are self-maintaining in that they originate from embryonic hematopoiesis are responsible for the efficient clearance and degradation of apoptotic cardiomyocytes. And that process is called efferocytosis. Now, efferocytosis is required for inflammation resolution and tissue repair. However, the underlying molecular mechanisms of this process really remain unknown. Dr. Greg Hundley: So, as such, listeners, these authors sought to identify the mechanisms of the continued clearance and degradation of phagolysosomal cargo by cardiac resident macrophages during myocardial infarction. Well, what did Dr. Ge and colleagues find? Several things. First, they identified legumine as a gene specifically expressed by cardiac resident macrophages, and legumine deficiency resulted in a considerable exacerbation in cardiac function, accompanied with the accumulation of apoptotic cardiomyocytes and a reduced index of in-vivo efferocytosis in the border area of infarcts. Furthermore, the formation of LC3 to dependent phagosome around secondary encountered apoptotic cardiomyocytes was disabled. In addition, legumine deficiency increased infiltration of MHC to high CCR2+ macrophages, and the enhancement of recruitment of MHC to low CCR2+ monocytes with downregulation of anti-inflammatory mediators, such as IL10 and TGF-beta, and upregulation of pro-inflammatory mediators, including interleukin-1-beta, Tumor Necrosis Factor alpha, IL6, and IFN-gamma. Dr. Greg Hundley: So, listeners, in summary, the results of this study directly link efferocytosis to wound healing in the heart and identify legumine as a significant link between acute inflammation resolution and cardiac function after infarction. Well, listeners, also in this issue, we have a wonderful On My Mind feature from Professor Camlet entitled “A Role for the Vascular Endothelium in Post-Acute COVID-19.” Well, next, we're going to head to our feature article on e-cigarette use and the risk of cardiovascular disease. Dr. Greg Hundley: Well, listeners, welcome to our feature discussion today. A very interesting topic. E-cigarette use and the risk of cardiovascular disease. And we have with us today the senior author of this particular manuscript, Dr. Andrew Stokes from the Boston University School of Public Health in Boston, Massachusetts. Welcome, Andrew. Andrew, to get started, can you describe some of the background information pertaining to your study and what was the hypothesis that you wanted to address? Dr. Andrew Stokes: Absolutely, and thank you for having me on the podcast. Despite the increasing popularity of electronic cigarettes, the long-term health effects of habitual e-cigarette use remain unclear. Most of the studies that have been conducted to date are either cross-sectional or they pertain to small clinical samples. The goal of the present study was to develop a longitudinal design to see if e-cigarette use at a point in time was linked to cardiovascular events over a multi-year follow-up period. Dr. Greg Hundley: Very nice. So, your specific hypothesis really pertained to e-cigarette use, correct? Dr. Andrew Stokes: That's right. As a novel product, information on e-cigarette use and its health effects is lacking, and so our goal was to see if e-cigarette use was associated with the incidence of clinical events. Dr. Greg Hundley: And so, can you describe for us your study population and your study design? Dr. Andrew Stokes: Absolutely. Data come from the Population Assessment of Tobacco and Health Study or the PATH Study, which is a nationally representative cohort study of the non-institutionalized population containing five annual waves of self-reported data collected between 2013 and 2019. The initial sample included over 30,000 US adults ages 18 years and older with oversampling of tobacco users. We excluded respondents who were lost to follow-up or who had a previous diagnosis of CVD or were missing baseline exposure information. Ultimately, we ended up with a sample of just over 20,000 individuals. Dr. Greg Hundley: Very nice. And so, what were your study results? Dr. Andrew Stokes: So, we had several key findings. One key finding was that, compared to people who only smoke cigarettes, people who smoke both traditional cigarettes and used e-cigarettes had no significant reduction in risk for heart attack, heart failure, or stroke, nor any cardiovascular disease outcome. This is significant because many e-cigarette users use both e-cigarettes and cigarettes in combination. Very few move to exclusive e-cigarette use. Additionally, we found that those who do move to e-cigarette use exclusively though, representing a very small fraction of the cohort, had some evidence of reduction in cardiovascular harm. However, these results for exclusive e-cigarette users were not statistically significant, indicating that additional studies with longer follow-up will be required before we can make any definitive conclusions about this group. Dr. Greg Hundley: Very nice. And did you notice any discrepancy in your results between either men versus women or between individuals that were younger in age versus those that may say be 50 years or older? Dr. Andrew Stokes: I think both sources of effect modification will be valuable directions for future research. Unfortunately, samples of e-cigarette users are quite small and incident events over follow-up are quite limited. Therefore, the present study did not pursue or explore these types of stratifications. Dr. Greg Hundley: Very good. So, sounds like more research to come forward. Well, Andrew, how do we put your results really in the context with other studies evaluating the harmful effects of e-cigarettes? Dr. Andrew Stokes: Of course. So, we know from toxicological studies that there are many constituents of e-cigarette aerosols that are concerning and have substantial toxicity. We know that the inhalation of e-cigarette aerosols among young healthy adults induce inflammation and oxidative stress. Population-based studies from cross-sectional data sources also suggest evidence of harm. What's needed are more longitudinal studies with longer follow-up periods and more incidence events so we can really parse this risk and identify the magnitude of these harms. Finally, we also need to understand better whether there's any harm reduction potential associated with e-cigarette use. E-cigarettes are currently not an FDA-approved cessation product. Therefore, we do not recommend their use despite preliminary evidence of potential harm reduction. We'll need further evidence before we can make any such conclusions. Dr. Greg Hundley: And Andrew, describe for us, and you've started to already, what series of studies are needed next to be performed in this sphere of research? Dr. Andrew Stokes: Right. So, it's difficult to really identify definitively the effects of e-cigarette use in the absence of randomized control trials. However, we can use observational data with target trial approaches to emulate the clinical trial that we would like to do if we were able to. So, the next step is really to look at transitions across products between cigarette and e-cigarette use and to associate those who switch products, such as from e-cigarettes to cigarettes or vice versa, to see if those switches are associated with any harm or harm reduction. Dr. Greg Hundley: Very good. Any specific racial or ethnic groups or even social determinants of health that may need to be targeted with some of these future studies? Dr. Andrew Stokes: That's a great question. So, what we know so far from preliminary research is that some groups are more likely to switch to e-cigarettes than other groups. Particularly among current combustible cigarette users, the rates of switching do vary by race and ethnicity. Thus, we need further research to understand why these patterns differ across subgroups and what their implications may be for health. Dr. Greg Hundley: Do you foresee any difficulty in trying to enroll participants from those other groups as you plan these studies moving forward? Dr. Andrew Stokes: The advantage of the current research design is that we're using a large secondary data set of survey participants who are enrolled in the Population Assessment of Tobacco and Health study. Therefore, we are not enrolling patients ourselves and the response rates are quite high in these surveys. Dr. Greg Hundley: Well, Andrew, we hear that some of the inhalants that are mixed with the inhaled nicotine can be flavors and perhaps have been approved by the FDA for consumption in the GI tract where, whatever these additives are, you would think might be broken down by the digestive system. But if they're inhaled and get into the lung tissue and the parenchyma, the alveoli, et cetera, do they perhaps have harmful effects that maybe we're not aware of? Dr. Andrew Stokes: Absolutely. E-cigarettes come in thousands of characterizing flavors including sweet flavors, tobacco flavors, and many other miscellaneous flavors. As we saw with the outbreak of lung injury associated with the use of e-cigarettes in 2019, inhaling flavors can have health effects that are unanticipated based on research in the GI tract, and therefore, as a next step in this research, we really need more work to investigate how different flavors are associated with the incidence of clinical events, whether cardiovascular or pulmonary conditions. Dr. Greg Hundley: Very nice. Well, listeners, we want to thank Dr. Andrew Stokes from the Boston University School of Health for bringing us this data from the PATH study, suggesting that combining smoking with e-cigarette use does not reduce cardiovascular events and that quitting both products is needed to ensure overall cardiovascular disease risk reduction. Dr. Greg Hundley: Well, on behalf of Carolyn and myself, we want to wish you a great week and we will catch you next week on the run. This program is copyright of the American Heart Association 2022. The 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, please visit ahajournals.org.
This week, join author Marco Valgimigli and Associate Editor Mark Link as they discuss the original research article "Amulet or Watchman Device for Percutaneous Left Atrial Appendage Closure: Primary Results of the SWISS-APERO Randomized Clinical Trial." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage fast as a journal and editors. We're your co-host, I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: I'm Dr. Greg Hundley, Associate Editor, Director of Poly Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, I've got a personal interest in this feature paper that's coming up. I've always been very intrigued with the left atrial appendant closure. Well guess what? This is the results of the Swiss-Apero Randomized Clinical Trial, comparing the Amulet with the Watchman device for percutaneous left atrial appendage closure, really interesting stuff coming right up, but let's hold everyone in suspense. As we go through some of the other favorite papers in today's issue. Would you like to go first? Dr. Greg Hundley: You bet Carolyn, this first paper really pertains to driving restrictions and earlier arrhythmia is in patients receiving a secondary prevention, implantable cardioverter defibrillator, and it comes to us from Dr. Christian Steinberg. So Carolyn, regulatory authorities of most industrialized countries recommend six months of private driving restriction after implantation of a secondary prevention ICD and these driving restrictions result in significant inconvenience in social implications. And so Carolyn, the purpose of this study was to assess the instance rate of appropriate device therapies in contemporary recipients of a secondary prevention ICD using a retrospective across three Canadian tertiary care centers enrolling 721 consecutive patients with new secondary prevention ICD implants between the years of 2016 and 2020. And they were followed for a median of 760 days. Dr. Carolyn Lam: Nice. An important question. So what did they find Greg? Dr. Greg Hundley: Right Carolyn. So they found that the cumulative incidents of arrhythmic syncope resulting in sudden cardiac incapacitation was 1.8% within the first 90 days, and subsequently dropped to 0.4% between 91 and 180 days after ICD insertion. So Carolyn the incidence rate of appropriate therapies resulting in sudden cardiac incapacitation in contemporary recipients of a secondary prevention ICD is much lower than previously reported and significantly declines after the first three months, lowering driving restrictions to months after the index cardiac event seems safe and revision of the existing guidelines should be considered in countries still adhering to a six month period. Dr. Carolyn Lam: Oh, love it Greg. Elegant study with clinically impactful results as with the next paper, I'm going to talk about a post talk analysis of the Danish trial in which authors led by Dr. Boas from Denmark and Dr. Bower from Austria and their colleagues. And what they did is they tested whether periodic repolarization dynamics or P R D, which is a marker of repolarization instability associated with increased sympathetic activity. Could indeed identify patients with non-ischemic cardiomyopathy that may benefit from prophylactic ICD implantation. So, 748 patients were included in this P R D sub-study. And they were included if they had a 24 hour whole term monitor recording at baseline with technically acceptable ECG signals during the night hours. P R D as a reminder of periodic repolarization dynamics was assessed using wavelet analysis according to previously validated models. Dr. Greg Hundley: Very interesting, Carolyn. So what did they find? Dr. Carolyn Lam: Periodic repolarization dynamics was independently associated with mortality. More over P R D was significantly associated with mortality in the control group, but not in the ICD group in this Danish trial. There was a significant interaction between P R D and effect of ICD implantation on mortality, such that patients with higher P R D had greater benefit in terms of mortality reduction with the ICD. Based on P R D the investigators could identify a new group of patients where prophylactic ICD implantation was associated with a significant absolute mortality reduction of 17.5% after eight years corresponding to a number needed to treat of only six. So this is the first sub-study of Danish to identify a marker on top of age, that can predict the treatment effect of prophylactic ICD implantation in patients with nonischemic cardiomyopathy. Dr. Greg Hundley: Very nice Carolyn. Well, my next paper comes to us from Dr. Kory Levine from the Washington University School of Medicine. And Carolyn recent studies have established that cc chemokine receptor type two are CCR2 marks the pro inflammatory subsets of monocytes, macrophages and dendritic cells that contribute to adverse left ventricular remodeling and heart failure progression. Now elucidation of the effector mechanisms that mediate adverse effects of CCR2 plus monocytes, macrophages and dendritic cells could yield important insights into therapeutic strategies to suppress myocardial inflammation. Dr. Carolyn Lam: Hmm, indeed. And so what did these author determine regarding the suppression of myocardial inflammation? Dr. Greg Hundley: So Carolyn this team utilized mouse models of re perfused myocardial infarction, and angiotensin two and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate cc chemokine ligand 17. They found that cc chemokine ligand 17 serves as a pro-inflammatory mediator of CCR2 macrophages and dendritic cells, and their results suggest that inhibition of cc chemokine ligand 17 may serve as an effective strategy to promote T-cell regulation recruitment, and thereby suppress myocardial inflammation. Dr. Carolyn Lam: Wow, thanks, Greg. That was beautifully summarized great stuff. Well, there are other exciting articles in today's issue. There's an exchange of letters between Doctors Nagareddy and Spear on “Interleukin One Alpha as a Central Regulator of Leukocyte Endothelial Adhesion in Myocardial Infarction and Chronic Kidney Disease. There's an ECG challenge by Dr. Yang entitled, “A Fatal Case of Y QRS tachycardia following Sintilimab treatment for lung cancer. It can happen.” There's an On My Mind paper by Dr. Faed on “CYP2C19 Genotyping in Anticoagulated Patients Post PCI: Should it be Routine?” Dr. Greg Hundley: Great, Carolyn and I've got a Research Letter from Professor Poo entitled “Efficient in Vivo Hemology- Directed Repair within Cardiomyocytes.” Well, how about we get onto that feature, discuss and learn more about Watchman devices and left atrial appendage closure. Dr. Carolyn Lam: Ooh, let's go. Dr. Greg Hundley: Well, listeners. Now we are onto the feature discussion today, and we're so privileged to have with us, Dr. Marco Valgimigli from Bern, Switzerland and our own Associate Editor, Dr. Mark Link from UT Southwestern. Welcome gentlemen. Well, Marco, we will start with you. Can you describe for us a little bit of the background material as to why you wanted to perform this study and what was the hypothesis that you wanted to address? Dr. Marco Valgimigli: But thank you so much for having me left atrial appendage closure is a therapeutic option for a patient who have formal indication to oral anti-population because of atrial fibrillation yet have some relative or absolutely contraindication to the treatment because mainly of high bleeding risk features, mainly because of previous bleeding complications. The two most frequently use device to accomplish that procedure are from one side Watchman, which is FDA approved. And on the other hand, the Amulet, which has been historically the device, which has been most frequently used in Europe, these two devices have been formally compared in one head to head study, which is Amulet IDE and also reported in the journal some month ago. However, it was important also to compare the new Watchman iteration, which the Watchman Flex, which was not part of the original Amulet IED, which only compared Amulet with Watchman 2.5. Dr. Marco Valgimigli: Before we set up this multicenter study, which recruited patients across eight centers and four European countries, we roughly screened 450 patients. And we ended up randomizing 222 to either of these two device being Amulet or Watchman. We set up this study to answer a superiority hypothesis, actually, of Amulet versus Watchman and we assume that Amulet would be superior compared to Watchman with the respect to the need for crossover to a known randomly allocated device or LA Patency at 45 day SCCTA. Dr. Greg Hundley: Very nice Marco. And so now describe for us your study design. And then what was your study population? Dr. Marco Valgimigli: That was an investigate initiated and conducted multicenter study, which took place across eight centers in four European countries. We screened roughly 450 patients to be able to randomize. Finally, 222 were evenly allocated to either of these two device. We selected patients with either fibrillation and form lead for oral anti-regulation to with relatively high chat box score on average four and very high has bled at least three or greater. In fact, 95% of our patient had prior bleeding before being considered for the study. Dr. Greg Hundley: Very good. And describe for us your results. Dr. Marco Valgimigli: So the superiority that we assumed was actually not met. The primary point, which was again, crossover to a known allocated device or patency rate at 45 day SCCTA was pretty much similar with rates being 68% in the Watchman group and 70% in the Amulet group. So basically no real difference. We did see though some interesting differences with respect to some key secondary endpoints. Dr. Greg Hundley: And describe those. Dr. Marco Valgimigli: So for example, the type of leaks that was recorded was actually different with respect to the two devices with intra device leaks, being much more frequent with Amulet and peri device leak, being much more frequent with Watchman. Also the rate of any or major procedure complications were slightly yet significantly higher in the Amulet group. And interestingly as well, we had some minor differences with respect to the device related thrombosis, which was numerically slightly higher in Watchman as compared to Amulet also, we performed at 45 day TEE, Transesophageal Echo and with that respect, we did see a slightly, yet numerically and significantly higher rate of peri device leak, detected at Transesophageal Echo with Watchman as compared to Amulet, which is in a way reflecting our SCCTA findings. Dr. Greg Hundley: Very nice. And so lots of results comparing these two devices for a left atrial appendage closure. So Mark, you see many papers come across your desk. What attracted you to this particular paper? Dr. Mark Link: Yeah, this is a very important clinical paper. The left atrial occlusion devices are going to continue to be used and with increasing frequency and there can be very beneficial. The Amulet is not approved in the United States. And so comparison between Watchmen and the Amulet are very important, both for the US and for Europe and the rest of the world. And that's why we were attracted to this paper, it's a nice randomized paper comparing the two most commonly use devices. Dr. Greg Hundley: Help us put the results of this study into the context in really how we might manage patients and how we're considering these devices, both from the European perspective and then also here in the US. Dr. Mark Link: I think what this study shows is that they're more or less equivalent and there may be theoretical reasons why one may be better than the other, but in this trial it didn't really come out. So I think the Amulet used more in Europe. The Watchman is used exclusively here, but I think this opens a window for the Amulet to come to America also and be approved by the FDA. Dr. Greg Hundley: Very good, well, coming back to you, Marco, where do you see is the next study that needs to be performed in this arena of research? Dr. Marco Valgimigli: I think now we have relatively effective device in accomplishing what we would like to accomplish. Namely sitting the LAA for preventing subsequent thromboembolic events. However, the procedure despite operators have been growing in terms of interest and experience is still associated with some degree of complications. Some of them are minor, but some of them are not. I think the next future, we should try to minimize those complication to the lowest possible event so that this procedure can potentially be also offered to a broader patient population, perhaps not just as a replacement of oral anticoagulation, but perhaps in association with oral anticoagulation to further minimize the stroke and other thrombotic events. Dr. Greg Hundley: And Mark, do you have anything to add here? Dr. Mark Link: Yes. I think what we need more information on is two things. One is procedural complications, procedural complications were higher with the Amulet device. And is that because it was a newer device and people are just learning how to use it, or is there something there that's really important? So that's one and then the really important one is long term clinical outcomes. This was a relatively short term trial when you're talking about strokes and anticoagulation. And so what I'd like to see is longer term follow up with both devices in the future. Dr. Greg Hundley: Well, thank you Mark. So now Marco, do you have any additional studies that you're performing really on this same dataset? Dr. Marco Valgimigli: Well, I actually fully agree with Mark, very long term follow up is absolutely mandatory in this patient population. And I'm very glad to announce that this Swiss Apero study that we have been discussing will actually continue. Continuing patients follow up five years so that we will have much more clinical information, at least comparative effectiveness, clinical information between two devices. And also we'll be performing at 13 months, a second SCCTA to better understand the dynamic nature of these peri device or inter device list, whether they actually do see over time and which of them may or not play a clinical role. Dr. Greg Hundley: Very nice well listeners, we want to thank Dr. Marco Valgimigli from Bern, Switzerland and our own associate editor, Dr. Mark Link from UT Southwestern for really bringing us this very important paper that compared the results of the Watchman, which is for primarily used in the US versus the Amulet, primarily used in Europe, a left atrial appendage closure device. Well, on behalf of Carolyn and myself, we want to wish you a great week and we will catch you next week on the run. Speaker 5: This program is copied right of the American Heart Association, 2022. The 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, please visit ahajournals.org.
This month on Episode 31 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the December 3 issue of Circulation Research. This episode also features a conversation with Drs Xavier Revelo, and Jop van Berlo from the University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis. Article highlights: Tong, et al. Alternative Mitophagy Protects Obesity Hearts Soetkamp, et al. Myofilament Phosphorylation in CDC Treated HFpEF Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hillaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'll be highlighting two articles presented in our December 3rd issue of CircRes, and I'll also speak with doctors, Xavier Revelo, and Jop van Berlo from University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis. Cindy St. Hilaire: The first article I want to share is titled, Alternative Mitophagy Protects the Heart Against Obesity-Associated Cardiomyopathy. The first doctor is Ming Ming Tong, and the corresponding author is Jun Sadoshima from Rutgers University. People with obesity or diabetes have an increased risk of developing cardiomyopathy, a condition which can eventually lead to heart failure. One of the major pathological features of obesity-related cardiomyopathy at the cellular level is a decrease in mitochondrial function. This decrease in mitochondrial function is likely due to a decrease in the canonical mitophagy pathway, which is a process by which dysfunctional mitochondria are degraded. However, a new process termed alternative mitophagy was recently discovered. When mice were fed a high fat diet for 24 weeks after only eight weeks, canonical mitophagy ceased. However alternative mitophagy steadily increased over the 24 weeks. Alternative mitophagy is regulated via the protein ULK1 and Rab9. The team went on to show that suppressing alternative mitophagy by knocking out ULK1, or expressing a loss of function, Rab9 mutant exacerbated the high fat diet induced cardiac dysfunction. Over expression of Rab9 in mouse hearts increased the alternative mitophagy pathway and protected the animals from cardiac dysfunction. These results suggest that pharmacological boosting of this ULK1 Rab9 mediated alternative mitophagy pathway might be a treatment strategy for preventing obesity related cardiomyopathy. Cindy St. Hilaire: The second article I want to share is titled Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure. The first doctor is Daniel Soetkamp and the corresponding author is Jenny Van Eyk from Cedar Sinai Medical Center. Weakness, fatigue and troubled breathing are among the symptoms experienced by someone suffering from heart failure with preserved ejection fraction, which is frequently called HFpEF. The pathology of the condition includes hypertrophy, fibrosis and stiffening of the heart and hyperphosphorylation of the cell sarcomere proteins. Because this hyperphosphorylation is a key contributor to HFpEF pathology, and because cardio sphere derived stem cells or CDCs have shown promise as a potential HFpEF treatment, this group investigated whether CDC treatment reduces phosphorylation levels of the sarcomere proteins in the heart. They found that administering CDCs to rats with HFpEF decreased the associated protein hyperphosphorylation, compared with that seen in untreated animals. Bioinformatic analysis revealed that protein kinase C or PKC is a prime suspect behind the phosphorylation. The authors suggest that CDCs alleviate HFpEF in part by reversing PKC-induced phosphorylation, and that PKC inhibition may be a desirable alternative treatment strategy, especially as it avoids regulatory issues associated with cell-based therapies. Cindy St. Hilaire: Today, I have Dr Xavier Revelo and Dr Jop van Berlo from the University of Minnesota and they're with me to discuss their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulates Angiogenesis. And this article is in our December 3rd issue of Circ Res. So, thank you both so much for joining me today. Jop van Berlo: Thanks for having us. Cindy St. Hilaire: Absolutely. So, your study is investigating the contribution of resident and monocyte drug of macrophages in cardiac remodeling, specifically in hypertrophy remodeling. So can you just introduce the topic of cardiac hypertrophy in humans, why that's not great to have, and then maybe tell us a little bit about what is known or what was known about the role of inflammatory cells in that hypertrophic remodeling. Jop van Berlo: Yeah, so absolutely. Cardiac hypertrophy is not a disease in and of itself in humans, but it is often a consequence of pathologies that can happen in patients, such as high blood pressure, hypertension or aortic valve stenosis, or if you've had a myocardial infarction the remaining myocardial may also become hypertrophic. We know that cardiac hypertrophy has downsides to it. People can develop sudden cardiac death when they have hypertrophic heart disease. We notice from population studies like the Framingham Heart Study and other studies, but it also increases the chance of developing heart failure later on. So even though cardiac hypertrophy by itself is not a disease, it is contributing to the cardiac pathology that can develop in patients and that can contribute to the development of heart failure. Cindy St. Hilaire: Great. So, what's the base level of knowledge of what is known regarding inflammatory cells in cardiac hypertrophy or cardiac hypertrophic remodeling? Xavier Revelo: So previous forecast focused on the role of infiltrating cells, specifically monocyte-derived macrophages, and generally these cells are pro-inflammatory and they aggravate the progression of heart failure. With Jop, we focus on, and what we think is exciting is the role of cardiac resident macrophages. And so, in our experiments, we decided to look at what's the role of these cardiac meta macrophages during pressure overload. Cindy St. Hilaire: That's a perfect segue to my next question, which is you obviously modeled this in mice, you used mice as your model and the method that you used to induce this hypertrophy is a technique called Transverse Aortic Constriction. So how does that actually work in a mouse and are there certain pros and cons to using that as a model for cardiac hypertrophy and, does it really recapitulate well, what happens in humans? Jop van Berlo: So, you're absolutely right that we use model systems to mimic what happens in humans and every model system has pros and cons to it. What we're trying to do here is to induce essentially acute cardiac pressure overload in a mouse model by inducing a constriction of the transverse aorta, right between the anomia and the left carotid artery. And we do this by ligating a needle on top of the transverse aorta that is of a specific size. And then we pull the needle out of the ligation and that immediately induces constriction. This is known to induce cardiac hypertrophy, and there are thousands of papers about this model as a model to induce cardiac hypertrophy. I think Howard Rockman was the first to publish this as a model of cardiac hypertrophy. Over the past decades, most of the research has focused on how cardiomyocytes within the heart respond to their stress and how they become hypertrophic. And I think what is new about our study is that instead of really focusing on the cardiomyocyte, we are focusing more on the non-cardiomyocyte compartments early after this stress is induced on the heart. Cindy St. Hilaire: That was one of the things I liked about this paper. We read about TAC a lot, the transaortic constriction model, but a lot of it is looking at either just the fibrotic cells or the scarring or the cardiomyocytes. So, this was, I thought a really nice unique take. So, one of the things I'm wondering is what are the functional differences between the systemic macrophages and these resident macrophages? I guess, resident to the cardiac tissue. And how does one tell the difference between these cells in the mice, but also in the humans? What is the human equivalent of those cells? Xavier Revelo: So, these cells, they rest in the macrophages in the cardiac tissue. One of the key differences from circulatory cells is the origin of the cells. In the heart, these cells self-renew, and they are from embryonic origins, as opposed to circulatory immune cells that come from the bone marrow. In terms of similarities between mice and humans, there are some markers that we can use to specifically study the cardiac resident macrophages. And these markers fortunately seem to be consistent between people and mice, which is advantages. Cindy St. Hilaire: That is good. That's always nice when it works out that way. So, you, you actually answered my next question, which was, are these residents macrophages a) able to self-replicate or are they from their own source? And so, regarding that developmental origin, how far apart are these lineages of the circulating monocytes versus the resident or the cardiac resident? How similar and how different, and how far back on the tree do they diverge? If we know it? Xavier Revelo: It's a complicated question. Jop van Berlo: And it's an active area of study right now, not just by us, but also by many other groups. Xavier Revelo: So, what we know is that regardless of origin, the cells are myeloid cells. So, they're the same lineage within the big family of immune cells. Having said that, the function of the cells is dictated by the origin, as well as the issue of residency. I forgot a second part of your question. Cindy St. Hilaire: I'm wondering how much they diverge functionally from the circulating monocytes? Xavier Revelo: They do. It seems like the tissue factors and the residency dictates the function of the cells in general. This is a general comment. Resident cells seem to have a protective role. Sometimes they help with the repair and healing as opposed to infiltrating cells that come into the tissue and they cause inflammation, generally they aggravate disease progression. Jop van Berlo: But what I also find fascinating about these resident macrophages is they are not only found in the heart, but they're also found in all organs, and they all come from developmental origins. But if you compare the macrophages between these different organs, they resemble the organ itself more than macrophages between organs and that's based on recent work where people have compared resident macrophages from different organs. And I think that's just fascinating how this develops in the heart, but also in other organs as a way to protect specific organs from potentially dangerous signals. Cindy St. Hilaire: Yeah, that's so interesting. So, it's almost like their niche, their new residential home, is really informing their function. So, there's some kind of back and forth between that environment and the cell itself. Jop van Berlo: That's what we presume, but I don't think we truly understand how the niche is important in dictating the function of these resident macrophages. And I think we need to do a lot more research into how the niche of tissue resident macrophages has formed and how that then dictates the differentiation of these resident macrophages to give rise to certain functionalities. Cindy St. Hilaire: Maybe you can summarize in a couple short sentences or so what, what your key findings were. Jop van Berlo: The main findings of our study is that very early after the induction of acute cardiac pressure overload, there is a high level of inflammation happening in the heart. And this allows the replication of resident macrophages and our study showed that these resident macrophages are really important for a protective mechanism within the heart to allow the heart to deal with this increased pressure in a heart. And what they do is they stimulate the formation of new block vessels, also known as angiogenesis and furthermore, they inhibit the formation of scar tissue or fibrosis, and we used different ways to substantiate these conclusions. Xavier Revelo: We studied cardiac-resident macrophages as one population. But one thing we learned in this study is that these macrophages are highly diverse. And so, using our techniques, we discover that within cardiac macrophages, we have 11 different subsets. And so, our future studies will be aiming at understanding the precise role of these different subsets that we think have different roles in pressure overload. Cindy St. Hilaire: One of the things I was thinking about is these 11 subsets, do they represent kind of end stage fully differentiated resident macrophages, meaning 11 different types, or are they kind of representing maybe the different stages that get to the one end type? Do we have a sense of what's going on? Xavier Revelo: I don't think it's completely understood my take on that is that these different subsets they can represent different activation states or functional subsets that we don't really understand why is that we have this diversity? Jop van Berlo: I think one of the aspects that we as a field need to work on is to better understand that complexity of immune cells that reside within an organ and associate that complexity to specific functionalities. And right now, the field is mostly lacking in technologies that allow us to do this. For example, we cannot culture these resident macrophages right now. We don't know the proper culturing conditions that allow us to test functional differences between subsets of macrophages. We don't have very good genetic tools to dissect these specific subsets of macrophages. And I think those are important areas that the field and us of course need to work on in the coming years. Cindy St. Hilaire: Every layer of discovery, just brings like 10 more layers complexity, or 11 more co-layers of complexity in this case. Jop van Berlo: Which is why we all love science! Cindy St. Hilaire: Exactly, exactly. It's a drug that, that keeps on giving. So, one of your experiments, I forget which number, I think figure five or six or something like that, but in wild type mice, you went on to use an anti CD115 antibody. And because that treatment others, as well as yourselves has shown depletes the resident macrophages. And, and one thing I thought was really interesting. I just want to hear how you unpack it. And that is in the wild type mice that were treated with the anti CD115 antibody. You found that the depletion of the resident macrophages exacerbated the adverse remodeling and it increased fibrosis, it decreased angiogenesis, but when you did the same thing in a CCR2 knockout mouse in that mouse, they don't have the circulating macrophages, but they also don't have the resident macrophages. They were protected from the increased fibrosis, but there was no change in the angiogenesis. And I was just wondering if you could unpack these results for me and kind of talk about the competing roles of the resident and the non-resident macrophage in this pathogenesis. Jop van Berlo So I think you highlight a really important experiment that we performed that try to dissect the protective versus damaging effects of different subsets of macrophages within the heart. We know that if you delete the receptor CCR2, that circulating monocytes cannot extravasate and enter the tissue in response to the cytokine CCL2 that is produced by the myocardium. So, using the CCR2 knockout, we essentially blocked the invasion of circulating monocytes into the myocardium to become monocyte-derived macrophages. And we knew from the literature that, especially the monocyte-derived macrophages, were pro fibrotic. So, we wanted to discern the effects on fibrosis between resident macrophages and monocyte derived macrophages. So, we were happy to indeed see that when we blocked extravasation of circulating monocytes and blocked them to become macrophages, that we indeed reduce the amount of fibrosis that we observed within the heart. I think the difficulty here that we observed that we don't have a very good explanation for right now are the effects on angiogenesis. And I think what this highlights is that there are many, many more complexities than just the resident and recruited macrophages on the development of angiogenesis because when we block tissue resident macrophages, are we actually depleting tissue resident macrophages? We didn't completely block the development of angiogenesis. We merely inhibited this by a little bit. And so, I think there are many more actions happening within the heart in response to stress than just the immune cells. And I think it highlights how complex a living organ really is. And we always try to do reductionist experiments to try to understand the functioning of specific aspects of that organ, but it's much more complex than just one cell type doing one thing and another cell type doing another thing. Xavier Revelo: One potential explanation to this complexity is the fact that when we deplete resident macrophages, the monocyte-derived macrophages, the infiltrating macrophages, they can replenish those resident macrophages. And so, whether there's a difference between the original resident macrophages compared to the replacing macrophages is unknown. And so, all these complexities can perhaps explain that different phenotypes that we observed in terms of angiogenesis. Cindy St. Hilaire: What do your findings suggest about potential therapies or you even potential therapeutic targets? Is it possible in a human to be able to target one or the other macrophage population? I know a lot of your experiments, because it's an experiment, you're targeting the depletion of macrophages before to see the effects, but are we able to possibly activate or stimulate their production, post MI for example? Xavier Revelo: Yeah, absolutely. So, thinking about cardiomyocyte independent interventions that can enhance the preparation process of any stressed heart, we could see potential in manipulating resident macrophages, specifically enhancing the functions of these resident macrophages that will help us heal and prevent fibrosis and enhance angiogenesis. So, we think that future studies need to look at what factors can be manipulated to enhance the function and survival of these resident macrophages. Jop van Berlo: One important aspect of our study that we don't highlight is that after this large increase in tissue resident macrophages, that we observed within the first week after cardiac pressure overload, these cells actually disappear. And right now, we don't really know the signals that are important for mediating that disappearing of cells. And we don't know this whether maintenance of these signals could improve longer beneficial effects of tissue resident macrophages. Cindy St. Hilaire: Interesting. I guess we know some questions you're going to start to ask in the future. Jop van Berlo: Absolutely. There's always more questions to answer in science. Cindy St. Hilaire: Well, great. Well, Dr Revelo, Dr van Berlo. Thank you so much for joining me today. Congrats on a wonderful paper and we look forward to these future studies. Jop van Berlo: Thank you. Xavier Revelo: Thank you. Cindy St. Hilaire: That's it for the highlights from the December 3rd issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at @CircRes and #DiscovererCircRes. Thank you to our guests, Dr Xavier Revelo and Jop van Berlo. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hillaire. 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, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or the American Heart Association for more information visit aha journals.org.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.277285v1?rss=1 Authors: Terauchi, R., Kohno, H., Watanabe, S., Saito, S., Watanabe, A., Nakano, T. Abstract: Retinal inflammation accelerates photoreceptor cell death (PCD) caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has previously been reported to show PCD rescue effect in retinal degeneration. The purpose of this study was to assess the effect of minocycline on Cx3cr1 and Ccr2 expression in retinal degeneration. Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice, which enabled observation of Cx3cr1 - and Ccr2 - expression pattern in inherited retinal degeneration, were used to test the effect of minocycline. Minocycline was systemically administered to Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice. For observing the effect of minocycline on Cx3cr1 and Ccr2 expression, administration was started on 4-week-old mice and continued for 2 weeks. To assess the PCD rescue effect, minocycline was administered to 6-week-old mice for 2 weeks. The expression pattern of Cx3cr1-GFP and Ccr2-RFP were observed on retinal and retinal pigment epithelium (RPE) flat-mounts. The severity of retinal degeneration was assessed on retinal sections. Minocycline administration suppressed Ccr2 expression in Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice as observed in retinal and RPE flat-mounts. On the contrary, Cx3cr1 expression was not affected by minocycline administration. Retinal degeneration is ameliorated in minocycline administered Mertk -/- Cx3cr1 GFP/+ Ccr2 RFP/+ mice. In conclusions, Minocycline suppression of Ccr2 expression correlates to amelioration of retinal degeneration. Copy rights belong to original authors. Visit the link for more info
This month on Episode 111 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the March 27 issue of Circulation Research and talks with Dr. Matthias Nahrendorf and Dr. Maximilian Schloss about their article Modifiable Cardiovascular Risk, Hematopoiesis and Innate Immunity. Article highlights: Liu et al. Genetics of Transposition of the Great Arteries Park et al. Mild Lipid Abnormalities and ASCVD in the Young Yan, et al. Gut Flora Adjusts Blood Pressure By Corticosterone Transcript Cindy St. Hilaire: Hello and 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. Today, I'm going to share with you articles selected from the March 27th issue of Circulation Research, as well as give you a hint of the cutting-edge ideas in the Compendium on atherosclerosis. We'll also have a discussion with Dr Maximilian Schloss and Matthias Nahrendorf about their article On Modifiable Cardiovascular Risk, Hematopoiesis And Innate Immunity. So, first the highlights. The first article I'm sharing with you is titled Exome-Based Case Control Analysis Highlights the Pathogenic Role of Ciliary genes and Transposition of the Great Arteries Exome-Based Case-Control Analysis Highlights the Pathogenic Role of Ciliary Genes in Transposition of the Great Arteries. The first authors are Xuanyu Liu and Wen Chen and the corresponding author is Zhou Zhou from Peking Union Medical College in Beijing, China. In normal healthy hearts, the aorta develops from the left ventricle and the pulmonary arteries from the right ventricle, but in the common congenital heart malformation called transposition of the great arteries or TGA, the plumbing of these two major vessels is switched. Thus, the pulmonary arteries arise from the left ventricle and the aorta from the right. This is a life-threatening condition, requires surgery in the earliest days of life and currently, the genetic etiology of this congenital disease is largely unknown. To identify the genetic drivers of transposition of the great arteries, the authors of this study performed whole exome sequencing of 249 TGA patients and, in 66 cases, they were actually able to do exome sequencing on their parents as well. The analysis identified 82 candidate genes in which the allele variant or mutation that was found in TGA patients was predicted to alter protein function. Interestingly, a quarter of these mutations or variants were found to be in genes that are involved in cilia function. So, the cilium is an organelle that's found on all eukaryotic cells and is in the shape of a slender protuberance that projects from the much larger cell body. Recently, cilia have been identified as playing a central role in the pathogenesis of congenital heart diseases, and it has been suggested that congenital heart disease may be a new class of ciliopathy. Transposition of the great arteries has been hypothesized to arise from disturbances in the left right patterning during embryo development, and cilia are required cellular organelles and they are essential for left-right axis determination in early development. These findings add to the growing body of evidence that has identified a role of cilia genes and congenital heart disease and may lead to future prenatal diagnostic screenings. The next article I want to highlight is titled Mildly Abnormal Lipid Levels, but Not High Lipid Variability, Are Associated with Increased Risk of Myocardial Infarction and Stroke in ‘Statin-Naive’ Young Population: A Nationwide Cohort Study. The first author is Jun-Bean Park and the corresponding author is Hyung-Kwan Kim from Seoul National University Hospital in Seoul in the Republic of Korea. High levels of lipids in the blood increase a person's risk of cardiovascular disease, and evidence suggests that this risk builds over lifetime. However, in young adults, and in this case, young adult means any individual between 20 and 39 years of age. In young adults, relatively little evidence is available that identifies individuals at high risk for atherosclerotic cardiovascular diseases, except for very high LDLC levels. Variability in lipid levels has recently emerged as a predictor of adverse clinical outcomes and lipid level variability may be causally linked with the atherosclerotic cardiovascular disease risk. This is because theoretically, high lipid levels can induce fluctuations in the atherosclerotic plaque composition. These fluctuations result in plaque instability and rupture and ultimately, plaque related clinical events, such as myocardial infarction. However, high lipid level variability may merely reflect other risk factors or confounders for atherosclerotic cardiovascular diseases, including unhealthy lifestyle and unrecognized comorbidities. This study therefore examined health data of close to two million Korean individuals aged 20 to 39. None of them had ever been treated for high cholesterol with statins nor had any of them suffered any myocardial infarctions or stroke. Over a four-year period, the subjects had undergone at least three lipid measurements as part of their general health assessments and then they were followed for a further four years or until death. The data showed that high baseline lipid levels was linked with an increased risk of adverse cardiovascular events, and in particular, myocardial infarctions. They also found that individuals who exhibited high lipid variability, so sometimes getting high readings, sometimes getting low readings, these individuals who exhibited high variability and lipid level measurements were not at any greater risk of such cardiovascular events. While such up and downs have previously been linked to cardiovascular disease, this study argues that perhaps statin use in other cohorts may have contributed to the variability and thus confounded research interpretation, an issue that was specifically avoided in this study. Together the results indicate that lipid in young adults can indeed indicate future cardiovascular risk and therefore suggest lipid-lowering strategies could be beneficial for this age group. The next article I want to share with you is titled Intestinal Flora Modulates Blood Pressure by Regulating the Synthesis of Intestinal-Derived Corticosterone in High Salt-Induced Hypertension. The first author is Xuefang Yan and the corresponding authors are Zhe Wang and Qunye Zhang from Shandong University in China. Hypertension is highly prevalent in the adult population all over the world and it is a major risk factor for heart disease and stroke. A high salt diet can help to drive hypertension pathogenesis, but complete details about the mechanisms by which high salt intake shapes vascular pathology are lacking. Recent studies show that fecal transfer from salt hypertensive to salt normotensive animals can lead to hypertension in the recipients, and this suggests that perhaps gut flora may play a role in hypertension. In the article by Yan and colleagues, they show that rats on a high salt diet have altered gut flora profiles and in particular that levels of the bacterium, Bacteroides fragilis, was reduced. Analysis of intestinal metabolites and substrates in high salt diet fed rats also showed that levels of arachidonic acid, which is produced by this bacterium, were low and levels of the stress hormone, corticosterone, which regulates blood pressure, were elevated. The team went on to show that supernatants from this bacterial culture could prevent corticosterone production in the intestinal tissue of high salt fed mice as could direct treatment with arachidonic acid. Moreover, both B. fragilis and arachidonic acid were found to be lower in the feces of humans with hypertension compared to that of healthy controls. The results suggest B. fragilis and arachidonic acid normally curb corticosterone production and could therefore be novel targets for hypertension treatment strategies. The last thing I want to mention before we switch to our interview is the Circulation Research Compendium on Atherosclerosis. The last compendium on this topic was back in 2016 and this new compendium provides the most cutting-edge ideas in the field. The topics highlighted in this compendium are polygenic scores to assess atherosclerotic risk, clinical perspectives, and basic implications, epigenetic reader proteins and cardiovascular transcriptional programs, sex as a biological variable in atherosclerosis, neutrophil extracellular traps in cardiovascular diseases, CD31 as a therapeutic target in athero, interleukin-1 and the inflammasome as therapeutic targets in cardiovascular disease, non-coding RNAs in vascular diseases, intracellular aspects of macrophage immunometabolism in atherosclerosis, single cell RNA sequencing in atherosclerosis, vaccination strategies and immune modulation in atherosclerosis and we have an update from the group leading the One Brave Idea. Please check out this compendium. All right. So, now we're going to switch over to our interview portion of the podcast. I have with me today Dr. Matthias Nahrendorf, who is a professor at the Center of Systems Biology at Massachusetts General Hospital Research Institute and Harvard Medical School and his research fellow, Dr. Maximilian Schloss. Today, we're going to be discussing the article Modifiable Cardiovascular Risk, Hematopoiesis, Innate Immunity, which is part of our Compendium on Atherosclerosis. Circulation Research puts together two to three compendiums annually and this current one is the Compendium on Atherosclerosis. We will have two additional compendiums planned for 2020. One on Obesity, Metabolic Syndrome and Cardiovascular Disease and that should come out in late May and another on Atrial Fibrillation scheduled for June. So stay tuned. So, thank you very much for being with me here today, Matthias and Maximilian. Matthias Nahrendorf: Thanks for having us. Maximilian Schloss: Thanks for having us. Cindy St. Hilaire: So, I really enjoyed this review article. I actually learned a lot. I also really liked your cartoons at the end, so maybe we can talk about those a little bit later, but what it's on is essentially the role of inflammation and cardiovascular disease and years of study, which have recently culminated in the completion of the CANTOS trial, have showed us that reducing inflammation can help reduce cardiovascular events. When we look at the factors that we know drive cardiovascular disease, it's things like bad diet choices, lack of exercise, stress, and inadequate or disrupted sleep and in this article you make the more nuanced argument that these modifiable factors are in fact influenced by the innate immunity. So, before we dig too deep into what you are really discussing in this article, could you maybe give us a brief introduction to the role of innate immunity and cardiovascular disease initiation and progression? Matthias Nahrendorf: Sure. Yeah. So, I think one very instructive experiment that had been done by more than one lab actually almost two decades ago, is stopping innate immune cells from migrating to atherosclerotic plaque by deleting the chemokine MCP-1 or the chemokine receptor CCR2 in mice that have a propensity to develop atherosclerosis. What became apparent is that these mice, despite having very high blood cholesterol levels, they don't really develop atherosclerosis. This really led the whole field now almost 20 years ago, to the insight that it's not only the hypercholesterolemia, it's also the immune system that contributes to the disease. So, innate immune cells, most numerous neutrophils and monocytes then in tissue also macrophages and they're meant to defend us against infections and they support healing. In this particular setting, they are probably doing more harm than good because they promote inflammation in areas where inflammation shouldn't be i.e., in the vessel wall. Maximilian Schloss: Yeah, I would add that what Matthias said is that basically it's all about a balance between necessary inflammation and too much inflammation. If we take, for instance, myocardial infarction as an example, we need a certain amount of inflammation, local inflammation. We need a recruitment of innate immune cells like neutrophils and monocytes and eventually macrophages, to do their job. For instance, phagocytizing a dying cardiomyocytes or inducing fibrosis. So in this example, we need inflammation, but what we see in different models where we can manipulate inflammation being at reducing or increasing inflammation, we can see that if we do either/or then wound healing and scar formation is impaired. I think that's all we are interested in studying not only the mechanisms, how inflammation can be increased or decreased, but also what is actually the perfect balance in view also of finding ways of improving outcomes in cardiovascular patients. Cindy St. Hilaire: One of the things in my research, so I focus on cardiovascular calcification, which is very hard to do in a mouse. They don't like to calcify similarly like they don't like to make plaque without a proper genetic background. Are there aspects of the mouse versus the human innate immune system that are very different? I mean I know specific receptors are slightly different, but in general, are they matched up pretty well or is there things that are quite different about them? Matthias Nahrendorf: I think the answer is both and there are very important parallels and then there are very important differences. So, one important difference is just if you look at sheer numbers and the contribution of immune cells in the blood and, possibly also in the plaque, can be quite different. So, recent studies that use unbiased profiling in human plaques, for instance, say that there's quite a lot of lymphocytes and we still have to understand whether this is due to the retrieval or if it says species difference or the situation, but I think there are important differences. On the other hand, I think that it really make sense to study mice because a lot of the important discoveries about the immune system in the setting have translated to humans. Cindy St. Hilaire: Like the IL-1 beta story. Matthias Nahrendorf: That's right. Yeah. Cindy St. Hilaire: So, actually one of the topics that you started out with in your article is on the role of hematopoiesis in cardiovascular disease. You had a beautiful paper years ago actually with my colleague at University of Pittsburgh, Partha Dutta, who's right down the hall from me, where you guys showed that myocardial infarction itself further exacerbates atherosclerotic plaques mid part through recruiting monocytes from the spleen and mobilizing the immune system. So, I'm wondering, what are the role of the cells when they get mobilized? You talk about these modifiable risk factors of stress and sleep interruption, unhealthy diet. So, how can these risk factors help or promote this mobilization of hematopoietic cells? Matthias Nahrendorf: Yeah. So, I think that early on when we thought about going down this road and studying these risk factors, even before going there, you realize that the cells that we're interested in, innate immune cells are very short lived. So they live on the order of hours or days. So, they're really produced just in time. That's different to lymphocytes and resident macrophages, which have much longer lifespans. So, this really triggered the insight that we should look at production and release because it's a just in time supply situation. So, what we were wondering is whether in the setting of cardiovascular disease, whether production rates are increased and we now know and a number of labs have studied hematopoiesis in this setting including Fil Swirski, Alan Tall, and some others. We now know that this is really the case, so hematopoiesis increases in chronic atherosclerosis. It increases in acute myocardial infarction and increases in heart failure. What we don't know is what mechanisms actually ramp up blood cell production and we're beginning to understand that the sympathetic nervous system is involved. But I think we only see the tip of the iceberg here. That's why we wanted to study modifiable risk factors, because if you look at others such as high cholesterol, once the insight was gained that lowering cholesterol is helpful, we had the statins which make a huge change. So, we hope to repeat that. Cindy St. Hilaire: Maximilian, one of the things that you brought up is this balance. The inflammation's a little bit good and then it's a little bit bad or a lot bad. So, where is that good and bad spectrum in terms of mobilizing hematopoiesis or hematopoietic cells? Maximilian Schloss: Yeah. I think that depends a bit on the disease type or we're talking about a chronic disease or an acute disease? For instance, to stay at the example of myocardial infarction, once cardiomyocytes become ischemic, they will release certain chemokines and cytokines into the blood, which then circulate to the bone marrow and tell the cells that leukocytes need to leave the bone marrow to enter the blood circulation system and then go to the heart to fulfill their very important functions there. Once the cells leave the bone marrow, the bone marrow need to reproduce themselves, then this process starts of hematopoiesis and there we can go back again to the concept of a balance. Of course, there is a certain beneficial physiological need of cell production, but one sees mechanisms so to say maybe go out of control and too many leukocytes are produced and released to the blood. Then that again impairs patient outcome. There are very many papers, clinical papers, who have shown that leukocyte counts after myocardial infarction have a certain U shape relationship with the outcome. That I think is best described that if leukocyte counts are very high, that they actually negatively correlate with the outcome of MI patients. If you look at the bone marrow specifically, there are certain mechanisms, which we know, and what we are more closely looking at now, what are actually the modifier of this process, what are the signals which tell these cells to secrete more hematopoietic factors or quiescence factors? I think that's what also the Review is a little bit about. Cindy St. Hilaire: Yeah, it's great. So, you were speaking about that kind of U-shaped curve in the release of these cells. Do we know based on some of the other things you spoke about, I guess I'm thinking about like diet or exercise or sleep in contributing to that release after an event like myocardial infarction. Is that known yet or has anyone looked into that? Matthias Nahrendorf: Yeah. So, I think we're in the very beginning of understanding what's happening acutely. There's more knowledge on the chronic side and this is what we've been working on. Often the things that influenced the chronic situation can be quite different from what happens acutely. So I think in general, we're just beginning to understand what happens in acute myocardial infarction. Well, we know for instance is that exercise doesn't compromise the release and supply of leukocytes that's necessary in acute infection or acute myocardial infarction. So, if the mouse or the individual was exercising before the event, that may reduce overall leukocyte levels, but not to a degree that it's harmful. Cindy St. Hilaire: Yeah. You can't exercise your way beyond a certain point. Matthias Nahrendorf: Maybe that's also possible. If you run more than one marathon a day, I'm sure that's… Cindy St. Hilaire: That will do something else. Matthias Nahrendorf: Yeah. Cindy St. Hilaire: Actually, so one of the interesting things that I saw in the article was when you were talking about diet and the role of diet in innate immunity, which is something I really never thought about, and you did bring up things like intermittent fasting. Can you discuss what's known at least scientifically about how that kind of diet timing can impact the immune system and therefore maybe cardiovascular disease? Matthias Nahrendorf: So, that's a very emerging field. There's very little known about this. I think it's very interesting because very relevant and a lot of people are excited about it, but it's basically, from what I know, it's mostly two papers that were published, I think both in Cell, and they say that intermittent fasting leads to a decline of cells that are in circulation. So, that's a very exciting observation. I think it's similar insight as to discovering that immune cell levels circulate the circadian rhythms, which had been discovered a while ago. So, I think there's definitely an impact and we're just beginning to understand why this is and what regulates it. Cindy St. Hilaire: Yeah, that segues nicely into the next thing I was wondering about and that is we all know not enough sleep, you get tired, your brain's not focused and stuff like that, but it really does impact the inflammatory system and also cardiovascular disease. So how is sleep involved in this innate immunity cardiovascular disease progression? Matthias Nahrendorf: The way we approached this was actually thinking about lifestyle factors and their impact on cardiovascular disease. Maybe a decade ago, Fil, who's our middle author on this Review, and I started thinking about lifestyle factors and what struck us is that the association of some of these risk cardiovascular events is really high. So, if you look at sleep or if you look at psychosocial stress, psychosocial stress has an odds ratio of 2.4 for premature myocardial infarction. That is right on scale with all these powerful risk factors that everybody knows about like hypertension, but then what isn't really clear or maybe not entirely, is whether or not these risk factors also act via the innate immune system and that's where we were coming from. I think at this point it's pretty clear that they do have an influence via the immune system. What I think what we've done is we uncovered a couple mechanisms that lead to the activation or dampening of inflammation depending on what you look at, but we don't really understand the broader network. I think there's a lot of work to be done looking into these pathways, which is exciting because I think that we can learn from nature what's dangerous and what's helpful. That this is how humans learn to fly. So, I think that observing what leads to cardiovascular disease, which behaviors are really harmful, will maybe lead us to new ways of mitigating it. Cindy St. Hilaire: Yeah. Also, I think all of this, it's interesting. We all went after smoking for decades, stop smoking, reduce cardiovascular risk and maybe it's stress and sleep is the next smoking. Matthias Nahrendorf: Smoking was so successful, right? I mean if you look 50 years back, it was promoted as this healthy thing that you should do. Then people really started to learn how bad it is and now we're at a time where smoking is declining and has declined and we see the results. Lung cancer is really on the decline. So, I think that's a good example how understanding health effects of behavior can be really helpful. Maximilian Schloss: I think one thing I would like to add is when you ask more general question about innate immunity and when we talk about sleep and sleeping habits, I think what's generally quite interesting to know is that the immune system or these leukocyte numbers in circulation, they oscillate quite dramatically over the course of a day in a healthy human being and also in mouse models. I think one aspect also among others to consider is when we have unhealthy steeping habits, like for example, going to bed late or being a shift worker, drinking for example before going to bed. Then this will also confuse a system on the circadian entrainment, which then subsequently will lead to other problems. I also think another thing is that what you were mentioning with the fasting is what we learned from this similar to these extreme circadian patterns seen when we fast or when a mouse is fasting, then monocyte levels drop into extreme low levels and these monocytes hone back into the bone marrow. I think this is interesting because it shows how dynamic actually a system like innate immune cells actually is. So, it's a very delicate system which responds to sleep disruption, exercise, diets in a very dramatic way. Cindy St. Hilaire: All right, I'm going to bed early tonight and eating a good dinner. Well, this was a wonderful Review. I really enjoyed reading it. I really do think it's introducing the next targets that we have to go after in modifying cardiovascular disease. Thank you so much for taking the time to speak with me today. Matthias Nahrendorf: Thank you. Maximilian Schloss: Thank you so much. Cindy St. Hilaire: That's it for our highlights from the March 27th and Compendium issue of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. Thank you to our guests, Max Schloss and Matthias Nahrendorf. I'm your host, Dr. Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most up-to-date and exciting discoveries in basic cardiovascular research.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 17/19
Interstitial lung diseases (ILD) are severe chronic lung diseases characterized by an increased deposition of extracellular matrix in the lung interstitial space, leading to a thickening of the alveolar walls and impairment of the gas exchange. One of the most common entities in this category is idiopathic pulmonary fibrosis (IPF) with a mean survival time of 2 to 3 years from diagnosis. Until now, there is no curative therapy available and the symptomatic anti- inflammatory treatment and oxygen supplementation cannot prevent the development of the end stage pulmonary fibrosis. The chemokine receptor CCR2 is important for leukocyte recruitment to inflamed tissues through interaction with CCL2 (MCP-1). The blockade of the CCR2/CCL2 pathway attenuated the development of pulmonary fibrosis in mouse models. However, CCR2+ T-lymphocytes acquired regulatory functions in experimental arthritis during the course of disease. Therefore, it is unknown whether CCR2+ T cells are involved in the pathogenesis of IPF or, on the contrary, represent an unsuccessful effort of the immune system to limit the disease. Observations in paediatric patients with different forms of ILDs suggested a role for CCR2+ T cells in pulmonary fibrosis. To characterize these T cells, flow cytometric studies were performed using the bleomycin mouse model of pulmonary fibrosis. The kinetic of CCR2+ T cells in BALF, lung tissue, and spleen following intratracheal administration of bleomycin (BLM) was assessed at time points between day 3 and day 21. To determine, if the constellation of naïve, central memory and effector memory T cells changes after BLM treatment, and to which of these subtypes CCR2+ T cells belong to, the cells were additionally stained for CD62L and CD44. For further characterization of CCR2+ T cells, chemokine receptor co-expression with CCR2 was investigated at the time point of the maximal presence of CCR2+ T cells. Total T cell numbers increased in BAL and lung tissue but not in spleen. Percentages of CD62LlowCD44hi effector memory T cells increased in lung tissue in the early phase of BLM induced fibrosis, while the CD62LhiCD44low naïve T cell population decreased. The percentage of CCR2+ T cells increased following BLM treatment with a maximum on day 12. The majority of CCR2+CD4+ T cells showed a Tem phenotype. CCR3, CCR4, CCR6, CXCR4, and CXCR5 expressing cells increased within the pulmonary CD4+ T cell population following bleomycin treatment. Among CD8+ T cells from treated mice, CCR5, CCR6, and CXCR5 positive cells were increased. CCR7 was highly co-expressed with CCR2 in saline and bleomycin treated mice, whereas co-expression of CCR3, CCR4, CCR6 and CXCR5 increased significantly in treated mice. The results indicate an activation of pulmonary T cell populations following bleomycin treatment. CCR2+CD4+ T cells probably take part on this T cell response as they exhibit an effector memory phenotype and increase following BLM treatment. In contrast, the stable percentages of the different T cell subtypes in spleens gave no hint for a systemic T cell reaction. The pattern of chemokine receptor expression argues against a Th1 polarization and towards a Th2, Th17 or TFH polarization of CCR2+ T cells.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 16/19
Der humane Mas-related gene X1 (hMrgX1)-Rezeptor ist ein G-Protein-gekoppelter Rezeptor, der selektiv in nozizeptiven Spinalganglienneuronen exprimiert wird. Eine spezifische Aktivierung des Rezeptors durch das Proenkephalin-Spaltprodukt BAM8-22 (bovine adrenal medulla 8-22) wird als schmerzhaft wahrgenommen. Damit stellt der hMrgX1-Rezeptor eine neue molekulare Zielstruktur für eine potentiell nebenwirkungsarme, analgetische Therapie dar. Trotz dieses Potentials sind die pro-algetischen Signalwege des hMrgX1-Rezeptors bislang nicht verstanden. Der MrgX1-Rezeptor entwickelte sich unter hohem positivem Selektionsdruck und kommt nur in Primaten vor. Trotzdem wurden die nicht-homologen MrgC-Rezeptoren der Nagetiere zur Analyse des hMrgX1-Rezeptors verwendet. Im Rahmen dieser Arbeit wurden daher zunächst vergleichende Ligandenprofile des hMrgX1- und der MrgC-Rezeptoren aus Maus und Ratte erstellt. Dabei wurden deutliche Unterschiede offensichtlich, da der hMrgX1-Rezeptor exklusiv von BAM8-22 aktiviert wurde, während die MrgC-Rezeptoren durch weitere Liganden, u. a. Spaltprodukte des Proopiomelanocortins, z. T. sogar effizienter aktiviert wurden. Zudem konnte gezeigt werden, dass die MrgC-vermittelte Ca2+-Mobilisation auf Grund einer β-Arrestin-abhängigen Rezeptorendozytose deutlich desensitisierte, während der hMrgX1-Rezeptor resistent gegenüber dieser Liganden-induzierten Regulation war. Daher können die MrgC-Rezeptoren der Nagetiere nicht als Modellsytem für den hMrgX1-Rezeptor verwendet werden, so dass in dieser Arbeit weiterführend Signalwege des hMrgX1-Rezeptors in Spinalganglienneuronen-ähnlichen F11-Zellen und primären Spinalganglienneuronen untersucht wurden. Dabei zeigte sich eine duale funktionelle Regulation des etablierten pro-algetischen TRPV1 (transient receptor potential cation channel vanilloid 1)-Ionenkanals. Zum einen sensitisierte der hMrgX1-Rezeptor den TRPV1 über einen etablierten, Proteinkinase C-abhängigen Signalweg. Zum anderen zeigte sich eine direkte hMrgX1-mediierte Aktivierung des TRPV1. Dieser Regulationsmechanismus wurde durch eine Phospholipase C (PLC)-β-induzierte Produktion des endogenen TRPV1-Liganden Diacylglycerol und durch die Degradation des tonisch TRPV1-inhibierenden PLC-β-Substrates Phosphatidylinositol-4,5-bisphosphat vermittelt. Neben der TRPV1-Modulation induzierte der hMrgX1-Rezeptor die Expression verschiedener Gene, deren zentrale Bedeutung bei der inflammatorischen und neuropathischen Schmerzchronifizierung etabliert ist. Einerseits wurde eine hMrgX1-induzierte Phosphorylierung der extracellular signal-regulated kinases 1/2 beobachtet, die in einer Aktivierung von serum response factor-abhängigen Reportergenkonstrukten und in der Induktion von c-Fos auf mRNA- und von early growth response protein 1 auf mRNA- und Proteinebene resultierte. Andererseits zeigte sich die transkriptionelle Ca2+/Calcineurin-abhängige Aktivierung des nuclear factor of activated t cells, die in der Induktion des CCR2 (chemokine receptor 2) auf mRNA- und Proteinebene resultierte. Somit konnte erstmalig ein physiologischer Induktor des CCR2 in Spinalganglienneuronen beschrieben werden. Weiterhin wurde nach der Etablierung der endogenen Proteinexpression des hMrgX1-Rezeptors in LAD2-Mastzellen eine BAM8-22-induzierte Freisetzung des CCR2-Agonisten chemokine ligand 2 ermittelt, so dass der hMrgX1-Rezeptor die parakrine Stimulation von nozizeptiven Spinalganglienneuronen durch Mastzellen fördern könnte. Diese Dissertation trägt somit zum besseren molekularen Verständnis akuter und chronischer pro-algetischer Funktionen des hMrgX1-Rezeptors bei und könnte damit die Entwicklung neuer analgetischer Wirkstoffe ermöglichen.
Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain.
Background: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods: SP-C-A116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results: Stable expression of SP-C-A116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-C-A116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-C-A116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4(+) lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-C-A116D on neighboring cells in the alveolar space. Conclusions: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06
T cells directed against brain antigens are generally held to play a crucial role in the initiation of multiple sclerosis (MS). This was deduced from experimental autoimmune encephalomyelitis (EAE). In this model for MS, T cells reactive for myelin antigens induced a severe paralytic disease upon transfer to healthy syngeneic recipients. Intriguingly, the disease does not start immediately upon transfer of the pathogenic effector T cells. Instead, as earlier studies have shown, the effector T cells attack their target organ only after having migrated in the periphery through secondary lymphoid organs. The aim of the project was to characterize the functional properties of these migrating encephalitogenic T cells during the course of EAE and to identify biological pathways which determine their migratory behaviour and pathogenic potential. To this end, average linkage hierarchical clustering, pathway and gene ontology (GO) analyses of transcriptomes from cultured and ex vivo-isolated myelin basic protein-reactive T cells (TMBP cells) were performed. At the time of transfer, encephalitogenic T cells in vitro are maximally activated, i.e. they exhibit a prominent upregulation of cell cycle genes such as cyclin A2 (CCNA2) and cyclin B2 (CCNB2) among others. In contrast, T cells isolated from spleen 3 days post transfer, downregulated activation markers such as interleukin 2 receptor (IL2R) and interferon γ (IFNγ), and at the same time upregulated migration specific genes such as CC-chemokine receptor 1 (CCR1), CC-chemokine receptor 2 (CCR2) and CC-chemokine receptor 5 (CCR5). Hierarchical cluster analysis revealed that several transcription regulators known for inhibiting cell cycle progression such as krüppel-like factor 4 (KLF4), B-cell translocation gene 2 (BTG2) and transducer of ERBB2, 1 (TOB1) were clustered together with cell cycle and migration genes. Overexpression of KLF4 in T cells not only inhibited G1/S phase progression of the cell cycle but additionally induced upregulation of CCR2 and CCR5. A novel tetraspan membrane protein called epithelial membrane protein (EMP1), was found to be up regulated in ex vivo-isolated effector T cells. Overexpression of EMP1 in encephalitogenic T cells influenced the migratory behaviour of effector T cells both in vitro and in vivo. EMP1 enhanced T cell motility within the extracellular matrix milieu in vitro and promoted T cell migration from the connective tissue to lymph nodes in vivo resulting in an accelerated onset of EAE. In conclusion, gene expression profiling of encephalitogenic T cells revealed interesting genome wide transcriptomic changes and established a correlation between cell cycle progression and cell migration. As a result, in silico analysis put forth several interesting candidate genes that hold promise as potential targets for therapeutic intervention.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 11/19
Summary and hypothesis Role of pro-inflammatory chemokines in diabetic nephropathy Beyond hemodynamic and metabolic abnormalities associated with diabetes, the role of inflammation in development and progression of diabetic nephropathy is well accepted. Recruitment and activation of macrophages in different renal compartment is considered to be hallmark of all inflammation in diabetic nephropathy. Although recruitment of macrophages to the renal compartment has been extensively studied, the exact mechanisms involved are still to be explored. The chemokine-chemokine receptor interactions are implicated to be mainly responsible for trafficking and infiltration of different monocytes and macrophages. Contribution of macrophages to the development of DN can be addressed in either by inhibiting chemokines or chemokine receptor associated with diabetes. We hypothesized that inhibition of CCL2 may inhibit macrophages infiltrating into different compartments in kidney and inhibition started at earlier stage of disease progression may show more beneficial effects than CCL2 blockade at late stage of DN. To address the involvement of additional chemokine receptors we hypothesized that blocking CCR5 and CCR2 simultaneously might have some additive or synergistic effects. Role of homeostatic chemokines in diabetic nephropathy Homeostatic chemokies are mainly involved in hematopoeisis, immune cell survival and adaptive immune responses. CXCL12 attracted our attention as it is being extensively studied and reported to be responsible for different functions like stem cell survival and homing and trafficking to different compartments. The role of CXCL12 in diabetic nephropathy has not been explored yet. CXCL12 is constitutively expressed by different renal cells. It may contribute to tissue repair and inhibit disease progression by stem cell recruitment or may cause increased tissue fibrosis and aggravate the disease. We hypothesized that CXCL12 plays role in development and progression of diabetic nephropathy. In order to address this question we used CXCL12 blocker in a mouse model of diabetic nephropathy.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 11/19
Mit der vorliegenden Studie wurde am Beispiel der diabetischen Nephropathie und der Nephropathie beim Alport-Syndrom die Rolle des Chemokins "macrophage chemoattractant protein-1" (Mcp-1) bei der Progression der chronischen Niereninsuffizienz untersucht, da es die damit assoziierte Rekrutierung von Makrophagen in die Niere vermittelt und in Korrelation zum Erkrankungsstadium verstärkt exprimiert bzw. verstärkt im Urin ausgeschieden wird. Mcp-1-defiziente diabetische Mäuse zeigen zudem eine verlangsamte Progression. Eine therapeutische Blockade von Mcp-1 sollte daher die Infiltration von Makrophagen reduzieren und mit geringerer Nierenschädigung einhergehen. Mcp-1 wurde bei uninephrektomierten db/db-Mäusen mit fortgeschrittener diabetischer Nephropathie und bei Kollagen-IV-a3-defizienten Mäusen mit autosomal-rezessiver Alport-Nephropathie mit einem neuartigen Antagonisten, dem Spiegelmer mNOX-E36-3’PEG blockiert. Bei uninephrektomierten db/db-Mäusen führte die achtwöchige Behandlung mit dem Spiegelmer zu einer Reduktion der glomerulären Makrophagen um 40 % und der tubulointerstitiellen Makrophagen um 53 %. Dies war assoziiert mit einer signifikanten Reduktion glomerulärer und tubulointerstitieller Schäden sowie einer Verbesserung der glomerulären Filtrationsrate (GFR). Daneben konnte die Expression von Mcp-1 und Ccr2 in der Niere signifikant reduziert werden. Bei Kollagen-defizienten Mäusen führte die sechs- bzw. dreiwöchige Behandlung zu einer Reduktion der glomerulären Makrophagen um 50 % und der tubulointerstitiellen Makrophagen um 30 %. Dies war jedoch weder mit einer Reduktion der Nierenschädigung oder der Expression von Mcp-1 und Ccr2, noch mit einer Verlängerung des Überlebens assoziiert. Zusammenfassend konnte gezeigt werden, dass die spät begonnene therapeutische Blockade von Mcp-1 durch das Spiegelmer mNOX-E36-3’PEG bei der db/db-Maus einen Schutz vor Glomerulosklerose bietet, die tubulointerstitielle Schädigung reduziert und die Nierenfunktion verbessert. Die Therapie mit anti-Mcp-1-Spiegelmeren könnte demnach das derzeitige Behandlungsregime der Diabetes-assoziierten chronischen Niereninsuffizienz um einen neuen Therapieansatz ergänzen und so die Progression verlangsamen. Beim Alport-Syndrom konnte die therapeutische Blockade von Mcp-1 durch das Spiegelmer mNOX-E36-3’PEG nicht vor Glomerulosklerose schützen, die tubulointerstitielle Schädigung nicht reduzieren, die Nierenfunktion nicht verbessern und das Überleben nicht verlängern. Ein Einfluss auf die Progression des Alport-Syndroms konnte nicht nachgewiesen werden. Die Therapie mit anti-Mcp-1-Spiegelmeren bietet demnach keine zusätzliche Behandlungsoption bei der hereditären Alport-Nephropathie.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 10/19
Die vorliegende Studie trägt zum besseren Verständnis des breiten klinischen Spektrums und des Fehlens universeller Serum- Marker der infektionsassoziierten Krankheitsaktivität der Lupus- Nephritis und möglicherweise anderer Formen der Immunkomplexnephritis bei. Exogener Kontakt mit TLR 7- Liganden hat die Entwicklung der Lupus- Nephritis bei jungen, gesunden MRL- Wildtyp und MRLlpr/lpr Mäusen nicht getriggert und hatte keinen signifikanten Effekt auf die Krankheitsaktivität bei diesen jungen Mäusen. Bei alten Lupus- Mäusen führte eine ähnliche Exposition jedoch zu einem merklichen Anstieg der Serumspiegel proinflammatorischer Zytokine und von IFNα, sowie zu einer Infiltration der Nierenglomeruli 18 Wochen alter MRLlpr/lpr Mäuse mit Makrophagen und einer (nicht signifikanten) Erhöhung der Autoantikörperspiegel. Diese Daten unterstützen die Theorie, dass dem Toll- like Rezeptor 7 eine Rolle bei den Mechanismen, die das Fortschreiten der autoimmunen Gewebsschädigung in MRLlpr/lpr Mäusen fördern, zukommt. Basierend auf den Ergebnissen der funktionellen Rolle von TLR 7 in Lupus- Mäusen und in Primärzellen, die aus Lupusmäusen isoliert wurden, sahen wir in der TLR 7- Blockade ein mögliches neues Ziel, um die schädlichen Effekte des Signalings über Immunkomplexe und endogene Liganden zu begrenzen. Es zeigte sich, dass die Blockade von TLR 7 und TLR 7 + TLR 9 die Gewebsschäden in Nieren und Lunge signifikant reduzieren konnte. Eine TLR 7 Antagonisierung mit dem Oligodeoxyribonukleotid IRS661 senkte die Menge an Autoantikörpern (insbesondere anti- SM, anti- dsDNA, IgG2a, IgG2b), entzündlichen Zytokine und Chemokinen im Serum, glomerulären Ablagerungen von IgG2a und Komplementfaktor C3c deutlich. Die Hemmung von TLR 7 reduzierte ebenfalls die CC- Chemokin- gesteuerten Makrophagen- und T- Zell- Infiltrate in den Nieren. Dies zeigte sich durch erniedrigte Spiegel von Ccr2, Ccr5, Ccl2 und Ccl5 in den Nieren behandelter Tiere. Diese Ergebnisse unterstützen das Konzept, dass endogene TLR 7- Liganden zur Pathogenese der Autoantikörperproduktion und der autoimmun vermittelten Gewebsschädigung des SLE beitragen. Die TLR 7- Blockade könnte ein neues therapeutisches Konzept beim Lupus erythematodes sein.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 10/19
The two chemokine receptors CCR2 and CCR5 play important roles in the recruitment and activation of monocytes/macrophages and T-lymphocytes at sites of infection and inflammation. To further examine their function, I cloned the two murine chemokine receptors Ccr2 and Ccr5 from genomic mouse DNA by a PCR-based cloning strategy and functionally expressed them in stably transfected CHO-cells. These cells were used to generate the first monoclonal antibodies against Ccr2 and Ccr5.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 08/19
Die Eigenschaft von Leukozyten, das Gefäßsystem zu verlassen und in das umliegende Gewebe auszuwandern, ist von essentieller Bedeutung für die Bekämpfung von Infektionen und darüber hinaus entscheidend für die Pathogenese des I/R-Schadens. Die Extravasation von Leukozyten stellt dabei einen kaskadenartig verlaufenden Prozess dar, welcher sich in die Schritte Rolling, Adhärenz, transendotheliale und interstitielle Migration gliedern lässt. Ein geeignetes Versuchsmodell, welches am M. cremaster der Maus in vivo alle Schritte des leukozytären Rekrutierungsprozesses während I/R zu analysieren erlaubt, liegt bisher nicht vor. Während die frühen Schritte des leukozytären Extravasationsprozesses weitgehend aufgeklärt sind, sind die Schritte der transendothelialen und interstitiellen Migration von Leukozyten unzureichend verstanden. In vitro Untersuchungen zeigen, dass das Molekül JAM-A in die Transmigration von Leukozyten involviert ist, jüngste in vivo Studien zeigen jedoch kontroverse Ergebnisse. Ferner gibt es zunehmend Hinweise darauf, dass die Chemokinrezeptoren Ccr1, Ccr2 und Ccr5 an der Extravasation von Leukozyten beteiligt sind. Welche Bedeutung diese Chemokinrezeptoren für die einzelnen Schritte des leukozytären Rekrutierungsprozesses bei Entzündung und I/R besitzen, ist bislang unklar. Die Ziele der vorliegenden Arbeit waren daher i) ein geeignetes Modell zur Untersuchung aller Schritte des leukozytären Rekrutierungsprozesses bei I/R am M. cremaster der Maus zu entwickeln, ii) die Bedeutung des Adhäsionsmoleküls JAM-A für die Transmigration von Leukozyten zu untersuchen und iii) die Rolle der Chemokinrezeptoren Ccr1, Ccr2 und Ccr5 für die einzelnen Schritte des leukozytären Rekrutierungsprozesses bei Entzündung und I/R zu analysieren. In unterschiedlichen Versuchsansätzen wurde mit Hilfe der RLOT-Intravitalmikroskopie am M. cremaster anästhesierter Mäuse die leukozytären Migrationsparameter untersucht. Zur Bestimmung des Phänotyps transmigrierter Leukozyten wurden immunhistochemische Färbungen von Paraffinschnitten durchgeführt. In einer ersten Versuchsreihe wurden die einzelnen Schritte des leukozytären Extravasations-prozesses systematisch in Abhängigkeit von Ischämiedauer und Reperfusionszeit untersucht. Die Ergebnisse zeigen, dass es bereits nach 30 min Ischämie und 120 min Reperfusion gegenüber schein-operierten Kontrolltieren zu einem starken Anstieg von Leukozyten-adhärenz und -transmigration kommt. Eine Verlängerung der Ischämiezeit auf 60 bzw. 90 min konnte keine Steigerung der Effekte erzielen. Diese Befunde waren der Ausgangspunkt für weitergehende Untersuchungen, welche die Mechanismen des leukozytären Rekrutierungs-prozesses näher charakterisieren sollen. In diesem Zusammenhang wurde in einer zweiten Versuchsreihe unter Verwendung von JAM-A-defizienten Mäusen die Bedeutung des Adhäsionsmoleküls JAM-A für die Leukozytenrekrutierung systematisch unter verschiedenen inflammatorischen Bedingungen analysiert. Unsere Daten belegen, dass die transendotheliale Migration von neutrophilen Granulozyten und Monozyten einer Stimulus-spezifischen Regulation durch JAM-A unterliegt. Ferner lassen die Ergebnisse unserer Untersuchungen in eJAM-A-defizienten Tieren darauf schließen, dass endotheliales JAM-A die Transmigration von neutrophilen Granulozyten und Monozyten zwar in der Initialphase entzündlicher Prozesse vermittelt, zu späteren Zeitpunkten jedoch keine Bedeutung mehr zu besitzen scheint. Schließlich deuten unsere Befunde darauf hin, dass leukozytäres JAM-A an den der interstitiellen Leukozytenmigration zugrunde liegenden Mechanismen beteiligt ist. In einer letzten Versuchsreihe wurde die Rolle der Chemokinrezeptoren Ccr1, Ccr2 und Ccr5 für die Rekrutierung von Leukozyten bei Chemokin-induzierter Entzündung und I/R untersucht. Es konnte gezeigt werden, dass diese Chemokinrezeptoren die Extravasation von neutrophilen Granulozyten und Monozyten bei Chemokin-induzierter Entzündung durch Effekte auf Adhärenz und (konsekutive) transendotheliale Migration mediieren und keinen Einfluss auf das interstitielle Migrationsverhalten transmigrierter Leukozyten besitzen. Des Weiteren ist es mittels durchflusszytometrischer Analyse gelungen, die Expression von Ccr2 und Ccr5 auf nativen neutrophilen Granulozyten nachzuweisen. Darüber hinaus konnte erstmals gezeigt werden, dass die Chemokinrezeptoren Ccr1, Ccr2 und Ccr5 zur Rekrutierung von neutrophilen Granulozyten und Monozyten in das postischämische Gewebe durch dynamische bzw. differentielle Regulation von Adhärenz und (konsekutiver) Transmigration beitragen.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 07/19
The global burden of chronic kidney diseases remains an ongoing medical challenge. Therapies that can halt or reverse advanced renal injury are not yet available. Increasing numbers of patients progress to the end-stage renal failure and require renal replacement therapy, the latter being associated with significant mortality, a lower quality of life, and high costs for national health systems. Thus, new treatment strategies that slow down, halt or even revert progressive renal damage are requested. Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocytes and macrophages recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocytes are the major source for proinflammatory and profibrotic cytokines and are therefore critical for mediating fibroblast proliferation, differentiation into myofibroblasts, matrix production, and tubular atrophy. Recent advances in the understanding of the molecular mechanisms that regulate renal leukocyte recruitment suggest chemokines and chemokine receptors as novel targets for specific pharmacological intervention. The aim of the present thesis was to investigate the role of chemokine receptor CCR1 for the progression of chronic kidney diseases, e.g. Alport disease and diabetic nephropathy. Two different animal models were used: Col4A3-deficient mice and type 2 diabetic db/db mice with advanced diabetic nephropathy. We blocked CCR1 in Col4A3-deficient mice with BX417, a small molecule CCR1 antagonist, and BL5923, a novel orally available antagonist with a high specificity for human and murine CCR1 in uninephrectomized type 2 diabetic db/db mice, respectively. Treatment with BX471 (25mg/kg) from weeks 6 to 10 of life improved survival of COL4A3- deficient mice, characterized by glomerulosclerosis and subsequent progressive tubulointerstitial injury, leading to fatal end-stage renal disease (ESRD). Improvement was associated with less interstitial macrophages, apoptotic tubular epithelial cells, tubular atrophy, interstitial fibrosis, and less globally sclerotic glomeruli. BX471 reduced total renal Ccl5 mRNA expression by reducing the number of interstitial CCL5-positive cells in inflammatory cell infiltrates. Intravital microscopy of the cremaster muscle in male mice identified that BX471 or lack of CCR1 impaired leukocyte adhesion to activated vascular endothelium and transendothelial leukocyte migration, whereas leukocyte rolling and interstitial migration were not affected. Furthermore, in activated murine macrophages, BX471 completely blocked CCL3-induced CCL5 production. When CCR1 was blocked with BL5923 (60mg/kg, b.i.d), the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with type 2 diabetes. Similarly, BL5923 orally administered from month 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, Tgf-β1, and collagen I-α1 when compared to untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for CCR1-dependent recruitment of interstitial macrophages for the progression of chronic kidney disease in Alport disease and diabetic nephropathy. These data identify CCR1 as a potential therapeutic target for Alport disease and late stage diabetic nephropathy or other progressive nephropathies associated with interstitial macrophage infiltrates.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 06/19
In der vorliegenden Arbeit wurde bronchoalveolären Lavage Flüssigkeit (BALF) von Patienten mit interstitiellen Lungenerkrankungen (23 Patienten) auf Chemokinkonzentrationen und Chemokinrezeptorexressionsmuster der Lymphozyten analysiert und die Ergebnisse mit Patienten, die an chronischer Bronchitis (6 Patienten) oder malignen Erkrankungen (9 Patienten) der Lunge erkrankt waren verglichen. Mittels ELISA wurden die Chemokinkonzentrationen und mittels Durchflusszytometrie der Anteil an Chemokinrezeptor-exprimierenden T-Zellen in der BALF bestimmt. Hierbei wurden die Chemokinkonzentrationen von MCP 1, TARC, MDC und RANTES und die Häufigkeit CCR2+, CCR5+, CCR4+ und CXCR3+ Zellen innerhalb der CD4+ und CD8+ T-Zellpopulationen gemessen. Es konnte gezeigt werden, dass bei interstitiellen Lungenerkrankungen im Vergleich zu den Kontrollgruppen die MCP-1 Konzentration knapp signifikant (p = 0,055) und die CCR2+CD4+T-Zellen signifikant erhöht waren. Im Zusammenhang mit Daten aus Kinder- und Erwachsenenstudien, in denen in der bronchoalveolären Lavageflüssigkeit erhöhte MCP-1 Werte und vermehrt CCR2+ T-Zellen nachgewiesen wurden, legen diese Ergebnisse eine wichtige Rolle der MCP 1/CCR2-Achse in der Pathogenese der interstitiellen Lungenerkrankungen nahe. Ebenso fanden sich bei interstitiellen Lungenerkrankungen signifikant mehr der TH2-assoziierten CCR4+ T-Zellen; bei dem TH1-assoziierten Rezeptor CXCR3+ ergab sich kein Unterschied. Gemeinsam mit ähnlichen Ergebnissen einiger Studien in Mausmodellen und humanen Studien weisen sie auf eine TH2-Polarisierung der T Zellen bei interstitiellen Lungenerkrankungen hin, welche hierbei einen profibrotischen Effekt haben sollen. Gleichzeitig konnten bei interstitiellen Lungenerkrankungen signifikant mehr CCR5+CD4+ und CCR5+CD8+ Zellen als in den Kontrollgruppen nachgewiesen werden. Da auch bei gesunden Menschen CCR5+ T-Zellen nachgewiesen werden konnten, postulieren wir, dass CCR5+ T-Zellen auch unabhängig von der Polarisierung der T-Zellen ein regulärer Bestandteil des bronchoalveolären Raumes im Rahmen einer normalen Immunreaktion sind. Insgesamt hat diese explorative Analyse aufgezeigt, dass sowohl die MCP 1/CCR2-Achse, als auch TH2-polarisierte T-Zellen ein potentielles Angriffsziel in der Behandlung interstitieller Lungenerkrankungen darstellen könnten. Die Ergebnisse sollten den Anstoß für ausführlichere Untersuchungen mit einem wesentlich größeren Patientenkollektiv geben.
Background: Interstitial lung diseases (ILD) are chronic inflammatory disorders leading to pulmonary fibrosis. Monocyte chemotactic protein 1 (MCP-1) promotes collagen synthesis and deletion of the MCP-1 receptor CCR2 protects from pulmonary fibrosis in ILD mouse models. We hypothesized that pulmonary MCP-1 and CCR2(+) T cells accumulate in pediatric ILD and are related to disease severity. Methods: Bronchoalveolar lavage fluid was obtained from 25 children with ILD and 10 healthy children. Levels of pulmonary MCP-1 and Th1/Th2-associated cytokines were quantified at the protein and the mRNA levels. Pulmonary CCR2(+), CCR4(+), CCR3(+), CCR5(+) and CXCR3(+) T cells were quantified by flow-cytometry. Results: CCR2(+) T cells and MCP-1 levels were significantly elevated in children with ILD and correlated with forced vital capacity, total lung capacity and ILD disease severity scores. Children with lung fibrosis had significantly higher MCP-1 levels and CCR2(+) T cells in bronchoalveolar lavage fluid compared to non-fibrotic children. Conclusion: The results indicate that pulmonary CCR2(+) T cells and MCP-1 contribute to the pathogenesis of pediatric ILD and might provide a novel target for therapeutic strategies.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 03/19
Epidemiologische Daten und Tiermodelle geben Hinweise auf eine verschlechterte Immunabwehr bei Karzinomen des Kopf- und Halsbereiches, wobei die genauen Wirkmechanismen bisher nicht aufgeschlüsselt werden konnten. In Untersuchungen konnte gezeigt werden, dass Karzinome im Kopf- und Halsbereich mit erhöhten Konzentrationen an Prostaglandinen im Serum einhergehen, ein anderer Autor wies eine verminderte Expression des Chemokinrezeptors CCR5 auf Monozyten nach Behandlung mit Prostaglandin nach. In der vorliegenden Arbeit konnte erstmalig aufgezeigt werden, dass es im Rahmen einer Karzinomerkrankung im Hals- und Kopfbereich zu strukturellen und funktionellen Defiziten der Monozytenfunktion kommt. Hierbei finden sich erniedrigte Expressionsraten an CCR5, CCR2 und des Adhäsionsmoleküls CD11b des β-Integrins Mac-1 sowie eine verminderte Adhäsionsfähigkeit der Monozyten an das interzelluläre Adhäsionsmolekül ICAM-1. Weiterhin konnte nachgewiesen werden, dass nach Inkubation der Monozyten in Serum gesunder Spender die verminderte Immunfunktion wieder verbessert wird. Parallel dazu zeigte sich, dass die oben beschriebenen Immundefekte auf Monozyten gesunder Spender durch Inkubation in Serum von Tumorpatienten künstlich herbeigeführt werden können. Dies legt den Schluss nahe, dass im Rahmen einer Karzinomerkrankung Metaboliten im Serum gelöst sind, die eine immunologische Tumorabwehr erschweren. Diese Erkenntnisse sind ein weiterer Schritt zum Verständnis der Tumorimmunologie und könnten dazu hilfreich sein, immunologische Therapieverfahren voranzubringen.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 03/19
Die Infiltration mit leukozytären Zellen spielt eine entscheidende Rolle in der Pathogenese glomerulärer Erkrankungen der Niere und wird durch sequentielle und sich überschneidende Interaktionen verschiedener Signalmoleküle gesteuert. In dieser Arbeit wurde die Adhäsionsmolekül- und Chemokinexpression von Mesangialzellen (MC) und glomerulären Endothelzellen (GEC untersucht) und deren Regulation durch verschiedene NF-kappaB Inhibitoren bestimmt. Die Zytokinstimulation der MC mit TNF-alpha induzierte die Transkription und Proteinexpression der Adhäsionsmoleküle ICAM-1 und VCAM-1 sowie der Chemokine GRO-alpha, IL-8, MCP-1, aber nicht Fractalkine. GRO-alpha und IL-8 wurden auf der MC Oberfläche über Heparanproteoglykane immobilisiert, MCP-1 und auch IL-8 als lösliche Moleküle sezerniert. Fractalkine hingegen war nur geringfügig induzierbar konstitutiv auf der MC Oberfläche exprimiert. Diese Induktion der Adhäsionsmoleküle und Chemokine durch Zytokinstimulation der MC war assoziiert mit einer vermehrten Monozytenadhäsion und Transmigration. Dabei vermittelte ICAM-1 und VCAM-1, der GRO-alpha Rezeptor CXCR2 und in geringerem Maße auch Fractalkine den festen monozytären Arrest, während der MCP-1 Rezeptor CCR2 die transendotheliale Chemotaxis von Monozyten auf aktivierte MC zu steuerte. Die transkriptionelle Hochregulation der untersuchten Moleküle wurde über eine Inhibition der IkappaB-alpha Degradation durch den Proteaseninhibitor TLCK, den Proteosomeninhibitor MG132 und durch die adenovirale IkappaB-alpha Überexpression gehemmt. Dies war assoziiert mit der Inhibition der monozytären Adhäsion auf aktivierten MC sowie der transendothelialen Migration auf stimulierte MC zu. Im Gegensatz zu MC wurde Fractalkine auf mRNA wie Proteinebene in transformierten GEC deutlich induziert, und sowohl CXCR2 also auch CX3CR vermittelten den Monozytenarrest auf aktivierten GEC unter Flussbedingungen. Die Relevanz dieser Ergebnisse konnte in einem akzelerierten Glomerulonephritis-Modell an der Ratte bestätigt werden, in dem die Blockade von CCR2 deutlich, und die Blockade von CXCR2 fast komplett die akute glomeruläre Makrophagenrekrutierung inhibierte. Dies unterstreicht die Bedeutung von CXCR2 für die Makrophageninfiltration in den frühen Phasen der NTN. Zusammenfassend erschließt sich ein mehrstufiges Modell, das die inflammatorische Monozytenrekrutierung durch funktionell spezialisierte Adhäsionsmoleküle und Chemokine zeigt.
To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune ( obstructive nephropathy) and immune-mediated ( lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8(+) cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets.
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